Steroid Transformation by Dactylium dendroides (Bulliard) Fries

16.05.20210

EUGENE L. DULANEY, W. J. McALEER, H. R. BARKEMEYER and CHARLES HLAVAC
Research Laboratories, Chemical Division, Merck & Co., Inc., Rahway, New Jersey Received for publication July 18, 1955

The transformation of 17-desoxysteroid to 17ahydroxysteroid has been reported recently by Meystre et al. (1954) and Meister et al. (1954). Trichothecium roseum Cda. was the organism used in both instances. Transformation of progesterone to 1 la, 17a-dihydroxyprogesterone by T. roseum has been obtained also in these laboratories. The present communication reports
steroid hydroxylation, including the introduction of 17a-hydroxyl, by Dactylium dendroides.

 

MATERIALS AND METHODS


 

Two cultures of Dactylium dendroides obtained from the Quartermaster Corps Culture Collection were studied. Both exhibited similar steroid transformation patterns on preliminary investigation and one was
selected for further study. Two media were used in these studies. A synthetic medium of the following composition was employed
for large scale growth from which 1 la, 17a-dihydroxyprogesterone was isolated: dextrose 50 g, (NH4)2HP04 7.5 g, K2HP04 1 g, KCI 0.5 g, MgS04*7H20 0.5 g, FeS04*7H20 0.01 g, ZnS04*7H20 0.01 g, and distilled H20 to 1 liter. For growth and steroid transformation studies in shaken Erlenmeyer flasks, the following medium was used: cerelose 50 g, edamine 20 g, corn steep liquor 5 ml, and H20 to 1 liter (Murray and Peterson, 1952). The pH was adjusted to 6.5 before sterilization. Vegetative growth, developed in the latter medium, was used for inoculum in all experiments.

In the shaken flask experiments 50 ml of medium was dispensed in 250-ml Erlenmeyer flasks and sterilized by autoclaving at 15 lb pressure for 17 minutes. These were inoculated with 5 ml of vegetative growth. The inoculated flasks were incubated at 28 C on rotary shakers moving at 220 rpm and describing a circle 1.5 inches in diameter. After 48 to 72 hours growth, the flasks were charged with 20 mg of steroid in 0.5 ml of dimethylformamide. The charged flasks were incubated further, individual flasks being removed at the desiredtime for assay. For assay, the contents of a flask were homogenized in a Waring blender in 100 ml of ethyl acetate for 10 minutes. The solvent was separated from the homogenized growvth by filtration and the filtrate extracted with three other 100-ml portions of ethyl acetate. The ethyl acetate extracts were combined, evaporated under reduced pressure to a volume of 12 ml and spotted on strips of Whatman No. 1 filter paper. Papergrams were developed using the solvent systems of Zaffaroni and associates (Zaffaroni et al., 1950; Burton et al., 1951; and Zaffaroni and Burton, 1951). The steroids were located by means of an ultraviolet light scanner (Haines and Drake, 1950), eluted in pure methanol, and the per cent transformations calculated from optical densities determined at 2400 A. The introduction of 1 a- and 17a-hydroxyls by Dactylium dendroides was confirmed by isolation and
characterization of 11a, 17a-dihydroxyprogesterone from conversions in synthetic medium. Approximately 28 liters of growth were incubated 78 hours following the addition of 24.4 g of progesterone. The broth and mycelium were extracted with portions of ethyl acetate. The 25 liters of ethyl acetate extract were reduced to 6 liters, washed well with saturated aqueous NaHCO3 and distilled H20, then further evaporated to a volume of 150 ml. This was shaken with 50 ml of acetone and placed at 4 C overnight after which 2 g of solids were removed by filtration. The filtrate was concentrated to near dryness and partitioned between 50 per cent
aqueous methanol and petroleum ether. The methanol fraction then was evaporated and extracted with ethyl acetate. The ethyl acetate fraction was further evaporated and placed at 4 C overnight. Papergrams of the crude crystals thus obtained showed la, 17adihydroxyprogesterone to be present along with progesterone, lca-hydroxyprogesterone, and a product near the mobility of 6# , 11a-dihydroxyprogesterone. The ethyl acetate solution of these crude crystals was streaked on sheets of Whatman No. 4 paper and

TABLE 1. Qualitative test of steroid transformation by Dactylium dendroides papergrams developed to separate the 1 la, 17adihydroxyprogesterone bands. These bands were cut out, eluted with methanol, the methanol removed, and the residue partitioned between ethyl acetate and H20. The ethyl acetate layer was treated with anhydrous Na2SO4, filtered, and the filtrate concentrated. The crystals thus obtained were recrystallized from ethyl acetate. This crystalline product was compared with an authentic reference sample of 1 la, 17a-dihydroxyprogesterone.

TABLE 2. Quantitative assay of steroid transformation by Dactylium dendroides

 

RESULTS AND DISCUSSION


 

Results of a study of transformation of six steroids by Dactylium dendroides are summarized in table 1. In this experiment, individual shaken Erlenmeyer flasks were incubated for 24 and 72 hours following steroid charge then pooled and assayed qualitatively.
The addition of an Ila or a 17a-hydroxyl to various steroids is indicated quite clearly. Apparently both the lla- and 17a-hydroxyls are added to progesterone and 11-desoxycorticosterone. The transformation of progesterone to 1la, 17a-dihydroxyprogesterone was proved conclusively by isolation of the 1la, 17a-dihydroxy derivative and comparing it with an authentic reference
sample of hla, 17a-dihydroxyprogesterone. The following comparisons show the isolated product and the reference la, 17a-diol to be identical: Mixed chromatograms: The isolation product could not be separated from the reference 1 la, 17adihydroxyprogesterone in  mixed papergrams. Sulfuric acid chromogen: That of the isolated product is identical with that of the reference 1la, 17adiol.
Melting point: Isolated product, 224-227 C; remelts 248-251 C; reference hla, 17a-diol, 225-227 C; remelts 249-252 C.
Ultraviolet absorption spectrum: Isolated product, XH2804 290 m,u, 370 m,u, 450 m,i, identical with that of reference 1la, 17a-diol.

Infra-red spectrum: Isolated product, ‘X .., 2.93 I,¾ 5.82 l,u 6.02 IA, 6.18 Iu; reference 1la,17a- diol, nujol 295 I,u 5.91 A), 6.0 ,g, 6.20 u. Specific rotation: Isolated products = [a]14 + 74 (C = 1.0 in methanol) The above results show the ability of Daclylium dendroides to introduce lla- or 17a-hydroxyl into various steroids. Some of these transformations have been measured quantitatively with the results given in table 2. The results in tables 1 and 2 reveal the effect of substrate specificity. It appears that under the limited
experitnental conditions employed, that substrates containing an 1 1a-hydroxyl are more easily transformed by the addition of a 17a-hydroxyl. The data supporting this thesis are not extensive. Steroid transformation has iiot been studied under a wide variety of conditions. Moreover, the number of steroids used as substrate is limited. Transformations of 11-keto steroids are not reported and a small number of substrates containing 1 13-hydroxyls were charged. The presence of a hydroxyl at carbon-21 has no effect on the further direction of hydroxylation. When 11, 17-desoxy steroids are charged, however, the 1 Iahydroxyl appears to be added first followed by the 17ahydroxyl. Thus, with progesterone as the substrate, one can detect i la-hydroxyprogesterone and 1ha, 17a, dihydroxyprogesterone as transformation products. This would indicate the transformation of progesterone to 1 la-hydroxyprogesterone and further conversion of this derivative to 1la, 17a-dihydroxyprogesterone. In table 2, progesterone is reported to be transformed to lca-hydroxyprogesterone in 30 per cent yield. If one assumes, however, that all of the 1ha, 17a-dihydroxyprogesterone was formed via lla-hydroxyprogesterone, then approximately 45 per cent of the charged progesterone must have been converted to lc1a-hydroxyprogesterone. Conversely, it might be argued that progesterone is also transformed to 17a-hydroxyprogesterone and this derivative transformed immediately to l la,17adihydroxyprogesterone. Thus formation of 17a-hydroxyprogesterone would be rate limiting and the 17ahydroxyprogesterone would be difficult to detect. The lla, 17a-dihydroxyprogesterone would be formed, simultaneously via the 1 la- and 17a-hydroxyprogesterone.

Transformation of 11-desoxycorticosterone, also, shows the presence of an 1la-hydroxyl favors 17ahydroxylation. The 11-desoxycorticosterone is transformed to epi-corticosterone. This derivative apparently is converted to compound epi F although this
latter product is present in low yield. Failure to detect 11-desoxy-17a-hydroxycorticosterone from charged 11- desoxycorticosterone and 17a-hydroxycorticosterone from corticosterone would indicate the addition of a 17a-hydroxyl to these substrates must occur at a low level, if at all under these conditions.

SUMMARY


 

Dactylium dendroides is capable of introducing an 1la-hydroxyl and/or a 17a-hydroxyl to a number of steroid substrates. The transformation pattern is determined by the steroid substrate. The presence of a hydroxyl at carboni-21 had no observed effect on the route of transformation. The presence of a hydroxyl with the a-configuration at carbon-il appears to favor
17a-hydroxylation under the experimental conditions employed. The 11 desoxy steroids, progesterone, 1 1-desoxycorticosterone, and 17a-hydroxyprogesterone, were converted to the lla-hydroxy derivatives.

 

REFERENCES


 

BURTON, R. B., ZAFFARONI, A., AND KEUTMANN, E. H. 1951
Paper chromatography of steroids. II. Corticosteroids and related compounds. J. Biol. Chem., 188, 763-771.
HAINES, W. J., AND DRAKE, N. A. 1950 Fluorescence scanner for evaluation of papergrams of cortical hormones. Federation Proc., 9, 180.
MEISTER, P. D., REINEKE, L. M., MEEKS, R. C., MURRAY, H.
C., EPPSTEIN, S. H., LEIGH OSBORN, H. M., WEINTRAUB, A., AND PETERSON, D. H. 1954 Microbiological transformation of steroids. XII. 17a-hydroxylation. J. Am.
Chem. Soc., 76, 4050-4051.
MEYSTRE, C., VISCHER, E., AND WETTSTEIN, A. 1954 Microbiological hydroxylation of steroids in the 17a and 21- positions. Microbiological reactions, fourth communication. Helv. Chim. Acta, 37, 1548-1553.
MURRAY, H. C., AND PETERSON, D. H. 1952 Oxygenation of steroids by mucorales fungi. U. S. Patent 2,602,769.
July 8.
ZAFFARONI, A., BURTON, R. B., AND KEUTMANN, E. H. 1950
Adrenal cortical hormones; analysis by paper partition chromatography and occurrence in the urine of normal persons. Science, 111, 6-8.
ZAFFARONI, A., AND BURTON, R. B. 1951 Identification of corticosteroids of beef adrenal extracts by paper chromatography. J. Biol. Chem., 193, 749-767.

 

Source:

https://aem.asm.org/content/aem/3/6/372.full.pdf?ck=nck


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