Amino Acid Analogues

CHAPTER 1

William Shive and Charles G. Skinner

I. Introduction 2 II. Amino Acid Antagonists 3 A. Aromatic Amino Acids 4 B. Leucine, Isoleucine, Valine, Alanine, and Glycine Analogues 11 C. Analogues of Sulfur-containing Amino Acids 15 D. Dicarboxylic Amino Acids and Their Amides 17 E. Hydroxy Amino Acids 22 F. Basic Amino Acids and Proline 23 III. Antagonisms among Amino Acids Essential for Protein Synthesis ... 26 A. Natural Antagonisms Involving Aromatic Amino Acids 27 B. Antagonisms Involving Aliphatic Amino Acids 28 C. Antagonisms of Polyfunctional Amino Acids 29 D. Mutation and Amino Acid Antagonisms 30 IV. Biological Studies Involving Amino Acid Inhibitors 31 A. Determination of Type of Inhibition 31 B. Antagonists in the Study of Biochemical Transformations In volving Amino Acids 32 C. Amino Acid Transport 34 D. Utilization of Peptides, Keto Acids, and Related Amino Acid Derivatives 37 E. Amino Acid Analogues in the Study of Protein Synthesis and Related Processes 39 F. Incorporation of Amino Acid Analogues into Proteins 42 G. Activation and Transfer of Amino Acid Analogues to Ribonucleic Acid 44 H. End Product Control Mechanisms 45 I. Enzymic Transformations Involving Amino Acid Analogues 47 J. Amino Acid Analogues and Chemotherapy 53 References 58 1 2 W. SHIVE AND C. G. SKINNER

I. INTRODUCTION

Although amino acids under certain conditions had been known for a long time to be toxic to growth of certain organisms, specific reversals of inhibitory effects by a particular metabolite have been observed only since 1935. For example, 0-alanine was observed to be potent as a growth- stimulating agent for yeast only in the absence of asparagine, the presence of which was essential for the development of the specific assay in the dis covery of pantothenic acid (1). The antagonism is mutual, since not only does asparagine prevent the conversion of ^-alanine to pantothenic acid, but β-alanine also exerts a toxic effect on yeast, which is prevented by asparagine or by aspartic acid (β). Ethionine, prepared and tested for its ability to replace methionine in stimulating the growth of rats on a cysteine- deficient diet, proved to be toxic, but methionine supplementation offset the apparent toxicity (8). In a study of 2- and 5-methyltryptophan as re placements for tryptophan in a deficient diet in the growth of rats, these compounds exerted a depressing effect upon growth, but this effect of 5-methyltryptophan was not noted with a complete diet (4). In a study of the amino acid requirement of Bacillus anthracis, Glad stone (5) observed that the organisms did not grow if leucine, isoleucine, or valine were singly omitted from the medium, but omission of all three amino acids did permit delayed growth. Further investigation indicated that the toxicity of valine could be counteracted by leucine ; that of leucine by valine; that of isoleucine by a combination of both valine and leucine; that of α-aminobutyric acid by valine; that of serine by threonine, or by valine and leucine combined but not singly; that of threonine by valine or serine but not by leucine or isoleucine; that of norleucine by a mixture of leucine and valine. Gladstone concluded that "it is possible that excess of one (amino acid) may 'block' the reaction or enzyme necessary, either for the synthesis of another of similar chemical composition, or for building it when synthesized into bacterial protoplasm." Although the concept of competitive enzymic inhibition by structural analogues of the substrate had been demonstrated with succinic de hydrogenase (£), and enzymic inhibition involving amino acids had been observed in such cases as ornithine inhibition of arginase (7) and the competitive inhibition of the hydrolysis of glycylglycine with intestinal peptidases by either alanine or glycine, the concept of a competitive en zymic relationship by growth inhibitory analogues and their corresponding amino acids was not prevalent until after the appearance of the report of Woods that sulfonamide drugs exerted their effect by competing with p-aminobenzoic acid for an essential enzymic site (#, 9). Subsequently, numerous compounds analogous in structure to the natural amino acids 1. AMINO ACID ANALOGUES 3 have been broadly tested as metabolic antagonists, and for most natural amino acids an appreciable number of such analogue inhibitors are known.

II. AMINO ACID ANTAGONISTS

The structural features which are essential for a metabolite antagonist include certain functional groups necessary for binding with an enzyme in the manner of the metabolite, and a distance between such groups similar to that in the metabolite. This type of structural similarity exists among many of the natural amino acids and accounts for the many natural antagonisms which exist among these metabolites. However, the high degree of specificity essential for the enzymes selecting specific amino acids for protein synthesis and other metabolic processes must result from struc tural features other than the α-amino and carboxyl groups. Outstanding among these features are the size and particular shape of the other a-substituent which, through steric assistance and steric hindrance, permits its binding on a specific site and prevents its binding other enzymic sites involving different amino acids. Other factors, such as the presence of additional functional groups and the degree of hydration of functional groups, have important roles in the specificity of action. The structural analogues of amino acids which have biological activity related to one of the natural amino acids are included in this section, and the relationship of structure and biological activity is discussed. In many instances, it is difficult from reported data to ascertain whether or not an analogue is indeed an inhibitor of a specific amino acid. Inhibitors are too frequently termed metabolite antagonists solely on the basis of structural similarity and some degree of metabolite reversal at the lowest effective concentration of the inhibitor. Competitive reversal of an inhibition, with a high degree of specificity by the metabolite, over as broad a range of concentrations as is feasible in the biological system is essential for the demonstration of metabolite antagonism of the competitive type. For non competitive inhibitors, it is essential to demonstrate that the analogues do inhibit an enzymic transformation of the corresponding metabolite. Studies with potential competitive antagonists should be attempted only under conditions such that the inhibition is the sole limiting process and nutritional and other factors do not appreciably limit the response of the biological system in the absence of the inhibitor. Reversing agents at the lowest inhibitory concentration of an amino acid analogue may be many in number, and reversing effects only at such a concentration are of doubt ful significance. The ability of either of two amino acids to reverse a com petitive antagonist cannot be ascribed to antagonism of both, since dual 4 W. SHIVE AND C. G. SKINNER inhibition would require the presence of both amino acids for competitive reversal of the toxicity. Accordingly, in this section, the amino acid ana logues have been classified as antagonists of a particular amino acid, and the effects of other reversing agents which may influence the inhibition are discussed in a subsequent section on inhibition analysis.

A. Aromatic Amino Acids

1. PHENYLALANINE ANALOGUES The variety of structural modifications which could be made with reten tion of structural similarity in the case of phenylalanine resulted in the preparation of numerous analogues. Many of these have been found to be competitive inhibitors of phenylalanine. Structural modifications which have been successful in producing phenylalanine antagonists include (a) replacement of the phenyl group by an isosteric ring, (b) placement of substituent groups in the side chain, (c) substitution on the phenyl group, and (d) replacement of the benzyl group by a nonaromatic group having an appropriate planarity. Representative of these types of analogues are 2-thiophenealanine (10-12), phenylserine (^-hydroxyphenylalanine) (13), 3-fluorophenylalanine (14), and 1-cyclopentenealanine (15).

(/ y-CH— CH-COOH

S CH2- CH—COOH

2-Thiophenealanine Phenylserine (β - Hydroxy phenylalanine)

I ^V-CH2— CH—COOH •CH2—CH—COOH

3-Fluorophenylalanine 1-Cyclopentenealanine

Aromatic heterocyclic groups which have been substituted in lieu of the phenyl group of phenylalanine to give active antagonists include the following: 2- and 3-thienyl (10, 16, 17), 2- and 3-furyl (18, 19), 2-pyrrolyl (20), 2- and 4-pyridyl (21, 22), 4-thiazolyl (22), and 4-pyrazolyl (22) groups. These analogues have been most frequently tested with micro- 1. AMINO ACID ANALOGUES 5 organisms such as Escherichia coli and Saccharomyces cerevisiae, but others have also been used, such as Mycobacterium tuberculosis with 2- and 3-thiophene- and 2-furanalanines (23, 24) and Streptococcus pyrogenes with 4-pyridinealanine (21). These analogues have also been used on insects, e.g., 2-thiophenealanine with Oniscus asellus (26), and in mammalian systems the toxicities of 2- and 3-thiophenealanine for rats have both been found to be reversed by phenylalanine (11, 17, 26). Tissue culture studies have also been carried out with some of these analogues (27). Pathological changes associated with feeding 2-thiophenealanine are similar to phenylalanine deficiency symptoms in the rats except that some of the changes in tissues have not been previously reported for phenyl alanine deficiencies (28). Replacement of the phenyl group of phenylalanine with larger aromatic substituents has not been particularly successful in producing effective antagonists. Neither 1- nor 2-naphthalenealanine inhibits the growth of E. coli; however, the 1- but not the 2-naphthalenealanine reverses the toxicity of 2-thiophenealanine for this organism (19), apparently by pre venting certain processes essential for the utilization of exogenous supple ments of either the analogue or phenylalanine. 6-Methoxy-4-quinoline- alanine inhibits the growth of S. pyrogenes, but the nature of the inhibition was not investigated (21). Among the various ring-substituted phenylalanine analogues, the fluorophenylalanines have been found to be the most effective antagonists of phenylalanine. Growth inhibitions of Neurospora crassa by 3-fluorophenylalanine (14), and of Pseudomonas aeruginosa (29), Lactobacillus arabinosus (30), S. cerevisiae (31), and E. coli (32) by 4-fluorophenylalanine were demonstrated to be competitively reversed by phenylalanine. Earlier, the toxicity of the 3-fluorophenylalanine for rats had been reported, and subsequently, similar studies on 2- and 4-fluorophenylalanines appeared (31, 83, 84)- The inability of phenylalanine to reverse completely the toxic action of the 4-fluoro analogue is ascribed in part to the formation of inorganic fluoride from the analogue. 4-Fluorophenylalanine is a phenyl alanine antagonist for chick-heart cell cultures (27). 4-Chlorophenylalanine is a weak growth inhibitor for E. coli (85) and competitively inhibits the incorporation of phenylalanine into protein of Staphylococcus aureus (36). The 2,4-dichloro analogue may displace phenylalanine in protein synthesis in S. cerevisiae (see Section IV, F). The aminophenylalanines are somewhat unusual in that the toxicity of p-aminophenylalanine, a potent growth inhibitor for E. coli, is reversed by either phenylalanine or tyrosine over a range of concentrations (21, 85), and ra-aminophenylalanine, a competitive antagonist of phenylalanine for E. coli (37), Lactobacillus casei, and Leuconostoc dextranicum (88) is also 6 W. SHIVE AND C. G. SKINNER

reported to be a competitive antagonist of lysine for S. cerevisiae (87). The p-(7-chloro-4-quinolylamino) and p-aminomethyl derivatives of phenylalanine have some toxicity for E. coli and S. pyrogenes, respectively (21, 85). The ring-substituted methyl and dimethyl analogues of phenylalanine, although not inhibitory to the growth of E. coli, have been found to pre vent the toxicity of thienylalanine (89, 40). The mode of action of these analogues apparently is to prevent assimilation of exogenous analogues or phenylalanine, since p-tolylalanine inhibits competitively the utilization of phenylalanine in reversing the toxicity of a peptide of 2-thienylalanine (see Section IV, D). o-Hydroxyphenylalanine (o-tyrosine) similarly appears to be an in hibitor of the utilization of exogenous but not endogenous phenylalanine since a mutant requiring phenylalanine, but not the parent strain of E. coli, is inhibited by the analogue. Another phenylalanine-requiring organism, Leuconostoc mesenteroides P-60, as well as an E. coli strain sensitive to tyrosine inhibition, was also inhibited by o-tyrosine (41). Among side chain modifications of phenylalanine, the ^-hydroxy ana logue (β-phenylserine) has been found to be a phenylalanine antagonist for E. coli (18), L. arabinosus (42), Lactobacillus brevis, and P. aeruginosa (29). 0-Phenylserine has a slight toxicity for rats (84) and inhibits chick heart tissue culture (27). Both diastereoisomers of phenylserine are in hibitory to L. brevis, but only ^reo-phenylserine is active against L. arabinosus. In the latter case the D-form is not reversed by phenylalanine to as great an extent as the L-Z/ireo-phenylserine (42). Of a number of fluoro and chloro derivatives of ^-phenylserine, only p-fluoro-/3-phenylserine was found to be an antagonist of phenylalanine, and it was similar to phenylserine in its biological effect upon the growth of E. coli (43, 44)- 2-Thiopheneserine slightly retards growth of E. coli and augments the effect of phenylserine (44a). iV-Phenylphenylalanine at moderately high concentrations exerts an inhibitory effect on growth of L. mesenteroides P-60 which is reversed by phenylalanine (45). When α-methylphenylalanine is used as the sole source of nitrogen, it is inhibitory to the growth of S. cerevisiae (46). The β-phenyl derivative of phenylalanine is a weak growth inhibitor of E. coli (85), α-amino-jS-phenylethanesulfonic acid has some antiviral activity (see Sec tion IV, J), and phenylalanine hydrazide has slight tuberculostatic ac tivity (47). m-Amino and ra-nitro-/3-phenylserines are not inhibitory to a number of strains of E. coli, but they do exert at high concentrations a reversing effect upon the growth inhibitions caused by p-amino- and p-fluorophenylalanine, m-nitrotyrosine, and β-phenylserine (82). 1. AMINO ACID ANALOGUES 7

In studies to determine the structural features essential for biological selection of phenylalanine from other amino acids in its utilization by L. dextranicum, a number of amino acids containing cycloalkenyl and alkenyl groups were synthesized. Many of these analogues have been found to be competitive antagonists of phenylalanine. Structural altera tions in the phenyl grouping which have produced competitive antag onists in their approximate order of decreasing effectiveness include 1-cyclohexenyl- (48), 1-cyclopentenyl- (15), l-(l-butenyl)- (49), trans-2- (1-butenyl)- (50), m-2-(2-butenyl)- (51), trans-l-(l-propenyl)- (49, 52,53), and m-l-(2-butenyl)alanine (49). In the above examples, where a cis or trans isomer is specified, the alternate isomer was inactive as a phenyl alanine antagonist. It is thus apparent that neither an aromatic group

CH3—CH NH2 CH=CH NH2 \ I / \ I CH—CH2—CH—COOH CH3 CH2CH2—CH—COOH / CH3 2-Amino-4-methyl-4-hexenoic acid m-Crotylalanine (Tiglylglycine) nor a cyclic group is essential for the enzymic binding, as indicated by the activity of 2-amino-4-methyl-4-hexenoic acid; and, in addition, neither branching of the chain nor the presence of a double bond in the 4-position is essential, as indicated by the activity of czs-crotylalanine. The minimal structural features necessary for enzymic binding of a phenylalanine analogue include (a) a planar configuration of the γ-carbon and its attached carbons, including the β-carbon of the alanine moiety, or (b) a molecular configuration which can assume such a planar conforma tion, and (c) sufficient size of the group attached to the β-position of the amino acid to assist sterically the analogue in binding at the site but not so large a group as to hinder interaction with the appropriate enzymic sites. Examples of similar derivatives which have not been found to be phenylalanine antagonists include 2-amino-4-ethyl-4-hexenoic acid (with the methyl and ethyl groups in a cis configuration) and 2-amino-4-methyl- 4-pentenoic acid (methallylglycine) (50) ; the lack of activity of the former analogue is attributed to steric factors which hinder the transition of non- planar conformations of the α-alkenyl substituent to planar configurations, and the inactivity of the latter derivative was attributed to the lack of sufficient size of the /3-substituent to assist sterically the binding of the analogue at the site of phenylalanine utilization. 8 W. SHIVE AND C. G. SKINNER

2. TYROSINE ANALOGUES Among previously reported antagonists, 3-fluorotyrosine appears to be one of the few analogues which specifically inhibit tyrosine utilization in a competitive manner. 3-Fluorotyrosine causes a growth inhibition in N. crassa which is competitively reversed by tyrosine (14). The analogue is also toxic for rats (33, 54) and mice (55). 3 \ 5-Difluorotyrosine and 3-fluoro-5-iodotyrosine are appreciably less toxic than the 3-fluoro analogue. 3,5-Diiodotyrosine displaces an amino acid, presumably tyrosine, in the synthesis of protein in S. cerevisiae (371). Certain potential tyrosine antagonists, such as p-aminophenylalanine (32, 35) and 3-nitro tyrosine (32), are inhibitory to microorganisms. How ever, these growth inhibitions are reversed not only by tyrosine but also by phenylalanine and by tryptophan, respectively, in such a manner as to suggest that tyrosine may not necessarily be specifically antagonized by these two compounds. p-Nitrophenylalanine though less effective has been compared with p-aminophenylalanine (56); however, its toxicity in a tyrosineless mutant, in contrast to its inactivity for the corresponding parent strain of E. coli, suggests that the analogue may inhibit utilization of exogenous tyrosine and possibly phenylalanine, but it does not ap preciably affect endogenous tyrosine (32).

-N

3-Fluorotyrosine 5-Hydroxy-2-pyridinealanine

An analogue containing the pyridine ring in place of the benzene ring of tyrosine, 5-hydroxy-2-pyridinealanine, is a specific and potent competitive antagonist of tyrosine in L. dextranicum (57) and is a moderately active growth inhibitor of E. coli. In the latter organism, the analogue is com petitively reversed by tyrosine but only in the presence of phenylalanine as a synergistic antagonist, suggesting a second pathway of tyrosine utilization. The weak inhibitory effects of p-methylthio- and p-ethylthiophenyl- alanine upon the growth of E. coli are reversed by tyrosine but not by phenylalanine (58). Growth inhibition of E. coli caused by £-(l,2-di- chlorovinyl)-L-cysteine is reversed by either tyrosine or phenylalanine (59), but the concentrations of the metabolites required for reversal are exceptionally large in comparison to the toxic level of the inhibitor. The toxicity of p-hydroxycinnamic acid for L. mesenteroides P-60 is also re ported to be prevented by tyrosine and phenylalanine (60). 1. AMINO ACID ANALOGUES 9

3. TRYPTOPHAN ANALOGUES Many of the effective inhibitory analogues of tryptophan contain substituents on the indole moiety, and the methyl-substituted tryptophans were the first reported (4, 61). 5-Methyltryptophan inhibits the growth of E. coli) however, tryptophan and indole, but not anthranilic acid, reverse the inhibition noncompetitively. These data suggest that the analogue prevents the biosynthesis of tryptophan at the stage of the conversion of anthranilic acid to a product replaceable by indole (61-63). Although tryptophan and indole reverse noncompetitively the growth inhibition by this analogue in two strains of Lactobacillus plantarum, anthranilic acid reverses the toxicity in a competitive manner. This suggests that a com petitive relationship of 5-methyltryptophan with an intermediate may exist at some stage in the biosynthetic pathway before the formation of the product replaced by indole. Tryptophan similarly reverses noncom petitively the toxicity of 4-methyltryptophan for E. coli, but indole appears to reverse the toxicity competitively and is inhibited in its interaction with serine to form tryptophan by cell-free enzyme preparations from the organism. However, 4-methyltryptophan also inhibits the formation of anthranilate by a mutant strain of E. coli, but does not inhibit the con version of anthranilate to indole in mutants accumulating indole (64). In contrast to the ability of tryptophan to reverse methyltryptophan non competitively in many organisms, growth inhibition by 4-methyltrypto phan of Streptococcus faecalis R, L. mesenteroides P-60, L. arabinosus 17-5, two strains of S. aureus, and one strain of E. coli is reversed competitively by tryptophan (65). In the order of increasing activity, 4-, 5-, 6-, and 7-methyltryptophan inhibit the growth of Bacterium typhosum (Salmonella typhosa), but 2- methyltryptophan had little, if any, inhibitory activity. For 4-methyl tryptophan, tryptophan appears to reverse the toxicity competitively over a narrow range of concentrations (66), but with the 2- and 5-analogues, which antagonize indole utilization, inhibition of tryptophan utilization is not so apparent (67). Because of the activity of fluoro analogues of phenylalanine and tyrosine, 5-fluorotryptophan was prepared as a tryptophan analogue (68) and ap pears to inhibit the conversion of anthranilic acid to a product replaceable by indole in E. coli (69, 70). 6-Fluorotryptophan also inhibits the utiliza tion of anthranilic acid in vitro. The condensation with phosphoribosyl- pyrophosphate is inhibited by the analogue as well as by tryptophan in a possible control mechanism (71). Replacement of a carbon of the indole ring with nitrogen has been successful in producing antagonists of tryptophan. DL-7-Azatryptophan is 10 W. SHIVE AND C. G. SKINNER

a competitive antagonist of L-tryptophan in promoting the growth of Tetrahymena pyriformis W (72, 73), and 2-azatryptophan (tryptazan) (74) is a competitive inhibitor of tryptophan utilization in yeast (75). Replace ment of the nitrogen of the indole ring by sulfur is also reported to result in a moderately active antagonist of tryptophan for L. arabinosus 17-5, jS-(2-benzothienyl)-o:-aminopropionic acid (76).

CH 3^^^v_.CH2- CHCOOH *V^r^ ^CH2- CHCOOH

5-Methyltryptophan 5-Fluorotryptophan.

NH2 -CHCOOH

Ν Ν Η

7 -A zat r y ptophan

Tryptophan reverses the toxicity of 3-indoleacrylic acid in such a manner as to indicate that the analogue prevents the formation of trypto phan from indole in E. coli and B. typhosum. Indole does not reverse the toxicity in a competitive manner, and serine exerts only a slight effect; however, indole accumulates in the presence of the inhibitor (77). Phenyl alanine in addition to metabolites related to tryptophan also reverses the toxicity of indoleacrylic acid (78). 1-Naphthaleneacrylic acid, styrylacetic acid, and cinnamic acid appear to exert growth effects analogous to indole acrylic acid (79). 3-Quinolineacrylic acid and indoleacrylic acid are re ported to be competitive inhibitors of tryptophan for L. arabinosus and L. mesenteroides P-60, and the corresponding 2- and 4-quinolyl derivatives are partially reversed by tryptophan (60). Some benzimidazole analogues of tryptophan are reported to be competitive antagonists, 2-benzimidazole- alanine and its β-methyl derivative for L. mesenteroides P-60, and the cor responding 5-methyl derivatives of these analogues for E. coli (80). a. Indole Analogues. In order of decreasing activity, 4-, 6-, 7-, and 5-methylindoles inhibit the growth of B. typhosum, and because of the greater activity of the 4- and 6-isomers, double hydrogen-bonding of the 1. AMINO ACID ANALOGUES 11

=N—H group to the enzyme was suggested (66). The 4-methylindole toxicity is reversed competitively by indole and noncompetitively by tryptophan. 2-Methyl- and 5-methylindole inhibit the utilization of indole, but only slightly inhibit tryptophan utilization (67). 7-Azaindole is reported to inhibit competitively the utilization of indole by mutants of Neurospora which require indole or tryptophan for growth (81). b. Anthranilic Acid Analogues. In E. coli, 4- and 5-fluoroanthranilic acid and 4- and 5-methylanthranilic acid appear to be antagonists of anthranilic acid. Indole and tryptophan are noncompetitive reversing agents (69, 82). In B. typhosum, 4- and 5-methylanthranilic acid, but not 6-methylanthranilic acid, inhibit growth and are reversed by anthranilic acid, indole, or tryptophan (83).

B. Leucine, Isoleucine, Valine, Alanine, and Glycine Analogues

A number of amino acids are homologues of glycine and thus differ only in the type of alkyl substituent, so that enzymes must differentiate be tween hydrogen, methyl, isopropyl, sec-butyl, and isobutyl groups. In many organisms, the enzymes are not sufficiently specific in their interac tions, so that leucine, isoleucine, and valine are frequently antagonists of one or both of the others, and glycine and alanine antagonisms have often been observed (see Section III, B). In addition, the close structural rela tionships of these amino acids results in specific inhibitory analogues of one of the amino acids being reversed to some extent by the others, through interference with the utilization of the analogue as well as through other biochemical interrelationships.

1. LEUCINE ANALOGUES Since biological selective mechanisms tend to eliminate isoleucine inter ference in leucine metabolism, it would be anticipated that the most of the effective leucine antagonists would be singly ^-substituted alanines. Methallylglycine (19, 84) and 2-amino-4-methylhexanoic acid (51) are among the most effective competitive antagonists of leucine. Both ana-

H2C NH2 CH3—CH2 NH2 \ C—CH2—CH—COOH CH—CH2—CH—COOH

CH3 CH3 Methallylglycine 2-Amino-4-methylhexanoie acid (2-Amino-4-methyl-4-pentenoic acid) 12 W. SHIVE AND C. G. SKINNER logues are competitive leucine antagonists for Ε. coli and L. dextranicum, and methallylglycine has been demonstrated to inhibit leucine utilization in S. cerevisiae. Cyclopentanealanine inhibits the utilization of leucine but not phenyl alanine, while 1-cyclopentenealanine is an antagonist of phenylalanine but not of leucine in L. dextranicum (15). Thus, extension of a group in the plane of the isopropyl group of leucine does not prevent interaction at the point of binding of leucine in its utilization; however, extension of the planar configuration of the isopropenyl group of methallylglycine prevents enzymic binding in place of leucine and permits binding in place of phenyl alanine. The selective mechanism differentiating between leucine and phenylalanine appears to be concerned not only with the size of the group but also the degree of planar configuration of the α-substituent. An inter mediate structure, 2-amino-4-methyl-4-hexenoic acid, which is the higher homologue of methallylglycine, retains (although with considerable loss in effectiveness) the ability to inhibit leucine utilization, but gains the ability to antagonize phenylalanine (51). 3-Cyclohexenealanine is also capable of antagonizing leucine utilization by L. dextranicum (85). Cyclo- propanealanine is reported to be an amino acid antagonist for E. coli, and although not specified, it presumably is a leucine antagonist (85a). A δ-chloro derivative of leucine, 2-amino-5-chloro-4-methylpentanoic acid, inhibits the germination of a leucineless strain of N. crassa, and leucine at similar concentrations prevents the inhibition (86). The growth- inhibitory effect of one of the diastereoisomers of 0-hydroxyleueine is pre vented by leucine in L. arabinosus (87). iV-Phenylleucine appears to be a weak inhibitor of leucine for L. arabinosus (45). The inhibitory effect of ethyl diazopyruvate upon growth of E. coli is reversed by amino acids such as leucine and isoleucine (88). Norleucine greatly reduces the utiliza tion of D-leucine by rats (89).

2. ISOLEUCINE AND VALINE ANTAGONISTS Among the effective and specific isoleucine antagonists are O-methylthreonine (90) and cyclopentaneglycine (91). O-Methylthreonine, but not O-methylallothreonine, is a competitive antagonist of isoleucine incorpora tion into proteins of ascites cells. Thus, for inhibitory activity, the O-methyl group must occupy the same steric position as the ethyl group of isoleucine.

CH3—Ο NH2 CH2—CH2 NH2 \ I CH—CHCOOH CH—CHCOOH / / CH3 CH2—CH2 O-Methylthreonine Cyclopentaneglycine 1. AMINO ACID ANALOGUES 13

Bridging the methyl groups of isoleucine with a methylene group results in an effective and specific antagonist of isoleucine for E. coli and many other organisms. 2- and 3-Cyclohexeneglycine, but not cyclohexaneglycine, are isoleucine antagonists for E. coli. Since the boat form of cyclohexane glycine is similar structurally to the boat conformation of the cyclohexene analogues, it appears that the active forms involve the half-chair structures of the cyclohexeneglycines (48, 92). The similarity of structure of valine and isoleucine not only results in mutual antagonisms, but analogues frequently are antagonists of both amino acids. The growth inhibition of E. coli caused by 2-cyclopentene- glycine is reversed competitively by a mixture of isoleucine and valine but not by either alone (93). In contrast, the saturated analogue is specifically an isoleucine antagonist. The differences in specificity has been attributed to a slight puckering of the cyclopentane ring and the more planar struc ture and slightly smaller size of the cyclopentene ring, which permits it to occupy the same enzymic position normally associated with the planar isopropyl group of valine. The conformation required of the sec-butyl group of isoleucine in binding with its enzymes need not be a planar con formation. ω-Dehydroisoleucine (2-amino-3-methyl-4-pentenoic acid), which is structurally similar to isoleucine as well as the cyclopentene- glycine, is also a potent dual antagonist of isoleucine and valine in L. arabinosus and in E. coli (53).

CH2=CH NH2 CI NH2 \ I \ I CHCH—COOH CH—CH—COOH / / CH3 CH3

ω-Dehydroisoleucine a-Amino-/3-chlorobutyric acid

Replacement of methyl groups of metabolites by chloro substituents has frequently produced antagonists; thus, both diastereoisomeric forms of a-amino-jS-chlorobutyric acid were found to be growth inhibitors of E. coli) and valine, isoleucine, and, less effectively, leucine reverse the inhibition. a-Amino-0-chlorobutyric acid prepared from allothreonine is a potent antagonist for valine incorporation into protein of rabbit reticulocytes in vitro, and the inhibition can be prevented by valine. The diastereoisomer, prepared from threonine, is only one-fifth as effective (94). Among a number of amino sulfonic acids which delay or prevent growth of several microorganisms, 2-methyl-l-amino-l-propanesulfonic acid caused inhibitions of growth of strains of Proteus and Staphylococcus which were reversed to some extent by valine. Glycine and alanine also partially re versed the inhibition of the analogue as well as that of other amino sulfonic acids (95). 14 W. SHIVE AND C. G. SKINNER

The toxicity of α-aminobutyric acid for Ε. coli is prevented by valine, isoleucine, and, very effectively over a limited range of concentrations, by leucine (96). The endogenous conversion of α-aminobutyric acid to a-ketobutyric acid may account for the effect of leucine, since a-ketobutyric acid inhibits growth of E. coli and is reversed in a seemingly competitive manner by α-ketoisovaleric acid but noncompetitively by the keto acid corresponding to leucine; thus, the biosynthesis of leucine is apparently blocked in this manner at the stage of utilization of α-ketoisovaleric acid (97). Since leucine only partially reverses the toxicity, a-aminobutyric acid must exert additional antagonisms related to isoleucine or valine since either amino acid reverses the toxicity over a range of concentra tions (96). Norvaline also causes growth inhibition of E. coli, but is re versed in a competitive manner only by a mixture of a group of amino acids, suggesting that more than one antagonism is involved (96). L-Penicillamine toxicity in E. coli is reversed by branched-chain amino acids; isoleucine is the most effective reversing agent, but the relationship is not competitive (98). One form of ^-hydroxyvaline is inhibitory to the growth of L. arabinosus and is reported to be an antagonist of valine (99). High concentrations of norleucine inhibit the utilization of alloisoleucine but not isoleucine by L. arabinosus (53). Inhibition of penicillin bio synthesis in resting mycelial preparations results with α-methylvaline, and L-valine reverses the inhibition and is used for penicillin synthesis, while D-valine exerts an inhibitory effect (100).

3. ALANINE AND GLYCINE ANALOGUES Antagonisms by natural amino acids have comprised most of the studies concerning inhibitions of alanine and glycine. However, a few other in hibitory analogues are known. The delay in growth of Proteus vulgaris caused by aminomethanesulfonic acid and 1-aminoethanesulfonic acid is reversed by glycine and alanine, respectively; however, the inhibitory effect of the latter analogue for S. aureus is reversed by glycine but not alanine (95). Reversals of growth inhibitions by aminosulfonic acid ana logues are generally not specific for particular amino acids. Aminomethane sulfonic acid inhibition of phage reproduction in E. coli is prevented by xanthine and presumably interferes with glycine conversion to purines (101).

CH CO NH — CH —S0 H 2 2 2 3 Οi NJ__H_

Aminomethanesulfonic Acid 4-Amino-3~isoxazolidone (Cycloserine, Oxamycin) 1. AMINO ACID ANALOGUES 15

The antibiotic, D-4-amino-3-isoxazolidone, is competitively reversed by D-alanine as a growth inhibitor for S. aureus (102), and has been found to be a competitive antagonist of the incorporation of the D-alanine into a uridine nucleotide necessary for cell wall synthesis in the organism (102a, 102b). The antibiotic is a competitive inhibitor of the interconversion of L- and D-alanine and of the conversion of D-alanine to D-alanyl-D-alanine, which is subsequently incorporated into the uridine nucleotide (103). The L-isomer of the antibiotic, which inhibits certain transaminase reactions involving L-alanine, prevents the incorporation of lysine and uracil into cellular materials in E. coli, and the inhibition is prevented by L-alanine (104). Thus, separate roles of the L- and D-forms of this antibiotic involve inhibition of essential roles of L- and D-alanine, respectively.

C. Analogues of Sulfur-Containing Amino Acids

1. METHIONINE ANTAGONISTS Shortly after the demonstration of the toxicity of ethionine and its re versal by methionine in rats (3), ethionine was found to be an antagonist of methionine in E. coli (105). Ethionine has since been found to be ef fective against a variety of organisms and also inhibits a variety of bio chemical roles of methionine. In rats, D- or L-ethionine causes growth in hibitions which are reversed by either configuration of methionine (106). Ethionine inhibits normal protein synthesis and incorporation of methio nine sulfur into cystine (107), causes fatty livers in fasting females (108,109) and castrated males but not in intact males and testosterone treated females (110), and slightly inhibits transmethylation from methionine to choline (111). Incorporation of ethionine into proteins of protozoa and mammal tissues (see Section IV, F) and conversion of ethionine in yeast to £-adenosylethionine (see Section IV, I) further demonstrate the broad spectrum of activities of this analogue. Of several other £-alkylhomocysteines, /S-isoamylhomocysteine was the most effective against Salmonella enteritidis and E. coli (112).

NH2 NH2 CH3—CH2—S—CH2—CH2—CH—COOH CH3—O—CH,—CH2—CH—COOH Ethionine Methoxinine

Methoxinine which contains an oxygen in place of the sulfur of methi onine is a methionine antagonist for E. coli and S. aureus (113). Methoxinine, though similar to methionine in reducing liver lipid, is toxic to rats, and methionine exerts a reversing effect upon the toxicity (114). The analogue also has antiviral activity (see Section IV, J). 16 W. SHIVE AND C. G. SKINNER

Early in studies of methionine antagonists, norleucine was shown to be an effective inhibitor of growth of E. coli and to be reversed by methionine (105) in a competitive manner (115) ; it is also an antagonist of methionine for Proteus morganii (116). In a mutant strain of E. coli, subinhibitory levels of norleucine increase the response to methionine suggesting that nor leucine replaces some of the functions of methionine or inhibits nonessen tial functions (115). It has been shown since that norleucine is indeed in corporated into proteins of E. coli (see Section IV, F). In certain micro organisms, D-norleucine is more effective as a growth inhibitor than L-norleucine (117).

CH2—CH2 NH2 CH=CH NH2 / \ I / \ I CH3 CH2—CH—COOH CH3 CH2—CH—COOH Norleucine cts-Crotylglycine

Interchange of a vinylene group and sulfur, which had been successful in altering aromatic rings of metabolites to produce antagonists, was ap plied to the sulfur-containing amino acids. A preparation of crotylglycine (2-amino-4-hexenoic acid), primarily the trans isomer (19, 84), was found to be inhibitory to E. coli and reversed by methionine and other amino acids. However, such preparations may contain impurities which affect the inhibition, and further investigation has shown that cis- but not Jrans-crotylglycine is an effective methionine antagonist (53). The results give information of value with regard to a conformation of methionine essential for its biological utilization. 2-Amino-5-heptenoic acid (crotylalanine) is a very weak growth inhibitor of one strain of E. coli, but the inhibition is specifically prevented by methionine (52). 2-Amino-4-pentenoic acid (allylglycine) inhibits the growth of both E. coli and S. cere visiae, but like norvaline the inhibition is not specifically reversed by methionine but by a group of amino acids (19). Replacement of the sulfur of methionine with selenium produces an analogue, selenomethionine, which is a competitive antagonist of methi onine in inhibiting the growth of Chlorella vulgaris, and methionine pre vents the incorporation of the selenium of selenomethionine into the organism (118). Selenomethionine replaces methionine in all of its roles in supporting growth of a mutant strain of E. coli (360), and growth of E. coli and S. cerevisiae in selenite-containing medium low in sulfur results in proteins containing selenomethionine and probably selenocystine (119,120). Growth inhibition of E. coli caused by ω-trifluoronorvaline is reversed by methionine as well as by leucine and valine (121). 2-Methylmethionine is reported to be a methionine antagonist (122), and both 2- and 4-methylmethionine, $-methyl-3-phenylcysteine, and 2-amino-4-(benzylsulfinyl)-n- valeric acid suppress multiplication of E. coli phage (123). Methionine 1. AMINO ACID ANALOGUES 17 sulfone suppresses the utilization of D-methionine by L. arabinosus (124) and is reported to interfere with methionine metabolism in erythrocyte formation (125) and in growth of E. coli (126). Growth inhibition of Ochromonas malhemansis caused by AS-hydroxymethylhomocysteine is reversed by methionine (127). A toxic principle in agenized flour which causes convulsions in animals results from the interaction of nitrogen trichloride and methionine; it has been synthesized by adding an NH grouping to the sulfur of methionine sulfoxide by treatment with hydrazoic acid (128, 129). This principle, methionine sulfoximine, has been found to be toxic for L. mesenteroides P-60, and the growth inhibition is reversed by high concentrations of methionine (ISO). Methionine also suppresses or delays the onset of con vulsion caused by the toxic principle in mice and rabbits (181, 182), and reverses the inhibitory effect of methionine sulfoximine on oxidase levels in liver of rats (188) and on the incorporation of methionine into tissues (184-186). However, since glutamine is a potent reversing agent for many inhibitory effects of methionine sulfoximime, including some that are not reversed by methionine, methionine sulfoximime cannot be considered to be a specific antagonist of methionine. The phytopathogenic toxin of Pseudomonas tabaci which causes wildfire disease of tobacco inhibits the growth of Chlorella, and methionine reverses the toxicity (187). Similarity in effects of the toxin, which appears to be the lactone of a-lactoylamino-/3-hydroxy-eaminopimelic acid (188), and those of methionine sulfoximime have been cited as further evidence of methionine antagonism (187).

2. OTHER SULFUR-CONTAINING AMINO ACIDS The utilization of a naturally occurring methionine derivative, the methyl sulfonium derivative of methionine, is inhibited competitively by the ethylsulfonium derivative of ethionine in E. coli (189), and the in hibitory effect of djenkolic acid in 0. malhemansis is reversed by homodjen- kolic acid (127). Only a few metabolic inhibitors have been related to cysteine or cystine. Inhibition of penicillin synthesis in resting mycelial suspensions by S-ethyl- cysteine is reversed by cystine (100). Cysteine and cystine are reported to be specific in their reversal of the growth inhibitory effects of methionine on a strain of E. coli (140).

D. Dicarboxylic Amino Acids and Their Amides

1. GLUTAMIC ACID ANALOGUES Since glutamine is one of the products of glutamic acid metabolism and can be a donor of a 7-glutamyl group, growth inhibitions by analogues of 18 W. SHIVE AND C. G. SKINNER

glutamic acid are frequently reversed more effectively by glutamine than glutamic acid, and it is often difficult to determine the nature of the antagonism. Structural modifications of glutamic acid which have resulted in effective antagonists include replacement of the 7-carboxyl by struc turally related groups, and the introduction of various substituent groups on the α-, β-, and γ-carbons. Replacement of the 7-carboxyl group of glutamic acid with a sulfoxide, sulfoximine, sulfonamide, or phosphonic acid group results in inhibitory analogues. Methionine sulfoxide and iS-benzylhomocysteine sulfoxide at moderately high concentrations are inhibitory to the growth of L. ara binosus, and the inhibition which is reversed by increased levels of glutamic acid cannot be demonstrated in the presence of glutamine (141)- These results suggest that the .analogues inhibit the biosynthesis of glutamine. Ethionine sulfoxide and homologous /S-alkylhomocysteine sulfoxides, as well as the corresponding sulfones and methionine sulfone, are appreciably less active than methionine sulfoxide (11$). One of the diastereoisomers of L-methionine sulfoxide is considerably more active than the other, indi cating stereospecificity of the inhibition (Ι/β). Enzyme preparations from S. aureus (144) and sheep brain (145) which synthesize glutamine from glutamic acid are competitively inhibited by methionine sulfoxide. 3-Amino-3-carboxypropanesulfonamide, the sulfonamide analogue of glutamine, at low concentrations inhibits growth of E. coli and multiplica tion of phage, and the inhibitory effects are reversed by glutamic acid or glutamine (146). The toxicity of methionine sulfoximine on growth of L. mesenteroides was found to be reversed not only by methionine but also by glutamine (147)' Glutamine synthesis and more effectively production of hydroxamate from glutamine was suppressed by methionine sulfoximine in sheep brain preparations (14$). Also, the inhibition by several levels of methi onine sulfoximine on the incorporation of amino acids into Ehrlich ascites cell protein was specifically prevented by a low level of glutamine (149). The normal increase in bound acetylcholine upon incubation of slices of cerebral cortex tissue is not observed with animals convulsed with methi onine sulfoximine, but glutamine as well as methionine reverses this effect (150). Methionine sulfoximine as well as methionine sulfoxide and ethionine relieve the ammonium ion inhibition of the formation of bound acetyl choline by rat brain preparations which presumably is caused by depletion of adenosine triphosphate used in glutamine synthesis (151). Relatively high concentrations of glutamine reverse the toxic effect of methionine sulfoximine for wheat embryos (152). On the basis of various data, methionine sulfoximine appears to inhibit in most organisms the conversion of glutamic acid to glutamine rather than the utilization of glutamine. 1. AMINO ACID ANALOGUES 19

7-Phosphonoglutamic acid and P-ethyl-7-phosphonoglutamic acid are strong inhibitors of glutamine synthetase; the latter derivative has an affinity forty times greater than that of the former analogue or glutamic acid for the enzyme. In contrast, P-phenyl-7-phosphonoglutamic acid and homocysteic acid have only slight affinities for the enzyme (153). δ-Hydroxylysine, a naturally occurring amino acid which contains an aminomethyl carbinol group in place of the carboxyl of glutamic acid, inhibits the incorporation of amino acids into proteins of Ehrlich ascites cells, and the inhibition is prevented by a small amount of glutamine (15If). δ-Hydroxylysine inhibits glutamine synthetase in rat brain and liver, but the inhibition is noncompetitive with respect to glutamate (155). Growth inhibitions of S. aureus by iV-7-glutamyl derivatives of ethylamine and ethanolamine are reversed by glutamic acid but not by glutamine (156). In contrast, amide-substituted glutamines are antagonists of glu tamine in other organisms as subsequently indicated.

NH2 OH NH2 I I I CH3—SO—CH2—CH2—CH—COOH HOOC—CH,—CH—CH—COOH Methionine sulfoxide /3-Hydroxyglutamic acid Among substituted derivatives of glutamic acid, 0-hydroxyglutamic acid, a growth inhibitor for L. arabinosus, was the first to be reported to be a competitive antagonist of glutamic acid (11$), and only one of the diastereoisomers has significant activity (157). A small amount of glu tamine reverses the inhibition noncompetitively (11$). a-Methylglutamic acid also inhibits glutamic acid utilization for growth of L. arabinosus; the utilization of glutamine is not inhibited by the analogue (157). These glutamic acid antagonists are more effective than the glutamine antag onists, 7-glutamohydrazide (7-glutamylhydrazine) and its acetone deriva tive, in inhibiting growth of L. arabinosus under conditions such that glutamine is not limiting, but the glutamine antagonists are otherwise more effective inhibitors (158). 7-Fluoroglutamic acid has been reported to be a growth inhibitor of M. tuberculosis but the nature of the inhibition was not determined (159). p-Nitrobenzoylglutamic acid is reported to be an antagonist for glutamic acid for L. casei (160).

2. GLUTAMINE ANALOGUES Two types of structural modifications of glutamine which have produced glutamine antagonists include replacement of the amide group with struc turally similar groups, and substitution of a sulfur or oxygen atom for the 3-methylene group. 20 W. SHIVE AND C. G. SKINNER

Among JV-substituted glutamines, iV-benzylglutamine is a competitive inhibitor of glutamine utilization in S. lactis (161), and growth inhibition of Trichomonas vaginalis by iV-ethylglutamine is reversed by glutamic acid but not by glutamine (162). 7-Glutamohydrazide (γ-glutamylhydrazine) and less effectively its acetone derivative are growth inhibitors of faecalis which are reversed by glutamine, and 7-glutamohydrazide prevents the deamination of glutamine during glycolysis (126, 168). Both stereoisomers of 7-glutamo hydrazide are inhibitory to growth of Mycobacterium ranae and Myco bacterium smegmatis (164).

Ο NH2 Ο NH2 II I II I NH2—NH—C—CH2CH2—CHCOOH NH2—C—O—CH2—CHCOOH 7-Glutamohydrazide O-Carbamoylserine

Ο NH2 Ο NH2 II I II I N2CH—C—O—CH2—CHCOOH NH2—C—S—CH2—CHCOOH Azaserine θ-Carbamoylcysteine In studies concerned with the role of the antibiotic, azaserine (O-diazoacetyl-L-serine), in preventing purine biosynthesis, it was found that azaserine inactivates the enzyme which catalyzes the condensation of glutamine and formylglycinamide ribotide to form formylglycinamidine ribotide and that glutamine can competitively delay the inactivation (165). These results suggest that azaserine interacts at the site of utilization of glutamine, but that once azaserine is complexed with the enzyme an in active protein derivative is formed. The chemically reactive diazo group has been depicted as initiating this process. 6-Diazo-5-oxo-L-norleucine (DON), a related antibiotic, has a similar effect and is more active (165). A derivative analogous to azaserine not containing the reactive group ing, O-carbamoylserine, was prepared as a possible competitive antagonist of glutamine, and the L-form was found to inhibit competitively the utilization of glutamine in S. lactis, L. arabinosus and E. coli (166). Of a number of 0-(substituted) carbamoylserines, only the methyl derivative caused inhibitions reversed by glutamine (167). In order to demonstrate that the introduction of a reactive chemical grouping could convert such an analogue to a noncompetitive antagonist, £-carbamoylcysteine, which contains a reactive thioester group, was prepared, and it was indeed found to inhibit growth of a number of organisms. The growth inhibitions were not appreciably affected by glutamine but were partially reversed by a number of end products capable of affecting competitive glutamine an- 1. AMINO ACID ANALOGUES 21 tagonists (168). This thioester analogue like azaserine has antitumor activity in mice (609), and, although it is somewhat less inhibitory, it similarly inactivates the enzyme forming the glycinamidine ribotide. A number of structural modifications of these active antagonists have been prepared and studied, including O-carbazylserine which is a com petitive antagonist of glutamine for S. lactis (169) and some ^-(substi tuted) carbamoy Icy steines which may be noncompetitive antagonists of glutamine (170).

3. ASPARTIC ACID AND ASPARAGINE ANALOGUES Structural modifications of aspartic acid which have produced effective antagonists include substitution of various groups in the a- and ^-positions and modifications involving the β-carboxyl group. β-Hydroxyaspartic acid was the first analogue found to be a competitive antimetabolite of aspartic acid. It is effective in a number of organisms including E. coli (171) and L. arabinosus (172). Recent work indicates that the en/^ro-L-/3-hydroxyaspartic acid is the biologically active diastereoisomer in many systems (173), and undergoes several enzymic reactions in a manner analogous to aspartic acid (Section IV). raeso-Di- aminosuccinic acid (171) and £/ireo-/3-methylaspartic acid (174) are also effective antagonists of aspartic acid for E. coli. 0-Methylaspartic acid appears to inhibit the utilization of aspartic acid in pyrimidine and aspara gine synthesis since a mixture of asparagine and dihydroorotic acid com pletely reverses the toxicity (174) >

OH NH2 CH3 NH2 NH2 II II I HOOC—CH—CH—COOH HOOC—CH—CH—COOH H03S—CH2—CH—COOH j8-Hydroxyaspartic 0-Methylaspartic Cysteic acid acid acid Among analogues involving modifications of the β-carboxyl group of aspartic acid, cysteic acid is an effective competitive inhibitor of aspartic acid utilization in E. coli, L. arabinosus, L. casei, and L. mesenteroides (175). A number of metabolic transformations have been studied with this analog (see Section IV, B). A sulfoxide, (+)$-methyl-L-cysteine sulfoxide, inhibits the utilization of aspartic acid in L. mesenteroides (176). Of the five aspartic antagonists listed above, all appear to occur in nature, at least in small amounts. α-Methylaspartic acid is an inhibitory analogue of aspartic acid in L. mesenteroides (177), and has an effect on certain enzymic transforma tions involving aspartic acid (481). 2-Thiohydantoin-5-acetic acid in hibits growth and lactic acid production in L. casei, and both aspartic 22 W. SHIVE AND C. G. SKINNER

acid and asparagine reverse the toxicity. The metabolism of aspartic acid appears to be inhibited by the analogue (178). Relatively few asparagine analogues have been found to have specific biological activities. β-Aspartohydrazide (0-aspartylhydrazine) is in hibitory to the growth of certain strains of Streptococcus, but the inhibitory effects are only diminished by asparagine and glutamine (126). Asparagine is more effective than aspartic acid in partially overcoming the toxicity of the mono- and dihydrazide of aspartic acid for E. coli (178a). Synthesis of an adaptive benzyl alcohol utilizing enzyme in Micrococcus urae is in hibited by β-aspartohydrazide, and reversal is obtained with the corre sponding amino acid (179). In contrast to α-methylaspartic acid, a-methyl- asparagine does not affect the growth of L. mesenteroides (177).

E. Hydroxy Amino Acids

1. SERINE AND THREONINE ANALOGUES Mutual antagonisms between threonine and serine have comprised most of the reports concerned with inhibition of the utilization of these amino acids. Since the first report of mutual antagonisms for B. anthracis (5) there have been many reports concerning inhibitions encountered with these two natural metabolites in many other organisms. Of a group of a-alkylserines, only α-methylserine inhibits growth of L. mesenteroides P-60 (180); however, other bacteria are unaffected by a-methylserine (19). Homoserine is also reported to inhibit serine utiliza tion for certain bacteria (19), but no details have been reported.

NH2 OH NH2 I I I HO—CH2—C—COOH CH3—CH2—CH—CH—COOH I CH 3 2-Amino-3-hydroxypentanoic a-Methylserine acid 2-Amino-3-hydroxypentanoic acid, the higher homologue of threonine, inhibits the growth of S. faecalis, and reversal of the toxicity is obtained with threonine. Only one of the two racemic diastereoisomers is active for S. faecalis, which suggests that this form has the steric configuration of threonine. Neither diastereoisomeric form inhibits the growth of L. ara binosus (99). An increase in the length of the carbon chain reduces the activity of the threonine analogue, since 2-amino-3-hydroxyhexanoic acid has only slight ability to inhibit the utilization of threonine for S. faecalis. The activity again in this case resides in only one of the two diastereo isomeric forms (87). 1. AMINO ACID ANALOGUES 23

F. Basic Amino Acids and Proline

1. LYSINE ANALOGUES For efficient lysine antagonism, it has been proposed that the distance between the two amino groups in the proposed analogue may have to be essentially the same as in lysine. €-C-Methyllysine (2,6-diaminoheptanoic acid) strongly inhibits the utilization of lysine in L. mesenteroides and AS. faecalis J whereas ornithine and homolysine are not toxic (181). Other structural alterations which have been found effective include the replace ment of the 4-methylene group of lysine by an oxygen or sulfur atom to produce the lysine antagonists, 4-oxalysine and 4-thialysine [/S-(jfr-amino- ethyl) cysteine] (182, 183). 4-Oxalysine antagonizes lysine utilization for E. coli as well as a number of lactobacilli, and 4-thialysine is a competitive inhibitor of lysine utilization in L. mesenteroides and L. arabinosus.

CH3 NH2 I I NH2—CH—CH2—CH2—CH2—CH—COOH e-C-Methyllysine

NH2 I NH2—CH2—CH2—O—CH2—CH—COOH 4-Oxalysine

H I NH2—CH2—C NH2 \ I CH—CH2—CH—COOH Zrans-4,5-Dehydrolysine Some cyclic lysine analogues have also been prepared; 3-aminocyclo- hexanealanine, but not 4-aminocyclohexaneglycine, is a competitive lysine antagonist for several lactobacilli (38). The activity of 3-aminomethyl- cyclohexaneglycine as a lysine antagonist for L. dextranicum indicated that /^-substitution did not account for the inactivity of 4-aminocyclo hexaneglycine (184)- More recently, cis- and transA, 5-dehydrolysine were prepared to determine if a structure in which the carbons corresponding to the β- and €-carbons of lysine are in a trans-like configuration might be essential for lysine antagonism. transA, 5-Dehydrolysine, but not the cis 24 W. SHIVE AND C. G. SKINNER

isomer, is an effective competitive inhibitor of lysine utilization for L. dextranicum, L. arabinosus, and S. lactis; thus, it appears that an essential conformation of lysine in its utilization involves a trans-like configuration of the β- and e-carbons (185). m-Aminophenylalanine is reported to inhibit utilization of lysine by S. cerevisiae, but for E. coli it is an antagonist of phenylalanine (87). δ-Hydroxylysine appears to inhibit glutamine synthesis (155) in certain biological systems; however, for L. mesenteroides, growth inhibition by δ-hydroxylysine is reversed by lysine (186). The natural metabolite, arginine, is also a competitive lysine antagonist for a lysineless strain of N. crassa (187). Supplements of 2-amino-6-hydroxyhexanoic acid fed to rats on a lysine- deficient diet (188) produce an anemia comparable to that observed in animals fed deaminized casein (189), and lysine reverses a number of toxic effects of the analogue (190). α-Aminoadipic acid also produces anemia in lysine-depleted rats (191). In addition, 2-amino-6-hydroxyhexanoic acid replaces lysine for certain lysine-requiring strains of Neurospora, but is inhibitory for others (192). a. a, e-Diaminopimelic Acid Analogues. Since this amino acid occurs only in bacteria and has been found not only to be a precursor of lysine but also to be essential for incorporation into the cell walls of certain bacteria, attempts have been made to prepare inhibitory analogues as chemotherapeutic agents (198). Growth inhibition by cystine of E. coli mutants re quiring diaminopimelic acid is overcome competitively by increasing concentrations of diaminopimelic acid, and lysis which occurs in the presence of lysine is prevented by lanthionine (194, 195). Lysis of such a mutant growing in limiting amounts of diaminopimelic acid can be pre vented by lanthionine, cystathionine, 7-methyldiaminopimelic acid, or 0-hydroxydiaminopimelic acid or by increasing the amount of diamino pimelic acid (196). Only one of the four racemic modifications of β-hydroxydiaminopimelic acid can replace diaminopimelic acid (197).

2. ARGININE ANTAGONISTS

Canavanine and homoarginine are inhibitory analogues of arginine. Canavanine, a natural amino acid discovered in jack beans, has been found to cause growth inhibitions reversed by arginine in Neurospora (198), a number of lactic acid bacteria (199), an arginineless E. coli strain (199), a variety of yeast strains (200-202), several species of green algae (202), avena coleoptile sections with growth induced by indoleacetic acid (208), carrot phloem expiant in tissue culture (204), and in many other systems. 1. AMINO ACID ANALOGUES 25

NH NH2 NH2—C—NH—Ο—CH2—CH2—CH—COOH Canavanine

NH NH2

NH2—C—NH—CH2—CH2—CH2—CH2—CH—COOH Homoarginine

Homoarginine is a growth inhibitor which is reversed by arginine in E. coli (206) as well as C. vulgaris and several other species of green algae (202). In E. coli, arginine reverses the toxicity of homoarginine in a com petitive manner over lower ranges of concentrations, but at higher con centration it prevents completely and noncompetitively the toxicity. These data suggest that homoarginine inhibits a function of arginine in its own biosynthesis or is transformed into an inhibitor with the rate becoming the limiting effect at higher concentrations. Inhibition of growth by canavanine is reversed not only by arginine but also by lysine in Neurospora and C. vulgaris, and by lysine and homo arginine in Torulopsis utilis (202). Similarly, lysine as well as arginine reverses homoarginine inhibition in C. vulgaris (202). The inhibitory analogue in such cases apparently must be acted upon before inhibiting the endogenous metabolite, and substances capable of preventing this action upon the analogue reverse the inhibition.

3. HISTIDINE ANALOGUES

In an extensive study of replacement of various heterocyclic radicals in lieu of the imidazole nucleus in histidine (206-209), a number of the analogues were found to have antihistamine activity, as determined by a spasmogenic effect on isolated guinea pig ileum and by depression of blood pressure in an anesthetized cat (210). However, of the analogues prepared and studied, only 2-thiazolealanine and l,2,4-triazole-3-alanine (209) were appreciably inhibitory to E. coli, and the toxicities were reversed by histidine.

NH2 NH2 HC (p— CH2 CH—COOH HÇ^ £-CH2CH-COOH S ΗΝ

2-Thiazolealanine 1,2,4-Triazole-3-alanine 26 W. SHIVE AND C. G. SKINNER

A combination of natural amino acids has been found to inhibit growth (211) by preventing histidine uptake in the mycelium of histidineless strains of N. crassa (212).

4. PROLINE ANTAGONISTS The structure of proline is such that a number of potential modifications of its structure would be anticipated to yield antimetabolites; however, only in a few instances has this been realized. The naturally occurring hydroxyproline possesses fungistatic activity for a number of organisms, e.g. Trichophyton mentagrophytes (218) and Trichophyton album (214), and it was subsequently shown that proline reverses these inhibitions (215). 4-Thiazolidinecarboxylic acid (4-thiaproline) inhibits growth of E. coli, and the toxicity is reversed to a limited extent by proline, and to a lesser extent by a number of other amino acids (216).

COOH

3,4-Dehydroproline

The only effective proline antagonist that has been reported is 3,4-dehydroproline (217). Dehydroproline inhibits growth of a number of lactic acid bacteria and E. coli, and the toxicities are competitively reversed by proline (218).

III. ANTAGONISMS AMONG AMINO ACIDS ESSENTIAL FOR PROTEIN SYNTHESIS

All of the amino acids which are usually essential for protein synthesis have been observed to exert at elevated concentrations growth-depressing effects upon some organisms. The extent of this type of effect is illustrated by a tabulation of seventeen amino acids which have been shown by various investigators to cause growth retardations in rats (219). Frequently, such responses have been demonstrated in diets deficient in some essential com ponent, so that the mechanisms of the inhibitory effects are frequently difficult to interpret; however, some of the many "amino-acid imbalances" result from specific antimetabolite action. Lactobacilli and other organisms requiring numerous specific amino acids for growth are particularly sus- 1. AMINO ACID ANALOGUES 27

ceptible to natural amino acid antagonisms, and mutant strains of a particular organism may become sensitive to such specific antagonisms or in certain cases become sensitive to several amino acids. Mechanisms of action of toxic levels of an amino acid include not only a specific antimetabolite effect upon the utilization of another amino acid but also inhibition of the biosynthesis of a specific amino acid; for example, tyrosine inhibits the biosynthesis of phenylalanine in a strain of E. coli (220), and the inhibitory effect of valine upon growth of E. coli is ascribed to an interference in the biosynthesis of isoleucine which appears to reverse the inhibition noncompetitively (221). In addition, amino acids may also exert an inhibitory control effect upon their own biosynthesis (see Sec tion IV, H). D-Amino acids have often been observed to have inhibitory effects upon growth of various organisms, and in many cases these toxicities have not been found to involve antagonisms of the corresponding L-amino acids (222-225).

A. Natural Antagonisms Involving Aromatic Amino Acids

Inhibitory effects by amino acids have implications in metabolic and nutritional diseases. That toxicities resulting from phenylalanine, or a derivative, are detrimental and cause the impairment of mental and neurological development in phenylketonuria (a genetic disease) is evident from the beneficial effects of diets which are low in phenylalanine in these patients (226, 227). Tyrosinase inhibition by phenylalanine which has been demonstrated in melanoma may also be the cause of diminished pigmentation of phenylketonurics (228). Phenylalanine inhibits tyrosine incorporation into liver protein, and either phenylalanine or phenylpyruvic acid inhibits the oxidation of tyrosine in rat liver (229). Pellagra is a nutritional disease in which amino acid imbalances appear to have a detrimental role. In rats, threonine is inhibitory to growth on a diet deficient in tryptophan and nicotinic acid, either of which reverse the growth retardation (230-288). Supplements of the second most limiting amino acid in the diet may cause a retardation of the growth which can be alleviated by the most limiting amino acid, such as tryptophan (234), but many of these effects are not the result of relatively specific antagonisms as in the case of threonine. Other antagonisms involving aromatic amino acids include tryptophan inhibition of phenylalanine utilization in S. faecalis R (235), reciprocal antagonisms of the keto acids corresponding to phenylalanine and tyrosine in the utilization of the keto acids by L. arabinosus and S. faecalis (236), growth-depressing effects of phenylalanine and tyrosine in rats which are 28 W. SHIVE AND C. G. SKINNER

prevented by threonine supplements (287), and growth inhibitions by phenylalanine and tyrosine in Streptococcus bovis and rats which are re versed by tryptophan (288).

B. Antagonisms Involving Aliphatic Amino Acids

Since the classic work on leucine, isoleucine, and valine antagonisms in B. anthracis, inhibitions involving one or more of these amino acids, or their keto acids, have been observed in numerous organisms including Pasteurella pestis (289), L. arabinosus (240), L. mesenteroides (240), mu tants of Neurospora (241, 242), mutants of E. coli (221, 248), and hiochi bacteria (244)· All six possible antagonisms among leucine, isoleucine, and valine have been observed in L. dextranicum (245). A number of these antagonisms, particularly leucine antagonism of isoleucine and valine, have been demonstrated in rats, and it appears probable that, if the amino acids were adjusted properly in the diet, all six antagonisms which have been observed with bacteria could be demonstrated in rats (219, 246). Leucine infused in dogs increases manyfold the excretion of isoleucine which suggests that leucine interferes with resorption of isoleucine in the kidney (247, 248), and intestinal absorption of isoleucine is reduced one- fourth by leucine in rats (249). Such effects may account in part for leucine antagonism of isoleucine in animals. Leucine inhibits the utilization of D-isoleucine in replacing L-isoleucine in promoting growth of L. arabinosus (250) and, in Brucella abortus leucine or isoleucine, prevents pantothenic acid synthesis from valine and ^-alanine (251). Glycine is frequently a reversing agent for D-amino acid inhibitions. Inhibition by D-serine of the growth of E. coli (252) and P. pestis (258), and elongation and inhibition of division of cells of Rhodospirillum rubrum caused by D-glutamic acid (254) is overcome by glycine supplements. D-Serine exerts an effect upon the utilization of β-alanine for pantothenic acid synthesis (255); however, this may be secondary to glycine antag onism, since pantothenic acid does not reverse concentrations of D-serine which are reversed by glycine. Purines partially overcome the toxicity of D-serine in P. pestis, indicating that the role of glycine in purine bio synthesis might be affected (258). When the interconversion of D- and L-alanine is prevented by vitamin B6 deficiency, mutual antagonisms of these two isomers have been observed. In vitamin B6-deficient S. faecalis the utilization of D-alanine, now known to be essential for cell wall syn thesis, is inhibited by glycine, and less effectively by threonine, serine, and β-alanine (256). D-Alanine inhibits the utilization of L-alanine in L. casei grown in the absence of vitamin B6, and high levels of glycine inhibit the 1. AMINO ACID ANALOGUES 29 utilization of D-alanine but do not inhibit the utilization of L-alanine (257). It is proposed that this inhibition of L-alanine concerns the penetration into the cell since peptides of L-alanine are not inhibited in their utilization. L-Alanine also reverses D-alanine inhibition of germination of B. anthracis and Bacillus subtilis spores (258, 259), and D-methionine inhibits the concentration of L-methionine into cells of Alcaligenes fecalis (260). In germination of B. subtilis, glycine, D-serine, D-cysteine, and D-a-aminobutyric acid (in order of decreasing activity) are also inhibitory but are much less active than D-alanine; and L-a-aminobutyric acid has significant activity in replacing L-alanine in stimulation of germination (259). The D-forms of a number of amino acids (serine, methionine, phenyl alanine, threonine, or histidine) inhibit cell division and growth in a species of Erwinia, and the D-serine inhibition was found to be reversed by D- and L-alanine, ammonium salts and p-aminobenzoic acid (261).

C. Antagonisms of Polyfunctional Amino Acids

Aspartic acid inhibition of growth of L. casei (262) and of L. arabinosus (2Jfi) is reversed by glutamic acid or more effectively by glutamine; for L. casei, the inhibition is also prevented by asparagine. A detailed study of this inhibition of L. arabinosus indicates that aspartic acid inhibits the utilization of exogenous glutamic acid for the biosynthesis of glutamine, citrulline, and proline, and that glutamine can perform the latter roles more effectively than glutamic acid in the inhibited system (263). Aspartic acid antagonism of glutamic acid also occurs in hiochi bacteria (264). Using the techniques of inhibition analysis (see Section IV, A), glutamic acid was demonstrated to inhibit the utilization of exogenous aspartic acid in the biosynthesis of lysine, threonine, and pyrimidines in either L. ara binosus or L. dextranicum (265). It is of interest that in preventing the biosynthesis of these end products glutamic acid inhibits growth of L. dex tranicum at a level far below that required for it to supply the growth requirement for glutamic acid or glutamine. L-Glutamine inhibition of growth of S. aureus is competitively reversed by L- but not by DL-glutamic acid, which suggests that D-glutamic acid is also an inhibitor of L-glutamic acid utilization. Folic acid and glutathione are more effective than L-glutamic acid in reversing the inhibition (266). Arginine specifically inhibits competitively the utilization of lysine by a lysineless mutant of N. crassa but does not inhibit the growth of the parent strain (187). Inhibition of growth of S. lactis caused by arginine is pre vented in a competitive manner by glutamine or less effectively by glu tamic acid. On the basis of growth studies, proline synthesis appears to be limiting under these conditions, and aspartic acid and arginine are syner- 30 W. SHIVE AND C. G. SKINNER

gistic in inhibiting growth, suggesting different sites of inhibition in the utilization of glutamate. Arginine appears to be primarily a glutamine antagonist in this system (267). D-Aspartic acid inhibits the oxidation of L-aspartic acid in Shigella flexneri 3 (268), and inhibition by D-aspartic acid of protein synthesis in cell suspensions of Pseudomonas saccharophila is reversed by L-aspartic acid (269). In cell-free preparations of the latter organism, D-aspartic acid inhibits the utilization of L-aspartic acid in the conversion of inosinic acid to adenosine-5'-phosphate, which accounts in part for the effect upon protein synthesis (269). D-Asparagine prevents the conversion of L-asparagine to /3-alanine in B. abortus (251). Mutual antagonisms of threonine and serine, which were originally observed with B. anthracis, have also been observed with other organisms. Threonine inhibition of serine utilization by Lactobacillus delbrueckii, L. casei, L. mesenteroides, and S. faecalis and serine inhibition of threonine utilization in the latter two organisms and L. arabinosus have been re ported (270). Supplements such as folic acid which promote the synthesis of serine by S. faecalis increase the amount of threonine required for the inhibition (271). Mutual antagonisms involving these amino acids also occur in hiochi bacteria (264), and D-serine inhibited competitively the stimulation by L-serine of the synthesis of pantothenic acid from valine and β-alanine by B. abortus (251). Reversal by proline of hydroxyproline toxicity for certain microorganisms has been previously mentioned.

D. Mutation and Amino Acid Antagonisms

Mutant strains of organisms which require an amino acid for growth are frequently sensitive to inhibition by a number of other amino acids. E. coli mutants requiring valine have been found to be inhibited by six different amino acids (272), and more than half of the amino acids occurring in proteins inhibit the utilization of the required amino acid in mutant strains of N. crassa requiring phenylalanine, tyrosine, or tryptophan for growth (278, 274) · A search for E. coli mutants sensitive to amino acid inhibitions resulted in the isolation of a number of strains sensitive to many different amino acids (275). In these mutants, valine inhibition is reversed by leucine or threonine; aspartic inhibition is reversed by valine or by a combination of valine, proline and glutamic acid; and serine inhibition is prevented by aspartic acid. In addition, a methionine inhibition is reversed by cysteine or cystine (140), and reversals of serine inhibition by glycine, cystine inhibition by methionine, lysine inhibition by methionine, and valine inhibition by leucine or isoleucine have also been reported (276). Mutual inhibitions involving homocysteine and threonine have also been found to occur in mutants of Neurospora (277). 1. AMINO ACID ANALOGUES 31

IV. BIOLOGICAL STUDIES INVOLVING AMINO ACID INHIBITORS

A. Determination of Type of Inhibition

In cell-free enzyme preparations, a convenient method of demonstrating the type of inhibition involves determination of the rate of the reaction over a range of substrate concentrations in the presence of constant amounts of the inhibitor and in the absence of the inhibitor. Under these conditions, in the most usual case, a plot of the reciprocal of the rate of the reaction vs. the reciprocal of the substrate concentration produces a straight line. For a competitive inhibition, the presence of the inhibitor changes the slope, but not the extrapolated intercept of the line at the origin. Non competitive inhibitors change both the slope and the intercept, uncom petitive inhibitors change the intercept but not the slope of the line (278). While this classic analysis can be applied with some modifications to studies involving the response of an intact organism, e.g., growth of the organism, it has been more convenient to determine the ratio of concen trations of inhibitor to metabolite necessary to obtain an appropriate inhibition for a specific experimental period (9). This ratio has been termed the inhibition ratio or index necessary for the specific degree of inhibition. An antagonist which competes with a metabolite for an enzyme site may interact with the enzyme to form either an inactive complex or a complex which undergoes the normal reaction to yield a product which is analogous to the normal enzymic product. The latter reaction may either produce an inactive product or one that can perform some of the functions of the natural product. In either case, competition for the same enzymic site results in partitioning of the enzyme into two parts, one part complexed with the analogue and the other complexed with the metabolite. The amount of free enzyme is usually negligible, particularly with increasing concentrations of the antagonist and metabolite. The proportion of the enzyme in its active form (that complexed with the metabolite) is deter mined by the ratio of the concentrations of the substrate (metabolite) to analogue and the ratio of the equilibrium constants of the complexes. Thus, the ratio of the concentrations of analogue to metabolite determines the degree of inhibition of the enzyme, which in turn determines the degree of inhibition for a defined experimental period, this ratio, the inhibition index, is essentially a constant, provided that the concentrations of ana logue and substrate are the only variables. Thus, the degree of inhibition caused by a competitive antimetabolite is not related directly to the con centration of the inhibitor but to the ratio of concentrations of inhibitor to the metabolite. To demonstrate a competitive inhibition, this ratio should be shown to be relatively constant over a 30-100-fold range of concentrations. In some systems, particularly with higher organisms, such 32 W. SHIVE AND C. G. SKINNER concentration ranges are not possible, so that the possibility for errors in interpretation is increased. Noncompetitive and uncompetitive inhibitors are not reversed by the substrates over any appreciable range in concen tration; thus, antagonism of a particular metabolite by an analogue that is not a competitive antagonist can be demonstrated only by its effect upon an enzymic reaction known to involve the specific metabolite.

B. Antagonists in the Study of Biochemical Transformations Involving Amino Acids

In addition to the effect of the analogue on the inhibited metabolite in a complex biological system, other substances may affect the degree of inhibition induced by a competitive antimetabolite. Agents other than the metabolite which are capable of reversing the inhibitory effects exerted by an antagonist include (a) any substance which causes the production of higher concentrations of the metabolite in the organism, (b) the normal metabolic product (or its equivalent) of the reaction catalyzed by the inhibited enzyme, (c) secondary reversing agents, such as end products, which decrease (spare) the amount of primary product which is needed by the organism to produce the observed response, (d) any substance which increases the effective concentration of the enzyme, and (e) any substance which increases the rate of destruction or decreases the rate of utilization of the inhibitor (879, 280). The effects of reversing agents of type (a) and (e) generally are limited in magnitude and are additive with that of the concentration of exogenous substrate or inhibitor. Thus, with appropriately high concentrations of metabolite, and consequently of inhibitor, the effects of these types of re versing agents no longer contribute appreciably to that of the exogenous substrate and inhibitor, so that the inhibition index determined under these conditions is unchanged. If the normal product (or its equivalent) of the inhibited reaction [type (b) reversing agent] is supplied to the organism from exogenous sources, either all concentrations of the analogues are reversed, or higher relative concentrations of the analogue inhibits a second enzyme system which utilizes the metabolite. The latter effect results in a higher inhibition index, which is related to the second enzyme having a different function and different equilibrium constants for its complexes. The presence of reversing agents of type (c) necessitates an increase in the inhibitor to metabolite concentration ratio in order to diminish the proportion of the enzyme in its active form, so that the rate of the forma tion of product is reduced below the initial rate to a level which again limits the response of the organism to the same initial degree of observed 1. AMINO ACID ANALOGUES 33 inhibition. To attain this same degree of inhibition, the increased levels of effective enzyme resulting from the addition of type (d) reversing agents require a higher concentration ratio of inhibitor to metabolite, so that the amount of enzyme in the active form is reduced to the initial level. An increase in inhibition index caused by addition of reversing agents of type (c) and (d) involves no change in equilibrium constants, which is in contrast to a change in equilibrium constants due to type (b) reversing agents. The applications of these techniques of inhibition analysis for determin ing the type of effects of various reversing agents have aided in the recog nition of a number of metabolite interrelationships and have been of ap preciable value in the study of biochemistry. When applied with discretion, such methods can give relatively accurate indications of metabolic trans formations. The simplicity of the technique is such that one needs merely to study the effect of an inhibitor and its reversing agents upon a single biological response, most frequently the growth of an organism. Through thr e use of this method, a number of new developments in bio chemistry were made independently, or concurrently with other methods. For example, cysteic acid as an inhibitor of aspartic acid has been used to demonstrate essential roles of aspartic acid in certain Lactobacilli in the biosynthesis of threonine through homoserine, of lysine, of pyrimidines, and of purines at a stage involving the synthesis of a conjugate of 5-amino- 4-imidazolecarboxamide (281-283). 0-Hydroxyaspartic acid and cysteic acid were employed to demonstrate an essential role of aspartic acid in the formation of β-alanine for the biosynthesis of pantothenic acid, and studies with cysteic acid limiting the biosynthesis of pantothenic acid in E. coli gave evidence for a role of pantothenic acid in the condensation of oxalacetate with an active acetate (284). Although not involving amino acid antagonists, a number of amino acid interrelationships have been established using metabolic antagonists (279), and these techniques were used to develop an assay for the isolation of the methylsulfonium derivative of methionine from natural sources (285). The ability of tryptophan and tyrosine to reverse phenylserine toxicity for E. coli only at low concentrations of phenylalanine suggests that these amino acids increase the endogenous level of phenylalanine. Since uni formly labeled tyrosine is not incorporated into phenylalanine, it has been suggested that tyrosine, by preventing its own biosynthesis, diverts a common precursor to increase phenylalanine synthesis. At least part of the effect of tryptophan could be similarly attributed to sparing of a common intermediate (18, 286, 287). Mercaptosuccinic acid and homoserine combined replace homocysteine or methionine in reversing growth inhibition of Vibrio comma by nor- 34 W. SHIVE AND C. G. SKINNER

leucine (288). The inability of cysteine to replace mercaptosuccinic may be the result of a permeability difference between the two, or the bio synthesis of homocysteine from mercaptosuccinic acid may not involve cysteine. An inhibition of an essential biosynthetic pathway by an amino acid analogue can lead to the discovery of a previously unknown metabolic transformation. For example, the incorporation of ureidosuccinic acid and orotic acid into the cytosine moiety of nucleic acids of some mammalian tissues was found to be depressed by 6-diazo-5-oxo-L-norleucine in com parison to the effect on incorporation into nucleic acid uracil and thymine. In studies with tumor slices in vitro, partial reversal of the inhibition with glutamine and lack of inhibition of cytidylic acid incorporation into nucleic acid suggested a role of glutamine in the amination of a derivative of uracil (289). Such a role was also indicated by amide-labeled nitrogen incorporation into the amino group of cytosine in HeLa cells (290) and subsequently confirmed by enzymic studies in which glutamine is required for transforming uridine nucleotides (291) to cytidine nucleotides. The utilization of analogues in place of the normal substrate to demon strate a mechanism, such as the classic work on fatty acid oxidation using ω-phenylderivatives, has been employed to demonstrate the mechanism of a step in biosynthesis of tryptophan. A tryptophan auxotroph which readily converts anthranilic acid to indole was shown to convert 4-methylanthranilic acid to 6-methylindole, so that the indole ring formation must occur through the 1-position (292).

C. Amino Acid Transport

The ability of peptides of L-alanine to circumvent the inhibitory effect of D-alanine upon the utilization of L-alanine by L. casei led to the sugges tion that analogues of metabolites may exert their effects upon the ab sorption process at the cell wall as well as upon essential biosynthetic processes within the cell (257). Individual amino acids are concentrated into cells by specific energy-requiring mechanisms (298-295). In E. coli, an amino acid concentrated into the cell rapidly equilibrates with exogenous amino acid and can be competitively displaced by certain structurally re lated analogues (294). Valine concentrated into the cell is displaced by isoleucine, leucine, norleucine, and threonine, and partially by methionine; other displacements include L-phenylalanine by p-fluorophenylalanine but not by D-phenylalanine, and methionine by norleucine. The concentration system appears to be stereospecific in at least some cases in E. coli (295). Thus, it appears that the external concentration ratio of analogue to amino acid may regulate that ratio in this pool. 1. AMINO ACID ANALOGUES 35

Considerable speculation concerning the effects of such a process upon the use of competitive inhibitors in the study of biochemistry has been made; however, it was early considered that the "ratio within the cel: l is a function of the ratio of concentration of analog to metabolite in the (ex ternal) medium" (279). An analogue which blocks solely an essential step in the utilization of a required metabolite from the extracellular environ ment could be reversed noncompetitively by any substance giving rise to endogenous metabolite, e.g., peptide of an inhibited amino acid (see Sec tion IV, D), which amounts to supplying the product of the inhibited system and is not inconsistent with inhibition analysis theory. An analogue which has the capability of interfering solely with exogenous metabolite utilization may prevent in a competitive manner the utilization of another metabolite antagonist which exerts internal effects. However, in such cases the analogue usually will be required in amounts exceeding that of the metabolite antagonist exerting the internal effects, and the inhibition index obtained in the presence of appropriately high concentrations of metabolite will not be affected as exemplified by a type (e) reversing agent. The concentration ratio of isoleucine necessary to reverse valine toxicity in E. coli K-12 correlates well with that necessary for displacement of the valine concentrated in the cell (295, 296). Competitive displacement in an essential concentration mechanism would result in a competitive relation ship, but it has been reported that isoleucine reverses valine toxicity non competitively and thus prevents the biosynthesis rather than utilization of isoleucine (221). However, in the latter study, the concentration range of testing was not large, so that a competitive relationship might exist at higher concentrations. Even so, the inhibitory effect of L-valine on multi plication of E. coli K-12 is actually due to an alteration of protein synthesis resulting in inactive enzymes and proteins containing an excess of valine (297). Since valine is activated, but is not transferred to s-RNA by a preparation of isoleucyl-s-RNA synthetase from another strain of E. coli (298), it is possible that in E. coli K-12 valine might also be transferred to the isoleucine-specific s-RNA and incorporated into protein in lieu of isoleucine. Selection of E. coli mutants resistant to L-canavanine or D-serine gave organisms with impaired concentration mechanisms for the uptake of arginine, lysine, and ornithine in the canavanine-resistant mutant, and glycine, D-serine, and L-alanine in the D-serine-resistant mutant (299). Resistance to inhibitory analogues may also produce organisms with im paired control mechanisms (see Section IV, H). An inducible tryptophan transport system in E. coli is inhibited by D-tryptophan, 5-methyltryptophan, the ethyl ester of tryptophan, indole, 5-methylindole, 5-methoxyindole, and serine and to some extent by 36 W. SHIVE AND C. G. SKINNER pyruvate, glucose, ribose, and 5-hydroxytryptophan (300). Aliphatic diamines inhibit the cellular uptake of lysine, arginine, and ornithine in Bacterium cadaveris and E. coli, and the inhibition of growth of a lysineless mutant of E. coli and S. aureus by the amines is ascribed to an inhibition of entry of the amino acids into the cell (301). In Candida utilis, two functionally distinct pools of amino acid exist. A concentrating pool accumulates exogenous amino acid to levels exceeding the medium level and is exchangeable with external amino acids. The concentrating pool becomes apparent only in the presence of external amino acid and is sensitive to osmotic shock. In addition, there is a con version pool in which amino acid interconversions occur and which is formed from endogenous sources in the absence of exogenous amino acids. This latter pool is insensitive to osmotic shock, does not exchange with exogenous amino acids, and furnishes the amino acids for protein synthesis (302). Transfer of amino acids from exogenous sources into the conversion pool does not appear to involve an equilibrium with the concentrating pool. In another yeast, S. cerevisiae, exogenous amino acid is utilized prefer entially to amino acid accumulated into the pool (303). In S. cerevisiae, the accumulation of valine is inhibited by isoleucine, methionine, phenyl alanine, and p-fluorophenylalanine, and the accumulation of phenylalanine is inhibited by methionine, isoleucine, valine, p-fluorophenylalanine, and D-phenylalanine. Only a part of previously accumulated phenylalanine can be displaced (803). Apparently, concentration mechanisms may involve more than one pathway; for example, in histidineless strains of N. crassa, the uptake of histidine was inhibited by a combination of either arginine or lysine with any one of a number of amino acids (especially tryptophan, methionine, tyrosine, or glycine for one strain (212). The same combinations corre spondingly produced growth inhibitions (211, 212). In contrast to E. coli, histidine which has been accumulated from the medium is not displaced from the mycelium of Neurospora by the inhibitory amino acids. Ν euro- spora like C. utilis has an expandable (or concentrating) pool of amino acids and an internal (conversion) pool (304). In S. aureus, canavanine suppresses uptake of arginine, but not lysine or other amino acids (305). D-Methionine displaces L-methionine concen trated by cells of A. faecalis (260). Histidine, tryptophan, phenylalanine, valine, and p-fluorophenylalanine decrease the concentration of L-tyrosine by rat brain slices which also concentrate D-tyrosine, L-a-methyltyrosine and tyramine (306). A number of other natural amino acids also appear to inhibit tyrosine accumulation in the brain (307). Cellular penetration of an amino acid independently of other metabolic transformations can be studied with α-aminoisobutyrie acid which is not appreciably metabolized in rat tissues (807a). 1. AMINO ACID ANALOGUES 37

D. Utilization of Peptides, Keto Acids, and Related Amino Acid Derivatives Biological activities of derivatives of amino acids which exceed that of the corresponding free amino acid have been observed for some time in intact cells. In many cases enhanced activity of the derivative results from an inhibition of the utilization of the free amino acid by either a naturally occurring analogue or a synthetic antimetabolite. Greater activity of pep tides in counteracting the inhibitory effects of amino acid antagonists have been observed with a variety of antagonist-amino acid pairs, and with several different organisms, as follows: D-alanine-L-alanine (L. casei) {257); 4-methyltryptophan-tryptophan (S. faecalis, S. aureus) (65); isoleucine- leucine (E. coli leucineless mutant) (308) ; isoleucine-valine (E. coli strains, L. arabinosus) (221, 245, 272); alanine-serine (L. delbrueckii) (309); 2-thiophenealanine-phenylalanine (E. coli, L. arabinosus, L. mesenteroides) (310-812); ethionine-methionine (L. mesenteroides) (311); canavanine- arginine (E. coli, L. arabinosus) (311); leucine-isoleucine (L. mesenteroides) (812); and alanine-glycine (L. mesenteroides) (312). Reversal by the cor responding peptides were noncompetitive in most cases, but in certain cases peptides with enhanced activity reverse in a competitive manner (245). The general nature of these effects gave an early indication that a different mode of utilization of peptides from that of free amino acids occurred within the cell, or that the inhibitory effect of the antagonist in volved a specific step (such as absorption of the amino acid) in the utiliza tion of exogenous amino acid but not in the utilization of peptides. Growth inhibitory effects of the two glycine peptides of 2-thiophene alanine upon E. coli are reversed in a competitive manner by the corre sponding glycine peptides of phenylalanine, whereas they are reversed in a noncompetitive manner by certain other peptides of phenylalanine; however, leucyl-2-thiophenealanine not only inhibits the utilization of leucylphenylalanine but of other peptides of phenylalanine as well (310). Growth of L. arabinosus in the presence of leucylphenylalanylglycine is inhibited by the corresponding 2-thiophenelalanine tripeptide, but it is not affected by the free amino acid analogue (2-thiophenealanine), leucyl- 2-thiophenealanine, or 2-thiophenealanylglycine, the three of which in hibit the utilization of phenylalanine and the two corresponding dipeptides, respectively (313). Glycylglycylphenylalanine effectively reverses the toxicity for E. coli of a mixture of 2-thiophenealanine and glycyl-2-thiophenealanine at concentrations which prevent measurable responses to a mixture of phenylalanine and glycylphenylalanine (314)- Some tripeptides of 2-thiophenealanine are also capable of inhibiting phenylalanine tri peptides of slightly different structure. From these results, it is apparent that the utilization of a tripeptide does not involve sites required for the utilization from the medium of the component amino acids or dipeptides which could be formed on hydrolysis. 38 W. SHIVE AND C. G. SKINNER

In L. mesenteroidesy a number of peptides of glycine and alanine as well as leucyltyrosine, but not the free amino acids, inhibit the utilization of glycylserine (but not of serine, the assimilation of which is inhibited by glycine, threonine, or alanine). However, greater specificity is required for peptides which are capable of inhibiting growth stimulated by glycylphenylalanine (315). Enhanced activity of α-keto acids and α-hydroxy acids over that of the corresponding free amino acid in reversing inhibitory growth effects have also been observed with a number of antagonist-amino acid pairs in several organisms, as follows: isoleucine and valine-leucine (α-ketoisocaproic acid) (L. dextranicum) (245), isoleucine-valine (α-ketoisovaleric acid) (L. ara binosus, E. coli) (221, 245), en/tf/iro-jft-phenylserine and m- and p-fluorophenylalanine-phenylalanine (phenylpyruvic acid or phenyllactic acid) (L. casei) (316)} and 2-thiophenealanine-phenylalanine (phenyllactic acid) (Lactobacillus mannitopoeus) (317). In L. casei, m-hydroxyphenylpyruvic acid inhibits growth stimulated by phenylalanine or phenyllactic acid more effectively than growth stimulated by phenylpyruvic acid (316). Studies upon growth inhibition of E. coli by mixtures of the competitive pairs, 2-thiophenealanine-phenylalanine, 2-thiophenepyruvic acid-phenylpyruvic acid, and glycyl-2-thiophenealanine-glycylphenylalanine, have demonstrated that the amino acids, the keto acids, and the peptides each have separate competitive sites of action, which are not involved in the utilization of the other two (318). Similarly, competitive relationships of methyl esters of amino acids have been demonstrated to involve separate sites for two competitive antagonisms (2-thiophenealanine-phenylalanine and isoleucine-valine) affecting the growth of L. arabinosus (319). Separate exclusive sites of utilization of an amino acid, its keto acid, and peptide were similarly demonstrated in the synthesis of the adaptive malic enzyme of L. arabinosus by demonstrating the specificity of free amino acid ana logue (2-thiophenealanine or isoleucine), the α-keto acid, or a peptide in inhibiting the corresponding free amino acid (phenylalanine or valine) or one of its corresponding derivatives (320). Cysteic acid and β-hydroxyaspartic acid exert synergistic inhibitory effects upon the utilization of aspartic acid for the synthesis of the induced malic enzyme, but neither inhibits the utilization of glycylasparagine for enzyme synthesis (172). Thus, two steps appear to be affected in the utilization of aspartic acid which are not common to the utilization of the peptide. There is general agreement that the assimilation of an exogenous amino acid by cells requires certain enzymic or "enzymic-like" steps before reach ing a common point in the utilization of peptides and other derivatives for protein synthesis. Whether the first common intermediate in the assimila tion of an amino acid and its derivative is an active complex or merely free amino acid within the cell has not been satisfactorily resolved. In the latter 1. AMINO ACID ANALOGUES 39 case, competitive analogue inhibition of cellular penetration is viewed as occurring with the amino acid without appreciable displacement of en dogenous amino acid formed from its derivatives within the cell. The ability of peptides to be utilized correlates well with the ability of a cell to hydrolyze the peptides, but this would be expected whether the cleavage produced solely free amino acid or an available active amino acyl group during hydrolysis (321). In certain organisms, the rate of concentration of peptides into the cellular pool of free amino acid has been found to be greater than the rate of accumulation of free amino acid (321, 322) ; how ever, external amino acid is preferentially utilized over pool amino acid in some organisms, and the concentration (expandable) pool may be a result of an essential step rather than being essential itself in protein synthesis, since exogenous amino acid incorporated into proteins does not equilibrate with the concentration pool. Furthermore, in E. coli, peptides do not prevent the accumulation of isotopically labeled amino acid (295), and analogues which competitively and rapidly displace amino acid from the pool do not interfere with the utilization of certain peptides. While the expandable pool itself does not appear to be an essential intermediate in protein synthesis, free amino acid in the conversion (internal) pool cannot be excluded as the common intermediate from assimilation of exogenous amino acid and its derivatives. A method of demonstrating specific inhibitions of the utilization of exogenous amino acid in contrast to that of biosynthetic amino acid is afforded by peptide analogues. Thus, inhibition of growth of E. coli by glycyl-2-thiophenealanine is reversed by phenylalanine; and either p-tolylalanine or 1-naphthalenealanine (which alone do not affect growth of E. coli) inhibits the growth response caused by phenylalanine in a com petitive manner. In contrast, these two analogues competitively reverse growth inhibition by 2-thiophenealanine but do so only at concentrations exceeding that of the inhibitor. These results indicate that p-tolylalanine or 1-naphthalenealanine prevent the assimilation of exogenous 2-thio phenealanine or phenylalanine (323). Phenylpyruvic acid is reported to inhibit competitively the growth of Achromobacter fischeri (Photobacterium fischeri) by preventing the utiliza tion of phenyllactic acid, a normal precursor of tyrosine and phenylalanine in this organism (324).

E. Amino Acid Analogues in the Study of Protein Synthesis and Related Processes

That induced enzyme synthesis occurs from free amino acids was early demonstrated by inhibiting enzyme synthesis with amino acid antagonists (325). Such inhibitions of induced enzyme synthesis have been demon- 40 W. SHIVE AND C. G. SKINNER strated in a number of organisms, as follows: 2-azatryptophan (tryptazan), 6-methyl-2-azatryptophan, 5-methyltryptophan, o-, m-, and p-fluorophenylalanine, and m-chlorophenylalanine (maltase in S. cerevisiae) (75, 325) ; ethionine, β-aspartohydrazide, and γ-glutamohydrazide (adaptive benzoic acid enzyme in M. urae) (179); β-hydroxyaspartic acid and cysteic acid (malic enzyme in L. arabinosus) (172); p-fluorophenylalanine (catalase in yeast and β-galactoside permease and maltase in E. coli) (326, 327); and β-phenylserine, 4- or 5-methyltryptophan, and 2-thiophenealanaine (β-galactosidase in E. coli) (327). Amino acid analogues have been used to demonstrate enzyme synthesis from free amino acids during the restoration of normal enzymic activity in cells deficient in some essential metabolite. The restoration of ornithine transcarbamylase activity to biotin-deficient cells of L. arabinosus in a medium containing biotin is inhibited by 4-oxalysine or 2-thiophenealanine, the effects of which are reversed by the corresponding amino acids (828). 5-Methyltryptophan inhibits protein synthesis stimulated by uracil in grapevine and olive trees (329). Studies concerning the dependence of nucleic acid synthesis upon amino acid metabolism, but not upon net protein synthesis, have shown that amino acid analogues can inhibit nucleic acid synthesis (330, 831). In E. coli, 2-thiophenealanine depresses both protein and ribonucleic acid syntheses with only slight effects on deoxyribonucleic acid synthesis (332) ; but in connective tissue cells from rat heart the analogue can, under ap propriate conditions, reduce the rate of ribonucleic acid synthesis without affecting protein synthesis (833). If added prior to the lag phase, the analogue prevents deoxyribonucleic acid synthesis in the tissue cells (334). p-Fluorophenylalanine, an analogue which is known to be incorporated into protein (see Section IV, F) at a linear rate, also permits a linear in crease in ribonucleic acid in a phenylalanineless E. coli mutant (335). Amino acid antagonists have been used to demonstrate the involvement of free amino acids in synthesis de novo of an enzyme released from re pression by end products; for example, 5-methyltryptophan inhibits the synthesis of aspartate transcarbamylase released from uracil repression in E. coli (836). In cell-free systems, inhibition by amino acid analogues has been used to indicate de novo synthesis. Substrate-antagonized inhibition by p-fluorophenylalanine occurs in cell-free preparations of E. coli Β synthesizing β-galactosidase (837). However, p-fluorophenylalanine inhibition of the increase of amylase activity in a soluble system from acetone-dried pigeon pancreas was not considered to involve de novo synthesis; but rather, synthesis from a precursor protein was proposed, since supplements of only arginine and threonine were required by the preparation (338). 1. AMINO ACID ANALOGUES 41

Other inhibitions of synthesis of enzymes and protein include azaserine and methionine sulfoximine inhibition of protease and amylase synthesis in mouse pancreas slices (339). ^-Amylase formation in the mycelium of Aspergillus oryzae is inhibited by ethionine, 7-glutamchydrazide, or nor leucine, and inhibition studies suggest that the enzyme formation may involve the internal free amino acids in the young mycelium, but not in old mycelium (340). Ethionine greatly inhibits the incorporation of methi onine into cellular proteins without inhibiting the formation of amylase by B. subtilis (341). 2-Thiophenealanine markedly inhibits antibody re sponses in rats (342). Amino acid antagonists may also reduce specific enzyme activities; e.g., 3-thiophenealanine and 2-amino-4-pentynoic acid reduce liver xanthine oxidase activity in rats (343). Variation in the ability of methionine sulfoximine, δ-hydroxylysine, and O-earbamoylserine to inhibit protein synthesis in tumor and normal tissues has been attributed to differences in the limiting effect of glutamine synthesis and to a balance of competing reactions for glutamine utiliza tion (344)- Methionine sulfoximine inhibition of enzyme formation in pancreas slices is reversed by glutamine (845). Antagonists of amino acids have been useful in the study of develop ment and differentiation in determining the stage at which de novo protein synthesis is essential to the processes; for example, tentacle regeneration in Hydra is reversibly inhibited by 2-thiophenealanine, which inhibits growth of tentacles and also decreases the tentacle number. The amino acid analogue is active only during the first four hours of the 18-hour period required for regeneration, which suggests that protein synthesis is essential to regeneration only during the early period of cell division (846). A role of phenylalanine in the differentiation in vitro of the cranial neural crest region of Ambystoma maculatum embryos has been demonstrated by reversal of specific inhibition of differentiation of ectomesenchyme by 2- and 3-thiophenealanine and 2-furanalanine (347). Phenyllactic acid in hibits differentiation in pigment cells. Phenylalanine, either supplied exogenously or synthesized by the archenteron roof mesoderm from exogenous precursors, reverses these inhibitions. Thus, it appears that the archenteron roof mesoderm is essential for normal differentiation of the neural" crest and may exert some control over the process (347). Amino acid analogues have also been found to cause characteristic developmental patterns in chick embryos (348, 849) and to affect protein synthesis in studies on differentiation in the fern gametophyte (350). Upon mechanical removal of flagella of Salmonella typhimurium, regeneration of flagella occurs, and in the presence of p-fluorophenylalanine, nonmotile cells are produced; certain other amino acid analogues do not produce this effect (851). Teratogenic effects of antimetabolites upon both avian and mam- 42 W. SHIVE AND C. G. SKINNER

malian embryos have been frequently reported (862, 853). Fetal resorption in rats is caused by antimetabolites, and use of the embryo-destroying properties of these substances for therapeutic abortion has shown promise (see Section IV, 5). Many amino acid analogues when added to a culture of an organism in the exponential growth phase reduce the growth to a linear rate, indicating that certain enzymes cease to be synthesized in an active form; however, protein synthesis frequently continues. In E. coli, protein precursors are still incorporated in the presence of analogues, but analogues differ in their effects upon specific activities; for example, in increasing order of activity, 2-thiophenealanine, 5-methyltryptophan, 4-fluorophenylalanine, and nor leucine diminish respiration; and although several analogues inhibit the synthesis of β-galactosidase, p-fluorophenylalanine and norleucine permit the synthesis of an active form of the enzyme. In contrast, p-fluorophenylalanine does inhibit the formation of active forms of β-galactoside permease and adaptation to maltose (327). These differences result from incorpora tion of analogues into proteins producing enzymes with varying degrees of activity.

F. Incorporation of Amino Acid Analogues into Proteins

The fact that analogues frequently undergo reactions simulating the metabolite to form analogous products has been recognized for some time. These analogue products may be inert or perform some or all of the func tions of the natural product. Vitamin analogues were among the first discovered to replace a natural essential metabolite, and the structural variations were of such a nature as to make it unlikely that the vitamin analogue was being converted to the vitamin. The demonstration that oxybiotin in replacing biotin in many organisms is incorporated as such and is not converted to biotin gave conclusive evidence for a complete analogue replacement and performance of all of the functions of the vitamin (854). A demonstration of this type of effect with amino acid analogues was hampered by the fact that amino acids perform numerous roles as components of proteins, and in addition they may have essential metabolic roles in other biosynthetic processes. Thus, it would be antici pated that complete replacement of all essential functions of an amino acid analogue would be rare; however, incorporation of an amino acid analogue even though it is inhibitory to growth, can be demonstrated readily with isotopically labeled derivatives. Ethionine was thus demon strated to be incorporated into the protein of rats fed the isotopically labeled analogue (355-357), and it has subsequently been shown to be incorporated into protein of T. pyriformis (858), as well as being a com petitive substrate in Ehrlich ascites tumor cells (359). 1. AMINO ACID ANALOGUES 43

Many other amino acids have also been found to be incorporated into proteins, and, depending upon the analogue structure and extent of re placement of the natural amino acid, the modified proteins possess varying degrees of activity and changes in physical properties. Of considerable interest is the fact that selenomethionine replaces methionine for an E. coli mutant in stimulating exponential growth in the absence of methi onine, with the formation of active enzymes containing selenomethionine in place of methionine (360). A considerable number of other analogues have been found to be in corporated into proteins in a variety of organisms as follows: p-fluorophenylalanine (L. arabinosus, E. coli, Saccharomyces italicus, Bacillus cereus, minced hen oviduct) (861-364, 873) ; o-fluorophenylalanine (Ehrlich ascites tumor cells, E. coli, minced hen oviduct) (359, 864, 865) ; m-fluorophenylalanine (E. coli) (865); 2-thiophenealanine (Ehrlich ascites tumor cell, connective tissue cells from rat heart, E. coli) (334, 859, 366) ; 2-azatryptophan (tryptazan) (E. coli) (367, 375); 7-azatryptophan (E. coli) (367, 876) ; norleucine (E. coli, casein of cow's milk) (866,367a) ; canavanine (S. aereus, Walker carcinosarcoma 256) (868-370); 3,5-diiodotyrosine (S. cerevisiae) and 2,4-dichlorophenylalanine (S. cerevisiae) (371); and certain halophenylcysteines (rats) (872). On the basis of the ability of 7-glutamohydrazide to stimulate protein synthesis in certain tumor tissues, it was proposed that the tumors may utilize the analogue in lieu of glu tamine (344)- Examples of inhibitory analogues which are not incorporated into proteins include 4- and 5-methyltryptophan (E. coli) (366, 367, 376). An analogue which acts as a competitive substrate and is incorporated into protein prevents the incorporation of only the corresponding natural amino acid; whereas, an inhibitory analogue which is not incorporated prevents protein synthesis, and this prevents incorporation of amino acids other than the corresponding natural amino acid (359, 874)- Incorporation of an amino acid analogue in place of the natural amino acid required by an organism usually results in a linear growth rate rather than in cessation of the usual exponential rate of growth and protein synthesis. Growth at a linear rate continues for a limited period, along with nucleic acid synthesis and other biosynthetic processes (362, 366- 368, 375, 876). In most instances a considerable number of constitutive enzymes are not synthesized in active forms, but in a number of cases modified enzymes (or proteins) which have biological activity are synthe sized. 7-Azatryptophan and 2-azatryptophan (tryptazan) permit doubling of the protein content of a tryptophanless strain of E. coli before growth is arrested, and, although a number of adaptive and constitutive enzymes are not synthesized in active forms under these conditions, either analogue permits the formation of increased activities of serine deaminase and 44 W. SHIVE AND C. G. SKINNER aspartic transcarbamylase (373, 375, 376). p-Fluorophenylalanine, in re placing phenylalanine for E. coli, prevents the formation of active forms of a number of induced enzymes, but the synthesis of an active β-galactosidase occurs with incorporation of p-fluorophenylalanine (362). The o- and ra-fluorophenylalanines similarly promote the formation of β-galactosidase (365), and all three fluorophenylalanines are effective in replacing phenylalanine in the induced formation of alkaline phosphatase in a phenylalanineless mutant of E. coli (377). In contrast, 2-thiophenealanine inhibits the formation of an active β-galactosidase (362). Ethionine is incorporated into the α-amylase of a methionine-requiring B. subtilis, and the crystalline modified enzyme has the same physico- chemical properties and enzyme activity as those of the normal protein (378). p-Fluorophenylalanine is incorporated into ovalbumin and lysozyme in minced hen oviduct (864) and into muscle protein, aldolase, glyceralde- hyde 3-phosphate dehydrogenase of rabbits (379). p-Fluorophenylalanine is incorporated effectively into exopenicillinase of B. cereus, which is formed at a depressed rate with impaired activity and a lower efficiency of reaction with specific antiexopenicillinase serum (863). p-Fluorophenylalanine and norleucine are incorporated into E. coli proteins which are not radically different molecular species and which are physicochemically similar to the protein normally synthesized. Each methionine incorporation site appears to have equal probability of ana logue substitution (380). Selection of phenylalanine over p-fluorophenylalanine no longer occurs after the two amino acids are incorporated into the internal (conversion) pool of yeast (381).

G. Activation and Transfer of Amino Acid Analogues to Ribonucleic Acid

Studies have been made concerning the specificity of a number of en zymes which convert specific amino acids to enzyme-bound amino acyl adenylates and transfer the amino acid to soluble ribonucleic acid, which in turn is involved in protein synthesis in ribosomes. Activation of an amino acid is experimentally determined either by formation of hydroxamate in the presence of hydroxylamine and adenosine triphosphate or by pyrophosphate exchange with adenosine triphosphate. Although activat ing enzymes are highly specific for one amino acid, a specific activating enzyme can utilize any of a large number of α-amino acyl adenylates and pyrophosphate for the formation of adenosine triphosphate (382). The pancreatic tryptophan-activating enzyme has been found to form adenyl ates with 5- and 6-fluorotryptophan, 7-azatryptophan, and 2-azatryptophan (tryptazan) ; and, although not activated, β-methyltryptophan, tryptophan hydroxamate, tryptamine, 5-hydroxytryptophan, 5-methyltryptophan, 1. AMINO ACID ANALOGUES 45

D-tryptophan, and 6-methyltryptôphan inhibit the activation of trypto phan (883). A tyrosine-activating enzyme from hog pancreas activates only 3-fluorotyrosine of a number of analogues, and tyrosine amide and tyramine inhibit the activation of tyrosine (384). α-Aminobutyric acid, threonine and its chloro analogue, a-amino-jS-chlorobutyric acid, and, less effectively, the chloro analogue of allothreonine undergo activation with the valyl-ribonucleic acid synthetase preparation from E. coli (298). Leucine, isoleucine, and alloisoleucine competitively inhibit the valine- activating enzyme (885). Of considerable interest is the fact that L-valyl adenylate is apparently formed by L-isoleucyl-ribonucleic acid synthetase, but it is not transferred to the soluble ribonucleic acid, D-Valine inhibits the pyrophosphate exchange reaction with both leucine and isoleucine in cell-free preparations from E. coli, and L-leucine or L-isoleucine appear to diminish the level of exchange of combinations with L-valine (386). A crude enzyme preparation from rat liver activating methionine does not activate ethionine and is not inhibited by ethionine [however, peptides of methi onine, particularly glycylmethionine, are utilized by the preparation, but evidence is not presented which indicates utilization before hydrolysis (387)]. This is the first case in which an analogue known to be incorporated into protein does not undergo the activation reaction of the corresponding amino acid. 2-Thiophenealanine undergoes activation but is not appreci ably effective in stimulation of net protein synthesis in E. coli (388).

H. End Product Control Mechanisms

The ability of metabolites to control their own biosynthesis was early recognized in growth inhibition studies. Demonstration of a close inter relationship of the biosynthesis of phenylalanine and tyrosine, now known to involve a common precursor, and the ability of tyrosine to inhibit the biosynthesis of phenylalanine led to the suggestion that tyrosine may serve in a mechanism of control of the biosynthesis of phenylalanine and related metabolites. It was also recognized that the implications of this type of biochemical control would be far-reaching (220). Culturing of an organism in the presence of a metabolite was frequently found to increase its sensitivity to a corresponding inhibitory analogue, so that the testing concentrations could be extended over a greater range (18, 171); for ex ample, successive transfers of a strain of E. coli in the presence of high concentrations of phenylalanine increased the sensitivity to growth in hibition by β-phenylserine more than tenfold without altering the amount of analogue necessary to inhibit the utilization of exogenous phenylalanine (389). Such effects were attributed to inhibition by the metabolite of its own biosynthesis. 46 W. SHIVE AND C. G. SKINNER

Isotopic competition methods have also demonstrated metabolite con trol of biosynthesis of the metabolite by the fact that exogenous isotopically labeled metabolites are preferentially incorporated, presumably by sup pression of endogenous biosynthesis (390-392). The first demonstration of end product inhibition of the formation of an intermediate in the biosynthetic pathway was the inhibitory effect of purines upon the synthesis of 5-amino-4-imidazolecarboxamide in E. coli (393). Subsequently, with the elucidation of metabolic pathways and the development of enzymic steps foTr these biosyntheses, more elaborate studies on end product control w ere possible. The mechanisms by which such biochemical controls are exerted include not only metabolite in hibition of enzymic activity, but also repression of the synthesis of enzymes involved in the biosynthesis. These inhibitions are most frequently exerted at the first stage in a biosynthetic sequence, but they may also occur in subsequent stages. Metabolite control of enzymic activity is sometimes referred to as "feedback" inhibition by analogy with the process of "nega tive feedback" in electrical systems. A considerable number of amino acids are now known to exert end product control upon steps in their own biosynthesis; for example, valine competitively inhibits the conversion of pyruvate to acetolactate in E. coli and Aerobacter aerogenes (394-, 395). Two distinct acetolactate-forming enzymes occur in A. aerogenes, only one of which is competitively inhibited by valine (396). Isoleucine competitively inhibits the deamination of threonine to α-ketobutyric acid and prevents overproduction of threonine deaminase (397). Histidine prevents the biosynthesis of iV-l-(5'-phosphoribosyl)-adenosine triphosphate from 5-phosphoribosyl-l-pyrophosphate and adenosine triphosphate in S. typhimurium and apparently in S. cere visiae (398-400). A histidine analogue, 2-thiazolealanine, similarly inhibits the biosynthesis of histidine (71). The ability of tryptophan analogues to inhibit the formation of anthranilic acid, and in certain organisms the conversion of anthranilic acid to indole, has been known for some time (see Section II, A, 3). Tryptophan itself inhibits the enzymic conversion of 5-phosphoskikimic acid and glutamine to anthranilic acid in A. aerogenes (401, 402), as well as condensation of anthranilic acid with 5-phospho- ribosylpyrophosphate in E. coli (71). In addition, analogues of tryptophan, 5-fluorotryptophan in the latter process and 5-methyltryptophan in the former reaction, have inhibitory activities like tryptophan. The formation of A^pyrroline-ô-carboxylic acid from glutamic acid by washed resting cells of an E. coli mutant is inhibited by proline (403). Cytidine-5'-phos phate and cytidine are competitive inhibitors of aspartic acid conversion to ureidosuccinic acid and presumably control the biosynthesis of pyrimidines in E. coli (404)- 1. AMINO ACID ANALOGUES 47

Repression of the synthesis of an enzyme essential for the biosynthesis of a metabolite may be caused by the metabolite itself. Methionine synthe tase, which converts homocysteine to methionine, is repressed in an E. coli mutant by methionine (405, 406). The inhibition of enzyme synthesis is not always specifically effected by the end products, since leucine, valine, phenylalanine, and threonine also inhibit production of methionine syn thetase. Tryptophan as well as indole, 7-methylindole, and 4- and 5-methyl tryptophan prevent the formation of tryptophan desmolase, which forms tryptophan from indole and serine (407). Ornithine transcarbamylase is repressed by arginine in E. coli, and release from repression permits the synthesis of many times the normal amount of enzyme (408-410). Other enzymes which are repressed in their synthesis by end products include acetylornithine transaminase (E. coli), repressed by arginine (411)) acetyl- ornithinase (E. coli) by arginine (412); arginosuccinase (E. coli) by arginine (413, 414)', and kidney arginine-glycine transamidinase (rats) by creatine (415). Five histidine biosynthetic enzymes were coordinately repressed so that histidine affected the level of each enzyme to the same extent (399, 416). A number of metabolites are synthesized from common precursors, and the ability of one of these end products to control the biosynthesis of a common intermediate would be detrimental to the supply of other end products. However, multiple metabolite control mechanisms appear to be involved in some biological systems. In cell-free preparations from E. coli, the formation of cyclic intermediates related to shikimic acid from phospho- enolpyruvate and erythrose-4-phosphate is partially inhibited by either phenylalanine or tyrosine, and a mixture of phenylalanine and tyrosine synergistically diminishes the activity of the preparation (417). E. coli also contains two different aspartokinases which are independently controlled both in activity and synthesis, one by lysine and the other by threonine. The lysine inhibition is noncompetitive, while that of threonine on the enzyme under its control is competitive (418). Such multiple control mechanisms apparently are of general significance.

I. Enzymic Transformations Involving Amino Acid Analogues A number of amino acid analogues undergo enzymic transformations analogous to the corresponding natural amino acid, while others inhibit various enzymic activities. Of over 650 known enzymes which were tabu lated in 1957 (419) about one-fourth were directly concerned with protein or amino acid interactions. It is beyond the scope of this chapter to itemize the specificity of all of the enzymic reactions which have been reported to occur with amino acids and related derivatives; however, representative examples, with special attention to the amino acid antagonists, are included. 48 W. SHIVE AND C. G. SKINNER

1. SPECIFIC BIOSYNTHETIC AND CLEAVAGE REACTIONS Some of the more interesting transformations involve biosynthetic processes; for example, β-hydroxyaspartic acid (an inhibitor of pyrimidine biosynthesis) is converted to the corresponding β-hydroxyanalogue of ureidosuccinate by aspartate transcarbamylase preparations from rat liver or Ehrlich ascites cells (420). Ethionine is converted with adenosine tri phosphate to £-adenosylethionine, the ethyl analogue of the active methyl- ating agent (£-adenosylmethionine), in yeast and rat liver preparations, and transethylation reactions with #-adenosylethionine have been re ported (421-423). α-Methyltryptophan, in inhibiting the conversion of tryptophan to kynurenine in a step in the biosynthesis of nicotinic acid, is converted to an analogue, presumably a-methylkynurenine (424, 425). The reaction of canavanine, a naturally occurring arginine antagonist, with fumaric acid to form canavanosuccinic acid is catalyzed by the enzyme arginosuccinase, which cleaves arginosuccinic acid reversibly to arginine and fumaric acid, and occurs in organisms crpable of synthesis of arginine from citrulline (426). It is of interest that canavanosuccinic acid inhibits the growth of L. arabinosus, and the inhibition is reversed by citrulline, arginine, or arginosuccinic acid (427). Canavanine is cleaved by an arginase preparation to canaline and urea (428), but inhibits arginine desimidase preparations from S. faecalis (429). An enzyme preparation from hog kidney was found to catalyze a transfer of the amidine moiety of canavanine to ornithine, forming arginine and canaline (480). α-Methylaspartic acid exerts an antimetabolite action in inhibiting the conversion of aspartic acid and citrulline to arginosuccinic acid by rat liver sections (4SI), but the antagonist has only a negligible effect on the glu tamic-pyruvic transaminase system (432). Although the growth of a particular E. coli, strain 4c, is not inhibited by 2-, 4-, 5-, or 7-methyltryptophan, these analogues do inhibit the increased production of nicotinamide activity caused by ornithine (433). The tryptophanase system of E. coli cleaves in increasing order of activity 4-, 6-, 5-, 2-, and 7-methyltryptophan to form the appropriate indole derivative (66). A study of the degradation of tryptophan and its benzene-substituted methyl and chloro derivatives by tryptophanase in acetone-dried E. coli and washed cell suspensions of E. coli suggested that the formation of the enzyme-substrate complex is facilitated by electron-attracting substances in the benzene ring of the tryptophan molecule (434)· A number of ana logues are known to inhibit this reaction, such as jS-3-indolepropionic acid, DL-a-amino-£-(3-indolyl)butyric acid (/S-methyltryptophan), 3-indoleacetic acid and even indole itself (485, 436). 5-Hydroxytryptophan is not de graded by tryptophanase of E. coli and rabbit liver; however, it does in- 1. AMINO ACID ANALOGUES 49 hibit degradation of tryptophan by the liver but not by the bacterial preparation (437).

2. TRANSAMINATION REACTIONS A summary concerning amino acids which participate in transamination reactions has recently been presented (438) ; among the analogues of amino acids occurring in proteins which have been reported to undergo trans amination are cyclohexanealanine (439, 440), cyclohexaneglycine (440), cysteic acid (440-444), cysteinesulfonic acid (44$), ethionine (447, 448, 440), 7-glutamyldimethylamide (iV,iV-dimethylglutamine) (449)) 7-gluta- mylmethylamide (449, 4&0), homocysteic acid (451), e-hydroxy-a-amino- caproic acid (489, 440), 7-methyleneglutamic acid (449), 7-methyl-glutamic acid (438, 449), 7-methylglutamine (449), norleucine (452, 449, 453), norvaline (447, 4&0, 454~456), phenylglycine (440), 0-phenylserine (457), 2-thiophenealanine (458, 440), and β-hydroxyaspartic acid (446, 4-59, 460). The transamination reaction with certain amino acid analogues is suf ficiently slow so that the transamination of the natural amino acid can be effectively inhibited in a competitive manner, e.g., 0-hydroxy-aspartic acid is a competitive inhibitor of the transamination of aspartic acid by aspartate-a-ketoglutarate transaminase (459, 460). Analogues which have been found to inhibit transamination reactions include α-methylglutamic acid (glutamic acid-pyruvic acid) (482) ; acetone- dicarboxylic acid, 2,3-diaminopropionic acid, and 2,3-diaminosuccinic acid (a-ketoglutaric acid-aspartic acid) (459, 460, 461); and isoserine, glycine, and ethanolamine (serine-pyruvic acid) (462). In addition, glutamate-oxalacetate transaminase is inhibited by β-fluorooxalacetate. A kinetic analysis of the data indicates that the inhibition is caused by competition with oxalacetate, and when 0-fluorooxalacetate is trans- aminated with aspartic acid, the resulting β-fluoroaspartic acid undergoes subsequent dehydrofluorination and deamination to yield oxalacetate (463).

3. DECARBOXYLATION REACTIONS Amino acid decarboxylases are usually sufficiently specific to be em ployed in the determination of the particular substrate. However, analogues are decarboxylated by these enzymes as follows: 0- and m-hydroxyphenylalanine (464)', 2,3-, 2,4-, 2,5-, 2,6-, and 3,5-dihydroxyphenylalanine (464~466); ery^ro-3-(3-hydroxyphenyl)serine, erythro- and £/ireo-3-(3,4- dihydroxyphenyl)serine (465, 467-469); and 2- and 6-methyl-3,4-dihydroxyphenylalanine (469) by mammalian 3,4-dihydroxyphenylalanine (dopa) decarboxylase; m-hydroxyphenylalanine (464, 470), 3-(3,4-di- 50 W. SHIVE AND C. G. SKINNER

hydroxyphenyl)serine (464), 2,3-, 2,4-, 2,5-, and 3,5-dihydroxyphenylalanine (slight activity by latter two) (465,466, 470), 4-hydroxy-3-methoxy- phenylalanine (465), 4-aminophenylalanine (471), 2- and 3-thiophenealanine (weakly active) (472), 3-aminotyrosine (472), and possibly phenylalanine (slight activity) (473) by tyrosine decarboxylase from S. faecalis; y-hydroxyarginine and canavanine by arginine decarboxylase from E. coli (474-476) ; δ-hydroxylysine slowly by lysine decarboxylase from B. cadaveris (477); and β-hydroxyglutamic acid slowly by glutamic decarboxylase of P. fischeri (478). Norvaline and α-aminobutyric acid are decarboxylated by P. vulgaris (479). Compounds which react slowly can inhibit the utilization ο f the normal substrate, e.g., 3,4-dihydroxyphenylalanine decarboxylation by dopa decarboxylase is inhibited by 3-(3-hydroxyphenyl)serine (480). Decar boxylase reactions can also be inhibited by compounds capable of reacting with carbonyl groups, such as hydrazides which inactivate the coenzyme, pyridoxal phosphate. Amino acid analogues frequently afford specific inhibitors, and one of the important decarboxylases for which inhibitors are of possible chemotherapeutic value is 3,4-dihydroxyphenylalanine (dopa) decarboxylase. One of the most widely studied analogues is the α-methyl derivative of 3,4-dihydroxyphenylalanine (481), which has been found to be inhibitory to dopa decarboxylase from pig kidney (482), to decrease blood pressure in dogs (483), and to be an effective inhibitor of dopa decarboxylase in the formation of serotonin, tryptamine, and tyramine from the corresponding amino acids in mammals (484, 485). It also lowers blood pressure in hypertensive patients and has transient sedative effects (484)- a-Methyl-3-hydroxyphenylalanine (482) and a-methyl-3,4-dihydroxyphenylalanine not only are decarboxylase inhibitors but in addition have an action which differentially affects serotonin and norepinephrine binding sites (486). Extensive variations in the structure of 3,4-dihydroxyphenylalanine have been studied (487). In a study of over 200 potential inhibitors of 3,4-dihy droxyphenyl- alanine(dopa) decarboxylase, the most effective competitive antagonists employed were 5-(3,4-dihydroxycinnamoyl)salicylic acid, 3-hydroxycinnamic acid, and caffeic acid (469). The most effective compounds contained the 3,4-dihydroxy or 3-hydroxycinnamoyl group attached to a substituent hydroxy, alkyoxy, alkyl, or aryl group, listed in order of decreasing activity. Mammalian dopa decarboxylase (hog kidney) is inhibited by 3,4'-dihydroxy- and 3,4,4'-trihydroxy-3'-carboxy-chalcones, l-(5-hydroxyindolyl- 3)-2- (3-carboxy-4-hydroxybenzoyl)ethylene, 2- (3,4-dihy droxyphenyl)- ethylamine, l-(3-hydroxyphenyl)-2-amino-l-propanol, norepinephrine, epinephrine, and 3-indoleacrylic acid. Studies on the first three com pounds indicate that the inhibitions are competitive. On the basis of 1. AMINO ACID ANALOGUES 51

the effects of 5-hydroxytryptophan or 3,4-dihydroxyphenylalanine upon the decarboxylation of the other, it has been suggested that only one enzymic site is involved and that this may have significance in the control of the production of the physiologically active amines (485, 488, 489). A number of analogues of tyrosine and 3,4-dihydroxyphenylalanine inhibit the decarboxylase activity and lower blood pressure in dogs (490), and certain pteridine compounds also inhibit dopa decarboxylase (491). 2,4-Dichlorophenylalanine and, less effectively, p-bromo- and p-chlorophenylalanine inhibit the decarboxylation of 3,4-dihydroxyphenylalanine or tyrosine by S. faecalis preparations, but the latter two analogues are more effective than the 2,4-dichloro derivative in inhibiting decarboxyla tion of phenylalanine (492). Tyrosine decarboxylase is somewhat inhibited by α-methylphenylalanine and a-methyltyrosine (424)· The amine products of decarboxylases and their analogues are frequently inhibitors of the enzymes. A considerable number of amines are known to inhibit dopa decarboxylase (498-495), and tyramine is a competitive inhibitor of tyrosine decarboxylase in S. faecalis (496). Phenylpyruvic acid, phenylacetic acid, and phenyllactic acid inhibit dopa decarboxylase of beef adrenal medulla, and the former two compounds as well as their p-hydroxy derivatives inhibit glutamic decarboxylase of rat brain (497, 498). It has been suggested that such inhibitions may con tribute to the disturbances in phenylpyruvic oligophrenia. Glutamic decarboxylase from E. coli is inhibited by α-methylglutamic acid, which appears to bind the enzyme and dissociate slowly, and slight inhibition by D-glutamic acid and methionine sulfoxide is observed (499). Mouse brain glutamic decarboxylase is inhibited to some extent by carbamylglutamic acid and several natural amino acids (500). Adipic, glutamic, and acetic acids also inhibit glutamic decarboxylase (501). 2,6-Diaminopimelic de carboxylase from A. aerogenes is inhibited by pyridine-2,6-dicarboxylic acid, a, a'-diamino-/3-hydroxypimelic acid, and glutamic acid (502). The decarboxylation of L-aspartic acid to alanine is inhibited by malonate or fumarate in Bacillus roseus fluorescens (508). D-Histidine and D-dihydroxyphenylalanine both inhibit decarboxylation of L-histidine, whereas, L-dopa is only about one-fourth as active as the D-form (504). Inhibitors of histidine decarboxylase include 2-thiophene alanine, 3-benzothiophenealanine, iV-sulfanilyl-4-aminobenzimidazole, and certain flavonoids (505-507).

4. OXIDATIVE REACTIONS Extensive studies of the rate of oxidation of 48 α-amino acids, including straight- and branched-chain and cyclic a-aminocarboxylic acids, aliphatic α-aminodicarboxylic acids, diaminocarboxylic acids, and amino acids con- 52 W. SHIVE AND C. G. SKINNER

taining some other substitueras, were carried out using D-amino acid oxidases from sheep kidney and N. crassa and L-amino acid oxidases from cobra venom and N. crassa. Biological actions of the enzymes were different both in optical specificity and many other properties. None of the enzymes attacked those analogues containing a tertiary a-carbon atom or β-substituted aspartic acids; 31 compounds were acted upon by kidney D-amino acid oxidase, 19 by the cobra venom L-amino acid oxidase, 33 by the N. crassa L-amino acid oxidase, and 23 by the N. crassa D-amino acid oxidase (508). L-Amino acid oxidase from rattlesnake venom catalyzed the oxidation of 22 amino acid analogues; the theoretical yield of oxygen was observed in most instances, but for 2-furanalanine, 4-aminophenylalanine, and crotylglycine more than the theoretical amount of oxygen was liberated (472). D-Amino acid oxidase from kidney oxidized the same derivatives at a somewhat slower rate (472). Venom amino acid dehy drogenase catalyzed the oxidation of sulfoxides of methionine, ethionine, and a number of other $-alkyl derivatives (509). iV-Methyl-a-amino acids (510, 511) are oxidatively demethylaminated by these oxidases. The action of D-amino acid oxidase on a number of amino acids is in hibited by amides and peptides of both the corresponding and different amino acids. For example, the action of kidney D-amino acid oxidase on leucine is inhibited by leucineamide, leucylglycine, leucylglycylglycine, glycylleucine, and some iV-alkyl derivatives (512). a-Methylmethionine blocks D-amino acid oxidase action on phenylalanine (122), and DL-(O-, m- and p-)chlorophenylalanines act as substrates for the D-amino acid oxidase of kidney and L-amino acid oxidase of venom (518). The inhibitory activities on amino acid oxidases of a number of optical antipodes for the corresponding natural substrate, as well as inhibitions by other naturally occurring amino acids of the same or different configuration, have been demonstrated in several instances; for example, D-aspartic acid inhibits the oxidation of L-aspartic acid in S. fiexneri (268), and D-amino acid oxidase acting on D-alanine is inhibited by D-lysine, but not by the L-isomer (514). The keto acid of methionine inhibits the oxidative deamination of D-methionine in E. coli (515). 5- and 7-Hydroxytryptophan inhibit com petitively the enzymatic oxidation of tryptophan (516) by the tryptophan peroxidase-oxidase system from a tryptophan-adapted Pseudomonas.

5. MISCELLANEOUS REACTIONS

D-Asparagine competitively inhibits deamination of L-asparagine by asparaginase (517). A number of α-iV-alkyl derivatives of L-asparagine which are not hydrolyzed by asparaginase from either rat or guinea pig 1. AMINO ACID ANALOGUES 53 livers or Pseudomonas fluorescens do inhibit the enzyme acting on L-as paragine, and the inhibitions are reported to be competitive (518). α-Methylglutamine inhibits glutamotransferase and glutamine cleav age (424). The decomposition of L-histidine by histidinase from rat liver is inhibited by a number of analogues, including D-histidine, histamine, and imidazole (519). 7-Hydroxyarginine serves as a substrate for arginase (4-76), and the arginase reaction has been found to be inhibited by α-amino acids of the L-configuration but not by D-amino acids (520)) α-aminovaleric acid is particularly inhibitory (521), and inhibitions by ornithine and lysine are reversed competitively (520, 522). Benzoyl-L-aspartic acid inhibits the activity of esterase from pig liver (523), and the optical specificity of acylase I from hog kidney is appreciably less for the iV-trifluoroacetyl amino acids (524) than for the corresponding acetyl or chloroacetyl derivatives (525). The action of carboxypeptidase on synthetic substrates has been the subject of several studies; for example, the action of pancreatic carboxy peptidase on carbobenzoxyglycylphenylalanine is inhibited by a number of analogues (527), and its action on peptides containing aromatic amino acid antagonists has also been described (528). Cysteic acid amide is hydrolyzed slowly relative to leucine amide by leucine aminopeptidase (526). Serine dehydrase is inhibited by 2,3-diaminopropionic acid (462).

J. Amino Acid Analogues and Chemotherapy

The number of amino acid analogues which have been found to possess significant chemotherapeutic activity is less than might have been antici pated. The inability to maintain sufficiently high blood levels, which would be essential for inhibitory effects with most of the competitive antagonists, would normally eliminate this type of analogue as a chemotherapeutic agent. However, a few competitive amino acid analogues have been re ported to possess some chemotherapeutic activity as subsequently indi cated; but the most effective agents have been discovered as antibiotics, and the modes of action of some of these compounds give new insight into types of amino acid antagonists which may have chemotherapeutic activity. The antibiotics O-diazoacetyl-L-serine (azaserine) and 6-diazo-5-oxo-L- norleucine (DON) exert their effects upon purine biosynthesis by spe cifically binding in place of glutamine and denaturing the enzyme which catalyzes the conversion of formylglycinamide ribotide to formylglycinamidine ribotide (see Section II, D, 2). A glutamine analogue, #-carbamoyl-L- cysteine, which contains a reactive thioester linkage rather than the chemically reactive diazo grouping as in azaserine and DON, is similarly 54 W. SHIVE AND C. G. SKINNER

a noncompetitive glutamine antagonist with antitumor activity (609). Azaserine and DON also exert inhibitory effects upon other enzymic reac tions of glutamine, such as the formation of phosphoribosylamine from 5-phosphoribosyl-l-pyrophosphate (529), the amination of xanthosine-5'- phosphate to guanosine-5'-phosphate (530), the amidation of deamido- diphosphopyridine nucleotide to diphosphopyridine nucleotide (531), and in the conversion of uridine nucleotides to cytidine nucleotides (291). Competitive reversals of inhibitions, or competitive delays of inactivation of the enzyme systems, by glutamine are usually observed. Another potential approach to chemotherapy with amino acid antag onists involves the inhibition of a growth factor which is essential for the pathogenic agent, but which is not essential in appreciable amounts for the host. Thus, the antibiotic, D-4-amino-3-isoxazolidone (cycloserine, oxamycin), inhibits the synthesis and utilization of D-alanine for the synthesis of a uridine nucleotide derivative essential for cell wall forma tion (see Section II, B, 3). However, the antibiotic effects of cycloserine may not necessarily be concerned only with D-alanine metabolism, since its effects on growth of certain organisms are not reversed by D-alanine. It is of interest to note that the corresponding leucine analogue of cyclo serine, 4-amino-5-isopropyl-3-isoxazolidone, inhibits the utilization of D- but not L-leucine in L. dextranicum (532). The "carrier molecule" concept has also been applied to the design of amino acid analogues of chemotherapeutic importance. A chemically re active group is introduced into a metabolite analogue which serves as a carrier to place the reactive group within the cell, so that it may interact with cellular constituents in such a manner as to interfere specifically with nucleic acid or protein metabolism of malignant cells (533). Such a reactive group is the nitrogen mustard grouping which has been introduced into the structure of several amino acids, including phenylalanine. The L-isomer of p-(bis-2-chloroethyl)aminophenylalanine is an effective cytotoxic agent (534, 535) ; however, there is no evidence that these alkylating agents are specific antagonists of the corresponding amino acids. The structural similarity of chloramphenicol to phenylalanine which slightly reverses its toxicity in E. coli and L. casei led to the proposal that it was a noncompetitive antagonist of phenylalanine (536). Protein syn thesis beyond assimilation of the free amino acids appears to be inhibited by chloramphenicol in bacterial but not in mammalian systems (537-539). Chloramphenicol does exert inhibitory effects upon metabolism of aro matic amino acids, e.g., the oxidation and deamination of phenylalanine, tyrosine, and phenylserine in Pseudomonas aeruginosa (540). Puromycin, which structurally resembles the terminal grouping of aminoacyl-sRNA, inhibits the transfer of leucine from the soluble ribo nucleic acid into protein (541). 1. AMINO ACID ANALOGUES 55

A variety of antimetabolites, including natural amino acids and their analogues, have been found to affect virus infections, but in most instances the activity has only been demonstrated in rigidly controlled test systems and not under normal physiological conditions. In only a few instances have metabolite reversal studies been carried out; but, in general, where a study was made, the anticipated natural metabolite reversed the inhibition. Natural amino acids have been demonstrated to posses+s antiviral ac tivity for the indicated virus as follows: L-leucine for T2r (E. coli phage) (542)) L-isoleucine (the D-form was ineffective) for tobacco mosaic (548)', L-serine for T2r+ (542) and Theiler's GD VII (mouse encephalomyelitis) (544)) threonine, glutamic acid, and cysteine for tobacco mosaic (545)', L-lysine for Theiler's GD VII (544, 546-549), influenza (PR8 and Lee), mumps (550), and tobacco mosaic (545); ornithine for influenza (PR8 and Lee) (550) and Theiler's GD VII (549); arginine for influenza (PR8 and Lee) and mumps (550) ; histidine for Theiler's GD VII (547, 549); and tryptophan for Theiler's GD VII (547, 549). A number of other amino acids, most of which had previously been demonstrated to inhibit microbial growth, have been found to be in hibitory for the indicated virus as follows: canavanine for Lee influenza (551); 0-, m-, and p-fluorophenylalanine for Theiler's GD VII (544)) p-fluorophenylalanine for polio virus (552, 553), influenza (554), fowl plague (555), western equine encephalomyelitis (556), and the foot-and- mouth virus (557); 2-thiophenealanine is a phenylalanine antagonist for Theiler's GD VII (544, 549), vaccinia (558), Lansing poliovirus (559), and psittacosis (560); 2-furanalanine and 1-naphthalenealanine for Theiler's GD VII (544)) L-2/ireo-phenylserine for influenza A (561); 5-methyl tryptophan for T2 and T4 bacteriophage (562, 563) ; 6-methyltryptophan for Lansing poliovirus (564, 565) and psittacosis (560); 7-azatyrptophan for bacteriophage (376); ethionine for Lansing poliovirus (559, 566- 568), influenza (PR8) (569, 570), and psittacosis (560); 3-amino-3-carboxypropanesulfonamide for T2 bacteriophage (146)) methionine sulfoxide for T2r bacteriophage (542); methoxinine for vaccinia (571) and in fluenza (PR8) (570) ; methionine sulfoximine for Theiler's GD VII (548) and Lansing poliovirus (572) methallylglycine (but not allylglycine) for Theiler's GD VII (548); norleucine for Theiler's GD VII (548) and to bacco mosaic (545) ; α-aminoadipic acid and α-ketoadipic acid for Theiler's GD VII (547, 544)) cysteic acid for Lansing poliovirus (559)) amino methanesulfonic acid for bacteriophage (101), vaccinia (571), and in fluenza (573)) l-amino-2-phenylethanesulfonic acid for influenza (573), bronchitis (574), and Lansing poliovirus (559)) 1-aminobutanesulfonic acid for bronchitis and influenza (574)) 1-aminophenylmethanesulfonic acid for vaccina (571) and influenza (PR8) (573)) and 1-amino-p-methoxy- phenylmethanesulfonic acid for influenza (573, 575). 56 W. SHIVE AND C. G. SKINNER

Excess dietary methionine causes decreased susceptibility of mice to Lansing poliomyelitis virus, and 6-methyltryptophan, which alone at high levels protects completely a significant number of mice, augments this effect of methionine (564). In contrast, 6-methyltryptophan is inef fective against Lansing poliomyelitis virus in tissue culture (559). Feeding mice, which were infected with semliki forest virus, either D-, L-, or DL- ethionine increased the rate of survival when they were inoculated intra- peritoneally, but not when they were inoculated intracerebrally. The ethionine-treated mice which survived the infection were usually immune to the virus, and methionine did not reverse the ethionine effect. Tryptophan which is an essential cofactor for absorption of T4 bacterio phage in E. coli Β is antagonized by indole which thus inhibits phage infec tion of E. coli cells (576-579). Because of the ability of metabolite analogues to inhibit cell division, many antimetabolites have been observed to affect the growth of rapidly proliferating systems, such as fetal growth and cancerous tissue. In certain instances these effects have been utilized in chemotherapy. Inhibitory effects on mitotic activity in various stages are observed with a number of amino acid analogues (580); for example, the following amino acid antagonists inhibit mitosis in the indicated systems, and in several in stances, the toxicities are reversed by the corresponding metabolite: azaserine in mouse sarcoma 180, various mouse ascites tumors, human carcinoma and mouse tissue culture (581-588); γ-glutamohydrazide in mouse sarcoma 180, human carcinoma and mouse tissue culture (588); o-, m- and p-fluorophenylalanine in mouse sarcoma 180, mouse embryo skin, and chick heart cells (584, 585) ; and p-fluorophenylalanine in mouse sarcoma T241 and mouse heart cells (584). 2-Thiophenealanine in Lewis sarcoma T241 produces highly polyploid cells in the shrinking tumor (586). An excess of either arginine, histidine, or lysine depresses mitotic activity in chick fibroblast cultures, and prophases accumulate in lysine- deficient medium (587); aspartic acid also inhibits mitosis (588). Reproduction in rats can be controlled with the aid of antimetabolites, such as azaserine, DON, and ethionine. Azaserine causes complete litter destruction after implantation without damaging effects to the mother, as evidenced by subsequently normal pregnancies and litters (589). It affects only the fetus (not the ovary, placenta, or pituitary) and is more effective in the presence of 6-mercaptopurine (590). Comparable effects were observed with 6-diazo-5-oxo-L-norleucine (DON), and repeated litter destruction gave no evidence of a cumulative toxicity (591). Ethionine causes a marked resorption of embryo and placenta, and the toxicity can be partially negated by methionine (592, 592a). Some studies have also been carried out using antimetabolites, e.g., azaserine, for therapeutic 1. AMINO ACID ANALOGUES 57 abortion in humans; and, especially in conjunction with 6-mercaptopurine, it was found to be effective in an early stage of pregnancy (593). A number of amino acid analogues have been studied in various cancer chemotherapy screening programs. Some of the more active derivatives include antibiotics, such as azaserine and DON (Volume II, Chapter 17), and synthetic analogues containing alkylating agents, such as the nitrogen mustard derivatives, using various amino acids as "carrier" molecules (Volume II, Chapter 23). r Other types of amino acid analogues which have been studied include ethionine (3), which has been reported to inhibit growth of various tumors (e.g. Jensen and T241 sarcoma) in rats, but its effectiveness varied with strain and sex of the host animals (594-598). Ethionine augments the effect of a folic acid antagonist on mouse mammary carcinoma (599), and it is incorporated into Ehrlich ascites protein (600). Recent studies have suggested that the inhibitory action of ethionine may also involve an indirect effect on the utilization of amino acids other than methionine (601). A study of oral ethionine therapy on six patients with advanced neoplastic disease, while being maintained on a low methionine diet, indi cated a number of toxic manifestations but no significant antitumor effects (602). Among a group of cystine-cysteine analogues (603-605) which were tested for antileukemic effects, one of the most effective was selenocystine (606). The antitumor properties of both 2- and 3-thiophenealanine have been studied; for example, 2-thiophenealanine showed little difference in toxicity for heart fibroblasts and cells of sarcoma T241 (458), but at a high dosage it did exert an inhibitory effect upon sarcoma T241 in mice (586, 595). Both phenylalanine and phenylpyruvic acid reversed 2-thio phenealanine toxicity, yet sarcoma T241 is practically unable to trans- aminate the keto acid (458, 586, 607). Feeding rats 3-thiophenealanine caused a loss in weight and a negative nitrogen balance, but gave a marked reduction in Jensen sarcoma transplants as well as antibody formation, and phenylalanine reversed this toxicity (608). The inhibitory effects of both 3-thiophenealanine and 2-furanalanine for sarcoma T241 were re versed by phenylalanine (595). In a study of 50 amino acid analogues against heart fibroblast cells and sarcoma T241, highly selective toxicities were rare. Of a group of halogen- ated phenylalanines, only the fluoro derivatives were inhibitory and reversed by phenylalanine; ethionine was fairly selective but was not reversed by methionine; and the toxicities of 6-methyl and 5-fluorotryptophan were competitively reversed by tryptophan (595). Other amino acids which have been found to be inhibitory include /S-carbamoyl-L-cysteine and O-carbazylserine for RC mammary adeno- 58 W. SHIVE AND C. G. SKINNER carcinoma (609). Of a number of sulfur-containing amino acids, including #-alkylcysteines, which were studied as antitumor agents, >S-ethyl-L- cysteine, £-(2-chloroethyl)-D-cysteine, and djenkolic acid were partially inhibitory for growth of certain fowl sarcomas in eggs (610). Several com pounds have been reported to reduce the growth of Novikof hepatoma in the rat as follows: 1-aminocyclopentane-l-carboxylic acid, 1-aminocyclo- hexane-l-carboxylic acid and its iV-methyl and iV-ethyl analogue, 6- hydroxynorleucine, alanylglycine, α-methylalanine, iV-methylaminophenyl- alanine, and 2-amino-5-(methylthio)-valeric and -caproic acid (611). 1-Aminocyclopentane-l-carboxylic acid (612) as well as some related compounds have also been found to reduce tumor growth in Walker rat carcinoma and ascites tumor (613). It is apparent from various studies that effective chemotherapy with amino acid analogues will require a more specific action than has been attained in the available analogues. Attainment of such a goal does seem possible as more knowledge is gained concerning the nature of the reac tions to be inhibited and the type of control essential for effective treat ment of particular diseases. It should be possible to design analogues which noncompetitively denature specific enzymes without appreciably affecting other enzymic processes.

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