New Reagents and Methods for the Synthesis of Internal and3′-Labeled DNA

Matthew H. Lyttle,* Troy A. Walton, Daren J. Dick, Timothy G. Carter, Jacob H. Beckman, andRonald 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 maskedby a levulinate protecting group are presented; N4-(2-(ethylene glycol-2-levulinate)ethyl)-5-methyl-5′-(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-cyanoethyldiisopro-pylphosphoramidite)-2′-deoxyuridine 3. Optimization of solid-phase-supported synthetic parametersfor 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-(6-trifluoroacetylaminohexyl)-3(E)-acrylamido)-5′-(4,4′-dimethoxytrityl)-3′-(2-cyanoethyldiisopropy-lphosphoramidite)-2′-deoxyuridine 2 (1), containing a masked amine at the end of an alkyl chainattached at the 5 position, was also revisited using new techniques developed for 3.

INTRODUCTION

Modern, high-speed synthesis of labeled DNA requiresthe fewest amount of steps conducted in the shortestpossible time. Synthetic methodology (2-4) has beencontinuously evolving to meet these challenges. Thesecurrent trends utilize direct attachment of rapidly react-ing reagents to solid-phase-immobilized nascent oligo-nucleotides, followed by cleavage from the support andpurification of the product in precisely the form desired.

While a variety of efficient strategies for solid-phasesynthesis of 3′ and 5′ terminal labeled structures areavailable, there are not many choices for solid-phase-supported internal labeling of DNA. Useful informationabout the preparation of the needed reagents as well asthe performance of the structures produced is limited.Internal labels are usually attached by solution-phaseactive ester coupling to alkylamines pendant on pyrima-dine bases (5). These procedures involve multiple puri-fications and other cumbersome methods not amenableto high throughput synthesis. Label derivatized nucleo-side phosphoramidites for internally modified DNA syn-thesis are commercially available (6), but at considerableexpense. Furthermore, coupling efficiencies of these havebeen low in our hands, perhaps due to the high molecularweight and polar nature of some of these compounds.Recently, palladium-assisted coupling of acetylenic com-pounds to iodinated pyrimidine nucleosides immobilizedon solid supports has been reported (7). Other currentwork details solid-phase photolytic deprotection of 2′amine oligonucleotides and subsequent on support at-tachment of various useful moieties (8). Hopefully thesemethods will achieve widespread application in this area.

We have developed solid-phase-supported methods inwhich phosphoramidites normally used to functionalizeonly the 5′ terminus of DNA can also be used to effectlabeling internally or at the 3′ terminus of the sequence.Removal of masking groups and the incorporation oflabels are done while the DNA is attached to the solid-

phase support, which facilitates the separation of taggedDNA products from excess label and deprotection re-agents.

Chang, Urdea and Horn (9) developed a synthesis ofnovel branching nucleotide structures in which a hy-droxyl functionality, attached via a hydrocarbon chainat N-4 of cytidine, was masked by a levulinate group andthen deprotected with a hydrazine cocktail after synthe-sis of the primary strand. Branching sequences were thenadded to the oxygen with standard phosphoramiditechemistry. Another phosphoramidite 4 based on 1,2,3-propanetriol containing a similarly masked oxygen iscommercially available (10). Neither of these structuresis desirable for internally labeled DNA which is expectedto anneal to a complementary strand at energies (Tms)close to that of the unmodified DNA sequences. Toaddress this issue we have developed the uridine deriva-tive 3 which has a hydroxyl at the end of a hydrocarbonchain, pendant at C-5, masked by a levulinate group.Groups attached at the C-5 position of uridine would beexpected to be less disruptive of double helix formationthan at the N-4 of cytidine (11). A companion paper (12)compares the performance of oligonucleotides made with1, 2, and 3 in practical applications involving annelationto complementary DNA structures.

MATERIALS AND METHODS

Aqueous ammonia, lithium chloride, and concentratedHCl were reagent grade from J. T. Baker. Dichlo-romethane (DCM), tetrahydrofuran (THF), ethyl acetate(EtOAc), acetonitrile, ethanol, methanol (MeOH), andpetroleum ether were Omnisolve grade from VWR.Sodium hydroxide, mercuric acetate, sodium bicarbonate,magnesium sulfate, 6-amino-1-hexanol, 1,6-hexanedi-amine, acryloyl chloride, methyl trifluoroacetate, levulinicacid, 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. DMTT, TET and HEX amidites were obtained from

* To whom correspondence should be addressed. Phone 415-883-8400; fax 415-883-8488. E-mail matt@biosearchtech.com.

1146 Bioconjugate Chem. 2002, 13, 1146−1154

10.1021/bc020011c CCC: $22.00 © 2002 American Chemical SocietyPublished on Web 07/19/2002

Annovis Corp. Potassium tetrachloropalladate was ob-tained from Alfa (J. M. Corp.). Controlled pore glass DNAsynthesis supports, 6-carboxyfluorescein amidite (6-FAM)and Tamra amidite (3), were obtained in house. All otherDNA synthesis reagents were as previously described(13). Elemental analyses were performed by DesertAnalytics (Tucson, AZ), and NMR work was performedby Acorn NMR (Livermore, CA). MALDI Mass spectrawere performed in house on a Bruker Biflex MALDI-TOF.

EXPERIMENTAL PROCEDURES

N4-(2-(Ethylene glycol-2-levulinate)ethyl)-5-meth-yl-5′-(4,4′-dimethoxytrityl)-3′-O-tert-butyldimethyl-silyl-2′-deoxycytidine, 7. A solution of 12 mL (20 g, 130mmol) of phosphorus oxychloride and 40 g (580 mmol) of1,2,4-triazole in 400 mL of dry CH3CN was chilled on iceto 0 °C under argon. Triethylamine (90 mL, 90 g, 900mmol) was added slowly dropwise with stirring over 0.5h. Next, 31 g (47 mmol) of 5′-(4,4′-dimethoxytrityl)-3′-O-tert-butyldimethysilylthymidine 5 (14) was dissolved in200 mL of dry CH3CN and added slowly dropwise over0.5 h. The solution was stirred and allowed to warm toroom temperature overnight. The solution was concen-trated to a gum under reduced pressure and dissolvedin 500 mL of EtOAc. Saturated aqueous NaHCO3 (300mL) was added, and the mixture was shaken andseparated. The organic phase was washed with another300 mL portion of satd aqueous NaHCO3 and then driedover MgSO4, filtered, and evaporated to give the N4-triazolide as a brown foam. This material was dissolvedin 150 mL of CH3CN and added dropwise with stirringto a solution of 30 mL (31.4 g, 300 mmol) of 2-(2-aminoethoxy)ethanol in 300 mL of CH3CN which hadbeen chilled to 0 °C on ice. The mixture was stirredovernight and allowed to warm to room temperature. Thesolution was reduced to a tar by rotary evaporation andthen redissolved in 300 mL of EtOAc. The solution waswashed with 300 mL of satd NaHCO3 and dried overMgSO4. Filtration, followed by reduction to a tar byrotary evaporation and high vacuum overnight, gave 24.3g of the N4-(2-(ethylene glycol)ethyl) adduct 6 as a foam.All of this material was dissolved in 300 mL of drypyridine and 12 mL of N-methylimidazole, 12 mL (13.6

g, 117 mmol) levulinic acid was added, and the solutionwas reduced to an oil by rotary evaporation. Dry CH3-CN, 200 mL, was added along with 12 mL (9.7 g, 77mmol) diisopropylcarbodiimide. The solution was allowedto stand overnight after thorough mixing. Crystals ofN,N′-diisopropylurea were removed by filtration, and thesolution was concentrated to a tar by rotary evaporation.The residue was dissolved in 300 mL of EtOAc, and theorganic phase was washed with 150 mL of 1 M citric acidfollowed by 150 mL of satd NaHCO3 and then dried overMgSO4 and filtered. The product was purified by columnchromatography on a 5 × 20 cm bed of activated silicagel 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 andheat. Once the product (Rf 0.5) began to elute from thecolumn, the MeOH in the column mobile phase wasincreased to 2%. Fractions containing pure product werepooled and concentrated by rotary evaporation and highvacuum to yield 28 g (70%) of 7 as a white foam. 1H NMR(δ, 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 forC46H61N3O10Si‚1/2MeOH: C, 64.94; H, 7.38. Found: C,64.59; H, 7.34.

N4-(2-(Ethylene glycol-2-levulinate)ethyl)-5-meth-yl-5′-(4,4′-dimethoxytrityl)-2′-deoxycytidine, 8. 7 (24g, 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. Thesolution was allowed to stand for 24 h. An aliquot of thesolution for TLC was prepared by mixing a few drops ofthe mixture with 1 mL of EtOAc and 1 mL of satdNaHCO3 in a test tube and spotting the upper phase. TheTLC (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 satdNaHCO3 and most of the THF was removed by rotaryevaporation. The residue was dissolved in 300 mL ofEtOAc and the organic phase washed with 300 mL of satdNaHCO3 followed by drying with MgSO4 and filtration.The solution was concentrated to a foam by rotary

Chart 1. Phosphoramidite Synthons for Internal Labeling

Modified Oligonucleotide Bases, Internally Dye-Labeled DNA Bioconjugate Chem., Vol. 13, No. 5, 2002 1147

evaporation, and the product was purified by columnchromatography on a 5 × 20 cm bed of activated silicagel eluted with a gradient of 1-6% MeOH over 8 L ofmobile phase and 1% pyridine in DCM. Fractions wereinspected by TLC as above, and those with pure productwere pooled and evaporated to yield 19 g (93%) of 8 as awhite foam. 1H NMR (δ, 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 forC40H47N3O10: 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-meth-yl-5′-(4,4′-dimethoxytrityl)-3′-O-(2-cyanoethyldiiso-propylphosphoramidite)-2′-deoxycytidine, 1. 8 (19g, 26 mmol) was dried by rotary evaporation frompyridine followed by high vacuum in a 500 mL round-bottom flask. A mixture of 8 g (27 mmol) of 2-cyanoethyltetraisopropylphosphorodiamidite and 480 mg of tetra-zole in 250 mL of CH3CN was prepared and added to theflask containing the nucleoside 8. The flask was stop-pered and allowed to stand for 2 h after thorough mixing.Inspection of the reaction mixture by TLC as aboveshowed complete conversion to a new product, Rf 0.4. Thereaction mixture was concentrated to a tar by rotaryevaporation and redissolved in 300 mL of EtOAc. Thesolution was washed with 200 mL of satd NaHCO3followed by drying with MgSO4 and filtration. Thesolution was concentrated to a tar by rotary evapora-tion and the product purified by column chromatog-raphy as above for 8. Fractions containing pure 1 werepooled and concentrated by rotary evaporation and highvacuum to yield 13.3 g (55%) of 1 as a white foam. 31PNMR (ppm, CDCl3): 149.756, 149.211. Anal. Calcd forC49H64N5O11P: 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-hexanedi-amine, 9. 1,6-Hexanediamine (100 g, 0.86 mol) wasdissolved in 800 mL of THF and chilled in an ice bath.Di-tert-butyl dicarbonate, 60 g (0.28 mol) was dissolvedin 400 mL of THF and added slowly over 1 h by adropping funnel. The solution was stirred for 2 h and thenfiltered. The solid was washed with 200 mL of THF, andthe combined filtrates were concentrated by rotaryevaporation to 70 g of an oil. The oil was redissolved in700 mL of CHCl3 and washed three times with 400 mLof water. The solution was concentrated by rotary evapo-ration and redissolved in 300 mL of THF, and 200 mL ofsatd Na2CO3 was added. The mixture was chilled in anice bath, and 30 mL (33.4 g, 0.37 mol) of acryloyl chloridewas added in 10 mL portions over 2 h. Solid Na2CO3 wasadded, as needed, to keep the pH of the aqueous layerabove 8. The mixture was stirred overnight and pouredinto a separatory funnel. EtOAc, 500 mL, was added, andthe lower aqueous layer was removed after the mixturewas shaken and allowed to separate. The organic phasewas washed with 500 mL of 0.5 M KH2PO4 followed by500 mL of satd NaHCO3 and dried over MgSO4. Thesolution was filtered and concentrated by rotary evapora-tion. The material was purified by column chromatogra-phy on a 15 × 50 cm bed of activated silica gel elutedwith a gradient of 0-4% MeOH over 18 L of mobile phaseand 1% pyridine in DCM. Fractions were inspected byTLC (Rf desired product 0.6, UV active, in 2% MeOH,2% pyridine in DCM), and those with pure product werepooled and evaporated to yield 39 g (52%) of 9 as a whitesolid. 1H NMR (δ, 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)-acryla-mido-5′-(4,4′dimethoxytrityl)-2′-deoxyuridine, 11.(This reaction must be done in a fume hood because H2S,which is a toxic and malodorous gas, is used to precipitatemetals during workup). Potassium tetrachloropalladate-(II), K2PdCl4 (40 g 122 mmol), was added over 1 h in 10g portions to a gently refluxing solution of LiCl (24 g,0.6 mol) in 1200 mL of THF. A solution of 9 (33 g, 122mmol) in 200 mL of THF was added slowly over 10 min.The reaction mixture became dark brown. The solutionwas stirred and refluxed for 20 min, and then 57 g (123mmol) of finely powdered 5-chloromercurate-2′-deoxyuri-dine (1) was added in small portions over 15 min. Thesolution became black and a silver mirror was depositedon the sides of the reaction vessel. Stirring and gentlereflux was continued overnight. The solution was allowedto cool, and H2S gas was bubbled into the solution. A trapcontaining Clorox was used to control the odor of the gas.Once the brown precipitate was no longer generated, H2Sdelivery was stopped and the solution was filtered toproduce a clear yellow solution. The solvent was removedby rotary evaporation and high vacuum overnight to give75 g of alkylated nucleoside 10 as a beige foam. A smallamount of the foam was purified by dissolution in MeOH,addition of water to a cloud point, and chilling. Crystalswere obtained, TLC Rf 0.4 (20% MeOH, 2% pyridine inDCM) which was UV (254 nm) active; the spot alsofluoresced blue when irradiated with 312 nm UV. Thebulk, unpurified material was dissolved in a mixture of900 mL of DCM and 100 mL of MeOH, and 100 mL oftrifluoroacetic acid was added. The solution was refluxedovernight during which a milky precipitate appeared. Thesuspension was concentrated to a gum by rotary evapo-ration, and high vacuum was applied to the material for24 h. The resulting solid was dissolved in 700 mL ofMeOH, and 60 mL of TEA was added, followed by 40 mLof methyl trifluroacetate. The solution was mixed andallowed to stand for 6 h and was concentrated to a solidby rotary evaporation. The solid was dried by rotaryevaporation with 500 mL of dry pyridine and was thenredissolved in 500 mL of dry pyridine. 4,4′-Dimethox-ytrityl chloride (25 g, 75 mmol) was added, and themixture was stirred for 4 h. A drop of the reactionmixture was dissolved in 1 mL of EtOAc and washed with1 mL of satd NaHCO3. TLC showed the appearance ofproduct (Rf 0.2, 10% MeOH, 2% pyridine in DCM) whichhad the same UV characteristics as above and alsobecame orange when the plate was sprayed with 10% H2-SO4 and heated. MeOH (20 mL) was added to quenchthe reaction, and the brown solution was concentratedto a gum by rotary evaporation. The material wasdissolved in 700 mL of DCM and washed with 500 mL of0.5 KH2PO4 solution followed by 500 mL of satd aqNaHCO3. The solution was dried over MgSO4, filtered,concentrated by rotary evaporation, and then purified bycolumn chromatography on a 15 × 150 cm bed ofactivated silica gel eluted with a gradient of 1-8% MeOHover 18 L of mobile phase and 1% pyridine in DCM.Fractions were inspected by TLC (Rf desired product0.2, UV active as above, in 10% MeOH, 2% pyridine inDCM), and those with pure product were pooled andevaporated to yield 16.5 g (17%) of 11 as a white foam.1H NMR (δ, 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.

1148 Bioconjugate Chem., Vol. 13, No. 5, 2002 Lyttle et al.

Calcd for C41H45F3N4O9‚C5H5N: 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)-acryla-mido-5′-(4,4′-dimethoxytrityl)-3′-(2-cyanoethyldiiso-propylphosphoramidite)-2′-deoxyuridine, 2. 11 (16.5g, 21 mmol) was dried by rotary evaporation from 200mL of dry pyridine followed by high vacuum in a 1000mL round-bottom flask. A mixture of 8.3 g (27 mmol) of2-cyanoethyl tetraisopropylphosphorodiamidite and 500mg of tetrazole (7.2 mmol) in 300 mL of dry CH3CN wasprepared and added to the flask containing the nucleoside11. The flask was stoppered and allowed to stand for 2 hafter thorough mixing. Inspection of the reaction mixtureby TLC (2% pyridine/EtOAc) showed complete conversionto a new product, Rf 0.5. The reaction mixture wasconcentrated to a tar by rotary evaporation and redis-solved in 300 mL of EtOAc. The solution was washedwith 200 mL of satd NaHCO3 followed by drying withMgSO4 and filtration. The solution was concentrated toa tar by rotary evaporation and the product purified bycolumn chromatography on a 5 × 20 cm bed of activatedsilica gel eluted with 2% pyridine in EtOAc. Fractionswere inspected by TLC (2% pyridine in EtOAc) andvisualized with 10% H2SO4 and heat. Fractions contain-ing pure 2 were pooled and concentrated by rotaryevaporation and high vacuum to yield 13.5 g (65%) of 2as 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 400mL 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 NaHCO3was added, as needed, to maintain a basic pH. Themixture was stirred overnight. EtOAc (600 mL) wasadded, and the mixture was partitioned. The organicphase was washed with 300 mL of 1 N HCl, and then300 mL of water followed by 300 mL of satd NaHCO3.The solution was dried over MgSO4, filtered, and evapo-rated to 53 g of a beige solid. The solid was dissolved in400 mL of EtOAc with heating and applied to a 15 × 50cm bed of activated silica gel packed and eluted withEtOAc. Fractions were inspected by TLC (Rf 0.25, 2%pyridine in EtOAc) and visualized with iodine vapor.Fractions containing pure material were pooled andconcentrated by rotary evaporation and high vacuum toyield 25.2 g of N-acryloyl-6-amino-1-hexanol as a whitesolid. A portion of this material (10 g, 58 mmol) wasdissolved in a mixture of levulinic acid (7 mL, 8 g, 68mmol), 5 mL of N-methylimidazole, and 200 mL of drypyridine. The solution was concentrated to a tar by rotaryevaporation and redissolved in 300 mL of dry CH3CN.Diisopropylcarbodiimide (10 mL, 8 g, 64 mmol) wasadded, and the mixture was allowed to stand overnightafter mixing. Solids were filtered off, and the solutionwas concentrated to a tar by rotary evaporation. Thematerial was dissolved in 300 mL of DCM and washedwith 200 mL of 1 N citric acid followed by 200 mL of satdNaHCO3 followed by drying with MgSO4 and filtration.The solution was concentrated to a tar by rotary evapora-tion and the product purified by column chromatographyon a 5 × 20 cm bed of activated silica gel eluted with 1:1petroleum ether: EtOAc. Fractions were inspected byTLC (1:1 petroleum ether: EtOAc) and visualized withiodine vapor. Fractions containing pure 13 (Rf 0.35) werepooled and concentrated by rotary evaporation and highvacuum to yield 6 g (38%) of 12 as a white solid. 1H NMR(δ, 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)-acryla-mido-5′-(4,4′dimethoxytrityl)-2′-deoxyuridine, 14.(This reaction must be done in a fume hood because H2S,which is a toxic and malodorous gas, is used to precipitatemetals during workup). Potassium tetrachloropalladate-(II), K2PdCl4 (12 g, 36 mmol), was added over 20 min inthree 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 10min. The reaction mixture became dark brown. Thesolution was stirred and refluxed for 20 min, and then17 g (37 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 wasdeposited on the sides of the reaction vessel. Stirring andgentle reflux was continued overnight. The solution wasallowed to cool, and H2S gas was bubbled into thesolution. A trap containing Clorox was used to controlthe odor of the gas. Once the brown precipitate was nolonger generated, H2S delivery was stopped and thesolution was filtered to produce a clear yellow solution.The solvent was removed by rotary evaporation and highvacuum overnight to give 28 g of alkylated nucleoside13 as a beige solid. A small amount of the foam waspurified by dissolution in MeOH, addition of water to acloud point, and chilling. Crystals were obtained, TLCRf 0.3 (20% MeOH, 2% pyridine in DCM), which was UV(254 nm) active; the spot also fluoresced blue whenirradiated with 312 nm UV. The bulk, unpurified mate-rial was dried by rotary evaporation with 300 mL of drypyridine and was then redissolved in 300 mL of drypyridine. 4,4′-Dimethoxytrityl chloride (10 g, 29 mmol)was added, and the mixture was stirred overnight. A dropof the reaction mixture was dissolved in 1 mL of EtOAcand washed with 1 mL of satd NaHCO3. TLC showedthe appearance of product (Rf 0.5, 6% MeOH, 2% pyridinein DCM) which had the same UV characteristics as 11and also became orange when the plate was sprayed with10% H2SO4 and heated. MeOH (10 mL) was added toquench the reaction, and the brown solution was con-centrated to a gum by rotary evaporation. The materialwas dissolved in 500 mL of DCM and washed with 400mL of 0.5 M KH2PO4 solution followed by 400 mL of satdNaHCO3. The solution was dried over MgSO4, filtered,concentrated by rotary evaporation, and then purified bycolumn chromatography on a 7 × 50 cm bed of activatedsilica gel eluted with a gradient of 1-5% MeOH over 10L of mobile phase and 1% pyridine in DCM. Fractionswere inspected by TLC (Rf desired product 0.5, UV activeas above, in 6% MeOH, 2% pyridine in DCM), and thosewith pure product were pooled and evaporated to yield6.8 g (24%) of 14 as a white foam. 1H NMR (δ, 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 C44H51N3O11‚1/2C5H5N: 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)-acryla-mido-5′-(4,4′dimethoxytrityl)-3′-(2-cyanoethyldiiso-propylphosphoramidite)-2′-deoxyuridine, 3. 14 (6.8g, 8.5 mmol) was dried by rotary evaporation from 100mL of dry pyridine followed by high vacuum in a 500 mLround-bottom flask. A mixture of 3.0 g (10 mmol) of

Modified Oligonucleotide Bases, Internally Dye-Labeled DNA Bioconjugate Chem., Vol. 13, No. 5, 2002 1149

2-cyanoethyl tetraisopropylphosphorodiamidite and 180mg of tetrazole (2.6 mmol) in 100 mL of dry CH3CN wasprepared and added to the flask containing the nucleoside14. The flask was stoppered and allowed to stand for 2 hafter thorough mixing. Inspection of the reaction mixtureby TLC (2% pyridine/EtOAc) showed complete conversionto a new product, Rf 0.6. The reaction mixture wasconcentrated to a tar by rotary evaporation and redis-solved in 200 mL of EtOAc. The solution was washedwith 100 mL of satd NaHCO3 followed by drying withMgSO4 and filtration. The solution was concentrated toa tar by rotary evaporation and the product purified bycolumn chromatography on a 3 × 20 cm bed of activatedsilica gel eluted with 2% pyridine in EtOAc. Fractionswere inspected by TLC (2% pyridine in EtOAc) andvisualized with 10% H2SO4 and heat. Fractions contain-ing pure 3 were pooled and concentrated by rotaryevaporation and high vacuum to yield 5.5 g (81%) of 3 asa 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 8750DNA synthesizers with standard conditions as previouslydescribed (13). Test sequences 5′-CGATCTGAXTAGCTY-3′, 5′-TTTTXTTTTT-3′ and 5′-TTTTTTTTTX-3′where Xwas the nucleotide residue resulting from the use of 1 or3 and Y were different natural bases were made at 1.0µm and 200 nm synthesis scales. For internal labeling,first a hydrazine solution of 160 µL of hydrazine hydrate(Aldrich, 50 wt % water) in 8 mL of pyridine and 2 mLof HOAc was prepared and well mixed. The DNAsequencer was halted after the base (before X) added andthe DMT was removed. After thorough washing withCH3CN, a solution of 200 µL of 100 mg/mL of 1 or 3 indry acetonitrile and 200 µL of 0.4 M S-ethyltetrazole indry acetonitrile were introduced onto the column by theDNA synthesizer. After 2-3 min, the coupling solutionwas washed off the column with acetonitrile, and oxida-tion, followed by capping, was performed. Manual cou-pling with syringes was also successfully performed (15).The hydrazine cocktail above was introduced into thecolumns; we did this with 200 µL of solution in 1 mLsyringes but it can also be done on the instrument. Afterthe support was well wetted with the solution, it wasallowed to stand 12-15 min and then was thoroughlywashed with CH3CN. Either TAMRA amidite or 6-FAMwas coupled next; the same procedures as above wereused. A 10 min coupling time was used for the TAMRAamidite. Oxidation and capping were performed after thecoupling solution was washed off the column with aceto-nitrile. The TAMRA coupling sample was exposed to thecapping solution for 5 min. Next, automated DNAsynthesis was continued to the 5′ terminus, and the finalDMT was removed. The CPG was placed into screw capeppendorf tubes, and 1 mL of ammonia for the fluoresceinDNA and 1 mL of the TAMRA deprotection cocktail forthe TAMRA DNA was added to each. The tubes wereheated at 55 °C for 18 h and cooled and the solutionsevaporated. Yields of labeled DNA at all synthesis scaleswere close to those without hydrazine treatment, indicat-ing negligible loss of material due to premature cleavageby the hydrazine reagent.

3′-HEX and TET CPG. DMT-O-ethylsulfonyl CPG(16) (15 g) was placed in a 300 mL coarse frit sinteredglass funnel atop a 2 L sidearm flask. 3% Dichloroaceticacid in CH2Cl2 (200 mL) was poured in, and the supportwas agitated briefly with a spatula. The solution becamebrightly orange. After 2 min, the solution was drainedand the step was repeated twice with 200 mL of fresh

3% dichloroacetic acid in CH2Cl2. The support was thenwashed three times with 200 mL of CH2Cl2, twice with100 mL of CH3CN, and once with 100 mL of pyridine.The support was transferred into a 250 mL round-bottomflask, and 1 g of 1 was added. 40 mL of dry pyridine wasadded, and the solvent was removed by rotary evapora-tion. A plug of glass wool was used to keep the CPG fromleaving the flask. Once dry, high vacuum was applied tothe flask for 18 h. A solution of 150 mL of 0.4 MS-ethyltetrazole in dry acetonitrile was prepared, andenough of this was added to the flask containing the CPGto make a slurry. The slurry was allowed to stand for 15min and was poured into a sintered glass funnel. Thesupport was washed three times with 100 mL of CH3-CN, and then 100 mL of amidite oxidizer solution (13)was added. After 5 min, the support was washed twicewith 100 mL of CH3CN. Next, 100 mL of a mixture ofacetic anhydride, N-methyl imidazole, and THF (1:1:8)was added to the support. After 15 min exposure, theacetylation solution was removed and the support waswashed three times with 100 mL of CH3CN, and a smallamount of this was dried. The DMT loading was 18 µm/g(17). The hydrazine cocktail above (100 mL) was mixedand added to the support. After 15 min, the supportwas washed three times with 100 mL of CH3CN andwas divided into two roughly equal portions. HEX andTET Amidite, 0.5 g respectively, were added to eachportion, and coupling, oxidation, and capping steps usingthese amidites proceeded as above. Test sequences 5′-TTTTTTTTTdC(N4-(2-(ethylene glycol)ethyl)-HEX)-3′ andthe analogous TET sequence were made; the HEX samplewas deprotected with concentrated ammonia for 18 h atroom temperature while standard conditions were usedfor the TET sample.

Anion exchange HPLC analyses were performed asfollows: 2-20 µL of the aqueous samples, depending onthe concentration, was injected onto a Dionex anionexchange column (4.6 × 250 mm); samples were elutedat 2 mL/min with aqueous buffers of (A) 0.025 M TrisHCl and 0.01 M TRIS, and (B) 0.025 M Tris HCl, 0.01 MTRIS, and 1.0 M NaCl, using a linear gradient of 1:0 to0:1 over 20 min, with UV detection at 260 nm. Reversephase HPLC as follows: 20 µL of the aqueous samplewas injected onto a HAISIL HL C18 5 µm particle sizecolumn (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 over15 min. UV detection at 260 nm. Samples were preparedfor 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 of1 and 3 had to be developed. (2) Easy and efficient solid-phase synthesis methods for incorporation of thesecompounds into DNA as well as subsequent label attach-ment had to be established. (3) Finally analytical tech-niques had to be devised to establish the purity andefficacy of the product DNA in practical applications.

Synthesis of Compounds 1-3. For 1, commerciallyavailable 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, whichwas converted into levulinate ester 7, obtained as a whitefoam. The use of the TBDMS group instead of thetrimethylsilyl transient protection used in (9) affordedcertain advantages: the 3′-OTBDMS triazolide was a

1150 Bioconjugate Chem., Vol. 13, No. 5, 2002 Lyttle et al.

stable compound which could be prepared in largequantities and stored cold, allowing many differentderivatives to be prepared from one preparation oftriazolide. Also, the 3′-OTBDMS group lent greatersolubility in organic solvents which facilitated extractionand isolation of more polar conjugates containing pendantmoieties such as biotin. Removal of the 3′-hydroxylmasking TBDMS group with TBAF gave alcohol 8,followed by phosphitylation with 2-cyanoethyl tetraiso-propylphosphorodiamidite and catalytic tetrazole gave 1.See Scheme 1. The principal advantage of the use of 1 iscost; a similar derivative without the 5-methyl group canbe made starting with more expensive deoxyuridine. Thiscompound should also be accessible from DMTdC(Bz), butmore steps would be required (18). The primary disad-vantage to the use of 1 are anticipated lower Tms of DNAmade with this residue internally than with a naturaldeoxycytidine in binding to a complimentary DNA strand.A companion paper compares structures made with 1with those made with 2 and 3 and bears this out. Forthis reason 1 is primarily used by us as a 3′ labelingtool.

Synthesis of 2. The Heck palladium-assisted couplingof organomercurials to olefins was first applied to nucleo-side modification by Bergstrom (19). There is a recentpaper about palladium-mediated reactions applied tonucleosides also coauthored by Bergstrom (20). We at-tempted first the literature synthesis (1) of 2. Formationof N-acryloyl-N′-trifluoroacetyl-1,6-hexanediamine by aone-pot mixing of 1,6-hexanediamine, acryloyl chloride,and trifluoroacetic anhydride gave a mixture of products

which gave the desired product in low yield. Purificationwas difficult with the conditions described, owing to closeRf values of products and contaminants. A palladium-(II)-mediated coupling reaction with the material and5-chloromercurated deoxyuridine also gave a mixture ofproducts, which afforded only a few percent overall yieldof the desired DMT nucleoside 11 when treated withDMT chloride followed by chromatography. Upon con-sideration of the low overall yield and high cost of thelithium tetrachloropalladate(II) used, we decided thatthis was not a cost-effective way to prepare 2. Analternative scheme was developed, see Scheme 2. 1,6-Hexanediamine was treated with di-tert-butyl dicarbon-ate, and the resulting 6-tert-butyloxycarbonamidyl-1-aminohexane was partially purified by extraction. Acryloylchloride was added to the compound under basic condi-tions to give 6-acrylolyl-N′-tert-butyloxycarbonyl-1,6-hex-anediamine, 9, which was obtained in high purity afterchromatography. 5-Chloromurcurated deoxyuridine wasprepared as per Ruth (1), and a palladium(II)-mediatedcoupling reaction with 9 gave, after several steps, theDMT nucleoside 11 in 24% yield (see Scheme 2). Signifi-cant savings in the cost of the palladium reagent wasachieved by refluxing a THF solution of potassiumtetrachloropalladate with lithium chloride prior to theaddition of 9 and the chloromurcurated nucleoside.Recent literature reports that the needed palladiumreagent can also be prepared by mixing PdCl2 and LiClin MeOH for 24 h (20) and this also works well, as longas all of the methanol is removed prior to dissolution inTHF. 11 was converted into 2 by phosphitylation with

Scheme 1. Synthesis of 1

Scheme 2. Synthesis of 2

Modified Oligonucleotide Bases, Internally Dye-Labeled DNA Bioconjugate Chem., Vol. 13, No. 5, 2002 1151

2-cyanoethyltetraisopropylphosphorodiamidite and cata-lytic tetrazole.

Synthesis of 3. A similar strategy to that used for 2was employed. 6-amino-1-hexanol was first reacted withacryloyl chloride to give 6-acryloylamido-1-hexanol, fol-lowed by esterification with levulinic acid to give 6-acry-loylamido-1-hexane levulinate 12, obtained as a whitesolid after chromatography. 12 was coupled to chloro-murcurated deoxyuridine by those methods used for 2to give 13, which gave 14 after treatment with DMTchloride. Conversion of 14 into phosphoramidite 3 wasaccomplished by the same method as for 2.

Labeled DNA Synthesis. Incorporation of 1 and 3into DNA was accomplished with standard methods andcoupling times (13) and removal of levulinate protectionwas carefully optimized; previously reported conditions(9) were too harsh (0.5 M hydrazine in 1:1 pyridine:HOAcfor 60 min), resulting in unwanted DMT loss and poorproducts, in our hands. We found that 0.25 M hydrazinein 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 DNAoligomerization scheme. For us, the optimum exposuretime was determined by an experimental system wherein1 was first coupled onto sulfonylethyl CPG (16) and thensubjected to various times of hydrazine exposure (2-12min). 6-FAM was then coupled onto the deprotectedhydroxyl, followed by oxidation and capping, and thenextension of the DNA fragment by nine more additioncycles of T amidite. The product 3′-nucleoside fluoresce-inated 10 mers were then cleaved with ammonia, andAX HPLC showed that the best results were obtainedwith 12 min of hydrazine exposure. The composition wasconfirmed by MALDI mass spectroscopy, calcd 3682,found 3687 amu. Longer exposure times gave moreextraneous products. The same conditions using 4 in lieuof 1 did not produce good results (data not shown).

Another question to be addressed was the order ofaddition. Was it best to build the whole DNA fragmentwith the internal levulinated base and then deprotect andadd the tag? This would be easier than the converse,

Figure 1. Time course for levulinoyl protecting group removal; A ) 5′T9C(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 minhydrazine, same other conditions; F ) FAM after chain assembly with 12 min hydrazine exposure.

1152 Bioconjugate Chem., Vol. 13, No. 5, 2002 Lyttle et al.

which was to deprotect the levulinated hydroxyl at theposition where it was added, add the tag, and thencontinue the synthesis out to the 5′ terminus. An ad-ditional experiment was done with the time course studyabove, which removed the OLev group with the 12 min(best) conditions after all 10 bases were added and added6-FAM as the last step. Figure 1 panel F shows clearly aresult inferior to addition of fluorescein amidite prior tobuilding the DNA fragment. 3′-HEX and TET CPGs weremade with these techniques and gave good results in the

synthesis of the analogous 10 mers. See Figures S1 andS2. Another comparison of the order of addition was donewith 5′-CGATCTGAXTAGCTT-3′ where X resulted fromthe incorporation of 1; good product purity was seen whenaddition of FAM occurred after addition of 1 and beforechain extension (MALDI m/e calcd 5182, found 5176).Addition of fluorescein after the completion of the se-quence gave other products in addition to that desired;incomplete FAM addition as well as double FAM additionwere seen by mass spectroscopy (see Figure 2) Similar

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

Modified Oligonucleotide Bases, Internally Dye-Labeled DNA Bioconjugate Chem., Vol. 13, No. 5, 2002 1153

results were obtained with 3 and FAM. The analogousinternally labeled DNA fragment was made with 3 anda TAMRA amidite (3) and also gave good results whenthe TAMRA amidite was added before the rest of thechain, m/e calcd 5288, found 5275 amu (see Figure S3).Finally, the question of potential base modification bythe conditions employed had to be addressed. CPGsamples containing DMT-ABz, -CBz, -CAc, -GiBu, -GDMF and-T were subjected to the conditions employed for TAMRAamidite incorporation which includes the hydrazinetreatment and a 5 min acetylation step before DMTremoval and cleavage with tert-butylamine cocktail (18h, 55 °C). The cleaved nucleosides were analyzed by RP-HPLC, and no extraneous peaks were seen compared tocontrols (data not shown).

CONCLUSION

Oligonucleotide protecting group chemistry first de-veloped by Van Boom for 5′-hydroxyl protection (21) andlater applied to side chain branching structures (9) hasnow been extended by us to allow the synthesis ofinternally dye-labeled DNA. New techniques and novelnucleoside phosphoramidite synthons have been devel-oped which should allow the rapid synthesis of internallylabeled structures. Utility of the new methods has beendemonstrated with a variety of currently used fluoro-phores. Significant advantages in speed and simplicityover widely used solution-phase coupling methods, usu-ally of an internally amine-labeled oligonucleotide withan active ester in aqueous solution, should be realized.

Supporting Information Available: Spectra and com-bustion analyses data. This material is available free ofcharge via the Internet at http://pubs.acs.org.

LITERATURE CITED

(1) Ruth, J. (1991) Oligodeoxynucleotides with reporter groupsattached to the base. Oligonucleotides and Analogues (Eck-stein, F., Ed.) pp 255-282, IRL Press, Oxford.

(2) Vinayak, R. (1999) A convenient, solid-phase coupling ofrhodamine dye acids to 5′ amino-oligonucleotides. Tetrahe-dron Lett. 40, 7611-7613.

(3) Lyttle, M. H., Carter, T. C., Dick, D. J., and Cook, R. M.(2000) A tetramethyl rhodamine (Tamra) phosphoramiditefacilitates solid-phase-supported synthesis of 5′-Tamra DNA.J. Org. Chem. 65, 9033-9038.

(4) Mullah, B., and Andrus, A. (1997) Automated Synthesis ofDouble Dye-Labeled Oligonucleotides using Tetramethyl-rhodamine (TAMRA) Solid Supports Tetrahedron Lett. 38,5751-5754.

(5) Rudert, W. A., Braun, E. R., Faas, S. J., Menon, R., Jaquins-Gerstl, A., and Trucco, M. (1997) Double Labeled FluorescentProbes for 5′ Nuclease Assays: Purification and PerformanceEvaluation. Biotechniques 22, 1140-1145.

(6) Glen Research has biotin, fluorescein, and TAMRA func-tionalized dU amidites.

(7) Kahn, S. I., and Grinstaff, M. W. (1999) Palladium(0)-Catalyzed Modificatons of Oligonucleotides during AutomatedSolid-Phase Synthesis J. Am. Chem. Soc. 121, 4704-4705.

(8) Hwang, J., and Greenberg, M. (2001) Synthesis of 2′-Modified Oligonucleotides via On-Column Conjugation. J.Org. Chem. 66, 363-369.

(9) Chang, C., Urdea, M. S., and Horn, T. (1996) N-4 ModifiedPyrimadine Deoxynucleotides and OligonucleonucleotideProbes 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 Nu-cleases by 5-Alkyl, 5(1-alkenyl)- and 5(1-alkynyl) pyri-madines. Nucleosides Nucleotides 18, 1929-1933.

(12) Walton, T., Lyttle, M., Dick, D., and Cook, R. (2002)Evaluation of New Linkers and Synthetic Methods forInternal 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 Syn-thesized with 2′-tert-butyldimethylsilyl Protected Cyanoeth-ylphosphoramidite Monomers. Nucleic Acids Res. 18, 3347-3355.

(14) Lyttle, M. H., Napolitano, E. W., Calio, B. L., and Kauvar,L. M. (1995) Mutagenesis Using Trinucleotide â-CyanoethylPhosphor-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′-ModifiedDNA. 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-amino-

hexyl)-2-deoxy-5′-O-(4,4′dimethoxytrityl) cytidine NucleosidesNucleotides 17, 1209-1213.

(19) Bergstrom, D. E., and Ogawa, M. K. (1978) C-5 Substitutedpyrimidine nucleosides. 2. Synthesis via olefin coupling toorganopalladium intermediates derived from uridine and 2′-deoxyuridine. 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 Nucleo-sides. 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 ofcomplementary DNA fragments via phosphotriester inter-mediates. Tetrahedron Lett. 17, 4875-4882.

BC020011C

1154 Bioconjugate Chem., Vol. 13, No. 5, 2002 Lyttle et al.