Large-scale synthesis of a key catalytic reagent for phosphorus protection in building blocks for isopolar phosphonate oligonucleotide preparation

Article abstract of DOI:10.1021/op000056d

A simple procedure for the large-scale synthesis of 4-methoxy-2-pyridinemethanol 1-oxide (5), a key compound in modern triester synthesis of oligonucleotides, employing Buechi R 152 evaporator as a reaction apparatus was elaborated. Two crucial reaction steps, both strongly exothermic, were satisfactorily handled. Our procedure provides a high-purity, light-and heat-stable product in a satisfactory yield.

Full text of DOI:10.1021/op000056d

Organic Process Research & Development 2000, 4, 473−476
Large-Scale Synthesis of a Key Catalytic Reagent for Phosphorus Protection in
Building Blocks for Isopolar Phosphonate Oligonucleotide Preparation
Dominik Rejman, Jirˇ´ı Erbs, and Ivan Rosenberg*
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic,
166 10 Prague 6, Czech Republic
Abstract:
A simple procedure for the large-scale synthesis of 4-methoxy-
2-pyridinemethanol 1-oxide (5), a key compound in modern
triester synthesis of oligonucleotides, employing Bu1chi R 152
evaporator as a reaction apparatus was elaborated. Two crucial
reaction steps, both strongly exothermic, were satisfactorily
handled. Our procedure provides a high-purity, light- and heat-
stable product in a satisfactory yield.
Introduction
A search for nucleophilic catalysts more powerful than
pyridine1 or N-methylimidazole2,3 in phosphotriester meth-
odology of oligonucleotide synthesis1,4 has led to the
introduction of various 4-substituted derivatives of pyridine
Figure 1.
ther investigation in the area of isopolar phosphonate
nucleotide analogues, the elaboration of a reliable synthesis
of 4-methoxy-2-pyridinemethanol 1-oxide (10) as the supe-
rior reagent for the protection of phosphonate moiety is
highly desirable. In addition, the usefulness of the compound
10 as the sugar hydroxyl-protecting group in ribonucleo-
sides16 as well as the starting compound for the preparation
of arylsulfonylhydrazones of 2-formylpyridine derivatives17,18
(as compounds with antineoplastic activities) is also well-
known.There are two procedures described in the litera-
ture16,19 for the synthesis of 4-methoxy-2-pyridinemethanol
1-oxide (10). One of them16 utilizes a simple reduction of
4-substituted 2-formylpyridine 1-oxides obtainable by a
laborious multistep synthesis, whereas the other,19 apparently
much simpler one, provided, in our hands, very variable
yields of low-purity product 10. Despite employing two
chromatographical steps on silica gel and crystallization we
obtained a light- and heat-sensitive gray product. Under the
conditions given, the synthesis of pure 10 in 10 g amounts
would thus be very problematic and laborious. Herein we
describe a procedure enabling the simple, reliable, and safe
large-scale synthesis of 4-methoxy-2-pyridinemethanol 1-ox-
ide (10).
N-oxide 1.5 These O-nucleophiles, when added to the reaction
mixture in the presence of arylsulfonyl chloride strongly
accelerate the condensation reaction between the phosphodi-
ester and the OH-component, leading to the formation of a
neutral phosphotriester species. The idea of intramolecular
catalysis using a phosphorus-protecting group based on a
pyridine N-oxide derivative was verified6 in practice by
introduction of a 4-methoxy-N-oxido-2-picolyl group. Since
that time, this group has also been successfully applied to
the protection of the phosphonate moiety in various nucleo-
side phosphonic acids 2-5 from which the internucleotide
linkage-modified oligonucleotides7-15 were synthesized by
phosphotriester-type condensation method. Considering fur-
* Author for correspondence. Telephone: (4202) 20183381. Fax: (4202)
3111733 or 24310090. E-mail: ivan@uochb.cas.cz.
(1) Reese, C. B. Tetrahedron 1978, 34, 3143.
(2) Efimov, V. A.; Reverdatto, S. V.; Chakhmakhcheva, O. G. Nucleic Acids
Res. 1982, 10, 6674.
(3) Efimov V. A.; Buryakova A. A.; Reverdatto S. V.; Chakhmakhcheva, O.
G.; Ovchinnikov, Yu. A. Nucleic Acids Res. 1983, 11, 8369.
(4) Zarytova, V. F.; Knorre, D. G. Nucleic Acids Res. 1984, 12, 2091.
(5) Efimov, V. A.; Chakhmakhcheva, O. G.; Ovchinnikov, Yu. A. Nucleic Acids
Res. 1985, 13, 3651.
(6) Efimov, V. A.; Buryakova, A. A.; Dubey, I. Y.; Polushin, N. N.;
Chakhmakhcheva, O. G.; Ovchinnikov, Yu. A. Nucleic Acids Res. 1986,
14, 6526.
(7) Szabo, T.; Kers, A.; Stawinski, J. Nucleic Acids Res. 1995, 23, 893.
(8) Efimov, V. A.; Choob, M. V.; Kalinkina, A. L.; Chakhmakhcheva, O. G.
Nucleosides Nucleotides 1997, 16, 1475.
(9) Efimov, V. A.; Buryakova, A. A.; Choob, M. V.; Chakhmakhcheva, O. G.
Nucleosides Nucleotides 1999, 18, 1393.
(10) Efimov, V. A.; Choob, M. V.; Buryakova, A. A.; Chakhmakhcheva, O. G.
Nucleosides Nucleotides 1999, 18, 1425.
(14) Rejman, D.; Liboska, R.; Endova´, M.; Kavenova´, I.; Tocˇ´ık Z.; Rosenberg,
I. Collect. Czech. Chem. Commun. (Symposium Series) 1999, 2, 92.
(15) Rejman, D.; Rosenberg, I. Collect. Czech. Chem. Commun. (Symposium
Series) 1999, 2, 96.
(11) Chakhmakhcheva, O. G.; Andrianov, M.; Buryakova, A. A.; Choob, M.
V.; Efimov, V. A. Nucleosides Nucleotides 1999, 18, 1427.
(12) Tocˇ´ık, Z.; Kavenova´, I.; Rosenberg, I. Collect. Czech. Chem. Commun.
(Symposium Series) 1999, 2,109.
(13) Rinnova´, M.; Rosenberg, I. Collect. Czech. Chem. Commun. (Symposium
Series) 1999, 2, 60.
(16) Mizuno, Y.; Endo, T.; Takahashi, A.; Inaki, A. Chem. Pharm. Bull. 1980,
28, 3041.
(17) Agraval, K. C.; Sartorelli, A. C. J. Med. Chem. 1978, 21, 218.
(18) Mizuno, Y.; Endo T.; Inoue, Y.; Tampo, H.; Takahashi, A.; Iigo, M.; Hoshi,
A.; Kuretani, K. Chem. Pharm. Bull. 1980, 28, 1584.
(19) Mizuno, Y.; Endo, T. J. Org. Chem. 1978, 43, 684.
10.1021/op000056d CCC: $19.00 © 2000 American Chemical Society and The Royal Society of Chemistry
Published on Web 09/22/2000
Vol. 4, No. 6, 2000 / Organic Process Research & Development
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473

Scheme 1
(recommended particle size >40 µm) in water to remove an
essential amount of crystallization-preventing coloured com-
pounds with high affinity towards reverse phase (a partial
regeneration of the reverse phase column can be achieved
by its successive washing with methanol, acetic acid, acetic
acid-chloroform mixture (1-1) and finaly with methanol).
The N-oxide 10 obtained in this purification step was
crystalized from chloroform-dichloromethane mixture as
described above.
Results and Discussion
The nitration of the starting, commercially available
2-picoline-N-oxide (6) (see Scheme 1) with fuming nitric
acid at elevated temperature performed according to the
modified procedure20 afforded pure 4-nitro-2-picoline-N-
oxide (7) in a good yield. Treatment of this compound with
excess of sodium methoxide in methanol gave the expected
4-methoxy-2-picoline-N-oxide (8)21 in nearly quantitative
yield. Subsequent acetylation of the methoxy derivative 8
by acetic anhydride in chloroform under the formation of
derivative 9 has been described19 to involve a silica gel
chromatography. We performed a large scale acetylation of
8 by stepwise addition of a solution of methoxy derivative
8 in glacial acetic acid to preheated acetic anhydride. This
reaction arrangement was safe enough to maintain a strongly
exothermic acetylation fully under control. The isolation of
a key acetoxymethyl derivative 9 was accomplished by
fractional distillation in vacuo and no chromatographic step
was needed. We experienced that a noncontrolled, exothermic
reaction having almost explosive character occurred when
addition of reagents is carried out in the reverse order.
The subsequent oxidation step19 of derivative 9 in aqueous
peroxoacetic acid was considerably modified. We used an
anhydrous solution of peroxoacetic acid in acetic acid
prepared from acetic acid, 30% aqueous hydrogen peroxide,
and acetic anhydride. In our procedure, the acetoxymethyl
derivative 9 was slowly added to a solution of peroxoacetic
acid at elevated temperature under strict temperature control.
If necessary, the temperature of the oxidation reaction
mixture can be efficiently and quickly decreased by direct
addition of dry ice. Evaporation of the reaction mixture
followed by hydrolysis of the acetoxy group in concentrated
aqueous ammonia afforded the crude N-oxide 10 which was
deionized on Dowex 50 (H+ form) and purified by a two-
step crystallization. The first one performed from hot ethyl
acetate allowed removal of an essential amount of a gummy
residue that prevent crystallization. The obtained brown
crystalline compound was recrystallized from a chloroform-
dichloromethane mixture in which both N-oxide 10 and the
brownish by-products were well soluble. The crystals were
washed with dichloromethane in which the coloured com-
pounds were much better soluble than N-oxide 10. Additional
recrystallization from the same mixture afforded a highly
pure 4-methoxy-2-pyridinemethanol 1-oxide (10).
The described simplified procedure afforded light- and
heat-stable product 10 in an excellent purity and a good yield.
Considering a shelf life of the compound 10, we stored it in
an amber bottle almost one year at room temperature without
any changes in its chemical reactivity and composition.
Experimental Section
All chemicals were obtained from commercial suppliers,
solvents were distilled prior to use. Chemical ractions were
carried out in a Bu¨chi R 152 evaporator. Melting points are
1
uncorrected. H and 13C NMR spectra were measured on a
Varian Unity 500 instrument (1H at 500 MHz, 13C at 125.7
MHz) in hexadeuteriodimethyl sulfoxide and were referenced
to the solvent signal (δH ) 2.50, δC ) 39.7). UV spectra
were recorded on Cary 100 (Varian). Analytical TLC was
performed on silica gel plates, Silufol (Kavalier, Czech
Republic) in ethyl acetate-acetone-ethanol-water (6:1:1:
0.5) (system H).
4-Nitro-2-picoline-N-oxide (7). A round-bottom flask (20
L) with 2-picoline-1-oxide (Fluka) (6) (1350 g, 12.37 mol)
and fuming nitric acid (8 L) (added at room temperature)
connected to a Bu¨chi R 152 evaporator in “reflux regime”
was heated at 80 °C under gentle rotation in a tetraethylene
glycol bath. The course of nitration was checked by TLC
on silica gel in system H. After 8 h of reaction (complete
conversion of starting material), nitric acid was distilled off
at reduced pressure (approximately 6 L), the residue was
cooled and diluted with ice-cold water (6 L), and the mixture
was again concentrated at reduced pressure (5 mbar). This
co-distillation procedure was repeated several times until pH
of the distillate exceeded the value of 4. The semisolid
residue was cooled in an ice bath, cold water (2 L) was
added, and crystals were filtered off and washed by ice-cold
water (4 × 2 L) until a neutral pH value of the filtrate was
achieved. The filtrates were combined, slowly neutralized
by solid sodium hydrogencarbonate to pH 7 (Warning: rapid
evolution of CO2), and the solution was concentrated in
vacuo (to the volume of 1 L). Cooling of this solution to 0
°C afforded, after filtration, a second crop of the crude
product. Both crystalline parts were combined and dissolved
under reflux in water (6 L), and the solution was filtered
through Celite and concentrated at reduced pressure (to
approximately 800 mL). During the concentration process,
a spontaneous crystalization took place. Crystals were filtered
off, washed quickly with ice-cold water (3 × 200 mL), and
dried in vacuo. Yield: 1603 g (10.40 mol, 84%) of
compound 7; the compound does not melt; sublimation
Additional ∼10% of the product 10 can be obtained by
the following process: Mother liquors containing N-oxide
10 and concentrated coloured by-products were subjected
to a filtration through a small column of C18 silica gel
(20) Kohl, B.; Sturm, E.; Senn-Bilfinger, J.; Simon, W. A.; Krueger, U. et al. J.
Med. Chem. 1992 3, 51049.
(21) Katada, Y. Z. Chem. Abstr. 1951, 9537.
474
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occurred upon heating (ref22 mp 154-156 °C).
After the addition, the reaction mixture was heated at the
same temperature for further 8 h. TLC in system H revealed
complete disappearance of the starting material and the
presence of a faster product. The solution was then subjected
to fractional distillation. Acetic acid and acetic anhydride
were distilled off at 30-56 °C/25 mbar, and the product at
138 °C/4-5 mbar. Yield, 1000 g (6.61 mol, 27%) of 9.
A small sample of the product 9 (100 mg, 0.55 mmol)
was deacetylated in 30% aqueous ammonia (10 mL) at room-
temperature overnight. The solution was evaporated in vacuo
and a semisolid residue dried at 50 °C (0.1 mbar) for 20 h.
The obtained crystalline sample of 2-hydroxymethyl-4-
methoxypyridine was subjected, instead of compound 9, to
all analyses; mp 67-70 °C (ref24 74-75 °C).
Elemental anal.: for calcd C6H6N2O3 (154.12) C 46.76,
H 3.92, N 18.18; found C 46.52, H 3.93, N 18.08.
HR FAB+ (PEG 100, methanol) calcd for C6H6N2O3
154.037842, found 155.045667 (M + H)+.
1H NMR: 2.42 s, 3H (CH3); 8.08 dd, 1H, J(H5,H3) )
3.3, J(H5,H6) ) 7.1 (H-5); 8.42 dd, 1H, J(H3,H5) ) 3.3,
J(H3,H6) ) 0.5 (H-3); 8.44 dd, 1H, J(H6,H5) ) 7.1,
J(H6H3) ) 0.5 (H-6).
13C NMR: 17.29 (CH3); 118.72 (C-5); 121.03 (C-3);
140.12 (C-6); 141.44 (C-4); 150.04 (C-2).
4-Methoxy-2-picoline-N-oxide (8). An ice-cold 2 M
solution of sodium methoxide in methanol [prepared from
metalic sodium (718 g, 31.2 mol) and anhydrous methanol
(15.6 L)] was added to 4-nitro-2-picoline-N-oxide (7) (1603
g, 10.40 mol), and the resulting suspension was stirred by
rotating on Bu¨chi R 152 evaporator at room temperature.
Nucleophilic displacement of the nitro group for methoxyl
was checked by TLC in system H. After 8 h, the suspension
was neutralized to pH 7 by adding of dry ice. The resulting
thick suspension was then evaporated to dryness, the solid
residue suspended in chloroform (6 L), and the suspension
transferred onto the chromatography column (150 × 1500
mm) which was eluted by chloroform. Ethyl acetate can be
used instead of chloroform for this disolving-washing
procedure. In this case, elution of the product is much slower
because of lower solubility of methoxy compound in ethyl
acetate. The effluent was concentrated in vacuo and the solid
residue dried at 2 mbar. Yield, 1430 g (10.28 mol, 99%) of
a TLC-pure product 8. For analytical purpose, a small sample
of this compound was crystallized from water-saturated ethyl
acetate); mp 56-60 °C (ref23 79.5-81 °C).
Elemental anal.: for C7H9NO2 (139.15) calcd C 60.42,
H 6.52, N 10.07; found C 60.39, H 6.60, N 10.11.
HR FAB+ (glycerol, methanol) calcd for C7H9NO2
139.063328, found 140.071154 (M + H)+.
1H NMR: 3.82 s, 3H (OCH3); 4.51 d, 2H, J(CH2,OH) )
4.5 (OCH2); 5.51 t, 1H, J(OH,CH2) ) 4.5 (OH); 6.90 dd,
1H, J(H5,H3) ) 2.6, J(H5,H6) ) 5.8 (H-5); 7.01 d, 1H,
J(H3,H5) ) 2.6 (H-3); 8.27 d, 1H, J(H6,H5) ) 5.8 (H-6).
13C NMR: 55.30 (OCH3); 64.23 (OCH2); 105.92 (C-5);
108.35 (C-3); 150.07 (C-6); 164.07 (C-2); 166.05 (C-4).
4-Methoxy-2-pyridinemethanol 1-oxide (10). Ice-cold
30% aqueous hydrogen peroxide (272 mL, 2.66 mol) was
slowly added to acetic anhydride (1.322 L, 14.00 mol) at 0
°C under stirring and then, within 2 h, the temperature was
gradually risen to 45 °C. Then, 2-acetoxymethyl-4-meth-
oxypyridine (9) (1000 g, 5.5 mol) was added dropwise under
stirring to the prepared solution of peroxoacetic acid, and
the reaction temperature was maintained at 45 °C.
Caution: The above-mentioned oxidation step is strongly
exothermic. Since during addition of the pyridine derivative
9 the temperature started to rise rapidly, pieces of dry ice
were added directly into the mixture to maintain the
temperature near 45 °C.
Elemental anal.: for C7H9NO2‚H2O (157.16) calcd C
53.49, H 7.05, N 8.91; found C 53.42, H 7.10, N 9.08.
HR FAB+ (glycerol, methanol) calcd for C7H9NO2
139.063328, found 140.071154 (M + H)+.
1H NMR: 2.32 s, 3H (CH3); 3.79 s, 3H (O CH3); 6.89
dd, 1H, J(H5,H3) ) 3.5, J(H5,H6) ) 7.2 (H-5); 7.10 d, 1H,
J(H3,H5) ) 3.5 (H-3); 8.11 d, 1H, J(H6,H5) ) 7.2 (H-6).
13C NMR: 17.82 (CH3); 56.23 (OCH3); 110.61 (C-5);
112.01 (C-3); 139.54 (C-6); 148.88 (C-2); 156.31 (C-4).
2-Acetoxymethyl-4-methoxypyridine (9). A solution of
4-methoxy-2-picoline-N-oxide (8) (1430 g, 20.28 mol) in
glacial acetic acid (3 L) was slowly added to acetic anhydride
(4.92 L) preheated to 90-100 °C. The reaction was carried
out in Bu¨chi R 152 evaporator in “reflux” regime.
Caution: This acetylation is a strongly exothermic
reaction, and therefore, the addition of compound 8 must
be strictly under control, if the large-scale experiment is
realised. We also tried to perform this reaction in reverse
order, that is, by step-by-step addition of acetic anhydride
to picolyl derivative 8. After addition of ∼10% of amount
of acetic anhydride, a noncontrolled exothermic, almost
explosiVe reaction started up and a substantial amount of
material was lost.
After addition, the reaction mixture was set aside at the
same temperature for 12 h. The solution was then concen-
trated in vacuo (45 °C in bath, 4-5 mbar). The crude
product, the 2-acetoxymethyl-4-methoxypyridine-N-oxide
was used for final deacetylation without further purification.
The residue was dissolved in concentrated aqueous ammonia
(2 L), and the resulting solution was set aside overnight at
room temperature and then concentrated in vacuo (50 °C in
bath, 8-10 mbar). The highly viscous, dark red-brown
residue was dissolved in water (4 L), and the resulting
solution was applied onto a column of Dowex 50 (H+) (5
L). The column was washed with water until UV absorbing
compounds were eluted, and then the column was washed
with 10% aqueous ammonia. This ammonia effluent was
evaporated to dryness, the solid residue extracted with hot
ethyl acetate (10 × 1 L), and the dark solution containing
gummy residue was filtered through Celite. The combined
filtrates were concentrated to approximately 3 L, and the
solution was set aside to crystallize in a refrigerator overnight.
Crystals were filtered off, washed by a small volume of
(22) Hom, R. K.; Chi, D. Y.; Katzenellenbogen, J. A. J. Org. Chem. 1996, 8,
2624.
(23) Bojarska, D. Recl. TraV. Chim. Pays-Bas 1959, 78, 981, 985.
(24) Schultz; Fedders Arch. Pharm. (Weinheim Ger.) 1977, 310, 128, 135.
Vol. 4, No. 6, 2000 / Organic Process Research & Development
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dichloromethane (100 mL), and dried in vacuo. Mother
liquors were concentrated in vacuo, the semisolid residue
was dissolved in water (2 L), and the dark solution was
pumped, after previous filtration through Celite, on a
chromatography column (25 × 500 mm) charged with
octadecyl silica gel (40-60 µm) at the flow rate of 15 mL/
min. Elution of the column with water (approximately 2 L)
afforded a light-brown effluent which was evaporated, the
solid residue was dried in vacuo and recrystallized from hot
ethyl acetate as described above to afford a further crop of
N-oxide 10. All of the obtained crude crystalline parts were
combined and dissolved in refluxing chloroform (5 mL/g),
and the solution, diluted with 2 vols of dichloromethane, was
set aside overnight in a refrigerator. The crystals were
collected, washed with small amount of cold dichlo-
romethane, and dried in vacuo. Recrystalization under the
same conditions afforded pure 4-methoxy-2-pyridinemetha-
nol 1-oxide (10) in the yield of 240 g (1.55 mol, 23%) as
light- and heat-stable white needles; mp 143-143.5 °C (ref19
135-137 °C).
UV spectra in neutral (50% aqueous methanol), λmax 261
nm, λmin 226 nm; in acidic (0.01 M HCl in 50% aqueous
methanol) λmax 261 nm, λmin 224 nm; in basic (0.01 M NaOH
in 50% aqueous methanol) λmax 261 nm, λmin 227 nm.
1H NMR: 3.83 s, 3H (OCH3); 4.54 d, 2H, J(CH2,OH) )
5.6 (OCH2); 5.71 t, 1H, J(OH,CH2) ) 5.6 (OH); 6.95 dd,
1H, J(H5,H3) ) 2.5, J(H5,H6) ) 8.0 (H-5); 7.06 d, 1H,
J(H3,H5) ) 2.5 (H-3); 8.13 d, 1H, J(H6,H5) ) 8.0 (H-6).
13C NMR: 56.25 (OCH3); 58.58 (OCH2); 108.24 (C-5);
110.50 (C-3); 139.61 (C-6); 152.50 (C-2); 156.89 (C-4).
Acknowledgment
This work was supported by Grant NL5441-3 of the Grant
Agency of Ministry of Health and Grant A405 5902 of the
Grant Agency of the Academy of Sciences, Czech Republic.
We are indebted to Dr. Milena Masoj´ıdkova´ and Dr. Jitka
Kohoutova´, both of this Institute, for measurements and
interpretation of NMR and mass spectra, respectively. Special
thanks are given to Dr. Zdeneˇk Tocˇ´ık of this Institute for
valuable discussion and help with the preparation of the
manuscript.
Elemental anal.: for C7H9NO3 (155.15) calcd C 54.19,
H 5.85, N 9.03; found C 53.93, H 5.85, N 9.09.
HR FAB+ (glycerol, methanol) calcd for C7H9NO3
155.058243, found 156.066068 (M + H)+.
Received for review May 25, 2000.
OP000056D
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