Coupling through Hydroxy, Amino, or Thiol Groups via a 12-Carbon Spacer Arm

Epoxy-activated Sepharose 6B

Epoxy-activated Sepharose 6B is used for coupling ligands that contain hydroxyl, amino, or thiol groups. Because of the long hydrophilic spacer arm, it is particularly useful for coupling small ligands such as choline, ethanolamine, and sugars. The preactivated matrix is formed by reacting Sepharose 6B with the bis-oxirane, 1,4 bis-(2,3-epoxypropoxy-)butane. The partial structure is shown in Figure 4.12.

Partial structure of Epoxy-activated Sepharose 6B

Fig 4.12. Partial structure of Epoxy-activated Sepharose 6B.

A stable ether linkage is formed between the hydrophilic spacer and the matrix. Free oxirane groups couple via stable ether bonds with hydroxyl-containing molecules such as sugars, via alkylamine linkages with ligands containing amino groups, and via thioether linkages with ligands containing thiol groups.

Chromatography medium characteristics

Characteristics of Epoxy-activated Sepharose 6B are shown in Table 4.11.

Table 4.11. Characteristics of Epoxy-activated Sepharose 6B medium

Product Composition pH stability1 Average particle size (µm)
Epoxy-activated Sepharose 6B Sepharose 6B reacts with 1,4 bis- (2,3 epoxypropoxy-) butane to form a stable ether linkage. Short term: 2 to 14 Long term: 2 to 14 90

1 Short term refers to the pH interval for regeneration, cleaning-in-place, and sanitization procedures. Stability data refers to the coupled medium provided that the ligand can withstand the pH. Long term refers to the pH interval over which the matrix is stable over a long period of time without adverse effects on its subsequent chromatographic performance.

Purification options

Purification options for Epoxy-activated Sepharse 6B are shown in Table 4.12

Table 4.12. Purification options for Epoxy-activated Sepharose 6B medium

Product Spacer arm Substitution (µmol/ml medium) Coupling conditions Maximum operating flow velocity (cm/h)1 Comments
Epoxy-activated Sepharose 6B 12-atom 19 to 40 epoxy groups pH 9 to 13, 1 h to several days, 20°C to 40°C 75 Supplied as a freeze-dried powder.

1 See Appendix 4 to convert flow velocity (cm/h) to volumetric flow rate (ml/min). Maximum operating flow is calculated from measurement in a packed column with a bed height of 10 cm and i.d. of 5 cm.

Purification example

Capture and purification of fucose-specific lectin from a crude plant extract using Epoxy- activated Sepharose 6B is shown in Figure 4.13.

Chromatography of a crude extract of Ulex europaeus

Fig 4.13. Chromatography of a crude extract of Ulex europaeus on fucose coupled to Epoxy-activated Sepharose 6B, column volume 11 ml. Extract was applied in 0.9% NaCl. Fucose-specific lectin was eluted with 5 ml fucose (50 mg/ml).

Alternative coupling solutions

Distilled water or aqueous buffers with sugars and carbohydrates are preferable. Carbonate, borate, or phosphate buffers can be used.

Sodium hydroxide may be used for solutions of high pH.

Organic solvents such as dimethylformamide (up to 50%) and dioxane (up to 50%) may be used to dissolve the ligand. The same concentration of organic solvent should be included in the coupling solution.

Coupling procedure

  1. Suspend the required amount of freeze-dried powder in distilled water (1 g freeze-dried powder gives about 3.0 ml of final medium volume).
  2. Wash immediately for 1 h on a sintered glass filter (porosity G3), using approximately 200 ml distilled water per gram freeze-dried powder, added in several aliquots.
  3. Dissolve the ligand in the coupling buffer to a final concentration of 0.5 to 10 mg/ml (for protein ligands) or transfer solubilized ligands into the coupling buffer using a desalting column (see Buffer exchange and desalting in Appendix 1). Adjust the pH of the aqueous phase.
  4. Use a medium: buffer ratio of 1:0.5 to 1:1, mix the medium suspension with the ligand solution for 16 h at 20°C to 40°C in a shaking water bath.
  5. Block remaining excess groups with 1 M ethanolamine for at least 4 h or overnight, at 40°C to 50°C.
  6. Wash away excess ligand with coupling solution followed by distilled water, 100 mM NaHCO3, 500 mM NaCl, pH 8.0, and 100 mM NaCl, 100 mM acetate, pH 4.0.

If organic solvents have been used, use pH paper to measure pH since solvents can damage pH electrodes.

Using the higher temperatures can decrease coupling times.

Do not use Tris, glycine, or other nucleophilic compounds as these will couple to the oxirane groups.

Do not use magnetic, stirrers as they can disrupt the Sepharose medium.

When a ligand contains more than one kind of group (thiol, amino and hydroxyl), the coupling pH will determine which of these groups is coupled preferentially. As a general rule, the order of coupling is e-amino > thiol > a-amino > hydroxyl although the exact result will depend on the detailed structure of the ligand.

The time of reaction depends greatly on the pH of the coupling solution, properties of the ligand, and the coupling temperature. The stability of the ligand and the carbohydrate chains of the matrix limit the maximum pH that can be used. Coupling is performed in the pH range of 9.0 to 13.0 as shown in Figure 4.14 and the efficiency of coupling is pH- and temperature-dependent (Fig 4.15).

pH dependence of coupling N-acetyl-D-galactosamine

Fig 4.14. pH dependence of coupling N-acetyl-D-galactosamine to Epoxy-activated Sepharose 6B. Carbonate/bicarbonate buffers were used in the pH range of 9.0 to 11.0, sodium hydroxide solution in the pH range of 12.0 to 14.0. Ligand concentrations: 5 mg/ml and 20 mg/ml.

Efficiency of coupling glycyl-leucine

Fig 4.15. Efficiency of coupling glycyl-leucine to Epoxy-activated Sepharose 6B.

Storage

Store the freeze-dried powder dry below 8°C.

Store the column in a solution that maintains the stability of the ligand and contains a bacteriostatic agent, see Appendix 8, or 20% ethanol in a suitable buffer.

The pH stability of the chromatography media when coupled to a ligand will depend upon the stability of the ligand.

Coupling other functional groups

EAH Sepharose 4B may be used as a starting material for coupling via alternative functional groups (Fig 4.16). Phenolic groups may be attached via diazonium derivatives (VII) or via the bromoacetamidoalkyl derivative (V) prepared by treating EAH Sepharose 4B with O-bromoacetyl-N-hydroxysuccinimide. This derivative also couples via primary amino groups. The spacer arm of EAH Sepharose 4B may be extended by reaction with succinic anhydride at pH 6.0 (VI) to form a derivative to which amino groups can be coupled by carbodiimide reaction. Carboxyl groups are coupled to EAH Sepharose 4B by the carbodiimide reaction (III). Thiol derivatives, prepared by reaction (IV), couple carboxyl groups in the presence of carbodiimide and the thiol ester bond may be cleaved specifically using hydroxylamine, thus providing a simple and gentle method for eluting the intact ligand-protein complex.

Reactions used to couple ligands to Sepharose

Fig 4.16. Reactions used to couple ligands to Sepharose.

Materials