Nucleic Acids Research

Sym posium Series No. 7 1980

F u rther studies on oligoribonucleotide synthesis

W.T.Markiewicz, E.Biała, R.W.A dam iak, K.G rzeskowiak, R .K ie rz e k , A.Kraszewski, i.Staw iński and M.Wiewiórowski

D epartm ent o f Stereochem istry of N atural Products, In stitu te o f O rganic Chemistry, Polish Academy of Sciences, Noskowskiego 12, 61-704 Poznan, Poland

ABSTRACT

Recent results concerning the synthesis of oligoribonucleotides via the phosTihotriester methods such as functionalization of ribonucleosides^ new pho— sphorylating agentsf 5 f~0~sulfonylation and chromatography on Sephadex LH-20 for monitoring the removal of internucleotide phosphotriester groupsf are pre— sentedc To show the efficiency of a new approach to the synthesis of oligori­ bonucleotides the pentamer /Up/^U was obtained^

INTRODUCTION Suitably protected nucleoside 3'-phosphotriester II, a key component for the synthesis of oligoribonucleotides,, proved to be useful for the preparation of medium-size oligonucleotidic chainsf due to the development of modified procedures for the removal of the 2-cyanoethyl group1 and the 2 f2 f2-.trichloro~ o 5 1 2 ethyl group as well as the application of analytical techniques / B»NMR and a Sephadex LH-20 chromatography /«

H0/ X0H

+

-isomer 2 ,-i

cci5gh2o/ xoch2ch2cm

I

II

B = pyrimidine or purine residue We have described previously the synthesis of an anticodon loop of tRHAme

6

containing a hypermodified nucleoside t A by a phosphotriester appro-

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115

Nucleic Acids Research

ach using the phosphotriester Il\ The key components of this type have been prepared*'

from commercially available mixtures of 5 E/2 S/-nucleotides as

starting mateialse The preparations howeverf of the above phosphotriester key components /il/ involved phosphomonoesters as intermediates and thus has some disadvanta— ges of the diester approach to oligoribonucleotide synthesiss although the ul­ timate syntheses of intemucleotidic bonds were achieved by the triester ap­ proach* We have been aware of these disadvantages and, in order to assure the easy access to the key components in a multigram scales we have carried out studies on the application of nucleosides as starting materials* Recently we have described a new type of silyl reagent which allows for the simultaneous protection of 3 % 5 ’—hydroxy functions of ribonucleosides^ namely 1 53-*dichlorotetraisopropyldisiioxane /TIFDSiCl^j III/.

i-Pr_SiCl 2I i-BCgSiCl

i-Er^SiO HO

OH

i-Er2SiO

OH

IV

RESULTS AND DISCUSSION We would like to present the last results concerning the application of 5 the tetraisopropyldisiloxane-1 s3-*diyl group for the synthesis of different key components. We took advantage of easy access to 3 % 5 f-0~protected ribonucleosides V to develope the procedures for their conversion into different ty­ pes of key components for oligoribonucleotide synthesis. The experiments cong

oeming the synthesis and use of key components X are the most advanced , After the introduction of a proper 2I~hydroxy protecting group, the si5 7 lyl group is selectively removed with fluoride anions , During the investi­ gation of different fluorides^for the cleavage of the silyl groups} we found that the use of an easily accessible triethylamine hydrogen fluoride /TEAHP/ simplifies the large-scale work-up procedures* The selective removal of the 5 f-0~dimethoxytrityi group in the presence of an acid-labile 2 f-0~tetrahydropyranyl group of ribonucleoside 3*-phosphoQ triester had been described previously f although the detailed studies on this

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Nucleic Acids Research

b i-Er2S i O ^ _ 0_ J

i-Kr2SiO-|^_0^

b ,

bcoh1

i~Er„SiOn

0. i-Ec^SiO

OH

i-BrgSiO

V

x i-Er„SiO -^ ,

OR

VI

OR

711

S '

COH.1 B

R = methojiytetrahydropyranyl R^= alkyl or aryl 2 R = aryl or alkyl

HO

OR

rai COR

HO

1

OR H

problem were not reported* We checked different acidic conditions for the re­ moval of the 5*-0-dime thoxytrity1 group from the key component X /B = uracil_Vyl/f changing the concentration of trifluoroacetic acidf the temperature and solvents /Table l/« The data collected in Table 1 show that the use of pu­ re chloroform as a solvent at 0°C favours the selective removal of diraethoxy-

Table 1* Selective removal of the 5s--0,”MM 0.075

/MT/U/mthp/-U/mthp/p/Cl Hi/ U/mthp/—U/mthp/—U>MM 0.045

0.045

/DMP/tJ/mthp/-U/mthp/p/ClPh,CNEt/ 0.195

65

/DKT/CJ/mthp/~U/mth p/-ll>MM o„0525

70

/DMT/U/mthp//—IT/mthp//jU>MM 0 .0 315

70

Abbreviations used: DMT - dimethoxytrityl, mthp - methoxytetrahydropyxanyl, MM - methoxymethylene, Cl Hi - p~chlorophenyl and CNEt - 2-cyanoethyl group. Each intemucleotidic phosphate is protected by p—chlorophenyl group and is abbreviated as +« Terminal phosphate group is represented by symbol p and its protective groups are specified in parantheses.

Nucleic Acids Research ride in tetrahydrofuran in the presence of trieihylamine at -70°C. This method

tool 3

-^°!U.

C1.L1 2

ciL1 I OR

XI

XII

R1OHs Gl^CCHgOH, p-chlorophenol, 4~tert«~butyl-2~chlorophenol R2OH; NCCH2CH2OH was found to be generally very efficient for the synthesis of any desired di­ alkyl, diaryl or alkyi-aryl phosphomonochloridates* The monofunctional phosphoryiating reagents which were synthe&ized in this way were successfully applied in the preparation of nucleoside phospho™ triesters /65-80$ yield/ 1 We solved the problem of phosphorylation on yet another route,. During these studies we have synthetized a new phosphoryiating reagent, phosphoryl 1 4tris-triazole/XIV/ 0 This allows us to overcome the disadvantages connected with the application of phosphodichloridates or phosphoryl chloride. Thus,

DMT-T-OH

+

,N=i OP/-K ]/.

0 ^ 1 IMT-T-OPOR I2 OR

.DMT-.T-.0P/-ir

XIV

XIII

XVa-f

Table 3« Synthesis of nucleoside 3*-phosphotriesters /XV/ from nucleosides Am/t

phosphoiyl tris-triazole /XIV/ and appropriate alcohols

R1 0H

r 2oh

product

iaolated yield %

CNEtOH

CHEtOH

X?a

?0

CHEtOH

EtOH

XVb

60

CHEtOH

hich2ococh2oh

XVc

61

CMEtOH

BrCHgCH/Br/CH^OH

XVd

6;

CHEtOH

C1,CCH„0H 3 HiCHgOCOCHgOH

XVe

15

XVf

60

rhCHgOCOGHgOn

120

Nucleic Acids Research phosphoryl tris-triazole was synthetized from POCl^ and triazole in the pre­ sence of triethylamine. %• means of this reagentf several key components ha­ ving various phosphate protective groups were obtained1^ /Table J/< Having in our hands tvro kinds of phosphoryiating agents 5, phosphoryl tris-triazole and monofunctional phosphorochloridates^ the synthesis of any des±~« red key component-.with different phosphate protective groups represents no difficulties at presents Some interesting observations were made during the experiments of the condensation of nucleoside 3 S“d.iesters bearing alkyl phosphate protective groups with 5 *~hydro3cy nucleosidic components^ We found that in all cases the condensation reaction was rather slow even when TFS-Te was used andE moreover, a high degree of sulfonation of the nucleosidic component was observed /ca 10 « 3 M o r e

detailed studies showed that the degree of sulfonation occurring

during the condensation depends strongly on the kind of phosphate protective group present in the phosphodiesters* Substituents which decrease the partial positive charge on the phosphorus atom /electron donating groups/ slowed down the rate of intemucleotidic bond formation, and as a consequence, considera­ ble amounts of sulfonated products were formed /Table 4/» These results prompted us to take a closer look at sulfonating proper­ ties of arylsulfonyl tetrazoles, which are known to be powerful condensing 0 tl SMT-T—OPOR l_ 0

+

XVI

0 II HCMT-OBz -- DKET-T-OPOT-OBz I OK XVII

+

ArS0,-T~0Bz 5

XVIII

XIX

Table 4* Composition of reaction mixtures after the condensation of phosphodiester XVI and 35~0~benzoylthymidine /XVIl/ entry

XVI

coupling agent

time /hr/

isolated yields $ XVIII XVII XIX

1

fcEt

TPS-Te

20

60

10

27

2

r=Bich2 ococh2

TPS-Te

20

60

10

17

3

R=CNEt

TFS-Te

20

50

15

4

RsCl^CCHg

TPS-Te

20

80

8

15 8

5

Hs=p-ClHi

TFS-Te

a

95

0

traces

6

R=IhCH20C0CH2

BST

96

60

39

traces

121

Nucleic Acids Research agents with very mild sulfonating properties. The comparison of TFS~Te and 2 (4-f6-triisoproEylbenzenesulfony1 chloride^ /TPS/ showed that both compounds have very similiar sulfonaxing properties when the sulfonation is the only possible reaction pathway. During the condensation, however, when two parallel reactions are possibles TPS exhibited significantly stronger sulfonating pro­ perties than TPS“Te1^. From the results presented here the following conclusion can be drawnt the degree of sulfonation depends not only on the kind of activating agent but alsos to a large extentf on the character of the phosphate protective group in the phosphodiester. The last problem ve would like to mention is the analytical method for studying xhe deprotection of the phosphotriester groups,, Difficulties encoun­ tered during the removal of the 2 ,i!sy-trichloroethyl intemucleotide phospha­ te protective groups from heptamers CCGAUAA and CCCAUt^AA^ forced us to search for a fast and efficient method of analysis of xhe phosphotriester phodiester transormation, As we reported very recently phadex LH-20 in a 0*-tfO$ gradient of methanol m

x

phos-

chromatography on Se­

0.02 M TEAB buffer pH 7*5 is

a very useful method for this purpose* Due to both the hydrophilic and lipo­ philic properties of this gel* xhe separation of incompletely deprotected heptamers, ±„e* intermediates still bearing some 2 (2 t2'*tnchloroexiiyl groups, was possible. At present tnis chromatography /e.g. Pig, 1 / serves in our la­ boratory as a method for monitoring the removal of intemucLaotide 2 f2f2-trichloroethyl and p-chlorophenyl groups,, It is a fast and reproducible method of isolation and purification of short partially protected oligoribonucleotide phosphodiesters of, at least, 90$ purity and is our choice for purification of oligonucleotides contaminated with zinc cations.

EXPERIMENTAL The syntheses of 1,3-dichlorotetraisopropyldisiloxane /ill/, J*,5r-0~ /tetraisopropyldisiloxane-1 ,3-diyl/nucleosides /V/ and 2'-0~methoxytetrahydronyranyluridine from VI were described previously . Phos-ohoryl tris-triazole /XIV/ was obtained in the reaction of P0C1, 3 / 1 eoval/, triazole /j eqval/ and Et^K /j eaval/ in dioxane as described in ref. 14* Phosphorylation of nucleoside XIII with the reagent XIV in dioxane, followed by the treatment with appropriate alcohols, afforded the desired nu­ cleoside phosphotriesters XVa-f in 60-75/“ yields1^.

122

Nucleic Acids Research F ig .

1 Chromatography on Sephadex LH-20 to monitor the progress

of deprotection of 25252-trichloroethyl groups from protected CCCAUAA by reductive elimination with the 2 zmc~acetylacetone~pyridine method . The elution profiles a-d correspond to 0„i)s 1 * 2 and 4 hrs of the depro­ tection respectivelys

Dialkyl phosphomonochloridates f X . I l / were prepared from POCl^ in THP at -70°C by stepwise addition of appropriate alcohols under anhydrous condi­ tions1"^ The phosphorylations using XII were carried out in acetonitriles using N-methylimidazole as a catalyst, or in pyridine The condensation of alkyl nucleoside 3*-phoaphodiesters /triethylammonium salts//XVl/ were carried out in pyridine in the presence of TFS-Te or BST as condensing agents

16 „ The experimental details concerning- the application

of chromatography on Sephadex LH-20 were described prevoiusly\ 2ff^-O-amethoxymethyleneuridine was obtained according to ref. 9.

spectra in CDCl^

were recorded on a Varian EM36O /60 KHz/ spectrometer using tetramethylsilane as an internal standard. TLC analysis was performed on Merck silica gel 60 F254 Pre-ooa'ted plates in the following systemsi chloroform containing metha­ nol /5-10$/, isopropanol-ammonium hydroxide-water 7 :2 :1 v/v. Short column chromatography on TLC silica gel type H /Merck/ in chloroform containing methanol /0~5$/ was used for separation of protected nucleosides and oligonu­ cleotides. Deprotected /Up/^U was separated on DE&E-Sephadex A25 column eluted with gradient 0-0,5 M NaCl aq, 0.01 K Tris-HCl buffer pH 7.5 with the use of LKB Uvicordi

the fidelity of 3*-5' bonds was checked as desribed before^.

123

Nucleic Acids Research Triethylamine hydrogen fluoride. /TEAHF/ was prepared according to a fol­ lowing procedure e To the solution of Et^N /4s 20 mlt JO mmoles/ in methanol /ca JO ml/ 40$ aq hydrofluoric acid /1,5 S f 30 mmoles/ was added /final pH 7 /. The solution was concentrated under the water pump pressure and then coevapo­ rated with anhydrous dioxane /30 ml/ to the oil* This oil was dissolved in THF to obtain 30 ml of ca 1 M solution of TEAHFe Finally, pH of the solution was adjusted to 7 by addition of Et^N if necessary. The removal of tetraisopropyldisiloxane-l „5—diyl groups^, In a typical reaction using TEAHP as a deprotecting agent nucleoside VI or VII /1 eqval/ was treated with 1 M solution of TEAHF in THP /3 eqval/e After the completion of the reaction /overnight/ the excess of TEAHF was decomposed by addition of aqueous sodium bicarbonate* The solution was concentrated and the residue was partitioned between chloroform and water* In the case of nucleosides VIII in™ soluble in chloroform the residue was coevaporated with pyridine to remove wa­ ter and after addition of pyridine the precipitated inorganic salts were fil­ tered

offt In both casesorganic layers were evaporated and nucleosides VIII

were isolated after a silica gelcolumn chromatography /S0-=90/c yield/* 5>~0~Dimethoxytrityl~2*-0~methoxytetrahydrowranvluridine /IXs B=uracil-1-yl/. Dimethoxytrityl chloride /1.352 gs 3«99 mmole/ was added to the solu­ tion of VIII /B=uracil-1-yl/ /1.302 gs 3*63 mmole/ in anhydrous pyridine /36 ml/e The reaction vessel was tightly stoppered and left at room temperature in darkness^ After 20 hr the reaction was quenched with waters partially con­ centrated and partitioned between chloroform and aqueous sodium bicarbonate. The aqueous layer was extracted twice with chloroform* The combined extracts were concentrated and after a silica gel column chromatography IX was obtained as a solid foam /2.033 gt 3.08 mmole, 84fo yield/. 1H-HMRs cf 7„77/dt1sJ=8 Hzs H-6/j

6.20/d, 1, J=6 Hz, H-1*/, 5.25/d, 1, J=e Hz, H-5/, 3.75/s, 6, 2xOCH

DMT/,

3.1 6/s, 3s OCHj ofmthp/ 1

of

4f mthp ring protons pto oxygen/*

5 1-Q-Dimethoxytrityl-2-Q-roethoxytetrahydropyranyIurldine 3*-/p-chlorophenyl.2-cyanoethyl/phosphate /X, B=uracil-1-yl/. Compound IX /fcuracil-l-yl/ /991 mg, 1 .5 mmole/ was dried by coevaporation with anhydrous pyridine and toluene and dissolved in anhydrous acetonitrile /1 . 5 ml/ and 1 -methylimidazoie /370 mg, 4.5 mmole/ was added. To this solution p-chlorophenyl,2-cyanoethyl phosphochloridate

acetonitrile /3 ml/. After ca 15 min

TLC showed that the reaction was completed. The reaction mixture was concen­ trated, and partitioned between chloroform and 0,5 M phosphate buffer pH 6.5, The chloroform layer was dried, concentrated and chromatographically pure X

124

Nucleic Acids Research was isolated after silica gel column chromatography and precipitation with hexane /986 mg{ 1.09 mmolet 72% yield/. 1H-NMR; cf 7,,73/d, 1* J=8 Hzs H~6/s 61 4,0/dt 1, J=8 Hz, H-5/f 6,,23/d, 1S J=1.5 Hz, H-1s/ { 4.4/t, 2, CH^GH^CN/,, 3.83 /af 6, 2xOCHj of BMT/f 3»22 and 3 »15 /2xs( 3s OCH^ of mthp of two diastereoisomers/, 2,83 /t, 2S CH CJ^CN/. The reaction of selective removal of the 5*-'0-dimethoxytrityl group from compound X /B = uracil«1-yl/ was studied using the starting concentration of X ca 0.01 Me The reactions were followed with TLC and the results are sum-, marized in Table 1c General procedure for the oligoribonucleotide synthesis5s-Deprotection,,The DMT group was removed from the fully protected mono/oligo/ ribonucleotide by treatment with 0.1% trifluoroacetic acid in chloroform /ca 85 ml per 1 mmole/ during ca 120 min. at 0°Ce After the completion of the rea­ ction 1 M phosphate buffer pH 7*5 /10 ml/ was added to quench the reaction,, The layers were separated and the aqueous layer was extracted with chloroform /2x10 ml/, The combined extracts were dried, concentrated and purified by the silica gel chromatography followed by precipitation with hexane. Yields 65-90^ 3£-Beprotection^ The 2-cyanoethyl group was removed from the terminal 3 ^triester according to the previously published procedure"' „ Coupling, 3s- and 5f- deprotected units were coupled in pyridine with TFS-Te according to the previously published procedure^,, The data for the coupling reactions are summarized in Table 2* ./Up/^I was deprotected by treatment with conc. ammonium hydroxide /1 6 hr/ at room temperature and 0.01 N HC1 aq /16 hr/, and chromatographed on DEAE~Se« phadex A25® The elution profile is shown in Fig, 2S The yield of total depro­ tection was ca 80$,

Fige 2

Separation of the deprotected /Up/^IT

on DEAE-Sephadex A25 column*

125

Nucleic Acids Research ACKNOWLEDGEMENTS This work was supported by Polish Academy of Sciences projects: MR I 12 1.7.11 and W 09.7.2.3.1. The authors would like to thank Mrs. B. Nowakowska and mgr. E« Gembiak for technical assistance. REFERENCES

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