Aliphatic Nitro- and Amino-monocarboxylic Acids and Related Compounds

Chapter 15

Aliphatic Nitro- and Amino-monocarboxylic Acids and Related Compounds H. D. LAW 1. Nitromonocarboxylic acids and their derivatives Culture filtrates from Aspergillus wentii Wehmer produce unusual growthregulating effects when sprayed on pea plants (P. W. Brian et al., Nature, 1965, 207: 998). The active component in the culture has been identified as 1-amino-2-nitrocyclopentane-l-carboxylic acid (abbreviated to ANCPA) (B. F. Burrows, S. D. Mills and W. B. Turner, Chem. Comm., 1965, 75 ; Burrows and Turner, J. chem. Soc., C, 1966, 255) and so it could clearly be discussed equally satisfactorily in the second part of this chapter. Its biological activity is perhaps a feature of its c~-amino-acid constitution since 1-aminocyclopentane carboxylic acid and 1-aminocyclohexane-l-carboxylic acid act in a similar way (L. J. Machlin, R. S. Gordon and F. Puchal, Nature, 1963, 198: 87). However, ANCPA is dealt with here because naturally occurring nitro-carboxylic acids are extremely rare and because much of the interest in the chemistry of ANCPA derives from the presence of the nitro group. The only naturally occurring nitro-carboxylic acid mentioned in Rodd's CCC, 2nd Edn., is also obtained from A. wentii. Hydrolysis of ANCPA with boiling water gives a mixture of two carboxylic acids and ammonia. Either carboxylic acid regenerates ANCPA when treated with ammonia:

NO2

NO2 major component

NO2 minor component

More extensive degradation occurs when ANCPA is hydrolysed with hot mineral acid. The products in this case are ammonia, carbon dioxide and [313]

314

15

NITRO- AND AMINO-MONOCARBOXYLICACIDS

glutaric acid. A reverse aldol reaction, the key step in this degradation, followed by the well-documented conversion of a nitro-group to a hydroxamic acid and subsequent "transhydroxylamination", accounts for this somewhat surprising result. The hydroxy- and unsaturated nitro-acids mentioned above decompose in a similar way: ANCPA 'heat aq'HClr ~

OH CO2H NO2

O

CO2H

I OH

~ ~.

LO ~ ~CO2H NO2

HOw "N / ~CO2H t OH

O CO2H

O

~"

I OH

HO2

CO2H

J OH

ANCPA has been synthesised by treating cyclopent-l-ene carboxylic acid with dinitrogen tetroxide in the presence of iodine and ammonolysing the resultant iodocarboxylic acid (T. E. Stevens and W. D. Emmons, J. Amer. chem. Soc., 1958, 80: 338).

2. Aliphatic aminocarboxylic acids and their derivatives The chemistry of amino acids and peptides has been reviewed annually over the last few years (H. D. Law, Ann. Repts. Chem. Soc., (London), 1965, 389; 1966, 517; 1967, 451; P. M. Hardy, ibid., 1968B, 509; 1969B, 491). In addition, the Chemical Society has now started a series of Specialist Periodical Reports which provides a more detailed year-by-year account of developments in this area (Chem. Soc. Specialist Periodical Rept.. "Amino acids, Peptides and Proteins", ed. G. T. Young, Vol. 1, 1969; Vol. 2, 1970; Vol. 3, 1971). Other reviews are mentioned in context below. Since the appearance of Rodd's CCC, 2nd Edn., peptides have been under intensive investigation, as indeed, they have for the last twenty years. Appropriate weighting is given in the following accounts.

(a) Syntbesis, stereocbemistry and resolution of amino acids A new general synthesis of c~-amino acids which starts from N,N-bis(trimethylsilyl)-glycine ethyl ester has been described; otherwise, most recent synthetic work has been concerned with the application of old and tried

2

315

SYNTHESIS OF AMINO ACIDS

routes to the synthesis of new amino acids. Asymmetric processes have come in for considerable attention, but synthesis of the racemic amino acid followed by its resolution is still the established pattern. The observation that the tritium labelling of optically active a-amino acids by gaseous tritium (Wilzbach procedure) causes racemization must be borne in mind when the synthesis of tritiated amino acids is undertaken (J. L. Garnett et al., Chem. Comm., 1969, 323).

(i) Synthesis of amino acids (1) By replacing halogen by amino groups. The photochlorination of isoleucine under acidic conditions - so-called "C-derivitization" (J. Kollonitsch, A. Rosegay and G. Doldouros, J. Amer. chem. Soc., 1964, 86: 1857) - opens a new route to the stereospecific synthesis of 3-methylprolines from isoleucine and alloisoleucine by intramolecular aminolysis (Kollonitsch, A. N. Scott and Doldouros, ibid., 1966, 88: 3624): CH2~C,~ t~~ e.~

~,,,,,'H

/ H3N

=CO2H

C,,''Me C12 ~ H 2 - - [ "~H ~.~.' CH~ c ......H J / h Cl H3N CO2H

OHO

,,,,,,,Me [..~ ~"~H ' Z'" N CO2H H

(2) By reduction of keto acid derivatives or keto acids. A degree of asymmetric synthesis is achieved in the catalytic reduction of c~-keto acids in the presence of D- or L-a-methylbenzylamine. For example, pyruvic acid hydrogenated in the presence of ~.-a-methylbenzylamine yields 91% L-alanine (R. G. Hiskey and R. C. Northrop, ibid., 1961, 83: 4798). Similarly, oximes prepared from laevo-menthyl esters of a-keto acids are reduced to some extent stereoselectively (K. Matsumoto and K. Harada, J. org. Chem., 1966, 31: 1956; 1967, 32. 1790, 1794; Nature, 1966, 212: 1571). The steric course of the 0xime reaction can be explained in terms of the conformation of the adsorbed molecule. Repulsion between the C - N and C - O will ensure that the molecule adopts the conformation shown. It is adsorbed with its less bulky side on the catalyst: R\ N [[ R

./C~

~ H2 C

/O._

...

...... ,"

.-

R,/C ......H 0

.... ........ ~

316

NITRO- AND AMINO-MONOCARBOXYLICACIDS

15

Benzylidene derivatives of L-menthyl-esters of c~-keto acids (Matsumoto and Harada, loc. cit.) and c~-methylbenzylamides of acetamidoacrylic acids(J. C. Sheehan and R. E. Chandler, J. Amer. chem. Soc., 1961, 83: 4795) show less optical discrimination upon reduction. Phenylglycine has been used in stereoselective reductive transaminations to affect the preparation of 40-60% optically-pure amino acids from c~-keto acids (Harada, J. org. Chem., 1967, 32: 1790). The condensation product between N-aminoanabasine and ethyl pyruvate can be reduced to c~-alanine with about 40% optical purity with zinc-hydrochloric acid (A. N. Kost, R. S. Sagitullin and M. A. Yurovskaja, Chem. and Ind., 1966, 1496)-

U

"C N H Me- C" CO2Et

(3) From carbonyl compounds by the Strecker synthesis. The Bucherer modification of the Strecker synthesis is often difficult to apply directly in the synthesis of c~,c~-dialkyl amino acids because of the resistance of the hydantoins to hydrolysis. This problem can be avoided by tosylating the hydantoins. The resulting 3-tosyl derivatives can be hydrolysed with alkali to the corresponding hydantoic acids which give the amino acids on treatment with dilute acid (K. Hiroi, K. Achiwa and S. Yamanda, Chem. pharm. Bull., Japan, 1968, 16: 444):

R\ /R H~/C'~ 0 OC~NH

R\ /R TosCl HI~//C'(~0

(i) OH~H20

OC

(ii) He

. N- Tos

R\ /R HN/C"cO2H H| [ NH" Tos ~ § CO"

R~ / R HaN/C~co2 H

s-Amino acids which possess allenic side chains have been prepared by the Strecker route, but the method is only satisfactory when the allenic aldehydes are fully substituted in the 2-position (D. K. Black and S. R. Landor, J. chem. Soc., C, 1968, 281). (4) By the condensation of aldehydes with compounds containing an active methylene group. 2-Benzylimidazol-4-one is an easily accessible

2

317

S Y N T H E S I S OF A M I N O ACIDS

starting material (H. Lehr, S. Karlan and M. W. Goldberg, J. Amer. chem. Soc., 1953, 75: 3640) for the preparation of fl-methylleucine (H. Kotake, T. Saito and K. Okubo, Tetrahedron Letters, 1968, 24: 2015). Presumably this ring system could be condensed with other carbonyl compounds to produce a range of amino acids: NH [[ Ph "CH 2 9 C" O E t

Gly- OEt

N--C=O # [ ~- Ph " C H 2 " C [ N--CH 2 H

9 (i) Pr ~. CO- Me (ii) HCI/EtOH

N--C--O Ph'CH2"C~/

H2 - P t O 2

[

N~C=CMePr i H2

PhC.

C

N--C=O #

Me~/Pri

[

.N?CH--CHMePr

' i

I NH 2 " CH 9 CO2H

(5) Displacement of halogen with compounds containing an active methylene group. In the presence of sodium bis(trimethylsi!yl)amide, N,N-bis(trimethylsilyl)glycine ethyl ester forms a sodio-derivative which reacts readily with alkyl halides. The trimethylsilyl groups are easily cleaved from the products with aqueous acid. This appears to constitute an entirely new general method for the synthesis of a-amino acids (K. Ruhlmann and G. Kuhrt, Angew. Chem., intern. Edn., 1968, 7: 809): (Me3Si)2 N . CH 2 . CO2R1

+ R2 . X (MeaSi)2N. CHR2. CO2RI

HO H20

9 9 CHR2 9 CO2 R1 H3N

The Schiff's base complex formed from glycine, salicylaldehyde and copper(II) can be alkylated with various alkyl halides to give Ca-substituted compounds. Yields vary, but a-alanine, and phenylalanine have been prepared effectively in this way (A. Nakahara, S. Nishikawa and J. Mitani, Bull. chem. Soc., Japan, 1967, 40: 2212). The photochemical alkylation of glycine has also been studied and, in this way, glycine residues in dipeptides have been reacted with isobutene, but-l-ene or toluene in the presence of acetone to give leucine, norleucine or phenylalanine residues respectively (D. Elad and J. Sperling, Chem. Comm., 1968, 655).

318

NITRO- AND AMINO-MONOCARBOXYLIC ACIDS

15

(6) S6renson synthesis. Formamido-malonate seems to be the reagent of choice in this approach. Thus, allenic amino acids have been prepared from the appropriate allenic bromides by displacement with diethyl formamidomalonate followed by deformylation and decarboxylation (D. K. Black and S. R. Landor, J. chem. Soc., C, 1968, 283). In the synthesis of hypoglycin A by this approach the decarboxylation is stereospecific. Only the (2S.4S) and (2R:4R) enantiomers are formed. Models show that the process must be under thermodynamic control (idem, ibid., p. 288): CH2=C=CH - CH2Br +

~

[ NH-CHO

2 ~ CH2=C=CH "CH2

CH212 (CO2Et)2 Cu-Zn complex NH" CHO

H2C =T',..~ H2C-~ L ~ - - - C H 2 - C(CO2Et)2 - - - - * NH- CHO

CH 2 "~H'CO2H NH 2

N-Benzyloxycarbonylaminomalonate has been described (D. A. Cox, A. W. Johnson and A. B. Mauger, J. chem. Soc., 1964, 5024). (7) Prebiotic synthesis. Work on the synthesis of amino acids under pseudo-primeval conditions continues to produce suggestive results. For example, when heated with water, hydrogen cyanide oligomers, such as diaminomaleonitrile, form peptides which apparently contain up to eleven different types of amino acids (R. E. Moser and C. N. Matthews, Experientia, 1968, 24: 658; Moser, A. R. Claggett and Matthews, Tetrahedron Letters, 1968, 1599). Observations which might have a bearing on the predominance of L-amino acids in nature include the following-D-tyrosine in aqueous solution is less stable than L-tyrosine under bombardment with polarised /3-particles from 9~ (A. S. Goray, Nature, 1968, 219: 338); L-amino acids are more effective than D-amino acids for stabilising nucleic acid helices (G. Manecke and G. Gfinzel, Naturwiss., 1968, 55: 84).

(ii) Stereocbemistry and resolution An improved dichrograph for studies of amino acids and proteins has been described (Y. P. Myer and L. H. MacDonald, J. Amer. chem. Soc., 1967, 89: 7142). Aqueous solutions of L-a-amino acids generally exhibit a positive Cotton effect with a peak at ~< 216 nm (J. C. Craig and S. K. Roy, Tetrahedron, 1965, 2 1 : 3 9 1 and references therein). However, various derivatives of amino acids are more useful for stereochemical determinations and dichroism studies of N-thiobenzoyl (G. C. Barrett, J. chem. Soc., 1965, 2825; C, 1966, 1771), N-dimedonyl (P. Crabb~ and B. Halpern, Chem. and Ind., 1965, 346; Crabb6, Halpern and E. Santos, Tetrahedron, 1968, 24:

2

STEREOCHEMISTRY

AND R E S O L U T I O N

319

4315), N-(2-pyridyl-N-oxide) (V. Tortorella and G. Bettani, Gazz., 1968, 98: 316), N-neopentylidene (Z. Badr et al., J. chem. Soc., C, 1966, 2047) and N-acryloyl (N. Sakota, J. chem. Soc., Japan, 1968, 89: 425) amino acids have all been reported, as well as studies of N-acryloyl amino acid osmate esters (Sakota, loc. cit.) and of various metal complexes of amino acids (C. J. Hawkins and P. J. Lawson, Chem. Comm., 1968, 177 - c o b a l t complexes). N.m.r. spectra at 220 M. Herz have been reported for all of the protein amino acids (B. Bak et al., J. mol. Spectroscopy, 1968, 26: 78). This technique is very useful for distinguishing diastereoisomers of amino acids which possess a second asymmetric carbon atom, for example, 4-substituted prolines (R. H. Andreatta, V. Nair and A. V. Robertson, Austral. J. Chem., 1967, 20: 2701). Enantiomeric c~-amino acid methyl esters can be distinguished on the basis of their n.m.r, spectra measured in (R)-(-)-2,2,2trifluoro-l-phenylethanol. Short-lived solvates, which involve an association between the ester link and the aromatic ring, are responsible for the different signals (W. H. Pirkle and S. D. Beare, J. Amer. chem. Soc., 1969, 91: 5150): H H ~ ~ ;""

CH3

.~~

." . ."

..---

0

//

C----C,~ / - ] - - _ _ \ OCH3

O\C....," F3C

H

The absolute configurations of c~-alkyl-c~-amino acids have been determined chemically by reference to the correlation established between (+)-isovaline and o(--)-quinic acid (K. Achiwa and S. Yamada, Chem. and pharm. Bull., Tokyo, 1966, 14: 537; S. Terashima, Achiwa and Yamada, ibid., pp. 572, 579): C_O2H =

Et~-C_-~NH 2 =

Me (+)-Isovaline

D L-Alanine benzenesulphonate may be resolved by recrystallization from 97% aqueous acetone if the mixture is seeded with one of the enantiomers. The other enantiomer can be obtained from the mother liquors (I. Chibata et al., Experientia, 1968, 24: 638). Other amino acids have been resolved by

320

NITRO- AND AMINO-MONOCARBOXYLIC

ACIDS

15

stereoselective precipitation with metal complexes; for example, the L-histidine complex precipitates first from a solution of DL-histidine and D~.-[Co(EDTA)] ~ ion in acidified aqueous ethanol (R. D. Gillard, P. R. Mitchell and H. L. Roberts, Nature, 1968, 217: 949). Racemic amino acids can also be resolved conveniently by the recrystallization of optically-active salts of their N-ortho-nitrophenylsulphenyl derivatives. The N-ortho-nitrophenylsulphenyl group can be cleaved subsequently by acidolysis (Von J. Konig, L. Nov~ik and J. Rudinger, Naturwiss., 1965, 52: 453). In an interesting and potentially important experiment tert-butyl N-trifluoroacetyl-(+)-alaninate was resolved by g.l.c, over Chromosorb W coated with cyclohexyl N-trifluoroacetyl-r.-valyl-~.-valinate (E. Gil-Av and B. Feibush, Tetrahedron Letters, 1967, 3345).

(b) Reactions of amino acids Studies of amino acid reactions have been mainly concerned with the roles of amino acids as constituents of peptides and proteins. Hence, many of the reactions and derivatives discussed in this section are of interest principally from the points of view of peptide degradation or synthesis.

(i) Reactions of the amino group (1) Reaction with ninhydrin and comparable reagents. Kinetic studies have established that the rate-determining step in the reaction between ninhydrin and amino acids is nucleophilic attack by the amine moiety, but, in the case of cysteine, competitive attack by the thiol group might be important (M. Friedman and C. W. Sigel, Biochemistry, 1966, 5: 478). Additional support for the Schiff's base, decarboxylation, hydrolysis, Schiff's base, enolisation mechanism for the reaction o f ninhydrin with a-amino acids (D. J. McCaldin, Chem. Reviews, 1960, 60: 39) is provided by studies of the reaction of the following compound with a-amino acids (H. Wittmann, W. Dreveny and E. Ziegler, Monatsh., 1968, 99: 1205, 1543): O

OH H

(2) Reaction with aldehydes. The thermal decarboxylation of Schiff's bases produced by the reaction of amino acids with ketones has been studied

2

R E A C T I O N S OF AMINO ACIDS

321

in some detail including cases in which the amine equivalent to the ketone is produced by transamination (A. F. A1-Sayyab and A. Lawson, J. chem. Soc., C, 1968, 406). Somewhat related, c~-pyrrolo-derivatives have been made by the reaction of amino acids with 2,5-diethoxytetrahydrofuran in the presence of sodium acetate in acetic acid (J. Gloede et al., Coll. Czech. chem. Comm., 1968, 33: 1307): N - C H R " CO2H

(3) Arylation. 2,4,6-Trinitrobenzene sulphonic acid reacts readily with c~-amino acids to make the corresponding 2,4,6-trinitrophenylamino acids. These derivatives can be used for the spectrophotometric estimation of amino acids, for example with the autoanalyser (J. Harmeyer, H.-P. Sallmann and L. Ayoub, J. Chromatog., 1968, 32: 258). 2,4-Dinitrophenyl derivatives of amino acids are important, of course, as the end products of Sanger's 2,4-dinitrofluorobenzene method for peptide N-terminal residue identification. It has long been appreciated that 2,4-dinitrophenyl-amino acids are unstable in light. Now it has been shown that the photolytic products are 2-substituted 6~ 1-oxides and 4-nitro-2-nitrosoaniline; the relative proportions of each depend upon the pH (D. J. Neadle and R. J. Pollitt, J. chem. Soc., C, 1967, 1764). N-(5-Nitro-6-methyl-2-pyridyl) derivatives of most of the protein amino acids and the reactions of fluoronitropyridines in general with amino acids have been described (Z. Talik and B. Brekiesz-Lewandowska, Roczniki Chem., 1967, 41: 2095; T. Talik and Z. Talik, Bull. Acad. polon. Sci., Ser. chim., 1968, 16: 13). 3,5-Dinitropyridyl derivatives of peptides are of interest and may prove valuable in that the hydrolytic cleavage of the terminal peptide bond in these compounds is facilitated by the pyridine nitrogen. Relatively mild conditions (6 M HCI, 30% formic acid, 60 ~ 8 - 1 0 h) suffice to liberate the 3,5-dinitropyridyl derivative of~the N-terminal amino acid. From the point of view of spectroscopic properties and stability, these derivatives are just as suitable as the classical 2,4-dinitrophenyl derivatives for terminal residue indentification. The derivatives are formed by the reaction of the amino acid or peptide with 2-chloro-3,5-dinitropyridine under mildly basic conditions; amino acids can be regenerated from their 3,5-dinitropyridyl derivatives by treatment with 2 M NaOH at 100 ~ (A. Signor, A. Previero and M. Terbojevich, Nature, 1965, 205: 596; Signor and L. Biondi, Ricerca Sci., 1964, 4(II-A): 165).

322

NITRO- AND AMINO-MONOCARBOXYLIC ACIDS

15

Mononitropyridyl peptides are cleaved even more readily in dilute acid and 2-fluoro-3-nitropyridine and 2-fluoro-5-nitropyridine are also advocated for terminal residue studies (Signor et al., J. org. Chem., 1967, 32: 1135; Europ. J. Biochem., 1969, 7: 328). With 0.1-0.2 M HC1 containing 20% formic acid at 100 ~ the 3-nitropyridyl-terminal residue is released completely in 30 min, whereas the 5-nitro-compound requires 24 h. Chromatographic systems for the direct identification of these derivatives have been described (E. Celon, Biondi and E. Bordignon, J. Chromatog., 1968, 35: 47). 7-Chloro-4-nitro-2,1,3-benzo-oxo-diazole forms derivatives with amino acids and peptides which fluoresce in visible light. Since the reagent is more stable and soluble in aqueous solution than dansyl chloride (see below) it may be useful for terminal residue studies (P. B. Ghosh and M. W. Whitehouse, Biochem. J., 1968, 108: 155). (4) Acylation and sulpbonylation. The acylation of amino acids is discussed in some detail in the section on peptide synthesis. Other points of interest are discussed in this section. a-Amino acids with primary amino groups give tars when treated with oxalyl chloride, whereas proline forms a crystalline anhydride.

OC~cJO 0

The tars are probably formed via oxazolones (W. R. Hearn and R. E. Worthington, J. org. Chem., 1967, 32: 4072). Selective acylation of the co-amino group of a,co-diamino acids can be achieved at pH 11 by the use of active esters, e.g. p-nitrophenyl acetate (J. Leclerc and L. Benoiton, Canad. J. Chem., 1968, 46: 1047). a,~-Diaminobutyric acid and a,3-diaminopropionic acid cannot be acylated selectively in this way. Toluene-p-sulphonyl (tosyl) derivatives of amino acids are valuable as N-protected forms in peptide synthesis. 5-Dimethylaminonaphthalene-1sulphonyl amino acids (dansyl amino acids) are now finding extensive use in terminal residue studies. The dansyl peptide is formed first by the reaction of the peptide with the sulphonyl chloride at mildly alkaline pH; after total hydrolysis, the terminal residue is obtained and identified as its N-dansyl derivative since the sulphonamide group is, of course, relatively resistant to hydrolysis. This approach is attractive because the fluorescence of the dansyl amino acids makes it approximately one hundred times more sensitive than

2

323

REACTIONS OF AMINO ACIDS

the dinitrofluorobenzene method (W. R. Gray and B. S. Hartley, Biochem. J., 1963, 89: 379; L. B. Smittie and Hartley, ibid., 1966, 101: 232. See also especially Gray in "Methods in Enzymology", Vol. II, ed. C. W. Hirs, Academic Press, New York, 1967). Peptides and amino acids can be recovered from the dansyl derivatives by sodium-liquid ammonia reduction at - 7 0 ~ (Z. Tamura, T. Tanimura and H. Yoshida, Chem. and pharm. Bull., Japan, t967, 15: 252).

(ii) Reactions of the carboxyl group (1) Amino acid amides. Amides of the N-benzyloxycarbonyl and N-phthalylamino acids can be dehydrated readily to the corresponding nitriles (B. Liberek, A. Nowicka and J. Szrek, Roczniki Chem., 1965, 39. 369), whereas tosyl derivatives, if the sulphonamide nitrogen bears a replaceable hydrogen atom, do not react in a clear-cut manner. N-Formylvalinamide does give the nitrile on treatment with toluene-p-sulphonylchloride, but perhaps an indirect mechanism is involved: For example, N-formyl-DL-valylglycinamide also gives a nitrile upon dehydration but this reaction proceeds via an intermediate isocyano compound (F. Sakiyama and B. Witkop, J. org. Chem., 1965, 30: 1905). pr i

pr i

I

I

OCHNHCHCONHCH2CONH 2

) CN" CHCONHCH2CONH 2

(4.68/~) Pr i

pr i

I

pr i

I

C H ~ C = N C H 2 CONH2

I

I

N~c/O

H

~

HCONHCHC~N

I

I

O\c/CH2

II

I

OCHNHCHCONHCH2CN (4.47/~)

NH

N-Benzyloxycarbonyl-asparagine is dehydrated readily with phosgene to give N-benzyloxycarbonyl-~-cyano-L-alanine (M. Wilchek, S. Ariely and A. Patchornik, ibid., 1968, 33: 1258).

(iii) Reactions involving both amino and carboxyl groups (1) Pbenyltbiobydantoins and related compounds. Hydantoins are important in degradative peptide chemistry as the end products of the Edman degradation, which remains the most important method for determining N-terminal residue sequences. An apparatus, the "protein sequenator", has been described in which the condensation of the peptide with isothiocyanate and the cleavage of the thiazolinones corresponding to the terminal

324

NITRO- AND AMINO-MONOCARBOXYLIC ACIDS

15

amino acid residues are carried out automatically (P. Edman and G. Begg, Europ. J. Biochem., 1967, 1: 80). The estimation of phenylthiohydantoins can be performed by an isotope dilution technique by using phenyl [3ss]isothiocyanate in the condensation step (G. L. Callewaert and C. A. Vernon, Biochem. J., 1968, 107: 728). Alternatively, mass spectrometry can be used for the identification of phenylthiohydantoins. When methyl isothiocyanate is used in the condensation step the addition of l SN enriched methyl-thiohydantoins to the thiohydantoins produced by the cleavage has facilitated their estimation by mass spectrometric measurements of the 14N/~SN ratio in the M e peak (F. F. Richards et al., Nature, 1969, 221: 1244). Fluoroescein isocyanates can be used to convert amino acids into fluorescent thiohydantoin derivatives. The fluorescence of these derivatives facilitates their detection so that the reagent might prove useful in Edman-type degradations (H. Maeda and H. Kawauchi, Biochem. biophys. Res. Comm., 1968, 31: 188). (2) 2,5-Oxazolidinediones (Leucb's anhydrides) and related compounds. These compounds are also very important cyclic derivatives of amino acids because of their significance in peptide chemistry. They are now used in the synthesis of controlled-sequence peptides as well as in the synthesis of polyamino acids (see p. 344).

(iv) Reactions of amino acids with metals In recent years there has been a spate of papers concerned with metal derivatives of amino acids, peptides and proteins (R. D. Gillard and S. H. Laurie, Chemical Specialist Periodical Report, Amino acids, Peptides and Proteins, Vol. I, 1968, p. 262, list in their own authoritative review, eleven other reviews of this subject which appeared in the period 1966-1968). It is confirmed that the complexes are generally of the form: XnM/Oxco

\l

. CHR H2N'

and that cis and trans arrangements of the ligands are possible. Thus, solid cis and trans forms of the [Cu(~.-Ala)2 ] complex have been isolated despite the fact that the isomers are labile (A. Dijkstra, Acta Cryst., 1966, 20. 588; R. D. Gillard et al., Chem. Comm., 1966, 155). Geometrical isomerism is even more complicated when three bidentate amino acids are arranged about a central octahedral metal ion. For a series of such isomers, relative stabilities in water seem approximately the same (Gillard, lnorg. Chim. Acta Rev., 1967, 1: 69).

2

INDIVIDUALa-AMINOACIDS

325

Some terdentate bindings are found when the amino acids involved have functional side-chains, as in the molybdenum-cysteine (J. R. Knox and C. K. Prout, Chem. Comm., 1968, 1227) and cobalt-histidine (Harding and Long, J. chem. Soc., A, 1968, 2554) complexes:

~----NH ~ O ~ j /O--CO OCH~c/ NfC~ I H2 H2 /CH H An empirical octant rule has been proposed for distinguishing bidentate and terdentate binding on the basis of circular dichroism measurements (K. M. Wellman et al., J. Amer. chem. Soc., 1968, 90: 805) and c.d. has also been used to relate crystal structure to solution structure (Gillard and Laurie, Chem. Comm., 1969, 489). Mixed complexes, in which more than one .type of amino acid (B. Sarkar, M. Bersohn, Y. Wigfield and T. 12. Chiang, Canad. J. Biochem., 1968, 46: 595) or different enantiomers (Gillard et al., J. chem. Soc., A, 1966, 201) participate have been studied and an advanced theory of the formation of mixed complexes in solution has been developed (D. D. Perrin and V. S. Sharma, ibid., 1968, 446 and refs. therein). The nature of the binding in metal peptide complexes has been extensively studied (H. C. Freeman, Adv. prot. Chem., 1967, 22: 258). Complexes of this type are of considerable potential importance because of the way in which they can promote peptide hydrolysis (J. P. Collman and D. A. Buckingham, J. Amer. chem. Soc., 1963, 85: 3039).

(c) Individual a-amino acids Methods available for the determination of a-amino acids are discussed in the book by S. Blackburn (Amino Acid Determination, Arnold, London, 1968). Ion-exchange chromatography is used routinely to separate the amino acid components of protein hydrolysates and biological fluids. The individual amino acids are usually estimated colorimetrically by the ninhydrin reaction. G.l.c. has also been studied intensively as a potential analytical tool for

326

NITRO- AND AMINO-MONOCARBOXYLIC ACIDS

15

amino acids and it is now possible to separate all of the protein amino acids in one experiment. It is necessary to convert the amino acids to volatile derivatives before the separation and Notrifluoroacetylamino acid n-butyl esters seem promising from this point of view (C. W. Gehrke, R. W. Zumwalt and L. L. Wall, J. Chromatog., 1968, 37. 398). The potential advantages of the g.l.c, method are speed and sensitivity; the major drawback is the necessity to convert the amino acids quantitatively to volatile derivatives before chromatography, although this drawback is reputedly not insurmountable. The separation of amino acids by t.1. chromatography has been reviewed (G. Patakai, Chromatog. Reviews, 1967, 9" 23). Two animal proteins, elastin and resilin, contain unusual amino acids which effectively cross-link the peptide chains. Lysinonorleucine [Ne-(5 amino-5-carboxypentyl)-lysine] (C. Franzblau et al., Biochem. biophys. Res. Comm., 1965, 21. 575), and the pyridinium derivatives, desmosine [I, R 1 = (CH2)2 "CH(NH2)'CO2 H, R 2 = (CH 2 )3 "CH(NH2 )'CH(NH2 )'CO2 H, R 3 = H, R 4 = (CH2)4"CH(NH2)'CO2H] and isodesmosine [I, R 1 = (CH2) 2" CH(NH2)'CO2H, R 2 = H, R 3 = (CH2)3"CH(NH2)'CO2H, R 4 = (CH2)4" CH(NH2)'CO2H], have been obtained from elastin. Young elastin, treated with alkali and reduced with borohydride, yields the open-chain compounds merodesmosine [II, R l = (CH2)2 "CH(NH2)-CO2 H, R 2 = (CH 2 )4-CH(NH 2)CO2 H] which supports the hypothesis that the elastin fibres are formed by an oxidative cross-linking process from lysine residues in the soluble precursor protein (J. Thomas et al., Nature, 1963, 200. 651; 1966, 209: 399). N*-(2-Amino-2-carboxyethyl)-ornithine has also been obtained from alkali-treated proteins (K. Ziegler, I. Melchert and C. Lurken, ibid., 1967, 214: 404). The cross-linking in resilin seems to be through tyrosine derivatives (S. S. Lehrer and G. D. Fasman, Biochemistry, 1967, 6. 757):

RI(""~

R1

R3 R (I)

R 1 "cH

I CH2 IR2 (II)

CH2

I

H2NCH

I

CO2H

CH2

I

HCNH2

I

C02H

Plants and microorganisms in particular are the sources of a large range of unusual amino acids (L. Fowden, Ann. Rev. Biochem., 1964, 33. 173)(see Table 1). Many of' these occur in the free state but others are bound, for example, as components of peptides; several unusual amino acids occur as

2

327

I N D I V I D U A L (X-AMINO ACIDS Table I SOME UNUSUAL NATURAL a-AMINO ACIDS

Structure

Source

Reference

L-2-amino-4,4-dichlorobutyric acid

Streptomyces spp.

R. R. Herr et al., Biochemistry, 1967, 6: 165.

L-2-amino-3-formylpent-3-enoic acid L-2-amino-3-hydroxymethylpent-3-enoic acid

Bankera fuligineoalba

R. P. Doyle and B. Levenberg, Biochemistry, 1968, 7: 2457.

2-amino-4-methylhexanoic acid 2-amino-4-methylhex-4-enoic acid 2-amino-6-hydroxy-4-methylhex-4-enoic acid

A esculus californica

L. A. Fowden and A. Smith, Phytochemistry, 1968, 7: 809; D. S. Millington and R. C. Sheppard, ibid., p. 1027.

L-(-)-2-amino-5-methylhex-4-enoic acid

Leuco c ort inarius bulbiger

2-amino-4-carboxy-hex-4-enoic acid

tulips

N:methyl-O-methyl-L-serine

Mycobacterium lra tyricu m and M. avium

O-ethyl(and propyl and butyl)-L-homoserine

Corynebacterium etbanolaminopbilum

(+)-S-(1-trans-propenyl)-L-cysteine S-oxide

Allium cepa

/~-acetamido-L-alanine

Acacia armata

2-amino-3-methylaminopropionic acid

Cycas circinalis

G. Dardenne and J. Casimir, Phytochemistry, 1968, 7: 1401. L. Fowden, Biochem. J., 1966, 98: 57. E. Vilkas, A. Rojas and E. Lederer, Compt. rend., 1965, 261: 4258. Y. Murooka and T. Harada, Agric. and biol. Chem., Japan, 1967, 31: 1035. J. F. Carson, R. E. Lundlin and T. M. Lukes, J. org. Chem., 1966, 31: 1634. A. S. Senevitarne and L. Fowden, Phytochemistry, 1968, 7: 1039. A. Vega and E. A. Bell, Phytochemistry, 1967, 6: 759. Herr et al., loc. cit.

L-2-amino-3-dimethylaminopropionic acid

Streptomyces spp.

L-~-N-acetyl-~/~-diaminopropionic acid

Acacia spp.

Senevitarne and Fowden, loc. cir.

328

15

N I T R O - AND A M I N O - M O N O C A R B O X Y L I C A C I D S Table 1 (continued)

Structure

Source

Reference

L-~-amino-~,-(guanylureido)valeric acid

red algae

K. I to and Y. Hashimoto, Nature, 1966, 211 : 417.

L-~-amino-e-amidinocaproic acid

Indigo fera spicata

M. P. H egarty and A. W. Pound, Nature, 1968, 217: 354.

e-N-methyl-L-lysine

histones

K. Hempel, H. W. Lauge and L. Birkhofer, Naturwiss., 1968, 5 5 : 3 7 and refs. therein.

elastin

C. Franzblau et al., Biochim. biophys. Res. Comm., 1965, 21: 575.

e-N, N-dimethyl-L-lysine e-N, IV,N-trime thyl-L-lysine

Ne-(5-amino-5-carboxypentyl)-L-lysine

3-hydroxyphenylglycine 3,5-dihydroxyphenylglycine

L. P. Miiller and H. R. Schiitte, Z. Naturforsch., 1968, 23b: 659.

4-hydroxymethyloL-phenylalanine

N. H. Sloane and S. C. Smith, Biochim. biophys. Acta, 1968, 158: 394.

3-hydroxy-L-kynurenine (III)

butterflies

T. Tokuyama et al., J. Amer. chem. Soc., 1967, 89: 1017.

(+)-(2S,4S)-~-(methylenecyclopropyl)-alanine (hypoglycin A)

Bligbia sapida

C. yon Holt and W. Leppla, Angew. Chem., 1958, 70: 25; A. John and W. G. Stoll, Helv., 1959, 42: 150; D. K. Black and S. R. Landor, J. chem. Soc., C, 1968, 281,283, and refs. therein; E. V. Ellington et al., J. chem. Soc., 1959, 80.

B-(methylenecyclopropyl)-B-methylalanine

A. californica

Fowden and Smith, loc. cit.

1-amino-2-nitrocyclopentanecarboxylic acid

A spergillus r en tii

Burrows, Mills and Turner, loc. cit.; Burrows and Turner, loc. cit.

329

I N D I V I D U A L O~-AMINO A C I D S Table 1-continued Structure

Source

Reference

N-( 3-amino-3-carboxypropyl)-/3-carboxypyridinium betaine

tobacco leaves

M. Noguchi, H. Sakuma and E. Tamaki, Arch. biochim. Biophys., 1968, 125: 1017.

0-(2,6~lihydroxypyrimidin-l-yl)alanine

pea seedlings

F. Lambein and R. van Parijs, Biochem. biophys. Res. Comm., 1968, 32: 474; E. G. Brown and B. S. Mangat, Biochim. biophys., Acta, 1969, 177: 427.

0-(2-amino-pyrimidin-4-yl)alanine (lathyrine)

Latbyrus spp.

E. A. Bell and R. G. Foster, Nature, 1962, 194: 91; B. J. Whitlock, S. H. Lipton and F. M. Strong, J. org. Chem., 1965, 30: 115.

erytbro-a-amino- 3-o xo- 5-iso xazolidineacetic acid (IV) (tricholomic acid)

Tricboloma muscarium

H. lwasaki et al., Chem. and pharm. Bull., Japan, 1965, 13: 753.

2-alanyl- 3-isooxazolin-5-one (V)

pea seedlings

F. Lambein et al., Biochem. biophys. Res. Comm., 1969, 37: 375.

[ 2( 3H)-oxazolonyl-(5) ] -glycine (VI)

Amanita muscaria

R. Reiner and C. H. Eugster, Helv., 1967, 50: 128.

claviciptic acid (VII)

ergot

J. E. Robbers and H. G. Floss, Tetrahedron Letters, 1969, 1857.

T-L-glutamyl derivatives, including T-glutamyl-2-amino-4-metbylbex-4-enoic acid (L. A. Fowden and A. Smith, Phytochemistry, 1968, 1. 809), N-(T-L-glutamyl)-l-amino-D-proline (H. J. Klosterman, G. L. Lamoureux and J. L. Parsons, Biochemistry, 1967, 6: 170), N-(T-L-glutamyl)-4-arninobutyric acid (P. O. Larsen, Acta chem. Scand., 1965, 19: 1071; A. P. Fosker and H. D. Law, J. chem. Soc., 1965, 7305), and T-glutamylbypoglycin A (Table 1). Other examples are mentioned by S. G. Waley (Adv. prot. Chem., 1966, 211). With one exception, naturally-occurring proline derivatives are not

330

15

NITRO- AND AMINO-MONOCARBOXYLIC ACIDS

mentioned in Table 1; they have been the subject of an authoritative review by A. B. Mauger and B. Witkop (Chem. Reviews, 1966, 66: 47). 0

NH2 I '~ COCH2CHCO2

II H

f HN H . ' ~ H

~t~~NH2 OH

/%. H02C (IV)

(Ill)

0 ][

H

NH ?

02 H

XN-CH 2 9CH- CO2H I. NH 2 (V)

CH" CO2H I NH 2 (VI)

H (VII)

The general methods of amino acid synthesis can usually be adapted adequately to the synthesis of unusual amino acids, but some observations which relate to specific examples are of particular interest.

(i) N-and O-Alkylated amino acids N~-Metbyl-L-bistidine has been prepared by the general method (P. Quitt, J. Hellerback and K. Vogler, Helv., 1963, 46: 327) involving methylation of the N~-benzyl derivative and subsequent hydrogenolysis (V. N. Reinhold, Y. Ishikawa and D. B. Melville, J. med. Chem., 1968, 11: 258). O-Alkyl tyrosine may be prepared by alkylation with alkyl halide in the presence of two equivalents of sodium hydroxide in dimethylsulphoxide solution. No N-alkylation is observed in this medium although some ester formation does occur (S. L. Solar and R. R. Schumaker, J. org. Chem., 1966, 31: 1996; C. A. Kingsbury, ibid., 1964, 29: 3262).

(ii) Hydroxy-amino acids The reaction of aryl-Grignard reagents with 4-carboalkoxyoxazolones followed by borohydride reduction and hydrolysis gives ~3-aryl-~-methyl serine derivatives. At low temperatures the main product is the erytbro diastereoisomer, but the erytbro and tbreo forms are easily interconverted

2

331

INDIVIDUAL ~-AMINO ACIDS

by treatment with thionyl chloride (S. H. Pines et al., ibid., 1968, 33. 1758, 1762). Me CO 2R ~ q \/ u/C'/o fC--O Me"

(i) ArMgX, Et20, ~ --70 ~

,CO2R HO~~/Ar

(ii) NaBH4 Ac" H N

Y H

C,O2 R H O ~ A r

Cl H

Me

--.,.'~ I ~ H3N --y H

Me

13-Hydroxy-a-amino acids have also been obtained by the ammonolysis of various oxiranes. The ammonolysis of cis-epoxysuccinic acid proceeds stereospecifically to give tbreo-f3-hydroxy-De-aspartic acid (H. Okai, N. Imamura and N. Izumiya, Bull. chem. Soc., Japan, 1967, 40: 2154) and aminolysis of the trans-epoxyacid with benzylamine is also stereospecific (Y. Liwschitz, Y. Rabinsohn and A. Haber, J. chem. Soc., 1962, 3589; J. Oh-Hashi and K. Harada, Bull. chem. Soc., Japan, 1967, 40: 2977). Less clear-cut is the ammonolysis of trans-epoxysuccinic acid which seems to give a 1:2 mixture of the tbreo and erytbro isomers (Okai et al., loc. cit.). %6,6'-Tribydroxyleucine has also been prepared via the hydrolysis of an oxirane obtained from L-asparagine (F. Weygand and F. Mayer, Ber., 1968, 101: 2065): 0 CONH2

I

CH 2

I

CF3'- CO- NH- CH- CO2Et

II

O0

I

Me2S--CH2

C" CH 2 9OAc . . . . . . . ~.

I

CH 2

!

CF 3 - CO" NH- CH- CO2Et O--CH 2

CH2OH

\/

C--CH 2 9OAc

] CF3- CO-NH- CH" (202Et

I

~

C(OH) 9CH2OH ] CH 2 H2N- CH- CO2H

threo-~-Hydroxybomolysine has been prepared by the hydrolysis of the photochlorination product of lysine (Y. Fujita, K. Kollonitsch and Witkop, J. Amer. chem. Soc., 1965, 87: 2030). Photochlorination is specific with respect to both position and configuration. (iii) Perfluoroamino acids 3,3,3-Trifluoroalanine may be prepared by the addition of isocyanato derivatives to N-acyltrifluoroacetaldimines. Asymmetric synthesis must

332

NITRO- AND AMINO-MONOCARBOXYLIC

15

ACIDS

occur to an extent because one diastereoisomer predominates (72:28) in acid hydrolysates of the adduct: CH- CF 3 It R " CO " N

R'NC ~

R'N=C~CH l I O~c//N

- CF 3

R'NHCOCHCF 3 ' [ ~"

HO2CCHCF 3 I

RCONH

NH 2

I

R

3,3,3-Trifluoroalanine can also be prepared from the reaction between the acetaldimine and vinyl magnesium bromide followed by oxidation and hydrolysis (Weygand et al., Angew Chem., intern. Edn., 1966, 5: 600). {3-Perfluoroalkylalanines can be prepared from the appropriate perfluorocarboxylic acid by the following sequence of reactions. The appropriate perfluorocarboxylic anhydride and ethyl diazoacetate react to give the perfluoroacyldiazoacetic ester. Photoaddition of this compound to acetonitrile produces an oxazolone which can be hydrogenolysed to give the N-acetyl amino acid ester, from which the required acid can be obtained by hydrolysis (W. Steglich et al., ibid., 1967, 6: 807): (RFCO)20

N2CH- CO2Et

RF " C==C--CO2Et / \ 0 "C//N

!

~ R F C O 9 C(N2) - C O 2 E t

CH 3CN

H2, AcOH PrO2 ~ R F ' C H 2 " C H ' C O 2 E t / NH- COCH3

conc.

o

HCI

90,1h

~ R F " CH2 " C H " C 0 2 H

I

~H3

CH3 (R F = p e r f l u o r o a l k y l )

3. Peptides and polypeptides Methods available for the determination of the structures of peptides and polypeptides have been comprehensively reviewed in "Methods in Enzymology", ed. C. H. W. Hirs, Vol. II, Academic Press, New York, 1967. Techniques in Protein Chemistry, by J. Leggett-Bailey, 2nd Edn., Elsevier, Amsterdam, 1967, is a useful laboratory guide on the same topic. M. Bodansky and M. A. Ondetti have produced an excellent text on "Peptide Synthesis" (Interscience, New York, 1966) and E. Schrbder and K. Lubke, a notable compendium of synthetic methods and syntheses, "The Peptides" (Academic Press, New York, 1965, 1966) which is likely to be the authoritative work on structure-activity relationships for the foreseeable future. An undergraduate text, "The Organic Chemistry of Peptides" (H. D. Law, Wiley, New York, 1970) which goes into the chemistry of peptide

3

PREPARATION OF PEPTIDES

333

degradation and synthesis in some detail is available. In recent years, the European Peptide Symposia have continued to be a source of inspiration to everyone concerned in this field and the reports of these symposia are invaluable repositories of information: "Proc. Seventh European Peptide Symp., Budapest, 1964", eds. V. Bruckner and K. Medzihradszky, Acta chim. acad. Sci. Hung., 1965, 44:5 et seq.; "Proc. Eighth European Peptide Symp., Noordwijk, The Netherlands, 1966", eds. H. C. Beyerman, A. van de Linde and W. Maassen van den Brink, North-Holland, Amsterdam, 1967; "Proc. Ninth European Peptide Symp., Orsay, France, 1968", ed. E. Bricas, North-Holland, Amsterdam, 1968; "Proc. Tenth European Peptide Symp., Aban Terme, Italy, 1969", ed. E. Scoffone, North-Holland, Amsterdam, 1970. In addition the Annual Reports of the Chemical Society and the Chemical Society Specialist Periodical Reports mentioned above should be consulted. Nomenclature is covered by IUPAC rules (IUPAC Combined Commission on Chemical Nomenclature: Tentative Rules for Abbreviated Nomenclature of Synthetic Polypeptides (Biochemistry, 1968, 7: 483;Arch. Biochem. Biophys., 1968, 123: 633; J. biol. Chem., 1968, 243: 2451); Tentative Rules for a One-letter Notation for Amino-acid Sequences, (Biochemistry, 1968, 7: 2703; Arch. Biochem. Biophys., 1968, 125: i-v; J. biol. Chem., 1968, 243: 3557).

(a) Preparation of peptides One of the most important considerations during the synthesis of peptides from optically-active amino acids is the preservation of optical purity. It has long been suspected that oxazolone formation is potentially one of the most serious sources of racemisation during peptide synthesis and this has now been confirmed (I. Antonovics and G. T. Young, J. chem. Soc., C, 1967, 595; W. H. McGahren and M. Goodman, Tetrahedron, 1967, 2017, 2031; Goodman, Tetrahedron Letters, 1969, 3473). Oxazolones form by the participation of acyl-substituents of carboxyl-activated amino acids in an intramolecular displacement of the activating group (X). ~:B R" c~/H\cH" R' CO" X

R" c//N'CH" R' 0

CO

13 "

R" c / / N \ c - R' 0

~0 ~

They are not produced by N-alkoxycarbonyl amino acids (R - alkyl.O) but they are produced by peptidylamino acids (R . . . . NH-CHR"-CO.NH. CHR'"). It seems that racemization via the oxazolone will generally be at least as fast and generally faster than the various ring-opening reactions

334

NITRO-AND

AMINO-MONOCARBOXYLIC

ACIDS

15

which the oxazolone can undergo, although the rate of ring opening does vary dependent on the strength of the nucleophile involved. To avoid oxazolone formation, peptide synthesis is ideally planned as a stepwise process which starts with the C-terminal acid, to which the other residues are added one unit at a time as their N-alkoxycarbonyl derivatives, or as other derivatives which cannot form oxazolones. However, in practice, large peptides are often constructed by the combination of smaller peptide fragments. These fragments are best selected so that their C-terminal residues, which are to be activated, either cannot (e.g. glycine) or will not (e.g. proline) racemize. Alternatively, coupling methods must be used which are racemization free, and there are very few such methods. In specific instances, racemization can also occur by a 3-elimination pathway (e.g. S-benzylcysteine) or by direct ionization (e.g. phenylglycine) (M. Bodanszky and A. Bodanszky, Chem. Comm., 1967, 591 and refs. therein), but, in general, these mechanisms are of minor importance in peptide synthesis. The determination of the optical purity of peptides is beyond the scope of this chapter, but it may be commented that the amount of racemization inherent in the use of a particular protecting group or coupling method has usually been estimated by the synthesis of modeldi- and tri-peptides and the resulting diastereoisomers have been separated by recrystallization, by t.l.c., by g.l.c, or by paper or ion-exchange chromatography. N.m.r. has also been employed to indicate the proportions of diastereoisomers in a diastereoisomeric mixture. (The Annual Reports of the Chemical Society and the Specialist Periodical Report provide original references.)

(i) Protecting groups The methods of functional group protection which have been most popular in peptide synthesis involve benzyloxycarbonyl-, tert-butyloxycarbonyl- and o-nitrophenylsulphenyl-amino groups; simple alkyl, tert-butyl, benzyl and p-nitrobenzyl esters; nitro-, tosyl- and protonated guanidino groups (arginine); benzyl ethers (serine) and thio esters (cysteine); and 1Vim -benzylimidazoles (histidine). Most of these methods, particularly those dealing with amino- and carboxyl-group protection, are likely to remain in common use, but a number of new protecting groups have recently been described (Table 2) and some of these promise to make their mark on the practice of peptide synthesis. Liquid hydrogen fluoride deserves special mention as a new medium for the cleavage of protecting groups. Many protecting groups are cleaved by this reagent, including the nitro-group from nitroguanidino moieties, and benzyl thio-ethers, and it will probably be used extensively despite the potential health hazard associated with its use.

Table 2 SOME PROTECTING GROUPS FOR PEPTIDE SYNTHESIS Derivatives

Formula

Method of removal

Amino l-(p-biphenyl)-l-methylethoxycarbonyl

p-C6H $ 9C6H4 9 CMe2 9O" CO-~ AcOH

o-nitrophenylsulphenyl

o-NO 2 "C6H4" S -

piperidino-oxycarbonyl /N" O" C ( ~ 6-nitroveratryloxycarbonyl MeM~

HCI-Et 20/HCN/Raney nickel/PhS H/sulphonic acid imides/etc.

Zinc or sodium dithionate/electrolytic reduction/H2,Pd,C

350 nm irradiation in ~ - - C H 2 9O "CO-- presence of NH2NH2

Special comments

References

N o problems with steric hindrance. Cleaved 3000x faster than tert-butoxycarbonyl

P. Sieber and B. Iselin, Helv., 1968, 51 : 614.

Usually gives crystalline amino acid derivatives as dicyclohexylammonium salts

G. C. Stelakatos A. Paganon and L. Zervas, J. chem. Soc., C, 1966, 1191 and refs.therein.

Resistant to HBr-AcOH/CF3CO2 H etc.

D. Stevenson and G. T. Young, Chem. Comm., 1967, 900. A. Patchornik, B. Amit and R. B. Woodward, "Proc. lOth European Peptide Symposium Italy, 1969, ed. Scoffone, North-Holland, Amsterdam, 1970.

Also cleaved by H2 ,Pd,C/HBr in CF3CO2H

NO2

carboxyJ 4-picolyl ester

cold alkali or reduction

Basic ester group facilitates isolation on ion-exchanger

R. Camble, R. Garner and Young, Nature, 1968, 217: 247; J. chem. Soc., C, 1969, 1911.

~v

Z O

hta t~q

Table 2 (continued) Derivatives

Formula

pdimethylaminoazobenzyl

Me2NC6H4N2C6H4CH20-

pentamethylbenzyl ester

Mes Cts" CH2" O -

~-methylthioethyl

p-tolyisulphonylethyl

p-methylthiophenyl

lmidazole ( bistidine ) 2,4-dinitrophenyl

MeSCH2CH 2 9O -

p-MeC6H4SO2CH2CH20--

Method of removal

cold CF3CO 2 H

(i) H2 O2-ammoninm molybdate (ii) OH e

Refexences

As above and is coloured

T. Wieland and W. Racky, Chimia (Switz.), 1968, 22: 375.

Useful particularly in depsipeptide synthesis for protection of a-hydroxy acids. Good crystallizing properties

F. H. C. Stewart, Austral. J. Chem., 1968, 21: 1327.

Very mild cleavage conditions

P. M. Hardy, H. N. Rydon and R. C. Thompson, Tetrahedron Letters, 1968, 2525 and refs. therein. A. W. Miller and C. J. M. Stirling, J. chem. Soc., C, 1968, 2612.

OH O

Converted to active sulphonyl ester when required

p - M e . C 6 H 4 9S - -

(NO2)2C6H3-

Special comments

EtSH, pH 8

O~

z

B. J. Johnson and P. M. Jacobs, Chem. Comm., 1968, 73. F. Chillemi and R. B. Merrifield, Biochemistry, 1969, 8: 4344; cf. S. Shaltiel, Binchem. biophys. Res. Comm., 1967, 27: 178.

o > D >

o o z o f3 > o N r"

E > f3

Table 2 (continued) Derivatives

Formula

Method of removal

Special comments

References

acetamidomethyi

MeCONHCH2 -

Hg20, pH 4

Stable to alkali and to acid, including liquid HF at 0 ~

D. F. Weber et al., Tetrahedron Letters, 1968, 3057.

benzyl

PhCH 2 -

Na/NH 3 liq. or anhydrous HF

Method fallen into disrepute due to sidereactions during Na/NH 3 liq. treatment of large peptide. Cleavage with HF might bring new lease of life

S. Sakakibara and Y. Shimonishi, Bull. chem. Soc., Japan, 1965, 38: 1412.

1-phenylcyclohexyl

~ P h

CF3CO2 H

W. Ki~nig, R. Geiger and W. Siedel, Ber., 1968, 101: 681.

2,2,2-trifluoro-N-benzyloxycarbonylaminoethyl

CF3CH-! NHCOOCH2 Ph

H2, Pd, C or liquid HF

F. Weygand et al., Ber., 1968, 101: 923.

benzyl

CH2Ph

H2, Pd, C

for latest developments see T. Miznguchi et al., J. org. Chem., 1968, 33: 903.

ten-butyl

Me3C--

H@

for latest developments see E. Wiinsch and F. Angelo, Ber., 1968, 101: 323.

Tbiol (cysteine)

Hydroxyl (serine and tbreonine)

rod "-.I

104

0o

Table 2 (continued) Derivatives

Formula

Method of removal

Special comments

CF3CO2H, room temp., 30 h

Stable to H2, Pd, C

References

Amide (asparagine and

z

giut~me~ 2,4-dimethoxybenzyl

OMe _

~

MeO 2,4,6-trimethoxybenzyi

CH 2 OMe

CF3CO2 H, room temp., 30 h

MeO~CH2--

Stable to H2, Pd, C

Weygand et al., Ber., 1968, 101: 3623, 3642.

P. Pietta, F. Chiilemi and P. Corbellini0 ibid., p. 3649.

/: o o z o > o

OMe 4,4'-dimethoxybenzhydryl

50% aq. acetic acid

Geiger and Seidel, Eighth European Peptide Syrup., Ioc. cit.

S f'l

3

PREPARATION

OF P E P T I D E S

339

(ii) Carboxyl activation All of the coupling methods which are in general use in peptide chemistry are methods of carboxyl activation and the few methods of amino-group activation that have been described (for recent examples see J. Gante, Angew. Chem., intern. Edn., 1966, 5: 315, 593) probably proceed indirectly through a carboxyl activated intermediate (Schrbder and Li~bke, loc. cit.). Broadly speaking, the most popular carboxyl-activating methods have involved four types of active intermediate: azides, mixed anhydrides, acylisoureas and activated esters. These remain important, but two additional methods which could prove significant have recently been described. One of these employs the classical Leuch's anhydrides (Ncarboxyanhydrides), the other, acyloxyphosphonium salts. (1) Azide method. This technique remains important, despite occasional interference from side reactions (e.g.E. Schnabel, Ann., 1962, 659: 168) because it proceeds without racemization. It has proved possible to induce racemization during azide coupling, but only under extreme conditions which would never be used in peptide synthesis (G. W. Anderson, J. E. Zimmerman and F. M. Callahan, J. Amer. chem. Soc., 1966, 88:1338). The azide method is therefore suitable for fragment condensation syntheses in which the C-terminal residue of a fragment is susceptible to racemization. It has been postulated that conformational rigidity induced by the type of electrostatic interaction shown below might inhibit oxazolone formation and hence account for the absence of racemization (I. Antonovics et al., Proc. Sixth European Peptide Symp., Athens, 1963, ed. L. Zervas, Pergamon, Oxford, 1965, 121): H,,,

~,R

N, I,

C. ~'~-

o c4" _o

]', _,/c', ^ .N :s ~ rt x,2b~, I : 0 Nle |

N

(2) Mixed anhydride method. Under the most favourable conditions, even C-terminal residues in peptides can be converted into mixed anhydrides by the acid chloride technique and coupled without racemization. For isobutylchloroformate, a 4-min activation at -15 ~ seems to be optimal using aliphatic tertiary amines which possess at least one N-methyl group, instead of the more traditional triethylamine (Anderson et al., J. Amer. chem. Soc., 1967, 89" 5012). The reagent, 2-ethoxy-N-ethoxycarbonyl-l,2-dihydroquinoline, affords mixed anhydrides on treatment with N-protected amino

340

15

NITRO-AND AMINO-MONOCARBOXYLIC ACIDS

acids. No racemization is detected during the subsequent coupling when the activation is carried out in the presence of the amino-component (A. Belleau and G. Malek, ibid., 1968, 90- 1651).

OEt I CO2Et

c

R

O=C] ~ / / I OEt + R-CO-O-CO.Et

(3) Acylisourea (diimide ) method. N,N'-Dicyclohexylcarbodi-imide reacts with N-protected amino acids to form the intermediate acylisourea which is aminolysed readily to form peptides. This technique is very simple since it only requires the addition of the diimide reagent to a mixture of the amino and carboxyl components in an inert solvent. The most troublesome side reaction is the formation of the N-acylurea by intramolecular rearrangement of the N-acylisourea, but this is only important when the nucleophilicity of the amino component is impaired, e.g. due to steric hindrance: R- CO-NH- R' + 1

RCO2H + C6H IlN=C=NC6 H 11

C6H 11NHCONHC6H ll

R'CO'? C6Hll 9N=C" NH" C6Hll R" CO I C6H 11NCONHC6H 11

This mechanism has been substantiated by kinetic studies (D. F. DeTar et al., ibid., 1966, 88. 1013, 1020, 1024) and O-acylisoureas similar to the postulated intermediate have been isolated (Doleschall and Lambert, Tetrahedron Letters, 1963, 1195). Guanidine formation by attack of the amine component on the diimide has also been postulated as a source of by-products (DeTar et al., loc. cit.; I. Muramatsu, T. Hirabayashi and A. Hagitani, C. A., 1963, 60: 12100c): CH2 H2NCHRCO2E t + C6HIINCNC6Hll

~.

HN/''CO ~C N C6H I1NH C6H II

3

PREPARATION OF PEPTIDES

341

Diimides are about the best dehydrating agents to use for the preparation of oxazolones so it is not surprising that appreciable racemization sometimes occurs when a diimide is used in the fragment condensation approach to peptide synthesis. However, provided two equivalents of N-hydroxysuccinimide are present in the reaction mixture, no racemization can be detected (E. Wi~nsch and F. Drees, Ber., 1966, 99: 110; F. Weygand, D. Hoffmann and WiJnsch, Z. Naturforsch., 1966, 21b: 426). N-Hydroxysuccinimide probably reacts very rapidly with the first formed acylisourea and converts it entirely to the N-hydroxysuccinimide ester. This type of ester is susceptible to aminolysis, but it does not form oxazolones (see below). The addition of 1-hydroxybenztriazole has been found even more effective in preventing racemization (B. W. Bycroft in Proc. Tenth European Peptide Symp., loc. cit.). (4) Activated ester metbod, p-Nitrophenyl (M. Bodanszky, Ann. N.Y. acad. Sci., 1960, 88(3): 655) and 2, 3, 5-trichlorophenyl (J. Pless and R. A. Boissonnas, Helv., 1963, 46: 1609; J. S. Morley, J. chem. Soc., C, 1967, 2410) esters are the most widely used esters of the type which is dependent for its activity on the acidity of the component phenol. These esters are generally prepared from the N-protected amino acid and the phenol by the use of dicyclohexylcarbodiimide as the condensing agent (D. F. Elliott and D. G. Smyth, Proc. chem. Soc., London, 1963, 18). Pentachloro-and pentafluoro-phenol form complexes with diimide which react with carboxyl components to make the corresponding active esters, but it is thought that the complex dissociates into the diimide and the phenol so that ester formation takes place in the usual way. This technique appears to give very little racemization (J. Kovacs, L. Kisfaludy and M. Q. Ceprini, J. Amer. chem. Soc., 1967, 89: 183):

( ) N.=N ( ) O I

C I / F S ) CI/F CI/F~ . C1/F CI/F The reactivity of another class of activated ester cannot be accounted for in terms of the acidity of the phenolic components. This class includes esters of N-hydroxypiperidine (J. H. Jones and G. T. Young, J. chem. Soc., C, 1968, 53 and refs. therein), catechol, 4,5-dichlorocatechol (idem, ibid., p. 436), 8-hydroxyquinoline (H. D. Jakubke and A. Voigt, Ber., 1966, 99: 2419), various halogenated 8-hydroxyquinolines (J akubke, Z. Chem., 1965, 5:453;

342

15

NITRO- AND AMINO-MONOCARBOXYLIC ACIDS

Jakubke and Voigt, Ber., 1966, 99: 2944), and N-hydroxysuccinimide (Anderson, Zimmerman and CaUahan, J. Amer. chem. Soc., 1963, 85: 3039). In this case, the reactivity of the esters is thought to be due to anchimeric assistance arising from the presence in the phenolic component of a group which facilitates the removal of a proton from the approaching amine and hence stabilises the reaction intermediate. For example, in the 8-hydroxyquinoline esters assistance could be visualised as follows:

0 r O=C

I

R

H N

I

0

H

I

o] ^ 'O--C~N =

9R'

I

H

R

0

o

"R'

I

O--C--NH

I

"R'

I

H

R

Oxazolone formation would not be facilitated in this way and this is substantiated by the finding that couplings of this type seem to proceed with complete absence of racemization. A variety of insoluble polymeric activated esters has been described for use in peptide synthesis including N-protected amino acid esters of 4-hydroxy-3-nitrostyrene/styrene copolymers, polyamino acids incorporating 3-nitrotyrosine (M. Fridkin, A. Patchornik and E. Katchalski, ibid., 1966, 88: 3164; idem, Israel J. Chem., 1966, 3: 69), 4,4'-dihydroxydiphenylsulphone/formaldehyde polymers (Th. Wieland and Ch. Birr, Angew. Chem., intern. Edn., 1966, 5: 310), 8-benzyloxy-5-vinylquinoline/divinylbenzene copolymers (Van G. Manecke and E. Haake, Naturwiss., 1968, 55: 343) and a polymeric form of N-hydroxysuccinimide (D. Laufer et al., J. Amer. chem. Soc., 1968, 20: 2696). (5) Isoxazolium salt method. This technique, which has clear affinities with both the diimide and activated ester methods, involves the addition of an isoxazolium salt, for example, N-ethyl-5-phenylisoxazolium-3'sulphonate, to the carboxyl component in the presence of a tertiary base. The enol esters which are formed undergo smooth aminolysis without racemization (R. B. Woodward and D. J. Woodman, ibid., 1968, 90:1371 and refs. therein):

so~'

1~//.N-Et ~H~(,,

HC~ C %NEt

:B

[R - m-SO~C6H 41

3

PREPARATION OF PEPTIDES

R\c//O

RIco2H

I

,

HC% C~NEt R~c//O

R.

~C//'0 I

H2C\

,

C'O'CO Rl II NEt

343

R~c/OH II HCNc" . O CORt II NEt

Rx(,/O" CO" Rl NH2R2 R~c/OH + RI'cO'NH'R2

HC~c"

HC. "CO" NHEt

O" CORI

i NH-

HC.. "CONHEt

Et

2-Ethyl-7-hydroxybenzisoxazolium salts react with N-protected amino acid sodium salts to give substituted catechol esters which undergo aminolysis, again without racemization (D. S. Kemp and S. W. Chien, ibid., 1967, 89: 2743): OH

~

O. /NEt

OH R'NHCHRCO20 ~ O C O C H R N H R ' , ~CONHEt

~

OCOCHRNHR' OH CONHEt

(6) Acyloxypbospbonium salt metbod. This approach to peptide synthesis has only been described recently and instances of its application are still limited in number. However, the preliminary results look extremely promising and this may well prove one of the most valuable coupling methods available. N-Protected amino acid acyloxyphosphonium salts, generated from the N-protected amino acids and a mixture of toluene-p-sulphonic anhydride and hexamethylphosphorotriamide, react readily with amino acid esters to yield optically pure protected peptides (G. Gawne, G. W. Kenner and R. C. Sheppard, ibid., 1969, 91: 5669):

(Me2N)3P=O

Tos-o. Tos-" I(Me2N)3P--O" @ Tos 1 (-TosOO) @

9

(Me2N)3P--O--P(NMe~!)a

RICO2O

' 9

~ RIco2P(NMe2)3

R2NH2 , RICONHR2

Triphenyl phosphine coupling methods probably involve similar mechanisms (T. Makaiyuma et al., ibid., 1968, 90: 4490; 1969, 91: 1554).

344

NITRO- AND AMINO-MONOCARBOXYLIC ACIDS

15

(iii) The use of cyclic derivatives involving both carboxyl and amino groups Leuch's anhydrides can be used in a stepwise manner in peptide synthesis provided the pH is accurately controlled and rapid mixing of reactants is achieved (R. G. Denkewalter et al., ibid., 1966, 88:3163;R. Hirschmann et al., J. org. Chem., 1967, 32: 3415). Due to CO2 transfer from the carbamate to the amino component, up to 1% of the starting material always remains after each stage of this method, no matter how much N-carboxyanhydride is employed. Other impurities are also introduced concomitantly because of the further reaction of the liberated peptide amino group. Nonetheless, peptides of up to five residues in length can be prepared by this method without isolating the various intermediates: CHR n-1 HN/"X'CO !

I

OC

O

NH2CHRnCO ~ ....

~

H N / C H R n - I " CONHCHRnCO~

Ho

l

~O o / C H R n-1 . CONHCHRnCO2H bubble N2 through mixture to remove CO2

C02H / C H R n-1 CONHCHRnCO2 H eNH 3 .

(i) adjust to pH 10.2 NHCHRn-2co (ii) + ] ] co o

/ C H R n - 2 CONHCHR n - 1CONHCHRnCO2H HN"

Thiazolidine-2,5-diones have advantages over N-carboxyanhydrides for the incorporation of glycine and histidine into peptides. In these cases, the N-carboxyanhydrides tend respectively to give the following by-products by way of the corresponding isocyanates (R. S. Dewey et al., J. Amer. chem. Soc., 1968, 90: 3254): o O2CCHRnNHCONHCH2CO2O

Y 0 The ready availability of pure pentapeptides greatly facilitates the preparation of much larger peptides, as exemplified by the synthesis of material with ribonuclease activity (see Table 3). In this synthesis, which

345

P R E P A R A T I O N OF P E P T I D E S Table 3 SYNTHESIS OF SOME NATURALLY-OCCURRING PEPTIDES Peptide

Source

Size

Reference

Fungisporin

mould metabolite

cyclic tetrapeptide

R. O. Studer, Experientia, 1969, 25: 898.

Ferrichrome

fungi

iron-binding cyclohexapeptide

W. Keller-Schierlein and B. Maurer, Heir., 1969, 52: 603.

Antamanide

Amanita pballoides

cyclic decapeptide

Th. Wieland et al., Angew. Chem., intern. Edn., 1968, 7: 204.

Caerulein

skin of Hyla caerula

10 residues

A. Anastasi, V. Erspamer and R. Endean, Arch. Biochem. Biophys., 1968, 125: 57.

15 residues

R. S arges and B. Witkop, J. Amer. chem. Soc., 1965, 87: 2011, 2020, 2027.

Gramicidin A and B

Gastrins

stomach (various species)

17 residues

K. L. Agarwal et al., Nature, 1968, 219: 614 and refs. therein.

Melittin (I and II)

bee venom

26 and 27 residues

K. Liibke and E. Schr/~der, Proc. Eighth European Peptide Symp.

Secretin

pig

27 residues

M. Bodanszky et al., Chem. and Ind., 1966, 1757.

Glucagon

Islets of Langerhans (various species)

29 residues

E. Wfinsch etal., Ber.,1968,101: 326,336,3418, 3659,3664.

Thyrocalcitonin

porcine thyroid

32 residues

W. Rittel et al., Helv., 1968, 51: 924; St. Guttman et al., ibid., 1968, 51: 1155. Human: P. Sieber et al., ibid., 1968, 51: 2057.

346

N I T R O - AND A M I N O - M O N O C A R B O X Y L I C A C I D S

15

Table 3 (continued) Peptide

Source

Size

Reference

Insulin

Islets of Langerhans (various species)

51 residues arranged in two chains

Orthodox: H. Zahn et al., Z. Naturforsch., 1963, 181>: 1130; P. G. Katsoyannis et al., J. Amer. chem. Soe., 1964, 86: 930; K. T. Kung et al., Sei. Sinica, 1965, 14: 1710. Solid Phase: A Marglin and R. B. Merrifield, J. Amer. chem. Sot., 1966, 88: 5051.

Ferredoxin

C. pasteurianum

55 residues

E. Bayer, G. Jung and H. Hagenmaier, Tetrahedron, 1968, 2 4 : 4 8 5 3.

Ribonuclease

Bovine pancreas

124 residues M 13,700

B. Gutte and R. B. Merrifield, J. Amer. chem. Sot., 1969, 91. 501; R. Hirschmann et al., ibid., p. 507.

involved the construction of a single chain of 124 residues, 40% of the peptide bonds were made by N-carboxyanhydrides or their thio-counterparts.

(iv) Solid-phase peptide synthesis In the discussion on the use of N-carboxyanhydrides in the stepwise synthesis of peptides, the concept of synthesis without isolation of intermediates was touched upon. Since the more orthodox approach, involving the characterisation of intermediates, would be prohibitively laborious in the synthesis of large proteins, considerable importance is to be attached to the new type of approach. The so-called "solid phase" synthesis introduced by R. B. Merrifield carries this philosophy to its logical conclusion. In this technique, mentioned briefly in Rodd's CCC, 2nd Edn., the C-terminal residue is attached by a benzyl-ester type linkage to an insoluble, high molecular weight polystyrene preparation. Further residues are added in a stepwise manner by the use of N,N'-dicyclohexyl-

3

B I O S Y N T H E S I S OF ,PEPTIDES AND P R O T E I N S

347

carbodiimide, generally with tert-butyloxycarbonyl protection of the ~oamino groups. No isolation is attempted until the completed peptide is finally cleaved from the peptidyl polymer, for example, by treatment with hydrogen bromide in trifluoroacetic acid. The method has formed the subject of a recent text by J. M. Stewart and J. D. Young ("Solid Phase Peptide Synthesis", Freeman, London, 1969). Many peptides have been prepared by this technique including insulin and ribonuclease (see Table 3). In the latter synthesis, 11,931 individual steps were carried out by an automated machine. Completely automated equipment for solid-phase synthesis is commercially available. Although intermediate peptides are not isolated in solid-phase synthesis there is a growing awareness of the need to monitor the acylation step and many experiments in this direction have been reported (L. C. Dorman, Tetrahedron Letters, 1969, 2319; Th. Wieland, Ch. Birr and H. Wissenbach, Angew. Chem., intern. Edn., 1969, 8: 764). Even so, it may ultimately prove, because of isolation difficulties, that the solid-phase method is best adapted for the synthesis of decapeptides, or similar oligopeptides, which are subsequently coupled together, like the intermediate peptides produced by the N-carboxy-anhydride method. In a modification to the solid-phase method, the C-terminal residue is attached to the resin via a tert-amyloxycarbonylhydrazide link; treatment with trifluoroacetic acid yields the hydrazide of the peptide which can be coupled to other peptides by the azide method (Su-sun Wang and Merrifield, J. Amer. chem. Soc., 1969, 91: 6488).

(v) Biosyntbesis of peptides and proteins The genetic code has been elucidated (F. H. C. Crick, Proc. Roy. Soc., B, 1967, 167: 334, and refs. therein; see also F. Sanger, Nature, 1969, 223: 1009) and a great deal is known about the individual stages of peptide synthesis (Annual Reviews of Biochemistry contains many references and, for the organic chemist, particularly interesting expositions have been given by R. Schwyzer in "Proc. Eighth European Peptide Symp." and by F. Gros and M. Revel in Proc. Ninth European Peptide Symp.). It is noteworthy that biosynthesis starts with the N-terminal amino acid residue and proceeds in a stepwise manner to the C-terminal residue. Perhaps oxazolone formation is avoided due to the steric constraints of the system, but it is interesting that the carboxyl groups of the individual residues are activated by ester formation through the 3'-hydroxyl group of a ribose moiety. The proximity of the 2'-hydroxyl reminds one of the racemization-resistant catechol-type esters discussed above.

348

15

NITRO- AND AMINO-MONOCARBOXYLIC ACIDS

It has been suggested that D-amino acids in peptides arise from the epimerization of L-amino acid residues in the peptides themselves (M. Bodanszky and D. Perlman, Science, 1969, 163: 352), and that the same mechanism prevails in all cases (B. W. Bycroft, Nature, 1969, 224: 595).

RCO f

H

COR1

I:I

HH

-H2 9 R

R" CO N

~"N~CR

R2

R2

+ H2

9co / ~ ~N

R2 1 II

o

COR1 RCO f

R2

~N~"-X.COR 1 H

(b) Naturally-occurring peptides Many peptides have been isolated (review: S. G. Waley, loc. cit.) and completely characterised by degradation and by synthesis, and several hundred peptides have been synthesised in studies aimed at the correlation of structure and biological activity (H. D. Law, Progress in Medicinal Chemistry, eds. Ellis and West, Vol. 4, p. 86, 1965, deals with peptide hormones; R. O. Studer, ibid., Vol. 5, p. 1, 1966, with peptide antibiotics). Some of the peptides which have been synthesised are listed in Table 3. In each case, reference given is for the synthesis, but this also serves as a key reference for the degradative work.

(c) Polypeptides or poly-~-amino acids Vol. 1 in the series Biological Macromolecules is devoted to polyp-amino acids (ed. G. D. Fasman, Arnold, London, 1967).

4. Hydroxylamino-carboxylic acids N-Alkylation of anti-benzaldoxime with a bromoester and hydrolysis of the resulting nitrone provides a general route for the synthesis of hydroxylamino acids (E. Buehler and G. B. Brown, J. org. Chem., 1967, 32: 265, who also list other synthetic routes; E. Bellasio et al., Ann. Chim., 1968, 58. 407).

5

HYDRAZINO-CARBOXYLIC ACIDS

Ph\

Ph~ "CH I1 + BrCHRCOzEt N

oO/

349

~

"CH II N o O~ o \ C H R C O 2 E t

HCI H20

HONHCHRCO2Et + PhCHO

5. Hydrazino-carboxylic acids The natural occurrence of 1-amino-proline has been referred to above (Table 1). N-Amino derivatives (hydrazino) of L-, D- and o L-histidine have been prepared by hydrazinolysis of the chlorides produced by the deamination of histidine (M. Sletzinger et al., J. med. Chem., 1968, 11: 261). The interesting azo-analogues of dipeptides, N-aminoguanylamino acids have been prepared by reacting S-methylisothiosemicarbazide hydroiodide with amino acid salts in boiling water or ethanol (J. Gante, Ber., 1968, 101: 1195): NH

II

H2N- NH- C- NH- CHR- CO~H