Chemical conjugation of DOTA and Cy5.5 to knottin peptide 2.5D. The peptide-based crosslinker Fmoc-Lys(ivDde)-Gly-Gly-Tyr was first synthesized and coupled to the N-terminus of the folded knottin peptide 2.5D using succinimide ester activation chemistry. Next, the Fmoc group was removed from the N-terminus of the crosslinker (i: 10% piperidine/DMF, 0.5 hr, RT), and the resulting compound was reacted with a 5-fold molar excess of an NHS ester-activated DOTA group (ii: DMF/2% DIEA, 0.5 hr, RT) to generate DOTA-Lys(ivDde)-Gly-Gly-Tyr-2.5D. Finally, the ivDde group was removed from the ε-amino group of Lys (iii: 2% hydrazine/DMF, 0.5 hr, RT) and the resulting compound was reacted with Cy5.5 NHS ester (iv: DMF/2% DIEA, 0.5 hr, RT) to give the final product: DOTA-Lys(Cy5.5)-Gly-Gly-Tyr-2.5D, which was used for NIRF imaging experiments, or radiolabeled with 64Cu and used for PET imaging experiments.