A solanesol-derived scaffold for multimerization of bioactive peptides

Abstract

A flexible molecular scaffold bearing varying numbers of terminal alkyne groups was synthesized in five steps from solanesol. R(CO)-MSH(4)-NH(2) ligands, which have a relatively low affinity for binding at the human melanocortin 4 receptor (hMC4R), were prepared by solid phase synthesis and were N-terminally acylated with 6-azidohexanoic acid. Multiple copies of the azide N(3)(CH(2))(5)(CO)-MSH(4)-NH(2) were attached to the alkyne-bearing, solanesol-derived molecular scaffold via the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. Control studies showed that the binding affinity of the triazole-containing ligand, CH(3)(CH(2))(3)(C(2)N(3))(CH(2))(5)(CO)-MSH(4)-NH(2), was not significantly diminished relative to the corresponding parental ligand, CH(3)(CO)-MSH(4)-NH(2). In a competitive binding assay with a Eu-labeled probe based on the superpotent ligand NDP-alpha-MSH, the monovalent and multivalent constructs appear to bind to hMC4R as monovalent species. In a similar assay with a Eu-labeled probe based on MSH(4), modest increases in binding potency with increased MSH(4) content per scaffold were observed.

Figure 1

(a) A portion of the DEPT 135 ¹³C NMR spectrum of 7 in CD₃OD (see Ref 8d). The lines from 76–79 ppm are due to methine carbons bearing an OH group. The line at 62.8 ppm is due to the two methylene carbons that bear OH groups. The relatively sharp line at 73.3 ppm is due to residual byproduct 3-methyl-2-butanol in this sample. (b) MS showing the distribution of products in the mixture of polyols 7. Peaks near 1572, 1590, 1608, and 1626 represent solanesol-derived heptadecaols, octadecadols, nonadecaols, and eicosaols, respectively.

Figure 2

MS showing the distribution of products in the mixture 9 resulting from alkylation of polyols 7. Peaks near 1888, 1968, 2048, 2128, 2208, 2288, and 2368 represent incorporation of 3, 4, 5, 6, 7, 8, and 9 alkyne units, respectively, onto the solanesol-derived scaffold.

Scheme 1. Synthesis of Polyol/Polyalkyne Scaffold 9^a–c

^aReagents: (a) PBr₃, ether (Ref. 12). (b) NaCH(CO₂Me)₂, EtOH. (c) LiAlH₄, ether. (d) disiamyborane, THF; H₂O₂, NaOH. (e) NaH, Br(CH₂)₄C≡CH (8). ^bProduct 7 resulting from anti-Markovnikov hydration is shown. It is assumed that mixtures of all possible stereoisomers of 7 are produced. ^cStructure 9 is a representative hexaalkyne scaffold. The sites of attachment of the 5-hexyn-1-yl groups shown in 9 are arbitrary.

Scheme 2. Solid Phase Synthesis^a

^aReagents: (a) piperidine. (b) Fmoc/tBu solid phase synthesis. (c) N₃(CH₂)₅(CO)OH, Cl-HOBt, DIC. (d) TFA/1,2-ethanedithiol/thioanisole/water (91/3/3/3).

Scheme 3. Synthesis of mixtures of multivalent constructs 15 and 16a–16e from solanesol-derived molecular scaffold 9 via CuAAC ^a

^aOnly one set of hexameric products from each mixture is depicted here for illustrative purposes. The sites of sidechain attachment shown are arbitrary.