DD272856A1 - METHOD FOR SIMULTANE SYNTHESIS OF PEPTIDES - Google Patents

Abstract

The invention relates to a method for the simultaneous synthesis of peptides. It has the task of developing a synthesis method that allows the simultaneous solid-phase synthesis of several peptides on a mmol-scale. According to the invention, a mechanically and chemically stable surface carrier is used, on which, after covalent binding of a suitable anchor group and carrier segmentation, several peptides are built up simultaneously by stepwise attachment of the corresponding amino acid residues. After completion of the synthesis, the peptides are either cleaved off the carrier and isolated or traegerfixiert immunologically tested. The field of application of the invention is peptide chemistry as well as biomedical basic research.

Description

Field of application of the invention

The invention relates to a method for the simultaneous solid-phase synthesis of several peptides in μηποΙ scale. The field of application of the invention is peptide chemistry as well as biomedical basic research.

Characteristic of the known state of the art

Due to the increasing demand for synthetic peptides, in particular in immunology and in molecular biology, the search for effective synthetic methods in peptide chemistry is currently increasing, whereby brain synthesis is of great importance. An essential prerequisite for this is the possibility of carrier segmentation for each peptide to be produced, which allows the optional combining, separating and sorting of the individual carrier segments. For the simultaneous synthesis of peptides by carrier segmentation, the following methods are known:

1. R. A. Houghten (EP 0196174A2) describes a method for the simultaneous solid-phase synthesis of peptides by segmentation of the carrier portions for each peptide to be prepared in polypropylene bags. The support used is the divinylbenzene-crosslinked polystyrene resin customary for solid-phase synthesis. This method involves a final cleavage of the peptides from the carrier; a carrier-fixed testiing is not described.

2. H. M. Geysen (PCT / AU 84/00038) describes a method for the simultaneous synthesis of peptides on an nmol scale on polyacrylic acid-coated polyethylene rods and their carrier-fixed immunological testing. A synthesis in the preparative sense (cleavage and isolation of the products) is not possible with this method. Disadvantages here are the limited possibilities, due to the nature of the carrier, of the analytical control of both the course of the synthesis and of the products.

Object of the invention

The aim of the invention is to provide a method for the simultaneous solid-phase synthesis of peptides while saving time and reagents.

Explanation of the essence of the invention

The invention has for its object to develop a synthesis method that allows the simultaneous synthesis of several peptides.

According to the invention, the process for simultaneous synthesis is carried out on mechanically and chemically stable surface carriers, to which anchor groups suitable for peptide synthesis are covalently bound. The peptide synthesis itself is carried out by stepwise attachment of the individual amino acid residues.

According to the invention, a mechanically and chemically stable surface carrier with hydroxyl groups, preferably cellulose paper, is used whose hydroxyl groups are activated with activated compounds, eg. B. N ° -protected amino acid chlorides are reacted. The peptide synthesis itself is carried out according to the general scheme of solid phase synthesis with the wiederhol times (x = number of amino acids) repeating cycle

Deprotection

ί I

Washing the wearer Washing the wearer

T 1

Attachment of the next amino acid,

all the protective group combinations known in solid-phase peptide synthesis (eg Boc / benzyl and Fmoc / Bj ¹ strategy) as well as activation possibilities for the amino acids (eg active esters, carbodiimides, symmetrical anhydrides) are applicable.

The starting amino acid is either coupled directly to the carrier or it becomes between the carrier and peptide a spacer, ζ. Β. an ω-amino acid, or a selectively cleavable linker, for example of the p-alkoxybenzyl start type, incorporated. Simultaneous synthesis is made possible by the segmentability of the> surface carrier used. The deprotection and all washing processes on η surface carriers ₍ n = number of peptides to be synthesized) are carried out together in one vessel. Before each coupling, the carriers are sorted and placed in reaction tubes containing the appropriate amino acid derivatives. After completion of the synthesis, the peptides are either cleaved from the carrier and isolated or, optionally after acetylation of the N-terminus, provided for carrier-fixed testing. The cleavage of the peptides takes place either from the selectively cleavable left or directly from the carrier. The method according to the invention has the following advantages:

1. The simultaneous solid-phase synthesis of peptides in common reaction and wash solutions saves time, chemicals and solvents.

2. The surface carrier used is inexpensive and chemically easily accessible.

3. It is both a carrier-fixed immunological testing of the synthesized peptides as well as their cleavage and isolation possible.

4. The method allows the analytical control of both the synthesis (turnover control) and the products (characterization after cleavage).

The invention will be explained in more detail with reference to embodiments.

embodiments

Example 1: Synthesis of paper base

Collulose paper, e.g. Shake Whatman 540 paper (β χ 6cm) wi-d in 3ml 1 N NaOH for 15min at room temperature, briefly dried between blotting paper, immediately in 3ml of a 5% Fmoc-amino acid chloride solution, eg. B. Fmoc-Ala-Cl or Fmoc-Gly-Cl, in toluene / acetone (1: 1 v / v) and shaken therein for 30min at room temperature. The paper is briefly dried between blotting paper, immediately immersed in 30% acetic acid / acetone and shaken for 3 minutes. The paper is then washed thoroughly with acetone, water, ethanol and dichloromethane (5 ml each, 3 min) and dried.

The degree of substitution thus produced surface carrier is 1-2μπιοΙΛ: ίΤ) ² paper.

Example 2: Simultaneous Synthesis of Peptides

2.1. A 6 × 6 cm piece of a surface support prepared according to Example 1 (Fmoc-Ala paper, 1.5 pmol / cm ² ) is shaken for 10 min in 3 mol 20% piperidine / DMF for Fmoc cleavage, with DMF and dichloromethane (2x 3 ml each 2min) washed and dried. It is then coupled for 10 min in preactivated Fmoc-e-aminocaproic acid HOBt ester (0.5 mmol / 2 ml DMF), washed with DMF and dichloromethane and dried. Unreacted amino groups are acetylated. The degree of substitution of the carrier is now 1.2 pmol / cm ² . After cleavage of the Fmoc group, the support is cut into 4 equal parts (3 x 3 cm each). The following 4 model peptides are simultaneously synthesized on these supports:

I: Tyr-Phe-Gly-Leu-eAcp-Ala-P

II: Tyr-Val-Pro-Lys-eAcp-Ala-P P = carrier

III: Tyr-Glu-Gly-Thr-eAcp-Ala-P

IV: Tyr-Lys-Gln-Ile-cAcp-Ala-P

For the couplings, pre-activated HOBt esters of Boc-Leu-OH, Boc-Thr (Bzl) -OH, Bcc-Ile-OH, Boc-Gly-OH, Fmoc-Lys (Boc) -OH, Boc-Gln-OH , Fmoc-Pro-OH, Boc-Phe-OH, BoC-Glu (BzI) -OH, Fmoc-Val-OH and Boc-Tyr-OH. The couplings are carried out simultaneously, should the same amino acid be coupled, also together in the same solution. Washing processes and deprotection occur together. The individual coupling cycles take place according to the following scheme:

a) Boc-amino acids:

1. 50% trifluoroacetic acid / dichloromethane (2% anisole) 3ml 15min

2. dichloromethane 2x 3ml2min

3. 5% diisopropylethylamine / dichloromethane 2x3..il2min

4. Dichloromethane Sort of carrier 2x 3ml 2min

5. Boc-amino acid HOBt ester (0.5mmol / 2mlDMF) 1-3h

6. DMF 2x 3ml 2min

7. Dichloromethane 3ml 2min

b) Fmoc-amino acids:

• 1. 20% piperidine / DMF 3ml 2min

2. DMF 2x 3ml2min

3. Dichloromethane Sort the carrier. 2x 3ml 2min

4. Fmoc-amino acid HOBt ester (0.5mmol / 2mlDMF) 1-3h

5. DMF 2x 3ml2min

6. Dichloromethane 2x 3ml 2min

The completeness of the couplings is checked by means of the qualitative ninhydrin test. After final Boc cleavage of I-IV III to cleave the benzyl groups with Bortrisftrif luoracetat) (1M in trifluoroacetic acid) for 1 h at room temperature, washed with trifluoroacetic acid and dichloromethane and dried. For the purpose of analytical characterization of the products, MV are split off in 3 ml of ethanolic ammonia solution (2 h at room temperature), the papers are washed with 3 ml of methanol and 10 mM water, and the combined cleavage and washing solutions are concentrated at 30 ° ± 1 to remove ammonia and methanol and lyophilized , The HPLC analysis of the lyophilizates (5-7 mg) (LiChrosorb C ₁₈ -SuuIe 10μιτι, gradient elution 5-100% AC N / 0.05 M KH ₂ PO ₄ pH 3.0) has major components of 65-70%, the It amino acid analysis with the target sequence are identical. 2.2. A 6 x 6 cm piece of a prepared according to Example 1 surface carrier (Fmoc Ala paper, 2 pmol / cm ² ) is after Fmoc elimination overnight in 2 ml of a 0.25 M solution of

+ HOBt coupled in DMF, washed with DMF and dichloromethane (2 x 3ml each 2min) and dried. Unreacted amino groups are acetylated. The degree of substitution of the carrier is now 1.8 pmol / cm ² . After Fmoc cleavage, the support is cut into 4 equal parts. The following 4 model peptides are simultaneously synthesized on these surface carriers:

V: Boc-Tyr-Ala-Lys (Boc) val-anchor Ala-P

Fl: Boc-Tyr-Thr (Bu ') - Pro-Val-anchor Ala-P

VII: Boc-Tyr-Lys (Boc) Pro-Val anchor Ala-P

VIII: Boc-Tyr-Thr (Bu ') - Leu-Val-anchor Ala-P

Anchor = -0-CHa- (§) -0-CH ₂ -CH ₂ -CO-

The synthesis of V-VIIi is carried out simultaneously as in Example 2.1. using preactivated HOBt esters of Fmoc-Pro-OH, Fmoc-Leu-OH, Fmoc-Lys (Boc) -OH, Fmoc-Thr (Bu ') - OH, Fmoc-Ala-OH and Boc-Tyr-OH. After completion of the synthesis, the papers for simultaneous cleavage of the Boc or Bu 'groups and the peptides from the carrier for 2 h in 2mol 50% trifluoroacetic acid / dichloro than (2% anisole) shaken. The papers are removed, washed with 2 ml each of 50% trifluoroacetic acid / dichloromethane and 10 ml of water, the combined cleavage and washing solutions to remove the dichloromethane at room temperature i.V. concentrated, the aqueous solutions extracted 2x with 5 ml of ether, concentrated again and lyophilized.

HPLC analysis of the lyophilizates (2-3 mg) (SIX-C, ₈ column, gradient elution 0-90% ACN / 0.05% TFA) has major components of 71-81% identical to the amino acid analysis of the target sequence are.

Example 3: Peptide Synthesis on Other Cellulose Sheet Carriers

Cellulose paper, e.g. B. Whatman 540 paper (5 χ 10cm) is activated according to DD-WP 220316 with cyanuric chloride. The paper w Vd was removed from its special packaging and immediately placed in 10 ml of a 5% solution of hexamethylenediamine in DMF and shaken therein for 3 h at room temperature. The paper is then washed with DMF, water, ethanol and dichloromethane (15 ml each for 3 min) and dried between blotting paper. Instead of DMF, other polar organic solvents or aqueous buffer systems can also be used.

The solid-phase synthesis of (i) Phe-Phe-Gly-Leu-Nle-P ([ΝΙο'Ί substance P (7-11)) according to Example 2.1 is carried out on 5 × 5 cm of the surface carrier produced in this way. described scheme using pre-activated Boc-amino acid HOBt-Es'.er (0.5mmol / 2ml DMF per coupling). After the 3rd and 4th coupling 1.5 × 5 cm are separated from the carrier and no longer treated, so that after completion of the synthesis both (i) and the truncated sequences (ii) Phe-Gly-Leu-Nle-P and (iii) Gly-Leu-Nle-F are present in approximately equal amounts. The peptides are then cleaved in 5 ml methanolic ammonia solution for 2 h at room temperature from the carrier, the papers washed with methanol and the combined cleavage and washings concentrated at 3O ⁰ C IV.

The cleaved peptides are then tested for their binding to anti-substance P antiserum, where (i) shows the expected binding, while the truncated sequences (ii) and (iii) are expected to have no cross-reactivity.

Example 4: Carrier-fixed immunological testing

On 3 χ 3cm of a surface carrier prepared according to Example 1 (Fmoc-Gly paper, 1.2 pmol / cm ² ) is as in Example 2.1. Fmoc-e-aminocaproic acid coupled. The solid phase synthesis of Arg-Pro-Lys-Pro-Acp-Ala-P takes place on this support after that in Example 2.1. described scheme using pre-activated Fmoc-amino acid HOBt ester. The carrier-fixed peptide reacts positively in a binding assay against the corresponding anti-peptide antiserum.

Claims (6)

1. A process for the simultaneous synthesis of peptides, characterized in that a plurality of peptides are synthesized simultaneously on a mechanically and chemically stable surface carrier having hydroxyl groups after covalent binding of suitable anchor groups. 2. The method according to claim 1, characterized in that are preferably used as surface carriers having hydroxyl groups cellulose papers. 3. Process according to claims 1 and 2, characterized in that to the hydroxyl groups of the surface carrier activated compounds, preferably N ^a -protected amino acid chlorides are covalently bound. 4. Process according to claims 1 to 3, characterized in that the synthesis of η peptides on η surface carriers by simultaneous reactions and washing processes in z.T. common reaction vessels takes place. 5. Process according to claims 1 to 4, characterized in that before the synthesis of a selectively cleavable left or a spacer can be bonded to the surface support. Process according to claims 1 to 5, characterized in that the synthesized peptides are either cleaved from the carrier and isolated or supplied to a carrier-fixed immunological assay.