facile synthesis of oligonucleotides on solid support using...
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Indian Journal of Chemi stry Vol. 428 , Jul y 2003 , pp. 1696- 1700
Facile synthesis of oligonucleotides on solid support using pyridine derivatives for amino and phosphate protection
Sarika Sinha & Ramendra K Singh*
Nucleic Acids Research Laboratory, Department of Chemistry, University of Allahabad, Allahabad 2 11002, India
Received 5 August 2002; accepted (revised}"4 February 2003
The picolinoyl group has been used for amino protection in the case of all the three deoxynucieosides-dC, dA and dG , and good yie lds of N-protected nucleosides are obtained. This pyridine derivative along with another pyridine deri vat ive, viz. (a-pyridyl) methyl, an autocata lyti c phosphate protecting group, has been used successfull y in the sy nthes is o f two oligonucleotides, d(T ACGTTTTGCT) and d(ACCGATATCGT) fo llowing solid phase methodology . The good y ie lds of these ll -mers (48 and 45%, respectively) are attributed to the greater solubilising effect generated due to the combi nat ion of these two groups. The structures of these o ligomers have been confirmed by enzy matic hydrol ys is with snake venom phosphodiesterase fo llowed by alkaline phosphatase.
The enormous utility of sequence-defined oligodeoxyribonucleotides (ODNs) in the field of their conformational studies l
.3
, biochemjcal processes4.7 and molecu
lar biologl l3 has generated immense curiosity for their smoother synthesis. The sequential synthesis of oligomers requires the prior protection of amino, sugar and phosphate functions by suitable groups. Here compatibility of amino and phosphate protecting groups is a very important factor since they remain intact throughout the course of synthesis. Several amino protecting groups have been used successfully in thi s laborator/ 4
-17 in combination with reported
phosphate protecting groups. But this time, the authors wish to report picolinoyl , a new amino protecting group along with an autocatalytic phosphate protecting group, (a-pyridyl ) methyl , designed and used in this laborator/ 8
. The picolinoyl group has been used for amino protection in the case of all the three deoxynucleosides, i.e., dC, dA and dG. Using these protecting groups, two oligomers, d(T ACGITITGCT) and d(ACCGATATCGT) were synthesized applying phosphotriester approach on controlled pore glass as solid support.
Results and Discussion The use of picolinoyl group following the classical
approach for amino protection developed by Khorana21 (peracylation approach) has shown marked improvement in the yield of N-protected nucIeosides. The mechani sm of N-acylation, which is facilitated in a bas ic solvent like pyridine, probably derives support from greater solubilising effect generated due to pi-
colinoyl group being pyridine deri vative. The solubili sing effect also eliminated the poss ibility of side reactions upto certain extent. The reactions during amino protection were neat and clean and resulted in higher yields of N-protected nucIeosides (Figure 1). Further, the selective O-deacylation with triethylamine-water-pyridine eliminates any possibility of depurination that occurs sometimes in the case of deoxyadenosine22 during O-deacylation at higher pH.
The optimum condition for N-deacylation was 25% ammonia for 30, 40 and 55 min in the case of dC, dA and dG, respectively at ambient temperature and hence the time for N-deacylation has been reduced significantly. Thus the removal of this new protecting group was under milder conditions, unlike the other amjno protecting groups removed at elevated temperature.
The efficiency of the indigeneously prepared phosphorylating reagent has been proved beyond a!1y doubt in an earlier pUblication l8
. But it deserves a mention here that no detectable di meri zation during phosphorylation was observed following the usual procedure23
.
Two oligonucleotides, viz. d(T ACGTTTTGCT) and d(ACCGA T A TCGT) have been synthesized using picolinoyl bearing monomers on solid support following the standard protocol and phosphotrieSter chemistry (Figure 2). The higher yields in the preparation of monomeric blocks and the synthesis of two oligomers, i.e., 48 and 45 % in the case of 'A' and 'B ', respectively (Figure 3), can be attributed to the higher solubilising effect generated due to the combination
SINHA et al. : FACILE SYNTHESIS OF OLIGONUCLEOTIDES ON SOLID SUPPORT 1697
OH
3- 5
3 . B = Cytosine ~
4 : B = Adenine R = ~.NI~-5 : B = Guanine
Figure 1- Protecti on of amino function on deoxyri bonucIeosides
+ e N' PI,o
1) H _ I U --+ iii 0 l11r '\J ---o·p - ONHflJ I H SHT I HII"
I
1'4. pic ... G 0
il H I _ II -+ id OHTro~-rO NHEtJ I HSNT I M. I ..
G"" "O T OR ( N-P ; 'O T O R
D H Tro \ l-o-~- o I_o c ICtI, J, OiN-®
" I '\j II \I OHTr O I- O-~-O~-OCiC H11 ' CHH-----®
\j I II II OR 0 0
OR 0 0
\
9. Iii H+ I i iJ fully prot. c to<! ,." I..,tid< ;
1 L HSNT/ H.lm
\) oxirrrrle iil omsnoniu.
19< \;1 H-+ . ! I iii full y prut. cI.d nucl.oti d. ; ! L rl SHT 1". 1 ..
I l J o_i.ate ii l J.mm onic.
DMTr - d (TpApCpGpTp Tp TpGpCpT )
~
DMT r- d ( ApCpCpGpApTpApTpCpGpT)
o R = I N ' CH,.
o ' M,el m = [~ MS NT = -d-s-~j --0 : LCAA- CPG as so lid support
~ ~'" W---- NO CH, 0 I
Figure 2 - Solid phase synthesis of oligonucleotides
of these two groups. The composition of these o ligomers was confirmed by their enzymatic hydrolysis with snake venom phosphodiesterase. HPLC analysis of hydrolysed products clearly establi shed the percentage character of each nucleoside. The retention time of each peak was confirmed by comparing it with authentic samples of respecti ve nucleosides and thi s also established that no base modification has taken place during the course of oli gomer synthesis or their purification.
These amino and phosphate protecting groups, the pyridine deri vati ves, were chosen with an aim that right from the preparation of the monomeric units upto the final steps of sy nthesis, pyridine is used as a solvent, and the greater solubili sing effect eliminated the possibilities of various side reactions and in-
creased the coupling yie lds with a reduced coupling time. This improved the overall yields of o ligomers indicating thereby the successful use of these groups in o li gonucleotide synthesis. A detailed survey of amino, sugar and phosphate protecting groups has been given in the review article published by Singh et. al,z4. The compatibility of the group in phosphoramidite approach of oligonucleotide synthes is is being tested.
Experimental Section Various nucleosides, dimethoxytrityl chloride
(DMTrCl), 1, 1 ,3,3-tetramethylguanidine, 4-nitrobenzaldoxime, mesitylenesulphonyl nitrotri azo le (MSNT), l-methylimidazo le (Melm) and longchain alkylamine-contro lled pore glass (LCAA-CPG) were pur-
1698 INDIAN J. CHEM. , SEC B, JUL Y 2003
~ A E T ~
<=> -0
"" ~ 4lJ u ~ 0
.L3 L... P
de d (,
'" .D d «
o 5 10 0 10 ,s 20 Retent ion t i m~ (min) Hetention tilDe (min)
Figure 3 - RPC purification of tritylated oligonucleotides (left-side graphs). Buffer A: triethylammonium acetate (0. 1 M, pH 7.0) and B: lriethylammonium acetate (0. 1 M, pH 7.0) in acetonitrile (80%). Gradient: 25-65% B over 40 min, flow 4 ml/min, temp. 35°C. Inset: detritylated oligonucleot ides after RPC purification. Enzym:;nically hydrolysed oligonucleotides (righI-side graphs), 6% buffer B over 20 min , flow rate 0.5 ml/min .
chased from Sigma Chemical Co., USA. , Biosearch, • London and Cruachem Chemical Co., Scotland. The
enzymes, snake venom phosphodiesterase (SVPD) (EC 3.1.4.1, Crotalus durissus) and alkaline phosphatase (EC 3.1.3.1., E. coli) were purchased from Sigma Chemical Co., USA.
Solvents were duly purified and dried before lise. TLC was done on silica gel G (Merck) plates and the plates were sprayed with Isch'erwood reagent, iodine and sulphuric acid (10% in methanol) for identification and differentiation of spots. LCAA-CPG was used as a support for solid phase synthesis, which was carried out on a dual column DNA bench synthesizer (Omnifit Ltd. , Cambridge). HPLC analysis was done on LKB Ultrapac TSK DEAE-3 SW column. UV ab-
sorption was measured on a Hitachi 220S spectrophotometer. NMR was recorded on a Bruker WM-400 model.
Preparation of N-picolinoyl deoxyribonucleosides. To each nucleoside (l mmole) , dried by coevaporation with anhydrous pyridine (3 x 4 mL) in vacuo and suspended in 'anhydrous pyridine (8 mL), picolinic anhydride (4.0 mmoles) was added. The flask was sealed and shaken in dark for 3 hr for completion of the reaction. The reaction mixture was evaporated to a gum in vacuo, poured into water (25 mL) and extracted with dichloromethane (3 x 5 mL). The organic phase was evaporated to a gum, which was subjected to hydrolysis with triethylamine in pyridine-water. After 30 min. , the mixture was ren-
SINHA et al.: FACILE SYNTHESIS OF OLIGONUCLEOTIDES ON SOLID SUPPORT 1699
Table I - Preparation and characterisation of N-picolinoyl dC/dA/dG
Comp Yield *Rr UV (MeOH) 'H NMR (CDCI 3)
d (%) A max (nm) 3 82 0.35 30 1,256 7.5 & 8.3 (d, H-5 , H-6), 7.0-8.4 (m, 4H), 6.25 (t , H-I '), 4.4 (m, H-3 /), 4.2 (m, H-4/),
3.7-3.9 (m, H-5'), 2.15-2 .6 (m, 2H, H-2/)
4 80 0.50 282, 230 8.25 & 9.05 (s, 2H , H-2, H-8), 7.2-8.5 (m, 4H), 7.02 (t, H-I '), 5.25 (m, H-3/), 4.6 (m, HA'), 4 .1 -4.35 (m. 2H, H-5 /), 2.7-3.3 (m, 2H, H-2/ )
5 75 0.55 263,23 1 8.05 (s, H-8), 7 .1 -8.4 (m, 4H), 6.4 (t, H- I ') , 5.5 (m, H-3/), 4.33 (m, HA'), 2.6-3.1 (m. 2H, H-2/)
dered dry by repeated evaporation with benzene in vacuo. This dried mass was subjected to silica gel column (2 x 25 cm) chromatography for purification. A gradient elution was done with CH2ClJMeOH to obtain the desired products. Data obtained are reported in Table I.
Removal of N-picolinoyl group. N-protected derivatives were treated with 25 % ammonia at ambient temperature. The reactioQ was quenched at certain time interva ls and the reaction mixture was analyzed for deprotected nucleosides by preparative TLC and estimated quantitatively by UV spectroscopy. The complete removal of picolinoyl group with 25% ammonia was obtai ned in 30, 40 and 55 min in the case of dC, dA and dG, respectively.
Preparation of 5' -O-dimethoxytrityl-N-picolinoyldeoxynucleosides (5'-O-DMTr-NPic-dNS). N-protected nucleosides ( 1 mmole each) were treated with DMTrCI (l.2 mmoles) in anhydrous pyridine at room temperature and the reaction mixture was stirred for 2.0-2.5 hr. After completion of the reaction , the mixture was poured into water (25 mL) and extracted with CH2Ci 2 (4 x 5 mL). The organic phase was dried over anhydrous Na2S04 and evaporated to a gum in vacuo. The products were purified on silica gel column (2 x 15 cm) using dichloromethane and methanol as e luents. The products, viz. 5'-0-DMTr-Npic -dC, 5'O~DMTr-NPic-dA and 5/-O-DMTr-Npic-dG were obtained in 92%, 94% and 95% yields, respectively .
Phosphorylation of 5'-O-dimethoxytrityl-N-picolinoyldeoxynucleosides. The phosphorylating reagent. viz. (a-pyridyl)methy lphosphoro-bis-triazolide and different phosphoryl ated derivatives of N,5'protected nucleosides, viz. 5/-O-DMTr-N4-pic-2'-dC-3'-( a-pyridyl)methylphosphoro-bis-triazolide, 5'-0-DMTr-T -3'-( a-pyridyl)methy lphosphoro-bis-triazolide, 5'-0-DMTr-N6 -pic-2'-dA-3' -( a-pyridy I)methylphos-
phoro-bis-triazolide and 5' -0-DMTr-N2 -pic-2'-dG-3'(a-pyridyl)methylphosphoro-bis-triazolide were ob-
. d d I' IS tame as reporte ear ler . Preparation of 5' -O-DMTr-T -3'-O-succinate.
Thi s unit was prepared by usual procedure ls
Synthesis and purification of oligonucleotides: d(TACGTTTTGCT) and d(ACCGATATCGT). 5'-0-DMTr-T-3'-0-succinate was linked to LCAACPG, as the solid support l9
. Loading of thi s nucleoside was found to be 38 J,!mol/g on the basis of trityl estimation2o. This derivatised support was taken in both the columns (100 mg each) of the DNA synthesizer and the wash cycle was followed in the following order:
Step Treatment Time (min)
Pyridine wash 3 2 Dichloromethane (DCM) wash 2 3 Dichloroacetic acid (3 % in DCM) l.5 4 Dichloromethane wash 2 5 Pyridine wash 3 6 Coupling 6
The cycles of wash and coupling were carried out till the synthesis of the respective undecamers w.as complete. The incoming nucleotide was used in 10-fold excess in two batches and preactivated with MSNT and methyl imidazole. After the final coupling reaction, the support was washed with dichloromethane, methanol and ether. The support was taken out of the column and dried. The oligomers attached to the solid support were treated with 0.5M solution of I, I ,3,3-tetramethylguanidinium and 4-nitrobenzaldox ime in dioxane-water ( 1:1 , v/v) for 16 hr at ambient temperature. The support was filtered off and the filtrate evaporated till dryness in vacuo. It was treated with 25% ammonia for I hr at room temperature.
1700 INDIAN 1. CHEM., SEC B, 1UL Y 2003
Ammonia was removed using water pump and the reaction mi xture was evaporated to dryness in vacuo.
The partially deprotected oligomers were subjected to purification by reverse phase HPLC using C 18 column . The tritylated o ligomers were separated and subjected to detritylation with 80% acetic acid for 30 mjn at room temperature and again purified on RPC column .
Enzymatic hydrolysis of oligomers. The oligomers (0.5 A260 units) dissolved in O.IM tri s- HCI buffer, pH 8.5 (0.7 mL) were first digested with SVPD ( 10 jlg, 37°C, 2 hr) and then with alkaline phosphatase (10 jlg, 37°C, I hr). The nucleosides were thus separated using O.IM triethylammonium acetate buffer (PH 7.0) and aceton itrile. A gradient elution was done using 6% buffer B over 20 min . After separation , quantification was made at 260 nm on the basis of peak areas and exti nction coeffi cient of the nucleosides (C260 : dA, 15400: dG , J 1700; dC, 7300 and dT, 8800).
Acknowledgement Financial ass istance from the Department of
Science & Technology, New Delhi is sincerely acknow ledged.
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