8
98 J . Org. Chem., Vol. 62, No. 4, 1997
Varila et al.
5′-O-Meth ylth ym id in e 3′-(P h en yl p h osp h a te) (12). δ
First-order rate constants, kobs, for the hydrolysis of 1a were
H
calculated by applying the integrated first-order rate equation
to the diminution of the signal area of the starting material.
First-order rate constants for the parallel reactions in acidic
solutions, viz. depyrimidination and phosphotriester hydroly-
sis, were determined by bisecting the values of kobs on the basis
of product distribution during the early stages of the reaction.
Under very acidic conditions, however, 2 is depyrimidinated
too rapidly to be accumulated as an intermediate, and hence
the rate rate constants of the parallel reactions could not be
determined accurately at pH < 2. The molar ratio of the
products accumulated, viz. 3, 4, and thymine, remained
constant at pH < 1, suggesting that all the reactions are first
order in hydronium ion concentration.
(500 MHz): 11.2 (1 H, NH), 7.54 (1 H, s, H6), 7.22-6.94 (5 H,
Ph), 6.11 (1 H, dd, J 1′,2′ and J 1′,2′′ ) 6.3, 7.9 Hz, H1′), 4.64 (1 H,
m, H3′), 4.02 (1 H, m, H4′), 3.48 (1 H, dd, J 5′,5′′ ) 10.6 Hz, J 5′,4′
) 3.2 Hz, H5′), 3.42 (1 H, dd, J 5′′,4′ ) 4.4 Hz, H5′′), 3.27 (3 H,
s, OCH
3
), 2.20 (1 H, m, H2′), 2.08 (1 H, m, H2′′), 1.77 (3 H, s,
(125.6 MHz, BCM irradiation): 12.2, 38.1, 58.4,
5-CH ). δ
3
C
72.4, 74.8 (d, J C,P ) 5.6 Hz), 84.0, 84.1 (d, J C,P ) 5.5 Hz), 109.5,
119.9 (d, J C,P ) 4.5 Hz), 121.7, 128.8, 135.9, 150.4, 154.0 (d,
J
C,P ) 6.4 Hz), 163.5. δ
3a . t : 32 min. δ
P
: -6.7 ppm.
(500 MHz): 11.3 (1 H, s, NH), 7.45-
1
R
H
7
.20 (6 H, H6 and Ph), 5.98 (1 H, d, J 4′,O4′ ) 6.9 Hz), 5.73 (1 H,
dd, J 1′,2′ and J 1′,2′′ ) 7.3, 5.8 Hz, H1′), 4.35-4.20 (2 H, m, H3′
and H5′), 4.17 (1 H, td, J 5′,5′′ ) J H,P ) 11.3 Hz, J 4′,5′′ ) 1.5 Hz,
H5′′), 3.79 (1 H, m, H4′), 3.18 (3 H, s, OCH
Meth a n olysis. The products of methanolysis were sepa-
rated on a semipreparative LiChrospher RP-18 column (10 ×
3
), 2.37 (1 H, m,
(125.6 MHz,
H2′), 2.22 (1 H, m, H2′′), 1.78 (3 H, s, H5). δ
C
2
50 mm, 5 µm), using mixtures of the above-mentioned acetic
1 13 31
BCM irradiation): 12.1, 36.5 (d, J C,P ) 8.2 Hz), 55.6, 64.3 (d,
acid buffer and acetonitrile as eluent. The H, C, and
P
J
8
C,P ) 5.5 Hz), 70.4 (d, J C,P ) 6.4 Hz), 79.8 (d, J C,P ) 7.3 Hz),
2.3, 110.3, 119.7 (d, J C,P ) 4.6 Hz), 125.2, 130.0, 135.5, 149.8
NMR spectra of the isolated compounds were recorded on a
J EOL GX 400 or a J EOL J NMA 500 spectrometer in DMSO-
(
d, J C,P ) 6.4 Hz), 151.1, 163.7.
3b. t : 37 min. MS: m/z 413. δ
NH), 7.5-7.2 (6 H, H6 and Ph), 5.85 (1H, d, J 4′,O4′ ) 6.7 Hz,
6
d at 300 K. The proton chemical shifts were measured from
1
R
H
(500 MHz): 11.3 (1 H,
3
1
internal TMS and the P shifts from external H
3 4
PO .
The base-catalyzed methanolysis of 1a was carried out in
4
5
4
3
′OH; at 353 K, this resonance turned into a broad singlet at
.65 ppm), 5.54 (1 H, dd, J 1′,2′ and J 1′,2′′ ) 12, 2.1 Hz, H1′),
.38 (2 H, m, H3′ and H5′), 4.19 (1 H, t, J ) 11.3 Hz, H5′′),
-
3
methanolic sodium methoxide (0.1 mol dm ). The reaction
was followed for 1 h at 298.2 K. Three products were initially
accumulated with HPLC retention times of 18.0, 29, and 33
3
.73 (1 H, m, H4′), 2.99 (3 H, s, OCH ), 2.5 (1H, m, H 2′,
min. The product at t
to two products (t 17.5 and 19.0 min). The products eluted
at 19, 29, and 33 min were isolated by HPLC and characterized
R
18.0 min was subsequently degraded
overlaps with the DMSO resonance), 1.87 (1H, H2′′), 1.77 (3
H, s, 5-CH ). δ (125.6 MHz, BCM irradiation): 12.0, 36.9 (d,
R
3
C
J
C,P ) 9.0 Hz), 55.7, 64.4 (d, J C,P ) 4.6 Hz), 70.7 (d, J C,P ) 7.3
1
31
by H and P NMR spectroscopy. They were assigned as
thymidine 3′-(dimethyl phosphate) (9, t 19 min), thymidine
Hz), 80.0 (d, J C,P ) 7.4 Hz), 81.3, 110.3, 119.5 (d, J C,P ) 4.6
Hz), 125.3, 130.1, 135.1, 149.8 (d, J C,P ) 6.4 Hz), 150.9, 163.7.
R
3
5
′-(methyl phenyl phosphate) (10, t
′-(methyl phenyl phosphate) (11, t
P
and S diastereomers, but no NMR spectral
R
33 min), and thymidine
29 min). 10 and 11 may
δ
P
-12.3 ppm.
P r od u cts a t High Cl or AcO- Con cen tr a tion . When
the hydrolysis of 1a was followed in an acetic acid/sodium
R
-
be mixtures of R
P
evidence for this was obtained.
-
3
-3
acetate buffer (0.02/0.02 mol dm ) containing 0.98 mol dm
Th ym id in e 3′-(Dim et h yl P h osp h a t e) (9).
δ
H
(400
sodium chloride, accumulation of thymidine 3′,5′-cyclic mono-
phosphate (2), thymidine 3′-(phenyl phosphate) (3), and thym-
ine was found to be accompanied by formation of an additional
product, having a slightly longer retention time (24 min). This
product was isolated by HPLC. It exhibited the following
MHz): 11.3 (1H, s NH), 7.67 (1H, s, H6), 6.18 (1H, dd, J 1′,2′
and J 1′,2′′ ) 6.4, 4.8 Hz, H1′), 4.91 (1H, m, H3′), 4.04 (1H, m,
H4′), 3.69 (6H, d, J P,H ) 11.2 Hz, 2 × POCH
3
), 3.51-3.22 (2
H5′, overlaps with the HDO resonance), 2.32 (2 H, m, H2′ and
H2′′), 1.77 (3 H, s, 5-CH ). δ 0.3 ppm.
Th ym id in e 3′-(Meth yl p h en yl p h osp h a te) (10). δ
MHz): 11.4 (1 H, NH), 7.66 (1H, H6), 7.4-7.2 (5 H, Ph), 6.18
1 H, t, J 1′,2′ ) J 1′,2′′ ) 6.7 Hz, H-1′), 5.06 (1 H, H3′), 4.08 (1 H,
m, H4′), 3.82 (3 H, d, J H,P ) 11.5 Hz, POCH ), 3.6 (2 H, m, H5′
and H5′′), 2.34 (2 H, m, H-2′ and H2′′), 1.76 (3 H, 5-CH ). δ
3
P
1
13
31
molecular ion signals on FAB MS and H, C and
P
H
(400
resonances in D O at 300 K and was on these bases assigned
2
as 5′-chloro-5′-deoxythymidine 3′-(phenyl phosphate) (14).
5′-Ch lor o-5′-deoxyth ym idin e 3′-(P h en yl ph osph ate) (14).
(
3
3
P
H
MS m/z: 416 (100), 418 (35%). δ (400 MHz): 7.41 (1 H, H6),
-
5.0 ppm.
7.3-7.05 (5 H, phenyl protons), 6.17 (1 H, t, J 1′,2′ ) J 1′,2′′ ) 7.2
H
Th ym id in e 5′-(Meth yl p h en yl p h osp h a te) (11). δ (400
Hz, H-1′), 4.80 (1 H, m, H-3′), 4.22 (1 H, dd, H-4′), 3.70 (1 H,
MHz): 11.3 (1 H, NH), 7.4-7.2 (6 H, H6 and Ph), 6.19 (1 H, t,
dd, J 5′,5′′ ) 12.7 Hz, J 4′,5′′ ) 3.9 Hz, H-5′′), 3.63 (1 H, dd, J 4′,5′
5.2 Hz, H-5 ), 2.40 (1 H, ddd, J 2′,2′′ ) 16 Hz, J 2′′,3′ ) 4.0 Hz,
H2′′), 2.32 (1 H, dd, J 2′,3′ ) 0 Hz, H-2′), 1.73 (1 H, 5-CH ). δ
(125.6 MHz): 12.4, 37.9, 44.8, 76.7, 84.8, 85.9, 112.6, 120.9,
125.3, 130.7, 138.2, 152.5, 167.3. δ : -5.9 ppm.
)
J
3
1′,2′ ) J 1′2′′ ) 6.6 Hz, H1′), 4.4-4.2 (3 H, H3′,H5′, and H5′′),
.93 (1 H, H4′), 3.79 (3 H, d, J H,P ) 11.3 Hz, POCH ), 2.05 (2
). δ : -5.0 ppm.
′
3
3
C
H, m, H2′ and H2′′), 1.71 (3 H, 5-CH
Three products, having retention times of 24, 32, and 37
min, were formed in methanolic trifluoroacetic acid (1.0 and
3
P
P
When the hydrolysis of 1a was followed in a concentrated
-
3
0
.1 mol dm ) and in methanolic triethanol amine/triethano-
-3
acetic acid/sodium acetate buffer (1.0/1.0 mol dm ) in the
-3
lammonium buffers (0.005/0.05 and 0.05/0.005 mol dm ) upon
d incubation in sealed tubes at 363.2 K. The first one (t 24
absence of additional electrolytes, another product having t
R
5
R
of 27 min was accumulated. It was isolated by HPLC and
min) was assigned as 5′-O-methylthymidine 3′-(phenyl phos-
phate) (12). The other two products were most likely formed
by opening of the sugar ring, and they were assigned as the
two diastereomers of structure 13, i.e., the phenyl esters of
assigned as 5′-O-acetylthymidine 3′-(phenyl phosphate) (15)
by FAB mass spectroscopy and 1H, C, and P NMR spec-
13
31
troscopy.
′-O-Acetylth ym id in e 3′-(p h en yl p h osp h a te) (15). MS
m/z: 440. δ (400 MHz): 7.35 (1 H, H6), 7.3-7.0 (5 H, Ph),
.14 (1 H, t, J 1′,2′ ) J 1′,2′′ ) 6.8 Hz, H1′), 4.77 (1 H, m, H3′),
.19 (1 H, dd, H4′), 4.14 (1 H, dd, J 5′,5′′ ) 12.3 Hz, J 4′,5′′ ) 3.3
5
(
2-deoxy-L- and -D-glycero-1-O-methyl-1-C-(1-thyminyl)-D-
H
erythro-pentitol) 3′,5′-cyclic monophosphate.
6
4
(
18) Harned, H. S.; Embree, N. D. J . Am. Chem. Soc. 1934, 56,
042-1044.
19) Harned, H. S.; Ehlers, R. W. J . Am. Chem. Soc. 1932, 54, 1350-
357.
Hz, H5′′), 4.03 (1 H, dd, J 4′,5′ ) 4.5 Hz, H5′), 2.42 (1 H, ddd,
2′,2′′ ) 16 Hz, J 2′′,3′ ) 4.1 Hz, H2′′), 2.33 (1 H, dd, J 2′,3′ ) 0 Hz,
H2′), 1.93 (3 H, s, OAc), 1.72 (3H, s, 5-CH ). δ (125.6 MHz):
12.4, 21.2, 38.4, 61.9, 76.4, 85.9, 86.4, 112.4, 120.9, 125.3, 130.7,
1
1
3
J
(
3
C
(
20) Bates, R. G.; Allen, G. F. J . Res. Nat. Bur. Stand. 1060, 64A,
43-346.
(
138.4, 152.5, 167.4, 171.3. δ : -6.0 ppm.
P
21) Cox, M. C.; Everett, D. H.; Landsman, D. A.; Munn, R. J . J .
Chem. Soc. B 1968, 1373-1379.
J O960693V