Letters in Peptide Science, 9: 211-219, 2002. KLUWER/ESCOM 9 2003 Kluwer Academic Publishers. Printed in the Netherlands.

211

A rapid coupling protocol for the synthesis of peptide nucleic acids

Christopher J. Vearing 1 & John V. Fecondo 2.
1 School of Engineering and Science, Swinburne University of Technology, Hawthorn, Victoria, Australia; 2 Department ofBiotechnology andEnvironmental Biology, RMIT University, Bundoora, Victoria, Australia (* Author for correspondence, e-mail: john.fecondo@rmit.edu.au, Fax: +61 3 9925 7110) Received 16 September2002; Accepted16 December2002 Key words: anti-sense/anti-gene oligonucleotide, peptide nucleic acid, PNA, SPPS

Summary
With the current interest in anti-sense and anti-gene technologies, an efficient, fast and less toxic synthesis protocol would be advantageous for the oligoraerisation of Peptide Nucleic Acids (PNA). Most of the methods currently in use for the t-Boc synthesis of PNA's use TFA/m-cresol, pyridine, piperidine and capping reagents. In this work, a rapid synthesis protocol has been adapted from an earlier published peptide synthesis method allowing a reduction in cycle time from around 30 min down to 16 min. By utilising quantitative deprotection with 100% TFA, a coupling time of 10 rain and a four-fold excess of monomer, this synthesis protocol has been used to synthesise a number of PNA's incorporating all four nucleotides of varying sequence, up to 17 residues in length. Abbreviations: DCM, dichloromethane; DECA, N,N-diethylcyclohexylamine; DIEA, N,N-diisopropylethylamine; DMF, N,N-dimethylformamide; HBTU, 2-benzotriazole-N,N,N',N~-tetramethyl-uronium-hexafluoro phosphate; mBHA, 4-methylbenzhydrylamine; TFA, trifluoroacetic acid; TFMSA, trifluoromethanesulfonic acid.

Introduction

Compounds that bind specifically to DNA or RNA, resulting in inhibition of the expression of a particular gene, are useful tools in molecular biology and gene therapy research [1]. This is primarily due to their potential as specific gene-targeting antisense (RNA binding) and antigene (DNA binding) drugs. Oligonucleotide analogues have attracted much attention as gene therapy agents since Stephenson and Zamecnik in 1978 [2] first demonstrated that an oligonucleotide could bind RNA and block the expression of a particular gene. Since then, problems such as non-specific binding, degradation within the cellular environment, lowered strength of binding and toxicity effects on the cell, have all hampered the development of DNA analogues as gene therapy agents [3]. These difficulties have been addressed by many oligonucleotide mimetics over the past years but more recently by the adaptation of PNA's from the DNA scaffold.

Peptide nucleic acids are DNA mimetics in which the ribose phosphodiester backbone has been replaced by N- (2-aminoethyl) glycine units (Figure 1). The nucleobases are attached to this pseudopeptide backbone using methylene carbonyl links, thus maintaining the structural configuration observed in native DNA and RNA [4]. This structural similarity and the unaltered nucleobases allow the PNA molecule to bind DNA or RNA in accordance with the Watson-Crick hydrogen bonding rules [5] while maintaining flexibility. The final oligomer comprises a neutral backbone that lacks repulsion from the negatively charged backbone of naturally occurring oligonucleotides [6] resulting in greatly increased melting temperatures and binding constants [5, 7-9]. Neither nucleases nor proteases recognise the structure and hence do not hydrolyse the molecule [10]. Moreover, PNA's have so far shown no toxicity at their effective concentrations during both in vitro and in vivo experiments [1119]. These observations support further investigation

212

:i
~

'",

o~Base
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N ~
~

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o

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I

DNA
into the precise functional mechanisms of PNA's and the refining of their potential as gene-specific agents. Given this interest in PNA's and their potential therapeutic applications, a need for faster, more efficient and cost effective methods for the synthesis of these molecules would be of value. One of the reasons a pseudopeptide backbone was originally incorporated into the design of PNA's was so that solid-phase peptide synthesis (SPPS) methods could be utilised [4]. These methods allow PNA's to be synthesised in milligram to gram quantities, using high efficiency coupling protocols, while permitting labelling at either the N or C termini, by means of a variety of established peptide chemistry methods [20]. The original homothymine PNA oligomers were assembled using pentafluorophenyl ester activation of the carboxyl group on the monomer [4], but attempting to include cytosine monomers demonstrated this assembly method to be inefficient [21] and a stepwise Merrifield [31] based solid-phase method was

PNA
employed. Utilisation of this in situ dicyclohexylcarbodiimide activation and a double coupling protocol allowed for the incorporation of all bases except adenine at high levels of coupling efficiency [22]. Previously however, use of N,N-diisopropylcarbodiimide as an activating agent in a double coupling procedure, had generated almost quantitative yield from the oligomerisation of a PNA containing all four nucleobases [23]. This method proved effective until the synthesis of larger (>15 mer) PNA's was investigated [24]. This extensive study into the synthesis of PNA's by Christensen et al. [24] reviewed the solvent systems, monomer concentration, activating agents and coupling reagents, capping reagents and acid neutralising compounds. The result was a manual synthesis method used to assemble a test 17-mer PNA using cycle times of less than 30 min, with minimal deletion by-products. A further study automated this optimised solid-phase t-Boc method, modifying the solvent sys-

Figure 1. Chemicalstructuresof DNA and PNA.Base = nucleobase(adenine,thymine,cytosineor guanine).

213

Rapid t-Boc PNA synthesis protocol

Figure 2. The rapid t-Bocsynthesiscycle. tern used, to obtain very clean products in high yield [25]. During the assembly of PNA's, two side reactions have been shown to occur, both resulting from excessive time between deprotection and coupling or when the growing PNA has prolonged exposure to a basic environment. These side reactions, the N-acyl transfer reaction [24] or cyclisation and ketopiperazine formation of the terminal residue [26] have both been implicated in the depletion of desired product. In the present study, a rapid t-Boc peptide synthesis protocol has been applied to the manual synthesis of PNA's leading to a reduction in cycle time to around 16 min. In addition, minimal deletion products are evident while elimination of many of the noxious washing solutions such as pyridine or piperidine and the necessity for a capping reagent have been removed. The required monomer concentration has also been reduced from seven-fold excess [25] to four-fold excess, thus leading to significant savings in reagent costs.

Materials and reagents

PNA monomers were obtained from PerSeptive Biosystems (Framingham, MA); Trifluoracetic acid, N,N-diisopropylethylamine and N,N-dimethylformamide were sourced from Auspep (Melbourne, Australia); 2-Benzotriazole-N,N,N',N'-tetramethyluronium-hexafluorophosphate (Richelieu Biotechnologies, Montreal, Canada); m-cresol and N,N-diethylcyclohexylamine (Sigma-Aldrich, St. Louis, MO); 4-methylbenzhydrylamine polystyrene resin containing 1% divinyl benzene (substitution of 0.24 meq/g) (Peptide Institute Inc., Osaka, Japan); 2,6-dihydroxyacetophenone (Fluka, St. Louis, MO). All other reagents used in this research were of analytical grade or higher. LG Scientific, Melbourne, Australia, assembled a custom-made reaction vessel designed for micro-scale syntheses. In order to achieve effective mixing on a micro-scale, a nitrogen mixing mechanism using a reaction vessel incorporating a #1 sintered glass frit disperser was used.

214

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Figure 3. Crude chromatogramsof a homopyrimidinePNA (CCTCTCT-Lys)(a) rapid t-Boc chemistry(b) PNA chemistryby Christensen et al. (1995) [24].

Methods
Synthesis o f the peptide nucleic acids

Assembly of the PNA's was carried out using 5/zmol of m-BHA polystyrene solid support (0.24 meq/g) using a SPPS method based upon the original technique developed by Schn61zer et al. (1992) [27]. Briefly, the monomers were deprotected using 100% TFA and

the resin washed twice with DME During the second wash, 20/zmole (or 4 fold excess) of the monomer was activated with 400/zl 0.085 M HBTU. The resin was drained and the activated monomer solution, plus 10% v/v neutralising base for in situ neutralisation, added to the resin for a 10 min coupling at a final monomer concentration of 0.05 M. The resin was washed again with DMF and drained prior to deprotection at the beginning of the next cycle (summarised in Figure 2).

215
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30

40

50

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Figure 4. Crude chromatogram of the PNA test sequence (H-CGGACTAAGTCCATTGC-Gly-NH2) (MW: calculated (observed); 4661.2 (4660.8)) synthesised using rapid t-Boc chemistry with (a) DIEA and (b) DECA as TFA neutralising agents.

216 A monomer concentration of 0.05 M was found to be the minimum required to achieve >80% purity of the sequences synthesised. Increasing the final monomer concentration to 0.1 M is likely to increase the yield and purity of synthesis, as shown by Christensen et al. (1995) [24] and would be recommended for difficult sequences, but this also increases the cost. Ninhydrin monitoring of the synthesis efficiency was prevented due to the small scale of the reactants. Cleavage of the product from the resin A low/high TFMSA cleavage protocol developed by Tam et al. (1986) [28] was used. Briefly, two TFA deprotections to remove the t-Boc protecting groups were carried out on ca. 20 mg dry PNAresin. The resin was vacuum dried and transferred to an Ultrafree| filter unit containing a 0.22 # m Durapore| membrane (Millipore, Bedford, MA) and cooled on ice for 10 min. Low TFMSA cleavage (5% v/v TFMSA) was performed at ambient temperature for 1 hr using 200/zl of freshly prepared low TFMSA solution (TFA: DMS: m-cresol: TFMSA, 11:6:2:1). The microcentrifuge device was spun at 6500 rpm for 5 min and the resin washed once with 100% TFA. 200 /zl of freshly prepared high TFMSA solution (TFMSA: TFA: m-cresol, 1:8:1) was added to the resin at 0 ~ and allowed to react at ambient temperature for 1 hr to cleave the PNA from the solid support. The PNA product was precipitated and washed with anhydrous diethyl ether. The crude PNA was dissolved in 0.1% v/v TFA for purification or in the case of mixed sequence PNA's or sequences with poor solubility, 1020% (v/v) acetic acid was used to obtain a solution for purification. HPLC analysis and purification The cleaved PNA product was analysed on reversed phase Brownlee T M HPLC Aquapore C18 columns, 30 mm 4.6 mm (Perkin-Elmer, CT). Detection was achieved using a 1000S Diode Array Detector (Applied Biosystems) or a 785A programmable absorbance detector (Applied Biosystems) combined in series with a 900 Series Interface data collection unit (PE Nelson), for analytical and purification testing respectively. Absorbance was recorded at 260 nm. All samples were purified using a gradient from 0-60% (v/v) acetonitrile, containing 0.1% (v/v) TFA, at a flow rate of 1 ml/min was used to separate and elute the sample components. The chromatograms were analysed using TurbochromT M software to isolate the pure PNA. MALDI-TOF mass spectrometry analysis on a Brtiker Reflex mass spectrometer was used to confirm molecular weights of the purified PNA's.

Results

The first attempt at synthesising a PNA using our rapid t-Boc solid-phase method (Figure 2) was tried with a hompyrimidine PNA (CCTCTCT-Lys) so that the final oligomer could be utilised for triplexing antisense work at a later date. Reversed phase HPLC analysis of the synthesised PNA (Figure 3a) revealed one major peak, indicative of an efficient synthesis and one minor peak. This minor peak corresponds to inefficient removal of the hydrophobic benzyloxycarbonyl groups resulting in an increased retention time. As a comparison, the same homopyrimidine sequence was synthesised using the chemistry developed by Christensen et al. [24]. The chromatogram obtained (Figure 3b) showed similar purity (~95%) and yield of product results as those observed with the PNA synthesised on the same apparatus using our rapid chemistry ("~90% pure). MALDI-TOF mass spectral analysis on the purified PNA showed that both chemistries had synthesised the desired PNA (data not shown). To test the rapid t-Boc chemistry further, a trial synthesis of the test sequence (H-CGGACTAAGTCCATTGC-Gly-NH2, [24]) was attempted with similar results. The chromatogram of the crude PNA (Figure 4a) indicated a relatively efficient synthesis containing few deletion sequences and the major product within a single peak resulting in ~70% purity. Mass spectral analysis of this purified major peak confirmed that the complete test sequence had been synthesised (data not shown). DECA had been employed by Christensen et al. [24] to reduce insoluble salt formation of the monomers with the solvents generally utilised during PNA synthesis (DMF and pyridine). When DECA was used with the rapid t-Boc peptide chemistry, the resulting crude chromatogram revealed multiple deletion sequences (Figure 4b) and provided only ~20% pure product. These deletion products also appeared to be in higher proportions than those observed from the synthesis of the test PNA using the rapid t-Boc methodology with DIEA (Figure 4a). More recent syntheses of PNA's, using this rapid t-Boc synthesis method with in situ DIEA neutralisation, have resulted in very clean products generally >80% pure (Figure 5).

217
Table 1.

Comparisonof published automatedand manual synthesis cycles Manual synthesis Automated synthesis (Christensen et al. (1995) [24]) (Kochet al. (1997) [25]) Manual synthesis rapid t-Boc chemistry TFA DMF DMF/DIEAa DMF 16 rain

Deprotection Washing Washing Coupling Washing Capping Washing Washing Total time

TFA/m -cresol DMF/DCM (1:1) Pyridine Pyridine/DMF/DECA a Pyridine Rapoport's reagent Pyridine DCM/DMF ( 1:1) Approx. 30 rain

TFA/m-cresol DCM x5 NMP x 5 Pyridine/NMP/DIEA b Pyridine Acetic mthydride/pyridine/NMP c Piperidine NMP 42 rnin

a HBTUutilised in couplingreactions as the activator,dissolvedin DME b HATU/HBTUused as activatorin combination. c Not utilised after the first guanine residue is incorporated. Discussion veloped by Christensen e t aL [24], showed two distinct peaks in both crude chromatograms (Figure 3). Following purification and mass spectral analysis, the early eluting peaks in both cases were found to contain the desired PNA. The later eluting peak contained PNA with residual benzyloxycarbonyl protecting groups, indicative of incomplete deprotection but not failed coupling. This was confirmed by data obtained following a second cleavage procedure, when the later eluting peak was no longer detectable, but the earlier peak had increased from ~70% yield to >90%. The resulting chromatogram now showed one major peak with mass spectral data confirming that the desired PNA had been synthesised. It was found that more efficient deprotection could be achieved by using freshly prepared cleavage solutions, leading to an improved recovery of the desired synthesis product. Previous studies indicated that the type of solvents typically used in rapid t-Boc peptide chemistry, in particular the wash steps in which 100% DMF was used, would have a deleterious effect on the synthesis of PNA's [24]. Contrary to this, but in keeping with common SPPS experimental practice [30], all of the syntheses to date have concluded with a clean synthesis, the desired PNA in good yield (Figure 5) and with mass spectral data on the purified product confirming the completed synthesis. The synthesis of homopyrimidine PNA's is generally considered to provide more pure syntheses products than the assembly of mixed sequence PNA's due to the difficulties they can cause, both during synthesis and post-cleavage, due to aggregation/insolubility. [Peter E. Nielsen, personal communication] Although the

In this work, an efficient cycle for the synthesis of PNA's is presented based on the rapid t-Boc peptide chemistry described by Schnrlzer e t al. [27]. The application of this rapid chemistry to the synthesis of PNA's was first thought useful to minimise the previously mentioned side reactions, while minimising the cost and time involved in PNA synthesis. These side reactions are caused by excessively long time intervals between deprotection and coupling or when the growing PNA has prolonged exposure to basic conditions [24, 26]. Incorporating fast and quantitative deprotection using 100% TFA, with in s i t u neutralisation and flow washing made possible by the reaction vessel designed for low-volume mixing, decreases the time factor and the necessary number of washes considerably. TFA also has excellent solvation properties [27, 29] that would disturb any previously formed intermolecular bonds within the growing oligomers maintaining easy access to the terminal residue for the incoming activated monomer. The use of the acid labile Boc/Z strategy and DMF instead of pyridine during the wash steps (Figure 2) decreases the hasicity of the environment throughout the synthesis and should help to maintain protonation and hence protection of the primary amine from any acyl transfer [25], until it is required for the reaction with the subsequent monomer. These steps combined should assist in decreasing the extent of undesired deletion sequences or termination of the synthesis. The trial syntheses on the homopyrimidine PNA's using the rapid t-Boc method and the chemistry de-

218 aggregation/insolubility post-cleavage can be alleviated somewhat by the addition of lysine residues at the C-terminal, our synthesis data obtained to date is in agreement with this premise by showing that homopyrimidine PNA purity is greater than mixed sequence syntheses. Although Christensen et al. [24] have performed an extensive solvent optimisation study, a selection of solvents regularly utilised in peptide chemistry such as DMF, NMP, pyridine and combinations thereof, were trialed with this in situ neutralisation approach, using the guanine monomer, which is known to be relatively insoluble. NMP gave good solvation when used in conjunction with DIEA as a neutralising agent, but only after 5 min. All other chemistries trialed, including those used by Christensen et al. (1995) [24] and Koch et al. (1997) [25] gave similar results within the 2 min activation time required. Previous studies have suggested that capping of unreacted terminal amino groups during the assembly of PNA molecules with Rapoport's reagent or acetic anhydride [24, 25] could result in a cleaner synthesis. A capping step using either of these reagents was not included with the rapid cycle in order to keep the chemistry simple and efficient while still achieving high purity products in a short cycle time. Moreover, it has been shown that acetic anhydride capping could have a deleterious effect on the synthesis if a piperidine wash was not incorporated immediately following capping [25]. Our original aim in trying to adapt the rapid t-Boc peptide chemistry to the synthesis of PNA's was to keep the chemistry uncomplicated, make it less toxic to both the environment and user, at a lower synthesis cost. By addition of further compounds into the reaction mixture, it was thought this could complicate the synthesis, when they appeared to be unnecessary. A comparison of various methods used to synthesise PNA's (Christensen et al. (1995) [24]; Koch et al. (1997) [25], see Table 1) highlights the differences between this rapid chemistry and other t-Boc chemistries currently in use. The main distinction between the chemistries appears in the time taken to complete each cycle and in the reagents used. The rapid t-Boc chemistry protocol nearly halves the time taken to complete a cycle, but also utilises less hazardous reagents than the previously reported m-cresol, pyridine and those used to synthesise Rapoport's reagent. This combined with the lower amounts of wash solutions used and the need for only a 4-fold excess of

! PNA+277 ITCCTCCTCC'I-I'CTC- Lys MW: 3750.8 (3751.4)

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~4~

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7 3~

2~

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10 20 30 40

Minutes
5. Crude chromatogram of PNA+277 (TCCTCCTCCTTCTC-Lys) (MW: calculated (observed); 3750.8 (3751.4))using the rapid t-Bocchemistrywith DIEAin situ neutralisation. Figure

219 monomer would suggest that this is a fast, reliable and cost effective method for the t-Boc synthesis of PNA's.
9. Ratilainen, T., Holm6n, A., Tuite, E., Nielsen, RE. and Nord6n, B., Biochemistry, 39 (2000) 7781. 10. Demidov, V.V., Potaman, V.N., Frank-Kamenetskii, M.D., Egholm, M., Buchardt, O., S6nnichsen, S.H. and Nielsen, EE., Biochem. Pharmacol., 48 (1994) 1310. 11. Simmons, C.G., Pitts, A.E., Mayfield, L.D., Shay, J.W. and Corey, D.R., Bioorg. Med. Chem. Lett., 7 (1997) 3001. 12. Basu, S. and Wickstrom, E., Bioconjug. Chem., 8 (1997) 481. 13. Tyler, B.M., McCormick, D.J., Hoshall, C.V., Douglas, C.L., Jansen, K., Lacy, B.W., Cusack, B. and Richelson, E., FEBS Letters, 421 (1998) 280. 14. Pooga, M., Soomets, U., Hallbdnk, M., Valkna, A., Saar, K., Rezaei, K., Kahl, U., Hao, J.-X., Xu, X.-J., Wiesenfeld-Hallin, Z., Htikfelt, T., Barffai, T. and Langel, 0., Nat. Biotechnol., 16 (1998) 857. 15. Farese-Di Giorgio, A., Pairot, S., Patino, N., Condom, R., Di Giorgio, C., Anmelas, A., Aubertin, A.-M. and Guedj, R., Nucleosides and Nucleotides, 18 (1999) 263. 16. Tyler, B.M., Jansen, K., McCormick, D.J., Douglas, C.L., Boules, M., Stewart, J.A., Zhao, L., Lacy, B.W., Cusack, B., Fauq, A. and Richelson, E., Proc. Natl. Acad. Aci. U.S.A, 96 (1999) 7053. 17. Chinnery, E, Taylor, R.W., Diekert, K., Lill, R., TurnbaU, D.M. and Lightowlers, R.N., Gene Ther., 6 (1999) 1919. 18. Fraser, G.L., Holmgren, J., Clarke, EB.S. and Wahlestedt, C., Mol. Phannacol., 57 (2000) 725. 19. Doyle, D.E, Braasch, D.A., Simmons, C.G., Janowski, B.A. and Corey, D.R., Biochemistry, 40 (2001) 53. 20. Hyrup, B. and Nielsen, EE., Bioorg. Med. Chem., 4 (1996) 5. 21. Egholm, M., Nielsen, EE., Buchardt, O. and Berg, R.H., J. Am. Chem. Soc., 114 (1992) 9677. 22. Dueholm, K.L, Egholm, M., Behrens, C., Christensen, L., Hansen, H.E, Vulpius, T., Petersen, K.H., Berg, R.H., Nielsen, EE. and Buchardt, O., J. Org. Chem., 59 (1994) 5767. 23. Egholm, M., Behrens, C., Christensen, L., Berg, R.H., Nielsen, EE. and Buchardt, O., J. Chem. Soc., Chem. Commun., 9 (1993) 800. 24. Christensen, L., Fitzpatrick, R., Gildea, B., Petersen, K.H., Hansen, H.E, Koch, T., Egholm, M., Buchardt, O., Nielsen, EE., Coull, J. and Berg, R.H., J. Pept. Sci., 1 (1995) 175. 25. Koch, T., Hansen, H.E, Anderson, E, Larsen, T., Batz, H.G., Otteson, K. and ~'um, H., J. Pept. Res., 49 (1997) 80. 26. Thomson, S.A., Josey, J.A., Cadilla, R., Gaul, M.D., Hassman, C.E, Luzzio, M.J., Pipe, A.J., Reed, K.L., Ricca, D.J., Wiethe, R.W. and Noble, S.A., Tetrahedron, 51 (1995) 6179. 27. Schn61zer,M., Alewood, P., Jones, A., Alewood, D. and Kent, S.B.H., Int. J. Pept. Protein Res., 40 (1992) 180. 28. Tam, J.P., Heath, W.E and Merrifield, R.B., J. Am. Chem. Soc., 108 (1986) 5242. 29. Milton, R. C. de L., Milton, S.C.E and Adams, P.A., J. Am. Chem. Soc., 112 (1990) 6039. 30. Kent, S.B.H., Ann. Rev. Biochem., 57 (1988) 957. 31. Merrifield, R.B., J. Am. Chem. Soc., 85 (1963) 2149.

Conclusions
A suitable synthesis apparatus and method was developed that enabled the assembly of various PNA sequences incorporating all four nucleotides. The resultant synthesis products are easily purified from within the major peak of the crude chromatograms in favourable proportions. Development of this more environmentally compatible protocol has revealed that faster cycle times along with fewer reagents can be used to synthesise PNA's, all of which are beneficial to an industrial application.

Acknowledgements
We thank Dr. Jeffrey J. Gorman and Dean Whelan from CSIRO Health Sciences and Nutrition, Parkville, Victoria, Australia for the MALDI-TOF Mass spectrometry analysis.

References
1. Gewirtz, A.M., Sokol, D.L. and Ratajczak, M.Z., Blood, 92 (1998) 712. 2. Stephenson, M.L. and Zameenik, P.C., Proc. Natl. Acad. Sci. U.S.A, 75 (1978) 285. 3. Milligan, J.E, Matteucci, M.D. and Martin, J.C., J. Med. Chem., 36 (1993) 1923. 4. Nielsen, P.E., Egholm, M., Berg, R.H. and Buchardt, O., Science, 254 (1991) 1497. 5. Egholm, M., Buchardt, O., Christensen, L., Behrens, C., Freier, S.M., Driver, D.A., Berg, R,H., Kim, S.K., Nord6n, B. and Nielsen, P.E., Nature, 367 (1993) 566. 6. Almarsson, 0., Bruice, T.C., Kerr, J. and Zuckermann, R.N., Proc. Natl. Acad. Sci. U.S.A, 90 (1993) 7518. 7. Schwarz, F.P., Robinson, S. and Butler, J.M., Nucleic Acids Res., 27 (1999) 4792. 8. Chakrabarti, M.C. and Schwarz, EP., Nucleic Acids Res., 27 (1999) 4801.