Table 3 The difference in normalised average areas for the 18O
enriched and natural abundance ions
ether extract (250 ml) of the reaction mixture was washed with
a saturated aqueous solution of salt (15 ml), added to ethanol
(20 ml) and evaporated to a volume of 10 ml. Ethanol (10 ml)
and water (20 ml) were added and the cooled solution was
adjusted to pH 10.7 with an ammonium hydroxide solution.
Na13CN (172 g, 3.44 mmol; 92% 13C) and NH4Cl (452 mg,
8.45 mmol) were added and the solution stirred at 4 ЊC for 10
min before the temperature was raised to 70 ЊC for 3 hours.
The mixture was cooled in ice, treated with concentrated HCl
(20 ml) and stirred at room temperature for three days. The
solution was boiled under reflux for 0.75 h and then cooled
and evaporated to dryness. Further concentrated HCl (20 ml)
was added and the solution boiled under reflux for three hours
and evaporated to dryness. Acidic hydrolysis and evaporation
of the solution were twice repeated, following which the residue
was azeotroped with water (50 ml) to give a solid.
A solution of the solid was chromatographed over a 3.5 × 1
inch column of Dowex 50W 8X ion exchange resin in the (H+)
form, which was eluted with 1 M NH4OH. Fractions containing
ninhydrin positive material were bulked, adjusted to pH 3.8
with HCl and evaporated to dryness. The residue was triturated
with ethanol and the resulting solid was recrystallised from
water (5 ml) to which boiling ethanol (70 ml) had been added,
giving a white solid (210 mg, 36% with respect to Na13CN),
νmax(KBr) 2976 (br), 2106, 1625 and 1500 (br) cmϪ1; δH (250
MHz, D2O) 1.60–2.00 (4H, m, 3-H2 and 4-H2), 3.00 (2H, t,
J 7.4, 5-H2), 3.74 (1H, q, J 6.0, 2-H); δC (100 MHz, D2O) 21.9
(4-C), 26.5 (3-C), 38.0 (5-C), 53.3 (d, J 53.5, 2-C), 173.2 (1-C);
m/z (FAB, thioglycerol) MH+ 134.
m/z
From [18O2]ornithine
Natural abundance
Difference
362
364
366
1.67
0.166
0.0375
1.67
0.124
0.003
0
0.042
0.035
45 min while the temperature was maintained below 5 ЊC and
the pH within the range 12–13 by the addition of a solution of
NaOH.
The reaction mixture was evaporated to dryness, redissolved
in DMF (10 ml) and reacted with benzyl bromide (15.5 mg,
0.1 mmol) for six days. Evaporation of the bulk of the
DMF left a residue which was triturated with ethyl acetate.
The ethyl acetate was decanted, the trituration of the solid
residue repeated and the ethyl acetate extracts combined and
evaporated to dryness. Recrystallisation (diisopropyl ether)
gave the product as white rosettes of needles (10 mg, 46%),
νmax(KBr) 3330, 1691 (br), 1533, 751 and 697 cmϪ1; δH (400
MHz, CDCl3) 1.40–1.96 (4H, m, 3-H2 and 4-H2), 3.08–3.25
(2H, m, 5H2), 4.37–4.48 (1H, m, 2-H), 4.65–4.80 (1H, m, NH),
5.08 (2H, s) and 5.10 (2H, s) and 5.15 (2H, m) (CH2Ph), 5.30–
5.40 (1H, m, NH) and 7.25–7.45 (15H, m, Ph); δC (100 MHz,
CDCl3) 25.79 (4-C), 29.96 (3-C), 40.43 (5-C), 53.59 (2-C), 66.70,
67.08 and 67.24 (C᎐O), 128.12–128.67 (2,3,4 and 5 phenyl),
᎐
135.20, 136.19, 136.56 (1-phenyl), 155.88, 156.37 (HNCO2)
and 171.93 (CO2); m/z 512 ([M18O2+NH4]+, 86%), 510
([M18O,16O+NH4]+, 14%).
(RS)-[1-13C,18O2]Ornithine dihydrochloride 13
Feeding of S-[18O2]ornithine dihydrochloride to S. clavuligerus
fermentations
RS-[1-13C]Ornithine (168 mg, 0.99 mmol) was dissolved in
H218O (0.25 ml, 12.5 mmol, 97% 18O) which had been acidified
by the passage of gaseous HCl for 30 seconds while atmos-
pheric moisture was excluded by a blanket of argon. The sealed
reaction vessel was heated to 70 ЊC for three days when the
solvent was removed under vacuum and the residue dried in
a desiccator to give a foam (194 mg). 13C NMR revealed that
the ornithine had been substituted with 18O in the following
pattern: [18O2] 75%, [18O,16O] 22% and [16O2] 2.7%. Exchange
with H218O (0.1 ml, 5 mmol, 97% 18O) was repeated to give a
buff coloured hygroscopic solid (172 mg, 83%), δH (400 MHz,
D2O) 1.70–2.07 (4H, m, 3-H2 and 4-H2), 3.04 (2H, t, J 7.5,
5-H2), 4.00 (1H, q, J 6.1, 2-H); δC (100 MHz, D2O) 23.7 (4-C),
27.8 (3-C), 39.8 (5-C), 53.5 (d, J 58.2, 2-C), 172.933 (87%,
[13C,18O2]) and 172.967 (13%, [13C,18O,16O]).
A sterile-filtered aqueous solution of S-ornithine dihydro-
chloride containing isotope labels in the ratio 86% [18O2] and
14% [18O,16O] (29.8 mg, 0.14 mmol) was fed to S. clavuligerus
SC2 fermentations (180 ml) during production phase and the
resulting clavulanate was isolated10 and purified as the benzyl
ester 12. 13C and H NMR and IR spectroscopy showed the
1
sample to be indistinguishable from benzyl clavulanate con-
taining natural isotopic abundance.
The benzyl clavulanate was treated with a mixture of N,O-
bis(trimethylsilyl)trifluoroacetamide–pyridine (1 : 1) for 30
minutes at room temperature and immediately injected onto a
BP1 capillary column (25 metre) which, after 2 min at 120 ЊC,
was heated to 280 ЊC at 8 ЊC minuteϪ1. The eluate from
the capillary was monitored by a Finnigan MAT TSQ70 mass
spectrometer operating in isobutane chemical ionisation mode.
The areas beneath the signals due to the molecular ions m/z 362
[M16O2+H]+, 364 [M18O,16O+H]+ and 366 [M18O2+H]+, were
determined by selected ion recording and averaged for five
separate analyses. Identical analyses were performed for benzyl
clavulanate of natural isotopic abundance and the maximum
variation within each set of five determinations was less
than 5% of the average. The difference in normalised average
areas for the 18O enriched and natural abundance ions were
determined and are shown in Table 3.
Feeding of (RS)-[1-13C,18O2]ornithine dihydrochloride 13 to S.
clavuligerus fermentations
RS-[1-13C]ornithine dihydrochloride containing isotope labels
in the ratio 87% [18O2] and 13% [18O] (29.4 mg, 0.14 mmol) was
fed to S. clavuligerus SC2 fermentations (180 ml) as before
and the resulting clavulanate was isolated and purified as the
benzyl ester, benzyl 3-(β-hydroxyethylidene)-7-oxo-4-oxa-1-
azabicyclo[3.2.0]heptane-2-carboxylate 12, δH (400 MHz,
CDCl3) 3.07 (1H, dd, J 16.7 and 0.6, 6β-H), 3.48 (1H, dd, J 16.7
and 2.9, 6α-H), 4.14–4,27 (2H, m, 9-H), 4.88 (1H, dt, J 7.0 and
1.3, 8-H), 5.09 (1H, m, 2-H), 5.18 and 5.22 (2H, ABq, 12.2,
CH2Ph), 5.70 (1H, dd, J 2.8 and 0.5, 5-H) and 7.30–7.42 (5H,
m, Ph); δC (100.614 MHz, CDCl3) 46.39 (6-C), 57.30 (9-C),
60.57 (d, J 67, 3-C13C), 60.59 (2-C), 67.66 (CH2Ph), 88.00
(5-C), 100.39 (8-C), 128.41, 128.72 and 134.78 (Ph), 153.38
(3-C), 166.979 (10-13C,18O ), 166.993 (10-13C,[᎐18O]), 167.015
RS-[1-13C]Ornithine
A procedure modified from Teplan and Marton17 was followed:
4,4-diethoxybutylamine (3 g, 18.6 mmol) dissolved in pyridine
(45 ml) was treated with benzoyl chloride (2.16 ml, 186 mmol)
in a dry atmosphere below 10 ЊC. After 2 hours, water (60 ml)
and ethyl acetate (400 ml) were shaken with the reaction
mixture and the ethyl acetate layer was separated, extracted
with a saturated aqueous salt solution, evaporated to dryness
and azeotroped with toluene to give an oil (4.5 g).
᎐
2
(10-13C,[-18O]), 167.029 (10-13C,16O2) and 174.25 (m, 7-C).
Acknowledgements
The oil (2 g, 7.5 mmol) was dissolved in THF (50 ml), cooled
in an ice bath and stirred with 2 M H2SO4 (10 ml) for 0.75 h. An
We thank our colleagues of SmithKline Beecham, Worthing,
for provision of the mutant S. clavuligerus dcl I 111, and DNA
J. Chem. Soc., Perkin Trans. 1, 2001, 1122–1130
1129