Automated RNA-synthesis with photocleavable sugar and nucleobase protecting groups

Article abstract of DOI:10.1055/s-1999-3098

A new synthetic method for the N-alkyloxycarbonylation of adenine and guanine nucleosides was developed and used for the preparation of RNA- phosphoramidites carrying photolabile sugar and nucleobase protecting groups. From these building blocks, a heptam

Full text of DOI:10.1055/s-1999-3098

930
LETTER
Automated RNA-Synthesis with Photocleavable Sugar and Nucleobase
Protecting Groups
Alfred Stutz, Stefan Pitsch*
Organisch-Chemisches Laboratorium der Eidgenössischen Technischen Hochschule, Universitätstrasse 16, CH-8092 Zürich, Switzerland
Fax +41(1)6321136
Received 29 January 1999
ester-linkage towards basic hydrolysis at pH-values,
Abstract: A new synthetic method for the N-alkyloxycarbonylation
where the enzymatic ligation step is carried out (t1/2 (pH
of adenine and guanine nucleosides was developed and used for the
7.5) = 22 min.).5
preparation of RNA-phosphoramidites carrying photolabile sugar
and nucleobase protecting groups. From these building blocks, a
heptameric oligoribonucleotide was prepared by automated synthe-
sis, followed by detachment from the solid support and photolytic
concept by excluding all enzymatic steps. We are plan-
deprotection under mild conditions. The presented strategy allows a
simple preparation of 3'-O-aminoacylated RNA-sequences.
Our retrosynthetic scheme for the preparation of 3’-O-
aminoacylated t-RNA analogues differs from the known
ning to attach aminoacylated RNA-fragments to the 3’-
end of truncated, chemically synthesized t-RNAs by
Key words: phosphoramidites, base-protecting groups, RNA-syn-
thesis, photolabile protection, aminoacylation
chemical ligation.8 The non-enzymatic ligation needs a
template which is provided by the 5’-region of the truncat-
ed t-RNA; its 3’-end serves thereby as primer. This dis-
connection requires the synthesis of an aminoacylated
A severe limitation in the chemical synthesis of oligonu-
riboheptanucleotide which is able to form at least three
cleotide analogues is the requirement to remove the com-
base-pairs during ligation.
monly used acyl-type nucleobase protecting groups with
Herein, we report a synthesis of novel phosphoramidite
building blocks which allow the synthesis of fully or par-
tially protected oligoribonucleotides that are deprotected
under very mild conditions and can serve as precursors for
the preparation of labile RNA-conjugates, such as 3’-ami-
noacylated oligoribonucleotides.9
strong nucleophiles such as NH3 or MeNH2. For instance,
some of the naturally occurring modified nucleosides, e.g.
pseudouridine and dihydrouridine are not stable under the
conditions required for deprotection of the standard base
protecting groups benzoyl (for cytidine and adenosine)
and isobutyryl (for guanosine). In this context, more labile
base protecting groups such as phenoxyacetyl have been
developed and extend the range of tolerated modifications
significantly.1 Another approach involves protection
groups such as allyloxycarbonyl which are removed under
nonnucleophilic conditions by the assistance of Pd0-com-
plexes.2
As starting materials for the introduction of the photo-
labile N-[(2-nitrobenzyl)oxy]carbonyl base protecting
groups we used 5’-O-dimethoxytritylated, 2’-O-[(2-ni-
trobenzyl)oxy]methylated nucleosides 5-7, which we pre-
pared from the corresponding base-acetylated compounds
1-3, employing a method described in our earlier publica-
tions.3,10 After removal of the acetyl base-protecting
groups of 5-7 by short treatment with MeNH2 in EtOH, in-
termediates 9-11 were obtained in quantitative yield
(Scheme 1a).
One of the mildest deprotection is offered by photolytic
removal of appropriate protecting groups. We had earlier
developed an efficient method for the synthesis of RNA
phosphoramidites carrying the photolabile 2’-O-[(2-ni-
trobenzyl)oxy]methyl sugar protecting group, which can
be removed under mild, weakly acidic conditions.3 In the
above mentioned context, we now wanted to extend the
scope of RNA-synthesis by preparing phosphoramidite
building blocks with photocleavable sugar and base pro-
tecting groups.
Specifically, we were interested in a general, nonenzy-
matic synthesis of 3’-O-aminoacylated t-RNA analogues
which allow the biosynthetic, ribosome-mediated incor-
poration of unnatural amino acids into proteins.4,5 So far,
such aminoacylated t-RNA analogues were synthesized
by enzymatic ligation of truncated t-RNAs with 3’-O-ami-
noacylated dimers. The latter compounds were prepared
by condensation of a weekly activated, N-protected
amino acid with a non- or partially protected ribodinu-
cleotide.4,5,6,7 A severe limitation is the sensitivity of the
Scheme 1a a: c(1-4) = 0.25 M, 1.1 eq. Bu2SnCl2, 4 eq. EtN(iPr)2 in
(ClCH2)2, 25 °C, 60 min; then 70 °C, addition of 1.1 - 1.6 eq. [(2-ni-
trobenzyl)oxy] methyl chloride3, 30 min, extr. (aq. NaHCO3/CH2Cl2),
SiO2-chrom. (5,6,8: EtOAc/hexane, 7: CH2Cl2/MeOH); b: c(5-
7) = 0.15 M in MeNH2 (8 M in EtOH), 25 °C, 30 min, evaporation.
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LETTER
Automated RNA-Synthesis with Photocleavable Sugar and Nucleobase Protecting Groups
931
Treatment of nucleosides 10 and 11 with Ac2O/DMAP in
pyridine led to a quantitative acetylation of the 3'-O-posi-
tion. After extractive workup these intermediates were
converted into the N-[(2-nitrobenzyl)oxy]carbonylated
derivatives 13 and 14 by subsequent treatment with
COCl2/DMAP and 2-nitrobenzyl alcohol/NEt3, respec-
tively, in CH2Cl2/pyridine. After extraction, selective re-
moval of the intermediate 3'-O-acetyl protecting groups
was achieved with NaOH in THF/MeOH/H2O and led, af-
ter extraction and purification by silica gel chromatogra-
phy, to the isolation of nucleosides 15 and 16 in 75% and
70% yield, respectively (Scheme 1b).
Following standard procedures, the protected nucleosides
8, 12, 15 and 16 were converted into the phosphoramidite
building blocks 17-20, which were suitable for the synthe-
sis of the corresponding oligonucleotides with a DNA
synthesizer. The solid support 21 loaded with the adenine
nucleoside was synthesized using a slightly modified,
known protocol by first preparing its succinic acid
monoester and then immobilizing it on aminoalkyl-
functionalized CPG-material (Scheme 1c).
Scheme 1b a: c(9) = 0.5 M in (ClCH2)2, 2 eq. [(2-nitrobenzyl)oxy]
chloroformate11, 5 eq. aq. Na2CO3 (1 M), 25 °C, 60 min, extr. (aq.
NaHCO3/CH2Cl2), SiO2-chrom. (EtOAc/hexane); b: c(10,11) = 0.25
M, 0.1 eq. DMAP, 1.2 eq. Ac2O in pyridine, 4 °C, 15 min, extr. (aq.
NaHCO3/CH2Cl2), evaporation; then dissolved in CH2Cl2 (0.5 M),
slow addition to: 1.5 eq. COCl2 (1.9 M in toluene), 0.1 eq. DMAP in
CH2Cl2/pyridine 5:1 (final c = 0.15 M), 25 °C, 10 min, then 4 (for 10)
or 6 eq. (for 11) 2-nitrobenzyl alcohol/NEt3 (1:1), 25 °C, overnight,
extr. (aq citric acid, aq. NaHCO3/CH2Cl2); c: c(13,14) = 0.03 M, 0.1
M NaOH in THF/MeOH/H2O (5:4:1), 25 °C, 2.5 min, extr. (aq.
NaHCO3/CH2Cl2), SiO2-chrom. (15: EtOAc/hexane, 16: CH2Cl2/Me-
OH).
Scheme 1c a: c(12,15,16,8) = 0.25 M, 1.3 eq. (2-cyanoethyl)(N,N-
diisopropylamino)chlorophosphite, 2.5 eq. EtN(iPr)2 in CH2Cl2,
25 °C, overnight, SiO2-chrom. (17,18,20: EtOAc/hexane, 19: EtOAc/
hexane/EtOH); b: c(21) = 0.25 M, 1 eq. succinic anhydride, 1.5 eq.
DMAP in CH2Cl2, 25 °C, 30 min, extr. (aq. citric acid/CH2Cl2), eva-
poration, then CPG (1-4 g/0.1 mmol), 3 eq. BOP, 3 eq. N-methyl imi-
dazole in CH3CN (c = 0.02 M), filtration.
Carbamate-formation of the cytosine-nucleoside 9 with
[(2-nitrobenzyl)oxy] chloroformate11 in a biphasic mix-
ture of aqueous Na2CO3/dichloroethane at 25 °C gave the
N4-protected derivative 12 in 85% yield. No conversion
was observed by applying the same reaction conditions to
the adenine- and the guanine-nucleosides 10 and 11, re-
spectively. Attempts to activate the reagent (in the ab-
sence of water) towards reaction with the nucleobase-
moieties by addition of pyridine, DMAP, Ag+-salts or
heating the reaction mixtures failed completely (some-
times partial formation of the 3'-O-carbonates occurred).
Instead, efficient decomposition of the reagent [(2-ni-
trobenzyl)oxy] chloroformate to (2-nitrobenzyl)chloride
was observed.12 In contrast, good yields of the base pro-
tected adenine and guanine nucleosides 15 and 16 were
obtained by the following reaction sequence, which was
carried out without purification of the intermediates.13
All mentioned compounds have been characterized by
NMR-spectroscopy and FAB MS-spectrometry and all
data unambiguously agree with the proposed structures.
Some of the relevant spectroscopic data are presented in
Table 1. All FAB-MS-spectra (pos. mode, glycerol as ma-
trix) showed beside the peak of the dimethoxytrityl-cation
(m/z = 303) the product peaks as most intense signals. 1H
NMR spectra of nucleotides 12-19 showed the typical sig-
nals of an additional 2-nitrobenzyl-group in the aromatic
region (data not shown) and the signals of an additional
CH2-group around d = 5.6 ppm, which could be attributed
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932
A. Stutz, S. Pitsch
LETTER
to the benzylic hydrogens. The 1H and the 13C NMR spec- detritylation assay were >99%. The removal of the phos-
tra showed that the signals of the nucleobase-moieties un- phate-protecting groups and the cleavage from the solid
derwent no significant changes upon exchange of the base support was carried out at 25° with 0.1 M NaOH in THF/
protecting groups, which illustrated the correct position of MeOH/H2O for a period of only 1.5 min followed by neu-
the [(2-nitrobenzyl)oxy]carbonyl groups.
tralization with aqueous Tris-HCl buffer (pH 7.4), which
resulted in complete precipitation of the water-insoluble
product 23 (Scheme 2).
The assembly of the heptameric oligoribonucleotide 22
from the phosphoramidite building blocks 17-19 and the
solid support 21 was carried out in the “trityl-on” mode on Prior to this reaction, the stability of the N-[(2-nitroben-
a “Gene Assembler” (Pharmacia) using a protocol which zyl)oxy]carbonylated ribonucleotides was investigated
we had developed in a different context10, but omitting under a variety of potential detachment conditions (Table
the capping step. The individual coupling yields by 2). In the presence of MeNH2, all three nucleoside
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LETTER
Automated RNA-Synthesis with Photocleavable Sugar and Nucleobase Protecting Groups
933
derivatives were degraded completely in a few minutes. In of this product-sequence 24 was unambiguously con-
contrast, they were stable for at least 10 h in NaOH/THF/ firmed by the following data. Its UV-spectrum was typical
H2O. In NH3/EtOH and NaOH/THF/MeOH/H2O they for an oligonucleotide. Its chromatographic behaviour on
showed an intermediate stability. Remarkable is the rela- two different HPLC-systems15 was identical with the cor-
tive difference in stability of the guanine derivative 16, responding authentic RNA-sequence16 as shown by coin-
which is the most stable in NH3/EtOH, but the least stable jection experiments. Furthermore, its MALDI-TOF
in NaOH/THF/MeOH/H2O.14
spectrum17 showed the expected mass of 2202 amu.
In Figure 1, the HPLC-trace of the crude, still protected
oligonucleotide 23 is presented. It confirms the good cou-
pling yields observed by the detritylation assay. Apart
from the dominant peak of the product, only small peaks
of earlier eluting, shorter or partially deprotected com-
pounds are visible. From the reaction mixture, sequence
23 was obtained in a yield of 50% after purification by
HPLC (Figure 2).
Figure 2 RP-HPLC of a): purified sequence 23, b) reaction mixture
after 2 h irradiation (Scheme 2, step c). HPLC-conditions: see Figure
1 (impurities marked with an asterisk are from the buffer).
In conclusion, a synthetic method for the introduction of
photocleavable base protecting groups into RNA-phos-
phoramidites was developed. The presented general meth-
od can potentially be used for the introduction of any
desired carbamoyl-group into ribo- and 2’-deoxyribonu-
cleosides, because they can be formed on the nucleobase
without need to preassemble the corresponding chlorofor-
mates (which are often sensitive by nature). Meanwhile,
we succeeded in efficiently transforming the intermediate,
protected heptamer 23 into a 3’-O-aminoacylated deriva-
tive, which subsequently could be deprotected under the
presented conditions18 and are now about to explore the
chemical ligation with truncated t-RNAs.
Figure 1 RP-HPLC of crude reaction mixture obtained after assem-
bly and detachment from the solid support (Scheme 2, steps a and
b). HPLC-conditions: Nucleosil 5C18 (5 x 200 mm), flow 1 ml/min,
detection at 260 nm, elution at 40 °C; eluent A: 0.1 M (Et3NH)OAc
in H2O (pH 8.0), eluent B: CH3CN.
The purified sequence 23 was subjected to photolysis in
aqueous sodium citrate buffer (pH 3.5)/tBuOH using
Pyrex-filtered light from a mercury-lamp. Additionally,
complete detritylation occurred under these acidic condi-
tions (Scheme 2). In Figure 2 HPLC traces of the starting
material 23 and the reaction mixture obtained after 2 h ir-
radiation are shown. They reveal clean formation of a
new, much earlier eluting ( = much more polar) product,
which was isolated by HPLC in 55% yield. The structure
Acknowledgement
We thank Patrick A. Weiss (Xeragon AG, Switzerland) for provi-
ding us with reagents, nucleoside precursors and the authentic
RNA-sequence. The ETH Zürich Research Council and the Alfred
Werner Foundation are gratefully acknowledged for financial sup-
port.
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934
A. Stutz, S. Pitsch
LETTER
of CO2 and chloride. The reagent is stable in biphasic reaction
mixtures, where chloride ions are extracted into the aqueous
phase (as realized in the preparation of 12 from 9).
(13) Preparation of 15 from 6: A solution of 1.175 g (1.51 mmol) 6
in 10 ml MeNH2-solution (8 M in EtOH) was kept 15 min at
25 °C. After evaporation and drying (5 h, 0.01 torr), the
product was dissolved in 6 ml pyridine, treated with 18 mg
(0.15 mmol) DMAP and 184 mg (1.8 mmol) Ac2O and stirred
15 min at 4 °C. After extraction (CH2Cl2/NaHCO3), drying
(MgSO4), evaporation and drying (15 h, 0.01 torr), the product
was dissolved in 3 ml CH2Cl2 and added during 20 min to a
suspension obtained from 1.17 ml (2.25 mmol) COCl2-
solution (1.9 M in toluene) / 18 mg (0.15 mmol) DMAP / 10
ml CH2Cl2 / 2 ml pyridine. After 10 min at 25 °C, a solution
of 915 mg (6 mmol) 2-nitrobenzyl alcohol and 607 mg (6
mmol) Et3N in 3 ml CH2Cl2 was added. The mixture was
stirred 15 h at 25 °C, extracted (CH2Cl2/10% aqueous citric
acid solution, then NaHCO3), dried (MgSO4) and evaporated.
This crude product was dissolved in 50 ml 0.1 M NaOH
solution (THF/MeOH/H2O 5:4:1), kept for 2.5 min at rt.,
extracted (CH2Cl2/NaHCO3), dried (MgSO4) and evaporated.
The residue was subjected to chromatography (30 g SiO2,
EtOAc/hexane (+2% NEt3) 1:1→ 9:1):1.00 g (75%) 15 as off-
white, solid foam.
References and Notes
(1) Gasparutto, D.; Livache, T; Bazin, H.; Duplaa, A.-M.; Guy,
A.; Khorlin, A.; Molko, D.; Roget, A.; Téoule, R. Nucl. Acids
Res 1992, 20, 5159.
(2) Hayakawa, Y.; Kato, H.; Uchiyama, H. K.; Noyori, R. J. Org.
Chem. 1986, 51, 2402. Hayakawa, Y.; Wakabayashi, S.; Kato,
H.; Noyori, R. J. Am. Chem. Soc. 1990, 112, 1691. Hayakawa,
Y.; Hirose, M.; Hayakawa, M.; Noyori, R. J. Org. Chem.
1995, 60, 925.
(3) Pitsch, S. Helv. Chim. Acta 1997, 80, 2286.
(4) Noren, C. J.; Anthony-Cahill, S. J.; Griffith, M. C.; Schultz, P.
G. Science 1989, 244, 182.
(5) Robertson, S. A.; Ellman, J. A.; Schultz, P. G. J. Am. Chem.
Soc. 1991, 113, 2722.
(6) Heckler, T. G.; Chang, L.; Zama Y.; Naka, T.; Chorghade, M.
S.; Hecht, S. M. Biochemistry 1984, 23, 1468. Baldini, G.;
Martoglio B.; Schachenmann, A.; Zugliani, C.; Brunner, J.
Biochemistry 1988, 27, 7951.
(7) The synthesis of longer conjugates is not reported.
(8) The chemical synthesis of the truncated t-RNAs allows the
incorporation of natural or unnatural modified nucleosides
and would extend the scope of the method.
(9) The preparation of a trimeric aminoacylated RNA-sequence
from a partially protected RNA-precursor has been reported.
Thereby, Fmoc ([(9-fluorenyl)methoxy]carbonyl), MTHP
(4-methoxytetrahydropyran-4-yl), BPOC ([2-(4-biphenylyl)-
2-propyloxy]carbonyl were employed as protecting groups for
the nucleobases, the sugar-moieties and the amino acid,
respectively. The tedious, stepwise deprotection procedure
yielded only 20-30%: Hagen, M. D.; Scalfi-Happ, C.; Happ,
E.; Chladek, S. J. J. Org. Chem. 1988, 53, 5040. Hagen, M. D.;
Chladek, S. J. J. Org. Chem. 1989, 54, 3189.
(14) These results are an indication of different mechanisms
operating under different reaction conditions. In the presence
of a strong base (such as NaOMe) formation of an isocyanate
species (elimination of 2-nitrobenzyl alcohol from the [(2-
nitrobenzyl)oxy]carbonyl-moiety) seems to precede the attack
of a nucleophile. Weak bases, but relatively strong
nucleophiles (such as NH3), seem to react by addition to the
carbonyl group, followed by elimination of 2-nitrobenzyl
alcohol.
(10) Wu, X.; Pitsch, S. Nucleic Acids Res. 1998, 26, 4315.
(11) At 4 °C, 10.4 ml of a COCl2-solution (1.9 M in toluene, 20
mmol) was treated slowly with a solution of 3.06 g (20 mmol)
2-nitrobenzyl alcohol in 20 ml THF. After 30 min at 4 °C and
2.5 h at 25 °C, it was evaporated (< 40 °C) and dried (2 h, 0.01
torr): crude [(2-nitrobenzyl)oxy] chloroformate as orange oil
(90% according to 1H NMR). IR (CHCl3): 3009, 1776, 1530,
1347, 1142; 1H NMR (300 MHz, CDCl3): d = 5.75 (s, 2H),
7.25 (m, 1H), 7.55-7.74 (m, 2H), 8.19 (d, 1H); 13C NMR (75
MHz, CDCl3): d = 69.6 (CH2), 124.7, 129.3, 130.0, 134.5 (4
CH), 138.1, 147.5, 150.8 (3 C).
(15) The coinjections were carried out on a reversed-phase column
(see Figure 1, 0 → 20% B (30 min) and on an ion-exchange
column (Nucleogel SAX, 10 mM phosphate (pH 6) → 10 mM
phosphate + 0.2 M KCl (30 min).
(16) Obtained from XERAGON AG (Zürich, Switzerland)
(17) Pieles, U.; Zürcher, W.; Schär, M.; Moser, H. E. Nucleic Acids
Res. 1993, 21, 3191.
(18) Stutz, A.; Kuntze, S.; Pitsch, S. in preparation.
(12) This decomposition occurred very fast in polar, aprotic
solvents, such as DMF, pyridine or N,N-dimethylacetamide; it
also occurred during attempts to purify the reagent by
distillation. The mechanism most probably involves attack of
a chloride anion at the benzylic position with concomitant loss
Article Identifier:
1437-2096,E;1999,0,S1,0930,0934,ftx,en;W04299ST.pdf
Synlett 1999, S1, 930–934 ISSN 0936-5214 © Thieme Stuttgart · New York