Amines with SATA

SATA has been used to form conjugates with avidin or steptavidin with excellent retention of activity (Chapter 23, Section 3.1). It also has been used in the formation of a therapeutically useful toxin conjugate with recombinant CD4 (Ghetie et al., 1990), to study syntaxin proteins (Amessou et al., 2007), to prepare bispecific antibodies (Lindorfer et al., 2001), and to make a unique polylysine conjugate as a vehicle for drug delivery (Sakharov et al., 2001). 

SATA is freely soluble in many organic solvents. In use, it is typically dissolved as a stock solution in DMSO, DMF, or methylene chloride, and then an aliquot of this solution is added to an aqueous reaction mixture containing the protein to be modified. 

The following protocol represents a generalized method for protein thiolation using SATA. For comparison purposes, contrast the variation of this SATA modification method as outlined in Chapter 20, Section 1.1 for use in the preparation of antibody-enzyme conjugates. 

Dissolve the protein to be thiolated at a concentration of l-5mg/ml in 50 mM sodium phosphate, pH 7.5, containing 1-10 mM EDTA. Other non-amine containing buffers such as borate, HEPES, and bicarbonate also may be used as the reaction medium. The effective pH for the NHS ester modification reaction is in the range of 7.0-9.0, but environments closer to neutrality will limit the hydrolysis of the ester. 

Dissolve the SATA reagent (Thermo Fisher) in DMSO at a concentration of 65 mM (15 mg/ml). Note DMSO should be handled in a fume hood. 

A versatile reagent for introducing sulfhydryl groups into proteins is SATA, N-suc-cinimidyl 5-acetylthioacetate (Duncan et al., 19S3). The active NHS ester end of SATA reacts with amino groups in proteins and other molecules to form a stable amide linkage (Fig. 54) (Chapter 2, Section 1.4). The modified protein then contains a 

SATA-Labeled Protein with Protected Sulfhydryl 

SATA has been used to form conjugates with avidin or steptavidin with excellent retention of activity (Chapter 13, Section 3.1). It has been used in the formation of a therapeutically useful toxin conjugate with recombinant CD4 (Ghetie etal., 1990A). 

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Oligonucleotides containing amine groups introduced by enzymatic or chemical means may be modified with SATA (Chapter 1, Section 4.1) to produce protected sulfhydryl derivatives. The NHS ester end of SATA reacts with a primary amine to form a stable amide bond. After modification, the acetyl protecting group can be removed as needed by treatment with hydroxylamine under mildly alkaline conditions (Fig. 401). The result is terminal sulfhydryl groups that can be used for subsequent labeling with thiol-reactive probes or activated-enzyme derivatives (Kumar and Malhotra, 1992). 

Figure 1.62 SATA can react with available amine groups in proteins and other molecules via its NHS ester end to form protected sulfhydryl derivatives. The illustrated protein is glutathione-S-transferase (E.C.2.5.1.18) (Ji et al., 1995).

Figure 1.65 SATP reacts with amine-containing proteins or other molecules via its NHS ester end to create protected sulfhydryl derivatives in a manner similar to that of SATA. Deprotection can be done with hydroxy-lamine to free the thiol.

Although amine-reactive protocols, such as SATA thiolation, result in nearly random attachment over the surface of the antibody structure, it has been shown that modification with up to 6 SATAs per antibody molecule typically results in no decrease in antigen binding activity (Duncan et al., 1983). Even higher ratios of SATA to antibody are possible with excellent retention of activity. 

Dissolve the antibody to be modified in 0.1 M sodium phosphate, 0.15M NaCl, pFl 7.2, at a concentration of 1-5 mg/ml. Note Phosphate buffers at various pFl values between 7.0 and 7.6 have been used successfully with this protocol. Other mildly alkaline buffers may be substituted for phosphate in this reaction, providing they don t contain extraneous amines (e.g., Tris) or promote hydrolysis of SATA s NHS ester (e.g., imidazole). 

Figure 20.6 Available amine groups on an antibody molecule may be modified with the NHS ester end of SATA to produce amide bond derivatives containing terminal protected sulfhydryls. The acetylated thiols may be deprotected by treatment with hydroxylamine at alkaline pH. Reaction of the thiolated antibody with a maleimide-activated enzyme results in thioether crosslinks.

The amine groups on these fragments also may be modified with thiolating agents, such as SATA or 2-iminothiolane, to create sulfhydryl residues suitable for coupling to maleimide-activated enzymes (Section 1.1, this chapter) (Figure 20.13). Amine groups further may be utilized... 

Figure 27.8 SATA may be used to modify a 5 -amine derivative of an oligonucleotide, forming a protected sulf-hydryl. Deprotection with hydroxylamine results in generation of a free thiol.

Figure 5 Covalent coupling of cyclic peptide moieties to human serum albumin (HSA). The depicted cyclic peptide, C SRNLIDC, in which C denotes the cyclizing cysteine residues, mimics the receptor binding site of PDGF-BB. First, a sulfhydryl group is introduced to the cyclic peptide by a reaction with succinimide-acetyl thioacetate (SATA). The primary amino groups of lysine in HSA are derivitized with maleimide-hexoyl-At-hydroxysuccinimide ester (MHS). Subsequently, the cyclic peptide is coupled to HSA. In this latter reaction, hydroxyl amine is used to remove the protecting acetate group from the sulfhydryl group of the cyclic peptide.

T. Sata, S. Nojima and K. Matsusaki, Anion exchange membranes prepared by amination of cross-linked membranes having chloromethyl groups with 4-vinylpyridine and trimethylamine, Polymer, 1999, 40, 7243. 

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