Synthesis and Binding Properties of Oligonucleotides Carrying

Bioconjugate Chem. 1999, 10, 1005−1012

1005

Synthesis and Binding Properties of Oligonucleotides Carrying Nuclear Localization Sequences Beatriz Garcı´a de la Torre, Fernando Albericio,† Ester Saison-Behmoaras,‡ Angela Bachi,§ and Ramon Eritja*,§ Centro de Investigacio´n y Desarrollo, C.S.I.C. Jordi Girona 18-26, E-08034 Barcelona, Spain, Department of Organic Chemistry, Universitat de Barcelona, Martı´ i Franque`s 1-11, E-08028 Barcelona, Spain, Museum National d’Histoire Naturelle, 43 rue Cuvier Paris Cedex, France, and European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany. Received April 20, 1999; Revised Manuscript Received August 5, 1999

The synthesis of oligonucleotides carrying nuclear localization peptide sequences is described using two strategies: first, oligonucleotides carrying a thiol group at the 5′ end were reacted with maleimido peptides; second, peptide and oligonucleotide were prepared stepwise on the same support, yielding oligonucleotide-3′-peptide conjugates. This second approach was thoroughly studied. Using amino acids and small peptides as model compounds, some side reactions were analyzed, detected, and minimized. Oligonucleotides complementary to Ha-ras gene and carrying nuclear localization peptides at the 3′ and 5′ ends were prepared. Melting temperature studies showed that duplexes containing nuclear localization peptides were more stable than duplexes with unmodified oligonucleotides. Moreover, oligonucleotide-peptide conjugates maintain a good mismatch discrimination when they bind to their target RNA.

INTRODUCTION

A large interest in oligonucleotide-peptide conjugates has arised. These conjugates are chimeric molecules constituted by oligonucleotides connected to peptide sequences and they are produced to add some of the biological and/or biophysical properties of peptides to synthetic oligonucleotides. They were first used to introduce multiple nonradioactive labels (Haralambidis et al., 1987, 1990) and as artificial sequence-specific nucleases (Corey and Schultz, 1987). Later, they were used as antisense oligonucleotides (Crooke and LeBleu, 1994). Conjugation of oligonucleotides to polylysine (Lamaitre et al., 1987), basic (Vive`s and LeBleu, 1997), hydrophobic (Juby et al., 1991), fusogenic (Bongartz et al., 1994; Soukchareun et al., 1995), and signal peptides (Arar et al., 1993; Ede et al., 1994; Reed et al., 1995; Zanta et al., 1999) and penetratin (Allinquant et al., 1995) has been performed in order to increase the cellular uptake or the intracellular delivery of antisense oligonucleotides. Moreover, binding oligonucleotides to certain peptide sequences improves the binding to complementary DNA (Tung et al., 1996), the hybridization speed (Corey, 1995), the binding to RNA (Tung et al., 1995) and proteins (Lin et al., 1995), and the resistance to nucleases (Robles et al., 1997). Finally, peptide sequences have been used for encoded combinatorial libraries (Tetzlaff et al., 1998). Two strategies can be followed to synthesize oligonucleotide-peptide hybrids. In the postsynthetic conjugation approach, the two moieties are independently * To whom correspondence should be addressed, Centro de Investigacio´n y Desarrollo. Phone: 34-93-4006145. Fax: 34-932045904. E-mail: [email protected]. † Universitat de Barcelona. ‡ Museum National d’Histoire Naturelle. § European Molecular Biology Laboratory.

prepared and thiols and maleimido groups are especially incorporated to link both molecules (Eritja et al., 1991a; Arar et al., 1993; Ede et al., 1994). In the stepwise approach, oligonucleotide-peptide conjugates are prepared by stepwise addition of amino acids and nucleobases in solid phase on the same solid support (de la Torre et al., 1994; Bergmann and Bannwarth, 1995; Soukchareun et al., 1995; Robles et al., 1997). In this case, the problem to be solved is the incompatibility of the standard schemes of protection. For example, at the end of the solid-phase peptide synthesis, a treatment with acid is usually required and can provoke partial depurination of DNA. In the synthesis of oligonucleotide 3′peptides, this effect could be prevented using Bocprotected amino acids with Fm or Fmoc groups for the protection of side chains, a base-labile linker, and standard phosphoramidites (de la Torre et al., 1994; Bergmann and Bannwarth, 1995; Soukchareun et al., 1995; Robles et al., 1997). Moreover, protected peptide fragments (Peyrottes et al., 1998) and protected oligonucleotide (McMinn and Greenberg, 1998, 1999) fragments have been used. In the present communication, we describe the synthesis of oligonucleotides carrying nuclear localization peptide sequences following two strategies and the binding properties of these conjugates. Our target oligonucleotide sequence is the dodecamer R5, complementary to a mutated Ha-ras oncogene (Duroux et al., 1995). The specific binding to mutated mRNA and not to the wildtype sequence accounts for the antiproliferative activity of R5 (Duroux et al., 1995). Thus, the discriminatory properties of the conjugates were analyzed. EXPERIMENTAL SECTION

Peptide syntheses. The maleimido peptide carrying the SV40 nuclear localization sequence (maleimidoben-

10.1021/bc990046l CCC: $18.00 © 1999 American Chemical Society Published on Web 09/28/1999

1006 Bioconjugate Chem., Vol. 10, No. 6, 1999

zoyl-Ala-Ala-Pro-Lys-Lys-Lys-Arg-Lys-Val-CONH2) was prepared on polystyrene supports using Fmoc amino acids, as described in Eritja et al. (1991a). Other peptide sequences were synthesized in a homemade manual synthesizer using PEG-PS functionalized with amino groups (PerSeptive Biosystems) as starting solid support. The NPE1 handle 2,4,5-trichlorophenyl 4-hydroxyethyl3-nitrobenzoate [3-fold excess, Eritja et al. (1991b)] was anchored in excellent yields to the amino-PEG-PS resin when HOBt was added (3-fold excess) using DMF as solvent. The Fm handle (Rabanal et al., 1992) was coupled to the amino-PEG-PS resin following Rabanal et al. (1995). The C-terminal-protected Boc amino acid derivative (5 equiv) was reacted with the hydroxyethyl group of the NPE and Fm resins with DCC (5 equiv), DMAP (0.5 equiv) and DMF as solvent. After 2 h of coupling, unreacted hydroxyl groups were acetylated with 10% acetic anhydride in pyridine. The aminohexyl handle MMT-NH-hexyl hemisuccinate (Will et al., 1995) was coupled to the amino-PEG-PS following Gupta et al. (1995). The elongation of peptide chains was performed in DMF using 5-fold excess of amino acid Boc-protected and 5-fold excess of PyBOP for 1 h. The peptide sequences were (A) Gln-Ala-[Lys(Fmoc)]4-Leu-Asp(Fm)-Lys(Fmoc) and (B) Ala-[Lys(Fmoc)]4-Leu-Asp(Fm)-Lys(Fmoc). For the incorporation of Boc-Gln-OH, DIPCI and HOBt (5fold excess) were used to avoid the formation of the nitrile. Synthesis and Solid-Phase Coupling of the Spacer. p-Nitrophenyl 6-(4,4′-dimethoxytrityloxy)hexanoate was prepared as has been described for the hydroxybutyrate derivative (Haralambidis et al., 1990). The Boc-peptide supports described above were treated with 50% TFA in DCM to remove the Boc protecting group of the last amino acid and were neutralized (5% DIEA in DCM). The resulting resins were swelled in DMF for 10 min, and then 5-fold excess of spacer dissolved in DMF was added. After 2 h, the support was washed with DMF five times, twice with DCM and once with MeOH. Finally, it was dried and stored for further use in oligonucleotide synthesis. Small aliquots of the supports carrying the peptide and the spacer were detritylated and treated with concentrated ammonia at 55 °C overnight. The resulting products were purified by reversed-phase HPLC, and the major products were collected and analyzed by MALDI-TOF mass spectrometry: HO-spacer-AK4LDK-CONH-(CH2)6-OH, MS (MALDI-TOF) [M] ) 1171.6 g/mol (M) 1171.4 g/mol calculated for C55H106N14O13); HO-spacer-QAK4LDK-CONH-(CH2)6OH, MS (MALDI-TOF) [M] ) 1299.7 g/mol (M) 1299.5 g/mol calculated for C60H114N16O15). Synthesis and Purification of OligonucleotidePeptide Conjugates. The conjugate carrying the SV40 nuclear localization sequence was prepared by conjugation of maleimido-benzoyl-Ala-Ala-Pro-Lys-LysLys-Arg-Lys-Val-CONH2 with 5′-thiolhexyl phosphate1 Abbreviations: AmHex, 6-aminohexylsuccinyl; Boc, t-butoxycarbonyl; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; DCC, N,N-dicyclohexylcarbodiimide; DCM, dichloromethane; DIEA, N,N-diisopropylethylamine; DIPCI, N,N′-diisopropylcarbodiimide; DMAP, N,N-(dimethylamino)pyridine DMF, N,N-dimethylformamide; DMT, dimethoxytrityl; DTT, dithiothreitol; Fm, fluorenylmethyl; Fmoc, fluorenylmethoxycarbonyl; HOBt, 1-hydroxybenzotriazol; MBS, N-succinimidyl 3-maleimido benzoate; MeOH, methanol; MMT, monomethoxytrityl; NPE, 2-(4-nitrophenyl)ethyl; PEG-PS, poly(ethylene glycol)-polystyrene; PyBOP, (benzotriazol-1-yloxy)trispyrrolidinophosphonium hexafluorophosphate, TEAA, triethylammonium acetate; TFA, trifluoroacetic acid.

de la Torre et al.

CACCGACGGCGC (Eritja et al., 1991a). The thiol group at the 5′ end of the oligonucleotide was incorporated by addition of the available (Glen Research) phosphoramidite of DMT-protected bis-(6-hydroxyhexyl) disulfide. The oligonucleotide was deprotected with a solution (1 mL) of 0.1 M DTT in concentrated ammonia at 55 °C overnight as described in Gottschling et al. (1998). The desired product was characterized by amino acid analysis and enzymatic digestion. VKRKKKPAA-maleimidobenzoyl-hexylthio-CACCGACGGCGC. Amino acid analysis: Arg 1.22 (1), Ala 1.81 (2), Pro 1.04 (1), Val 1.00 (1), Lys 3.89 (4). Enzymatic analysis: dC 5.6 (6), dG 4.0 (4), dA 2.3 (2). Yield (1 µmol scale synthesis): 14 OD units at 260 nm. The conjugates with the nucleoplasmine nuclear localization sequence were synthesized following a stepwise method. The sequences were (I) 5′ T5-spacer-QAK4LDK, (II) 5′ T5-spacer-AK4LDK, (III) 5′ CAC CGA CGG CGCspacer-QAK4LDK, and (IV) 5′ CAC CGA CGG CGCspacer-AK4LDK. Oligonucleotides were synthesized by a DNA synthesizer (Applied Biosystems 392) using standard 2-cyanoethyl phosphoramidites on 1 µmol scale. The phosphoramidites were dissolved in dry DCM (0.1 M), and a modified cycle was used. Coupling time was increased to 5 min, capping and oxidation times to 1 min, and detritylation time to 2 min (4 × 30 s). The last DMTprotecting group was not removed. The average coupling yield was >98%/step. The solid supports containing the oligonucleotide-peptide conjugates were washed with acetonitrile, treated with a 0.5 M DBU solution in acetonitrile for 5 min, washed with acetonitrile and dried. The resulting supports were finally treated with concentrated aqueous ammonia-dioxane 10:1 overnight at 55 °C. After filtration of the solid supports, the solutions were evaporated to dryness, the residues dissolved in water, and the hybrids purified using a standard twostep HPLC purification. HPLC conditions were as follows: column, Nucleosil 120-10 C18 (250 × 4 mm); 20 min linear gradient from 15 to 80% B and 5 min 80% B (DMT on conditions); 20 min linear gradient from 0 to 60% B (DMT off conditions); flow rate 1 mL/min; mobile phase A was 20 mM TEAA in water and B (1:1) acetonitrile: water. In the first step, truncated sequences were separated from the product containing DMT, and in the second step the desired conjugates were isolated after removal of the DMT with 80% acetic acid. Characterization of the conjugates was made by amino acid analysis after 6 N HCl hydrolysis and nucleoside analysis after enzyme digestion. Moreover, the purified products were analyzed by electrospray and MALDI-TOF mass spectrometry: T5-spacer-AK4LDK-CONH-(CH2)6-OH, MS (MALDI-TOF) [M] ) 2692.3 g/mol (M) 2692.2 g/mol calculated for C105H171N24O48P5); T5-spacer-QAK4LDKCONH-(CH2)6-OH, MS (MALDI-TOF) [M] ) 2820.3 g/mol (M ) 2820.3 g/mol calculated for C110H179N26O50P5); R5 dodecamer-spacer-AK4LDK-CONH-(CH2)6-OH, MS (electrospray) [M] ) 4848.5 g/mol (M) 4851.3 g/mol calculated for C169H252N62O83P12); R5 dodecamer-spacer-QAK4LDKCONH-(CH2)6-OH, MS (electrospray) [M] ) 4979.0 g/mol (M) 4979.4 g/mol calculated for C174H260N64O85P12). Amino acid analysis of the R5 dodecamer-spacer-QAK4LDK-CONH-(CH2)6-OH: Leu 1.00 (1), Asp 1.07 (1), Glu 1.23 (1), Lys 5.48 (5), Ala 0.86 (1). Nucleoside composition for R5 dodecamer-spacer-QAK4LDK-CONH-(CH2)6OH: dC 5.5 (6), dG 4.0 (4), dA 2.2 (2). The average yield of the R5 dodecamer conjugates synthesis (1 µmol scale synthesis) was 12 OD units at 260 nm. Mass Spectrometry Analysis. Mass measurements were performed on a Bruker REFLEX MALDI time-of-

Oligonucleotides with Nuclear Import Sequences

flight mass spectrometer (Bruker-Franzen, Bremen, Germany) using R-cyano-4-hydroxy-cinnamic acid as matrix and the fast evaporation technique for sample preparation (Jensen et al., 1997). Alternatively, the samples were dissolved in 50% acetonitrile and analyzed by nano electrospray on a triple quadrupole mass spectrometer on negative mode (API III, PE-Sciex, Ontario, Canada). Evaluation of the Optical Purity of the Amino Acids. Boc amino acids were anchored on NPE-PEG-PS as described above. After removal of the Boc-protecting group, resins were treated with concentrated aqueous ammonia-dioxane 10:1 and left overnight at 50 °C. After filtration of the solid supports, the solutions were evaporated to dryness. A total of 0.2 mL of the residue was dissolved in 0.1 M NaHCO3 and added to 0.2 mL of 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (FDAA) 10 mM in acetone freshly prepared. The mixture was kept for 1 h at 40 °C with frequent shaking. After cooling, 0.2 mL of 0.2 N HCl was added, and the resulting solution was analyzed by HPLC on a Nucleosil 120-10 C18 column (250 × 4 mm) using a 30 min gradient from 10 to100% B at a flow rate of 1 mL/min, detection at λ ) 340 nm. The mobile phase A was 20 mM TEAA in water and B acetonitrile:water (1:1). Standards were prepared following the same procedure using commercially available unprotected amino acids in L- and D-form. The amino acids studied were Phe, Leu, Val, and the dipeptide GlyVal. Carboxylic Acid versus Carboxamide Formation during Treatment of NPE and Fm Solid Supports with Ammonia. To study the formation of carboxylic acid and/or amide at the C-terminal position of peptides when NPE and Fm supports were used, model pentapeptides (H-Tyr-Gly-Gly-Phe-Leu-OH and H-Tyr-Gly-GlyPhe-Leu-NH2) were prepared following standard Fmoc methodology. The same sequence (H-Phe-Gly-Gly-PheLeu) was assembled on PEG-PS supports carrying the NPE and Fm linkers using Boc amino acids as described above. Aliquots of the peptide supports were treated with several solutions: (a) concentrated ammonia at 55 °C, (b) 20% piperidine in DMF at room temperature, and (c) 0.1 M tetrabutylammonium fluoride in tetrahydrofuran at room temperature. At different times, the supports were filtered out and the resulting solutions were evaporated to dryness. The residues were dissolved in water and analyzed by HPLC using the same conditions as for the racemization studies. The retention time of H-TyrGly-Gly-Phe-Leu-OH was 20 min and the peptide amide eluted at 24 min. Stability of the Side Chain of Trifunctional Amino Acids during Oligonucleotide Synthesis Conditions. Dipeptides Boc-Gln-Phe, Boc-Asp(Fm)-Phe, and Boc-Lys(Fmoc)-Phe were synthesized on PEG-PS supports with the AmHex linker as described above. Aliquots of these supports were treated for 1 h at room temperature with the following solutions: (a) a 1:1 mixture of the oligonucleotide synthesis capping solutions [Cap A, acetic anhydride/lutidine/tetrahydrofuran (1:1:8) and Cap B: 0.1M N-methylimidazole in tetrahydrofuran]; (b) 0.05 M iodine solution in tetrahydrofuran, H2O/pyridine (7: 2:1); and (c) a 1:1 mixture of 0.1 M solution of A phosphoramidite in acetonitrile and 0.4 M tetrazol in acetonitrile. Thereafter, the supports were washed with acetonitrile, treated with a 0.5 M DBU solution in acetonitrile for 5 min, washed again with acetonitrile, and dried. The resulting supports were finally treated with concentrated ammonia at 55 °C overnight. The supernatants were concentrated to dryness and the

Bioconjugate Chem., Vol. 10, No. 6, 1999 1007

residues were analyzed by HPLC and mass spectrometry. The desired dipeptide was obtained and no side products were found. Boc-Gln-Phe-CONH-(CH2)6-OH MS (electrospray) [M + H] ) 493.4 g/mol (M ) 492.6 g/mol calculated for C25H40N4O6); Boc-Asp-Phe-CONH-(CH2)6-OH MS (electrospray) [M] ) 479.3 g/mol (M ) 479.5 g/mol calculated for C24H37N3O7); Boc-Lys-Phe-CONH-(CH2)6-OH MS (electrospray) [M+H] ) 493.5 g/mol (M) 492.6 g/mol calculated for C26H44N4O5). Melting Studies. Melting experiments were carried out by mixing equimolar amounts of two dodecamer strands dissolved in a solution containing 0.15 M NaCl and 0.05 N Tris-HCl buffer, pH 7.5. Duplexes were annealed by slow cooling from 80 to 4 °C. UV absorption spectra and melting curves (absorbance vs temperature) were recorded in 1 cm path-length cells using a Varian Cary 13 spectrophotometer with a temperature controller and a programmed increase of 0.5 °C/min. Melts were run on duplex concentrations of 4 µΜ at 260 nm. Binding of Oligonucleotide-Peptide Conjugates to Ha-ras RNA (Gel Shift Assay). 27-mer RNA (4 nM) carrying the sequence complementary to the R5 dodecamer (5′ GUG GUG GGC GCC GUC GGU GUG GGC AAG 3′) and wild-type RNA sequence (5′ GUG GUG GGC GCC GGC GGU GUG GGC AAG 3′) were radioactively labeled and incubated with unlabeled oligonucleotides in a buffer containing 40 mM Tris (pH 7.9) and 100 mM KCl. After preincubation for 15 min at 37 °C, glycerol loading buffer (80% glycerol, 0.1% xylene cyanol, 0.1% bromophenol blue) was added, and samples were electrophoresed on a 15% native polyacrylamide gel at 37 °C, using 0.05 M Tris-borate (pH 9). Hybridization to the 5′-end-labeled 27 mer (32P) RNA was analyzed by quantitation of gel shifts using particle detection (Phosphorimager). The fraction of duplex is the ratio of particle counts of the double-stranded oligonucleotide-RNA complex (arrow) referred to the total (single and double stranded). RESULTS

Synthesis of Oligonucleotide-Peptide Conjugates Using the Postsynthetic Conjugation Approach. Two types of oligonucleotide-peptide conjugates were prepared. The conjugate carrying the SV40 nuclear localization sequence was synthesized by conjugation of maleimido-benzoyl-AAPKKKRKV-CONH2 with 5′-thiolhexyl phosphate-CACCGACGGCGC, as described in Eritja et al. (1991a) (see Figure 1). The thiol group at the 5′ end of the oligonucleotide was incorporated by addition of a phosphoramidite derivative of bis-(6-hydroxyhexyl) disulfide. The maleimido peptide carrying the SV40 nuclear localization sequence (maleimidobenzoylAAPKKKRKV-CONH2) was prepared on polystyrene supports using Fmoc amino acids (Eritja et al., 1991a). Preliminary Studies on the Synthesis of Oligonucleotide-Peptide Conjugates Using the Stepwise Approach. Oligonucleotide-peptide conjugates carrying the nucleoplasmine nuclear localization sequence (QAKKKKLDK) were synthesized by the stepwise approach. Polyethylenglycol-polystyrene (PEG-PS) was selected as solid support since it gave the best results for the coupling of both amino acids and nucleoside phosphoramidites (de la Torre et al., 1994). Figure 2 shows the pathway followed to synthesize oligonucleotide-peptide conjugates on the same support (de la Torre et al., 1994). To avoid the use of strong acids in the presence of the oligonucleotide, the peptide part was first synthesized using the acid-labile Boc group to protect the R-amino function. The protective groups of the side

1008 Bioconjugate Chem., Vol. 10, No. 6, 1999

de la Torre et al.

Figure 1. Synthesis of oligonucleotide-peptide conjugates following the postsynthetic conjugation approach.

Figure 4. Ammonolysis of 2-substituted ethyl esters: Nucleophillic attack versus β-elimination mechanisms yielding peptides with carboxylic acid or carboxamide group at the C-terminus.

Figure 2. Synthesis of oligonucleotide-peptide conjugates following the stepwise conjugation approach.

Figure 3. Base labile linker molecules used to connect the first amino acid to the solid support. P, polymer [poly(ethylene glycol)-polystyrene]; NPE, 2-(2-nitrophenyl)ethyl linker; Fm, fluorenylmethyl linker; AmHex, 6-aminohexylsuccinyl linker.

chain of the amino acids (Fmoc for Lys and Fm for Asp) and the linker of the first amino acid to the support were base labile so that they would be removed at the same time as the protective groups of the nucleobases. To that end, three base-labile linkers (see Figure 3) were examined: the 2-(2-nitrophenyl)ethyl (NPE, Eritja et al., 1991b), the fluorenylmethyl [Fm, Rabanal et al. (1992, 1995)], and the 6-aminohexylsuccinyl [AmHex, Will et al. (1995)]. The linkage between the amino acid and the NPE and the Fm linkers is excised by strong bases such

as piperidine or DBU following a β-elimination reaction which releases the free carboxylic function of the amino acid or peptide. In the AmHex linker, the excised bond is a succinyl ester which yields the amino acid or peptide with an aminohexylamide group at the C-terminal. Prior to the synthesis of oligonucleotide-peptide conjugates, racemization of the C-terminal amino acid was studied. Racemization Studies on the C-Terminal Amino Acid in Base Deprotection Conditions. The synthetic protocol implies the use of basic conditions in the removal of the protective groups. Oligonucleotides are usually treated with concentrated aqueous ammonia for 16 h at 55 °C. To check the stability of the peptide part in such conditions, several amino acids and a dipeptide were attached to the support and analyzed after ammonia treatment. The possible racemization of the C-terminal amino acid due to the anchoring method or the cleavage conditions was carefully studied. Boc-valine, Boc-leucine, and Boc-phenylalanine were incorporated to the NPE support (Albericio et al., 1991). After removal of the Boc group, the amino acyl-NPE supports were treated with concentrated ammonia. The resulting amino acids were reacted with Marfey’s reagent (Adamson et al., 1992) to obtain the diastereoisomers, which were analyzed by HPLC. The free L-amino acids were obtained and no racemization was found (detection limit 0.1%), in agreement with Bray et al., who showed that there was no racemization when deblocking peptides with ammonia vapors (Bray et al., 1994). Moreover, the dipeptide Gly-L-Val was prepared on the NPE-resin, the support was treated with concentrated ammonia, and the product was reacted with Marfey’s reagent. Likewise, no racemization was found, but a second product, thought to be, the dipeptideamide (Gly-L-Val-NH2), was detected. This side product coeluted with a synthetic sample of the peptide amide. In addition to the expected β-elimination reaction of the ester, there was direct nucleophillic attack of ammonia to the ester, which resulted in the formation of the amide (Figure 4). Carboxylic Acid vs Carboxamide Formation during Deprotection. The sequence H-Tyr-Gly-Gly-Phe-Leu (Leuenkephaline) was selected in order to study this reaction and, therefore, synthesized on NPE and Fm handles. Independently, H-Tyr-Gly-Gly-Phe-Leu-OH and H-Tyr-

Oligonucleotides with Nuclear Import Sequences

Gly-Gly-Phe-Leu-NH2, used as controls, were synthesized following conventional solid-phase peptide protocols. Aliquots of the pentapeptide-NPE and Fm supports were treated with concentrated ammonia, 20% piperidine in DMF, and 0.1 M tetrabutylammonium fluoride. The resulting solutions were analyzed by HPLC. When concentrated ammonia at 55 °C was used, the ratio carboxylic acid:carboxamide was 50:50 on the NPE support and 75:25 on the Fm support. When 20% piperidine in DMF and 0.1 M tetrabutylammonium fluoride in tetrahydrofuran were used, only the carboxylic acid was found. In the deprotection of larger molecules, such as oligonucleotide-peptide conjugates, treatments with piperidine and, especially, fluoride were less efficient than treatment with ammonia. These results are not consistent with those described by Robles et al. (1997), who found that in fluoride solutions, oligonucleotide-peptide conjugates were released. Here, poly(ethylene glycol)polystyrene was used as solid support, whereas Robles et al. (1997) used polystyrene. Therefore, poly(ethylene glycol) may inhibit the fluoride-catalyzed reaction. As poly(ethylene glycol) improves the coupling of phosphoramidites (de la Torre et al., 1994), we focused on the linker AmHex (Figure 3), which has been used in the synthesis of peptide nucleic acid chimeras on poly(ethylene glycol)polystyrene supports (Will et al., 1995). The succinate bond is easily cleaved by concentrated ammonia, yielding the peptide with the N-(6-hydroxyhexyl)amide group at the C-terminus. This linker also allows the removal of the Fm (Asp), Fmoc (Lys), and cyanoethyl (phosphate) groups with a nonnucleophillic base (such as DBU) before ammonia deprotection. At this stage, a DBU treatment will minimize or prevent the formation of Asn from Asp(Fm) and the alkylation of lysine residues by the acrylonitrile formed during the phosphate deprotection described by Tetzlaff (Tetzlaff et al., 1998). Stability of the Side Chains of Trifunctional Amino Acids in Oligonucleotide Synthesis Conditions. The stability of the peptide moiety in oligonucleotide synthesis conditions was also studied. Hydrophobic amino acids are stable in oligonucleotide synthesis conditions (Soukchareun et al., 1995; Robles et al., 1997; Tetzlaff et al., 1998), but to our knowledge, the behavior of trifunctional amino acids has not been thoroughly studied. To test the stability of the trifunctional amino acids belonging to the nucleoplasmine sequence, Boc-[Gln, Asp(Fm) and Lys(Fmoc)]-Phe dipeptides were prepared on PEG-PS supports with the AmHex linker. The Boc-dipeptide supports were treated with oligonucleotide synthesis reagents such as capping (acetic anhydride, N-methylimidazol), oxidation (iodine), and coupling (phosphoramidite and tetrazol) solutions for 1 h at room temperature. After DBU treatment and ammonia deprotection, the products were analyzed by HPLC and mass spectrometry. Only the expected dipeptides were found. To determine whether activated phosphoramidites can react with peptide bonds and/or with the primary amide group of Gln., aliquots treated with phosphoramidite and tetrazole were also treated with iodine and 2% trichloroacetic acid in DCM between the phosphoramidite treatment and deprotection. A small amount of phosphoramidite may react with peptides, as, after treatment with 2% trichloroacetic acid, a red solution was observed. The amount of DMT released from the peptide supports was 12, 15, and 14% for Boc-Lys(Fmoc)-Phe, Boc-Asp(Fm)-Phe, and Boc-GlnPhe, respectively. No extra peak was observed on HPLC or MS analysis. Thus, the small amount of phosphoramidite addition on the peptide supports does not yield detectable side products.

Bioconjugate Chem., Vol. 10, No. 6, 1999 1009

Synthesis of the Oligonucleotide-Peptide Conjugates Using the Stepwise Approach. The following molecules were prepared: (I) 5′ TTTTT-spacer-Gln-AlaLys-Lys-Lys-Lys-Leu-Asp-Lys, (II) 5′ TTTTT-spacer-AlaLys-Lys-Lys-Lys-Leu-Asp-Lys, (III) 5′ CACCGACGGCGCspacer-Gln-Ala-Lys-Lys-Lys-Lys-Leu-Asp-Lys, and (IV) 5′ CACCGACGGCGC-spacer-Ala-Lys-Lys-Lys-Lys-Leu-AspLys. The pentanucleotide sequence was used to find the optimal coupling conditions. The dodecamer sequence is complementary to the mutated Ha-ras oncogene. The peptide sequence is the nuclear localization sequence found in nucleoplasmine. In one set of experiments (II and IV), the last glutamine was not included so that the influence of unprotected glutamine in the purity of the oligonucleotide-peptide conjugates could be studied. Although our results on model dipeptides (see above) indicated that there were no side-reactions on glutamine, the formation of N-acyl phosphoramidates has been described as side reaction of glutamine and chlorophosphines (Robles et al., 1995). First, peptide sequences were prepared on the PEG-PS support containing the aminohexyl linker (AmHex, Figure 3). Once the peptide was synthesized, a spacer molecule was added to connect the oligonucleotide by conversion of the last amino group of the peptide into an hydroxyl group protected with a DMT group. For this purpose, we used the active ester of the O-DMT protected derivative of the 6-hydroxy hexanoic acid [Figure 2, Haralambidis et al. (1990)]. After the addition of the linker, oligonucleotide synthesis was studied in the pentathymidine sequence I. Unexpectedly, the addition of the phosphoramidites was strongly influenced by the solvent used during the coupling reaction. We tried different combinations of acetonitrile, pyridine, DMF, and DCM. The highest coupling efficiencies (10 7.2 9

a In the modification column: no ) unmodified R5 dodecamer, 5′-peptide ) VKRKKKPAA-R5 dodecamer and 3′-peptide ) R5 dodecamer-QAKKKKLDK.

DISCUSSION

There is a growing interest in oligonucleotide-peptide conjugates, due to their potential use as oligonucleotide probes and antisense inhibitors of gene expression. The incorporation of peptide sequences opens the possibility of enhancing some of the biophysical and biological properties of oligonucleotides. Signal sequences (Arar et al., 1993), penetratin (Allinquant et al., 1995), and fusogenic peptides (Bongartz et al., 1994) are among the most interesting peptide sequences introduced into oligonucleotides because of their biological properties. The aim of this study is the synthesis of oligonucleotide-peptide conjugates carrying nuclear localization sequences. Previously, oligonucleotide-peptide conjugates carrying nuclear localization sequences were found, inactive, in the freshwater ciliate Paremecium (Reed, 1995). Recently, it has been shown that a single nuclear localization signal sequence in a 3.3 kbp luciferase gene induces a strong transfection enhancement (Zanta, 1999). We studied the effect of the peptide sequence upon the binding properties of oligonucleotides, especially in mismatch discrimination. Nuclear localization sequences are rich on basic amino acids, especially lysine, that may hinder the synthesis of oligonucleotide-peptide conjugates (Tetzlaff et al., 1998). For the synthesis of the conjugates, we used two approaches. First, we prepared the oligonucleotide and the peptide on separate supports following well-known methods. A thiol group was introduced at the 5′ end of the oligonucleotide and a maleimido group at the Nterminal position of the peptide to ensure the formation of a defined oligonucleotide-peptide conjugate (Eritja et al., 1991; Arar et al., 1993; Ede et al., 1994). Such conjugates carry the peptide moiety at the 5′ end of the oligonucleotide, which is attached to the N-terminal residue of the peptide. Likewise, other conjugates can be prepared by changing the position of the thiol and the maleimido group. Nevertheless, the most common connection between peptide and oligonucleotide in the postsynthetic conjugation approach is the N-terminus f 5′ end connection since the connecting molecules are coupled at the end of the synthesis preventing the exposure of the connecting molecules to peptide and oligonucleotide synthesis conditions. Most of the oligonucleotide-peptide conjugates described in the literature are prepared following the postsynthetic conjugation approach, which allows the use of standard conditions in the assembly of both polymers. The synthesis of oligonucleotide-peptide conjugates following the stepwise approach is an attractive choice in the large-scale production of conjugates. In this approach, the building blocks are not the standard Boc/ Bzl or Fmoc/tBu-protected amino acids and the connection between peptide and oligonucleotide is N-terminus to the 3′ end because of the normal C f N and 3′ f 5′ sense of synthesis. It is also easier to assemble first the peptide using Boc chemistry without the oligonucleotide

since oligonucleotides can undergo depurination during the TFA treatments needed for the removal of the Boc group. To avoid further acid treatments in the presence of oligonucleotides, the side chains of the amino acids are protected with base-labile groups. Whether the conditions used during oligonucleotide synthesis and deprotection provoke side reactions in the peptide part was also studied. To that end, we used model compounds and exhaustive characterization to analyze the possibility of racemization and the modification of the side chain of trifunctional amino acids in synthesis and deprotection conditions. The use of a special 6-aminohexyl linked prevented the formation of a mixture of products containing carboxylic acid/carboxamide at the C-terminus. This side reaction may also take place on Fm protected Asp and Glu. Moreover, alkylation of lysine residues by acrylonitrile (Telzlaff, 1998) is avoided by a two-step deprotection protocol that removes acrylonitrile before ammonia treatment, but does not prevent small amounts of several products eluting near the desired product (see Figure 5b). Nevertheless, these products can be easily separated from the desired compound. In agreement with several authors (Bermann and Bannwarth 1995, Souckchareun et al., 1995; Robles et al., 1997), we found that both postsynthetic conjugation and stepwise approaches produce the desired oligonucleotide-peptide conjugates with similar yields and purity. All oligonucleotide-peptide conjugates carrying nuclear localization sequences hybridized with complementary sequences with a slightly higher stability than unmodified sequences. This is in agreement with the results obtained with conjugates carrying basic amino acids (Harrison and Balasubramanian, 1998; Zhu et al., 1993). On the contrary, the binding of conjugates carrying nuclear localization sequences to the target 27-mer RNA was 10-fold less efficient (see Table 1) and several complexes were found. This indicates that melting studies, although they are needed to screen high-affinity compounds, cannot be used for predicting RNA binding because of the secondary structure of RNA. To understand the binding of oligonucleotide analogues to their target RNA, further structural studies ahould be carried out. However, and despite the less efficient binding, conjugates carrying the nuclear localization sequence discriminate single-mutated RNA from wild-type RNA. CONCLUSION

In summary, we showed that oligonucleotide-peptide conjugates carrying nuclear localization peptides can be synthesized using either a postsynthetic conjugation or a stepwise approach. These conjugates show a slightly higher affinity to complementary DNA but a lower affinity to target RNA than ummodified oligonucleotides, although they maintain a good mismatch discrimination. ACKNOWLEDGMENT

This work was supported by funds from CICYT (PB 92-0043) and E.E.C.C. Biomed and Health Program (BMH1-CT93-1669). LITERATURE CITED (1) Adamson, J. G., Hoang, T., Crivici, A., and Lajoie, G. A. (1992) Use of Marfey’s reagent to quantitate racemization upon anchoring of amino acids to solid supports for peptide synthesis Anal. Biochem. 202, 210-214. (2) Albericio, F., Giralt, E., and Eritja, R. (1991) NPE-resin, a new approach for the solid-phase synthesis of protected

1012 Bioconjugate Chem., Vol. 10, No. 6, 1999 peptides and oligonucleotides. II Synthesis of protected peptides. Tetrahedron Lett. 32, 1515-1518. (3) Allinquant, B., Hantraye, P., Mailleux, P., Moya, K., Bouillot, C., and Prochiantz, A., (1995) Downregulation of amyloid precursor protein inhibits neurite outgrowth in vitro J. Cell. Biol. 128, 919-927. (4) Arar, K., Monsigny, M., and Mayer, R. (1993) Synthesis of oligonucleotide-peptide conjugates containing a KDEL signal sequence. Tetrahedron Lett. 34, 8087-8090. (5) Bergmann, F., and Bannwarth, W. (1995) Solid phase synthesis of directly linked peptide-oligodeoxynucleotide hybrids using standard synthesis protocols. Tetrahedron Lett. 36, 1839-1842. (6) Bongartz, J. P., Aubertin, A. M., Milhaud, P. G., and Lebleu, B. (1994) Improved biological activity of antisense oligonucleotides conjugated to a fusogenic peptide. Nucleic Acids Res. 22, 4681-4688. (7) Bray, A. M., Jhingran, A. G., Valerio, R. M., and Maeji, N. J. (1994) Simultaneous multiple synthesis of peptide amides by the multipin method. Application of vapor-phase ammonolysis J. Org. Chem. 59, 2197-2203. (8) Corey, D. R. (1995) 4800-fold acceleration of hybridization by chemically modified oligonucleotides. J. Am. Chem. Soc. 117, 9373-9374. (9) Corey, D. R., and Schultz, P. G. (1987) Generation of a hybrid sequence-specific single-stranded deoxyribonuclease Science 238, 1401-1403. (10) Crooke, S. T., and Lebleu, B. (1993) Antisense Research and Applications, CRC Press Inc., Boca Raton, FL. (11) Duroux, I., Godard, G., Boidot-Forget, M., Schwab, G., He´le`ne, C., and Saison-Behmoaras, T. (1995) Rational design of point mutation-selective antisense DNA targeted to codon 12 of Ha-ras mRNA in human cells. Nucleic Acids Res. 23, 3411-3418. (12) de la Torre, B. G., Avin˜o´, A., Tarraso´n, G., Piulats, J., Albericio, F., and Eritja, R., (1994) Stepwise solid-phase synthesis of oligonucleotide-peptide hybrids. Tetrahedron Lett. 35, 2733-2736. (13) Ede, N. J., Tregear, G. W., and Haralambidis, J. (1994) Routine preparation of thiol oligonucleotides: application to the synthesis of oligonucleotide-peptide hybrids. Bioconjugate Chem. 5, 373-378. (14) Eritja, R., Pons, A., Escarceller, M., Giralt, E., and Albericio, F. (1991a) Synthesis of defined peptide-oligonucleotide hybrids containing a nuclear transport signal sequence. Tetrahedron 47, 4113-4120. (15) Eritja, R., Robles, J., Ferna´ndez-Forner, D., Albericio, F., Giralt, E., and Pedroso, E. (1991b) NPE-resin, a new approach for the solid-phase synthesis of protected peptides and oligonucleotides. I Synthesis of the supports and their application to oligonucleotide synthesis. Tetrahedron Lett. 32, 1511-1514. (16) Gottschling, D., Seliger, H., Tarraso´n, G., Piulats, J., and Eritja, R. (1998) Synthesis of oligodeoxynucleotides containing N4-mercaptoethylcytosine and their use in the preparation of oligonucleotide-peptide conjugates carrying c-myc Tagsequence. Bioconjugate Chem. 9, 831-837. (17) Gupta, K. C., Kumar, P., Bhatia, D., and Sharma, A. K. (1995) A rapid method for the functionalisation of polymer supports for solid-phase oligonucleotide synthesis. Nucleosides Nucleotides 14, 829-832. (18) Haralambidis, J., Duncan, L., and Tregear, G. W. (1987) The solid-phase synthesis of oligonucleotides containing a 3′peptide moiety. Tetrahedron Lett. 28, 5199-5202. (19) Haralambidis, J., Duncan, L., Angus, K., and Tregear, G. W. (1990) The synthesis of polyamide-oligonucleotide conjugate molecules. Nucleic Acids Res. 18, 493-499. (20) Harrison, J. G., and Balasubramanian, S. (1998) Synthesis and hybridization analysis of a small library of peptideoligonucleotide conjugates. Nucleic Acids Res. 26, 3136-3145. (21) Jensen, O. N., Mortensen, P., and Mann, M. (1997) Identification of the components of simple protein misxture by high-accuracy peptide mass mapping and database searching. Anal. Chem. 69, 4741-4750.

de la Torre et al. (22) Juby, C. D., Richardson, C. D., and Brousseau, R. (1991) Facile preparation of 3′ oligonucleotide-peptide conjugates Tetrahedron Lett. 32, 879-882. (23) Lemaitre, M., Bayard, B., and LeBleu, B. (1987) Specific antiviral activity of a poly(L-Lys) conjugated oligodeoxyribonucleotide sequence complementary to vesicular stomatitis virus N protein mRNA initiation site. Proc. Natl. Acad. Sci. U.S.A. 84, 648-652. (24) Lin, Y., Padmapriya, A., Morden, K. M., and Jayasena, S. D. (1995) Peptide conjugation to an in vitro-selected DNA ligand improves enzyme inhibition. Proc. Natl. Acad. Sci. U.S.A. 92, 11044-11048. (25) McMinn, D. L., and Greenberg, M. M. (1998) Postsynthetic conjugation of protected oligonucleotides containing 3′-alkylamines. J. Am. Chem. Soc. 120, 3289-3294. (26) McMinn, D. L., and Greenberg, M. M. (1999) Convergent solution-phase synthesis of a nucleopeptide using a protected oligonucleotide. Bioorg. Med. Chem. Lett. 9, 547-550. (27) Peyrottes, S., Mestre, B., Burlina, F., and Gait, M. J. (1998) The synthesis of peptide-oligonucleotide conjugates by a fragment coupling approach. Tetrahedron 54, 12513-12522. (28) Rabanal, F., Giralt, E., and Albericio, F. (1992) A new fluorene-derived anchor for solid-phase synthesis of protected peptides. Tetrahedron Lett. 33, 1775-1778. (29) Rabanal, F., Giralt, E., and Albericio, F. (1995) Tetrahedron 51, 1449-1458. (30) Reed, M. W., Fraga, D., Schwartz, D. E., Scholler, J., and Hinrichsen, R. D. (1995) Synthesis and evaluation of nuclear targeting peptide-antisense oligodeoxynucleotide conjugates. Bioconjugate Chem. 6, 101-108. (31) Robles, J., Pedroso, E., and Grandas, A. (1995) Peptideoligonucleotide hybrids with N-acylphosphoramidate linkages. J. Org. Chem. 60, 4856-4861. (32) Robles, J., Maseda, M., Beltra´n, M., Concernau, M., Pedroso, E., and Grandas, A. (1997) Synthesis and enzymatic stability of phosphodiester-linked peptide-oligonucleotide hybrids. Bioconjugate Chem. 8, 785-788. (33) Soukchareun, S., Tregear, G. W., and Haralambidis, J. (1995) Preparation and characterization of antisense oligonucleotide-peptide hybrids containing viral fusion peptides. Bioconjugate Chem 6, 43-53. (34) Tetzlaff, C. N., Schwope, I., Bleczinski, C. F., Steinberg, J. A., and Richert, C. (1998) A convenient synthesis of 5′amino-5′-deoxythymidine and preparation of peptide-DNA hybrids. Tetrahedron Lett. 39, 4215-4218. (35) Tung, C. H., Wang, J., Leibowitz, M. J., and Stein, S. (1995) Dual-specificity interaction of HIV-1 TAR RNA with Tat peptide-oligonucleotide conjugates. Bioconjugate Chem. 6, 292-295. (36) Tung, C. H., Breslauer, K. J., and Stein, S. (1996) Stabilization of DNA triple-helix formation by appended caitonic peptides. Bioconjugate Chem. 7, 529-531. (37) Vive`s, E., and LeBleu, B. (1997) Selective coupling of a highly basic peptide to an oligonucleotide. Tetrahedron Lett. 38, 1183-1186. (38) Will, D. W., Breipohl, G., Langner, D., Knolle, G., and Uhlmann, E. (1995) The synthesis of polyamide nucleic acids using a novel monomethoxytrityl protecting-group strategy. Tetrahedron 51, 12069-12082. (39) Zanta, M. A., Belguise-Valladier, P., and Behr, J. P. (1999) Gene delivery: A single nuclear localization signal peptide is sufficient to carry DNA to the cell nucleus. Proc. Natl. Acad. Sci. U.S.A. 96, 91-96. (40) Zhu, T., Wei, Z., Tung, C.-H, Dickerhof, W. A., Breslauer, K. J., Georgopoulos, D. E., Leibowitz, M. J., and Stein, S. (1993) Oligonucleotide-poly-L-ornithine conjugates: binding to complementary DNA and RNA. Antisense Res. Dev. 3, 265-275.

BC990046L