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Strategies for the Synthesis of Labeled Peptides

Labeled peptides are frequently used by researchers for binding studies, to determine substrate specificity, and for receptor cross-linking studies. Many researchers would like to synthesize biotin, FITC, nanoparticle, or drug-labeled peptide. It is suggested that a new strategy, using Rink amide 4-methylbenzhydrylamine resin coupled with Fmoc-Lys(Dde)-OH, be used. The major advantage of this approach is that other labels such as fluorescein, dansyl groups, methyl coumarin, and potentially fluorophores and quenchers used for fluorescence resonance energy transfer (FRET) can be directly incorporated into peptides.

Two approaches can be used to generate labeled peptides: the peptide can be synthesized using labeled amino acids, or the label can be added after peptide synthesis has been completed. When a peptide is labeled after synthesis, there occasionally are problems with the location of the label, depending on the composition of the peptide. The use of labeled amino acids during synthesis usually ensures the correct positioning of the label.

This study focused on the synthesis of side-chain-labeled peptides--specifically, a peptide with a C-terminal biotin-labeled lysine. H-His-Ala-Phe-Gly-Gly-Glu-Ala-Lys(biotin)-NH2

Using of Fmoc-Lys(biotin)-OH was an obvious choice for direct assembly of this sequence. However, our goal is to create a method for introduction of a variety of different labels other than biotin. We used a side-chain protected lysine residue orthogonal to Fmoc/t-Bu that employs Rink amide MBHA resin coupled with Fmoc-Lys(Dde)-OH. In this approach, first the peptide is synthesized, then the label is added for coupling to the Lys, followed by cleavage and deprotection.

Peptide Synthesis Method:

Rink amide MBHA resin was coupled with Fmoc-Lys(Dde)-OH using HBTU/HOBt/DIEA activation. All remaining residues except the N-terminal His were incorporated as Fmoc-amino acids with the same activation chemistry, using Boc for Lys, and OtBu for Glu as the side-chain protecting groups. The N-terminal His was incorporated as t-Boc-His. After completion of the synthesis, the resin was washed twice with N, N-dimethylformamide (DMF). The Dde side-chain protection was removed by treatment with 2% hydrazine in DMF (7 mL/0.1 mmoles of the protected peptide resin; 2 times, 5 minutes each). The resin was then washed three times with DMF followed by two washes with DMF: dimethyl sulfoxide (DMSO; 1:1, v/v). A 10-fold molar excess of biotin was dissolved in 5 mL of DMF: DMSO (1:1). It was necessary to warm the mixture and vortex it for several minutes to dissolve the biotin completely. The biotin solution was then treated with 2.1 mL of 0.45-M HBTU/HOBt in DMF followed by 0.3 mL of diisopropylethylamine (DIEA). The activated biotin solution was added to the resin, and the mixture was stirred overnight. The resin was washed with DMF: DMSO (1:1; 3 times) followed by dichloromethane: methanol (1:1; 2 times). After the resin was thoroughly dried, the peptide was cleaved and deprotected with Reagent-K.