Preparation of bifunctional silica polymer support for the synthesis of 3 '-labeled oligonucleotides

Preparation of a silica polymer support with a linker arm containing protected amino and hydroxy groups is described. 0-Benzotriazol-l-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) in the presence of 1-hydroxybenzotriazole (HOBT) was used as an efficient activating reagent at several steps of the polymer synthesis. This bifunctional support is suitable for the preparation of 3'-amino-modified oligonucleotides for post-synthetic conjugation, as well as for the direct solid phase synthesis of 3'-labeled oligonucleotides. The fluorescein-modified silica polymer was synthesized.

Introduction.Modified oligonucleotides are widely used for DNA sequencing, as nucleic acids probes, PCR primers, gene expression inhibitors, etc. [1 ].In the past years non-radioactive labeling, i. e. covalent attachment of fluorescent, chemiluminescent, enzy matic or affinity reporter groups to DNA fragments, has evolved into the preferred method of DNA detection for a wide variety of applications and is believed to replace the use of traditional isotopic labels in the future [2][3][4].From this point of view, the development of efficient new approaches and procedures for oligonucleotide labeling is still in great demand.There are two general approaches to the preparation of labeled oligonucleotides [2,5,6].The first one is based on the synthesis and isolation of oligonucleotides, functionalized with amino or mercapto groups, with their post-synthetic conjugation to corresponding molecules.In another method, reporter groups are introduced during solid phase oligonuc leotide synthesis followed by the deblocking and purification of resulting conjugate.In this case, the key factor is stability of the attached group during oligonucleotide synthesis and deprotection.Recently, the polymer supports containing reporter groups at tached via special linkers that allow the elongation of oligonucleotide sequences resulting in the formation of 3'-labeled oligomers have been described, and some of them are currently commercially available (see e. g. [7]).Here we report the preparation of a new silica-based support for the solid phase synthesis of 3'-labeled oligonucleotides.This polymer support can be also used for peptide synthesis.
Polymer 5 was treated with 5 ml of 20 % piperidine in DMF for 30 min, washed with DMF (5x3 ml) and treated with the solution of dipivaloyl-FITC 7 (56 mg, 0.1 mmol) in 3 ml DMF for 24 h at room temperature in the dark with gentle agitation.Support 6 was filtered off, washed with DMF (5x3 ml) and acetonitrile and unreacted amino groups were capped with 5 ml of acetonitrile solution of acetic anhydride (945 pi, 10 mmol) and Melm (795 pi, 10 mmol) for 30 min.Silica was washed with CH 3 CN, CHC1 3 , MeOH and ether (3 x 3 ml each) and dried in vacuo.
Fluorescein content was determined as follows: an aliquot (few mg) of the polymer 6 was treated with 200 pi of cone.NH 4 OH in the sealed tube overnight at room temperature, the ammonia solution was removed, polymer washed with 0.25 M TEAB buffer, pH 8 (4 x 200 pi) with centrifugation, and absorbance of combined washings was determined at 494 nm (for FITC conjugates, e 494 = 75000 [4]).The dye loading was 28 ^mol/g.Results and Discussion.Polymer supports mo dified with fluorescent dyes including fluorescein and rhodamine have been described in the literature [13][14][15] and many are commercially available.To prepare the polymer support for the synthesis of 3'-labeled oligonucleotides, a bifunctional linker gro up should be used allowing the attachment of both reporter molecule and the first nucleotide of the oligonucleotide sequence to the polymer.The linkers are usually based on the structures like 2-substituted 1,3-propanediols.We have used 3-aminopropane-l,2diol 1 for the preparation of the linker (Scheme).This starting synthon contains amino group that can react with many electrophilic reagents in the presence of OH-groups, and then primary hydroxyl can be pro tected selectively to leave free secondary OH for the linker attachment to a solid support.The amino group of starting trifunctional compound 1 was protected with Fmoc group by the reaction with Fmoc-chloride in DMF in the presence of diisopropylethylamine, and then N-protected intermediate was tritylated with DMTrCl in pyridine at primary hydroxyl.1,3-Pro tected aminodiol 2 was converted into 2-O-succinate 3 by reaction with succinic anhydride in pyridine in the presence of methylimidazole as a catalyst.The use of Melm instead of 4-dimethylaminopyridine (DMAP) improved: the yield of succinate (62 %) since Fmoc group is sensitive to strongly basic DMAP.When DMAP was used, the reported yield was 55 % [8].NMR spectram of succinate 3 showed that methylene protons in both CH 2 0 and CH 2 N frag ments of aminopropanediol are not equivalent forming ABX systems with tertiary CH proton.Before we have observed the same nonequivalence of methylene pro tons in symmetrical 1,3-ditrityl glycerol (unpublished results).
N,0-protecte<l succinate reagent 3 was attached to the silica supjjort containing aminoalkyl groups.The polymer used for the preparation of modified support was previously described Silochrom-2 silica with the aminopropyl-succinate-ethylenediamine lin ker [9].Usually nucleoside succinates are linked to polymers as activated esters or in the presence of coupling reagent!: like carbodiimides or arylsulfonylchlorides.In the present work, the coupling of succinate to amnated polymer was accomplished using highly efficient uronium coupling reagent HBTU in the rfresence of HOBT.A variety of phosphonium and uronium reagents have been used in coupling reactions in peptide synthesis [16].Onium salts are now very popular since they are stable, non-hygroscopic and easy to use coupling reagents.There are numerous examples of the use of these reagents in oligonucleotide synthesis, including nuc leoside attachment to solid supports [8,14,17,18] and oligonucleotide functionalization or labeling [19][20][21].We have recently described the synthesis of silica polymer and nucleoside immobilization at this support using onium salts in the presence of HOBT [22].In the present work, the succinate reagent 3 activated with HBTU reagent was coupled to the aminoalkyl linker of the silica.At this point, the support 4 is ready for the attachment of reporter groups, however we have decided to perform the elongation of spacer arm to improve the yield of conjugation of fluorophores, as well as the yield of the first coupling reaction of the oligonucleotide chain synthesis.N-Fmoc-protected amino acids were used previously for the spacer elongation [23].We have utilized N-protected /3-alanine for this purpose.Fmoc protection was removed from the polymer 4 by piperidine treatment.Then Fmoc-/?-alanine was attached to the free NH 2 linker group in the presence of the same HBTU/HOBT activating reagent.Fmoc group loading was 33 /tmol/g.The silica support 5 containing N,0-protected linker can be used for the preparation of oligonuc leotides functionalized with the aliphatic amino group at the 3'-end for post-synthetic labeling.After detritylation of the support, the oligonucleotide synthesis can be performed on the polymer followed by deprotection of amino group.Deblocked and removed from the support, the aminoalkyl-modified oligonucleotides can be subsequently labeled with any suitable reagent developed for the modification of amino groups inclu ding fluorescent dyes, intercalating agents, chemical nucleases, etc.However, it was interesting to prepare a support for the direct solid phase synthesis of 3'-labeled oligonucleotides.For this reason, the linker amino group was deblocked and coupled to the amine-reactive isothiocyanate derivative of the fluo rescein dye, FITC.To avoid side reactions at phenolic hydroxyls of fluorescein, they were protected with pivaloyl chloride [12,13].Diacylated FITC lactone 7 is almost colourless and nonfluorescent since the chromophore system of the dye is changed.However, its deacylation upon ammonolysis leads to the reco very of the normal fluorescein structure.Dipivaloyl FITC was efficiently linked to the aminated polymer support in DMF, and remaining NH 2 groups were capped with acetic anhydride.The dye content in the resulting polymer 6 was found to be 28 fimol/g.After detritylation, the silica support with a free hydroxy group is suitable for the'solid phase oligonucleotide synthesis to produce 3'-fluorescein labeled oligo nucleotides.
It should be stressed that silica polymer 5 is the afunctional support containing both protected OH and NH 2 functions and therefore can be also used for the solid phase synthesis of peptides at the amino linker.The peptide chain elongation can be performed after deprotection of the polymer amino group.The similar polymer supports have been used for the synthesis of peptides and peptide-oligonucleotide con jugates [24][25][26][27].Even the direct synthesis of pep tide-oligonucleotide hybrids seems to be possible on this support, with the peptide chain elongation on the aminoalkyl part of the linker followed by the oligo nucleotide synthesis at the OH-linker arm.
Hence, the silica support containing the protected or dye-labeled amino group has been prepared with the use of uronium coupling reagent HBTU.The experiments on the synthesis of modified oligo nucleotides on the proposed silica polymer support are in progress, and the results will be published else where.