Polymer Journal, Vol. 13, No. 12, pp 1151-1154 (1981)

SHORT COMMUNICATION

Convenient Preparation of Poly(L-histidine) by the Direct Polymerization of L-Histidine or Nim_Benzyl-L-histidine with Diphenylphosphoryl Azide

Takumi NARUSE, Bun-ichiro NAKAJIMA, Akihiro TSUTSUMI, and Norio NISHI

Department of Polymer Science, Faculty of Science, Hokkaido University, Nishi 8-chome, Kita 10-jo, Kita-ku, Sapporo 060, Japan.

(Received August 14, 1981)

KEY WORDS Polymerization I Diphenylphosphoryl Azide (DPPA) I L- Histidine I N'm-Benzyl-L-histidine I Poly(L-histidine) I Poly(N'm-benzyl-L­ histidine) I IR Spectrum I 13C NMR Spectrum I Intrinsic Viscosity I

Poly(L-histidine) is a very interesting poly(a­ histidine) by the polymerization of L-histidine with amino acid) as a synthetic functional polymer or as iodine-phosphonic acid esters was unsuccessful. The a model for the functional biopolymer such as simplification of the synthetic route for poly(L­ enzymes. It is known, however, that poly(L­ histidine) is still necessary. histidine) can not be prepared by the Fuchs­ Diphenylphosphoryl azide (DPPA) has been used Farthing method 1 which is very popular for prepar­ as a convenient reagent for racemization-free pep­ ing poly(a-amino acid)s. A synthetic route with tide synthesis, since it was synthesized by Shioiri et several steps involving the synthesis of NCA (a• a!. in 1972.6 Recently, we reported that this reagent amino acid N-carboxyanhydride) by the rather can also be used successfully for the polymerization classical Leuchs method/ have been used for the of amino acids or peptides. 7 -to A polypeptide can preparation ofpoly(L-histidine) as shown in Scheme easily be prepared by simply stirring the solution or I-(A)? Among the many successful attempts for the the suspension of amino acid or peptide in a suitable direct polycondensation of amino acids,4 •5 that of solvent with DPPA and a tertiary amine such as Guilly et a/. 5 for the one-step preparation ofpoly(L- triethylamine. One of the merits ofDPPA in peptide

Bzl-Cl, Na Z-Cl (A) H-His-OH H-His(Bzl)-OH ->Z-His(Bzl)-OH NH3 NaOH

PCI5 Polymerization ->(-COCI)

Poly[His(Bzl)) NH3

. DPPA p I (H. ) (B) H-Hts-OH o y ts Base H-His(Bzl)-OH DPPA Poly[His(Bzl)) Base NH3 Scheme I. Synthetic routes of poly(L-histidine) (A) with the NCA method proposed by Patchonl.ik et al./ and (B) with DPPA method: Bzl, benzyl; Z, benzyloxycarbonyl.

1151 T. NARUSE et a/.

synthesis is that the protection of imidazole group is In the present study, the one-step preparation of not necessary for the preparation of histidine­ poly(L-histidine) by the polymerization of L­ containing peptide/ 1 since the reaction proceeds via histidine with DPPA was investigated to simplify acyl azide12 similarly as in the case of the well­ the preparative method for poly(L-histidine). The known azide coupling method. polymerization of N im_benzyl-L-histidine to give

4000 3000 Wave Number (cm-1) Figure 1. Infrared absorption spectra ofpoly(L-histidine)s prepared by (a) DPPA method, and (b) NCA method.3

(a)

crJ. ct3 Dioxane

(b)

200 150 100 50 0 ppm from TMS

Figure 2. 13C NMR spectra of poly(L-histidine)s in aqueous solution (pD 3) measured at 15.04 MHz: repetition time, 2s; (a) DPPA method; (b) NCA method. 3

1152 Polymer J., Vol. 13, No. 12, 1981 Convenient Preparation of Poly(L-histidine) with DPPA

poly(N im_benzyl-L-histidine) has also been reported. ular weight peptides by the thin-layer chromatog­ The synthetic routes proposed in the present study raphies carried out on the mother liquor. However, are shown in Scheme I-(B). this yield of poly(L-histidine) is no worse than the To stirred suspensions of L-histidine (500 mg) in overall yield by the NCA method, since the syn­ DMSO or some other solvent were added DPPA thetic route via NCA requires several reaction steps. and triethylamine or another base. These mixtures The polymerizations of N im_benzyl-L-histidine were stirred for I h at 5-8°C followed by stirring were also investigated. The polymers were pre­ under various reaction conditions. Variation in the cipitated with 5% aqueous citric acid saturated with reactions was made by changing the amounts of sodium chloride. The sirupy precipitates were col­ solvent or reagent, reaction time and other reaction lected and washed with 5% aqueous citric acid conditions. The polymers were precipitated by the ( x 3), water ( x 3), warm 5% aqueous sodium bicar­ addition of saturated aqueous sodium chloride bonate ( x 3), and then with water ( x 3) by de­ (I 00 ml). The sirupy precipitates were collected and cantation. The residues were treated with ether to washed with water ( x 3), 5%,aqueous sodium bicar­ give the powders which were centrifuged, washed bonate ( x 3), and with water ( x 3), by decantation. with ether and then dried in vacuo. The results were The residues were treated with acetone to give the compared with each other as in the case of the powders which were centrifuged and washed with polymerizations of L-histidine. Consequently, the acetone ( x 3), methanol ( x 3), and ether ( x 1), and optimum reaction conditions were determined as then dried in vacuo. follow: Reaction time of one day at room tempera­ The IR and 13C NMR spectra of poly(L­ ture employing 0.6 ml of DMSO, 1.3 equiv mol of histidine) thus obtained were found to show the DPPA and 2.3 equiv mol of triethylamine to 300 mg same patterns as those of poly(L-histidine)3 pre­ of Nim_benzyl-L-histidine, followed by the reaction pared by the NCA method shown in Figures I and for another one day with the additional same 2. amount of solvent and reagents, and final heating at The reaction conditions, such as a reaction time 90°C for one hour at the end of the polymerization. of two days at room temperature or a reaction time Poly(N im_benzyl-L-histidine), which shows an in­ of six days at 5-8°C using I ml of DMSO, 1.3 trinsic viscosity ([ry]) of 0.12 in DCA at 25°C, can equiv mol of DPPA and 2.3 equiv mol of tri­ be obtained in a 46% yield by these optimum ethylamine to 500 mg of L-histidine, were con­ conditions. sidered to be most suitable. Under these reaction TheIR spectrum of poly(Nim_benzyl-L-histidine) conditions, poly(L-histidine) of [ry]=0.16 (in DCA thus obtained showed the same pattern as that of at 25°C) can be obtained in 24-25% yield directly poly(Nim_benzyl-L-histidine)3 prepared by NCA from L-histidine. The cause of the low yield is method as shown in Figure 3. considered due to the removal of very low molec- The amino acid analyses and the silica-gel thin-

3500 3000 1700 1500 1000 500 Wave Number (cm-1) Figure 3. Infrared absorption spectra of poly(N'm-benzyl-L-histidine)s prepared by (a) DPPA method, and (b) NCA method.3

Polymer J., Vol. 13, No. 12, 1981 1153 T. NARUSE et ai. layer chromatographies, carried out on two dif­ ported by a. Grant-in-Aid for Scientific Research ferent solvent systems (upper layer of 1-butanol­ from the Ministry of Educations, Science and acetic acid-water=4: I: 5 and 1-butanol-pyridine­ Culture of Japan. acetic acid-water= IS: 10:3: 12), for the acid hy­ drolysates ofpoly(L-histidine) and poly(Nim_benzyl­ REFERENCES L-histidine) showed only starting amino acids. This, along with the IR and NMR spectra, suggest the I. A. C. Farthing and R. J. W. Reynolds, Nature, 165, absence of side reactions induced by the Curtius 647 (1950). rearrangement which sometimes accompanies azide 2. H. Leuchs, Bericht., 37, 857 (1906). 3. A. Patchornik, A. Berger, and E. Katchalski, J. Am. coupling. Chern. Soc., 79, 5227 (1957). It was found that poly(L-histidine) and poly(Nim_ 4. F. Higashi, K. Sano, and H. Kakinoki, J. Poiym. benzyl-L-histidine) could be prepared directly from Sci., Poiym. Chern. Ed., 18, 1841 (1980). L-histidine and N im_benzyl-L-histidine, respectively, 5. L. L. Guilly, A. Brack, and G. Spach, Macromoi. by the DPPA method. The procedures for the Chern., 179, 2829 (1978). polymerization reaction or working up are simple 6. T. Shioiri, K. Ninomiya, and S. Yamada, J. Am. and easy. This novel method may be recommended Chern. Soc., 94, 6203 (1972). 7. N. Nishi, B. Nakajima, N. Hasebe, and J. Noguchi, as a convenient synthetic method for poly(L­ Int. J. Bioi. Macromoi., 2, 53 (1980). histidine). 8. B. Nakajima and N. Nishi, Poiym. J., 13, 183 (1981). Further investigation will be made for obtaining 9. B. Nakajima, K. Hirata, N. Nishi, and J. Noguchi, poly(L-histidine) of higher molecular weight in Int. J. Bioi. Macromoi., 3, 46 (1981). higher yield. In particular, polymerization re­ 10. N. Nishi, Peptide Chemistry 1978, Protein Research actions using polymer matrices must be investi­ Foundation, Osaka, 1979, p 151. 11. T. Shioiri and S. Yamada, Chern. Pharm. Bull., 22, gated in further detail. 859 (1974). 12. T. Shioiri and S. Yamada, Chern. Pharm. Bull., 22, Acknowledgment. The present work was sup- 855 (1974).

1154 Polymer J., Vol. 13, No. 12, 1981