An Efficient, Convenient Solid-Phase Synthesis of Amino Acid

Feb 18, 2006 - Truman Memorial Veterans' Hospital, Columbia, Missouri 65201. ... Columbia, Columbia, MO 65211, Phone: (573) 814-6000, ext. 3703,...
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Bioconjugate Chem. 2006, 17, 551−558

551

An Efficient, Convenient Solid-Phase Synthesis of Amino Acid-Modified Peptide Nucleic Acid Monomers and Oligomers Baghavathy S. Balaji,† Fabio Gallazzi,‡ Fang Jia,† and Michael R. Lewis*,†,§,|,⊥ Department of Veterinary Medicine and Surgery, Molecular Biology Program, Department of Radiology, and Nuclear Science and Engineering Institute, University of MissourisColumbia, Columbia, Missouri 65211, and Research Service, Harry S. Truman Memorial Veterans’ Hospital, Columbia, Missouri 65201. Received July 22, 2005; Revised Manuscript Received December 12, 2005

An efficient and highly versatile method for the synthesis of amino acid-modified peptide nucleic acid (PNA) monomers is described. By using solid-phase Fmoc techniques, such monomers can be assembled readily in a stepwise manner and obtained in high yield with minimal purification. Protected neutral hydrophilic, acidic, and basic amino acids were coupled to 2-chlorotrityl chloride resin. Following Fmoc removal, innovative conditions for the key step, reductive alkylation with N-Fmoc-aminoacetaldehyde, were developed to circumvent problems encountered with previously reported methods. Activation and coupling of pyrimidine and purine nucleobases to the resulting secondary amines afforded amino acid-modified PNA monomers. The mild reaction conditions utilized were compatible with sensitive and labile functional groups, such as tert-butyl ethers and tert-butyl esters. PNA monomers were obtained in 36-42% overall yield and very high purity, after cleavage and purification. Using standard solid-phase Fmoc chemistry, two of these monomers were incorporated with high coupling efficiency into a variety of modified PNA oligomers, including four tetradecamers designed to target bcl-2 mRNA. Such modified oligomers have the potential to enhance water solubility and cell portability, while maintaining hybridization affinity and promoting favorable biodistribution properties.

INTRODUCTION (PNAs)1

Peptide nucleic acids are DNA-like molecules having unique binding properties to either single-stranded DNA and * To whom correspondence should be addressed: Michael R. Lewis, Ph.D., Department of Veterinary Medicine and Surgery, College of Veterinary Medicine, 379 E. Campus Dr., University of Missouris Columbia, Columbia, MO 65211, Phone: (573) 814-6000, ext. 3703, Fax: (573) 814-6551, e-mail: [email protected]. † Department of Veterinary Medicine and Surgery, University of MissourisColumbia. ‡ Molecular Biology Program, University of MissourisColumbia. § Department of Radiology, University of MissourisColumbia. | Nuclear Science and Engineering Institute, University of Missouris Columbia. ⊥ Harry S. Truman Memorial Veterans’ Hospital. 1Abbreviations: % ID/g, percent injected dose per gram of tissue; bcl-2, B-cell lymphoma/leukemia-2 gene; bcl-XL, large splice variant of B-cell lymphoma/leukemia-X gene; Bhoc, benzhydryloxycarbonyl; Bn, benzyl; Boc, tert-butyloxycarbonyl; Bz, benzoyl; Cbz, benzyloxycarbonyl; DCE, dichloroethane; DCM, dichloromethane; DhbtOH, 3-hydroxy-1,2,3-benzotriazin-4(3H)-one; DIC, N,N′-diisopropylcarbodiimide; DIPEA, diisopropylethylamine; DMF, N,N-dimethylformamide; DMT, 4,4′-dimethoxytriphenylmethyl; EDCI, 1-ethyl-3-[3(dimethylamino)propyl]carbodiimide; ESI-MS, electrospray ionization mass spectrometry; Fmoc, (9H-fluoren-9-ylmethoxycarbonyl); FmocCl, Fmoc chloride; Fmoc-OSu, Fmoc succinimidyl carbonate; GS, serine-derivatizedbenzyl-protected guanine PNA residue; HATU, 2-(7aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; HBTU, O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; HOBT, 1-hydroxybenzotriazole; IBX, o-iodoxybenzoic acid; NMP, 1-methyl-2-pyrrolidinone; PNA, peptide nucleic acid; PyBOP, benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate; TD, aspartic acid derivatized thymine PNA residue; TK, lysine-derivatized thymine PNA residue; TS, serine-derivatized thymine PNA residue; TBTU, O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate; tBu, tert-butyl; TFA, trifluoroacetic acid; THF, tetrahydrofuran; TMOF, trimethyl orthoformate.

RNA or double-stranded DNA (1, 2). In recent years, much attention has been focused on synthetic (3-5), biomedical (69), and bioconjugate (10-12) applications of PNAs. The major advantage of using PNAs for DNA and RNA hybridization and targeting is due to the presence of an uncharged, achiral, acyclic amide backbone composed of (2-aminoethyl)glycine units. This structural feature can give rise to considerable conformational flexibility, as well as stability toward degradation by nucleases and proteases (2). These properties, coupled with high sequence specificity (13, 14), make PNAs important molecules for various biological applications such as gene silencing (7, 15, 16). However, the poor water solubility and extremely low cellular uptake of PNAs necessitated incorporation of modifications that can enhance cell portability (17-19). We recently synthesized and evaluated several fluorescent and radiometal-labeled cell-permeating peptide-PNA conjugates for targeting the B-cell lymphoma/leukemia-2 (bcl-2) gene at the mRNA expression level (20, 21). While the use of a cellpermeating peptide resulted in efficient internalization of bcl-2 antisense PNA in tumor cells in vitro, such a peptide delivery vehicle is likely to have little or no in vivo specificity. We subsequently prepared an 111In-labeled anti-bcl-2 PNA conjugated to a peptide receptor ligand designed for in vivo tumor targeting (22). When labeled with 111In, this PNA-peptide conjugate is zwitterionic but has sufficient water solubility for in vitro and in vivo applications. However, evaluation of a corresponding PNA conjugate complementary to the large splice variant of the B-cell lymphoma/leukemia-X gene (bcl-XL) was limited by its extremely poor water solubility (23). At present, it is not possible to predict the water solubility of a PNA-peptide conjugate a priori. Nielsen, Buchardt, and co-workers showed that replacing some of the glycine residues of PNA with hydrophilic or charged amino acids (24) improved water solubility. The incorporation of positively charged amino acids, such as D-lysine, afforded modified PNAs with superior

10.1021/bc0502208 CCC: $33.50 © 2006 American Chemical Society Published on Web 02/18/2006

552 Bioconjugate Chem., Vol. 17, No. 2, 2006

hybridization affinity for single-stranded DNA (24). However, a recent in vivo study indicated that radiometal-labeled PNAs accumulate in the kidneys of tumor-bearing mice (25). Conjugation of these PNAs to a tumor-targeting peptide improved renal clearance (26), but kidney uptake persisted out to 24 h postinjection. Another recent report demonstrated that a 64Culabeled antisense PNA, conjugated to a cell-permeating peptide with high positive charge density, could be used to image mouse tumor xenografts, albeit with renal uptake as high as 280% ID/g (27). On the basis of these findings, we reasoned that judicious incorporation of neutral hydrophilic and/or negatively charged residues may enhance water solubility and cell portability of radiolabeled PNAs, while maintaining hybridization affinity and promoting renal clearance. Though our goal was to make thymine monomers (1, 2) as our lead compounds, we also synthesized additional monomers with a basic amino acid (3, lysine) and containing a purine nucleobase (4, guanine), to show the versatility and robustness of this methodology. There are a number of strategies available for synthesis of side chain-modified PNA monomers (10, 28-33). Modifications can be made either on the glycine unit or on the aminoethyl unit. When oligomers are synthesized from these modified monomers, the presence of bulky side chains on the aminoethyl unit may pose difficulties during coupling reactions and may also exert steric hindrance on base pair formation. Moreover, preparation of N-protected, 2-substituted aminoacetaldehyde requires two steps from the commercially available amino acid, sometimes affording low yields and/or unstable products (34, 35). Another approach uses a Mitsunobu reaction between the N-protected amino acid and N-protected β-amino alcohols (28). In this method, every step requires chromatographic purification, and the incorporation of carboxylic acid functionalities such as aspartic acid is not compatible with reduction using Vitride or sodium borohydride. Thus, the present work focused on modifications of the glycine unit, where most of the appropriately protected starting materials are commercially available, and a simple N-protected aminoacetaldehyde can be employed for synthesis of the aminoethyl unit. A very recent report describes the solution phase synthesis of a single, modified PNA monomer having glutamic acid as the amino acid derivative (33). The formation of the pseudopeptide backbone was attempted using two methods, reductive N-alkylation or Mitsunobu condensation. It was found that the second method afforded higher yields and fewer byproducts. However for this route, the amine nitrogen on the pseudopeptide backbone had to be protected with an N-o-nitrobenzenesulfonyl group. Recently it was shown that modified PNA monomers and oligomers can be synthesized readily on a solid support, using Fukuyama-Mitsunobu alkylation of the desired R-amino acid, followed by acylation with the desired nucleobase-acetic acid derivative (36). However, in this case even thymine has to be protected in order to be used in the synthesis. The overall goal of the present work was to develop a robust synthetic method for introduction of amino acid-modified residues into PNA oligomers. The construction of oligomers with these modifications first required an efficient synthesis of appropriate PNA monomers. We report herein a facile solidphase synthesis of various PNA monomers prepared from different amino acid derivatives and containing either a pyrimidine or a purine as the nucleobase. We initially chose to synthesize modified derivatives of thymine for two reasons. First, protected nucleobase-acetic acid derivatives for adenine, guanine, and cytosine are not readily available from commercial sources. Second, the thymine derivative requires no protecting group using the conditions employed here. In addition, PNA hybridization stability can be highly

Technical Notes

sensitive to amino acid modifications, particularly at internal sequence positions (24). Judicious incorporation of a neutral or negatively charged hydrophilic side chains at or near the end of a duplex structure, such as the C-terminal thymine complimentary to the start codon of mRNA, may yield more water soluble PNA oligomers without abrogating target binding. The solid-phase strategy described here affords higher yields and higher purity crude products than analogous solution-phase methods. Unlike previously reported solid-phase methods, the mild conditions utilized are compatible with 9H-fluoren-9ylmethoxycarbonyl (Fmoc) PNA and peptide chemistry. To show the generality of the newly developed method, we also synthesized a lysine derivative of thymine and a serine derivative of guanine. In addition, we showed that two of these monomers can be incorporated efficiently into a variety of PNA oligomers in various sequence positions.

EXPERIMENTAL PROCEDURES General. All reagents were HPLC or peptide synthesis grade. N-R-Fmoc-L-Ser(OtBu)-OH, N-R-Fmoc-L-Asp(OtBu)-OH, N-RFmoc-L-Lys(Boc)-OH, and 2-cholorotrityl chloride resin were obtained from Novabiochem (San Diego, CA). N-R-Fmocethanolamine and thymine-1-acetic acid were purchased from Aldrich (Milwaukee, WI). PNA monomers Fmoc-A(Bhoc)-OH, Fmoc-C(Bhoc)-OH, Fmoc-G(Bhoc)-OH, and Fmoc-T-OH, as well as Fmoc-XAL PEG PS resin, were obtained from Applied Biosystems (Foster City, CA). All laboratory glassware was washed with a mixed acid solution (37) and thoroughly rinsed with ultrapure water (18 MΩ-cm resistivity). Manual reaction vessels were obtained from Chemglass, Inc. (Vineland, NJ). 1H NMR analysis of purified monomers was performed using a Bruker DRX 300 MHz spectrometer (Bruker BioSpin, Westmont, IL), and elemental analysis was performed by Atlantic Microlab (Norcross, GA). ESI-MS analyses were performed on a Finnigan TSQ7000 mass spectrometer (Thermo Finnigan, San Jose, CA). A Waters (Milford, MA) NovaPak C18 column (3.9 × 300 mm) was used for LC-MS analysis. Analytical and preparative reversed-phase HPLC were performed on a Waters 600 chromatograph equipped with a Waters Delta 600 pump, a Rheodyne 7725i injector, Waters 2487 dual wavelength UV detector, Waters 600 controller, and the Empower Pro software package (Build No. 1154). A Phenomenex (Torrance, CA) C-18 Jupiter column (4.6 × 250 mm, 5 µm, 300 Å) was used for analytical HPLC. For preparative HPLC, a Phenomenex Jupiter Proteo column (21.2 × 250 mm, 10 µm, 90 Å) was used. The flow rate was maintained at 1.0 mL/min for analytical runs and at 3.0 mL/min for preparative purification. The wavelengths used for UV detection were 214 and 280 nm for analytical HPLC and 280 nm for preparative HPLC, respectively. Eluents used in all runs consisted of solvent A (0.1% TFA/H2O) and solvent B (0.1% TFA/CH3CN). Two different gradients were used: (1) “Analytical Gradient”: sequential linear from 30% to 80% solvent B in 40 min (step 1) and 80% to 95% solvent B in 10 min (step 2), or 30% to 80% solvent B in 40 min (step 1) and 80% to 95% solvent B in 20 min (step 2); or (2) “Prep Gradient” (optimized for preparative purification of monomers): sequential linear from 35% to 65% solvent B in 60 min (step 1), 65% to 80% solvent B in 30 min (step 2), and 80% to 100% solvent B in 10 min (step 3). Monomer Synthesis. Syntheses of PNA monomers having serine, aspartic acid, or lysine derivatives were performed manually, using Fmoc chemistry and 2-chlorotrityl chloride resin at a substitution level of 0.6 mmol/g. All reactions were carried out at room temperature. Amino Acid Coupling. In a typical experiment, 350 mg of the resin was charged to the reaction vessel. The resin was

Technical Notes

washed with DCM (4 × 3 mL), DMF (2 × 3 mL), and DCM/ NMP (1:1, 2 × 3 mL). A preactivated mixture of 215 mg (561 µmol) of N-R-Fmoc-L-Ser(OtBu)-OH or 230 mg (559 µmol) of N-R-Fmoc-L-Asp(OtBu)-OH or 262 mg (560 µmol) of N-RFmoc-L-Lys(Boc)-OH and 389 µL (289 mg, 2.24 mmol) of DIPEA, dissolved in 3 mL of 1:1 DCM/NMP, was charged to the reaction vessel, and a stream of argon was used for mixing. After 45 min the activation and coupling reactions were repeated, and the resin was washed with DCM/NMP (1:1, 3 × 3 mL), DCM/CH3OH/DIPEA (17:2:1, 2 × 3 mL), and DMF (3 × 3 mL). A solution of 25% piperidine in 3 mL of DMF was charged to the reaction vessel and Fmoc removal was carried out for 30 min. The deprotection reaction was repeated, and the resin was washed with DMF (3 × 3 mL) and TMOF (2 × 3 mL). N-Fmoc-aminoacetaldehyde Coupling. o-Iodoxybenzoic acid (IBX) was synthesized according to the literature procedure (38). N-Fmoc-ethanolamine was oxidized using IBX (39), and the corresponding aldehyde, N-Fmoc-aminoacetaldehyde, was obtained as a single pure product in 90-95% yield. The aldehyde (81 mg, 282 µmol) was dissolved in TMOF (3 mL) and charged to the resin. A small amount (∼0.3-0.5 mL) of DMF was added to increase the solubility of the aldehyde. After 40 min, a few drops of acetic acid were added, followed by sodium (triacetoxy)borohydride (60 mg, 283 µmol), and the resulting suspension was mixed under argon for 20 min. The resin was washed with TMOF (2 × 3 mL) and DMF (3 × 3 mL). Nucleobase Coupling. In the case of the L-serine monomer, thymine-1-acetic acid (81 mg, 440 µmol) or benzyloxypurine9-acetic acid (132 mg, 441 µmol) was activated with 71 mg of DhbtOH (436 µmol) and 69 µL of DIC (55 mg, 437 µmol) in 3 mL of DMF. This mixture was added to the resin, and the resulting suspension was mixed under argon for 6-8 h. The activation and coupling steps were repeated using benzyloxypurine-9-acetic acid. For the L-aspartic acid monomer, the activation and coupling steps were also repeated. Then the resin was washed with DMF (3 × 3 mL) and DCM (3 × 3 mL). Cleavage and Purification of S-3-tert-Butyloxy-2-{2N[(9H-fluoren-9-ylmethoxycarbonyl)aminoethyl]-2-[(thymine1-acetyl)-amino]}-propanoicAcid(1), S-3-tert-Butyloxycarbonyl2-{2N-[(9H-fluoren-9-ylmethoxycarbonyl)aminoethyl]2-[(thymine-1-acetyl)-amino]}-propanoic Acid (2), S-6tert-Butyloxycarbonylamino-2-{2N-[(9H-fluoren-9-ylmethoxycarbonyl)aminoethyl]-2-[(thymine-1-acetyl)-amino]}-hexanoic Acid (3), and S-3-tert-Butyloxy-2-{2N-[(9H-fluoren-9-ylmethoxycarbonyl)aminoethyl]-[2-(2-amino-6-benzyloxypurine-9-acetyl)-amino]}-propanoic Acid (4). A small amount of each resin was removed from the reaction mixture and washed with DMF, DCM, and CH3OH. The final products were cleaved from the resin using 0.5% TFA in DCM for 30 min and precipitated using diethyl ether. The precipitate was dissolved in CH3CN:H2O (1:1) and analyzed by HPLC (“Analytical Gradient”) to confirm completion of nucleobase coupling. The remainder of the resin was then washed, and the final products were cleaved and isolated by precipitation as described above. The side chain-modified PNA monomers were purified by either preparative HPLC (“Prep Gradient”) or flash chromatography on silica gel, using DCM:CH3OH (10:1) as the mobile phase. After purification, 60.0 mg (42.2%) of 1 (TS) was obtained as a white powder. 1H NMR (300 MHz, CDCl3) δ 1.08 (s, 9H), 1.76 (s, 3H), 2.87 (br, 1H), 3.37-3.40 (m, 2H), 3.50 (s, 2H), 3.76-3.82 (m, 2H), 3.88-3.90 (m, 1H), 4.11-4.17 (m, 2H), 4.37 (d, 1H), 4.58 (d, 1H), 4.69 (br, 1H), 6.15 (s, 1H), 6.78 (s, 1H), 7.20-7.23 (m, 2H), 7.30-7.33 (m, 2 H), 7.49-7.52 (m, 2H), 7.66-7.70 (m, 2H), 10.2 (br, 1H). ESI-MS m/z calcd for C31H36N4O8 (M + H)+ ) 593.7, found 537.2 (M + H - tBu)+,

Bioconjugate Chem., Vol. 17, No. 2, 2006 553

593.2 (M + H)+, 615.1 (M + Na)+. Anal. (C31H36N4O8‚ CH2Cl2): C, H, N. Chromatographic purification afforded 48.0 mg (36.8%) of 2 (TD) as a white powder. 1H NMR (300 MHz, CDCl3) δ 1.40 (s, 9H), 1.77 and 1.85 (2s, 3H), 2.78-2.87 (m, 2H), 3.01-3.20 (m, 2H), 3.46 (s, 1H), 3.57 (s, 1H), 3.79-3.82 (d, 1H), 3.95 (br, 1H), 4.09-4.20 (m, 1H), 4.37 (d, 1H), 4.52-4.54 (m, 1H), 4.72 (d, 1H), 5.83 (s, 1H), 6.81 and 6.86 (2s, 1H), 7.19-7.21 (m, 2H), 7.30-7.37 (m, 2 H), 7.48-7.54 (m, 2H), 7.62-7.73 (m, 2H), 10.62 (br, 1H). ESI-MS m/z calcd for C32H36N4O9 (M + H)+ ) 621.7, found 565.1 (M + H - tBu)+, 621.4 (M + H)+, 643.3 (M + Na)+. Anal. (C32H36N4O9‚CH2Cl2‚1/2 (CH3CH2)2O): C, H, N. Chromatographic purification afforded 68.0 mg (38.0%) of 3 (TK) as a white powder. 1H NMR (300 MHz, CDCl3) δ 1.31 (t, 1H), 1.46 and 1.49 (2s, 9H), 1.59 (s, 1H), 1.76 (s, 1H), 1.82 and 1.86 (2s, 3H), 1.92-2.03 (m, 1H), 2.72 (br, 1H), 2.94 (s, 1H), 3.10 (s, 2H), 3.28 and 3.37 (m, 2H), 3.49 (s, 1H), 3.67 (br, 1H), 3.82 (m, 1H), 4.20 (d, 1H), 4.39, (d, 1H), 4.66 (d, 1H), 4.82 (m, 1H), 6.12 (s, 1H), 6.98 (s, 1H), 7.27-7.29 (m, 2H), 7.35-7.42 (m, 2 H), 7.53-7.62 (m, 2H), 7.70-7.82 (m, 2H), 10.52 (br, 1H). ESI-MS m/z calcd for C35H43N5O9 (M + H)+ ) 678.7, found 579.2 (M + H-Boc)+, 700.1 (M + Na)+. Anal. (C35H43N5O9‚CF3CO2H): C, H, N. Chromatographic purification afforded 64.0 mg (36.2%) of 4 (GS) as a white powder. 1H NMR (300 MHz, CDCl3) δ 1.141.17 and 1.22-1.23 (d, 9H), 2.84-3.04 (m, 1H), 3.08-3.20 (m, 1H), 3.45-3.79 (m, 2H), 3.80 (t, 1H), 3.91 (s, 1H), 4.06 (s, 1H), 4.12-4.25 (m, 2H), 4.31 (s, 1H), 4.55 (dd, 1H), 4.90 and 4.96 (s, 1H), 5.05 (q, 1H), 5.49-5.57 (m, 2H), 6.30 (s, 1H), 7.26-7.29 (m, 4H), 7.32-7.41 (m, 4H), 7.42-7.49 (m, 2H), 7.51-7.58 (m, 2H), 7.64-7.71 (m, 1H), 7.73-7.78 (m, 2H), 10.62 (br, 1H). ESI-MS m/z calcd for C38H41N7O7 (M + H)+ ) 708.8, found 708.3 (M + H)+. Anal. (C38H41N7O7‚2 CF3CO2H): C, H, N. Synthesis of Model PNA Tetramers (5-11). Syntheses of PNA tetramers using 2 were performed according to a previously published procedure (20), using Fmoc-XAL PEG PS resin at a substitution level of 0.15 mmol/g. Compound 2 was incorporated into tetramers 5 (AAGTD; TD ) aspartic acid derivatized thymine residue), 6 (GATDA), 7 (ATDCG), 8 (TDACA), 9 (ATDGC), 10 (AGATD), and 11 (TDCAA) on a 1 µmol scale. After cleavage, deprotection, precipitation with diethyl ether, and lyophilization, 0.8-1.0 mg (68-86%) of 5-11 were obtained as white powders. ESI-MS (5) m/z calcd (M + 2H)2+ ) 592.4, found 592.4; (6) m/z calcd (M + 2H)2+ ) 593.4, found 592.2; (7) m/z calcd (M + 2H)2+ ) 581.4, found 580.2; (8) m/z calcd (M + 2H)2+ ) 573.4, found 572.2; (9) m/z calcd (M + 2H)2+ ) 580.9, found 580.4; (10) m/z calcd (M + 2H)2+ ) 592.9, found 592.6; (11) m/z calcd (M + 2H)2+ ) 572.9, found 572.4. Synthesis of bcl-2 Antisense Tetradecamers (12-15). Compounds 1 and 2 were each incorporated into sequence positions 1 or 8 of four bcl-2 antisense PNA tetradecamers: 12 (CCAGCGTGCGCCATS; TS ) serine derivatized thymine residue), 13 (CCAGCGTSGCGCCAT), 14 (CCAGCGTGCGCCATD), and 15 (CCAGCGTDGCGCCAT). The syntheses were carried out on a 5 µmol scale, using Fmoc-XAL PEG PS resin at a substitution level of 0.15 mmol/g and modifications of a method reported previously (20). After cleavage, Bhoc deprotection, and precipitation with diethyl ether, the N-terminal Fmoc-protected tetradecamers were purified by preparative reversed-phase HPLC, using a sequential linear gradient of 5% to 15% solvent B in 10 min (step 1), 15% to 55% solvent B in 40 min (step 2), 55% to 75% solvent B in 30 min (step 3), 75% to 85% solvent B in 20 min (step 4), 85% to 95% solvent B in 20 min (step 5), and 95% to 100% solvent B in 10 min

554 Bioconjugate Chem., Vol. 17, No. 2, 2006

Technical Notes

Scheme 1

(step 6). The fractions corresponding to the major products were collected, pooled, and lyophilized. Fmoc removal was carried out in 25% piperidine in DMF for 30 min at room temperature, after which the products were isolated by precipitation with diethyl ether and dried to give 1.2-1.3 mg (6.3-6.8%) of 1215 as white powders. ESI-MS (12) m/z calcd (M + 3H)3+ ) 1268.9, (M + 4H)4+ ) 951.7; found, 1268.2, 951.7; (13) m/z calcd (M + 3H)3+ ) 1268.9, (M + 4H)4+ ) 951.7; found, 1268.4, 951.6; (14) m/z calcd (M + 3H)3+ ) 1278.2, (M + 4H)4+ ) 958.7; found, 1277.8, 958.5; (15), m/z calcd (M + 3H)3+ ) 1278.2, (M + 4H)4+ ) 958.7; found, 1277.3, 958.3.

RESULTS AND DISCUSSION Initially the solution-phase synthesis previously reported for similar amino acid-modified PNA monomers (11) was attempted. To obtain the desired products, protection and deprotection of the C-terminus was mandatory, most steps required chromatographic purification of the intermediates, and the overall yields were 95% by reversed-phase HPLC analysis

Technical Notes

Figure 5. Analytical reversed-phase HPLC chromatograms of serine derivatized tetradecamers 12 (CCAGCGTGCGCCATS, top) and 13 (CCAGCGTSGCGCCAT, bottom), using a sequential linear gradient of 5% to 25% solvent B in 20 min (step 1), 25% to 45% solvent B in 10 min (step 2), and 45% to 95% solvent B in 10 min (step 3). The peaks at a retention times of 26.9 min (top) and 22.2 min (bottom) represent the desired products.

Figure 6. Analytical reversed-phase HPLC chromatograms of aspartic acid derivatized tetradecamers 14 (CCAGCGTGCGCCATD, top) and 15 (CCAGCGTDGCGCCAT, bottom), using a sequential linear gradient of 5% to 25% solvent B in 20 min (step 1), 25% to 45% solvent B in 10 min (step 2), and 45% to 95% solvent B in 10 min (step 3). The peaks at a retention times of 16.8 min (top) and 16.3 min (bottom) represent the desired products.

(data not shown). The high purity of the tetramer preparations indicated that the coupling efficiency of 2 was comparable to that of commercially available glycine monomers. For each of the tetramers, ESI-MS analysis was consistent with the structures of the desired products. Finally, four tetradecamers, complementary to the translational start sequence of bcl-2 mRNA, were synthesized using compounds 1 and 2. A serine (TS) or an aspartic acid (TD)derivatized thymine residue was incorporated at the C-terminus (position 1) or coupled to a guanine residue in position 8 of the respective tetradecamers: CCAGCGTGCGCCATS (12), CCAGCGTSGCGCCAT (13), CCAGCGTGCGCCATD (14), and CCAGCGTDGCGCCAT (15). After cleavage, Bhoc deprotection, HPLC separation, Fmoc removal, and precipitation, reversed-phase HPLC analysis of 12 (Figure 5, top), 13 (Figure

Technical Notes

5, bottom), 14 (Figure 6, top), and 15 (Figure 6, bottom) indicated that the purity of each of the modified oligomers was approximately 90%. ESI-MS of the major products was consistent with the structures of the respective tetradecamers. Minor impurities (∼10%) present in the tetradecamer preparations may represent truncated species not removed by preparative reversed-phase HPLC. However, these syntheses demonstrated that two of these amino acid-modified monomers (TS and TD) can be incorporated with high efficiency into PNAs sufficiently long for statistically unique targeting in the human RNA pool (52).

CONCLUSION We have developed a convenient, efficient, and high yielding synthesis of amino acid-modified PNA monomers requiring minimal purification using solid-phase techniques. Amino acids represented neutral hydrophilic (serine), acidic (aspartic acid), and basic (lysine) residues, and the nucleobases included a pyrimidine (thymine) and a purine (protected guanine), to afford a wide variety of compounds. Furthermore, we have synthesized several PNA tetramers and bcl-2 antisense tetradecamers in which two of these modified residues have been incorporated into various sequence positions, with coupling efficiencies comparable to those of commercially available glycine analogues. Studies evaluating the water solubility and DNA hybridization properties of PNAs containing these amino acid modifications are currently in progress.

ACKNOWLEDGMENT This work was supported by NIH Grant CA103130 from the National Cancer Institute. The authors also acknowledge the Department of Veterans Affairs, for providing resources and the use of facilities at the Harry S. Truman Memorial Veterans’ Hospital in Columbia, MO. Supporting Information Available: ESI mass spectra and 1H

NMR spectra of compounds 1-4, as well as ESI mass spectra of compounds 12-15. This material is available free of charge via the Internet at http://pubs.acs.org.

LITERATURE CITED (1) Nielsen, P. E., Egholm, M., Berg, R. H., and Buchardt, O. (1991) Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide. Science 254, 1497-1500. (2) Nielsen, P. E., and Haaima, G. (1997) Peptide nucleic acid (PNA). A DNA mimic with a pseudopeptide backbone. Chem. Soc. ReV. 26, 73-78. (3) Uhlmann, E., Peyman, A., Breipohl, G., and Will, D. W. (1998) PNA: synthetic polyamide nucleic acids with unusual binding properties. Angew. Chem., Int. Ed. 37, 2796-2823. (4) Di Giorgio, C., Pairot, S., Schwergold, C., Patino, N., Condom, R., Farese-Di Giorgio, A., and Guedj, R. (1999) Liquid-phase synthesis of polyamide nucleic acids (PNA). Tetrahedron 55, 19371958. (5) Maison, W., Schlemminger, I., Westerhoff, O., and Martens, J. (2000) Multicomponent synthesis of novel amino acid-nucleobase chimeras: a versatile approach to PNA-monomers. Bioorg. Med. Chem. 8, 1343-1360. (6) Nielsen, P. E. (2000) Peptide nucleic acids: on the road to new gene therapeutic drugs. Pharmacol. Toxicol. 86, 3-7. (7) Wang, G., and Xu, X. S. (2004) Peptide nucleic acid (PNA) binding-mediated gene regulation. Cell Res. 14, 111-116. (8) Doyle, D. F., Braasch, D. A., Simmons, C. G., Janowski, B. A., and Corey, D. R. (2001) Inhibition of gene expression inside cells by peptide nucleic acids: effect of mRNA target sequence, mismatched bases, and PNA length. Biochemistry 40, 53-64. (9) Kaihatsu, K., Huffman, K. E., and Corey, D. R. (2004) Intracellular uptake and inhibition of gene expression by PNAs and PNA-peptide conjugates. Biochemistry 43, 14340-14347.

Bioconjugate Chem., Vol. 17, No. 2, 2006 557 (10) Sforza, S., Tedeschi, T., Corradini, R., Ciavardelli, D., Dossena, A., and Marchelli, R. (2003) Fast, solid-phase synthesis of chiral peptide nucleic acids with a high optical purity by a submonomeric strategy. Eur. J. Org. Chem., 1056-1063. (11) Hamzavi, R., Dolle, F., Tavitian, B., Dahl, O., and Nielsen, P. E. (2003) Modulation of the pharmacokinetic properties of PNA: preparation of galactosyl, mannosyl, fucosyl, N-acetylgalactosaminyl, and N-acetylglucosaminyl derivatives of aminoethylglycine peptide nucleic acid monomers and their incorporation into PNA oligomers. Bioconjugate Chem. 14, 941-954. (12) Matysiak, S., Reuthner, F., and Hoheisel, J. D. (2001) Automating parallel peptide synthesis for the production of PNA library arrays. BioTechniques 31, 896-904. (13) Egholm, M., Buchardt, O., Christensen, L., Behrens, C., Freier, S. M., Driver, D. A., Berg, R. H., Kim, S. K., Norden, B., and Nielsen, P. E. (1993) PNA hybridizes to complementary oligonucleotides obeying the Watson-Crick hydrogen-bonding rules. Nature 365, 566-568. (14) Lomakin, A., and Frank-Kamenetskii, M. D. (1998) A theoretical analysis of specificity of nucleic acid interactions with oligonucleotides and peptide nucleic acids (PNAs). J. Mol. Biol. 276, 57-70. (15) Pooga, M., and Langel, U. (2001) Targeting of cancer-related proteins with PNA oligomers. Curr. Cancer Drug Targets 1, 231239. (16) Mier, W., Eritja, R., Mohammed, A., Haberkorn, U., and Eisenhut, M. (2003) Peptide-PNA conjugates: targeted transport of antisense therapeutics into tumors. Angew. Chem., Int. Ed. 42, 1968-1971. (17) Koppelhus, U., and Nielsen, P. E. (2003) Cellular delivery of peptide nucleic acid (PNA). AdV. Drug DeliVery ReV. 55, 267-280. (18) Li, X., Zhang, L., Lu, J., Chen, Y., Min, J., and Zhang, L. (2003) Signal peptide mimics conjugated to peptide nucleic acid: a promising solution for improving cell membrane permeability. Bioconjugate Chem. 14, 153-157. (19) Braasch, D. A., and Corey, D. R. (2002) Novel antisense and peptide nucleic acid strategies for controlling gene expression. Biochemistry 41, 4503-4510. (20) Gallazzi, F., Wang, Y., Jia, F., Shenoy, N., Landon, L. A., Hannink, M., Lever, S. Z., and Lewis, M. R. (2003) Synthesis of radiometal-labeled and fluorescent cell-permeating peptide-PNA conjugates for targeting the bcl-2 proto-oncogene. Bioconjugate Chem. 14, 1083-1095. (21) Lewis, M. R., Jia, F., Gallazzi, F., Wang, Y., Zhang, J., Shenoy, N., Lever, S. Z., and Hannink, M. (2002) Radiometal-labeled peptidePNA conjugates for targeting bcl-2 expression: preparation, characterization, and in vitro mRNA binding. Bioconjugate Chem. 13, 1176-1180. (22) Jia, F., Gallazzi, F., Lever, S. Z., Hannink, M., and Lewis, M. R. (2005) Synthesis and in Vitro evaluation of 111In-DOTA-anti-bcl2-PNA-Tyr3-octreotate in chronic lymphocytic leukemia cells. J. Labeled Cpd. Radiopharm. 48, S286. (23) Gallazzi, F., Jia, F., Balaji, B. S., Hannink, M., Lever, S. Z., and Lewis, M. R. (2004) Synthesis of radiometal-labeled, receptortargeted anti-bcl-xL PNA conjugates. Abstract Number NUCL-4, 228th American Chemical Society National Meeting, Philadelphia, PA. (24) Haaima, G., Lohse, A., Buchardt, O., and Nielsen, P. E. (1996) Peptide nucleic acids (PNAs) containing thymine monomers derived from chiral amino acids: hybridization and solubility properties of D-lysine PNA. Angew. Chem., Int. Ed. Engl. 35, 1939-1941. (25) Rao, P. S., Tian, X., Qin, W., Aruva, M. R., Sauter, E. R., Thakur, M. L., and Wickstrom, E. (2003) Tc-99m-peptide-peptide nucleic acid probes for imaging oncogene mRNAs in tumours. Nucl. Med. Commun. 24, 857-863. (26) Tian, X., Aruva, M. R., Qin, W., Zhu, W., Duffy, K. T., Sauter, E. R., Thakur, M. L., and Wickstrom, E. (2004) External imaging of CCND1 cancer gene activity in experimental human breast cancer xenografts with 99mTc-peptide-peptide nucleic acid-peptide chimeras. J. Nucl. Med. 45, 2070-2082. (27) Sun, X., Fang, H., Li, X., Rossin, R., Welch, M. J., and Taylor, J.-S. (2005) MicroPET imaging of MCF-7 tumors in mice via unr mRNA-targeted peptide nucleic acids. Bioconjugate Chem. 16, 294305. (28) Falkiewicz, B., Kolodziejczyk, A. S., Liberek, B., and Wisniewski, K. (2001) Synthesis of achiral and chiral peptide nucleic acid (PNA) monomers using Mitsunobu reaction. Tetrahedron 57, 7909-7917.

558 Bioconjugate Chem., Vol. 17, No. 2, 2006 (29) Dueholm, K. L., Petersen, K. H., Jensen, D. K., Egholm, M., Nielsen, P. E., and Buchardt, O. (1994) Peptide nucleic acid (PNA) with a chiral backbone based on alanine. Bioorg. Med. Chem. Lett. 4, 1077-1080. (30) Zhang, L., Min, J., and Zhang, L. (1999) Studies on the synthesis and properties of new PNA analogs consisting of L- and D-lysine backbones. Bioorg. Med. Chem. Lett. 9, 2903-2908. (31) Falkiewicz, B. (2002) Application of Mitsunobu reaction to solidphase peptide nucleic acid (PNA) monomer synthesis. Nucleosides Nucleotides Nucleic Acids 21, 883-889. (32) Li, Y., Jin, T., and Liu, K. (2001) Synthesis and binding affinity of a chiral PNA analogue. Nucleosides Nucleotides Nucleic Acids 20, 1705-1721. (33) Prokhorov, D. I., Kirillova, Y. G., Boyarskaya, N. P., Tevyashova, A. N., Esipova, O. V., Zvonkova, E. N., and Shvets, V. I. (2005) Synthesis of thymine-containing monomers of negatively charged peptide nucleic acids. Pharm. Chem. J. 39, 323-328. (34) Lubell, W. D., and Rapoport, H. (1987) Configurational stability of N-protected R-amino aldehydes. J. Am. Chem. Soc. 109, 236239. (35) Jurczak, J., and Golebiowski, A. (1989) Optically active Nprotected R-amino aldehydes in organic synthesis. Chem. ReV. 89, 149-164. (36) Viirre, R. D., and Hudson, R. H. E. (2001) Optimization of a solid-phase synthesis of a PNA monomer. Org. Lett. 3, 3931-3934. (37) Thiers, R. E. (1957) Contamination in trace element analysis and its control. Methods Biochem. Anal. 5, 273-335. (38) Frigerio, M., Santagostino, M., and Sputore, S. (1999) A userfriendly entry to 2-iodoxybenzoic acid (IBX). J. Org. Chem. 64, 4537-4538. (39) More, J. D., and Finney, N. S. (2002) A simple and advantageous protocol for the oxidation of alcohols with o-iodoxybenzoic acid (IBX). Org. Lett. 4, 3001-3003. (40) Leggio, A., Liguori, A., Napoli, A., Siciliano, C., and Sindona, G. (2000) New strategies for an efficient removal of the 9-fluorenylmethoxycarbonyl (Fmoc) protecting group in the peptide synthesis. Eur. J. Org. Chem., 573-575. (41) Carpino, L. A., and Han, G. Y. (1972) 9-Fluorenylmethoxycarbonyl amino-protecting group. J. Org. Chem. 37, 3404-3409.

Technical Notes (42) Sheppard, R. (2003) The fluorenylmethoxycarbonyl group in solidphase synthesis. J. Pept. Sci. 9, 545-552. (43) Greene, T. W., and Wuts, P. G. M. (1999) Protection for the carbonyl group. ProtectiVe Groups in Organic Synthesis, 3rd ed., pp 299-302, Chapter 4, John Wiley & Sons, New York. (44) Falkiewicz, B., Kowalska, K., Kolodziejczyk, A. S., Wisniewski, K., and Lankiewicz, L. (1999) Synthesis of new chiral peptide nucleic acid (PNA) monomers by a simplified reductive amination method. Nucleosides Nucleotides 18, 353-361. (45) Davis, P. W., and Swayze, E. E. (2000) Automated solid-phase synthesis of linear nitrogen-linked compounds. Biotechnol. Bioeng. 71, 19-27. (46) Chan, W. C., and White, P. D. (2003) Basic procedures. Fmoc Solid-Phase Peptide Synthesis. A Practical Approach (Chan, W. C., and White, P. D., Eds.) p 56, Chapter 3, Oxford University Press, New York. (47) Knorr, R., Trzeciak, A., Bannwarth, W., and Gillessen, D. (1989) New coupling reagents in peptide chemistry. Tetrahedron Lett. 30, 1927-1930. (48) Neuner, P., and Monaci, P. (2002) New Fmoc pseudoisocytosine monomer for the synthesis of a bis-PNA molecule by automated solid-phase Fmoc chemistry. Bioconjugate Chem. 13, 676-678. (49) Wang, W., and Nan, F. (2003) First total synthesis of leucamide A. J. Org. Chem. 68, 1636-1639. (50) Carpino, L. A. (1993) 1-Hydroxy-7-azabenzotriazole. An efficient peptide coupling additive. J. Am. Chem. Soc. 115, 4397-4398. (51) Dueholm, K. L., Egholm, M., Behrens, C., Christensen, L., Hansen, H. F., Vulpius, T., Petersen, K. H., Berg, R. H., Nielsen, P. E., and 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. (52) Summerton, J. (1999) Morpholino antisense oligomers: the case for an RNase H-independent structural type. Biochim. Biophys. Acta 1489, 141-158. BC0502208