Hybridization and cellular uptake properties of lipophilic oligonucleotide-dendrimer conjugates

Dendrimeric compounds were conjugated to oligonucleotides in order to improve their cellular uptake. Second and third generation lipophilic dendrimers were covalently attached either to the 5’-or 3’-end of oligonucleotides. Thermal denaturation experiments showed that the attachment of a third generation dendrimer leads to a substantial decrease in binding affinity. The considerably smaller second-generation dendrimers, however, are well tolerated. Fluorescence measurements revealed that the presence of a second-generation dendrimer leads to a marked increase in the cellular uptake of oligonucleotides


Introduction
There is considerable pharmaceutical interest in the use of synthetic oligonucleotides that intervene in the process of gene expression, such as antisense oligonucleotides 1 or siRNAs. 2 Antisense oligonucleotides (ODNs) are designed to bind to mRNA in a sequence-specific manner.Subsequently, the bound mRNA is either degraded by RNaseH, or the mRNA is prevented from nuclear or ribosomal processing. 3Antisense oligonucleotides have been pursued as a major class of new pharmaceuticals for a variety of human diseases, including cancer and viral infections. 4Several limiting factors were encountered in the practical application of oligonucleotides.6][7][8] Numerous efforts have been made to increase the cellular uptake of antisense oligonucleotides by covalently attaching various chemical groups.Examples are conjugates with cationic compounds, such as poly-(L)-lysine 9 or polyamides, 10 lipophilic conjugates, such as cholesterol, 11 long chain alcohols, 12 phospholipids, 13 aromatic compounds, 14 as well as polyethylene glycol. 15Furthermore, the effect of attachment of these compounds at different position of the oligonucleotides including the bases, the backbone and the 3 ' -or 5 ' end was investigated.Uekama and coworkers reported that gene transfer activity was significantly improved by conjugates of α-, β-, and γ-cyclodextrins with polyamidoamine starburst dendrimers. 16Most recently, lipophilic conjugates of siRNAs were used for gene-specific knockdown. 17In an attempt to increase the cellular concentration of oligonucleotides, we synthesized lipophilic dendrimeric building blocks and incorporated them into oligonucleotides.The influence of the dendrimer moiety on the oligonucleotide conjugates can readily be controlled by the size, the type and the number of the end groups.The dendrimeric compounds could act as 'nests' for a part of the conjugated oligonucleotide, and thereby improve their cellular uptake while still allow proper binding to its target sequence.The present paper describes the synthesis of various 5′-and 3′-oligonucleotide-dendrimer conjugates, their hybridization properties and uptake by T24 cells.

Synthesis of the oligonucleotide dendrimer conjugates
The dendrons 1 and 2 were prepared following the method of Frėchet. 18Due to the large size of the dendrimers, adverse effects on the hybridization had to be expected.Therefore, we devised different linker moieties.The conversion of dendrimers 1 and 2 into activated N-hydroxy succinimide (NHS-) esters is shown in Scheme 1. NHS-esters 3 and 4 were obtained after the elongation of the second-generation dendron 1 and the third-generation dendron 2 with a benzyl linker group using p-bromomethylbenzoic acid.For the synthesis of 3′-oligonucleotide-dendrimer conjugates, a dendrimer-derived solid support was prepared.The procedure is illustrated in Scheme 2 for the second-generation dendrimer 1.The elongation of dendron 1 with p-bromomethylbenzoic acid in the presence of NaH in refluxing THF leads to the compound 5.The activated NHS-ester 6 was coupled to 3amino-1,2-propanediol. Compound 7 was then tritylated with 4,4′-dimethoxytrityl chloride (DMTCl).Succinylation of compound 8 was followed by activation with p-nitrophenol and coupling to the controlled pore glass (CPG) solid support to yield the dendrimer-derived solid support product 11 with a loading of 30µmol/g.With the necessary activated ester and solid support in hands, the oligonucleotide conjugates 12 to 14 (shown in Table 1) and the fluoresceine labeled oligonucleotides 15 to 18 (see later, Scheme 3) were prepared.Incorporation of the elongated and N-hydroxy succinimide activated dendrimers to 5′-aminomodified oligonucleotide in DMF/dioxane/water at 40°C led to the oligonucleotide-dendrimer conjugates 12 and 13.The corresponding 3′-oligonucleotidedendrimer conjugate 14 was assembled using the appropriate solid support 11 and standard phosphoramidite chemistry. 19Conjugates 15 to 18 contained a 5′-fluoresceine label, which was attached post-synthetically, through a 5'-aminolinker.After cleavage from the solid support and deprotection with aqueous ammonium hydroxide (55°C, overnight), the oligonucleotides were purified by reverse phase HPLC.Typical overall yields for a 1 µmole synthesis of reverse phase HPLC-purified oligonucleotide-dendrimer conjugates ranged from 20-25%.The purity and identity of the obtained conjugates were verified by capillary gel electrophoresis and by matrixassisted laser desorption/ionization mass spectrometry.

Hybridisation properties of dendrimer conjugates
The oligonucleotide-dendrimer conjugates 12, 13, and 14 were tested for their hybridization properties against a fully complementary RNA 15mer.As indicated in Table 1, thermal denaturation studies showed a marked drop (∆Tm = -9.2°C) in the melting temperature (Tm) for the conjugate 13 containing the G 3 -dendrimer.The corresponding G 2 -dendrimer conjugate 12, however, showed only a slightly reduced Tm (∆Tm = -0.8°C)compared with the unmodified oligonucleotide.The conjugate 14, bearing the second-generation (G 2 ) dendrimer on the 3'-end showed even a slightly higher affinity (∆Tm = +3.0°C)compared with the unmodified oligonucleotide.All Tm curves showed a single cooperative transition (see Suppl.Material).
These data indicate that the G 2 -dendrimer does not interfere significantly with the hybridization process.

Cellular uptake of oligonucleotide dendrimer conjugates
The influence of the dendrimer part on cellular uptake of the oligonucleotides was studied using fluoresceine-labeled PO (phosphodiester) and PS (phosphorothioate) second-generation dendrimer-conjugates (17 and 18) and the control oligonucleotides, fluoresceine-labeled PO and PS control oligonucleotides (15 and 16).Fluorescence experiments revealed that covalent attachment of lipophilic dendrimers resulted in a substantial increase in the cellular association of oligonucleotides.Fluorescence microscopy was used to establish the intracellular accumulation of the oligonucleotide conjugates.Figure 2 shows the cellular uptake results obtained with T-24 cell cultures.A large increase in the cellular uptake was observed in the case of dendrimer conjugated oligonucleotides, both the phosphodiester and the phosphorothioate derivative.Quantitation of the fluorescence (Figure 3) showed that the fluorescence was enhanced 11fold in the case of fluoresceine-labelled PO-oligonucleotide-dendrimer conjugate (17).The effect was even more pronounced in the case of the fluoresceine-labelled PS oligonucleotide-dendrimer conjugate (18) with a 15-fold enhancement of uptake.Thus, the dendrimer part of the conjugates acts as a lipophilic anchor and facilitates the penetration of the oligonucleotides through the cellular membrane.

Conclusions
We have shown that defined lipophilic oligonucleotide-dendrimer conjugates containing secondgeneration dendrimer building blocks at 5'-and 3'-terminus can be efficiently synthesized by use of N-hydroxy succinimide esters and by use of a dendrimer derived solid support.While the third generation conjugates show a reduction in Tm, the hybridization properties of the secondgeneration dendrimer conjugates are very similar to the unconjugated oligonucleotides.The dendrimers have a pronounced influence on the cellular uptake of the oligonucleotides as demonstrated by fluorescence microscopy of labeled conjugates.Since our studies demonstrate that the second-generation (G2) 3'-oligonucleotide-dendrimer conjugates hybridize to their complementary nucleic acid targets and are taken up well by cells, these conjugates are of value as antisense agents.

Experimental Section
General Procedures.Solvents used were puriss, except for THF, which was purified by fresh distillation over Na/benzophenone.Commercially available reagents were used as received, without any further purification.Analytical TLC were performed on commercial Merck silica gel 60 F 254 .Flash chromatography was carried out using siliga gel 60 (230-400 mesh). 1 H NMR spectra were recorded on a Brucker AMX (400 MHz) spectrometer using tetramethylsilane (TMS) as internal standard, chemical shifts are reported in ppm (δ) referenced to TMS, coupling constants J in Hz.The dendrons 1 and 2 were synthesized as described. 18Oligonucleotides were prepared by automated synthesis on a 1.0 µmol scale on a DNA synthesizer (Applied Biosystems inc.394A-08).The crude oligonucleotide-dendrimer conjugates were purified by HPLC using a semi-preparative RP-C18 column [Hypersil TM , 5 µm particle size; 50 mM triethylammonium acetate (TEAA, pH 7.0).After concentration of the product-containing fractions, the purity was checked by capillary gel electrophoresis.The identity of the obtained oligonucleotide-denrimer conjugates was assessed by matrix-assisted laser desorption/ionization mass spectrometry.

Table 1 .
Sequences of oligonucleotide dendrimer conjugates 12 -14 synthesized and investigated by thermal denaturation experiments.Melting temperature (Tm) values of dendrimer conjugates were determined against the 15mer oligoribonucleotide 5′-UAUAUAUAUAAAAAA; total strand concentration was 2µM, pH 7.4 in phosphate buffer (∆Tm values are given as the difference in Tm relative to the reference oligonucleotide).Conjugate Sequence Tm [°C] ∆Tm [°C] (reference) 5' TTTTTCTCTCTCTCT 3'