Practical Protein Chemistry - A. Darbre 1989

Methods of Solid-Phase Amino Acid Sequence Analysis
Resin Synthesis
Polystyrene-Based Resins

The Starting Material for the synthesis of modified polystyrene-based resins is a styrene-1% divinylbenzene copolymer (Biobeads S-XI, Biorad). Schemes for the synthesis of several resins are shown in Fig. 12.1.

Polymers intended for solid-phase sequence analysis must possess two essential characteristics:

1) the Functional groups of the polymer must be accessible to Peptides and Reagents;

2) the resin must be chemically and mechanically stable under Edman Degradation conditions.

12.3.1.1. Swelling Capacity. To determine the swelling degree of a resin, the height of the resin bed (in a small flat-bottomed test tube) is measured before and 1 h after The addition of the solvent [27]. The ratio of the swollen to dry resin volume (V_s/V_d) must be at least 3:5 to allow reagents to penetrate into the interior of the hydrophobic polymer particles. Following the synthesis of polystyrene-based resins, their swelling capacity should be compared with the values presented in Table 12.1. Beads of an ideal support for sequence analysis should be small in size and have a narrow particle diameter distribution, which is crucial for chromatographic Column packing; they must also maintain a constant swelling volume and resistance to all Reagents and Solvents used in the sequencing Procedure. However, the resins most suitable for polypeptide Structure analysis swell significantly in pyridine, DMF, 1,2-dichloroethane, and TFA. Since these resins only partially satisfy the ideal requirements outlined above—and changes in their volume during structure determination lead to column clogging or inefficient penetration of reagents and solvents into the pores of the support—the resins are "diluted" with Glass beads (30-fold dilution by weight) prior to column packing (see Section 12.5.2).

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FIG. 12.1. Synthesis of cross-linked polystyrene-based Supports. 1 — cross-linked polystyrene, 2 — nitropolystyrene, 3 — aminopolystyrene, 4 — chloromethylated polystyrene, 5 — ethylenediaminopolystyrene, 6 — triethylenetetraminopolystyrene, 7 — nitrochloromethylpolystyrene, 8 — aminoethylenediaminopolystyrene. The preparation of compounds 3 and 8 is described in the text; for the Synthesis of the other supports, see reference [29].

12.3.1.2. Synthesis of Aminopolystyrene. Several modifications have been introduced to the original procedure [28, 30].

Synthesis of Nitropolystyrene. 5 g of polystyrene (Biobeads S-XI, minus 400 mesh) is thoroughly washed sequentially with benzene, chloroform, dioxane, and methanol, using 200 mL of each solvent; the duration of each wash is

Table 12.1. Swelling capacity of polystyrene-based resins^a

^a The dry (V_D) and swollen (V_S) resin volumes and the V_S/V_D ratio were determined according to Larsen [27]: after centrifugation, the volume of 100 mg of resin was measured, treated with 2 mL of each solvent, mixed, incubated for 60 min at room Temperature, centrifuged, and the swelling volume was measured.

^b The synthesis is described in Section 12.3.1.2.

^c The synthesis is described in Section 12.3.1.3.

30 min. The mixture is then washed with methanol on a metal sieve (60 µm) to remove large particles, and the beads are dried in vacuo. Yield: ~4 g. The resin is added in small portions over 15 min with stirring to fuming (90%) nitric acid (55 mL) precooled to –3°C, maintaining the temperature at or below –3°C. The mixture is stirred for 1 h at 0°C, poured onto 500 mL of ice, and the resin is collected on a sintered glass funnel. It is washed alternately with dioxane (four 200-mL portions) and Water (four 200-mL portions), followed by methanol (two 200-mL portions). The product is sieved (60 µm) to remove large resin particles, and the nitropolystyrene is dried in vacuo. Yield: 6–7 g.

Synthesis of Aminopolystyrene. A suspension of 6.3 g of nitropolystyrene in 115 mL of DMF is stirred at 140°C (oil bath). 48 g of SnCl₂·2H₂O in 40 mL of DMF is added slowly (exothermic reaction), and the reaction mixture is then kept at 140°C for 30 min. The mixture is cooled to 100°C, and 41 mL of concentrated Hydrochloric acid is added. The temperature is maintained at 95°C for 1 h, and the resin is collected on a sintered glass funnel. It is washed with 2 M HCl (two 200-mL portions) and water (two 200-mL portions), and sieved through a 100-µm screen to separate coarse resin particles. The washes with 2 M HCl and water are repeated, followed by a DMF–triethylamine mixture (3:1, two 200-mL portions) until chloride ions are absent in the washings. The product (a dark green resin) is washed with water and sieved again through a 100-µm screen. It is washed with methanol (two 200-mL portions) and re-sieved through a 60-µm screen. The yellowish-brown resin is dried in vacuo at 60°C. Yield: 5.5 g.

Storage of the Resin. Aminopolystyrene retains its properties and characteristics at room temperature for only one month. Small quantities can be stored under nitrogen in sealed ampoules at –20°C. Under these conditions, the resin remains stable for up to one year.

Swelling Capacity. The polymer swells readily in DMF and TFA, and to a lesser extent in pyridine and methanol (Table 12.1).

12.3.1.3. Synthesis of Triethylenetetraminopolystyrene (TETA-Polystyrene). TETA-polystyrene is prepared According to the Procedures described in References [24, 30]. Chloromethylated polystyrene particles (Biobeads S-XI) are washed with benzene, chloroform, dioxane, and methanol, and dried in vacuo. 1 g of freshly prepared resin is stirred with 15 mL of triethylenetetramine (TETA) for 30 min at room temperature, and then heated in an oil bath at 115°C for an additional 90 min. The resin is collected on a sintered glass funnel, washed thoroughly with 20 mL of triethylamine for 30 min at room temperature, and filtered. It is washed alternately with methanol and water (three 200-mL portions of each solvent), washed once more with water, and centrifuged. The resin is boiled in 50 mL of 1 M hydrochloric acid for 2 h to remove traces of unreacted TETA, and filtered. It is washed with water, triethylamine, water, and methanol (two 1-mL portions of each solvent). The product is dried in vacuo overnight. Yield: ~1 g.

Store as for aminopolystyrene. In terms of stability, TETA-polystyrene is inferior to aminopolystyrene. After several months of storage, its use in Edman degradation leads to an increased Background level of impurities.

Swelling capacity is good (Table 12.1). The resin should be washed immediately before use with the following solvents: DMF (two 1-mL portions), methanol (two 1-mL portions), triethylamine (15 mL for 20 min), water, and methanol; dry in vacuo.