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Bioconjugate Chem. 2002, 13, 11461154

New Reagents and Methods for the Synthesis of Internal and 3-Labeled DNA
Matthew H. Lyttle,* Troy A. Walton, Daren J. Dick, Timothy G. Carter, Jacob H. Beckman, and Ronald M. Cook
Biosearch Technologies, Inc., 81 Digital Drive, Novato, California 94949. Received January 22, 2002

The syntheses of two new nucleoside phosphoramidites containing a hydroxyl functionality masked by a levulinate protecting group are presented; N4-(2-(ethylene glycol-2-levulinate)ethyl)-5-methyl5-(4,4-dimethoxytrityl)-3-O-(2-cyanoethyldiisopropylphosphoramidite)-2-deoxycytidine 1 and 5-(N(6-O-levulinoyl-1-aminohexyl)-3(E)-acrylamido)-5-(4,4-dimethoxytrityl)-3-(2-cyanoethyldiisopropylphosphoramidite)-2-deoxyuridine 3. Optimization of solid-phase-supported synthetic parameters for incorporation of these into DNA, removal of the levulinate group by exposure to dilute hydrazine, and subsequent attachment of dye labels is described. Synthesis of the known compound 5-(N-(6trifluoroacetylaminohexyl)-3(E)-acrylamido)-5-(4,4-dimethoxytrityl)-3-(2-cyanoethyldiisopropylphosphoramidite)-2-deoxyuridine 2 (1), containing a masked amine at the end of an alkyl chain attached at the 5 position, was also revisited using new techniques developed for 3.

INTRODUCTION

Modern, high-speed synthesis of labeled DNA requires the fewest amount of steps conducted in the shortest possible time. Synthetic methodology (2-4) has been continuously evolving to meet these challenges. These current trends utilize direct attachment of rapidly reacting reagents to solid-phase-immobilized nascent oligonucleotides, followed by cleavage from the support and purification of the product in precisely the form desired. While a variety of efficient strategies for solid-phase synthesis of 3 and 5 terminal labeled structures are available, there are not many choices for solid-phasesupported internal labeling of DNA. Useful information about the preparation of the needed reagents as well as the performance of the structures produced is limited. Internal labels are usually attached by solution-phase active ester coupling to alkylamines pendant on pyrimadine bases (5). These procedures involve multiple purifications and other cumbersome methods not amenable to high throughput synthesis. Label derivatized nucleoside phosphoramidites for internally modified DNA synthesis are commercially available (6), but at considerable expense. Furthermore, coupling efficiencies of these have been low in our hands, perhaps due to the high molecular weight and polar nature of some of these compounds. Recently, palladium-assisted coupling of acetylenic compounds to iodinated pyrimidine nucleosides immobilized on solid supports has been reported (7). Other current work details solid-phase photolytic deprotection of 2 amine oligonucleotides and subsequent on support attachment of various useful moieties (8). Hopefully these methods will achieve widespread application in this area. We have developed solid-phase-supported methods in which phosphoramidites normally used to functionalize only the 5 terminus of DNA can also be used to effect labeling internally or at the 3 terminus of the sequence. Removal of masking groups and the incorporation of labels are done while the DNA is attached to the solid* To whom correspondence should be addressed. Phone 415883-8400; fax 415-883-8488. E-mail matt@biosearchtech.com.

phase support, which facilitates the separation of tagged DNA products from excess label and deprotection reagents. Chang, Urdea and Horn (9) developed a synthesis of novel branching nucleotide structures in which a hydroxyl functionality, attached via a hydrocarbon chain at N-4 of cytidine, was masked by a levulinate group and then deprotected with a hydrazine cocktail after synthesis of the primary strand. Branching sequences were then added to the oxygen with standard phosphoramidite chemistry. Another phosphoramidite 4 based on 1,2,3propanetriol containing a similarly masked oxygen is commercially available (10). Neither of these structures is desirable for internally labeled DNA which is expected to anneal to a complementary strand at energies (Tms) close to that of the unmodified DNA sequences. To address this issue we have developed the uridine derivative 3 which has a hydroxyl at the end of a hydrocarbon chain, pendant at C-5, masked by a levulinate group. Groups attached at the C-5 position of uridine would be expected to be less disruptive of double helix formation than at the N-4 of cytidine (11). A companion paper (12) compares the performance of oligonucleotides made with 1, 2, and 3 in practical applications involving annelation to complementary DNA structures.
MATERIALS AND METHODS

Aqueous ammonia, lithium chloride, and concentrated HCl were reagent grade from J. T. Baker. Dichloromethane (DCM), tetrahydrofuran (THF), ethyl acetate (EtOAc), acetonitrile, ethanol, methanol (MeOH), and petroleum ether were Omnisolve grade from VWR. Sodium hydroxide, mercuric acetate, sodium bicarbonate, magnesium sulfate, 6-amino-1-hexanol, 1,6-hexanediamine, acryloyl chloride, methyl trifluoroacetate, levulinic acid, 2-(2-aminoethoxy)ethanol, phosphorus oxychloride, triethylamine, 1,2,4-triazole, iron sulfide, triethylamine, and tetrabutlyammonium fluoride were from Aldrich. Chromatographic silica was from Grace Chemical Co. Deoxyuridine was obtained from Crystal Chem. Co. DMT T, TET and HEX amidites were obtained from

10.1021/bc020011c CCC: $22.00 2002 American Chemical Society Published on Web 07/19/2002

Modified Oligonucleotide Bases, Internally Dye-Labeled DNA


Chart 1. Phosphoramidite Synthons for Internal Labeling

Bioconjugate Chem., Vol. 13, No. 5, 2002 1147

Annovis Corp. Potassium tetrachloropalladate was obtained from Alfa (J. M. Corp.). Controlled pore glass DNA synthesis supports, 6-carboxyfluorescein amidite (6-FAM) and Tamra amidite (3), were obtained in house. All other DNA synthesis reagents were as previously described (13). Elemental analyses were performed by Desert Analytics (Tucson, AZ), and NMR work was performed by Acorn NMR (Livermore, CA). MALDI Mass spectra were performed in house on a Bruker Biflex MALDI-TOF.
EXPERIMENTAL PROCEDURES

N4-(2-(Ethylene glycol-2-levulinate)ethyl)-5-methyl-5-(4,4-dimethoxytrityl)-3-O-tert-butyldimethylsilyl-2-deoxycytidine, 7. A solution of 12 mL (20 g, 130 mmol) of phosphorus oxychloride and 40 g (580 mmol) of 1,2,4-triazole in 400 mL of dry CH3CN was chilled on ice to 0 C under argon. Triethylamine (90 mL, 90 g, 900 mmol) was added slowly dropwise with stirring over 0.5 h. Next, 31 g (47 mmol) of 5-(4,4-dimethoxytrityl)-3-Otert-butyldimethysilylthymidine 5 (14) was dissolved in 200 mL of dry CH3CN and added slowly dropwise over 0.5 h. The solution was stirred and allowed to warm to room temperature overnight. The solution was concentrated to a gum under reduced pressure and dissolved in 500 mL of EtOAc. Saturated aqueous NaHCO3 (300 mL) was added, and the mixture was shaken and separated. The organic phase was washed with another 300 mL portion of satd aqueous NaHCO3 and then dried over MgSO4, filtered, and evaporated to give the N4triazolide as a brown foam. This material was dissolved in 150 mL of CH3CN and added dropwise with stirring to a solution of 30 mL (31.4 g, 300 mmol) of 2-(2aminoethoxy)ethanol in 300 mL of CH3CN which had been chilled to 0 C on ice. The mixture was stirred overnight and allowed to warm to room temperature. The solution was reduced to a tar by rotary evaporation and then redissolved in 300 mL of EtOAc. The solution was washed with 300 mL of satd NaHCO3 and dried over MgSO4. Filtration, followed by reduction to a tar by rotary evaporation and high vacuum overnight, gave 24.3 g of the N4-(2-(ethylene glycol)ethyl) adduct 6 as a foam. All of this material was dissolved in 300 mL of dry pyridine and 12 mL of N-methylimidazole, 12 mL (13.6

g, 117 mmol) levulinic acid was added, and the solution was reduced to an oil by rotary evaporation. Dry CH3CN, 200 mL, was added along with 12 mL (9.7 g, 77 mmol) diisopropylcarbodiimide. The solution was allowed to stand overnight after thorough mixing. Crystals of N,N-diisopropylurea were removed by filtration, and the solution was concentrated to a tar by rotary evaporation. The residue was dissolved in 300 mL of EtOAc, and the organic phase was washed with 150 mL of 1 M citric acid followed by 150 mL of satd NaHCO3 and then dried over MgSO4 and filtered. The product was purified by column chromatography on a 5 20 cm bed of activated silica gel eluted with 1% MeOH and 1% pyridine in DCM. Fractions were inspected by TLC in 5% MeOH, 1% pyridine in DCM, and visualized with 10% H2SO4 and heat. Once the product (Rf 0.5) began to elute from the column, the MeOH in the column mobile phase was increased to 2%. Fractions containing pure product were pooled and concentrated by rotary evaporation and high vacuum to yield 28 g (70%) of 7 as a white foam. 1H NMR (, CDCl3): 7.8 (s, 1H), 7.5-7.2 (m, 11H), 6.8 (d, 4H), 6.4 (t, 1H), 5.4 (t, 1H), 4.5 (q, 1H), 4.2 (dd, 2H), 3.8 (s, 6H), 3.75 (m, 2H), 3.7 (m, 4H), 3.5 (dd, 1H), 3.2 (dd, 1H), 2.8 (t, 2H), 2.6 (t, 2H), 2.4 (m, 1H), 2.2 (m, 1H), 2.1 (s, 3H), 1.5 (s, 3H), 0.8 (s, 9H), 0.0 (d, 6H). Anal. Calcd for C46H61N3O10Si1/2MeOH: C, 64.94; H, 7.38. Found: C, 64.59; H, 7.34. N4-(2-(Ethylene glycol-2-levulinate)ethyl)-5-methyl-5-(4,4-dimethoxytrityl)-2-deoxycytidine, 8. 7 (24 g, 28 mmol) was dissolved in a solution of 200 mL of THF, 60 mL of 1 N TBAF (in THF), and 10 mL of HOAc. The solution was allowed to stand for 24 h. An aliquot of the solution for TLC was prepared by mixing a few drops of the mixture with 1 mL of EtOAc and 1 mL of satd NaHCO3 in a test tube and spotting the upper phase. The TLC (same mobile phase and visualization as above) showed complete conversion to a new lower spot (Rf 0.15). The reaction mixture was quenched with 20 mL of satd NaHCO3 and most of the THF was removed by rotary evaporation. The residue was dissolved in 300 mL of EtOAc and the organic phase washed with 300 mL of satd NaHCO3 followed by drying with MgSO4 and filtration. The solution was concentrated to a foam by rotary

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evaporation, and the product was purified by column chromatography on a 5 20 cm bed of activated silica gel eluted with a gradient of 1-6% MeOH over 8 L of mobile phase and 1% pyridine in DCM. Fractions were inspected by TLC as above, and those with pure product were pooled and evaporated to yield 19 g (93%) of 8 as a white foam. 1H NMR (, CDCl3): 7.8 (s, 1H), 7.5-7.2 (m, 11H), 6.8 (d, 4H), 6.4 (t, 1H), 5.4 (t, 1H), 4.5 (s, 1H), 4.2 (dd, 2H), 4.1 (s,1H), 3.8 (s, 6H), 3.75 (m, 2H), 3.7 (m, 4H), 3.5 (dd, 1H), 3.4 (m, 2H), 2.7 (t, 2H), 2.6 (m, 3H), 2.25 (m, 1H), 2.2 (s, 3H), 1.5 (s, 3H). Anal. Calcd for C40H47N3O10: C, 65.83; H, 6.49; N, 5.76. Found: C, 65.74; H, 6.68; N, 6.41. N4-(2-(Ethylene glycol-2-levulinate)ethyl)-5-methyl-5-(4,4-dimethoxytrityl)-3-O-(2-cyanoethyldiisopropylphosphoramidite)-2-deoxycytidine, 1. 8 (19 g, 26 mmol) was dried by rotary evaporation from pyridine followed by high vacuum in a 500 mL roundbottom flask. A mixture of 8 g (27 mmol) of 2-cyanoethyl tetraisopropylphosphorodiamidite and 480 mg of tetrazole in 250 mL of CH3CN was prepared and added to the flask containing the nucleoside 8. The flask was stoppered and allowed to stand for 2 h after thorough mixing. Inspection of the reaction mixture by TLC as above showed complete conversion to a new product, Rf 0.4. The reaction mixture was concentrated to a tar by rotary evaporation and redissolved in 300 mL of EtOAc. The solution was washed with 200 mL of satd NaHCO3 followed by drying with MgSO4 and filtration. The solution was concentrated to a tar by rotary evaporation and the product purified by column chromatography as above for 8. Fractions containing pure 1 were pooled and concentrated by rotary evaporation and high vacuum to yield 13.3 g (55%) of 1 as a white foam. 31P NMR (ppm, CDCl3): 149.756, 149.211. Anal. Calcd for C49H64N5O11P: C, 63.28; H, 6.94; N, 7.53. Found: C, 62.99; H, 6.61; N, 7.82. N-Acrylolyl-N-tert-butyloxycarbonyl-1,6-hexanediamine, 9. 1,6-Hexanediamine (100 g, 0.86 mol) was dissolved in 800 mL of THF and chilled in an ice bath. Di-tert-butyl dicarbonate, 60 g (0.28 mol) was dissolved in 400 mL of THF and added slowly over 1 h by a dropping funnel. The solution was stirred for 2 h and then filtered. The solid was washed with 200 mL of THF, and the combined filtrates were concentrated by rotary evaporation to 70 g of an oil. The oil was redissolved in 700 mL of CHCl3 and washed three times with 400 mL of water. The solution was concentrated by rotary evaporation and redissolved in 300 mL of THF, and 200 mL of satd Na2CO3 was added. The mixture was chilled in an ice bath, and 30 mL (33.4 g, 0.37 mol) of acryloyl chloride was added in 10 mL portions over 2 h. Solid Na2CO3 was added, as needed, to keep the pH of the aqueous layer above 8. The mixture was stirred overnight and poured into a separatory funnel. EtOAc, 500 mL, was added, and the lower aqueous layer was removed after the mixture was shaken and allowed to separate. The organic phase was washed with 500 mL of 0.5 M KH2PO4 followed by 500 mL of satd NaHCO3 and dried over MgSO4. The solution was filtered and concentrated by rotary evaporation. The material was purified by column chromatography on a 15 50 cm bed of activated silica gel eluted with a gradient of 0-4% MeOH over 18 L of mobile phase and 1% pyridine in DCM. Fractions were inspected by TLC (Rf desired product 0.6, UV active, in 2% MeOH, 2% pyridine in DCM), and those with pure product were pooled and evaporated to yield 39 g (52%) of 9 as a white solid. 1H NMR (, CDCl3): 6.5-6.0 (m, 3H), 5.9 (d, 1H), 5.8 (d, 1H), 3.3 (m, 2H), 3.0 (m, 2H), 1.5-1.3 (m, 17H).

Anal. Calcd for C14H26N2O3: C, 62.19; H, 9.69; N, 10.36. Found: C, 62.43; H, 9.83; N, 10.42. 5-(N-(6-Trifluoroacetylaminohexyl)-3(E)-acrylamido-5-(4,4dimethoxytrityl)-2-deoxyuridine, 11. (This reaction must be done in a fume hood because H2S, which is a toxic and malodorous gas, is used to precipitate metals during workup). Potassium tetrachloropalladate(II), K2PdCl4 (40 g 122 mmol), was added over 1 h in 10 g portions to a gently refluxing solution of LiCl (24 g, 0.6 mol) in 1200 mL of THF. A solution of 9 (33 g, 122 mmol) in 200 mL of THF was added slowly over 10 min. The reaction mixture became dark brown. The solution was stirred and refluxed for 20 min, and then 57 g (123 mmol) of finely powdered 5-chloromercurate-2-deoxyuridine (1) was added in small portions over 15 min. The solution became black and a silver mirror was deposited on the sides of the reaction vessel. Stirring and gentle reflux was continued overnight. The solution was allowed to cool, and H2S gas was bubbled into the solution. A trap containing Clorox was used to control the odor of the gas. Once the brown precipitate was no longer generated, H2S delivery was stopped and the solution was filtered to produce a clear yellow solution. The solvent was removed by rotary evaporation and high vacuum overnight to give 75 g of alkylated nucleoside 10 as a beige foam. A small amount of the foam was purified by dissolution in MeOH, addition of water to a cloud point, and chilling. Crystals were obtained, TLC Rf 0.4 (20% MeOH, 2% pyridine in DCM) which was UV (254 nm) active; the spot also fluoresced blue when irradiated with 312 nm UV. The bulk, unpurified material was dissolved in a mixture of 900 mL of DCM and 100 mL of MeOH, and 100 mL of trifluoroacetic acid was added. The solution was refluxed overnight during which a milky precipitate appeared. The suspension was concentrated to a gum by rotary evaporation, and high vacuum was applied to the material for 24 h. The resulting solid was dissolved in 700 mL of MeOH, and 60 mL of TEA was added, followed by 40 mL of methyl trifluroacetate. The solution was mixed and allowed to stand for 6 h and was concentrated to a solid by rotary evaporation. The solid was dried by rotary evaporation with 500 mL of dry pyridine and was then redissolved in 500 mL of dry pyridine. 4,4-Dimethoxytrityl chloride (25 g, 75 mmol) was added, and the mixture was stirred for 4 h. A drop of the reaction mixture was dissolved in 1 mL of EtOAc and washed with 1 mL of satd NaHCO3. TLC showed the appearance of product (Rf 0.2, 10% MeOH, 2% pyridine in DCM) which had the same UV characteristics as above and also became orange when the plate was sprayed with 10% H2SO4 and heated. MeOH (20 mL) was added to quench the reaction, and the brown solution was concentrated to a gum by rotary evaporation. The material was dissolved in 700 mL of DCM and washed with 500 mL of 0.5 KH2PO4 solution followed by 500 mL of satd aq NaHCO3. The solution was dried over MgSO4, filtered, concentrated by rotary evaporation, and then purified by column chromatography on a 15 150 cm bed of activated silica gel eluted with a gradient of 1-8% MeOH over 18 L of mobile phase and 1% pyridine in DCM. Fractions were inspected by TLC (Rf desired product 0.2, UV active as above, in 10% MeOH, 2% pyridine in DCM), and those with pure product were pooled and evaporated to yield 16.5 g (17%) of 11 as a white foam. 1 H NMR (, CDCl3): 7.9 (s, 1H), 7.7 (t, 1H), 7.4 (d, 1H), 7.3 (m, 5H), 7.1 (m, 1H), 6.8 (d, 4H), 6.6 (d, 1H), 6.4 (t, 1H), 5.4 (broad s, 1H), 4.5 (s, 1H), 4.1 (s, 1H), 3.75 (s, 6H), 3.5 (dd, 1H), 3.3 (m, 3H), 3.2-3.0 (m, 2H), 2.5 (m, 1H), 2.3 (m, 1H), 1.5 (m, 2H), 1.4-1.2 (m, 6H). Anal.

Modified Oligonucleotide Bases, Internally Dye-Labeled DNA

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Calcd for C41H45F3N4O9C5H5N: C, 63.27; H, 5.77; N, 8.02. Found: C, 63.47; H, 5.92; N, 8.13. 5-(N-(6-Trifluoroacetylaminohexyl)-3(E)-acrylamido-5-(4,4-dimethoxytrityl)-3-(2-cyanoethyldiisopropylphosphoramidite)-2-deoxyuridine, 2. 11 (16.5 g, 21 mmol) was dried by rotary evaporation from 200 mL of dry pyridine followed by high vacuum in a 1000 mL round-bottom flask. A mixture of 8.3 g (27 mmol) of 2-cyanoethyl tetraisopropylphosphorodiamidite and 500 mg of tetrazole (7.2 mmol) in 300 mL of dry CH3CN was prepared and added to the flask containing the nucleoside 11. The flask was stoppered and allowed to stand for 2 h after thorough mixing. Inspection of the reaction mixture by TLC (2% pyridine/EtOAc) showed complete conversion to a new product, Rf 0.5. The reaction mixture was concentrated to a tar by rotary evaporation and redissolved in 300 mL of EtOAc. The solution was washed with 200 mL of satd NaHCO3 followed by drying with MgSO4 and filtration. The solution was concentrated to a tar by rotary evaporation and the product purified by column chromatography on a 5 20 cm bed of activated silica gel eluted with 2% pyridine in EtOAc. Fractions were inspected by TLC (2% pyridine in EtOAc) and visualized with 10% H2SO4 and heat. Fractions containing pure 2 were pooled and concentrated by rotary evaporation and high vacuum to yield 13.5 g (65%) of 2 as a white foam. 31P NMR (ppm, CDCl3): 149.462, 149.363. Anal. Calcd for C50H62F3N6O10P: C, 60.35; H, 6.28; N, 8.45. Found: C, 60.32; H, 6.32; N, 8.57. N-Acryloyl-O-levulinoyl-6-amino-1-hexanol, 12. 6-Amino-1-hexanol (50 g, 427 mmol) was dissolved in 400 mL of THF, and 200 mL of satd NaHCO3 was added. Acryloyl chloride (caution: lachrymator) (35 mL, 39 g, 430 mmol) was added dropwise over 1 h. Solid NaHCO3 was added, as needed, to maintain a basic pH. The mixture was stirred overnight. EtOAc (600 mL) was added, and the mixture was partitioned. The organic phase was washed with 300 mL of 1 N HCl, and then 300 mL of water followed by 300 mL of satd NaHCO3. The solution was dried over MgSO4, filtered, and evaporated to 53 g of a beige solid. The solid was dissolved in 400 mL of EtOAc with heating and applied to a 15 50 cm bed of activated silica gel packed and eluted with EtOAc. Fractions were inspected by TLC (Rf 0.25, 2% pyridine in EtOAc) and visualized with iodine vapor. Fractions containing pure material were pooled and concentrated by rotary evaporation and high vacuum to yield 25.2 g of N-acryloyl-6-amino-1-hexanol as a white solid. A portion of this material (10 g, 58 mmol) was dissolved in a mixture of levulinic acid (7 mL, 8 g, 68 mmol), 5 mL of N-methylimidazole, and 200 mL of dry pyridine. The solution was concentrated to a tar by rotary evaporation and redissolved in 300 mL of dry CH3CN. Diisopropylcarbodiimide (10 mL, 8 g, 64 mmol) was added, and the mixture was allowed to stand overnight after mixing. Solids were filtered off, and the solution was concentrated to a tar by rotary evaporation. The material was dissolved in 300 mL of DCM and washed with 200 mL of 1 N citric acid followed by 200 mL of satd NaHCO3 followed by drying with MgSO4 and filtration. The solution was concentrated to a tar by rotary evaporation and the product purified by column chromatography on a 5 20 cm bed of activated silica gel eluted with 1:1 petroleum ether: EtOAc. Fractions were inspected by TLC (1:1 petroleum ether: EtOAc) and visualized with iodine vapor. Fractions containing pure 13 (Rf 0.35) were pooled and concentrated by rotary evaporation and high vacuum to yield 6 g (38%) of 12 as a white solid. 1H NMR (, CDCl3): 6.3-6.25 (dd, 1H), 6.2 (broad s, 1H), 6.15-

6.05 (dd, 1H), 5.6 (dd, 1H), 4.0 (t, 2H), 3.3 (q, 2H), 2.75 (t, 2H), 2.55 (t, 2H), 2.2 (s, 3H), 1.7-1.5 (m, 4H), 1.4-1.3 (m, 4H). Anal. Calcd for C14H23NO4: C, 62.43; H, 8.61; N, 5.20. Found: C, 61.91; H, 8.94; N, 5.74. 5-(N-(6-O-Levulinoyl-1-aminohexyl)-3(E)-acrylamido-5-(4,4dimethoxytrityl)-2-deoxyuridine, 14. (This reaction must be done in a fume hood because H2S, which is a toxic and malodorous gas, is used to precipitate metals during workup). Potassium tetrachloropalladate(II), K2PdCl4 (12 g, 36 mmol), was added over 20 min in three 4 g portions to a gently refluxing solution of LiCl (8 g, 194 mol) in 800 mL of THF. A solution of 12 (10 g, 37 mmol) in 200 mL of THF was added slowly over 10 min. The reaction mixture became dark brown. The solution was stirred and refluxed for 20 min, and then 17 g (37 mmol) of finely powdered 5-chloromercurate-2deoxyuridine (1) was added in small portions over 15 min. The solution became black, and a silver mirror was deposited on the sides of the reaction vessel. Stirring and gentle reflux was continued overnight. The solution was allowed to cool, and H2S gas was bubbled into the solution. A trap containing Clorox was used to control the odor of the gas. Once the brown precipitate was no longer generated, H2S delivery was stopped and the solution was filtered to produce a clear yellow solution. The solvent was removed by rotary evaporation and high vacuum overnight to give 28 g of alkylated nucleoside 13 as a beige solid. A small amount of the foam was purified by dissolution in MeOH, addition of water to a cloud point, and chilling. Crystals were obtained, TLC Rf 0.3 (20% MeOH, 2% pyridine in DCM), which was UV (254 nm) active; the spot also fluoresced blue when irradiated with 312 nm UV. The bulk, unpurified material was dried by rotary evaporation with 300 mL of dry pyridine and was then redissolved in 300 mL of dry pyridine. 4,4-Dimethoxytrityl chloride (10 g, 29 mmol) was added, and the mixture was stirred overnight. A drop of the reaction mixture was dissolved in 1 mL of EtOAc and washed with 1 mL of satd NaHCO3. TLC showed the appearance of product (Rf 0.5, 6% MeOH, 2% pyridine in DCM) which had the same UV characteristics as 11 and also became orange when the plate was sprayed with 10% H2SO4 and heated. MeOH (10 mL) was added to quench the reaction, and the brown solution was concentrated to a gum by rotary evaporation. The material was dissolved in 500 mL of DCM and washed with 400 mL of 0.5 M KH2PO4 solution followed by 400 mL of satd NaHCO3. The solution was dried over MgSO4, filtered, concentrated by rotary evaporation, and then purified by column chromatography on a 7 50 cm bed of activated silica gel eluted with a gradient of 1-5% MeOH over 10 L of mobile phase and 1% pyridine in DCM. Fractions were inspected by TLC (Rf desired product 0.5, UV active as above, in 6% MeOH, 2% pyridine in DCM), and those with pure product were pooled and evaporated to yield 6.8 g (24%) of 14 as a white foam. 1H NMR (, CDCl3): 7.9 (s, 1H), 7.7 (t, 1H), 7.4 (d, 1H), 7.3 (m, 5H), 7.1 (m, 1H), 6.8 (d, 4H), 6.6 (d, 1H), 6.4 (t, 1H), 5.4 (broad s, 1H), 4.5 (s, 1H), 4.1 (s, 1H), 4.0 (t, 2H), 3.75 (s, 6H), 3.4 (m, 1H), 3.3 (m, 1H), 3.1 (m, 2H), 2.7 (t,2H), 2.5 (m, 3H), 2.2 (m, 1H), 2.1 (s, 3H), 1.5 (m, 2H), 1.4-1.2 (m, 6H). Anal. Calcd for C44H51N3O111/2C5H5N: C, 66.76; H, 6.44; N, 5.86. Found: C, 66.52; H, 6.37; N, 5.91. 5-(N-(6-O-Levulinoyl-1-aminohexyl)-3(E)-acrylamido-5-(4,4dimethoxytrityl)-3-(2-cyanoethyldiisopropylphosphoramidite)-2-deoxyuridine, 3. 14 (6.8 g, 8.5 mmol) was dried by rotary evaporation from 100 mL of dry pyridine followed by high vacuum in a 500 mL round-bottom flask. A mixture of 3.0 g (10 mmol) of

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2-cyanoethyl tetraisopropylphosphorodiamidite and 180 mg of tetrazole (2.6 mmol) in 100 mL of dry CH3CN was prepared and added to the flask containing the nucleoside 14. The flask was stoppered and allowed to stand for 2 h after thorough mixing. Inspection of the reaction mixture by TLC (2% pyridine/EtOAc) showed complete conversion to a new product, Rf 0.6. The reaction mixture was concentrated to a tar by rotary evaporation and redissolved in 200 mL of EtOAc. The solution was washed with 100 mL of satd NaHCO3 followed by drying with MgSO4 and filtration. The solution was concentrated to a tar by rotary evaporation and the product purified by column chromatography on a 3 20 cm bed of activated silica gel eluted with 2% pyridine in EtOAc. Fractions were inspected by TLC (2% pyridine in EtOAc) and visualized with 10% H2SO4 and heat. Fractions containing pure 3 were pooled and concentrated by rotary evaporation and high vacuum to yield 5.5 g (81%) of 3 as a white foam. 31P NMR (ppm, CDCl3): 149.677, 149.329. Anal. Calcd for C53H68N5O12P: C, 63.78; H, 6.87; N, 7.02. Found: C, 63.75; H, 6.91; N, 7.13. DNA Synthesis. DNA was made on Biosearch 8750 DNA synthesizers with standard conditions as previously described (13). Test sequences 5-CGATCTGAXTAGCTY3, 5-TTTTXTTTTT-3 and 5-TTTTTTTTTX-3where X was the nucleotide residue resulting from the use of 1 or 3 and Y were different natural bases were made at 1.0 m and 200 nm synthesis scales. For internal labeling, first a hydrazine solution of 160 L of hydrazine hydrate (Aldrich, 50 wt % water) in 8 mL of pyridine and 2 mL of HOAc was prepared and well mixed. The DNA sequencer was halted after the base (before X) added and the DMT was removed. After thorough washing with CH3CN, a solution of 200 L of 100 mg/mL of 1 or 3 in dry acetonitrile and 200 L of 0.4 M S-ethyltetrazole in dry acetonitrile were introduced onto the column by the DNA synthesizer. After 2-3 min, the coupling solution was washed off the column with acetonitrile, and oxidation, followed by capping, was performed. Manual coupling with syringes was also successfully performed (15). The hydrazine cocktail above was introduced into the columns; we did this with 200 L of solution in 1 mL syringes but it can also be done on the instrument. After the support was well wetted with the solution, it was allowed to stand 12-15 min and then was thoroughly washed with CH3CN. Either TAMRA amidite or 6-FAM was coupled next; the same procedures as above were used. A 10 min coupling time was used for the TAMRA amidite. Oxidation and capping were performed after the coupling solution was washed off the column with acetonitrile. The TAMRA coupling sample was exposed to the capping solution for 5 min. Next, automated DNA synthesis was continued to the 5 terminus, and the final DMT was removed. The CPG was placed into screw cap eppendorf tubes, and 1 mL of ammonia for the fluorescein DNA and 1 mL of the TAMRA deprotection cocktail for the TAMRA DNA was added to each. The tubes were heated at 55 C for 18 h and cooled and the solutions evaporated. Yields of labeled DNA at all synthesis scales were close to those without hydrazine treatment, indicating negligible loss of material due to premature cleavage by the hydrazine reagent. 3-HEX and TET CPG. DMT-O-ethylsulfonyl CPG (16) (15 g) was placed in a 300 mL coarse frit sintered glass funnel atop a 2 L sidearm flask. 3% Dichloroacetic acid in CH2Cl2 (200 mL) was poured in, and the support was agitated briefly with a spatula. The solution became brightly orange. After 2 min, the solution was drained and the step was repeated twice with 200 mL of fresh

3% dichloroacetic acid in CH2Cl2. The support was then washed three times with 200 mL of CH2Cl2, twice with 100 mL of CH3CN, and once with 100 mL of pyridine. The support was transferred into a 250 mL round-bottom flask, and 1 g of 1 was added. 40 mL of dry pyridine was added, and the solvent was removed by rotary evaporation. A plug of glass wool was used to keep the CPG from leaving the flask. Once dry, high vacuum was applied to the flask for 18 h. A solution of 150 mL of 0.4 M S-ethyltetrazole in dry acetonitrile was prepared, and enough of this was added to the flask containing the CPG to make a slurry. The slurry was allowed to stand for 15 min and was poured into a sintered glass funnel. The support was washed three times with 100 mL of CH3CN, and then 100 mL of amidite oxidizer solution (13) was added. After 5 min, the support was washed twice with 100 mL of CH3CN. Next, 100 mL of a mixture of acetic anhydride, N-methyl imidazole, and THF (1:1:8) was added to the support. After 15 min exposure, the acetylation solution was removed and the support was washed three times with 100 mL of CH3CN, and a small amount of this was dried. The DMT loading was 18 m/g (17). The hydrazine cocktail above (100 mL) was mixed and added to the support. After 15 min, the support was washed three times with 100 mL of CH3CN and was divided into two roughly equal portions. HEX and TET Amidite, 0.5 g respectively, were added to each portion, and coupling, oxidation, and capping steps using these amidites proceeded as above. Test sequences 5TTTTTTTTTdC(N4-(2-(ethylene glycol)ethyl)-HEX)-3 and the analogous TET sequence were made; the HEX sample was deprotected with concentrated ammonia for 18 h at room temperature while standard conditions were used for the TET sample. Anion exchange HPLC analyses were performed as follows: 2-20 L of the aqueous samples, depending on the concentration, was injected onto a Dionex anion exchange column (4.6 250 mm); samples were eluted at 2 mL/min with aqueous buffers of (A) 0.025 M Tris HCl and 0.01 M TRIS, and (B) 0.025 M Tris HCl, 0.01 M TRIS, and 1.0 M NaCl, using a linear gradient of 1:0 to 0:1 over 20 min, with UV detection at 260 nm. Reverse phase HPLC as follows: 20 L of the aqueous sample was injected onto a HAISIL HL C18 5 m particle size column (4.6 150 mm); samples were eluted at 1 mL/ min with buffers of (A) 0.1M TEAA, 5% acetonitrile, (B) acetonitrile, with a linear gradient of 1:0 to 0:1 A:B over 15 min. UV detection at 260 nm. Samples were prepared for MALDI mass spectral analysis as per Bruker.
RESULTS AND DISCUSSION

To develop this technology in an efficient manner, several endeavors had to be successfully completed: (1) Reliable and cost-effective processes for the synthesis of 1 and 3 had to be developed. (2) Easy and efficient solidphase synthesis methods for incorporation of these compounds into DNA as well as subsequent label attachment had to be established. (3) Finally analytical techniques had to be devised to establish the purity and efficacy of the product DNA in practical applications. Synthesis of Compounds 1-3. For 1, commercially available DMT-T was treated with TBDMS-Cl to give 5 (14) in 84% yield. 5 was treated with 2-(2-aminoethoxy)ethanol after activation as per Chang (9) to give 6, which was converted into levulinate ester 7, obtained as a white foam. The use of the TBDMS group instead of the trimethylsilyl transient protection used in (9) afforded certain advantages: the 3-OTBDMS triazolide was a

Modified Oligonucleotide Bases, Internally Dye-Labeled DNA


Scheme 1. Synthesis of 1

Bioconjugate Chem., Vol. 13, No. 5, 2002 1151

Scheme 2. Synthesis of 2

stable compound which could be prepared in large quantities and stored cold, allowing many different derivatives to be prepared from one preparation of triazolide. Also, the 3-OTBDMS group lent greater solubility in organic solvents which facilitated extraction and isolation of more polar conjugates containing pendant moieties such as biotin. Removal of the 3-hydroxyl masking TBDMS group with TBAF gave alcohol 8, followed by phosphitylation with 2-cyanoethyl tetraisopropylphosphorodiamidite and catalytic tetrazole gave 1. See Scheme 1. The principal advantage of the use of 1 is cost; a similar derivative without the 5-methyl group can be made starting with more expensive deoxyuridine. This compound should also be accessible from DMTdC(Bz), but more steps would be required (18). The primary disadvantage to the use of 1 are anticipated lower Tms of DNA made with this residue internally than with a natural deoxycytidine in binding to a complimentary DNA strand. A companion paper compares structures made with 1 with those made with 2 and 3 and bears this out. For this reason 1 is primarily used by us as a 3 labeling tool. Synthesis of 2. The Heck palladium-assisted coupling of organomercurials to olefins was first applied to nucleoside modification by Bergstrom (19). There is a recent paper about palladium-mediated reactions applied to nucleosides also coauthored by Bergstrom (20). We attempted first the literature synthesis (1) of 2. Formation of N-acryloyl-N-trifluoroacetyl-1,6-hexanediamine by a one-pot mixing of 1,6-hexanediamine, acryloyl chloride, and trifluoroacetic anhydride gave a mixture of products

which gave the desired product in low yield. Purification was difficult with the conditions described, owing to close Rf values of products and contaminants. A palladium(II)-mediated coupling reaction with the material and 5-chloromercurated deoxyuridine also gave a mixture of products, which afforded only a few percent overall yield of the desired DMT nucleoside 11 when treated with DMT chloride followed by chromatography. Upon consideration of the low overall yield and high cost of the lithium tetrachloropalladate(II) used, we decided that this was not a cost-effective way to prepare 2. An alternative scheme was developed, see Scheme 2. 1,6Hexanediamine was treated with di-tert-butyl dicarbonate, and the resulting 6-tert-butyloxycarbonamidyl-1aminohexane was partially purified by extraction. Acryloyl chloride was added to the compound under basic conditions to give 6-acrylolyl-N-tert-butyloxycarbonyl-1,6-hexanediamine, 9, which was obtained in high purity after chromatography. 5-Chloromurcurated deoxyuridine was prepared as per Ruth (1), and a palladium(II)-mediated coupling reaction with 9 gave, after several steps, the DMT nucleoside 11 in 24% yield (see Scheme 2). Significant savings in the cost of the palladium reagent was achieved by refluxing a THF solution of potassium tetrachloropalladate with lithium chloride prior to the addition of 9 and the chloromurcurated nucleoside. Recent literature reports that the needed palladium reagent can also be prepared by mixing PdCl2 and LiCl in MeOH for 24 h (20) and this also works well, as long as all of the methanol is removed prior to dissolution in THF. 11 was converted into 2 by phosphitylation with

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Figure 1. Time course for levulinoyl protecting group removal; A ) 5T9C(N4linker-O-lev)-3 only; B ) 2 min hydrazine exposure, then FAM, then T9 addition; C ) 4 min hydrazine, same other conditions; D ) 8 min hydrazine, same other conditions; E ) 12 min hydrazine, same other conditions; F ) FAM after chain assembly with 12 min hydrazine exposure.

2-cyanoethyltetraisopropylphosphorodiamidite and catalytic tetrazole. Synthesis of 3. A similar strategy to that used for 2 was employed. 6-amino-1-hexanol was first reacted with acryloyl chloride to give 6-acryloylamido-1-hexanol, followed by esterification with levulinic acid to give 6-acryloylamido-1-hexane levulinate 12, obtained as a white solid after chromatography. 12 was coupled to chloromurcurated deoxyuridine by those methods used for 2 to give 13, which gave 14 after treatment with DMT chloride. Conversion of 14 into phosphoramidite 3 was accomplished by the same method as for 2. Labeled DNA Synthesis. Incorporation of 1 and 3 into DNA was accomplished with standard methods and coupling times (13) and removal of levulinate protection was carefully optimized; previously reported conditions (9) were too harsh (0.5 M hydrazine in 1:1 pyridine:HOAc for 60 min), resulting in unwanted DMT loss and poor products, in our hands. We found that 0.25 M hydrazine in 4:1 pyridine:HOAc for 12-15 min gave the best results. This mixture and time is closer to the levulinate removal

conditions originally described by Van Boom, et al. (21) for 5 deprotection during a phosphate triester DNA oligomerization scheme. For us, the optimum exposure time was determined by an experimental system wherein 1 was first coupled onto sulfonylethyl CPG (16) and then subjected to various times of hydrazine exposure (2-12 min). 6-FAM was then coupled onto the deprotected hydroxyl, followed by oxidation and capping, and then extension of the DNA fragment by nine more addition cycles of T amidite. The product 3-nucleoside fluoresceinated 10 mers were then cleaved with ammonia, and AX HPLC showed that the best results were obtained with 12 min of hydrazine exposure. The composition was confirmed by MALDI mass spectroscopy, calcd 3682, found 3687 amu. Longer exposure times gave more extraneous products. The same conditions using 4 in lieu of 1 did not produce good results (data not shown). Another question to be addressed was the order of addition. Was it best to build the whole DNA fragment with the internal levulinated base and then deprotect and add the tag? This would be easier than the converse,

Modified Oligonucleotide Bases, Internally Dye-Labeled DNA

Bioconjugate Chem., Vol. 13, No. 5, 2002 1153

Figure 2. Order of label addition comparison with the synthesis of 5-CGATCTGAC(N4linker-O-FAM)TAGCTT-3 by (left) MALDI mass spectra and (right) AX HPLC. Top: No FAM coupling control. Middle: whole fragment synthesis, then OLev removal and FAM addition at the end. Bottom: OLev removal and FAM addition after addition of base 7, then chain completion.

which was to deprotect the levulinated hydroxyl at the position where it was added, add the tag, and then continue the synthesis out to the 5 terminus. An additional experiment was done with the time course study above, which removed the OLev group with the 12 min (best) conditions after all 10 bases were added and added 6-FAM as the last step. Figure 1 panel F shows clearly a result inferior to addition of fluorescein amidite prior to building the DNA fragment. 3-HEX and TET CPGs were made with these techniques and gave good results in the

synthesis of the analogous 10 mers. See Figures S1 and S2. Another comparison of the order of addition was done with 5-CGATCTGAXTAGCTT-3 where X resulted from the incorporation of 1; good product purity was seen when addition of FAM occurred after addition of 1 and before chain extension (MALDI m/e calcd 5182, found 5176). Addition of fluorescein after the completion of the sequence gave other products in addition to that desired; incomplete FAM addition as well as double FAM addition were seen by mass spectroscopy (see Figure 2) Similar

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Lyttle et al.
(5) Rudert, W. A., Braun, E. R., Faas, S. J., Menon, R., JaquinsGerstl, A., and Trucco, M. (1997) Double Labeled Fluorescent Probes for 5 Nuclease Assays: Purification and Performance Evaluation. Biotechniques 22, 1140-1145. (6) Glen Research has biotin, fluorescein, and TAMRA functionalized dU amidites. (7) Kahn, S. I., and Grinstaff, M. W. (1999) Palladium(0)Catalyzed Modificatons of Oligonucleotides during Automated Solid-Phase Synthesis J. Am. Chem. Soc. 121, 4704-4705. (8) Hwang, J., and Greenberg, M. (2001) Synthesis of 2Modified Oligonucleotides via On-Column Conjugation. J. Org. Chem. 66, 363-369. (9) Chang, C., Urdea, M. S., and Horn, T. (1996) N-4 Modified Pyrimadine Deoxynucleotides and Oligonucleonucleotide Probes Synthesized Therewith. US Patent 5580731. (10) Clontech, Inc., cat. no. 5251. (11) Otvos, L., Sagi, J., Sagi, G., and Szemzo, A. (1999) Base Modified Oligonucleotides. II. Increase of Stability to Nucleases by 5-Alkyl, 5(1-alkenyl)- and 5(1-alkynyl) pyrimadines. Nucleosides Nucleotides 18, 1929-1933. (12) Walton, T., Lyttle, M., Dick, D., and Cook, R. (2002) Evaluation of New Linkers and Synthetic Methods for Internal Labeled Oligonucleotides Bioconjugate Chem. 13, 1155-1158. (13) Wang, W. W., Lyttle, M. H., and Borer, P. N. (1990) Enzymatic and NMR Analysis of Oligoribonucleotides Synthesized with 2-tert-butyldimethylsilyl Protected Cyanoethylphosphoramidite Monomers. Nucleic Acids Res. 18, 33473355. (14) Lyttle, M. H., Napolitano, E. W., Calio, B. L., and Kauvar, L. M. (1995) Mutagenesis Using Trinucleotide -Cyanoethyl Phosphor-amidites. Biotechniques 19, 274-280. (15) Lyttle, M. H., Adams, H., Hudson, D., and Cook, R. M. (1997) Versatile Linker Chemistry for Synthesis of 3-Modified DNA. Bioconjugate Chem. 8, 193-198. (16) Biosearch Technologies, Inc. (17) Extinction coefficient of DMT cation 70000 @ 498 nm; 3% DCA in DCM. (18) Hovinen, J. (1998) A simple synthesis of N-4 (6-aminohexyl)-2-deoxy-5-O-(4,4dimethoxytrityl) cytidine Nucleosides Nucleotides 17, 1209-1213. (19) Bergstrom, D. E., and Ogawa, M. K. (1978) C-5 Substituted pyrimidine nucleosides. 2. Synthesis via olefin coupling to organopalladium intermediates derived from uridine and 2deoxyuridine. J. Am. Chem. Soc. 100, 8106-8112. (20) Ahmadian, M., Klewer, D. A., and Bergstrom, D. E. (2000) Palladium-mediated C-5 substitution of Pyrimidine Nucleosides. Current Protocols in Nucleic Acid Chemistry (Beaucage, S., and Jones, R., Eds.) 1.1.1-1.1.18. (21) Van Boom, J. H., and Burgers, P. M. (1976) Synthesis of complementary DNA fragments via phosphotriester intermediates. Tetrahedron Lett. 17, 4875-4882.

results were obtained with 3 and FAM. The analogous internally labeled DNA fragment was made with 3 and a TAMRA amidite (3) and also gave good results when the TAMRA amidite was added before the rest of the chain, m/e calcd 5288, found 5275 amu (see Figure S3). Finally, the question of potential base modification by the conditions employed had to be addressed. CPG samples containing DMT-ABz, -CBz, -CAc, -GiBu, -GDMF and -T were subjected to the conditions employed for TAMRA amidite incorporation which includes the hydrazine treatment and a 5 min acetylation step before DMT removal and cleavage with tert-butylamine cocktail (18 h, 55 C). The cleaved nucleosides were analyzed by RPHPLC, and no extraneous peaks were seen compared to controls (data not shown).
CONCLUSION

Oligonucleotide protecting group chemistry first developed by Van Boom for 5-hydroxyl protection (21) and later applied to side chain branching structures (9) has now been extended by us to allow the synthesis of internally dye-labeled DNA. New techniques and novel nucleoside phosphoramidite synthons have been developed which should allow the rapid synthesis of internally labeled structures. Utility of the new methods has been demonstrated with a variety of currently used fluorophores. Significant advantages in speed and simplicity over widely used solution-phase coupling methods, usually of an internally amine-labeled oligonucleotide with an active ester in aqueous solution, should be realized.
Supporting Information Available: Spectra and combustion analyses data. This material is available free of charge via the Internet at http://pubs.acs.org. LITERATURE CITED
(1) Ruth, J. (1991) Oligodeoxynucleotides with reporter groups attached to the base. Oligonucleotides and Analogues (Eckstein, F., Ed.) pp 255-282, IRL Press, Oxford. (2) Vinayak, R. (1999) A convenient, solid-phase coupling of rhodamine dye acids to 5 amino-oligonucleotides. Tetrahedron Lett. 40, 7611-7613. (3) Lyttle, M. H., Carter, T. C., Dick, D. J., and Cook, R. M. (2000) A tetramethyl rhodamine (Tamra) phosphoramidite facilitates solid-phase-supported synthesis of 5-Tamra DNA. J. Org. Chem. 65, 9033-9038. (4) Mullah, B., and Andrus, A. (1997) Automated Synthesis of Double Dye-Labeled Oligonucleotides using Tetramethylrhodamine (TAMRA) Solid Supports Tetrahedron Lett. 38, 5751-5754.

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