Preparation of 1′-c deuterated synthons for RNA synthesis by H-phosphonate method aiming at two-dimensional NMR secondary structure studies

Article abstract of DOI:10.1135/cccc19960389

1′-C Deuterated H-phosphonate synthons were prepared via a 12-step procedure starting from [1′-²H]ribose. The procedure included nucleosidation of 1′-O-acetyl-2′,3′,5'-O-tribenzoyl[1′- ²H]ribose with appropriately protected nucleobases and preparation of nucleoside-H-phosphonates by slightly modified described procedures. The automated RNA synthesis of 5′-G* C*U*A*U*UUAU-3′ and 3′-AC* G*A*U*A*AAGU-5′ was performed on a Gene Assembler Plus DNA-synthesizer. These specifically deuterated oligoribonucleotides were subsequently compared with the corresponding nondeuterated sequences using 2D-NMR NOESY spectra. Specific deuterium incorporation resulted in the expected simplification of spectral pattern.

Full text of DOI:10.1135/cccc19960389

1’-C Deuterated Synthons for RNA Synthesis
389
PREPARATION OF 1′-C DEUTERATED SYNTHONS FOR RNA SYNTHESIS
BY H-PHOSPHONATE METHOD AIMING AT TWO-DIMENSIONAL NMR
SECONDARY STRUCTURE STUDIES
a
a
a
b
Lubos ARNOLD , Martina PRESSOVA , David SAMAN , Martin VOGTHERR
b
and Stefan LIMMER
a Institute of Organic Chemistry and Biochemistry,
Academy of Sciences of the Czech Republic, 166 10 Prague 6, Czech Republic
b Laboratorium für Biochemie,
Universität Bayreuth, D-95440 Bayreuth, Germany
Received July 19, 1995
Accepted September 21, 1995
1′-C Deuterated H-phosphonate synthons were prepared via a 12-step procedure starting from [1′-2H]ribose.
The procedure included nucleosidation of 1′-O-acetyl-2′,3′,5′-O-tribenzoyl[1′-2H]ribose with
appropriately protected nucleobases and preparation of nucleoside-H-phosphonates by
slightly modified described procedures. The automated RNA synthesis of 5′-G*C*U*A*U*UUAU-3′ and
3′-AC*G*A*U*A*AAGU-5′ was performed on a Gene Assembler Plus DNA-synthesizer. These spe-
cifically deuterated oligoribonucleotides were subsequently compared with the corresponding non-
deuterated sequences using 2D-NMR NOESY spectra. Specific deuterium incorporation resulted in
the expected simplification of spectral pattern.
Key words: [1′-2H]Ribose; Nucleoside-H-phosphonates; Oligoribonucleotides, deuterated.
The elucidation of the structure of RNA molecules with molecular weight of ca 8 000 Da
and more (about 25 nucleotides) using the standard 2D-NMR method is successful only
in rare cases1–5. Usually a severe overlap of cross peaks in the spectra prevents an
unequivocal assignment (and quantitative peak intensity determination) of proton reso-
nance signals. These difficulties can be overcome by using isotope-labelled nucleotides,
especially 13C-ribose- and 15N-base-labelled ones6–8, and employing three-dimensional
NMR methods. This approach is demanding with respect to both the production of the
labelled nucleotides and the NMR experiments which require sophisticated NMR
equipment and long measuring cycles9,10
.
The alternative approach we used is based on the fact that for the assignment the
most important region in the NOESY spectra is that containing NOE contacts between
the ribose 1′ and base aromatic (H-6, H-8, and H-2) protons (chemical shifts at about
6.2–5.0 ppm for the 1′ protons and 8.5–6.9 ppm for the aromatic protons). In this
“coupling box” a continuous assignment path can be drawn connecting all the nucleo-
tides of one strand which are located in regions with approximately regular helical
Collect. Czech. Chem. Commun. (Vol. 61) (1996)

390
Arnold, Pressova, Saman, Vogtherr, Limmer:
geometry. If the 1′ protons are replaced by deuterons, the corresponding interaction is
eliminated and hence the cross peak vanishes from the NOESY spectrum. In most of
the functionally interesting RNA molecules regions with well-defined secondary struc-
ture (helical stems) are combined with regions lacking any pronounced secondary
structure. It is obvious that in most cases only the non-helical regions (loops, bulges,
pseudo-knots, etc.) without distinct regular base pairing patterns need to be charac-
terized in more detail.
Recently, Chattopadhyaya11 and co-workers concentrated on non-specific introduc-
tion of deuterium into the ribose moiety and on the use of appropriate partly labelled
2
nucleoside synthons for oligonucleotide synthesis. However, the relative H isotopic
enrichment at the 1′ position reached only ca 20%, which is clearly not sufficient to
suppress efficiently the 1′-aromatic cross peaks and consequently to reduce the reson-
ance overlap in this crucially important coupling box.
We decided to use specifically 1′-deuterated ribose to prepare the ribonucleotide
H-phosphonate synthons by chemical synthesis.
The synthetic procedure leading to 1′-deuterated nucleoside-H-phosphonates
(Schemes 1–4), first optimized on non-deuterated compounds, included the following
1
steps (intermediates at all stages were monitored by H NMR spectroscopy (Tables I–V)
mainly with respect to the presence and content of deuterium in the 1′ position:
1. Stereoselective protection of [1′-2H]ribose (1), realized according to the published
procedure11 comprising preparation of 1-O-methyl-D-ribofuranose, separation of mix-
ture of stereoisomers by crystallization, benzoylation of the β-isomer leading to 2,3,5-
tri-O-benzoyl-β-D-ribofuranose and acetylation of the latter yielding 1-O-acetyl-
2,3,5-tri-O-benzoyl-β-D-ribofuranose (2) (Scheme 1, Table I).
2. Preparation of appropriately protected nucleobases according to the known proce-
dures: benzoylation12 of adenine leading to N6-benzoyladenine, acetylation of guanine
followed by reaction with diphenylcarbamoyl chloride13 leading to N2-acetyl-O6-(di-
phenylcarbamoyl)guanine, trimethylsilylation14 of uracil leading to O2,O4-bis(tri-
methylsilyl)uracil and acetylation of cytosine followed by trimethylsilylation14 leading
to N4-acetyl-O2-(trimethylsilyl)cytosine.
3. Nucleosidation starting with 2,3,5-tri-O-benzoyl-1-O-acetyl[1-2H]ribose and sub-
sequent hydrolysis with saturated methanolic ammonia (Scheme 2, Table II); the con-
densation14 with N6-benzoyladenine gave [1′-2H]adenosine (3), condensation15 with
N4-acetyl-O2-(trimethylsilyl)cytosine [1′-2H]cytidine (4), condensation16 with N2-acetyl-
O6-(diphenylcarbamoyl)guanine gave [1′-2H]guanosine (5), condensation15 with O2,O4-
bis(trimethylsilyl)uracil afforded [1′-2H]uridine (6).
4. Step-by-step protection17–20 comprising preparation of 5′-O-DMTr-N-protected nu-
cleosides 7–10, silylation leading to mixture of 2′ (11–14) and 3′ (15–18) isomers, and
their chromatographic separation (Scheme 3, Tables III and IV).
Collect. Czech. Chem. Commun. (Vol. 61) (1996)

1’-C Deuterated Synthons for RNA Synthesis
391
OCOCH3
HO
C6H5COO
O
O
a, b, c
OH,D
D
OH
C6H5COO
OCOC6H5
OH
1
2
a) MeOH, H2SO4; b) benzoylchloride, pyridine; c) CH COOH, (CH3CO)2O, H2SO4
3
SCHEME 1
OCOCH
D
B
D
C H COOO
HO
3
6
5
O
O
a, b
C H COO
OH
OCOC H
OH
6
5
6
5
3-6
2
c, d
B
D
DMTrO
O
OH
OH
7-10
B
B
3
4
5
6
7
8
adenine-9-yl
cytosine-1-yl
guanine-9-yl
uracil-1-yl
N6-dimethylaminomethyleneadenine-9-yl
N4-benzoylcytosine-1-yl
N2-dimethylaminomethyleneguanine-9-yl
uracil-1-yl
9
10
a) N6-benzoyladenine, CH CN, SnCl ; or N4-acetyl-O2-(trimethylsilyl)cytosine,
3
4
(CH Cl) , SnCl ; or N2-acetyl-O6-(diphenylcarbamoyl)guanine,
2
2
4
N,O-bis(trimethylsilyl)acetamide),(CH Cl) , toluene, CF SO Si(CH ) ;
2
2
3
3
3 3
or O2,O4-di(trimethylsilyl)uracil, (CH Cl) , SnCl ;
2
2
4
b) NH OH, MeOH
4
c) in case of 3 and 5 N,N-dimethylformamide dimethylacetal;
in case of 4 benzanhydride, methanol, ethanol
d) 4,4’-dimethoxytriphenylmethyl (DMTr-Cl) chloride, pyridine
SCHEME 2
Collect. Czech. Chem. Commun. (Vol. 61) (1996)

392
Arnold, Pressova, Saman, Vogtherr, Limmer:
B
D
B
D
DMTrO
+
B
D
DMTrO
DMTrO
O
O
O
a
OH
OTBDMS
TBDMSO
OH
HO
HO
11-14
7-10
15-18
a) tert-butyldimethylsilyl chloride (TBDMS-Cl), imidazole, pyridine
B
N6-dimethylaminomethyleneadenine-9-yl
11, 15
12, 16
13, 17
14, 18
N4-benzoylcytosine-1-yl
N2-dimethylaminomethyleneguanine-9-yl
uracil-1-yl
SCHEME 3
B
B
D
DMTrO
DMTrO
O
O
a
D
OTBDMS
OTBDMS
O
HO
11-14
O P H
Et3NH
O
19-22
a) PCl3, imidazole, Et3N, CH2Cl2
B
N6-dimethylaminomethyleneadenine-9-yl
N4-benzoylcytosine-1-yl
19
20
21
22
N2-dimethylaminomethyleneguanine-9-yl
uracil-1-yl
SCHEME 4
Collect. Czech. Chem. Commun. (Vol. 61) (1996)

1’-C Deuterated Synthons for RNA Synthesis
393
5. Phosphitylation of the 2′-O-silylated intermediates 11–14 according to the publish-
ed procedure21 leading to the following nucleoside triethylammonium H-phosphonates
(Scheme 4, Table V), or 2′-O-(tert-butyldimethylsilyl)-5′-O-(4,4′-dimethoxytrityl)[1′-
2H]-N6-dimethylaminomethyleneadenosine-3′-H-phosphonate (19), or 2′-O-(tert-butyl-
dimethylsilyl)-5′-O-(4,4′-dimethoxytrityl)[1′-2H]-N4-benzoylcytidine-3′-H-phosphonate
(20), or 2′-O-(tert-butyldimethylsilyl)-5′-O-(4,4′-dimethoxytrityl)[1′-2H]-N2-dimethyl-
aminomethyleneguanosine-3′-H-phosphonate (21), or 2′-O-(tert-butyldimethylsilyl)-5′-
O-(4,4′-dimethoxytrityl)[1′-2H]uridine-3′-H-phosphonate (22).
Automated synthesis of RNA fragments was performed on an automatic synthesizer
according to a modified synthesizer program22 with coupling time 3 min on the 1.5 µmol
scale. The four following oligoribonucleotides were synthesized: non-deuterated Rz-1
and Rz-2, and deuterated Rz-1 and Rz-2.
Non-deuterated
11 12 13 14 15 16 17 18 19
Rz-1
Rz-2
5′-
G C U A U U U A U -3′
3′- A C G A U A A A G U -5′
10 9
8 7 6 5 4 3 2 1
Deuterated
11 12 13 14 15 16 17 18 19
Rz-1
Rz-2
5′-
G* C* U* A* U* U U A U -3′
3′- A C* G* A* U* A* A A G U -5′
10 9
8
7
6
5
4 3 2 1
EXPERIMENTAL
The nucleobases were purchased from Fluka, D-[1-2H]ribose (isotopic enrichment >98%) was from
Omicron Chemicals (South Bend, IN, U.S.A.). Pyridine (dry), acetonitrile (distilled from P2O5 and
CaH2), dichloromethane (dry), N,N-dimethylformamide dimethylacetal, 4,4′-dimethoxytriphenylmethyl
chloride, tert-butyldimethylchlorosilane, imidazole (puriss.), triethylamine (puriss.), 1-adamantane-
carbonyl chloride, tetrabutylammonium fluoride and dichloroacetic acid were Fluka products and
were used without further purification. Phosphorus trichloride (2 M solution in dichloromethane) was
purchased from Aldrich. DEAE-Sephacel and Sephadex G-15 were obtained from Pharmacia. TLC
was carried out on Silufol UV254 (Kavalier) using the following eluents: S1, ether–acetone 95 : 5; S2,
chloroform–methanol 7 : 3; S3, ether–acetone 9 : 1; S4, 2-propanol–ammonia–water 7 : 1 : 2; S5,
chloroform–methanol 8 : 2; S6, chloroform–methanol–triethylamine 95 : 4.5 : 0.5; S7, toluene–ethyl
acetate 2 : 1; S8, chloroform–methanol 9 : 1. The automated RNA synthesis was performed on a
Gene Assembler Plus DNA-synthesizer (Pharmacia). Ultraviolet spectra were measured on a UV-VIS
Collect. Czech. Chem. Commun. (Vol. 61) (1996)

394
Arnold, Pressova, Saman, Vogtherr, Limmer:
spectrometer Unicam 8625. 1H NMR spectra of intermediates and H-phosphonate synthons were
measured on a Varian UNITY-500 (499.8 MHz) spectrometer in hexadeuteriodimethyl sulfoxide
(99.8 at.% D, Aldrich); for standardization the central line of the non-deuterated solvent (δ 2.50 ppm)
was used. 2D-NMR spectra of oligonucleotides were measured on a Bruker DRX 500 instrument
equipped with an Aspect X-32 computer. 500 MHz-NOESY spectra in D2O buffer solution were re-
corded at 16 °C using standard procedures with mixing times of 300 ms. The spectra were acquired
with 4 096 complex data points in the domain t2 and 512 complex data points in the domain t1 and a
recycle delay of 1.5 s employing zero-filling to 1 024 data points in the F1 frequency dimension in
the Fourier-transformed spectra (no zero filling in F2).
For the 2D-measurements, RNA (8–13 mg) was dissolved in 0.5 ml D2O buffer solution contain-
ing 0.1 mol l–1 NaCl, 10 mmol l–1 sodium phosphate, pH 6.5. The resonances were referenced to
internal 2,2-dimethyl-2-silapentane-5-sulfonate. RNA annealing was achieved by heating the samples
at 80 °C for five minutes and subsequent cooling to room temperature.
1-O-Acetyl-2,3,5-tri-O-benzoyl[1-2H]ribose (2)
The title compound was prepared according to the published procedures11 starting from [1-2H]ribose
(15 g, 0.1 mol); yield 20.5 g (41%) after recrystallization from 1-propanol.
TABLE I
1
Spectral H NMR parameters (δ, ppm; J, Hz) of starting ribose 1 and compound 2
Compound
Parameter
1(a)
1(b)
2a
δ(H-2)
δ(H-3)
δ(H-4)
δ(H-5a)
δ(H-5b)
δ(1-OH)
δ(2-OH)
δ(3-OH)
J(2,3)
3.37 dd
3.72 dt
3.49 dt
3.28 dd
3.61 dd
6.04 s
4.76 d
5.02 d
2.7
3.21 dd
3.71 dt
3.54 dt
3.44 dd
3.59 dd
6.30 s
4.73 d
4.62 d
2.9
5.73 d
5.86 dd
4.84 dt
4.50 dd
4.72 dd
–
–
–
4.9
J(3,4)
3.9
3.3
7.1
J(4,5a)
J(4,5b)
J(5a,5b)
J(2,OH
J(3,OH)
3.9
6.6
4.2
7.8
3.3
3.4
11.2
10.7
12.2
7.1
5.6
6.8
5.1
–
–
a Other proton signals: 1.93 s (OCOCH3); 7.52–8.03 m (OBz).
Collect. Czech. Chem. Commun. (Vol. 61) (1996)

1’-C Deuterated Synthons for RNA Synthesis
395
N6-Benzoyladenine
N6-Benzoyladenine was prepared according to the published procedure12 starting from adenine (75 g,
0.55 mol); yield 94.5 g (60%) after recrystallization from ethanol, m.p. 242–242.5 °C.
N4-Acetyl-O2-(trimethylsilyl)cytosine
N4-Acetyl-O2-(trimethylsilyl)cytosine was prepared according to the published procedure14 starting
from N4-acetylcytosine (7.65 g, 50 mmol); yield 11.25 g (100%) of the crude product.
TABLE II
1
Spectral H NMR parameters (δ, ppm; J, Hz) of 1′-deuterated nucleosides
Compound
Parameter
3a
4b
5c
6d
δ(H-2)
4.60 dd
4.14 dt
3.96 q
3.55 ddd
3.67 ddd
5.43 d
5.18 d
5.41 dd
7.34 bs
–
3.93 dd
3.92 dte
3.80 dte
3.53 ddd
3.64 ddd
5.26 d
5.03 t
4.96 t
7.15 bs
–
4.39 dd
4.08 dt
3.86 dt
3.52 ddd
3.61 ddd
5.39 d
5.12 d
5.04 t
7.93 bs
10.63 bs
4.9
4.02 bt
3.96 dt
3.83 bq
3.54 ddd
3.61 ddd
5.38 d
5.09 d
5.12 t
–
δ(H-3)
δ(H-4)
δ(H-5a)
δ(H-5b)
δ(2-OH)
δ(3-OH)
δ(5-OH)
δ(NH2)
δ(NH)
11.19 bs
4.6
J(2,3)
5.1
5.2e
J(3,4)
3.2
4.7e
3.5
3.6
J(4,5a)
J(4,5b)
J(5a,5b)
J(2,OH)
J(3,OH)
J(5a,OH)
J(5b,OH)
3.7
3.4
4.1
3.8
3.9
3.2
4.1
3.8
12.2
12.2
12.1
12.2
6.6
4.6
7.1
4.4
5.1
5.3
5.3
5.3
6.1
5.6
4.6
4.9
5.8
5.4
5.4
5.4
Other proton signals: a H-2, 8.34 s; H-8, 8.13 s. b H-5, 5.69 d (7.5); H-6, 7.83 d (7.5). c H-8, 7.93 s.
d H-5, 5.63 bd (8.0); H-6, 7.86 d (8.0). e Chemical shifts and coupling constants determined by means
of simulative-iterative calculations.
Collect. Czech. Chem. Commun. (Vol. 61) (1996)

396
Arnold, Pressova, Saman, Vogtherr, Limmer:
N2-Acetyl-O6-(diphenylcarbamoyl)guanine
N2-Acetyl-O6-(diphenylcarbamoyl)guanine was prepared according to the published procedures13
starting from guanine (60.44 g, 0.40 mol); yield 35.53 g (21%) after recrystallization from ethanol.
O2,O4-Bis(trimethylsilyl)uracil
O2,O4-Bis(trimethylsilyl)uracil was prepared according to the published procedure14 starting from
uracil (5.6 g, 50 mmol); yield 12.27 g (96%), b.p. 107–109 °C/13 Pa.
[1′-2H]Adenosine (3)
N6-Benzoyladenine was condensed with 1-O-acetyl-2,3,5-tri-O-benzoyl[1-2H]ribose by the known
method14. The obtained N6-benzoyl-2′,3′,5′-tri-O-benzoyl[1′-2H]adenosine (RF 0.53, S1) deprotected
by treatment with saturated methanolic ammonia for 24 h to give the title compound (RF 0.46, S2).
TABLE III
1
Spectral H NMR parameters (δ, ppm; J, Hz) of fully protected 1′-deuterated nucleoside-2′ isomers
Compound
Parameter
11a
12b
13c
14d
δ(H-2)
4.87 d
4.24 ddd
4.10 q
3.24 dd
3.28 dd
5.15 d
–
4.17 d
4.21 ddd
4.07 ddd
3.35 dd
3.39 dd
5.16 d
11.25 bs
0.10 s
0.13 s
0.89 s
4.4
4.57 d
4.18 dt
4.05 dt
3.23 dd
3.30 dd
5.09 d
11.33 s
–0.05 s
0.01 s
0.80 s
5.2
4.21 d
4.07 q
3.99 ddd
3.24 dd
3.31 dd
5.13 d
11.38 s
0.05 s
0.06 s
0.85 s
5.1
δ(H-3)
δ(H-4)
δ(H-5a)
δ(H-5b)
δ(3-OH)
δ(NH)
δ(CH3-Si)
–0.06 s
–0.17 s
0.73 s
5.1
δ((CH3)3C-Si)
J(2,3)
J(3,4)
4.3
8.1
5.4
5.6
J(4,5a)
5.1
2.1
2.9
2.6
J(4,5b)
4.2
3.9
5.5
4.1
J(5a,5b)
J(3,OH)
10.7
10.8
10.5
10.8
6.1
6.4
5.9
6.1
Other proton signals: a H-2, 8.34 s; H-8, 8.36 s; =CH, 8.90 s; N(CH3)2, 3.13 s, 3.19 s; DMT, 3.73 s,
6.83–6.85 m, 7.17–7.42 m. b H-5, 7.99 dd (1.7, 7.4); H-6, 8.39 d (7.4); DMT, 3.76 s, 6.90–6.94 m,
7.24–7.42 m, 7.50–7.54 m, 7.60–7.64 m. c H-8, 7.91 s; =CH, 8.46 s; N(CH3)2, 3.03 s, 3.06 s; DMT,
3.73 s, 6.83–6.87 m, 7.23–7.40 m. d H-5, 5.30 dd (2.0, 8.0); H-6, 7.75 d (8.0); DMT, 3.74 s, 6.88–6.92 m,
7.12–7.38 m.
Collect. Czech. Chem. Commun. (Vol. 61) (1996)

1’-C Deuterated Synthons for RNA Synthesis
397
[1′-2H]Cytidine (4)
N4-Acetyl-O2-(trimethylsilyl)cytosine was condensed with 1-O-acetyl-2,3,5-tri-O-benzoyl[1-2H]ribose
by the method described by Hrebabecky15. The obtained N4-acetyl-2′,3′,5′-tri-O-benzoyl[1′-2H]-
cytidine (RF 0.15, S1) was deprotected by treatment with saturated methanolic ammonia for 24 h to
give the title compound (RF 0.13, S2).
[1′-2H]Guanosine (5)
N2-Acetyl-O6-(diphenylcarbamoyl)guanine was condensed with 1-O-acetyl-2,3,5-tri-O-benzoyl[1-2H]-
ribose by the method described by Jenny et. al.16. The obtained N2-acetyl-2′,3′,5′-tri-O-benzoyl-O6-
(diphenylcarbamoyl)[1′-2H]guanosine (RF 0.60, S3) was deprotected by treatment with saturated
methanolic ammonia for 24 h to give the title compound (RF 0.46, S4).
TABLE IV
1
Spectral H NMR parameters (δ, ppm; J, Hz) of fully protected 1′-deuterated nucleoside-3′ isomers
Compound
Parameter
15a
16b
17c
18d
δ(H-2)
4.88 dd
4.52 t
4.03 q
3.11 dd
3.35 dd
5.36 d
–
4.07 t
4.28 dd
4.05 dt
3.22 dd
3.51 dd
5.48 d
11.26 bs
–0.07 s
0.02 s
0.74 s
4.6
4.57 dd
4.31 t
3.94 dt
3.15 dd
3.28 dd
5.36 d
11.33 bs
–0.01 s
0.05 s
0.80 s
5.1
4.10 t
4.15 t
3.91 dt
3.16 dd
3.34 dd
5.34 d
11.36 bs
–0.03 s
0.02 s
0.77 s
5.6
δ(H-3)
δ(H-4)
δ(H-5a)
δ(H-5b)
δ(2-OH)
δ(NH)
δ(CH3-Si)
0.04 s
0.08 s
0.84 s
5.6
δ((CH3)3C-Si)
J(2,3)
J(3,4)
4.4
7.8
5.1
5.4
J(4,5a)
4.8
3.4
5.3
4.4
J(4,5b)
4.3
2.5
3.8
3.2
J(5a,5b)
J(2,OH)
10.5
6.4
10.9
10.5
10.7
5.6
6.1
5.9
Other proton signals: a H-2, 8.35 s; H-8, 8.40 s; =CH, 8.90 s; N(CH3)2, 3.12 s, 3.19 s; DMT, 3.72 s,
6.80–6.84 m, 7.17–7.35 m. b H-5, 7.99 dd (1.2, 7.3); H-6, 8.48 d (7.3); DMT, 3.75 s, 6.89–6.93 m,
7.15–7.41 m, 7.49–7.53 m, 7.60–7.64 m. c H-8, 7.96 s; =CH, 8.44 s; N(CH3)2, 3.03 s, 3.07 s; DMT,
3.72 s, 6.81–6.85 m, 7.19–7.35 m. d H-5, 5.37 bd (8.0); H-6, 7.77 d (8.0); DMT, 3.73 s, 6.87–6.91 m,
7.19–7.38 m.
Collect. Czech. Chem. Commun. (Vol. 61) (1996)

398
Arnold, Pressova, Saman, Vogtherr, Limmer:
[1′-2H]Uridine (6)
O2,O4-Bis(trimethylsilyl)uracil (2.86 ml, 10.5 mmol) was condensed with 1-O-acetyl-2,3,5-tri-O-ben-
zoyl[1-2H]ribose (4.77 g, 9.5 mmol) by the method described by Hrebabecky15. The obtained 2′,3′,5′-
tri-O-benzoyl[1′-2H]uridine (RF 0.66, S1) was deprotected by treatment with saturated methanolic
ammonia for 24 h to give the title compound (RF 0.15, S5).
2′-O-(tert-Butyldimethylsilyl)-5′-O-(4,4′-dimethoxytrityl)-N6-dimethylaminomethylene[1′-2H]-
adenosine (11)
Step-by-step protection of the nucleoside was performed according to the published procedure17 start-
ing from [1′-2H]adenosine (2.67 g, 10 mmol); the yield 1.19 g (16%) of the title compound as a
chromatographically pure solid foam (RF 0.55, S6).
TABLE V
1
Spectral H NMR parameters (δ, ppm; J, Hz) of nucleoside-H-phosphonate synthons
Compound
Parameter
19a
20b
21c
22d
δ(H-2)
5.04 d
4.72 ddd
4.31 dt
3.32 m
3.32 m
–
4.31 d
4.63 ddd
4.20 dt
3.39 m
3.39 m
11.24 bs
0.11 s
0.14 s
0.86 s
4.2
4.77 d
4.79 dt
4.18 ddd
3.23 dd
3.32 dd
11.26 bs
–0.09 s
0.01 s
0.77 s
5.1
4.31 d
4.59 dt
4.15 dt
3.27 dd
3.35 dd
11.37 bd
0.05 s
0.08 s
0.83 s
4.9
δ(H-3)
δ(H-4)
δ(H-5a)
δ(H-5b)
δ(NH)
δ(CH3-Si)
–0.07 s
–0.25 s
0.67 s
4.9
δ((CH3)3C)
J(2,3)
J(3,4)
2.7
7.5
4.4
4.2
J(4,5a)
4.4
2.9
2.6
2.7
J(4,5b)
4.4
2.9
5.9
4.2
e
e
J(5a,5b)
J(H-3,31P)
–
–
10.4
10.7
10.3
10.1
9.8
10.4
Other proton signals: a H-2, 8.29 s; H-8, 8.33 s; =CH, 8.90 s; N(CH3)2, 3.13 s, 3.19 s; DMT, 3.73 s,
6.81–6.84 m, 7.19–7.34 m. b H-5, 7.06 d (7.6); H-6, 8.37 d (7.6); DMT, 3.75 s, 6.89–6.93 m, 7.19–7.44 m,
7.49–7.53 m, 7.60–7.64 m. c H-8, 7.90 s; =CH, 8.58 s; N(CH3)2, 3.03 s, 3.07 s; DMT, 3.73 s, 6.80–6.84 m,
7.19–7.38 m. d H-5, 5.25 dd (1.8, 8.1); H-6, 7.71 d (8.1); DMT, 3.74 s, 6.87–6.91 m, 7.19–7.34 m. e Value
not determined.
Collect. Czech. Chem. Commun. (Vol. 61) (1996)

1’-C Deuterated Synthons for RNA Synthesis
399
N4-Benzoyl-2′-O-(tert-butyldimethylsilyl)-5′-O-(4,4′-dimethoxytrityl)[1′-2H]cytidine (12)
Step-by-step protection of the nucleoside was performed according to the published procedures18–20
starting from [1′-2H]cytidine (2.43 g, 10 mmol); yield 3.2 g (42%) of the title compound as a chro-
matographically pure solid foam (RF 0.29, S7).
2′-O-(tert-Butyldimethylsilyl)-5′-O-(4,4′-dimethoxytrityl)-N2-dimethylaminomethylene[1′-2H]-
guanosine (13)
Step-by-step protection of the nucleoside was performed according to the published procedure17 start-
ing from [1′-2H]guanosine (2.83 g, 10 mmol); yield 1.35 g (18%) of the title compound as a chroma-
tographically pure solid foam (RF 0.26, S6).
2′-O-(tert-Butyldimethylsilyl)-5′-O-(4,4′-dimethoxytrityl)[1′-2H]uridine (14)
Step-by-step protection of the nucleoside was performed according to the published procedure18,19
starting from [1′-2H]uridine (2.44 g, 10 mmol); yield 1.65 g (25%) of the title compound as a chro-
matographically pure solid foam (RF 0.32, S7).
Nucleoside-H-phosphonates
Phosphitylation of all appropriately protected nucleosides was carried out by the slightly modified
Froehler method17,21 with 85–95% yield of triethylammonium H-phosphonates:
2′-O-(tert-Butyldimethylsilyl)-5′-O-(4,4′-dimethoxytrityl)-N6-dimethylaminomethylene[1′-2H]adenosine-3′-
H-phosphonate (19) (96%), RF 0.20, S8.
N4-Benzoyl-2′-O-(tert-butyldimethylsilyl)-5′-O-(4,4′-dimethoxytrityl)[1′-2H]cytidine-3′-H-phosphonate
(20) (92%), RF 0.25, S8.
2′-O-(tert-Butyldimethylsilyl)-5′-O-(4,4′-dimethoxytrityl)-N2-dimethylaminomethylene[1′-2H]guanosine-3′-
H-phosphonate (21) (85%), RF 0.20, S8.
2′-O-(tert-Butyldimethylsilyl)-5′-O-(4,4′-dimethoxytrityl)[1′-2H]uridine-3′-H-phosphonate (22) (86%),
RF 0.25, S8.
Automated Synthesis of RNA Fragments
The H-phosphonates were dissolved in pyridine–acetonitrile (1 : 1) to a concentration of 0.1 mol l–1
each, 1-adamantanecarbonyl chloride was dissolved in the same mixture to a concentration of 0.4 mol l–1.
The automated oligoribonucleotide synthesis was performed according to a modified synthesizer
protocol22 with coupling time 3 min on 1.5 µmol scale. Cleavage of the crude oligoribonucleotide
from the CPG-support and deprotection were carried out by the successive treatment with saturated
methanolic ammonia (24 h) and 1 M TBAF in DMF (24 h) at room temperature. Work-up of the
deprotection mixtures was carried out on a DEAE-Sephacel column (1 × 2 cm) using 0.01 and 1 M LiCl
as eluents, and followed by desalting on G-15 Sephadex column (2 × 40 cm). Two separate syntheses
of each fragment afforded 150 OU for non-deuterated Rz-1, 140 OU for non-deuterated Rz-2, 149 OU
for deuterated Rz-1 and 145 OU for deuterated Rz-2.
RESULTS AND DISCUSSION
The described method of chemical synthesis provided [1′-2H]-protected nucleoside-H-
phosphonate synthons with isotopic enrichment >98% in the position 1′. This was con-
Collect. Czech. Chem. Commun. (Vol. 61) (1996)

400
Arnold, Pressova, Saman, Vogtherr, Limmer:
firmed by 1H NMR spectra of compounds 2–22 (Tables I–V), where no residual signal
of 1′-1H proton was observed.
The effect of 1′-ribose deuteration on the NOESY spectra of the duplex formed by
the oligoribonucleotide strands Rz-1 and Rz-2 is demonstrated in Fig. 1 where a part of
the 1′-ribose-base aromatic proton cross peak region (frequently referred to as “finger-
print region”) is displayed.
The corresponding spectral sections for the non-deuterated (a) and the partly deute-
rated samples (b) are compared there. Since unpurified, crude products have been used
for the NMR studies, a few cross peaks, due to impurities (in particular truncated frag-
7.0
a
δ, ppm
7.2
7.4
7.6
7.8
8.0
8.2
6.0
5.8
5.6
5.4
5.2
δ, ppm
FIG. 1
Part of the NOESY spectrum (mixing time 300 ms, 4K data points in frequency domain F2, 1K data
points in domain F1, relaxation delay 1.5 s) of the duplex formed by the oligoribonucleotide strands
Rz-1 and Rz-2 (see text), containing the cross peaks resulting from 1′-ribose-base aromatic proton
interactions. a Spectrum of the non-deuterated sample. The assignment path for the Rz-1 strand is
indicated by solid lines. For numbering see text. b The same part of the NOESY spectrum as in a
for the partially deuterated sample (nucleotides 5–9 in Rz-2 and nucleotides 11–15 in Rz-1). The
missing cross peaks due to NOE contacts between the nucleotides of the Rz-1 strand with reference
to a are marked
Collect. Czech. Chem. Commun. (Vol. 61) (1996)

1’-C Deuterated Synthons for RNA Synthesis
401
ments) are also seen. Nevertheless, it is immediately evident that the number of cross
peaks in the spectrum of the partially deuterated sample is considerably reduced as it
was expected. The missing cross peaks are due to NOE interactions involving 1′-ribose
protons of (specifically deuterated) residues 5–9, and 11–15 (numbering according to
the sequence scheme, vide supra), whereas all cross peaks resulting from interactions
between nucleotides 1–4, and 16–19 are still present (cf. Fig. 1b). Moreover, the strong
cross peaks originating from the pyrimidine (H-5)–(H-6) interactions – both for the
non-deuterated and the partially deuterated residues – are found in the spectrum. They
can be removed, too, by deuteration of the pyrimidine C-5 position which is intended
in a further step of this work.
The reduction of the number of cross peaks in the fingerprint region (as well as the
1′–2′region) can facilitate the resonance assignment considerably, in particular for dis-
tinctly greater RNA molecules (>20 nucleotides) with extended helical stem regions.
Usually, these stems need not to be analyzed extensively since they do not display
severe deviations from regular helical geometry (at least if they contain only regular
7.0
δ, ppm
b
7.2
7.4
7.6
7.8
8.0
8.2
6.0
5.8
5.6
5.4
5.2
δ, ppm
FIG. 1
(Continued)
Collect. Czech. Chem. Commun. (Vol. 61) (1996)

402
Arnold, Pressova, Saman, Vogtherr, Limmer:
Watson–Crick base pairs). Rather, regions with non-regular arrangement of the nucleo-
tides, as, e.g., in loops, bulges, pseudoknots etc., need to be structurally characterized
in greater detail. To this end, in the first step of an NMR structure determination a safe
assignment of the resonances of non-exchangeable protons has to be achieved. As men-
tioned already before, especially for RNA molecules with more than 20 nucleotides the
cross peak overlap in the NOESY spectrum region being most important for the reson-
ance assignment can be substantially diminished making use of the above-described
deuterium labelling. Accordingly, the resonance assignment can be simplified, in par-
ticular, if additionally the number and/or position of the partially deuterated residues is
varied along the sequence region of major interest.
Of course, the synthetic procedure presented in this work can also be used to produce
specifically 13C-labelled synthons starting from correspondingly 13C-labelled ribose.
This would be interesting in view of a detailed structural and conformational charac-
terization of certain particularly important sites in a given RNA sequence by means of
multidimensional heteronuclear NMR spectroscopy.
This work was supported by the Volkswagen-Stiftung and the Deutsche Forschungsgemeinschaft,
Projekt Sp 243/6-1.
REFERENCES
1. Szewczak A. A., Moore P. B., Chan Y.-L., Wool I. G.: Proc. Natl. Acad. Sci. U.S.A. 90, 9581
(1993).
2. Puglisi J. D., Wyatt J. R., Tinoco I., jr.: J. Mol. Biol. 214, 437 (1990).
3. Chastain M., Tinoco I., jr.: Biochemistry 31, 12733 (1992).
4. Borer P. N., Lin Y., Wang S., Roggenbuck M. W., Gott J. M., Uhlenbeck O. C., Pelczer I.:
Biochemistry 34, 6488 (1995).
5. Moore P. B.: Acc. Chem. Res. 28, 251 (1995).
6. Batey R. T., Inada M., Kujawinski E., Puglisi J. D., Williamson J. R.: Nucleic Acids Res. 20,
4515 (1992).
7. Nikonowicz E. A., Sirr A., Legautt P., Jucker F., Baer L. M., Pardi A.: Nucleic Acids Res. 20,
4507 (1992).
8. Michnicka M. J., Harper J. W., King G. C.: Biochemistry 32, 395 (1993).
9. Nikonowicz E. A., Pardi A: J. Mol. Biol. 232, 1141 (1993).
10. Allain F. H.-T., Varani G.: J. Mol. Biol. 250, 333 (1995).
11. Foldesi A., Nilson F. P. R., Glemarec C., Gioeli C., Chattopadhyaya J.: Tetrahedron 48, 9033
(1992).
12. Ness N. K. in: Synthetic Procedures in Nucleic Acid Chemistry (W. W. Zorbach and R. S.
Tipson, Eds), Vol. 1, p. 183. Interscience, New York 1968.
13. Zou R., Robins M. J. : Can. J. Chem. 65, 1436 (1987).
14. Hrebabecky H., Dockal J., Holy A.: Collect. Czech. Chem. Commun. 59, 1408 (1994).
15. Hrebabecky H., Holy A.: Carbohydrate Res. 216, 179 (1991).
16. Jenny T. F., Benner S. A.: Tetrahedron Lett. 33, 6619 (1992).
Collect. Czech. Chem. Commun. (Vol. 61) (1996)

1’-C Deuterated Synthons for RNA Synthesis
403
17. Arnold L., Smrt J., Zajicek J., Ott G., Schiesswohl M., Sprinzl M.: Collect. Czech. Chem.
Commun. 56, 1948 (1991).
18. Schaller H., Weinmann G., Lerch B., Khorana H. G.: J. Am. Chem. Soc. 85, 3821 (1963).
19. Usman N., Ogilvie K. K., Jiang M.-J., Cedergren R. J.: J. Am. Chem. Soc. 109, 7845 (1987).
20. Otter B. A., Fox J. J. in: Ref.12, Vol. 1, p. 285.
21. Froehler B. C., Ng P. G., Matteucci M. D.: Nucleic Acids Res. 14, 5399 (1986).
22. Ott G., Arnold L., Smrt J., Sobkowski M., Limmer S., Hofmann H-P., Sprinzl M.: Nucleosides
Nucleotides 13, 1069 (1993).
Collect. Czech. Chem. Commun. (Vol. 61) (1996)