Vol. 6o PEPTIDE ESTER REDUCTION 173 from solution. The complexes with the toluene-p- 2. Quantitative reduction of the ester groups sulphonyl derivatives are particularly insoluble. with lithium aluminium hydride (LiAlH4) was Where homogeneous reaction mixtures are possible, impossible in many cases owing to the formation of reduction of the peptide carbonyl becomes con- insoluble aluminium complexes. siderable even at low temperatures, thus presenting 3. Both LiAlH4 and aluminium hydride (ARH3) a serious drawback to the use of LiAlH4 in the reduce the peptide bond. Greater control of the peptide and protein field. reaction, however, is possible with the latter A solution of AlH3 in tetrahydrofuran ivi the reagent. presence of a little aluminium chloride has been 4. Lithium borohydride (LiBH4) is the most found useful for the preparation of a number of suitable reagent for reducing peptide esters. ,-hydroxyamides in good yield. Best results are Homogeneous reaction mixtures are usually ob- obtained when the reducing solution is added to the tained and the minimum ofside reactions take place. ester gradually. In the preparation of ,B-hydroxy- I wish to express my thanks to Dr A. C. Chibnall, F.R.S., amides from the ethyl esters of glycylglycine and for his advice and interest in this work which was carried out L-leucylglycylglycine using this reagent at a temper- during tenure of an Imperial Chemical Industries Research ature of - 40° losses due to amide reduction Fellowship. amounted to 5-10%. LiBH4 is the best of the three reducing agents REFERENCES described, in that most of the peptide derivatives Bailey, J. L. (1950). J. chem. Soc. p. 3461. are freely soluble in a tetrahydrofuran solution of Finholt, A. E., Bond, A. C. & Schlesinger, H. I. (1947). the reagent and reduction of the ester group occurs J. Amer. chem. Soc. 69, 1199. at room temperature. Where suspensions are Fromageot, C. & Jolles, P. (1952). Biochim. biophy8. Acta, 9, obtained it is possible to reflux the mixture with 287. little risk of amide reduction. Karrer, P. & Nicolaus, B. J. R. (1952). Helv. chim. acta, 35, 1581. Krynitsky, J. A., Johnson, J. E. & Carhart, H. W. (1948). SUMMARY Analyt. Chem. 20, 311. McChesney, E. W. & Swann, W. K. (1937). J. Amer. chem. 1. ,B-Hydroxyamides corresponding to a number Soc. 59, 1116. of peptide esters and their toluene-p-sulphonyl Wilson, E. J. & Pacsu, E. (1942). J. org. chem. 7, derivatives have been prepared. 126. Systematic Degradation of Peptides from the Carboxyl End BY J. LEGGETT BAILEY* Department of Biochemistry, University of Cambridge (Received 21 October 1954) Chemical methods are available for operating on pendently by Waley & Watson (1951) and Tibbs both the amino end and the carboxyl end ofproteins (1951). Both groups of workers concluded that and peptides. The amino end has been more some modification in technique is necessary before amenable to the techniques oforganic chemistry and the method can be used on proteins. The recently the majority of degradative methods relate to this reported procedure ofKenner, Khorana & Stedman end group. Of the methods for the identification (1953) using diphenyl phosphoroi8othiocyanatidate and systematic removal of amino acids at the brings about cyclization under very mild conditions, carboxyl end of peptide chains, that of Schlack & but it has yet to be applied to a protein. A new Kumpf (1926) has received the most attention. This approach to the problem of degradation at. the procedure which converts the C-terminal amino acid carboxyl end was made possible when Chibnall & into a thiohydantoin has been re-investigated inde- Rees (1951) showed that the C-terminal ester groups * Present address: The Rockefeller Institute for Medical in insulin methyl ester could be reduced quanti- Research, 66th Street and York Avenue, New York 21, tatively with lithium borohydride (LiBH4) to U.S.A. produce C-terminal amino alcohols (I -+ II). 174 J. L. BAILEY I955 Acid or R.CO.NH.CHR'.CO.NH.CHR'.CH2OH RCO.NH.CHR'.COO.CH2.CHR".NH3 + Acid chloride fi-Hydroxyaiide (II) fl-Amino ester salt (III) t Reduction ; Reduction . R. CO. NH. CHR'. CO. NH. CHR. COOCH3 (I) RCO . NH . CHR' . CH2OH + HOCH2 CHR' . NH2 (IV) The selective removal of C-terminal amino alcohols migration in an aliphatic system made use of from polypeptides reduced in this way appeared phosphorus pentachloride and thionyl chloride; one possible by a procedure involving two steps. Under instance of the use of phosphorus oxychloride, certain conditions employing acids or acid chlorides, however, is cited later by Bergmann & Brand fi-hydroxyamides (N-peptidylaminoalcohols (II)) (1923). The formation of oxazolines from ,B- may be converted into the corresponding fl-amino hydroxyamides and the mechanism of ring-opening esters (III). Reductive cleavage of the ,B-amino has been discussed by Attenburrow, Elliott & esters with LiBH4 would liberate the C-terminal Penny (1948), Goldberg & Kelly (1948) and Fry amino alcohols (IV). (1950). A recent review of the subject with special CH2-CHR' Acid H,C-HCHR' Acid C" -2CHR" I ONO NH OHl NH Base Base NHa (V) R' (VI) R' (VII) co POC13 ClOH,-CHR' OH SH NH CIH CIHR x \- I ) (V) R' s vNH+ Cl el IV (IX Base 101- lel (VIIII) fH2 l ]Rv NHt al NHI 00 [H I R' (VII) (X) R' In acid media N, 0 migration of acyl groups reference to N, 0 migration at the serine residues in (V -> VII) is generally considered to occur via an proteins has been made by Elliott (1952). Many intermediate hydroxyoxazolidine (VI). Reagents substituted 2-oxazolines (VIII) have been isolated used for this purpose have been concentrated by various workers as stable salts, which usually sulphuric and hydrochloric acids (Bettzieche, 1925) give the salts of the corresponding fl-amino esters and ethanolic hydrogen chloride (Phillips & (VII) on treatment with water or dilute acid. In the Baltzly, 1947). Experiments carried out in this presence of alkali the fl-amino esters (VII) revert laboratory using methanolic hydrogen chloride and almost quantitatively to the original hydroxy- boron trifluoride have been only partially successful amides (V). and this type of reagent was abandoned. l-COhloroethylamides (X) can arise by the action On the other hand, dehydration of the peptide of CO- on the intermediate chlorophosphates (IX) or hydroxyamides (V) to oxazolines (VIII) using by ring cleavage of the oxazolines (VIII). This phosphorus oxychloride and subsequent opening of undesirable secondary reaction can be kept to a the ring with dilute acid gave high yields of the minimum by avoiding the more reactive acid ,8-amino ester salts (VII). The original studies of chlorides such as thionyl chloride. Bergmann, Brand & Dreyer (1921) of N, 0-acyl ,B-Hydroxamides can be prepared in high yield Vol. 6o SYSTEMATIC DEGRADATION OF PEPTIDES 175 frompeptide esters by reductionwithmetalhydrides conditions: (a) treatment with BF3-formic acid (0-2 ml.; (Bailey, 1955). The present paper deals with the b.p. 79-83°/15 mm.) for 2 hr. at 600; (b) treatment with preparation of P-amino esters from peptide fi- BF3-acetic acid (0-2 ml.; b.p. 141°/760 mm.) for 3 hr. at hydroxyamides, without isolation of the inter- 700; (c) treatment with SnCl4-formic acid (1:3, by vol.) for 4 hr. at 800. mediate oxazoline derivatives, and subsequent In the case of N-L-leucylglycylaminoethanol (5 mg.) best reduction of these esters to liberate the terminal results were obtained by warming the compound with BF3- amino alcohols. In conjunction with the methods formic acid (0-1 ml.) for 1 hr. at 600. Although the main now available for the detection and estimation of spot on the chromatogram corresponded to the fl-amino amino alcohols on the micro scale (Grassmann, ester (R,, 0-28; solvent system, propanol-water-formic Hormann & Endres, 1953; Fromageot, Jutisz, acid), the presence of other faint ninhydrin spots indicated Privat de Garilhe & Suquet, 1954; Chibnall & Rees, secondary products. Treatment ofthe fi-hydroxyamide with unpublished) the above principles might constitute 97 5 % H2SO4 at 210 for 3 days as described by Elliott (1952) a workable stepwise degradation of peptides from gave no ,B-amino ester. the carboxyl end. Acyl migration via intermediate oxazolines EXPERIMENTAL AND RESULTS N-Glycylaminoethanol and SOC12 at room temperature yielded glycyl-,B-chloroethylamide (Rp, 0-45) and un- All melting points are uncorrected. changed hydroxyamide. Interaction of a solution of L- Aminoethanol (Ethanolamine). This was estimated by leucylglycylaminoethanol in nitromethane with SOC12 at oxidation with periodic acid. The resulting formaldehyde - 100 or POCl3 at room temperature gave unchanged hydr- (2 moles/mole of ethanolamine) was determined colori- oxyamide, the fl-amino ester and ,B-chloroethylamide (Rp, metrically using chromotropic acid according to the modi- 0.44). When the reactions were carried out in dimethyl- fied micro method of Rees (unpublished). formamide, the fl-chloroethylamides were themain products. O-Glycylaminoethanol dihydrochloride. The following When the N-toluene-p-sulphonyl derivatives of the synthesis was effected. ,B-Nitroethanol (1-5 ml.) was satur- hydroxyamides were used, excellent yields of corresponding ated with anhydrous HCI at - 200 with rigorous exclusion fl-amino ester hydrochlorides were obtained. of moisture. N-Carboxyglycine anhydride (100 mg.) was N-Toluene-p-sulphonyl-0-glycylaminoethanol hydrochlor- added to the solution and the mixture kept at - 40° during ide. N-Toluene-p-sulphonylglycylaminoethanol (44 mg.) 1 hr. when the temperature was allowed to rise slowly to was dissolved in acetonitrile (0.3 ml.) containing SOC12 room temperature. On dilution with ether crystals of (0.05 ml.; 4 mol. prop.) at - 100. At the end of 2 hr. the O-glycine fl-nitroethanol ester hydrochloride separated, m.p. excess SOC12 was removed in vacuo at - 100 and the residue 118-119°; yield 55 mg. (Found: N, 15-1.