Dombi György

Kovács Kálmán

Penke Botond

English

University of Szeged

INVESTIGATIONS ON THE STABILITY OF THE Nim-BOC* PROTECTING GROUP OF HISTIDINE By B. PENKE, GY. DOMBI and K. KOVÁCS Institute of Organic Chemistry, Attila József University, Szeged (Received January 10, 1973) The optimal conditions of synthesizing №,Nim-di-i-butyloxycarbonyl-histidine were elabo-rated and the stability of the N i m-BOC protecting group was studied under the conditions of solid phase peptide synthesis. According to our results, the conditions for using this protecting group are rather restricted. In the solid phase peptide synthesis, the nitrogen of the histidine imidazole has to be protected in a suitable way. The protecting group should satisfy the following requirements: ready availability, stability to the removal conditions of the N-pro-tecting groups in the solid phase peptide synthesis, and selective removal of the pro-tecting group without racemization. YAMASHIRO et al. [1] used Nz-BPOC-Nim-BOC histidine and Na,Nim-di-BOC--histidine (II) for synthesizing a sequence of human growth hormone (HGH). As the literature gives contradictory data concerning the synthesis of I I and the stability of the Nim-BOC protecting group, it seemed justified to clear up these prob-lems in a series of experiments. According, to literature [1—3], histidine and /-butyloxy-carbonyl-azide were brought into reaction, giving the following results under the conditions used: a.) H—His—OH 4- BOC—N3 20 " ^ ¡ Г 5 * BOC—His—OH [2] 1 m m o l e 2 x 2 m m o l e 1 , 9 2 % b.) H—His—OH + BOC—N3 •^рнзд-Г 1 BOC—His (BOC) ОН Ш 1 m m o l e 4 m m o l e I I , 2 3 % c.J H—His—OH + BOC—N3 20 °MgodayS~ BOC—His (BOC) OH [3] . 1 m m o l e 3 m m o l e П , 5 0 % . Our experiments showed that the divergences between the quoted data could be explained by the sensibility of the Nim-BOC protecting group to higher tempera-tures and to basicity of the medium. Under the conditions given in [2] I I always forms in small quantities, but at the pH value used it hydrolyses to I, Indeed, I I can be obtained with the best yield by the method of FRIDKIN [4] in consequence * The abbreviations used conform with the standards of IUPAC-IUB. BPOC=biphe nyl-isopropyl-oxycarbonyl. 166 B. PENKE, GY. DOMBI AND K. KOVÁCS of the comparatively low temperature (20 °C) and the low basicity of the medium (MgO). YAMASHIRO'S method gives I I with a yield of only 20—25%; thus the in-creased temperature and basicity prove to be unfavourable for the reaction. Similarly contradictory data are found in literature concerning the synthesis of i n and I V . SCHRÖDER and GIBIAN[5] considered the oil obtained by the reaction of histi-dine methylester and BOC-azide as pure III , because it transforms with hydrazinehy-drate into homogeneous BOC-histidine-hydrazide. According to WÜNSCH and ZWICK [6] the product formed by the reaction of histidine methylester and 2 equiv. BOC-azide consists of a mixture of I I I and IV, which they were able to separate by fractional crystallization. Our results show — in good accordance with HANFORD et al. [7] — that by the reaction of histidine methylester and 1 equiv. BOC-azide both I I I and IV are formed; they can be easily separated by column chromatography on silicagel. After synthesizing compounds I — I V , we have studied the stability of both BOC-protecting groups of histidine under various reaction conditions. Our results can be summarized as follows: la). Trifluoroacetic acid (100%, 90% and 50% in CH2C12) splits both №-and N ,m-BOC protecting groups of histidine quantitatively, without by-products, in 10 to 15 min. lb). IN HBr in acetic acid splits both №- and Nim-BOC groups totally in 10 to 15 min, giving, however, significant quantities of Nim-acetyl-histidine methylester from I I I and IV as by-product. Addition of 20 to 30% water to the reaction mixture prevents this acetylation. lc). The №-BOC group of I—-IV is cleaved with N HCl in acetic acid in about 60 min. The cleavage of the Nim-BOC protecting group of I I and IV is only partial in this time, but from I I also I, from IV also I I I are formed and the cleavage reaction is not completed even in 24 hours. The following reactions and equilibrium may be supposed: BOC—His (BOC)—OR I BOC—His—OR Formation of Nim-BOC-histidine and Nim-BOC-histidine methylester can be demonstrated by thin layer chromatography. 2a). By hydrolysis both I I I and IV are converted into I. The Nim-BOC group is sensible to bases to a degree, that in a buffer solution of pH 9.2 IV transforms quan-titatively into I I I in an hour. (At pH 8 no hydrolysis of the Nim-BOC group occurs.) 2b). The Nim-BOC protecting group is also cleaved by primary amines. Thus from IV with equiv. benzylamine at 20°C I I I and about 5% N-benzyl-carbamic acid z-butylester, with equiv. n-butylamine I I I and 10% N-n-butyl-carbamic acid r-butylester are formed. Amino acid esters and amino acid amides split Nim-BOC group very slowly. IV transforms with NH3 in methanol into Na-BOC-histidine amide, with hydrazine hydrate to BOC-His-N2H3 [5, 6], by splitting off the Nim-BOC protecting group. 3. The Nim-BOC group is sensitive to higher temperatures, so about the half of IV transforms into I I I in 10% methanolic solution by refluxing for 1 hour. This, — H—His (BOC)—OR I ~ H—His—OR R = H or CH3 STABILITY OF THE N l m - B O C PROTECTING GROUP OF HISTIDINE 167 as well as the sensitivity to bases mentioned above, may explain the failure in syn-thesizing I I I and IV, and the unexpectedly low yields [1]. Summing up our results, it can be stated that №,Nim-di-/-butyloxycarbonyl-histidine (II) can be obtained with good yield only at room temperature and at pH 7.5—8.5. The cleavage of the N^-BOC group is not selective, acidolysis also splits the Nim-BOC group partially or totally. This group is sensitive to nucleophilic attack, too, therefore under the reaction conditions of the solid phase peptide synthesis — during and after the neutralization step—it can react also with the N-terminal amino group by transform-ing it to a BOC-product. A further problem in the solid phase peptide synthesis is the forming of Nim-aminoacyl derivatives of histidine, mentioned in [1], as the N im-BOC group is split by any kind of acidolysis step. Therefore the №-BOC-N im--trityl-histidine [2] and/or Na-BOC-Nim-dinitrophenyl-histidine [8] are to be used for synthesizing the ACTH sequence 5—10. This synthesis shall be dealt with in a further paper. Experimental Melting points were measured by a BOETIUS "Mikroheiztisch"; the values gi-ven are uncorrected. IR spectra were measured with a Unicam SP 200 spectrophoto-meter. For thin layer chromatography Silicagel G (Merck) adsorbent was applied in a layer thickness of 0.25 mm. The following solvent systems were used: System A.: n-butanol—acetic acid—-water 4:1:1 System B.: ethylacetate—pyridine—acetic acid—water 60:20:6:11 N"-t-Butyloxycarbonyl-L-histidine (I) and Nx,Nim-di-t-butyloxycarbonyl-L-histidine (II) a) From 24.2 g (100 mmole) L-histidine hydrochloride by the method of FRIDKIN [4] 14.2 g (40 mmole) II, m.p. 155—157 °C (DCHA-salt) was obtained. In this reaction also I forms; it can be separated from I I by chromatography on silicagel column; with system B as solvent. b) By the method of YAMASHIRO [1], from 15.5 g (100 mmole) L-histidine, 9.05 g (25%) I I was obtained; m.p. 154—157 °C (DCHA-salt). From the aqueous phase, after evaporation (pH 5.6) and extraction with ethanol, 3.5 g (13.7%) I could be separated, m.p. 178—180 °C. c) The method of LOSSE [2] yielded 9 .1 g ( 51 .2%) I from 7 .5 g (50 mmole) L-histidine. N"-t-Butyloxycarbonyl-L-histidine methylester ( I II) and N*,Nim-di-t-butyloxycarbo-nyl L-histidine methylester (IV) I I I and IV were synthesized as described by SCHRODER and GIBIAN [5], from 48.4 g (200 mmole) L-histidine methylester-dihydrochloride. A mixture of I I I and IV was obtained in the form of an amorphous solid; the components were separated by chromatography on silicagel column. 13.0 g (17.6%) IV was eluated with CHC13— acetone 7:3, and then 12.6 g (24%) I I I with CHC13—methanol 9:1. I l l : m.p. 121—122 °C, v™r 3460 (NH), 2970 (CH), 1750 and 1700 (C=0) , 1680 (urethane), 1390and 1360(/-butyl),.1150cm-1 (CO) (broad) Rf0.50,R?0.75. Anal.: C12H19N304 1 6 8 B. PENKE, GY. DOMBI A N D K. KOVÁCS (269.32); Calc.: 53.52 H 7.11 Found: C 53.30H6.93. IV: m.p. 127—128°C, 9390 (NH), 2980 (CH) 1760 and 1710 (C=0) , 1695 (urethane) 1385 and 1340 0-butyl) 1160 (broad) (CO) cm-1 ; RfA 0.65, Rf 0.95. Anal.: C17H2703N6 (369.43) Calc.: C 55.27 H 7.37 Found: C 55.52 H 7.14. Reaction of compounds I — I V with trifluoroacetic acid Compounds I — I V (50 mg each) were reacted with 1 ml trifluoroacetic acid (100%; 90% and 50%, respectively, in CH2C12). Samples were taken from the reac-tion mixtures in 5min intervals and the cleavage of the protecting group determined by thin layer chromatography. After 10—15 min reaction time the BOC protecting groups of I — I V were quantitatively split off, evaporation of the mixtures and addi-tion of ether gave from I and I I histidine trifluoroacetate (hygroscopic crystals, R* 0.10, Rf 0.05); from I I I and IV histidine methylester-di-trifluoroacetate (m.p. 112—115 °C, recrystallized from ethanol—ether, Rf 0.25, Rf 0.10). Reaction of compounds I — I V with 2 N HBr in acetic acid a) 50 mg I — I V were reacted with 1 ml 2N HBr in acetic acid. The progress of the reaction was followed as described above. After 10—15 min the BOC protect-ing groups were split off. From I and II, beside histidine-hydrobromide (Rf 0.10, Rf 0.05), also a small quantity of Nim-acetyl-histidine (Rf 0.25, Rf 0.15); from I I I and IV, beside histidine methylester-dihydrobromide (m.p. 199—200 °C, RfA 0.20, Rf 0.10), a considerable amount of Nim-acetyl-histine methylester (Rf 0.50, Rf 0.40) were obtained. The acetylated histidine derivatives could be isolated from reaction mixtures by ethereal precipitation of the hydrobromides. Identification of the N im--acetyl-histidine derivatives was based on the respective Rf values and IR spectra. b) The same reaction in the presence of 30% water yielded from I and I I only histidine-hydrobromide, from I I I and IV only histidine methylester-dihydrobro-mide; no traces of acetylated derivatives were found. Reaction of compounds I — I V with N HCl in acetic acid 50 mg of I — I V was solved in 1 ml N HCl in acetic acid and the progress of the reaction followed by thin layer chromatography. In 60 min I was converted into histidine, I I I into histidine methylester, which could be isolated from the reaction mixtures as hydrochlorides. The N im-BOC protecting group of I I and IV was not totally cleaved in 60 min; after 24 hours standing from the reaction mixture of I I histidine-hydrochloride (m.p. 255 °C, Rf 0.10, Rf 0.05) and I, from that of IV histidine methylester-dihydrochloride (m.p. 202—204 °C; Rf 0.25, Rf 0.10) and I I I could be isolated. As mentioned, the presence of some N'm-BOC-histidine and Nim-BOC-histidine methylester, respectively, could be detected by thin layer chro-matography. Reaction of I I I and IV with NaOH 1 mmole of I I I or IV was solved in 2 ml methanol and 1 ml NaOH was added. Thin layer chromatography of the reaction mixtures showed that both I I I and I V were converted into I in 1 5 — 2 0 min; this could separated from the reaction mixture with 85% yield by LOSSE'S method [2]. STABILITY OF THE N , m - B O C PROTECTING GROUP OF HISTIDINE 1 6 9 Reaction of I I I and IV with ammonia 50 mg I I I or IV was treated with methanolic ammonia solution. In 2 hours the Nin,-BOC group of IV was quantitatively cleaved and the methyl ester group partly converted into amide. After 24 hours standing, about 50% №-BOC-histidine--amide (R® 0.30) could be separated from both reaction mixtures by fractional crys-tallization or silicagel column chromatography. I Q = H, R = H II Q = (CH3)3C—O—CO R = H m Q = H, R = CH3 IV Q = (CH3)3 C—O—CO R = CHS Fig. 1. Histidine derivatives used for testing the stability of the protecting groups. Reaction of IV with primary amines 50 mg TV, solved in 1 ml abs. dimethylformamide, was reacted with equivalent л-butylamine or benzylamine. After 2—3 hours the Nim-BOC-group of IV was cleaved and only III , beside a small quantity of N-benzyl-carbamic acid /-butyl ester or N-n-butyl-carbamicacid-Z-butyl ester, could be found in the reaction mixture. References [1] Yamashiro, D., J. Blake, С. H. Li: J. Amer. Chem. Soc. 94, 2855 (1972). [2] Losse, G., U. Krychowsky: J. prakt. Chem. 312, 1097 (1970). [3] Schnabel, E. et al: Annalen 716, 175 (1968). [4] Fridkin M., H. J. Goren: Can. J. Chem. 49, 1578 (1971). [5] Schroder E„ H. Gibian: Annalen 656, 190 (1962). [6] Wunsch E., A. Zwick: Chem. Ber. 97, 2497 (1964). [7] Hanford, О. B. et al.: J. Org. Chem. 33, 4251 (1968). [8] Chillemi F., R. B. Merrifield: Biochemistry 8, 4344 (1969). ' ИССЛЕДОВАНИЕ СТАБИЛЬНОСТИ м1т-ТРЕТ-БУТИЛЬНОЙ ГРУППЫ ГИСТИДИНА Б. Пэнкэ, Д. Домби и К. Ковач Разработаны оптимальные условия синтеза №,Ы1т-ВОС-гистидина и исследована ста-бильность N l m-BOC защитной группы в условиях твердофазного синтеза. Нами было дока-зано, что эта защитная группа применяется только в определённых случаях. COOR I N гг—Н2С—СН \ у NH—С—О—С(СН3)3 N II I О 

Investigations on the stability of the Nim-Boc protecting group of histidine

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Investigations on the stability of the Nim-Boc protecting group of histidine

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