Antibiotic A-35512B aglycone, prepared from A-35512 factor B by acid hydrolysis, is a useful antibacterial agent.

SUMMARY OF THE INVENTION 
The A-35512 antibiotics are closely related, glycopeptide antibiotics. 
A-35512 factor B is the major component of the A-35512 antibiotic complex. 
The A-35512 antibiotics, including A-35512 factor B, are described in the 
co-pending application of Michel and Higgens titled ANTIBIOTIC A-35512 AND 
PROCESS FOR PRODUCTION THEREOF, Ser. No. 689,274, filed herewith this even 
date. The A-35512 complex is produced by culturing a novel strain of 
Streptomyces candidus, NRRL 8156, under submerged aerobic fermentation 
conditions until a substantial level of antibiotic activity is produced. 
The A-35512 complex can be separated from the filtered fermentation broth 
by absorption on a resin column, elution of the column with an aqueous 
alcohol solution, and evaporation of the eluates to obtain the A-35512 
complex. A-35512 factor B can be separated from the complex and further 
purified by chromatography. A-35512 factor B occurs in the A-35512 
complex and is recovered from the complex in the form of a dihydrochloride 
salt. The novel compound of this invention is produced from A-35512 factor 
B dihydrochloride by mild acid hydrolysis. Mild acid hydrolysis removes 
glucose, fucose, mannose, and rhamnose from A-35512 factor B. The novel 
compound of this invention, therefore, is arbitrarily designated herein as 
A-35512B aglycone. 
A-35512B aglycone is recovered from the hydrolysis reaction in the form of 
a hydrochloride. A-35512B aglycone hydrochloride may be converted to its 
free base (ionic-chlorine free) form by, for example, treatment with an 
ion-exchange resin. In addition to the free base and dihydrochloride form, 
other pharmaceutically-acceptable salts of A-35512B aglycone are also 
useful.

DETAILED DESCRIPTION OF THE INVENTION 
The following paragraphs describe the physical and spectral properties of 
A-35512B aglycone. 
A-35512B Aglycone 
A-35512B aglycone hydrochloride is a white amorphous compound having the 
following approximate percentage elemental composition: 
Carbon, 54.29%; hydrogen, 4.34%; 
NITROGEN, 7.40%; CHLORINE, 5.02%; 
OXYGEN, 28.95% (BY DIFFERENCE). 
The infrared absorption spectrum of A-35512B aglycone hydrochloride in 
Nujol mull is shown in the accompanying drawing. The most significant 
absorption peaks occur at the following frequencies (cm.sup..sup.-1): 3440 
(shoulder), 3340 (shoulder), 3215 (strong), 2950 (shoulder), 2910 
(strong), 2840 (strong), 2640 (shoulder), 1735 (weak), 1655 (strong), 1590 
(medium), 1500 (strong), 1460 (strong), 1378 (medium), 1365 (shoulder), 
1298 (medium), 1215 (medium), 1155 (medium), 1120 (shoulder), 1105 (weak), 
1060 (weak), 1040 (weak), 1008 (medium), 925 (weak), 875 (weak), 765 
(shoulder), and 718 (weak). 
Electrometric titration of A-35512B aglycone hydrochloride in 66 percent 
aqueous dimethylformamide indicates the presence of three titratable 
groups with pK.sub.a values of approximately 7.5, 9.25, and 11.0 and the 
possible presence of two additional groups with pK.sub.a values greater 
than 11.0. 
A-35512B aglycone hydrochloride has a molecular weight of about 1282, as 
determined by titration. 
A-35512B aglycone hydrochloride has the following specific rotations: 
[.alpha.].sub.D.sup.25 -178.degree. (c 5, CH.sub.3 OH) 
[.alpha.].sub.365.sup.25 -716.8.degree. (c 5, CH.sub.3 OH) 
The ultraviolet absorption spectra of A-35512B aglycone hydrochloride show, 
in acidic and neutral methanol, an absorption maximum at 282 nm 
(E.sub.1cm.sup.1% 102.62) and, in basic methanol, an absorption maximum at 
302 nm (E.sub.1cm.sup.1% 182.09). 
A .sup.13 C nuclear-magnetic-resonance spectrum of A-35512B aglycone in 
DMSO-d.sub.6 at 90.degree. C. has the following characteristics: 
______________________________________ 
No. PPM Height (%) 
______________________________________ 
1 187.8 37.2 
2 172.0 40.9 
3 170.7 45.2 
4 170.1 46.9 
5 169.6 47.7 
6 168.4 57.6 
7 166.7 52.5 
8 157.4 49.9 
9 156.6 45.5 
10 155.7 55.8 
11 155.6 71.5 
12 155.4 56.7 
13 154.5 50.5 
14 149.3 43.0 
15 138.8 38.8 
16 136.9 54.3 
17 136.3 40.2 
18 135.2 31.7 
19 134.7 28.4 
20 133.8 40.9 
21 128.2 102.9 
22 126.2 77.2 
23 123.0 57.5 
24 121.3 38.1 
25 117.7 44.4 
26 117.0 31.5 
27 108.6 31.0 
28 106.7 47.9 
29 105.7 81.2 
30 103.2 26.5 
31 93.6 33.4 
32 74.9 39.8 
33 71.8 33.9 
34 68.9 40.2 
35 63.2 43.1 
36 60.4 33.6 
37 57.1 57.8 
38 55.2 33.1 
39 53.2 30.1 
40 51.8 36.0 
41 40.7 43.4* 
42 39.9 60.9* 
43 39.1 43.9* 
44 23.8 55.7 
45 17.1 47.8 
46 0.0 43.7 
______________________________________ 
*DMO-d.sub.6 
The .sup.13 C nmr spectrum indicates that A-35512B aglycone continues to 
retain the 3-amino-2,3,6-trideoxy-3-C-methyl-L-xylo-hexopyranose moiety 
(one of the sugars present in A-35512 factor B). 
Amino-acid analysis of further-acid-hydrolyzed A-35512B aglycone 
hydrochloride indicates that A-35512B aglycone contains glycine and at 
least three complex amino-acid residues. The structure of one of these 
amino-acid residues appears to be: 
##STR1## 
A-35512 aglycone hydrochloride is soluble in water and methanol, but is 
insoluble in less polar organic solvents such as benzene, chloroform, 
acetone, diethyl ether, ethyl acetate, toluene, hexane, acetonitrile and 
dioxane. 
A-35512B aglycone hydrochloride has an approximate R.sub.f value of 0.80 on 
cellulose thin-layer chromatography (aluminum support), using a 
1-butanol:pyridine:acetic acid:water (15:10:3:12) solvent system. 
Bioautography, using Sarcina lutea, is a preferred detection method. 
A-35512B aglycone hydrochloride has an approximate R.sub.f value of 0.26 on 
silica gel thin-layer chromatography using a methanol:chloroform:conc. 
NH.sub.4 OH (3:2:1) solvent system. 
A-35512B aglycone (free base) is a white amorphous compound having the 
following approximate percentage elemental composition (average): 
Carbon, 52.65%; Hydrogen, 4.57%; Nitrogen, 6.91%; 
Chlorine, 2.94%; Oxygen, 27.04%; Ash, 4.70%. 
The infrared absorption spectrum of A-35512B aglycone (free base) in KBr 
pellet exhibits significant absorption peaks at the following frequencies 
(cm.sup..sup.-1): 3360 (strong), 3260 (shoulder), 2940 (shoulder), 1735 
(shoulder), 1660 (strong), 1598 (medium), 1510 (strong), 1440 (medium), 
1295 (weak), 1215 (medium), 1165 (medium), 1122 (weak), 1070 (weak), 1018 
(strong), 940 (weak), and 920 (weak). 
Electrometric titration of A-35512B aglycone (free base) in 66% aqueous 
dimethylformamide indicates the presence of five titratable groups with 
pK.sub.a values of approximately 6.2, 8.2, 10.1, 11.4, and 12.4 and the 
possible presence of one or two additional groups with pK.sub.a values 
greater than 12.5. 
A-35512B aglycone (free base) has the following specific rotation: 
[.alpha.].sub.D.sup.25 -64.5.degree. (c 3, DMSO). 
The ultraviolet absorption spectra of A-35512B aglycone (free base) show, 
in neutral and acidic methanol, an absorption maximum at 282 nm 
(E.sub.1cm.sup.1% 43.65) and, in basic methanol, an absorption maximum at 
301 nm (E.sub.1cm.sup.1% 67.46). 
A-35512B aglycone (free base) has the same approximate R.sub.f values 
earlier-described for A-35512B aglycone hydrochloride. 
In addition to the free base and hydrochloride forms of A-35512B aglycone, 
other pharmaceutically-acceptable acid-addition salts of A-35512B aglycone 
are also part of this invention. "Pharmaceutically-acceptable" salts are 
salts in which the toxicity of the compound as a whole toward warm-blooded 
animals is not increased relative to the non-salt form. Representative and 
suitable salts of A-35512B aglycone include those salts formed by standard 
reaction with both organic and inorganic acids such as, for example, 
sulfuric, phosphoric, acetic, succinic, citric, lactic, maleic, fumaric, 
palmitic, cholic, pamoic, mucic, D-glutamic, d-camphoric, glutaric, 
glycolic, phthalic, tartaric, lauric, stearic, salicylic, methanesulfonic, 
benzenesulfonic, sorbic, picric, benzoic, cinnamic and like acids. 
A-35512B aglycone hydrochloride is prepared by mild acid hydrolysis of 
A-35512 factor B dihydrochloride. The following paragraphs describe the 
physical and chemical characteristics of A-35512 factor B. 
A-35512 FACTOR B 
A-35512 factor B is a white, amorphous, basic compound. The approximate 
empirical formula for A-35512 factor B is C.sub.97-99 H.sub.101-105 
N.sub.8-9 O.sub.46-48 Cl. A-35512 factor B has the following average 
percentage elemental composition: 
Carbon, 53.97%; hydrogen, 4.75%; 
nitrogen, 5.25%; oxygen, 34.29%; 
chlorine, 1.59% 
This elemental composition is in particular agreement with a preferred 
empirical formula of C.sub.98 H.sub.104 N.sub.9 O.sub.47 Cl (Calcd: C, 
53.60; H, 4.75; N, 5.74; O, 34.30; Cl, 1.61). An alternative preferred 
empirical formula is C.sub.98 H.sub.103 N.sub.8 O.sub.47 Cl (Calcd: C, 
54.00; H, 4.75; N, 5.15; O, 34.50; Cl, 1.60). 
The ultraviolet absorption (uv) spectra of A-35512 factor B show, in acidic 
and neutral methanol, an absorption maximum at 282 nm (.epsilon. 15000) 
and, in basic methanol, an absorption maximum at 292 nm (.epsilon. 16000), 
calculated using a molecular weight of 2000. The uv spectra of A-35512 
factor B also show end-absorption at 225 nm. 
A-35512 factor B has the following specific rotations: 
[.alpha.].sub.D.sup.25 -123.degree. (c 1, H.sub.2 O) 
[.alpha.].sub.365.sup.25 -446.degree. (c 1, H.sub.2 O) 
Electrometric titration of A-35512 factor B in 66 percent aqueous 
dimethylformamide indicates the presence of four titratable groups with 
pK.sub.a values of approximately 7.15, 8.81, 10.20, 12.00 and the possible 
presence of another group with a pK.sub.a value greater than 13.50. 
The apparent molecular weight of A-35512 factor B, as determined by 
titration, is about 2143. 
A-35512 factor B dihydrochloride is a white crystalline compound (from 50 
percent aqueous methanol). Although A-35512 factor B dihydrochloride is 
hygroscopic and does not exhibit a distinct melting point, a thermogram 
showed weight loss beginning at 25.degree. C., resulting in a 7.4 percent 
loss at 121.degree. C.; at 135.degree. C. another loss occurred, resulting 
in decomposition. 
A-35512 factor B dihydrochloride has the following approximate percentage 
elemental composition (average): 
Carbon, 52.57%; hydrogen, 4.80%; 
nitrogen, 5.66%; oxygen, 32.86%; 
chlorine, 4.51% 
This elemental composition is in particular agreement with another 
alternative empirical formula of C.sub.98 H.sub.103 N.sub.9 O.sub.47 
Cl.sup.. 2HCl (Calcd: C, 51.93; H, 4.65; N, 5.57; O, 33.20; Cl, 4.65). 
The infrared absorption spectrum of A-35512 factor B dihydrochloride in KBr 
pellet exhibits significant absorption maxima at the following frequencies 
(cm.sup..sup.-1): 3420 (strong), 3300 (shoulder), 2950 (weak), 1752 
(weak), 1675 (strong), 1630 (shoulder), 1605 (strong), 1520 (strong), 1470 
(weak), 1440 (weak), 1410 (weak), 1345 (shoulder), 1312 (medium), 1225 
(medium), 1180 (weak), 1135 (weak), 1080 (strong), and 1020 (weak). 
The uv spectra of A-35512 factor B dihydrochloride show, in acidic and 
neutral methanol, an absorption maximum at 282 nm (.epsilon. 12,000) and, 
in basic methanol, an absorption maximum at 292 nm (.epsilon. 14,000), 
calculated using a molecular weight of 2000. The uv spectra of A-35512 
factor B dihydrochloride also show end-absorption at 225 nm. 
A-35512 factor B dihydrochloride has the following specific rotations: 
[.alpha.].sub.D.sup.25 -128.degree. (c 1, H.sub.2 O) 
[.alpha.].sub.365.sup.25 -475.degree. (c 1, H.sub.2 O) 
Electrometric titration of A-35512 factor B dihydrochloride in 66 percent 
aqueous dimethylformamide indicates the presence of four titratable groups 
with pK.sub.a values of approximately 7.15, 8.87, 10.30, and 12.10 and the 
possible presence of another group with a pK.sub.a greater than 13.1. 
The apparent molecular weight of A-35512 factor B dihydrochloride, as 
determined by titration, is about 2027. 
A .sup.13 C nuclear-magnetic-resonance spectrum of A-35512 factor B 
dihydrochloride in D.sub.2 O has the following characteristics: 
______________________________________ 
NO. PPM Height (%) 
______________________________________ 
2 173.0 4.1 
3 171.9 3.7 
4 171.6 3.3 
5 171.0 5.8 
6 170.8 5.0 
7 169.6 3.6 
8 159.0 4.1 
9 157.9 4.4 
10 157.5 3.7 
11 156.6 4.8 
12 155.6 6.1 
13 155.3 4.2 
14 154.9 3.3 
15 154.3 4.2 
16 151.7 3.3 
17 144.3 3.1 
18 136.7 3.5 
19 136.2 4.9 
20 135.4 4.0 
21 135.2 4.4 
22 133.6 4.2 
23 133.3 4.1 
24 129.8 1.7 
25 129.3 3.0 
26 128.8 2.6 
27 127.6 1.5 
28 126.1 3.9 
29 124.2 5.6 
30 122.4 1.4 
31 122.0 4.4 
32 120.7 3.3 
33 116.5 2.7 
34 109.5 0.8 
35 108.2 1.1 
36 107.7 2.7 
37 104.5 1.7 
38 101.8 2.9 
39 100.9 1.6 
40 98.2 1.0 
41 76.9 1.2 
42 76.1 1.8 
43 74.1 2.0 
44 73.5 2.7 
45 72.7 2.4 
46 72.3 4.0 
47 71.0 7.1 
48 70.3 2.5 
49 69.7 2.5 
50 67.4 74.7* 
51 64.6 1.2 
52 62.0 1.5 
53 58.0 1.3 
54 56.8 1.7 
55 55.4 3.9 
56 54.3 2.5 
57 24.5 2.0 
58 17.9 3.0 
59 17.2 2.0 
60 16.3 2.5 
______________________________________ 
*dioxane standard 
A-35512 factor B dihydrochloride, crystallized from methanol-water, has the 
following characteristic X-ray powder diffraction pattern (Cu.sup.+.sup.+ 
radiation, 1.5405 .lambda., nickel filter, d= interplanar spacing in 
angstroms): 
______________________________________ 
Relative 
d Intensity 
______________________________________ 
17.15 100 
12.90 80 
10.85 70 
9.25 70 
8.87 60 
8.22 50 
7.86 50 
6.93 40 
6.20 40 
5.62 40 
5.04 05 
4.02 02 
3.54 02 
______________________________________ 
Amino-acid analysis of acid-hydrolyzed A-35512 factor B dihydrochloride 
indicates that A-35512 factor B contains at least five amino-acid 
residues, one of which is glycine. 
Analyses of its acid-hydrolysis products indicate that A-35512 factor B 
dihydrochloride contains the following sugars: glucose, fucose, mannose, 
rhamnose, and 3-amino-2,3,6-trideoxy-3-C-methyl-L-xylo-hexopyranose. 
A-35512 factor B dihydrochloride has at least one hydroxyl group capable of 
esterification. 
A-35512 factor B dihydrochloride is soluble in water, is partially soluble 
in alcohols such as methanol and ethanol, and is insoluble in other less 
polar organic solvents, such as benzene, chloroform, acetone, diethyl 
ether, ethyl acetate, toluene, hexane, acetonitrile and dioxane. 
A-35512 factor B dihydrochloride is stable for as much as 72 hours in 
aqueous solutions having a pH of from about 3 to about 10. 
A-33512 factor B is produced by culturing Streptomyces candidus NRRL 8156 
under submerged aerobic conditions in a suitable culture medium until 
substantial antibiotic activity is produced. A-35512 factor B is recovered 
by various isolation and purification procedures used in the fermentation 
art. 
The culture medium employed to grow Streptomyces candidus NRRL 8156 
(Northern Regional Research Center, U.S. Department of Agriculture, 
Agricultural Research Service, Peoria, Ill. 61604) can be any one of a 
number of media. For economy in production, optimal yield, and ease of 
product isolation, however, certain culture media are preferred. Thus, for 
example, a preferred carbohydrate source in large-scale fermentation is 
sucrose, although glucose, tapioca dextrin, fructose, mannitol, maltose, 
lactose, and the like can also be employed. A preferred nitrogen source is 
soluble meat peptone, although soybean flour, pork-blood meal, amino acids 
such as glutamic acid, and the like are also useful. Among the nutrient 
inorganic salts which can be incorporated in the culture media are the 
customary soluble salts capable of yielding zinc, sodium, magnesium, 
calcium, ammonium, chloride, carbonate, sulfate, nitrate and like ions. 
Essential trace elements necessary for the growth and development of the 
organism should also be included in the culture medium. Such trace 
elements commonly occur as impurities in other constituents of the medium 
in amounts sufficient to meet the growth requirements of the organism. 
It may be necessary to add small amounts (i.e. 0.2 ml/l.) of an antifoam 
agent such as polypropylene glycol to large-scale fermentation media if 
foaming becomes a problem. 
For production of substantial quantities of A-35512 factor B, submerged 
aerobic fermentation in tanks is preferred. Small quantities of A-35512 
factor B may be obtained by shake-flask culture. Because of the time lag 
in antibiotic production commonly associated with inoculation of large 
tanks with the spore form of the organism, it is preferable to use a 
vegetative inoculum. The vegetative inoculum is prepared by inoculating a 
small volume of culture medium with the spore form or mycelial fragments 
of the organism to obtain a fresh, actively growing culture of the 
organism. The vegetative inoculum is then transferred to a larger tank. 
The A-35512-producing organism can be grown at temperatures between about 
20.degree. and about 40.degree. C. Optimum A-35512 production appears to 
occur at temperatures of about 30.degree.-34.degree. C. 
As is customary in aerobic submerged culture processes, sterile air is 
blown through the culture medium. For efficient growth of the organism the 
volume of air employed in tank production is preferably above 0.1 volume 
of air per volume of culture medium per minute (V/V/M). For efficient 
production of the A-35512 antibiotics the volume of air employed in tank 
production is preferably about 0.25 V/V/M. 
Production of the A-35512 antibiotics, including A-35512 factor B, can be 
followed during fermentation by testing samples of the broth or of 
extracts of the mycelial solids for antibiotic activity against organisms 
known to be sensitive to the antibiotics. One assay organism useful in 
testing these antibiotics is Bacillus subtilis ATCC 6633. The bioassay is 
preferably performed by paper-disc assay on agar plates containing a 
nutritionally-limited medium. 
Following its production under submerged aerobic fermentation conditions, 
A-35512 factor B can be recovered from the fermentation medium by methods 
employed in the fermentation art. The antibiotic activity produced during 
fermentation of an A-35512-producing organism generally occurs in the 
filtered broth. Maximum recovery of A-35512 factor B is accomplished, 
therefore, by an initial filtration to remove the mycelial mass. The 
filtered broth can be purified to give the A-35512 complex by a variety of 
techniques. A preferred technique involves adsorption of the filtered 
broth on a polyamide column and elution of the column with water and 
aqueous alcohol mixtures. The eluted fractions can be combined on the 
basis of thin-layer-chromatographic behavior to give purified A-35512 
factor B. 
A-35512 factors A, B, C, E, and H and minor factors F and G are 
conveniently separated by paper chromatography using a 
1-butanol:pyridine:acetic acid:water (15:10:3:12) solvent system. 
Bioautography, using Sarcina lutea, is a preferred detection method. The 
approximate R.sub.f values of the A-35512 factors in this system are given 
in Table I. 
Table I 
______________________________________ 
A-35512 Factor R.sub.f Value 
______________________________________ 
Factor A . 2HCl 0.21 
Factor B . 2HCl 0.34 
Factor C . 2HCl 0.46 
Factor E . HCl 0.64 
Factor F 0.81 
Factor G 0.93 
Factor H . HCl 0.15 
______________________________________ 
Further purification of A-35512 factor B includes additional adsorption and 
extraction procedures. Adsorptive materials such as alumina, silica gel, 
ion-exchange resins and the like can be advantageously used. 
The A-35512 factors occur in the fermentation broth as hydrochlorides. The 
preferred polyamide separation procedure provides A-35512 factor B as a 
dihydrochloride. 
A-35512B aglycone is prepared by mild acid hydrolysis of A-35512 factor B. 
A-35512 factor B is most readily available in its dihydrochloride form. 
A-35512 factor B dihydrochloride is, therefore, a preferred starting 
material for the preparation of A-35512B aglycone. A-35512 factor B or 
other A-35512 factor B acid-addition salts may also be used. The acid 
hydrolysis is carried out according to standard procedures. Although any 
one of a number of acids may be used, hydrochloric acid is a preferred 
acid for the preparation of A-35512B aglycone. When hydrochloric acid is 
used, the A-35512B aglycone will be recovered as a hydrochloride salt. The 
hydrolysis is preferably carried out in water under reflux for a period of 
from about one to about two hours. Longer reaction times result in 
degradation of the aglycone to give less active, and, later, inactive 
products. Optimum reaction times for specific reaction conditions can be 
determined by checking reaction aliquots for bioactivity. 
A-35512B aglycone and its pharmaceutically-acceptable salts inhibit the 
growth of certain pathogenic microorganisms, particularly gram-positive 
bacteria. 
The activity of A-35512B aglycone, as measured by conventional 
disc-diffusion assay (6 mm pads dipped in solutions containing 1 mg of 
test compound per ml of solution and placed on agar plates seeded with 
test organism), is summarized in Table II. 
TABLE II 
______________________________________ 
Zone of Inhibition (mm) 
A-35512 aglycone 
Test Organism (free base) 
______________________________________ 
Staphylococcus aureus 
19 
Bacillus subtilis 13 
Sarcina lutea 14 
Bacillus subtilis* 22 
______________________________________ 
*With nutritionally-limited agar 
Table III summarizes minimal inhibitory concentrations (MIC's) at which 
A-35512 B aglycone hydrochloride is active against selected Staphylococcus 
aureus strains, as determined by standard agar-dilution tests. 
TABLE III 
______________________________________ 
Test Organism MIC (mcg/ml) 
______________________________________ 
Staphylococcus aureus 3055* 
0.5 
Staphylococcus aureus H290* 
0.5 
Staphylococcus aureus V92* 
0.5 
Staphylococcus aureus V104* 
0.25 
Staphylococcus aureus 3074** 
0.25 
Staphylococcus aureus H43** 
0.5 
Staphylococcus aureus V57** 
0.5 
Staphylococcus aureus H356** 
1.0 
Staphylococcus aureus 3130*** 
0.5 
Staphylococcus aureus 3131**** 
0.5 
______________________________________ 
*penicillin-G susceptible 
**penicillin-G resistant 
***penicillin-G resistant; methicillin resistant 
****penicillin-G resistant; methicillin resistant; clindamycin resistant 
Table IV gives additional agar-dilution test results for A-35512B aglycone 
hydrochloride against several Group-D streptococci. 
TABLE IV 
______________________________________ 
Test Organism MIC (mcg/ml) 
______________________________________ 
Streptococcus sp. Shrigley 
1.0 
Streptococcus sp. Mitis 1.0 
Streptococcus sp. 12253F 1.0 
Streptococcus sp. SS992 1.0 
Streptococcus sp. 9933 1.0 
Streptococcus sp. 9913 1.0 
Streptococcus sp. 282 1.0 
Streptococcus sp. 238 1.0 
______________________________________ 
A-35512B aglycone has shown in vivo antibacterial activity against 
experimental bacterial infections. When two subcutaneous doses of A-35512B 
aglycone hydrochloride were administered to mice in illustrative 
infections, the observed activity was measured as an ED.sub.50 value 
[effective dose in mg/kg to protect 50 percent of the test animals; see W. 
Wick, et al., J. Bacteriol. 81, 233-235 (1961)]. The ED.sub.50 values 
observed for A-35512B aglycone hydrochloride are given in Table V. 
TABLE V 
______________________________________ 
Test Organism ED.sub.50 
Infecting Challenge 
______________________________________ 
Streptococcus pyogenes C203 
5.8 2570 .times. LD.sub.50 (ip) 
Streptococcus pneumoniae 
7.0 374 .times. LD.sub.50 (ip) 
Staphylococcus aureus 3055 
1.04 370 .times. LD.sub.50 (ip) 
______________________________________ 
In order to illustrate more fully the operation of this invention, the 
following examples are provided. 
EXAMPLE 1 
Preparation of A-35512 Factor B 
A. shake-flask Fermentation of A-35512 
A lyophilized pellet of Streptomyces candidus NRRL 8156 was dissolved in 
1-2 ml. of sterilized water. This solution was used to inoculate a Bacto 
yeast malt extract (ISP No. 2, Difco Laboratories, Detroit, Michigan) agar 
slant. 
The inoculated slant was incubated at 30.degree. C. for about seven days. 
The mature slant culture was covered with water (2 ml) and scraped with a 
sterile pipette to loosen the spores. A portion (0.1 ml) of this water 
suspension of spores was used to inoculate another ISP No. 2 agar slant. 
This inoculated slant was incubated at 30.degree. C. for about seven days. 
The mature slant culture was covered with water (5 ml) and scraped with a 
sterile pipette to loosen the spores. A portion (2.5 ml) of the resulting 
suspension of spores was used to inoculate 50 ml. of a vegetative medium 
having the following composition: 
______________________________________ 
Ingredient Amount 
______________________________________ 
Trypticase Soy Broth 30 g 
(Baltimore Biological 
Laboratories, 
Cockeysville, Md.) 
Water (deionized) q.s. 1 liter 
______________________________________ 
The inoculated vegetative medium was incubated in a 250-ml Erlenmeyer flask 
at 30.degree. C. for 48 hours on a shaker rotating through an arc two 
inches in diameter at 250 rpm. 
This incubated vegetative medium (0.5 ml) was used to inoculate 50 ml of a 
production medium having the following composition: 
______________________________________ 
Ingredient Amount (g/l.) 
______________________________________ 
Tapioca dextrin 25.0 
Glucose 10.0 
NH.sub.4 NO.sub.3 2.5 
KCl 1.5 
MgSO.sub.4 1.1 
FeCl.sub.2 . 4H.sub.2 O 
.03 
ZnCl.sub.2 .03 
KH.sub.2 PO.sub.4 0.1 
L-Glutamic acid 1.0 
DL-citrulline 0.1 
CaCO.sub.3 5.0 
Deionized water q.s. 1 liter 
______________________________________ 
The inoculated production medium was incubated in a 250-ml Erlenmeyer flask 
at 32.degree. C. for 8-10 days on a shaker rotating through an arc two 
inches in diameter at 250 rpm. 
B. tank Fermentation of A-35512 
In order to provide a larger volume of inoculum, 20 ml of incubated 
vegetative medium prepared as described above was used to inoculate 400 ml 
of a second-stage vegetative growth medium having the same composition as 
that of the vegetative medium. This second-stage medium was incubated in a 
2-liter flask for 24 hours at 32.degree. C. on a shaker rotating through 
an arc 2 inches in diameter at 250 rpm. 
Incubated second-stage vegetative medium (800 ml) thus prepared was used to 
inoculate 100 liters of sterile production medium. The inoculated 
production medium was allowed to ferment in a 165-liter fermentation tank 
for about 8-10 days at a temperature of 32.degree. C. The fermentation 
medium was aerated with sterile air at the rate of 0.25 V/V/M and was 
stirred with conventional agitators at 200 rpm. 
C. separation of A35512 Antibiotic Complex 
Whole fermentation broth (250 gal.), obtained as described in Section B, 
was filtered, using a filter aid (Hyflo Super-cel, a diatomaceous earth, 
Johns-Manville Products Corp.) at broth pH (pH 6.8-7.2). The clear 
filtrate thus obtained was passed through a column containing 10 ml. of 
polymeric adsorbent (Amberlite XAD-4, Rohm and Haas Co.) per 100 ml. broth 
filtrate at a rate of 150 ml. per minute. Fractions thus obtained were 
monitored for biological activity using a standard disc assay against 
Sarcina lutea. The biologically inactive effluent was discarded. The 
column was washed with water (1/8 of the broth volume) at a rate of 150 
ml. per minute. The inactive water wash was discarded. 
The column was then eluted with a 50 percent aqueous methanol solution (600 
liters) at a rate of 200 ml. per minute. The eluate, containing the 
A-35512 antibiotic complex, was concentrated under vacuum to a volume of 
15 liters, containing about 200 grams at A-35512 antibiotic complex per 
liter. 
D. separation of A-35512 Factor B 
The A-35512 antibiotic complex (about 3000 grams dissolved in 15 liters of 
methanol), obtained as described in Section C, was chromatographed on a 
polyamide column (Woelm, 100 liter). The column was eluted with deionized 
water at a flow rate of about 80-120 ml. per minute. 
Fractions were monitored using cellulose thin-layer chromatography or paper 
chromatography, n-butanol: pyridine:acetic acid:water (15:10:3:12) solvent 
system, and Sarcina lutea bioautography. 
The first 100 liters of eluate were discarded. The flow rate was then 
changed to about 160-200 ml. per minute, and 12-liter fractions were 
collected. Twenty fractions were collected in this manner. 
At this point the eluting solvent was changed to a water-methanol gradient 
using the following procedure: 
A container holding 360 liters of methanol was siphoned into a container 
holding 120 liters of water. In the water container the mixing solution 
was stirred and fed into the column. Twenty-four fractions (24 liters 
each) were collected at a flow rate of 200-300 ml/minute. 
On the basis of bioautography results, groups of fractions were combined 
and evaporated to dryness under vacuum to give A35512 factor B 
dihydrochloride and the following enriched mixtures of factors: 
______________________________________ 
Vol. 
Fractions (liters) Factor(s) Weight 
______________________________________ 
1 - 10 120 A + H 192 g. 
11 - 24 216 B 269 g. 
25 - 31 168 B + C 590 g. 
32 - 44 312 C,E,F,G 224 g. 
______________________________________ 
E. purification of A-35512 Factor B 
Partially purified A-35512 factor B dihydrochloride (400 g.), obtained as 
described in Section D, was dissolved in 1.2 liters of 50 percent aqueous 
methanol and chromatographed on an alumina column prepared as follows: 
Acidic aluminum oxide (10 kg., M. Woelm) was stirred in a 50 percent 
aqueous methanol solution. After allowing the mixture to stand, the 
supernatant solution was decanted and discarded. The alumina was again 
stirred with 50 percent aqueous methanol and packed into a column having a 
diameter of 13.5 cm. The alumina column was washed with 50 percent aqueous 
methanol until a clear effluent was obtained. 
The column was eluted with 50 percent aqueous methanol at a flow rate of 
about 8-10 ml/minute, collecting fractions having a volume of about 
240-300 ml. Fractions were monitored by thin-layer bioautography as 
described in Section D. On the basis of this data, fractions were combined 
and yielded purified A-35512 factor B dihydrochloride as follows: 
______________________________________ 
Fractions Weight 
______________________________________ 
17 - 21 9.6 g. 
22 - 29 72.0 g. 
30 - 37 117.0 g. 
______________________________________ 
Each of these was crystallized from a concentrated 50 percent aqueous 
methanol solution at 4.degree. C. A-35512 factor B dihydrochloride thus 
purified contains about 4.6 percent chlorine. A solution of A-35512 factor 
B dihydrochloride in 66 percent aqueous dimethylformamide has a pH of 
about 6.5. 
EXAMPLE 2 
A-35512 factor B is prepared by the method of Example 1 except that the 
vegetative medium described in Section A is maintained in liquid nitrogen 
by the following procedure: 
In a small (13-.times. 100-mm) sterilized screw-cap tube is placed in 2 ml. 
of a suspension agent having the following composition: 
______________________________________ 
Ingredient Amount 
______________________________________ 
Glycerol 20% 
Lactose 10% 
Water (deionized) 70% 
______________________________________ 
To this suspension agent is added 2 ml. of a 48-hour-incubated vegetative 
medium prepared as described in Example 1. The mixed solution is frozen 
and maintained in the gas phase of a liquid-nitrogen tank. 
Vegetative medium thus stored is thawed for use in shake or tank 
fermentation by placing the vial in a 43.degree. C. water bath. A portion 
of the thawed solution (1 ml.) in the vial is used to inoculate 50 ml. of 
a vegetative medium having the same composition as that described in 
Example 1, Section A. The inoculated vegetative medium is used, as 
described in Example 1, either for shake-flask fermentation or to provide 
a larger inoculum for tank fermentation. 
EXAMPLE 3 l 
The fermentation is carried out according to the method of Example 1, but 
using a shake-flask/tank production medium having the following 
composition: 
______________________________________ 
Ingredient Amount (g/l.) 
______________________________________ 
Tapioca dextrin 75.0 
Molasses 40.0 
Soluble meat peptone 
15.0 
MgSO.sub.4 . 7H.sub.2 O 
0.5 
CaCO.sub.3 2.0 
Water q.s. 1 liter 
______________________________________ 
EXAMPLE 4 
Preparation of A-35512B Aglycone 
A-35512 factor B dihydrochloride (5.0 g), obtained as described in Example 
1, was dissolved in water (200 ml). This solution was acidified with 4 N 
hydrochloric acid (14 ml). The resulting solution was refluxed for two 
hours. The solution then was cooled and evaporated under vacuum to about 
3/4 its original volume. Hydrochloric acid (6 N) was added dropwise to 
this solution until precipitation was complete. The resulting precipitate 
was separated by filtration and dried to give 3.56 g. of crude A-35512B 
aglycone hydrochloride. 
The filtrate was concentrated and analyzed. The filtrate contained glucose, 
fucose, mannose and rhamnose. 
The crude A-35512B aglycone was purified by chromatography on acid-washed 
alumina (Woelm, Grade I), using a water:methanol (1:9) solvent system. 
Elution of the column was monitored by cellulose thin-layer 
chromatography. The eluted fractions containing A-35512B aglycone were 
combined and evaporated under vacuum to give 398 mg. of partially-purified 
product. Comparison thin-layer chromatographic results, using a ninhydrin 
spray for detection, confirmed that non-bioactive impurities were still 
present. 
A portion of this partially-purified A-35512B aglycone (100 mg) was further 
purified by chromatography over polyamide (4 g; Machery, Nagel & 
Co.MN-SC-6, distributed by Brinkmann Instruments Co.; &lt;0.07 mm), eluting 
with water. This column was also monitored by cellulose thin-layer 
chromatography as earlier described. The eluted fractions containing 
A-35512B aglycone were combined and lyophilized to give 64 mg. of purified 
A-35512B aglycone hydrochloride. (Total yield of 5.08% from starting 
A-35512 factor B). 
EXAMPLE 5 
Preparation of A-35512B Aglycone Free Base 
A-35512B aglycone hydrochloride (90 mg), obtained as described in Example 
4, was dissolved in 30 ml of methanol-water (1:1). This solution was 
neutralized with ion-exchange resin [3.5 ml, Bio-Rad AG-3-4X (OH.sup.-)]. 
The resulting solution was stirred for 15 minutes at room temperature. The 
resin was then removed by filtration. The filtrate was concentrated under 
vacuum, maintaining the temperature at less than 60.degree. C., to about 
half volume, and then lyophilized to give 68 mg of A-35512B aglycone free 
base.