Highly efficient C-8 oxidation of substituted xanthines with substitution at the 1-, 3-, and 7- Positions using biocatalysts

Article abstract of DOI:10.1039/a900089e

A bacterial consortium consisting of strains belonging to the genus Klebsiella and Rhodococcus quantitatively converts 1-, 3- and 7-substituted xanthines to their respective 8-oxo compounds.

Full text of DOI:10.1039/a900089e

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Highly efficient C-8 oxidation of substituted xanthines with
substitution at the 1-, 3-, and 7- positions using biocatalysts
K. M. Madyastha*a,b and G. R. Sridhara
a Department of Organic Chemistry, Indian Institute of Science, Bangalore-56012, India
b Chemical Biology Unit, Jawaharlal Nehru Center for Advanced Scientific Research,
Bangalore-560 064, India
Received (in Cambridge) 5th January 1999, Accepted 29th January 1999
A bacterial consortium consisting of strains belonging to the genus Klebsiella and Rhodococcus quantitatively
converts 1-, 3- and 7-substituted xanthines to their respective 8-oxo compounds.
The C-8 oxidation of purine DNA bases is one of the major
forms of oxidative base damage and this chemical modification
Results and discussion
A mixed culture grown on caffeine 1 transformed quantitatively
has biological implications particularly in DNA replication,
mutagenicity and aging.1,2 In fact patients with leukemia excrete
higher levels of 8-oxoguanine in urine than normal humans
suggesting the significance of C-8 oxidation of purine bases.3
It is also known that 8-oxo derivatives of alkyl and substi-
tuted xanthines are biologically active and have numerous
applications in the formulations of drugs and cosmetics.
8-Oxocaffeine (1,3,7-trimethyluric acid) has been classified as
a good radical scavenger and a potent antioxidant in model
systems.4 Studies carried out in vitro have demonstrated that
8-oxoxanthine (uric acid) protects erythrocytes against damage
by singlet oxygen.5 Uric acid has also been shown to inhibit
lipid peroxidation,6 protect against oxidant damage to DNA,7
and act as a scavenger of hydroxyl radicals.8 In fact it has been
suggested that uric acid can serve as an important physiological
antioxidant against oxidative injury and thus can play a role in
the prevention of aging and cancer. It is interesting to note that
8-oxomethylxanthines and their derivatives are used as one of
the components in obesity-treating pharmaceuticals, cosmetic
skin and antidandruff preparations.9–11 However, details of
these studies are not available as they are covered by patents.
To explore further the utility of various C-8 oxidized alkyl-
xanthines and related compounds, studies have been carried out
to develop synthetic routes to such compounds.12,13
theophylline (1,3-dimethylxanthine, 2), theobromine (3,7-
dimethylxanthine, 3) and paraxanthine (1,7-dimethylxanthine,
4) to their corresponding C-8 oxidized compounds 22–24 (Table
1 and Scheme 1). The HPLC analyses revealed that the conver-
O
O
R3
R3
N
N
R1N
R1N
O
N
O
N
N
O
N
H
R2
R2
21 -37
1 -17
NH2
N
NH2
H
H
N
N
N
N
O
N
H
N
N
38
18
O
H
N
HN
No C-8 oxidation
8-Oxocaffeine is one of the metabolites of caffeine in the
mammalian system.14 Although several xanthines substituted at
the 1-, 3-, and 7- positions (caffeine analogues) such as pent-
oxyfylline, lisofylline, enprofylline, are used as drugs,15,16 the
corresponding 8-oxo derivatives have never been prepared so
far and hence these compounds are not available for biological
evaluation. Since many of these substituted xanthines are water
soluble, we were interested to explore the possibility of using
biocatalysts as a reagent to selectively carry out C-8 oxidation
in these compounds. A search for such a microbial system led to
the isolation of a naturally occurring bacterial consortium con-
sisting of strains belonging to the genus Klebsiella and Rhodo-
coccus capable of utilizing caffeine (1, 1,3,7-trimethylxanthine)
as the sole source of carbon and energy. Mixed cultures nor-
mally display a variety of degradative activities against sub-
stances of natural origin and in recent years they have been
successfully used in the synthesis of valuable products.17,18
We report here that the mixed culture isolated by the enrich-
ment culture technique using caffeine 1 as the carbon source,
selectively and efficiently carries out C-8 oxidation of adenine
and various 1-, 3- and 7-substituted xanthines. Many of the
8-oxo compounds (uric acids) prepared in the present investig-
ation were hitherto unknown.
N
H2N
N
19
Scheme 1
sions were quantitative and the isolated yields were 95–97%.
The 8-oxo compounds 22–24 formed were fully characterized
by comparison (NMR, MS, HPLC) with authentic samples and
also the spectral characteristics agreed with the earlier reports
for these compounds.19,20 When caffeine 1 was used as the sub-
strate, it was rapidly metabolized and although all the substrate
1 was transformed, the amount of the 8-oxo compound (1,3,7-
trimethyluric acid, 21) present in the incubation medium after
12 h of incubation was significantly low (35%, on the basis of
HPLC analysis). However, incubation carried out in the pres-
ence of N-methylmaleimide (see Experimental section) resulted
in the quantitative conversion of 1 into 21. This suggests that
enzymes involved in the further metabolism of 21 are inhibited
by the thiol-group blocking reagent. The spectral character-
istics (NMR, MS, HPLC) of compound 21 were in good
agreement with the authentic sample of 1,3,7-trimethyluric acid
21.
J. Chem. Soc., Perkin Trans. 1, 1999, 677–680
677

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Table 1 C-8 oxidation of substituted xanthines with substitution at
accumulation (70%, Table 1) of a metabolite which was identi-
fied as 8-oxoadenine 38 based on spectral analyses.21 Under
these experimental conditions, we could not demonstrate the
formation of 8-oxoguanine from gaunine. It is quite possible
that guanine is metabolized following a different pathway. It is
interesting to note that the mixed culture does not accept
adenosine 20 as the substrate.
the 1-, 3-, and 7-positions using biocatalysts
Substrate
R1
R2
R3
Product
1a
2
CH3
CH3
H
CH3
CH3
CH3
CH3
H
CH3
CH3
CH3
CH3
H
21
22
23
24
25
26
27
28
29
30
31
3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
Enzymatic hydroxylation at the C-8 position of xanthine
derivatives is effected either by a mixed function oxidase or
xanthine oxidase.22 We have noticed that cell-free extract pre-
pared from the mixed culture readily carries out C-8 oxidation
of various substituted xanthines in the presence of PES (phen-
azine ethosulfate)† (unpublished observation). So it is quite
possible that the C-8 oxidation is mediated by xanthine oxidase.
However, xanthine oxidases isolated from various microbial
sources have never been shown to accept trimethyl- and
dimethylxanthines as substrates.23,24 The exceptional ability of
the mixed culture to convert 1,3,7-trimethylxanthine and vari-
ous 1-, 3-, and 7- substituted xanthines to their corresponding
8-oxo derivatives in quantitative yields clearly distinguishes the
xanthine oxidase from this mixed culture from previously
reported xanthine oxidases from various microbial sources.
The results of the present investigation document a micro-
bial method for carrying out selectively and efficiently the C-8
oxidation of substituted xanthines (1–17). In all the cases, no
N-oxide derivatives were formed suggesting that the organism is
not capable of addition of O2 onto the nitrogen atom(s) of the
imidazole or purine ring. In all the cases, the products formed
were free from the substrates and there were no side products
and hence did not require any further purification. Considering
the fact that derivatives of uric acids have numerous applica-
tions, the scope for the biocatalyst mediated process for the
preparation of these compounds appears to be unlimited.
4
5
CH3CH2
CH3CH2CH2
CH3CH2CH2CH2
CH3CH2CH2CH2CH2 CH3
CH2CH2OH
PhCH2
6
7
8
9
CH3
CH3
10
11
CH3COCH2CH2CH2- CH3
CH2
12
13
14
15
16
17
CH3COCH2
CH3
CH3
CH3
CH3
CH3CH2CH2
CH3CH2CH2
CH3
CH3
CH3
CH3
H
32
33
34
35
36
37
CH2᎐᎐CHCH2
᎐
CH᎐CCH2
᎐
CH2᎐᎐CHCH2CH2
H
CH3
CH3
a Incubation of 1 and 18 were carried out in the presence of
N-methylmaleimide (1 mM). Details are given in the Experimental
section.
In order to investigate the specificity of this bacterial con-
sortium for inserting an oxygen atom at the C-8 position, we
synthesized a range of 1-, 3-, and 7- substituted xanthines 5–17
(Table 1) and used them as substrates. It was observed that
caffeine 1 grown cells convert quantitatively analogues of
theobromine 3 with N-1-H replaced by various groups such as
alkyl (5–8), hydroxyethyl (9), benzyl (10), 5-oxohexyl (11),
2-oxopropyl (12), allyl (13), propynyl (14) and but-3-enyl (15) to
their corresponding C-8 oxidized compounds (25–35, Table 1).
The compounds 25–35 (Table 1) were fully characterized by
various spectral analyses (see Experimental section) and as far
as we know these uric acids 25–35 were hitherto unknown. The
optimal size of alkyl substituent at the N-1 position appears to
be pentyl and any substitution higher than pentyl, for example
1-hexyl-3,7-dimethylxanthine was not accepted by the mixed
culture as a substrate. However, pentoxyfylline 11 with a
–(CH2)4COCH3 substitution at the N-1 position was readily
accepted by the bacterial consortium and quantitatively con-
verted into its corresponding C-8 oxidized product 31 (Table 1).
It is interesting to note that theophylline 2 analogues prepared
by replacing N-7-H with alkyl substitution higher than methyl,
for example, ethyl, propyl and butyl, were not accepted by the
mixed culture as substrates, possibly due to the proximity of the
bulky substituents to the C-8 position. However, the bacterial
consortium readily accepted 3-propylxanthine 16 (enprofylline)
and 3-propyl-1,7-dimethylxanthine 17 as substrates and both
these compounds (16, 17) were converted quantitatively into
their corresponding 8-oxo derivatives 36, 37. The compounds
36 and 37 appear to be hitherto unknown and have been fully
characterized by various spectral analyses (see the Experi-
mental section). These results suggest that replacement of the
N-3 methyl in caffeine 1 with a higher alkyl group such as pro-
pyl has no effect on the C-8 oxidation, whereas a substituent at
the N-7 position with an alkyl group higher than methyl is not
accepted by the mixed culture as a substrate.
Experimental
IR spectra were recorded using a Perkin-Elmer spectrometer.
NMR studies were carried out at 90 MHz using a JEOL FT-90
spectrometer or at 300 MHz using a JEOL JNM-LA 300 FT
spectrometer. J values are quoted in Hz and chemical shifts are
reported relative to TMS. Mass spectra were determined using
a JEOL JMS-DX 303 spectrometer. HPLC analysis was carried
out on a Shimadzu CR7A instrument, on a reversed phase
ODS column using either NaOAc (0.1 M)–CH3OH–CH3CN
(70:20:10, v/v) or H2O–CH3OH–CH3CN (70:10:20, v/v) and
the solvent system and eluents (1 ml minϪ1) were monitored
with a UV detector at 254 nm. TLC analyses were performed on
silica gel GF254 plates (0.5 mm) developed with CHCl3–CH3OH
(92:8, v/v).
Materials
Caffeine 1, theophylline (1,3-dimethylxanthine, 2), theobromine
(3,7-dimethylxanthine, 3), paraxanthine (1,7-dimethylxanthine,
4), 1,3-dimethyluric acid, 22, 3,7-dimethyluric acid 23, 1,7-
dimethyluric acid 24, 1,3,7-trimethyluric acid 21, adenine 18,
and guanine 19 were purchased from Sigma. Chloroacetone
was purchased from Fluka. Enprofylline (3-propylxanthine, 16)
was a generous gift from Dr Hans Jurgen Federsel, Astra Pro-
duction Chemicals AB, Sweden. Chlorohexane was prepared
following the method reported earlier.25 Other bromides used in
the present study were prepared according to the standard
procedure given in Vögel.26
Since purine bases (18, 19) have considerable structural
similarities with caffeine 1, we were interested in finding
out whether the mixed culture has the ability to carry out C-8
oxidation of these compounds. Although caffeine 1 grown
cells readily metabolized both adenine 18 and guanine 19,
the HPLC analyses of the incubation mixture indicated the
absence of any 8-oxo derivatives of 18 and 19. However,
incubation of 18 carried out in the presence of N-methyl-
maleimide (see the Experimental section) resulted in the
Synthesis of N-1 substituted theobromines (5–15)
Substituted theobromines 5–15 were synthezised as reported
earlier.27 A mixture of theobromine (1.0 g, 5.55 mmol), sodium
hydroxide (8 cm3, 3.2 mmol), alkyl/allyl/propynyl/but-3-enyl/
† The IUPAC name is N-ethylphenazin-5-ium ethylsulfate.
678
J. Chem. Soc., Perkin Trans. 1, 1999, 677–680

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hydroxyethyl/benzyl bromides (11 mmol) and propan-2-ol (12
cm3) was taken in a sealed tube and heated at 120 ЊC for 24 h.
The reaction mixture was allowed to cool to room temperature
and extracted with chloroform (50 cm3 × 4). The organic layer,
evaporated and purified by column chromatography over silica
gel using CHCl3–CH3OH (97:3, v/v), yielded pure 5–15 and
yields varied from 45–83%.
uric acids (21–37) varies from 95–97% (Table 1). The isolated
yield of 8-oxoadenine 38 was only 70%. The spectral character-
istics of the 8-oxo compounds 25–38 are given below.
1-Ethyl-3,7-dimethyluric acid (25). Mp 295 ЊC (CHCl3–
MeOH); νmax (Nujol)/cmϪ1 1640, 1680, 1695 (–NC᎐O–); δ (300
᎐
H
MHz, CDCl3) 1.2 (3H, t, J 7.2, CH3CH2–), 3.5 (3H, s, –NCH3),
3.58 (3H, s, –NCH3), 4.0 (2H, q, J 7.2, CH2CH3); δC(75 MHz,
CDCl3) 14.2, 30.0, 30.8, 41.2, 99.0, 136.7, 150.9, 153.7, 154.0;
m/z 224 (Mϩ, 65%), 153 (40%), 82 (100%), 67 (55%); HRMS:
found Mϩ, 224.0900. C9H12N4O3 requires 224.0909.
Synthesis of 3-propyl-1,7-dimethylxanthine (17)
The compound 17 was prepared following the published pro-
cedure.28 To a stirred suspension of 3-propylxanthine (0.97 g, 5
mmol) and anhydrous K2CO3 in 8 cm3 of DMF was added
dropwise 15 mmol of methyl iodide. The reaction mixture was
heated at 35 ЊC for 4 h and the volatile material removed under
vacuum. The product was isolated by adding water and
extracted with ethyl acetate. The crude product was purified by
column chromatography over silica gel using CHCl3–CH3OH
(97:3, v/v) and yielded pure 17. Following the same pro-
cedure,28 N-7-substituted theophyllines were prepared using
theophylline 2 and alkyl halide.
1-Propyl-3,7-dimethyluric acid (26). Mp 280 ЊC (CHCl3–
MeOH); νmax (Nujol)/cmϪ1 1640, 1680, 1695 (–NC᎐O–); δ (300
᎐
H
MHz, CDCl3) 0.97 (3H, t, J 7.2, CH3CH2CH2–), 1.67 (2H, m,
CH3CH2CH2–), 3.5 (3H, s, –NCH3), 3.56 (3H, s, –NCH3), 3.9
(2H, t, J 7.2, –NCH2–); δC(75 MHz, CDCl3) 12.4, 22.3, 29.0,
31.2, 41.4, 98.9, 136.7, 150.0, 153.0, 154.5; m/z 238 (Mϩ, 100%),
219 (Mϩ Ϫ H2O, 50%), 196 (Mϩ Ϫ C3H7, 70%), 153 (60%), 82
(75%); HRMS: found Mϩ, 238.1061. C10H14N4O3 requires
238.1065.
Microorganism and growth media
1-Butyl-3,7-dimethyluric acid (27). Mp 268–270 ЊC (CHCl3–
MeOH); νmax (Nujol)/cmϪ1 1640, 1680, 1700 (–NC᎐O–); δ (300
᎐
H
The mixed culture used in this study was isolated from the
garden soil using caffeine 1 as the carbon source. It was shown
to be a mixed population of two bacterial strains. Based on
various morphological, cultural and biochemical character-
istics, the two organisms were identified as belonging to the
genus Klebsiella and Rhodococcus. The mixed culture was main-
tained at 3 ЊC on nutrient agar slants containing 0.04% of
caffeine 1 and 0.1% glucose. It was also maintained regularly in
a liquid mineral salts medium29 containing 0.04% caffeine 1 and
0.1% glucose. The starter culture was prepared by transferring
an aliquot (5 cm3) from the maintenance culture to a 100 cm3
sterilized liquid mineral salts medium (pH 7.2) containing
0.04% caffeine 1 and 0.1% glucose and incubating this on a
rotary shaker (220 rpm) at 29–30 ЊC for 36 h. Although the
mixed culture accepts caffeine 1 as the sole source of carbon,
the growth rate is slow. Hence to enhance the growth rate, 0.1%
glucose was added to the medium.
MHz, CDCl3) 0.95 (3H, t, J 7.2, CH3(CH2)3–), 1.4–1.6 (4H, m,
CH2CH2–CH2–N–), 3.5 (3H, s, –NCH3), 3.57 (3H, s, –NCH3),
3.97 (2H, t, J 7.2, –NCH2-); δC(22.5 MHz, CDCl3) 13.8, 20.2,
28.8, 30.0, 30.8, 41.4, 99.0, 136.0, 150.0, 153.0, 153.7; m/z 252
(Mϩ, 100%), 225 (Mϩ Ϫ H2O, 55%), 196 (Mϩ Ϫ C4H9, 95%),
153 (90%), 82 (80%); HRMS: found Mϩ, 252.1218. C11H16N4O3
requires 252.1222.
1-Pentyl-3,7-dimethyluric acid (28). Mp 265 ЊC (CHCl3–
MeOH); νmax (Nujol)/cmϪ1 1640, 1680, 1695 (–NC᎐O–); δ (200
᎐
H
MHz, CDCl3) 0.97 (3H, t, J 7.2, CH3–(CH2)4–), 1.35 (4H, m,
CH3CH2CH2), 1.6 (2H, m, –CH2CH2CH2–N–), 3.5 (3H, s,
–NCH3), 3.56 (3H, s, –NCH3), 3.97 (2H, t, J 7.2, –NCH2–);
δC(75 MHz, CDCl3) 13.9, 22.3, 27.6, 29.0, 29.5, 33.4, 41.0,
107.0, 141.0, 148, 151, 155; m/z 250 (Mϩ, 70%), 180 (Mϩ Ϫ
C5H11, 100%); HRMS: found Mϩ, 250.1435. C12H18N4O2
requires 250.1429.
General procedure for biotransformation
1-(2-Hydroxyethyl)-3,7-dimethyluric acid (29). Mp 255 ЊC
(CHCl3–MeOH); νmax (Nujol)/cmϪ1 3300 (–CH2OH), 1640,
A batch of 15 flasks containing 100 cm3 of sterile mineral salts
medium29 (pH 7.2) containing 0.1% glucose and 0.04% of 1
were inoculated from a 36 h old culture (5 cm3, A660 = 1.1) and
incubated on a rotary shaker at 29–30 ЊC for 36 h. At the end of
the incubation period, the cells were harvested by centri-
fugation (3000 g for 20 min), washed well with phosphate buffer
(0.03M, pH 7.2) and suspended in the same buffer (~1.5 g wet
weight in 100 cm3). To this cell suspension (100 cm3), substrate
(2–17, 100 mg) was added and incubated on a rotary shaker for
12 h at 29–30 ЊC. In the case of caffeine 1, adenine 18, and
guanine 19, the incubation mixture also contained N-methyl-
maleimide (1 mM) and the rest of the conditions were the same
as that used for substrates 2–17. At the end of the incubation
period, the assay mixture was acidified to pH 5–6 and centri-
fuged. An aliquot from the supernatant was subjected to HPLC
analyses which indicated the absence of substrate and its com-
plete conversion to the corresponding C-8 oxidized product
(21–37). For isolating the biotransformed product, the super-
natant was extracted with CHCl3–CH3OH (2:1, v/v), the
organic phase dried over Na2SO4 and solvent removed under
reduced pressure. The residue was passed through a small pad
of silica gel, eluting with CHCl3–CH3OH (95:5, v/v) to obtain
pure 8-oxo compounds (uric acids 21–37, 8-oxoadenine 38)
which were completely characterized by various spectral analy-
ses (IR, NMR, MS). Although the HPLC analyses indicated
the complete conversion (100%) of the substrates (1–17) to the
corresponding C-8 oxidized products, the isolated yields of the
1680, 1690 (–NC᎐O–); δ (90 MHz, DMSO-d ) 3.58 (6H, s,
᎐
H
6
2 × NCH3), 3.8 (2H, t, J 7.2, –CH2–OH), 4.15 (2H, t, J 7.2,
–CH2N–); δC(22.5 MHz, DMSO-d6) 29.0, 30.8, 45.6, 59.0, 97.0,
136.1, 150.2, 153.3, 153.9; m/z 240 (Mϩ, 70%), 196 (Mϩ Ϫ CH2-
CH2OH, 60%), 153 (65%), 82 (100%); HRMS: found Mϩ,
240.0852. C9H12N4O4 requires 240.0858 (Found: C, 44.89;
H, 4.92; N, 23.12. C9H12N4O4 requires C, 45.0, H, 5.0, N,
23.37%).
1-Benzyl-3,7-dimethyluric acid (30). Mp 315 ЊC (CHCl3–
MeOH); νmax (Nujol)/cmϪ1 1640, 1680, 1695 (–NC᎐O–); δ (200
᎐
H
MHz, CDCl3) 3.4 (3H, s, –NCH3), 3.5 (3H, s, –NCH3), 5.0 (2H,
s, –NCH2–), 7.2–7.4 (5H, m, aromatic); δC(22.5 MHz, DMSO-
d6) 28.0, 30.8, 43.0, 97.0, 126.9, 127.3, 127.6, 136.9, 137.5,
149.8, 151.6, 152.5; m/z 286 (Mϩ, 100%), 195 (30%), 91 (C6H5,
75%); HRMS: found Mϩ, 286.2896. C14H14N4O3 requires
286.2899 (Found: C, 58.64; H, 4.81; N, 19.48. C14H14N4O3
requires C, 58.74; H, 4.89; N, 19.58%).
1-(5-Oxohexyl)-3,7-dimethyluric acid (31). Mp 223–225 ЊC
(CHCl3–MeOH); νmax (Nujol)/cmϪ1 1700 (–C᎐O–), 1640, 1680
᎐
(–NC᎐O–); δ (300 MHz, CDCl ) 1.65 (4H, m, –CH CH ), 2.1
᎐
H
3
2
2
(3H, s, CH3–CO–), 2.5 (2H, t, J 7.2, –CO–CH2–), 3.5 (3H, s,
–NCH3), 3.57 (3H, s, –NCH3), 3.9 (2H, t, J 7.2, –NCH2–);
δC(22.5 MHz, CDCl3) 21.0, 27.4, 28.9, 29.7, 30.8, 41.6, 43.0,
99.2, 136.0, 150.2, 153.3, 153.5, 208.0; m/z 294 (Mϩ, 100%), 196
J. Chem. Soc., Perkin Trans. 1, 1999, 677–680
679

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(Mϩ Ϫ C6H11O, 95%), 153 (70%), 82 (55%); HRMS: found Mϩ,
294.1295. C13H18N4O4 requires 294.1325 (Found: C, 52.88; H,
6.04; N, 18.95. C13H18N4O4 requires C, 53.06; H, 6.12; N,
19.04%).
8-Oxoadenine (38). Mp 301 ЊC (decomposes)(MeOH); νmax
(Nujol)/cmϪ1 1640, 1680, 1695 (–NC᎐O–) 3310 (–NH ); δ (300
᎐
2
H
MHz, DMSO-d6) 8.02 (H-2), 6.22 (–NH2); m/z 152 (Mϩ ϩ 1,
25%), 136 (Mϩ ϩ 1 Ϫ NH2, 100%), 109 (30%), 54 (25%), 44
(50%). The spectral characteristics agreed with the earlier
report for 8-oxoadenine.21
1-(2-Oxopropyl)-3,7-dimethyluric acid (32). Mp 240 ЊC
(CHCl3–MeOH); νmax (Nujol)/cmϪ1 1700 (–C᎐O–), 1640, 1680
᎐
(–NC᎐O–); δ (90 MHz, CDCl ) 2.1 (3H, s, CH CO–), 3.5 (3H,
᎐
H
3
3
Acknowledgements
s, –NCH3), 3.57 (3H, s, –NCH3), 4.7 (2H, s, –NCH2–); δC(75
MHz, CDCl3) 28.0, 30.6, 33.8, 47.8, 98.2, 136.1, 150.0, 153.3,
153.5, 204.0; m/z 252 (Mϩ, 100%), 209 (Mϩ Ϫ CH3CO, 75%),
196 (Mϩ Ϫ C3H5O, 85%); HRMS: found Mϩ, 252.0852.
C10H12N4O4 requires 252.0858.
We thank Professor S. B. Sulia, Department of Microbiology,
Bangalore University, for identifying the strains. Financial
assistance from DBT (New Delhi, India), CSIR (New Delhi,
India) and JNCASR (Bangalore, India) is gratefully acknow-
ledged. Financial support to G. R. S. by the CSIR is greatly
appreciated.
1-Allyl-3,7-dimethyluric acid (33). Mp 305–306 ЊC (CHCl3–
MeOH); νmax (Nujol)/cmϪ1 1640, 1690 (–NC᎐O–); δ (200 MHz,
᎐
H
CDCl3) 3.5 (3H, s, –NCH3), 3.56 (3H, s, –NCH3), 4.59 (2H, td,
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172035h.
᎐
2
CH᎐CH ); δ (22.5 MHz, DMSO-d ) 28.2, 30.9, 42.5, 97.8,
᎐
2
C
6
116.5, 133.0, 136.8, 149.5, 151.8, 152.3; m/z 236 (Mϩ, 100%),
219 (Mϩ Ϫ H2O, 15%), 153 (40%), 82 (45%); HRMS: found
Mϩ, 236.0900. C10H12N4O3 requires 236.0909.
1-Prop-2-ynyl-3,7-dimethyluric acid (34). Mp 276 ЊC (CHCl3–
MeOH); νmax (Nujol)/cmϪ1 1640, 1680 (–NC᎐O–); δ (300 MHz,
᎐
H
CDCl ) 2.1 (1H, t, J 2.4, HC᎐C–CH ), 3.5 (3H, s, –NCH ), 3.59
᎐
3
2
3
(3H, s, –NCH3), 4.6 (2H, d, J 2.7, –NCH2–); δC(22.5 MHz,
CDCl3) 29.7, 30.3, 34.0, 71.4, 79.1, 97.0, 136.0, 150.7, 153.0,
153.8; m/z 234 (Mϩ, 100%), 153 (25%), 84 (45%), 82 (85%), 67
(57%); HRMS: found Mϩ, 234.0749. C10H10N4O3 requires
234.0752.
1-But-3-enyl-3,7-dimethyluric acid (35). Mp 262 ЊC (CHCl3–
MeOH); νmax (Nujol)/cmϪ1 1640, 1690 (–NC᎐O–); δ (90 MHz,
᎐
H
12 R. Saladino, C. Crestini, R. Bernini, E. Mincione and R. Ciafrino,
Tetrahedron Lett., 1995, 36, 2665.
CDCl ) 2.4 (2H, q, CH ᎐CH–CH –CH ), 3.5 (3H, s, –NCH ),
᎐
3
2
2
2
3
3.58 (3H, s, –NCH3), 4.0 (2H, t, J 7.2, –N–CH2–CH2), 5.0 (1H,
d, J 10, cis CH᎐CH ), 5.2 (1H, d, J 17, trans CH᎐CH ), 5.8 (1H,
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1995, 36, 2633.
14 J. L. Ferrero and A. H. Neims, Life Sci., 1983, 33, 1173.
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16 C. G. A. Persson and G. Kjellin, Acta. Pharmacol. Toxicol., 1981,
49, 313.
17 I. Goldberg and C. L. Cooney, Appl. Environ. Microbiol., 1981,
41(1), 148.
18 J. E. Makins, G. Holt and K. D. Macdonald, J. Gen. Microbiol.,
1980, 119, 397.
19 W. Pfleiderer, Liebigs Ann. Chem., 1974, 2030.
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25 W. V. Yikang and Per Ahlberg, Synthesis, 1994, 463.
26 Vögel’s Text book of Practical Organic Chemistry, 5th edition, eds.
B. S. Furniss, A. J. Hannaford, P. W. G. Smith and A. R. Tatchell,
Longman Publishers Pvt. Ltd, UK, 1989.
᎐
᎐
2
2
tdd, J 17, 10.3, 4.5, CH᎐CH ); δ (22.5 MHz, CDCl ) 28.0, 30.7,
᎐
2
C
3
31.8, 40.3, 97.8, 116.6, 135.1, 136.4, 149.5, 151.5, 152.5; m/z 250
(Mϩ, 40%), 196 (Mϩ Ϫ C4H7, 100%), 153 (45%), 82 (50%);
HRMS: found Mϩ, 250.1061. C11H14N4O3 requires 250.1065
(Found: C, 52.73; H, 5.58; N, 22.18. C11H14N4O3 requires C,
52.8; H, 5.6; N, 22.4%).
3-Propyluric acid (36). Mp 279 ЊC (CHCl3–MeOH); νmax
(Nujol)/cmϪ1 1640, 1680 (–NC᎐O–); δ (300 MHz, DMSO-d )
᎐
H
6
0.95 (3H, t, J 6.6, CH3CH2–), 1.64 (2H, m, CH3CH2CH2–), 3.9
(2H, t, J 7.5, –NCH2–), 11.0 (1H, s, –NH); δC(75 MHz,
DMSO-d6) 10.9, 21.5, 44.5, 98.9, 137.7, 151, 153.4, 153.9;
m/z 210 (Mϩ, 100%), 168 (Mϩ Ϫ C3H7, 95%), 137 (Mϩ Ϫ
C2H2NO2, 75%); HRMS: found Mϩ, 210.0748. C8H10N4O3
requires 210.0752.
3-Propyl-1,7-dimethyluric acid (37). Mp 282 ЊC (CHCl3–
MeOH); νmax (Nujol)/cmϪ1 1640, 1680 (–NC᎐O–); δ (90 MHz,
᎐
H
27 C. Wang, J. Wang and C. Chan, Bull. Inst. Chem., Acad. Sin., 1981,
28, 69.
28 J. W. Daly, W. L. Padgett and M. T. Shamim, J. Med. Chem. 1982,
25, 197.
29 W. Seubert, J. Bacteriol., 1960, 79, 426.
CDCl3) 1 (3H, t, J 7.2, CH3CH2–), 1.6 (2H, m, CH3CH2CH2–),
3.4 (3H, s, –NCH3), 3.5 (3H, s, –NCH3), 3.9 (2H, t, J 7.2,
–NCH2–); δC(75 MHz, CDCl3) 10.8, 21.6, 28, 28.9, 46.0, 99.2,
135.7, 150.0, 153.0, 153.9; m/z 238 (Mϩ, 100%), 196
(Mϩ Ϫ C3H7, 70%), 152 (Mϩ Ϫ C3H4NO2, 50%), 139 (65%);
HRMS: found Mϩ, 238.1059. C10H14N4O3 requires 238.1065.
Paper 9/00089E
680
J. Chem. Soc., Perkin Trans. 1, 1999, 677–680