Squalene-derived flexible linkers for bioactive peptides

Abstract

A regiochemical and stereochemical mixture of flexible linkers bearing terminal azide functionality was synthesized in two steps from squalene and was used to connect two high affinity NDP-alpha-MSH ligands or two low affinity MSH(4) ligands. The ligands were N-terminally acylated using N-hydroxysuccinimidoyl 5-hexynoate and were subsequently attached to the linker via copper-catalyzed 'click' 3+2 cyclization of the azide and alkyne moieties. In vitro biological evaluations showed that the binding affinity to the human melanocortin 4 receptor was not diminished for most linker-ligand combinations relative to the corresponding parental ligand. Statistical and cooperative binding effects were observed for dimeric constructs containing the low affinity ligand MSH(4), but not for dimeric NDP-alpha-MSH constructs, presumably due to slow off rates for this high affinity ligand.

Figure 1

Monovalent and divalent compounds on a squalene-derived scaffold prepared via “click” attachment of alkynylated ligands. The sites of ligand attachment shown are arbitrary.

Scheme 1

Reagents: (a) BH₃, THF; H₂O₂, NaOH. (b) NaH, DMF; Br(CH₂)₆N₃ (2). The sites of attachment of the 6-azidohexyl moieties to the squalane scaffold shown are arbitrary. Mixtures of all possible stereoisomers of 1 and egioisomers of 3 and 4 are produced.

Scheme 2

Reagents: (a) Et₃N, DMF, H₂O.