Synthesis of Oligonucleotide Inhibitors of DNA (Cytosine-C5) Methyltransferase Containing...

ANTISENSE amp NUCLEIC ACID DRUG DEVELOPMENT 11369ndash378 (2001)Mary Ann Liebert Inc

Synthesis of Oligonucleotide Inhibitors of DNA (Cytosine-C5)Methyltransferase Containing 5-Azacytosine Residues

at Specific Sites

RAMON GUumlIMIL GARCIacuteA1 ADAM S BRANK2 JUDITH K CHRISTMAN23 VICTOR E MARQUEZ4

and RAMON ERITJA15

ABSTRACT

The incorporation of 5-azacytosine residues into DNA causes potent inhibition of DNA (Cytosine-C5) methyl-transferases The synthesis of oligodeoxyribonucleotides incorporating single or multiple 5-aza-29-deoxycyti-dine residues at precise sites was undertaken to generate an array of sequences containing the reactive 5-aza-cytosine base as specific target sites for enzymatic methylation Preparation of these modified oligonucleotidesrequires the use of 2-(p-nitrophenyl)ethyloxycarbonyl (NPEOC) groups for the protection of exocyclic aminofunctions These groups are removed under mild conditions thus avoiding conventional protocols that aredetrimental to the integrity of the 5-azacytosine ring

INTRODUCTION

5-AZACYTOSINE (ZCYT OR Z) IS A CYTOSINE ANALOG whereinthe carbon at position 5 of the pyrimidine ring is replaced by

a nitrogen atom Both ribonucleoside and 29-deoxyribonucleo-side (ZCyd and ZdCyd) versions containing this modified baseare potent cytotoxic agents that have been used for the treat-ment of sickle cell anemia myelodysplastic syndrome leuke-mia and several cancers with varying degrees of success(Pinto and Zagonel 1993 Goldberg et al 1993 Momparler etal 1997 Rochette et al 1994) As result of their incorporationinto DNA these drugs have a unique mechanism of action ofdemethylating DNA that affects gene regulation (Bender et al1998 Christman 1984 Christman et al 1985 Creusot et al1982) In order to help elucidate the molecular basis of the in-hibitory properties of ZCyt in DNA we have undertaken thepreparation of oligodeoxyribonucleotides containing ZCyt Thehydrolyic instability of the triazine ring is well documented(Beisler 1978 Lin et al 1981) and precludes the use of stan-dard phosphoramidite protocols particularly during treatmentwith ammonia

Preparation of oligonucleotides containing the more stable

56-dihydro-5-azacytosine base has been described but conver-sion of the reduced base to ZCyt was not efficient enough toyield the desired oligonucleotides in acceptable amounts (God-dard and Marquez 1988) Previous studies (Avintildeoacute et al 1995)using the H-phosphonate derivative of ZdCyd without protec-tion of the exocyclic amino group showed that the triazine ringwas stable to nonnucleophilic bases (such as 18-diazabici-clo[540]undec-7-ene DBU) in anhydrous pyridine conditionsunder which the p-nitrophenylethyl (NPE) (Scheme 1) and p-nitrophenylethoxycarbonyl (NPEOC) (Scheme 1) groups de-scribed by Pfleidererrsquos group (Himmelsbach et al 1984) are ef-ficiently removed Unfortunately the H-phosphonate methodwas practical only for the prepartion of short oligonucleotidesequences When oligonucleotides of 15ndash20 bases were pre-pared the coupling efficiency of the NPEOC-protected H-phosphonate monomers dropped significantly To overcomethis problem we decided to use a modified approach of themore conventional phosphoramidite methodology

In this paper we describe the preparation of the NPEOC-pro-tected phosphoramidite of ZdCyd and its use in the preparationof longer oligonucleotides containing ZCyt A short descriptionof this work was reported previously (Eritja et al 1997)

1European Molecular Biology Laboratory D-69117 Heidelberg Germany2Department of Biochemistry and Molecular Biology and 3UNMCEppley Cancer Center University of Nebraska Medical Center Omaha NE

69198-45254Laboratory of Medicinal Chemistry Center for Cancer Research National Cancer Institute at Frederick Frederick MD 217025Present address Institut de Biologiacutea Molecular de Barcelona CSIC E-08034 Barcelona Spain

369

MATERIALS AND METHODS

29-Deoxy-N4-[2-(4-nitrophenyl)ethoxycarbonyl]-5-azacytidine (2)

29-Deoxy-5-azacytidine (228 mg 1 mmol) was dried after threesuccessive evaporations of a solution in anhydrous pyridine Theresidue was dissolved in anhydrous dimethylformamide (DMF)and treated with 053 ml (25 mmol) hexamethyldisilazane Afterbeing stirred for 1 hour at ambient temperature the solution wasconcentrated and the residue was dried by three cycles of evapo-ration from toluene (5 ml) yielding a white foam (thin-layer chro-matography [TLC] Retention factor (Rf) 066 20 ethanol indichloromethane) The residue was thrice dissolved in 5 ml anhy-drous pyridine and concentrated to dryness before finally beingdissolved in 30 ml anhydrous pyridine To this solution 350 mg(15 mmol) of 2-(p-nitrophenyl)ethyl chloroformate dissolved in 5ml dichloromethane was added and after 30 minutes of magneticstirring at ambient temperature the reaction was judged completeby TLC (Rf 093 10 ethanol in dichloromethane) The solutionwas concentrated to dryness and traces of pyridine were removedby three cycles of evaporation of a toluene solution (5 ml) Theresidue was dissolved in chloroform and the organic solution waswashed with 1 M sodium bicarbonate dried (Na2SO4) and con-centrated to dryness

Removal of the trimethylsilyl groups in DMF solutions Theresidue from the described reaction was dissolved in DMFTHF(tetrahydrofurane) (31) and the resulting solution was stirredfor 3 days at ambient temperature After the solvents were re-moved the residue was triturated with ether to form a whitesolid that was purified by column chromatography on silica gel(3ndash10 step gradient of ethanol in chloroform) to give 180mg (043 mmol 43) of the desilylated product [TLC (10ethanol in chloroform) Rf 5 053 (5 ethanol in chloroform)Rf 5 012] 1H-NMR (DMSO-d6) d (ppm) 103 (wide s 1HNH) 841 (s 1H H-6) 771 (d 2H J 5 86 Hz NPEOC) 718(d 2H J 5 86 Hz NPEOC) 556 (t 1H H-19) 486 (d 1H 39-OH) 471 (t 1H 59-OH) 392 (t 2H CH2 NPEOC) 38 (m 1HH-39) 345 (m 1H H-49) 317 (m 2H H-59) 264 (t 2H CH2

NPEOC) 18 (m 2H H-29) 13C-NMR (CDCl3) d (ppm) 1630(C-4) 1571 (C-6) 1535 (CO NPEOC) 1507 (C-2) 1467 (C-NO2) 1455 (Cq Ar) 1299 (CH Ar) 1235 (CH Ar) 879 (C-49) 867 (C-19) 677 (C-39) 654 (CH2) 404 (C-29) 346(CH2) Anal Calcd for C17H19N5O8 (Found C 481 H 47N 164 requires C 485 H 46 N 166)

Removal of the trimethylsilyl groups with TAS-F Alterna-tively the silyl protected product resulting from a 4 mmolscale reaction was treated with a solution of tris(dimethy-

lamino) sulfonium difluorotrimethylsilicate (TAS-F) (12mmol 33 mmol) in DMF (50 ml) After 2 hours at ambienttemperature the solution was concentrated to dryness andthe residue was purified by column chromatography on silicagel as described to give the same product as before (135 g32 mmol 80)

29-Deoxy-59-O-dimethoxytrityl-N4-[2-(4-nitrophenyl)ethoxycarbonyl]-5-azacytidine (3)

29-Deoxy-N4-[2-(4-nitrophenyl)ethoxycarbonyl]-5-azacyti-dine (163 g 387 mmol) was dried after repeated evaporationof a solution in anhydrous pyridine and reacted withdimethoxytrityl chloride (144 g 426 mmol) in pyridine (20ml) After being stirred for 3 hours at ambient temperature thesolution was concentrated to dryness Traces of pyridine wereremoved by three cycles of evaporation of a toluene solution (5ml) The residue was dissolved in dichloromethane and the or-ganic solution was washed with 1 M sodium bicarbonate aque-ous solution dried (Na2SO4) and concentrated to dryness Theresidue was purified by column chromatography on silica geleluted with a 0ndash5 step gradient of ethanol in chloroform togive 201 g (278 mmol 71) of 3 [TLC (10 ethanol indichloromethane) Rf 5 040] IR (cm21) 3400 2960 17701700 1610 1510 1350 1250 1220 1180 1030 830 1H-NMR(CDCl3) d (ppm) 860 (s 1H H-6) 816 (d 2H Ar NPEOC)743 (d 2H Ar NPEOC) 71ndash73 (m 9H Ar DMT) 681 (d4H Ar DMT) 616 (t 1H H-19) 44 (m 3H H-39 CH2

NPEOC) 42 (m 1H H-49) 377 (s 6H Me DMT) 34 (m2H H-59) 31 (t 2H CH2 NPEOC) 28 (m 1H H-29) 23 (m1H H-29) 13C-NMR (CDCl3) d (ppm) 1631 (C-4) 1584 (CqDMT) 1568 (C-6) 1534 (CO NPEOC) 1502 (C-2) 1467(C-NO2) 1451 (Cq Ar) 1442 (Cq DMT) 1354 (Cq DMT)1352 (Cq DMT) 1297 (CH Ar) 1278 (CH DMT) 1267(CH DMT) 1235 (CH Ar) 1131 (CH Ar) 877 (C-49) 869(C-19) 868 (Cq DMT) 712 (C-39) 655 (CH2) 631 (C-59)550 (CH3) 419 (C-29) 345 (CH2)

NN-Diisopropylamino-2-(p-nitrophenyl)ethoxychlorophosphine

In a 1-L round-bottom flash 175 ml (200 mmol) phospho-rous trichloride was dissolved in 500 ml anhydrous acetoni-trile and kept under argon atmosphere The solution wascooled over an icebath and treated dropwise with a solution of2-(p-nitrophenyl)ethanol (334 g 20 mmol) dissolved in 100ml acetonitrile The reaction mixture was gradually warmed toambient temperature and stirred for 1 hour The mixture wasconcentrated to give a yellowish oil that was used in the nextstep without further purification The crude 2-(p-nitro-phenyl)ethoxy dichlorophosphine obtained was dissolved in500 ml acetonitrile and the solution was cooled with an ice-bath To this solution 62 ml (44 mmol) NN-diisopropy-lamine dissolved in 100 ml acetonitrile was added and afterthe addition the mixture was gradually warmed to ambienttemperature after 6 hours of stirring The white precipitatewas filtered and the filtrate was concentrated to dryness togive 5 g (15 mmol 75) of a yellowish oil that was used di-rectly in the preparation of all phosphoramidites without fur-ther purification 31P-RMN dP (75 MHz CDCl3) 17774 ppm(90 pure by phosphorus NMR)

GUumlIMIL GARCIacuteA ET AL370

Scheme 1 The NPEOC and NPE protecting groups

Synthesis of 2-(p-nitrophenyl)ethyl phosphoramidites ofthe natural bases and 5-methyl-29-deoxycytidine

The appropriate 59-DMT-NO-(NPEOC NPE)-protected 29-deoxynucleoside (2 mmol) was dried after several coevapora-tions with dry acetonitrile The residue was dissolved in 50 mlanhydrous tetrahydrofuran containing 1 ml (6 mmol) diiso-propylethylamine The solution was cooled over an icebath and1 g (3 mmol) NN-diisopropylamino-2-(p-nitrophenyl)ethoxychlorophosphine was added After 1 hour of magnetic stirring atambient temperature the solution was concentrated to drynessThe residue was dissolved in dichloromethane and the organicsolution was washed with 1 M sodium bicarbonate and satu-rated sodium chloride solution dried (Na2SO4) and concen-trated to dryness The residue was purified by column chro-matography on silica gel with ethyl acetatedichloromethane(11) The column was packed with the solvent mixture contain-ing 1 triethylamine and eluted with the mixture without tri-ethylamine The appropriate fractions were collected and con-centrated to dryness

39-O-NPE phosphoramidite of DMT-T Yield 83 (141 g167 mmol) TLC (ethyl acetatedichloromethane 11) Rf 5

085 31P-RMN dP (75 MHz CDCl3) 14405 14443 ppm

39-O-NPE phosphoramidite of DMT-C(NPEOC) Yield76 (155 g 152 mmol) TLC (ethyl acetatedichloromethane11) Rf 5 078 31P-RMN dP (75 MHz CDCl3) 14462 ppm

39-O-NPE phosphoramidite of DMT-G(NPE NPEOC)Yield 79 (190 g 152 mmol) TLC (ethylacetatedichloromethane 11) Rf 5 093 31P-RMN dP (75 MHzCDCl3) 14398 ppm

39-O-NPE phosphoramidite of DMT-A(NPEOC) Yield 77(161 g 154 mmol) TLC (ethyl acetatedichloromethane 11)Rf 5 085 31P-RMN dP (75 MHz CDCl3) 14337 ppm

39-O-NPE phosphoramidite of DMT-5-methyl-C(NPEOC)Yield 83 (079 g 076 mmol starting from 092 mmol) TLC(ethyl acetatedichloromethane 11) Rf 5 089 31P-RMN dP (75MHz CDCl3) 14442 ppm

29-Deoxy-59-O-dimethoxytrityl-N4-[2-(4-nitrophenyl)ethoxycarbonyl]-5-azacytidine 3-O-(2-(p-nitrophenyl)ethoxy)-NN-diisopropyl phosphoramidite (4)

29-Deoxy-59-O-dimethoxytrityl-N4-[2-(4-nitrophenyl)ethoxycarbonyl]-5-azacytidine (1 g 138 mmol) was dissolvedin anhydrous tetrahydrofuran (35 ml) containing 069 ml (414mmol) diisopropylethylamine The solution was cooled in anicebath and 204 mmol NN-diisopropylamino-2-(p-nitro-phenyl)ethoxy chlorophosphine was added After 1 hour ofmagnetic stirring at ambient temperature the solution was concentrated to dryness The residue was dissolved indichloromethane and the organic solution was washed with 1M sodium bicarbonate and saturated sodium chloride solutionsdried (Na2SO4) and concentrated to dryness The residue waspurified by column chromatography on silica gel with ethyl ac-

etatehexane (21) The column was packed with the solventmixture containing 1 triethylamine and eluted with the samesolvent mixture without triethylamine Yield 78 (10 g 108mmol) TLC (ethyl acetatehexane 21) Rf 5 064 (5 ethanolin dichloromethane1 1 triethylamine) Rf 5 093 31P-RMNdP (75 MHz CDCl3) 1457 ppm

Oligodeoxynucleotide syntheses

Oligonucleotide sequences were prepared in a 1-mmol scaleusing phosphoramidite 4 and the 2-(4-nitrophenyl)ethyl phos-phoramidites of the natural bases protected with the NPE andNPEOC groups Controlled-pore glass (CPG) supports havingthe NPENPEOC-protected nucleosides attached through anoxalyl linkage were used (Alul et al 1991 Avintildeoacute et al 1996)Some phosphoramidites were not very soluble in acetonitrileand were dissolved in dry dichloromethane to obtain 01 M so-lutions Standard cycles were used with a slight modification ofcoupling times that were increased from 30 seconds to 2 min-utes Coupling efficiences were 96 as measured by DMTanalysis Deprotection was carried out by treatment of the oli-gonucleotide supports with a 05 M DBU solution in pyridinecontaining 5 mg thymine for 15 hours at ambient temperatureThe resulting solutions were neutralized with acetic acid andconcentrated to dryness The residues were dissolved in waterand desalted with a Sephadex G-25 or G-10 column

Sequences IndashV (Table 1) were prepared with the last DMTgroup on Purification of these oligonucleotides was performedby high-performance liquid chromatography (HPLC) using thefollowing solutions solvent A 5 acetonitrile (ACN) in 100mM triethylammonium acetate pH 65 solvent B 70 ACNin 100 mM triethylammonium acetate pH 65 Columns PRP-1 (Hamilton) 250 3 10 mm Flow rate 3 mlmin and a 30-minute linear gradient from 10 to 80 B Yield (OD units at260 nm after HPLC purification) DMT heptamer I 22 OD (45mg) DMT octamer II 63 OD (018 mg) DMT 15-mer III 15OD (045 mg) DMT 24-mer IV 25 OD (075 mg) DMT 24-mer V 23 OD (80 mg) DMT oligonucleotides IndashV weretreated with 1 ml acetic acid for 30 minutes at ambient temper-ature The acetic acid was removed by extractions with etherand the oligonucleotides without the DMT group were purifiedby HPLC using similar conditions as before but with a 30-minute linear gradient from 0 to 50 B Yield (OD units at 260nm after HPLC purification) heptamer I 09 OD (27 mg) oc-tamer II 2 OD (60 mg) 15-mer III 6 OD (018 mg) 24-mer IV75 OD (022 mg) 24-mer V 08 OD (24 mg)

Oligonucleotide sequences III and VndashXV (Table 2) were pre-pared removing the last DMT group on the synthesizer and puri-fied by HPLC Yield (OD units at 260 nm after HPLC purifica-tion) 15 OD (045 mg) of sequence III 10 OD (03 mg) ofsequence V 8 OD (024 mg) of sequence VI 14 OD (042 mg) ofsequence VII 5 OD (015 mg) of sequence VIII 9 OD (027 mg)of sequence IX 27 OD (081 mg) of sequence XI 15 OD (045mg) of sequence XII 3 OD (90 mg) of sequence XIII 45 OD(135 mg) of sequence XIV 55 OD (165 mg) of sequence XVFive independent syntheses (1 mmol each) of sequence X wereperformed yielding respectively 24 35 45 42 and 21 OD

Selected oligonucleotides were characterized by mass spec-trometry using electrospray ionization under neutral conditionsHeptamer I (59-TAGZTGA-39) expected 2120 found 21204

5-AZACYTOSINE OLIGODEOXYRIBONUCLEOTIDES 371

(M) plus sodium adducts in addition to peaks at 21105 (M-10)and 18308 (M-290) Octamer II (59-TAZGZTGA-39) expected2643 found 26424 (M) plus sodium adducts in addition to apeak at 26324 (M-10) Fifteen-mer III (59-GCAATGGAZC-CTCTA-39) expected 4537 found 4537 (M) in addition topeaks at 4527 (M-10) and 4236 (M-290) plus sodium adductsSequence of 15-mer III carrying the DMT group (59-DMT-GCAATGGAZCCTCTA-3 9) expected 4840 found 48393(M) with no M-10 and M-290 peaks observed Characteriza-tion of 24-mer oligonucleotides using MALDI-TOF was notpossible because of fragmentation during acquisition

Characterization of oligonucleotides by gel electrophoresis

ZCyt-oligonucleotide XII and the corresponding oligonucleo-tide containing cytosine instead of ZCyt were enzymatically radi-olabeled with 32P at the 59-end using TR kinase and g32P-ATP (Sambrook et al 1980) These oligonucleotides weresuspended in 10 mM Tris-HCl pH 78 1 mM EDTA (TE) andanalyzed directly (ss-ZCyt-ODN) or annealed duplexes (obtainedby incubation at 60degC for 60 minutes followed by slow coolingto 37degC) with their complementary strands Annealing conditionswere determined from melting temperature analysis and did notlead to alteration in the inhibitory capacity of the oligonu-cleotides (JK Christian and AS Brank unpublished observa-tions) ssODN (20 ng) or 40 ng dsODN were suspended in 10 ml

double-distilled water and mixed with 40 ml glacial acetic acidfollowed by 60 minutes incubation at 37degC For HhaI digestion20 ng ssCyt-ODN or 40 ng dsCyt-ODN XII were incubated at37degC for 60 minutes with 60 U HhaI in 50 ml NEBuffer 4 (NewEngland Biolabs Beverley MA) Samples in glacial acetic acidwere vacuum dried in a Savant Speed-Vac and resuspended in 10ml distilled water containing 025 xylene cyanol 025bromphenol blue and 30 glycerol All other samples werebrought to the same final concentrations of tracking dyes andglycerol prior to electrophoresis Gels (8 M ureandash20 acry-lamide) were formed and preelectophoresed for 30 minutes at200 V in 90 mM Tris-borate pH 80 2 mM EDTA (TBE) Sam-ples were also electrophoresed at 200 V for 30 minutes or untilthe tracking dye reached the end of the gel Radiolabeled ODN invacuum-dried gels were detected and quantified by PhosphorIm-ager (Molecular Dynamics Sunnyvale CA) analysis

Melting experiments

Equimolar solutions of each complementary pentadecamer(Table 3) dissolved in 50 mM Tris-HCl pH 75 015 M NaClwere mixed The resulting solutions were heated to 90degC andallowed to cool slowly to ambient temperature and sampleswere kept in the refrigerator overnight UV absorption spectraand melting experiments (absorbance vs temperature) wererecorded in 1 cm path-length cells using a spectrophotometerequipped with a temperature controller with a programmed step


TABLE 1 AZACYTOSINE (Z) OLIGONUCLEOTIDES PREPARED USING

TWO HPLC STEPS (DMT-ON AND DMT-OFF)

OD units afterOD units after first second HPLC

Sequences (59 reg 39) HPLC (DMT-on) (DMT-off)

I TAGZTGA 25 09II TAZGZTGA 63 2

III GCAATGGAZCCTCTA 15 6IV ATTGCGCATTCCGGATCZGCGATC 25 75V ATTGCGCATTCCGGATCCGZGATC 23 08

TABLE 2 OLIGONUCLEOTIDE SEQUENCES HAVING 5-AZACYTOSINE

PREPARED USING ONE-STEP PURIFICATION BY HPLCa

Sequence (59 reg 39) OD units after HPLC

III GCAATGGAZCCTCTA 15V ATTGCGCATTCCGGATCCGZGATC 10

VI ATTGZCCATTCCGGATCCGCGATC 8VII ATTGCCCATTCZGGATCCGCGATC 14

VIII ATTGZCCATTCZGATCZGZGATC 5IX ATTGCCCATTZCGGATCCGCGATC 9X TGTCAGZGCATGG 24 35 45 42 21

XI TGTCAGCGZATGG 27XII TGTCAGZGCATGGATGGTTATAAT 15

XIII ATTGCCMATTCCGGATCZGCGATC 3XIV ATGAGCMAAAAAAAAAAZGGCTCA 45XV ATGAGZMAAAAAAAAAACGGCTCA 55

aZ 5-azacytosine M 5-methylcytosine

increase of 05degCmin Melting curves were measured at 260nm on duplex concentration of 4 mM

RESULTS AND DISCUSSION

Preparation of the NPEOC-protected phosphoramiditeof ZCyd

We first attempted to prepare the phosphoramidite deriva-tive of 59-O-dimethoxytrityl-ZdCyd without protection of theexocyclic amino group The product was obtained in moderateyields However coupling of this compound to a growing oli-gonucleotide chain using standard phosphoramidite condi-tions resulted in severe branching after the addition of the Zd-Cyd monomer (data not shown) For this reason protection ofthe exocyclic amino group was judged necessary and theNPEOC group was selected for this purpose ZdCyd was pre-

pared as described (Piskala and Sorm 1978) and introductionof the NPEOC at the exocyclic position was performed by thetransient protection method (Ti et al 1982) (Scheme 2) First reaction of ZdCyd (1) with hexamethyldisilazane produced the silylated nucleoside which was treated with 2-(4-nitro-phenyl)ethoxycarbonyl chloroformate to give the N4-NPEOCderivative of 3959-O-bis(trimethylsilyl)-ZdCyd The removalof the trimethylsilyl groups without destroying the base wasthe critical step in this approach Use of tetrabutylammoniumfluoride ammonium fluoride sodium ethoxide p-toluensul-fonic acid or acetic acid effectively removed the silyl groupsbut also caused breakdown of the base However a gradualbut complete removal of the silyl groups without detectableside reactions was achieved after maintaining the solution ofthe silylated nucleoside in DMF for 4 days at ambient temper-ature Using this method the NPEOC-protected nucleoside(2) was obtained in 43 yield after silica gel purification Fi-nally it was discovered that the trimethylsilyl groups could beremoved more efficiently using TAS-F (Scheidt et al 1998)This source of fluoride was able to effectively remove thetrimethylsilyl groups and the desired product 2 was obtainedin 80 yield after silica gel purification

Reaction of 2 with DMT-Cl followed by reaction with theappropriate chlorophosphine gave the desired phosphoramidite(4) in good yields During all these reactions special care wastaken to eliminate traces of pyridine before any aqueousworkup Failure to do so resulted in complete degradation of thenucleoside

The rest of the phosphoramidites corresponding to the nat-ural bases protected with the NPE and NPEOC groups (Scheme3) were prepared following previously described protocols(Himmelsbach et al 1984 Schirmeister et al 1983 Avintildeoacute andEritja 1994 Diacuteaz et al 1997) including the NPE-phosphor-


TABLE 3 MELTING TEMPERATURES (degC) OF DUPLEXES

CONTAINING 5-AZA-29-DEOXYCYTIDINE (Z) BASE PAIRSa

59-GCAATGGAXCCTCTA-3 939-CGTTACCTYGGAGAT-5 9

X 5 Z X 5 C

Y 5 G 50 59Y 5 A 44 47Y 5 C 45 45Y 5 T 43 46

a015 M NaCl 005 M Tris-HCl buffer pH 75

Scheme 2 Preparation of the phosphoramidite of 5-aza-29-deoxycytidine DMT dimethoxytrityl NPEOC 2-(p-nitrophenyl)ethoxycarbonylTMS trimethylsilyl

amidite of 5-methyl-29deoxycytidine (M) protected with theNPEOC group (Ferrer et al 1996)

Synthesis of oligodeoxyribonucleotides containing 5-azacytosine (ZCyt)

Oligonucleotide sequences ranging from 7 to 24 nt (IndashXV)(Tables 1 and 2) were prepared Heptamer I and octamer II aretwo small sequences that were used for full characterization anddetailed analysis Pentadecamer III and mismatched variants(Table 3) were synthesized to study the hybridization propertiesand stability of the oligonucleotides carrying ZCyt Oligonu-cleotide sequences with 24 bases (IVndashIX XIIndashXV) were de-signed to contain target sequences for DNA (Cytosine-C5)methyltransferases Comparable sequences had been used pre-viously for characterization of the inhibitory properties ofoligonucleotides containing 56-dihydro-azacytosine (Marquezet al 1999 Sheikhnejad et al 1999) Finally 13-mer X andXI with similar target sequences for methylation were pre-pared for future cocrystallization studies with the MHhaI abacterial DNA (Cytosine-C5) methyltransferase All oligonu-cleotides were synthesized in 1 mmol scale using phosphor-amidite 4 and the NPE phosphoramidites of the natural bases

protected with the NPE and NPEOC groups (Scheme 3) CPGsupports having the NPENPEOC-protected nucleosides at-tached through an oxalyl linkage were used (Alul et al 1991)The oxalyl bond is efficiently cleaved by DBU solutions withhigher efficiency (Avintildeoacute et al 1996) than the previously de-scribed NPE linkage (Eritja et al 1992) Coupling efficienciesjudged by the absorbance of the DMT cation released duringdetritylation were higher than 96

Initially syntheses of sequences IndashV were performed To fa-cilitate purification the last DMT group on the oligonucleotidewas left on Deprotection was carried out by treatment of oligo-nucleotide supports with a 05 M DBU solution in pyridine con-taining 5 mg thymine for 15 hours at ambient temperature (Avintildeoacuteand Eritja 1994 Avintildeoacute et al 1996) Purification of the finalproducts was performed by HPLC using the standard DMT-onand DMT-off protocols HPLC chromatograms were similar tothose of unmodified oligonucleotides but yields were lower andvariable (08ndash75 OD units) (Table 1) For comparison one mayexpect about 30ndash40 OD units for a 20-mer in a 1 mmol synthesisLower yields could be attributed to less efficient cleavage fromthe linkage oligonucleotide support (Avintildeoacute et al 1996) or to theobserved drop (50 or more) in the recovery of product after re-moval of the DMT with acetic acid (Table 1) This loss of prod-


Scheme 3 Preparation and structures of the phosphoramidites and solid supports used in this work

uct was undoubtedly due to the instability of the 5-azacytosinering which underwent partial decomposition during the aceticacid treatment used for removal of the DMT group

Characterization of the products obtained was attempted byenzymatic digestion and mass spectrometry Enzymatic diges-tion was used first to characterize oligonucletodies I and II Af-ter enzyme treatment no ZdCyd was detected even thougholigonucleotides I and II do not contain any dC that couldcoelute with ZdCyd and mask its presence Thus it appearedthat the most probable cause for the absence of detectable ZCydwas nucleoside degradation during incubation at pH 85 for en-zymatic digestion (Beisler 1978 Lin et al 1981)

Electrospray ionization mass spectrometry analysis underneutral conditions gave the expected molecular weights ofoligonucleotides IndashIII thus confirming both the integrity of theoligonucleotides and that indeed degradation of 5-aza-29-de-oxycytidine happened during enzymatic digestion Special carewas taken to avoid acids or bases during acquisition of the spec-tra that could cause the degradation of the sensitive base Al-though electrospray ionization under neutral conditions gaveclear molecular ion peaks for oligonucleotides IndashIII along withpeaks corrsponding to the sodium adducts it failed to give theexpected molecular ion peaks for the 24-mer In addition to theexpected molecular weights analysis of purified oligonu-cleotides IndashIII also showed the presence of small amounts oftwo other components one having 10 atomic mass units (amu)less than the expected mass (M-10) and a second unknown peak290 amu less (M-290) The M-10 product was presumed to re-sult from the opening of the hydrated ZCyt ring with subse-quent loss of a formyl group Such spontaneous ring openingand loss of a formyl group have been observed in ZCyt deriva-tives (Beisler 1978 Lin et al 1981) and in 5-aza-29-deoxycy-tidine-thymine dimers (Goddard and Marquez 1989) The M-290 product on the other hand corresponded to anoligonucleotide that lacked 5-aza-29-deoxycytidine phosphatealtogether As coupling yields with 5-aza-29-deoxycytidinephosphoramidite were high and the capping step should haveprevented the elongation of unreacted sequences the presenceof this product can be explained by the instability of the ZdCydwhich degrades under either oxidation or detritylation condi-tions We believe that the amount of this side compound wasvery small at the onset However during the HPLC purificationand especially during acetic acid treatment the oligonucleotidecarrying Z was partially degraded Therefore the two-step pro-tocol of HPLC purification leads to an enrichment of the impu-rity relative to the desired product We confirmed this hypothe-sis by observing that oligonucleotides coming from DMT-offprotocols and purified once by HPLC were more homogeneousby gel electrophoresis and ion exchange HPLC than oligonu-cleotides coming from a DMT-on sequence that were treatedwith acetic acid and repurified Moreover mass spectrometricanalysis of DMT oligonucleotide III showed the absence of M-10 and M-290 peaks whereas after removal of the DMT withacetic acid and HPLC purification both M-10 and M-290 peakswere observed No spectra of 24-mer oligonucleotides were ob-tained using MALDI-TOF most probably because the largeroligonucleotides fragmented during spectrum adquisition

Stability studies with oligonucleotides III and XII were mon-itored by gel electrophoresis (Figs 1 and 2) This techniqueconfirmed that ZCyt oligonucleotides were stable in water in

neutral pH (for at least 48 hours) Overnight treatment withconcentrated ammonia at 55degC caused complete breakdown ofthe oligonucleotides into two shorter sequences as would beexpected if scission occurred at ZCyt residue (Fig 1) Productscoming from the acetic acid treatment did not stain with ethid-ium bromide (Fig 1) Therefore to obtain a better picture of theeffect of acetic acid on ZCyt oligonucleotides 59-32P-radiola-beled single-stranded (ss) and double-stranded (ds) oligonu-cleotides formed after annealing with a complementary strandwere incubated in 80 acetic acid at 37degC for 30ndash60 minutesand analyzed by electrophoresis on 8 M urea-20 polyacryl-amide gels The result of a typical treatment is shown in Figure2 The mobility of the acetic acid-treated oligonucleotide XII(Fig 2 lanes 1ndash3) was compared with that of an oligonucleo-tide of identical sequence but with a cytosine replacing ZCyt(Cyt oligonucleotide) before (Fig 2 lanes 45) or after cleavagewith HhaI (Fig 2 lane 6) The Cyt oligonucleotide treated withHhaI serves as a control for any breakdown of a standard oligo-nucleotide during acetic acid treatment Because the ZCyt inODN XII is in the recognitioncleavage site for HhaI thecleaved Cyt oligonucleotide serves as a molecular weightmarker for an oligonucleotide 8 bases long As can be seenwhether treated as a ss oligonucleotide or after annealing withthe complementary strand little general breakdown of full-length oligonucleotide XII occurs during 60 minutes of treat-ment Only a small proportion of the oligonucleotide appears tobe converted to fragments of the size (6 bases long) expectedfor scission at the ZCyt residue (Fig 2 compare lanes 23 withlane 6) These results suggest that even if opening of the ZCytring occurs decomposition to a form that results in the scissionof the phosphodiester bond does not occur rapidly under acidicconditions These results are in contrast with the reported insta-bility of ZCyd and ZdCyd (Beisler 1978 Lin et al 1981)


FIG 1 Effect of ammonia and acetic acid on the integrity of oligonu-cleotides containing Z Reactions containing 01 OD units of oligonu-cleotide III were incubated overnight with concentrated ammonia atambient temperature (lane 2) and at 55degC (lane 3) and with 80 aceticacid (1 hour) at ambient temperature (lane 4) Following these treat-ments the samples were concentrated to dryness Samples were ana-lyzed on a 20 acrylamide7 M urea gel Bands were visualized withethidium bromide and UV shadowing (lane 4 did not stain well withethidium bromide) Lane 1 shows the migration of untreated oligonu-cleotide III and lane 5 shows the migration of a control oligonucleotidethat has the same sequence as that of oligonucleotide III but containscytosine instead of ZCyt

which leads us to conclude that ZCyt is more stable inside theoligonucleotide polymer than as a free nucleoside Although itcan be argued that the direct decomposition of the base couldnot be detected by gel analysis the results obtained with theammonia treatment indicate that we could detect the breakdownof the abasic DNA site resulting from the elimination of ZCyt

Because of the poor recovery of oligonucleotide containingZCyt after treatment with acetic acid oligonucleotide se-quences III and VIndashXV were prepared without the last DMTgroup and purified directly using reverse HPLC DMT-off con-ditions (Table 2) Purity of the oligonucleotides was monitoredby gel electrophoresis and in all cases they appeared to be ho-mogeneous Three oligonucleotide (XIII XIV and XV) alsocontained 5-methyl-cytosine (M) In these cases the appropri-ate monomer carrying the NPEOC group was used

A strong correlation between yield of oligonucleotide and thenature of the nucleoside present at the 39-end was observed(Table 2) Thus oligonucleotides V VI VII VIII IX XII andXIII all of which have pyrimidines at the 39-end were obtainedin poorer yields (3ndash15 OD units) Oligonucleotides havingpurines at the 39-end on the other hand were obtained in muchbetter yields 24ndash45 OD units of oligonucleotide sequenceshaving a G at the 39-end (X and XII) and 15ndash55 OD units ofoligonucleotides having an A at the 39-end (III XIV and XV)The proposed mechanism of cleavage for the oxalyl bond is a

base-catalyzed intramolecular attack of the amide group ontothe ester bond at the 39-end of the oligonucleotide (Avintildeoacute et al1996) Thus it is possible that different steric or inductive ef-fects related to the nature of base might influence the reactivityof the oxalyl group In our specific case ease of cleavage of theoxalyl group with DBU followed the order G A T C Atthe 59-end a similar base-dependent reactivity has been ob-served for DMT groups toward acid cleavage Indeed it is wellknown that the lability of the DMT groups toward acid cleav-age follows the order G A T 5 C (Atkinson and Smith1984) To improve the yields the hydroquinone-OO-diaceticacid (Q-linker) (Pon and Yu 1997) was investigated Thislinker is resistant to DBU solutions but cleavable with verymild nucleophils Unfortunately after treatment with DBU abrief exposure with a mild source of fluoride ions (ie triethyl-amine trihydrofluoride in N-methylpyrrolidone or DMSO)failed

Melting experiments

The base-pairing properties of 5-azacytosine were measuredon a duplex formed by two complementary pentadecamers inwhich ZCyt was paired with the four natural bases (Table 3) Asexpected ZCyt formed the strongest base pair with G althoughthe ZG base pair is 9degC less stable than the natural CG basepair Mismatches of Z are also less stable than C mismatches(around 3degC) except for ZC which is as stable as the CC mis-match The strong decrease in the melting temperature observedfor the ZG base pair could be due to the 5-azacytosine ringrsquosbeing involved in rapid equilibrium between the syn and anticonformations in which the former rotamer is unable to hydro-gen bond effectively with G via the carbonyl group at C-2 It isalso likely that if the oligonucleotide is heated to 90degC duringthe preparation of the duplex the ZCyt may undergo ring open-ing and loss of formate To estimate the impact of this potentialdecomposition during heating five consecutive melting curveswere recorded We observed that the melting curve reproducedwell but a 1degC decrease in Tm was observed after each heatingcycle Thus we can conclude that a small decomposition ofZCyt may occur during each cycle and that the melting temper-atures reported in Table 3 may be at least 1degC lower than thereal melting temperature

In this paper we describe the preparation of the phosphor-amidite derivative of 5-aza-29-deoxycytidine protected with theNPEOC group This phosphoramidite allows the preparation ofoligonucleotides containing ZCyt provided that mild nonhy-drolytic conditions are used during the removal of theNPENPEOC groups The extreme lability of the Z interferesnot only with the preparation of the protected phosphoramiditeand the removal of the protecting groups but also with the pu-rification of the modified oligonucleotides In spite of thesedrawbacks we have successfully prepared several oligonu-cleotides containing one or more ZCyt units that have the ap-propriate length and purity required for biologic studies A re-port studying the inhibitory properties of these ZCyt-modifiedoligonucleotides against DNA methyltransferase has been sub-mitted (Brank et al 2001) We have observed that yields ofoligonucleotides are dependent on the nature of the nucleosidepresent at the 39-end probably due to a lower efficiency in thecleavage from the oligonucleotide-support linkage Therefore a


FIG 2 Electrophoretic (8 M urea-20 polyacrylamide gel) analysisof ZCyt and Cyt oligonucleotides after exposure to 80 acetic acid at37degC for 60 minutes Lane 1 ss-ZCyt-ODN (control no treatment)lane 2 ss-ZCyt-ODN (acid treated) lane 3 ds-ZCyt (acid treated) lane4 ss-Cyt-ODN (acid treated) lane 5 ds-Cyt-ODN (acid treated) lane6 ds-Cyt-ODN incubated with HhaI Cleavage with this restriction en-donuclease yields a radiolabeled fragment 8 bases long A radiolabeledscission product 59 to ZCyt would be 6 bases long Experimental detailsare presented in Materials and Methods

good alternative for a future investigation is the use of photola-bile linkers (Greenberg and Gilmore 1994) The results pre-sented here are of special interest for the preparation of oligonu-cleotides carrying sensitive molecules such as mutagenic orreactive bases (Avintildeoacute et al 1995) or labile internucleotidebonds (Vivegraves et al 1999)

ACKNOWLEDGMENTS

We are thankful to Dr Matthias Wilm and Gitta Neubauerfor obtaining mass spectra Partial support for this work in-cludes grant support from the DAMD Breast Cancer Program17-98-1-8215 to JKC and fellowship support from the Gradu-ate College of UNMC to ASB

REFERENCES

ALUL RH SINGMAN CN ZHAN G and LETSINGER RL(1991) Oxalyl-CPG A labile support for the synthesis of sensitiveoligonucleotide derivatives Nucleic Acids Res 19 1527ndash1532

ATKINSON T and SMITH M (1984) Solid-phase synthesis ofoligodeoxyribonucleotides by the phosphite-triester In Oligonu-cleotide Synthesis A Practical Approach MJ Gait ed (IRL PressOxford) pp 35ndash81

AVINtildeOacute A and ERITJA R (1994) Use of NPe-protecting groups forthe preparation of oligonucleotides without using nucleophiles dur-ing the final deprotection Nucleosides Nucleotides 13 2059ndash2069

AVINtildeOacute A GUumlIMIL GARCIacuteA R DIacuteAZ A ALBERICIO F andERITJA R (1996) A comparative study of supports for the synthe-sis of oligonucleotides without using ammonia Nucleosides Nucleo-tides 15 1871ndash1889

AVINtildeOacute A GUumlIMIL GARCIacuteA R MARQUEZ VE and ERITJAR (1995) Preparation and properties of oligodeoxynucleotides con-taining 4-O-butylthymine 2-fluorohypoxanthine and 5-azacytosineBioorg Med Chem Lett 5 2331ndash2336

BEISLER JA (1978) Isolation characterization and properties of alabile hydrolysis product of the antitumor nucleoside 5-azacytidineJ Med Chem 21 204ndash208

BENDER CM PAO MM and JONES PA (1998) Inhibition ofDNA methylation by 5-aza-29-deoxycytidine suppresses the growthof tumor cell lines Cancer Res 58 95ndash101

BRANK AS ERITJA R GUumlIMIL GARCIacuteA R MARQUEZ Vand CHRISTMAN JK (2001) Inhibition of HhaI DNA (Cytosine-C5) methyltransferase by oligodeoxyribonucleotides containing 5-aza-29-deoxycytidine Examination of the intertwined roles of co-factor target transition state structure and enzyme conformation Inpress

CHRISTMAN JK (1984) DNA methylation in Friend erythro-leukemia cells The effects of chemically induced differentiation andof treatment with inhibitors of DNA methylation Curr Top Micro-biol Immuno 108 49ndash78

CHRISTMAN JK SCHNEIDERMAN N and ACS G (1985) For-mation of a highly stable complexes between 5-azacytosine-substi-tuted DNA and specific non-histone nuclear proteins Implicationsfor 5-azacytidine-mediated effects on DNA methylation and gene ex-pression J Biol Chem 260 4059ndash4068

CREUSOT F ACS G and CHRISTMAN JK (1982) Inhibition ofDNA methyltransferase and induction of Friend erythroleukemia celldifferentiation by 5-azacytidine and 5-aza-29-deoxycytidine J BiolChem 257 2041ndash2048

DIacuteAZ AR ERITJA R and GUumlIMIL GARCIacuteA R (1997) Synthesisof oligodeoxynucleotides containing 2-substituted guanine deriva-

tives using 2-fluoro-29-deoxyinosine as common nucleoside precur-sor Nucleosides Nucleotides 16 2035ndash2051

ERITJA R MARQUEZ VE and GUumlIMIL GARCIacuteA R (1997)Synthesis and properties of oligonucleotides containing 5-aza-29-de-oxycytidine Nucleosides Nucleotides 16 1111ndash1114

ERITJA R ROBLES J AVINtildeOacute A ALBERICIO F and PE-DROSO E (1992) A synthetic procedure for the preparation ofoligonucleotides without using ammonia and its application for thesynthesis of oligonucleotides containing O-4-alkyl thymidinesTetrahedron 48 4171ndash4182

FERRER E FAgraveBREGA C GUumlIMIL GARCIacuteA R AZORIacuteN Fand ERITJA R (1996) Preparation of oligonucleotides containing5-bromouracil and 5-methylcytidine Nucleosides Nucleotides 15907ndash921

GODDARD AJ and MARQUEZ VE (1988) Synthesis of 29-de-oxy-56-dihydro-5-azactyosine Its potential application in the syn-thesis of DNA containing dihydro-5-aza and 5-azacytosine basesTetrahedron Lett 29 1767ndash1770

GODDARD AJ and MARQUEZ VE (1989) Synthesis of 29-de-oxy-56-dihydro-5-azacytosine and 5-azacytosine at specific CpGsites Nucleosides Nucleotides 8 1015ndash1018

GOLDBERG J GRYN J RAZA A BENNETT J BROWMANG BRYANT J and GRUNWALD H (1993) Mitoxantrone and5-azacytidine for refractoryrelapsed anII or cmI in blast crisis Aleukemia intergroup study Am J Hematol 43 286ndash290

GREENBERG MM and GILMORE JL (1994) Cleavage ofoligonucleotides from solid-phase supports using o-nitrobenzyl pho-tochemistry J Org Chem 59 746ndash753

HIMMELSBACH F SCHULZ BS TRICHTINGER TCHARUBALA R and PFLEIDERER W (1984) The p-nitro-phenylethyl (NPE) group A versatile new blocking group for phos-phate and aglycone protection in nucleosides and nucleotides Tetra-hedron 40 59ndash72

LIN KT MOMPARLER RL and RIVARD GH (1981) High-performance liquid chromatographic analysis of chemical stability of5-aza-29-deoxycytidine J Pharm Sci 70 1228ndash1232

MARQUEZ VE GODDARD A ALVAREZ E FORD HCHRISTMAN JK SHEIKHNEJAD G BRANK AMARASCO CJ SUFFRIN JR OrsquoGARA M and CHENG X(1999) Oligonucleotides containing 56-dihydro-5-azacytosine atCpG sites can produce potent inhibition of DNA cytosine-C5-methyltransferase without covalently binding to the enzyme Anti-sense Nucleic Acid Drug Dev 9 415ndash421

MOMPARLER RL COTE S and ELIOPOULOS N (1997) Phar-macological approach for optimization of the dose schedule of 5-aza-29-deoxycytidine (decitabine) for the therapy of leukemia Leukemia11 175ndash180

PINTO A and ZAGONEL V (1993) 5-Aza-29-deoxycytidine(decitabine) and 5-azacytidine in the treatment of acute myeloid leu-kemias and myelodysplastic syndromes Past present and futuretrends Leukemia 7(Suppl 1) 51ndash60

PISKALA A and SORM F (1978) 4-Amino-1-b-D-ribofuranosyl-S-triazin-2(1H)-one (5-azacytidine) In Nucleic Acid Chemistry Im-proved and New Synthetic Procedures Methods and TechniquesLB Townsend and RS Tipson eds (John Wiley amp Sons NewYork) Part one pp 443ndash449

PON RT and YU S (1997) Hydroquinone-OO9-diacetic acid (ldquoQ-linkerrdquo) as a replacement for succinyl and oxalyl linker arms in solidphase oligonucleotide synthesis Nucleic Acids Res 25 3629ndash3635

ROCHETTE J CRAIG JE and THEIN SL (1994) Fetal hemoglo-bin levels in adults Blood Rev 8 213ndash224

SAMBROOK J FRITSCH EF and MANIATIS T (1980) Molec-ular Cloning A Laboratory Manual (Cold-Spring Harbor Labora-tory Press New York) pp 1059ndash1061

SCHEIDT KA CHEN H FOLLOWS BC CHEMLER SRCOFFEY DS and ROUSH WR (1998) Tris(dimethylamino)sul-


fonium difluorotrimethylsilicate a mild reagent for the removal ofsilicon protecting groups J Org Chem 63 6436ndash6437

SCHIRMEISTER H HIMMELSBACH F and PFLEIDERER W(1993) Nucleosides Part LI The 2-(4-nitrophenyl)ethoxycarbonyl(npeoc) and 2-(24-dinitrophenyl)ethoxycarbonyl (dnpeoc) groupsfor the protection of hydroxy functions in ribonucleosides and 29-de-oxyribonucleosides Helv Chim Acta 76 385ndash401

SHEIKHNEJAD G BRANK A CHRISTMAN JK GODDARDA ALVAREZ E FORD H Jr MARQUEZ VE MARASCOCJ SUFRIN JR OrsquoGARA M and CHENG X (1999) Mecha-nism of inhibition of DNA (Cytosine-C5)-methyltransferases byoligodeoxyribonucleotides containing 56-dihydro-5-azacytosine JMol Biol 285 2021ndash2034

TI GS GAFFNEY BL and JONES PA (1982) Transient protec-tion Efficient one-flask syntheses of protected deoxynucleotides JAm Chem Soc 104 1316ndash1319

VIVEgraveS E DELLrsquoAQUILA C BOLOGNA JC MORVAN F

RAYNER B and IMBACH JL (1999) Lipophilic pro-oligonu-cleotides are rapidly and efficiently internalized in HeLa cells Nu-cleic Acids Res 27 4071ndash4076

Address reprint requests toDr Ramon Eritja

Institut de Biologiacutea Molecular de Barcelona CSICJordi Girona 18-26E-08034 Barcelona

Spain

E-mail recgmacidcsices

Received July 13 2001 accepted in revised form October 92001


MATERIALS AND METHODS

29-Deoxy-N4-[2-(4-nitrophenyl)ethoxycarbonyl]-5-azacytidine (2)

29-Deoxy-5-azacytidine (228 mg 1 mmol) was dried after threesuccessive evaporations of a solution in anhydrous pyridine Theresidue was dissolved in anhydrous dimethylformamide (DMF)and treated with 053 ml (25 mmol) hexamethyldisilazane Afterbeing stirred for 1 hour at ambient temperature the solution wasconcentrated and the residue was dried by three cycles of evapo-ration from toluene (5 ml) yielding a white foam (thin-layer chro-matography [TLC] Retention factor (Rf) 066 20 ethanol indichloromethane) The residue was thrice dissolved in 5 ml anhy-drous pyridine and concentrated to dryness before finally beingdissolved in 30 ml anhydrous pyridine To this solution 350 mg(15 mmol) of 2-(p-nitrophenyl)ethyl chloroformate dissolved in 5ml dichloromethane was added and after 30 minutes of magneticstirring at ambient temperature the reaction was judged completeby TLC (Rf 093 10 ethanol in dichloromethane) The solutionwas concentrated to dryness and traces of pyridine were removedby three cycles of evaporation of a toluene solution (5 ml) Theresidue was dissolved in chloroform and the organic solution waswashed with 1 M sodium bicarbonate dried (Na2SO4) and con-centrated to dryness

Removal of the trimethylsilyl groups in DMF solutions Theresidue from the described reaction was dissolved in DMFTHF(tetrahydrofurane) (31) and the resulting solution was stirredfor 3 days at ambient temperature After the solvents were re-moved the residue was triturated with ether to form a whitesolid that was purified by column chromatography on silica gel(3ndash10 step gradient of ethanol in chloroform) to give 180mg (043 mmol 43) of the desilylated product [TLC (10ethanol in chloroform) Rf 5 053 (5 ethanol in chloroform)Rf 5 012] 1H-NMR (DMSO-d6) d (ppm) 103 (wide s 1HNH) 841 (s 1H H-6) 771 (d 2H J 5 86 Hz NPEOC) 718(d 2H J 5 86 Hz NPEOC) 556 (t 1H H-19) 486 (d 1H 39-OH) 471 (t 1H 59-OH) 392 (t 2H CH2 NPEOC) 38 (m 1HH-39) 345 (m 1H H-49) 317 (m 2H H-59) 264 (t 2H CH2

NPEOC) 18 (m 2H H-29) 13C-NMR (CDCl3) d (ppm) 1630(C-4) 1571 (C-6) 1535 (CO NPEOC) 1507 (C-2) 1467 (C-NO2) 1455 (Cq Ar) 1299 (CH Ar) 1235 (CH Ar) 879 (C-49) 867 (C-19) 677 (C-39) 654 (CH2) 404 (C-29) 346(CH2) Anal Calcd for C17H19N5O8 (Found C 481 H 47N 164 requires C 485 H 46 N 166)

Removal of the trimethylsilyl groups with TAS-F Alterna-tively the silyl protected product resulting from a 4 mmolscale reaction was treated with a solution of tris(dimethy-

lamino) sulfonium difluorotrimethylsilicate (TAS-F) (12mmol 33 mmol) in DMF (50 ml) After 2 hours at ambienttemperature the solution was concentrated to dryness andthe residue was purified by column chromatography on silicagel as described to give the same product as before (135 g32 mmol 80)

29-Deoxy-59-O-dimethoxytrityl-N4-[2-(4-nitrophenyl)ethoxycarbonyl]-5-azacytidine (3)

29-Deoxy-N4-[2-(4-nitrophenyl)ethoxycarbonyl]-5-azacyti-dine (163 g 387 mmol) was dried after repeated evaporationof a solution in anhydrous pyridine and reacted withdimethoxytrityl chloride (144 g 426 mmol) in pyridine (20ml) After being stirred for 3 hours at ambient temperature thesolution was concentrated to dryness Traces of pyridine wereremoved by three cycles of evaporation of a toluene solution (5ml) The residue was dissolved in dichloromethane and the or-ganic solution was washed with 1 M sodium bicarbonate aque-ous solution dried (Na2SO4) and concentrated to dryness Theresidue was purified by column chromatography on silica geleluted with a 0ndash5 step gradient of ethanol in chloroform togive 201 g (278 mmol 71) of 3 [TLC (10 ethanol indichloromethane) Rf 5 040] IR (cm21) 3400 2960 17701700 1610 1510 1350 1250 1220 1180 1030 830 1H-NMR(CDCl3) d (ppm) 860 (s 1H H-6) 816 (d 2H Ar NPEOC)743 (d 2H Ar NPEOC) 71ndash73 (m 9H Ar DMT) 681 (d4H Ar DMT) 616 (t 1H H-19) 44 (m 3H H-39 CH2

NPEOC) 42 (m 1H H-49) 377 (s 6H Me DMT) 34 (m2H H-59) 31 (t 2H CH2 NPEOC) 28 (m 1H H-29) 23 (m1H H-29) 13C-NMR (CDCl3) d (ppm) 1631 (C-4) 1584 (CqDMT) 1568 (C-6) 1534 (CO NPEOC) 1502 (C-2) 1467(C-NO2) 1451 (Cq Ar) 1442 (Cq DMT) 1354 (Cq DMT)1352 (Cq DMT) 1297 (CH Ar) 1278 (CH DMT) 1267(CH DMT) 1235 (CH Ar) 1131 (CH Ar) 877 (C-49) 869(C-19) 868 (Cq DMT) 712 (C-39) 655 (CH2) 631 (C-59)550 (CH3) 419 (C-29) 345 (CH2)

NN-Diisopropylamino-2-(p-nitrophenyl)ethoxychlorophosphine

In a 1-L round-bottom flash 175 ml (200 mmol) phospho-rous trichloride was dissolved in 500 ml anhydrous acetoni-trile and kept under argon atmosphere The solution wascooled over an icebath and treated dropwise with a solution of2-(p-nitrophenyl)ethanol (334 g 20 mmol) dissolved in 100ml acetonitrile The reaction mixture was gradually warmed toambient temperature and stirred for 1 hour The mixture wasconcentrated to give a yellowish oil that was used in the nextstep without further purification The crude 2-(p-nitro-phenyl)ethoxy dichlorophosphine obtained was dissolved in500 ml acetonitrile and the solution was cooled with an ice-bath To this solution 62 ml (44 mmol) NN-diisopropy-lamine dissolved in 100 ml acetonitrile was added and afterthe addition the mixture was gradually warmed to ambienttemperature after 6 hours of stirring The white precipitatewas filtered and the filtrate was concentrated to dryness togive 5 g (15 mmol 75) of a yellowish oil that was used di-rectly in the preparation of all phosphoramidites without fur-ther purification 31P-RMN dP (75 MHz CDCl3) 17774 ppm(90 pure by phosphorus NMR)


Scheme 1 The NPEOC and NPE protecting groups






















Melting experiments





























X 5 Z X 5 C

Y 5 G 50 59Y 5 A 44 47Y 5 C 45 45Y 5 T 43 46





















Melting experiments






ACKNOWLEDGMENTS


REFERENCES








































Spain

























Melting experiments





























X 5 Z X 5 C

Y 5 G 50 59Y 5 A 44 47Y 5 C 45 45Y 5 T 43 46





















Melting experiments






ACKNOWLEDGMENTS


REFERENCES








































Spain















X 5 Z X 5 C

Y 5 G 50 59Y 5 A 44 47Y 5 C 45 45Y 5 T 43 46





















Melting experiments






ACKNOWLEDGMENTS


REFERENCES








































Spain






















Melting experiments






ACKNOWLEDGMENTS


REFERENCES








































Spain





ACKNOWLEDGMENTS


REFERENCES








































Spain












Spain




Synthesis of Oligonucleotide Inhibitors of DNA (Cytosine-C5) Methyltransferase Containing...

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Transcript of Synthesis of Oligonucleotide Inhibitors of DNA (Cytosine-C5) Methyltransferase Containing...