research article synthesis of oligonucleotide conjugates...

Hindawi Publishing CorporationJournal of ChemistryVolume 2013 Article ID 469470 8 pageshttpdxdoiorg1011552013469470

Research ArticleSynthesis of Oligonucleotide Conjugates andPhosphorylated Nucleotide Analogues An Improvement toa Solid Phase Synthetic Approach

Valeria Romanucci Armando Zarrelli Lorenzo De NapoliCinzia Di Marino and Giovanni Di Fabio

Department of Chemical Sciences University of Napoli ldquoFederico IIrdquo Via Cintia 4 80126 Napoli Italy

Correspondence should be addressed to Giovanni Di Fabio difabiouninait

Received 13 January 2013 Revised 20 April 2013 Accepted 23 April 2013

Academic Editor Alvaro Somoza

Copyright copy 2013 Valeria Romanucci et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

An improvement to our solid phase strategy to generate pharmacologically interesting molecule libraries is proposed here Thesynthesis of new 119900-chlorophenol-functionalised solid supports with very high loading (018ndash022meqg for control pore glass(CPG) and 025ndash050meqg for TG) is reported To test the efficiency of these supports we prepared nucleotide and oligonucleotidemodels and their coupling yields and the purity of the crude detached materials were comparable to previously available resultsThese supports allow the facile and high-yield preparation of highly pure phosphodiester and phosphoramidate monoesternucleosides conjugated oligonucleotides and other yet unexplored classes of phosphodiester and phosphoramidate molecules

1 Introduction

Oligonucleotides (ODNs) and nucleotides represent classesof potential therapeutic agents with a broad spectrum ofpharmacological activities Desired improvements of certainproperties such as cell-specific delivery cellular uptakeefficiency intracellular distribution and target specificitycan be achieved by chemical modifications Conjugationof ODNs to other molecules (eg proteins and peptidessaccharides fluorophores and photoprobes inhibitors andvitamins) that provide the conjugate with a desired novelproperty offers a feasible way to address these requirements[1ndash16] With regard to nucleosides however many researchgroups have developed a prodrug approach to deliver bio-logically active nucleosides into cells in the form of maskedcharged monophosphate derivatives a variety of different51015840-phosphorothioate and 51015840-phosphodiester nucleoside ana-logues have been prepared for this purpose [17ndash21]

Organic chemists investigating these fields must pre-pare many types of pure modified compounds in sufficientquantities The methods used for the preparation of thesemolecules fall into two major categories solution and solid

phase approaches The solid phase method in associationwith combinatorial chemistry approaches has proven usefulfor the synthesis of a large number of these analogues Anadvantage of the solid-supported method compared withconjugation or derivatisation in solution is that it is less labo-rious among other advantages In fact on a solid supportthe unreacted compounds are usually used in considerableexcess and the possible by-products can be removed by sim-ple washing The development of a broad array of reactionsin the solid phase has increased the scope and potentialof this method allowing the synthesis of large libraries ofcompounds endowed with a diverse series of molecularmotifs [26ndash29] Various combinatorial approaches have beensuccessfully adopted for the generation of a wide range ofoligomeric and smallmolecule libraries for biological screensMoreover the combinatorial approach has allowed the rapidscreening of a plethora of different substrates acceleratinglead identification and resulting in fundamental develop-ments in biomedicinal chemistryWithin this framework thelow loading of the solid supports has proven to be a limitationof this method as it strongly inhibits the amount of targetsthat can be obtained

2 Journal of Chemistry

Current solid phase methods for the synthesis of ODNconjugates include the utilisation of prefabricated labels pre-viously converted into the corresponding phosphoramiditeor H-phosphonate derivatives and elaborate supports bear-ing an appropriate linker to incorporate the conjugatingresidue generally employed as a postsynthetic modificationof the ODNs [30] In both strategies stringently appliedpurifications (in the first approach for the reactive phospho-rylated derivatives of the labels in the second for the prepa-ration of the linker or in the final step) are typically requiredto isolate the desired conjugatedmolecule in a pure form [31ndash34] Althoughmanymethods have been reported for the solidphase synthesis of conjugated ODNs the same cannot be saidfor solid phase synthesis of modified nucleotides A varietyof different 51015840-phosphorylated 51015840-phosphoramidate and 51015840-phosphorothioate nucleoside analogues have been preparedand evaluated for their biological activity [17ndash21]

As part of our continuing effort towards the synthesis ofnew solid supports that are useful for generating pharmaco-logically interestingmolecule libraries [22ndash25 35ndash40] of highquality in large quantities we present here an improvement ofour solid phase strategy discussed above Aiming to achievethe synthesis of a solid support with a higher load thanthat currently available and that is also compatible withphosphoramidite and phosphotriester chemistry we deviseda straightforward and efficient synthetic protocol to preparea new support in which the loading of the o-chlorofunctionalgroup is very high (020ndash050meqg)

2 Experimental

21 General NMR spectra were recorded in CDCl3and

CD3OD with a Bruker WM 400 spectrometer The chemical

shifts (120575) are given in ppm and referenced to the residualsolvent signal (726 and 331 ppm resp) and coupling constants (119869) are in Hz 31P NMR spectra were recorded at16198MHz using D

3PO4as an external standard For ESI-

MS analysis a Waters Micromass ZQ instrumentmdashequippedwith an electrospray sourcemdashwas used in the negative modeMALDI TOF mass spectrometric analyses were performedon a PerSeptive Biosystems Voyager-DE Pro MALDI massspectrometer in the linearmode HPLC analysis and purifica-tion were performed on an Agilent Technologies 1200 seriesinstrument equippedwith aUVdetectorThe crudematerialsof 5 and 6 were analysed by HPLC on a C18 PhenomenexLUNA column (5 120583m 100 times 250mm) eluted with a lineargradient of CH

3CN in H

2O flow rate = 08mLmin and

detection at 120582 = 260 nm The crude material of 9 wasanalysed by HPLC on a Nucleogel SAX column (Macherey-Nagel 1000ndash846) buffer A 20mM KH

2PO4aq solution

pH 70 containing 20 (vv) CH3CN buffer B 10M KCl

20mM KH2PO4aq solution pH 70 containing 20 (vv)

CH3CN linear gradient from 0 to 100 B over 30min flow

rate 08mLmin and detection at 120582 = 260 nm The crudematerial was purified by gel filtration chromatography ona Sephadex G25 column eluted with H

2OEtOH (4 1 vv)

LCAA-CPG and TentaGel amino supports were purchasedfrom Link Technologies and Novabiochem respectively

The nucleotide phosphoramidites the activator solution(045M tetrazole in CH

3CN) and the oxidiser solution

(01M I2THFH

2Opyridine) were purchased from Link

Technologies

22 Synthesis of Supports 1a and 1b Support 1a 250mgof LCAA-CPG-NH

2(010meqg 002mmol) was reacted at

rt overnight with a mixture of 1095mg (025mmol) of N-120572-Fmoc-3-chloro-L-tyrosine 515mg (025mmol) of DCCI45 120583L (025mmol) of DIEA and 380mg (025mmol) ofN-hydroxybenzotriazole (HOBtsdotH

2O) dissolved in 3mL of

anhydrous pyridine After exhaustivewashingwithDCMandEt2O the support was dried under reduced pressure and then

treated with 20 piperidine in DMF three times for 5minThe coupling and Fmoc removal were repeated twice morein similar conditions According to the Kaiser test [41] theincorporation of the linker was always in the range of 65ndash85 corresponding to 019ndash025meqg After capping theunreacted amino functional groups with Ac

2Opyridine (1 1

vv) for 1 h at rt the support was treated with conc aqammonia (28) at 50∘C for 1 h After exhaustivewashingwithCH3OH DCM and Et

2O the resulting support 1a was dried

under reduced pressureSupport 1b 250mg of TG-NH

2LL (029meqg 007

mmol) was reacted at rt overnight with a mixture of 3175mg (072mmol) of N-3-chlorotyrosine acid 1500mg (072mmol) of DCCI 1260120583L of DIEA and 1100mg (15mmol)of N-hydroxybenzotriazole (HOBtsdotH



treated with 20 piperidine in DMF three times for 5minThe coupling and Fmoc removal were repeated twice more insimilar conditionsThe incorporation of the linkerwas alwaysin the range of 65ndash85 corresponding to 050ndash075meqgaccording to the Kaiser and Fmoc tests After capping theunreacted amino functional groups with Ac

2Opyridine (1 1


2O the resulting support 1b was dried

under reduced pressure

23 Synthesis of Supports 4a 4b and 8a Support 4a 11mL(05mmol) of a commonly used ldquoactivator solutionrdquo (045M tetrazole in CH

3CN) was added to 008mmol of 51015840-O-(2-

cyanoethyl)-NN-diisopropylphosphoramidite-31015840-O-(441015840-di-dimethoxytriphenylmethyl)-thymidine and 250mg (022meqg 005mmol) of support 1a After 1 h the support wasexhaustively washed with CH

3CN and treated (3 times)

with 5mL of a commonly used ldquooxidiserrdquo solution (I2

pyridineH2OTHF) for 5min After exhaustive washing

with CH3CN DCM and Et

2O the resulting support 3a

was dried under reduced pressure Incorporation yieldsof the nucleotides were always in the range of 82ndash99(018ndash022meqg) as determined by a quantitative DMTtest performed on dried and weighed samples of support 3aAfter the standard capping procedure with Ac

2Opyridine

(1 1 vv) the 2-cyanoethyl group from the phosphate was

Journal of Chemistry 3

then removed by treatment with 20 piperidine in DMF for5min at rt (3 times) resulting in support 4a

Support 4b 49mL (22mmol) of a commonly usedldquoactivator solutionrdquo (045M tetrazole in CH

3CN) was added

to 035mmol of the 51015840-O-(2-cyanoethyl)-NN-diisopro-pylphosphoramidite-31015840-O-(441015840-dimethoxytriphenylmethyl)-21015840-deoxyribonucleoside and 250mg (055meqg 014mmol)of support 1b After 1 h the support was exhaustively washedwith CH

3CN and treated (5 times) with 5mL of a commonly

used ldquooxidiserrdquo solution (I2pyridineH

2OTHF) for 5min

After exhaustive washing with CH3CN DCM and Et

2O

the resulting support was dried under reduced pressure Thecomplete oxidation of the phosphite triester into a phosphatetriester leading to support 3b was monitored by 31P NMRof the resin suspended in CDCl

3 As is typical a relevant

upfield shift of the signal at 137 ppm to two signals centredat 120575 minus65 ppm was observed After a standard cappingprocedure with Ac

2Opyridine (1 1 vv) the 2-cyanoethyl

group was removed from the phosphate by treatment with20 piperidine in DMF for 5min at rt (5 times) resulting insupport 4b Incorporation yields of nucleotides calculatedafter capping were always in the range of 75ndash91 (040ndash050meqg) as determined by a quantitative DMT testperformed on dried and weighed samples of support 4bThe total deprotection of the phosphates was confirmed bya characteristic upfield shift in the signal of the 31P NMRspectrum of the solid support suspended in CDCl

3

Support 8a this support was obtained by following theprocedure described for the synthesis of support 4a using51015840-O-(441015840-dimethoxytriphenylmethyl)-31015840-O-(2-cyanoethyl)-NN-diisopropylphosphoramidite-thymidine starting fromsupport 1a

24 Synthesis of Thymidine Analogue 5 30mg (040meqg0012mmol) of dried support 4b was washed and swelledin anhydrous pyridine and then reacted with a mixture of46mg (012mmol) of cholesterol and 36mg (012mmol) ofMSNT in 500120583L of anhydrous pyridine for 12 h at rt Afterexhaustive washing with pyridine CH

3OH DCM and Et

2O

the target analogues were detached from the support by concaq ammonia treatment at 50∘C for 5 h The crude materialof 5 was analysed by HPLC on a C18 Phenomenex LUNAcolumn (5120583m 100 times 250mm) eluted with a linear gradient(from 10 to 100 B over 30min A = H

2O B = CH

3CN) flow

rate = 08mLmin detection at 120582 = 260 nm 119905119877= 165min

and HPLC purity 88 (see Figure 1) 1H NMR (CD3OD

400MHz) 120575 779 (1H s H-6 T) 635 (1H dd 119869 = 64 64HzH-11015840 T) 532 (1H m H-6 cholesterol residue) 450 (1H mH-31015840 T) 404ndash390 (3H overlapped signals H-41015840 and H

2-51015840

T) 365 (1H s H-3 cholesterol residue) 230 (2H m H2-21015840

T) 220ndash065 (complex signals of cholesterol residue) and194 (3H s CH

3T) ppm 31P NMR (CD

3OD 16198MHz)

120575 26 ppm ESI-MS119898119911 68851 [(MndashH)minus]

25 Synthesis of Thymidine Analogue 6 Synthesis of 6starting from 4a 50mg (022meqg 0011mmol) of driedsupport 4a was washed and swelled in anhydrous pyridineThe support was then treated with 1mL of a freshly prepared

0100200300400500600700

mAU

0 25 5 75 10 125 15 175min

Figure 1 SAX-HPLC profile of crude detached 5 (PhenomenexLUNA 5120583mC18 100times250mm) eluted with a linear gradient from10 to 100 B in 30min A = H

2O B = CH

3CN detection at 120582 =

260 nm flow rate 08mLmin

tosyl chloride solution (02MTsCl 04MNMIm in pyridine)for 15min at rt to generate the active ester followed byaddition of 1mL of the amine solution (045M in pyridine)with appropriate washing steps in between [42] This proce-dure was repeated six times After exhaustive washing withpyridine CH

3OHDCM and Et

2O the resulting support was

dried under reduced pressure The conjugation yields wereevaluated by the DMT cation test on a weighed sample ofthe support after ammonia treatment (28 NH

4OH 50∘C

5 h) The conjugation yields were always in the range of 65ndash75 The target analogue 6 was detached from the supportby conc aq ammonia treatment at 50∘C for 5 h The crudematerial was analysed byHPLC on aC18 Phenomenex LUNAcolumn (5120583m 100 times 250mm) eluted with a linear gradient(from 0 to 100 B over 30min A = H

2O B = CH

3CN) flow

rate = 08mLmin detection at120582= 260 nm 119905119877= 136min and

HPLC purity 91Synthesis of 6 starting from 4b 30mg (040meqg

0012mmol) of dried support 4b was washed and swelledin anhydrous pyridine The support was then treated with2mL of a freshly prepared tosyl chloride solution (02MTsCl 04M NMIm in pyridine) for 15min at rt to generatethe active ester followed by the addition of 1mL of thebutylamine solution (045M in pyridine) with appropriatewashing steps in between This procedure was repeated tentimes After exhaustive washings with pyridine CH

3OH

DCM and Et2O the resulting support was dried under

reduced pressure The conjugation yields were evaluated bythe DMT cation test on a weighed sample of the supportafter ammonia treatment (28 NH

4OH 50∘C 5 h) The

conjugation yields were always in the range of 85ndash90 Thetarget analogue 6 was detached from the support by concaq ammonia treatment at 50∘C for 5 h The crude materialwas analysed by HPLC on a C18 Phenomenex LUNA column(5 120583m 100 times 250mm) eluted with a linear gradient (from0 to 100 B over 30min A = H

2O B = CH

3CN) flow rate

= 08mLmin detection at 120582 = 260 nm 119905119877= 136min and

HPLC purity 86 1HNMR (CD3OD 400MHz) 120575 789 (1H

s H-6 T) 635 (1H dd J = 64 64HzH-11015840 T) 452 (1HmH-31015840 T) 403 (1H bs H-41015840 T) 396 (2H bs H

2-51015840 T) 286 (2H

m CH2-NH) 233-218 (2H m H

2-21015840 T) 195 (3H s CH

3T)


PO

O O

POminusO

X OR

P

O

O

Ominus

Nu(DMT)

Site forconjugation

ODN chainassembly

= labeling group

O N

O

B

OH

POminusO

X OR

CD

OHLinker

R

R

O

Cl

POOCE

R

∙ Oxidation phosphite to phosphate∙ CE removal∙ Second coupling with

∙ Condensation with R-XHby phosphotriestermethodology

∙ Deprotectionand detachment


∙ Pre- or postassemblyconjugation

X = O NHNu(ODMT) = 5998400-ODMT-3998400-phosphoramidite-nucleoside

or 5998400-phosphoramidite-3998400-ODMT-nucleoside

R = labeling group

∙ First coupling with(by phosphoramidite methodology)

N(ipr)2Ominus

NO2

CD = 120572CD 120573CD Me-120573CD and HP-120573CD

(by phosphotriester methodology)

Scheme 1 Solid phase methodology to obtain phosphodiester phosphoramidate monoester nucleoside analogues [22] oligonucleotideconjugates [23 24] and monofunctionalised CDs [25]

146 (2H m CH3-CH2-CH2-CH2-NH) 132 (2H m CH

3-

CH2-CH2-CH2-NH) and 089 (3H t 119869 = 72Hz CH

3butyl

residue) ppm 31P NMR (CD3OD 16198MHz) 120575 110 ppm

ESI-MS119898119911 37624 [(MndashH)minus]

26 Synthesis of Oligonucleotide Conjugated 9 50mg(022meqg 0011mmol) of dried support 8a was washedand swelled in anhydrous pyridine The support was thentreated with 1mL of a freshly prepared tosyl chloridesolution (02M TsCl 04M NMIm in pyridine) for 15minat rt to generate the active ester followed by the additionof 1mL of the butylamine solution (045M in pyridine)with appropriate washing steps in between This procedurewas repeated six times After exhaustive washings withpyridine CH

3OH DCM and Et

2O the resulting support

was dried under reduced pressure Starting from 25mg ofsupport a 10-mer oligodeoxyribonucleotide was assembledby the automated standard phosphoramidite procedure [43](DMT off) using commercially available phosphoramiditenucleosides Detachment from the support and deprotection

were achieved by treatment with conc aq ammonia solution(28 6 h 55∘C) and the crude material thus released wasthen purified by a simple gel filtration chromatography on aSephadex G25 column eluted withH

2OEtOH (4 1 vv)The

purity of the isolated compounds was then checked by ionexchange HPLC analysis and their identities determined byMALDI-TOFmass spectrometry 119905

119877= 312min MALDI-TOF

119898119911 304063 [(MndashH)+] (obsv) 303855 (calcd)

3 Results and Discussion

31 Synthesis of Supports following the New Strategy We haverecently reported a simple solid phasemethodology to obtainphosphodiester and phosphoramidate monoester nucleosideanalogues and 51015840- and 31015840-ODN conjugates in extremelypure form by using standard phosphotriester chemistry(Scheme 1) Initially inspired by Pedrosorsquos procedure previ-ously developed for the solid phase synthesis of cyclic ODNs[36] we prepared a small library of thymidine analoguesconjugated at the 51015840-position with a set of representative


COOH

NHFmoc

HO

Cl

HN N

HNHAc

O

O

OCH2

OHCl

OHCl

OHCl

NH

HN

O

HN

Cl

OHO

ThyOCEO

P

ODMT

PO

O

Cl

HN

O

ThyO

2

O

HN

1a 1b

o-chlorophenollinker

3 times∙ DCC DIEA in DMF rt 2h

∙ 20 piperidine in DMFCH2 CH2

NH2

= CPG TG

∙ Ac2Opyridine 11 (vv) 30998400 rt

∙ NH4OH conc 1 h 50∘C

CH2(ipr)2N

∙ 1H-tetrazole 1 h rt

∙ I2pyridineH2O 5998400 rt

OR998400998400

OR998400

3a 3b R998400 = DMT R998400998400 = CE

4a 4b R998400 = Ac R998400998400 = H

∙ 1 DCA in DCM 5998400 rt


∙ 20 piperidine in DMF rt

a = CPG b = TG

+

Scheme 2 The strategy to obtain CPG or TG with high loading of o-chlorophenol residues

alcohols and amines [22] Later a number of 51015840- or 31015840-ODN and 31015840-oligoribonucleotide conjugates incorporating avariety of labels covalently linked through a phosphodiesteror a phosphoramidate bond were synthesised and charac-terised [23 24 37] In all cases ad hoc derivatised solidsupports to which the first nucleoside unit was attachedthrough a phosphate linkage have been exploited (Scheme 1)The key step in our strategy is the derivatisation of the solidsupport (TG CPG) with a 3-chloro-4-hydroxyphenylaceticlinker onto which the nucleotide is attached through aphosphate triester linkage Due to the structure of the linkerafter cleavage from the support the HPLC analyses of thereleased nucleotides and ODNs showed only the singledesired product in all cases High purity can be obtainedbecause only the nucleoside or ODN linked to the supportthrough a phosphotriester or phosphoramidate diester bondis cleaved from the resin after ammonia treatment whereasthe nucleoside or ODN anchored through a phosphodiesterbond that is the unreacted ODN chain is not affected underthe same conditions

Recently this approachwas extended to the regioselectivesolid phase synthesis of cyclodextrins (CDs) tethered to

a variety of labels through a stable phosphodiester linkageat the C-6 position (Scheme 1) [25] The new support basedon the Novagel resin anchored with an o-nitrophenol linker(046meqg) allowed the detachment of the desired productsunder conditions milder than those required for the supportwith the o-chlorophenol linkerThese results have shown thatanchoring the o-chloro- or o-nitrophenol linker to a suitablematrix allows us to extend our methodology to all moleculeswhose phosphoramidites are either commercially available oreasily realised through standard recognised chemistry

To exploit the advantages of the regioselective release ofthe o-chlorophenol support here we report the synthesis of asupport derivatised with an o-chlorophenol linker that showshigher loading than those previously reported and is usefulfor large scale synthesesThekey stepwas the derivatisation ofcommonly used (LCAA-CPGandTG) solid supports withN-120572-Fmoc-3-chloro-L-tyrosine (3-Cl-Tyr) Unlike the 3-chloro-4-hydroxy-phenylacetic linker used previously the 3-chloro-L-tyrosine linker not only contains an o-chlorophenol skele-ton but also simultaneously has amino and acidic functionalgroups which allow a versatile elongation of the peptidechain with a resulting increase of the functionalisation of


POH

NOminus O

ThyO

OH

n-Bu

PO

Ominus O

ThyO

OH

PO

OOminus

Cl

HN O

ThyO

OAc4a 4b

O

HN

O

n-BuHN POminus

OO TCTCTCTCTC

5

6

9

P OOOR

Cl

HN

O

ThyDMTO

O

O

HN

∙ MSNT cholesterol 12 h rt


∙ TsCl NMIm 15998400 rt

∙ n-Bu-NH2 15998400 rt



∙ DNA assembly3998400ndash5998400 direction(DMT off)


∙ n-Bu-NH2 15998400 rt

7a R = CE

8a R = H

∙ 20 piperidine

in DMF rt

3998400

5998400

Scheme 3 The feasibility tests of new supports for the solid phase synthesis of nucleotide analogues and oligonucleotide conjugates

the OH groups In an initial series of experiments wesynthesised supports (LCAA-CPG (load 010meqg) or TG(load 029meqg) amino supports) with a homopeptide (3-Cl)-Tyr

3following an Fmoc protocol leading to 1a and 1b

respectively (Scheme 2) The peptide chain was preparedusing DCCHOBt as coupling agents with each monomeraddition monitored by the Kaiser test the yields were alwaysin the range of 65ndash85 corresponding to 019ndash025meqg for1a and 050ndash075meqg for 1b

To test the efficiency of these supports in the synthesis ofphosphodiester and phosphoramidate monoester nucleosideanalogues we followed two different methods as previ-ously reported In preliminary tests we chose to synthesisethe cholesteryl phosphodiester and butylamino phospho-ramidate of thymidine as nucleotide models Initially the51015840-phosphoramidite thymidine derivative 2 was anchoredto matrices (CPG and TG) by exploiting classical phos-phoramidite chemistry After conversion of the phosphiteto phosphate triesters affording supports 3a and 3b theincorporation of the nucleotide as determined by the DMTtest was always in the range of 018ndash022meqg for LCAA-CPG (3a) and 025ndash050meqg starting from a TG aminoresin (3b) Compared with our previous work here we havedoubled resin loading (008ndash010meqg and 019ndash022meqgresp)

To obtain the phosphodiester thymidine derivative 5(Scheme 3) support 4b was reacted with MSNT and choles-terol in pyridine at rt for 12 h To prepare the phosphorami-date thymidine derivative support 4awas treated three timeswith p-tosyl chloride in pyridine and then reacted with the

butylamine dissolved in pyridine As expected the conju-gation efficiency was always in the range of 70ndash80 lead-ing to supports with 013ndash018meqg and 018ndash040meqgloading for 4a and 4b respectively These yields could beindirectly evaluated by DMT tests on weighed samples of thesupport after ammonia treatment determining the amountof unconjugated material left on the solid support In factonly nucleosides linked to the support through a phosphotri-ester or phosphoramidate diester linkage are easily removedupon basic treatment (28 NH

4OH 50∘C 5 h) whereas

nucleosides anchored through a phosphodiester bond are notcleaved from the resin under the same conditions AfterDMTremoval and detachment from the supports the obtainedcrude material was analysed by RP-HPLC and the profilesshowed a single major peak with an area (85ndash91) similarto values reported previously (Figure 1)The identity of 5 and6was determined by 1H 31P NMR and ESI-MS experimentsthat were conducted directly on the crude detached materialAs expected starting from 30mg of support 4a or 4b thetarget nucleotides were recovered as discrete compounds in2ndash4mg and 4ndash9mg quantities respectively in a highly pureform

To demonstrate the reliability of the CPG supports for theautomatic synthesis of ODNs automated assembly has beenexplored for the synthesis of the ODN chains adopting theelongation directions (31015840ndash51015840) Starting from support 8a a 10-mer was synthesised (Scheme 3) and after ammonia cleavageand deprotection (6 h 50∘C) ion exchange HPLC analysis ofthe released ODN showed a single product corresponding tothe desired compound9Thepurity of the isolated compound


was then checked by HPLC and its identity was determinedbyMALDI-TOFMSanalysis In a typical experiment startingfrom 35mg of functionalised support 8a with an average015meqg incorporation of the conjugating residue 150ndash200OD units of pure ODNs were isolated after gel filtration

4 Conclusions

In conclusion we have reported the synthesis of a newo-chlorophenol-functionalised solid support characterisedby a higher loading of hydroxyl phenol functions thanpreviously achievable (018ndash022meqg to CPG and 025ndash050meqg to TG) This support allows the facile and high-yield preparation of phosphodiester and phosphoramidatemonoester nucleosides aswell as other yet unexplored classesof phosphodiester and phosphoramidate molecules To testthe efficiency of this support we prepared model thymidineanalogues conjugated at the 51015840-position to cholesterol andn-butylamine through phosphodiester and phosphoramidatebridges respectively In all cases the coupling yields andpurity of crude detached materials were comparable to ourprevious results and twice as much target was obtained dueto the loading being doubled on average Based on thesepreliminary studies the method is efficient and very reliableThis synthetic approach proposed here can be a starting pointfor the development of a preparative method for obtainingnew phosphodiester and phosphoramidate nucleotides andoligonucleotide conjugates Further studies are currentlyin progress to optimise the yields of 3-chloro-L-tyrosineincorporation on the matrix and to evaluate the relationshipbetween the loading ofmatrices with 3-chloro-L-tyrosine andthe structure of the targets as well as the HPLC purity of thecrude detached material

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

This study has been supported by AIPRAS Onlus (Asso-ciazione Italiana per la Promozione delle Ricerche sullrsquoAm-biente e la Saluta umana)The authors thank CIMCF (Centrodi Metodologie Chimico-Fisiche) and Universita degli Studidi Napoli ldquoFederico IIrdquo for the MS and NMR facilities Theyalso acknowledge Tecno Bios for grants in support of thisinvestigation

References

[1] T Aboul-Fadl ldquoAntisense oligonucleotides the state of the artrdquoCurrentMedicinal Chemistry vol 12 no 19 pp 2193ndash2214 2005

[2] S T Crooke ldquoProgress in antisense technologyrdquo AnnualReviews of Medicine vol 55 pp 61ndash95 2004

[3] N M Dean and C F Bennett ldquoAntisense-oligonucleotide-based therapeutics for cancerrdquo Oncogene vol 22 pp 9087ndash9096 2003

[4] H Grosshans and W Filipowicz ldquoMolecular biology theexpanding world of small RNAsrdquo Nature vol 451 pp 414ndash4162008

[5] D R Corey ldquoRNA learns from antisenserdquo Nature ChemicalBiology vol 3 pp 8ndash11 2007

[6] J F Lee G M Stovall and A D Ellington ldquoAptamer therapeu-tics advancerdquo Current Opinion in Chemical Biology vol 10 pp282ndash289 2006

[7] T Da Ros G Spalluto M Prato T Saison-Behmoaras ABoutorine and B Cacciari ldquoOligonucleotides and oligonu-cleotide conjugates a new approach for cancer treatmentrdquoCurrent Medicinal Chemistry vol 12 no 1 pp 71ndash88 2005

[8] E Uhlmann and J Vollmer ldquoRecent advances in the develop-ment of immunostimulatory oligonucleotidesrdquo Current Opin-ion in Drug Discovery and Development vol 6 no 2 pp 204ndash217 2003

[9] E De Clercq ldquoHighlights in the discovery of antiviral drugs apersonal retrospectiverdquoThe Journal ofMedicinal Chemistry vol53 pp 1438ndash1450 2010

[10] Y Richter and B Fischer ldquoNucleotides and inorganic phos-phates as potential antioxidantsrdquo Journal of Biological InorganicChemistry vol 11 pp 1063ndash1074 2006

[11] C Simons Nucleoside Mimetics Their Chemistry and BiologicalProperties Gordon and Breach Science Singapore 2001

[12] N Usman and L M Blatt ldquoNuclease-resistant syntheticribozymes developing a new class of therapeuticsrdquo Journal ofClinical Investigation vol 106 no 10 pp 1197ndash1202 2000

[13] X Tan C K Chu and F D Boudinot ldquoDevelopment andoptimization of anti-HIV nucleoside analogs and prodrugsa review of their cellular pharmacology structure-activityrelationships and pharmacokineticsrdquo Advanced Drug DeliveryReviews vol 39 no 1ndash3 pp 117ndash151 1999

[14] T S Mansour and R Storer ldquoAntiviral nucleosidesrdquo CurrentPharmaceutical Design vol 3 pp 227ndash264 1997

[15] J Balzarini ldquoMetabolism and mechanism of antiretroviralaction of purine and pyrimidine derivativesrdquo Pharmacy Worldand Science vol 16 no 2 pp 113ndash126 1994

[16] D M Huryn and M Okabe ldquoAIDS-Driven nucleoside chem-istryrdquo Chemical Reviews vol 92 pp 1745ndash1768 1992

[17] S L Chang G W Griesgraber P J Southern and C RWagner ldquoAmino acid phosphoramidatemonoesters of 3rsquo-azido-3rsquo-deoxythymidine relationship between antiviral potency andintracellular metabolismrdquo The Journal of Medicinal Chemistryvol 44 no 2 pp 223ndash231 2001

[18] S C Tobias and R F Borch ldquoSynthesis and biological studiesof novel nucleoside phosphoramidate prodrugsrdquoThe Journal ofMedicinal Chemistry vol 44 no 25 pp 4475ndash4480 2001

[19] CMcGuigan R N Pathirana NMahmood K G Devine andA J Hay ldquoAryl phosphate derivatives of AZT retain activityagainst HIV1 in cell lines which are resistant to the action ofAZTrdquo Antiviral Research vol 17 no 4 pp 311ndash321 1992

[20] W Zhou S Upendran A Roland Y Jin and R P IyerldquoNucleotide libraries as a source of biologically relevant chemi-cal diversity solution-phase synthesisrdquo Bioorganic and Medici-nal Chemistry Letters vol 10 no 11 pp 1249ndash1252 2000

[21] Y Jin A Roland W Zhou M Fauchon J Lyaku and R P IyerldquoSynthesis and antiviral evaluation of nucleic acid-based (NAB)librariesrdquo Bioorganic and Medicinal Chemistry Letters vol 10no 17 pp 1921ndash1925 2000

[22] L De Napoli G Di Fabio J DrsquoOnofrio and D MontesarchioldquoAn efficient solid phase synthesis of 5rsquo-phosphodiester and


phosphoramidate monoester nucleoside analoguesrdquo ChemicalCommunications no 20 pp 2586ndash2588 2005

[23] M Gaglione N Potenza G Di Fabio et al ldquoTuning RNA inter-ference by enhancing siRNAPAZ recognitionrdquo ACS MedicinalChemistry Letters vol 4 no 1 pp 75ndash78 2012

[24] L Moggio L De Napoli B Di Blasio et al ldquoSolid-phasesynthesis of cyclic PNA and PNA-DNA chimerasrdquo OrganicLetters vol 8 no 10 pp 2015ndash2018 2006

[25] G Di Fabio G Malgieri C Isernia et al ldquoNovel syntheticstrategy for monosubstituted cyclodextrin derivativesrdquo Chem-ical Communication vol 48 pp 3875ndash3877 2012

[26] S Booth P H H Hermkens H C J Ottenheijm and D ReesldquoSolid-phase organic reactions III a review of the literature Nov96ndashDec 97rdquo Tetrahedron vol 54 pp 15385ndash15443 1998

[27] S Kobayashi ldquoNew methodologies for the synthesis of com-pound librariesrdquo Chemical Society Reviews vol 28 pp 1ndash151999

[28] F Balkenhohl C von dem Bussche-Huennefeld A Lanskyand C Zechel ldquoCombinatorial synthesis of small organicmoleculesrdquo Angewandte Chemie International Edition vol 35pp 2288ndash2337 1996

[29] P A Tempest and RW Armstrong ldquoCyclobutenedione deriva-tives on solid support toward multiple core structure librariesrdquoJournal of the American Chemical Society vol 119 pp 7607ndash7608 1997

[30] H Lonnberg ldquoSolid-phase synthesis of oligonucleotide conju-gates useful for delivery and targeting of potential nucleic acidtherapeuticsrdquo Bioconjugate Chemistry vol 20 pp 1065ndash10942009

[31] P Virta J Katajisto T Niittymaki and H Lonnberg ldquoSolid-supported synthesis of oligomeric bioconjugatesrdquo Tetrahedronvol 59 pp 5137ndash5174 2003

[32] D A Stetsenko and M J Gait ldquoA convenient solid-phasemethod for synthesis of 3rsquo-conjugates of oligonucleotidesrdquoBioconjugate Chemistry vol 12 no 4 pp 576ndash586 2001

[33] D L McMinn T J Matray and M M Greenberg ldquoEfficientsolution phase synthesis of oligonucleotide conjugates usingprotected biopolymers containing 3rsquo-terminal alkyl aminesrdquoJournal of Organic Chemistry vol 62 no 21 pp 7074ndash70751997

[34] J D Kahl D L McMinn and M M Greenberg ldquoHigh-yielding method for on-column derivatization of protectedoligodeoxynucleotides and its application to the convergentsynthesis of 5rsquo3rsquo-bis-conjugatesrdquo Journal of Organic Chemistryvol 63 no 15 pp 4870ndash4871 1998

[35] J DrsquoOnofrioMDeChampdore LDeNapoli DMontesarchioand G Di Fabio ldquoGlycomimetics as decorating motifs foroligonucleotides solid-phase synthesis stability and hybridiza-tion properties of carbopeptoid- oligonucleotide conjugatesrdquoBioconjugate Chemistry vol 16 no 5 pp 1299ndash1309 2005

[36] E Alazzouzi N Escaja A Grandas and E Pedroso ldquoAstraightforward solid-phase synthesis of cyclic oligodeoxyri-bonucleotidesrdquoAngewandte Chemie vol 36 no 13-14 pp 1506ndash1508 1997

[37] J DrsquoOnofrio D Montesarchio L De Napoli and G Di FabioldquoAn efficient and versatile solid-phase synthesis of 5rsquo- and 3rsquo-conjugated oligonucleotidesrdquo Organic Letters vol 7 pp 4927ndash4930 2005

[38] L De Napoli G Di Fabio J DrsquoOnofrio and D MontesarchioldquoNew nucleoside-based polymeric supports for the solid phasesynthesis of ribose-modified nucleoside analoguesrdquo Synlett no11 pp 1975ndash1979 2004

[39] M de Champdore L De Napoli G Di Fabio A Messere DMontesarchio and G Piccialli ldquoNew nucleoside based solidsupports Synthesis of 5rsquo3rsquo-derivatized thymidine analoguesrdquoChemical Communication pp 2598ndash2599 2001

[40] G Di Fabio A De Capua L De Napoli et al ldquoA new strategyfor the solid-phase synthesis of glycoconjugate biomoleculesrdquoSynlett no 3 pp 341ndash344 2001

[41] E Kaiser R L Colescott C D Bossinger and P I Cook ldquoColortest for detection of free terminal amino groups in the solid-phase synthesis of peptidesrdquoAnalytical Biochemistry vol 34 no2 pp 595ndash598 1970

[42] P W Davis and S A Osgood ldquoA new method for introducingamidate linkages in oligonucleotides using phosphoramiditechemistryrdquo Bioorganic and Medicinal Chemistry Letters vol 9no 18 pp 2691ndash2692 1999

[43] F Eckstein Oligonucleotides and Analogues A PracticalApproach IRL Press Oxford UK 1991

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy


Carbohydrate Chemistry

International Journal of


Journal of

Chemistry


Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of


Chromatography Research International


Applied ChemistryJournal of



Theoretical ChemistryJournal of


Journal of

Spectroscopy

Analytical ChemistryInternational Journal of


Journal of


Quantum Chemistry


Organic Chemistry International

ElectrochemistryInternational Journal of



CatalystsJournal of


Current solid phase methods for the synthesis of ODNconjugates include the utilisation of prefabricated labels pre-viously converted into the corresponding phosphoramiditeor H-phosphonate derivatives and elaborate supports bear-ing an appropriate linker to incorporate the conjugatingresidue generally employed as a postsynthetic modificationof the ODNs [30] In both strategies stringently appliedpurifications (in the first approach for the reactive phospho-rylated derivatives of the labels in the second for the prepa-ration of the linker or in the final step) are typically requiredto isolate the desired conjugatedmolecule in a pure form [31ndash34] Althoughmanymethods have been reported for the solidphase synthesis of conjugated ODNs the same cannot be saidfor solid phase synthesis of modified nucleotides A varietyof different 51015840-phosphorylated 51015840-phosphoramidate and 51015840-phosphorothioate nucleoside analogues have been preparedand evaluated for their biological activity [17ndash21]

As part of our continuing effort towards the synthesis ofnew solid supports that are useful for generating pharmaco-logically interestingmolecule libraries [22ndash25 35ndash40] of highquality in large quantities we present here an improvement ofour solid phase strategy discussed above Aiming to achievethe synthesis of a solid support with a higher load thanthat currently available and that is also compatible withphosphoramidite and phosphotriester chemistry we deviseda straightforward and efficient synthetic protocol to preparea new support in which the loading of the o-chlorofunctionalgroup is very high (020ndash050meqg)

2 Experimental

21 General NMR spectra were recorded in CDCl3and

CD3OD with a Bruker WM 400 spectrometer The chemical

shifts (120575) are given in ppm and referenced to the residualsolvent signal (726 and 331 ppm resp) and coupling constants (119869) are in Hz 31P NMR spectra were recorded at16198MHz using D

3PO4as an external standard For ESI-

MS analysis a Waters Micromass ZQ instrumentmdashequippedwith an electrospray sourcemdashwas used in the negative modeMALDI TOF mass spectrometric analyses were performedon a PerSeptive Biosystems Voyager-DE Pro MALDI massspectrometer in the linearmode HPLC analysis and purifica-tion were performed on an Agilent Technologies 1200 seriesinstrument equippedwith aUVdetectorThe crudematerialsof 5 and 6 were analysed by HPLC on a C18 PhenomenexLUNA column (5 120583m 100 times 250mm) eluted with a lineargradient of CH

3CN in H

2O flow rate = 08mLmin and

detection at 120582 = 260 nm The crude material of 9 wasanalysed by HPLC on a Nucleogel SAX column (Macherey-Nagel 1000ndash846) buffer A 20mM KH

2PO4aq solution

pH 70 containing 20 (vv) CH3CN buffer B 10M KCl

20mM KH2PO4aq solution pH 70 containing 20 (vv)

CH3CN linear gradient from 0 to 100 B over 30min flow

rate 08mLmin and detection at 120582 = 260 nm The crudematerial was purified by gel filtration chromatography ona Sephadex G25 column eluted with H

2OEtOH (4 1 vv)

LCAA-CPG and TentaGel amino supports were purchasedfrom Link Technologies and Novabiochem respectively

The nucleotide phosphoramidites the activator solution(045M tetrazole in CH

3CN) and the oxidiser solution

(01M I2THFH

2Opyridine) were purchased from Link

Technologies

22 Synthesis of Supports 1a and 1b Support 1a 250mgof LCAA-CPG-NH

2(010meqg 002mmol) was reacted at

rt overnight with a mixture of 1095mg (025mmol) of N-120572-Fmoc-3-chloro-L-tyrosine 515mg (025mmol) of DCCI45 120583L (025mmol) of DIEA and 380mg (025mmol) ofN-hydroxybenzotriazole (HOBtsdotH



treated with 20 piperidine in DMF three times for 5minThe coupling and Fmoc removal were repeated twice morein similar conditions According to the Kaiser test [41] theincorporation of the linker was always in the range of 65ndash85 corresponding to 019ndash025meqg After capping theunreacted amino functional groups with Ac

2Opyridine (1 1


2O the resulting support 1a was dried

under reduced pressureSupport 1b 250mg of TG-NH

2LL (029meqg 007

mmol) was reacted at rt overnight with a mixture of 3175mg (072mmol) of N-3-chlorotyrosine acid 1500mg (072mmol) of DCCI 1260120583L of DIEA and 1100mg (15mmol)of N-hydroxybenzotriazole (HOBtsdotH



treated with 20 piperidine in DMF three times for 5minThe coupling and Fmoc removal were repeated twice more insimilar conditionsThe incorporation of the linkerwas alwaysin the range of 65ndash85 corresponding to 050ndash075meqgaccording to the Kaiser and Fmoc tests After capping theunreacted amino functional groups with Ac

2Opyridine (1 1


2O the resulting support 1b was dried

under reduced pressure

23 Synthesis of Supports 4a 4b and 8a Support 4a 11mL(05mmol) of a commonly used ldquoactivator solutionrdquo (045M tetrazole in CH

3CN) was added to 008mmol of 51015840-O-(2-

cyanoethyl)-NN-diisopropylphosphoramidite-31015840-O-(441015840-di-dimethoxytriphenylmethyl)-thymidine and 250mg (022meqg 005mmol) of support 1a After 1 h the support wasexhaustively washed with CH

3CN and treated (3 times)

with 5mL of a commonly used ldquooxidiserrdquo solution (I2

pyridineH2OTHF) for 5min After exhaustive washing

with CH3CN DCM and Et

2O the resulting support 3a

was dried under reduced pressure Incorporation yieldsof the nucleotides were always in the range of 82ndash99(018ndash022meqg) as determined by a quantitative DMTtest performed on dried and weighed samples of support 3aAfter the standard capping procedure with Ac

2Opyridine

(1 1 vv) the 2-cyanoethyl group from the phosphate was




3CN) was added




2OTHF) for 5min


2O






3



3OH DCM and Et

2O


2O B = CH

3CN) flow




2-51015840



3T) ppm 31P NMR (CD

3OD 16198MHz)



0100200300400500600700

mAU

0 25 5 75 10 125 15 175min


2O B = CH




3OHDCM and Et



4OH 50∘C


2O B = CH

3CN) flow




3OH



4OH 50∘C 5 h) The


2O B = CH

3CN) flow rate




2-51015840 T) 286 (2H

m CH2-NH) 233-218 (2H m H

2-21015840 T) 195 (3H s CH

3T)


PO

O O

POminusO

X OR

P

O

O

Ominus

Nu(DMT)

Site forconjugation

ODN chainassembly

= labeling group

O N

O

B

OH

POminusO

X OR

CD

OHLinker

R

R

O

Cl

POOCE

R








R = labeling group


N(ipr)2Ominus

NO2





3-


3butyl




3OH DCM and Et




2OEtOH (4 1 vv)The







COOH

NHFmoc

HO

Cl

HN N

HNHAc

O

O

OCH2

OHCl

OHCl

OHCl

NH

HN

O

HN

Cl

OHO

ThyOCEO

P

ODMT

PO

O

Cl

HN

O

ThyO

2

O

HN

1a 1b




NH2

= CPG TG



CH2(ipr)2N



OR998400998400

OR998400

3a 3b R998400 = DMT R998400998400 = CE

4a 4b R998400 = Ac R998400998400 = H




a = CPG b = TG

+







POH

NOminus O

ThyO

OH

n-Bu

PO

Ominus O

ThyO

OH

PO

OOminus

Cl

HN O

ThyO

OAc4a 4b

O

HN

O

n-BuHN POminus

OO TCTCTCTCTC

5

6

9

P OOOR

Cl

HN

O

ThyDMTO

O

O

HN




∙ n-Bu-NH2 15998400 rt





∙ n-Bu-NH2 15998400 rt

7a R = CE

8a R = H

∙ 20 piperidine

in DMF rt

3998400

5998400













4 Conclusions




Acknowledgments


References























































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of




3CN) was added




2OTHF) for 5min


2O






3



3OH DCM and Et

2O


2O B = CH

3CN) flow




2-51015840



3T) ppm 31P NMR (CD

3OD 16198MHz)



0100200300400500600700

mAU

0 25 5 75 10 125 15 175min


2O B = CH




3OHDCM and Et



4OH 50∘C


2O B = CH

3CN) flow




3OH



4OH 50∘C 5 h) The


2O B = CH

3CN) flow rate




2-51015840 T) 286 (2H

m CH2-NH) 233-218 (2H m H

2-21015840 T) 195 (3H s CH

3T)


PO

O O

POminusO

X OR

P

O

O

Ominus

Nu(DMT)

Site forconjugation

ODN chainassembly

= labeling group

O N

O

B

OH

POminusO

X OR

CD

OHLinker

R

R

O

Cl

POOCE

R








R = labeling group


N(ipr)2Ominus

NO2





3-


3butyl




3OH DCM and Et




2OEtOH (4 1 vv)The







COOH

NHFmoc

HO

Cl

HN N

HNHAc

O

O

OCH2

OHCl

OHCl

OHCl

NH

HN

O

HN

Cl

OHO

ThyOCEO

P

ODMT

PO

O

Cl

HN

O

ThyO

2

O

HN

1a 1b




NH2

= CPG TG



CH2(ipr)2N



OR998400998400

OR998400

3a 3b R998400 = DMT R998400998400 = CE

4a 4b R998400 = Ac R998400998400 = H




a = CPG b = TG

+







POH

NOminus O

ThyO

OH

n-Bu

PO

Ominus O

ThyO

OH

PO

OOminus

Cl

HN O

ThyO

OAc4a 4b

O

HN

O

n-BuHN POminus

OO TCTCTCTCTC

5

6

9

P OOOR

Cl

HN

O

ThyDMTO

O

O

HN




∙ n-Bu-NH2 15998400 rt





∙ n-Bu-NH2 15998400 rt

7a R = CE

8a R = H

∙ 20 piperidine

in DMF rt

3998400

5998400













4 Conclusions




Acknowledgments


References























































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


PO

O O

POminusO

X OR

P

O

O

Ominus

Nu(DMT)

Site forconjugation

ODN chainassembly

= labeling group

O N

O

B

OH

POminusO

X OR

CD

OHLinker

R

R

O

Cl

POOCE

R








R = labeling group


N(ipr)2Ominus

NO2





3-


3butyl




3OH DCM and Et




2OEtOH (4 1 vv)The







COOH

NHFmoc

HO

Cl

HN N

HNHAc

O

O

OCH2

OHCl

OHCl

OHCl

NH

HN

O

HN

Cl

OHO

ThyOCEO

P

ODMT

PO

O

Cl

HN

O

ThyO

2

O

HN

1a 1b




NH2

= CPG TG



CH2(ipr)2N



OR998400998400

OR998400

3a 3b R998400 = DMT R998400998400 = CE

4a 4b R998400 = Ac R998400998400 = H




a = CPG b = TG

+







POH

NOminus O

ThyO

OH

n-Bu

PO

Ominus O

ThyO

OH

PO

OOminus

Cl

HN O

ThyO

OAc4a 4b

O

HN

O

n-BuHN POminus

OO TCTCTCTCTC

5

6

9

P OOOR

Cl

HN

O

ThyDMTO

O

O

HN




∙ n-Bu-NH2 15998400 rt





∙ n-Bu-NH2 15998400 rt

7a R = CE

8a R = H

∙ 20 piperidine

in DMF rt

3998400

5998400













4 Conclusions




Acknowledgments


References























































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


COOH

NHFmoc

HO

Cl

HN N

HNHAc

O

O

OCH2

OHCl

OHCl

OHCl

NH

HN

O

HN

Cl

OHO

ThyOCEO

P

ODMT

PO

O

Cl

HN

O

ThyO

2

O

HN

1a 1b




NH2

= CPG TG



CH2(ipr)2N



OR998400998400

OR998400

3a 3b R998400 = DMT R998400998400 = CE

4a 4b R998400 = Ac R998400998400 = H




a = CPG b = TG

+







POH

NOminus O

ThyO

OH

n-Bu

PO

Ominus O

ThyO

OH

PO

OOminus

Cl

HN O

ThyO

OAc4a 4b

O

HN

O

n-BuHN POminus

OO TCTCTCTCTC

5

6

9

P OOOR

Cl

HN

O

ThyDMTO

O

O

HN




∙ n-Bu-NH2 15998400 rt





∙ n-Bu-NH2 15998400 rt

7a R = CE

8a R = H

∙ 20 piperidine

in DMF rt

3998400

5998400













4 Conclusions




Acknowledgments


References























































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


POH

NOminus O

ThyO

OH

n-Bu

PO

Ominus O

ThyO

OH

PO

OOminus

Cl

HN O

ThyO

OAc4a 4b

O

HN

O

n-BuHN POminus

OO TCTCTCTCTC

5

6

9

P OOOR

Cl

HN

O

ThyDMTO

O

O

HN




∙ n-Bu-NH2 15998400 rt





∙ n-Bu-NH2 15998400 rt

7a R = CE

8a R = H

∙ 20 piperidine

in DMF rt

3998400

5998400













4 Conclusions




Acknowledgments


References























































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of



4 Conclusions




Acknowledgments


References























































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

research article synthesis of oligonucleotide conjugates...

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