BIOSYNTHESIS OF PENICILLINS

I. BIOLOGICAL PRECURSORS FOR BENZYLPENICILLIN (PENICILLIN G)

BY OTTO K. BEHRENS, JOSEPH CORSE,* REUBEN G. JONES, MARJORIE J. MANN, QUENTIN F. SOPER, F. R. VAN

ABEELE, AND MING-CHIEN CHIANGt

(Prom the Lilly Research Laboratories, Indianapolis)

(Received for publication, February 28, 1948)

Early work on the structure of penicillin quickly yielded information concerning the identity of the fragments which may be obtained through hydrolytic cleavage of penicillin. Such knowledge made possible a system- atic study of the course of penicillin biosynthesis. It seemed probable that the fermentative production of penicillin might be limited by the capacity of the mold to form adequate amounts of essential intermediates. To test this thesis a comprehensive study was begun to determine whether degradation products, proposed metabolic intermediates, or similar sub- stances might be capable of stimulating the production of penicillin by the mold by acting as precursors. In this search we have been successful; the penicillin yield may be substantially increased by making certain additions to the media.

Studies carried out in the Northern Regional Research Laboratory (1) had previously shown that the addition of small amounts of phenylacetic acid to the medium stimulated the production of penicillin in surface cul- tures but had little effect on the yields obtained in submerged cultures. An effort by these workers to demonstrate an influence on the type of peni- cillin produced was unsuccessful (1) .l

This effect of phenylacetic acid was regarded in many quarters as a prob- able stimulation by a plant hormone-like substance. In contrast, we held it likely that phenylacetic acid acted as a precursor in surface culture, and it was suggested that some other substances would fulfill a similar function for submerged cultures. A considerable number of derivatives of phenyla- cetic acid have been found to be effective in stimulating the production of penicillin in submerged cultures. As has been pointed out, phenylacetic

* Present address, University of California, Los Angeles, California. t Present address, Department of Chemistry, National University of Peking,

Peiping, China. 1 It is evident that direct utilization of phenylzcetic acid by the mold for penicillin

formation will lead to the formation of benzylpenicillin, in which the acyl portion of the molecule is the phenylacetyl group.

751

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752 PRECURSORS FOR BENZYLPENICILLIN

acid was not effective in submerged cultures with mold strains tested at the inception of this work. However, following the introduction of new and higher yielding strains, many compounds were retested, and it was found that phenylacetic acid is utilized by some of these strains (e.g. Penicillium chrysogenum Q-176, X1612).

Early efforts to obtain leads concerning the types of compounds that could influence penicillin production were made with strain NRRL 1976. For these tests a suboptimal synthetic medium was used, designed to supply sources of energy for the mold but not to allow optimal penicillin formation. Control flasks usually produced about 15 to 20 units of penicillin per ml.2 in 48 hours. The test compound was added to such a medium in 0.0008 M

concentration. Complete conversion of the precursor to penicillin would yield a broth assaying 475 units per ml.; utilization of only one form of a racemic compound would yield one-half this value. A washed suspension of the mold, which had been grown in corn steep medium, served as the inoculum. This test method was designated the “low” (L) method.

It was realized, however, that a stimulation of yield on such a medium could be interpreted as being due to improvement of a suboptimal medium rather than to the effect of a direct precursor. For this reason a second test, designated the “high” (H) method was used. The medium was more complete. It included corn steep’ solids, and normally yielded 100 to 120 units per ml. In general, the two methods gave comparable results; i.e., compounds that stimulated production in Method L also stimulated pro- duction when tested by Method H. Additionally, it was proposed to use substances containing isotopic elements to obtain direct proof that the stimulating compounds actually functioned as penicillin precursors. These latter experiments are described in Paper II of this series.

Table I presents the effect of a number of compounds on the yield of penicillin. More extensive data may be found in the monograph on penicillin (2).

Marked specificity was evident in the ability of the mold to utilize com- pounds. This specificity applied to both the acyl and amide portions of the test compounds. It has been determined that differences in the acyl portion of the molecule are of importance because of limitations in the mold’s metabolic ability to incorporate these groups into the penicillin molecule. We may therefore conclude that under the conditions used in these tests the mold probably does not form penicillins which contain ben- zoyl or fl-phenylpropionyl groups. However, in Table I it is seen that a y-phenylbutyryl compound provided good stimulation in yield. The peni-

2 Assays were obtained by the plate method with Staphylococcus aureus, strain 209P. We acknowledge with thanks the numerous assays performed by Dr. J. M. McGuire. The method is described in detail in the penicillin monograph (2).

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BEERENS, CORSE, JONES, MANN, SOPER, VAN ABEELE, CHIANG 753

cillm formed when N-(2-hydroxyethyl)--y-phenylbutyramide was used as the precursor was isolated and identified as benzylpenicillin. This was interpreted as evidence that phenylbutyryl compounds are degraded by the mold with the loss of 2 carbon atoms. Proof of this interpretation is pro- vided in Paper IV in this series (3).

TABLE I

Eflect of Various Compounds on Penicillin Production

The tests were performed with strain NRRL 1976. Compounds were added at 0.0008 Y concentration. The numerical values represent the ratio, units in test container to units in control container. The following values were obtained with the “high” method.

- N-Phenylacetyl-L-valine ........................................... 1.42 Phenylacetic acid + DL-valine ..................................... 1.0 N-Phenylacetyl-D-valine ........................................... 1.0 N-Phenylacetyl-L-alanine .......................................... 1.0 N-Phenylacetylglycine + N-acetyl-DL-valine ....................... 1.0 N-Benzoyl-DL-valine ............................................... 1.0 N-&Phenylpropionyl-nn-valine ..................................... 1.0 Nq-Phenylbutyryl-nn-valine ....................................... 1.26 N-(2-Hydroxyethyl)-phenylacetamide ............................... 1.57 N-(2-Aminoethyl)-phenylacetamide ................................. 1.56 2-Aminoethyl phenylacetate hydrochloride .......................... 1.26 N-Allylphenylacetamide ............................................ 1.48 N-Crotylphenylaceta~de .......................................... 1.94 Phenylacetylated pancreatic digest of casein (125 mg. 70). .......... 1.46 N-&Methylallylphenylacetamide ................................... 1.24 Nq,r-Dimethylallylphenylacetamide ............................... 1.21 N-(2-Acetoxyethyl)-phenylacetamide ............................... 1.41 N-(2-Butyroxyethyl)-phenylacetamide. ............................. 1.46 N-(2-Isocaproxyethyl)-phenylacetamide . ........................... 1.29 N-(2-Ethoxyethyl)-phenylacetamide. ............................... 1.20 N-(2-Pentenyl)-phenylacetamide ................................... 1.28 N-(2-Ethyl-2-hydroxybutyl)-phenylacetamide ....................... 1.0 N-(2-Hydroxy-2-phenylethyl)-phenylacetamide ..................... 1.0 Butyl nr,-cu-phenylacetylamino-n-valerate ........................... 1.15 N-(2-Hydroxy-2-methylpropyl)-phenylacetamide. ................... 1.1

-

The reasons for the marked specificity of the nitrogen moiety have not been determined. It has become evident that in these compounds this portion of the molecule is not directly incorporated into the penicillin. Its function, therefore, appears to be an indirect one and may be concerned with the availability of the acyl group to the mold.

The effect of variation in the concentration of a precursor is illustrated in Table II. At concentrations of 0.01 per cent or less, it was still possible

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754 PRECURSORS FOR BENZYLPENICILLIN

to demonstrate, by the differential assay method,3 that the precursor affected the type of penicillin produced.

Many of the compounds which were tested as precursors for benzylpeni- cillin do not appear to have been previously described. They are presented in Table IV.

EXPERIMENTAL

Methods of Testing Precursors

Low (L) Method-The vegetative inoculum was grown in cotton-stop- pered 1 liter Erlenmeyer flasks containing 200 ml. of a culture medium con- sisting of 20 gm. of lactose, 20 gm. of corn steep solids (American Maize Products), 0.5 gm. of monopotassium phosphate, 0.25 gm. of magnesium sulfate heptahydrate, 2 gm. of sodium nitrate, and 0.02 gm. of zinc sulfate heptahydrate per liter. Seeding was accomplished with 0.5 ml. of a spore suspension of Penicillium not&urn, strain NRRL 1976. As a source of

TABLE II E$ect of N-Phenylacetyl-nL-valine on Strain NRRL 19Y6

Concentration Molarity Units per ml.

ser cent Control 0 119

0.01 0.00038 129 0.02 0.00076 195 0.04 0.00152 182

spores the mold was grown on Moyer and Coghill’s sporulation medium (4) in test-tube slant cultures. Suspensions of the spores were prepared by adding 10 ml. of water and brushing off the spores with a platinum wire. The culture was incubated for 3 to 5 days at 24” with continuous agitation on a reciprocating shaker (3 inch stroke, 100 strokes per minute). At the end of this period the broth was removed from the well formed pellets by sterile filtration. The pellets were washed once with sterile water and were suspended in 100 ml. of a medium containing 1 gm. of monopotassium phosphate, 1 gm. of dipotassium phosphate, 1 gm. of magnesium sulfate heptahydrate, 2 gm. of sodium nitrate, 10 gm. of lactose, and 0.01 gm. of zinc sulfate heptahydrate per liter and adjusted to pH 6.5.

The pellets and medium from one flask were subdivided into four equal portions and were placed in 300 ml. Erlenmeyer flasks. To one of these flasks, used as a control, were added 5 ml. of 0.02 M phosphate buffer, pH

a The differential assay is the ratio of antibacterial activity for Bacillus subtilis to Staphylococcus aureus compared with that for pure benzylpenicillin, which is defined as 1.00.

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BEHRENS, CORSE, JONES, MANN, SOPER, VAN ABEELE, CHIANG 755

6.5, and 15 ml. of water. To the other three flasks were added the three sets of constituents to be tested. Each of the substances (0.00004 mole) to be tested in a given flask was dissolved in 5 ml. of 0.02 M phosphate buffer, pH 7.0, and 5 ml. of water, and was adjusted to pH 6.5. This test solution was introduced into the Erlenmeyer flask through a Seitz filter, followed by 10 ml. of water. The set of flasks was shaken at 24” and samples were re- moved at suitable intervals for testing. Duplicate experiments were per- formed.

High (H) Method-100 ml. of medium (containing 25 gm. of lactose, 20 gm. of American Maize corn steep solids, 2 gm. of calcium carbonate, and 0.044 gm. of zinc sulfate heptahydrate per liter) in a 1 liter Erlenmeyer flask were inoculated with 1.0 ml. of a spore suspension of strain NRRL 1976. The flask was shaken for 2 days at 24”. 10 ml. of the germinated spores were then introduced into each of a series of 300 ml. Erlenmeyer flasks containing 40 ml. of a medium, double the concentration of constitu- ents in the original flask, to which had been added 25 ml. of water and 5 ml. of 0.02 M phosphate buffer, pH 6.5, containing 6.4 X 10e6 mole of the test substance. Control flasks without addition of precursors generally yielded 100 to 120 units per ml. in 4 or 5 days.

Penicillin from N-(%Hydroxyethyl)-y-phenylbutyramide-The precursor (166 mg.) was added to a broth containing 25 gm. of lactose, 20 gm. of corn steep solids, 2 gm. of calcium carbonate, and 0.044 gm. of zinc sulfate heptahydrate per liter. 225 ml. of broth in each 1 liter Erlenmeyer flask were inoculated with 0.5 ml. of a spore suspension of Penicillium notatum, strain NRRL 1976. The flasks were shaken at 25” for 5 days and har- vested. 5.4 liters of cold filtered broth, 150 units per ml., were extracted at pH 2.2 with 3.2 liters of cold amyl acetate. The amyl acetate was sepa- rated (in some instances the emulsion separated on standing for 3 hour; in other cases use was made of a small Sharples supercentrifuge), and the sodium salt was prepared by stirring with three 50 ml. portions of 0.1 N

sodium bicarbonate solution. The pH values of the aqueous extracts were in the range 6.9 to 7.2. An ethereal solution of the penicillin was prepared by acidifying the chilled aqueous solution to pH 2.1 with 10 per cent phos- phoric acid and extracting with three 50 ml. portions of alcohol-free ether. Assay of a sample of the ethereal solution indicated the presence of 455,000 units of penicillin.

A column was prepared for chromatographic purification of the penicillin. 50 gm. of dry silica (a suitable material may be obtained from the G. Freder- ick Smith Chemical Company, Columbus, Ohio) were thoroughly mixed with 42.5 ml. of 1.5 M potassium phosphate buffer, pH 6.2. The silica, which still retained a dry appearance, was suspended in ether and trans- ferred to a glass tube 1 inch in diameter. Glass wool was used at the bottom of the tube to retain the silica. Additional ether was used in transferring the silica and a few pounds of air pressure were applied to obtain proper

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756 PRECWRRORS FOR B~~NzYLPENICILLIN

packing. Care was exercised to keep excess ether on the column at all times. The ethereal solution of the penicillin was carefully poured into the column, followed by successive 100 ml. portions of ether containing 0.5, 1, 1.5, 2, 2.5, and 3 per cent of methanol. The ether effluent was collected in 100 ml. portions. At the conclusion of the development of the column the ether was allowed to drain from the silica. The silica was removed in 3 inch cuts and the penicillin was recovered from the portions of silica and ether by extraction with 0.067 M phosphate buffer of pH 7.0. Assays on the various fractions indicated the distribution shown in Table III.

Fractions S-6, S-7, and S-8, containing 60 per cent of the recovered units, were combined, extracted with three 40 ml. portions of cold chloroform at pH 2.2, and were further purified by use of a chloroform-phosphate buffer (pH 6.2) column. The development of the column was carried out with successive 100 ml. portions of chloroform containing 1, 2, and 3 per cent

TABLE III Distribution of Penicillin from N-(2-Hydroxyethyl)-y-phenylbutyramide on Ether-

Buffer (pH 6.9) Chromatographic Column

Fraction - I Units Fraction

-- s-1 14,600 s-10 s-2 5,000 s-11 s-3 5,000 F-7 s-4 None F-6 s-5 5,400 F-5 S-6 92,000 F-4 s-7 152,000 F-3 S-8 42,600 F-2 s-9 47,000 F-l

Units

40,000 28,000 40,400 7,500 6,600 5,000

None “ “

methanol. 218,000 units were recovered in a single sharp band. The penicillin was extracted from the buffer solution with ether at pH 2.2 and the sodium salt was prepared with 0.1 N sodium hydroxide solution. The aqueous solution was dried from the frozen state, and the resulting dry so- dium salt was treated with 1 ml. of acetone. Partial solution followed by reprecipitation occurred. This precipitate was washed with several portions of dry acetone and was crystallized from 2 ml. of 90 per cent acetone by addition of 4 ml. of dry acetone. It was recrystallized in a similar manner.

Analysis-Sodium r-phenylpropylpenicillin, ClsH21N204SNa Calculated. C 56.23, H 5.51, N 7.31

Sodium benzylpenicillin, CIQH1,N20aSNa Calculated. C 53.92, H 4.80, N 7.83 Found. “ 53.47, “ 4.51, “ 7.85

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BEHRENS, CORSE, JONES, MANN, SOPER, VAN ABEELE, CHIANG 757

The material assayed 1630 units per mg. and gave a differential assay value of 0.99. These properties indicated that the product was benzylpenicillin.

Preparation of Phenylacetylated Protein Hydrolysates

Phenylacetylated Corn Steep Solids-20 gm. of American Maize corn steep solids were dissolved in 35 ml. of water. The solution was cooled well, made alkaline with 5 N sodium hydroxide solution, and 10 ml. of phenylacetyl chloride and 45 ml. of 2.5 N sodium hydroxide solution were added in por- tions with vigorous stirring over a period of 3 hour. After an additional 4 hour the mixture was acidified with 5 N hydrochloric acid and was shaken with several portions of ether. The ether extracts were combined, evapo- rated to dryness in vacua, and the residue was dissolved in 50 ml. of ethyl acetate and precipitated with 50 ml. of petroleum ether. The oily precipi- tate was returned to the acid aqueous solution, which was then adjusted to pH 6.5 with 5 N sodium hydroxide solution.

Phenylacetylated Casein and Liver Hydrolysates-A pancreatic casein di- gest, a papain digest of casein, an acid-hydrolyzed casein, and a pancreatic digest of liver were each phenylacetylated in the manner described above for corn steep solids. 10 gm. of digest in 15 ml. of water were treated with 10 ml. of phenylacetyl chloride and alkali.

The above hydrolysates were prepared in the following manner. Pancreatic Digest of Liver4 kilos of freshly ground liver were incubated

at 37” for 6 days with 800 gm. of freshly ground pancreas in a total volume of 16 liters. 53 gm. of sodium carbonate were added at the beginning of the reaction. Toluene and chloroform were added as preservatives. At the end of the incubation period, hydrochloric acid was added in an amount equivalent to that of the sodium carbonate, and the mixture was heated to 90-94” for 15 minutes. After the addition of 25 gm. of Nuchar, the mix- ture was filtered, and the filtrate was evaporated to dryness in vacua.

Pancreatic Digest of Casein- gm. of casein were digested with 150 gm. of minced pancreas in a total volume of 3 liters. 19 gm. of sodium carbonate were added at the beginning of the reaction. The subsequent procedure was the same as that described for the liver digest above, except that no decolorizing carbon was used.

Acid-Hydrolyzed Casein-Casein (300 gm.) was hydrolyzed by refluxing for 21 hours with 5 volumes of 26 per cent sulfuric acid. The sulfuric acid was removed quantitatively by addition of barium hydroxide. The pre- cipitated barium sulfate was removed by filtration and was thoroughly washed with hot water. The aqueous solution was decolorized with carbon and was evaporated to dryness in vacua.

Papain Digest of Case&-United States patent 2,364,008 of E. H. Stuart. Preparation of Miscellaneous Precursors-“Penillic acid” was prepared

by treating 500 unit commercial penicillin with dilute hydrochloric acid

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753 PRECURSORS FOR BENZYLPENICILLIN

according to the directions for preparation of penillic acid from crystalline penicillin (2). The neutralized solution was used for the test.

“Penicilloic acid” was prepared from 500 unit commercial penicillin by treatment with dilute sodium hydroxide solution at pH 11.5 for 2 hours. The neutralized solution was used for the test.

Preparation of Benzylpenicillin Precursors-A number of different meth- ods (as designated in Table IV) was employed in the preparation of the various compounds.

Method A-A methyl or ethyl ester of the acyl portion of the molecule was heated at 100-150” for several hours with a slight excess of the appropriate amine. The mixture was cooled and the product was recrystallized from appropriate solvents such as alcohol-ether, ethyl acetate, benzene-petro- leum ether, etc. Those products which did not crystallize were purified as follows: The solution of the compound in a solvent such as ether or ethyl acetate was washed successively with dilute acid, dilute alkali, and water. The solution was dried, and the solvent removed, finally by heating in a high vacuum.

Method B-The regular Schotten-Baumann method was applied to the preparation of these compounds. N-Alkylphenylacetamides were purified as described under Method A. N-Phenylacetylamino acids were purified by precipitation from acidified solution, followed by recrystallization from appropriate solvents.

Method C-A suspension of 22.5 gm. of N-allylphenylacetamide (Table IV) in 1800 ml. of water at 0” was treated dropwise with a solution of 10 gm. of potassium permanganate in 300 ml. of water. The mixture was filtered and the filtrate was evaporated to dryness in vacua. The residue, 25.7 gm., was recrystallized from ethylene dichloride to yield 12.5 gm. of N-(2,3-dihydroxypropyl)-phenylacetamide containing 1 molecule of water of hydration.

Method D-A mixture of 29.6 gm. (0.4 mole) of trimethylenediamine, 34 gm. (0.25 mole) of phenylacetic acid, and 0.3 mole of 4 N hydrochloric acid was heated to 250” during 1 hour. The melt was cooled, dissolved in 300 ml. of water, and the solution was filtered and made alkaline with sodium hydroxide solution. The aqueous solution was extracted with ether and the ether extract was dried and evaporated, leaving N-(3-aminopropyl)- phenylacetamide as a crystalline solid.

Method E-To a solution of 27 gm. (0.2 mole) of phenylacetamide in 200 ml. of dioxane were added 7.8 gm. of potassium. After all of the metal had reacted, 18.1 gm. of p-methylallyl chloride were added, and the solution was heated under a reflux for 4 hours. The mixture was filtered and the filtrate was evaporated in vacua. The residue was taken up in warm benzene. The solution was filtered, evaporated, and the residue recrystallized several

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BEHRENS, CORSE, JONES, MANN, SOPER, VAN ABEELE, CHIANQ 759

TABLE IV Benzylpenicillin Precursors

Compound

N-(2-Hydroxyethyl)-phenyl- acetamide

N-Allylphenylacetamide N-(2-Methoxyethyl)-phenyl-

acetamide N-(l-Hydroxyisopropyl)-phenyl-

acetamide N-(2-Hydroxypropyl)-phenyl-

acetamide N-(1,3-Dihydroxyisopropyl)-

phenylacetamide N-(2,3-Dihydroxypropyl)-

phenylacetamide N-(3-Aminopropyl)-phenyl-

acetamide N-Crotylphenylacetamide N-@-Methylallyl)-phenylacet-

amide a-Phenylacetylamino-n-butyric

acid N-(2-Acetoxyethyl)-phenylacet-

amide N-(2-Hydroxyethyl)-phenacetur-

amide N-(2-Hydroxyethyl-a-methyl-

propyl)-phenylacetamide N-(1-Hydroxy-2-butyl)-phenyl-

acetamide N- (2-Hydroxybutyl) -phenyl-

acetamide N-(l,l-Dimethyl-2-hydroxy-

ethyl)-phenylacetamide N-(2-Hydroxyethyl)q-phenyl-

butyramide N-Ethyl-N-(2-hydroxyethyl)-

phenylacetamide N-(2-Ethoxyethyl)-phenylacet-

amide N,N-Di-(2-hydroxyethyl)-

phenylacetamide N-(2-Aminoethyl)+phenylbu-

tyramide hydrochloride

kthod I Prep .ration

A

B “

A

“

“

C

D

B E

B

F

A

“

I‘

“

“

“

“

B

A

G

Nitrogen

M.P. F$;- Found

~-- “C. )W cenl per Cf%f

61 - 62 7.81 7.70

53 - 55 8.00 7.92 Oil 7.25 6.93

80 - 81 7.25 7.20

49 - 52 7.25 7.18

129 -132 6.69 6.89

38 - 40 6.69 6.11

97 -100 16.08 15.63

57 - 59 * 46.5- 48

124 -126 6.33 6.44

75 - 78 6.33 6.30

143 -144 11.86 12.01

67 - 69 6.27 6.75

54 - 56 6.76 6.82

57 - 59 6.76 6.81

Oil 6.76 6.68

“ 6.76 7.40

72 - 73 6.76 6.82

47 - 48 6.76 6.79

Oil 6.27 6.75

95 - 98 11.57 11.78

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TABLE IV-Concluded ,-..- -._,_

Compound

a-(Phenylacetylamino)-@,&di- methylacrylic acid

N-(~,r-Dimethylallyl)-phenyl- acetamide

N-(2-Pentenyl)-phenylacetamide N-Phenylacetyl-on-norvaline N-Phenylacetyl-nn-penicill-

amine N-Phenylacetyl-L-penicillamine N-Phenylacetyl-n-penicillamine N-Phenylacetyl-nn-@-hydroxy-

valine N-Phenylacetyl-on-valine amide N-Phenylacetyl-nn-N-methyl-

valine N-Phenylacetyl-nn-valine

methyl ester N-Phenylacetylglucosamine N-(2-Ethyl-2-hydroxybutyl)-

phenylacetamide N-(2-Butyroxyethyl)-phenyl-

acetamide N-Phenylacetyl-nL-@,p-diethyl-

alanine N-(r-Phenylbutyryl)-nn-valine N-(2-Hydroxyethyl)-cz-phenyl-

acetylaminoisovaleramide N-(2-Isocaproxyethyl)-phenyl-

acetamide N-(2-Phenylethyl)-phenylacet-

amide N-(2-Hydroxy-2-phenylethyl)-

phenylacetamide Butyl nn-oc-phenylacetylamino-

n-valerate

kthod i prep ration

H

E

B “ I

J ‘I

K

A B

L

K B

F

B

‘6

M

F

A

B

N

Nitrogen

M.p.

“l”,:i Found --

“C. per c‘mt per cent 176 -177 6.01 5.99

66 - 68

65 - 66 6.89 7.39 136 -138 5.96 5.95 125 -127 5.24 5.21

132 -134 5.24 5.24 132 -134 5.24 5.20 119 -121 5.57 5.53

195 -197 11.97 11.92 104 -105 5.62 5.24

52 - 53 5.62 5.73

214 -217 4.71 4.83 85 - 86 5.95 5.99

45 - 48 5.62 5.60

98 -100 5.3 5.6

110 -112 5.32 5.28 Oil 10.07 10.04

‘I 5.06 5.05

83 - 86 5.86 5.84

94 - 96 5.49 5.69

27 - 28 4.81 4.75

* ClzHrsNO, calculated, C 76.15, H 7.99; found, C 76.09, H 7.61.

times from benzene-petroleum ether to yield 7.5 gm. of N-(&methylallyl)- phenylacetamide.

Anal&s--CrzH1bNO. Calculated. C 76.15, H 7.99 Found. “ 76.47, “ 8.25

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BEHRENS, CORSE, JONES, MANN, SOPER, VAN ABEELE, CHIANG 761

N-(7 ,r-Dimethylallyl)-phenylacetamide was prepared in the same man- ner with y , y-dimethylallyl chloride.

Analysis-CnH1,NO. Calculated. C 76.81, H 8.43 Found. ‘I 77.01, “ 8.74

Method F-A solution of N-(2-hydroxyethyl)-phenylacetamide (Table IV) in dry pyridine was treated with 1 equivalent of the appropriate acid anhydride. The mixture was heated at 60” for several hours and then poured into ice water. The resulting oils, which soon solidified, were re- crystallized from alcohol-water.

Method G-Ethyl y-phenylbutyrate was heated with exdess ethylenedia- mine for several hours. The volatile constituents of the mixture were removed by heating in vacua. The residue was dissolved in an alcohol- ether mixture, and the solution was treated with dry hydrogen chloride to precipitate N-(Baminoethyl)-y-phenylbutyramide hydrochloride.

Method H-A solution of 1.5 gm. of N-phenylacetyl-nL+hydroxyvaline (Table IV) in 5 ml. of acetic anhydride was heated at 70” for 1 hour, and then evaporated to dryness in vacua. To the residue were added 5 ml. of water and 6 ml. of acetone. The solution was heated under a reflux for 1 hour. After removal of the acetone by distillation, a crystalline product separated. The material was collected on a filter, washed with 1: 1 chloro- form-petroleum ether, and recrystallized from 15 ml. of acetone to yield 1 .O gm. of ar-phenylacetylamino-/? ,P-dimethylacrylic acid.

Method I-N-Phenylacetyl-S-benzyl-nn-penicillamine was prepared from phenylacetyl chloride and S-benzylpenicillamine by the Schotten-Baumann method. It was recrystallized from ethylene dichloride, m.p. 66-68”.

Anal~si~-C~~H~~N0& Calculated, N 3.9; found, N 3.7

A solution of 5 gm. of the N-phenylacetyl-S-benzyl-nn-penicillamine in 200 ml. of liquid ammonia was treated with sodium in small pieces until a blue color persisted. The excess sodium was neutralized with a little am- monium chloride and the ammonia was allowed to evaporate. The residue was dissolved in water and warmed under a vacuum to remove any residual ammonia. The N-phenylacetyl-nn-penicillamine was precipitated with hydrochloric acid and recrystallized from ethyl acetate-petroleum ether.

Method J-A solution of 21.0 gm. (0.059 mole) of N-phenylacetyl-S- benzyl-DL-penicillamine in 50 ml. of methanol was treated with a solution containing 26.9 gm. (0.059 mole) of brucine. The crystals which separated on standing were collected, washed with a little absolute alcohol, and air- dried. They weighed 20.5 gm., m.p. 108-109”. After three recrystalliza- tions from absolute alcohol, there were obtained 15.9 gm. of crystals of

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762 PRECURSORS FOR BENZYLPENICILLIN

constant rotation, m.p. 117-119”, [o~]E’r = -19.0” (in 1 per cent abso- lute alcohol solution). This salt, 14.9 gm., yielded 6.4 gm. of crude N-phenylacetyl-S-benzyl-L-penicillamine which after three recrystalliza- tions from 3 : 1 water-alcohol gave 5.5 gm. of pure acid of constant rotation; m.p. 141-142”, [~r]i*.~ = -7.5” (in 2 per cent absolute alcohol solution).

The filtrate from the first crop of the brucine salt was evaporated in vucuo to dryness. 200 ml. of water were added and the crystals, 20 gm., were collected on a filter and recrystallized from 4: 1 water-alcohol to yield 15.8 gm. of salt of constant rotation, m.p. 10%ill”, [oL]:‘.~ = -3.75” (in 4 per cent absolute alcohol solution). From 14.8 gm. of this salt there were ob- tained 5.6 gm. of. N-phenylacetyl-S-benzyl-n-penicillamine, m.p. 141-142’, [or]:*.’ = $7.5” (in 2 per cent absolute alcohol solution).

The N-phenylacetyl-S-benzylpenicillamines were converted to the re- spective N-phenylacetyl-n-penicillamine and N-phenylacetyl-n-penicilla- mine as described under Method G, above.

Method K-These compounds were prepared by the Schotten-Baumann method with sodium bicarbonate.

Method ,%---Phenylacetyl-nn-valine was esterified with diazomethane in ether. The ester was recrystallized from ethyl acetate-petroleum ether.

Method M-Phenylacetylvaline methyl ester was dissolved in 10 parts of methanol saturated at 0” with ammonia. After standing at room tempera- ture for 4 days the solution was chilled. The crystalline amide was col- lected and recrystallized from alcohol-water.

Method N-10 gm. of nn-a-amino-n-valeric acid xvere esterified with 200 ml. of dry n-butanol saturated with hydrogen chloride. The resulting ester hydrochloride was phenylacetylated with 11.5 ml. of phenylacetyl chloride and dilute Na=#ZO, solution. The resulting oil was crystallized by cooling in a dry ice bath. Recrystallization from petroleum ether yielded 2.7 gm. of product.

Resolution of Phenylacetyl-DL-valine

Phenylacetyl-nn-valine, 23.5 gm. (0.1 mole), was dissolved in 50 ml. of methanol and a solution of 46.7 gm. (0.1 mole) of brucine in 100 ml. of methanol was added slowly to it, with constant stirring. The solution was allowed to stand in a refrigerator overnight and no crystallization of the salt occurred. Most of the solvent was evaporated under reduced pressure and the oily mass left was dissolved in 400 to 450 ml. of hot water. After cooling, a crop of colorless needles was obtained which weighed 55.0 gm. and melted at 105-108”.

The crystals were recrystallized five times from water; m.p. 105”,

bl, 29.6 = -9.0” (2 per cent absolute ethanol solution). The yield was 20.1 gm.

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BEHRENS, CORSE, JONES, MANN, SOPER, VAN ABEELE, CHIANG 763

14 gm. of this salt were suspended in 100 ml. of water. Excess of dilute sodium hydroxide solution was added, and the precipitate of brucine was filtered off and washed several times with water. The filtrate was made acid by adding an excess of concentrated hydrochloric acid. The precipi- tate was filtered and washed twice with water and air-dried overnight; yield 4.7 gm., m.p. 139-140”. Two recrystallizations from alcohol-water (1: 3) gave the pure product, 3.2 gm., m.p. 139-140”, [~r]:‘~ = +9.6”, in 4 per cent absolute ethanol. It gave no depression in a mixed melting point determination with an authentic sample of phenylacetyl-D-valine.

The filtrate from the first crop of brucine salt was evaporated to dryness under reduced pressure. The residue was dissolved in 100 ml. of water and decolorized with a little charcoal. It was evaporated again to dryness under reduced pressure, 100 ml. of ether were added, and the solid collected. It weighed 15.0 gm., m-p. 105-108”. Four recrystallizations from water gave a product of constant rotation; yield 6.0 gm., m.p. 106-107”, [cx]:.~ = -19.6” (2 per cent absolute ethanol solution).

The salt was converted into the free acid in the same manner as the iso- merit compound. From 6.0 gm. of the salt, 1.7 gm. of crude phenylacetyl- n-valine were obtained, m.p. 130-132”. After four recrystallizations from alcohol-water (1: 3), 0.42 gm. of a product of constant rotation was ob- tained; m.p. 137-138”, [a]:*.’ = -9.0” (in 4 per cent absolute ethanol solution).

We are happy to acknowledge helpful discussions and suggestions con- tributed by Professor H. E. Carter of the University of Illinois.4 The authors also express their gratitude to Dr. G. H. A. Clowes and Dr. E. C. Kleiderer for their interest in this work. The microanalyses were per- formed by W. L. Brown and H. L. Hunter.

SUMMARY

1. Methods have been described for evaluating compounds as precursors for benzylpenicillin.

2. Addition to media of certain compounds containing the phenylacetyl group or of certain compounds which may be converted biologically to con- tain this group has resulted in increased production of benzylpenicillin.

3. Benzylpenicillin has been isolated after use of N-(2-hydroxyethyl)-r- phenylbutyramide as a precursor.

4. Numerous compounds which have been tested as benzylpenicillin precursors (cf. (2)) have been prepared.

4 Valuable technical assistance has been given by Charlotte Harris, John O’Brien, and Dorothea Huff.

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764 PRECURSORS FOR BENSYLPENICILLIN

BIBLIOGRAPHY

1. Coghill, R. D., Moyer, A. J., and Ward, G. E., Reports to the Committee on Medical Research, Nos. 16,18-20, Nov. 20,1943-July 5,1944. Moyer, A. J., and Coghill, R. D., J. Bact., 53, 329 (1947).

2. Behrens, 0. K., in Clarke, H. T., Johnson, J. R., and Robinson, R., The chemistry of penicillin, Princeton, chapter 19, in press.

3. Behrens, 0. K., Corse, J., Edwards, J. P., Garrison, L., Jones, R. G., Soper, Q. F., Van Abeele, F. R., and Whitehead, C. W., J. Biol. Chem., 176,793 (1948).

4. Moyer, A. J., and Coghill, R. D., J. Back, 61,57 (1946).

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R. Van Abeele and Ming-Chien ChiangJones, Marjorie J. Mann, Quentin F. Soper, F.

Otto K. Behrens, Joseph Corse, Reuben G.BENZYLPENICILLIN (PENICILLIN G)

BIOLOGICAL PRECURSORS FOR BIOSYNTHESIS OF PENICILLINS: I.

1948, 175:751-764.J. Biol. Chem. 

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