Transformation of organic compounds by...
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Transformation of Organic
D. Perlman Squibb Institute for Medical Research
New Brunswick, New Jersey
Compounds by Microorganisms
During the past quarter century an illcreasing number of chemists have been employing microorganisms t o carry out desired changes in organic molecules; changes which in many instances can be accomplished only with considerable difficulty by the standard techniques of organic chemistry. It is the purpose of this review t o classify some of the transformations in which microbial processes have been of some use, and to indicate some of the practical limitations which have been encountered. This discussion will be confined t o those changes where the basic structure of the molecule is left intact and will not mention the purely synthetic microbial activities. As the literature is voluminous and space is limited, only twelve types of transformations will be mentioned and only a few examples will be presented. Other examples of the types of transformations mentioned in the following pages can be found in the summary prepared by Stodola (1). Information on the mechsnisms of the reactions and possible intermediates can be found in the monographs by Gale (2)and by Oginsky and Umbreit (3). The types of chemical reactions carried out by microorganisms which will be discussed include: (1) Ozidations where oxygen is added to the molecule or hydra-
gen is removed. (2) Rednetions where hydrogen is added to the molecule or oxygen is removed. (3) Specific hydrolyses where water is added to a certsin linkage followed by splitting of the molecule. ( 4 ) Decarbozplation where carbon dioxide is eliminated from the molecule. Deomination where amino groups are removed from the molecule resulting in formation of a keto acid (oxidative demnination), formation of a double bond (desaturation), or replacement with hydrogen (reductive desmination). Amination where an amino group is introduced into the molecule. Phosphorylation where phosphate esters me added directly to the molecule. Transfer reactions where glyooside residues, amino acid residues, or phosphate residues are transferred from one molecule to another without anuarent uarticiuation of water in the reaction. ( 9 ) Raeemization where optically active compounds are eonverted into the enentiomorphs. (10) Dehydrations where water is eliminated from the molecule. (11) Demethylalion where a mrthyl group is eliminated from the molecule. (12) Condensations where two aldehydes m e joined.
..
Examples of these types of modifications of organic compounds by microorganisms and simplifications of the chemistrv involved are nresented in Table 1. Presented in part before the Division of Medicinal Chemistry at the 128th Meeting of the American Chemical Society, Minneapolis, Septemher, 1955, and in March, 1958, before the School of Pharmacy, University of Wisconsin.
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Many of these examples represent processes which have gained industrial importance, and these are marked by a footnote in the table. Among these are the followingoxidations: the conversion by bacteria or Aspergillus niger of glucose to gluconic acid is a process used for the annual production of about 2500 tons of the acid; the conversion by Acetobacter subozydans of sorbitol to sorbose, a sugar used as an intermediate in the production of the nearly 1000 tons of ascorbic acid produced every year; the introduction of the hydroxyl function in the 11 position of the cyclopentanophenanthrene nucleus by a number of fungi including Rhizopus nigricans and Curvularia lunata resulting in intermediates used in the production of thousands of pounds of hydrocortisone; and the conversion of hydrocortisone to prednisolone by bacterial action. Hydrolyses important in the food industries are those which use microbial enzymes including the processes where sucrose is inverted t o fructose and glucose, starch is hydrolyzed to maltose and to dextrins, lactose is hydrolyzed to galartose and glucose, and dextrins are hydrolyzed to maltose. Of more interest t o the practical organic chemist are the hydrolyses catalyzed by enzymes including the splitting of N-acetylated L-amino acids (but not acylated n amino acids) by fungal acylase (39)and the conversion of saponins to diosgenin by fungal enzymes (19). The production of lysine by microbial decarboxylation of diaminopimelic acid has recently assumed commercial importance, as has a process for the production of glutamic acid where microbial aminatiou of ar-keto&taric acid is the important step. Practically all of the L-ephedrine produced for medicinal purposes is made by a process in which yeast is used to condense benzaldehyde with acetaldehyde to form phenylacetylcarbinol which in turn is converted to L-ephedrine by catalytic reduction with methyl-amine. Undesired microbial transformations which are of some concern t o the chemist are the conversion of glycerol to acrolein (accountine for the "weo~erv" . .. " taste of some whiskies), .. and the racemization of lysine and lactic acids as a result of microbial enzymes produced by contaminating bacteria in fermentation processes used for the production of these chemicals. While some of the other transformations mentioned in Table 1 have reached commercial scale, many of them have been of more interest t o the chemist in preparing new compounds. The recent interest in the relationship of structure to biological activity in the corticosteroid series has resulted in many reports on the microbial transformations of these rather complex organic molecules. Among the oxidations reported are processes for the introduction of hydroxyl groups in
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positions la, 28. 68, 7a, 8,9a, 10, lla, 118, 12p, 14a, 15a, 15p, 16a, 17a, 19, and 21 of certain pregnene derivatives, and removal of hydrogen from positiou 1 (2). Microorganisms can also be used t o remove side chains (leaving a hydroxyl) or to insert an oxygen into the D ring as well as to oxidize a hydroxyl to a ketone or reduce a ketone t o a hydroxyl. These microbial systems often have remarkable stereospecific it.^ and oxidize or reduce only the D isomer when presented with a D, L mixture. This specificity was used by Vischer (40) in his process for the synthesis of the biologically important steroid, aldosterone. A consideration of these and other problems encountered in studying microbial modification of steroids is the subject of an extensive review by Eppstein et al. (41). I n studying the use of microorganisms to transform organic substances a certain amount of knowledge of the techniques of microbiology is needed. If the interest lies in reproducing results reported from other laboratories, care must be exercised in carrying out the fernlentations exactly as described using the same strain of microorganism, conditions, and medium for growth of microorganism, and recovery of the desired material. Experience has shown that when different microbial cultures are used with the same substrate (or substance to be transformed) varying results can be expected, and if the conditions for growth of the organisms vary from those reported as optimal, different transformations are likely to occur. For example, in one laboratory a strain of Streptomyces jradiae was used in the conversion of Reichstein's compound S to hydrocortisone (62) while in another laboratory the same strain grown under what were apparently slightly different conditions produced epihydrocortisone instead of hydrocortisone (45). The addition to the growth medium (intentionally or un-
intentionally) of traces of metallic ions such as zinc will lead t o the formation of different metabolic products from progesterone by the mold, Aspergillus ochraeeus (44). Another difficulty encountered is that the desired transformation product is often converted to other products if the incubation period with the microbial cells is extended. This may result in the accumulation of undesired products which make isolation of the prized material more difficult and lower the yield of the desired product ( ~ ) .All of these factors should be considered in planning programs utilizing microbial processes for the transformation of organic substances and the carrying out of certain reactions. In addition to the knowledge of microbial techniques, some acquaintance with the analytical chemistry of organic compounds is helpful in determining quickly the extent of transformation so that highest conversion rates of added material to desired product can be secured. Filter paper partition chromatography and the techniques using liquid-liquid counter current separations have been especially useful in studying the transformations of steroids (41). The examples listed in Table 1 represent only a few in the literature. Stodola (1) mentions that he has found more than 1400 examples of the use of microbial systems to carry out transformation of organic compounds. Microbial modification of such substances as alkaloids (nicotine, yohimbioe, and reserpine), vitamins (nicotinic acid, pyridoxine, the cobalamins, and thiamin) and cancer antagonists (substituted purines) has led to the isolation of new and potentially useful chemotherapeutic agents as well as to a better understanding of the relationship of chemical structure and biological activity. I t is evident that by utilizing microbial systems to carry out reactions the chemist has added another weapon to the arsenal at his disposal.
Literature Cited ( 1 ) STODOLA, F. H., "Chemical Trrtnsformatians by Mieroorganisms," John Wiley R Sons, Inc., New Yark, 1958. ( 2 ) GALE,E. F., "The Chemical Activities of Bacteria," 3rd ed., University Tutorisl Press, Ltd., 1952. (3) OGIXSKY, E. L., AND UMBREIT, W. W.,"An Introduction to Barterial Physiology," W. H. Freeman & Co., Ssn Francisco, 1954. ( 4 ) PERLXAN,D., Abstracts of 119th ACS Meeting, April, 1951, p. 24A. ( 5 ) MOYER,A. J., ET AL.,Ind. Eng. Chem., 29, 777 (1937). ( 6 ) WELLS,P: A,, ET AL.,Znd. Eng. Chem., 29, 1385 (1937). ( 7 ) SHELL,G. M., AND KITA,D. A,, J . Am. Chem. Sor., 77, 763
(23) (24) (25j (26) (27) (28) (29)
(11) MASN,K. G., ETAL., Appl. n l i m b i ~ l .3, , 14 (1955) B. M., AND SHULL,G. M., J . Am. Chem. Soe., 7 7 , (12) BLOOM, 5i67 (1955). J . Am. Chem. Soe.. 75. 5764 (1953) (13) FRIED.J.. ETAL.. ~ ~ -- \,- ~! - - , -
HARARY, I., J . Biol. Chem., 227, 823 (1957). FISCHER, F. G., AND WIEDEMANN, O., Ann., 513,260 (1934). NEUBERG, C., AND WELDE,E., Bioehem. Z., 62, 477 (1914). FARBER, E., AND NORD,F. F., Biochem. Z., 112, 313 (1920). ROTHROCK. J. W.. ET AT,.. A ~ z h Biochem. . Biovhvs.. 57.. 151 (1955). ABR~HAM, E. P., A N D CHAIN,E. B., Nature, 146,837 (1940). IMAIZUMI, M . , J. Biochem. (Japan), 27, 227 (1938). CASIDA,L. E., U.S. Pat,ent 2,771,396 (1956).
SLOANE, N . H., ET AL., J . B i d . Cham., 193, 453 (1051) HOROWITZ. N . H.. J. Biol. Chem.. 154. 1415~~(1944). ~~, GALE,E.F . , ~ i o c L mJ., . 32, 1583 (1638). STICKLAND, I.. H., Biochem. J . , 28, 1746 (1934). QUASTEL, J . H., AND WOOLF, B., Biochem. J . , 20,545 (1926). OTSUKA, S. I . , ET AL., J . Gen. Appl. Microbial., 3,35 (1957). SMYTHE, C. V., AND ROBB,L. A,, U.S. Pittent 2,606,899 1 10*1\ \A.l"",.
(30) TACHIBANA, S., ET AL., Int. S,vm. on Enzyme Chem. (Tokyo), paper 116 (1957). W. J., AND THORNE, C. B., J . B i d . Chem., 210, (31) WILLIAMS, 203 (19.541. \ - ~ - - , .
(32) MEISTER,A., Ado. in Enzymology, 16, 185 (1955). C. E., AND LELDIR, L. F., Arch. Biochem. Biophys., (33) CARDINI, 45.55 (1953). (34) HUANG,H. T., ET AT,., J . Am. Chem. SOC.,80, 1006 (1958). H., AND KITAHARA, K., Biochem. J., 31, 909 (35) KATAGIRI, (19371.
(39) ' ~ T E U B E R ~C., AND MANDL, I., Enzymologia, 14, 128 (1951). U.S. Patent 2.511.867 (1950).
.
.
(41)
EPP~EIN,
s
,
H.,
.
ET AL.,
, . .
.
Vitamins and Hormones, 14, 259
(1956). D. R., ET AL., J . 4 m . Chem. Soc., 7 4 , 2381 (42) COLINGSWORTH, (1952). (43) FRIED,J., ET AL., Recent Pmgr. Hormone Res., 11, 157 (1955). E. L., ET AL., Mycoloyia, 67, 464 (1955). (44) DULANEY, G. E., ET AL., J . Dad., 74, 684 (1957). (45) PETERSOP,
Volume 36, Number 2, February 1959
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Table 1.
Some Types of Modification of Orgonic Compounds b y Microorganisms
Type of transformation Ozidations H
I
-C-OH
-
1
-C=O
1
I
- I c 1c I
- L O
Glucose to gluconic acida
H b O H
Reichstein's compound S to hydrocortisone"
I
I
-LA-
A
Corticosterone to hydrocortisone
(8)
Progesterone to deoxycarticosterone
(9)
Hydrocortisone to prednisolane*
II
Roichstein's compound S to cortisone
-LA- -J-J+
I
1
Nicotinic acid to Ghydroxynicotinic acid
(16)
Cinnamyl deohal to hydrocinnamyl alcohol
(16)
H
g --e.
-
Acetol to optically active propylene glycol
I
H Hydrolyis H H -L O - b
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(10)
0
+ -C-
I
I 0 I
(4)
OH
I
H H H C
Phenethsnal to phenaeetaldehyde
-C O H
-A-H
I
-LO
-
H-A-H
Reference
H
-.
H H
Example
I
-
H -L o H
I 0 I
H
+ HO-A-
Journal o f Chemical Education
Dioscorea saponins to diosgenin"
Examule
Tvne of transformation
Reference
Penicillin to penicilloic acid"
Sterol ester to free sterol Decarbozylations H H O
-
H-4-4-4-oH
I
H H H-A-LH
Diaminopimelic acid to lysinea
I
p-Aminohenzoic acid t o paminophenol
*-Amino acids to a-keta acids
Aspartid acid to succinic acid 2 aramino acids 0 0
-
d!d-oH
++
Glycine plus almine to pyrovic acid plus propionic acid
H 0 ? - o H I
H H
I
H
-
O
-c-&-LoH
H A 0 - b L - - O H
Asoartic acid to fumaric acid
I
H
-
Phosphorylation sugar phosphate sugar Transjw ~eactions transglycosylation transpeptidetion transamination transphosphoryletion Racemizabions warnino acids n-hydroxy acids Dehydration OH OH OH
1
-
1
H H H Demethylation H
I
-c-0-L
k
-C-H
OH H-c=cAH
I
I
I
Glycerol t o acroleinl
H H H
-
1
-c-OH
p-Methoxyhenzoie acid to
I
Condensation 0
II
Riboflavin to rihoflavinglucosides Glutamine to glutamylglutamic acid a-Ketoglubaric aoid plus alanine to glutsmic aeid plus pyruvic soid Gdaotosamine plus adenosine-triphosphab t o galactosamine-1phosphate and adenosinedephosphrtte Lysines Lactic acid'
H - L & - L
I
Adenosine to adenosinetriphosphate
0
+ H-!L
- ALL 0 OH
Bcnealdehyde plus acetnldehvde to phenylacetylearbinol"
" Carried out on an industrial scde. Volume 36, Number 2, February 1959
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