Trityl mass-tags for encoding in combinatorial

7999 Oxford University Press

Nucleic Acids Symposium Series No. 42

107-108

Trityl mass-tags for encoding in combinatorial oligonucleotide synthesis Mikhail S. Shchepinov*, Rod Chalk and Edwin M. Southern

Department of Biochemistry, University of Oxford, South Parks Road, Oxford 0X1 3QU, UK

INTRODUCTION The combinatorial approach to simultaneous synthesis of large numbers of chemical compounds, first used for screening in electronic materials (1), is an important development in biological chemistry. The main types of combinatorial libraries are: spatially addressable (parallel) (2) and 'one-bead-one compound' (3) libraries. The former contain high numbers of oligonucleotide, peptide, or other probes in defined positions on a surface, whereas the second type comprises pools of compounds (initially) attached to a separate insoluble beads. Some applications need complete libraries of all possible oligonucleotides of a defined length. It is difficult to analyse small quantities of short nucleic acids directly; the split-andmix strategy for synthesis of these libraries creates the problem of encoding of each compound, i.e. marking each chemical step applied to a bead, so that the sequence and/or composition of the members of the library selected by an assay can be identified. Encoding methods include spatial arraying; splitand-mix- compatible graphical encoding, trivially known as the 'tea-bag' method; chemical encoding (for example peptide libraries encoded with oligonucleotide tags readable by sequencing; small chemical libraries encoded using different secondary amines, which at the end of the synthesis are cleaved, converted to dansyl derivatives and analysed using HPLC or similar chromatography-based methods, including binary coding; spectrometric encoding, including massspectrometric, fluorescent and NMR detection; electronic encoding, which is based on the use of radio-frequency micro

chips imbedded into polymer supports used for combinatorial synthesis; and physical encoding, based on the properties of the carriers, such as their shape and density (reviewed in ref 4). The characteristic signal of the DMTr+ cation (303 Da) is always present on mass-spectra of DMTr-containing compo unds, suggesting that derivatives of trityl group with different masses could serve as unique mass-tags in combinatorial syn thesis. We describe a new encoding method based on the high desorption rate of triphenylmethyl-based tags in the conditions of laser desorption/ionisation TOF MS. It is desirable to reduce the number of chemical and separation steps involved in identification of tags, or completely omit them. This can be readily achieved with trityl-based tags, the ether bond of which is easily cleaved by the 337 nm laser irradiation during laser desorption-TOF analysis (5). The resulting ions are detectable without any matrix. Alternatively, the trityls can be released by acidic treament and detected by laser desorption/ionisation TOF analysis with or without matrix. These properties make trityl-based tags promising for encoding in strategies not involving strong acids, such as oligonucleotide and peptide synthesis and small molecule combinatorial libraries. RESULTS AND DISCUSSION A dimethoxytrityl group bearing an NHS-activated carboxyl function was previously used for reversible labelling of oligonucleotides and for other applications (6).We used this approach to synthesise trityl groups which are more stable to acid (monomethoxy and without methoxy groups (Scheme 1)). The masses of commercially available primary amines which would withstand the conditions of oligonucleotide synthesis and are relatively inexpensive lie mainly in the range of 50-250 Da. For some applications it is desirable to have several hundred mass-tags. The resolution of the tags in TOF massspectrometry is satisfactory with >2 Da difference between the masses of tags. To increase the amount of tags using the same pool of amines, we made an additional trityl containing two NHS-activated carboxyl groups, which allows attachment of two amines giving a new series of mass-tags (Fig 1). To introduce a tagging moiety during oligonucleotide synthesis, we made phosphoramidite synthon based on propanediol structure, which provides reactivity similar to the standard A, C, G and T phosphoramites. It was mixed (~5 mol%) with standard A, C, G and T phosphoramidites prior to oligonucleotide syn

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ABSTRACT Combinatorial libraries of oligonucleotides on beads were synthesised by a split-and-mix strategy using 5'-DMTr- or 5'-Fmoc- nucleoside phosphoramidites. Trityl moieties with different masses were used to encode for the bases coupled at each step in the synthesis of oligonucleotides selected by hybridisation from the libraries. Tags orthogonal to the nucleotides added were produced by coupling amines of different MW to an activated carboxyl group(s) on the trityl moiety. Tags can be released from the support by laser irradiation and measured directly by TOF without matrix. Alternatively, they may be released by an acidic treatment and then analysed by (MA)LDI-TOF.

108 Nucleic Acids Symposium Series No. 42

1. repeat split and mix strategy till the final length of tetramer library is achieved and then deprotect 2. pick out the beads selected by a hybridisation test with unknown tetramer (for example, CTAA)

analyse tags in MALDI-TOF MS

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thesis. Assuming the stepwise yield of oligonucleotide synthesis to be about 99%, for an 8-mer library we would have ca 60% of all sites of the beads occupied by full length oligonucleotides. The concentration of the first tag (5% of all initial sites) would be about two-fold greater than that of the last tag (5% of the remaining 60% of the sites), which still makes it possible to detect all of them in the same mixture. Oligonucleotide synthesis was carried out on a 4-column ABI machine. After each oxidation step, the columns were removed and treated with different amines, then the beads were combined, mixed and split again. After deprotection beads were selected by hybridisation with Cy5-labelled oligonucleotide. The size of TentaGel Rapp-beads (-0.3 mm) allows for manual removal of positively identified (blue) beads from the pool. Selected beads were detritylated and the mixtures of tags released analysed by mass-spectrometry. To eliminate the problem of gradual loss of encoding MMTr-based tags during the detritylation step in oligonucleotide synthesis, we have also

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used 5'-Fmoc-protective strategy (7), thus omitting the use of acidic conditions altogether. After each oxidation step, the columns were removed from the synthesizer, and the beads were treated with corresponding amines, washed with acetonitrile and then treated with 0.1 M DBU in acetonitrile to remove Fmoc-protection. The tags encoding for 9-mer oligonucleotide synthesised using this strategy were detected using (MA)LDITOF analysis. For longer sequences, the 3'-methylphosphoramidites of 5'-Fmoc-protected nucleosides could be preferably used instead of cyanoethoxy phosphoramidites, to prevent the untimely loss of the CNEt- group due to the treatment with amines and DBU. We have shown that trityl-based mass-tags, consisting of a highly stabilised trityl carbocation, which fly extremely well in the positive mode of a LDI process, linked to a mass-modifying moiety, represented by essentially any residue initially containing a reactive group to react with a trityl-based pro-tag, can be used for encoding in combinatorial chemistry. The tags describing the sequence/composition of biopolymer attached to a bead are introduced after each chemical transformation which the bead is subject to. The tags are easily detectable using (MA)LDI-TOF analysis. The scheme and reagents we suggest are very simple as compared to other methods of encoding. REFERENCES

Fig. 1. Mass-spectrum of a mixture of trityl-based tags obtained by treatment of the trityl bearing 2 NHS-activated carboxyl groups with different amines.

1. HanakJJ. (1970) J.Mater.Sci. 5, 964-971. 2. Southern,E.M., Maskos.U. and Elder.J.K. (1992) Genomics, 13, 1008-1017. 3. Furka,A., Sebestyen,F., Asgedom.M. and Dibo.G. Highlights of Modern Biochemistry, Proceedings of the 14th International Congress of Biochemistry (1988) VSP: Ultrecht, The Netherlands, 5, 47. 4. Czamik,A.W. (1997) Current Opinion in Chem. Biol. 1, 60-66. 5. Herz,M.L. (1975) J. Amer. Chem. Soc, 97, 6777-6785. 6. Gildea,B.D., CoullJ.M. and Koster.H. (1990) Tetr. Lett., 31, 7095-7098. 7. Gioeli.C. and ChattopadhyayaJ.B. (1982) J. Chem. Soc. Chem. Commun. 672-674.

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Scheme 1. General principle of the split-and-mix strategy. Boxed are the tags used in this work.

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