Synthesis of multiply labelled ribonucleosides for sequence-specific labelling of oligo-RNA

Article abstract of DOI:10.1002/jlcr.503

The synthesis of ribonucleotide blocks multiply labelled with ²H, ¹³C and ¹⁵N for solid support synthesis of sequence specifically labelled RNA is described. Labels were introduced in the ribose ring (¹³C), C5 position of pyrimidine nucleobases (²H) and exocyclic amino groups (¹⁵N) and serve as multiple probes for studying the various physicochemical consequences of physiologically important RNA folding by high-resolution multi-nuclear NMR spectroscopy.

Full text of DOI:10.1002/jlcr.503

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Labelled Cpd Radiopharm 2001; 44: 763–783.
DOI: 10.1002/jlcr.503
Research Article
Synthesis of multiply labelled ribonucleosides
for sequence-specific labelling of oligo-RNA
1,
2,y
3
.
Jan Milecki *, Andras Foldesi , Artur Fischer ,
Ryszard W. Adamiak3 and Jyoti Chattopadhyaya2
1 Faculty of Chemistry, A. Mickiewicz University, Grunwaldzka 6,
!
60-780 Poznan, Poland
2 Department of Bioorganic Chemistry, Box 581, Biomedical Center,
University of Uppsala, SE-751 23 Uppsala, Sweden
3 Institute of Bioorganic Chemistry, Polish Academy of Sciences,
!
Noskowskiego 12/14, 61-704 Poznan, Poland
Summary
The synthesis of ribonucleotide blocks multiply labelled with H, 13C and 15N
for solid support synthesis of sequence specifically labelled RNA is described.
Labels were introduced in the ribose ring (13C), C5 position of pyrimidine
nucleobases (2H) and exocyclic amino groups (15N) and serve as multiple
probes for studying the various physicochemicalconsequences of physiool gi-
cally important RNA folding by high-resolution multi-nuclear NMR spectro-
scopy. Copyright # 2001 John Wiley & Sons, Ltd.
2
2
Key Words: ribonucleoside; multiple labelling; stable isotopes; H; 13C; 15N;
oligo-RNA
Introduction
Labelling of biologically important macromolecules like proteins and
nucleic acids with stable NMR active isotopes has contributed
immensely to our understanding of the influence of the biopolymer
structure and function.1–4 The initialapproach was to albelthese
*Correspondence to: J. Milecki, Faculty of Chemisty, Adam Mickiewicz University, Grunwaldzka 6,
60-780 Poznan, Poland. E-mail: janmil@amu.edu.pl
y On leave from the Department of Analytical Chemistry, University of Pecs, Hungary.
Copyright # 2001 John Wiley & Sons, Ltd.
Received 26 March 2001
Revised 6 June 2001
Accepted 12 June 2001

764
J. MILECKI ET AL.
molecules via enzymatic means, which gives mainly uniformly labelled
molecules.5–9 Although such uniform labelling has led to many
important results, this method has its intrinsic limitations in that no
segmental labelling can be easily achieved, and the problems of
overcrowding of resonances become acute (specially for large RNA
with repeating sugar and aglycon units) as the molecular size increases.
Such labelling, although very productive,5–9 has serious limitations.
Specifically labelled oligonucleotides10–18 appear to be the best means
for studies of this kind as problems associated with crowding of spectral
lines are overcome and the potential of modern NMR methods can be
fully exploited.19–21
Since the preparation of labelled oligonucleotides involves consider-
able expense and labor,22–26 it would be of great value to collect as many
label-related data as possible from one synthesized oligomer.
Multiple labelling of nucleosides has already been used with great
success.22,27–33 Usually multiple labelling was limited to one part of the
nucleoside moiety, either the ribose or base. The nucleoside with the
label introduced into both of these parts will serve as an even more
versatile component for producing multilabelled RNA for NMR
studies. The label arrangements we report here are as follows: 13C
labelling of all ribose carbon atoms, deuteriation of the C5 of
pyrimidines and 15N labelling of the exocyclic amino groups of A, C
and G residues. All of these labels should be introduced at a level close
of 100% isotopic abundance in order to avoid interference from the
stray signals.
Experimental
All reagents were from Aldrich and, unless otherwise stated, were used
without further purification. Labelled 15NH4Cl(99% isotopic purity)
was from Martek Biosciences Corp. (USA), labelled KC15N (98%
isotopic purity) was from Aldrich. Adenosine deaminase was purchased
from Sigma (cat. no. A1030). The 1-O-acetyl-2,3,5-tri-O-toluoyl-b-d-
[1,2,3,4,5-13C5]-ribofuranose for the synthesis of labelled nucleosides
was synthesized according to our earlier procedure.34 Methylene
chloride, ethyl acetate, cyclohexane, hexane, acetone, methanol and
ethanol were distilled prior to use. Triethylamine and N,N-diisopropyl-
ethylamine (DIPEA) were distilled from CaH2 and stored over
(
molecular sieves (4 A). Toluene and pyridine were refluxed over CaH2
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MULTIPLY ISOTOPE LABELLED RIBONUCLEOSIDES
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for 6 h, distilled with exclusion of moisture and stored over molecular
(
sieves (4 A). THF was refluxed over CaH2 under nitrogen and distilled
immediately before use with rigorous exclusion of moisture. NMR
spectra were recorded on a JeolGX 270 spectrometer operating at
270.2 MHz for 1H, 67.9 MHz for 13C, 27.0 MHz for 15N and 109.4 MHz
for 31P. Chemicalshifts ( d) are reported in ppm relative to TMS
(internalstandard) for 1H and 13C, to [15N]-nitromethane for 15N and
H3PO4 (external) for 31P spectra.
Mass spectra were recorded with AMD 604 spectrometer, using FAB
technique with nitrobenzylacloholmatrix.
6-Chloro-N9-(2-tetrahydropyranyl)-purine (2). Anhydrous ethylacet-
ate (40 ml) was warmed to 508C and 6-chloropurine (1) (1.75 g,
11.31 mmol) and p-toluenesulfonic acid (68 mg, 0.026 mmol) were
added. The mixture was stirred and 2,3-dihydro-2H-pyran (1.35 ml,
1.24 g, 14.76 mmol) was added dropwise over 30 min, maintaining the
reaction temperature between 55 and 608C. The solution was stirred for
an additional hour during which time it was allowed to cool to room
temperature. Concentrated aqueous ammonia (2.3 ml) was added and
the solution stirred for 5 min. It was then extracted with water
(2 Â 20 ml). The ethyl acetate solution was dried over anhydrous
MgSO4 and the solvent removed in a rotary evaporator. The remaining
syrupy residue was purified by chromatography on SiO2 using a
gradient of methanol (0–4%) in methylene chloride giving 2 (2.43 g,
1
10.18 mmol, 90%). H-NMR (CDCl3): 8.76 (s, 1H), 8.35 (s, 1H), 5.81
(m, 1H), 3.81–4.20 (m, 2H), 1.50–2.24 (m, 6H); MS (FAB) 239.1; 241.0
(MH+).
N9-(2-Tetrahydropyranyl)-[6-15N]-adenine (3). A mixture of 2
(1.06 g, 4.44 mmol), 15NH4Cl(0.475 g, 8.89 mmo),l and NaHCO
3
(1.116 g, 13.29 mmol) in DMSO (6.75 ml) was sealed in a 15 ml vial
which was kept in an oven at 808C for 6 days. The cooled (08C) reaction
vial was opened carefully and the resulting suspension was poured into a
big Petri dish. The major part of the DMSO was removed by hot air
flushing. The residue was suspended in methylene chloride, transferred
onto a silica column and 0–4% gradient of methanol in methylene
chloride was applied to elute the pure product. Appropriate fractions
were collected and after removing solvents 3 was obtained (0.921 g,
1
4.20 mmol, 95%). H-NMR (CDCl3): 8.37 (s, 1H), 8.05 (s, 1H), 6.36
(d, JN2H ¼ 89:8 Hz, 2H) NH2, 5.72 (m, 1H), 3.78 (m, 2H), 1.63–2.14
(m, 6H). 13C-NMR (CDCl3): 155.4 (d, JN2C ¼ 20:1 Hz, C-6), 152.7,
149.2, 138.2, 119.4 (d, JN2C ¼ 4:0 Hz, C-5), 81.8, 68.7, 31.8, 24.8, 22.7.
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15N-NMR (CDCl3): À298.5. HRMS (FAB+) found 221.11590,
calculated for MH+ (C10H14ON415NÞ 221.11678.
[6-15N]-Adenine (4). To 3 (0.921 g, 4.18 mmol) dissolved in 1,4-
dioxane (15 ml) 0.01 m HCl(30 m)l was added and the pH was adjusted
to 1.5 with 1 m HCl solution. After 24 h the reaction mixture was diluted
with water (100 ml) and concentrated to 30 ml to remove dioxane
(repeated three times). It was neutralized with Dowex-1 (HCOÀ3 form)
(100 ml). The product was eluted from the resin with water (2 l) and
1
concentrated to 4 as yellowish solid that was not further purified. H-
NMR (DMSO-d6): 12.62 (br. s, 1H) NH-9, 8.13, 8.14 (2 Â s, 2H) H-2 &
H-8, 7.16 (d, JN2H ¼ 89:7 Hz, 2H), NH2. 13C-NMR (DMSO-d6): 155.2
(d, JN2C¼ 20:1 Hz, C-6), 152.4, 151.4, 139.4, 117.4. 15N-NMR (DMSO-
d6): À299.1. HRMS (FAB+) found 137.05843, calculated for MH+
(C5H6N415NÞ 137.05931.
N6-Benzoyl-[6-15N]-adenine (5). A mixture of 4 (4.18 mmol) and
benzoic anhydride (1.91 g 8.46 mmol) was heated at 1608C for 2 h, then
cooled, dissolved in ethanol (20 ml) and heated under reflux for 30 min.
The reaction mixture was concentrated to dryness and then purified by
short column chromatography on silica gel (gradient 0–10% of
methanol in methylene chloride) giving 5 (0.776 g, 3.24 mmol, 77%
1
from 3). H-NMR (DMSO-d6): 12.42 (br. s, 1H) NH-9, 11.55 (br. d,
JN2H¼ 72 Hz, 1H) NH-6, 8.76, (s, 1H), 8.53, (s, 1H), 8.14 (d, 2H), 7.68
(m, 1H), 7.58 (m, 2H). 13C-NMR (DMSO-d6): 166.5 (d, JN2C¼ 12:4 Hz,
C-6), 151.0, 145.9, 144.7 (br., C-4), 132.6, 128.4, 128.4, 114.5 (br., C-5).
15N-NMR (DMSO-d6): À243.5. HRMS (FAB+) found 241.08349,
calculated for MH+ (C12H10ON415NÞ 241.08557.
9-(b-d-Ribfuranosyl)- [2-amino-6-(N-methoxy)- amino]-[2-15N]-pur-
ine (8). Potassium 15N-cyanide (2 g, 30.23 mmol) was dissolved in
anhydrous methanol (500 ml), cooled to 08C and bromine (1.55 ml,
4.84 g, 30.26 mmol) was added with stirring. Stirring and cooling was
continued for 3 h, when solid 627 (5.62 g, 19.86 mmol) was added. The
suspension was stirred at 08C for 1 h and at room temperature for an
additional 8 h, and the solid became gradually dissolved. Volatiles were
evaporated and the residue was dried in vacuo. The crude 7 (12.5 g) was
dissolved in dry DMF (75 ml), and triethylamine (7 ml, 5.08 g,
50.2 mmol) was added. The flask was flushed with argon and the
mixture was left for 45 min in darkness. Then iodomethane (3 ml, 6.84 g,
48.2 mmol) was added and the mixture was left in darkness for 5 h. The
solvent was evaporated and the dry residue was dissolved in 0.25 n
NaOH (300 ml). After 1 h the base was neutralized with 1 m HCl
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(pH 7.5). An equal volume of ethanol was added, and the solution was
heated at 608C for 5 h. The solution was concentrated to 30 ml and was
applied onto column of Dowex 50 (H+) resin (6 Â 20 cm). The column
was washed with 0.05 m HCl(1)l, water (1 1) and then the product was
eluted with 3% aqueous ammonia (1 l). Evaporation of the ammonia
1
fraction gave 8 (4.5 g, 14.36 mmol, 72%) as a light brown foam. H-
NMR (DMSO-d6): 7.73 (s, 1H), 6.43 (br. d, JN2H¼ 78 Hz, 2H), 5.62 (d,
J¼ 5:7 Hz, 1H), 4.33 (m, 1H), 4.07 (m, 1H), 3.81 (m, 1H), 3.72 (s, 3H),
3.54 (m, 2H). 15N-NMR (DMSO-d6): –247.7 ppm. HRMS (FAB+)
found 314.13754, calculated for MH+ (C11H17N515NO5Þ 314.12295.
[2-15N]-Guanosine (9). The 15N labelled 9-(b-d-ribfuranosyl)purine
derivative 8 (4.1 g, 13.1 mmol) was dissolved in 0.1 m phosphate buffer at
pH 7.4 (300 ml) and adenosine deaminase (720 units) was added. The
reaction mixture was gently shaken at 378C for 90 h. After keeping at
08C overnight, the precipitate was filtered and washed with small
1
amount of water to afford guanosine 9 (2.61 g, 9.21 mmol, 69%). H-
NMR (D2O): 8.06 (s, 1H), 6.04 (br. d, 1H), 4.83 (t, 1H), 4.49 (t, 1H),
4.35 (m, 1H), 3.83 (m, 1H), 3.54 (m, 1H). 15N-NMR (D2O): À228.7. MS
(FAB+) 285.1 (MH+).
[2-15N]-Guanine (10). [2-15N]-Guanosine 9 (2.61 g, 9.1 mmol) was
suspended in 1 m HCland was stirred at 100 8C for 90 min. The reaction
mixture was cooled, neutralized with 1 m NaHCO3 (pH 7.5) and left
overnight at 48C. The precipitate was filtered, washed with cold water
and dried to give guanine 10 (1.05 g, 6.90 mmol, 76%). MS (FAB+)
found 153.05504, calculated for MH+ (C5H6N415NOÞ 153.05422.
N2-Isobutyryl-[2-15N]-guanine (11). Thoroughly dried (1008C, 1
torr) labelled guanine 10 (1.05 g, 6.90 mmol) was suspended in
dimethylacetamide (15 ml), isobutyric anhydride (3.5 ml, 3.34 g,
21.1 mmol) was added and the mixture was heated at 1508C for 2.5 h.
The solution was cooled and evaporated to dryness. The residue was co-
evaporated with methanol and recrystallized from ethanol-water. The
product 11 (1.15 g, 5.17 mmol, 75%) was separated as buff-coloured
1
crystals. H-NMR (DMSO-d6): 13.14 (br. s, 1H), 12.08 (s. 1H), 11.56
(br. d, JN2H¼ 79 Hz, 1H), 8.04 (s, 1H), 2.76 (sept, J¼ 6:8 Hz, 1H), 1.12
(d, 6, J¼ 6:8 Hz, 6H). 13C-NMR (DMSO-d6): 180.0 (d, JN2C¼ 10:4 Hz,
C(O)iBu), 147.3 (d, JN2C¼ 22 Hz, C-2), 34.7; 18.9. 15N-NMR (DMSO-
d6): À244.7. HRMS (FAB+) found 223.09620, calculated for MH+
(C9H12N415NO2Þ 223.09613.
N2-Isobutyryl-O6-diphenylcarbamoyl-[2-15N]-guanine (12). Dried 11
(1.15 g, 5.17 mmol) was suspended in dry DMF (6 ml), acetic anhydride
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(1.1 ml, 1.19 g, 11.65 mmol) was added and the mixture was heated at
1008C for 45 min. The clear solution was evaporated to dryness and the
solid residue was suspended in dioxane (15 ml) and stirred overnight.
Crystals were filtered and dried (1.16 g). The acetyl derivative was co-
evaporated with pyridine (3 Â 10 ml), suspended in pyridine (16 ml),
DIPEA (1.55 ml, 1.15 g, 8.8 mmol) and diphenylcarbamoyl chloride
(1.12 g, 4.8 mmol) were added and stirring was maintained at room
temperature for 1.5 h. Water (1.5 ml) was added, and after 10 min the
solution was evaporated. The residue was boiled with ethanol-water
(16 ml) and after cooling crystalline product 12 (1.72 g, 4.12 mmol, 79%)
was collected. 1H-NMR (DMSO-d6): 13.53 (br. s, 1H), 10.56
(d, JN2H¼ 89 Hz, 1H), 8.47 (s, 1H), 7.25–7.51 (m, 10H), 2.78 (sept,
J¼ 6:8 Hz, 1H), 1.09 (d, J¼ 6:8 Hz, 6H). 13C-NMR (DMSO-d6): 174.7
(d, JN2C¼ 10 Hz, C(O)iBu), 152.2 (d, JN2C¼ 21:5 Hz, C-2), 150.3
(6-C(O)O), 141.9; 129.6, 129.3, 127.5, 34.6, 19.2. 15N-NMR (DMSO-
d6): À235.7. HRMS (FAB+) found 418.16335, calculated for MH+
(C22H21N515NO3Þ 418.16455.
Synthesis of 20,30,50-tri-O-(4-toluoyl) [10,20,30,40,50-13C5]-nucleosides
13a–d were carried out according to the reported procedure34.
20,30,50-Tri-O-toluoyl-N6- benzoyl-[(10,20,30,40,50-13 C5)-(6-15N)]-aden-
osine (13a). Labelled acylated ribose (2.34 g, 4.25 mmol) and compound
5 (1.22 g, 5.1 mmol) gave 13a (2.35 g, 3.14 mmol, 74% on the basis of
1
labelled sugar) as a foam. H-NMR (CDCl3): 8.97 (d, JN2H¼ 89 Hz,
1H), 8.73 (s, 1H), 8.17 (s, 1H), 8.02–7.15 (m, 17H), 6.55 (br. d,
JC2H¼ 166 Hz, 1H), 6.37 (br. d, JC2H¼ 156 Hz, 1H), 6.22 (br. d,
JC2H¼ 158 Hz, 1H), 4.83 (br. d, JC2H¼ 151 Hz, 1H), 4.90 (br. d,
JC2H¼ 153 Hz, 1H), 4.68 (br. d, JC2H¼ 148 Hz, 1H). 13C-NMR
(CDCl3): 165.3 (d, JC2N¼ 16:5 Hz, C(O)Bz), 149.6 (d, JC2N¼ 19:8 Hz,
C-6), 86.6 (d, JC2C¼ 43:6 Hz, C-10); 80.9 (dd, JC2C¼ 38:9 Hz,
JC2C¼ 43:1 Hz, C-40), 73.6 (dd, JC2C¼ 37:5 Hz, JC2C¼ 43:6 Hz,
C-20); 71.2 (dd, JC2C¼ 37:5 Hz, JC2C¼ 38:9 Hz, C-30), 63.2, (d, JC2C
¼
43:1 Hz, C-50). 15N-NMR (CDCl3): À248.0. HRMS (FAB+)
found 732.26871, calculated for MH+ (C3615C5H36N415NO8Þ
732.26994.
20,30,50-Tri-O-(4-toluoyl)-N2-isobutyryl-O6-(diphenylcarbamoyl)-[(10,
20,30,40,50-13C5)-(2-15N)]-guanosine (13b). Labelled acylated ribose
(1.98 g, 3.58 mmol) and compound 12 gave 13b (1.95 g, 2.15 mmol,
60%) as a foam. 1H-NMR (CDCl3): 8.20 (d, JH2N¼ 89 Hz, 1H), 8.07 (s,
1 H, H-8), 7.95–7.13 (m, 22H), 6.34 (br. d, JC2H¼ 151 Hz, 1H), 6.30 (br.
d, JC2H¼ 158 Hz, 2H) 4.86 (br. d, JC2H¼ 149 Hz, 2H), 4.68 (br. d,
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1
JC2H¼ 147 Hz, 1H), 2.47, 2.41, 2.37 (3 Â s, 3 Â 3 H, toluoyl). C-NMR
(DMSO-d6): 174.7 (d, JN2C¼ 10 Hz, C(O)iBu), 152.2 (d,
JN2C¼ 21:5 Hz, C-2), 150.3 (6-C(O)O), 141.9, 129.6, 129.3, 127.5, 87.0
(d, JC2C¼ 43:6 Hz, C-10), 80.9 (dd, JC2C¼ 37:1 Hz;JC2C¼ 43:7 Hz,
C-40), 74.1 (dd, JC2C¼ 43:6 Hz, JC2C¼ 39:5 Hz, C-20), 71.4 (dd,
JC2C¼ 39:5 Hz, JC2C¼ 37:1 Hz C-30), 63.6 (d, JC2C¼ 43:7 Hz, C-50),
34.6, 19.2, 15N-NMR (CDCl3): À238.7.
20,30,50-Tri-O-(4-toluoyl)-[10,20,30,40,50-13C5]-uridine (13c). Labelled
acylated ribose (8.32 g, 15.10 mmol) and uracil (2.03 g, 18.20 mmol) gave
1
13c (7.28 g, 12.07 mmol, 80%) as white foam. H NMR(CDCl3) : 8.79
(s, 1H), 7.95–7.13 (m, 13 H, touoyl, H-6), 6.30 (br. d, JC2H¼ 167 Hz,
1H) H-l0, 5.89 (br. d, JC2H¼ 163 Hz, 1H) H-30, 5.66 (m, 1H) H-20, 5.58
(d, JH62H5¼ 6 Hz, 1H) H-5, 4.74 (br. d, JC2H¼ 150 Hz, 1H) H-50, 4.62
(m, 1H) H-40, 4.55 (br. d, JC2H¼ 153 Hz, 1H) H-500, 2.41, 1.39, 2.36
(3 Â s, 3 Â 3 H, toluoyl). 13C-NMR (CDC13): 87.6 (d, JC2C ¼43:7 Hz,
C-l0), 80.8, (dd, JC2C¼ 39:5 Hz, JC2C ¼ 43:1 Hz, C-40), 73.6 (dd,
JC2C¼ 43:7 Hz, JC2C¼ 40:5 Hz, C-20), 71.1 (dd, JC2C¼ 40:5 Hz,
JC2C¼ 39:5 Hz, C-30), 63.6, (d, JC2C¼ 43:1 Hz, C-50).
20,30,50-Tri-O-(4-toluoyl)-N2-isobutyryl-[(10,20,30,40,50-13C5)-(2-15N)]
-guanosine (13d). The diphenylcarbamoyl derivative 13b (3.63 g,
4.00 mmol) was dissolved in 90% CF3COOH and left at room
temperature for 15 min. Volatiles were evaporated, the remaining oil
was dissolved in CH2Cl2 (20 ml) and washed with NaHCO3, water,
dried (MgSO4) and evaporated to a foam, which was purified by column
chromatography (methylene chloride with 0–2% methanol) to yield 13d
1
(2.37 g, 3.32 mmo1, 83%). H-NMR (CDCl3): 9.3 (d, JN2H¼ 91 Hz,
1H), 8.0–7.14 (m, 13 H, toluoyl, H-8), 6.55 (br. d, JC2H¼ 157 Hz, 1H),
6.33 (br. d, JC2H¼ 160 Hz, 1H), 6.16 (br. d, JC2H¼ 167 Hz, 1H), 4.85
(m, 3H), 2.67 (sept, J¼ 6:9 Hz, 1H), 2.41, 2.39 (2 Â s, 3+6 H, toluoyl),
1.31 (d, J¼ 6:9 Hz, 6H). 13C-NMR (CDCl3): 165.2 (d, JN2C¼ 16:5 Hz,
C(O)iBu), 155.3, 149.6 (d, JN2C¼ 19:7 Hz, C-2), 147.8, 138.4, 122.6,
87.7 (d, JC2C¼ 44:1 Hz, C-l0), 80.2 (dd, JC2C¼ 35:9 Hz, JC2C¼ 39:2 Hz,
C-40), 73.1 (dd, JC2C¼ 43:6 Hz, JC2C¼ 39:5 Hz, C-20), 70.8 (dd,
JC2C¼ 39:5 Hz, JC2C¼ 37:1 Hz, C-30), 62.9 (d, JC2C¼ 43:7 Hz, C-50.
15N-NMR (CDCl3): À245.3. HRMS (FAB+) found 714.28223,
calculated for MH+ (C3313C5H38N415NO9) 714.28049.
N6-Benzoyl-[(10,20,30,40,50-13C5)-(6-15N)]-adenosine (14a). Com-
pound 13a (4.96 g, 6.63 mmol) was dissolved in ethanol-pyridine
(20+20 ml), 2 m NaOH and ethanol(24+24 m)l were added and the
mixture stirred for 6 min at room temperature. Dowex 50 resin
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(pyridinium form) was added to neutralize the base (approx. 80 ml). The
suspension was filtered, the resin was washed with ethanol and pyridine
(100+100 ml) and the filtrate was evaporated. The solid residue was
triturated with diethylether (2 Â 30 ml) and methylene chloride
(2 Â 30 ml) and filtered. The product 14a (2.49 g, 99%) was obtained
1
as a white solid and it was used without further purification. H-NMR
(DMSO-d6): 11.26 (br, 1H), 8.78, 8.74 (2 Â s, 2 Â 1H), 6.06 (br. d,
JC2H¼ l65 Hz), 4.68 (br. d, JC2H¼ 142 Hz), 4.20 (br. d, JC2H¼ 144 Hz),
4.00 (br. d, JC2H¼ 149 Hz), 3.70 (2 Â m, JC2H¼ 152 Hz), 3.60 (2 Â M,
JC2H¼ 145 Hz); 13C-NMR (DMSO-d6): 165.5 (d, JC2N¼ 12:9 Hz, C(O)
benzoyl), 152.4, 151.8, 150.6 (d, JC2N¼ 19:6 Hz, C6), 143.3, 133.6,
132.6, 128.5, 126.0, 87.7 (d, JC2C¼ 42:0 Hz, Cl0), 85.8 (dd,
JC2C¼ 40:1 Hz, JC2C¼ 37:0 Hz, C40), 73.7 (dd, JC2C¼ 41:7 Hz,
JC2C¼ 37:6 Hz, C20), 70.4 (JC2C¼ 37:4 Hz, 41.0 Hz, C30), 61.3 (d,
JC2C¼ 40:1 Hz, C50) 15N-NMR (DMSO-d6): À242.1. HRMS (FAB+)
found 378.14430, calculated for MH+ (C1213C5H18N415NO5Þ
378.14445.
N2-Isobutyryl-[(10,20,30,40,50-13C5)-(2-15N1)]-guanosine (14d). The
title compound 14d (0.898 g, 2.49 mmol, 74%) was obtained from 13d
(2.40 g, 3.37 mmol) as described for 14a. It was used without further
1
purification. H NMR (DMSO-d6): 12.2 (br. s, 1H), 11.7 (d, JN2H
¼
91 Hz), 8.28 (s, 1H), 5.82 (br. d, JC2H¼ l69 Hz, 1H), 4.45 (br. d,
JC2H¼ 152 Hz, 1H), 4.16 (br. d, JC2H¼ 151 Hz, 1H), 3.93 (br. d,
JC2H¼ 150 Hz, 1H), 3.60 (2 Â m, 2H), 2.78 (sept, J¼ 6:9 Hz, 1H), 1.13
(d, J¼ 6:9 Hz, 6H); 13C NMR (DMSO-d6): 180.5 (d, JN2C¼ 11:5 Hz,
C(O)Bz), 155.2, 149.1, 148.4 (d, JN2C¼ 21:8 Hz, C2), 138.0, 120.2, 86.8
(d, JC2C¼ 42:7 Hz, Cl0), 85.5 (dd, JC2C¼ 38:1 Hz, JC2C¼ 43:6 Hz, C40),
74.2 (dd, JC2C¼ 43:3 Hz, JC2C¼ 39:7 Hz, C20), 70.4 (dd,
JC2C¼ 39:7 Hz, JC2C¼ 38:1 Hz, C30), 61.3 (d, JC2C¼ 43:7 Hz, C50);
15N-NMR (DMSO-d6): À245.0. HRMS (FAB+) found 360.15612,
calculated for MH+ (C913C5H20N415NO6Þ 360.15500.
[(10,20,30,40,50-13C5)-(5-2H)]-Uridine (15). Compound 13c (7.24 g,
11.99 mmol) was dissolved in methanolic ammonia (23% w/w, 190 ml)
and was left for 60 h at ambient temperature. The solvent was
evaporated and the residue was partitioned between dichloromethane
and water. The water phase was washed once with dichloromethane and
evaporated to dryness. The residue (14c) was dissolved in D2O (15 ml),
evaporated to dryness, and the procedure was repeated. After dissolving
the solid in D2O (20 ml), anhydrous potassium carbonate (1.88 g,
13.60 mmol) was added and the solution was heated in an oil bath
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(958C) with protection from atmospheric moisture. Deuteriation was
followed by NMR. After 34 h the dark solution was cooled, water
(50 ml) was added and the base was neutralized with Dowex 50
(pyridinium form). The resin was washed with water, methanoland
pyridine (100 mleach) and the eulates were evaporated to give
(2.93 g, 11.72 mmol, 97%) as a brown foam which was used for the next
15
1
steps without further purification. H-NMR (D2O): 7.42 (s, 1H), 5.91
(br. d, JC2H¼ 167 Hz, 1H), 5.72 (trace, 0.04H), 4.34 (br. d,
JC2H¼ 156 Hz, 1H), 4.27 (br. d, JC2H¼ 146 Hz, 1H), 4.12 (br. d,
JC2H¼ 154 Hz, IH), 3.94 (br. d, JC2H¼ 141 Hz, 1H), 3.79 (br. d,
JC2H¼ 141 Hz, 1H). 13C-NMR (D2O): 89.5 (d, JC2C¼ 43:1 Hz, C-l0),
84.4 (dd, JC2C¼ 38:8 Hz, JC2C¼ 38:8 Hz, C-40), 73.8 (dd,
JC2C¼ 37:8 Hz, JC2C¼ 42:5 Hz, C-20), 69.5 (dd, JC2C¼ 38:3 Hz,
JC2C¼ 42:0 Hz, C-30), 60.9 (d, JC2C¼ 42:0 Hz, C-50). MS (FAB+)
found 251.00985, calculated for MH+ (C413C5H12DN2O6) 251.10029.
20,30,50-Tri-O-acetyl-O4-(2-nitrophenyl)-[(10,20,30,40,50-13C5)-(5-2H)]-
uridine (16). Uridine 15 (2.1 g, 8.39 mmol) was acetylated with acetic
anhydride (6 ml, 6.49 g, 63 mmol), to give tri-O-acetyl derivative35
(2.60 g, 6.91 mmol), which was dissolved in dry dichloromethane
(60 ml). Triethylamine (8.45 ml, 6.13 g, 60.2 mmol), mesitylenesulfonyl
chloride (4.0 g, 18.29 mmol) and DMAP (0.125 g, 1.02 mmol) were
added to the solution. After stirring at room temp. for 1 h, 2-
nitrophenol(3.95 g, 28.39 mmo)l and DABCO (0.14 g) were added
and stirring was continued for 2 h. The reaction mixture was quenched
with satd. NaHCO3, the organic layer was separated, the water phase
was extracted with methylene chloride (2 Â 50 ml). The combined
extracts were dried (MgSO4) and evaporated. Chromatography (silica
gel, methylene chloride À3% methanol) gave 16 (3.08 g, 6.19 mmol,
74%) as light yellow foam. 1H-NMR (CDCl3): 8.14–7.31 (m, 5H), 6.28
(d, J=7.4 Hz, ꢀ0.04H), 6.10 (br. d, JC–H=172 Hz, 1H), 5.35 (2 Â m,
JC–H=149 Hz, 2H), 4.39 (2 Â m, JC–H=148 Hz, 3H), 2.16, 2.10 (2 Â s,
6+3H). 13C-NMR (CDCl3): 89.2 (d, JC–C=44.0 Hz, C-l0), 80.1 (dd,
JC–C=39.9 Hz, JC–C=42.5 Hz, C-40), 73.8 (dd, JC–C=39.7 Hz,
JC–C=39.1 Hz, C-20), 69.9 (dd, JC–C=42.6 Hz, JC–C=39.9 Hz, C-30),
62.9 (d, JC–C=43.6 Hz, C-50). HRMS (FAB+) found 498.14911, calcd.
for MH+ (C1613C5H21DN3O11) 498.14829.
N4-Acetyl-[(10,20,30,40,50-13C5)-(4-15N)-(5-2H)]-cytidine (18). La-
belled 15NH4Cl(3.0 g, 55 mmo)l and K 2CO3 (7.62 g, 55.13 mmol) were
suspended in DMSO (45 ml) and the mixture heated in a pressure flask
at 808C for 30 min. After cooling in ice, the flask was opened and 4-O-
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(2-nitrophenyl)-uridine 16 (2.44 g, 4.89 mmol) was added. The closed
flask was heated at 808C for 45 h. After cooling the content of the flask
was purged with nitrogen, which was trapped in 10% HCl. (After
evaporating this solution 1 g of labelled 15NH4Clwas recovered). The
DMSO solution was filtered, the solid was washed with pyridine
(150 ml) and the filtrates were concentrated to approx. 70 ml and
evaporated three times with water (150 ml). The remaining solution was
applied onto a column of Dowex 50 (H+) (130 ml). The column was
washed with 0.05 m HCl (500 ml), water (500 ml) and 3% NH3 (750 ml).
The ammonia solution was evaporated, leaving cytidine 17 (1.3 g) as a
yellowish foam. The foam was dissolved in DMF (25 ml), acetic
anhydride (0.51 ml, 0.55 g, 53.6 mmoles) was added and the whole was
left for 24 h. Solvent was evaporated, the residue boiled with methanol
40 ml) and cooled. Crystals were filtered and dried to furnish acetylated
1
cytidine 18 (1.18 g, 82%). H-NMR (DMSO-d6): 8.36 (s, 1H); 5.91
(d, JC2H¼ 175 Hz, 1H), 4.29 (d, JC2H¼ 140 Hz, 1H), 4.20 (2 Â m,
JC2H¼ 146 Hz, 2H), 3.94 (2 Â m, JC2H¼ 144 Hz, 2H); 2.25 (s, 3H).
13C NMR (DMSO-d6): 90.5 (d, JC2C¼ 42:5 Hz, C-50), 84.0 (dd,
JC2C¼ 39:4 Hz, JC2C¼ 41:1 Hz, C-40), 74.1 (dd, JC2C¼ 37:9 Hz,
JC2C¼ 39:1 Hz, C-20), 69.4 (dd JC2C¼ 38:4 Hz, JC2C¼ 41:1 Hz, C-30),
60.9 (d, JC2C¼ 42:1 Hz, C-50) 15N-NMR: À233.5. HRMS (FAB+)
found 293.12307 calculated for MH+ (C613C5H15DN215NO6Þ
293.12386.
50-O-Dimethoxytrityl-N6-benzoyl-[(10,20,30,40,50-13C5)-(6-15N)]-aden-
osine (19a). Adenosine 14a (2.46 g, 6.52 mmol) was evaporated with
pyridine (3 Â 15 ml), dissolved in pyridine (30 ml) and dimethoxytrityl
chloride (2.54 g, 7.5 mmol) was added. After 18 h the solvent was
evaporated, the residue was dissolved in methylene chloride, washed
with sat. NaHCO3, then water and dried with MgSO4. Evaporation of
solvent and chromatography (methylene chloride-3% methanol) gave
1
the product 19a (3.37 g, 4.96 mmol, 76%) as a yellow foam. H-NMR
(CDCl3): 9.15 (d, JN2H¼ 90 Hz, 1H), 8.67 (s, 1H), 8.24 (s, 1H),
8.01–6.71 (m, 18H), 6.08 (br. d, JC2H¼ 167 Hz, 1H), 4.93 (m, 1H), 4.48
(br. d, JC2H¼ 154 Hz, 1H), 4.40 (br. d, JC2H¼ 146 Hz, 1H), 3.74
(s, 6H), 3.46 (m, 1H), 3.33 (m, 1H). 13C-NMR (CDCl3): 165.9
(d, JN2C¼ 13 Hz, C(O)15NH), 150.6 (d, JN2C ¼ 19:5 Hz, C-6), 90.4
(d, JC2C¼ 41:7 Hz, C-l0), 85.8 (dd, JC2C¼ 37:1 Hz, JC2C¼ 43:1 Hz,
C-40), 75.6 (dd, JC2C¼ 41:6 Hz, JC2C¼ 37:6 Hz, C-20), 72.2 (dd,
JC2C¼ 37:6 Hz, JC2C¼ 37:2 Hz, C-30), 63.5 (d, JC2C¼ 43:2 Hz, C-50);
15N-NMR (CDC13): À248.8; MS (FAB) 680.1 (MH+).
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MULTIPLY ISOTOPE LABELLED RIBONUCLEOSIDES
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50-O-Dimethoxytrityl-N4-acetyl-[(10,20,30,40,50-13C5)-(4-15N)-(5-2H)]-
cytidine (19b). Compound 18 (1.175 g, 4.03 mmol) was dimethoxytri-
tylated as described for adenosine 14a to yield 19b (1.70 g, 2.86 mmol,
1
71%). H-NMR(CDCl3): 9.01 (br. d, JH2N¼ 89 Hz, 1H), 8.21 (s, 1H),
5.87 (br. d, JC2H¼ 173 Hz, 1H), 4.42 (br. d, JC2H¼ 147 Hz, 3H), 3.42
(m, 2H). 13C-NMR (CDCl3): 93.6 (d, JC2C¼ 40:0 Hz, C-10), 86.0 (dd,
JC2C¼ 39:9 Hz, JC2C¼ 42:6 Hz, C-40), 77.1 (dd, JC2C¼ 40:0 Hz,
JC2C¼ 37:4 Hz, C-20), 71.8 (dd, JC2C¼ 37:4 Hz, JC2C¼ 39:9 Hz, C-30),
62.9 (d, JC2C¼ 42:6 Hz, C-50); 15N-NMR (CDCl3): À233.0. HRMS
(FAB+) found 595.25532, calculated for MH+ (C2713C5H33DN215NO8Þ
595.25444.
50-O-Dimethoxytrityl-[(10,20,30,40,50-13C5)-(5-2H)]-uridine (19c). Com-
pound 15 (0.90 g, 3.60 mmol) was dimethoxytritylated as described for
1
14a, giving 19c (1.65 g, 2.98 mmol, 83%) as yellow foam. H-NMR
(CDCl3): 7.96 (s, 1H), 5.88 (br. d, JC2H¼ 172 Hz, 1H), 5.33 (trace,
ꢀ0.04H), 4.42 (br. d, JC2H¼ 151 Hz, 1H), 4.34 (br. d, JC2H¼ 152 Hz,
1H), 4.19 (br. d, JC2H¼ 150 Hz, 1H), 3.50 (2 Â m, JC2H¼ 150 Hz, 2H).
13C-NMR (CDCl3): 90.7 (d, JC2C¼ 41:1 Hz, C-l0), 84.0 (dd,
JC2C¼ 37:9 Hz, JC2C¼ 43:1 Hz, C-40), 75.6 (dd, JC2C¼ 41:1 Hz,
JC2C¼ 39:4 Hz, C-20), 69.9 (dd, JC2C¼ 39:4 Hz, JC2C¼ 37:9 Hz, C-30),
62.0 (d, JC2C¼ 43:1 Hz, C-50). HRMS (FAB+) found 553.23192,
calculated for MH+ (C2513C5H29DN2O8) 553.23087.
50- O- Dimethoxytrityl-[(10,20,30,40,50-13C5)-(2-15N)]-N2-isobutyrylgu-
anosine (19d). Compound 14d (1.37 g, 3.82 mmol) was dimethoxytrity-
lated as described for 14a, giving 19c (2.31 g, 2.75 mmol, 72%) as yellow
foam. 1H-NMR (CDCl3): 7.70 (s, 1H), 5.84 (br. d, JC2H¼ 171 Hz, 1H),
5.28 (br. d, JC2H¼ 151 Hz, 1H), 4.56 (br. d, JC2H¼ 152 Hz, 1H), 4.29
(br. d, JC2H¼ 151 Hz, 1H), 3.49 (m, 1H), 3.09 (br. d, JC2H¼ 155 Hz,
1H), 1.90 (m, 1H), 0.93, 0.65 (2 Â d, J¼ 7 Hz, 2 Â 3H). 13C-NMR
(CDCl3): 147.3 (d, JN2C¼ 18 Hz, C-2), 90.1 (d, JC2C¼ 43:5 Hz, C-l0)
85.2 (dd, JC2C¼ 38:0 Hz, JC2C¼ 43:6 Hz, C-40), 72.6 (dd, JC2C
¼
0
43:5 Hz, JC2C¼ 39:6 Hz, C-2 ), 71.3 (dd, JC2C¼ 39:6 Hz, JC2C¼ 38:1 Hz,
C-30), 63.7 (d, JC2C¼ 43:7 Hz, C-50);. 15N-NMR (CDCl3): À245.3.
HRMS (FAB+) found 662.27886, calculated for MH+
(C3013C5H37N415NO8Þ 662.28558.
50- O- Dimethoxytrityl-20- O -t-butyldimethylsilyl-N6-benzoyl-[(10,20,30,
40,50-13C5)-(6-15N)]-adenosine (20a). Dried 19a (3.52 g, 5.17 mmol) was
dissolved in THF (40 ml), and pyridine (0.95 ml) was added, followed by
silver nitrate (1.475 g, 8.68 mmol). The mixture was stirred for 20 min
and t-butyldimethylchlorosilane (1.36 g, 9.02 mmol) was added. The
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reaction mixture was stirred in darkness for an additional6 h. The
resulting suspension was filtered through a pad of Celite, washed with
methylene chloride and the filtrates were evaporated. The oily residue
was dissolved in methylene chloride, the solution was washed with
NaHCO3, dried with MgSO4 and evaporated. Chromatography (silica
gel, benzene-ethyl acetate, gradient 12–18%) gave the desired com-
pound 20a (2.16 g, 2.73 mmol, 53%) together with the 30-OSi isomer
1
(0.45 g, 11%). H-NMR (CDCl3): 8.98 (d, JN2H¼ 89 Hz, 1H), 8.74
(s, 1H), 8.24 (s, 1H), 8.04–6.80 (m, 18H), 6.11 (br. d, JC2H¼ 166 Hz,
1H), 5.02 (br. d, JC2H¼ 149 Hz, 1H), 4.37 (br. d, JC2H¼ 151 Hz, 1H),
4.28 (br. d, JC2H¼ 150 Hz, 1H), 3.78 (s, 6H), 3.55 (2 Â m, 1H), 3.42
(2 Â m, 1H), 0.85 (s, 9H), 0.147, À0.005 (2 Â s, 2 Â 3H). 13C-NMR
(CDCl3): 164.6 (d, JN2C¼ 13:2 Hz, C(O)Bz), 149.7 (d, JN2C¼ 20 Hz,
C-6), 88.5 (d, JC2C¼ 43:1 Hz, C-l0), 84.3 (dd, JC2C¼ 36:9 Hz,
JC2C¼ 43:1 Hz, C-40), 75.8 (dd, JC2C¼ 43:1 Hz, JC2C¼ 37:4 Hz, C-20),
71.6 (dd, JC2C¼ 37:4 Hz, JC2C¼ 36:9 Hz, C-30), 63.3 (d, JC2C¼ 43:1 Hz,
C-50); 15N-NMR (CDCl3): À252.8. MS (FAB) 793.1 (MH+).
50-O-Dimethoxytrity1-20-O-t-butyldimethylsilyl-N4-acety1-[(10,20,30,40,
50-13C5)-(4-15N)-(5-2H)]-cytidine (20b). Compound 19b (2.13 g,
3.58 mmol) was silylated as described for 20a, giving 20b (1.32 g,
1.86 mmol, 52%) as a colorless foam. 1H-NMR (CDCl3): 9.62 (d, JN2H
¼ 89 Hz, 1H, NH-4), 8.45 (s, 1H, H6), 7.45–6.85 (m, 13H), 5.95 (br. d,
JC2H¼ 176 Hz, 1H), 4.40 (br. d, JC2H¼ 149 Hz, 1H), 4.32 (br. d,
JC2H¼ 156 Hz, 1H), 4.12 (2 Â m, 1H) 3.81 (s, 6H), 3.61 (2 Â m, 1H),
3.51 (m, 1H), 0.95 (s, 9H), 0.30 (s, 3H), 0.19 (s, 3H); 13C-NMR (CDCl3):
170.8 (d, JN2C¼ 11:3 Hz, COCH3), 162.9 (d, JN2C¼ 18:3 Hz, C-4),
155.4 (C-2), 97.5 (br., low intensity, C-5), 90.8 (d, JC2C¼ 42:0 Hz, C-10),
83.3 (dd, JC2C¼ 39:7 Hz, JC2C¼ 43:3 Hz, C-40), 76.7 (dd, JC2C¼ 42:0 Hz,
JC2C¼ 37:9 Hz, C-20), 69.2 (dd, JC2C¼ 37:9 Hz, JC2C¼ 39:7 Hz, C-30), 61.5
(d, JC2C¼ 43:3 Hz, C-50). 15N-NMR (CDCl3): À233.6. MS (FAB) 709
(MH+).
50- O- Dimethoxytrityl - 20-O -t -butyldimethylsilyl-[(10,20,30,40,50-13C5)-
(5-2H)]-uridine (20c). Compound 19c (1.68 g, 3.05 mmol) was silylated
as described for 20a, giving 20c (1.36 g, 2.04 mmol, 67%) as colorless
foam. 1H-NMR (CDCl3): 7.91 (s, 1H, H6), 7.40–6.83 (m, 13H), 5.95 (br.
d, JC2H¼ 173 Hz, 1H), 4.35 (br. d, JC2H¼ 151 Hz, 2H), 4.07 (m, 1H),
3.80 (s, 6H), 3.50 (2 Â m, JC–H=146 Hz, 2H), 0.93 (s, 9H), 0.19, 0.16
(2 Â s, 2 Â 3H). 13C-NMR (CDCl3): 102 (br., low intensity, C-5), 88.8 (d,
JC2C¼ 43:1 Hz, C-l0), 76.4 (dd, JC2C¼ 43:1 Hz, JC2C¼ 36:9 Hz, C-20),
70.5 (dd, JC2C¼ 36:9 Hz, JC2C¼ 41:0 Hz, C-30), 83.6 (dd, JC2C
¼
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41:0 Hz, JC2C¼ 43:1 Hz, C-40), 62.4 (d, JC2C¼ 43:1 Hz, C-50). MS
(FAB) 667.1 (MH+).
50- O- Dimethoxytrityl - 20-O-t-butyldimethylsilyl-N2-isobutyryl-[(10,20,
30,40,50-13C5)-(2-15N)]-guanosine (20d). Compound 19d (2.29 g,
3.47 mmol) was silylated as described for 20a, giving 20d (1.54 g,
1
1.98 mmol, 57%) as a colorless foam. H-NMR (CDCl3):7.70 (s, 1H),
7.55–6.76 (m, 13H), 5.72 (br. d, JC2H¼ 165 Hz, 1H), 5.30 (br. d,
JC2H¼ 144 Hz, 1H), 4.37 (br. d, JC2H¼ 151 Hz, 1H), 4.22 (br. d,
JC2H ¼ 150, 1H), 3.74 (s, 6H), 3.55 (m, 1H), 3.00 (2 Â m,
JC2H¼ 142 Hz, 1H), 1.29–1.15 (m, 1H), 0.83 (s, 9H), 0.78, 0.50 (2 Â d,
J ¼ 7 Hz, 2 Â 3H), 0.017, À0.19 (2 Â s, 2 Â 3H). 13C-NMR (CDCl3):
160.9 (d, JN2C¼ 16 Hz, C(O)iBu), 149.8 (d, JN2C¼ 19 Hz, C-2), 90.5 (d,
JC2C¼ 43:6 Hz, C-l0), 86.1 (dd, JC2C¼ 39:9 Hz, JC2C¼ 43:6 Hz, C-40),
74.2 (dd, JC2C¼ 43:6 Hz, JC2C¼ 39:4 Hz, C-20), 72.3 (dd, JC2C¼ 39:4 Hz,
JC2C¼ 39:9 Hz, C-30), 63.2 (d, JC2C¼ 43:6 Hz, C-50). 15N-NMR
(CDCl3): À245.0. MS (FAB) 776.2 (MH+).
50-O-Dimethoxytrityl-20-O-t-butyldimethylsilyl-[10,20,30,40,50-13C5]-nu-
clcoside phosphoramidites (21a–d). General procedure: The nucleoside
20a–d (1 mmol) was dissolved in dry THF (5 ml) under nitrogen, DIPEA
(0.35 ml, 0.259 g, 2 mmol) was added, followed by dropwise addition of
2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (0.90 ml, 0.945 g,
4 mmol). The reaction proceeded at room temperature for 2–4 h. After
complete disappearance of substrate 20a–d (TLC), methanol(1 m)l was
added and after 10 min the reaction mixture was diluted with methylene
chloride (35 ml), washed with NaHCO3, sat. NaCland dried with
MgSO4. Solvents were evaporated and the crude phosphoramidite was
chromatographed on silica gel. Appropriate fractions were combined,
evaporated, dissolved in minimal amount of methylene chloride
containing 1% Et3N and added dropwise to vigorously stirred
anhydrous hexane at À708C. The precipitated solid was centrifuged,
the supernatant was decanted and the solid dried at 0.5 torr for 48 h.
50- O- Dimethoxytrityl - 20-O-t-butyldimethylsilyl-N6-benzoyl-[(10,20,30,
40,50-13C5)-(6-15N)]-adenosine 30-O-(2cyanoethyl N,N-diisopropylpho-
sphohramidite) (21a). Nucleoside 20a (1.12 g, 1.41 mmol) was converted
into phosphoramidite 21a (0.91 g, 0.92 mmol, 65%). Chromatography:
cyclohexane-3% Et3N with gradient of methylene chloride from 20% to
35%. 31P-NMR(CDC13): 151.8, 151.6, 149.9, 149.6.
50- O- Dimethoxytrityl-20-O-t-butyldimethylsilyl-N4-acetyl-[(10,20,30,40,
50-13C5)-(4-15N)-(5-2H)]-cytidine 30-O-(2-cyanoethyl N,N-diisopropyl-
phosphohramidite) (21b). Nucleoside 20b (0.98 g, 1.38 mmol) was
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converted into phosphoramidite 21b (0.78 g, 0.85 mmol, 62%).
Chromatography: cyclohexane-3% Et3N with gradient of methylene
chloride from 10% to 35%. 31P-NMR (CDCl3): 150.9, 150.8, 149.9,
149.7.
50- O - Dimethoxytrityl - 20-O-t-butyldimethylsilyl -[(10,20,30,40,50-13C5)-
(5-2H)]-uridine 30-O-(2-cyanoethyl N,N-diisopropylphosphoramidite)
(21c). Nucleoside 20c (0.83 g, 1.24 mmol) was converted into phos-
phoramidite 21c (0.82 g, 0.95 mmol, 76%). Chromatography: cyclohex-
ane-3% Et3N with gradient of methylene chloride from 20% to 35%.
31P-NMR (CDCl3): 150.8, 150.4, 150.2.
50- O - Dimethoxytrityl -20-O-t-butyldimethylsilyl-N2-isobutyryl - [(10,20
30,40,50-13C5)-(2-15N)]-guanosine 30-O-(2-cyanoethyl N,N-diisopropyl-
phosphohramidite) (21d). Nucleoside 20d (1.21 g, 1.56 mmol) was
converted into phosphoramidite 21d (0.99 g, 1.01 mmol, 65%). Chro-
matography: cyclohexane-3% Et3N with gradient of methylene chloride
from 20 to 50%. 31P-NMR (CDCl3): 151.6, 149.5, 149.3.
Results and discussion
Recently we have reported34 the synthesis of ribonucleosides with [13C5]-
labelled ribose residues and introduced them into the TAR–RNA
oligonucleotide at specifically chosen sites. The aim of the present study
was to extend this approach to construct nucleoside building blocks,
which are labelled with different isotopes in different residues of the
molecule. Considering the fact that the cost and availability of the label
is the limiting factor for the synthesis, we chose the following synthetic
procedures.
Since in the case of purine nucleosides the introduction of the 15N
label is a multi-step process with moderate yields,29,36–38 performing
these reactions on 13C labelled nucleoside leads to the loss of the
valuable 13C label. This prompted us to choose an optimal convergent
approach in which the 15N-labelled aglycon is prepared first followed by
coupling to the [13C5]-labelled ribose moiety.
In the case of pyrimidine nucleosides the introduction of deuterium
into the C5 position of the uridine is very simple and efficient.39–41
Moreover, the 13C labelled, deuteriated uridine is the starting material
for the 4-[15NH2]-labelled cytidine. The conversion of the uridine into
cytidine and 15N labelling occur in the same reaction. This means that
one coupling reaction of 13C-ribose and uracilis the source of two
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appropriately labelled nucleosides: uridine and cytidine. Hence we have
chosen the linear approach.
The [13C5]-labelled acylated ribose was prepared in 42% total yield
starting from commercially available [13C6]-d-glucose via our earlier
reported procedure.34
The synthesis of the [6-15N]-adenine aglycon was performed accord-
ing to Scheme 1.
Amination of 1 gave in our hands inconsistent results of complicated
reaction mixtures and thereby low yields of the desired [6-15N]-adenine
(4). Hence the 6-chloropurine 1 was first converted to its 9-
tetrahydropyranyl( a=b) derivative 242 (mimic of the nucleoside). After
blocking of the N9 nitrogen atom, the reaction of 2 in the system
15NH4Cl/KHCO3/DMSO29 smoothly afforded the labelled adenine
derivative 3. From this compound the tetrahydropyranylgroup was
removed with acidic treatment and the resulting labelled nucleobase 4
was benzoylated to give derivative 5. The N6-[6-15N]-benzoyladenine (5)
was used in the coupling reaction with the appropriate uniformly [13C5]-
labelled ribose derivative.34
The [2-15N]-labelled guanine (10) was synthesized according to
Scheme 2, by modification of the procedure reported by Jones.27 We
have found that laborious and costly preparative HPLC for the
Scheme 1.
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J. MILECKI ET AL.
Scheme 2.
Scheme 3.
purification of intermediates could be omitted without reducing yields.
Enzymatic deamination of intermediate 8 was carried out on crude
reaction product without affecting the enzyme efficiency. Thus,
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MULTIPLY ISOTOPE LABELLED RIBONUCLEOSIDES
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Scheme 4.
adenosine 1-N oxide 627 upon treatment with 15N labelled cyanogen
bromide (prepared in situ from the labelled KC15N and bromine) gave
intermediate 7. Further reaction with triethylamine followed by
treatment with iodomethane and heating brought about a Dimroth
rearrangement and led to 2-amino-6-methoxyamino-[2-15N]-purine
riboside 8. After enzymatic deamination of the C6 position with
adenosine deaminase, [2-15N]-guanosine (9) was obtained. This nucleo-
side was cleaved with hot 1 m HClgiving the desired agylcon 10. This
was subsequently reacted with isobutyric anhydride to give 11 and
further converted into O6-diphenylcarbamoyl-N2-isobutyryl-[2-15N]-
guanine (12), which was used in the coupling reaction.
The coupling reactions of 15N labelled purine aglycons and uracil
with uniformly 13C labelled ribose were performed with the well-known
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Scheme 5.
43
.
Vorbruggen procedure , giving the multilabelled nucleosides 13a–c.
The diphenylcarbamoyl protection was selectively removed from
guanosine 13b via our earlier published procedure.44
The 13C-labelled uridine 13c was deprotected with methanolic
ammonia to the free nucleoside 14c. It was first evaporated with excess
of D2O to exchange the hydroxylhydrogen atoms for deuterium, then it
was heated in D2O with potassium carbonate at 958C for 34 h which
resulted in a >96 at% exchange of the H5 for deuterium. Negligible
(57%) exchange of the H6 also occurred. Part of this deuterated
uridine 15 was acetylated and converted into the O4-(2-nitrophenyl)
derivative 16. A subsequent treatment of 16 with 15NH4Cl/K2CO3/
DMSO at 808C for 45 h yielded cytidine 17, a nucleoside labelled with
13C at ribose ring, deuterium at C5 position of the pyrimidine ring and
15N at the 4-exocyclic amino group.
The labelled cytidine was acetylated at [4-15NH2], giving derivative
18, which was used for the synthesis of the oligonucleotide synthesis
block.
The oligo-RNA synthesis blocks 21a–d were obtained according to
Scheme 5, using earlier published procedures.34
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Conclusions
The above procedures enabled the efficient synthesis of multiply labelled
oligoribonucleotide building blocks with different labels in the ribose
2
(13C) and base (15N, H) moieties of the molecule. To the best of our
knowledge it is the first report on specific labelling with multiple
isotopes both in base and ribose units by chemical means. The labelled
blocks enable the construction of oligomers carrying selectively
positioned labels which can serve as objects of simultaneous study of
different aspects of RNA structure,45–48 e.g. signalassignment, sugar-
phosphate backbone geometry, sugar pucker, as well as base hydrogen
bonding.
The deuteriation of the C5 position of pyrimidines contributes to the
reduction of line overcrowding in the diagnostically important H10
region.
In non-helical regions (bulges and loops) of an oligonucleotide, an
exocyclic NH2 becomes one of the primary targets of interactions with
metal ions and other molecules.49–53 All three bases (A, C, G) bearing
such group are 15NH2 labelled. The presence of the 15N labelled
exocyclic amino group gives the potential to study the interactions
involving specific sites of the oligonucleotide by placing labelled units in
such sites. Although this synthetic work was aimed mainly at RNA
oligomers for NMR studies, 15N labelled exocyclic NH2 groups are also
advantageous in FTIR54,55 and Raman56,57 spectroscopy studies of
RNA association and hydrogen bonding.
Acknowledgements
This work was supported by a grant from the State Committee for
Scientific Research, Republic of Poland (7T09A09720). Thanks are due
to the Swedish Board for TechnicalDeveolpment (NUTEK), the
Swedish NaturalScience Research Council(NFR), the Swedish
Research Councilfor Engineering Sciences (TFR) and the Carl
Tryggers Styftelse (to AF) for generous financial support.
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