MXPA01009209A - Method for acylating peptides and novel acylating agents - Google Patents
Method for acylating peptides and novel acylating agentsInfo
- Publication number
- MXPA01009209A MXPA01009209A MXPA/A/2001/009209A MXPA01009209A MXPA01009209A MX PA01009209 A MXPA01009209 A MX PA01009209A MX PA01009209 A MXPA01009209 A MX PA01009209A MX PA01009209 A MXPA01009209 A MX PA01009209A
- Authority
- MX
- Mexico
- Prior art keywords
- glp
- ester
- peptide
- arg26
- reactive
- Prior art date
Links
- 102000004196 processed proteins & peptides Human genes 0.000 title description 10
- 108090000765 processed proteins & peptides Proteins 0.000 title description 10
- 150000002148 esters Chemical class 0.000 claims abstract description 29
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 25
- 239000001257 hydrogen Substances 0.000 claims abstract description 22
- 229910052739 hydrogen Inorganic materials 0.000 claims abstract description 22
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 22
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 22
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims abstract description 18
- 125000003277 amino group Chemical group 0.000 claims abstract description 17
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 17
- 239000000203 mixture Substances 0.000 claims abstract description 12
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 claims abstract description 11
- 125000004435 hydrogen atoms Chemical group [H]* 0.000 claims abstract description 11
- 230000003637 steroidlike Effects 0.000 claims abstract description 11
- 125000004185 ester group Chemical group 0.000 claims abstract description 6
- SECXISVLQFMRJM-UHFFFAOYSA-N n-methylpyrrolidone Chemical compound CN1CCCC1=O SECXISVLQFMRJM-UHFFFAOYSA-N 0.000 claims abstract description 5
- 125000004400 (C1-C12) alkyl group Chemical group 0.000 claims abstract 2
- 125000000217 alkyl group Chemical group 0.000 claims description 20
- 150000001875 compounds Chemical class 0.000 claims description 16
- 239000011541 reaction mixture Substances 0.000 claims description 14
- 125000003342 alkenyl group Chemical group 0.000 claims description 10
- 102400000326 Glucagon-like peptide 2 Human genes 0.000 claims description 8
- 101710013993 Mcpt10 Proteins 0.000 claims description 8
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- 108010090776 Glucagon-like peptide 1(7-37) Proteins 0.000 claims 1
- DTHNMHAUYICORS-KTKZVXAJSA-N 107444-51-9 Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(N)=O)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC=1N=CNC=1)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=CC=C1 DTHNMHAUYICORS-KTKZVXAJSA-N 0.000 abstract description 13
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- 238000006243 chemical reaction Methods 0.000 description 17
- HEMHJVSKTPXQMS-UHFFFAOYSA-M sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 15
- CSCPPACGZOOCGX-UHFFFAOYSA-N acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 14
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- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 5
- 230000000875 corresponding Effects 0.000 description 5
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- 238000002844 melting Methods 0.000 description 5
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- RWRDLPDLKQPQOW-UHFFFAOYSA-N tetrahydropyrrole Substances C1CCNC1 RWRDLPDLKQPQOW-UHFFFAOYSA-N 0.000 description 5
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- IDLFZVILOHSSID-OVLDLUHVSA-N corticotropin Chemical compound C([C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](C(C)C)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)NC(=O)[C@@H](N)CO)C1=CC=C(O)C=C1 IDLFZVILOHSSID-OVLDLUHVSA-N 0.000 description 2
- 229940105423 erythropoietin Drugs 0.000 description 2
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- 101710038873 glc-1 Proteins 0.000 description 2
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- 229950002350 secretin human Drugs 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 239000011877 solvent mixture Substances 0.000 description 2
- NHXLMOGPVYXJNR-ATOGVRKGSA-N somatostatin Chemical compound C([C@H]1C(=O)N[C@H](C(N[C@@H](CO)C(=O)N[C@@H](CSSC[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CC=2C3=CC=CC=C3NC=2)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(=O)N1)[C@@H](C)O)NC(=O)CNC(=O)[C@H](C)N)C(O)=O)=O)[C@H](O)C)C1=CC=CC=C1 NHXLMOGPVYXJNR-ATOGVRKGSA-N 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
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- 125000001424 substituent group Chemical group 0.000 description 2
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- OOEQVYXYLUGKCX-UHFFFAOYSA-N 1-(benzotriazol-1-yl)hexadecan-1-one Chemical compound C1=CC=C2N(C(=O)CCCCCCCCCCCCCCC)N=NC2=C1 OOEQVYXYLUGKCX-UHFFFAOYSA-N 0.000 description 1
- ASOKPJOREAFHNY-UHFFFAOYSA-N 1-hydroxybenzotriazole Chemical compound C1=CC=C2N(O)N=NC2=C1 ASOKPJOREAFHNY-UHFFFAOYSA-N 0.000 description 1
- 125000003903 2-propenyl group Chemical group [H]C([*])([H])C([H])=C([H])[H] 0.000 description 1
- 101710044881 GHRH Proteins 0.000 description 1
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- 239000000095 Growth Hormone-Releasing Hormone Substances 0.000 description 1
- AHLPHDHHMVZTML-BYPYZUCNSA-N L-ornithine Chemical compound NCCC[C@H](N)C(O)=O AHLPHDHHMVZTML-BYPYZUCNSA-N 0.000 description 1
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-hydroxy-Succinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 1
- 229960003104 Ornithine Drugs 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
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- 125000000539 amino acid group Chemical group 0.000 description 1
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- 238000007707 calorimetry Methods 0.000 description 1
- 150000001718 carbodiimides Chemical class 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 1
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- 150000001991 dicarboxylic acids Chemical class 0.000 description 1
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- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
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- 125000000959 isobutyl group Chemical group [H]C([H])([H])C([H])(C([H])([H])[H])C([H])([H])* 0.000 description 1
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- 238000000034 method Methods 0.000 description 1
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 1
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- 125000004108 n-butyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 125000001280 n-hexyl group Chemical group C(CCCCC)* 0.000 description 1
- 125000004123 n-propyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
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Abstract
The present invention provides a method for acylating one or more amino groups (typically arising from lysine) of a peptide (or protein), e.g. GLP-1, the method comprising (a) reacting a peptide (or protein) having at least one free amino group with an acylating agent of general formula (I) wherein n is 0-8;R1 is COOR4;R2 is a lipophilic moiety, e.g. selected from C3-39-alkyl, C3-39-alkenyl, C3-39-alkadienyl and steroidal residues;R3 together with the carboxyl group to which R3 is attached designa te a reactive ester or a reactive N-hydroxy imide ester;and R4 is selected from hydrogen, C1-12-alkyl and benzyl,under basic conditions in a mixture of an aprotic polar solvent and water (e. g. N-methyl-2-pyrrolidone and water);and (b) if R4 is not hydrogen, saponifying the acylated peptide ester group (COOR4) under basic conditions (e.g. at a pH in the range of 7-14);in order to obtain an N-acylated peptide (or an N-acylated protein).
Description
METHOD FOR ACILING NOVEDOUS PICTILS AND ACILATION AGENTS
FIELD OF THE INVENTION The present invention relates to a method for introducing one or more acyl groups into a peptide or a protein. More particularly, the present invention relates to an improved method of acylation of the e-amino group of a lysine residue contained in a naturally occurring GLP-1 or analogue thereof. Additionally the present invention relates to compounds useful as acylating agents in the method.
BACKGROUND OF THE INVENTION Peptides are widely used in medical practice, and because they can be produced by a recombinant DNA technology, their importance is expected to increase also in the years to come. When the original peptides or analogs thereof are used in medical therapy, it is generally found that they have a broad spectrum of acceptance. A broad spectrum of acceptance of a therapeutic agent in disadvantage in cases where it is desired to maintain a high blood level thereof for a prolonged period of time since they will then be
REF: 132581 necessary repeated administrations. Examples of peptides which in their original form have a broad acceptance spectrum are: ACTH, corticotropin releasing factor, angiotensin, calcitonin, exendin, 3-exendin 4-exendin, insulin, glucagon, glucagon-like peptide 1, -Glucagon-like peptide, insulin-like growth factor-1, insulin-like growth factor-2, gastric inhibition peptide, growth hormone release factor, pituitary adenylate cyclase activating peptide, secretin, enterogastrin, somatostatin, somatotropin, somatomedin, parathyroid hormone, thrombopoietin, erythropoietin, hypothalamus releasing factors, prolactin, thyroid stimulating hormones, endorphins, enkephalins, vasopressin, oxytocin, opioids and their analogues, superoxide dismutase, interferon, asparaginase , arginase, arginine deaminase, adenosine deaminase and ribonuclease. The introduction of lipophilic acyl groups into naturally occurring peptides or analogs thereof leads to acylated peptides which have a long-acting profile with respect to the native peptide (or unmodified analogue). This phenomenon has been fully described and demonstrated in the previous request of the current applicant, O98 / 08871, which i.a. describes the acylation of GLP-1 and the like; and WO98 / 08872, which i.a. describes the acylation of GLP-2 and the like and WO99 / 43708, which i.a. describes the acylation of exendin and analogues. Additionally, it has been suggested that the inclusion of a group, which may be negatively charged, e.g. a group of carboxylic acid adjacent to the lipophilic group may have a greater advantage. European Patent Application No. 92107035.5 (Kurary Co) describes the reactive monoesters of long chain dicarboxylic acids for the introduction of long chain carboxylic acids into proteins. The introduction of lipophilic acyl groups in GLP-1 via mono- or dipeptide separators can be especially interesting and has been suggested and exemplified in WO98 / 08871. Aspartic acid and glutamic acid were mentioned as appropriate linkers. However, just as the mono- and dipeptide separators include a carboxylic acid group, protection steps and subsequent deprotection were also considered necessary. The deprotection was carried out under acidic conditions, which to a certain degree lead to the destruction of the peptide. (GLP-1). Thus, alternative methods for the preparation of these variants are desirable.
Thus, the essence of the present invention is to provide an alternative method for the introduction of lipophilic groups within the peptides via a-amino-α, β -dicarboxylic acid separators Such a method will facilitate the preparation of modified peptides wherein the carboxylic acid groups with charge are introduced in the vicinity of the lipophilic groups, but without directly influencing the lipophilic groups.
SUMMARY OF THE INVENTION The present invention provides a method for acylating one or more amino groups of a peptide (or protein), the method comprising: (a) reacting a peptide (or protein) having at least one free amino group with an acylating agent of the general formula (I)
where n is 0-8;
R1 is COOR4; R2 is a lipophilic moiety, e.g. selected from alkyl of 3 to 39 carbon atoms; alkenyl of 3 to 39 carbon atoms; alkadienyl of 3 to 39 carbon atoms and steroidal residues; R3 together with the carboxyl group to which R3 is attached designates a reactive ester or a reactive N-hydroxy imide ester; and R 4 is selected from hydrogen, alkyl of 1 to 12 carbon atoms and benzyl, under basic conditions in a mixture of a polar aprotic solvent and water; and (b) if R4 is not hydrogen, saponifying the ester group of the acylated peptide (COOR4) under basic conditions; to obtain an N-acylated peptide (or an N-acylated protein). It has been found that saponification of the acylated peptide ester (wherein R is an alkyl or benzyl group) under basic conditions is possible only without racemization or with minimal racemization of the various amino acid fragments of the peptide and the separator. The present applicant has found certain advantages over previously used acidic hydrolysis with respect to the purity and suppression of side products, e.g. degradation products.
It has also been found that acylation using the acylating agent as free acid (where R is hydrogen) under basic conditions essentially leads directly to the desired product, the acylated peptide, without side products and without the need for a deprotection step. The present invention also provides novel compounds useful as acylating agents in the aforementioned method, such novel compounds have the general formula I
where n is 0-8; R is COOH; R is a lipophilic moiety, e.g. selected from alkyl of 3 to 39 carbon atoms; alkenyl of 3 to 39 carbon atoms; alkadienyl of 3 to 39 carbon atoms and steroidal residues;
R together with the carboxyl group to which R is attached designates a reactive ester or a reactive N-hydroxy imide ester.
DETAILED DESCRIPTION OF THE INVENTION Peptides and Proteins It is generally believed that the present invention is useful for the introduction of lipophilic acyl groups into any peptide (or protein) in order to reduce the proportion of a widely accepted spectrum in vi. Examples of such peptides and proteins are ACTH, corticotropin releasing factor, angiotensin, calcitonin, exendin and analogs thereof, insulin and its analogs, glucagon and its analogs, glucagon-like peptide 1-and analogs thereof, 2- Glucagon-like peptide and analogs thereof, insulin-like growth factor-1, insulin-like growth factor-2, gastric inhibitor peptide, growth hormone releasing factor, pituitary adenylate cyclase activating peptide secretin, enterogastrin, somatostatin, somatotropin, somatomedin, parathyroid hormone, thrombopoietin, erythropoietin, hypothalamus release factors, prolactin, thyroid stimulating hormones, endorphins, enkephalins, vasopressin, oxytocin, opioids and analogues thereof, superoxide dismutase, interferon, asparaginase, arginase, arginine deaminase, adenosine deaminase and ribonuclease. It should be understood that the peptide (or protein) must carry at least one free amino group, such amino group is the N-terminal amino group or a side chain amino group. Of particular interest are the amino groups of lysine and the amino acid residues of ornithine. The method is particularly relevant for the N-acylation of the e-amino group of lysine residues. It should also be understood that the peptide or protein in question may comprise two or more pendant amino groups which may be N-acylated according to the present invention. It is now believed that the present invention is especially suitable for the modification of GLP-1 and analogs thereof. Examples of GLP-1 and analogs that can be N-acylated according to the present invention are GLP-1 and truncated analogues, such as Arg26-GLP-1 (7-37).; Arg 3 -GLP-1 (7-37); Lys36-GLP-1 (7-37); Arg26'34Lys36-GLP-I (7-37); Arg26'34Lys38-GLP-1 (7-38); Arg26'34Lys39-GLP-I (7-39); Arg26'34Lys40-GLP-I (7-40); Arg26Lys36-GLP-I (7-37); Arg34Lys36-GLP-I (7-37); Arg26Lys39-GLP-1 (7-39); Arg34Lys40-GLP-I (7-40); Arg26 > 34Lys36'39-GLP-I (7-39); Arg26'34Lys36.40-GLP-I (7-40); Gly8 Arg26-GLP-1 (7-37); Gly8 Arg34-GLP-K7-37); Gly8 Lys36-GLP-1 (7-37); Gly8 Arg26'34Lys36-GLP-I (7-37); Gly8 Arg26'34Lys39-GLP-I (7-39); Gly8 Arg26'34Lys40-GLP-I (7-40); Gly8 Arg26Lys36-GLP-I (7-37); Gly8 Arg34 Lys36-GLP-1 (7-37); Gly8 Arg26 Lys39-GLP-1 (7-39); Gly8 Arg34Lys40-GLP-I (7-40); Gly8 Arg26'34Lys3 '39 -GLP-I (7-39); Gly8 Arg26 > 34Lys36 > 40-GLP-I (7-40); Arg2ß'34Lys38-GLP-I (7-38); Arg26 »34Lys39-GLP-I (7-39); Arg26J3 Lys40-GLP-1 (7-40); Arg26 > 34Lys41-GLP-I (7-41); Arg26'34Lys42-GLP-I (7-42); Arg2ß, 34Lys 3-GLP-I (7-43); Arg26.34Lys44-GLP-I (7-44); Arg26 / 34Lys45-GLP-1 (7-45); Arg26.34Lys38-GLP-I (1-38); Arg26'34Lys39-GLP-I (1-39'26.34)
; Arg26 34Lys40-GLP-I (1-401: Arg26 34Lys41-GLP-I (1-41): Arg Lys42-GLP-1 (1-42); Arg26.}. 34Lys43-GLP-I (1-43); Arg26'34 Lys44-GLP-1 (1-44); Arg26'34Lys45-GLP-1 (1-45); Arg26 ^ 34Lys38-GLP-1 (2-38); Arg26'34Lys39-GLP-1 (2); -39); Arg26-> 34Lys40-GLP-I (2-40);
Arg26; 34Lys41-GLP-1 (2-41); Arg26'34Lys42-GLP-I (2-42); Arg26 34 Lys43-GLP-1 (2-43); Arg26 ^ 34Lys44-GLP-1 (2-44); Arg26 34Lys45-GLP-1 (2-45); Arg26'34Lys38-GLP-I (3-38); Arg26'34Lys39-GLP-I (3-39);
-. 26.34t 40"t, -, / ._- ,. .. 26,34t 41 _t _. , -, ... - 26,34
Arg 'Lys-GLP-1 (3-40); Arg 'Lys-GLP-1 (3-41); Arg Lys42-GLP-1 (3-42); Arg26J34Lys43-GLP-I (3-43); Arg26- > 34Lys44-GLP-1 (3-44); Arg26'34Lys45-GLP-I (3-45); Arg26'34Lys38-GLP-I (4-38); 26 34 Arg6'34Lys-GLP-1 (4-39); Arg ^ 6 > '34T L.ysc 4 400 26.34 -GLP-1 (4-40¡ Arg
Lys41-GLP-1 (4-41); Arg26 > 34Lys42-GLP-I (4-42); Arg26'34Lys43-GLP-1 (4-43); Arg26.34Lys4-GLP-I (4-44); Arg26'34Lys45-GLP-I (4-45); Arg26.34Lys38-GLP-I (5-38); Arg26 > 34Lys39-GLP-I (5-39); Arg26'34
40 26.34 .. 41 26.34-. 42
Lys-GLP-1 (5-40); Argb, 4Lys-GLP-1 (5-41); Arg26 '4Lys -GLP- 26.34t. 43 1 (5-42); Arg26'34Lys43-GLP-I (5-43); Arg26'3 Lys 4-GLP-I (5-44);
26.34t. 45 Arg ^ '^ Lys ^ -GLP-l (5-45); Arg26'34Lys38-GLP-I (6-38); Arg26'34 Lys39-GLP-1 (6-39); Arg2ß'34Lys40-GLP-I (6-40); Arg26 > 34Lys41-GLP-1 (6-41); Arg26'34Lys42-GLP-I (6-42); Arg26'34Lys43-GLP-I (6-43);
Arg26'34Lys44-GLP-I (6-44); Arg26 '34Lys45-GLP-I (6-45); Arg26Ly s38 - GLP-1 (1-3I Arg34Lys38-GLP-I (1-38); Arg26 34Lys36 / 38-GLP-I (1-38); Arg26Lys38-GLP-I (7-38); Arg34Lys38-GLP- l (7-38); Arg26'34 Lys36,38-GLP-1 (7-38); Arg26'34Lys38-GLP-l (7-38); Arg26Lys39-GLP-1 (1-39); Arg34Lys39-GLP; -l (1-39); Arg26> 34Lys36'39-GLP-1 (1-39); Arg26Lys39-GLP-1 (7-39); Arg34Lys39-GLP-1 (7-39); Arg26'34Lys36 ' 39-GLP-1 (7-39); Arg26-GLP-I (7-37); Arg34-GLP-I (7-37), Lys36-GLP-I (7-37), Arg26'34Lys36-GLP- l (7-37), Arg26Lys36-GLP-I (7-37), Arg34 Lys36-GLP-K7-37), Gly8 Arg26-GLP-1 (7-37), Gly8 Arg3-GLP-1 (7-37) ), Gly8Lys36-GLP-l (7-37), Gly8 Arg26- > 34Lys36-GLP-I (7-37), Gly8 Arg26Lys36-GLP-I (7-37); Gly8 Arg34 Lys36-GLP-1 (7-37); Arg26Lys38-GLP-1 (7-38), Arg2d 34Lys38-GLP-I (7-38), Arg26 > 34Lys36'38-GLP-I (7-38), Gly8Arg26Lys38-GLP-I (7-38); Gly8 Arg26'34Lys36 > 38-GLP-I (7-38); Gly8Arg26'34Lys36 | 38-GLP-I (7-38); Gly8, Arg26'34, Glu37, Lys38-GLP-K7-38), Arg26Lys39-GLP-I (7-39), Arg26'34 Lys36'39-GLP-1 (7-39), Gly8Arg26Lys39-GLP-I ( 7-39); Gly8Arg26'34 Lys36'39-GLP-1 (7-39); Arg34Lys40-GLP-l (7-40), Arg26'34Lys3ß'40-GLP-l (7-40),
Gly8Arg3 Lys40-GLP-l (7-40) and Gly8Arg26'34Lys36 > 40-GLP-I (7-40).
Each of these GLP-1 analogs and truncated analogues constitute alternative embodiments of the present invention. It is now believed that the present invention is also especially suitable for the modification of GLP-2 and analogs thereof. Examples of GLP-2 and analogs that can be N-acylated according to the present invention are analogs of GLP-2 and truncated analogs, such as Lys 20 GLP-2 (1-33); Lys20Arg30GLP-2 (1-33); Arg30Lys34GLP-2 (1-34);
Arg30Lys35GLP-2 (1-35); Arg30'35Lys20GLP-2 (1-35); and Arg35GLP-2 (1-35). Each of these GLP-2 analogs and truncated analogs constitute alternative embodiments of the present invention.
It is now believed that the present invention is also especially suitable for the modification of exendin and analogs thereof. Examples of exendin and analogs that can be N-acylated according to the present invention are exendin analogs and truncated analogs such as 3-exendin and 4-exendin. Each of these exendin analogs and truncated analogues constitute alternative embodiments of the present invention. In a further embodiment of the present invention the N-acylation takes place in the e-amino group of the lysine residues. The effect of GLP-1 and its analogs are fully described in WO98 / 08871. The effect of GLP-2 and its analogs are fully described in WO98 / 08872. The effect of exendin and its analogues are fully described in
WO99 / 43708.
Acylation Agent In the method according to the invention, a peptide (or protein) which has at least one free amino group is reacted with an acylating agent of the general formula I
The integer n in the formula is preferably 0-8, in particular 0-6 which corresponds e.g. to aspartic acid, glutamic acid, etc. Preferably, n is 0-4 such as 0-2, e.g.
0 (aspartic acid) or 1 (glutamic acid). Each of these integers and ranges constitute alternative embodiments of the present invention.
R in formula i represents a free acid group
(COOH) or an ester group (COOR 4). In cases where R1 is 4 an ester group, R is selected from groups that can be
eliminated (as the corresponding alcohols) by hydrolysis under basic conditions. Examples of such groups are alkyl of 1 to 12 carbon atoms, e.g. methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-butyl, 2-methyl-1-propyl, 2-methyl-2-propyl (tert-butyl), 1-hexyl, etc., and benzyl . Each of these groups constitute alternative embodiments of the present invention. 2 R in formula I represents the lipophilic portion a
be incorporated into the peptide or protein. Such a lipophilic moiety is generally selected from alkyl of 3 to 39 carbon atoms, alkenyl of 3 to 39 carbon atoms, alkadienyl of 3 to 39 carbon atoms and steroidal residues. Specific examples of alkyl of 3 to 39 carbon atoms are heptyl, nonyl, undecanyl, tridecanyl, pentadecanyl, heptadecanyl and nonadecanyl. Each of these lipophilic portions constitute alternative embodiments of the present invention. The substituent or lipophilic portion is characterized by having a solubility in water at 20 ° C in the range of from 0.1 mg / 100 ml of water to about 250 mg / 100 ml of water, preferably in the range of about 0.3 mg / 100 ml of water at approximately 75 mg / 100 ml of water. For example octanoic acid (C8) has a solubility in water at 20 ° C of 68 mg / 100 ml, decanoic acid (CIO) has a solubility in water at 20 ° C of 15 mg / 100 ml and octadecanoic acid ( C18) has a solubility in water at 20 ° C of 0.3 mg / 100 ml. Each of these ranges in the lipophilic substituent constitutes alternative embodiments of the present invention. The terms "C3-39 alkyl", "C3-39 alkenyl" and "C3-39 alkadienyl" are intended to cover the straight and branched chain, preferably the straight, saturated, monounsaturated chain and unsaturated, respectively, hydrocarbon radicals of 3 to 39 carbon atoms.
Specific examples of C3-39 alkyl are heptyl, nonyl,
undecanyl, tridecanyl, pentadecanyl, heptadecanil and nonadecanil. When used herein, the term "steroidal residue" is intended to define a lipophilic group which
together with the carbonyl group to which R is attached is derived
of a steroid carboxylic acid, i.e. a tri-, tetra- and
pentacyclic, a C16-36 hydrocarbon totally saturated or
partially unsaturated Examples of such R-C groups (= 0) - are lithocolyl, deoxycoloyl and coloyl. Among the lipophilic groups mentioned above
the alkyl of C_25, alkenyl of 7-25 / alkadienyl of C-25 and
Steroidal waste is particularly relevant. Examples of particular interest are heptyl, nonyl, undecanyl, tridecanyl, pentadecanyl, heptadecanyl, nonadecanoyl, litocoloyl, deoxycholoyl and colony. Each of these lipophilic groups constitute alternative embodiments of the present invention.
R in formula I together with the carboxyl group at
3, which R is attached, denotes a reactive ester or a reactive N-hydroxy-imide ester. Each of these esters constitutes alternative embodiments of the present invention. Reactive esters and reactive N-hydroxy imide esters are well known in the art of organic chemistry (especially in the chemistry of peptides as functional groups, which are used in the acylation of amino, thio and hydroxy groups. Within the context of the present invention, the term "reactive ester or an N-hydroxy imide ester" is intended to define a functionalized ester of a carboxylic acid group suitable for acylating an amine, preferably a primary amine. It should thus be understood that the selectivity for the acylation of primary amines is preferred over the acylation of hydroxy and thio groups. Esters of N-hydroxy-imide are especially preferred.
Examples of reactive esters are esters of 1-hydroxybenzotriazole and derivatives. A number of highly effective reagents are known, e.g. 2- (lH-benzotriazol-lyl) -1, 1,3,3-tetramethyluronium tetrafluoroborate, for the formation of such activated esters of carboxylic acids. Such reactive esters are typically formed in-itself in the presence of a base, e.g. an organic base such as trialkylamine. Examples of the imide part of N-hydroxy imide esters are specifically described in European patent application No. 92107035.5, p. 13, line 3 to p. 17, line 10 (which are incorporated here as a reference). Particularly interesting examples of imide parts are succinimide, phthalimide, etc. Each of these imides constitutes alternative embodiments of the present invention. The reactive N-hydroxy imide esters of the formula I can be prepared by condensation of the acid
corresponding (i.e. the protected N-acylated a-carboxy-4 diacid (R is not hydrogen)) with an equimolar amount
(e.g. 0.95-1.05 mol, preferably 1.0 mol) of the N-hydroxy imide of the corresponding imide. The N-acylated a-carboxy diacid protected on the other hand is prepared
typically from the corresponding protected a-carboxy,
the a-amino acid and a benzotriazole ester of the portion
lipophilic This benzotriazole ester can, e.g. prepared from hydrochloric acid and benzotriazole or from the free acid and benzotriazole by DCC coupling as described in WO 98/02460 (Examples 1-3). Condensation is commonly carried out under dehydration conditions, e.g. in the presence of a coupling reagent such as the carbodiimide coupling reagent (e.g. dicyclohexylcarbodiimide (DCC)). The coupling reagent when present is preferably added in equimolar amounts with respect to the acid. The reaction is typically carried out in an aprotic polar solvent such as anhydrous tetrahydrofuran (THF), anhydrous dimethylformamide (DMF), anhydrous acetone, anhydrous dichloromethane, anhydrous dioxane, anhydrous dimethylacetamide or anhydrous N-methyl-2-pyrrolidine (NMP). The reaction is commonly carried out at a temperature in the range of 0-50 ° C, e.g. 5-30 ° C such as room temperature, for a period of 1-96 hours such as 4-36 hours. A possible group of reagents and conditions is as follows: N-hydroxy-imide
(e.g. succinimide or phthalimide) and the acid in question were dissolved in an approximate molar ratio of 1: 1 in anhydrous THF or anhydrous DMF (or a mixture thereof) and an equimolar amount of DCC was added to the solution. After completion of the reaction between the N-hydroxy-imide and the acid, the product was isolated and purified using conventional means such as filtration (filtration of precipitated dicyclohexylurea (DCU) when DCC is used as a coupling reagent), chrysatization, recrystallization , chromatography, etc. A possible purification route includes the removal of the precipitated coupling reagent used by filtration, evaporation of the solvent under reduced pressure, resuspension of the product, e.g. in acetone, filtration, crystallization by addition of a non-polar solvent, e.g. hexane and optionally recrystallization and / or washing. The product can be used directly as the acylation reagent of formula I in the method according to the invention. In the case where the acylation reagent of the
Formula I is going to be used as free a-carboxylic acid
(R 4 = hydrogen), a compound of the formula I wherein R 4 is
a group that can be eliminated is converted to the corresponding compound 4 wherein R is hydrogen. The protective group
The carboxylic acid can be a benzyl group which can be removed by catalytic hydrogenation or an allyl group which can be selectively removed. A benzyl protecting group can be removed by catalytic hydrogenation in a polar aprotic solvent, e.g. in acetone at room temperature using palladium on carbon and hydrogen. The reaction can be carried out in a closed vessel with a hydrogen atmosphere (commonly 0.1-10 atm.) With vigorous stirring. The reaction commonly ends within 0.5-12 hours depending on the quality of the palladium catalyst. Apply a conventional procedure. It is considered that the compounds of the formula I wherein
R is hydrogen are novelty such as and, well, those
compounds constitute a special aspect of the present invention. Accordingly, the present invention also provides novel compounds of the general formula I
where n is 0-8;
R1 is COOH; 2 R is a lipophilic portion, preferably
selected from C3-39 alkyl; C3-39 alkenyl;
C3-39 alkadienyl and steroidal residues;
R 3 together with the carboxyl group to which R3 is attached
denotes a reactive ester or a reactive N-hydroxy imide ester.
Reaction Conditions The reaction between the acylating agent of the formula I and the peptide or protein is carried out under basic conditions in a mixture of a polar aprotic solvent and water. The acylating agent of the formula I is typically used in slight excess with respect to the number of amino groups of the peptide to be acylated. The ratio is typically 1: 1 to 1:20 with an excess of the acylating agent, preferably 1: 1.2 to 1: 5, taking into account the number of amino groups in the peptide. It is to be understood that the peptide can be completely N-acylated or only partially N-acylated depending on the amount of acylating agent used and the reaction conditions. It is preferred that the N-acylation be substantially stoichiometric. Typically, the aprotic polar solvent is selected from anhydrous tetrahydrofuran (THF), anhydrous dimethylformamide (DMF), acetone, dichloromethane, dimethylsulfoxide (DMSO), dioxane, dimethylacetamide and N-methyl-2-pyrrolidine and mixtures thereof, among which dimethylformamide, dimethylsulfoxide, dimethylacetamide and N-methyl-2-pyrrolidine are preferred and N-methyl-2-pyrrolidine is especially preferred. The ratio between the polar aprotic solvent and water (eg N-methyl-2-pyrrolidine and water) is typically 1:10 to 10: 1, in particular 1: 5 to 5: 1, especially 1: 1 to 3: 1 . The temperature is typically maintained in the range of -10-50 ° C, preferably in the range of 0-25 ° C. It is important that the pH value of the solvent mixture be in the range of 7-14, such as 9-13, preferably in the range of 9.5 to 12.5, so that the reaction proceeds smoothly. The result with respect to yield and purity is normally optimal when the pH value of the solvent mixture is in the range of 10-12. The desired pH value is obtained by the addition of alkalimetal hydroxides, e.g. sodium hydroxide and potassium hydroxide, and / or organic bases such as trialkylamines (e.g. triethylamine, N, N-diisopropylethylamine, etc.). As a typical example, the reaction in step (a) is carried out using the protein and the acylating agent of the formula I in a molar ratio of 1: 1 to 1: 5. The peptide is typically pre-dissolved in water at -10-30 ° C such as 0-25 ° C and the pH is adjusted to the desired level using an alkalimetal hydroxide (e.g., sodium hydroxide or potassium hydroxide). The pH value can be further adjusted using acids such as e.g. acetic acid and bases e.g. trialkylamine, but the temperature is preferably within the above range. The aprotic polar solvent (or a mixture of solvents) is then added. The acylating agent is subsequently added. Generally the reaction is allowed to run to completion (it can be monitored by HPLC) which is typically obtained in 0.2 to 4 hours, such as 0.2 to 1 hour, before the addition of water and an acid, e.g. acetic acid, at a pH of 6.5 to 9.0. The product is commonly isolated and purified by HPLC, or is precipitated by isoelectric pH or is hydrolyzed (step (b)) before purification. When an acylating agent of the formula I is used in
4 where R is hydrogen, the peptide is obtained directly or
N-acylated protein that carries lipophilic portions and groups
free carboxylic acids So, the variant where R is
Hydrogen represents a preferred embodiment of the method of the present invention.
Alternatively, i.e. when the group R is alkyl of
C? _i2 or benzyl, the ester of the N-acylated peptide (or ester of
protein) is saponified under basic conditions in such a way as to obtain the N-acylated peptide or the N-acylated protein. Sapponification is commonly carried out in a 0.01-4.0 M solution of an alkalimetal hydroxide, e.g. sodium hydroxide or potassium hydroxide. The pH of the solution is typically 10-14. The reaction is generally allowed to proceed for 0.1-12 hours, preferably 0.5-4 hours at 0-40 ° C such as around room temperature. After the reaction, the product is purified, e.g. by precipitation at an isoelectric pH and / or by preparative HPLC. Thus, the variant wherein R is C1-12 alkali or benzyl represents another preferred embodiment of the method of the present invention. The present invention also relates to the following aspects: Aspect 1: a method for acylating an amino group of a peptide or protein, the method comprising: (a) reacting a peptide having at least one free amino group with an agent acylating general formula I
where n is 0-8;
R1 is COOR4;
R is a lipophilic portion;
R 3 together with the carboxyl group to which R3 is attached
designates a reactive ester or a reactive N-hydroxy imide ester; and 4 R is selected from hydrogen, alkyl from 1 to 12
carbon and benzyl atoms, under basic conditions in a mixture of a polar aprotic solvent and water; Y
(b) if R is not hydrogen, saponify the ester group 4 of the acylated peptide (COOR) under basic conditions;
in order to obtain an N-acylated peptide. Aspect 2. A method of compliance with aspect 1, in
4 where R is hydrogen.
Aspect 3. A method of compliance with aspect 1, in
4 wherein R is selected from C? -8 alkyl and benzyl.
Aspect 4. A method of compliance with any of the
aspects 1-3, wherein R together with the carboxyl group to which R is attached designates a reactive N-hydroxy imide ester. Aspect 5. A method according to any of aspects 1-4, wherein the mixture of the aprotic solvent and water is a 1: 5 to 5: 1 mixture of N-methyl-2-pyrrolidone and water. Aspect 6. A method of conformance to any of aspects 1-5, wherein the pH of the reaction mixture in step (a) is in the range of 9 to 13. Aspect 7. A method of compliance with any of aspects 1-6, wherein the temperature of the reaction mixture in step (a) is in the range of 0 to 50 ° C. Aspect 8. A method according to any of aspects 3 to 7, wherein the ester of the acylated peptide is saponified to a pH value in the range of 10 to 14. Aspect 9. A method of compliance with any of the aspects above where R is selected from C3-39 alkyl, C3-39 alkenyl, C3-39 alkadienyl and steroidal residues. Aspect 10. A compound of the formula I
where n is 0-8;
R1 is COOH;
R is a lipophilic portion; Y
R 3 together with the carboxyl group to which R3 is attached
denotes a reactive ester or a reactive N-hydroxy imide ester. Appearance 11. A compound in accordance with aspect 10, in
where n is 0 or 1 and R 3 together with the carboxyl group to which R3
is attached designates a reactive N-hydroxy-imide ester. Aspect 12. A compound according to any of the 2 aspects 10-11, wherein R is selected from alkyl of
C3-39, C3-39 alkenyl, C3-39 alkadienyl and residues
steroidal
EXAMPLES Preparation of starting materials Example 1
Preparation of the α-benzyl ester of N-hexadecanoyl-glutamic acid.
The a-benzyl ester of glutamic acid (4.75 g, 20.0 mmol)
was suspended in N-methyl-2-pyrrolidone (100 ml) at 20-25 ° C. Triethylamine (2.53 g, 25.0 mmol) and subsequently 1-hexadecanoylbenzotriazole (7.5 g, 20.0 mmol) was added. The reaction mixture was stirred at 20-25 ° C for 22 hours. To the resulting solution was added 0.2M hydrochloric acid (250 ml). The resulting suspension was cooled to 0 ° C for 3 hours. The product was isolated by filtration, washed with water (50 ml x 4) and dried at constant weight under reduced pressure and at 40 ° C. Yield: 9.15 g (96%) of white material, melting point at 90.0 ° C (peak value) determined by Differential Calorimetry (DSC).
Example 2 Preparation of the N-hexadecanoyl-glutamic acid a-methyl ester. Under the reaction conditions similar to those described in Example 1, the N-hexadecanoylglutamic acid a-methyl ester was prepared, using 8.06 g (50.0 mmol) of the α-methyl ester of glutamic acid. Yield: 17.70 g (88%) of white material, melting point at 95.4 ° C (peak value) determined DSC.
EXAMPLE 3 Preparation of the α-N-Hydrosuccinimide ester of the α-benzyl ester of N-hexadecanoylglutamic acid The α-benzyl ester of N-hexadecanoylglutamic acid (23.78g, 50.0 mmol) was dissolved in tetrahydrofuran (200 ml) at 20-25 ° C. N-hydroxysuccinimide (5.75 g, 50.0 mmol) and subsequently dicyclohexylcarbodiimide (10.32 g, 50.0 mmol) was added. The reaction mixture was stirred at 20-25 ° C for 20 hours. The resulting suspension was filtered and the filtrate evaporated to dryness under reduced pressure. The crystalline residue was dissolved in acetone (100 ml) at 40 ° C and filtered until it was clear. N-heptane (300 ml) was added to the filtrate. The resulting suspension was stirred for 4 hours at 20-25 ° C, then cooled to 0 ° C for 1/2 hour. The product was isolated by filtration, washed with n-heptane (50ml x 3), and dried at constant weight under reduced pressure at 40 ° C. Yield: 23.75 g (83%) of white material, melting point at 98.6 ° C (peak value) determined by DSC. Example 4 Preparation of the? -N-Hydrosuccinimide ester of N-hexadecanoylglutamic acid a-methyl ester.
Under similar reaction conditions as those described in Example 3, the? -N-hydrosuccinimide ester of N-hexadecanoylglutamic acid a-methyl ester was prepared using d.OOg (20 mmol) of N-hexadecanoylglutamic acid a-methyl ester. Yield: 6.45g (65%) of white material, melting point at 106.0 ° C (peak value) determined by DSC.
Example 5 Preparation of the N-hexadecanoylglutamic acid ester-N-hydrosuccinimide. The? -N-hydrosuccinimide ester of the α-benzyl ester of N-hexadecanoylglutamic acid (5.73 g, 10.0 mmol) was dissolved in acetone (100 ml) at 20-25 ° C. 10% palladium on charcoal was added (approximately 0.25 g as dry material). The suspension was stirred under hydrogen until the hydrogen consumption was stopped (290 ml of hydrogen, 45 minutes). The catalyst was removed by filtration, and the filtrate was evaporated to dryness under reduced pressure at 20-25 ° C. The residue was dissolved in acetone (25 ml) at 20-25 ° C and clarified by filtration. N-Heptane (200 ml) was added to the filtrate. The resulting suspension was stirred at 20-25 ° C for 1 hour. The product was isolated by filtration, washed with n-heptane (50 ml x 2) and dried to constant weight under reduced pressure at 40 ° C. Yield: 4.20 (87%) of white material, melting point at 100.8 ° C (peak value) determined by DSC
Preparation of Acylated GLP-1 Analogs Example 6 Preparation of Arg Lys - [N-e- (? -Glu (N-hexadecanoyl))] -GLP- 17-37
Arg 34-GLP-17-37 (5.0 g of frozen so-precipitated peptide material, ca. 0.15 mmol) was dissolved in water (25 ml) at 0-5 ° C. The pH of the solution was adjusted to 12.5 by the addition of 1.0 M sodium hydroxide solution (2.25 ml). After 2 minutes N-methyl-2-pyrrolidone (50 ml) and 1.0 M acetic acid (1.25 ml) were added, maintaining the temperature at 15 ° C. Triethylamine (0.2 ml) and subsequently N-hexadecanoylglutamic acid ester-N-hydrosuccinimide (97.0 mg, 0.20 mmol) were added. After 30 minutes water (50 ml) was added at 15 ° C, and the pH was adjusted to 8.0 by the addition of 1.0 M acetic acid (1.70 ml).
Yield: The reaction mixture was shown to contain 77% (per area) of Arg Lys - [N-e- (? -Glu (N-hexadecanoyl))] -GLP-1 by analytical RP-HPLC. The final purification of the product was obtained by column chromatography.
Example 7 Preparation of Arg Lys - [Ne- (? -Glu-OMe (N-hexadecanoyl))] - 7-37 GLP-1 Under similar reaction conditions to those described in Example 6, Arg34-GLP-17" 37 was acylated using β-N-hydrosuccinimide ester of N-hexadecanoylglutatic acid α-methyl ester Yield: The reaction mixture was shown to contain 64% (per area) of Arg34Lys26- [Ne- (β-Glu (N-hexadecanoyl) )] -GLP-17-37 by analytical RP-HPLC The product could be isolated as a precipitate by adjusting the pH of the reaction mixture to 6.0 using 1M acetic acid.Alternatively the reaction mixture could be used directly as described in the subsequent Example 8
Example 8 Preparation of Arg 34Lys26- [N-e- (? -Glu (N-hexadecanoyl))] -GLP-, 7-37
The reaction mixture containing the product obtained in Example 7 was subjected to basic hydrolysis by adjusting the pH to 12-13 using 1M sodium hydroxide. The temperature of the reaction mixture was maintained at 8-18 ° C. After 2 hours the reaction was complete and the pH of the reaction mixture was adjusted to 7.45 by the addition of 1M acetic acid.
Yield: The reaction mixture was shown to contain 65% (per area) of Arg Lys - [N-e- (β-Glu (N-hexadecanoyl))] -GLP-1 by analytical RP-HPLC.
The final purification of the product was obtained by column chromatography.
Example 9 The following compounds are analogous preparations to the compound of Example 6 and the final purification of the product is obtained by column chromatography. Arg26-34Lys36- (Ne- (? -Glu (N-hexadecanoyl))) -GLP-17"36, Arg26Lys34- (Ne- (? -Glu (N-hexadecanoyl))) -GLP-17 37, and Gly, Arg26'34, Glu37, Lys38- (Ne- (? -Glu (N-hexadecanoyl))) -GLP-l '
It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention
Claims (14)
- R1 is COOR4; 2 R is a lipophilic portion; 3 . 3 R together with the carboxyl group to which R is attached designates a reactive ester or a reactive N-hydroxy imide ester; and R4 is selected from hydrogen, C1-12 alkyl and benzyl, under basic conditions in a mixture of a polar aprotic solvent and water; and 4 (b) if R is not hydrogen, saponifying the ester group 4 of the acylated peptide (COOR) under basic conditions; in order to obtain an N-acylated peptide.
- 2. A method according to claim 1, characterized in that R is hydrogen.
- 3. A method according to claim 1, characterized in that R is selected from C 1-8 alkyl and benzyl
- 4. A method of compliance with any of the claims 1 to 3, characterized in that R together with the 3 carboxyl group to which R is attached designates a reactive N-hydroxy imide ester.
- 5. A method according to any of claims 1 to 4, characterized in that the mixture of the aprotic solvent and water is a 1: 5 to 5: 1 mixture of N-methyl-2-pyrrolidone and water.
- 6. A method according to any of claims 1 to 5, characterized in that the pH of the reaction mixture in step (a) is in the range of 9 to 13.
- 7. A method according to any of claims 1 to 6, characterized in that the temperature of the reaction mixture in step (a) is in the range of 0 to 50 ° C.
- 8. A method according to any of claims 3 to 7, characterized in that the ester of the acylated peptide is saponified at a pH value in the range of 10 to 14.
- 9. A method according to any of the preceding claims characterized in that R is selected from C3_39 alkyl, C3-39 alkenyl, C3-39 alkadienyl and steroidal residues.
- 10. A method according to any of the 2 preceding claims characterized in that R is selected from C7-25 alkyl.
- 11. A method according to any of the preceding claims characterized in that the peptide is selected from GLP-I (7-37) and analogs thereof, exendin and analogs thereof and GLP-2 (1-34) and analogs thereof.
- 12. A method according to any of the preceding claims, characterized in that the peptide is selected from 3-exendin, 4-exendin, Arg26-GLP-1 (7-37), Arg3-GLP-1 (7-37), Val8GLP -l (7-37), Thr8GLP-l (7-37), Met8GLP-l (7-37), Gly8GLP-l (7-37), Val8GLP-l (7-36) amide, Thr8GLP-l (7 -36) amide, Met8GLP-l (7-36) amide, and Gly8GLP-I (7-36) amide.
- 13. A compound of the formula I characterized in that n is 0-8; R1 is COOH; R is a lipophilic portion; R 3 together with the carboxyl group to which R3 is attached denotes a reactive ester or a reactive N-hydroxy imide ester.
- 14. A compound according to claim 3, characterized in that n is 0 or 1 and R together with the carboxyl group 3 to which R is attached designates a reactive N-hydroxyimide ester. 15 A compound in accordance with any of the 13 to 14, characterized in that R is selected from C3-39 alkyl, C3-39 alkenyl, alcadienil of 03-39 and steroidal residues.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP99610019.4 | 1999-03-17 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| MXPA01009209A true MXPA01009209A (en) | 2002-05-09 |
Family
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