WO1998055501A1 - Procede ameliore de glycosylation specifique de site - Google Patents
Procede ameliore de glycosylation specifique de site Download PDFInfo
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- 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
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- protein
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- 238000000034 method Methods 0.000 title claims abstract description 29
- 230000013595 glycosylation Effects 0.000 title claims abstract description 10
- 238000006206 glycosylation reaction Methods 0.000 title claims abstract description 10
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 63
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 47
- 229920001184 polypeptide Polymers 0.000 claims abstract description 37
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 34
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 34
- 125000002883 imidazolyl group Chemical group 0.000 claims abstract description 33
- 150000001720 carbohydrates Chemical group 0.000 claims abstract description 25
- 125000000524 functional group Chemical group 0.000 claims abstract description 18
- -1 p-nitrophenyl ester Chemical class 0.000 claims abstract description 8
- AHLPHDHHMVZTML-BYPYZUCNSA-N L-Ornithine Chemical compound NCCC[C@H](N)C(O)=O AHLPHDHHMVZTML-BYPYZUCNSA-N 0.000 claims abstract description 6
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims abstract description 5
- 239000004472 Lysine Substances 0.000 claims abstract description 4
- AHLPHDHHMVZTML-UHFFFAOYSA-N Orn-delta-NH2 Natural products NCCCC(N)C(O)=O AHLPHDHHMVZTML-UHFFFAOYSA-N 0.000 claims abstract description 4
- UTJLXEIPEHZYQJ-UHFFFAOYSA-N Ornithine Natural products OC(=O)C(C)CCCN UTJLXEIPEHZYQJ-UHFFFAOYSA-N 0.000 claims abstract description 4
- 229960003104 ornithine Drugs 0.000 claims abstract description 4
- 239000002253 acid Substances 0.000 claims description 5
- 210000000987 immune system Anatomy 0.000 claims description 5
- 229960005486 vaccine Drugs 0.000 claims description 4
- 239000000556 agonist Substances 0.000 claims description 2
- 239000005557 antagonist Substances 0.000 claims description 2
- 238000011161 development Methods 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims description 2
- 230000003278 mimic effect Effects 0.000 claims description 2
- 230000012846 protein folding Effects 0.000 claims description 2
- 230000017854 proteolysis Effects 0.000 claims description 2
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 abstract description 10
- BLCJBICVQSYOIF-UHFFFAOYSA-N 2,2-diaminobutanoic acid Chemical group CCC(N)(N)C(O)=O BLCJBICVQSYOIF-UHFFFAOYSA-N 0.000 abstract 1
- 125000002252 acyl group Chemical group 0.000 description 29
- 235000018102 proteins Nutrition 0.000 description 27
- 238000006243 chemical reaction Methods 0.000 description 19
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 19
- 235000014633 carbohydrates Nutrition 0.000 description 18
- 150000002148 esters Chemical class 0.000 description 13
- 230000007704 transition Effects 0.000 description 13
- 235000014304 histidine Nutrition 0.000 description 11
- 230000003197 catalytic effect Effects 0.000 description 10
- 125000000487 histidyl group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C([H])=N1 0.000 description 10
- 125000004432 carbon atom Chemical group C* 0.000 description 9
- 229960002885 histidine Drugs 0.000 description 9
- 239000000126 substance Substances 0.000 description 9
- 238000012546 transfer Methods 0.000 description 9
- 150000001413 amino acids Chemical class 0.000 description 8
- 230000000087 stabilizing effect Effects 0.000 description 8
- 229940024606 amino acid Drugs 0.000 description 7
- 235000001014 amino acid Nutrition 0.000 description 7
- 125000006239 protecting group Chemical group 0.000 description 7
- 238000006276 transfer reaction Methods 0.000 description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- 230000004001 molecular interaction Effects 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 239000003054 catalyst Substances 0.000 description 5
- 230000007062 hydrolysis Effects 0.000 description 5
- 238000006460 hydrolysis reaction Methods 0.000 description 5
- 235000018977 lysine Nutrition 0.000 description 5
- 239000000758 substrate Substances 0.000 description 5
- BTJIUGUIPKRLHP-UHFFFAOYSA-N 4-nitrophenol Chemical compound OC1=CC=C([N+]([O-])=O)C=C1 BTJIUGUIPKRLHP-UHFFFAOYSA-N 0.000 description 4
- 238000005481 NMR spectroscopy Methods 0.000 description 4
- 108091028043 Nucleic acid sequence Proteins 0.000 description 4
- 230000027455 binding Effects 0.000 description 4
- 238000010276 construction Methods 0.000 description 4
- 229910052739 hydrogen Inorganic materials 0.000 description 4
- 239000001257 hydrogen Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 230000010933 acylation Effects 0.000 description 3
- 238000005917 acylation reaction Methods 0.000 description 3
- 125000000217 alkyl group Chemical group 0.000 description 3
- 238000007306 functionalization reaction Methods 0.000 description 3
- 150000002460 imidazoles Chemical class 0.000 description 3
- 238000010348 incorporation Methods 0.000 description 3
- 230000003993 interaction Effects 0.000 description 3
- 125000003588 lysine group Chemical class [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 3
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- ANMYWFDSHIYRRL-VMPITWQZSA-N (e)-2-(4-nitrophenyl)but-2-enedioic acid Chemical compound OC(=O)\C=C(\C(O)=O)C1=CC=C([N+]([O-])=O)C=C1 ANMYWFDSHIYRRL-VMPITWQZSA-N 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- 108020004511 Recombinant DNA Proteins 0.000 description 2
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 2
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 125000004429 atom Chemical group 0.000 description 2
- 239000013626 chemical specie Substances 0.000 description 2
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- 229910052736 halogen Chemical group 0.000 description 2
- 150000002367 halogens Chemical group 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 230000002209 hydrophobic effect Effects 0.000 description 2
- 230000016784 immunoglobulin production Effects 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 229920005989 resin Polymers 0.000 description 2
- 239000011347 resin Substances 0.000 description 2
- 230000006641 stabilisation Effects 0.000 description 2
- 238000011105 stabilization Methods 0.000 description 2
- YBADLXQNJCMBKR-UHFFFAOYSA-M (4-nitrophenyl)acetate Chemical compound [O-]C(=O)CC1=CC=C([N+]([O-])=O)C=C1 YBADLXQNJCMBKR-UHFFFAOYSA-M 0.000 description 1
- 125000001431 2-aminoisobutyric acid group Chemical group [#6]C([#6])(N*)C(*)=O 0.000 description 1
- ZSYMMQSHFBERCN-UHFFFAOYSA-N 3,3-diaminobutanoic acid Chemical compound CC(N)(N)CC(O)=O ZSYMMQSHFBERCN-UHFFFAOYSA-N 0.000 description 1
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 1
- WKBOTKDWSSQWDR-UHFFFAOYSA-N Bromine atom Chemical compound [Br] WKBOTKDWSSQWDR-UHFFFAOYSA-N 0.000 description 1
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- PXGOKWXKJXAPGV-UHFFFAOYSA-N Fluorine Chemical compound FF PXGOKWXKJXAPGV-UHFFFAOYSA-N 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- OTXBNHIUIHNGAO-UWVGGRQHSA-N Leu-Lys Chemical compound CC(C)C[C@H](N)C(=O)N[C@H](C(O)=O)CCCCN OTXBNHIUIHNGAO-UWVGGRQHSA-N 0.000 description 1
- 244000137850 Marrubium vulgare Species 0.000 description 1
- 108010067902 Peptide Library Proteins 0.000 description 1
- 101710172711 Structural protein Proteins 0.000 description 1
- 239000004809 Teflon Substances 0.000 description 1
- 229920006362 Teflon® Polymers 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
- 230000007815 allergy Effects 0.000 description 1
- FUOOLUPWFVMBKG-UHFFFAOYSA-N alpha-amino-isobutyric acid Natural products CC(C)(N)C(O)=O FUOOLUPWFVMBKG-UHFFFAOYSA-N 0.000 description 1
- 230000009435 amidation Effects 0.000 description 1
- 238000007112 amidation reaction Methods 0.000 description 1
- 238000010640 amide synthesis reaction Methods 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 125000004103 aminoalkyl group Chemical group 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 150000008064 anhydrides Chemical class 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 235000009697 arginine Nutrition 0.000 description 1
- 125000000637 arginyl group Chemical class N[C@@H](CCCNC(N)=N)C(=O)* 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- GDTBXPJZTBHREO-UHFFFAOYSA-N bromine Substances BrBr GDTBXPJZTBHREO-UHFFFAOYSA-N 0.000 description 1
- 229910052794 bromium Inorganic materials 0.000 description 1
- 150000001719 carbohydrate derivatives Chemical class 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- 239000000460 chlorine Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000000994 depressogenic effect Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000009881 electrostatic interaction Effects 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 229910052731 fluorine Inorganic materials 0.000 description 1
- 239000011737 fluorine Substances 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 229910052740 iodine Inorganic materials 0.000 description 1
- 239000011630 iodine Substances 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 108010034529 leucyl-lysine Proteins 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 229920005615 natural polymer Polymers 0.000 description 1
- 150000007523 nucleic acids Chemical group 0.000 description 1
- 239000012038 nucleophile Substances 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- KHPXUQMNIQBQEV-UHFFFAOYSA-N oxaloacetic acid Chemical compound OC(=O)CC(=O)C(O)=O KHPXUQMNIQBQEV-UHFFFAOYSA-N 0.000 description 1
- 125000000636 p-nitrophenyl group Chemical group [H]C1=C([H])C(=C([H])C([H])=C1*)[N+]([O-])=O 0.000 description 1
- 238000010647 peptide synthesis reaction Methods 0.000 description 1
- 230000010287 polarization Effects 0.000 description 1
- 210000004896 polypeptide structure Anatomy 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 238000001542 size-exclusion chromatography Methods 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 229920001059 synthetic polymer Polymers 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 238000007079 thiolysis reaction Methods 0.000 description 1
- 238000005809 transesterification reaction Methods 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
Classifications
-
- 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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Abstract
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 |
---|---|---|---|
SE9702188-5 | 1997-06-06 | ||
SE9702188A SE9702188D0 (sv) | 1997-06-06 | 1997-06-06 | Improved method for site-selective glycosylation |
Publications (2)
Publication Number | Publication Date |
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WO1998055501A1 true WO1998055501A1 (fr) | 1998-12-10 |
WO1998055501A9 WO1998055501A9 (fr) | 1999-07-15 |
Family
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Application Number | Title | Priority Date | Filing Date |
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PCT/SE1998/001008 WO1998055501A1 (fr) | 1997-06-06 | 1998-05-28 | Procede ameliore de glycosylation specifique de site |
Country Status (3)
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AU (1) | AU8044498A (fr) |
SE (1) | SE9702188D0 (fr) |
WO (1) | WO1998055501A1 (fr) |
Cited By (2)
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)
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) |
-
1997
- 1997-06-06 SE SE9702188A patent/SE9702188D0/xx unknown
-
1998
- 1998-05-28 AU AU80444/98A patent/AU8044498A/en not_active Abandoned
- 1998-05-28 WO PCT/SE1998/001008 patent/WO1998055501A1/fr active Application Filing
Patent Citations (1)
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)
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)
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 |
---|---|
WO1998055501A9 (fr) | 1999-07-15 |
SE9702188D0 (sv) | 1997-06-06 |
AU8044498A (en) | 1998-12-21 |
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