SMPB

Succinimidyl-4-(p-maleimidophenyl)butyrate (SMPB), is a heterobifunctional analog of MBS containing an extended cross-bridge (Chapter 5, Section 1.6). The crosslinker has an amine-reactive NHS ester on one end and a sulfhydryl-reactive maleimide group on the other. Conjugates formed using SMPB thus are linked by stable amide and thioether bonds. 

As in the case of MBS, discussed previously, SMPB was found to be more effective than aliphatic crosslinkers in producing immunotoxin conjugates with ricin that have high yields of cytotoxicity (Myers et al., 1989). This was attributed to the reagent s aromatic ring structure. A comparison with SPDP produced immunotoxin conjugates concluded that SMPB formed more stable complexes that survive in serum for longer periods (Martin and Papahadjopoulos, 1982). 

The method for the preparation of immunotoxins with SMPB is identical to that used for MBS (above). Since the thioether bonds formed with sulfhydryl-containing molecules are non-cleavable, A-chain isolates or single-chain toxin molecules can not be conjugated with antibodies with retention of cytotoxicity. Only intact A-B toxin molecules may be used with this crosslinker, since the A chain still is capable of being reductively released from the complex. 

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Figure 5.7 SMPB may be used in a two-step procedure to conjugate an amine-containing molecule to a sulfhy-dryl compound, forming amide and thioether bonds, respectively.

SMPB contains a hydrophobic cross-bridge and relatively nonpolar ends, which allows the reagent to permeate membrane structures. Due to its water-insolubility, it must be dissolved in an organic solvent prior to adding an aliquot to a reaction mixture. The solvents DMF and DMSO work well for this purpose. A concentrated stock solution prepared in these solvents allows for easy addition of a small amount to a conjugation reaction. Long-term storage in these solvents is not recommended due to slow water pickup and possible hydrolysis of the NHS ester end. 

A water-soluble analog to SMPB, called sulfo-SMPB [sulfosuccinimidyl-4-(p-maleimidophenyl) butyrate] contains a negatively charged sulfonate group which lends considerable hydrophilic-ity to the molecule (Thermo Fisher). Sulfo-SMPB may be added directly to aqueous reaction... 

The most common type of heterobifunctional reagent used for the activation of lipid components includes the amine- and sulfhydryl-reactive crosslinkers containing an N-hydroxysuccinim-ide (NHS) ester group on one end and either a maleimide, iodoacetyl, or pyridyl disulfide group on the other end (Chapter 5, Section 1). Principle reagents used to effect this activation process include SMCC (Chapter 5, Section 1.3), MBS (Chapter 5, Section 1.4), SMPB (Chapter 5, Section 1.6), SIAB (Chapter 5, Section 1.5), and SPDP (Chapter 5, Section 1.1). Other... 

Figure 22.11 A sulfhydryl-reactive lipid derivative may be prepared through the reaction of SMPB with PE to produce a maleimide-containing intermediate. Sulfhydryl-containing molecules then may be coupled to the phospholipid via stable thioether linkages.

N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) also may be used to activate pure PE lipids in a similar manner to SMPB. The result will be a derivative containing pyridyl disulfide groups rather than maleimide groups (Figure 22.12). Pyridyl disulfides react with sulfhydryls to form disulfide linkages. Either the standard SPDP or the long-chain version, LC-SPDP, may be employed in the following protocol. 

Figure 22.16 SMPB-activated liposomes may be modified with peptide hapten molecules containing cysteine thiol groups. The resultant immunogen may be used for immunization purposes to generate an antibody...

Prepare a liposome suspension, containing MPB-PE, at a total lipid concentration of 5 mg/ ml in 0.05 M sodium phosphate, 0.15 M NaCl, pH 7.2. Activation of DPPE with SMPB is... 

Figure 22.27 SMPB-activated liposomes may be used to couple thiol-containing protein molecules, forming stable thioether linkages.

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