CHAPTER 1
Amino-acids
By B. W. BYCROFT
The presentation of this chapter conforms, in general, to the pattern established in last year's Report. New amino-acids with novel structures continue to be isolated from natural sources and the chemistry of amino-acids still attracts considerable attention from all branches of the subject. The emphasis remains on a-amino-acids and it is only for these that a comprehensive coverage has been attempted. Biochemical aspects have been covered only where they relate directly to the chemistry.
1 Naturally Occurring Amino-acids
A. Introduction. — The literature on non-protein naturally occurring amino-acids has been reviewed up to the end of 1968 in a valuable article which also discusses their probable origin and function. Speculation concerning the origin of D-amino-acids continues: the observation that for amino-acids containing more than one centre of chirality inversion of configuration occurs only at the α-centre has led to the rule of α-epimerisation. Amino-acids have been isolated during the year which conform to the rule, together with one apparent exception (see below). Two possible explanations to account for the origin of D-amino-acids, both of which accommodate most of the known information relating to D-amino-acids and are in accord with the above described rule, have been proposed.
B. New Free Occurring Amino-acids. — The continuing usefulness of physical methods in structural elucidation is apparent, and several new amino-acids have been characterised during the year. Those whose structure has been confirmed by synthesis are included in the list of newly synthesised amino-acids in section 2. Especially noteworthy is the characterisation by spectroscopic methods of the novel amino-acid (1), which is produced by Claviceps species inhibited with ethionine and is probably an intermediate in the biosynthesis of lysergic acid. Several other amino-acids have also been isolated from fungi or micro-organisms. The mushrooms Morchella esculenta and related species produce cis-3-amino-L-proline. β-Methylene-L-norvaline has been obtained from Lactarius helvus,s and the closely related allenic amino-acid (2) occurs in Amanita solitaria? A new sulphur-containing amino-acid (3) from Xeromus subtomentosus has been reported, but this is probably a modified dipeptide derived from glutamic acid and cysteine. The antimetabolic antibiotic (4), produced by an unidentified Streptomyces species, has been shown to be active against Gram-positive organisms.
A number of new plant amino-acids have been described. The phyto-chemical similarities between Aesculus and Blighia species have been extended: exo-3,4-methanoproline and cis-α-(carboxycyclopropyl)glycine (5) occur in Aesculus parviflora, while the trans-isomei of (5) is found in Blighia sapida. The structures of three new acetylenic amino-acids (6), (7), and (8) from the seeds of Euphoria longan have been determined and the structure of the pyrimidine amino-acid isolated from pea seedlings has been revised to (9). Selenomethylselenocysteine has been shown to be the principal selenium-containing amino-acid in Astragalus bisulcatus.
C. New Amino-acids from Peptide Hydrolysates. — Hydrolysis of the antibiotic monamycin affords the novel piperidazine amino-acid (10), the structure and absolute configuration of which have been confirmed by a direct correlation with L-ornithine, as well as the structurally related D-amino-acids (11) and (12) (opposite absolute configuration). In addition, D-isoleucine, an exception to the rule of α-epimerisation, has also been isolated from the acid hydrolysate. In contrast, the acid hydrolysates of the antibiotics aspartocin and enduracidin yield L-threo- and D -erythro]-αβ-diaminobutyric acid and enduracididine (13) and ollo-enduracididine (14) respectively; both the D-amino-acids are in accord with the rule. The structure, but not the stereochemistry, of the basic amino-acid stendomycidine (15) has been determined, and the close similarity between it and the previously isolated guanidine amino-acids capreo-mycidine and viomycidine is noteworthy. Further details on the chemistry of viomycidine have been published. Hydrolysis of diatom cell walls affords cis-4-3,4,trans-3,4-dihydroxy-L-proline, the structure of which has been confirmed by an X-ray crystallographic analysis (see section 3). N-(2-Hydroxyethyl) alanine was isolated from the alkali hydrolysate of a phospholipid obtained from rumen protozoa.
D. Occurrence of Known Amino-acids. — β-Cyanoalanine, the neurotoxin, is the product of cyanide fixation and an intermediate in asparagine biosynthesis in a number of Lathyrus and Vicia species. Canaline (L-γ-aminoxy-α-aminobutyric acid) has been obtained from the unripe seeds of Astragalus suricus and trans-3- hydroxy-L-proline from the seeds of Delonix regia. Nα-Acetyl-L-arginine has been reported to be present in cattle brain.
2 Chemical Synthesis and Resolution of Amino-acids
A. Introduction. — The majority of new syntheses, with notable exceptions, were performed by variations of well established routes or involved elaboration of available amino-acids. Therefore, only those syntheses which have points of particular interest will be discussed and the remainder merely mentioned or incorporated into the list of newly synthesised amino-acids.
B. Protein Amino-acids. — New syntheses have been reported for DL-lysine, DL-tryptophan, DL-cystine, d- and L-cysteine, and D-phenylalanine, and the advances in asymmetric synthesis of α-amino-acids reviewed. Both forms of alanine have been obtained from oxaloacetic acid by an asymmetric transamination, and an elegant asymmetric synthesis (Scheme 1) of optically pure L-aspartic acid has been reported. The detailed studies, first reported last year, on the resolution of glutamic acid and alanine by fractional crystallisation have been extended.
C. Other Naturally Occurring Amino-acids. — Interest in L-dopa (3,4-dihydroxyphenylalanine) continues: syntheses from L-tyrosine using crystalline β-tyrosinase and from N-blocked L-tyrosine derivatives employing micro-organisms have been published. DL-Indospicine, the heptatotoxic amino-acid, first reported last year, and all the four isomers of tricholomic acid (16) have been synthesised. Synthetic DL-α-amino-δ -(guanylureido)-n-valeric acid is identical with gigartinine in all respects except optical activity, and the structures of β-methylene-norvaline, the sulphur-containing amino-acid (3), and N-2,3-dihydroxybenzoyl-serine have all been confirmed by synthesis. Reduction of (17) followed by hydrolysis and methylation affords mainly DL-threo-N,β-dimethyl-leucine, indicating that the predominant geometrical isomer of (17) is that shown. A new synthesis of L-αβ-diaminopropionic acid has been described.
D. α-Alkyl-α-amino-acids. — A brief preliminary description of a new route to α-amino-acid orthoesters and hence a-amino-acids has appeared: the three-step synthesis (Scheme 2) involves conversion of a nitrile to an imino-ester (18), chlorination of the latter to an N-chloroimidate (19), and alkoxide treatment of (19) to form the α-amino orthoester. The synthesis was unsuccessful when α-disubstituted acetonitriles were employed, and it is suggested that the reaction proceeds through the intermediacy of (20). A convenient synthesis of the novel racemic amino-acid (21) from cyclo-heptatrienylium tetrafluoroborate and dimethyl formamidomalonate has been reported. Further syntheses of 3- and 4-substituted α-glutamic and 4-substituted-α-aminoadipic acid derivatives have also been described."
The reaction of (22) with hydrobromic acid followed by silver nitrate affords (23), which can be resolved by stereospecific enzymic deacylation to give (S)-α-amino-δ-nitrovaleric acid, an important intermediate in the synthesis of ferrichrome. Reduction of (24), prepared by cyanomethylation of either the D- or L- form of αβ-diaminopropionic acid, yields the D- and L- forms of 4-azalysine.' This method has also been used to make [6-14C]-L-4-azalysine.
Interest continues in substituted phenylalanines because of their potential biological activity, and a number of new derivatives have been prepared by standard methods. An extensive study of the application of the Meerwein reaction for a general synthesis (Scheme 3) of substituted phenylalanines has been reported. Twenty-one substituted aniline derivatives were investigated and it was observed that for those with electron-withdrawing groups in para, and, particularly, ortho positions, yields in the ammonolysis step were low. A similar observation was made in an attempted synthesis of pentafluorophenylalanine via the Meerwein arylation route: the intermediate α-bromo-acid afforded trans-4-amino-2,3,5,6-tetrafluorocinnamic acid rather than the phenylalanine on treatment with ammonia. Pentafluorophenylalanine was ultimately synthesised by the azlactone method.
E. α-Dialkyl-α-amino-acids. — The oxazole (25), readily prepared from the corresponding azlactone and benzoyl chloride, rearranges on warming in pyridine to the azlactone (26), which on reduction gives a mixture of erythro- and threo-N-benzoyl-2-methyl-3-phenylserine esters. Separation of the isomers was effected by chromatography. The advantage of this method over a similar synthesis, reported last year, is that it provides access to those amino-acids whose aromatic moiety is more readily available as the carboxylic acid chloride than as a Grignard reagent.
An interesting conversion of (S)-2-methyl-3-phenylpropionic acid to (R)-α-methylphenylalanine has been described. The key step in this sequence was the photolysis of the azidoformate (27). This generates a nitrene which then undergoes a stereospecific intramolecular insertion to give (28). A number of bicyclic α-amino-acids of the type (29) have been synthesised using a Diels-Alder addition of cyclopentadiene to substituted α-nitrocinnamates.
F. α-Amino-acids with Aliphatic Hydroxy-groups in the Side-chain. — A new synthesis of either the threo- or the erythro- form of β-hydroxy-DL-aspartic acid is shown in Scheme 4. The starting materials, threo- and erythro-β-furylserine, are readily prepared from furfural and glycine and the oxidation step proceeds in tolerable yield. Resolution of erythro-β- hydroxyaspartic acid has been achieved via the N-benzyl derivative. Amination of erythro-β-methoxy-α-bromohexiLnoic acid proceeds with retention of configuration when ammonia is used, but treatment with sodium azide followed by reduction results in inversion. This reaction was employed for the synthesis of threo- and erythro-β-hydroxylysines. The erythro- but not the threo- form of α-amino-β-hydroxy-γ-benzyloxybutyric acid can be resolved enzymatically. Resolution of the latter can be accomplished using the method of Vogler.
The preparation of α-methylserines was discussed in the previous section.
G. N-Substituted-α-amino-acids. — Interest continues on N-hydroxy-α-amino-acids, and further work describes the synthesis of a number of optically active forms, not previously reported, by the action of hydro-xylamine on α-bromo-esters. A number of N-β-hydroxyethyl-α -amino-acids have been synthesised by a modified Strecker procedure. This syn thesis is of interest since the first naturally occurring derivative of this type has been reported. A more convenient reduction of 2-pyrrolecarboxylic acid to 3,4-dehydroproline is possible using a mixture of hypophosphorous and hydriodic acids as the reducing agent.
H. α-Amino-acids Containing Sulphur or Selenium. — Alkylation of homo-cysteine thiolactone with a primary alkyl halide in the presence of sodium methoxide provides a more facile route to S-alkyl-DL-homocysteines, since it avoids the use of sodium in liquid ammonia necessary in the usual route. L-Selenomethionine (31) and L-selenoethionine (32) have been prepared from L-α-amino-γ-bromobutyric acid (30) using the method outlined in Scheme 5.
I. A List of α-Amino-acids which have been Synthesised for the First Time. —
J. Labelled Amino-acids. — The Strecker synthesis still continues to be an important route to labelled amino-acids: synthesis of [1-14C]-DL-glutamic acid, [1-14C] -DL-ornithine, and [1-14C]-DL-arginine using this method and starting with 3-cyanopropionaldehyde has been reported. A study of the reaction of α-ketocarboxylic acids with radioactive cyanide has shown that decarboxylation of the intermediate α cyano-α-hydroxyacetic acid is much more rapid than hydrolysis. The reaction can therefore be employed for labelling, and [1-14C] -DL-alanine and [1-14C]-DL-glutamic acid have been synthesised from pyruvic and a-oxoglutaric acids respectively. An alternative synthesis of [1-14C]-L-asparagine and [4-14C]-L-aspartic acid from [4-C]-β-L-cyanoalanine has been described, and the preparation and enzymic resolution of o-hydroxy-[2-14C]-DL-phenylalanine, starting from ethyl-[2-14C]-acetamidocyanoacetate published. Syntheses of [4-14C]-DL-tyrosine, [β-14C, 15N, 14CH3]-DL-N-α-methyltryptophan, and [6-14C]-L-4-azalysine have also been reported.
Further studies on enzymic transamination have resulted in an improved synthesis of [15N]-L-aspartic acid and [15N]-L-glutamic acid. Efficient utilisation of [15N]ammonium chloride is claimed, allowing preparation of any desired NN ratio. [αββ-2H 3]-L-Glutamic acid has been obtained by incubating L-glutamic acid with deuterium oxide in the presence of pig-heart glutamate-oxaloacetate aminotransferase and catalytic amounts of pyridoxal-5'-phosphate and oxaloacetic acid. However, under similar conditions with glutamate-pyruvate aminotransferase in the presence of pyruvic acid, [α-2H]-L-glutamic acid is produced. Chemical synthesis of glutamic acid specifically labelled in the α and β positions with either deuterium or tritium has also been reported. The absolute configuration of enzymatically tritiated glycine has been determined using D-amino-acid oxidase. The enantiomer prepared by incubating [α-3H2]glycine with serine hydroxymethylase in the absence of formaldehyde is (R)[2-3H]glycine. A synthesis of [2,3,3,4,4, 5,5-2H7]-DL-lysine, but without experimental details, has been reported, and the preparations of tritium-labelled N-methylated lysine derivatives and tritiated phenylalanine have been published.
3 Physical and Stereochemical Studies of Ammo-acids
A. Determination of Absolute Configuration. — Perhaps the most interesting development in this field has been the reported application of n.m.r. spectroscopy for the determination of absolute configuration. The spectra of the enantiomers of a given α-amino-acid methyl ester have been shown to differ appreciably in optically active 2,2, 2-trifluorophenylethanol. This spectral non-equivalence is ascribed to strong solvent-solute interactions resulting in the formation of transitory diastereomeric solvates. It has been demonstrated that the method is widely applicable, not only to the determination of absolute configuration but also to the determination of optical purity. It is claimed that the method is more versatile than o.r.d. and c.d. in correlating absolute configuration. The decision as to whether or not this claim is justified must await wider application of the technique.
Chemical correlation with L-ornithine has established that the novel amino-acid (10) possesses the configuration (S) at C-3, and chemical considerations, such as facile lactone formation (Scheme 6), and n.m.r. data have been used to assign the (S) configuration at C-5. The identification and configurational assignment of D-isoleucine, isolated from mona-mycin, was achieved by oxidation to the corresponding enantiomer of α-methylbutyraldehyde. N.m.r. studies have enabled an assignment of the relative and hence the absolute configuration of L-cis -3-aminoproline (33) by a comparison of the coupling constant (J2,3) with those of cis- and trans-3 -hydroxyproline. The β-hydroxyasparagine isolated from human urine has been identified as erythro-β-hydroxy-L-asparagine. Hydrolysis of the antibiotic actinoidine yields p -hydroxyphenylglycine and 3-chloro-4-hydroxyphenylglycine (34), both of which have been shown to have the configuration (R).
The determination of the absolute configuration of stereospecifically tritiated glycine was reported during the discussion on labelled amino-acids in section 2.
B. Crystal Structures of Amino-acids. — (See also Chapter 2, part II, section 1.) The crystal structures of L-cysteic acid, L-valine hydrochloride, L-ornithine hydrochloride, DL-acetyl-leucine-N-methyl-amide, and L-azetidine-2-carboxylic acid have been described and the cell dimensions and space groups of L-tyrosine and L-tryptophan have been published. A comparison of the β-synthesis and heavy-atom synthesis in the structure determination of L-arginine mono-hydrobromide monohydrate has also been reported.
