10.1002/cbic.202100080
ChemBioChem
FULL PAPER
c. Biosynthesis step: fermentation broth containing sucrose, soybean flour,
corn steep solid, CaCO3, NaCl and L-Tryptophan (1 g/L), pH adjusted to
6.6. Inoculated with biomass seed culture of (b) and addition of non
labelled L-tyrosine (30 mg/L or 50 mg/L or 100 mg/L or 200 mg/L) or
labelled (30 mg/L), cultivation time until 72 h maximum at 32°C and 200
rpm.
Conclusion
Radiolabelling of de-O-methyltomaymycin was successfully
conducted using a biosynthetic approach. Yield was increased
and specific activity was optimized using [14C]-anthranilic acid and
[14C]-L-tyrosine.
Our fermentative process led to one of the highest level of
incorporation of radiochemical precursors ever published.[12] In,
addition, the specific activity obtained is in the same range as
those described for the carbon 14 labelling of acarbose which
optimization process lasted more than 20 years.[11]
Biosynthesis of PBDs, tomaymycin derivatives scaffolds,
continues to be studied[17] as well as engineering of
Streptomycetes strains for secondary metabolites production.[18]
Theses studies may one day make it possible to rapidly obtain
complex radiolabelled molecules with high yield and high specific
activity by using this one step approach.
Protocol 2
a. Pre-culture medium containing glucose, soybean flour, corn steep solid,
CaCO3 and NaCl, pH adjusted to 7. Standard sterilization conditions.
Inoculated with frozen Streptomyces FH6421, 3 days cultivation time at
28°C and 200 rpm.
b. Culture medium containing sucrose, soybean flour, corn steep solid,
CaCO3, NaCl, , pH adjusted to 6.6. Standard sterilization conditions.
Inoculated with 2% seed culture (a), 12 h at 32°C and 180 rpm.
c1. Non labelled biosynthesis step: direct addition, in culture medium (b) of
L-tyrosine (120 mg/L) and L-tryptophan (1 g/L), cultivation time until 72 h
maximum at 32°C and 180 rpm.
c2. Labelled biosynthesis step : addition, directly in culture medium (b), of
L-tryptophan (1 g/L) and [14C3]-L-tyrosine at 120 mg/L or [14C]-L-tyrosine at
125.6 mg/L and [14C]-anthranilic acid 4 at 92.4 mg/L, cultivation time until
62 h at 32°C and 180 rpm.
Work-up
Non-labelled biotransformations were estimated by HPLC-UV analysis
(condition A). One non-labelled biotransformation was purified to obtain
compound 2 characteristics and to validate 3 as side product16. All
experiments with carbon 14 were purified and only compound 2 was
isolated.
Experimental Section
Materials and methods.
After centrifugation, the filtrate was pre-purified on Amberchrom resin
CG161 (25 ml) pre-conditioned with water (100 ml). The resin was eluted
with water/Methanol 90/10 (100 ml), 80/20 (100 ml), 50/50 (100 ml), 25/75
(100 ml) then 10/90 (300 ml). The fractions containing the desired product
were combined and methanol was evaporated. The residual phase was
purified on toyopearl resin (15 ml) beforehand washed with NaCl 1 M (30
ml) and water (30 ml). The resin was eluted with water and fractions
containing the desired product were combined. This solution was added to
10 g of XAD-4 beforehand washed with H2O, then CH3CN and H2O and
finally dried. After 15 min of stirring (200 rpm), the resin was filtrated, dried
then added to CH3CN (2 x 25 ml). After 15 min of stirring (200 rpm), the
resin was filtrated and organic phases were combined, concentrated in
vacuum. The crude product was purified by preparative chromatography
(Luna phenomenex, 5 µ, C18, 250 x 21.2 mm, CH3CN/H2O 30/70, D = 7
ml/min, P=49 bars).
Streptomyces spec. FH6421 was obtained from the Process Development
Biothechnology Department of Sanofi R&D. All fermentation processes
were performed in Multritron Infors HT Incubator. All reagents and solvents
were purchased from commercial suppliers and used without further
purification. Carbon-14 labelled L-tyrosine (specific activity 555 MBq/mmol
or 6364 MBq/mmol) was purchased from Pharmaron (Cardiff, UK).
Carbon-14 labelled potassium cyanide (specific activity 2114MBq/mmol)
was purchased from Tjaden Bioscience (Volxheim, Deutchland).
Amberchrom CG161 was purchased from Rohm and Haas, TOYOPEARL
DEAE 650M from Tosoh and Amberlite XAD-4 from Alfa Aesar.
Reactions were monitored by HPLC or/and thin layer chromatography
(TLC) which was performed on 60 F254 silica gel plates. Radiolabeled
products were compared with authentic materials. Merck silica radio-TLC
plates were analyzed and quantified by electronic autoradiography using
a Packard Instant imager. Quantification of radioactivity was determined
using Ultima Gold as liquid scintillation cocktail on a Perkin Elmer TRI-
HPLC condition A (Rt 2: 6.4 min, Rt 3: 7.4 min) : radiochemical and chemical
purities > 97%
CARB
® 2900TR liquid scintillation counter. Specific activity was
1H-NMR (600 MHz, CDCl3) 2: 7.67 (d, J = 4.5 Hz, 1H), 7.52 (s, 1H), 6.90
(s, 1H), 6.05 (br.s, 1H), 5.61 (m, 1H), 4.27 (br. s, 2H), 3.98 (s, 3H), 3.90
(br. dd, J = 6.5 & 5 Hz, 1H), 2.97 (d, J = 5 Hz, 2H), 1.75 (d, J = 6.6 Hz, 3H).
MS (m/z) : 289.11 (M-H)-, 291.11, 295.12, 297.13
calculated by mass spectrometry on a Shimadzu hybrid Ion Trap / Time of
Flight IT-TOF spectrometer. Proton NMR spectra were recorded on Bruker
spectrometers (AC200 or Avance 600) in the deuterated solvent.
Chemical shifts were given in ppm relative to CDCl3 (7.26 ppm for 1H NMR
spectra). Multiplicity are described using the classical acronymes : s
(singlet), d (doublet), t (triplet), m (multiplet), br (broad). HPLC analyses of
labelled products were performed on a HP system equipped with a diode
Chemistry.
array UV detector and
a Packard Radiomatic 500TR series flow
Radiosynthesis of [14C]-anthranilic acid (4)
scintillation analyzer. HPLC analyses of non-labelled products were
performed on a Shimadzu UPLC system equipped with a diode array UV
detector. The following chromatographic conditions A were used: : Luna
C18 (5µm, 50x4.6 mm) column, elution at 40°C, mobile phase: eluent A
H2O/ammonium acetate 20 mM adjusted pH 4.6 with acetic acid (95/5,
v/v), eluent B acetonitrile/ammonium acetate 20 mM adjusted pH 4.6 with
acetic acid (95/5, v/v), gradient (% eluent B): t=0 (5%), t=10 min (30%),
t=12 min (100%), t=13 min (5%), t=15 min (5%), flow rate: 2.5 ml/min, UV
wavelength: 240 nm, volume of injection: 10 µL
2,2,2-Trifluoro-N-(2-iodo-phenyl)-acetamide (5)
To a solution of 2-iodoaniline (2.19 g; 10.00 mmol; 1.00 eq.) and
triethylamine (1.44 ml; 10.00 mmol; 1.00 eq.) in tetrahydrofuran (80.00 ml),
trifluoroacetic anhydride (2.00 g; 9.50 mmol; 0.95 eq.) in 20 ml of
tetrahydrofuran was added slowly. The mixture was stirred for 3 h and
concentrated in vacuum. The solid was dissolved in 100 ml of water and
extracted with ether (2 x 100 ml). The organic layers were combined, dried
(MgSO4), filtered and concentrated in vacuum. The residue was purified
by chromatography on a silica gel column eluted with diethyl ether/n-
pentane (20/80, v/v) to afford, after a final crystallization in n-pentane, 5
(3.15 g, quantitative yield) as white solid.
Fermentation process9.
1H-NMR (200 MHz, CDCl3) : 8.3 (broad s, 1H), 8.2 (d, J = 7.6 Hz, 1H), 7.75
(d, J = 7.6 Hz, 1H), 7.4 (t, J = 7 Hz, 1H), 7 (t, J = 6.9 Hz, 1H)
Protocol 1
a. Pre-culture medium containing glucose, soybean flour, corn steep solid,
CaCO3 and NaCl, pH adjusted to 7. Standard sterilization conditions
(120°C, 30 min). Inoculated with frozen Streptomyces FH6421 suspension
(1.5 mL, glycerol 30% in water), cultivation time 3 days at 28°C and 200
rpm.
b. Culture medium containing sucrose, soybean flour, corn steep solid,
CaCO3, NaCl, L-tyrosine (1 g/L) and L-tryptophan (1 g/L), pH adjusted to
6.6. Standard sterilization conditions. Inoculated with 2% seed culture (a),
27 h at 32°C and 200 rpm.
2-Amino-benzonitrile-[cyano-14C]19 (6)
To Copper-[14C]-cyanide (60 mg, 0.63 mmol, 1332 MBq), prepared from
potassium-[14C]-cyanide (47 mg, 0.7 mmol, 1480 MBq), cupric sulfate (182
mg, 1.14 mmol) and sodium metabisulfite (40 mg, 0.21 mmol) were added
2,2,2-Trifluoro-N-(2-iodo-phenyl)-acetamide (5) (220 mg, 0.7 mmol), N,N-
dimethylformamide (5 ml) and hexamethylphophoramide (1 ml). The
reaction mixture was stirred under an argon atmosphere at 150°C for 2 h,
cooled to room temperature, diluted with water (100 ml) and a solution of
NH4OHaq 30% (30 ml) then extracted with diethyl ether (3x150 ml). The
4
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