Name: 4-succinimidyl-oxycarbonyl-α-(2-pyridyldithio)toluene
Brand: BOC Sciences
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Synonyms

(2,5-dioxopyrrolidin-1-yl) 4-[(pyridin-2-yldisulfanyl)methyl]benzoate;

IUPAC Name

Canonical SMILES

C1CC(=O)N(C1=O)OC(=O)C2=CC=C(C=C2)CSSC3=CC=CC=N3

InChI

InChI=1S/C17H14N2O4S2/c20-15-8-9-16(21)19(15)23-17(22)13-6-4-12(5-7-13)11-24-25-14-3-1-2-10-18-14/h1-7,10H,8-9,11H2

InChIKey

VNWUUFREFCAFEV-UHFFFAOYSA-N

Appearance

Soild powder

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Room temperature

4-succinimidyl-oxycarbonyl-α-(2-pyridyldithio)toluene, often referred to as SPDP, serves as a versatile cross-linking reagent with multifaceted applications in diverse biochemical settings. Here are four key applications of SPDP, presented with high perplexity and burstiness:

Protein-Protein Conjugation: SPDP emerges as a pivotal tool for covalently joining distinct proteins, opening avenues to investigate intricate protein-protein interactions. Through the establishment of steadfast disulfide bonds between proteins, researchers orchestrate functional protein complexes tailored for biochemical assays. This method is indispensable in unraveling the nuanced functional dynamics of protein associations within cellular processes.

Antibody Labeling: Delving into the realm of antibody labeling, SPDP plays a pivotal role in affixing antibodies with a spectrum of tags like enzymes, fluorescent dyes, or radioactive labels. This strategic maneuver enables the creation of antibody conjugates instrumental in diagnostic assays, flow cytometry, and imaging modalities. The precision inherent in the conjugation process upholds the biological activity of the antibody, ensuring the generation of dependable results.

Drug Delivery Systems: Embarking on the frontier of targeted drug delivery, SPDP facilitates the construction of tailored drug delivery systems by tethering therapeutic agents to targeting molecules, such as antibodies or peptides. This innovative approach bolsters the precise conveyance of drugs to specific cells or tissues, enhancing therapeutic efficacy while mitigating side effects. Such targeted systems prove exceptionally valuable in oncology and other domains of precision medicine.

Surface Immobilization: Leveraging the prowess of SPDP, researchers can immobilize biomolecules on solid substrates for a myriad of bioanalytical and biosensing endeavors. By affixing proteins, peptides, or other species onto surfaces, scientists fashion biosensors indispensable for detecting specific analytes. This capability is pivotal in realms spanning diagnostics, environmental surveillance, and biotechnological exploration, fostering the development of high-throughput screening methodologies.

1. Bioconjugation of ribonuclease A: a detailed chromatographic and mass spectrometric analysis of chemical modification by a cross-linking reagent

G H Lang, L S Nichols, D E Richardson, D H Powell, J R Eyler, T J Méndez, J V Johnson Bioconjug Chem . 2000 Mar-Apr;11(2):182-94. doi: 10.1021/bc990117g.

The modification of ribonuclease A with the heterobifunctional cross-linker, 4-succinimdyloxycarbonyl-methyl-alpha-[2-pyridyldithio]-toluene (SMPT) is described. RNase A has 11 potential sites of modification by the SMPT reagent. Tracking the two-dimensional separation and proteolytic digestion of SMPT-modified RNase A with ESI/FTICR-MS and HPLC/ESI/QIT-MS demonstrates the detailed information about number of SMPT modifications and sites of modification that can be obtained by application of these techniques. Analysis of native and modified RNase A tryptic digests by ESI/FTICR-MS resulted in the identification of the sites of modification. Semiquantitative results of the reactivity of certain lysine residues toward the coupling reagent SMPT are presented. Two sites (lysines 1 and 37) are highly reactive, while three sites (lysines 41, 61, and 104) appear to be unreactive toward SMPT under the conditions used. Experimental results demonstrate that quantitative comparison of relative intensities of peptide sequences of different charge states is not possible. No correlation was found between number of basic residues and sensitivity to detection. Digestion of the modified and unmodified RNase A by subtilisin followed by examination by HPLC/ESI/QIT-MS and MS(n) enabled further investigation of modification on lysines 1 and 7, including modification at the epsilon- and alpha-amino positions on lysine 1.

2. The GLP large scale preparation of immunotoxins containing deglycosylated ricin A chain and a hindered disulfide bond

E S Vitetta, P Knowles, V Ghetie, J W Uhr, P Thorpe, M A Ghetie J Immunol Methods . 1991 Sep 13;142(2):223-30. doi: 10.1016/0022-1759(91)90110-2.

The large scale preparation of two second generation immunotoxins containing murine monoclonal antibodies and deglycosylated ricin A chain is described. The procedure for the preparation of immunotoxins consists of the derivatization of antibody with SMPT and reduction of dgA with DTT followed by their reaction to establish a hindered interchain disulfide bond. The purification of the immunotoxin includes affinity chromatography on Blue-Sepharose to remove the free antibody and gel filtration on Sephacryl S-200HR to remove any high molecular weight material and free dgA. The two immunotoxins were prepared by GLP procedures and tested for yield, composition, purity, sterility and biological activity.

3. Quantitative analysis of derivatized proteins prepared with pyridyl disulfide-containing cross-linkers by high-performance liquid chromatography

B H Woo, D H Na, K C Lee Bioconjug Chem . 1999 Mar-Apr;10(2):306-10. doi: 10.1021/bc980029g.

Determination of the introduced moieties into derivatized proteins is an essential step in the preparation and quality control of chemically defined immunoconjugates. For the derivatized proteins using pyridyl disulfide-containing cross-linkers such as N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) and 4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pyridyldithio)tolu ene (SMPT), the derivatization degree (ratio of pyridyl disulfide moieties to protein) has been traditionally determined by measuring the absorbance of both the derivatized protein and 2-thiopyridone (2-TP) released from the dithiothreitol (DTT) treatment (spectrophotometric method). This method, however, causes several problems including false high and low determinations of the protein and 2-TP, respectively, low selectivity, poor reproducibility, and relatively large amounts of sample consumption. A quantitative determination method of the derivatization ratios using bovine serum albumin derivatized with SPDP and SMPT as the model system has been developed. The concentration of protein and 2-TP released from the DTT treatment of derivatized proteins was determined directly without consideration of different reagents used and their concentrations. The present HPLC method was proved to be better in terms of accuracy, selectivity, and reproducibility with micro sample consumption. Moreover, this HPLC method can be directly applied to all derivatized proteins prepared with pyridyl disulfide-containing cross-linkers.