Heterobifunctional cross-linking

Another approach towards conjugation is the use of cross-linkers containing two different reactive groups. Compared with homobifunctional cross-linkers, heterobifunctional linkers offer the possibility to prepare better-defined conjugates, since peptide-peptide and carrier-carrier conjugation can be prevented. Convenient procedures are based on couplings of thiols to sulphydryl-reactive groups. 

Cysteine-containing peptides can be conjugated to sulphydryl-reactive proteins, provided that the cysteine is not essential for the biological activity. If the peptide is devoid of Cys, this amino acid can be added N- or C-terminally during synthesis. Depending on the linker attached to the solid phase, C-terminal incorporation is not recommended to avoid possible racemization of Cys (17). 

The active ester AT-succinimidyl 3-(2-p3 ridyldithio)propionate (SPDP) is the classical reagent to modify proteins with pyridyldithiopropyl (PDP) groups (20). If the protein to be modified contains many free Cys-residues this creates the risk of carrier-carrier conjugation. In this case it is possible to block the free thiols with iodo- or bromoacetic acid (or amide) before reaction with SPDP (or to use a different conjugation method, as described in Section 3.2). 

Instead of using Cys-containing peptides it is convenient to use S-acetylmercaptoacetyl (SAMA) peptides for conjugation purposes. Unlike Cys, SAMA is not sensitive towards (air) oxidation during purification of the peptide. SAMA can be introduced into deprotected peptides in solution by reaction with N-succinimidyl S-acetylmercaptoacetate (18, 24). Selective introduction of SAMA can be achieved at the end of solid phase synthesis by N-terminal modification (Chapter 6) of the side-chain protected and resin-bound peptide with pentafluorophenyl 5-acetylmercaptoacetate (SAMA-OPfp) and 1-hydroxybenzotriazoIe (25). The thiol group of SAMA-peptides can be liberated by reaction with hydroxylamine. This S-deacetylation can be performed in the conjugation reaction mixture (25,26), i.e. in the presence of the sulphydryl-reactive protein Protocol 2). 

If a conjugate is required for immunization experiments, it is convenient to prepare not only the SAMA- or Cys-peptide, but also the corresponding l)iotinyl-peptide or an N-acetylated MAP-8 (Section 4). The biotinyl-peptide can be bound to (strept)avidin-coated microtitre plates to determine the antipeptide titre of the sera obtained. The MAP-8 can be coated directly onto regular plates. 

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Chitosan is the main structural component of crab and shrimp shells. Chitosan contains both reactive amino and hydroxyl groups, which can be used to chemically alter its properties under mild reaction conditions. Al-acyl chitosans were already reported as blood-compatible materials. UV irradiation grafting technique was utilized to introduce obutyrylchitosan (OBCS) onto the grafted SR film in the presence of the photosensitive heterobifunctional cross-linking agent. The platelet adhesion test revealed that films grafted on OBCS show excellent antiplatelet adhesion. 

Jaffe, C.L., Lis, H., and Sharon, N. (1980) New cleavable photoreactive heterobifunctional cross-linking reagents for studying membrane organization. Biochemistry 19, 4423. 

Lewis, R.V., Roberts, M.F., Dennis, E.A., and Allison, W.S. (1977) Photoactivated heterobifunctional cross-linking reagents which demonstrate the aggregation state of phospholipase A2. Biochemistry 16, 5650-5654. 

Singh, V., Mavila, A.K., and Kar, S.K. (1993) Comparison of the cytotoxic effect of hormonotoxins prepared with the use of heterobifunctional cross-linking agents N-succinimidyl 3-(2-pyridyldithio)propionate and N-succinimidyl 6-[3-(2-pyridyldithio)propionamido]-hexanoate. Bioconjugate Chem. 4, 473M82. 

Yan, M., Cai, S.X., Wybourne, M.N., and Keana, J.F.W. (1994) N-Hydroxysuccinimide ester functionalized perfluorophenyl azides as novel photoactivatable heterobifunctional cross-linking reagents. The covalent immobilization of biomolecules to polymer surfaces. Bioconjugate Chem. 5, 151-157. 

Zara, J.J. et al. (1991) A carbohydrate-directed heterobifunctional cross-linking reagent for the synthesis of immunoconjugates. Anal. Biochem. 194, 156-162. 

Frisch B, Boeckler C, Schuber F. Synthesis of short polyoxyethylene-based heterobifunctional cross-linking reagents. Application to the coupling of peptides to liposomes. Bioconjugate Chem 1996 7 180. 

Boeckler C, et al. Immunogenicity of new heterobifunctional cross-linking reagents used in the conjugation of synthetic peptides to liposomes. J Immunol Meth 1996 191 1. 

Table4.2. Selected homo-and heterobifunctional cross-linking reagents...

Modification Reagent that can create functionalities able to couple with reactive group 2 of the heterobifunctional cross-linking agent... 

Figure 19 Heterobifunctional cross-linking agents used in multistep protocols result in the best control over the products formed.

Heterobifunctional cross-linking reagents also may be used to site-direct a conjugation reaction toward particular parts of target molecules. Amines may be coupled on one molecule while sulfhydryls or carbohydrates are targeted on another molecule. Directed coupling often is important in preserving critical epitopes or active sites within macromolecules. For instance, antibodies may be coupled to other proteins... 

Figure 155 The general design of a heterobifunctional cross-linking agent includes two different reactive groups at either end and an organic cross-bridge of various length and composition. The cross-bridge may be constructed of chemically cleavable components for selective disruption of conjugates.

N-Succinimidyl 3-(2-pyridyldithio)propionate (SPDP) may be the most popular heterobifunctional cross-linking agent available. The activated NHS ester end of SPDP... 

Succinimidyloxycarbonyl-a-methyl-a-(2-pyridyldithio)toluene (SMPT) is a heterobifunctional cross-linking agent that contains an amine-reactive NHS ester on one end and a sulfhydryl-reactive pyridyl disulfide group on the other. SMPT is therefore an analog of SPDP that differs only in its cross-bridge which contains an aromatic ring... 

A relatively new set of heterobifunctional cross-linking agents now are available that contain a carbonyl-reactive group on one end and a sulfhydryl-reactive functional... 

N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) is by far the most popular heterobifunctional cross-linking agent used for immunotoxin conjugation (Chapter 5, Section 1.1). The activated NHS ester end of SPDP reacts with amine groups in one of the two proteins to form an amide linkage. The 2-pyridyldithiol group at the other end... 

Succinimidyloxycarbonyl-oi-methyl-oi-(2-pyridyldithio)toluene (SMPT) is a heterobifunctional cross-linking agent similar to SPDP that contains an amine-reactive NHS... 

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