First synthesis of C8-arylamine adducts of 2′-deoxyadenosine and incorporation of the phosphoramidite into an oligonucleotide

Article abstract of DOI:10.1055/s-2006-950411

The synthesis of C8-adducts of 2′-deoxyadenosine 7a,b with carcinogenic arylamines p-anisidine 4c and 4-aminobiphenyl 4b, their conversion into the phosphoramidites and successful incorporation into oligonucleotides are described. Georg Thieme Verlag Stut

Full text of DOI:10.1055/s-2006-950411

LETTER
2411
First Synthesis of C8-Arylamine Adducts of 2¢-Deoxyadenosine and
Incorporation of the Phosphoramidite into an Oligonucleotide
D
M
NA Damage by Carc
a
inogenic
A
i
rylamin
k
es e I. Jacobsen, Chris Meier*
Department of Chemistry, Organic Chemistry, Faculty of Mathematics, Informatics and Natural Sciences, University of Hamburg,
Martin-Luther-King-Platz 6, 20146 Hamburg, Germany
Fax +49(40)428382495; E-mail: chris.meier@chemie.uni-hamburg.de
Received 7 July 2006
identified. In the past, several studies discussed the bio-
chemical consequences of C8-arylamine-dG-adducts
Abstract: The synthesis of C8-adducts of 2¢-deoxyadenosine 7a,b
with carcinogenic arylamines p-anisidine 4c and 4-aminobiphenyl
‘
damaged’ by carcinogens like 2-aminofluorene 4a, 4-
4
b, their conversion into the phosphoramidites and successful in-
6
aminobiphenyl 4b or p-anisidine 4c (Figure 1).
corporation into oligonucleotides are described.
Key words: Pd-cross coupling, amination, nucleoside adducts, C8-Arylamine adducts are known to be the most persis-
3
phosphoramidites, oligonucleotides
tent DNA-adducts. In contrast to dG-adducts, no reports
have been published so far on how dA-adducts behave in
biochemical processes and how dA-adducts contribute to
Covalent modification of DNA by electrophiles alters the the possible induction of chemical carcinogenesis. To
structure as well as the fidelity of DNA replication. If study the mutagenic effects, structure and DNA-repair of
these DNA damages are not repaired, they may lead to these lesions, an efficient synthesis of adducted dA-nucle-
mutations and finally may play an important role in the in- oside phosphoramidites would be the prerequisite for the
1
duction of chemical carcinogenesis. Poly- and monocy- access to site-specifically modified oligonucleotides. In
7
clic aromatic amines belong to the class of chemical contrast to C8-dG adducts, no methodology has been re-
carcinogens that form covalently bonded adducts with ported either for the chemical synthesis of the arylamine
2
,3
DNA after metabolic activation. The ultimate carcino- dA-adduct phosphoramidites or the post-synthetic oligo-
gen is an arylnitrenium ion generated by cytochrome P450 nucleotide modification. Post synthetic procedures as re-
oxidation of the arylamine to the hydroxylamine, fol- ported for C8-arylamine-dG modified oligonucleotides
4
lowed by esterification and solvolysis. The predominant might be possible in principle but only oligonucleotides
site of reaction is the C8-position of 2¢-deoxyguanosine bearing one modified C8-dA are accessible by this strate-
dG) leading to so-called C8-adducts like 1 (about 80% of gy in extremely low chemical yields.8
(
5
the adducts found in in vivo studies). Moreover, the cor-
responding C8-arylamine adducts of 2¢-deoxyadenosine 2
9
10
In 1999, Lakshman and Johnson introduced C-N bond
formation by palladium-catalyzed reactions in the synthe-
sis of N -aryl-2¢-deoxyguanosine and N -aryl-2¢-deoxyad-
have been found in these studies to a minor extent
2
6
2
(
>10%). In addition, N -adducts of dG 3 have been
11
enosine adducts. Schoffers used comparable procedures
O
NH2
O
N
Aryl
HN
N
N
N
N
N
N
NH
NH2
Aryl
HN
N
NH
N
N
NH
HN
HO
O
HO
HO
O
O
Aryl
OH
OH
OH
1
2
3
NH2
MeO
NH2
NH2
4a
4c
4b
2
Figure 1 C8-arylamine-dG adduct 1, C8-arylamine-dA adduct 2 and N -arylamine adducts of dG 3 found in in vivo studies and three
carcinogens 4a,b,c
SYNLETT 2006, No. 15, pp 2411–2414
1
8
.0
9
.2
0
0
6
Advanced online publication: 08.09.2006
DOI: 10.1055/s-2006-950411; Art ID: G22606ST
©
Georg Thieme Verlag Stuttgart · New York

2
412
M. I. Jacobsen, C. Meier
LETTER
for the preparation of C8-arylamine-adenosine adducts. Because the exocyclic amino group of adenine should be
However, neither phosphoramidites nor oligonucleotides protected during oligonucleotide synthesis, the regularly
were prepared. We reported on the first synthesis of phos- used N-benzoyl group was introduced (BzCl in pyridine)
1
2
phoramidites of C8-arylamine-dG adducts. Here we re- in 73% (X = OMe) and 87% yields (X = Ph), respectively.
port on a highly convincing synthesis of C8-arylamine-2¢- Treating this material with TBAF in tetrahydrofuran led to
deoxyadenosine adducts, the first efficient conversion the deprotection of the 2¢-deoxyribose hydroxyl groups in
into the corresponding 3¢-phosphoramidites and their site- almost quantitative yields (compounds 9a,b). Then the 5¢-
selective incorporation into oligonucleotides.
dimethoxytrityl (DMTr) group was introduced. The yields
in these reactions were surprisingly moderate (42% and
46%). As side products, 3¢,5¢-di-DMTr-dA adducts were
isolated in about 30% yield. Attempts to optimize the re-
gioselectivity failed for unknown reasons. Finally, 5¢-
DMTr-protected adducts were converted into the
phosphoramidites using 2-cyanoethyl-N,N,N¢,N¢-tetra-
Starting from 2¢-deoxyadenosine (dA; 5), bromination in
1
3
acetate buffer (pH 5), led to the formation of 8-Br-dA. In
contrast to dG, the reaction can be accomplished in large
scales in good yields. In the case of dG, bromination was
achieved with NBS. However, this method failed in the
case of dA. 8-Br-dA was then protected by 1,3-dichloro-
isopropylphosphordiamidite and 4,5-dicyanoimidazole
1
3
,1,3,3-tetraisopropyldisiloxane (TIPDS-Cl ) to give
2
7,18
(
DCI).1 The phosphoramidites 10a,b were isolated af-
¢,5¢-protected 8-Br-dA 6 in excellent yield (98%,
1
4
ter chromatography on silica gel in 69% and 62% yields,
respectively. To prove the suitability of these compounds
to work as building blocks in the automated DNA-synthe-
sis, the phosphoramidite 10a was incorporated into a
mixed DNA sequence (Scheme 1). For the incorporation
of amidite 10a the standard coupling protocol of the DNA
synthesizer was used. In contrast to the corresponding dG
adducts,19 the coupling efficiency of p-anisidine-dA
phosphoramidite (10a) was almost quantitative [>97%;
the same stands for the following phosphoramidite (+1
position)]. After deprotection with ammonia, the oligo-
nucleotide was purified by RP18-silca gel column chro-
matography using triethylammonium buffer (pH 6.9)-
acetonitrile gradients. The final product was characterized
by ESI–MS spectrometry and the sequence was con-
firmed by an enzymatic degradation assay and analysis of
the ratio of the four nucleosides (Figure 2).20
Scheme 1). In the key step, the formation of the C8-
arylamine dA adducts 7a,b, the C-N bond was formed
with the aromatic amine in the presence of Pd (dba)3
2
(
10 mol%), rac-BINAP (30 mol%) and Cs CO as
2 3
1
5,16
base.
As arylamines the borderline carcinogen p-anisi-
dine (4c) and the strong carcinogen 4-aminobiphenyl (4b)
were used. C8-Arylamine adducts 7 were isolated in 70%
(
7a) and 60% yield (7b). However, lower amounts of the
catalyst also led to lower yields of the adducts
Scheme 1). The fully deblocked adduct 8a was accessi-
(
ble from 7a by desilylation using tetra(n-butyl)ammoni-
um fluoride (TBAF) in tetrahydrofuran.
This proves that 2¢-deoxyadenosine arylamine adducts
can now be prepared as reference compounds for analyti-
cal purposes.
NH2
NH2
X
NH2
MeO
NH2
N
N
N
N
N
N
N
N
1
) Br2, acetate
buffer, pH 5,
r.t., 3 h;
N
Br
N
N
N
3) Ar-NH2 4b,c
rac-BINAP
Pd (dba)
N
H
N
N
N
N
H
HO
O
N
O
2) TIPS-Cl2,
Si
O
O
2
3
O
Si
O
pyridine, r.t., 16 h
Cs2CO3
TBAF,
THF,
HO
O
O
OH
70%
O
1,2-DME,
90 °C , 48 h
O
r.t., 12 h
Si
Si
OH
5
6
8a
60–75%
X = OMe 7a
X = Ph 7b
X
NHBz
X
NHBz
MeO
6
) DMTrCl, pyridine,
r.t., 16 h;
N
4) a) BzCl, pyridine,
r.t., 3 h;
N
N
N
N
N
N
N
N
H
7
) NC(CH2)2OP [N(i-Pr)2]2
DCI, CH2Cl2, MeCN,
r.t., 30 min
N
H
b) C5H5N–NH3–H2O
) TBAF, THF,
r.t., 12 h
NH
3'-d[ATCTT(A)AGATG]-5'
5
DMTrO
HO
O
O
26–29%
68–78%
O
P
OH
NC
O
N(i-Pr)2
X = OMe 9a
X = Ph 9b
X = OMe 10a
X = Ph 10b
Scheme 1 Synthesis of phosphoramidites 10a,b and the incorporation of 10a into an oligonucleotide
Synlett 2006, No. 15, 2411–2414 © Thieme Stuttgart · New York

LETTER
DNA Damage by Carcinogenic Arylamines
2413
(
9) Lakshman, M. K.; Hilmer, J. H.; Martin, J. Q.; Keeler, J. C.;
Dinh, Y. Q. V.; Ngassa, F. N.; Russon, L. M. J. Am. Chem.
Soc. 2001, 123, 7779.
(
(
10) (a) De Riccardis, F.; Bonala, R. R.; Johnson, F. J. Am. Chem.
Soc. 1999, 121, 10453. (b) De Riccardis, F.; Johnson, F.
Org. Lett. 2000, 2, 293.
11) Schoffers, E.; Olsen, P. D.; Means, J. C. Org. Lett. 2001, 3,
4221.
(
(
12) Meier, C.; Gräsl, S. Synlett 2002, 802.
13) Guy, A.; Duplaa, A.-M.; Harel, P.; Téoule, R. Helv. Chim.
Acta 1988, 71, 1566.
(14) Markiewicz, W. T.; Samek, J.; Smrt, J. Tetrahedron Lett.
1
980, 21, 4523.
6
(
15) N -Benzoyl-8-N-(4-methoxyphenylamino)-3¢,5¢-O-[1,1,3,3-
tetrakis(isopropyl)-1,3-disiloxanediyl]-2¢-deoxyadenosine
6
(
[
7a) and N -benzoyl-8-N-(4-aminobiphenyl)-3¢,5¢-O-
1,1,3,3-tetrakis(isopropyl)-1,3-disiloxanediyl]-2¢-deoxy-
adenosine (7b): N -Bz-8-bromo-3¢,5¢-O-(TIPDS)-2¢-dA 6
6
Figure
2
Enzyme degradation assay of the oligonucleotide
ATCTT(A*)AGATG
(500 mg, 0.87 mmol), Cs CO (426 mg, 1.31 mmol), tris(di-
2
3
benzylideneacetone)dipalladium(0) [Pd (dba) ; 80.0 mg,
2
3
8
7.0 mmol], racemic-2,2¢-bis(diphenylphosphino)-1,1¢-
binaphthyl (BINAP; 163 mg, 0.26 mmol) and p-anisidine
4c; 215 mg, 1.74 mmol) or 4-aminobiphenyl (4b; 295 mg,
.74 mmol) were solubilized in anhyd 1,2-dimethoxyethane
In conclusion, we have developed a complete synthesis
of C8-arylamine-2¢-deoxyadenosine phosphoramidites
(
1
1
0a and 10b that are suitable for standard automated
DNA-synthesis. Due to this method site-selective in-
corporation of such adducts could be performed in any se-
quence for the first time. Now, the effects of chemically
modified dA-bases within oligonucleotides can be studied
and compared to the effects of the corresponding dG
adducts. This may provide new significant insights into
the importance of this type of DNA damage for the possi-
ble induction of the chemical carcinogenesis. Moreover,
fully unprotected C8-arylamine-dA adducts were pre-
pared which can be used in analytical studies as reference
compounds. Work along this field is currently underway
in our laboratories.
(30 mL) in an inert gas atmosphere and stirred at 90 °C until
the reaction was complete (TLC analysis). After cooling to
r.t., sat. NaHCO solution (1 mL) was added. After addition
3
of brine (10 mL) the layers were separated and the aqueous
layer was extracted with EtOAc (3 × 10 mL). The combined
organic layers were washed with brine (3 × 10 mL) and once
with a mixture of brine (10 mL) and H O (2 mL). The
2
organic layer was dried (Na SO ) and the solvent was
2
4
removed in vacuo. Purification by chromatography on silica
gel, eluting with 20% EtOAc in hexane afforded 7a (374 mg,
0.61 mmol; 70%) and 7b (345 mg, 0.52 mmol; 60%) as
light-yellow foams.
1
(
16) 7a: H NMR (400 MHz, CDCl ): d = 8.16 (s, 1 H), 7.48 (m,
3
2
H), 7.41 (br s, 1 H), 6.88 (m, 2 H), 6.31 (dd, J = 3.8, 7.8
Hz, 1 H), 5.34 (br s, 2 H), 4.90 (dd, J = 7.9, 15.2 Hz, 1 H),
.16 (dd, J = 3.4, 9.2 Hz, 1 H), 3.98 (dd, J = 5.0, 12.5 Hz, 1
4
Acknowledgment
H), 3.90 (ddd, J = 3.4, 4.9, 7.0 Hz, 1 H), 3.79 (s, 3 H), 3.06
(ddd, J = 3.6, 8.1, 13.4 Hz, 1 H), 2.59 (ddd, J = 7.9, 13.4 Hz,
This work was supported by the Deutsche Forschungsgemeinschaft
1
3
(DFG; ME 1161/5-1).
1 H), 0.89–1.13 (m, 28 H). C NMR (101 MHz, CDCl ):
3
d = 155.8, 152.5, 149.4, 132.5, 121.5, 117.4, 114.5, 85.3,
8
3.8, 70.5, 61.9, 55.7, 38.8, 17.0–17.6, 12.6–13.5. HRMS
References and Notes
+
(
FAB): m/z [M + H] calcd for C H N O Si : 615.3147;
29 46 6 5 2
1
found: 615.3155. 7b: H NMR (400 MHz, CDCl ): d = 8.19
3
(
(
(
1) Garner, R. C. Mutat. Res. 1998, 402, 67.
2) Neumann, H. G. J. Cancer Res. Clin. Oncol. 1986, 112, 100.
3) Beland, F. A.; Kadlubar, F. F. Environ. Health Perspect.
(
(
(
s, 1 H), 7.78 (br s, 1 H), 7.69 (m, 2 H), 7.58 (m, 4 H), 7.44
m, 2 H), 7.33 (m, 1 H), 6.35 (dd, J = 3.8, 7.5 Hz, 1 H), 5.59
s, 2 H), 4.88 (dd, J = 7.9, 15.2 Hz, 1 H), 4.16 (dd, J = 3.6,
1985, 62, 19.
1
2.6 Hz, 1 H), 4.04 (dd, J = 4.8, 12.5 Hz, 1 H), 3.95 (ddd,
(4) (a) Guengerich, F. P. Drug Metab. Rev. 2002, 34, 607.
J = 3.0, 4.4, 7.3 Hz, 1 H), 3.07 (ddd, J = 3.6, 8.0, 13.4 Hz),
(
(
b) Meier, C.; Boche, G. Chem. Ber. 1990, 123, 1691.
c) Famulok, M.; Boche, G. Angew. Chem., Int. Ed. Engl.
2
.59 (ddd, J = 7.8, 13.4 Hz, 1 H), 0.91–1.12 (m, 28 H).
1
3
C NMR (101 MHz, CDCl ): d = 156.7, 152.4, 149.7, 148.3,
3
1989, 28, 468; Angew. Chem. 1989, 101, 470.
140.9, 132.5, 121.5, 117.4, 114.5, 85.3, 83.8, 70.5, 61.9,
(
5) Tureky, R. J.; Rossi, S. C.; Welti, D. H.; Lay, J. O.;
Kadlubar, F. F. Chem. Res. Toxicol. 1992, 5, 479.
55.7, 38.8, 17.0–17.6, 12.6–13.5. HRMS (FAB): m/z [M +
+
H] calcd for C H N O Si : 661.3354; found: 661.3377.
3
4
48
6
4
2
(
(
6) Tureky, R. J.; Markovic, J. Chem. Res. Toxicol. 1994, 7, 752.
7) Meier, C.; Gräsl, S.; Detmer, I.; Marx, A. Nucleosides,
Nucleotides Nucleic Acids 2005, 24, 691.
6
(
17) N -Benzoyl-8-N-(4-methoxyphenylamino)-5¢-O-dimeth-
oxytrityl-2¢-deoxyadenosine-3¢-yl-b-cyanoethyl-N,N¢-
6
diisopropylphosphoramidite (10a) and N -benzoyl-8-N-(4-
aminobiphenyl)-5¢-O-dimethoxytrityl-2¢-deoxy-adenosine-
(
8) (a) Abuaf, P.; Hingerty, B. E.; Broyde, S.; Grunberger, D.
Chem. Res. Toxicol. 1995, 8, 369. (b) Shibutani, S.; Suzuki,
N.; Grollman, A. P. Biochemistry 1998, 37, 12034.
3¢-yl-b-cyanoethyl-N,N¢-diisopropylphosphor-amidite
6
(
10b): N -Bz-8-N-(4-methoxyphenylamino)-5¢-O-DMTr-2¢-
(
c) Shibutani, S.; Fernandes, A.; Suzuki, N.; Zhou, L.;
Johnson, F.; Grollman, A. P. J. Biol. Chem. 1999, 274,
7433.
6
dA (150 mg, 0.19 mmol) and N -Bz-8-N-(4-aminobi-
phenyl)-5¢-O-DMTr-2¢-dA (150 mg, 0.18 mmol), respec-
tively, were dissolved in anhydrous CH Cl (3 mL) and
2
2
2
anhydrous MeCN (3 mL) in an inert gas atmosphere and
Synlett 2006, No. 15, 2411–2414 © Thieme Stuttgart · New York

2
414
M. I. Jacobsen, C. Meier
LETTER
HRMS (ESI): m/z [M + Na] calcd for C H N O :
+
treated subsequently with a solution of 4,5-dicyano-
imidazole in MeCN (0.25 M, 1.5 mL, 0.38 mmol) and 2-
cyanoethyl-N,N,N¢,N¢-tetraisopropylphosphordiamidite
2
4
24
6
5
1001.4093; found: 1001.4120. 9b: 2 diastereomers + 2
1
rotamers. H NMR: (400 MHz, C D ): d = 9.87 (m, 2 H),
6
6
(
86 mg, 0.29 mmol). After stirring for 30 min at r.t., the
8.94 (s, 1 H), 8.92 (s, 1 H), 8.85 (s, 1 H), 8.84 (s, 1 H), 8.60
(m, 2 H), 8.44 (s, 1 H), 8.42 (s, 1 H), 8.24 (s, 1 H), 8.21 (s, 1
H), 8.03 (m, 4 H), 7.92 (m, 4 H), 7.86 (m, 4 H), 7.69 (m, 12
H), 7.49–7.63 (m, 20 H), 7.40 (m, 12 H), 7.32 (m, 12 H),
7.22 (m, 10 H), 7.04–7.18 (m, 12 H), 6.97 (m, 2 H), 6.85 (m,
8 H), 6.77 (m, 10 H), 5.87–6.03 (m, 2 H), 5.17–5.34 (m, 2
H), 4.99–5.15 (m, 2 H), 4.54–4.66 (m, 2 H), 4.41 (m, 2 H),
3.88 (m, 4 H), 3.74 (m, 2 H), 3.57–3.66 (m, 10 H), 3.31 (m,
12 H), 3.30 (m, 12 H), 3.00–3.16 (m, 8 H), 2.28–2.62 (m, 4
reaction was stopped by adding of MeOH (0.5 mL). The
solution was diluted with CH Cl (50 mL) and washed with
5
dried and concentrated to dryness. The residue was purified
by chromatography on silica gel, eluting with CH Cl , 2%
Et N and 0–3% MeOH. The products were redissolved in
2
2
% aq NaHCO followed by brine. The organic layer was
3
2
2
3
CH Cl and further washed with H O to give 10a (130 mg,
2
2
2
0
.13 mmol; 69%) and 10b (116 mg, 0.11 mmol; 62%) as
light-yellow solids.
18) 9a: 2 diastereomers + 2 rotamers. H NMR (400 MHz,
C D ): d = 9.55 (m, 2 H), 9.18 (s, 1 H), 9.16 (s, 1 H), 8.90
1
3
H), 1.70–1.97 (m, 10 H), 1.01–1.10 (m, 48 H). C NMR
1
(
(101 MHz, C D ): d = 159.2, 149.7, 141.1, 136.0, 135.9,
6
6
134.9, 132.6, 131.9, 130.8, 130.6, 129.0, 129.0, 128.7,
128.6, 127.9, 127.1, 127.0, 119.7, 117.6, 117.0, 116.3,
113.6, 113.5, 87.0, 86.8, 85.8, 59.2, 59.0, 58.0, 58.0 54.8,
45.4, 45.3, 43.7, 43.6, 24.8, 24.8, 24.7, 24.6, 23.7, 22.9, 22.8,
6
6
(
m, 2 H), 8.67 (s, 1 H), 8.66 (s, 1 H), 8.29 (s, 1 H), 8.27 (s, 1
H), 7.91 (m, 2 H), 7.82 (m, 5 H), 7.75 (m, 3 H), 7.64–7.70
m, 4 H), 7.54–7.62 (m, 6 H), 7.43–7.53 (m, 8 H), 7.32 (m,
2 H), 7.32 (m, 5 H), 7.08–7.16 (m, 8 H), 6.87–7.06 (m, 22
H), 6.64–6.80 (m, 24 H), 5.99–6.13 (m, 2 H), 5.91 (m, 2 H),
.25 (m, 2 H), 5.07 (m, 4 H), 4.50–4.64 (m, 2 H), 4.41 (m, 2
H), 3.81–3.93 (m, 4 H), 3.61–3.75 (m, 4 H), 3.44–3.62 (m,
6 H), 3.22–3.43 (m, 36 H), 2.28–2.53 (m, 4 H), 1.68–2.00
(
1
3
1
22.8, 22.8, 20.6, 20.1, 20.1, 19.5, 19.4. P NMR (202 MHz,
C D ): d = 149.37, 149.11, 148.69, 148.38
(0.31:0.37:0.17:0.15). MS (FAB): m/z [M + H] calcd for
6
6
+
5
C H N O : 1025.4480; found: 1025.4.
2
9
26
6
4
1
(19) Böge, N.; Gräsl, S.; Meier, C. J. Org. Chem. 2006,
submitted.
1
3
(
m, 12 H), 1.11-1.23 (m, 48 H). C NMR (101 MHz, C D ):
6 6
d = 165.9, 165.1, 159.2, 159.2, 159.9, 154.1, 152.6, 151.1,
(20) Enzyme degradation: Oligonucleotide (3 mg) was dissolved
1
1
1
1
8
5
2
1
50.1, 149.4, 146. 1, 145.9, 145.3, 144.2, 142.0, 136.9,
36.8, 136.0, 135.8, 135.0, 132.7, 132.6, 130.9, 130.8,
30.5, 128.6, 127.1, 127.0, 124.0, 123.4, 121.6, 121.5,
17.8, 117.6, 115.4, 114.6, 113.6, 113.5, 111.7, 86.9, 86.8,
6.0, 85.4, 82.5, 82.3, 64.9, 63.4, 63.2, 59.2, 59.1, 59.0, 58.8,
8.6, 55.0, 54.9, 54.9, 43.6, 43.6, 37.5, 37.2, 30.5, 30.2, 24.8,
in a NaOAc buffer (100 mL, pH 5.3, 30 mM). ZnSO solution
4
(5 mL, 20 mM) and the nuclease P1 (3 units, from peni-
cillium citrinum; Roche Nr. 91095923/30) were added and
incubated for 4 h at 37 °C. Then a tris(hydroxy-
methyl)aminomethane–HCl buffer (20 mL; pH 8.7; 50 mM)
and 4 units of the alkaline phosphatase (Roche Nr.
49048126/15) were added and incubated overnight at 37 °C.
3
1
4.8, 24.7, 20.2, 20.1, 20.1. P NMR (202 MHz, C D ): d =
6
6
49.20, 148.95, 148.84, 148.20 (0.30:0.33:0.20:0.17).
Synlett 2006, No. 15, 2411–2414 © Thieme Stuttgart · New York