15
SYNTHESIS OF
N,
13
C LABELLED PURINES
Maria CHIRIAC, D. AXENTE, N. PALIBRODA
National Institute of Research & Development for Isotope
and Molecular Technologies
3400 Cluj-Napoca 5, P. O. Box 700, ROMANIA
The heteronuclear NMR spectroscopy in the structural analysis a proteins and
nucleic acids requires labelling with 15N and 13C. These studies using oligonucleotides,
specifically labelled with 15N and 13C, may provide valuable informations regarding nucleic
acid structure, drug binding and nucleic acid - protein interaction.
For this purpose, in the last years we prepared some purines labelled with 15N and
13
C in different positions, which were asked by Institute Pasteur from Paris, where the
labelled purines will be used in enzymatic syntheses of nucleotides -15N,13C.
This paper presents 15N and 13C labelling of the following purines:
1) [6 - 15N] ADENINE
2) [1,3,7 - 15N, 8 - 13C] XANTHYNE
3) [1,3,6,7 - 15N, 8 - 13C] ADENINE
1) [6 - 15N] ADENINE synthesis is presented in Scheme 1.
15
Cl
NH2
N
N
15
N
NH3, n - C4H9OH
1500C, 48 h
N
N
H
N
N
N
H
Scheme 1
A flask containing 1 l of 15N enriched ammonia ( ~ 99 at. % 15N) was cooled to 780C, and 40 ml of 1-butanol was added. The resulting solution was heated with 2 g of 6chloro-purine (a) in a sealed tube at 1500C for 48 hours. Solvent was removed in vacuum,
and the dark gray-green residue was taken up in 350 ml methanol and boiled with Dareo for
2 hr. Removal of solvent after filtration gave 1,5 g pure adenine - 15N (b). Yield: 76% (based
on 15NH3) [1].
The structure and the purity of [6 - 15N] Adenine was confirmed using mass
spectrometry and IR spectroscopy, and the isotopic label was determined by MS on the
molecular compound.
1
2) [1,3,7 -
15N,
8-
13C]
XANTHYNE synthesis is presented in Scheme 2.
Na2CO3
KCN + ClCH2COOH
NC - CH2 - COOH + KCl
40 -
500C
15NH
O
15NH
2
O
C
C
NaOH 40%
H15N
H15N
20 -250C
CN
CH2
H
CN
O 15N
H2
[II]
[I]
15N
O
2CO
Na15NO2, NaOH (2%)
H2SO4, 00C
O
C
15NOH
H15N
O
C
15NH
15N
NH2
2
H15N
Na2SO4
NH4OH
15N
NH
O
H
H
[III]
[IV]
H13COOH 85%
O
O
15
H15N
O
15
15
N
C13H
NaOH 10%
900C
N
N
H
H
[VI]
NH13CHO
H15N
O
15
N
H
NH2
[V]
[I] - Cyanoacetylurea - [1, 3 - 15N2]
[II] - Iminobarbituric - [1, 3 - 15N2] acid
[III] - Iminovioluric - [1, 3 - 15N2] acid
[IV] - 4,5 - diaminouracil [1, 3, 7 - 15N3]
[V] - 4 - Amino, 5 - formylaminouracil - [1, 3, 7 - 15N3 , 8 - 14C]
[VI] - Xanthyne - [1, 3, 7 - 15N3 , 8 - 14C]
Scheme 2
2
Chloroacetic acid (3.46 g) was dissolved in a minimum amount of water and
neutralised by addition of anhydrous sodium carbonate. 1.97 g KCN in 10 ml water were
added to the sodium chloroacetate solution and the temperature allowed to rise to 600C. The
reaction continued until the temperature ceased to rise, them the mixture was allowed to
stand for twelve hours at room temperature. The resultant pale yellow solution was acidified
and the excess of HCN, water and HCl were removed under reduced pressure at 500C. The
residue was taken up in absolute alcohol and the solvent removed.
A suspension of urea - 15N2 (1.86 g) in freshly distilled acetic anhydride was added
to the dried residue and was then added to the solution and upon cooling a crystalline solid
was isolated, that proved to be cyanoacetylurea - 1,3 - 15N2 (I), in 82% yield relative to urea.
The melting point of the product is 211 - 20C. [2].
The cyclisation of I to II was made with 40% NaOH solution. The mixture was
stirred for 2.5 hours, keeping the temperature at 20-250C. Then the reaction mixture was
neutralised with HCl 1:1 and allowed to stand for twelve hours in a refrigerator. The final
product (2.72 g) II, was isolated by filtration. [3].
For the nitrosation compound II was dissolved in 2% NaOH solution and 2.18 g of
sodium nitrite - 15N was added. The mixture was cooled to 00C and concentration H2SO4
was added drop wise until the solution become dark violet.
After neutralization with ammonium solution the red precipitate obtained was
isolated by filtration, yielding 3.23 g (84%). [4].
To the suspension of III in water were added 2.4 ml of 19% ammonium solution and
15.6 g sodium hydrosulfide. The mixture was heated to 550C until the solution was
decoloured entirely. After cooling, the product was filtrated and washed with water (the
yield was 2.5 g of IV, 96%).
To the suspension of IV in 10 ml of water 1.5 ml of formic acid - 13C, 85% were
added and the mixture was refluxed for 60 min. Them, the reaction mixture was allowed to
stand for twelve hours at room temperature. The product was filtered, washed with water
and dried at 6o-700C, yielding 2.86 g of V (91%).
Compound V was suspended in 10 ml of water and 10% NaOH solution was added
drop wise to pH = 9. Then, the reaction mixture was heated to 900C and kept at this
temperature until the solution become clear. After cooling to 00C, the solution was
neutralised with HCl 1:1 and allowed to stand for twelve hours in the refrigerator. Then, the
product was filtered yielding 2 g of xanthyne (1,3,6,7 - 15N3 , 8 - 13C) (78%). [5].
3) [1,3,6,7 - 15N4 , 8 - 13C] ADENINE
Because of the possibility to transform purines one into another, we prepared
labelled adenine in the mentioned position, using [1,3,7 - 15N, 8 - 13C] xanthyne as starting
material.
The steps of [1,3,6,7 - 15N, 8 - 13C] adenine synthesis, is presented on Scheme 3.
For xanthyne chlorination to 2,6 - Dichloropurine, a mixture of [15N, 13C] xanthyne and
“pyrophosphoryl chloride” [6] are heated in a sealed glass tube at 1650C, for 19 hours. After
cooling, the brown solution obtained was concentrate under reduced pressure, and then,
form the sirupy residue the chlorinate purine was extracted with ether (yiled = 43%).
The amination of chloropurine was made by heating a mixture of 2,6 Dichloropurine [15N, 13C], with 15NH3 in water, in a sealed glass tube at 1000C for 6 hours
(yield = 92%). [7].
The dechlorination of 2 - Cl - 6 - aminopurine is realised by catalytic hydrogenation
on 5% Palladium - charcoal (at atmospheric pressure and 300C) and [1,3,6,7 - 15N, 8 - 13C]
adenine is obtained in 80% yield [8].
3
O
Cl
15
C
N
C
15
H N
POCl3
1650C,
C
O
15
15
C13H
C
C
15
Cl
1000C,
N
C
N
red. H2
C H
6 hr
C
C
15
NH2
C
15
13
Cl
N
H
N
H
15
NH2
C
15
N
[II]
15
15NH
3
15
C
19 hr
N
H
N
H
[I]
N
N
N
H
[III]
15
N
C
15
N
C13H
Pd/C
HC
C
15
N
N
H
[IV]
[I] - [1,3,7 - 15N, 8 - 13C] Xanthyne
[II] - 2,6 - dichloropurine [1,3,7 - 15N, 8 - 13C]
[III] - 2-chloro, 6-aminopurine [1,3,6,7 - 15N, 8 - 13C]
[IV] - [1,3,6,7 - 15N, 8 - 13C] Adenine
Scheme 3
The structure of adenine - [15N, 13C] synthesised by us, was confirmed by mass
spectrometry and by NMR analysis [9].
The 15N labelled compounds, (15NH2)2CO, Na15NO2 and 15NH3 used as starting
material for the synthesis of labelled xanthyne and adenine, were obtained form H15NO3 99
at. % 15N, produced at the National Institute of Research & Development for Isotopic and
Molecular Technologies, Cluj-Napoca, Romania. For the 13C labelling of xanthyne, formic 13
C acid 99 at. % 13C from Sigma, was used.
REFERENCES
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[2] J.W. Triplett, S.W. Moch, S.L. Smith, D. A. Digenis, J. of Lab. Comp., vol. XIV, No.
1, p. 35 (1978).
[3] D.I. Hmelevski, E.I. Abranova, Zhur. Obschei Khim., 28, p. 1970 (1958).
[4] H. Biltzu, W. Schmidt, Lieb. Ann., 431, p. 94 (1923).
[5] W. Traube, Chem. Ber., 34, p. 3035 (1900).
[6] G.B. Eliot, G. H. Hitchings, J. of Amer. Chem. Soc., Vol. 78, p. 3508 (1956).
[7] E. Fischer, A. Ach. Ber., 30, p. 2239 (1897).
[8] H. Bredereck, H. Herlinger, I. Graudums, Ber., 95, p. 54, (1962).
[9] Maria Chirac, D. Axente, N. Palibroda, J. of Lab. Comp. and Radiopharm., 42, 377385, (1999).