Phenol → L-Glutamic Acid

Participants

Reaction Steps

Synthesis of Diamine- and Gd-Substituted Aβ Derivatives. Aβ1-30, with the sequence Ahx (Fmoc-6-aminohexanoic acid)-DAEFRHDSGYEVHHQKLVFFAEDVGSNKGA (SEQ ID NO:5), was synthesized on an ABI 433 (Foster City, Calif.) peptide sythesizer using HBTU activation and the manufacturer's suggested synthesis protocols. The starting resin was Ala-NovaSyn TGA (Calbiochem-Novabiochem, San Diego, Calif.). Glutamic acid residues 3, 11, and 22 were synthesized with N-α-Fmoc-L-glutamyl-δ-N-(4-aminobutyl)carbamic acid tert-butyl ester (3b), and aspartic acid residues 7 and 23 were synthesized with N-α-Fmoc-L-aspartyl-γ-N-(4-aminobutyl)carbamic acid tert-butyl ester described above. After completion of the synthesis and final Fmoc deprotection, diethylenetriaminepentaacetic acid anhydride (DTPA) was added to the N-terminal Ahx residue by dissolving 120 mg of the DTPA in 2 ml of DMSO/8 ml DMF and reacting the DTPA solution with the peptide-resin, which had been washed previously with DIEA/DCM. The coupling of DTPA was allowed to proceed overnight at RT. Completion of the reaction was verified by a negative ninhydrin reaction. The Aβ1–30 peptide was then cleaved from the resin support using 5% crystalline phenol, 5% water, 2.5% triisopropylsilane, and 87.5% TFA for two hours at RT. The peptide was purified by reverse-phase HPLC on a C18 Jupiter column (250 mm×21.2 mm, Phenomonex Corp) using a gradient system of 0.1% aqueous TFA containing 80% acetonitrile. The calculated mass weight 3390 amu of for Aβ1–30 and 4231 amu for DTPA-[N-4ab/Q-4ab]Aβ30 was confirmed by electrospray ionization mass spectrometry (Sciex API 165). The element Gadolinium (Gd) was chelated at equal mole concentration to the DTPA functional group of the Aβ1–30 peptide using Gd(III) chloride hexahydrate (Sigma) in water at RT for one hour (now designated as Gd[N-4ab/Q-4ab]Aβ30) (FIG. 1). All Aβ peptides were labeled with 125I/131I using a modified chloramine T procedure as described previously (Poduslo, J. F., et al., Neurobiol. Dis. 4:27–34, 1997).

Provenance & Evidence