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US20040053241A1 - Catalytically active peptides - Google Patents

Catalytically active peptides Download PDF

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US20040053241A1
US20040053241A1 US10/258,286 US25828603A US2004053241A1 US 20040053241 A1 US20040053241 A1 US 20040053241A1 US 25828603 A US25828603 A US 25828603A US 2004053241 A1 US2004053241 A1 US 2004053241A1
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peptide
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reaction
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Lars Baltzer
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ModPro AB
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ModPro AB
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Publication of US20040053241A1 publication Critical patent/US20040053241A1/en
Priority to US11/396,556 priority Critical patent/US7364889B2/en
Priority to US12/015,161 priority patent/US20090182119A9/en
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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
    • C07K1/113—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 without change of the primary structure
    • C07K1/1136—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 without change of the primary structure by reversible modification of the secondary, tertiary or quarternary structure, e.g. using denaturating or stabilising agents
    • 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
    • C07K1/006—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length of peptides containing derivatised side chain amino 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
    • C07K1/107—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
    • 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

Definitions

  • the present invention relates to novel peptides that can catalyze site-selective acyl transfer.
  • acyl transfer reactions involve the transfer of an acyl group (the residue of an organic acid after removal of the carboxyl hydroxy group) either internally within a chemical species or from one chemical species to another. Examples are amide formation, transesterification and hydrolysis.
  • acyl transfer reactions can be catalyzed by imidazole in aqueous solution, the imidazole, 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).
  • the invention thus relates to a catalytically active peptide comprising an imidazole function in position i flanked by at least one functional group to be amidated in position i+3+4k, where k is an integer equal to or higher than ⁇ 1 or in position i ⁇ 4 ⁇ 4n, wherein n is an integer equal to or higher than 0, characterized in that it also comprises at least one activating group in position i+4+4n or i ⁇ 3 ⁇ 4n, respectively, wherein n is as above.
  • the reaction between a His residue and an ester is a two-step reaction where the first and rate-limiting step is the formation of an acyl intermediate under the release of the leaving group. In the second step the acyl intermediate reacts with the most potent nucleophile available to it.
  • the reaction product is the carboxylic acid and the reaction is hydrolysis. In the presence of 10 vol % trifluoroethanol the reaction product is the corresponding trifluoroethyl ester 4 .
  • a catalytically active peptide By using a catalytically active peptide according to the present invention it is possible to site-selectively form amide bonds between lysine side chains and acyl groups of active esters. Anything that can be transformed into an active ester will thus be transferable to a specific position on the surface of a protein or peptide comprising a structure according to the invention.
  • Peptides, proteins, PNAs, carbohydrate derivatives, drugs, inhibitors are examples of such transferable substances.
  • lysine residues of a protein or peptide with a structure according to the invention can be acylated, one after the other, in a controlled way so that complex sites, or epitopes, can be designed that are formed from several different ligands, and the concept can also be exploited in a combinatorial approach to form a large number of different binding sites.
  • the binding of a protein or peptide with a structure according to the invention to a surface by a covalent bond can be accomplished in a controlled way using the same chemical reaction that is used to bind the different ligands.
  • the bond forming reaction catalyzed by a catalytically active peptide according to the invention can be performed in an aqueous solution by adding the active ester to a solution of a protein or peptide with a structure according to the invention, preferably at pH 6 and room temperature. After the reaction is complete, any excess ester and the leaving group are washed away, and a new ester can be added.
  • the formation of e.g. a complex protein receptor or a binding site can thus be accomplished using very simple step-wise chemistry, and it does not depend on the use of expensive or hazardous coupling agents because the reactivity and site selectivity is encoded into the protein or peptide with a structure according to the invention.
  • the site selective incorporation of substituents requires that optimized concentrations of active esters are used in the reaction. If a peptide is subjected to prolonged treatment with a large excess of ester then the selectivity will decrease, but if the peptide is subjected to an optimized concentration of ester then the intramolecular competition will ensure that the optimum yield of selectively functionalized peptide is obtained.
  • the optimum concentrations and reaction times differs between different lysine side chains because the different geometrical relationship between His and Lys residues gives rise to acyl transfer reactions that are not equal.
  • the experimental conditions described for the present invention have been standardized for comparison between different sites and can be further optimized.
  • the introduced substituents are also known to influence the incorporation at a neighboring site because of its size and other molecular properties, such as for example charge and polarity.
  • the protein that has been bound to a surface by a covalent bond is a homodimer then only one of the peptides is covalently linked, and the other peptide can be replaced to form a heterodimer.
  • the functionalized protein is readily removed from the surface and replaced with a second one in a very short period of time (a few seconds or minutes), so that a new reaction can be performed.
  • targets are proteins and other bio-molecules that are extracted from, e.g., cell lysates. Combinatorially composed surfaces can then be used in the search of the “unknown proteins” that are not readily predictable form the genes in the cell, perhaps because they have been posttranslationally modified, so that their function is not predictable from the DNA sequence.
  • An added value is the capability of constructing tailor-made affinity columns form purification and isolation.
  • the receptor or binding site that were used for example to find a specific protein from a cell lysate can be constructed in large enough quantities to be used for its purification and isolation.
  • One object of the present invention is to provide a chemical structure element with improved capability of catalyzing an acyl transfer reaction. There is therefore provided a chemical structure element comprising backbone structure with a pendant imidazole function.
  • the structure element is a molecule, 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 complexes.
  • the catalytically active peptides according to the present invention is suitable for use in an improved method of performing an acyl transfer type reaction using an imidazole based catalyst. There is therefore provided 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.
  • 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 complexes.
  • the catalytically active peptides according to the present invention can be obtained by a method comprising transforming a host organism with a recombinant DNA construct comprising a vector and a DNA sequence encoding said protein or peptide, culturing the host organism to express said protein or peptide, and isolating the latter from the culture.
  • Another object of the present invention is to provide a vector comprising a nucleic acid sequence encoding the above protein or peptide.
  • the invention 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 present invention is based on peptides that increases the imidazole type catalytic activity in acyl transfer reactions by providing the imidazole function on a backbone structure with a pendant flanking group (or groups) or chain on one or both sides of the imidazole function, which flanking group or groups can interact with the imidazole-acyl complex formed such that the transition complex is stabilized.
  • the reaction rate for the desired acyl transfer reaction such as an amidation, trans-esterification, hydrolysis or thiolysis, will be increased considerably thereby. While esters are the currently preferred substrates, e.g. amide and anhydride substrates can also be used.
  • imidazole function is to be interpreted broadly, and is meant to encompass any imidazole-based structure that possesses the desired catalytic activity.
  • the imidazole group can consequently be modified in various ways.
  • An advantageous imidazole function for many purposes is based on the amino acid histidine ( ⁇ -amino-4-(or 5)-imidazolepropionic acid).
  • One or both of the available carbon atoms of the imidazole function can, for example, be independently substituted with alkyl or halogen.
  • the imidazole group can 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.
  • flanking group or groups can 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 can be pendant proton donating parts of other amino acids, e.g. selected from lysines, ornithines, arginines and/or further histidines.
  • the chemical structure element supporting the catalytic imidazolyl function should preferably have some type of rigidity, such as secondary structure, in 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 interactions 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.
  • compositions can have a variety of compositions readily apparent to the skilled person in the light of the present invention, and can thus be included in or be part of other types of structures, such as a carbohydrate, a natural or synthetic polymer, etc.
  • the size of the chemical structure is not either limiting, and it can, e.g., be a peptide of as few as, say, five amino acids.
  • a functional arrangement can readily be designed for each particular situation by the skilled person after having read the present description.
  • the transition complex can react with such a flanking chain in an intra-molecular reaction.
  • an intramolecular reaction can be used for selectively functionalizing peptides, proteins and other molecules.
  • polypeptides embodying the present invention can 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 can, for example, be referred to EP-B1-282 042 which discloses the preparation by recombinant technology of fusion proteins which contain neighboring His-residues.)
  • the above described selectivity of the reaction center can 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 can be another functional group in an appropriate position.
  • An important aspect of site-selective functionalization is the introduction of carbohydrates site-selectively into proteins and peptides. This is accomplished by modifying the carbohydrate in question to contain an ester function. Carbohydrates play an important role in the recognition in immunological, inflammatory and other processes. They can enhance the immunogenicity of proteins and peptides. They also protect proteins from proteolytic degradation and affect protein folding. Site-selective introduction of carbohydrates can therefore be used for antibody production and vaccine development and the systematic study of the role of carbohydrates. It can also be used to protect drugs from degradation.
  • the reaction can 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 regenerated, it can also be designed to participate in the active site of an engineered catalyst.
  • the peptide to be functionalized is dissolved in a buffer solution at pH 5.85 and the first ester is added at a concentration that has been estimated from the comparison between the pseudo first-order rate constant of the peptide catalyzed reaction (calculated from the second-order rate constant and the peptide concentration) and the background reaction.
  • the second-order rate constant is 0.039 M ⁇ 1 s ⁇ 1
  • the pseudo first-order rate constant is 3.9 ⁇ 10 ⁇ 5 s ⁇ 1
  • a factor of 3.9 larger than that of the background reaction.
  • a substrate ester is insoluble in water, it can be introduced into the sequence during the solid phase peptide synthesis using orthogonal protection group strategies.
  • an allyl protection group can be used for a lysine residue which can be selectively removed before the peptide is cleaved from the resin, and the lysine can be reacted with the hydrophobic substituent using standard carbodiimide coupling reagents.
  • 2,4-dinitrophenyl esters will, for example be incorporated very efficiently at pH 4-5 due to their inherent high reactivity. This will allow the introduction of several esters simultaneously into the reaction vessel, and the reaction is then controlled by a change of pH. For example, a mixture of three esters are added to a peptide, which is held at pH 4. One ester is a 2,4-dinitrophenyl ester, which reacts readily at pH 4 and is thus incorporated at the most reactive Lys. Then the pH is raised to pH 5 and the N-hydroxisuccinimide ester reacts and is incorporated into the second most reactive Lys site. Finally the pH is raised to 6 and the p-nitrophenyl ester is incorporated at the third most reactive site.
  • the incorporation is performed at the surface of a solid support, e.g. a gold plate in a Biacore instrument. Then the first reaction is used to form a bond between the active ester attached to the surface using e.g. thiol derivatives that are well known to bind to gold, and the most reactive Lys. Then an ester substrate is introduced into the reaction chamber and reacted to form an amide at the second most reactive site, and so on.
  • a solid support e.g. a gold plate in a Biacore instrument.
  • the first reaction is used to form a bond between the active ester attached to the surface using e.g. thiol derivatives that are well known to bind to gold, and the most reactive Lys.
  • an ester substrate is introduced into the reaction chamber and reacted to form an amide at the second most reactive site, and so on.
  • FIG. 1 is a modeled structure of a monomeric hairpin helix-loop-helix motif showing the positions of the residues that form the catalytic network; the sequences are those of the peptides described below; the arrows indicate the acyl migration pathways from His-11 to Lys-7, Lys-10, Lys-14 and to Lys-34.
  • a model protein the 84-residue helix-loop-helix homodimer described and characterized in detail elsewhere 6,7 , FIG. 1, was redesigned to have Ser residues I ⁇ 3, i+4 and I+8 relative to a His(i), and with Lys residues in positions that could NOT be acylated directly by the acylimidazole intermediate on the His side chain.
  • the resulting 42-residue peptides given in table II below, were synthesized on a PerSeptive Biosystems Pioneer automated peptide synthesizer using a Fmoc-PAL-PEG-PS polymer, standard commercial protocols and Fmoc protection group chemistry, purified using reversed phase HPLC on a semipreparative Kromasil column using 36-38% isopropanol in 0.1% trifluoroacetic acid and identified using electrospray mass spectrometry (ESMS). The mean residue ellipticities at 222 nm and 300 ⁇ M concentration were measured and typical values were ⁇ 19000 deg cm 2 dmol ⁇ 1 . The geometric relationship between the amino acids is shown in FIG. 1.
  • the proteins at 1 mM concentration in aqueous solution and pH 5.1 were reacted with an excess, typically 40-60%, of mono-p-nitrophenyl fumarate (I).
  • the excess amounts of substrate were needed since background hydrolysis wastes some of the ester, and the excess amount was estimated from the relative magnitudes of the second-order rate constants 8 , Table 1.
  • the resulting proteins were analyzed by HPLC, using analytical columns, and by ESMS (MW of the monomeric peptide S-I for example is 4333, found 4333, and that of the corresponding monofumarylated peptide is 4431, found 4431), with and without prior tryptic cleavage to identify the site of amidation as described earlier 2 .
  • Lys (i+4) is exclusively amidated under the reaction conditions used here. If a His is flanked by a lysine residue in a neighboring helix in such a conformation that the Lys has a similar geometric relationship to the His, as has a Lys(i+4) or Lys(i ⁇ 3) to a His(i), then this Lys will be amidated by the His in a direct acylation reaction.
  • S-I SEQ. ID. No. 2
  • S-II SEQ. ID. No. 3
  • S-III SEQ. ID. No. 4
  • S-IV SEQ. ID. No. 7
  • Orn-34 occupies such a position and in the absence of a Lys in position 15 Orn-34 will be preferentially amidated by His-11.
  • FIG. 1 When His-11 was flanked by Ser-15 As in S-I (SEQ. ID. No. 2), FIG. 1, the degree of acylation of Orn-34 was enhanced considerably in comparison with that obtained with the sequence S-II (SEQ. ID. No. 3), where the Ser was in position 8, showing that acylation is mediated by Ser-15. In these peptides no other Lys residues WERE in positions to accept acyl groups by direct transfer from His-11, and no amidations of residues other than Orn-34 were observed. When His-11 was flanked by Lys-14, in the peptide S-IV, FIG.
  • Ser-15 can simply hydrogen bond to the developing oxyanion in the transition state of the amidation reaction or accept a hydrogen bond from Lys-14 and increase the population of conformers where Lys-14 is in a reactive conformation.
  • the low binding energy of hydrogen bonds involving uncharged species in aqueous solution 9 makes both of these alternative explanations less likely.
  • flanking residue of a His(i) is a serine (i+4) then the acyl group is captured by the hydroxyl group to form an ester, but as the ester is not the thermodynamically most stable species the acyl group migrates further to finally “park” at the side chain of a lysine (i+3 relative to the His, i ⁇ 1 relative to the Ser) or it is hydrolyzed if no Lys is available for acylation.
  • Orn-34 is preferentially amidated by His-11 in S-I, S-II, S-III and S-IV so Orn-34 was replaced by Ala in the sequences S-IIIb and S-IIIc.
  • S-IIIb contains Ser-15 whereas in S-IIIc Ser-15 has been replaced by Ala-15.
  • Lys-10 and Lys-14 are amidated, whereas in S-IIIc amidation of Lys-10 and Lys-14 is not detected.
  • Ser-15 was supplemented by a Ser-19, i.e. i+8 relative to the histidine, while Lys-14 was replaced by a glutamate, to form the peptide S-VIII, FIG. 1. Since Ser-15 can function as an acylating agent for Lys-14, i.e. in an i, i ⁇ 1 pathway as described above, but probably not in an i, i+3 pathway, acyl migration from Ser-15 to Ser-19 is necessary in order to accomplish amidation via Ser-19.
  • acylation of Lys-10 is observed when Ser-19 is incorporated (S-VIII) but abolished when Ser-19 is replaced by Ala (S-IX). Consequently, acyl groups can migrate from serine to serine in helical segments in an i, i+4 pathway. Acyl groups can therefore migrate long distances over protein surfaces or in protein cavities provided that the appropriate groups are organized in a way to accommodate the structural requirements for intramolecular transesterification.
  • the reaction suggests that acyl intermediates in proteolytic cleavage of peptides and esters can escape nucleophiles by fast migration to other sites where the hydrolysis reaction is the most efficient. It also suggests that this reaction can be used to posttranslationally modify proteins and that perhaps phosphoryl groups can be transferred over protein surfaces to the final site of protein phosphorylation.
  • the rate of transacylation cannot be measured as it is not rate limiting, but the degree of amidation of each available side chain is clearly dependent on the relative magnitude of its rate of acylation suggesting that different side chains can be amidated in a stepwise fashion determined by the relative geometries, distances, pKa values etc of each-site.
  • the reactivity of the protein site is controlled by the pKa values of the His residues 10 and by the reactivity of the leaving group 11 but the partitioning of acyl groups between different sites is determined by the structure of the protein. Hydrolysis also competes with intramolecular acyl migration and a fraction of the reactants is clearly lost, although a very small one.
  • the discovered reaction provides the opportunity to use a simple one-step reaction in aqueous solution to form new proteins with tailor made properties.
  • the introduction of several ligands in a controlled way that recognize and bind known or unknown proteins can prove to be an important one in the upcoming era of proteomics.

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US12/015,161 US20090182119A9 (en) 2000-05-05 2008-01-16 Catalytically active peptides

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SE0001698A SE0001698D0 (sv) 2000-05-05 2000-05-05 Site-selective acyl transfer
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PCT/SE2001/000988 WO2001085906A2 (fr) 2000-05-05 2001-05-07 Peptides d'action catalytique

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Cited By (1)

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Publication number Priority date Publication date Assignee Title
US20060234291A1 (en) * 2001-11-21 2006-10-19 Lars Baltzer Site selective acylation

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SE0200968D0 (sv) * 2002-03-26 2002-03-26 Lars Baltzer Novel polypeptide scaffolds and use thereof
CN104163768B (zh) * 2013-05-17 2016-08-31 中国科学院理化技术研究所 可见光催化酰基迁移制备苯胺衍生物的方法

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US6171819B1 (en) * 1996-05-14 2001-01-09 A + Science Invest Ab Acyl transfer with stabilized transition complex using catalyst with catalytic imidazole (e.g. histidine) function

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SE9702188D0 (sv) * 1997-06-06 1997-06-06 Holdingbolaget Vid Goeteborgs Improved method for site-selective glycosylation
US6204041B1 (en) * 1997-09-09 2001-03-20 The Board Of Trustees Of The University Of Illinois Deregulation of glutamine PRPP amidotransferase activity
SE9804137D0 (sv) * 1998-11-30 1998-11-30 A & Science Invest Ab New peptides

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US6171819B1 (en) * 1996-05-14 2001-01-09 A + Science Invest Ab Acyl transfer with stabilized transition complex using catalyst with catalytic imidazole (e.g. histidine) function

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060234291A1 (en) * 2001-11-21 2006-10-19 Lars Baltzer Site selective acylation
US7514222B2 (en) * 2001-11-21 2009-04-07 Modpro Ab Site selective acylation

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AU2001258963A1 (en) 2001-11-20
SE0001698D0 (sv) 2000-05-05
JP2004503470A (ja) 2004-02-05
WO2001085756A3 (fr) 2002-03-14
US20080177032A1 (en) 2008-07-24
US20090182119A9 (en) 2009-07-16
US7364889B2 (en) 2008-04-29
EP1283873A2 (fr) 2003-02-19
US20040161815A1 (en) 2004-08-19
US7230072B2 (en) 2007-06-12
EP1283872B8 (fr) 2009-12-16
DE60140117D1 (de) 2009-11-19
ATE445008T1 (de) 2009-10-15
EP1283872A2 (fr) 2003-02-19
WO2001085756A2 (fr) 2001-11-15
JP2003532738A (ja) 2003-11-05
WO2001085906A2 (fr) 2001-11-15
EP1283872B1 (fr) 2009-10-07
US20060165712A1 (en) 2006-07-27
WO2001085906A3 (fr) 2002-10-03
AU2001256910A1 (en) 2001-11-20

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