Cystamine reduction

The following protocol is useful for the modification of proteins with cystamine with subsequent reduction to create the free sulfhydryl. 

Reduction of the cystamine-labeled oligo using a disulfide reducing agent releases 2-mer-captoethylamine and creates a thiol group for conjugation (Figure 27.6). DNA probes labeled in this manner have been successfully coupled with SPDP-activated alkaline phosphatase (Chapter 26, Sections 1.2 and 2.5), maleimide-activated horseradish peroxidase (HRP) (Chapter 26, Section 1.1), NHS-LC-biotin (Chapter 11, Section 1 and Chapter 27, Section 2.3), and the fluorescent tag AMCA-HPDP (Chapter 9, Section 3 and Chapter 27, Section 2.5). 

Figure 27.6 The 5 -phosphate group of oligonucleotides may be labeled with cystamine using the EDC/imid-azole reaction. This results in the formation of an amine-terminal spacer containing an internal disulfide group. Reduction of the disulfide provides a route to creating a free thiol for further derivatization.

For reduction of the cystamine disulfides, add 20 pi of 1.0 M DTT and incubate at room temperature for 15 minutes. This will release 2-mercaptoethylamine from the cystamine modification site and create the free sulfhydryl on the 5 terminus of the oligonucleotide. 

Lactose Modified with Cystamine at Its Reducing End via Reductive Amination... 

Cystamine Labeled Oligonucleotide (after reduction to sulfhydryl)... 

More recently, thiolated dextran was synthesised by modification of dextran with 4-nitrophenyl chloroformate in DMSO/pyridine in the presence of DMAP at 0 °C yielding a carbonate (content 6%), which can be substituted with cystamine. Subsequent reduction yields the thiolated dextran containing between 1 and 4% thiol moieties (Fig. 57) [370]. 

A putative y-amide derivative was also prepared from MTX by reaction with cystamine in water solution in the presence of EDC hydrochloride, followed by reduction of the disulphide bond with excess 2-mercaptoethanol [328]. The resulting thiol (VIII.220) was then allowed to react with... 

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