Synthesis of HyBeacons and dual-labelled probes containing 2A-fluorescent groups for use in genetic analysis† Neil Dobson, a David G. McDowell, b David J. French, b Lynda J. Brown, c John M. Mellor a and Tom Brown* a a Department of Chemistry, University of Southampton, Highfield, Southampton, UK SO17 1BJ. E-mail: tb2@soton.ac.uk; Fax: +44 (0)23 80592991; Tel: +44 (0)23 80592974 b LGC Ltd, Queens Road, Teddington, UK TW11 0LY c Oswel Research Products Ltd, Biological and Medical Sciences Building, University of Southampton, Boldrewood, Bassett Crescent East, Southampton, UK SO16 7PX Received (in Cambridge, UK) 13th March 2003, Accepted 3rd April 2003 First published as an Advance Article on the web 7th May 2003 An FMOC-protected 2A-hydroxyethyl uridine phosphor- amidite has been used to synthesise fluorescein-labelled HyBeacon probes and “FAM-ROX” dual-labelled fluoro- genic oligonucleotides. Many genetic diseases are linked to single nucleotide polymor- phisms occurring at a significant frequency within the popula- tion (1% or more), so the ability to rapidly detect SNPs is of great value. Commonly used homogeneous real-time PCR- based methods of genetic analysis such as Taqman™ 1 Scorpi- ons 2 and Molecular Beacons 3 are used to analyse SNPs on platforms such as the Roche LightCycler™. In a drive towards inexpensive, high throughput genetic analyses we have devel- oped the novel “HyBeacon” system. 4,5 HyBeacons™ are hybridisation probes consisting of a single-stranded oligonu- cleotide containing a fluorophore attached to a nucleoside within the DNA sequence. When the probe anneals to a complementary target and forms a duplex, an increase in fluorescence is observed, the precise origin of which is the subject of ongoing research (Fig. 1). As the probe possesses no secondary structure, hybridisation to the target is faster and more efficient than in other genetic analysis systems, and post-PCR melting analysis becomes possible. Furthermore, HyBeacons do not require enzymic activation, so they can be used in conjunction with rapid PCR cycling conditions. 6 Finally, HyBeacon synthesis is relatively simple and inexpensive, as the probe does not contain a fluorescence quencher. In previous work in this field 4,5 we incorporated fluorescent dyes in the major groove of DNA, via the 5-position of deoxyuridine. The next logical step was to investigate the properties of HyBeacons with the fluorophore in the minor groove. The results of the major groove studies 4,5 indicated that the most efficient HyBeacons would result from the use of a short linker between the nucleotide and the dye moiety, so we based our strategy on 2A-hydroxyethyluridine. In previous studies oligonucleotides have been labelled at the 2A-position of uridine using specific dye-labelled phosphoramidites, 7–9 or by solution-phase derivatisation of a 2A-amino dU residue after solid-phase synthesis. 9 However, our aim was to develop efficient general solid-phase approaches based on phosphor- amidites that can be prepared in large quantities and used in oligonucleotide synthesis in conjunction with other commer- cially available dye phosphoramidites. 10 The synthesis of the required phosphoramidite monomer is shown in Scheme 1‡. N(3)-BOM uridine 1 11 was protected with Markiewicz’s reagent to give 2, which was reacted with methyl bromoacetate in the presence of sodium hydride to give 3 in 72% yield. Reduction of the ester with sodium borohydride proved unsatisfactory with our substrate (28% yield), the major product being a tetrahydrouridine derivative obtained by reduction of the heterocycle. However, removal of the BOM protecting group from the nucleobase before reduction prevented this side- reaction. The BOM-protecting group was cleaved with Pd/C and reduction of the ester using sodium borohydride gave 4 (65% over 2 steps). Reprotection of N-3 was necessary to give a clean reaction in the next step in which the 2A-hydroxy group was protected with FMOC to give 5 (86%). Deprotection of the BOM group, and removal of TIPDS using HF–pyridine in THF, followed by tritylation gave 6 in 80% yield. Finally, phosphity- lation of the 3A-hydroxy group under argon afforded the phosphoramidite 7 in good yield. Monomer 7 can be introduced into synthetic oligonucleotides at thymine sites and used as a point of attachment for a variety of fluorescent dyes amidites. In order to evaluate its utility, the HyBeacon probe§ 2DC64C* was synthesised 4 (Scheme 2). Standard protocols were employed, with an extended reaction time of 5 min for 7, which coupled at 98%. We did not remove the 5A-DMT protecting group from the oligonucleotide at this stage. After oligonucleotide assembly the synthesis column was removed and treated with piperidine (20% in DMF) for 10 min to remove the FMOC group from the 2A-protected uridine residue. The column was then returned to the DNA synthesiser, 6-carboxyfluorescein phosphoramidite was coupled to the free 2A-hydroxy group and the 5A-DMT group was removed. The oligonucleotide was then cleaved from the resin, deprotected in † Electronic supplementary information (ESI) available: experimental details and real-time PCR. See http://www.rsc.org/suppdata/cc/b3/ b302855k/ Fig. 1 HyBeacon. Scheme 1 (i) 1,3-Dichloro-1,1,3,3-tetraisopropyldisiloxane (1.2 eq.) imida- zole (1.1 eq.), pyridine rt 7 h, 91%; (ii) BrCH 2 CO 2 Me (5 eq.), NaH (2.2 eq.), DMF, 25 °C, 2 h, 72%; (iii) 20 wt% Pd/C, THF : methanol (1 : 1), H 2 (g), RT, 86%; (iv) NaBH 4 , methanol, tert-butanol, rt, 76%; (v) benzyl chloromethyl ether, DBU, DMF, rt, 82%; (vi) FmocCl, pyridine, rt, 86%; (vii) 20 wt% Pd/C, 2 M HCl (aq), THF : methanol (1 : 1), H 2 (g), rt, 62%; (viii) HF–pyridine, THF, rt, 54%; (ix) DMTrCl (3 eq.), pyridine, rt, 3 h, 80%; (x) 2-cyanoethyldiisopropyl chlorophosphoramidite, DIPEA, THF, 72%. This journal is © The Royal Society of Chemistry 2003 1234 CHEM. COMMUN. , 2003, 1234–1235 DOI: 10.1039/b302855k