Claims
- 1. A process for preparing Antibiotic GE 2270 factor A having the following characteristics, in the non-salt form:
- A) ultraviolet absorption spectrum, which exhibits the following absorption maxima:
- ______________________________________ E.sub.1 cm.sup.1% Lambda max (nm)______________________________________0.1 M HCl 245 (shoulder) 3100.1 M KOH 245 (shoulder) 313Phosphate buffer pH 7.4 245 (shoulder) 314Methanol 244 (shoulder) 265 310______________________________________
- B) infrared absorption spectrum in nujol mull which exhibits the following absorption maxima (cm.sup.-1): 3700-3060; 3060-2660 (nujol); 1650; 1590-1490; 1490-1420 (nujol); 1375 (nujol); 1310; 1245; 1210; 1165; 1090; 1060; 1020; 970; 930; 840, 810, 750, 720 (nujol), 700;
- The main functional I.R. absorption bands of this spectrum can be attributed as:
- ______________________________________.nu., (cm.sup.-1) Assignment______________________________________3600-3100 .nu.NH, .nu.OH1650 amide I (.nu.C.dbd.O)1545 heterocyclic .nu.C.dbd.C and .nu.C.dbd.N1525, 1495 amide II (.delta.NH)1250, 1205 aromatic .delta.CH870 heterocyclic .gamma.CH745, 700 aromatic .gamma.CH______________________________________
- .sup.1 H-NMR spectrum which is shown in FIG. 3 and exhibits the following groups of signals (in ppm) at 500 MHz recorded in DMSO-d.sub.6 (hexadeuterodimethylsulfoxide) using TMS as the internal standard (0.00 ppm); the number of protons for each signal is reported between parenthesis:
- 9.02 (1); 8.68 (1); 8.70 (1); 8.57 (1); 8.50 (1); 8.43 (1); 8.37 (1); 8.26 (1); 8.25 (1); 7.4-7.20 (9); 6.96 (2); 6.02 (1); 5.30-5.18 (3); 5.01 (1); 4.97 (2); 4.80 (1); 4.56 (1); 4.30 (1); 4.26 (1); 3.98 (1); 3.81 (1); 3.79 (1); 3.38 (3); 2.72 (1); 2.58 (3); 2.48 (3); 2.16 (1); 2.13 (1); 1.96 (2); 1.88 (1); 1.34 (1); 0.87 (3); 0.84 (3);
- D) .sup.13 C-NMR spectrum which is reported in FIG. 4 of the accompanying drawings exhibiting the following groups of signals (ppm) at 125 MHz in DMSO-d.sub.6 with TMS as the internal reference (0.00 ppm), Q means quaternary carbon atoms or C.dbd.O groups;
- 173.69, Q; 171.10, Q; 169.83, Q; 169.51, Q; 168.45, Q; 168.26, Q; 167.84, Q; 165.68, Q; 164.75, Q; 161.40, Q; 161.23, Q; 160.46, Q; 160.29, Q; 159.35, Q; 153.42, Q; 150.31, Q; 150.11, Q; 149.41, Q; 146.93, Q; 144.73, Q; 143.75, Q; 142.10, Q; 141.78, Q; 141.33, CH; 140.97, Q; 139.53, Q; 128.68, CH; 127.99, 2[CH]; 127.67, Q; 127.67, CH; 126.88, CH; 126.76, 2/[CH]; 123.17, CH; 118.66, CH; 116.42, CH; 73.81, CH; 69.41, CH.sub.2 ; 67.97, CH; 67.36, CH.sub.2 ; 60.12, CH; 58.63, CH.sub.3 ; 58.24, CH; 55.41, CH; 48.15, CH; 47.03, CH.sub.2 ; 41.19, CH.sub.2 ; 37.60, CH.sub.2 ; 34.06, CH; 29.76, CH.sub.2 ; 25.85, CH.sub.3 ; 24.28, CH.sub.2 ; 18.49, CH.sub.3 ; 17.98, CH.sub.3 ; 11.99, CH.sub.3 ;
- E) retention-time (R.sub.t) of 14.9 min when analyzed by reverse phase HPLC under the following conditions:
- column: Ultrasphere ODS (reverse phase silanized silica gel; 5 micrometer) Altex (Beckman) 4.6 mm (i.d.).times.250 mm
- pre-column: Brownlee Labs RP 18 (octadecylsilane silica gel; 5 micrometer)
- eluent A: acetonitrile:18 mM sodium phosphate 70:30 (v/v), adjusted to pH 7.0
- eluent B: acetonitrile:18 mM sodium phosphate 10:90 (v/v), adjusted to pH 7.0
- elution mode: linear gradient of eluent A in eluent B from 45% to 70% in 20 min
- flow rate: 1.8 ml/min
- U.V. detector: 254 nm
- internal standard: Chloramphenicol (R.sub.t =3.7 min)
- F) elemental analysis, after the sample has been previously dried at about 140.degree. C. under inert atmosphere, which indicates the following composition: carbon, hydrogen, nitrogen, sulfur;
- G) R.sub.f value of 0.37 in the following chromatographic system: dichloromethane:methanol, 9:1 (v/v) using silica gel plates (silica gel 60F.sub.254, Merck Co)
- Visualization: U.V. light at 254 nm, yellow spot with iodine vapors or bioautography using B. subtilis ATCC 6633 on minimal Davis medium; internal standard: chloramphenicol (Rf 0.56)
- H) FAB-MS analysis showing the lowest mass isotope of the protonated molecular ion at m/z 1290.3.+-.0.1 dalton. All other peaks above 800 m/z mass units (not counting isotope peaks) in the spectrum were lower than 20% of the molecular ion, upon analysis with a Kratos MS-50 double focusing mass spectrometer under the following experimental conditions: Xe fast atom bombardment at 6 Kv; glycerol matrix; positive ionization mode
- I) an aminoacid analysis of the hydrochloric hydrolysate showing the presence of the following natural aminoacids: glycine, (L)proline and (L)serine, under the following experimental conditions:
- the sample is hydrolyzed at 105.degree. C. for 20 hours in the presence of 6N HCl containing 1% phenol and then derivatized in two steps as follows:
- a) formation of the n-propyl esters of the carboxylic acid functions with 2M HCl in anhydrous pronapol (90.degree. C., 1 h), and followed by drying under nitrogen;
- b) conversion of the free amino groups to amides with pentafluoropropionic anhydride/anhydrous dichloromethane, 1/9 (v/v) at room temperature for 1 h followed by drying under nitrogen;
- the derivatized residue so obtained is dissolved in dichloromethane and analyzed by GC-MS using a HP5985B system under the following conditions: column: chiral n-propionyl-L-valine t-butylamide polysiloxane coated fused silica capillary column (25 m.times.0.2 mm i.d.; C.G.C. ANALYTIC); temperature program 80.degree. C. for 4 min, then 4.degree. C./min
- L) Ionization studies
- No ionizable functions are detected by titration with 0.1N HCl and 0.1N NaOH in Methylcellosolve/water; a weak basic function is revealed by titration with 0.1N HClO.sub.4 in a non-aqueous medium (acetic acid);
- M) Specific rotation
- [alpha].sub.D.sup.20 =+140.8; absolute ethanol, at a concentration of about 5 gr/l,
- which comprises cultivating Planobispora rosea ATCC 53773 or a GE 2270 producing variant or mutant under submerged aerobic conditions in the presence of assimilable sources of carbon, nitrogen and inorganic salts and recovering the produced antibiotic therefrom.
- 2. A process according to claim 1 in which Planobispora rosea ATCC 53773 is cultivated.
Priority Claims (1)
Number |
Date |
Country |
Kind |
8821160 |
Sep 1988 |
GBX |
|
CROSS-REFERENCE TO RELATED APPLICATION
This is a divisional of application Ser. No. 401,278, filed Aug. 31, 1989, now abandoned.
Non-Patent Literature Citations (1)
Entry |
CA 89(25):210992p 1978. |
Divisions (1)
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Number |
Date |
Country |
Parent |
401278 |
Aug 1989 |
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