EP1373190A2 - Method for the production of carboxylic acid amides - Google PatentsMethod for the production of carboxylic acid amides
- Publication number
- EP1373190A2 EP1373190A2 EP20020735153 EP02735153A EP1373190A2 EP 1373190 A2 EP1373190 A2 EP 1373190A2 EP 20020735153 EP20020735153 EP 20020735153 EP 02735153 A EP02735153 A EP 02735153A EP 1373190 A2 EP1373190 A2 EP 1373190A2
- Grant status
- Patent type
- Prior art keywords
- method according
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
- C07K1/08—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents using activating agents
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C213/00—Preparation of compounds containing amino and hydroxy, amino and etherified hydroxy or amino and esterified hydroxy groups bound to the same carbon skeleton
- C07C213/02—Preparation of compounds containing amino and hydroxy, amino and etherified hydroxy or amino and esterified hydroxy groups bound to the same carbon skeleton by reactions involving the formation of amino groups from compounds containing hydroxy groups or etherified or esterified hydroxy groups
A process for the preparation of carboxamides
The invention relates to a process for the preparation of carboxylic acid amides, particularly peptides.
Carboxamides are usually through the formation of an amide bond CO-N between the carbonyl group of an acid component, such as a carboxylic acid, and the nitrogen atom of an amine component, for example a primary or secondary amine. This bond formation but is referred to as condensation, hereinafter referred to as a clutch.
Of particular importance is the coupling for the peptide synthesis. The aim of the peptide synthesis is the construction of peptides from amino acids such that precisely the desired order of the amino acid building blocks is maintained, achieving a high yield (efficiency) and epimerization - or racemization in the case only of an existing asymmetric carbon atom - are largely or completely avoided during the reaction in favor of higher product purity.
It starts with the synthesis of dipeptides, and as in all the following
Steps is to be ensured that by the two functional groups of the amino acids (or peptides) only occurs in the reaction. shown by blocking the respective other groups by protecting groups, hereinafter referred to symbolically as PG 'and PG' is achieved so that the acylation of the free amino group of an amino acid of the general formula AS,
HN (R) -CH (R) -COOH, (R, R = H, optionally substituted alkyl or aryl group; for example, AS = tryptophan, lysine, asparagine, serine, etc.) only by the carboxy group of another amino acid can take place. Accordingly, the first step of peptide synthesis is the synthesis of partially protected amino acids 1, PG "-N (R") - CH (R ") - COOH, and 2, FFN (R) -CH (R) -CO-PG ' . these are brought in the second step, the coupling step with each other in response, but for which an activation of the carboxy group is required. For this purpose, one converts these usually first in an upstream activation step with activating reagents intermediately in a labile, and especially reactive form, usually an electron-poor species such as an anhydride, ester or halide. This favors nucleophilic attack of the amine component 1.
The reaction of the partially protected, activated components 1 and 2 in the copper plungsschritt in the presence of coupling reagents (condensation reagents) finally leads to the desired dipeptide PG "-N (R") -CH (R ") - CO-N (R,) - CHt ^ CO-PC.
The third step of the peptide synthesis consists in the treatment with the removal of the protective groups, for which specific reagents are introduced, for example,
Hydrohalic acids (and / or trifluoroacetic acid in the case of PG "-Schutz- groups such as the benzyloxycarbonyl (PG '= Cbz), the t-butoxycarbonyl (Boc), the fluoren-9-ylmethoxycarbonyl (Fmoc), triphenylmethyl of Trt) or nitrobenzenesulfenyl group (Nps). The PG' protecting groups of the carboxyl group, for example methyl, ethyl, benzyl, 4-nitrobenzyl and tert-butyl esters, are similar and cleaved by alkaline saponification.
The isolable after removal of one or both protecting groups dipeptide can be used in analog to be carried out reaction steps as the basis for the synthesis of higher (longer) peptides. Depending on the structure of the peptide it may be advantageous to use segment couplings, such as a dodecapeptide - held consecutively in 11 coupling steps - prepare by linking three tetrapeptides, or even be necessary to develop specific synthesis strategies.
Most peptide syntheses are carried out as solid phase synthesis. Under
Solid-phase synthesis or solid phase technique refers to modes of operation in which at least one reactant - is present in solid phase-bound form - in the case of peptide synthesis, the acid or amine component, such as an amino acid or a peptide. This is done by immobilisation of the reactant on suitable carriers such as synthetic resins of various compositions. In this case, either the carboxy group of the amino or the carboxy
Terminus (short: C-terminal / terminal) of the peptide or the amino group of the amino acid or the amino-terminus (short: N-terminus / terminal) of the peptide to be fixed on the carrier. The dosage, reprocessing and separation of reaction products is greatly facilitated. The most well-known solid-phase peptide synthesis introduced by Merrifield and is now largely automated Merrifield technique.
In solid phase synthesis, the C-terminal amino acid unit of the peptide to be synthesized via its carboxy group is usually attached to the insoluble support. All functional groups of the amino acid side chains have to be provided with permanent protecting groups that are stable to the reaction conditions of the subsequent couplings. The temporary protecting group, the first masks the α-amino group during the loading of the carrier is subsequently removed. An excess of a second amino acid wherein the carboxy group of this amino acid is activated with an activating reagent for the amide bond formation is introduced. Following the coupling of the excess is removed by a washing process of reagents and the protective group of the N-terminus of the dipeptide, before the third amino acid is added. This process is repeated until the desired peptide sequence is assembled. In a final step, the peptide is cleaved from the support and the side chain protecting groups are removed. In general, the nature of the side chains and the immobilization are coordinated so that the deprotection and release of the peptide can be made from the solid phase in one step. In addition to the solid-phase technique of dispensing with the immobilization and transition across heterogeneous mixtures such as dispersions, especially suspensions, until a homogeneous phase or a combination of solid and liquid phase technique increases.
As activating reagents have in the coupling of amino acids 1 and 2, with R '= R "= H in addition to classical reagents for the formation of acid chlorides, mixed anhydrides, Pentafluorophenylestem etc. Combinations of differently substituted carbodiimides such as DCC (N, N'-dicyclohexylcarbodiimide) , DIC (di diisopropylcarbodiimide), or EDC (N-ethyl-N '- (3-dimethylaminopropyl) -carbodi- imide-HCl), and a benzotriazole such as HOBt (1-hydroxybenzotriazole) or aza- benzotriazole as HOAt (7-aza -l-hydroxybenzotriazole) proven.
For the special activating combinations DCC / HOAt and DIC / HOAt is known that the use of a weak base such as collidine for
Activation of the acid component and the addition of a stronger base during the coupling step may can greatly improve the efficiency and speed of the overall reaction (LA Carpino, A. El-Faham, Tetrahedron 1999, 55, 6813-6830).
The yield of the clutch to gain with increasing length (height) of the product peptide is significantly more important. So after 4 couplings represents a coupling yield of 95% per coupling for the preparation of a pentapeptide, starting from a first amino acid, a yield of 81%, based on the first amino acid. In the production of the decapeptide yield after 9
Couplings however, only 63%.
Low coupling yields usually occur when hindered
Amino acids complicate the clutch. This is for example the case with amino acids with bulky side chains such as valine (AS: R = iso-propyl), or isoleucine (AS: R = .sert-butyl), or iV-alkyl amino acids, ie, amino acids with N-alkyl, for example, N-methyl amino acids (AS: R '= Me).
N-alkyl amino acids are part of a variety of biologically active peptides (JM Humphrey, AR Chamberlin, Chem. Rev. 1997, 97, 2243-2266). Although numerous highly active activating reagents such as the iminium derived from HOBt or HOAt or uronium salts, such as BOP-Cl or the expensive HATU, are known so far succeeded yet no breakthrough to the problem of solid-phase coupling of hindered N-alkyl amino acids. For this reason it was previously instructed to circumvent this problem by using segment coupling and selective methylation at the resin, such as in the total synthesis of cyclo peptide cyclosporin A, which are methylated 7 of 11 amide bonds ((a) RM Wenger, He / v Chim Acta, 1983, 66, 2672;.. (b) WJ Colucci, RD Tung, JA Petri, DA Rich, J Org Chem 1990, 55, 2895;.. (c) P. Raman, SS Stokes, YM Angell , GR Flentke, DA Rich, J. Org. Chem. 1998, 63, 5734 to 5735).
Li et al activating reagents BEMT and BEP and their use in the total synthesis of cyclosporin described O ((a) P. Li were J. Cheng Xu,
Tetrahedron Leu. 1999, 40, 8301 to 8304; (B) P. Li, J. Cheng Xu, Chem. Lett. 2000, 204-205; (C) P. Li, j. Cheng Xu, J. Org. Chem. 2000, 65, 2951-2958). When used as a solid-activating BEMT and BEP are clearly superior to the reagent ΗATU, but still not efficient enough for use in the solid phase coupling N-methylated amino acids. Especially in the case of
Coupling two N-methyl amino acids with bulky side chains, for example Fmoc- MeVal-OH to MeVal-peptidyl resin, the yields are generally low own According to experiments with less than 30%.
From WO 00/02898 a method for peptide synthesis is well known with the activating triphosgene and collidine, which with bulky side chains higher coupling yields as HATU, BOP-Cl, TFFH or other reagents provides for the coupling of N-methyl amino acids (E. Falb, T. Yechezkel, Y. Salitra, C. Gilon, J peptide Res. 1999, 53, 507-517).
The coupling of Fmoc-protected amino acids to N-alkyl-amino acids which are linked to a peptidyl-resin Rink amide residue, this is done as follows: A Fmoc-protected amino acid is reacted with 1, 65 eq. (Eq. = Molar equivalents) of triphosgene and 14 eq. collidine reacted in THF and, after 1 min activation time
1 h of reaction at 50 ° C with the bound on the peptidyl-resin residue amino acid.
This method also has a number of disadvantages. Although the coupling of sterically hindered N-methyl amino acids succeeds well, the higher for the synthesis of
However, peptides essential coupling yields of over 90% can not be achieved. The required high reaction temperature is impractical for solid phase synthesis and leads to an increased extent to Νebenreaktionen. The Rink amide resin used requires strongly acidic deprotection conditions which result in the case of many N-methylated peptides known to decompose
(J. Urban, T. Vaisar, R. Shen, MS Lee, Int. J. Peptide Protein Res. 1996, 47, 182- 189). It is an object of the present invention is to provide a process for the preparation of carboxamides which enables the coupling of sterically hindered amino acids in high yields.
The object is achieved by a special activating reagent in combination with the use of certain bases both together with the acid component in the activation step as well as together with the amine component for the coupling step.
The invention relates to a process for the preparation of carboxylic acid amides, particularly peptides, having from an acid component in the form of at least one carboxyl group-containing compound and an amine component in the form of at least one primary or secondary amino group-containing compound, in which
(I) the amine component is initially introduced together with a coupling base in the form of an organic base having at least one nitrogen atom in a solvent,
(Ii) the acid component with an activating reagent in form of a carbonate of the formula I,
O = C (OX) 2 (I)
having both the same or different, separate or joined together, electron-withdrawing groups X,
whose monohalide of the formula II,
O = C (-OX) Y (II) in which has as in formula I X have the same meaning and Y represents a halogen atom,
or its dihalide of the formula III,
O = CYY '(III)
are in the Y and Y 'independently represent each a halogen atom,
and an activation base in the form of an organic base is added with at least one nitrogen atom in a solvent,
(Iii) the mixture, the acid component as in (ii) to which the amine component-containing mixture according to (i) is given.
The inventive method is highly efficient and allows couplings that took place with the previously known methods only in very low yields and therefore were not suitable in particular for the solid phase synthesis of peptides. Thus, efficient solid and liquid phase synthesis is possible by the many naturally occurring peptides with N-alkylated amino acids and hindered the first time. The process is simple to carry out, the reaction proceeds very rapidly and is completely epimerization even at high steric hindrance. Also possible to dispense with the harmful heating in the reaction procedure. Furthermore, it allows the use of cheaper coupling reagents such as
Triphosgene, while most of the known modern activating reagents are very expensive. Thus, the method is also economically universally for forming amide bonds in the solid phase as well as in liquid phase or solution, especially in cases where the conventional methods for forming amide bonds are not efficient enough. The present method is suitable especially for the preparation of many biologically active N-alkylamides, whose preparation is often problematic and expensive reagents required. Likewise, the present process for the production of numerous N-methylated cyclopeptidischer biologically active natural products such as cyclosporins tentoxins, dolastatins, jaspamides, Didemnide, nodularins and a number of other representatives of (JM Humphrey, AR Chamberlin provides, Chem. Rev. 1997 97 , 2243-2266). It can also be used for functionality screening of peptides by - are installed instead of a new non-N-alkyl-substituted amino acids, N-methyl amino acids - for the targeted elimination of specific hydrogen bonds. N-methyl peptides are also more hydrophobic and more stable to proteolytic enzymes, which can improve their bio availability and their therapeutic potential.
The acid component and / or the amine component is preferably an amino acid or a peptide, the rest of carboxyl and / or amino groups are protected.
The acid component and the amine component are used in a ratio, based on the molar amount of at least 1 to 1, preferably inserted from 1: 1 to 10: 1, especially from 1 to 1 to 5: 1.
In a preferred embodiment of the method either the acid component and the amine component are the same or different amino acids, or is an amino acid, the acid component and the amine component is a peptide or an amino acid, the amine component and the acid component a peptide, wherein on at least one carboxy group and at least one primary or secondary amino group, existing more carboxyl or primary or secondary amino groups are protected.
It is particularly preferred that the amino group of the amine component, a secondary amino group and / or bonded to the α-carbon atom of the acid component amino group is a secondary amino group, in particular the amino group of amine component and the protected peptide-linked amino group or the acid component are both N-alkylated, preferably independently of one another N-alkylated with a methyl, ethyl, propyl, iso-propyl, Cyclolhexyl- or benzyl group or one of these groups which is substituted with one or more amino and / or carboxy groups, where these amino and carboxy groups are in turn protected by appropriate protective groups.
When the activating reagent in form of a carbonate of Formula I, the unit (OX) 2 in the case of a separate electron withdrawing groups X, two separate groups
-OX, whereas in case it represents interconnected electron-withdrawing groups X a unit -OX XO, for example in a 1,3-di- oxolan-2-one derivative.
As activating reagent is usually used a carbonate of the formula I in which one or both groups X are, independently from each other for a group -N Y CH n, where n one of the numbers 1, 2 or 3 and Y n for one, two or three identical or different halogen atoms is, stand, or a halogenated 1,3-dioxo lan-2-one derivative, the four hydrogen atoms in 4 and 5 position in whole or in part by one, two, three or four identical or different halogen atoms are substituted or a monohalide of the formula II in which X is a group CH 3. n Y n, where n represents one of the numbers 1, 2 or 3 and Y n is one, two or three identical or different halogen atoms, stand, or a dihalide of the formula III.
Preferred halogen atoms are fluorine, chlorine and bromine, in particular chlorine, and in the case of two or three bound to a carbon atom of halogen atoms, these are preferably the same. Accordingly, the groups X in formulas I and II, independently of one another, in particular one of the groups CC1 3, CF 3, CBr 3, CHC1 2, CHF 2, CHBr 2, CHI 2, CH 2 C1, CH 2 F or CH 2 Br and are forthcoming ferred dihalides of the formula III O = CF 2, CCl 2 = O (phosgene), and O = CBr. 2 Preferably, the activating triphosgene (O = C (-OCCl) 2, bis (tri chloromethyl) carbonate, BTC), diphosgene (O = C (-OCCl 3) Cl), phosgene (O = CCl 2) and / or 4,4,5, 5-tetrachloro-l, 3-dioxolan-2-one, and particularly preferably triphosgene.
The acid component and the activator are typically at a ratio, based on the molar amount employed of at least 1 to 1, preferably from 1: 1 to 4: 1, in particular from 2: 1 to 3: 1. When using triphosgene as the activating reagent is a ratio of 3: 1, diphosgene of 2: more preferably 1: 1 and with phosgene and halogenated dioxolanones of Figure 1.
The coupling base and the activating base are usually independently selected from the group consisting of pyridine, and the mono- or poly-alkyl-substituted pyridine derivatives, preferably from the collidines, 2,4,6-tri-tert.-butylpyridine, 2,6-di-tert-butylpyridine, 2,6 -Ditert-butyl-4-methylpyridine, 2,6-
Dimethylpyridine, 2,3,5,6-tetramethylpyridine, 2-methylpyridine and pyridine, or from the group of trialkyl amines, preferably diisopropylethylamine (DIEA), tri- isopropylamine, N-methylmorpholine, triethylamine. The collidines are the different trimethylpyridines and Ethylmethylpyridine, such as 2,3,5-collidine and in particular 2,4,6-collidine. Equally, mixtures of two or more bases may be used.
The coupling efficiency can be increased by the careful selection of coupling and / or activating base. In a preferred embodiment of the method, the coupling base used together with the amine component
2,4,6-collidine, pyridine, triethylamine or a sterically hindered trialkylamine, preferably a hindered trialkylamine, especially diisopropylethylamine or triisopropylamine, more preferably diisopropylethylamine. In a likewise preferred embodiment of the method, the activation base used together with the acid component is a sterically hindered 2,6-mono- or poly-alkyl-substituted pyridine derivative, preferably butylpyridine, 2,4,6-collidine, 2,4,6-Tritert-, di-tert-butylpyridine, 2,6-di-tert-butyl-4-methylpyridine, 2,6-di-methylpyridine or 2,3,5,6-tetramethylpyridine, particularly preferably 2,4,6-collidine.
In a further preferred embodiment of the method guide the two foregoing preferred embodiment are combined together. Which reminds the
Increasing Kupplungseffϊzienz especially high when a sterically with the sterically hindered pyridine derivative, preferably 2,4,6-collidine or 2,4,6-tri-tert-butyl pyridine, in particular 2,4,6-collidine, as activating base hindered trialkylamine, in particular DIEA, is combined as a coupling base. 2,4,6-tri-tert butylpyridine the coupling efficiency increases as a rule, less than 2,4,6
Collidine, however, results in a homogeneous solution during the activation in THF.
The coupling base and / or the activation base are usually used in a ratio to the amine component, based on the molar amount of at least 2 to 1, preferably from 4 to 1 to 30 to 1, in particular from 8: 1 to 20: 1, more preferably from 12 to 1 to 16 to 1, are used.
The solvent in (i) and (ii) are independently selected from the liquid at the process conditions of organic and inorganic solvents, usually from tetrahydrofuran, 1,4-dioxane, tetrahydropyran,
Ethylene glycol dimethyl ether, diethylene glycol dimethyl ether, trichloromethane, 1,3-di- chloropropane, 1,2-dichloroethane, nitromethane or a mixture of two or more thereof, preferably tetrahydrofuran, 1,4-dioxane, tetrahydropyran, ethylene glycol dimethyl ether or diethylene glycol dimethyl ether or a mixture of two or more thereof , in particular tetrahydrofuran.
Particularly preferred are the solvents as in (i) and (ii) are identical.
In a particularly preferred embodiment of the method lung base as couplings DIEA as activating reagent triphosgene as the activating Base 2,4,6-
And collidine as the solvent in each of tetrahydrofuran (THF) was used, and in particular, based on the amine component, 8 eq. DIEA, 1.15 eq. Triphosgene and 10 eq. 2,4,6-collidine. The acid component is preferably present in a ratio, based on the molar amount of triphosgene used of 3 to 1, that is, based on the amine component, about 3.5 eq. the acid component.
The process is usually carried out at a temperature of 15 to 30 ° C, preferably from 18 to 25 ° C, especially from 20 to 22 ° C. According to (ii) 3 minutes is sufficient, preferably from 10 s to 2 min, particularly 30 sec to 1 min. After the addition of (iii) is preferably allowed for a period of 5 min to 4 h to react. Here is usually shaken or stirred.
In a particular embodiment of the method, the amine component or the acid component is reversibly bound to a solid phase, preferably on to a resin, in particular to a trityl resin, Wang-polystyrene resin or Rink amide-MBHA resin, and particularly preferably TCP resin:
Which are based on a benzyl alcohol to a support Wang resins and Sasrin resins are less suitable for the coupling of sterically hindered amino acids, as on the
Dipeptide a very high tendency may occur in the formation of diketopiperazines, which is associated with high yield losses. Particularly suitable for the sequential structure of N-methyl peptides is the TCP (trityl chloride-polystyrene) resin, a by the company RepChem Goldammer & Clausen (D-72076 Tübingen) available trityl. The TCP resin is an excellently balanced resin, in terms of stability and removability, and prevented by the bulky trityl linker the diketopiperazine formation at the dipeptide. The TCP resin is preferably such as that available from the company RepChem Goldammer & Clausen commercial product with little or even not contaminated with Friedel-Crafts Νebenprodukten which may prove to be problematic in solid phase synthesis.
The inventive method is ideally suited for the attachment of an N-methylated Fmoc protected amino acid to an N-methylated or non-methylated amino group of a peptidyl-resin suitable.
In carrying out the process as a liquid phase synthesis is used in the
Activation is preferably at least twice the amount of N-protected amino acid, based on the molar amount of activator used to prevent unreacted activator may take a decomposing effect on the coupling reaction if necessary.
In a particularly preferred embodiment, the liquid-phase synthesis, are based on the molar amount of the amine component,
(I) 1 eq. Amine component, in particular carboxy-protected amino acid or peptide, together with a clutch base, in particular DIEA, in a
in particular THF, is submitted solvent,
(Ii) 1.1 eq. Acid component, in particular Fmoc-protected amino acid with an activating reagent, in particular triphosgene, and an activation approximately base, in particular 2,4,6-collidine, in a solvent, in particular
THF, is added, (iii) optionally the mixture, the acid component as in (ii) to which the amine component-containing mixture according to (i),
said coupling base and activation base in an amount of at least 2 eq., preferably 4-30 eq., in particular 8-20 eq., particularly preferably 12 to 16 eq., for example, of 14 eq., can be used.
The liquid-phase synthesis is also highly sterically hindered cases, such as the coupling of MeVal with MeVal, extremely rapidly, in high yields and epimerization.
The present method lends itself to automation, for example in a peptide synthesizer.
Example 1 solid-phase coupling with triphosgene
Starting (from the resin-bound N-methylated amino acids mile He =
L-isoleucine) or MeLeu (Leu = L-leucine) were permethylated pentapeptide MeVal-MeVal-Sar-MeVal-Melle (Val = L-valine, Sar = L-sarcosine) or the permethylated tetrapeptide MeLeu-MeLeu-MeVal -MeLeu prepared by four and three clutches and thereby obtained in a product purity of about 98%.
Based on the amino functions present on the resin was carried out with the following surpluses of each coupling (eq = molar equivalent.): Fmoc-amino acid: 5 eq. Triphosgene: 1.65 eq. Collidine: 14 eq.
DIEA: 14 eq.
The concentration of the reaction solution was, based set to the Fmoc amino acid to 0.14 mol /. 1 By triphosgene as a stock solution in anhydrous tetrahydrofuran (THF (abs)) was prepared by adding 13.7 mg triphosgene per 1 ml
THF (abs) were dissolved. From this solution 7.14 ml was required per mmol of Fmoc-amino acid.
Procedure: The dried, pre-assigned resin (TCP resin by the company RepChem, sleeps about 0.4 mmol of amine per gram of resin component) was dissolved in a
Plastic syringe with plastic frit and plugs with DIEA / THF (abs) pretreated. To this was added per 100 mg of resin, added 100 .mu.l of THF (abs) and 100 .mu.l DIEA to the resin. The preswelling time up to the addition of the other reagents ranged from 5 to 10 minutes.
The Fmoc-amino acid (5 eq.) Was dissolved in in a polypropylene test tubes with caps in the triphosgene / THF solution (1.65 eq. Triphosgene / 7.14 ml per mmol of amino acid) while swirling. Once the amino acid was completely dissolved, 2,4,6-collidine (14 eq. 371 ul per mmol of amino acid), or 2,4,6-tri-tert-butyl pyridine (14 eq.) Was added, wherein in the case of collidine came to the formation of a colorless precipitate. The mixture was s converted pivots for about 30 to 60 in order to mix all of the reagents and to activate the amino acid.
Then the suspension was added with a Pasteur pipette to the pre-swollen resin. The syringe was capped and shaken on a shaker for 5 to 30 min at 20 ° C. Thereafter, the resin was washed successively with THF, methanol (MeOH), dimethylformamide (DMF), MeOH, dichloromethane (DCM), MeOH each washed 3 times.
To illustrate the performance of the method following model coupling was performed using three different coupling methods
performed (HATU / DIEA / DCM, BEMT / DIEA / DCM and triphosgene / collidine / DIEA / THF). As a model system, the coupling of two sterically hindered N-methylated amino acids (MeVal) has been selected, said serving as the amine component as part of the tripeptide MeVal-Melle-Sar Menal the resin (P) was bound. As the acid component respectively were 5 eq. Fmoc-MeVal used and the reaction is stopped after 30 min in each case.
In the key step, the following three different coupling conditions were employed:
1) HATU / DIEA / DCM, 30 min.
2) BEMT / DIEA / DCM, 30 min.
3) triphosgene collidine / DIEA / THF, 30 min.
Subsequently, the dipeptide with 1% trifluoroacetic acid (TFA) in dichloromethane (DCM) was cleaved from the resin.
Fmoc- eval - -Meval - mile - Sar - P
Fmoc -Meval ■ -Meval - mile - Sar-OH
As the basis of the HPLC chromatograms of the crude products (Fig. 1) is visible, performs the reagent HATU commonly used for problem clutches in the case shown to no transactions with BEMT the conversion is not satisfactory. In contrast, achieved when using the method according to the invention already after 30 minutes a high conversion. Repetition of this clutch under the same conditions lead to quantitative product formation. In the present case could be explicitly demonstrated that no epimerization take place, ie it is solely formed by the L-tetrapeptide (see Example 3).
Fig. I: HPLC chromatograms of the reaction products of the three reactions under various conditions (UV detection, λ = 214 nm). The curves for the environmental move with you BEMT and triphosgene are added in perspective shown (ie the baseline of all three curves starts at 0 AU).
50 mixture of L- and D-FmocMeVal coupled to the tripeptide, the resulting mixture of the two diastereomeric tetrapeptides DLLL- and LLLL isomer by RP: To find out if 2 epimerization occurred in the model coupling of example, a 50 was HPLC (Reverse phase high Pressure Liquid Cromatography) and separated by means of UV detection of the chromatogram
Diastereomeric mixture recorded (Fig. 2). RP-HPLC under the same conditions, the HPLC chromatograms (UV detection) of the isolated DLLL-isomer and the product of the coupling of pure L- FmocMeVal (Fig. 4) were recorded (Fig. 3). In the latter case, the signal of the LLLL diastereomer was exclusively found (Fig. 4), that there was no epimerization on.
Fig. 2: RP-HPLC chromatogram of a mixture of DLLL- and LLLL isomer (mAU = 10 "3 absorbance units).
Fig. 3: RP-HPLC chromatogram of the DLLL isomer.
Fig. 4: RP-HPLC chromatogram of the LLLL isomer.
A) Example 4: Liquid-phase synthesis of a dipeptide
The protected N-methyl-L-amino acids FmocMeVal-OH and MeVal-OBn were coupled to the dipeptide FmocMeVal -MeVal-OBn.
These were 1.1 eq. of Fmoc-amino acid in THF in the presence of 14 eq. 2,4,6-collidine with 0.5 eq. Triphosgene were added and the resulting activation solution into the solution of 1 eq. the carboxy-protected amino acid and 14 eq. DIEA in THF.
The reaction took 5 minutes to complete the reaction. There were no by-products to even took place epimerization.
Example 5 Synthesis of cyclosporin O
First, as a preliminary step, the linear undecapeptide having the sequence
H-Nva-Sar-MeLeu-Val-MeLeu-Ala-D-Ala-MeLeu-MeLeu-MeVal-MeLeu- (TCP resin)
built on TCP-resin and that after cleavage from the resin for cyclosporin O (IV) is cyclized.
1) Construction of linear Undecapeptid- precursor:
The assignment of 150 mg of TCP-resin was carried out with 55 mg of Fmoc-MeLeu-OH (3 eq) in
77 ul DCM with 3 eq of DIEA for 3 hours. Then the undecapeptide was set up with ten clutches. After each coupling the chloranil test or the Kaiser test was performed (Fmoc solid phase synthesis, WC Chan, PD White (ed.), Oxford University Press, 2000, p 61 ff). In case of a negative test result was coupled again.
In addition to the inventive couplings with triphosgene as activating clutches (HOAt) were carried out in special synthesis sections in addition to the activating combination of diisopropylcarbodiimide (DIC) and hydroxyazabenzotriazole. The use of these reagents was carried out in the following manner: 3 eq. the N-protected amino acid were each dissolved in a solution of 3 eq. HOAt and 3 eq. DIEA dissolved in CH 2 C1. 2 were 3 eq to this solution. DIC given. After 5 minutes 10-bis pre-activation, the reaction solution was added to the peptidyl resin. The reaction time was 12 hours. The clutches with triphosgene were carried out as described in Example 1, the reaction time was respectively 2.5 or 3 hours.
The couplings were broken down as follows performed, among others, the activating reagent and the reaction time is given in hours:
1.) MeVal-MeLeu- (TCP resin): triphosgene, 3 h:
IR checking shows Fmoc band, chloranil test positive • ^ recoupling on triphosgene, 3 h: chloranil test negative
2.) MeLeu- -Meval: triphosgene, 3 h:
Chloranil test is negative, HPLC: 100% area product peak
3.) MeLeu - MeLeu: triphosgene, 3 h:
Chloranil test is negative, HPLC: 99% area product peak 4) D-Ala-> MeLeu: triphosgene, 3 h:
Chloranil test positive, HPLC:. 10% area product peak ■ »subsequent coupling with HOAt / DIC, 12 h: chloranil test is negative, HPLC: 95% area product peak
5.) Ala-D-Ala: triphosgene, 3 h:
Kaiser test pos. HPLC: 80% area product peak ■ subsequent coupling with HOAt / DIC, 12 h: Kaiser test is negative, HPLC: 98% area product peak
6.) MeLeu-> Ala: triphosgene, 3 h: Kaiser test negative
7.) ^ Val- MeLeu: triphosgene, 3 h: chloranil test positive, HPLC: 60% area product peak
• * ■ subsequent coupling with HOAt / DIC, 12 h: chloranil test negative
8.) MeLeu -> Val: triphosgene, 3 h: Kaiser test negative
9.) Sar MeLeu: triphosgene, 2.5 h: chloranil test negative
10.) NVA> Sar: triphosgene, 2.5 hours:
Chloranil test is negative, HPLC: 96% area product peak
The tetrapeptide Fmoc-MeLeu-MeLeu-MeVal-MeLeu-OH was obtained at a purity of over 99% according to the invention exclusively by couplings with triphosgene, which were carried out as described in Example. 1 Only the first clutch had it be repeated once. The HPLC spectrum of the tetrapeptide is shown in Fig. 5.
According to the invention the coupling of the 5th residue (Fmoc-D-Ala-OH) with triphosgene, HPLC indicated only 10% conversion. However, the reaction could by additional
Use of HOAt / DIC coupling are led to complete conversion.
The tenth clutch shows that the coupling of the invention with triphosgene a non-N-methylated amino acid to a N-methylated can proceed almost quantitatively.
(FIG 6 s HPLC spectrum..) With the combination of the invention with triphosgene clutches and couplings with HOAt / DIC linear unprotected Undecapeptid- precursor of cyclosporin was dissolved in a HPLC yield of 90% was obtained.
2) cyclization and purification
For the removal of the linear peptide from the resin hexafluoroisopropanol was used. The crude peptide was after freeze-drying directly and without further
Workup cyclized. The cyclization was carried out in dichloromethane with HOAt, EDCI and DIEA over a period of 16 hours. (. HPLC spectrum: see Fig. 7) The crude yield was about 75 to 80%.
3) calculation of yield and purity control
The loading of the resin with the first amino acid was 0.4 mmol / g resin. There were used 150 mg of resin, an amount of 60 .mu.mol peptide on the resin corresponds to (theoretical yield of linear precursor of cyclosporine O (M = 1160.60) assuming a conversion of 100% per step: 69.6 mg). After carrying out the entire sequence of a crude product yield of linear (M = 1178.62) was obtained from 36.0 mg (30.5 .mu.mol, 50.8%) was obtained.
Subsequently, 10.0 mg were cyclized (8.5 .mu.mol) of crude product. The yield was 8.1 mg (7.0 .mu.mol, 82.4%). The total yield of crude product of
thus cyclosporin O is 41.8%.
The obtained 8.1 mg of crude product was purified by preparative HPLC to give 2.9 mg (2.5 .mu.mol, 35.7% of crude product) was obtained. The overall yield in the production of cyclosporin in relation to the first resin loading was thus 14.9%.
This yield is within the limits of most liquid-phase syntheses were obtained for different cyclosporin analogs. Η-NMR confirmed the identity of the synthetic cyclosporin O with the natural substance. There were no
Diastereomers found. The mass spectrum of the purified O cyclosporine is shown in Fig. 8.
Fig. 5: HPLC chromatogram of the Fmoc-protected tetrapeptide Fmoc-MEL MeL MeV MeL-OH (UV detection: λ = 214 nm)
Fig. 6: HPLC chromatogram of the deprotected linear Undecapeptides (UV detection: λ = 214 nm)
Fig. 7: HPLC chromatogram of the crude product of the cyclization reaction to
Cyclosporin O (UV detection: λ = 214 nm).
Fig. 8: Mass spectrum of the purified cyclosporin O. Example 6 Synthesis of A Omphalotin
With the inventive method for the first time succeeded in the total synthesis of omphalotin A. omphalotin A is a Cyclododecapeptid with a high nematicidal action, especially against the important plant-pathogenic
Nematode Meloidogyne incognita (A. Mayer, H. Anke, O. Stemer, Nat Prod Lett 1997, 10, 25-32;... O. Sterner, W. Etzel, A. Mayer, H. Anke, Prod Nat.. Lett 1997, 10, 33-38;. WO 97/20857).
First, as a preliminary step, the linear dodecapeptide having the sequence
built on TCP resin and cyclized this after cleavage from the resin for Omphalotin A (V).
1) Construction of linear dodecapeptide precursor
The assignment of 2 g of TCP resin was carried out with 733 mg of Fmoc-Melle-OH (3 eq) in 15 mL DCM with 1 mL of DIEA (3 eq) over 3 hours. It has been found with a loading of 0.56 mmol / g.
Then the dodecapeptide was built with eleven clutches. After each coupling the chloranil test or the Kaiser test was performed (Fmoc solid phase synthesis, WC Chan, PD White (ed.), Oxford University Press, 2000, p 61 ff). In case of a negative test result was coupled again. In
Complement to the inventive coupling with triphosgene were special synthesis sections couplings with diisopropylcarbodiimide (DIC) and hydroxy azabenzotriazole (HOAt) performed. The use of these reagents was carried out in the following manner: 3 eq. the N-protected amino acid was dissolved in a solution of 3 eq. HOAt and 3 eq. DIEA dissolved in CH 2 CI. 2 were 3 eq to this solution. DIC given. After 5-10 min. Preactivation the reaction solution was added to the peptidyl resin (reaction times as indicated).
The couplings were broken down as follows performed, among others, the activating reagent and the reaction time is given in hours:
1) coupling MeVal> mile: triphosgene (as Example 1), 2.5 h: chloranil test negative.
2.) coupling Sar- ^ MeVal: triphosgene (as Example 1), 4 h:
Chloranil test negative
3.) coupling -Meval - San triphosgene (as Example 1), 3 h: chloranil test negative 4.) MeVal -Meval: triphosgene (as Example 1), 3 h:
Chloranil test is negative, HPLC 96% area product peak (after Fmoc deprotection).
5.) Ile -Meval: HOAt / DIC, 16 h:
Chloranil test positive, HPLC: 80% area product peak - "post-coupling with HOAt / DIC, 20 h: Kaiser test negative
6.) -Meval - He: triphosgene (as Example 1), 1 hour:
Kaiser test negative, HPLC: 95% area product peak.
7.) Trp ^ -Meval: HOAt, h 18:
Chloranil test minimally positive, HPLC: 93% area product peak.
8.) Sar> Trp: triphosgene (as Example 1), 1 hour:
Kaiser test negative, HPLC: 93% area product peak.
9.) MeIle-> Sar: triphosgene (as Example 1), 1 h: chloranil test is negative, HPLC: 96% area product peak.
10.) Val »Mile: HOAt / DIC, 16 h:
Chloranil test positive, HPLC: 85% area product peak, ■ post-coupling with HOAt / DIC, 15 h: chloranil test negative, HPLC: area product peak> 90% (integration not possible as peak at the stop of the detector)
11.) Sar> Val: triphosgene (as Example 1), 1 hour:
Kaiser test negative, HPLC: 93% area product peak.
(All HPLC purities are for Fmoc-deprotected peptides, λ = 214 nm) 2) cyclization and purification
For the removal of the linear peptide from the resin hexafluoroisopropanol was used. The crude peptide (HPLC spectrum: s 9, ESI mass spectrum:.. S Fig..
10) was cyclized by freeze-drying directly and without further workup. There are four solid phase runs 1.29 g (0.965 mmol) of crude product used in linear dodecapeptide for the cyclization. The cyclization was performed in dichloromethane with HOAt, EDCI and DIEA over a period of 16 hours transit. (. HPLC spectrum: see Fig. 11) The crude yield was about 78%.
3) calculation of yield and purity control
The loading of the resin with the first amino acid was 0.56 mmol / g of resin. There are used a total of 2.0 g of resin, an amount of 1.12 mmol peptide on
Corresponds resin (theoretical yield of linear precursor of A Omphalotin (M = 1318.77) assuming a conversion of 100% per step: 1.48 g). After carrying out the entire sequence of a crude product yield of linear (M = 1336.78) was obtained from 1.336 g (1.0 mmol, 89.2%) was obtained.
Then 1.29 g (0.965 mmol) of crude product, divided into four equal batches were cycled. The yield was 1.11 g (0.842 mmol; 87.2%). thus the overall yield of crude product of omphalotin A is 77.8%.
70 mg of crude product was purified by preparative HPLC to give 24.5 mg
were obtained (HPLC spectrum: s Fig. 12.) (35.0% of the crude product 18.6 .mu.mol). The overall yield in the production of Omphalotin A with respect to the first resin loading was thus 27.2%.
Η-NMR spectroscopy confirmed the identity of the synthetic Omphalotin A with the natural substance (Fig. 13) (NMR data published in: O. Sterner, W. Etzel, A. Mayer, H. Anke, Nat Prod Lett 1997th.. , 10, 33-38). The mass spectrum of the purified Omphalotin A is shown in Fig. 14.
4) Racemisierungskontrolle of omphalotin A (OMA)
The racemization were carried out by the method of König (W. King, I. Benecke, N. Lucht, E. Schmidt, J. Schulze, S. Sievers, J. Chromagogr. 1983, 279, 555-562). To this was derivatized after total hydrolysis of the peptide in DCI / D 2 O with isopropyl isocyanate and measured the derivatives on a chiral GC-phase with GC / MS. The hydrolysis in deuterated solvent allows the
Adjustment of results for the percentage of epimerization, which was caused by the hydrolysis itself, since these derivatives contain a deuterium atom, and due to their order 1 Da mass shifted in the GC / MS analytical system can be neglected.
N-Me-D-MeVal N-Me-D-Melle N-Me-D-Melle
Natural Products 0.86% 1.76% -
Linear dodecapeptide 0.66% 0.97% -
OmA crude 0.76% 15.4% 30%
OmA purified 0.31% 6.9% 14%
The analysis of the epimerization of clearly shows that the coupling method is almost completely without racemization se. However, during the cyclization strong epimerization occurs. The resulting false diastereomer can be purified by column is not completely separated and thus contaminate the
Final product (see FIG. HPLC chromatograms of Fig. 11 and 12, the wrong diastereomer eluted even with isocratic run as a shoulder shortly after the Omphalotin A). Fig. 9: HPLC chromatogram of the linear, deprotected dodecapeptide (crude product after cleavage from the resin; Example 6).
Fig. 10: ESI mass spectrum of the linear, deprotected dodecapeptide (crude product after cleavage from the resin; Example 6).
FIG. 11: HPLC chromatogram of the crude product of the cyclization reaction (Example 6).
Fig. 12: HPLC chromatogram of the purified final product, Omphalotin A
Fig. 13: Η-NMR spectrum (700 MHz, CD 3 OD) of the purified final product, Omphalotin A (Example 6).
Fig. 14: ESI mass spectrum of the purified final product, Omphalotin A (Example 6).
Example 7 Improved Synthesis of Omphalotin A (V)
To avoid the problem of the epimerization of the C-terminal amino acid during cyclization, the optically active amino acid Fmoc-sarcosine was coupled as the first amino acid on the carrier polymer. Thereto, 200 mg of TCP resin (substitution 1.04 mmol / g) were added to a solution of 1 eq Fmoc-Sar-OH and 3 eq DIEA in DCM (abs) and the suspension was shaken for 3 h.
Thereafter was added about 0.5 mL of methanol to the suspension in order to cap the remaining Tritylchloridfunktionen as a methyl ether. An Fmoc determination gave an occupancy of 0.58 mmol / g. Taking into account the theoretical mass increase of the resin of about 40 mg, this means that on the synthetic resin 0.14 mmol Fmoc-sarcosine were immobilized. In relation to this amount of substance following reagents surpluses were used for BTC coupling:
Fmoc amino acid: 3.5 eq
Triphosgene: 1.15 eq
Collidine: 10.0 eq
DIEA 8.0 eq
Triphosgene was prepared as a stock solution having a concentration of 61.5 mmol / 1 (corresponding to 18.27 mg of BTC per milliliter of THF (abs)), by the per mmol
Amino acid 5.36 ml was used. This results in an amino acid concentration of 0.19 mol /. 1
The basic procedure for the triphosgene is analogous to Example 6, with two exceptions:
1) The addition of the now almost halved amount of DIEA to the deprotected peptidyl resin was carried out until immediately before addition of the activating solution (Fmoc-amino acid, triphosgene, collidine in THF (abs)).
2.) The Fmoc-deprotection with piperidine the peptidyl resin was kept as short as possible time, ie the Fmoc-protected resin was 1 x 3 min, and 1 x 8 min with 20% piperidine / DMF.
Both of these measures should with the contact of the Fmoc-deprotected resin
minimize base to prevent a base-catalyzed nucleophilic attack of the free amino terminus of the trityl ester of sarcosine and cyclizing the resulting cleavage. This side reaction, which leads to the level of the dipeptide to the above-mentioned formation of diketopiperazines is responsible for the failure of earlier attempts at synthesis of omphalotin A and shorter peptide segments with sarcosine as a C-terminal amino acid using the triphosgene, presumably.
In some cases, the application of HOAt coupling to the application, which was carried out as in Example 6 was.
Furthermore, the HATU coupling has been used in some cases. In this procedure was as follows: to be coupled Fmoc-amino acid (3.5 eq) was weighed together with HATU (3.5 eq) and in a minimum amount of DCM (abs) / DMF (abs) (1: 1) solved. To the solution was added DIEA (7 eq) and left to stand for 15 minutes for preactivation. Thereafter, this solution was pre-swollen directly to the in DMF (abs), optionally deprotected peptidyl resin and shaken for the specified time. The reaction solution was filtered off with suction and the resin washed with DMF, DCM, DMF, DCM, MeOH (3x each) and dried.
The changes described allowed the synthesis of optically high purity Omphalotin A starting with Fmoc-Sar occupied TCP resin. In this case, each of the three occurring in the target molecule Sarcosine was used as the C-terminal amino acid, resulting in the following three different linear precursor molecules:
a.) H-Trp-MeVal-Ile-MeVal-MeVal-Sar-MeVal-Melle-Sar-Val-Melle-Sar-OH b.) H-Val-Melle-Sar-Trp-MeVal-Ile-MeVal-MeVal -SAr-MeVal-Melle-Sar-OH c.) H-MeVal-Melle-Sar-Val-Melle-Sar-MeVal-Trp-Ile-MeVal-MeVal-Sar-OH
While the linear dodecapeptides incurred in all three cases in very satisfactory purities cyclization was quantitative only in cases a) and b)..; in case c.) in the final product were still about 20% of the linear peptide present.
By way of example the synthesis variant is to be shown on the linear dodecapeptide b.) Here. Synthesis course:
1.) Clutch miles -> Sar: triphosgene, 3 h, chloranil test negative. 2.) coupling -Meval -> Mile: triphosgene, 3 h, chloranil test negative. 3.) coupling Sar -> -Meval: triphosgene, 2.5 h, chloranil test negative.
4.) coupling -Meval -> Sar: triphosgene, 3 h, chloranil test negative.
HPLC purity:> 98%. 5.) coupling -Meval -> -Meval: h triphosgene 3, chloranil test weakly positive
Post-coupling triphosgene 3 hours, chloranil test negative. HPLC purity:> 95%.
6.) Coupling Ile -> -Meval: HATU, h 20, chloranil test weakly positive
Post-coupling HATU 3 hours, chloranil test weakly positive
Post-coupling HOAt 16 hours, chloranil test negative. HPLC purity: 90%. 7.) coupling MeV -> Ile: triphosgene, 3 h, HPLC purity: 90%. 8) Coupling Trp -> MeV: HATU 4 h, HPLC purity: 89%.
9) Coupling Sar -> Trp: triphosgene, 4 h, HPLC purity: 86%. 10.) coupling mile -> Sar: triphosgene, 4 h, HPLC purity: 83%. 11) coupling Val -> mile: HATU, 5 h, HPLC purity: 80%.
All indicated HPLC purities apply to Fmoc-protected peptide
(Λ = 214 nm). The prolonged reaction times of most BTC couplings result primarily from the parallel reaction procedure for the simultaneous synthesis of all peptides mentioned three linear precursor. A reduction of the coupling efficiency by reducing the amounts of reagents used was not observed.
The RacemisierungskontroUe the synthesized according to Example 7 Omphalotin A (OMA) was carried out as described in Example 6. FIG. N-Me-D-MeVal N-Me-D-Melle
Linear dodecapeptide 1.2% 2.4%
OmA crude product 1.5% 3.2%
As expected, here is the proportion of N-Me-D-Melle in crude cyclized not increased significantly compared with the linear precursor peptide, since it is not involved in the sierungsreaktion cyclical. These values and the peak sharpness of the cyclization product in the HPLC chromatograms (Fig. 15 and 16) indicate that the
Product purity of the synthesized according to Example 7 Omphalotin A must be significantly better than that of the product obtained according to Example 6. Fig. The HPLC chromatogram of the purified compound (Figure 17) confirmed this. Purification by preparative HPLC afforded the highly pure product in an overall yield of 21%.
Fig. 15: HPLC chromatogram of the Fmoc-deprotected, linear Dodekapeptides (Example 7).
Fig. 16: HPLC chromatogram of the crude product of the cyclization
Fig. 17: HPLC chromatogram of the purified optically pure Omphalotin A (Example 7).
Priority Applications (7)
|Application Number||Priority Date||Filing Date||Title|
|DE2001157882 DE10157882A1 (en)||2001-03-28||2001-11-26||A process for the preparation of carboxamides|
|PCT/EP2002/003153 WO2002076927A3 (en)||2001-03-28||2002-03-21||Method for the production of carboxylic acid amides|
|Publication Number||Publication Date|
|EP1373190A2 true true EP1373190A2 (en)||2004-01-02|
Family Applications (1)
|Application Number||Title||Priority Date||Filing Date|
|EP20020735153 Withdrawn EP1373190A2 (en)||2001-03-28||2002-03-21||Method for the production of carboxylic acid amides|
Country Status (3)
|US (1)||US6982315B2 (en)|
|EP (1)||EP1373190A2 (en)|
|WO (1)||WO2002076927A3 (en)|
Families Citing this family (5)
|Publication number||Priority date||Publication date||Assignee||Title|
|DE602005004153T2 (en)||2004-07-16||2008-12-24||Lonza Ag||The method for peptide synthesis|
|US7696166B2 (en)||2006-03-28||2010-04-13||Albany Molecular Research, Inc.||Use of cyclosporin alkyne/alkene analogues for preventing or treating viral-induced disorders|
|US7696165B2 (en)||2006-03-28||2010-04-13||Albany Molecular Research, Inc.||Use of cyclosporin alkyne analogues for preventing or treating viral-induced disorders|
|GB2472563B (en) *||2009-04-28||2013-02-27||Univ Leicester||Method of preparing hairpin and cyclic polyamides|
|CN104744570A (en) *||2013-12-31||2015-07-01||深圳先进技术研究院||Synthesis method of cyclosporins|
Family Cites Families (3)
|Publication number||Priority date||Publication date||Assignee||Title|
|JPH0112751B2 (en) *||1979-05-02||1989-03-02||Haruo Ogura|
|US5948693A (en) *||1994-09-01||1999-09-07||Wisconsin Alumni Research Foundation||Solid phase synthesis of immunosuppressive agents|
|DE19545463A1 (en) *||1995-12-06||1997-06-12||Bayer Ag||Organic chemical compound and processes for their preparation|
Non-Patent Citations (1)
|See references of WO02076927A2 *|
Also Published As
|Publication number||Publication date||Type|
|Najjar et al.||Solid Phase Peptide Synthesis. VI. The Use of the o-Nitrophenylsulfenyl Group in the Synthesis of the Octadecapeptide Bradykininylbradykinin|
|Isidro-Llobet et al.||Amino acid-protecting groups|
|US5811515A (en)||Synthesis of conformationally restricted amino acids, peptides, and peptidomimetics by catalytic ring closing metathesis|
|DeGrado et al.||Solid-phase synthesis of protected peptides on a polymer-bound oxime: preparation of segments comprising the sequence of a cytotoxic 26-peptide analog|
|Falb et al.||In situ generation of Fmoc‐amino acid chlorides using bis‐(trichloromethyl) carbonate and its utilization for difficult couplings in solid‐phase peptide synthesis|
|US6008058A (en)||Cyclic peptide mixtures via side chain or backbone attachment and solid phase synthesis|
|US3832337A (en)||Peptide enzyme inhibitors|
|Pietta et al.||Preparation and use of benzhydrylamine polymers in peptide synthesis. II. Synthesis of thyrotropin releasing hormone, thyrocalcitonin 26-32, and eledoisin|
|Kunz et al.||Solid phase synthesis of peptides and glycopeptides on polymeric supports with allylic anchor groups|
|Jackson et al.||Template-constrained cyclic peptides: design of high-affinity ligands for GPIIb/IIIa|
|Wang||Solid phase synthesis of protected peptides via photolytic cleavage of the. alpha.-methylphenacyl ester anchoring linkage|
|Guichard et al.||Preparation of N‐Fmoc‐protected β2‐and β3‐amino acids and their use as building blocks for the solid‐phase synthesis of β‐peptides|
|Lundquist IV et al.||Improved solid-phase peptide synthesis method utilizing α-azide-protected amino acids|
|Albericio||Orthogonal protecting groups for Nα‐amino and C‐terminal carboxyl functions in solid‐phase peptide synthesis|
|Lambert et al.||The synthesis of cyclic peptides|
|US4350627A (en)||Biologically active peptides|
|US5635598A (en)||Selectively cleavabe linners based on iminodiacetic acid esters for solid phase peptide synthesis|
|Brady et al.||Large-scale synthesis of a cyclic hexapeptide analog of somatostatin|
|Benoiton et al.||2-Alkoxy-5 (4 H)-oxazolones from N-alkoxycarbonylamino acids and their implication in carbodiimide-mediated reactions in peptide synthesis|
|Li et al.||1-Ethyl 2-halopyridinium salts, highly efficient coupling reagents for hindered peptide synthesis both in solution and the solid-phase|
|Kaul et al.||Systematic study of the synthesis of macrocyclic dipeptide β-turn mimics possessing 8-, 9-, and 10-membered rings by ring-closing metathesis|
|Stewart||The synthesis and polymerization of peptide p-nitrophenyl esters|
|US20030105103A1 (en)||Peptide beta-turn mimetic compounds and processes for making them|
|Biron et al.||Convenient synthesis of N-methylamino acids compatible with Fmoc solid-phase peptide synthesis|
|Stewart||Poly-L-alanylglycyl-L-alanylglycyl-L-serylglycine: A synthetic model of Bombyx mori Silk Fibroin|
|17P||Request for examination filed||
Effective date: 20031028
|AX||Extension or validation of the european patent to||
Countries concerned: ALLTLVMKROSI
|AK||Designated contracting states:||
Kind code of ref document: A2
Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE TR
Inventor name: JUNG, GUENTHER
Inventor name: RUDOLPH, JOACHIM
Inventor name: THERN, BERND
|RAP1||Transfer of rights of an ep application||
Owner name: LANXESS DEUTSCHLAND GMBH
|17Q||First examination report||
Effective date: 20071217
|18D||Deemed to be withdrawn||
Effective date: 20080429