A sucrose-derived scaffold for multimerization of bioactive peptides

Abstract

A spherical molecular scaffold bearing eight terminal alkyne groups was synthesized in one step from sucrose. One or more copies of a tetrapeptide azide, either N(3)(CH(2))(5)(CO)-His-DPhe-Arg-Trp-NH(2) (MSH4) or N(3)(CH(2))(5)(CO)-Trp-Met-Asp-Phe-NH(2) (CCK4), were attached to the scaffold via the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. Competitive binding assays using Eu-labeled probes based on the superpotent ligands Ser-Tyr-Ser-Nle-Glu-His-DPhe-Arg-Trp-Gly-Lys-Pro-Val-NH(2) (NDP-α-MSH) and Asp-Tyr-Met-Gly-Trp-Met-Asp-Phe-NH(2) (CCK8) were used to study the interactions of monovalent and multivalent MSH4 and CCK4 constructs with Hek293 cells engineered to overexpress MC4R and CCK2R. All of the monovalent and multivalent MSH4 constructs exhibited binding comparable to that of the parental ligand, suggesting that either the ligand spacing was inappropriate for multivalent binding, or MSH4 is too weak a binder for a second 'anchoring' binding event to occur before the monovalently-bound construct is released from the cell surface. In contrast with this behavior, monovalent CCK4 constructs were significantly less potent than the parental ligand, while multivalent CCK4 constructs were as or more potent than the parental ligand. These results are suggestive of multivalent binding, which may be due to increased residence times for monovalently bound CCK4 constructs on the cell surface relative to MSH4 constructs, the greater residence time being necessary for the establishment of multivalent binding.

Figure 1

A space-filling representation of one component of Olestra (6). This representation © 1997 by Daniel J. Berger and may be copied without limit if its use is for non-profit educational purposes.

Figure 2

Superposition of cis-3-pentene (7) and 1,4-dimethyl-1H-1,2,3-triazole (8). The methyl carbon-to-methyl carbon distances are 6.04 Å and 5.00 Å for 7 and 8, respectively. The conformers of 7 and 8 used in this comparison were generated using Spartan '02 v1.0.5 for Macintosh.

Figure 3

MALDI-TOF mass spectrum of 14e showing the distribution of products. The numbers indicate the number of attached MSH4 ligands. The insert shows the anticipated statistical product distribution assuming attachment of 4.1 ligands per scaffold. For peak identification and for spectra of 13, 14a–14e, and 15a see the Supplementary Data.

Scheme 1

Synthesis of Multivalent Constructs 3.

Scheme 2

Synthesis of sucrose derivative 9.

Scheme 3

Solid Phase Synthesisa ^aReagents: (a) piperidine. (b) Fmoc/tBu solid phase synthesis. (c) N₃(CH₂)₅CO₂H, Cl-HOBt, DIC. (d) trifluoroacetic acid/triisopropylsilane/thioanisole/water (91/3/3/3).

Scheme 4

Synthesis of multivalent compounds 13, 14a–14e, and 15a–d via CuAAC.a ^aThe positions of ligand attachment are presumed to be random.