For the synthesis of folate-containing conjugates 10-15, an extra lysine was added at the N-terminal of the peptidyl backbone, and cyanine dye 1 and folate were coupled to its α-and ε-amine groups, respectively. A literature procedure was first attempted. However, this strategy led to folate conjugation at both its α-and γ-carboxylate groups, giving an inseparable mixture of labeled isomers (Supplementary Figure ). To overcome this issue, a protected glutamic acid residue, Fmoc-Glu(OtBu)-OH, was first coupled to the peptide, followed by coupling with pteroic acid. Since no difference was found in the endocytosis efficiency of conjugates where the folate is labelled through either its α-or γ-carboxylic group, we opted for the reaction of the α-carboxylate due to the ready availability of the reagent. As a protecting group for the extra lysine side chain to be conjugated with folate, 4-methyltrityl (Mtt) was chosen for conjugates 10-14. In this way, the protecting group on the N-terminal lysine could be selectively cleaved with conditions orthogonal to the other protecting groups on the peptidyl resin. However, the synthesis of compound 15 proved to be more challenging.