Bioconjugate Chem. 2005, 16, 1038−1044
1038
Oligonucleotides Conjugated to Short Lysine Chains Johannes Winkler, Ernst Urban, and Christian R. Noe* Department of Medicinal Chemistry, Universita¨t Wien, Althanstrasse 14, 1090 Wien, Austria. Received November 10, 2004; Revised Manuscript Received April 15, 2005
A new method for synthesizing oligonucleotide peptide conjugates by an in-line approach is presented. A phosphorothioate oligonucleotide with the sequence of bcl-2 targeted Oblimersen by employing a modified 2′-amino-2′-desoxy-uridine nucleotide bearing a succinyl linker at the 2′ position was prepared. The carboxyl group was protected for solid-phase synthesis as the benzyl ester. Ester cleavage was afforded by a phase transfer reaction using palladium nanoparticles as catalyst and cyclohexadiene as hydrogen donor. Short tails of up to three lysyl residues were conjugated to the oligonucleotide by an inverse stepwise peptide synthesis. The conjugates were characterized by HPLC, mass spectrometry, and circular dichroism. Influence of lysyl tails on CD spectra were minimal. Melting profiles revealed only minimal destabilizing effects on duplexes by conjugation of peptides.
INTRODUCTION
The aim of the antisense strategy (1) is the sequence specific inhibition of a gene by hybridization of a singlestranded oligonucleotide to the complementary mRNA in order to prevent the expression of the corresponding protein product. Besides the inhibition of translation by binding to the mRNA to give double-stranded nucleic acid, activation of the RNA degrading RNAse H is a key effect of antisense substances (2, 3). In addition to this pharmacodynamic effect, which is determined by the nucleotide sequence of the antisense oligonucleotide, sufficient pharmacokinetic properties of these substances are essential for achieving the desired effect. Unmodified phosphodiester oligonucleotides lack sufficient stability against degradation by mainly exo- but also endonucleases and are therefore not suited for therapy. Numerous modifications have been introduced and tested for their binding affinity and biological properties (4, 5). Initially, modifications of the phosphodiester backbone were the main field of activity. Phosphorothioate oligonucleotides, often called antisense molecules of the first generation, possess better stability against enzymatic degradation. Although their hybridization properties are poorer than that of natural oligonucleotides, their advantageous pharmacokinetic characteristics and the ability to activate RNAse H made them a promising new class of antisense molecules (6). The first approved antisense drug, fomivirsene (7), and most of the substances currently in various phases of clinical trials (8) are phosphorothioate oligonucleotides. Oblimersen sodium is one of the most progressed antisense drugs in clinical trials (9). A large multicenter phase III study against malignant melanoma, in which Oblimersen was administered in combination with the standard dacarbazine treatment, was recently concluded. Despite an increase in progression-free survival (PFS), Genta withdrew its appeal for approval after a review by the FDA advisory committee, because median survival, the primary end-point, was not prolonged statisti* To whom correspondence should be addressed. Phone: +431 4277 55103, Fax: +431 4277 9551, E-mail: christian.noe@ univie.ac.at.
cally significantly (10). Still other phase III trials against several cancer types are ongoing. Oblimersen is a phosphorothioate oligonucleotide targeted to the first six codons of the open reading frame of Bcl-2 (11). Bcl-2 is an important inhibitor of apoptosis (12) and has been shown to be overexpressed in a large numbers of tumors. Because of its phosphorothioate backbone, a 5′-TC dinucleotide, and two distinct CpG motifs, some non-antisense effects may contribute to the antiproliferative action of Oblimersen. A sequencespecific true antisense effect has been shown in some in vitro and in vivo experiments (13-15), while others reported that the cytostatic properties are independent from the antisense effect (16, 17). The second generation of antisense oligonucleotides bears modifications at the 2′-position (18-20). Oligonucleotides with an alkoxy substituent in position 2′ proved to be stable against DNA or RNA cleaving enzymes. This increased stability has been explained by the steric hindrance, which inhibits the attack of nucleases at the phosphate groups. Base pairing properties decrease with increasing length of alkyl substituent. 2′Ethylene glycol-modified oligonucleotides showed better base pairing properties (6, 21), and the 2′-methoxy ethyl group is particularly suited. As a drawback, 2′-O-substitued oligonucleotides proved to be unable to catalyze enzymatic degradation of the complementary RNA by activation of RNAse H (22). Hence, so-called gapmere oligonucleotides, bearing modified nucleotides at both the 3′ and 5′ end, but with an unmodified region of at least five nucleotides between, were introduced. They provide good stability against exonucleases while the unmodified region is sufficient to activate RNAse H (23). Zwitterionic antisense oligonucleotides are seen as the third generation. Introduction of a 6-aminohexyl group at the 2′ position not only increases stability against enzymatic degradation, but also minimizes the electrostatic repulsion of the sense and antisense strands leading to improved hybridization affinity (24). Other more rigorous modifications include substitution of the polyanionic phosphate backbone by peptides (PNA), change of the ribose moiety to morpholino groups (MNA) (25), and introducing a methylene bridge between oxygen
10.1021/bc049729d CCC: $30.25 © 2005 American Chemical Society Published on Web 07/02/2005
Technical Notes
2′ and carbon 4′ of the carbohydrate to increase rigidity of the backbone (locked nucleic acids, LNA) (26, 27). Lately, the focus in improving oligonucleotide properties has been shifted toward conjugation of ligands (28). This approach allows a directed change of molecule characteristics enhancing binding affinity or pharmacokinetic parameters such as hydrophobicity and improvements in biodistribution or cellular uptake. A large number of ligands has successfully been ligated to oligonucleotides, among them lipophilic molecules such as cholesterol (29) and lipids (30), vitamin B6 (31), a synthetic ligand to the asialoglycoprotein receptor for tissue and cell-specific targeting to parenchymal liver cells (32), and various proteins (28). Conjugates of oligonucleotides with penetratins (33) or signal peptides acquire increased uptake rates due to the membrane dislocation properties of these peptides. Conjugation of a PNA to a monoclonal antibody of the rat transferrin receptor via the biotin streptavidin complex enabled the transport through the blood-brain barrier (34). Attachment of arginine rich SV40 nuclear localization signal efficiently transported DNA as well as PNA (35, 36). Conjugates with either a 35 amino acid basic sequence of the HIV Tat protein or a 16 amino acid basic sequence from the Drosophila antennapedia protein improved cellular uptake of antisense phosphorothioate oligonucleotides without loss of specificity to target RNA (37, 38). It has been firmly established that an increase in cellular uptake of oligonucleotides can be achieved by conjugation or complexation of the oligonucleotide to poly-L-lysine (39-42). A tail of one, two, or four lysine moieties at the 5′-end of a PNA oligonucleotide improved cellular uptake (43). The extent of uptake was dependent on the number of conjugated lysine monomers. The affinity to the target sequence was not increased nor was the nuclear translocation process influenced. It was concluded that the lysine chain improved the transport through the cell membrane or enhanced the release from endosomes. Herein, we report the conjugation of oligonucleotides with short lysyl chains. The majority of oligonucleotide peptide conjugates have been prepared by linkage at either the 3′ or the 5′ end of the oligonucleotide (28). The two components were either synthesized independently by distinct solid-phase syntheses and ligated afterward or on the same solid phase by sequential stepwise synthesis. Commonly used linkers are disulfides (44-46), maleimides, thioethers, and amides (28). In an in-line synthesis, first described by Haralambidis (47), the peptide and oligonucleotide are synthesized sequentially on automatic synthesizers. Using an acid labile amino protection group, a possibility for stepwise peptide synthesis at the 5′-end of an oligonucleotides prepared on the same solid support was described (48). Conjugation at the 2′ position of the carbohydrate moiety of the oligonucleotide has previously been achieved by use of the 6-aminohexyl (49, 50) and 4-aminopropyl linkers (51). Ligands have been coupled to the primary amino group by amide bonds or the use of a chloroformate or carbonate to give a carbamate or by an active ester to give an amide (49). Recently, a procedure for the attachment of amines to an allyl-protected carboxyl linker attached at the 2′-position of uridine has been published (52). The allyl ester was removed by treatment with Pd(0) and morpholine. Subsequently, several small peptides were coupled to the free carboxyl group. Our approach relies closely on the previously published procedure for a fluoresceine oligonucleotide conjugate (53). We derivatized 2′-amino-2′-desoxy-uridine with succinic anhydride to give the hemisuccinate. Protection of
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the carboxyl group for the solid-phase synthesis was achieved by preparing the corresponding benzyl ester. This nucleotide building block was incorporated in an oligonucleotide at the 3′ terminus. Conjugation of fluorescein was done after deprotection of the carboxyl group by a catalytic phase transfer reaction with palladium nanoparticles and 1,4-cyclohexadiene as the hydrogen donor. After establishing this simple and convenient procedure for conjugating ligands at the 2′ position of a 3′-terminal uridine nucleoside of an oligonucleotide, we decided to expand this method into the field of oligonucleotide peptide conjugates. For the preparation of oligonucleotide peptide conjugates, protein synthesis had to be achieved in N to C, the inverse direction. We designed a suitably protected lysine synthon to build up short peptide strands at the hemisuccinate. In addition to the synthesis, we present data of circular dichroism spectroscopy and binding affinity to the sense strand. EXPERIMENTAL PROCEDURES
Reagents were used in standard quality for synthesis. Acetonitrile was heated over potassium carbonate and distilled, followed by heating over calcium hydride and distillation. Dichloromethane was dried over phosphorus pentoxide and distilled. Melting points were measured on a Kofler melting point apparatus and are uncorrected. NMR spectra were recorded on a Bruker Avance 200 MHz or a Varian Unity 300 MHz machine. Shifts are reported relative to the solvent peak (CHCl3 in CDCl3: δ 7.26 and 77.00), coupling constants are in Hz. Spin multiplicities are given with the following abbreviations: s singlet, d doublet, t triplet, q quartet, m multiplet. Thin-layer chromatography (TLC) was performed using silica gel 60-F254 precoated aluminum plates by Merck. Spots were visualized by UV. N()-Trifluoracetyl-L-lysine Benzyl Ester 2. N()Trifluoracetyl-L-lysine (1, 300 mg, 1.24 mmol) was triturated with p-toluenesulfonic acid monohydrate (480 mg, 2.52 mmol) and 7 mL of benzyl alcohol. The resulting paste was dissolved in 18 mL of benzene and heated to reflux for 18 h. The arising water was removed from the reaction by a Soxhlet apparatus filled with molecular sieve (4 Å). After cooling, the solvent was removed in vacuo. The reaction product was crystallized by adding 10 mL of diethyl ether. After cooling at 4 °C overnight, the precipitate was collected by filtration and dried in vacuo to give the p-toluenesulfonic acid salt of N()trifluoracetyl-L-lysine benzyl ester (2, 570 mg, 91%). The free base was gained by extraction with saturated sodium bicarbonate solution. Tosylate: 1H NMR (CDCl3, 200 MHz): δ ) 7.70 (d, J ) 7.8, 2H, Ar-H (tos)), 7.37 (s, 5H, Ar-H (bzl)), 7.21 (d, J ) 7.8, 2H, Ar-H (tos)), 5.29 (d, J ) 12.1, 1H, CH2-Ph (1)), 5.22 (d, J ) 12.6, 1H, CH2-Ph (2)), 4.06 (t, J32 ) 5.8, 1H, H-2), 3.20 (t, J56 ) 6.6, 1H, H-6), 2.35 (s, 3H, CH3), 1.88 (m, 2H, H-3), 1.56-1.29 (m, 4H, H-4, H-5). 13C NMR (CDCl , 50 MHz): δ ) 170.35 (C-1), 143.42 (C-4 3 (tos)), 141.76 (ArC-1 (tos)), 136.41 (ArC-1 (bzl)), 129.84, 129.80 and 129.70 (ArC-2,6, ArC-3,5, ArC-4 (bzl), ArC3,5 (tos)), 126.92 (ArC-2,6 (tos)), 117.53 (q, J ) 286, CF3), 69.08 (CH2-bzl), 53.85 (C-2), 40.12 (C-6), 31.03 (C-3), 29.24 (C-5), 23.03 (C-4), 21.30 (CH3). Fp: 144-145 °C. C22H27F3N2O6S: calcd C 52.37 H 5.39 N 5.55 S 6.36 found C 52.22 H 5.53 N 5.52 S 6.39. 2: 1H NMR (CDCl3, 200 MHz): δ ) 7.27 (s, 5H, ArH), 5.07 (s, 2H, CH2-Ph), 3.38 (m, 2H, H-2), 3.25 (d, J6′6 ) 6.4, 1H), 3.19 (d, J66′ ) 6.4, 1H), H-6′), 1.91 (bs, 2H, NH2), 1.75-1.13 (m, 6H, H-3, H-4, H-5). 13C NMR
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(CDCl3, 50 MHz): δ ) 175.52 (COObzl), 135.47 (ArC-1), 128.55, 128.39 and 128.26 (ArC-3,5, ArC-2,6, ArC-4), 115.87 (q, J ) 286, CF3), 66.73 (CH2-Ph), 53.96 (C-2), 39.45 (C-6), 33.64 (C-3), 28.04 (C-5), 22.45 (C-4). Loading of Solid Support. TentaGel-NH2 (230 mg, 60 µmol free NH2-groups), purchased from Fluka, was loaded with succinyl modified uridine 3 and N,N′diisopropylcarbodiimide, 1-hydroxy-1H-benzotriazole, and 4-(dimethylamino)pyridine as described earlier (53). Degree of loading was around 190 µmol/g. Oligonucleotide Synthesis. Standard phosphoroamidites and tetrazole solution were obtained from Carl Roth GmbH & Co. KG. Oligonucleotide syntheses were carried out on a ABI 392 Applied Biosystems DNA/RNASynthesizer in standard 1 µmol scale in DMT1-on mode. Coupling of the first nucleotide to solid-supported modified nucleoside was prolonged to 2 min. Sulfurization was done with tetraethyldiuram disulfide, which was purchased from Sigma and recrystallized (54). For the synthesis of phosphorothioates, the sulfurization step was performed before capping. 1H-Tetrazol was used as activation reagent. Other reagents used for DNA synthesis were obtained in analytical quality and purified before use. Preparation of Oligonucleotide-Lysine Conjugates. The synthesis column was removed from the synthesizer and opened. The resin-bound oligonucleotide was transferred to an 5 mL reaction tube of a Quest 210 manual synthesizer. Cleavage of the benzyl protecting group was achieved by phase-transfer hydrogenation with PVP-stabilized palladium nanoparticles (0.7 mL) (53) and 1,4-cyclohexadiene (0.05 mL) as hydrogen donor. Cyclohexadiene was added to the PVP-stabilized suspension of palladium, and the resulting solution was microfiltrated (0.22 µm) to ensure no precipitated palladium was applied to the resin. The reaction mixture was stirred at room temperature for 3 h. The resin was washed three times with methanol and three times with DMF. Prior to coupling, the carboxyl group was activated by a solution of 1-hydroxy-1Hbenzotriazole (0.4 mg, 3.0 µmol) and N,N′-diisopropylcarbodiimide (1.1 mg, 8.7 µmol) in 1 mL of DMF. The mixture was stirred for 3 h, and the solution was again filtered off. Coupling was done by adding a solution of 1-hydroxy-1H-benzotriazole (0.4 mg, 3.0 µmol), N,N′diisopropylcarbodiimide (0.4 mg, 8.7 µmol), and lysine building block 2 (2.0 mg, 6.0 µmol) in 1 mL of DMF and stirring for 18 h. The resin was washed three times with DMF and three times with water. For the preparation of conjugates with two and three lysines, the deprotection and coupling procedures were repeated. For cleavage and deprotection of oligonucleotides, 2 mL of concentrated ammonia was added and the mixture was heated to 55 °C for 18 h. The solution was filtered, and the solvent was evaporated in vacuo. Purification and Analyses of Oligonucleotides. Oligonucleotide concentrations were determined by measuring OD260 in a Hitachi U3000 spectrophotometer. Molar extinction coefficents were calculated as the sum of nucleotides (A: 15400, G: 11700, C: 7300, T: 8800, U: 9950). Initial purifications of DMT-on oligonucleotides were done with Poly-Pak columns obtained from Carl Roth GmbH & Co. KG according to manufacturer except for elution, which was done with 20% acetonitrile. Analytical 1Abbreviations: DMT, dimethoxytriphenylmethyl; PVP, poly(vinylpyrrolidone); CD, circular dichroism; DIC, N,N′-diisopropylcarbodiimide; HOBt, 1-hydroxy-1H-benzotriazole.
Technical Notes Scheme 1. Synthesis and Structure of Solid-PhaseBound Starter Nucleotide 4a
a Reagents and conditions: (a) benzyl succinyl ester, DIC, HOBt, THF, rt; (b) succinic anhydride, pyr, 30 °C.
HPLC was performed on a Nucleosil CC 250/4 100-5 C18 column with the following gradient system: A: 0.1 M triethylammonium acetate in water, B: 0.1 M triethylammonium acetate in 80% acetonitrile, linear gradient 10-40% B in 0-30 min, flow rate 1 mL/min. If the purity after Poly-Pak-purification was deemed insufficient, preparative HPLC was performed on a LiChrosphere 100 RP-18 using the same gradient as for analytical HPLC. Mass spectrometric analysis was done on a Kratos seq MALDI mass spectrometer. Sample preparation was done following the method described by Pieles et al. (55). One microliter of sample (100 pmol/µL in water) was briefly vortexed with 10 µL of a 0.5 M solution of 2,4,6trihydroxyacetophenone in ethanol and 5 µL of a 0.1 M solution of di-ammonium hydrogen citrate. One microliter of this mixture was spotted on the target and allowed to air-dry. The mass spectrometer was run in the negative ion and reflectron mode, and spectra were usually obtained by summing up 100 single laser pulses. Circular dichroism spectrometry was performed on a Jasco J-810 spectropolarimeter equipped with a Neslab RTE 7 thermostatic unit. Oligonucleotides were diluted to a 9 µM solution in 0.15 M NaCl and 0.01 M Tris-HCl (pH 7.0) buffer. CD spectra were collected from 320 to 210 nm using a quartz cuvette (Hellma 100-QS) with a path length of 1 mm. Duplexes were measured after heating the equimolar mixture of complementary strands to 50° for 10 min and subsequent slow cooling to room temperature. Melting temperatures (Tm) of the duplexes were determined in the Jasco J-810 by slowly heating from 30 to 90 degrees (50 °C/h) and recording CD at 248 nm or OD at 260 nm as a function of temperature. Melting curves showed cooperative form and Tm were obtained from the maxima of the first derivative plots. Temperatures given are that of the cuvette holder. RESULTS
Conjugation of cationic peptides has been shown to improve properties of antisense oligonucleotides and enhance their efficacy. The synthesis of nucleoside building block 4 was described earlier (Scheme 1). It proceeds straightforward from 2′-amino-2′-desoxy-uridine by reac-
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Technical Notes Scheme 2. Lysine Building Blocka
a Reagents and conditions: (a) benzyl alcohol, p-toluenesulfonic acid, benzene, reflux.
Figure 1. HPLC of oligonucleotide trilysine conjugate 7.
Scheme 3. Structure of Oligonucleotide Conjugates 5 (n ) 1), 6 (n ) 2), and 7 (n ) 3)
Figure 2. CD spectra of single strands of 4 (+), 5 (2), 6 (O), 7 (×), and Oblimersen (9).
Table 1. HPLC Retention Times and Melting Temperatures of Oligonucleotide Duplexes with the Complementary DNA Strands
antisense-DNA Oblimersen 4 4 5 6
HPLC retention time (min)
Tm (°C)
19.13 17.95 18.06 18.37 18.56
78.7 ( 0.1 69.5 ( 1.3 65.5 ( 0.8 67.4 ( 0.1 67.6 ( 1.0 68.2 ( 0.4
tion with succinic acid mono benzyl ester with an excellent selectivity for the amino group (53). For peptide synthesis, a suitable lysine derivative had to be protected at the carboxyl group and the -amino function. We decided that the trifluoracetyl group was well suited for masking the terminal amino group, because it can be cleaved in the same step as the nucleic base protecting groups (56). Because attachment is conducted after complete assembly of the oligonucleotide, the reported replacement of the trifluoracetyl group by the acetyl capping reagent (57) is of no importance. Additionally, no cleavage of peptide bonds was reported to occur using concentrated ammonia for deprotection (58, 59). Easily available N()-trifluoracetyl-L-lysine (1) was reacted with benzyl alcohol under catalysis of p-toluenesulfonic acid to give benzyl ester 2 (Scheme 2). A fluorescent dye was successfully coupled to the succinic linker by using a 3-fold excess of DIC and HOBt as catalysts (53). Coupling of 2 to solid bound deprotected modified nucleoside monomer using the same conditions gave only poor yields of 30-40% as judged by HPLC analysis. Thus, we used an optimized protocol for inverse solid-phase peptide synthesis (60). After a prior activation step with an 8-fold excess of DIC and a 3-fold excess of HOBt, coupling was achieved with 3-fold excesses each of DIC, HOBt, and 2. This procedure resulted in good yields when tried with solid bound nucleoside monomer. Consequently, it was employed for conjugating short lysine tails to an 18mer all-phosphorothioate oligonucleotide (4) with a sequence according to that of
Oblimersen (5′-TCTCCCAGCGTGCGCCAU-3′). Conjugates containing one (5), two (6), and three (7) lysyl residues were prepared (Scheme 3). Purification was achieved using Poly-Pak-cartridges and the standard protocol. HPLC analysis with a reversed phase column showed clear differences in retention times of the three products and unconjugated oligonucleotide. With increasing number of lysyl residues, the retention times were prolonged as can be seen in Table 1. Due to the broad peaks of phosphorothioate oligonucleotides, no reliable measurement of coupling yields could be determined. For example, Figure 1 shows the HPLC chromatogram of trilysyl oligonucleotide 7. Structures were verified by MALDI mass spectrometry. CD spectra of the conjugation products were recorded and compared to that of Oblimersen and unconjugated succinyl modified oligonucleotide 4. All single stranded oligomers gave relatively weak CD curves (Figure 2) with only a well-defined positive band around 280 nm that showed small variability for all substances. In the far UV region of 200-250 nm, a small positive band could be observed at 220 nm, while generally negative CD values were recorded in that area. No straightforward relation of spectra with lysyl substitution could be detected. Duplexes with complementary unmodified phosphodiester DNA were prepared by mixing of single strands in equimolar amounts in 0.01 M Tris buffer (pH 7.0) with 0.15 M concentration of NaCl. To ensure completion of duplex binding, the mixture was heated to 50 °C for 10 min and subsequently cooled. The formation of duplexes was verified by comparing the spectra of single strands and duplexes recorded at 10 °C (53). All oligonucleotides gave nearly identical CD curves (Figure 3). Comparison of the CD values at 200 nm shows smaller results for lysine substituted oligonucleotides, although there is no simple correlation with the number of substitutions. To determine hybridization affinity and duplex stability, melting profiles were recorded either using CD value at 248 nm or UV absorption at 260 nm. Both methods gave cooperative curves with identical melting temperature. A minimum of four distinct profiles were recorded for each oligonucleotide. Melting temperature of modified oligonucleotides were generally lower than that of control
1042 Bioconjugate Chem., Vol. 16, No. 4, 2005
Figure 3. CD spectra of duplexes of 4 (+), 5 (2), 6 (O), 7 (×), and Oblimersen (9) with sense-DNA.
Oblimersen. Lysyl-substituted substances gave higher melting points than unconjugated oligonucleotide 4. DISCUSSION
Our method for preparing oligonucleotide conjugates proved to be easy and convenient for the synthesis of a fluoresceine conjugate (53). Herein, we show that it is also a versatile method and can be employed for conjugation of at least short peptides in an in-line approach. Sequential solid-phase synthesis of first the oligonucleotide and then a peptide chain of up to three lysyl residues has been achieved. Preparation of lysyl building block was straightforward. Protection of Lys gamma amino function was accomplished as trifluoracetyl amide. This group is cleaved in the same deprotection step as nucleic base masking groups with concentrated ammonia. Because the trifluoracetyl-protected amine is introduced after complete oligonucleotide synthesis, no side products due to transamination caused by the capping reagent are generated (57). The carboxyl function was protected as a benzyl ester, because a cleavage protocol by phase transfer-catalyzed reaction has been established for succinyl linker modified oligonucleotide (53). The deprotection reaction was completed after 3 h at room temperature. Initially, we tried coupling the lysyl building block to solid-bound monomer by direct reaction with DIC and HOBt, but the yields of around 40% were disappointing. Therefore, we decided to use a protocol published for the inverse N to C peptide synthesis (60), which resulted in nearly quantitative yields. Prior to coupling, free carboxyl group was activated with a large excess of DIC (8-fold) for 3 h. Coupling was performed with a 3-fold excess of DIC, HOBt, and lysine building block 2 with a reaction time of 18 h. No further optimization attempts were made, although switching to other reagents such as HATU/TMP may result in shorter coupling times (61, 62). Peptide synthesis in the so-called inverse direction usually results in poorer yields than synthesis in established C to N direction. Additionally, a certain extent of racemization is often imminent. Therefore, for preparing conjugates of 4 with larger peptides, distinct syntheses of the components followed by ligation seems to be the favorable option. But for peptides consisting of up to three amino acids, the danger of racemization can be neglected. Even very short cationic peptides have an enhancing influence on the properties of antisense oligonucleotides, especially on cell uptake (43). Conjugates of modified oligonucleotide 4, bearing the sequence of Oblimersen, targeted to apoptosis inhibitor bcl-2 with one, two, and three lysyl residues were prepared by this procedure. HPLC analyses gave broad peaks because of the diastereomeric attributes of phosphorothioate oligonucleotides. Although exact quantification of coupling yields was therefore impossible, successful conjugation with good yields was proved by changes in retention times. No shoulders on the peaks could be
Technical Notes
detected. Coinjection of unsubstituted conjugate 4 with lysyl modified conjugates 5-7 revealed the presence of two different substances. MALDI analyses confirmed the successful conjugation, as the expected masses were found. The mass of mono- or dilysyl substituted oligonucleotide could not be found in the MALDI mass spectrum of 7. CD spectra of single strands were quite similar in their shape showing a B-DNA type curve. Phosphorothioate oligonucleotides generally give lower intensities in CD than phosphodiester DNA (53, 63). Positive cotton effect at 280 nm, an indicator of base stacking, was practically identical in all substances. No characteristic curve was observed in the far UV region (200-250 nm) indicating no pronounced secondary structure. No clear influence of lysine substitution could be detected. At neutral pH, poly-L-lysine gives a typical random coil CD spectrum with low intensities in the wavelength region over 210 nm, whereas at lower wavelength, an intense negative band centered at 195 nm is observed (64). Although CD values of lysyl-substituted oligonucleotides at 200 nm are lower than that of Oblimersen, there is no correlation with the number of lysyl residues. Measurement at this wavelength was considerably noisy due to the increasing absorption of sodium chloride. No substantial influence on the CD curve is caused by the introduction of short lysyl chains, because the concentration of conjugates 5-7 is too low for a reliable measurement of peptide secondary structure. With regard to the short peptide strand, the concentration of conjugate 7 is around 3 µg/mL. A typical concentration for recording peptide CD spectra is 500 µg/ mL (60). CD spectra of duplexes with sense DNA exhibited less differences between 5-7 and Oblimersen. A well defined helical structure with characteristics of B-DNA is obviously caused by the DNA strand. Differences between conjugates and Oblimersen are minimized. Again, lysyl substitution does not seem to influence CD values. The 18 mer oligonucleotide presumably is extremely dominant concerning CD spectroscopy over the short lysine tail with a maximum of three amino acids. Melting point determination was done by temperaturedependent measurement of either CD value or UV absorption. Oblimersen gives a melting temperature of 69.5 °C, 9.2 °C lower compared to phosphodiester oligonucleotide (53). Unconjugated oligonucleotide 4 shows a decrease of 4 °C. Substitution of the free carboxyl group with lysine increases the duplex stability. Melting point of 7 is only 1.3 °C lower than that of Oblimersen and very similiar to that of a fluoresceine conjugated oligonucleotide (53). Differences between 5, 6, and 7 are small and partly within standard deviation, but a tendency toward duplex stabilization by an increasing number of lysine monomers can be observed. This effect can be explained by partial neutralization of the negatively charged phosphate backbone by cationic terminal amino groups of lysine. The electrostatic repulsion of the two strands is thus decreased. This stabilizing effect evidently overcomes the destabilizing effect of the increasing steric bulk in the minor groove. On the other hand, the increasing steric hindrance seems to be responsible for the somewhat lower increase in melting temperature than expected. In conclusion, our previously reported procedure for preparing oligonucleotide conjugates has been shown to be suitable for preparing conjugates with short peptide tails in an in-line approach. Increasing length of the oligolysyl chain resulted in both better base pairing ability, reflecting the high potential of conjugated basic
Technical Notes
peptides to improve the therapeutical properties of nucleic acid analogues. The conjugation of oligolysyl chains to the 3′-terminal 2′-position altered physical and biological properties only to a minimal extent and is therefore a technique to specifically enhance oligonucleotides characteristics by ligating functional or cationic peptides or proteins. LITERATURE CITED (1) Zamecnik, P. C., and Stephenson, M. L. (1978) Inhibition of Rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide. Proc. Natl. Acad. Sci. U.S.A. 75, 280-284. (2) Crooke, S. T. (2000) Potential roles of antisense technology in cancer chemotherapy. Oncogene 19, 6651-6659. (3) Crooke, S. T. (1999) Molecular mechanisms of action of antisense drugs. Biochim. Biophys. Acta 1489, 31-44. (4) Urban, E., and Noe, C. R. (2003) Structural modifications of antisense oligonucleotides. Farmaco. 58, 243-258. (5) Freier, S. M., and Altmann, K. H. (1997) The ups and downs of nucleic acid duplex stability: structure-stability studies on chemically modified DNA: RNA duplexes. Nucleic Acids Res. 25, 4429-4443. (6) Levin, A. A. (1999) A review of the issues in the pharmacokinetics and toxicology of phosphorothioate antisense oligonucleotides. Biochim. Biophys. Acta 1489, 69-84. (7) Orr, R. M. (2001) Technology evaluation: fomivirsen, Isis Pharmaceuticals Inc/CIBA vision. Curr. Opin. Mol. Ther. 3, 288-294. (8) Tamm, I., Dorken, B., and Hartmann, G. (2001) Antisense therapy in oncology: new hope for an old idea? Lancet 358, 489-497. (9) Bayes, M., Rabasseda, X., and Prous, J. R. (2004) Gateways to clinical trials. Methods Find Exp Clin Pharmacol. 26, 473503. (10) Frantz, S. (2004) Lessons learnt from Genasense’s failure. Nat Rev Drug Discovery 3, 542-543. (11) Klasa, R. J., Gillum, A. M., Klem, R. E., and Frankel, S. R. (2002) Oblimersen Bcl-2 antisense: facilitating apoptosis in anticancer treatment. Antisense Nucleic Acid Drug Dev. 12, 193-213. (12) Adams, J. M., and Cory, S. (1998) The Bcl-2 protein family: arbiters of cell survival. Science. 281, 1322-1326. (13) Jansen, B., Wacheck, V., Heere-Ress, E., SchlagbauerWadl, H., Hoeller, C., Lucas, T., Hoermann, M., Hollenstein, U., Wolff, K., and Pehamberger, H. (2000) Chemosensitisation of malignant melanoma by BCL2 antisense therapy. Lancet 356, 1728-1733. (14) Wacheck, V., Krepler, C., Strommer, S., Heere-Ress, E., Klem, R., Pehamberger, H., Eichler, H. G., and Jansen, B. (2002) Antitumor effect of G3139 Bcl-2 antisense oligonucleotide is independent of its immune stimulation by CpG motifs in SCID mice. Antisense Nucleic Acid Drug Dev. 12, 359367. (15) Dias, N., and Stein, C. A. (2002) Potential roles of antisense oligonucleotides in cancer therapy. The example of Bcl-2 antisense oligonucleotides. Eur. J. Pharm. Biopharm. 54, 263-269. (16) Raffo, A., Lai, J. C., Stein, C. A., Miller, P., Scaringe, S., Khvorova, A., and Benimetskaya, L. (2004) Antisense RNA down-regulation of bcl-2 expression in DU145 prostate cancer cells does not diminish the cytostatic effects of G3139 (Oblimersen). Clin Cancer Res. 10, 3195-3206. (17) Lai, J. C., Benimetskaya, L., Santella, R. M., Wang, Q., Miller, P. S., and Stein, C. A. (2003) G3139 (oblimersen) may inhibit prostate cancer cell growth in a partially bis-CpGdependent non-antisense manner. Mol Cancer Ther. 2, 10311043. (18) Manoharan, M. (1999) 2′-carbohydrate modifications in antisense oligonucleotide therapy: importance of conformation, configuration and conjugation. Biochim. Biophys. Acta 1489, 117-130.
Bioconjugate Chem., Vol. 16, No. 4, 2005 1043 (19) Wagner, E., Oberhauser, B., Holzner, A., Brunar, H., Issakides, G., Schaffner, G., Cotten, M., Knollmueller, M., and Noe, C. R. (1991) A simple procedure for the preparation of protected 2′-O-methyl- or 2′-O-ethylribonucleoside 3′-O-phosphoramidites. Nucleic Acids Res. 19, 5965-5971. (20) Cotten, M., Oberhauser, B., Brunar, H., Holzner, A., Issakides, G., Noe, C. R., Schaffner, G., Wagner, E., and Birnstiel, M. L. (1991) 2′-O-methyl, 2′-O-ethyl oligoribonucleotides and phosphorothioate oligodeoxyribonucleotides as inhibitors of the in vitro U7 snRNP-dependent mRNA processing event. Nucleic Acids Res. 19, 2629-2635. (21) Werner, D., Brunar, H., and Noe, C. R. (1998) Investigations on the influence of 2′-O-alkyl modifications on the base pairing properties of oligonucleotides. Pharm. Acta Helv. 73, 3-10. (22) Lima, W. F., and Crooke, S. T. (1997) Binding affinity and specificity of Escherichia coli RNase H1: impact on the kinetics of catalysis of antisense oligonucleotide-RNA hybrids. Biochemistry. 36, 390-398. (23) Monia, B. P., Lesnik, E. A., Gonzalez, C., Lima, W. F., McGee, D., Guinosso, C. J., Kawasaki, A. M., Cook, P. D., and Freier, S. M. (1993) Evaluation of 2′-modified oligonucleotides containing 2′-deoxy gaps as antisense inhibitors of gene expression. J. Biol. Chem. 268, 14514-14522. (24) Manoharan, M., Prakash, T. P., Barber-Peoc′h, I., Bhat, B., Vasquez, G., Ross, B. S., and Cook, P. D. (1999) N-(2Cyanoethoxycarbonyloxy)succinimide: A New Reagent for Protection of Amino Groups in Oligonucleotides. J. Org. Chem. 64, 6468-6472. (25) Summerton, J. (1999) Morpholino antisense oligomers: the case for an RNase H-independent structural type. Biochim. Biophys. Acta 1489, 141-158. (26) Braasch, D. A., and Corey, D. R. (2001) Locked nucleic acid (LNA): fine-tuning the recognition of DNA and RNA. Chem. Biol. 8, 1-7. (27) Braasch, D. A., Liu, Y., and Corey, D. R. (2002) Antisense inhibition of gene expression in cells by oligonucleotides incorporating locked nucleic acids: effect of mRNA target sequence and chimera design. Nucleic Acids Res. 30, 51605167. (28) Tung, C.-H., and Stein, S. (2000) Preparation and Applications of Peptide-Oligonucleotide Conjugates. Bioconjugate Chem. 11, 605-618. (29) Letsinger, R. L., and Chaturvedi, S. (1998) Tailored hydrophobic cavities in oligonucleotide-steroid conjugates. Bioconjugate Chem. 9, 826-830. (30) Shea, R. G., Marsters, J. C., and Bischofberger, N. (1990) Synthesis, hybridization properties and antiviral activity of lipid-oligodeoxynucleotide conjugates. Nucleic Acids Res. 18, 3777-3783. (31) Stein, S., and Zhu, T. (1997) Preparation of vitamin B6peptide and vitamin B6-peptide-oligonucleotide conjugates. Methods Enzymol. 280, 51-58. (32) Maier, M. A., Yannopoulos, C. G., Mohamed, N., Roland, A., Fritz, H., Mohan, V., Just, G., and Manoharan, M. (2003) Synthesis of antisense oligonucleotides conjugated to a multivalent carbohydrate cluster for cellular targeting. Bioconjugate Chem. 14, 18-29. (33) Derossi, D., Chassaing, G., and Prochiantz, A. (1998) Trojan peptides: the penetratin system for intracellular delivery. Trends Cell Biol. 8, 84-87. (34) Pardridge, W. M., Boado, R. J., and Kang, Y.-S. (1995) Vector-mediated delivery of a polyamide (\“peptide\”) nucleic acid analog through the blood-brain barrier in vivo. Proc. Natl. Acad. Sci. U S A. 92, 5592-5596. (35) Branden, L. J., Mohamed, A. J., and Smith, C. I. E. (1999) A peptide nucleic acid-nuclear localization signal fusion that mediates nuclear transport of DNA. Nat. Biotechnol. 17, 784787. (36) Benimetskaya, L., Guzzo-Pernell, N., Liu, S. T., Lai, J. C., Miller, P., and Stein, C. A. (2002) Protamine-fragment peptides fused to an SV40 nuclear localization signal deliver oligonucleotides that produce antisense effects in prostate and bladder carcinoma cells. Bioconjugate Chem. 13, 177-187. (37) Astriab-Fisher, A., Sergueev, D., Fisher, M., Shaw, B. R., and Juliano, R. L. (2002) Conjugates of Antisense Oligonucleo-
1044 Bioconjugate Chem., Vol. 16, No. 4, 2005 tides with the Tat and Antennapedia Cell-Penetrating Peptides: Effects on Cellular Uptake, Binding to Target Sequences, and Biologic Actions. Pharm. Res. 19, 744-754. (38) Fisher, A. A., Ye, D., Sergueev, D. S., Fisher, M. H., Shaw, B. R., and Juliano, R. L. (2002) Evaluating the specificity of antisense oligonucleotide conjugates. A DNA array analysis. J. Biol. Chem. 277, 22980-22984. (39) Leonetti, J. P., Degols, G., and Lebleu, B. (1990) Biological activity of oligonucleotide-poly(L-lysine) conjugates: mechanism of cell uptake. Bioconjugate Chem. 1, 149-153. (40) Degols, G., Leonetti, J. P., Benkirane, M., Devaux, C., and Lebleu, B. (1992) Poly(L-lysine)-conjugated oligonucleotides promote sequence-specific inhibition of acute HIV-1 infection. Antisense Res. Dev. 2, 293-301. (41) Clarenc, J. P., Degols, G., Leonetti, J. P., Milhaud, P., and Lebleu, B. (1993) Delivery of antisense oligonucleotides by poly(L-lysine) conjugation and liposome encapsulation. AntiCancer Drug Des. 8, 81-94. (42) Ginobbi, P., Geiser, T. A., Ombres, D., and Citro, G. (1997) Folic acid-polylysine carrier improves efficacy of c-myc antisense oligodeoxynucleotides on human melanoma (M14) cells. Anticancer Res. 17, 29-35. (43) Sazani, P., Kang, S. H., Maier, M. A., Wei, C., Dillman, J., Summerton, J., Manoharan, M., and Kole, R. (2001) Nuclear antisense effects of neutral, anionic and cationic oligonucleotide analogs. Nucleic Acids Res. 29, 3965-3974. (44) Eritja, R., Pons, A., Escarceller, M., Giralt, E., and Albericio, F. (1991) Synthesis of defined peptide-oligonucleotide hybrids containing a nuclear transport signal sequence. Tetrahedron 47, 4113-4120. (45) 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. (46) Corey, D. R., Munoz-Medellin, D., and Huang, A. (1995) Strand invasion by oligonucleotidesnuclease conjugates. Bioconjugate Chem. 6, 93-100. (47) Haralambidis, J., Chai, M., and Tregear, G. W. (1987) Preparation of base-modified nucleosides suitable for nonradioactive label attachment and their incorporation into synthetic oligodeoxyribonucleotides. Nucleic Acids Res. 15, 4857-4876. (48) Zaramella, S., Yeheskiely, E., and Stromberg, R. (2004) A method for solid-phase synthesis of oligonucleotide 5′-peptideconjugates using acid-labile alpha-amino protections. J. Am. Chem. Soc. 126, 14029-14035. (49) Manoharan, M., Tivel, K. L., and Cook, P. D. (1995) Lipidic nucleic acids. Tetrahedron Lett. 36, 3651-3654. (50) Manoharan, M., Inamati, G., Tivel, K. L., Conklin, B., Ross, B. S., and Cook, P. D. (1997) 2′- and 3′- cholesterol-conjugated adenosine and cytosine nucleoside building blocks: synthesis of lipidic nucleic acids. Nucleosides Nucleotides 16, 11411143. (51) Hwang, J.-T., and Greenberg, M. M. (2001) Synthesis of 2′-Modified Oligodeoxynucleotides via On-Column Conjugation. J. Org. Chem. 66, 363-369.
Technical Notes (52) Kachalova, A., Zubin, E., Stetsenko, D., Gait, M., and Oretskaya, T. (2004) Oligonucleotides with 2′-O-carboxymethyl group: synthesis and 2′-conjugation via amide bond formation on solid phase. Org. Biomol. Chem. 2, 2793-2797. (53) Winkler, J., Urban, E., Losert, D., Wacheck, V., Pehamberger, H., and Noe, C. R. (2004) A novel concept for ligand attachment to oligonucleotides via a 2′-succinyl linker. Nucleic Acids Res. 32, 710-718. (54) Vu, H., and Hirschbein, B. L. (1991) Internucleotide phosphite sulfurization with tetraethylthiuram disulfide. Phosphorothioate oligonucleotide synthesis via phosphoramidite chemistry. Tetrahedron Lett. 32, 3005-3008. (55) Pieles, U., Zurcher, W., Schar, M., and Moser, H. E. (1993) Matrix-assisted laser desorption ionization time-of-flight mass spectrometry: a powerful tool for the mass and sequence analysis of natural and modified oligonucleotides. Nucleic Acids Res. 21, 3191-3196. (56) Gilbert, M. (1999) Optimierung von Antisense-Oligonukleotiden - zwitterionische Nukleinsa¨ uren und Tra¨ gersystemvariationen, Shaker Verlag, Aachen, Germany. (57) Griffey, R. H., Monia, B. P., Cummins, L. L., Freier, S., Greig, M. J., Guinosso, C. J., Lesnik, E., Manalili, S. M., Mohan, V., Owens, S., Ross, B. R., Sasmor, H., Wancewicz, E., Weiler, K., Wheeler, P. D., and Cook, P. D. (1996) 2′-Oaminopropyl ribonucleotides: a zwitterionic modification that enhances the exonuclease resistance and biological activity of antisense oligonucleotides. J. Med. Chem. 39, 5100-5109. (58) 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. (59) de La Torre, B. G., Albericio, F., Saison-Behmoaras, E., Bachi, A., and Eritja, R. (1999) Synthesis and binding properties of oligonucleotides carrying nuclear localization sequences. Bioconjugate Chem. 10, 1005-1012. (60) Henkel, B., Zhang, L., and Bayer, E. (1997) Investigations on solid-phase peptide synthesis in N-to-C direction (inverse synthesis). Liebigs Annalen/Recueil. 2161-2168. (61) Gutheil, W. G., and Xu, Q. (2002) N-to-C solid-phase peptide and peptide trifluoromethylketone synthesis using amino acid tert-butyl esters. Chem. Pharm. Bull. 50, 688691. (62) Zaramella, S., Yeheskiely, E., and Stromberg, R. (2004) A method for solid-phase synthesis of oligonucleotide 5′-peptideconjugates using acid-labile alpha-amino protections. J. Am. Chem. Soc. 126, 14029-14035. (63) Clark, C. L., Cecil, P. K., Singh, D., and Gray, D. M. (1997) CD, absorption and thermodynamic analysis of repeating dinucleotide DNA, RNA and hybrid duplexes [d/r(AC)]12.[d/ r(GT/U)]12 and the influence of phosphorothioate substitution. Nucleic Acids Res. 25, 4098-4105. (64) Greenfield, N. J., and Fasman, G. D. (1969) Computed circular dichroism spectra for the evaluation of protein conformation. Biochemistry. 8, 4108-4116.
BC049729D
