New Reagents for the Introduction of Reactive Functional Groups into

Chandrasekhar V. Miduturu and Scott K. Silverman. The Journal of Organic ... Zbigniew Skrzypczynski and Sarah Wayland. Bioconjugate Chemistry 2004 15 ...
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Bioconjugate Chem. 2003, 14, 642−652

New Reagents for the Introduction of Reactive Functional Groups into Chemically Synthesized DNA Probes Zbigniew Skrzypczynski* and Sarah Wayland Third Wave Technologies, 502 S. Rosa Road, Madison, Wisconsin 53719. Received December 19, 2002; Revised Manuscript Received March 10, 2003

An efficient and versatile preparative approach is described, allowing for the preparation of DNA probes modified with an aldehyde group at the 3′- or 5′-end. The developed synthetic strategy allows for the preparation of a new family of phosphoramidites and solid supports compatible with the automated synthesis of modified oligonucleotide probes. These new reagents were prepared from intermediates 3 and 3a, obtained from the commercially available aleuritic acid 1. It was demonstrated that the new phosphoramidite reagents also could be used as new types of cleavable linkers. A new and efficient method for the production of 5′ aldehyde-labeled DNA probes was developed.

INTRODUCTION

The discovery of the phosphoramidite method, which enables automated synthesis of natural and modified DNA molecules (1-4), has stimulated the development of numerous reagents and methods to introduce a specific modification or functional group at a selected position within a synthesized oligonucleotide (5-19). Some phosphoramidite labeling reagents are commercially available. However, escalating interest in the use of chemically modified, synthetic oligonucleotides in the disciplines of biology, medicine, and biotechnology (20-30) has intensified the need for less expensive and more broadly applicable labeling reagents. Despite its popularity and efficiency, automated oligonucleotide synthesis does not always address all synthetic requirements. Efforts are focused increasingly on the development of new postsynthetic strategies for the preparation of oligonucleotide conjugates with other molecules and biological moieties, as well as on new protocols for immobilizing DNA onto solid surfaces. In our search for new options to address our synthesis goals, we focused on commercially available aleuritic acid (DL-erythro-9,10,16-trihydroxypalmitic acid) 1 (Figure 1) as a valuable starting material for the preparation of new versatile labeling reagents. The structural features of aleuritic acid 1 offer ample opportunities for the preparation of new types of phosphoramidite reagents that can introduce both a carboxyl group and a masked aldehyde group into a synthesized DNA probe or can be used for the preparation of a new family of cleavable linkers. Carboxyl and aldehyde groups play particularly significant roles in the postsynthetic modification of oligonucleotide probes (31-47), the preparation of oligonucleotide conjugates (48-59), and the immobilization of DNA probes onto amino-modified solid surfaces (60-70). Several recent papers highlighting the use of DNA probes modified with different functional groups at either their 3′- or 5′-terminus (60, 65, 68) demonstrate the superiority of the aldehyde group in postsynthetic conjugation and immobilization techniques compared to * To whom correspondence should be addressed: [telephone (608) 273-8933; fax (608) 273-8618; e-mail zskrzypczynski@ twt.com].

Figure 1. Structure of aleuritic acid 1.

other functional groups. We outline an efficient and flexible synthetic strategy that leads to the preparation of new phosphoramidite reagents, new cleavable linkers, and a new type of solid supportsall of which are useful in the preparation of DNA probes modified with an aldehyde group. MATERIALS AND METHODS

All new compounds, aldehyde-labeled DNA probes, and products of their conjugation with molecules containing a reactive primary amino group were characterized by HPLC and mass spectrometry (MS). HPLC analyses were performed with a Hitachi D-7000 Interface, L-7100 gradient pump, and L-7400 UV detector using a Varian Omnisphere 5 C18 column (250 × 4.6 mm) and 100 mM TEAA, pH 7/acetonitrile. 1H NMR spectra were recorded on a Varian UNITYINOVA instrument. MS analysis of all DNA-containing species was performed using a PerSeptive Biosystems Voyager-DE Biospectrometry Workstation V800520. MS analysis of small molecules was performed using an Applied Biosystems/MDS Sciex API 365 LC/MS/MS triple quadrapole with an electrospray ionization source. Automated oligonucleotide synthesis was performed using a PerSeptive Biosystems Expedite Nucleic Acid Synthesis System. Column chromatography purification used 70 × 230 mesh, 60 Å silica gel (Aldrich, Milwaukee, WI). Analytical TLC was carried out on EM Science F254 glass-backed fluorescence indicator plates. DNA synthesis reagents and columns were purchased from Applied Biosystems (Foster City, CA). Unless otherwise noted, reagents were purchased from Aldrich and used without further purification. Solvents were dried over activated 3 Å molecular sieves. Nap-10 columns were purchased from Amersham Pharmacia Biotech (Uppsala, Sweden). Synthesis of 2. A 250 mL round-bottom flask was charged with 2.2 g (7.2 mmol) aleuritic acid 1 (Lancaster

10.1021/bc025657j CCC: $25.00 © 2003 American Chemical Society Published on Web 04/10/2003

New Reagents for Chemical DNA Modification

Synthesis Inc, Windham, NH) and 100 mL of pyridine. Solid 4,4′-dimethoxytrityl chloride (3.2 g, 9.4 mmol) was added slowly and mixed for 1 h. The reaction was concentrated by rotary evaporation, and column chromatography purification (5%methanol/5%triethylamine/ methylene chloride) resulted in an off-white solid product with a yield of 4.0 g, 90%. Rf ) 0.35 (10% methanol/5% triethylamine/methylene chloride). 1H NMR, CD3CN, 400 MHz, δ 7.41 (m, 2H), 7.29 (m, 6H), 7.22 (t, 1H), 6.86 (m, 4H), 3.76 (s, 6H), 3.60 (m, 2H), 2.99 (t, 2H), 2.83 (m, 2H), 2.27 (t, 2H), 1.6-1.2 (aliphatic, 22H) ppm. ESI-MS for C37H50O7 (M + K)+: calculated 646; found 646. Synthesis of 3. Combined 2 (4.0 g, 6.6 mmol), N,Ndiisopropylethylamine (8.5 g, 66 mmol), N-methylimidazole (1.6 g, 20 mmol), 60 mL of tetrahydrofuran, and 60 mL of pyridine in a 250 mL round-bottom flask. With stirring at 0 °C under argon flow, acetic anhydride (3.4 g, 33 mmol) was added via syringe. After 1 h, the reaction mixture was concentrated by rotary evaporation. The crude reaction material was dissolved in methylene chloride (100 mL), washed with saturated sodium chloride (100 mL), and purified by column chromatography (5% methanol/5% triethylamine/methylene chloride). Compound 3 was obtained as a yellow oil (3.7 g, 81%). Rf ) 0.5 (5% methanol/5% triethylamine/methylene chloride). 1 H NMR, CD3CN, 400 MHz, δ 7.43 (m, 2H), 7.30 (m, 6H), 7.21 (t, 1H), 6.85 (m, 4H), 4.93 (m, 2H), 3.76 (s, 6H), 2.98 (t, 2H), 2.20 (t, 2H), 2.00 (s, 3H), 1.99 (s, 3H), 1.50 (m, 8H), 1.25 (m, 14H) ppm. ESI-MS for C41H54O90 (M + K)+: calculated 730; found 730. Synthesis of 3a. Dissolved 3 (see above, 1.6 g, 2.3 mmol) in 100 mL of tetrahydrofuran. Solid N-hydroxysuccinimide (0.30 g, 2.6 mmol) and 1,3-dicyclohexylcarbodiimide (0.72 g, 3.5 mmol) were added to the mixture, stirred at room temperature for 16 h under a drying tube, and concentrated by rotary evaporation. Crude material 3a was purified by column chromatography (50% ethyl acetate/50% hexane), resulting in a white solid with a yield of 1.1 g, 60%. Rf ) 0.62 (75% ethyl acetate/25% hexane). 1H NMR, CD3CN, 400 MHz, δ 7.43 (m, 2H), 7.30 (m, 6H), 7.23 (m, 1H), 6.85 (m, 4H), 4.93 (m, 2H), 3.76 (s, 6H), 2.99 (t, 2H), 2.75 (s, 4H), 2.58 (t, 2H), 2.00 (s, 3H), 1.99 (s, 3H), 1.6-1.2 (aliphatic, 22H) ppm. ESI-MS for C45H57NO11 (M + K)+: calculated 827; found 827. Synthesis of 4. A 100 mL round-bottom flask was charged with compound 3 (0.92 g, 1.3 mmol), 4-(dimethylamino)pyridine (210 mg, 1.7 mmol), 1,6-hexanediol (1.6 g, 13 mmol), and 50 mL of tetrahydrofuran. Solid 1,3dicyclohexylcarbodiimide (3.0 g, 14.3 mmol) was added, and the reaction mixture was stirred at room temperature under a drying tube for 16 h. Rotary evaporation and purification by column chromatography (2% triethylamine/49% ethyl acetate/49% hexane) yielded a white solid, 0.59 g, 59%. Rf ) 0.58 (75% ethyl acetate/25% hexane). 1H NMR, CD3CN, 400 MHz, δ 7.42 (m, 2H), 7.29 (m, 6H), 7.23 (t, 1H), 6.85 (m, 4H), 4.93 (m, 2H), 4.02 (t, 2H), 3.76 (s, 6H), 3.47 (q, 2H), 2.99 (t, 2H), 2.44 (t, 1H), 2.25 (t, 2H), 2.00 (s, 3H), 1.99 (s, 3H), 1.6-1.2 (aliphatic, 30H) ppm. ESI-MS for C47H66O10 (M + K)+: calculated 830; found 830. Synthesis of 5. 4 (0.59 g, 0.74 mmol), N,N-diisopropylethylamine (0.12 g, 0.89 mmol), and 4-(dimethylamino)pyridine (45 mg, 0.37 mmol) were combined in 50 mL of tetrahydrofuran. Solid diglycolic anhydride (0.13 g, 1.1 mmol) was added to the reaction mixture and stirred for 3.5 h under a drying tube. The solvent was evaporated, and purification by column chromatography (70 × 230 mesh, 60A silica, 5% methanol/5% triethylamine/methylene chloride) yielded a pale yellow oil (0.44 g, 70%).

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Rf ) 0.31 (5% methanol/5% triethylamine/methylene chloride). ESI-MS for C51H70O14 (M + H)+: calculated 906; found 906. Synthesis of 6. Four grams of lcaa-CPG (Glen Research, Sterling, VA, #20-0001, 1000A, 69 µmol/g) was added to a 50 mL round-bottom flask. Compound 5 (0.36 g, 0.36 mmol) was dissolved in 20 mL of pyridine and added to the CPG. 4-(Dimethylamino)pyridine (24 mg, 0.20 mmol), triethylamine (0.16 g, 1.6 mmol), N-hydroxysuccinimide (91 mg, 0.79 mmol), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.30 g, 1.6 mmol) were added, and the reaction mixture was vortexed at RT for 16 h. Additional aliquots of 1-ethyl-(3dimethylaminopropyl)carbodiimide hydrochloride (total of 0.60 g, 3.2 mmol) and 5 (total of 0.10 g, 0.01 mmol) were added to the reaction slurry to achieve a final loading of at least 20 µmol 5/g CPG. The support was filtered, washed with acetonitrile, and dried with argon flow. The material was capped with an equal mixture of 6% 4-(dimethylamino)pyridine in acetonitrile and 2/3/5 (acetic anhydride/2,4,6-collidine/acetonitrile) (100 mL total volume) for 2 h. The support was filtered, washed with pyridine, methanol, and methylene chloride, and dried overnight under vacuum. By measuring the absorbance at 504 nm of a solution containing a known mass of CPG and a known volume of 3% dichloroacetic acid/ methylene chloride, to determine the concentration of the released trityl cation, the amount of 5 conjugated to the CPG was calculated to be 22.8 µmol/g CPG. Synthesis of 10a. A 100 mL round-bottom flask was charged with material 3a (0.52 g, 0.66 mmol), N,Ndiisopropylethylamine (0.13 g, 0.99 mmol), 6-amino-1hexanol (0.093 g, 0.80 mmol), and 50 mL of acetonitrile. The reaction mixture was stirred for 45 min at RT under a drying tube and concentrated by rotary evaporation. The reaction product was purified by column chromatography (5% methanol/5% triethylamine/methylene chloride), yielding a pale yellow oil, 0.44 g, 84%. Rf ) 0.47 (5% methanol/5% triethylamine/methylene chloride). 1H NMR, CD3CN, 400 MHz, δ 7.42 (m, 2H), 7.29 (m, 6H), 7.23 (t, 1H), 6.85 (m, 4H), 6.28 (s, broad, 1H), 4.93 (m, 2H), 3.76 (s, 6H), 3.47 (q, 2H), 3.10 (q, 2H), 2.98 (t, 2H), 2.51 (t, 1H), 2.05 (t, 2H), 2.00 (s, 3H), 1.99 (s, 3H), 1.61.2 (aliphatic, 30H) ppm. ESI-MS: calculated for C47H67NO9 (M + K)+ 829; found 829. Synthesis of 11a. Material 10a (0.40 g, 0.51 mmol) was coevaporated three times with 20 mL of acetonitrile and dissolved in 4 mL of methylene chloride. 2-Cyanoethyl N,N,N′,N′-tetraisopropylphosphorodiamidite (0.19 g, 0.61 mmol) was added, followed by the addition of a solution of 1H-tetrazole in acetonitrile (28 mg, 0.40 mmol/2 mL) with vigorous swirling. The reaction mixture was vortexed at RT for 2 h. Methylene chloride (25 mL) was added to the reaction to increase the volume, and the crude solution was washed with 25 mL of 5% sodium bicarbonate/0.5% triethylamine. The organic layer was dried over magnesium sulfate for 10 min, filtered, concentrated, and coevaporated twice with (20 mL) acetonitrile. The residue was dissolved in acetonitrile (15 mL) and dried over several granules of calcium hydride. The product solution was aliquoted (five aliquots of 3 mL, 100 µmol) into amber Expedite bottles, concentrated by aspiration vacuum, then dried overnight under vacuum in a desiccator containing phosphorus pentoxide (0.49 g, 98%). Rf ) 0.48 (5% triethylamine/dioxane). Synthesis of 10b. The method for the synthesis of 10a was followed using 3a (0.18 g, 0.22 mmol), N,N-Diisopropylethylamine (0.04 g, 0.29 mmol), PEG3400-amine (Shearwater Corporation, Huntsville, AL, 0.50 g, 0.15

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mmol), and 50 mL of acetonitrile. The crude product was purified by column chromatography (2% methanol/5% triethylamine/methylene chloride). Compound 10b was obtained as a white solid with a yield higher than the theoretical value due to the hygroscopic character of the material. Rf ) 0.29 (5% methanol/5% triethylamine/ methylene chloride). ESI-MS: calculated for C195H363NO85 (M + H)+ 4080, product displayed a PEG mass dispersion centered around 4100. Synthesis of 11b. The method for the synthesis of 11a was followed using 10b (0.40 g, 0.01 mmol), 2-Cyanoethyl N,N,N′,N′-tetraisopropylphosphorodiamidite (0.04 g, 0.12 mmol), and 1H-tetrazole in acetonitrile (5 mg, 0.08 mmol/1 mL). The product yield was 0.23 g, 56%. Rf ) 0.69 (5% triethylamine/dioxane). Synthesis of Mono-dimethoxytrityl-1,12-dodecanediol. Combined 1,12-dodecanediol (4 g, 20 mmol), N,N-diisopropylethylamine (1.3 g, 9.9 mmol), and 100 mL of THF. 4,4′-Dimethoxytrityl chloride (2.2 g, 6.6 mmol) was added slowly as a solid. After 1 h, the reaction was concentrated by rotary evaporation, and the product was purified by column chromatography (25% ethyl acetate/ 75% hexane), yielding a colorless oil, 3.3 g, 99%. Rf ) 0.39 (50% ethyl acetate/50% hexane). ESI-MS: calculated for C33H44O4 (M + K)+ 543, found 543. Synthesis of Dodecanediol Phosphoramidite. The method for the synthesis of 11a was followed using monodimethoxytrityl-1,12-dodecanediol (0.60 g, 1.2 mmol, 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphorodiamidite (0.43 g, 1.4 mmol), and 1H-tetrazole in acetonitrile (64 mg, 0.92 mmol/3 mL). The product yield was 0.80 g, 96%. Rf ) 0.42 (75% ethyl acetate/25% hexane). Cleavage and Deprotection of Aleuritic AcidConjugated Oligonucleotides Leading to the Formation of 7, 12a, and 12b. After synthesis of the aleuritic acid DNA conjugate on a 1µmol scale, the product was cleaved and deprotected in a solution of 20% 0.4 M sodium hydroxide/methanol (1 mL) for 3 h at 55 °C. The slurry was filtered, and the filtrate was neutralized with 4 µL of 80% acetic acid and desalted on a NAP10 column. Oxidation of Aleuritic Acid-Conjugated Oligonucleotides (7, 12a, and 12b) Leading to the Preparation of Aldehyde-Modified Probes (8, 13a, and 13b). A 1 µmol synthesis of aleuritic acid-DNA conjugate was dissolved in 0.9 mL of 100 mM sodium phosphate buffer, pH 7.6, and 0.1 mL of 100 mM sodium periodate in 100 mM sodium phosphate buffer, pH 7.6. The reaction solution was incubated in the dark for 30 min at RT, then purified on a NAP-10 column. Coupling of Aldehyde-Modified Oligonucleotides to Amine-Containing Compounds. Preparation of 9a, 9b, and 15. A solution of amine (25 µmol) was prepared in 1 mL of reaction buffer (for synthesis of 9a: 25 mM sodium borate, pH 9.5; for synthesis of 9b: 8% DMSO/25% methanol/25 mM sodium borate buffer, pH 9.5; for synthesis of 15: 10% methanol/30% acetonitrile/ 25 mM sodium borate buffer, pH 9.5). The aldehydelabeled oligonucleotide (1 µmol) was dissolved in the appropriate amine-containing reaction buffer, and 10 µL of 5 M sodium cyanoborohydride in 1 N sodium hydroxide was added. The pH of the reaction solution was adjusted between 9 and 10 with either 80% acetic acid or 1 N sodium hydroxide, and the reaction solution was incubated at RT. The crude reactions were monitored by HPLC analysis and, upon completion (3 h), were purified on NAP-10 columns. The products (9a, 9b, and 13a) were analyzed by MS to confirm product formation.

Skrzypczynski and Wayland

Figure 2. Synthesis of the NHS ester of the selectively protected aleuritic acid 3a.

Protocol for the Preparation of the 5′-AldehydeModified DNA Probes using C18 Oligonucleotide Purification Cartridge. The purification was performed using Supelco, Supelclean Envi-18, 1 g, 12 mL tubes. All additions were allowed to completely enter the gel bed before initiating the next step. The resin was equilibrated with one wash each of 5 mL of methanol, 5 mL of water, and 5 mL of 0.5 M sodium chloride. The DNA sample was loaded onto the column in an equal volume of water and 0.5 M sodium chloride (3 mL total volume). The column was washed with 5 mL of 0.5 M sodium chloride, followed by a 1 mL addition of 100 mM sodium periodate solution which was allowed to incubate on the column for 15 min. The column was washed with 5 mL of water, then 1.5 mL of 10% methanol/water to elute the cleaved fragments. The final product was eluted with 1.5 mL of methanol/water 1:1. RESULTS AND DISCUSSION

Attachment of Aleuritic Acid Moiety to the 3′Loci of the DNA Probes. Synthesis of the Aleuritic Acid CPG. In recent years, the chemical literature has reported numerous methods for synthesizing different phosphoramidite reagents used to introduce single or multiple functional groups at the 3′ or 5′ terminus of a synthesized DNA oligonucleotide (5-19). In contrast, far fewer methods are reported for utilizing a specifically modified solid support to synthesize 3′-modified oligonucleotides (13, 71-82), most likely due to the synthetic inconveniences associated with the preparation of such reagents. We found that the approach of synthesizing a solid support modified with an aleuritic acid moiety represents a very attractive way to introduce a carboxyl or masked aldehyde group into the 3′-end of a synthesized DNA oligonucleotide, leaving the 5′-teminus available for other modifications. In the first step of our synthetic strategy, we prepared selectively protected derivatives of aleuritic acid 3 and 3a that can serve as intermediates for further preparations of modified solid supports or for the synthesis of new phosphoramidite reagents (Figure 2). Known differences in the reactivity between the primary and secondary hydroxyl groups (83) facilitated the

New Reagents for Chemical DNA Modification

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Figure 3. Preparation of the aleuritic acid CPG 6.

synthesis and chromatographic isolation of the aleuritic acid derivative 2 with a DMT-protected primary hydroxyl group. In the subsequent step of the procedure, derivative 2 was treated with acetic anhydride in pyridine and converted into intermediate compound 3, in which both the primary and the secondary hydroxyl groups were selectively protected with the DMT and the acetyl groups. In the next step, the fully protected compound 3 was converted into the N-hydroxysuccinimide (NHS) ester 3a. Synthesized compounds 3 and 3a can serve as a key intermediates for the synthesis of a variety of reagents used in the introduction of the modification into DNA probes. Figure 3 illustrates the use of the compound 3 in the preparation of a new CPG material modified with the aleuritic acid moiety. In the initial step, compound 3 was converted into the 1,6-hexanediol monoester 4. In the next step, the free hydroxyl group of the ester 4 was reacted with diglycolic anhydride (85) to form the diglycolic monoester 5. Finally, the diglycolic monoester 5 was coupled to the lcaa-CPG support, yielding solid support 6 modified with the aleuritic acid moiety. In all synthetic steps leading to the production of the solid support 6, standard reaction conditions and coupling protocols were employed (1, 2, 75, 84, 85). The efficiency of the coupling reaction of the diglycolic monoester 5 to the lcaa-CPG solid support was estimated by measuring the concentration of the DMT cation released from the support 6 after treatment with 3% dichloroacetic acid in dichloromethane. Typically, the observed loading of the DMT-protected aleuritic acid on the synthesized solid support 6 was in the range of 22-26 µmol/g. To examine the applicability of the prepared solid support 6 to the synthesis of 3′-modified DNA probes, we tested this material under the conditions of automated oligonucleotide synthesis. To perform the synthesis, an appropriate amount of the solid support 6, containing 1 µmol of the attached aleuritic acid, was loaded into cartridges compatible with the PerSeptive Biosystems Expedite automated DNA synthesizer. The standard phosphoramidite coupling protocol was applied for the synthesis of the DNA probes. In the first experiment, we synthesized a 3′-modified dinucleotide 7 (n ) 2) as a

Figure 4. Structure of the dT dinucleotide 7 (n ) 2), modified at its 3′-end with the aleuritic acid moiety.

convenient model material for the study of cleavage and deprotection conditions that would lead to formation of a final product with a deprotected carboxyl group and fully deprotected vicinal hydroxyl groups (Figure 4). We expected the relatively low molecular weight of compound 7 (n ) 2) to facilitate the reversed-phase (RP) HPLC analysis of the reaction product, given that any partially deprotected derivatives of the desired final product would be more easily separated and detected. In our initial experiments with compound 7 (n ) 2), we followed the standard protocol widely used for the cleavage and deprotection of chemically synthesized oligonucleotides (concentrated ammonia, 6 to 12 h at 55 °C) (1-4). RP HPLC analysis (data not shown) of the generated reaction product revealed that such deprotection conditions yielded multiple compounds regardless of the length of contact of the synthesized material with concentrated ammonia. The disappearance of certain compounds by use of extended incubation in concentrated ammonia suggested that the crude product of the cleavage and deprotection reaction contained incompletely deprotected final material. The nature of other compounds present in the crude material was not studied. We achieved formation of a uniform and fully deprotected product 7 (n ) 2) by replacing the concentrated ammonia with a solution of 80% methanol/20% 0.2 M NaOH/H2O and incubating the protected compound 7 (n ) 2) for 3 h at 55 °C (conditions recommended by GlenResearch to cleave and deprotect DNA probes synthesized on 3′carboxylate photolabile CPG; see also ref 75). When this deprotection protocol was applied to the synthesis of longer, 3′-labeled oligonucleotides 7 (n ) 10 and n ) 20), we synthesized the desired products with efficiency and purity comparable to those achieved in the synthesis of

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Figure 5. (A) RP HPLC of the crude unmodified dT20 cleaved from the solid support with concentrated ammonia. (B) RP HPLC of the 3′-modified probe 7 (n ) 20) cleaved from the solid support 6 with a solution of 80% methanol/20% 0.2 M NaOH/H2O for 3 h at 55 °C.

Figure 6. Sodium periodate oxidation of the aleuritic acid moiety attached to the 3′-loci of the probes 7 (n ) 2, 10, 20).

unmodified dT oligomers of the same length. Figure 5 compares the RP-HPLC analytical profile of crude material 7 (n ) 20), synthesized on solid support 6 and deprotected with adapted protocol, to that of the crude 3′-unmodified dT-20-mer synthesized and deprotected according to the standard protocols. All synthesized compounds 7 (n ) 2, 10, 20) labeled at the 3′-end with the aleuritic acid moiety were isolated by RP HPLC, and their identities were confirmed by MS. As mentioned above, the structural features of the aleuritic acid attached to the synthesized oligonucleotide probe allow for the efficient introduction not only of a carboxyl group, but also a masked aldehyde group. Vicinal diols can be readily oxidized by a solution of sodium periodate under very mild conditions. This reaction leads to the bond cleavage between carbon atoms bearing vicinal hydroxyl groups and to the formation of two molecules containing aldehyde functional groups (36, 40). The two newly formed molecules may be identical or different, depending on the symmetry of the oxidized vicinal diol. Such a synthetic approach was reported in a number of cases describing the preparations of aldehyde-modified DNA probes and oligonucleotide bioconjugates, as well as in the immobilization protocols of DNA probes onto solid surfaces (31-70). We selected previously synthesized compounds 7 (n ) 2, 10, and 20) as models to study the conditions of the sodium periodate oxidation of vicinal hydroxyl groups present in the structure of aleuritic acid linked to the 3′-ends of the synthesized DNA probes 7 (Figure 6).

Oxidation of compounds 7 (n ) 2, 10, 20) was performed with 100 mM sodium periodate in 100 mM sodium phosphate buffer, pH 7.5, at room temperature (46, 97). The corresponding 3′-aldehyde labeled DNA probes 8 (n ) 2, 10, 20) were formed quickly and efficiently. Monitoring the progress of the oxidation reaction by RP HPLC revealed that the full conversion of compounds 7 (n ) 10, 20) into the corresponding 3′aldehyde labeled derivatives 8 (n ) 10, 20) occurred in less than 5 min (Figure 7). The structure of the oxidation products 8 (n ) 2, 10, 20), isolated by RP HPLC, was confirmed by MS. To demonstrate the utility of synthesized 3′-aldehydelabeled probe 8 (n )10) as a starting material in the postsynthetic conjugation protocols, we used this material as a substrate in the reductive amination reaction with 1-pyrene methylamine and 4,7,10-trioxa-1,13-tridecanediamine (Figure 8). The conjugation of material 8 (n ) 10) to water soluble 4,7,10-trioxa-1,13-tridecanediamine proceeded with excellent yield under standard reaction conditions (97), producing conjugate 9a. The conjugation reaction of the 1-pyrene methylamine to material 8 (n ) 10), leading to the formation of the conjugate 9b, was, however, negatively affected by the low solubility of the 1-pyrene methylamine hydrochloride in aqueous media. The use of a solvent system containing 25% methanol and 8% of DMSO was necessary to achieve 60% coupling yield of the 1-pyrene methylamine to the 3′-aldehyde labeled probe 8 (n ) 10). Phosphoramidites of Aleuritic Acid Derivatives. The synthesis of new reagents allowing for the introduction of an aldehyde group into DNA probes has been reported recently (40, 41, 43, 44, 55, 68). This research indicates a renewed interest in the synthesis and application of the aldehyde group not only in the preparation of modified DNA probes, but also in the immobilization of modified oligonucleotides onto solid surfaces (60, 63, 65, 68, 70). During the course of our study, we have found that the aleuritic acid intermediate 3a serves as a convenient intermediate in the synthesis of a new family of structurally diverse phosphoramidites 11, capable of introducing a masked aldehyde group into a synthesized DNA probe (Figure 9). Group R in the synthesized phosphoramidites 11 can be selected from a variety of organic molecules, depending on the length of the linker or its desired chemical, physical or spectral properties (86-94). As an example, we synthesized two new phosphoramidites: phosphoramidite 11a, R ) C6H12, and phosphoramidite 11b, R ) PEG, MW 3400. Both phosphoramidites 11a and 11b

New Reagents for Chemical DNA Modification

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Figure 7. RP HPLC analysis of the 3′-aleuritic acid-labeled probes 7: (A) compound 7 (n ) 10); (B) compound 8 (n ) 10); (C) compound 7 (n ) 20); and the RP HPLC analysis of the crude product of their oxidation with sodium periodate after 5 min; (D) compound 8 (n ) 20). All analytical runs were done using 2%/min acetonitrile gradient.

Figure 8. Reductive amination coupling reaction of the 3′aldehyde-labeled probe 8 (n ) 10) to 4,7,10-trioxa-1,13-tridecanediamine and 1-pyrene methylamine.

were successfully applied as reagents in the automated synthesis of two different 5′-modified DNA probes, 12a

Figure 9. The synthesis of the phosphoramidites 11a and 11b.

(n ) 2), R ) C6H12 and 12b (n ) 10), R ) PEG, MW 3400. Both DNA probes 12a and 12b were subsequently oxidized to the 5′-aldehyde-modified probes 13a and 13b (Figure 10). During the course of these experiments, phosphoramidites 11a and 11b were proven to be fully compatible with the standard phosphoramidite protocol for automated DNA synthesis. With phosphoramidite 11b, however, a lower coupling yield was observed. Lower coupling efficiencies of phosphoramidite reagents used to introduce long linker molecules have been reported previously (86, 94). The synthesis of the phosphoramidite 11b deserves special attention. Due to their chemical and physiological properties, conjugates of poly(ethylene glycol) (PEG) with other biologically active molecules (enzymes, peptides, and oligonucleotides) represent a very important class of bioconjugates (98). Recent chemical literature reports many synthetic efforts leading to the preparation of such

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Figure 10. Synthesis of the 5′-aldehyde-modified DNA probes 13a and 13b.

Figure 11. Reductive amination coupling of the 5′-aldehyde modified probe 13a to the bis(3-aminopropyl)-terminated polytetrahydroduran 14.

molecules (91-94). In our experiments, we demonstrated that the PEG-modified oligonucleotide probe 12b, containing a masked aldehyde group, could be synthesized using phosphoramidite 11b in conjunction with the standard phosphoramidite protocol for automated oligonucleotide synthesis (Figure 10). The effectiveness of the reductive amination reaction, leading to the formation of the 3′-amino labeled compound 9a, prompted us to test the efficiency of the reductive amination reaction between the 5′-aldehyde labeled compound 13a and bis(3-aminopropyl)-terminated polytetrahydroduran 14, (Average Mn ca. 1100) (Figure 11). The desired reaction product 15, in which the primary amino group was linked to the 5′-loci of the oligonucleotide by a long, nonlipophilic polymeric linker, was formed with excellent yield. MS analysis of the HPLC-purified material 15 revealed the presence of products with mass equal to the sum of the masses of the compounds 13a and 14. Synthetic flexibility in the preparation of struc-

turally different phosphoramidites 11 and the possibility of their combined use with phosphoramidites of other reagents opens new avenues allowing for the synthesis of structurally diverse DNA probes modified with the aldehyde group. Such probes can be used in the immobilization protocols of DNA probes onto solid surfaces (60-70) and in the preparation of specific DNA conjugates with performance dependent upon the character and the length of the linker attached the oligonucleotide moiety (86, 88). Aleuritic Acid Phosphoramidites as Cleavable Linkers. Synthesized phosphoramidite 11a (R ) C6H12) can be used not only as a reagent for the introduction of an aldehyde group into the 5′-end of the DNA probe, but also as a new type of cleavable linker (95), useful in the preparation of specific DNA probes or conjugates. We tested the efficiency of this approach by the synthesis of three model compounds 16-18, each containing two units that were linked by phosphoramidite 11a (R ) C6H12) (Figure 12). The difference in size and chemical character between compounds 16-18 and the products of their sodium periodate oxidation facilitated RP HPLC monitoring of the progress of cleaved product formation. At room temperature, the sodium periodate oxidation of the vicinal diols of the aleuritic acid linker in probes 16-18 was fast (less than 15 min) and led to the formation of the dT10 DNA probes modified with the aldehyde group at the 5′- or 3′-end. Oligonucleotides modified with the aldehyde group and formed as products of the sodium periodate oxidation of compounds 16-18 were isolated by RP HPLC (data not shown), and their identity was confirmed by MS. The efficiency and ease with which the aleuritic acid linker

Figure 12. Synthesized compounds 16, 17, and 18. 5′-Fluorescein phosphoramidite (FAM) was purchased from Glen Research (Sterling, VA).

New Reagents for Chemical DNA Modification

Figure 13. Preparation of the 5′-aldehyde-modified probe 13a via sodium periodate oxidation of the 5′-aleuritic acid-modified probe 17 absorbed on the C18-OPC cartridge.

was cleaved by the sodium periodate solution led us to the development of a new, efficient method for the production of DNA probes labeled with the aldehyde group at the 5′ end, without time-consuming chromatographic purification of the final material. The method is conceptually similar to the “DMT-ON” purification protocol that is widely used in the separation of the “5′-DMTON,” full-length DNA sequences from truncated fragments formed during the automated solid-phase synthesis of oligonucleotides (96). The “DMT-ON” purification method uses commercially available C18-oligonucleotide purification columns capable of retaining oligonucleotides modified with lipophilic (e.g., DMT) groups, while allowing elution of unmodified and truncated DNA fragments with an appropriate buffer. In the next step of the “DMTON” purification protocol, an acid solution is applied to the column, which leads to the cleavage of the DMT group from the retained full-length sequence. Subsequently, the full-length 5′-deprotected product is eluted from the column with another buffer addition. Generally, this purification protocol works well for 5′-DMT-protected oligonucleotides. However, the inherent instability of the DMT protecting group can occasionally cause its partial loss and, in consequence, the full-length product may be eluted along with shorter fragments in the initial step of the purification. In our method, we adapted the “DMT-ON” purification protocol with a minor modification to the preparation and purification of the 5′-aldehyde-modified DNA probe synthesized with phosphoramidite 11a (R ) C6H12). To ensure the presence of the lipophilic group at the 5′-loci of the synthesized material, we used phosphoramidite of the DMT-protected 1-dodecanol to incorporate the desired, more stable (in comparison to the DMT group), moiety into the DNA probe 17. In the initial step of the developed method, crude product 17 (R ) C12H25) was applied to the C18-OPC cartridge (Supelco, Supelclean Envi-18), and all truncated and unmodified oligonucleotide fragments were eluted according to the standard purification protocol (see Experimental Section). Next, 1 mL of the 100 mM solution of sodium periodate was applied on the column. After the sodium periodate solution was fully absorbed, the column was left for 15 min at room temperature. This step led to the oxidation and cleavage of the aleuritic acid of the 5′-modified DNA probe 17 still retained on the column and to the formation of the 5′-aldehyde-modified DNA probe 13a. Subsequently, the sodium periodate solution was removed by washing the column with deionized water. Finally, the desired 5′-aldehyde-labeled full-length product 13a was efficiently eluted using a methanol:water (1:1) solution. An HPLC analysis of the eluted material 13a demonstrated its high purity (Figure 13). MS analysis of the HPLC isolated material 13a confirmed its structure.

Bioconjugate Chem., Vol. 14, No. 3, 2003 649

Figure 14. RP HPLC of the material 13a eluted from the C18OPC column. All analytical runs were done using 2%/min acetonitrile gradient.

Figure 14 illustrates the RP HPLC analytical profile of the 5′-aldehyde-modified probe 13a eluted from the C18-OPC cartridge. In conclusion, we have presented an efficient and versatile synthetic strategy that allows for the preparation of a broad range of new reagents. These reagents can be used in the synthesis of aldehyde-modified DNA probes and for the preparation of new types of cleavable linkers. We demonstrated that the aldehyde-labeled DNA probes, synthesized according to the presented method, can serve as valuable starting materials in conjugation reactions with other organic moieties containing primary amino groups. The mild reaction conditions, leading to either the introduction of the aldehyde group in DNA probes or the cleavage of the aleuritic acid linker, facilitate the preparation of aldehyde-labeled materials, even in the presence of sensitive organic groups. A simple method to prepare DNA probes modified at their 5′- end with the aldehyde group has been developed. ACKNOWLEDGMENT

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