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WO1998055501A1 - Procede ameliore de glycosylation specifique de site - Google Patents

Procede ameliore de glycosylation specifique de site Download PDF

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Publication number
WO1998055501A1
WO1998055501A1 PCT/SE1998/001008 SE9801008W WO9855501A1 WO 1998055501 A1 WO1998055501 A1 WO 1998055501A1 SE 9801008 W SE9801008 W SE 9801008W WO 9855501 A1 WO9855501 A1 WO 9855501A1
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site
polypeptide
group
protein
function
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WO1998055501A9 (fr
Inventor
Lars Baltzer
Per Ahlberg
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A+ Science AB
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A+ Science Invest AB
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Priority to AU80444/98A priority Critical patent/AU8044498A/en
Publication of WO1998055501A1 publication Critical patent/WO1998055501A1/fr
Publication of WO1998055501A9 publication Critical patent/WO1998055501A9/fr
Anticipated expiration legal-status Critical
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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
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
    • C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • the present invention relates to an improved method for site-selective glycosylation of folded polypeptides and proteins.
  • acyl transfer reactions involve the transfer of an acyl group (the residue of an organic acid after removal of the carboxyl hydroxy group) either in- ternally within a chemical species or from one chemical species to another. Examples are amide formation, trans- esterification and hydrolysis.
  • acyl transfer reactions may be catalyzed by imidazole in aqueous solution, the imid- azole, which is a strong nucleophile, forming an intermediary reactive complex with the acyl group.
  • polymer-supported imidazoles have been used as acyl transfer catalysts (see e.g. Skjujins, A., et al., Latv. PSR Zinat. Akad. Vestis, Kim. Ser. 1988 (6), 720-5). It has further been shown that small peptides containing a histidine (His) residue (an amino acid which contains an imidazolyl group) may have hydrolytic activity.
  • the above described imidazole induced catalytic activity in acyl transfer reactions may be increased considerably if the imidazolyl moiety is provided in a chemical structure flanked on one or both sides by a group of such a nature and position that it is capable of stabilizing the transition complex formed between the imidazolyl group and the acyl group in question.
  • the flanking group or groups should be capable of molecular interaction with the acyl complex, such as by hydrogen bonding, electrostatic or hydrophobic interactions or van der Waal forces (intramolecular polarization) .
  • the increased catalytic activity may be used in combination with inter olecular as well as intramolecular reactions in solution, with and without stereospeciflty . In the latter case it is possible to make site selective functionalization of peptides and other molecules. Such site selective functionalization will inter alia permit site selective immobilization of molecules, such as bio- molecules, e.g. antibodies or other proteins or polypep- tides .
  • One of the objects of the above mentioned patent ap- plication is to provide an improved method of performing an acyl transfer type reaction using an imidazole based catalyst.
  • an improved method of performing a chemical reaction involving an acyl transfer mechanism in the presence of an imidazole-based catalyst which can form a transition complex with the acyl group is characterized in that the imidazole function is provided by a chemical structure element comprising an imidazolyl group flanked on one or both sides by a group capable of stabilizing the transi- tion complex by molecular interaction with the acyl group. This molecular interaction may be selected from hydrogen bonding, electrostatic interaction and hydropho- bic interaction.
  • the chemical structure element constitutes or is part of a larger structure having a functional group in such a neighboring position that it can be site-specifically functionalized through the acyl transfer via the above intermediary complex.
  • Another object of the above mentioned application is to provide a chemical structure element with improved capability of catalyzing an acyl transfer reaction.
  • a chemical structure element comprising backbone structure with a pendant imidazole function, which element is characterized m that the lmida- zole function s flanked on one or both sides on said backbone structure by a pendant group capable of stabilizing the transition complex by molecular interaction with the acyl group.
  • the structure element is a mole- cule, such as a peptide or protein, comprising a function in such a neighboring position that it can be site- specifically functionalized through the acyl transfer via the above intermediary complex.
  • Yet another object of the above mentioned applica- tion is to provide a method of producing by genetic engineering a protein or peptide constituting or comprising a structure element having an imidazole function flanked on one or both sides by a transition complex stabilizing group. It therefore provides a method of producing a pro- tern or peptide which constitutes or comprises an imidazole function-containing structure element as defined above, which method comprises transforming a host organism with a recombmant DNA construct comprising a vector and a DNA sequence encoding said protein or peptide, cul- turing the host organism to express said protein or peptide, and isolating the latter from the culture.
  • the structure element comprises a functional group in a such a neighboring position to the imidazole function that the function can be site-specifically functionalized through acyl transfer catalyzed by the imidazole function.
  • Still another object of the above mentioned application is to provide a vector comprising a nucleic acid sequence encoding the above protein or peptide.
  • the application therefore provides a recombinant DNA construct comprising a vector and a DNA sequence encoding a protein or peptide which constitutes or comprises an imidazole function-containing structure element as defined above.
  • the DNA sequence also encodes a specific functional group in a such a neighboring position to the imidazole function that the functional group can be site-specifically functionalized through acyl transfer catalyzed by the imidazole function.
  • the above mentioned application is based on the concept of increasing the imidazole type catalytic activity in acyl transfer reactions by providing the imi ⁇ dazole function on a backbone structure with a pendant flanking group or chain on one or both sides of the imidazole function, which flanking group or groups can mter- act with the lmidazole-acyl complex formed such that the transition complex is stabilized.
  • the reaction rate for the desired acyl transfer reaction such as an amidation, trans-este ⁇ fication, hydrolysis or thiolysis, will be increased considerably thereby.
  • esters are the cur- rently preferred substrates, e.g. amide and anhydride substrates may also be contemplated.
  • imidazole function is to be interpreted broadly, and is meant to encompass any lmidazole-based Structure that possesses the desired catalytic activity.
  • the imidazole group may consequently be modified in vari ⁇ ous ways.
  • An advantageous imidazole function for manv purposes is based on the amino acid histidine ( ⁇ -am ⁇ no-4- (or 5) -lmidazolepropionic acid).
  • One or both of the available carbon atoms of the imidazole function may, for example, be independently substituted with alkyl or halogen.
  • the imidazole group may also be substituted in 1- position with alkyl.
  • Alkyl has preferably 1 to 6 carbon atoms, especially 1 to 4 carbon atoms, e.g. methyl or ethyl.
  • Halogen includes fluorine, chlorine, bromine and iodine .
  • the flanking group or groups may comprise a link or chain of, e.g., 1 to 6, preferably 1 to 4 atoms, usually carbon atoms, connected to a terminal functional group or other group capable of the required molecular interaction with the acyl transition complex.
  • the flanking chain or chains may be pendant proton donating parts of other ammo acids, e.g. selected from lysmes, ornithmes, argmines and/or further histid es.
  • the chemical structure element supporting the cata- lytic imidazolyl function should preferably have some type of rigidity, such as secondary structure, m order to localize the flanking group or groups with respect to the imidazolyl function in an optimal geometric relationship for the desired transition complex-stabilizing m- teractions to take place.
  • the chemical structure element is a so-called designed polypeptide with a stabilized secondary structure, e.g. ⁇ -helical coiled coils.
  • Designed helical peptides are, for instance, described in J. W. Bryson et al., Science, 270, 935 (1995).
  • the structure element is, however, not limited to a peptide. On the contrary, it may have any of a variety of compositions readily apparent to the skilled person in the light of the present invention, and may thus be included in or be part of other types of struc- tures, such as a carbohydrate, a natural or synthetic polymer, etc.
  • the size of the chemical structure is not either limiting, and it may, e.g., be a peptide of as few as, say, five amino acids.
  • a functional arrangement may readily be designed for each particular situation by the skilled person after having read the present description.
  • the transition complex may react with such a flanking chain in an intramolecular reaction.
  • an intramolecular reaction may be used for selectively functionalizing peptides, proteins and other molecules.
  • An example of such an intramolecular reaction is outlined in the reaction scheme below.
  • the imidazolyl structure is part of a histidine (His) residue and the aminoprcpyl chain is part of an crnithine (Orn) residue, both included in a designed ⁇ -helical polypeptide at a distance of four carbon atoms from each ether (i.e. at positions "i” (His) and "i+4" (Orn) ) , the His and Orn residues thereby being located on the same side of the helix (4 carbon atoms in each coil) .
  • "I” represents an active ester, here specifically mono-p-nitrophenylfumarate.
  • RA-42 an example of such an ⁇ -helical polypeptide is RA-42, the supersecondary structure of which is schematically shown in Fig. 1A.
  • RA-42 has 42 amino acids, and His-15, Orn-15 and Orn-34 residues.
  • the reaction starts with an initial attack of the imidazole residue of His on the active ester to form an acyl intermediate with release of p-nitrophenol.
  • the acyl intermediate is stabilized by the ornithine side chain which may flex towards the acyl complex to interact therewith through hydrogen bonding between the protonated amino group and the developing oxyanion of the acyl group.
  • the acyl group is then transferred from the his- tidine residue to the ornithine residue, free histidine being regenerated.
  • Exemplary groups for i+4 acylation are, in addition to ornithine mentioned above, lysine and 1,3-diamino- butyric acid, while i-3 acylation may be exemplified by lysine .
  • functional groups both at position i+4 and position i-3, and the functional groups in this case are e.g. lysines. It is also possible to use structural elements with more than one imidazolyl function in positions i, j , k etc., and these functions may then be flanked on one side or on both sides by functional groups, preferably at positions i+4, j+4, k+4 etc., and i-3, j-3, k-3 etc., re- spectively.
  • acyl transfer reactions using e.g. functionalized helix-loop-helix motifs designed from simple principles of transition state binding, the favorable complex stabilization being obtained by the introduction of e.g. positively charged hydrogen bond donors that interact with negatively charged substrates in a predictable way.
  • the main binding interaction in the transition state is that to the developing oxyanion of the ester functional group. That is shown by the fact that p-nitrophenylacetate, that has no negatively charged functional group, is catalyze ⁇ with almost the same efficiency as mono-p-nitrophenyl- fumarate.
  • polypeptides embodying the present invention may be produced by recombinant DNA technology (genetic engineering) . Such techniques are well known and to the skilled person and will not be described herein. (It may, for example, be referred to EP-Bl-282 042 which discloses the preparation by recombinant technology of fusion proteins which contain neighboring His-residues . )
  • the above described selectivity of the reaction center may be used to introduce new functionality in e.g. folded polypeptides .
  • the stabilizing flanking group (s) need, of course, not be the one to be functionalized through the acyl transfer but may be another functional group in an appropriate position.
  • site-selective immobilization is schematically illustrated below where a designed polypeptide of the above-mentioned helix-loop-helix type, which has a catalytic histidine residue in a stabilizing relationship with a flanking aminoalkyl chain, is immobilized via the amino function to an ester function (R ] _OCO) of a solid support.
  • the reaction is carried out at such pH conditions that all amino functions are almost completely protonated and thereby unavailable for direct reaction with the ester function.
  • Immobilization to a solid support may, of course, be effected the other way round, i.e. by providing the histidine residue and the amino function on the solid support and the ester function on the peptide.
  • the reaction may also be used to introduce residues that will not survive under the reaction conditions of peptide synthesis or that will not be reactive enough due to steric hindrance. Novel branched polypeptide structures are also possible if amino acid residues or peptides can be introduced. Since the histidine is regener- ated, it can also be designed to participate in the active site of an engineered catalyst.
  • the present invention is based on the same principal as the above mentioned application, but it is related to a considerable improvement of site-selective glycosyla- tion of folded polypeptides and proteins.
  • the pres- ent invention is related to a method for site-selective glycosylation of folded polypeptides and proteins comprising an imidazole function in position i flanked by functional groups in positions i+4 and/or 1-3, wherein a carbohydrate residue is incorporated into the polypeptide or protein by reacting a p-nitrophenyl ester of the carbohydrate with the polypeptide or the protein, wherein said carbohydrate residue lacks protecting groups.
  • Fig. 1 is a schematic illustration of a helix-loop- helix structure of a designed polypeptide, namely RA-42, with its designed reaction center indicated.
  • site- selective incorporation of carbohydrates into folded proteins is accomplished by reacting a p-nitrophenyl ester of the non-protected carbohydrate to be inserted with a folded polypeptid protein.
  • non-protected carbohydrate used here, as well as in the description below and in the appended claims, refers to a carbohydrate lacking protective groups.
  • the present invention relates to a method for site-selective glycosylation of folded polypeptides and proteins comprising an imidazole function, preferably a histidine, in position i flanked by functional groups in positions i+4 and/or 1-3.
  • an imidazole function preferably a histidine
  • a carbohydrate is incorporated into the polypeptide or protein by reacting a p-nitrophenyl ester of the nonprotected carbohydrate with the polypeptide or the protein.
  • the polypeptides or proteins which may be glycosylated according to the present invention comprises an imidazole function, preferably histidine, flanked by functional groups.
  • the position of the imidazole function is designated as i, and the functional groups are positioned three carbon atoms upstream the histidine, i.e. in position i-3, and/or four carbon atoms downstream the histidine, i.e. in position i+4.
  • the flanking group or groups may comprise a link or chain of, e.g., 1 to 6, preferably 1 to 4 atoms, usually carbon atoms, connected to a terminal functional group or other group capable of the required molecular interaction with the acyl transition complex.
  • the flanking chain or chains may be pendant proton donating parts of other amino acids, e.g. selected from lysines, ornithines, arginines and/or further histidines.
  • the method according to the invention can be used to develop vaccines, to produce antibodies, to mimic components of the immune system, and to construct antagonists and agonists for components of the immune system.
  • the method of the invention can also be used site- selective immobilizations and in the construction of functionalized polypeptides, such as novel catalysts, introduction of co-factors etc.
  • peptide libraries with peptides having a secondary or tertiary structure for specific binding of e.g. a substrate or a receptor; construction of polypeptides for specific non-covalent binding of endogenous substances in the blood circulation; in allergy diagnoses (and clinics) as well as immunology; construction of molecules having topologies for antibody production; and vaccine production.
  • polypeptide RA-42 The amino acid sequence of polypeptide RA-42 is shown in the Sequence Listing provided at the end of the description. The residues presented underlined in bold are the ones designed to constitute the catalytical binding site.
  • Aib is ⁇ -aminoisobutyric acid and Nle is norleucin.
  • the polypeptide is a helix-loop-helix motif. In solution the peptides dimerize to form four-helix bundles, but for simplicity only the monomer is shown.
  • LA-42 used in the example below, is identical to RA-42 except that the Orn-15 is replaced by Lys-15.
  • polypeptide used in the example below was synthesized on an automated peptide synthesizer (Biosearch 9600), using t-BOC protection groups and phenylacetami- domethyl (PAM) linked resins. It was cleaved from the resins by anhydrous HF on a Teflon vacuumline (Peptide
  • the starting p-nitrophenyl ester was that of tetra-O-acetyl-1- (2-carboxyethyl-l-thio) - ⁇ -D- galactopyranose. Since this ester contains protective groups, the first step was removal of these groups.
  • the lyophilized reaction mixture was redissolved in water and extracted with 3 * 3 ml of dichloromethane to remove free p-nitrophenol and was then purified by re- versed-phase HPLC using a 5 ⁇ m Shandon C8 column with 25% acetonitrile in water at pH 4 as the eluent and a flow rate of 1 ml/mm.
  • the chromatogram was recorded by moni- tormg the absorbance at 250 nm.
  • One fraction with a retention time of 5.5 minutes was shown by 1 H NMR-analysis to contain the product ester.
  • This product ester was then reacted with the polypeptide LA-42, containing a reactive site with a his- tidme in position 11 and a lysme in position 15. This reaction was shown, by electrospray mass spectrometry, to lead to the incorporation of 1- (2-carboxyethyl-l-thio) - ⁇ - D-galactopyranose into the folded polypeptide.
  • G-P-V-D 20 23 G-Aib-R-A-F-A-E-F-Qm-K-A-L-Q-E-A-Nle-Q-A-Aib ' 2 24

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  • Proteomics, Peptides & Aminoacids (AREA)
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Abstract

L'invention concerne un procédé amélioré permettant la glycosylation spécifique de site de polypeptides et de protéines pliés ayant une fonction imidazole, de préférence histidine, en position i flanquée d'au moins un groupe fonctionnel, tel que la lysine, l'ornithine et/ou un résidu d'acide diaminobutyrique aux positions i+4 et/ou i-3. Selon ce procédé, un résidu glucidique est incorporé au polypeptide ou à la protéine par réaction d'un ester de p-nitrophényle d'un glucide non protégé avec le polypeptide ou la protéine.
PCT/SE1998/001008 1997-06-06 1998-05-28 Procede ameliore de glycosylation specifique de site Ceased WO1998055501A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU80444/98A AU8044498A (en) 1997-06-06 1998-05-28 Improved method for site-selective glycosylation

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
SE9702188A SE9702188D0 (sv) 1997-06-06 1997-06-06 Improved method for site-selective glycosylation
SE9702188-5 1997-06-06

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WO1998055501A1 true WO1998055501A1 (fr) 1998-12-10
WO1998055501A9 WO1998055501A9 (fr) 1999-07-15

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001085756A3 (fr) * 2000-05-05 2002-03-14 A & Science Invest Ab Transfert d'acyle a selection de site
WO2003044042A1 (fr) * 2001-11-21 2003-05-30 Modpro Ab Acylation selective de site

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997043302A1 (fr) * 1996-05-14 1997-11-20 A+Science Invest Ab Transfert d'acyle avec complexe de transition stabilise utilisant un catalyseur a fonction imidazole catalytique (par exemple l'histidine)

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997043302A1 (fr) * 1996-05-14 1997-11-20 A+Science Invest Ab Transfert d'acyle avec complexe de transition stabilise utilisant un catalyseur a fonction imidazole catalytique (par exemple l'histidine)

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BIOCHEMISTRY, Volume 26, 1987, THOR J. BORGFORD et al., "Site-Directed Mutagenesis Reveals Transition-State Stabilization as a General Catalytic Mechanism for Aminoacyl-tRNA Synthetases", pages 7246-7250. *
THE JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 252, No. 5, March 1977, COLIN D. HUBBARD et al., "Mechanisms of Acylation of Chymotrypsin by Phenyl Esters of Benzoic Acid and Acetic Acid", pages 1633-1638. *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001085756A3 (fr) * 2000-05-05 2002-03-14 A & Science Invest Ab Transfert d'acyle a selection de site
WO2001085906A3 (fr) * 2000-05-05 2002-10-03 A & Science Invest Ab Peptides d'action catalytique
JP2003532738A (ja) * 2000-05-05 2003-11-05 モドプロ アーベー 部位選択的アシル基転移
US7230072B2 (en) 2000-05-05 2007-06-12 Modpro Ab Site-selective acyl transfer
US7364889B2 (en) 2000-05-05 2008-04-29 Modpro Ab Catalytically active peptides
WO2003044042A1 (fr) * 2001-11-21 2003-05-30 Modpro Ab Acylation selective de site
US7514222B2 (en) 2001-11-21 2009-04-07 Modpro Ab Site selective acylation

Also Published As

Publication number Publication date
SE9702188D0 (sv) 1997-06-06
AU8044498A (en) 1998-12-21
WO1998055501A9 (fr) 1999-07-15

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