Biochimica et Biophysica Acta, 491 (1977) 253-264

© Elsevier/North-Holland Biomedical Press BBA 37608 REACTIONS OF P O L Y F U N C T I O N A L A M I N O ACIDS W I T H N,N'-CARB O N Y L D I I M I D A Z O L E IN AQUEOUS SOLUTION - O L I G O P E P T I D E FORMATION

K. W. EHLER, E. GIRARD and L. E. ORGEL The Salk Institute ]'or Biological Studies, San Diego, Calif. 92112 (U.S.A.)

(Received October 8th, 1976)

SUMMARY 1. Serine reacts with N,N'-carbonyldiimidazole in aqueous solution at 0 °C to yield Na-[imidazolyl-(l)-carbonyl]-L-serine. This compound slowly transforms to L-2oxo-oxazolidine-5-carboxylic acid. We found that L-2-oxo-oxazolidine-5-carboxylic acid was stable to hydrolysis under a variety of conditions and did not oligomerize to yield peptides. Threonine was found to react in an analogous manner with N , N ' carbonyldiimidazole, yielding Na-[imidazolyl-(1)-carbonyl]-L-threonine and L-(÷)trans-5-methyl-2-oxo-oxazolidine-5-carboxylic acid. 2. Histidine reacts with N,N'-carbonyldiimidazole in aqueous solution at 0 °C to yield a variety of histidine-containing intermediates. These slowly transform to give 7-carboxy-imidazole-[1,5c]-tetrahydropyrimidin-5-one in up to 90 ~ yield. 3. T~e polymerization of 7-carboxy-imidazo-[1,5c]-tetrahydropyrimidin-5-one in imidazo|e buffer at 30 ° gives excellent yields of oligohistidines.

INTRODUCTION We have previously shown that carbonyldiimidazole (I) reacts with glycine in aqueous solution to generate N-[imidazolyl-(1)-carbonyl]-glycine [1]. At pH 7.0-7.3, 0 °C this intermediate polymerizes slowly via an N-carboxyanhydride [1, 2]. We have now found parallels between the chemistry of the N-carboxyanhydrides formed from polyfunctional amino acids and the chemistry of products (e.g. (II)) formed from (I) and these amino acids. Hirschmann et al. [3] investigated the reactions of N-carboxyanhydrides of a number of polyfunctional amino acids in aqueous solution at 0-2 °C. They showed [3] that the treatment of amino acids or peptides with the N-carboxyanhydride of serine at pH 10.2, 0 °C, did not lead to the incorporation of serine into peptides. They attributed this to the ease with which the N-carboxyanhydride of serine is known to rearrange to L-2-oxo-oxazolidine-5-carboxylic acid (IIIa) [4]. Hirschmann et al. also showed that at pH 7.5, the N-carboxyanhydride of histidine decomposed to give about equal amounts of histidine and 7-carboxy-imidazo-[1,5c]-tetrahydropyrimidin5-one (IV) [3].

254

N~,,/N-- C-- N~ . N 0 (T)

N'~ .N-C-NH-CH--CO:#-~ ~ ~ R~HOH (TT} (a,R=H; b,R =CHa) H

COL~.I .

(TIT) (o,R=H;b,R=CHa)

OyN'~

I

C02H

('IV)

In this report we show that L-serine, L-threonine and L-histidine react with (I) in aqueous solution to give the same cyclic intermediates that are formed from their N-carboxyanhydrides. Furthermore, we confirmed that no peptides are formed from serine and theronine. We do, however, obtain excellent yields of oligohistidines from (IV) at 30 °C in imidazole buffer. We believe that this work will provide the basis for a practical synthesis of oligohistidines and of random peptides rich in histidine. EXPERIMENTAL

Materials and analytical methods N,N'-Carbonyldiimidazole was purchased from Sigma; ethyl acetate, AR, from Mallinckrodt; L-histidine, free base, from Mann Research Lab; leucine aminopeptidase from Worthington (Lot LAPC-N5P-569); L-histidyl-L-histidine acetate and L-seryl-L-serine from Vega-Fox Biochemicals; imidazole from Matheson, Coleman, and Bell; phosgene from Matheson; L-serine and L-threonine from Calbiochem; Sephadex G-10 from Pharmacia; and carboxymethylcellulose (Microgranular, CM32) from Whatman. Radioactive L-[U-14C]histidine was purchased from Amersham-Searle and radioactive L-[a-:4C]serine and L-[a-~4C]threonine from Schwarz-Mann. The color reagents used to visualize functional groups were ninhydrin spray, chlorine-starch-iodide spray, and sulfanilamide spray [1]. Paper electrophoresis was done on Whatman 3MM paper, using Varsol as coolant. The buffers were: I, 0.05 M formic acid adjusted to pH 2.7 with conc. ammonia; II, 0.03 M potassium phosphate, pH 7.1 ; III, 0.05 M sodium borate, pH 8.5. Mobility values for all relevant compounds are given in Table I. Electrophoretograms of radioactive samples were cut into strips and passed through a Baird-Atomic RSC-363 scanner with integrator. The yields were determined as the percentage of the total radioactivity after correcting for background. Microanalysis was done by Midwest Microlab, Ltd., Indianapolis, Ind., 46226. Nuclear magnetic resonance spectra were recorded on a Jeolco-110 megahertz spectrometer. Infrared spectra were recorded on a Perkin-Elmer model 2378 grating infrared spectrophotometer. Melting points were done with capillary tubes on a MelTemp apparatus and are uncorrected.

255 TABLE 1 ELECTROPHORETIC MOBILITIES OF THE PRODUCTS FROM THE REACTION OF SERINE, THREONINE OR HISTIDINE WITH CARBONYLDIIMIDAZOLE

Serine Threonine (IIa)d (Ilia)e (llb) f (lllb) g Serylserine Histidine Histidylhistidine (IV)"

I Rm a

II R~ b III R m e

-- 1.0 Jr 1.0 --0.4 --8.2 --0.2 --6.1 +2.5 + 1.0 + 1.1 --0.3

0.0 0.0 -- 1.0 --1.4 -1.0 --1.3 0.0 0.0 -- I. 1

0.0 -- 1.0 -- 1.8

a Mobilities of derivatives given relative to parent amino acid; conditions, 52 V/cm, 1 h for serine, threonine and their derivatives; 70 V/cm, 1 h for histidine and its derivatives. b Mobilities given relative to (IIa); conditions, 52 v/cm, 1 h. c Mobilities given relative to histidylhistidine; conditions 70 V/cm, 1 h. d (lla) = Na-[imidazolyl-(l)-carbonyl]-L-serine. e (Ilia) = L-2-oxo-oxazolidine-5-carboxylicacid. f (lib) = Na-[imidazolyl-(1)-carbonyl]-L-threonine. z (lllb) = L-(..)-trans-5-methyl-2-oxo-oxazolidine-5-carboxylic acid. h (IV) -- 7-carboxy-imidazo-[1,5c]-tetrahydropyrimidin-5-one.

Analysis of the reactions of polyfunctional amino acids in imidazole buffer with carbonyldiimidazole (I) 250 bd o f 0.08 M (a-14C)-labeled a m i n o acid (0.125 Ci/mol, 0.02 m m o l ) in 0.4 M imidazole buffer at p H 6.8 was placed in a test tube. Solid c a r b o n y l d i i m i d a z o l e (I) (0.02 m m o l o r 0.04 m m o l ) was a d d e d to the tube a n d dissolved as r a p i d l y as possible at 0 °C. T h e resulting solutions always h a d a p H o f a b o u t 7.1. A l i q u o t s o f 5/A were t a k e n at v a r i o u s times a n d analyzed in systems I a n d II o r III. E l e c t r o p h o r e t o g r a m s in system I o f a reaction mixture f r o m serine a n d one equivalent o f ( I ) at the beginning o f the reaction a n d after 3 a n d 21 h at 0 °C are shown in Fig. 1. This system is used to determine the yield o f (IIIa). System II is used to d e t e r m i n e the a m o u n t o f u n r e a c t e d serine (see Table I), b u t does n o t p e r m i t the d e t e r m i n a t i o n o f ( I l i a ) , since (IIa) is t r a n s f o r m e d to ( I l i a ) d u r i n g the electrophoresis. R e a c t i o n mixtures f r o m t h r e o n i n e a n d (I) show a l m o s t identical e l e c t r o p h o r e t i c p a t t e r n s to those f r o m serine. E l e c t r o p h o r e t o g r a m s in system I o f a reaction mixture f r o m histidine a n d two equivalents o f (I) at the b e g i n n i n g o f the reaction a n d after 21 a n d 140 h at 0 °C are shown in Fig. 2. This system p r o v e d m o s t reliable for following the t r a n s f o r m a t i o n o f n u m e r o u s intermediates, e.g. those in p e a k s A a n d B, to (IV). H o w e v e r , it failed to resolve histidine f r o m A ; system II was used for this purpose. System I I I p r o v e d m o s t reliable for the analysis o f oligohistidines (see Fig. 3). All intermediates a n d p r o d u c t s showed positive reactions to sulfanilamide a n d n i n h y d r i n sprays. The n a t u r e o f the i n t e r m e d i a t e s giving p e a k s A a n d B is t a k e n u p in the discussion.

256

[g)

*

~' r'-----~

*

(hi

|,)

( m a)

A

5tA i

,ill

Fig. 1. Radioscans of electrophoretograms in system 1 of a reaction mixture from serine and one equivalent of carbonyldiimidazole at (a) the beginning of the reaction and after (b) 3 h and (c) 21 h at 0 °C. The electrophoreses were run for 1 h in a varsol tank with a gradient of 52 V/cm. Percentage yields are indicated by numbers at the bottom of the peaks. In this and all remaining figures, the origin is indicated by an arrow and markers by stars.

Isolation of L-2-oxo-oxazolidine-5-carboxylic acid (Ilia) from the reaction of L-serine in imidazole buffer with (I) A solution of L-serine (2.62 g, 25 mmol) in water (25 ml) at pH 5.1 was cooled to 0 °C. An equimolar amount of carbonyldiimidazole (I) (4.05 g, 25 mmol) was added at once, and the solution stirred vigorously. After 10 rain, all of (I) had dissolved and the solution was at pH 7.5. Electrophoresis in systems I and II showed the presence of 90 ~o compound (Ilia) after 96 h at 5 °C. The solution, which had a pH of 7.6, was acidified to pH 1 with 6 M HC1 (approx. 8 ml) at 0 °C. The residue obtained after lyophilization was extracted with hot ethyl acetate (5 × 25 ml). The ethyl acetate solution was dried with anhydrous sodium sulfate, evaporated to dryness, and the resulting solid was recrystallized twice from hot ethyl acetate/ligroin to give 291 mg of product (8 ~). The product was identified by mixture melting point (115-116 °C) with a sample prepared by the procedure of Kaneko et al. [5]. The two samples also gave identical infrared spectra. N MR, ppm ([C2Hs]2SO): 13.2 (1 H, broad, exchanges with 2H20),

257 (o)

+

i (b)

(c)

Fig. 2. Radioscans of electrophoretograms in system I of a reaction mixture from histidine and two equivalents of carbonyldiimidazole at (a) the beginning of the reaction, and after (b) 21 h and (c) 140 h at 0 °C. The electrophoreses were run for 1 h in a varsol tank with a gradient of 70 V/cm. Percentage yields are indicated by numbers at the bottom of the peaks. 8.20 (1 H, s, exchanges with H20), 4.25 (1 H, m), and 4.28 (1 H, m). The synthetic material co-migrated in electrophoresis systems I and II with the major product obtained from (I) and serine in aqueous solution, and like that material failed to give a color reaction with either sulfanilamide or ninhydrin sprays, but gave a positive reaction with chlorine/starch/iodide spray.

Isolation of L-(+)-trans-5-methyl-2-oxo-oxazolidine-5-carboxylic acid from the reaction of L-threonine in imidazole buffer with (I) A procedure very similar to that used for preparation of (IIIa) from serine and (I) gave 170 mg of product (9 ~). The product was identified by mixture melting point (135-136 °C) with a sample prepared by the procedure of Inui and Kaneko [6]. The two samples also gave identical infrared spectra. N M R , p p m ([C2H312SO): 13.2 (1 H, broad), 8.20 (1 H, s), 4.60 (1 H, quintuplet), 4.00 (1 H, d), 1.4 (3 H, d). This material was found to co-migrate in electrophoresis systems I and II with the main product obtained

258

(b)

Fig. 3. Radioscan of electrophoretograms in system III of a solution of 7-carboxy-imidazo-[l,5c]tetrahydropyrimidin-5-one (IV), obtained from a reaction of 0.08 M histidine with 2 equivalents of (I). (IV) was formed in 90~ yield over a period of 65 h at 0 °C. The solution was then maintained at 30 °C for (a) 7 h, (b) 22 h, and (c) 136 h. Electrophoreses were run for 1 h in a varsol tank with a gradient of 70 V/cm. f r o m (I) and threonine, and like that material failed to give a color reaction with either sulfanilamide or ninhydrin sprays but gave a positive reaction with chlorine/ starch/iodide spray.

Study of the hydrolysis of L-2-oxo-oxazolidine-4-carboxylic acid (Ilia) in aqueous solution Three solutions o f (Ilia) (13.1 mg, 0.1 mmol) in 1 ml o f 0.4 M imidazole buffer were brought respectively to p H 5, 7, 9 and kept at 30 °C. System I was used to follow these reactions (see Table I). After 4 days at 30 °C, the cyclic carbamate was essentially unchanged. However, after 3 days at 65 °C partial hydrolysis o f the carbamate to L-serine occurred at each o f the p H values. N o peptides were detected.

Synthesis of 7-carboxy-imidazo[1,5c-]-tetrahydropyrimidin-5-one (IV) e-Histidine free base (7.8 g, 50 mmol) in 100 ml o f 0.5 M K O H at 0 °C was treated with a solution of approximately 6.2 g (60 mmol) o f phosgene in 12 ml o f toluene over anhyd potassium carbonate. The reaction was stirred rapidly at 0-5 °C for 3 h. Then the reaction mixture was poured into a separatory funnel. The layers were separated and the toluene layer washed with three 20-ml portions o f deionized

259 water, the washes combined with the aqueous layer, and taken to pH 3.5. Between pH 4.0 and 3.5 a precipitate formed, which was collected, washed with deionized water (20 ml) and ethyl acetate (3 × 20 ml) and dried in vacuum at room temperature (1 mmHg) for 24 h. The yield of white amorphous solid was 2.7 g (30~). This substance does not melt, but changes to a yellow plastic substance from 190-200 °C. If the substance in a m p capillary is placed on a Mel-temp block at 195 °C, the substance changes to a yellow plastic substance from 199-200 °C; infrared maxima (cm -1) nujol: 1760 (carbamyl imidazole carbonyl), 1570 (carboxylate anion), 3460 (secondary amide); N M R (ppm) ([CZH3]2SO): 8.6-9.4 (1 H, broad, exchange w. 2H20 ), 8.52 (1 H, s), 8.20 (1 H, m, exchanges w. 2H20), 6.4-7.4 (broad, exchanges w. 2H20, and shifts to 4.2~.5), 3.0-3.5 (3 H, broad, m). The sample polymerizes during the measurement. Thus unambiguous assignment of the other aromatic proton, which is under the 6.4-7.4 water peak, is difficult; (2H20): 9.04 (I H, s), 7.56 (1 H, s), 4.58 (1 H, t, J = 7 Hz), 3.56 (2 H, d, J = 7 Hz). Polymerization occurred during sample preparation since heating of the sample was necessary to dissolve it. All of the assignments given are for peaks arising out of complicated multiplets. (Found: C, 44.56; H, 4.10; N, 22.50. CTH6N303 0.5 H20 requires C. 44.45; H, 3.73; N, 22.16). The synthetic material co-migrates in systems I and Ill with the major product obtained from (I) and histidine in aqueous solution at 0 °C.

Chromatographic separation of oligohistidines The basic electrophoresis system III proved excellent for the analytical separation of oligohistidines (Fig. 3c). For preparative purposes we used the following procedure. A sample of L-histidine (93.0 mg, 0.6 mmol) in a graduated, thick-walled centrifuge tube was dissolved in 0.1 M aq. acetic acid. The pH was adjusted to 6.86.9 with 2 M NaOH to give approximately 0.075 M L-histidine. Carbonyldiimidazole (196 mg, 1.2 mmol) was added at 0 °C. After 15 min at 0 °C the solution was brought to 30 °C. After 8 days at 30 °C, the solution was evaporated on a Buchler evapo-mix apparatus at 0.1 mmHg, 30 °C. The resulting gel-like material was dissolved in 1 ml of de-aerated 0.2 M aq. acetic acid and applied to a Sephadex G-10 column (40-120 #m; radius, 0.4 cm; height, 45.7 cm) prepared in de-aerated 0.2 M aqueous acetic acid, which was also the eluent. The first 30 ml of eluate (about one column volume) contained 85-90 ~ of the oligohistidines with less than 1 ~o imidazole, as determined by electrophoresis in system I. The material that eluted from the column later, together with the imidazole, did not contain oligomers longer than the trimer . . . . The oligohistidine-containing fraction was lyophilized (0.5 mmHg), dissolved in deionized water (de-aerated) brought to pH 6.5 with 2 M NaOH, and applied to a carboxymethyl cellulose column [7, 8] (carboxyl form; radius, 0.5 cm; height, 51.2 cm). The material was eluted using a convex gradient (mixing flask, 500 ml of deionized water; addition flask 500 ml of de-aerated 0.2 M ammonium acetate, pH 7.0). Elution of His4 and higher oligomers required substitution of 0.4 M ammonium acetate as indicated in Fig. 4. The tubes containing the separate fractions were combined and lyophilized (0.05 mmHg) to yield extremely hydroscopic powders. These powders were dissolved in sufficient deionized water (50-200/A) to give solutions 2050 mM in total histidine. When examined by electrophoresis in system III, the frac-

260

0 n hl

30 5

< .J Z

~ 20 _J

3

~-0 I.-HIS

z io bZ W > I-