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Bioconjugate Chem. 2002, 13, 676−678
TECHNICAL NOTES New Fmoc Pseudoisocytosine Monomer for the Synthesis of a Bis-PNA Molecule by Automated Solid-Phase Fmoc Chemistry Philippe Neuner* and Paolo Monaci Istituto di Ricerche di Biologia Molecolare P. Angeletti, Via Pontina Km 30,600, 00040 Pomezia (Roma), Italy . Received August 10, 2001; Revised Manuscript Received December 5, 2001
The synthesis of N-[2-(N-9-fluorenylmethoxycarbonyl)aminoethyl]-N-(2-N-(benzyloxycarbonyl)isocytosin-5-ylacetyl)glycine monomer and its incorporation into a PNA molecule via automated Fmoc solid-phase chemistry is described.
Scheme 1a
INTRODUCTION
Peptide nucleic acid (PNA) is a polynucleic acid consisting of natural or modified nucleobases linked to a peptide backbone (1). PNA exhibits a remarkable specificity and very high affinity in recognizing complementary DNA or RNA strands (2). Here we focus on a dimeric PNA molecule (bis-PNA) consisting of a homopyrimidine PNA oligomer (C/T) connected via a flexible linker (2aminoethoxy-2-ethoxyacetic acid) to a second PNA sequence (J/T, where J is pseudoisocytosine). This bis-PNA has been shown to form highly stable triple helix conjugates (PNA)2/DNA by strand invasion in a P-loop structure form. The formation and stability of the triple strand complex is only slightly influenced by pH in the neutral range, mainly due to the stabilizing effect of the protonated N1 position of the J residue at physiological pH (3). Early synthesis of PNA was essentially based on the manual assembly of monomers via tert-butyloxycarbonyl (Boc) chemistry on solid support (4). More recently, the design and preparation of sets of fluorenylmethoxycarbonyl (Fmoc)/acid labile PNA building blocks, the development of instrumentation, and optimization of synthesis protocols have led to the rapid and efficient synthesis of PNA probes by the mild and well-known Fmoc chemistry (5, 6). However, the only reported strategy for synthesising a bis-PNA-containing pseudoisocytosine still relies on the Boc chemistry (7). The lack of an Fmoc automated synthesis procedure for the preparation of this molecule led us to attempt the synthesis of the N-[2-(N-9-fluorenylmethoxycarbonyl)aminoethyl]-N-(2-N-(benzyloxycarbonyl)isocytosin-5-ylacetyl)glycine monomer, which is fully compatible with Fmoc chemistry, commercial PNA building blocks, and synthesizer. EXPERIMENTAL PROCEDURES
Synthesis of the required tert-butyl N-[2-(N-9-fluorenylmethoxycarbonyl)aminoethyl]glycine 1 and N2-(benzyloxycarbonyl)isocytosin-5-ylacetic acid 2 were carried out as described in the literature (5, 7). The base acetic acid 2 was reacted with 1, leaving the intermediate 3 in * Correponding author. Tel: + 39 6 91 093 335; fax: + 39 6 91 093 225; e-mail:
[email protected].
a Reagents: (a) N-methylmorpholine, HOBt, HBTU/DMF; (b) TFA/DCM.
good yield after flash chromatography (8). Removal of the tert-butyl ester with TFA in DCM gave 4 (Scheme 1) without significant loss of the N2-benzyloxycarbonylprotecting group (9). A key aspect of the synthesis of the bis-PNA using Fmoc (JCbz) was removal of the N2-benzyloxycarbonyl protecting group of the pseudoisocytosine. In a standard protocol, treatment of the synthesis resin with a TFA/ m-cresol (4:1) mixture efficiently released the polymer from the solid support and cleaved the cytosine benzhydryloxycarbonyl (Bhoc) blocking group. In a preliminary experiment, this deprotection ‘cocktail’ proved to be too mild to cleave the pseudoisocytosine benzyloxycarbonyl (Cbz), even after overnight treatment of the polymer (data not shown). Thompson et al. chose hydrogen fluoride (HF) in their work to cleave the N-benzyloxycarbonyl group (5). As an alternative, we chose a single treatment with a mixture of TFMSA/TFA/m-cresol to release and efficiently cleave the blocking group from the polymer. RESULTS AND DISCUSSION
The solid-phase synthesis of the bis-PNA (NH2-LLTCTCTCTC-LLL-JTJTJTJT-CONH2) was performed on a PerSeptive Biosystems Expedite 8909 automated synthesizer using the manufacturer’s protocols. All reagents including Fmoc C (Bhoc), Fmoc T, Fmoc AEEA spacer (L ) 2-aminoethoxy-2-ethoxyacetic acid), and HATU were
10.1021/bc0100758 CCC: $22.00 © 2002 American Chemical Society Published on Web 04/04/2002
Bioconjugate Chem., Vol. 13, No. 3, 2002 677
Figure 1. A. Preparative RP-HPLC trace of the crude products from bis-PNA synthesis. Peaks 1, 2, and 3 correspond to the products of the unsuccessful synthesis (data not shown). Peak 4 corresponds to the expected bis-PNA (H2N-LL-TCTCTCTCLLL-JTJTJTJT-CONH2). B. MS analysis of peak 4. M (calcd) for C198H274N78O71 ) 4882.87, M (experimental) ) 4882.00.
obtained from PerSeptive Biosystems. The PNA monomers were dissolved in NMP at a 0.2 M concentration. Synthesis using either XAL or PAL columns synthesis (2 µmol scale) were investigated and showed no significant difference in yield. After oligomerization, the resin was dried and transferred to a screw-cap vial. The vial was cooled on ice, and 200 µL of a mixture TFMSA/TFA/ m-cresol (18:73:9) was added. Reaction proceeded at room temperature for 90 min. Solid support was separated by filtration and the filtrate poured into 10 volumes of icecold Et2O. Centrifugation gave a pellet, which was purified by HPLC. Preparative RP C18 HPLC purification was performed using a SymmetryPrep C18 (7.8 × 150 mm) reversephase column (Waters). Eluant A: 99.9% water, 0.1% TFA; eluant B: 10% water, 89.9% acetonitrile, 0.1% TFA; a linear gradient of 5-30% eluant B over 25 min at a flow rate of 2.0 mL/min monitored at 260 nm (Figure 1). The expected bis-PNA eluted at 21.8 min. The average yield was usually 20-25% (1-1.25 mg) of purified oligomer Final product was analyzed for purity by mass determination on a LCQ quadrupole ion trap mass spectrometer Finningan-MAT (San Jose, CA). Labeling of the Bis-PNA in Solution. The bis-PNA synthesized above was routinely labeled in solution with various dyes (Biotin, Fluorescein, Rhodamine) according to the general protocol described here: To 12 OD (optical density) units of bis-PNA in ultrapure water was added a solution of 1 mg of N-succinimidyl ester dye (Fluka) in 100 µL of DMF, followed by 20 µL of a 10% NMM (aq). The solution was thoroughly vortexed, and the reaction proceeded for 3 h in the dark. The volume was taken to 0.5 mL by adding ultrapure water, and the reaction mixture was purified by RPHPLC using conditions described above. An average yield of 40-60% of the labeled PNA was easily obtained. In conclusion, we have described the synthesis of a new PNA pseudoisocytosine building block with orthogonal protecting groups (Fmoc/Cbz) and its use in the synthesis of a bis-PNA on an automated synthesizer. It was our experience that the versatile and economical automated assembly of the bis-PNA molecule described here facilitates the collinear synthesis of PNA-peptide chimeras by standard Fmoc chemistry (6). To our knowledge, no detailed description of the synthesis of the monomer and
Figure 2. Mass spectrum of bis-PNA Fluo-HN-LL-TCTCTCTCLLL-JTJTJTJT-CONH2 (L ) 2-aminoethoxy-2-ethoxyacetic acid). M (calcd) for C219H284N78O77 ) 5241.18; M (experimental) ) 5240.00.
its incorporation into a PNA fragment by Fmoc chemistry has been previously reported. Until now, our attempts to protect the N2 exocyclic of the pseudoisocytosine with a mild acid-labile protecting group (Boc and Bhoc) using the standard methodologies have resulted in poor yields (10). We are currently investigating other strategies and new blocking groups. ACKNOWLEDGMENT
We wish to thank J. Clench for linguistic editing and G. Bifolchetti for photographic work. LITERATURE CITED (1) Nielsen, P. E.; Egholm, M.; Berg, R. H.; Buchardt, O. (1991)Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide. Science 254, 1497-1500. (2) Knudsen, H.; Nielsen, P. E. (1996) Antisense properties of duplex and triplex forming PNAs. Nucleic Acid Res. 24, 495500. (3) Huhn, H.; Demidov, V. V.; Nielsen, P. E.; Frank-Kamenetskii, M. D. (1999) An experimental study of mechanism and specificity of peptide nucleic acid (PNA) binding to duplex DNA. J. Mol. Biol. , 286, 1337-1345. (4) Dueholm, L. K.; Egholm, M.; Berhens, C.; Christensen, L.; Hansen, H. F.; Vulpius, T.; Petersen, K. H.; Berg, H. R.; Nielsen, E. P.; Buchardt, O. (1994) Synthesis of peptide nucleic acid monomers containing the four natural nucleobases: Thymine, cytosine, adenine and guanine and their oligomerization. J. Org. Chem. 59, 5767-5773. (5) Thompson, S. A.; Josey, J. A.; Cadilla, R.; Gaul, M. D.; Hassman, C. F.; Luzzio, M. J.; Pipe, A. J.; Reed, J. L.; Ricca, D. J.; Wiethe, R. W.; Noble, S. A. (1995) Fmoc mediated synthesis of peptide nucleic acids. Tetrahedron 22, 61796194. (6) Mayfield, L. D.; Corey, D. R. (1999) Automated synthesis of peptide nucleic acids and peptide nucleic acid-peptide conjugates. Anal. Biochem. 268, 401-404. (7) Egholm, M.; Christensen, L.; Dueholm, K. L.; Buchardt, O.; Coull, J.; Nielsen, P. E. (1995) Efficient pH-independent equence-sepcific DNA binding by pseudoisocytosine-containing bis-PNA. Nucleic Acid Res. 23, 217-222. (8) To a stirred solution of 2 in DMF (1.5 mmol/10 mL) were added 1.5 equiv of N-methylmorpholine, 1.5 equiv of Nhydroxybenzotriazole (HOBt), followed by 1 equiv of 2-(1Hbenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluoro-
678 Bioconjugate Chem., Vol. 13, No. 3, 2002 phosphate (HBTU) at 0 °C. After 10 min, 0.9 equiv of 1 was added in one portion, and the mixture was stirred overnight after which dichloromethane was added. The organic layer was washed with half-saturated aqueous sodium bicarbonate, half-saturated aqueous potassium hydrogen sulfate, and brine, dried (Na2SO4), filtered, and evaporated under reduce pressure. The crude product was purified on silica gel flash chromatography using a gradient of 2-6% methanol in dichloromethane. Average synthetic yield: 55-65% of 3 as a white solid. 1H NMR (CDCl3) (two rotamers) δ: 1.30 (s, 4.5H), 1.40 (s, 4.5H), 3.15 (s, 1.2H), 3.25 (s, 0.8H), 3.40-3.70 (m, 4H), 3.95 (s, 1H), 4.15-4.45 (m, 4H), 5.20 (s, 2H), 7.25-7.35 (m, 10H), 7.45 (s, 0.45H), 7.50 (s, 0.55H), 7.60 (m, 2H), 7.75 (m, 2H). MS (FAB+) m/z: 682.20 (M + H).
(9) To a stirred solution of 3 was added (10 mL/mmol of 3) trifluoroacetic acid (TFA) dropwise at 0 °C. The reaction was followed by TLC and completed after 5-6 h. The solvent was evaporated under reduce pressure and coevaporated twice with acetonitrile and freeze-dried. Average synthetic yield: 85% of 4 as a white solid. 1H NMR (DMSO) (two rotamers) δ: 3.20-3.50 (m 6H), 3.95 (s 1H) 4.30 (m 3H), 5.25 (s 2H), 7.25-7.40 (m 10H), 7.55 (s 0.45H), 7.60 (s 0.55H), 7.65 (d, J ) 7 Hz, 2H), 7.90 (d, J ) 7 Hz, 2H). MS (FAB+) m/z: 626.15 (M + H). (10) Coull, J. M.; Egholm, M.; Hodge, R. P.; Ismail, M.; Rajur, S. Patent application WO 96/40685.
BC0100758
