METHOD OF SOLID-STATE PEPTIDE SYNTHESIS USING A NOVEL POLYMERIC SUPPORT

Abstract

A method of solid-state synthesis of peptides that provides a polymeric solid-state synthesis support, attaches a first amino acid to said support to form the first amino acid molecule of the desired peptide chain, attaches additional amino acids to form the desired peptide chain, characterized in that the polymeric solid-state synthesis support is a styrenic polymer containing at least 50 mol % divinylbenzene monomer units, the styrenic polymer being functionalized with chloromethyl and/or benzyloxybenzyl alcohol and/or amino moieties, and having porosity 60-90% and medium pore diameter ranging from 10 to 80 nm. A functionalized styrenic polymer is claimed.

Claims

1. A method of solid-state synthesis of peptides comprising the steps of providing a polymeric solid-state synthesis support, attaching a first amino acid to said support to form the first amino acid molecule of the desired peptide chain, attaching additional amino acids to form the desired peptide chain, characterized in that the polymeric solid-state synthesis support is a styrenic polymer containing at least 50 mol % divinylbenzene monomer units, said styrenic polymer being functionalized with chloromethyl and/or benzyloxybenzyl alcohol and/or amino moieties, and having a porosity of 60 to 90% and medium pore diameter of 10 to 80 nm.

2. The method of claim 1, wherein the styrenic polymer has a surface area of the pore walls 200 to 600 m.sup.2/g.

3. The method of claim 1, wherein the styrenic polymer contains at least 60 mol % divinylbenzene monomer units.

4. The method of claim 1, wherein the styrenic polymer contains at least 70 mol % divinylbenzene monomer units.

5. The method of claim 1, wherein the polymeric solid-state synthesis support is obtainable by a method comprising the step of radical polymerization of a monomeric mixture comprising at least 50 mol % divinylbenzene and a solvent selected from aromatic hydrocarbons, heterocycloalkanes, and halogenated aliphatic hydrocarbons, said solvent being in a volume amount of twice to ten times the volume of the monomer, and said solvent optionally comprising up to 20 vol. % of a component selected from liquid alkanes and fatty acids.

6. A styrenic polymer containing at least 50 mol % divinylbenzene monomer units, said styrenic polymer being functionalized with chloromethyl and/or benzyloxybenzyl alcohol and/or amino moieties, and having porosity 60 to 90% and medium pore diameter ranging from 10 to 80 nm.

7. The polymer of claim 6, wherein the styrenic polymer has a surface area of the pore walls from 200 to 600 m.sup.2/g.

8. The polymer of claim 6, wherein the styrenic polymer contains at least 60 mol % divinylbenzene monomer units.

9. The polymer of claim 6, wherein the styrenic polymer contains at least 70 mol % divinylbenzene monomer units.

Description

EXAMPLES OF CARRYING OUT THE INVENTION

Example 1

[0020] A clear, homogeneous mixture of 6.0 g (6.6 cm.sup.3) of divinylbenzene (technical grade, 80%), 60 cm.sup.3 of tetrahydrofurane, 6 cm.sup.3 of water and 165 mg of the initiator 2,2-azobis(2-methylpropionitrile) was kept in a closed vessel at 100 C. for 48 hours. After cooling to room temperature, a white, opaque cylinder of relatively soft polymer was removed and dried at room temperature for 10 days. The polymer (hereafter referred to as A) was then ground with pestle in a mortar and dried at 100 C. overnight. Inverse steric chromatography evaluation of its morphology after its re-expansion in tetrahydrofurane showed a pore volume equal to 8.1 cm.sup.3/g (corresponds to the porosity 89%), medium pore diameter (average value weighed by volumes of individual pore fractions) 50 nm and pore wall surface area 640 m.sup.2/g.

Example 2

[0021] A solution of sodium chloride (40 g) and poly(vinyl alcohol) (5 g) in water (600 cm.sup.3) was poured into a stainless steel autoclave equipped with a heating mantle, a propeller stirrer and connected to a circulator water bath. A clear, homogeneous mixture of 30.0 g (33 cm.sup.3) of divinylbenzene (tech. grade 80%), 240 cm.sup.3 of toluene, 60 cm.sup.3 of n-heptane and 0.60 g of the initiator 2,2-azobis(2-methylpropionitrile) as the initiator was added to the aqueous solution inside the autoclave. The autoclave was closed and the content stirred at room temperature for 1 hour. The temperature in the reactor was then raised to 85 C. and kept at this level for 6 hours while the mixture was stirred and for further 48 hours without stirring. After cooling to room temperature the reactor was opened and white polymer beads (hereafter referred to as B) were recovered upon filtration, then washed repeatedly with methanol and dried on air and then in an oven at 100 C. overnight. ISEC evaluation of its morphology after its re-expansion in tetrahydrofuran showed a pore volume of 4.6 cm.sup.3/g (considering the skeletal density of the polymer matrix close to 1 g/cm.sup.3 it corresponds to the porosity 82%), medium pore diameter 40 nm and pore wall area 560 m.sup.2/g.

Example 3

[0022] The first step for transforming the mesoporous polymers into supports for the solid state peptide synthesis is their functionalization with chloromethyl groups. In a typical procedure, 22 g of the mesoporous polymer B from Example 2 was suspended in 250 cm.sup.3 n-heptane and stirred at room temperature for 1 hour. The reagent for the chloromethylation (110 cm.sup.3), which was prepared the day before by mixing of 58 cm.sup.3 of dimethoxymethane, 5 cm.sup.3 methanol and 47 cm.sup.3 acetylchloride, and anhydrous stannic chloride (20 cm.sup.3) were added to the suspension. After 15 min the temperature was raised to 40 C. and the reaction let to proceed for another hour. The reaction was quenched by the addition of 200 cm.sup.3 of a 1:1 (v:v) mixture of ethanol and water. The product was separated upon filtration and repeatedly washed with ethanol until the filtrate was free of chlorides (negative silver nitrate test). Then it was dried at 105 C. overnight. The final polymer contained 6.7 wt. % chlorine. ISEC evaluation of its morphology after its re-expansion in tetrahydrofuran showed a pore volume of 2.6 cm.sup.3/g corresponding to the porosity 72%, medium pore diameter 39 nm and pore wall area 260 m.sup.2/g.

Example 4

[0023] The chloromethylated styrenic polymer from Example 3 was transformed into the so-called Wang type support, functionalized with 4-benzyloxybenzyl alcohol moieties (hereafter referred to as pDVB-4-benzyloxybenzyl alcohol) which is one of possible functionalization useful and desirable for the solid state peptide synthesis. For this purpose 13 g of the styrenic polymer from Example 3 were suspended in 100 cm.sup.3 of N,N-dimethylformamide (DMF) and stirred at 65 C. After 2 hours 6 g of 4-hydroxybenzyl alcohol and 4 g of sodium methoxide were added to the suspension. The mixture was further stirred at the same temperature for 5 hours. After cooling to room temperature the reaction mixture was diluted with methanol and the polymer was recovered upon filtration, thoroughly washed with ethanol and dried at 105 C. overnight. The elemental analysis of the final polymer showed that chlorine dropped from 6.7 wt. % of the polymer from Example 3 to 2.7 wt. %. The mass balance of chlorine indicates that approximately 1.1 mmol/g of the original chloromethyl groups were converted into the desired Wang functionality. ISEC evaluation of its morphology after its re-expansion in tetrahydrofuran showed a pore volume of 2.5 cm.sup.3/g corresponding to the porosity 71%, medium pore diameter 39 nm and pore wall area 256 m.sup.2/g.

Example 5

[0024] The chloromethylated styrenic polymer from Example 3 was transformed in a support functionalized with amino groups (hereafter referred to as pDVB-ethylendiamine), which is another kind of functionalization useful and desirable for the solid state peptide synthesis. For this purpose, the chloromethylated styrenic polymer B from Example 3 (5 g) was suspended in DMF (100 cm.sup.3). After 45 min DMF was filtered and the swollen polymer was put in contact with a solution of ethylenediamine (1.26 cm.sup.3) dissolved in the smallest amount of DMF required to keep the polymer wet. The reaction was then let to proceed overnight with stirring. The day after, the polymer was washed five times with DMF (50 cm.sup.3), then with ethanol and dried at 105 C. overnight. ISEC evaluation of its morphology after its re-expansion in tetrahydrofuran showed a pore volume of 2.6 cm.sup.3/g corresponding to the porosity 72%, medium pore diameter 39 nm and pore wall area 260 m.sup.2/g.

Example 6

[0025] Peptide syntheses were performed according the following procedure: For the attachment of the first amino acid to pDVB-4-benzyloxybenzyl alcohol the carboxylic function of the first N-Fmoc protected amino acid (Fmoc-AA-OH) was activated through the formation of a symmetric anhydride. 6 equivalents of Fmoc-AA-OH (with respect to the hydroxyl moiety of pDVB-4-benzyloxybenzyl alcohol) were dissolved in the minimum amount of anhydrous DMF and treated with 3 eq (0.11 cm.sup.3) N,N-diisopropylcarbodiimide (DIC). After 30 min the reaction mixture was transferred to a reaction vessel containing the polymer support swollen in DMF, and 0.05 eq (0.001 g) of 4-dimethylaminopyridine was added as a catalyst. The reaction was let to proceed for 1 hour, then the excess of reagents was removed by filtration and the polymer was washed 5-times with DMF and 5-times with dichloromethane (DCM). Deprotection of the amino function of the peptide on the solid support was achieved by a double treatment with a 20% piperidine solution in DMF, followed by the removal of the excess of reagents by filtration and washing procedure. The coupling of the next incoming Fmoc-AA-OH was performed through the HBTU C-activation method (2-fold excess of Fmoc-AA-OH, N,N,N,N-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU) and 1-hydroxybenzotriazole (HOBt); 5-fold excess N,N-diisopropylethylamine (DIPEA), 45 minutes reaction time). The coupling step was followed by a washing procedure. The process was repeated for each new residue in the peptide chain. Once the sequence has been completed, the peptide was cleaved from the polymeric support.

Example 7

[0026] Synthesis of a model tetrapeptide Fmoc-Leu-Leu-Val-Phe-OH (SEQ ID NO. 1) on pDVB-4-benzyloxybenzyl alcohol from Example 4, using acetonitrile as the solvent. To pDVB-4-benzyloxybenzyl alcohol from Example 4 Fmoc-Phe-OH was attached as the C-Terminal amino acid and the synthesis of the peptide proceeded according to Example 5, using acetonitrile as the solvent for all the steps of the procedure. The amount of reagents used are reported in Table 1.

TABLE-US-00001 TABLE 1 pDVB-4-benzyloxybenzyl alcohol 0.1002 g position in the sequence residue amount - g 3 Val (3eq) 0.042 2 Leu (3eq) 0.044 1 Leu (3eq) 0.044 For each coupling: HBTU (3eq) = 0.047 g HOBt (3eq) = 0.017 g; DIPEA (6eq) = 0.042 cm.sup.3

[0027] After cleavage from the solid support, using trifluoroacetic acid/triisopropylsilane/water (95:2.5:2.5 v/v), the desired product was obtained with a selectivity of 98%. The same synthesis run on a commercial styrenic Wang resin loaded with Fmoc-Phe-OH, the same amounts of reagents and the same solvent did not get to the target product: the Electron Spray Ionizatio Mass Spectrometry analysis of the products evidenced the presence of truncated and/or deletion sequences.

Example 8

[0028] pDVB-ethylendiamine from example 5 was suspended in DMF and put in contact with the activated form of Fmoc-Rink linker in presence of HOBt and N-methylmorpholine (NMM). 0.125 g of Fmoc-Rink linker were dissolved in DMF (1.5 cm.sup.3) in presence of 2 equivalents of HOBt (0.032 g), 2 equivalents of diisopropylcarbodimmide (DIC) (0.035 cm.sup.3) and 2.5 equivalents of NMM (0.025 cm.sup.3), the reaction was let to proceed for 4 hours with stirring. The excess of reagents was removed by filtration and the polymer (hereafter referred to as pDVB-Rink) washed 5 times with DMF (2 cm.sup.3) and further five times with DCM (2 cm.sup.3), then dried under vacuum.

[0029] The C-terminal residue of the sequence was atteached to pDVB-Rink according to the standard procedure for coupling, and the loading of the first residue was determined through the estimation of the Fmoc quantity. The amount of each reagent is reported in Table 2.

TABLE-US-00002 TABLE 2 pDVB-Rink 0.115 g C-terminal residue introduction Fmoc-Phe-OH 0.25 g (3eq) HBTU (3eq) 0.24 g HOBt (3eq) 0.08 g DIPEA (6eq) 0.22 cm.sup.3 Measured loading of the first residue 0.51 mmol/g Position in the sequence Residue Amount - g 3 Val (3eq) 0.059 2 Leu (3eq) 0.062 1 Leu (3eq) 0.062 For each coupling: HBTU (3eq) = 0.067 g HOBt (3eq) = 0.024 g; DIPEA (6eq) = 0.060 cm.sup.3

[0030] After cleavage from the solid support using trifluoroacetic acid/triisopropylsilane/water (95:2.5:2.5 v/v), the desired product was obtained with a selectivity of 93%.

[0031] The same synthesis was carried out using acetonitrile as the solvent, leading to comparable results.

Example 9

[0032] Synthesis of the fragment [65-74] of the Acyl Carrier Protein H-Val-Gln-Ala-Ala-Ile-Asp-Tyr-Ile-Asn-Gly-OH ([65-74]ACP) (SEQ ID NO. 2). The peptide [65-74]-ACP was synthesized on pDVB-4-benzyloxybenzyl from Example 4; the C-terminal amino acid glycine was introduced according to Example 5, using DMF as the solvent. The amount of reagents employed are reported in Table 3:

TABLE-US-00003 TABLE 3 pDVB-4-benzyloxybenzyl alcohol 0.1994 g Position in the sequence Residue amount - g 9 Asn (4eq) 0.2009 8 Ile (4eq) 0.1187 7 Tyr (4eq) 0.1544 6 Asp (4eq) 138.26 5 Ile (4eq) 0.1187 4 Ala (4eq) 0.1046 3 Ala (4eq) 0.1046 2 Gln (4eq) 0.2052 1 Val (4eq) 0.1140 For each coupling: HBTU (4eq) = 0.127 g; HOBt (4eq) = 0.045 g; DIPEA (8eq) = 0.115 cm.sup.3

[0033] The synthesis was run without any optimization of the coupling conditions, nevertheless the desired product was obtained in good yield (43%) and selectivity (88%). Similar results were obtained also using acetonitrile as the solvent for all steps of the synthesis on pDVB-4-benzyloxybenzyl alcohol.

[0034] The same sequence was prepared also starting from either commercial styrenic Wang resin or Wang resin based on polyethylene glycol. In the first case only a small amount of the target peptide was obtained along with truncated and deletion products while in the latter we got the desired peptide with a good selectivity (85%) but in poor yield (7.5%).