1. 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
V Singh, A K Mavila, S K Kar Comparative StudyBioconjug Chem. 1993 Nov-Dec;4(6):473-82.doi: 10.1021/bc00024a009.
With the aim of developing cytotoxic hybrid molecules which can be selectively targeted to specific cells in the gonads, a single chain ribosome-inactivating protein, gelonin, was conjugated to ovine luteinizing hormone (oLH) with the use of heterobifunctional cross-linking agents N-succinimidyl3-(2-pyridyldithio)-propionate (SPDP) and long-chain SPDP. Four hormonotoxins were synthesized having a variable spacer arm between oLH and gelonin. The spacer arms in C200A, C210A, C220A, and C230A were 13.6, 22.4, 22.4, and 31.2 A long, respectively. Extensive physiochemical and biochemical analysis revealed a 1:1 molar ratio of the ingredients in its oLH-S-S-gelonin conjugates. The linkage occurred through the epsilon-NH2 group of the alpha-subunit of oLH as judged from RP-HPLC analysis. The hormonotoxins retained substantial receptor binding ability, steriodogenic activity, and immunoreactivity of oLH and gelonin to their respective antibodies. Hormonotoxins bind to Leydig tumor cells via oLH, leaving gelonin free as judged by competitive displacement analysis. The hormonotoxins internalized to a sufficient degree to effectively inhibit protein synthesis. Upon comparison, immunoreactivity, receptor binding steroidogenic activity, and cytotoxicity of oLH-S-S-gelonin conjugates prepared with the use of only LC-SPDP (C230A, 31.2-A spacer arm) and by using both SPDP and LC-SPDP (C210A and C220A, 22.4-A spacer arm) were found to be comparable with that of conjugate prepared with SPDP alone (C200A, 13.6-A spacer arm). Therefore, it may be concluded that the cytotoxicity of oLH-based hormonotoxin remained unaffected with the use of long-chain spacer arms which are believed to be used generally to avoid steric hindrance.
2. 3-(2-pyridyldithio)propionic acid hydrazide as a cross-linker in the formation of liposome-antibody conjugates
S M Ansell, P G Tardi, S S Buchkowsky Bioconjug Chem. 1996 Jul-Aug;7(4):490-6.doi: 10.1021/bc960036+.
Liposome antibody conjugates are potentially useful as a means of targeting drugs to specific tissues. A new protocol for the conjugation of IgG to maleimide-containing liposomes was developed using 3-(2-pyridyldithio)propionic acid hydrazide (PDPH) as a cross-linker. Periodate-oxidized antibody was treated with PDPH to yield a hydrazone derivative. Deprotection with DTT produced a thiolated antibody which was then conjugated to liposomes containing N-[4-(p-maleidophenyl)butyryl]-1,2-sn-distearoylphosphatidyleth anolamine. The liposome-antibody conjugates were found to have in vitro properties similar to those of conjugates formed by the traditional 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester (SPDP) protocol but were cleared less rapidly in circulation. The PDPH protocol presents a viable alternative to SPDP, particularly for antibodies sensitive to amine modification.
3. Protein thiolation and reversible protein-protein conjugation. N-Succinimidyl 3-(2-pyridyldithio)propionate, a new heterobifunctional reagent
J Carlsson, H Drevin, R Axén Biochem J. 1978 Sep 1;173(3):723-37.doi: 10.1042/bj1730723.
A heterobifunctional reagent, N-succinimidyl 3-(2-pyridyldithio)propionate, was synthesized. Its N-hydroxysuccinimide ester group reacts with amino groups and the 2-pyridyl disulphide structure reacts with aliphatic thiols. A new thiolation procedure for proteins is based on this reagent. The procedure involves two steps. First, 2-pyridyl disulphide structures are introduced into the protein by the reaction of some of its amino groups with the N-hydroxysuccinimide ester sie of the reagent. The protein-bound 2-pyridyl disulphide structures are then reduced with dithiothreitol. This reaction can be carried out without concomitant reduction of native disulphide bonds. The technique has been used for the introduction of thiol groups de novo into ribonuclease, gamma-globulin, alpha-amylase and horseradish peroxidase. N-Succinimidyl 3-(2-pyridyldithio)propionate can also be used for the preparation of protein-protein conjugates. This application is based on the fact that protein-2-pyridyl disulphide derivatives (formed from the reaction of non-thiol proteins with the reagent) react with thiol-containing proteins (with native thiols or thiolated by, for example, the method described above) via thiol-disulphide exchange to form disulphide-linked protein-protein conjugates. This conjugation technique has been used for the preparation of an alpha-amylase-urease, a ribonuclease-albumin and a peroxidase-rabbit anti-(human transferrin) antibody conjugate. The disulphide bridges between the protein molecules can easily be split by reduction or by thiol-disulphide exchange. Thus conjugation is reversible. This has been demonstrated by scission of the ribonuclease-albumin and the alpha-amylase-urease conjugate into their components with dithiothreitol. N-Succinimidyl 3-(2-pyridyldithio)propionate has been prepared in crystalline form, in which state (if protected against humidity) it is stable on storage at room temperature (23 degrees C).
