Synthesis and in Vitro Evaluation of PNA−Peptide−DETA Conjugates

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Bioconjugate Chem. 2004, 15, 576−582

Synthesis and in Vitro Evaluation of PNA-Peptide-DETA Conjugates as Potential Cell Penetrating Artificial Ribonucleases Lene Petersen,†,§ Martijn C. de Koning,†,§ Petra van Kuik-Romeijn,‡ Jimmy Weterings,† Christine J. Pol,‡ Gerard Platenburg,‡ Mark Overhand,† Gijsbert A. van der Marel,† and Jacques H. van Boom*,†,‡ Leiden Institute of Chemistry and ProSensa B.V., Gorlaeus Laboratories, University of Leiden, P.O. Box 9502, 2300 RA Leiden, The Netherlands. Received December 4, 2003; Revised Manuscript Received February 24, 2004

We report the synthesis of novel artificial ribonucleases with potentially improved cellular uptake. The design of trifunctional conjugates 1a and 1b is based on the specific RNA-recognizing properties of PNA, the RNA-cleaving abilities of diethylenetriamine (DETA), and the peptide (KFF)3K for potential uptake into E. coli. The conjugates were assembled in a convergent synthetic route involving native chemical ligation of a PNA, containing an N-terminal cysteine, with the C-terminal thioester of the cell-penetrating (KFF)3K peptide to give 12a and 12b. These hybrids contained a free cysteine sidechain, which was further functionalized with an RNA-hydrolyzing diethylenetriamine (DETA) moiety. The trifunctional conjugates (1a, 1b) were evaluated for RNA-cleaving properties in vitro and showed efficient degradation of the target RNA at two major cleavage sites. It was also established that the cleavage efficiency strongly depended on the type of spacer connecting the PNA and the peptide.

INTRODUCTION

Peptide nucleic acids (PNAs)1 are nonionic DNA mimics composed of repeating N-(2-aminoethyl)glycine units, whose secondary amino atoms are anchored through methylenecarbonyl linkers to the natural nucleobases. PNAs are resistant to nuclease and protease digestion and bind exceptionally tight and with high affinity to complementary DNA and RNA (1-3). The latter features have led to the successful in vitro application of PNA as antisense agents (4-8). A few years ago we reported the design and synthesis of an artificial ribonuclease (see Figure 1) composed of a PNA strand attached to a diethylenetriamine (DETA) moiety via a urethane bond (9). In vitro studies revealed that this hybrid almost completely degraded a complementary 32P-labeled RNA strand within 24 h at 40 °C and pH 7. This encouraging result was an incentive to study the potential ribonucleolytic activity of this type of artificial ribonuclease in a biological system. However, the in vivo * Corresponding author. Tel: +31 (0)71 5274274. Fax: +31 (0)71 5274307. E-mail: [email protected]. † Leiden Institute of Chemistry. ‡ ProSensa B.V. § Authors contributed equally to this study. 1 Abbreviations: ACN, acetonitrile; Boc, tert-butyloxycarbonyl; Bhoc, benzhydryloxycarbonyl; BOP, benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate; DCM, dichloromethane; DETA, diethylenetriamine; DiPEA, N,N-diisopropylethylamine; DMF, N,N-dimethylformamide; Fmoc, 9-fluorenylmethyloxycarbonyl; HATU, O-(7-azabenzotriazol-1-yl)-N,N,N′,N′tetramethyluronium hexafluorophosphate; HOBT, N-hydroxybenzotriazole; LC-MS, liquid chromatography-mass spectrometry; MALDI TOF, matrix-assisted laser desorption ionization time-of-flight; NMP, N-methylpyrrolidone; PNA, peptide nucleic acid; PyBOP, benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate. RP-HPLC, reversed phase high performance liquid chromatography; TCEP, tris(2-carboxyethyl)phosphine; TFA, trifluoroacetic acid; THF, tetrahydrofuran; TIS, triisopropylsilane; TMS, tetramethylsilane.

Figure 1. PNA-DETA conjugate (PNA marked in bold) synthesized by Verheijen et al. (9). The target RNA was cleaved at the sites indicated, the major cleavage taking place after C-17 and C-19.

evaluation of PNA is severely hampered by its poor membrane permeability (10). This obstacle can in principle be circumvented by attachment of lipophilic groups such as adamantyl (11, 12) or phosphonium cation (13) to the PNA or by conjugation of PNA to membranepermeable peptides (14-17). For instance, Good et al. investigated (18, 19) the invasive properties of a peptide with the sequence (KFF)3Ksa cationic peptide that had previously been shown to permeate bacterial membranes (20, 21)sattached through a spacer to the N-terminus of an antisense PNA. Markedly higher cellular uptake of the conjugate was found as judged by the dramatically increased antisense activity of the PNA-peptide conjugate compared to “naked” PNA. A recent study by Geller et al. (22) also demonstrated that antisense phosphorodiamidate morpholino oligomers (PMOs) showed highly improved bactericidal effects when bound covalently to the peptide (KFF)3KC. It occurred to us that trifunctional constructs 1a or 1b, as depicted in Figure 2, containing an antisense PNA, a DETA moiety, and the (KFF)3K peptide may display enhanced cellular uptake and site-specific nuclease activity in E. coli. We here report the synthesis and the in vitro nucleolytic properties of conjugates 1a and 1b, having different types of spacers connecting the (KFF)3K peptide to the N-terminus of the PNA.

10.1021/bc034219p CCC: $27.50 © 2004 American Chemical Society Published on Web 04/28/2004

PNA−Peptide−DETA Conjugates

Figure 2. Structure of new trifunctional artificial ribonucleases 1a and 1b containing an RNA-recognizing PNA strand, a DETA moiety for RNA cleavage, and a cellular delivery peptide connected to the PNA via a spacer. EXPERIMENTAL PROCEDURES

General Remarks. All reagents were of analytical or peptide synthesis grades and used as obtained from the suppliers. All solvents were of HPLC grade or peptide synthesis grade. Analytical LC-MS was conducted on a JASCO system using an Alltima C18 analytical column (5 µm particle size, flow: 1.0 mL/min). The absorbance was measured at 214 and 254 nm. The solvent system was A: 100% Water, B: 100% ACN, C: 0.5% TFA. Gradients of B in A containing 10% C were applied over 15 min. Mass spectra were recorded on a Perkin-Elmer Sciex API 165 equipped with an electrospray interface (ES). MALDI-TOF-MS spectra were recorded on a Voyager-DE PRO mass spectrometer (PerSeptive Biosystems) using a 4-hydroxy-R-cyanocinnamic acid matrix (10 mg/mL 0.2% TFA in ACN/Water, 1/1). Purifications were conducted using a BioCAD “Vision” automated HPLC system (PerSeptive Biosystems), supplied with a semipreparative Alltima C18 column (5 µm particle size, running at 4.7 mL/min). Solvent system: A: 100% Water, B: 100% ACN, C: 1% TFA. Gradients of acetonitrile (B) in A containing 10% C were applied over 3 column volumes (CV). A TitroLine alpha machine or Merck Universalindikator pH 1-10 paper was used to measure the pH of buffers. The peptide and PNA fragments were prepared using an ABI 433A (Applied Biosystems) automatic peptide synthesizer. 1H NMR and 13C NMR spectra were recorded with a Bruker AC-200 spectrometer (200/50.1 MHz). Chemical shifts are given in ppm (δ) relative to tetramethylsilane (1H NMR) as an internal standard or to the peak of the used solvent (13C NMR). Synthesis. (KFF)3K-Spacer-Thioester 7a. To a suspension of 4-sulfamylbutyryl safety-catch resin 2 (1 g, loading capacity ) 1.1 mmol/g, 1.1 mmol) in DMF were added DiPEA (1.92 mL, 11 mmol) and N-Fmoc-Gly-OH (1.64 g, 5.5 mmol). The reaction mixture was shaken for 20 min and then cooled to -40 °C, and PyBOP (2.86 g, 5.5 mmol) was added. The mixture was shaken for 1 h at -40 °C and then 5 h at -20 °C. The resin was removed by filtration and washed with DMF (3×), MeOH (3×), and DCM (3×) and then dried under a flow of air. The loading of the resin (0.4 mmol/g) was determined by standard Fmoc quantification. The peptide synthesis was carried out automatically on an ABI 433a peptide synthesizer on a 50 µmol scale using an excess (5 equiv) of the amino acid building blocks N-Fmoc-Phe-OH, N-FmocLys(Boc)-OH, and N-Boc-Lys(Boc)-OH, the activating agents BOP/HOBT, DiPEA as a base, and Ac2O/DiPEA for capping. After complete synthesis, the resin was treated with trimethylsilyldiazomethane (1 M in THF/ hexane, 1:1, 3 mL) and shaken for 2 h. The solvents were removed, and the resin was washed with THF (3×), DMF (3×), and THF (3×) and resubjected to trimethylsilyldiazomethane (0.66 M in THF/hexane, 2:1, 3 mL) for 45 min. The solvents were removed, and the resin was washed with THF (3×) and DMF (3×), and treated with

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a solution of ethyl 3-mercaptoproprionate (2 mmol, 1 M in DMF, 2 mL) containing sodium thiophenolate (2.5 mg, 0.02 mmol). The mixture was shaken for 2 days, and the resin was removed by filtration. The resin was additionally washed with DMF (3×), and the combined filtrates were evaporated under reduced pressure. The residue was treated with 95% TFA/H2O (5 mL) and shaken for 1.5 h, and the product 7a was precipitated from Et2O and collected by centrifugation. LC-MS: 10-55% B, tR ) 13.4 min; ES MS: found/calcd: 1687.1/1687.1 (M + H)+, 843.9/ 844.1 (M + 2H)2+. (KFF)3K-Spacer-Thioester 7b. 4-Sulfamylbutyryl safety-catch resin 2 was loaded with the N-Fmocprotected diethyleneglycol linker 3, using the same method as described above, to give immobilized 4b. After determination of the loading by standard Fmoc quantification (0.31 mmol/g), the peptide 7b was synthesized according to similar procedures as described for thioester 7a. LC-MS: 10-55% B, tR ) 14.2 min; ES MS: found/ calcd: 1676.4/1676.1 (M + H)+, 838.2/838.6 (M + 2H)2+. Synthesis of Cys(StBu)-PNA 11. PNA 11 was synthesized automatically on an ABI 433a peptide synthesizer on a 10 µmol scale using an excess (5 equiv) of the Fmoc/Bhoc-protected PNA monomers or Fmoc/StBuprotected cysteine (0.11 M monomer concentration) for the couplings on tentagel resin containing the Fmocprotected Rink amide linker (loading 0.2 mmol/g). Monomers were preactivated (1 min) with HATU (4.9 equiv) and a mixture of DiPEA/lutidine as base (0.2 M DiPEA, 0.3 M lutidine in NMP). The coupling time was 30 min. Ac2O/lutidine/NMP (5:6:89) was used for capping. After complete synthesis, the resin was treated with TFA/TIS/ H2O (90:5:5, 5 mL) for 2 h at ambient temperature, and the product 11 was precipitated from Et2O and used without further purification. LC-MS: 5-35% B, tR ) 12.9min; ES MS: found/calcd: 1439.4/1439.5 (M + 2H)2+, 959.8/960.0 (M + 3H)3+, 720.2/720.3 (M + 4H)4+. Synthesis of PNA-Peptide Conjugates 12a, 12b. Crude PNA 11 (approximately 2 µmol) and crude peptide 7a or 7b (approximately 4 µmol) were dissolved in a degassed buffer solution (400 µL) containing 6 M guanidine‚HCl, 0.1 M tris(2-carboxyethyl)phosphine‚HCl (TCEP), and 0.1 M Na2HPO4. The pH was adjusted to 7.5 using 2 M NaOH. Thiophenol was added (4%, 16 µL), and the solution was shaken overnight. Complete consumption of PNA 11 was verified by LC-MS analysis. 1,4Dithiothreitol was added, and the desired conjugate was purified by RP HPLC. PNA-peptide conjugate 12a: LCMS: 5-55% B, tR ) 12.4 min; ES MS: found/calcd: 2171.2/2171.3 (M + 2H)2+, 1448.0/1447.9 (M + 3H)3+, 1086.6/1086.2 (M + 4H)4+, 869.4/869.1 (M + 5H)5+, 724.6/ 724.4 (M + 6H)6+. MALDI TOF MS: found/calcd: 4341/ 4342 (M + H)+. PNA-peptide conjugate 12b: LC-MS: 5-55% B, tR ) 12.3 min ES MS: found/calcd: 2166.2/ 2165.8 (M + 2H)2+, 1444.2/1444.2 (M + 3H)3+, 1083.6/ 1083.4 (M + 4H)4+. MALDI TOF MS: found/calcd: 4329/ 4333 (M + H)+. ({2-[tert-Butoxycarbonyl-(2-tert-butoxycarbonylaminoethyl)amino]ethylcarbamoyl}methyl)carbamic Acid 9H-Fluoren-9-ylmethyl Ester 14. To a solution of (2-aminoethyl)-(2-tert-butoxycarbonylaminoethyl)carbamic acid tert-butyl ester 13 (2.73 g, 9.0 mmol) in DMF (35 mL) were added N-Fmoc-glycine (3.48 g, 11.7 mmol), BOP (5.17 g, 11.7 mmol), HOBT (1.58 g, 11.7 mmol), and DiPEA (4.65 mL, 27 mmol). After 45 min, TLC analysis (4/1 EtOAc/PE, v/v) indicated complete consumption of the starting material. The mixture was concentrated in vacuo, and the residue was taken up in EtOAc. After being washed with 10% KHSO4, 10% NaHCO3, and finally with brine, the organic layer was

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dried with MgSO4, filtered, and evaporated under reduced pressure. The residue was applied to a silica gel column and eluted with a gradient of EtOAc in PE (50 f 100%), to yield 4.1 g (7.0 mmol, 78%) of the desired product as a white foam. 1H NMR (CDCl3): δ 7.43-7.25 (8H, CH arom. Fmoc), 4.40 (2H, d, CH2 Fmoc), 4.24 (1H CH Fmoc), 3.87 (2H, bd, CH2), 3.39 (4H, bs, 2 × CH2), 3.28 (4H, bs, 2 × CH2), 1.46 (9H, s, tBu Boc), 1.42 (9H, s, tBu Boc). 13C{1H} NMR (CDCl3): δ 169.5 (CdO Fmoc), 154.2, 153.1 (CdO Boc), 127.7, 127.0, 125.0, 119.9 (CH arom Fmoc), 79.2, 80.8 (Cq, tBu), 67.1 (CH2 Fmoc), 48.1, 44.4, 39.2 (CH2), 47.0 (CH Fmoc), 28.3 (tBu Boc). ES MS: 583.4 (M + H)+. (2-tert-Butoxycarbonylaminoethyl)-{2-[2-(2-chloroacetylamino)acetylamino]ethyl}carbamic Acid tertButyl Ester 15. Compound 14 (0.5 mmol, 0.292 g) was dissolved in 20% piperidine in DMF (4 mL). After 15 min the reaction mixture was concentrated and coevaporated with toluene (3×). The residue was dissolved in dioxane (7.5 mL) to which a solution of chloroacetic acid anhydride (0.170 g, 1 mmol) in dioxane (3 mL) was added dropwise. The reaction mixture was stirred for another 1 h and then concentrated to dryness in vacuo. The residue was taken up in EtOAc and washed with 0.1 M NaOH (2×). The organic layer was dried with MgSO4, filtered, and evaporated under reduced pressure. The product was purified by silica gel column chromatography (80 f 100% EtOAc in PE) to yield 0.163 g (0.37 mmol, 75%) of compound 15. 1H NMR (CDCl3): δ 4.09 (2H, s, ClCH2), 3.99 (2H, bs, CH2 Gly), 3.39 (4H, bs, 2 × CH2), 3.29 (4H, bs, 2 × CH2), 1.46 (9H, s, tBu), 1.44 (9H, s, tBu). 13 C{1H} NMR (CDCl3): δ 168.4 (CdO amide), 166.5 (Cd O amide), 158.0, 156.1 (CdO Boc), 80.4, 79.5 (Cq, tBu), 48.0, 47.3, 42.9, 42.3, 39.3, 39.2 (6 × CH2), 28.3 (2 × tBu). ES MS: 437.2 (M + H)+. N-{[2-(2-Aminoethylamino)ethylcarbamoyl]methyl}-2-chloroacetamide 16. To a solution of compound 15 (0.140 g, 0.32 mmol) was added TFA (4 mL), and the reaction mixture was stirred for 15 min. TLC analysis indicated complete disappearance of the starting compound into a baseline product. The mixture was concentrated and coevaporated with toluene under reduced pressure to yield a light yellow oil. The residue was dissolved in water and divided over 10 eppendorfer vessels (∼30 µmol/vessel) and lyophilized. 1H NMR (CD3OD): δ 4.15 (2H, s, ClCH2), 3.93 (2H, s, CH2 Gly), 3.573.51 (2H, m, CH2), 3.39-3.35 (4H, m, 2 × CH2), 3.263.21 (2H, m, CH2). 13C{1H} NMR (CD3OD): δ 174.8, 173.3 (CdO, amide), 50.5, 47.0, 45.4, 44.7, 38.5, 38.1 (6 × CH2). ES MS: 236.9 (M + H)+. Synthesis of PNA-Peptide-DETA Conjugates 1a, 1b. The PNA-peptide conjugates 12a or 12b (approximately 0.2 µmol) were dissolved in an aqueous buffered solution (300 µL) containing 0.05 M tris(2-carboxyethyl)phosphine (TCEP) and 0.1 M Na2HPO4. Compound 16 (30 µmol) was dissolved in the same buffer solution (150 µL) and added. After adjustment of the pH to 8 (2 M NaOH), the solution was shaken overnight. The progress of the reaction was monitored by LC-MS analysis. RP HPLC gave homogeneous 1a or 1b. PNA-peptide-DETA conjugate 1a: LC-MS: 5-55% B, tR ) 11.9min; ES MS: found/calcd: 2272.2/2271.5 (M + 2H)2+, 1514.4/1514.6 (M + 3H)3+, 1136.6/1136.2 (M + 4H)4+, 909.2/909.2 (M + 5H)5+, 758.0/757.8 (M + 6H)6+. MALDI TOF MS: found/ calcd: 4544/4542 (M + H)+, 2272/2271 (M + 2H)2+. PNApeptide-DETA conjugate 1b: LC-MS: 5-50% B, tR ) 12.9 min; ES MS: found/calcd: 2266.2/2265.9 (M + 2H)2+, 1511.0/1510.9 (M + 3H)3+, 1133.4/1133.5 (M + 4H)4+, 906.8/907.0 (M + 5H)5+, 756.4/756.0 (M + 6H)6+. MALDI TOF MS: found/calcd: 4528/4531 (M + H)+.

Petersen et al.

Synthesis of PNA 17. The N-terminal Fmoc group in resin 10 (18 mg, 3.5 µmol) was cleaved with 20% piperidine/NMP followed by thorough washing of the resin with NMP and DCM. The resin was treated with Ac2O/lutidine/NMP (5:6:89, 4 mL) for 2 min and then washed with NMP (3×) and DCM (3×). Treatment with TFA/TIS/H2O (90:5:5, 4 mL) for 2 h at ambient temperature led to the desired product 17, which was precipitated from Et2O and purified by RP-HPLC. LC-MS: 5-35% B, tR ) 14.9min; ES MS: found/calcd: 1460.8/ 1460.5 (M + 2H)2+, 973.8/974.0 (M + 3H)3+. MALDI TOF MS: found/calcd: 2921/2920 (M + H)+. Synthesis of PNA-DETA 18. PNA 17 (approximately 0.5 µmol) was dissolved in a buffered solution (200 µL) containing 0.1 M tris(2-carboxyethyl)phosphine (TCEP) and 0.1 M Na2HPO4. The mixture was shaken for 1 h followed by addition of the DETA derivative 16 (30 µmol) dissolved in the same buffer solution (200 µL). After adjustment of the pH to 8 (2 M NaOH), the solution was shaken overnight and the product was purified by RP HPLC. LC-MS: 5-35% B, tR ) 10.6min; ES MS: found/ calcd: 1517.2/1516.5 (M + 2H)2+, 1011.4/1011.3 (M + 3H)3+. MALDI TOF MS: found/calcd: 3032/3032 (M + H)+. In Vitro RNA Degradation Studies. All buffers were made from highly purified Milli Q water and were sterilized before use. The radioactively labeled RNA 25mer and the conjugate to be tested (1a, 1b, 12b, or 18) were mixed in a Tris‚HCl buffer (10 mM, pH 7) containing NaCl (100 mM) and EDTA (0.1 mM) to give the following concentrations: [RNA] ) 60 nM, [conjugate] ) 2 µM, as determined by A260 units. The samples (70 µL) were incubated at 40 °C for 16 h. After incubation, the RNA was immediately precipitated by addition of 3 M NaOAc (pH 5, 7 µL), EtOH (225 µL), and 10 µg µL-1 tRNA (1 µL). The precipitated RNA was recovered by centrifugation, dried, and then redissolved in 5 µL of H2O and 5 µL of loading buffer. The solutions were heated at 80 °C for 1 min, centrifuged, and analyzed on a 20% denaturing polyacrylamide electrophoresis gel. The gel was exposed to a phosphor screen (Molecular Dynamics), and the intensity of the RNA fragments was quantified by scanning the exposed screen on the Personal Molecular Imager FX System (Bio-Rad) followed by computer analysis with Quantity One software (Bio-Rad). RESULTS AND DISCUSSION

The synthesis of the target compound 1a (Scheme 2) entails two distinct stages. First, PNA-peptide conjugate 12a (Scheme 1) is prepared by native chemical ligation (23, 24) of the separately prepared peptide thioester 7a and the S-tert-butylthio-protected cysteine-functionalized PNA derivative 11. The advantage of using chemical ligation is that the resulting PNA-peptide conjugate 12a contains an exposed sulfhydryl group which can be directly used for the installation (see Scheme 2) of the DETA moiety 16 in the second step, to afford the target compound 1a. Peptide thioester 7a, as depicted in Scheme 1, was prepared according to protocols developed by Pessi et al. (25). Thus, commercially available 4-sulfamylbutyryl safety-catch resin 2 was loaded with N-Fmoc-Gly-OH (5 equiv) using the coupling reagent PyBOP at temperatures ranging from -40 °C to -20 °C. Fmoc deprotection with piperidine was followed by BOP/HOBT-mediated condensation of the resulting free amine with N-Fmoc5-aminopentanoic acid to give resin 4a. Peptide elongation was executed using standard solid-phase Fmoc chemistry employing the amino acid building blocks N-Fmoc-Phe-OH and N-Fmoc-Lys(Boc)-OH. Final elon-

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Scheme 1a

a Reagents and conditions: (i) TMS-CHN , THF/DMF, (ii) HSCH CH CO Et, NaSPh, (iii) 95% TFA/H O, (iv) 20% piperidine, (v) 2 2 2 2 2 TFA/TIS/H2O (90:5:5), (vi) 0.1 M TCEP, 6 M guanidine‚HCl, 0.1 M Na2HPO4, pH 7.5, 4% (v/v) thiophenol.

gation with N-Boc-Lys(Boc)-OH led to the immobilized peptide 5a. Cleavage of the peptide from the resin was achieved by subsequent alkylation of the sulfonamide in 5a using trimethylsilyldiazomethane (25) and substitution of resulting 6a with excess ethyl 3-mercaptopropionate in the presence of a catalytic amount of sodium thiophenolate. Cleavage of all Boc protective groups from the released peptide thioester with 95% TFA/H2O afforded compound 7a, which was sufficiently pure, as gauged by LC-MS analysis, for the crucial native chemical ligation step. The decameric PNA derivative 11, having a sequence complementary to the start codon region of the mRNA of the essential E. coli acpP gene and the N-terminal S-tert-butylthio protected cysteine residue, was assembled (see Scheme 1) on a solid support according to standard PNA synthesis protocols following Fmoc strategy (26). Thus, removal of the Fmoc group on the commercially available Rink Amide-Tentagel resin 8 with piperidine was followed by sequential elongation with Fmoc/benzhydryloxycarbonyl (Bhoc)-protected PNA building blocks 9 using the activating agent HATU (27) and a mixture of the bases DiPEA and lutidine (28). The elongation process was terminated by coupling with N-Fmoc-S-tert-butylthio-protected cysteine to give immobilized PNA oligomer 10. The resin was subjected to TFA, resulting in deblocking of the Bhoc protective

groups and concomitant release from the resin to afford the required major product Cys(StBu)-PNA 11 as judged by LC-MS analysis. It is worth mentioning that the cysteine S-tert-butylthio protecting group is resistant to TFA treatment, thus preventing scavenging of released benzhydryl cation by the thiol group (26). At this stage, conjugate 12a was assembled by native chemical ligation of the crude peptide thioester 7a with the crude PNA derivative 11. Accordingly, Cys(StBu)PNA 11 and an excess of peptide thioester 7a were dissolved in a denaturing phosphate buffer (0.1 M Na2HPO4, pH 7.5, 6 M guanidine‚HCl) containing the watersoluble reducing agent tris3(2-carboxyethyl)phosphine (TCEP, 0.1 M) for the in situ unmasking of the cysteine tert-butylthio protective group. Additionally, an excess of thiophenol (4% (v/v)) was used as a ligation enhancer (Scheme 1). LC-MS analysis, after 16 h incubation at ambient temperature (Figure 3), revealed complete conversion of the PNA derivative 11 and the corresponding tertbutylthio deprotected analogue, into the conjugate 12a. Furthermore, we observed impurities originating from the synthesis of the PNA fragment 11 (peaks a, Figure 3) and byproducts having a mass corresponding to a possible intramolecular lactamization of the excess peptide 7a (peaks b and c, Figure 3). RP-HPLC purification of the cude ligation product afforded homogeneous 12a,

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Figure 3. Top: HPLC chromatogram of the native chemical ligation mixture between thioester 7a and Cys(S-tBu)-PNA 11 after overnight incubation. The product 12a is seen as a distinct peak together with byproducts from the PNA synthesis (peak a) and presumably lactamized peptide thioester 7a (peaks b, c). Bottom: ES MS and MALDI-TOF MS (insert) of the purified product 12a. Table 1. MALDI TOF MS Values for the Conjugates compound

spacer

18 12a 12b 1a 1b

(CH2)4C(O)NHCH2 (CH2CH2O)2CH2 (CH2)4C(O)NHCH2 (CH2CH2O)2CH2

MALDI (found) MALDI (calcd) (M + H)+ (M + H)+ 3032 4341 4329 4544 4528

3032 4342 4331 4542 4531

the identity of which was ascertained by LC-MS analysis and MALDI-TOF mass spectrometry (Figure 3, Table 1).

Having the PNA-peptide conjugate 12a in hand, we focused attention on the construction of the DETA moiety 16 required for the intended condensation with the conjugate 12a (Scheme 2). The synthesis of chloride 16 commenced with a BOP/HOBT-mediated coupling of the known di-Boc-protected diethylenetriamine 13 (9) with N-Fmoc-glycine to afford the Fmoc/Boc-protected compound 14 in 78% yield. Subsequent removal of the Fmoc group in 14 with piperidine and treatment of the resulting free amine with chloroacetic anhydride led to conversion of 14 into the chloride 15 in 75% yield. Removal of the Boc protective groups with TFA gave the target compound 16 as the TFA salt in a near quantitative yield. Condensation of PNA-peptide conjugate 12a with an excess of DETA derivative 16 (see Scheme 2) was executed under buffered conditions (0.1 M Na2HPO4) at pH 8. The buffer contained an additional amount of TCEP to prevent dimerization of 12a by disulfide formation. LC-MS analysis (Figure 4), after 16 h incubation at room temperature, showed complete consumption of all starting material 12a and formation of the product 1a. HPLC purification afforded homogeneous 1a as judged by LC-MS and MALDI-TOF analysis (Figure 4, Table 1). With the objective to study the influence of the type of spacer connecting the PNA and the peptide units, compound 1b containing an ethylene glycol linker was prepared. To this end, the safety-catch resin 2 (see Scheme 1) was loaded with the Fmoc-protected ethylene glycol spacer 3 to give immobilized 4b which was processed further, as described for 7a, to give peptide thioester 7b. Compounds 12b and 1b were assembled starting from 7b according to similar procedures as described earlier for the synthesis of 12a and 1a (Schemes 1 and 2, Table 1). Additionally, compound 18, lacking the (KFF)3K peptide, was synthesized to serve as a positive control (Scheme 3). Thus, the Fmoc group on immobilized PNA 10 was removed with piperidine followed by acetylation of the resulting free amine with Ac2O. The resin was then treated with TFA/TIS/H2O (90:5:5), leading to simultaneous cleavage of the Bhoc protection groups and release of the desired PNA derivative 17, which was purified by RP-HPLC. Finally, 17 and chloride 16 were condensed

Scheme 2a

a Reagents and conditions: (i) Fmoc-Gly-OH, BOP, HOBT, DiPEA; 78%, (ii) 20% piperidine/DMF, (iii) (ClCH CO) O, 75% (two 2 2 steps), (iv) 50% TFA/DCM, (v) 16, buffer (0.05 M TCEP, 0.1 M Na2HPO4, pH 8).

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to a 20% denaturing polyacrylamide electrophoresis gel, and quantified. A representative gel is depicted in Figure 5 (left) whereas the quantification is shown as the average result of three separate experiments (Figure 5, right). Gratifyingly, incubation of the RNA with the conjugates 1a and 1b as well as the PNA-DETA 18, lacking the (KFF)3K peptide sequence, led to the formation of two major RNA degradation products stemming from cleavage at the CA and the UA sites marked in Figure 5. The cleavage sites were determined by comparison of the mobility of the bands with those of a T1 digest of the RNA (Figure 5) and a basic hydrolysis ladder (not shown). The intensity of the fragment spots revealed that incubation of the RNA with PNA-DETA 18 resulted in significant cleavage (20%) of the RNA compared to the negative controls (i.e. PNA-peptide conjugate 12b and RNA without any conjugate added), which only showed background cleavage of approximately 3.5%. An unexpected difference between the hydrolysis efficiency of conjugates 1a and 1b was observed. Conjugate 1a showed an even improved cleavage efficiency (30%) compared to the positive control 18 (20%), whereas conjugate 1b cleaved only 10% of the target RNA. This difference in hydrolysis ability renders conjugate 1a three times more potent than conjugate 1b. Figure 4. Top: HPLC chromatogram of the reaction mixture after overnight reaction of compounds 12a and 16. The product 1a is seen as a distinct peak. Bottom: MALDI-TOF MS of the pure product 1a.

as described earlier for the conversion of 12a into 1a to afford the desired product 18, which could be readily purified by HPLC. Having the requisite compounds in hand, we turned our attention to the in vitro degrading abilities of both PNA-peptide-DETA conjugates 1a,b, the PNA-DETA 18 and the PNA-peptide conjugate 12b as a negative control. To this end, the compounds were incubated with the 25-mer 32P-labeled RNA having a sequence resembling part of the E. coli acpP mRNA. After 16 h at 40 °C and pH 7, the RNA fragments were precipitated, applied

CONCLUSIONS

We have reported a straightforward route to the synthesis of unprecedented, highly functionalized PNA derivatives 1a and 1b based on native chemical ligation between the membrane permeable peptide thioesters 7a or 7b and the cysteine-containing antisense PNA derivative 11. The exposed cysteine side-chain residue in the resulting PNA-peptide conjugates was exploited for the installation through a thioether bond of a DETA-based RNA cleaving agent. In addition, we have demonstrated that the two trifunctional conjugates 1a and 1b, differing only in the type of spacer connecting the PNA and the peptide, both hydrolyzed the complementary 25-mer RNA at two major cleavage sites under physiological conditions. The re-

Scheme 3a

a Reagents and conditions: (i) 20% Piperidine/NMP, (ii) Ac O/Lutidine/NMP (5:6:89), (iii) TFA/TIS/H O (90:5:5), (iv) 16, 0.1 M 2 2 Na2HPO4, 0.1 M TCEP, pH 8.

Figure 5. Top: Major cleavage sites of the target 25-mer RNA. The PNA binding region is marked underlined. Left: Autoradiograph for the cleavage of the 5′-labeled RNA 25-mer, after 16 h at pH 7 and 40 °C of compounds 1a, 12b, 18, 1b, RNA (no additives), T1 (T1 digestion); [RNA]0 ) 60 nM, [Conjugates] ) 2 µM. Right: Average cleavage efficiency (in % degradation of mRNA) over three experiments.

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markable difference in the cleaving efficiency of the two conjugates (1a is three times more potent than 1b) indicate that the choice of spacer may have a great influence on the properties of the conjugate (29). On the other hand, the presence of the peptide does not seem to have a decisive effect on the cleavage capabilities. Although the RNA hydrolysis activity of conjugates 1a,b is somewhat lower than the earlier by us reported PNADETA conjugate (9), it is still considerably improved compared to the DNA-DETA conjugate of Komiyama and co-workers (30). Other amine-based RNA-cleaving moieties, e.g. cyclen which efficiently hydrolyzed the HIV-1 TAR-RNA (31), can be easily implemented in our synthetic strategy and may further enhance the cleavage activity. Currently, the bacterial uptake of the conjugates 1a,b and their effects on cell proliferation is in progress, and these results will be reported in due course. ACKNOWLEDGMENT

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