Practical Protein Chemistry - A. Darbre 1989

Methods of solid-phase amino acid sequencing
Coupling methods
Coupling via homoserine lactone

This coupling utilizes TETA-polystyrene [24, 33] as well as Glass (APS or β-APS).

12.4.3.1. Advantages, limitations, and attachment Procedure.

Advantages.

1. For homoserine-containing Peptides, coupling yields are 80—90%.

2. The complete sequence of such peptides, including the C-terminal residue, can be determined.

3. Apart from the cleaved Amino Acids, the solution contains few other impurities, provided that the support was stored in ampoules under nitrogen at —20 °C and thoroughly washed immediately before use.

4. This method allows the Separation of homoserine-containing peptides from the C-terminal peptide lacking homoserine in Cyanogen bromide Cleavage products.

Limitations.

1. TETA-polystyrene is relatively unstable and cannot be stored for long periods.

2. TETA-polystyrene must be thoroughly washed before use.

3. When working with Lysine-containing peptides, this support is less effective than aminopolystyrene and aminopropyl glass due to an increased Background of impurities. It has been reported that treating the peptidyl-polymer with N-acetylimidazole immediately after peptide-to-polymer coupling reduces the number of false ninhydrin-positive spots during the identification of PTH-amino acids.

Attachment procedure. Support capacity is 1 nmol/mg of resin. 50 mg of TETA resin or 100 mg of 3-aminopropyl glass is washed with DMF (4 times with 2 mL); in the case of TETA resin, it is additionally washed with triethylamine (2 times with 1 mL). Finally, wash with methanol (2 times with 2 mL) and DMF (2 times with 1 mL).

Peptide lactonization. The peptide is dried in vacuo over phosphorus pentoxide. Distilled anhydrous TFA (50—100 µL per 5—20 nmol of peptide; 0.5 mL per 50—200 nmol) is added, and the mixture is left for 1 h at room Temperature under nitrogen. The product is dried on a rotary evaporator or in a concentrator (Speed Vac Concentrator, Savant), then in vacuo over potassium hydroxide. The TFA Treatment is repeated for 15 min.

Attachment. The peptide lactone is dissolved in a mixture of 175 µL DMF + 25 µL triethylamine; if the peptide does not dissolve, triethylamine is added to a concentration of 20%. Solubility is monitored by taking 2—5 µL aliquots. Under nitrogen, the peptide solution is added to the pre-washed TETA resin (or to 20 mg of aminopropyl glass). The tube containing the peptide is rinsed with DMF (2 times with 100 µL), and the rinses are added to the reaction mixture. Triethylamine (50 µL) is added, the mixture is sonicated, incubated with gentle agitation under nitrogen for 90 min at 45 °C, washed with DMF and methanol, and centrifuged. The supernatant is monitored for peptide content.

Capping. To the support, 200 µL of FITC solution in acetonitrile (1:5, v/v) and 200 µL of buffer No. 1 (Sec. 12.2.6) are added. The mixture is stirred under nitrogen for 30 min at 30 °C. Centrifuge and wash the resin with DMF (2 mL) and methanol (2 times with 2 mL). Dry the resin in vacuo.