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DACB was synthesized by covalently conjugating iodoacetyl-deferoxamine and N-cysteinyl-biotin by the following Scheme II. ##STR20## Solid deferoxamine mesylate was added to methanol for a concentration of 20 mM, followed by addition of NaOH to reach a final concentration of 20 mM NaOH. The solution was heated and maintained at 65°. Solid iodoacetic acid N-hydroxysuccinimide ester (NHS-IA) was added to the deferoxamine solution to a final molarity of 50mM NHS-IA and incubated for at least one hour at 65°. The resulting iodoacetyl-deferoxamine (IA-DFO) was purified by HPLC with an EM LiChroCART reverse phase column using a linear gradient of 0-10 ml 100% Buffer A (0.025M phosphate, pH 6, 2 mM NTA) and 10-60 ml 0-100% Buffer B (0.025M phosphate, pH 6, 2 mM NTA, 75% methanol). IA-DFO, but not unconjugated DFO, has an absorbance at 260 nm and thus was purified by measuring the absorbance of the eluate and collecting the fraction with absorbance at 260 nm.

1. temperatureThe solution was heated
2. temperaturemaintained at 65°
3. waitincubated for at least one hour at 65°
4. customThe resulting iodoacetyl-deferoxamine (IA-DFO) was purified by HPLC with an EM LiChroCART reverse phase column
5. customthus was purified
6. customcollecting the fraction with absorbance at 260 nm