Synthesis of guanosine-3′-(5-bromo-4-chloroindol-3-YL)-phosphate (G-3′-BCIP)

Article abstract of DOI:10.1135/cccc19960427

N-2-Benzoyl-5′-O-dimethoxytrityl-2′-O- tetrahydropyranylguaaosine (5) and 1-acetyl-5-bromo-4-chloroindol-3-yl-3-phosphorodichloridate (7) were synthesized and coupled to give the title compound 9.

Full text of DOI:10.1135/cccc19960427

Short Communication
427
SYNTHESIS OF GUANOSINE-3′-(5-BROMO-4-CHLOROINDOL-3-YL)-
PHOSPHATE (G-3′-BCIP)
Abdelfattah HAIKAL*
Institute voor Moleculaire Biologie,
Vrije Universiteit Brussel, Paardenstraat 65, Sint-Genesius-Rode, Belgium
Received December 27, 1994
Accepted November 19, 1995
N-2-Benzoyl-5′-O-dimethoxytrityl-2′-O-tetrahydropyranylguanosine (5) and 1-acetyl-5-bromo-4-chlo-
roindol-3-yl-3-phosphorodichloridate (7) were synthesized and coupled to give the title compound 9.
Key words: Identification of ribonuclease activity.
Chromogenic substrates are routinely used for spectroscopic analysis of enzyme activity1,2.
The 5-bromo-4-chloroindol-3-yl group may be a particularly useful group in identifying
ribonuclease activity. Specific ribonucleases should cleave the bond between the phos-
phate and the chromophore. The oxidation of the resulting 5-bromo-4-chloroindol-3-ol
to a dimeric product results3 in an important absorbance increase between 600 and 700 nm.
For this reason a synthetic route to the title compound has been developed.
Synthesis of G-3′-BCIP was performed as illustrated in Scheme 1. N-2-Benzoyl-5′-
O-dimethoxytrityl-2′-O-tetrahydropyranylguanosine 5 was synthesized starting
from N-2-benzoylguanosine4 1 by protecting the 3′,5′ hydroxyl groups using 1,3-di-
chloro-1,1,3,3-tetraisopropyldisiloxane5 in the presence of anhydrous pyridine, to pro-
duce product 2 in good yield after workup and silica gel chromatography. Compound 2
was treated with 3,4-dihydro-2H-pyran (DHP) in anhydrous dioxane in the presence of
p-toluenesulfonic acid6 yielding a mixture of the tetrahydropyranyl isomers 3. This
mixture was used in the next step without purification. It was treated with potas-
sium fluoride–tetraethylammonium bromide mixture to cleave the 3′,5′ silyl protecting
group as described by Kamimura et al.7. After workup and silica gel chromatography
compound 4 was obtained in a good yield. Treatment of product 4 with 4,4′-dimethoxy-
trityl chloride (DMTCl) in the presence of anhydrous pyridine7 gave the product 5 after
purification by silica gel chromatography. 3-Acetoxy-1-acetyl-5-bromo-4-chloroindole was
selectively deacetylated at the 3-position8 with 80% sulfuric acid to give 6 which was
* Permanent address: Chemistry Department, Faculty of Science, Zagazig University, Zagazig, Egypt.
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428
Haikal:
O
N
O
N
N
N
N
N
HN
HO
HN
BzHN
BzHN
iPr2
Si
O
O
O
(iPr2SiCl)2O
O
Si
iPr2
O
HO OH
OR
1
2, R = H
R = THP
DHP
3,
O
O
N
N
N
HN
HN
RHN
N
N
N
BzHN
RO
HO
O
O
7
O
Cl
HO O
O P O OH
O
O
Br
N
R1
4, R = H
DMTCl
R = DMTO
5,
R = Bz, R1 = COCH3
R = H, R1= H
8,
9,
NH4OH
Cl
OR
Br
N
COCH3
6, R = H
R = P(O)Cl2
POCl3
7,
SCHEME 1
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429
phosphorylated as described3 yielding compound 7. Coupling of 7 with 5 in dry py-
ridine catalyzed by 1,2,4-triazole at room temperature was monitored by TLC and after
40 min quenched by triethylammonium hydrogencarbonate (TEAB). The crude product
was subjected to silica gel chromatography but a partial decomposition of the trityl
group during the column chromatography was observed, so all the fractions containing
the required product were collected together, evaporated to dryness and treated with
80% acetic acid to affect detritylation and to cleave the tetrahydropyranyl group9. Fi-
nally the benzoyl and the acetyl groups were cleaved using concentrated ammonium
hydroxide9–11 to give compound 9. All the synthesized products were characterized
1
using H and 31P NMR spectra and mass spectra, and treatment with ribonuclease T1
for the final product.
EXPERIMENTAL
1H NMR spectra were measured on Bruker AC (250 MHz) spectrometer, chemical shift values (δ,
ppm; J, Hz) were reported relative to CDCl3 (7.30), (CD3)2SO (2.50) or D2O (4.81) signals. Proton
decoupled 31P NMR spectra were measured on Bruker WP 80 (32.37 MHz). Chemical shifts are
given in ppm downfield from 85% aqueous H3PO4 as an external standard. Fast Atom Bombardment
(FAB) mass spectra were recorded on a Kratos instrument Model MS-890. TLC was performed on
silica gel F254 (Merck). Detection of spots was effected by UV light, then charring with 10% sulfuric
acid in 95% ethanol. Preparative HPLC was carried out using a VYDAC C18 column (1 × 25 cm).
Pyridine was distilled from CaH2 and stored over potassium hydroxide.
N-2-Benzoyl-3′,5′-di-O-(tetraisopropyldisiloxane-1,3-diyl)guanosine (2)
1,3-Dichloro-1,1,3,3-tetraisopropyldisiloxane (2.71 g, 8.6 mmol) was added to a solution of N-2-
benzoylguanosine (3 g, 7.75 mmol) in anhydrous pyridine (25 ml). The reaction mixture was stirred
at room temperature overnight, then evaporated under reduced pressure. The residue was partitioned
between chloroform (60 ml) and the same volume of saturated aqueous sodium hydrogencarbonate,
the chloroform layer was washed with water (2 × 30 ml), dried over anhydrous sodium sulfate and
finally purified over silica gel column chromatography using chloroform–methanol (97 : 3 v/v), giving
1
the compound 2 (3.8 g, 78%), RF = 0.54 (chloroform–methanol 9.5 : 0.5). Mass spectrum, m/z: 629. H
NMR spectrum: 8.06–7.51 m, 6 H (arom.); 5.91 s, 1 H (H-1′); 4.48 q, 1 H (H-3′); 4.33 dd, 1 H (H-2′); 4.03
m, 3 H (H-4′,H-5′,H-5″); 1.06 m, 28 H (TIPDS protons). For C29H43N5O7Si2 (629.3) calculated:
55.30% C, 6.88% H, 11.12% N; found: 55.45% C, 6.82% H, 11.23% N.
N-2-Benzoyl-2′-O-tetrahydropyranylguanosine (4)
To a solution of compound 2 (1.5 g, 2.4 mmol) in dioxane (15 ml) containing p-toluenesulfonic acid
monohydrate (0.12 g), 3,4-dihydro-2H-pyran (4.05 ml) was added and stirred for 6 h at room tem-
perature. After neutralization with concentrated ammonium hydroxide, filtration of ammonium tosylate,
and washing with dichloromethane, the washings and filtrate were evaporated under reduced pressure.
To the residue (compound 3) dissolved in acetonitrile (40 ml), KF (1.1 g, 18.7 mmol), Et4NBr (3.9 g,
18.7 mmol) and H2O (1 ml) were added and the mixture was stirred for 45 min at 50 °C and then
concentrated. The successive extractive workup and silica gel chromatography gave 4 (1.02 g, 91%)
as a mixture of two diastereoisomers. RF 0.30 and 0.40 (chloroform–methanol 9 : 1). Mass spectrum,
m/z: 471. 1H NMR spectrum of the fraction with lower RF: 8.2 s, 1 H (H-8); 7.98–7.47 m, 5 H
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Haikal:
(arom.); 5.99 d, 1 H, J(1′,2′) = 6.65 (H-1′); and the characteristic pattern of ribofuranose. For
C22H25N5O7 (471.2) calculated: 56.05% C, 5.34% H, 14.85% N; found: 56.25% C, 5.42% H, 14.73% N.
N-2-Benzoyl-5′-O-dimethoxytrityl-2′-O-tetrahydropyranylguanosine (5)
Compound 4 (0.8 g, 1.7 mmol) was co-evaporated with anhydrous pyridine (3 × 10 ml), dissolved in
anhydrous pyridine (15 ml), to this solution 4,4′-dimethoxytrityl chloride (0.68 g, 2.03 mmol) was
added and the reaction mixture was stirred at room temperature and monitored by TLC. When no
starting material was present (after one hour), water (3 ml) was added followed by extraction with
chloroform; the chloroform layer was dried over anhydrous sodium sulfate and evaporated. The
residue was chromatographed on a silica gel column eluted with chloroform–methanol (9.7 : 0.3 v/v)
giving compound 5 (1.1 g, 84%, two diastereoisomers) with RF 0.43 and 0.33 (chloroform–methanol
9.5 : 0.5). Mass spectrum, m/z: 773. 1H NMR spectrum for the isomer with lower RF: 8.0 s, 1 H
(H-8); 7.69–6.92 m, 18 H (arom.); 5.91 d, 1 H, J(1′,2′) = 7.19 (H-1′); 3.60 s, 6 H (2 × OCH3).
1-Acetyl-5-bromo-4-chloro-3-hydroxyindole (6)
Sulfuric acid (80%, 2.5 ml) was added dropwise into a beaker containing 3-acetoxy-1-acetyl-5-
bromo-4-chloroindole (0.5 g, 1.5 mmol), then stirred at room temperature for one hour and poured
into ice-water, stirring for 30 min, the solid was filtered, washed with a solution of 0.1 M sodium
acetate until neutral and then with water and dried over P2O5 giving compound 6 (0.4 g, 92%). Mass
spectrum, m/z: 288. 1H NMR spectrum: 8.38 d, 1 H; 7.81 d, 1 H; 4.34 s, 2 H (keto-enolic protons);
2.31 s, 3 H.
1-Acetyl-5-bromo-4-chloroindol-3-yl-3-phosphorodichloridate (7)
This product was synthesized from 3-hydroxyindole 6 exactly as described before3 giving the same
physical data.
Guanosine 3′-O-(5-Bromo-4-chloroindol-3-yl)phosphate (G-3′-BCIP, 9)
To a solution of compound 5 (0.35 g, 0.48 mmol) and 1,2,4-triazole (0.164 g, 2.4 mmol) in dry py-
ridine (3 ml), compound 7 (0.588 g, 1.43 mmol) was added and the reaction was stirred under argon
for 40 min and quenched with 1 M TEAB (2 ml) and evaporated to dryness under reduced pressure. The
residue was applied to a silica gel column eluted with chloroform–methanol–triethylamine (93 : 4 : 3).
All the fractions containing the product (even partially detritylated) were collected together, evapo-
rated and dissolved in 80% acetic acid (15 ml), stirred 1 h at room temperature, evaporated to dry-
ness and co-evaporated with water (3 × 10 ml) to eliminate all traces of acetic acid, followed by
dissolving the residue in concentrated ammonium hydroxide (15 ml). Then it was left at 3 °C for 15 h
and evaporated. The product was purified by flash chromatography on silica gel column using mix-
ture of butanol–water–methanol (75 : 15 : 10) and re-purified by preparative HPLC using a linear
gradient of 1 M ammonium acetate (pH 6) (A) and acetonitrile (B) (from 100% of A to 100% of B
in 25 min). The ammonium acetate which contaminated the product was removed by dissolving the
product in water (3 ml) and applied to a column of Sephadex A-25 (20 ml), washed with water (60 ml)
and the product eluted with TEAB (0.3 mol l–1, 50 ml), evaporated under reduced pressure and co-
evaporated with water (3 × 10 ml) to eliminate traces of TEAB. The residue was dissolved in water
1
(3 ml) and lyophilized to give the product 9 (30 mg, 10%). 31P NMR spectrum (D2O): 1.6. H NMR
spectrum (D2O): 7.97 s, 1 H (H-8); 7.38–7.23 m, 3 H (arom.); 5.76 d, 1 H, J(1′,2′) = 3.32 (H-1′);
4.26 m, 1 H (H-3′); 4.08 t, 1 H (H-2′); 3.95 dd, 1 H (H-4′); 3.86 dd, 2 H (H-5′ and H-5″). The
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triethylammonium salt of 9 was submitted to ion exchange chromatography using a column (15 ml)
of Dowex 50W-X4 (Na+ form, 200–400 mesh), using water (30 ml) as eluent followed by lyophiliz-
ation of the water phase. Mass spectrum (high resolution, m/z): For C18H16BrClN6NaO8P calculated:
611.9537; found: 611.9539. This product was further characterized by incubation with ribonuclease
T1 for 3 h giving a blue pigments as expected.
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