Shortening the lipohilic tail
To overcome the solubility problem of the oligonucleotide-catalipids conjugate 13 a new generation of catalipids was synthesized based on the substructure of MOH (8) (Fig. 18). Catalipids -as well as oligonucleotide-catalipid conjugates- with shorter alkyl chains are supposed to have a better solubility in the required ligation buffer.
Figure 18
In order to use NMR spectroscopy to study chemical characteristics
millimolar concentrations are required. Thus the synthesis of MC6H
(18), MC8H (19) and MC10H (20) was carried out in
solution to arrive at a sufficient amount of products. Catalipids 18 to
20 are accessible by coupling hexanoic-, octanoic- and decanoic
carboxylic acids to histamine (Figure 18). After purification using
column chromatography, 18 to 20 were analyzed via NMR- and MALDI-MS
spectroscopy. The reaction conditions elaborated to synthesize the
oligonucleotide-catalipid conjugate 13 were optimized for the new
catalipids MC6H (18), MC8H (19), MC10H
(20): In a typical experiment 1 µmol of catalipid dissolved in
chloroform was transferred into an Eppendorf tube and the solvent was
removed by evaporation. To the catalipid coated Eppendorf tube 25
µl buffer (0.1 M HEPES, pH 7.2, 20 % acetonitrile) was added.
According to the length of the alkyl chains different solubilities of
catalipids in buffered solution were found: MC6H (18) was
soluble after sonification for 15 minutes at RT. In the case of MC8H
(19) and MC10H (20)) an addition of 5 to 10 % DMF and
incubation at 45 °C for one hour was necessary to dissolve these
catalipids with longer alkyl chains. To the dissolved catalipid 70 nmol
Cy5-labeled pentamer 3'-phosphate (Cy5-TTCCGp 14) in buffer
(0.1 M HEPES, pH 7.2, 20% acetonitrile) containing 0.1 M EDC was added
and incubated for 2 hours at 30 °C. Purification and desalting of
oligonucleotide-catalipids conjugates was performed as described
previously for 13.
Activated oligonucleotide-catalipid conjugates using catalipids 18, 19
and 20 could be obtained in good yields. The RP-HPLC chromatogram (Fig.
19) and the MALDI-MS spectra (Figure 20) of the MC8H-
conjugate (21) are exemplarily shown.
Figure 19
Figure 20