Synthesis and structure assignments of amide protected nucleosides

Volume 16 Number 8 1988

Nucleic Acids Research

Synthesis and structure assignments of amide protected nucleosides and their use as

phosphoramidites in deoxyoligonucleotide synthesis Matthias Mag and Joachim W.Engels Institut fiir Organische Chemie, Johann Wolfang Goethe-Universitit Frankfurt, Niederurseler Hang, 6000 Frankfurt am Main 50, FRG Received January 7, 1988; Revised and Accepted March 18, 1988

ABSTRACT The syntheses of several amide protected deoxyguanosine- as well as thymidine nucleosides are described. These compounds were synthesized according to the Mitsunobu reaction and Michael addition. In contradiction to previous studies we have discovered that the Michael addition gives only products derived from N-alkylation. The occurence of N- or 0-alkylation was assigned by means of two dimensional IH,13C-COLOC-NMR spectroscopy. Further, we have found that the Mitsunobu reaction used for the protection of the amide function of dG is limited to alcohols without acidic hydrogen atoms. Amide protected phosphoramidites (15, 16) were used for the preparation of deoxyoligonucleotides with a large number of guanine and thymine bases using two different coupling times. We have shown that there is no experimentally detectable difference in the quality of the products if the starting monomer is amide protected or not.

INTRODUICTION Thymine and guanine are known to undergo base modification during oligo(deoxy)nucleotide synthesis by the phosphotriester

approach.'-5 Reese4

5 and Hata3 7 for example were able to show that guanosine derivatives were sulfonylated with condensing reagents at 06_, while thymine was vulnerable to sulfonylation at the 04-position. There are in addition reports of phosphor-

ylation by activated nucleotides. Recent studies from Jones8 and Ogilvie9'10 have shown that modifications of thymine and guanine also occurred during the phosphoramidite procedure. Therefore, protection of the amide function in guanine and thymine residues seems to be an obvious necessity also for the phosphite method. To prevent base modification and depurination of the guanine and thymine moiety various protecting groups are described by several authors.'-12 All these protecting groups require for their introduction fully protected sugar residues. This leads to © IRL Press Limited, Oxford, England.

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Nucleic Acids Research for introducing appropriate multi-step procedures aride protecting groups which is very time consuming. Here we report the results of our investigation for the preparation of amide protected phosphoramidites and their usefulness in automated solid phase deoxyoligonucleotide synthesis.

RESULTS AND DISCUSSION Michael Addition As previously described" we tried t;- overcome this probler of multi-step reactions. Therefore we prepared several -rotected thymidine derivatives by a one-flask reaction acrording to a The protected thymidines base catalyzed Michael type additionl 4 the Michael acceptors to a pyridine were obtained by adding solution of thymidine or DMTr-thym.idine in the presence of tetrabutylamrmmonium hydroxide (FIGURE 1.). The yield of the reaction of acrylonitrile with thyridine is modest (45%, product mixture) whereas the yield with DMTr-thymidine as starting material is nearly quantitative. All other derivatives were or DMTrfrom thymidine obtained in high yield starting thymidine, respectively. Whereas acrylonitrile and phenylvinylsulfone are commercially available we had to synthesize the 4.

0

N-CH2CH2R2

R1FO

0

N

NH

-

HOO

R10

OCH2CH2R2 HO

a

COMP. R1

R2

1 2 3 4 5

CN S02 C6 Hs CN S02 C6 H5 SO2 C6 4 NO2

H H DMTr DMTr H

FIGURE 1.

Scheme

YIELD

for

45% 80%

85% 88% 93%

the preparation of N-protected

thymidine

derivatives using a base catalyzed Michael type addition. 3526

Nucleic Acids Research (the nitrophenylvinylsulfone in a five step procedure preparation protocol is available by the authors on request). Tesser and coworkers'3 inferred from infra-red spectra of the putative 04 -(4-nitrophenylsulfonylethyl)-thymidine derivative that alkylation occurred at the 04- and not at the N3position. This result stands in contradiction with our attempts for the cleavage of the cyanoethyl- and phenylsulfonylethylgroup. To fully deprotect either of the derivatives 1 or 2 treatment with conc. ammonia for one week at 55'C was necessary. The deblocking of 5 can be easily achieved by aqueous ammonia at 55 C within 2.5 hours. Based on these results we argued that the Michael type reaction proceeds in direction of pathway I to give the Nalkylated product (FIGURE 1.) In order to furnish evidence for the structure of the N- or 0-alkylated products we used 2-D-13CNMR techniques. Two dimensional heteronuclear chemical-shift correlation spectroscopy (H,X-COSY)16 has proven to be a good way for assigning which proton is bonded to which 13C-nucleus, whereas the experiment is not effective for determining long range couplings. Simple scalar coupled proton spin systems are easily identified by two-dimensional homonuclear chemical-shift correlation ('H,'H-COSY)17 or 'H-Relayed-'H,'H-COSY'8 experiments. But spin systems which are separated by hetero- or quaternary C-atoms are much more difficult to assign. We attempted to solve our assignments by means of the COrrelation spectroscopy via LOng range Couplings (COLOC)'9. In the conventional H,X-COSY the evolution period and the polarization transfer are separated. There is a considerable magnetization loss due to proton relaxation in tL and in the delay A, and carbon relaxation in the delay A2 . In the COLOC experiment the evolution is included in the Ai delay. It reduces the critical time in which the proton transverse relaxation t2 takes place from ti+ AL to only A,. This is possible if the half of the evolution time is smaller than A,. It is thus possible to induce 'H,"3C-couplings in a high selectivity over three bonds (3JCH). In practice, it is of great interest to determine threebond 'H,'3C connectivities. It allows the "bridging" of nonprotonated carbons, and nuclei other than carbon, for example oxygen and nitrogen. All scalar coupled proton spin systems were identified from 'H,'H-COSY-NMR spectra and the non quarternary C-atoms were 3527

Nucleic Acids Research

_o-

6 (ppm1

300MHz-1H,13C-COLOC-NMR of 04-(4-nitrophenylethyl)thymdine (6), b) 300MHz-'H,13C-COLOC-NMR of N3-(4-nitrophenylsulfonylethyl)-thymidine (5), both spectra were recorded in D6-

FIGURE 2. a)

DMSO at 298K in 0,25 M solutions.

previously determined via 'H,13C-COSY. The distinction between the two possible alkylated forms (N- or 0-alkyl) can be easily achieved by the heteronuclear 2D-COLOC-NMR experiment in the following way: For 0-alkylation: The NMR-spectrum should show only one signal for the C-4/a-H connectivity. For N-alkylation: In order to prove our assumption there should be two connectivities observable, and C-4/a-H C-2/a-H respectively. FIGURE 2b. shows the COLOC-NMR of 5. Both possible resonances of the N-alkylated form are detectable. For the compounds 3 and 4 the results were similar, they also have the N-alkylation pattern. To check if the COLOC experiment allowes

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Nucleic Acids Research

0

N AN

HO

NH2

HO R

ICOMP.

RI

R2

11

7

H

C6H4NO2

i8

OH

C6 H4NO2

FIGURE 3. Scheme for the preparation sulfonylethyl)-2 -(deoxy)guanosine.

of

N1-(4-nitrophenyl-

0-alkylated nucleosides we have assigned the to determine structure of 6. The spectrum of 6 is shown in FIGURE 2a. Only the C-4/a-H resonance is detectable. The absence of the C-2/a-H connectivity indicates the 0-alkyl structure of 6. In the same manner as' described for the thymidine moiety guanosine and deoxyguanosine were reacted with 4-nitrophenylbetween order to distinguish vinylsulfone20 (FIGURE 3.). In the two possible regioisomers structural assignments were also done by the two dimensional heteronuclear COLOC-NMR experiment. As in the case of thymidine N-alkylation occurs at the guanine moiety. The two possible 3JcH-couplings (C-2/a-H and C-6/a-H) in the COLOC-NMR for compound 7 and 8, are detectable respectively. These results are in accordance with those described in the literature2l122 for amides and lactams. We have found that N-alkylation occurs under basic conditions also for nucleoside lactam functions. This regioselectivity can be associated with formation of the carboxamide anion in which alkylation occurs directly (the course of the reaction is controlled by the greater nucleophilicity of the nitrogen center). We next explored the use of this Michael-type reaction to prepare the amide protected cyanoethyl derivative. But the 3529

Nucleic Acids Research 'H/ppm COM. POS. a-CH2

3 4 5 6 7 8

N3 N3 N3 04

N'

N'

a-C

ESTIM. a-C

'3C/ppm C2

C4

C5

C6

110,3 110,2 108,5 102,9

134,3 134,3 135,0 141,2

36,6 35,0 34,6 66,3

33,1+6 33,5+3 33,1+6 66,8+1

149,5

115,8

149,4

115,9

156,1 156,2

34,7 34,7

33,2+6 33,2+6

4,26 4,27 4,12 4,54

150,5 150,2 150,0 154,7

163,0 163,0 162,3 169,4

4,34 4,31

153,5 153,5

from lactam 1. 'H-chemical shift data of the a-CH2 protected nucleosides. The 13C-chemical shifts of the important C-atoms are shown. For the 13 C o-C also the estimated data are given. TABLE

reaction of acrylonitrile with deoxyguanosine gave no alkylation product. Phenylvinylsulfone also failed as a Michael acceptor. Furthermore our attempts for the protection of the exocyclic amino function of 7 with isobutyrylchloride were negative. The use of N2-isobutyryl-2'-deoxyguanosine as starting material for the reaction with 4-nitrophenylvinylsulfone was also unsuccessBoth reactions probably did not occur due to sterical ful. hindrance at the reaction centers. The '3C-NMR spectra of the alkylated products 3-5 are very similar to each other (TABLE 1). This indicates that they have the same alkylation pattern. Whereas the proton shift difference of the N-CH2 methylene group from 5 and the O-CH2 from 6 is only 0,4ppm, the 13C chemical shifts vary significantly (5o-CH2-5NCH2=31ppm) owing to the different electronegativity of nitrogen All other 13C resonances of 3-5 in TABLE 1 differ and oxygen. for about 4-8 ppm compared with those from 6. The 13C spectra of The proton chemical shift 7 and 8 are nearly identical. Nland (9, -CH2: 4,80 ppm; 10, 06_ between 8) (7, difference -CH2: 4,81 ppm) methylene groups is 0,5 ppm. As in the case of the N-alkyl thymidine derivatives the 13C chemical shift of the a-CHz group of 7 and 8 is in the region of circa 35 ppm. In experimental results we estimated the 13C addition to the chemical shifts for the compounds 3-5, 7 and 8 (TABLE 1). They were computer generated and calculated with the help of known 13 C chemical shift data which are included in a 13 C-database2 3 Our estimated data are in total agreement with the experimental data. The empirical experimental and estimated data demonstrate the possibility to distinguish between the N-CH2R or O-CH2R

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Nucleic Acids Research COMP. R YIELD _ 9 80% CE H NO2 10 S-Cb H4 NO02 50% -SOa Cb H4 NO2 0% -- SOZ C6 H.5 0

NH N

HO

-CN0*

lOa* SOC6H4NO,

93%

OCH2CH2R

0

JJ .

HO

N

ii

v f

NNb

NNHib

~~~~~~~~~~9-10a HO

FIGURE 4. Scheme for the preparation of 06-potected deoxyguanosine derivatives by using the transient protection/ Mitsunobu-alkylation sequence. Reagents: i, trimethylsilyl-imidazole in dioxane; ii, RCH2CH2OH, triphenyl phosphine and diethylazodicarboxylate; iii, 1M pyridinium hydrofluoride in pyridine. *: compound 10a was yielded after oxidation of 10 with NaIO4. alkylated derivatives with the one dimensional 3C-NMR spectrum. Experimental and estimated data show that the 13C chemical shift from N-CH2 always is in the area of 35 ppm whereas the O-CH2 signal appears at circa 66 ppm. The Mitsunobu Reaction For the preparation of 06-protected guanosine derivatives we expected that the Mitsunobu reaction24 offers a good chance for the introduction of several protecting groups25. The nucleosides 9 and 10 were synthesized with the Mitsunobu reaction using the transient protection method of Jones and coworkers26 (FIGURE 4.). Therefore N2-isobutyryldeoxyguanosine was reacted in dioxane with N-(trimethylsilyl)-imidazol to give the 3',5'-Osiliyl protected derivative. After addition of 3.5eq each of triphenylphosphine, the appropiate alcohol and diethyl azodicarboxylate 06-alkylation occured during one hour. Using this transient protection/Mitsunobu alkylation sequence only the compounds 9 and 10 were obtained. Compound 10 was also prepared by the sulfonylation/displacement route from Jones27 and was found to be identical with the compound prepared by the Mitsunobu reaction. Due to the electron withdrawing groups of 2-(phenylsulfonyl)-ethanol, 2-(4-nitrophenylsulfonyl)-ethanol, and 3-hydroxypropionitrile (FIGURE 4.) the proposed alkoxyintermediate2 8 undergoes 1-elimination much faster phosphorane than the desired 06-alkylation. Furthermore the dehydration of alcohols with acidic adjacent hydrogens under Mitsunobu conditions is a well known phenomenon24 28.

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Nucleic Acids Research

Bx

HO

Ho

HO

| 15-

11-14

p

NCCH2CH2

11 12 13 14 15

Bx

DMTrO

DMTrO

0

B

X

T

N3 -CH2 CH2 S02 R N' -CH2 CH2 S02 R 06 -CH2 CH2R

dGt b dGi b dGi b T

06-CH2zCH2SOR ITP -CH2 CH2 SO2 R

-

N B

16

17 18 19 R=

la

X

C6-CH2CH2R dC.tb 06 -CH2 CH2 SOR dGi b T

dGlb -C6 H4 NO2|

of fully protected phosphoraTnidites. FIGURE 5. Preparation Reagents: i, DMTr-C1, TEA, DMAP in pyridine; ii, tetrazole and N2ib: 2-cyanoethoxy-bis-(N,N-diisopropyl-amino)-phosphine. isobutyryl

The 4-nitrophenylethyl group can be cleaved by 1,8 diazabicyclo(5.4.0)undec-7-ene in aprotic solvents such as pyridine within two hours at room temperature. The deprotection of the 4nitrophenylthioethyl derivatives is more complicated because it is necsessary to oxidize this group before the cleavage by ammonia can be accomplished (2hours, 55°C). The oxidation2 9 was performed with NaIO4 at r.t. yielding compound 10a. Preparation of fully protected phosphoramidites The compounds 5, 7, 9 and 10a were dimethoxytritylated30 (11-14) as shown in FIGURE 5. and converted to the 3-cyanoethoxy Our phosphoramidites (15-19) according to known procedures3' unsuccessful were 12 compound phosphitylating for attempts we failed to isolate the expected products from the complex reaction mixture. Deoxyoliaonucleotide Synthesis To test the efficiency of fully protected phosphoramidites in automated oligonucleotide synthesis we prepared several tetracosanucleotides. For the protection of the guanine moiety we used the 4-nitrophenylethyl group whereas p-nitrophenylsulfonylethyl was used in the case of the aglycon of thymidine. because

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Nucleic Acids Research REAGENT

FUNCTION

3% TCA/CH2Cl2 Activated amidites

Detritylation to column Condensation to column Capping to column Oxidation

Ac2O/DMAP/THF I2/H20/luthidine

CYCLE I

CYCLE II

50s

loos

5s 30s los 4s 15s 30s

5s 300s

los 120s 23s 60s

TABLE 2. Synthesis cycles used for automated deoxyoligonucleotide synthesis. The same oligonucleotides were also synthesized without amide protected phosphoramidites for comparison of the results. For elongation of the oligonucleotide chain, we have used two different synthesis cycles as shown in TABLE 2. After ammonia deprotection the crude mixtures were directly loaded on a lmm 12%/7M urea polyacrylamide gel and visualized by UV-shadowing. TABLE 3. shows the average yield per step and the overall yield which were both determined by trityl colour quantitation at 498nm. FIGURE 6. shows the results of the oligonucleotide syntheses. In the case of the oligothymidine syntheses the results were very good for the sequences prepared with the phosphoramidite 15 for both synthesis cycles (lane 1: cycle II, coupling time 300s; lane 2: cycle I, 30s). Nearly the same results were obtained with amide unprotected thymidine phosphoramidite. The sequence which was performed with the synthesis

OLIGO NUMBER

SEQUENCE PREPARED

PHOSPHORAMIDITE

CYCLE USED

AVERAGE YIELD

1 2 3 4

T24 T24 T24 T24

15 15 18 18

II I I II

5 6 7 8 9

d(TG)12 d(TG)12 d(TGG)8 d(TG)12 d(TG)12

18, 18, 18, 15, 15,

99% 99% 99% 99% 94% 98% 94% 97% 97%

19 19 19 16 16

I II I II I

OVERALL YIELD 90 88 89 88 39 48 27 49 52

TABLE 3. Prepared sequences and average coupling yields of the syntheses with amide unprotected and protected diisopropylaminephosphoramidites. The average and overall yield was determined by trityl colour quantitation at 498nm.

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Nucleic Acids Research

FIGURE 6. 12% Polyacrylamide/7M urea electrophoresic gel of the deoxyoligonuclectides 1-9; 4650 Vh, 250 V

cycle I (lane 3, coupling time 30s) was also very good as expected for this pyrimidine sequence. Only one strongly abscrbing spot was detectable. In lane 4 only a little smear was present if the coupling time was increased to 300s (cycle II). As in the case of the oligothymidines only one strongly absorbing and well defined band was observed for the oligo-nucleotide 5 (dG content 50%) by using cycle I (coupling time 30s) . By increasing the coupling time to 300s (cycle II) the polyacrylamide gel shows a smear of faster moving products. This smear indicates some type of chain degradation which is documented in the literature9 I 0 If the dG content was increased up to 67% (lane 7) the gel shows well defined failure bands but the expected 24mer appeared as the dominant product. Compared to the d(TG)1} oligonucleotide 5 no better gel pattern could be observed from the syntheses which were performed with the 06-4-nitrophenylethyl protected deoxyguanosine phosphoramidite with either cycle I or II. The HPLC determination is in agreement with the polyacrylamide gel electrophoresis results as shown in FIGURE 7. for the oligodeoxynucleotides 3, 5 and 6. The chromatogram of crude 3 clearly demonstrates that this oligodeoxynucleotide appears to be a homogeneous material under the used reaction conditions 3534

i.

Nucleic Acids Research

--1'~.

I '-1 I - ----T I - I -I : . jKtS--- -II

A_

T

I

bX

1 IIII I4
- (30Onl, 0°C). Filtration and drying afforded the phosphoramidites (15-19) as white powders. 5' -O-Dimethoxytri tyl -Ne - (4-ni trophenylsul fonyl ethyl) -thymdine3 '- (2-cyanoethyl -N,N-diisopropylamido-phosphi te 15 Yield: 85%, M: 957,8, Rf : 0, 8 (CHCl3/MeOH, 9:1, v:v) , UV (MeOH): 255 nm (E=16900); 234 nm (E=22800), 31P-NMR (CDC1.C): 149,42 and 149,47 5 '-O-Dimethoxytrityl-ft -isobutyryl-06 - (4-nitrophenyl ethyl) -2'deoxyguanosine-3 '- (2-cyanoe thyl) -N,N-diisopropylamido-phosphi te 16 Yield:

89%, M: 989,1,

UV (MeOH):

270 nm (E-31200);

234 ni

(e=27900) , Rf: 0,62 and 0,69 (CH2Cl2/ethyl acetate/ triethylamine, 45:45:10, v:v) 31P-NMR (CDCl3): 149,3 and 149,4 5 '-O-Dime thoxytri tyl -N2 -isobutyryl _Q6 - (4-ni trophenylsulfoxyethyl) -2 '-deoxyguanosine-3 '- (2-cyanoe thyl) -N,N-diisopropyl ami dophosphite 17 Yield: 70%, M: 1036,6, Rf: 0,69 CHCla/MeOH, 9:1, v:v), 3IPNMR (CDC13): 144,7 and 144,9

ACKNOWLEDGEMENTS We would like to thank Rainer Schmidt for providing the 2cyanoethyl-bis-(N,N-diisopropylamino)-phosphine and the amide unprotected 2-cyanoethyl-N,N-diisopropylamino deoxynucleoside phosphoramidites. Furthermore we have to thank Ingeborg Claes for expert technical assistance by performing the oligonucleotide syntheses and Dr.G. Zimmermann for his great help by recording and interpretation of the COLOC-NMR spectra.

1. 2. 3. 4.

5. 6. 7.

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Sung, W.L. (1982) J. Org. Chem., 47, 3623 Sung , W.L. and Narang, S.A. (1982) Can. J. Chem., 60, 111 Daskalov, H.P.; Sekine, M . and Hata, T. (1980) Tetrahedron Lett., 21, 3899 Reese, C. B. and Ubasawa, A. (1980) Tetrahedron Lett., 21, 2265 Reese, C.B. and Skone, P.A. (1984) J. Chem. Soc.Perk. Trans. I, 1263 Reese, C.B. and Richards, K.H. (1985) Tetrahedron Lett., 26, 2245 Sekine, M.; Matsuzaki, J.; Satoh, M. and Hata, T. (1982) J. Org. Chem., 47, 571

Nucleic Acids Research 8. 9.

10.

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14.

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