JP2005075765A - New non-natural histidine analog amino acid - Google Patents

New non-natural histidine analog amino acid Download PDF

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JP2005075765A
JP2005075765A JP2003307192A JP2003307192A JP2005075765A JP 2005075765 A JP2005075765 A JP 2005075765A JP 2003307192 A JP2003307192 A JP 2003307192A JP 2003307192 A JP2003307192 A JP 2003307192A JP 2005075765 A JP2005075765 A JP 2005075765A
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histidine
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Kazumasa Tahira
和誠 多比良
Yutaka Ikeda
豊 池田
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University of Tokyo NUC
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Abstract

<P>PROBLEM TO BE SOLVED: To obtain a new non-natural amino acid having structure different from that of a naturally occurring amino acid in order to obtain protein having new properties. <P>SOLUTION: β-(1,2,3-Triazol-4-yl)-DL-alanine being a new non-natural histidine analog is obtained. The new amino acid is transferred to protein (chitin-binding domain) by using histidine-requiring Escherichia coli. The non-natural histidine analog provides a new tool for supplying a new function to protein. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

本発明は、下記の化学式

Figure 2005075765

からなる構造を有することを特徴とするアミノ酸に関する。即ち、本発明は新規の非天然ヒスチジンアナログである、β-(1,2,3-トリアゾール-4-イル)-DL-アラニンに関する。 The present invention has the following chemical formula:
Figure 2005075765
It relates to an amino acid characterized by having a structure consisting of That is, the present invention relates to β- (1,2,3-triazol-4-yl) -DL-alanine, which is a novel unnatural histidine analog.

天然のタンパク質は２０種類のアミノ酸から構成されており、それら２０種類のアミノ酸中には酸として機能するもの、塩基として機能するもの、疎水性のもの、親水性のものなどがある。それらが巧みな配列と高次構造を構成することにより、タンパク質は様々な機能を担っている。このアミノ酸配列を人工的に設計することにより、またランダム変異によりin vivo又はin vitro選択などを行うことにより、タンパク質をエンジニアリングすることが最近盛んに行われている。   Natural proteins are composed of 20 types of amino acids, and these 20 types of amino acids include those that function as acids, those that function as bases, those that are hydrophobic, and those that are hydrophilic. Proteins are responsible for various functions because they constitute clever sequences and higher order structures. Recently, protein engineering has been actively performed by artificially designing this amino acid sequence, or by performing in vivo or in vitro selection by random mutation.

しかしながら、天然に存在するアミノ酸だけではアミノ酸の化学的性質（酸性・塩基性の強さ）に限界があるため、酵素活性のpH依存性や温度依存性の向上または変化を試みても限界があると考えられる。そこで近年天然に存在しないアミノ酸の合成なども試みられており、その一環として、米国などを中心にヒスチジンのアナログなどが報告されている。下記の実施例において使用したヒスチジン(化合物1)及びそのアナログ(化合物2〜5)の構造を図１に示す。   However, since only naturally occurring amino acids have limited amino acid chemical properties (acidity and basicity), there are limits to attempts to improve or change the pH dependence and temperature dependence of enzyme activity. it is conceivable that. Therefore, in recent years, attempts have been made to synthesize amino acids that do not exist in nature, and as part of this, histidine analogs have been reported mainly in the United States. The structures of histidine (compound 1) and analogs (compounds 2 to 5) used in the following examples are shown in FIG.

例えば図1の化合物(3)のアミノ酸については１９５５年にアメリカで特許が取得されており（米国特許番号 2,719,849：特許文献１）高血圧の薬の中間体として有用であると言われている。また、図1の化合物(4)のアミノ酸についてはN-methyl-D-aspartate(HMDA) receptor の拮抗薬としての可能性が示唆されている（Bioorganic & Medical chemistry letters (1993) 3 (1) 43-48.：非特許文献１）。   For example, the amino acid of the compound (3) in FIG. 1 was patented in the United States in 1955 (US Pat. No. 2,719,849: Patent Document 1) and is said to be useful as an intermediate for drugs for hypertension. In addition, the amino acid of compound (4) in Fig. 1 has been suggested as a potential antagonist of N-methyl-D-aspartate (HMDA) receptor (Bioorganic & Medical chemistry letters (1993) 3 (1) 43 -48 .: Non-patent document 1).

米国特許番号 2,719,849US Patent No. 2,719,849 Bioorganic & Medical chemistry letters (1993) 3 (1) 43-48.Bioorganic & Medical chemistry letters (1993) 3 (1) 43-48.

そこで本発明者は、タンパク質の機能を改変するための新たなツールを得るために、天然には存在しないアミノ酸を設計して合成することを考えた。よって天然に存在するアミノ酸とは異なった化学構造を有する、新規な非天然のアミノ酸を得ることが本発明の課題である。更に、そのようにして得られた非天然のアミノ酸をタンパク質中に導入することにより、タンパク質に新たな機能を付与する途を開くこともまた本発明の課題である。   Therefore, the present inventor has considered to design and synthesize amino acids that do not exist in nature in order to obtain new tools for modifying protein functions. Therefore, it is an object of the present invention to obtain a new unnatural amino acid having a chemical structure different from that of naturally occurring amino acids. Furthermore, it is also an object of the present invention to introduce a new function to the protein by introducing the unnatural amino acid thus obtained into the protein.

よって本発明により、新規な非天然ヒスチジンアナログであるβ-(1,2,3-トリアゾール-4-イル)-DL-アラニンが与えられた。当該アミノ酸は、ヒスチジンよりも酸として強いアナログを提供するものである。また、大腸菌によるシステムを用いて、当該新規アミノ酸をタンパク質（キチンバインディングドメイン）に導入することに成功した。   Thus, according to the present invention, β- (1,2,3-triazol-4-yl) -DL-alanine, a novel unnatural histidine analog, was provided. The amino acid provides an analog that is stronger as an acid than histidine. In addition, the novel amino acid was successfully introduced into a protein (chitin binding domain) using a system using E. coli.

ヒスチジンは酸解離定数が７であり、様々なタンパク質において酸・塩基触媒として働いており、ヒスチジンのイミダゾール基は種々の酵素において重要な役割を果たしている。本発明の非天然ヒスチジンアナログは、天然のヒスチジンのイミダゾール環（窒素原子２つ）をトリアゾール環（窒素原子３つ）にしたものであって構造が非常によく似ている。よって、本発明の非天然ヒスチジンアナログをタンパク質中に導入しても、その構造をほとんど歪めないと考えられる。本発明の新規ヒスチジンアナログに含まれるトリアゾール環はイミダゾール環に比べて酸性が強く（pKa=2）、酸触媒としての機能が強い。よって、タンパク質中においてヒスチジンが酸として働いている部分に本発明のヒスチジンアナログを導入すれば、その活性が上がると考えられる。天然のヒスチジン（化学式２）と本発明の新規ヒスチジンアナログ（化学式３）の構造を示す。   Histidine has an acid dissociation constant of 7 and acts as an acid / base catalyst in various proteins, and the imidazole group of histidine plays an important role in various enzymes. The non-natural histidine analog of the present invention is a structure in which the imidazole ring (two nitrogen atoms) of natural histidine is changed to a triazole ring (three nitrogen atoms), and the structure is very similar. Therefore, even when the non-natural histidine analog of the present invention is introduced into a protein, the structure is hardly distorted. The triazole ring contained in the novel histidine analog of the present invention is more acidic (pKa = 2) than the imidazole ring and has a strong function as an acid catalyst. Therefore, if the histidine analog of the present invention is introduced into the protein where histidine acts as an acid, its activity is considered to increase. The structures of natural histidine (Chemical Formula 2) and the novel histidine analog of the present invention (Chemical Formula 3) are shown.

Figure 2005075765
Figure 2005075765

Figure 2005075765
Figure 2005075765

本発明の非天然ヒスチジンアナログであるβ-(1,2,3-トリアゾール-4-イル)-DL-アラニンは、好ましくは図２に示す合成スキームにより合成される。しかし合成スキームはそれに限定されるものではなく、化学合成の分野で通常に行われる種々の改変した方法を用いることも当然に可能である。よって当業者の創意工夫によって種々の合成方法の改変や改良を行うことも、本発明の範囲内であると解されるべきである。   Β- (1,2,3-triazol-4-yl) -DL-alanine, an unnatural histidine analog of the present invention, is preferably synthesized according to the synthetic scheme shown in FIG. However, the synthetic scheme is not limited thereto, and it is naturally possible to use various modified methods usually performed in the field of chemical synthesis. Therefore, it should be understood that various synthesis methods can be modified or improved by the ingenuity of those skilled in the art within the scope of the present invention.

また、下記の実施例において、本発明の非天然ヒスチジンアナログであるβ-(1,2,3-トリアゾール-4-イル)-DL-アラニンを、ヒスチジン要求性の大腸菌の系を用いて、タンパク質であるキチンバインディングドメインに導入している。本発明の非天然ヒスチジンアナログを導入するタンパク質はキチンバインディングドメインに限定されるものではなく、その目的に応じて種々のタンパク質中に本発明の非天然ヒスチジンアナログを導入することが可能である。   Further, in the following examples, β- (1,2,3-triazol-4-yl) -DL-alanine, which is an unnatural histidine analog of the present invention, is converted into a protein using a histidine-requiring Escherichia coli system. It has been introduced into the chitin binding domain. The protein for introducing the non-natural histidine analog of the present invention is not limited to the chitin binding domain, and the non-natural histidine analog of the present invention can be introduced into various proteins according to the purpose.

非天然アミノ酸を導入するために利用できる方法はいくつか存在する(Liu, D. R., Schultz, P. G. (1999) Proc. Natl. Acad. Sci. USA, 96, 4780-4785； Kochendoerfer, G. G., Kent, S. B. (1999) Curr. Opin. Chem. Biol., 3, 665-671; Sisido, M., Hohsaka, T. (1999) Bull. Chem. Soc. Jpn., 72, 1409-1425)。また、天然のアミノ酸の他にいくつかのアナログをタンパク質に導入した例が報告されている(Koide, H., Yokoyama S., Katayama, Y., Muto, Y., Kigawa, T., Kohno, T., Takusari, H., Oishi, M., Takahashi, S., Tsukumoto, K et al. (1994) Biochemistry., 33, 7470-7476; Koide, H., Yokoyama S., Kawai, G., Ha JM., Oka, T., Kawai, S., Miyake, T., Fuwa, T., Miyazawa, T. (1988) Proc. Natl. Acad. Sci. USA.., 85, 6237-6241; van Hest, J. C. M., Kiick, K. L., Tirrell, D. A. (2000) J. Am. Chem. Soc., 122, 1282-1288)。そして、翻訳されたタンパク質について検討されたところ、非天然のアミノ酸は天然のアミノ酸を類似した構造を示したという報告がされている(van Hest, J. C. M., Kiick, K. L., Tirrell, D. A. (2000) J. Am. Chem. Soc., 122, 1282-1288)。   There are several methods available to introduce unnatural amino acids (Liu, DR, Schultz, PG (1999) Proc. Natl. Acad. Sci. USA, 96, 4780-4785; Kochendoerfer, GG, Kent, SB (1999) Curr. Opin. Chem. Biol., 3, 665-671; Sisido, M., Hohsaka, T. (1999) Bull. Chem. Soc. Jpn., 72, 1409-1425). In addition to the natural amino acids, some analogs have been introduced into proteins (Koide, H., Yokoyama S., Katayama, Y., Muto, Y., Kigawa, T., Kohno, T., Takusari, H., Oishi, M., Takahashi, S., Tsukumoto, K et al. (1994) Biochemistry., 33, 7470-7476; Koide, H., Yokoyama S., Kawai, G., Ha JM., Oka, T., Kawai, S., Miyake, T., Fuwa, T., Miyazawa, T. (1988) Proc. Natl. Acad. Sci. USA .., 85, 6237-6241; van Hest, JCM, Kiick, KL, Tirrell, DA (2000) J. Am. Chem. Soc., 122, 1282-1288). When the translated protein was examined, it was reported that unnatural amino acids showed similar structures to natural amino acids (van Hest, JCM, Kiick, KL, Tirrell, DA (2000) J Am. Chem. Soc., 122, 1282-1288).

以下の実施例は、本発明の範囲をいかなる意味においても限定するものではない。なお、本実施例において使用した試薬、および使用した実験条件は以下のとおりである。また下記の実施例１の合成例において各化合物に付与した番号は、図２の合成スキームにおける化合物の番号である。
（ａ）DCC (2.2当量), MeOH (1.5当量), CH2Cl2, 室温, 12時間
（ｂ）LiBH4 (0.5当量), THF還流, 3時間
（ｃ）TBDMSCl (1.5当量), imidazole (2.0当量), DMF, 室温, 24時間
（ｄ）n-BuLi (0.95当量), トリメチルシリルジアゾメタン (1.2当量), Et2O, 0℃, 3時間
（ｅ）TBAF (2.2当量), THF, 室温, 2時間
（ｆ）PDC (3.5当量), DMF, 室温, 24時間
（ｇ）TFA, CH2Cl2, 室温, 12時間, Dowex 50WX-200. The following examples do not limit the scope of the invention in any way. The reagents used in this example and the experimental conditions used are as follows. Moreover, the number given to each compound in the synthesis example of Example 1 below is the number of the compound in the synthesis scheme of FIG.
(A) DCC (2.2 equivalents), MeOH (1.5 equivalents), CH 2 Cl 2 , room temperature, 12 hours (b) LiBH 4 (0.5 equivalents), THF reflux, 3 hours (c) TBDMSCl (1.5 equivalents), imidazole ( 2.0 eq), DMF, room temperature, 24 h (d) n-BuLi (0.95 eq), trimethylsilyldiazomethane (1.2 eq), Et 2 O, 0 ° C., 3 h (e) TBAF (2.2 eq), THF, room temperature, 2 hours (f) PDC (3.5 equivalents), DMF, room temperature, 24 hours (g) TFA, CH 2 Cl 2 , room temperature, 12 hours, Dowex 50WX-200.

（実施例１：β-1,2,3-(トリアゾール-4-イル)-DL-アラニンの合成
(1) 2L-tert-ブトキシカルボニルアミノ-3-シアノ-プロピオン酸メチルエーテル：化合物7の合成
N-α-Boc-L-アスパラギン(化合物6)（1.54 g, 6.65 mmol） を CH2Cl2 (15 mL) に溶解させDCC (3.02 g, 14.6 mmol)を加えた。室温で６時間撹拌した後、メタノール(400μL, 9.98 mmol)を加え、さらに６時間撹拌した。 不溶のジシクロヘキシルウレアをろ過して除き、5% NaHCO3 で洗浄した。その後、飽和食塩水と水で洗いエバポレーションで溶媒を除き、カラムで精製して化合物7を1.06 g (70%) 得た。
1H NMR (CDCl3):δ1.41 (s, 9H, C(CH3)3); 2.92 (dd, J = 16.8, 5.3, 2H, CH2CN); 3.79 (s, 3H, CH3); 4.49 (m, 1H, CH2CH); 5.55 (m, 1H, NH). 13C NMR (CDCl3) :δ21.9, 28.4, 50.4, 53.5, 81.1, 116.5, 155.1, 169.7. IR (KBr): 3350 br (NH); 2253 (CN); 1739 (C=O, エステル); 1678 (C=O, カルバメート). HRMS found 229.1179. Calcd for (MH+) C10H17N2O4: 229.1187. Example 1: Synthesis of β-1,2,3- (triazol-4-yl) -DL-alanine
(1) 2L-tert-butoxycarbonylamino-3-cyano-propionic acid methyl ether: Synthesis of compound 7
N-α-Boc-L-asparagine (Compound 6) (1.54 g, 6.65 mmol) was dissolved in CH 2 Cl 2 (15 mL), and DCC (3.02 g, 14.6 mmol) was added. After stirring at room temperature for 6 hours, methanol (400 μL, 9.98 mmol) was added, and the mixture was further stirred for 6 hours. Insoluble dicyclohexylurea was filtered off and washed with 5% NaHCO 3 . Thereafter, the mixture was washed with saturated brine and water, the solvent was removed by evaporation, and the residue was purified by a column to obtain 1.06 g (70%) of Compound 7.
1 H NMR (CDCl 3 ): δ1.41 (s, 9H, C (CH 3 ) 3 ); 2.92 (dd, J = 16.8, 5.3, 2H, CH 2 CN); 3.79 (s, 3H, CH 3 ) ; 4.49 (m, 1H, CH 2 CH); 5.55 (m, 1H, NH). 13 C NMR (CDCl 3 ): δ21.9, 28.4, 50.4, 53.5, 81.1, 116.5, 155.1, 169.7. IR (KBr ): 3350 br (NH); 2253 (CN); 1739 (C = O, ester); 1678 (C = O, carbamate). HRMS found 229.1179.Calcd for (MH + ) C 10 H 17 N 2 O 4 : 229.1187.

(2) 2L- tert-ブトキシカルボニルアミノ-3-シアノ-プロパノール：化合物8の合成
2L-タート-ブトキシカルボニルアミノ-3-シアノ-プロピオン酸メチルエーテル(化合物7)(1.48 g, 6.51 mmom) をTHF (6.8 mL)に溶解させLiBH4 (70.8 mg, 3.23 mmol) を窒素雰囲気下で加え、３時間還流させて反応させた。３時間後、冷却して2 M KHSO4を用いて酸性にした（pH4）。ろ過した後、溶媒をエバポレーションで除いた。残留物をCHCl3 で抽出した後に水で洗浄した。エバポレーションで溶媒を除き、カラムで精製して化合物8を1.17 g (90%) 得た。
1H NMR (CDCl3):δ1.44 (s, 9H, C(CH3)3); 2.22 (br s, 1H, CH2OH); 2.71 (m, 2H, CH2CN); 3.77 (dd, J = 10.9, 4.1, 2H, CH2OH); 3.93 (m, 1H, CH2CH); 5.12 (m, 1H, NH). 13C NMR (CDCl3):δ20.3, 28.5, 49.0, 62.9, 80.6, 117.9, 155.7. IR (KBr)/ cm-1: 3339 br (NH); 2253 (CN); 1695 (C=O). HRMS found MH+ 201.1264. C9H17N2O3 requires 201.1238. (2) 2L-tert-butoxycarbonylamino-3-cyano-propanol: Synthesis of Compound 8
2L-tert-Butoxycarbonylamino-3-cyano-propionic acid methyl ether (compound 7) (1.48 g, 6.51 mmom) was dissolved in THF (6.8 mL) and LiBH 4 (70.8 mg, 3.23 mmol) was dissolved in a nitrogen atmosphere. The mixture was further refluxed for 3 hours to be reacted. After 3 hours, it was cooled and acidified with 2 M KHSO 4 (pH 4). After filtration, the solvent was removed by evaporation. The residue was extracted with CHCl 3 and washed with water. Removal of the solvent by evaporation and purification by column gave 1.17 g (90%) of compound 8.
1 H NMR (CDCl 3 ): δ1.44 (s, 9H, C (CH 3 ) 3 ); 2.22 (br s, 1H, CH 2 OH); 2.71 (m, 2H, CH 2 CN); 3.77 (dd , J = 10.9, 4.1, 2H , CH 2 OH); 3.93 (m, 1H, CH 2 CH); 5.12 (m, 1H, NH) 13 C NMR (CDCl 3):. δ20.3, 28.5, 49.0, 62.9, 80.6, 117.9, 155.7. IR (KBr) / cm -1 : 3339 br (NH); 2253 (CN); 1695 (C = O). HRMS found MH + 201.1264. C 9 H 17 N 2 O 3 requires 201.1238.

(3) 2L-tert-ブトキシカルボニルアミノ-3-シアノ-プロピル-tert -ブチルジメチルシリルエーテル：化合物9の合成
2L-tert-ブトキシカルボニルアミノ-3-シアノ-プロパノール(化合物8)(1.17g, 5.86 mmol)をDMFに溶解させ、tert-ブチルジメチルクロロシラン (1.32 g, 8.79 mmol)とイミダゾール (798 mg, 11.72 mmol)を加え一日撹拌した。溶媒をエバポレーションで除き、残留物をCHCl3 に溶解させた後、水で洗浄した。エバポレーションで溶媒を除きカラムで精製して化合物9を1.44 g (78%) 得た。
1H NMR (CDCl3) :δ0.06 (s, 6H, Si(CH3)2); 0.87 (s, 9H, SiC(CH3)3); 1.42 (s, 9H, C(CH3)3); 2.61 (d, J = 6.6, 2H, CH2CN); 3.68 (dd, J = 10.1, 5.0, 2H, CH2OSi); 3.92 (m, 1H, CH2CH); 4.83 (m, 1H, NH). 13C NMR (CDCl3) :δ5.7, 18.0, 19.9, 25.7, 28.2, 48.6, 63.1, 79.6, 117.2, 154.8. IR (KBr) : 3358 br (NH); 2251 (CN); 1718 (C=O). HRMS found 314.2034. Calcd for (MH+) C15H30N2O3Si1: 314.2024. (3) Synthesis of compound 9: 2L-tert-butoxycarbonylamino-3-cyano-propyl-tert-butyldimethylsilyl ether
2L-tert-Butoxycarbonylamino-3-cyano-propanol (Compound 8) (1.17 g, 5.86 mmol) was dissolved in DMF, tert-butyldimethylchlorosilane (1.32 g, 8.79 mmol) and imidazole (798 mg, 11.72 mmol) ) Was added and stirred for one day. The solvent was removed by evaporation and the residue was dissolved in CHCl 3 and washed with water. Removal of the solvent by evaporation and purification by column gave 1.44 g (78%) of compound 9.
1 H NMR (CDCl 3 ): δ0.06 (s, 6H, Si (CH 3 ) 2 ); 0.87 (s, 9H, SiC (CH 3 ) 3 ); 1.42 (s, 9H, C (CH 3 ) 3 ); 2.61 (d, J = 6.6, 2H, CH 2 CN); 3.68 (dd, J = 10.1, 5.0, 2H, CH 2 OSi); 3.92 (m, 1H, CH 2 CH); 4.83 (m, 1H 13 C NMR (CDCl 3 ): δ5.7, 18.0, 19.9, 25.7, 28.2, 48.6, 63.1, 79.6, 117.2, 154.8. IR (KBr): 3358 br (NH); 2251 (CN); 1718 (C = O). HRMS found 314.2034. Calcd for (MH + ) C 15 H 30 N 2 O 3 Si 1 : 314.2024.

(4) 2DL-tert-ブトキシカルボニルアミノ -(4-トリメチルシリル-1,2,3-トリアゾール-5-イル)-プロピル-tert-ブチルジメチルシリルエーテル：化合物10の合成
2.73 ml (4.34 mmol) の n-ブチルリチウム (1.59 M ヘキサン溶液) を トリメチルシリルジアゾメタン (2.0 M ヘキサン溶液, 2.74 mL, 5.48 mmol) のジエチルエーテル (28 mL)溶液に、0 ℃で滴下した。 その後２０分間攪拌し、更に化合物9 (1.44 g, 4.57 mmol) のジエチルエーテル (14 mL)溶液を0 ℃で滴下し、3時間撹拌した。３時間後、飽和塩化アンモニウム水溶液を加え、ジエチルエーテルで抽出した。ジエチルエーテル層を水で洗い、その後、エバポレーションで溶媒を除きカラムで精製して化合物10を1.47 g (75%) 得た。
1H NMR (CDCl3) :δ0.02 (s, 6H, Si(CH3)2); 0.35 (s, 9H, Si(CH3)3); 0.86 (s, 9H, SiC(CH3)3); 1.34 (s, 9H, C(CH3)3); 3.00 (dd, J = 15.0, 5.4, 2H, CH2C=C); 3.62 (dd, J = 9.9, 6.0, 2H, CH2OSi); 3.95 (m, 1H, CH2CH); 5.34 (m, 1H, NH). 13C NMR (CDCl3) :δ 5.5, -1.0, 18.2, 25.9, 27.5, 28.3, 52.2, 58.1, 64.3, 79.0, 155.6. IR (KBr) : 3134 br (NH); 1716 (C=O); 1687 (C=N). HRMS found MH+ 429.2678. Calcd for C19H41N4O3Si2: 429.2715. (4) 2DL-tert-butoxycarbonylamino- (4-trimethylsilyl-1,2,3-triazol-5-yl) -propyl-tert-butyldimethylsilyl ether: Synthesis of Compound 10
2.73 ml (4.34 mmol) of n-butyllithium (1.59 M in hexane) and trimethylsilyldiazomethane (2.0 M in hexane, 2.74 mL, 5.48 mmol) Was added dropwise at 0 ° C. to a diethyl ether (28 mL) solution. Thereafter, the mixture was stirred for 20 minutes, and a solution of compound 9 (1.44 g, 4.57 mmol) in diethyl ether (14 mL) was added dropwise at 0 ° C., followed by stirring for 3 hours. After 3 hours, a saturated aqueous ammonium chloride solution was added, and the mixture was extracted with diethyl ether. The diethyl ether layer was washed with water, and then the solvent was removed by evaporation, followed by purification with a column to obtain 1.47 g (75%) of Compound 10.
1 H NMR (CDCl 3 ): δ0.02 (s, 6H, Si (CH 3 ) 2 ); 0.35 (s, 9H, Si (CH 3 ) 3 ); 0.86 (s, 9H, SiC (CH 3 ) 3 ); 1.34 (s, 9H, C (CH 3 ) 3 ); 3.00 (dd, J = 15.0, 5.4, 2H, CH 2 C = C); 3.62 (dd, J = 9.9, 6.0, 2H, CH 2 OSi ); 3.95 (m, 1H, CH 2 CH); 5.34 (m, 1H, NH) 13 C NMR (CDCl 3):. δ 5.5, -1.0, 18.2, 25.9, 27.5, 28.3, 52.2, 58.1, 64.3, 79.0, 155.6. IR (KBr): 3134 br (NH); 1716 (C = O); 1687 (C = N) .HRMS found MH + 429.2678.Calcd for C 19 H 41 N 4 O 3 Si 2 : 429.2715.

(5) 2DL-tert-ブトキシカルボニルアミノ-(1,2,3-トリアゾール-4-イル)-プロパノール：化合物11の合成
2DL-tert-ブトキシカルボニルアミノ -(4-トリメチルシリル-1,2,3-トリアゾール-5-イル)-プロピル-tert-ブチルジメチルシリルエーテル(化合物10) (1.46 g, 3.23 mmol) をTHF (7.1 mL)に溶解させ、TBAF (1.0 M THF 溶液, 7.1 mL, 7.1 mmol)を加え2 時間撹拌した。エバポレーションでTHFを除き、アンモニウム塩をイオン交換カラム（Dowex 50WX-200 イオン交換樹脂）で除き、カラムで精製して化合物11を得た(81 %)。
1H NMR (CDCl3) :δ1.32 (s, 9H, C(CH3)3); 2.98 (d, J = 6.6, 2H, CH2C=C); 3.57 (d, J = 3.9, 2H, CH2OH); 3.88 (m, 1H, CH2CH); 5.48 (m, 1H, NH); 7.55 (s, 1H, C=CH).13C NMR (CDCl3) :δ 28.5, 52.2, 63.4, 80.1, 110.0, 156.4. IR (KBr)/ cm-1: 3312br (NH / OH); 1685 (C=O / C=N). HRMS found MH+ 243.1455. C10H19N4O3 requires 243.1453. (5) 2DL-tert-butoxycarbonylamino- (1,2,3-triazol-4-yl) -propanol: Synthesis of Compound 11
2DL-tert-butoxycarbonylamino- (4-trimethylsilyl-1,2,3-triazol-5-yl) -propyl-tert-butyldimethylsilyl ether (Compound 10) (1.46 g, 3.23 mmol) in THF (7.1 mL TBAF (1.0 M THF solution, 7.1 mL, 7.1 mmol) was added and stirred for 2 hours. The THF was removed by evaporation, the ammonium salt was removed with an ion exchange column (Dowex 50WX-200 ion exchange resin), and purification was performed on the column to obtain Compound 11 (81%).
1 H NMR (CDCl 3 ): δ1.32 (s, 9H, C (CH 3 ) 3 ); 2.98 (d, J = 6.6, 2H, CH 2 C = C); 3.57 (d, J = 3.9, 2H , CH 2 OH); 3.88 ( m, 1H, CH 2 CH); 5.48 (m, 1H, NH); 7.55 (s, 1H, C = CH) 13 C NMR (CDCl 3):. δ 28.5, 52.2, 63.4, 80.1, 110.0, 156.4. IR (KBr) / cm -1 : 3312br (NH / OH); 1685 (C = O / C = N). HRMS found MH + 243.1455. C 10 H 19 N 4 O 3 requires 243.1453.

(6) N-α-Boc-β-(1,2,3-トリアゾール-4-イル)-DL-アラニン：化合物12の合成
2DL-tert-ブトキシカルボニルアミノ-(1,2,3-トリアゾール-4-イル)-プロパノール (化合物11) (634 mg, 2.62 mmol) をDMF (25 mL)に溶解させ、PDC (3.44 g, 9.17 mmol)とセライト(5.1 g)を加えて一日撹拌した。不要物をセライトで除き、エバポレーションで溶媒を除き、化合物12を658 mg（98%）得た。
HRMS found M+ 256.1164. C10H16N4O4 requires 256.1123. (6) N-α-Boc-β- (1,2,3-triazol-4-yl) -DL-alanine: Synthesis of compound 12
2DL-tert-butoxycarbonylamino- (1,2,3-triazol-4-yl) -propanol (Compound 11) (634 mg, 2.62 mmol) was dissolved in DMF (25 mL) and PDC (3.44 g, 9.17 mmol) and Celite (5.1 g) were added and stirred for one day. Unnecessary substances were removed with Celite, and the solvent was removed by evaporation to obtain 658 mg (98%) of Compound 12.
HRMS found M + 256.1164. C 10 H 16 N 4 O 4 requires 256.1123.

(7) β-1,2,3-(トリアゾール-4-イル)-DL-アラニンの合成
N-α-Boc-β-(1,2,3-トリアゾール-4-イル)-DL-アラニン(化合物12) (658 mg, 2.57 mmol)をCH2Cl2 に溶解させTFAとアニソールを加え、室温で12 時間撹拌した。溶媒をエバポレーションで除き、ジエチルエーテルで結晶化させるとTFAの塩が得られた。この塩をDowex 50WX-200 ion-exchange-resinで脱塩し、β-1,2,3-(トリアゾール-4-イル)-DL-アラニンを612 mg（98%）得た。
mp 244-245℃, 1H NMR (D2O) :δ 3.25 (m, 2H, CH2); 3.95 (dd, J = 6.8, 5.2, 1H, CH); 7.69 (s, 1H, C=CH). IR (KBr) 3100〜2000 br (NH2); 1597 (C=O); 1549 (C=N). Anal. Calcd for C5H8N4O2: C, 38.45; H, 5.17; N, 35.89. Found: C, 38.47; H, 5.12; N, 35.46. (7) Synthesis of β-1,2,3- (triazol-4-yl) -DL-alanine
N-α-Boc-β- (1,2,3-triazol-4-yl) -DL-alanine (Compound 12) (658 mg, 2.57 mmol) was dissolved in CH 2 Cl 2 and TFA and anisole were added. Stir at room temperature for 12 hours. Removal of the solvent by evaporation and crystallization with diethyl ether gave the salt of TFA. This salt was desalted with Dowex 50WX-200 ion-exchange-resin to obtain 612 mg (98%) of β-1,2,3- (triazol-4-yl) -DL-alanine.
mp 244-245 ° C, 1 H NMR (D 2 O): δ 3.25 (m, 2H, CH 2 ); 3.95 (dd, J = 6.8, 5.2, 1H, CH); 7.69 (s, 1H, C = CH IR (KBr) 3100-2000 br (NH 2 ); 1597 (C = O); 1549 (C = N). Anal. Calcd for C 5 H 8 N 4 O 2 : C, 38.45; H, 5.17; N, 35.89. Found: C, 38.47; H, 5.12; N, 35.46.

（実施例２：非天然ヒスチジンアナログのタンパク質への導入）
調製できるタンパク質の量や簡便性、経済性を考え、非天然アミノ酸のタンパク質への導入法として大腸菌の系を用いることを試みた。大腸菌は通常、自身でヒスチジンを合成し、タンパク質中に取り込んでいるが、ヒスチジン欠損株はこの機能を持たない。そこで本発明者らは、今回合成したヒスチジン類似体の構造がヒスチジンに極めてよく似ているため、培地にこれら非天然アミノ酸を加えるだけで導入できないかと考えて検討を行った。そこで本発明者は、キチンバインディングドメインというタンパク質を選択し、このタンパク質に本発明のβ-1,2,3-(トリアゾール-4-イル)-DL-アラニンを導入することを試みた。 (Example 2: Introduction of non-natural histidine analog into protein)
Considering the amount of protein that can be prepared, convenience, and economy, we tried to use the E. coli system as a method for introducing unnatural amino acids into proteins. E. coli normally synthesizes histidine itself and incorporates it into proteins, but histidine-deficient strains do not have this function. Therefore, the present inventors investigated whether the structure of the histidine analog synthesized this time was very similar to that of histidine, and therefore could be introduced simply by adding these unnatural amino acids to the medium. Therefore, the present inventor selected a protein called a chitin binding domain and tried to introduce β-1,2,3- (triazol-4-yl) -DL-alanine of the present invention into this protein.

（プラスミドの構築）
通常のインバースPCRを用いて発現ベクター断片を調製した。１μｇの合成DNAプライマーを、高発現させるためにtacプロモーターを含んでいる市販のpGEX-4T-3（ファルマシア、アプサラ、スウェーデン）と混合し、Ex.TaqDNAポリメラーゼと共にPCR反応を行った。ここで用いたセンスとアンチセンスのプライマーは、それぞれ5’-GCCAAAGCATATGGGATCCCCGAATTCCCG-3’と 5’-CCGGGATCCCTCTTCATATTTTTCTTCAAGA-3’であった。センスプライマーはEcoRIサイトを有し、アンチセンスプライマーはBamHIサイト（下線）を含んでいた。1%アガロースゲル電気泳動上でPCR産物（ca. 4kbp）が単離され、BamHI とEcoRIで消化された。消化された産物はフェノール／クロロフォルム抽出によって精製し、エタノール沈殿した。 (Plasmid construction)
Expression vector fragments were prepared using conventional inverse PCR. 1 μg of the synthetic DNA primer was mixed with commercially available pGEX-4T-3 (Pharmacia, Apsara, Sweden) containing a tac promoter for high expression, and a PCR reaction was performed with Ex. Taq DNA polymerase. The sense and antisense primers used here were 5'-GCCAAAGCATATGGGATCCCC GAATTC CCG-3 'and 5'-CCG GGATCC CTCTTCATATTTTTCTTCAAGA-3', respectively. The sense primer had an EcoRI site and the antisense primer contained a BamHI site (underlined). A PCR product (ca. 4 kbp) was isolated on 1% agarose gel electrophoresis and digested with BamHI and EcoRI. The digested product was purified by phenol / chloroform extraction and ethanol precipitated.

プラスミドベクターpHEX-CBDを下記の様に構築した。市販のCBD発現ベクターであるpTYB1（ニューイングランドバイオラブス、ビバリー、MA）をEx.TaqDNAポリメラーゼ（タカラ）と共にPCR反応を行った。センスとアンチセンスプライマーの配列はそれぞれ、5’-GCGGGATCCACGACAAATCCTGGTGTATC-3’ と5’-CGGAATTCTCATTGAAGCTGCCACAAGG-3’であった。PCR産物を４％のポリアクリルアミドゲル電気泳動上で単離し、EcoRIとBamHIで消化した。消化した産物をフェノール／クロロホルム抽出で精製し、エタノール沈殿した。沈殿したDNA断片を上記の発現ベクター断片のEcoRIとBamHIサイトに結合させた。   Plasmid vector pHEX-CBD was constructed as follows. A commercially available CBD expression vector pTYB1 (New England Biolabs, Beverly, MA) was subjected to a PCR reaction with Ex. Taq DNA polymerase (Takara). The sequence of the sense and antisense primers was 5'-GCGGGATCCACGACAAATCCTGGTGTATC-3 'and 5'-CGGAATTCTCATTGAAGCTGCCACAAGG-3', respectively. PCR products were isolated on 4% polyacrylamide gel electrophoresis and digested with EcoRI and BamHI. The digested product was purified by phenol / chloroform extraction and ethanol precipitated. The precipitated DNA fragment was bound to the EcoRI and BamHI sites of the above expression vector fragment.

pHEXプラスミドと上記のpHEX-CBDプラスミドを操作するために、大腸菌JM109株を使用した。各々のプラスミドで大腸菌株を形質転換し、アンピシリンンを含んでいるLB上に細菌を置いた。3mlのYT培地中でシングルコロニーを培養し、プラスミドミニプレップキット（キアゲン、ヒルデン、ドイツ）を用いてプラスミドを単離した。プラスミドの配列をDNA配列解析によって確認した。   In order to manipulate the pHEX plasmid and the above pHEX-CBD plasmid, E. coli strain JM109 was used. E. coli strains were transformed with each plasmid and the bacteria were placed on LB containing ampicillin. Single colonies were cultured in 3 ml YT medium and plasmids were isolated using a plasmid miniprep kit (Qiagen, Hilden, Germany). The sequence of the plasmid was confirmed by DNA sequence analysis.

（キチンバインディングドメインの発現）
ヒスチジン要求性のバクテリア発現宿主であるUTH780コンピーテント細胞を、pHEX-CBD発現ベクターで形質転換した。その形質転換体を、20 μg/mlのアンピシリンを添加した5mlの最小培地(１リットルあたり、Fe(NH4)2(SO4)3 10 mg, MgSO4・6H2O 55 mg, KH2PO4 4.4 g, K2HPO4・3H2O 6.0 g, NH4Cl 1.0 g, グルコース6 g, チアミン10 mg, ヒスチジン20 mg, pH7) 中で、600nmにおける濁度（A600）が0.6になるまで37℃でインキュベートした。 (Expression of chitin binding domain)
UTH780 competent cells, which are histidine-requiring bacterial expression hosts, were transformed with the pHEX-CBD expression vector. The transformant was added to 5 ml of minimal medium (Fe (NH 4 ) 2 (SO 4 ) 3 10 mg, MgSO 4 · 6H 2 O 55 mg, KH 2 PO per liter, supplemented with 20 μg / ml ampicillin. 4 4.4 g, K 2 HPO 4 · 3H 2 O 6.0 g, NH 4 Cl 1.0 g, glucose 6 g, thiamine 10 mg, histidine 20 mg, pH 7), the turbidity at 600 nm (A 600 ) is 0.6 Incubated at 37 ° C.

3000rpmで10分間遠心分離することによって細菌を回収し、M9培地で２回洗浄し、ヒスチジンを欠く最小培地5mL中に再懸濁した。細胞を37℃で20分間震盪することにより細胞内のヒスチジンを実質的に除去した。そしてヒスチジン又はヒスチジン同等物を培地に添加した。イソプロピルβ-D-チオガラクトシド（IPTG、最終濃度0.4mM）を添加することにより誘導を行い、細菌を更に37℃で４時間培養し、3000rpmで10分間遠心分離することにより回収した。細菌のペレットを、1mlの0.1%(v/v)Triton X-100を含むPBSバッファー（pH 7.4）中に再懸濁し、氷上でソニケートした（50W, 15秒間５回）。ライゼートを12000rpmで1分間遠心分離し、上清中のキチンバインディングドメインタンパク質を下記のようにして検出した。   Bacteria were collected by centrifuging at 3000 rpm for 10 minutes, washed twice with M9 medium and resuspended in 5 mL of minimal medium lacking histidine. The cells were shaken at 37 ° C. for 20 minutes to substantially remove intracellular histidine. And histidine or histidine equivalent was added to the medium. Induction was performed by adding isopropyl β-D-thiogalactoside (IPTG, final concentration 0.4 mM), and the bacteria were further cultured at 37 ° C. for 4 hours and collected by centrifugation at 3000 rpm for 10 minutes. The bacterial pellet was resuspended in 1 ml of PBS buffer (pH 7.4) containing 0.1% (v / v) Triton X-100 and sonicated on ice (50 W, 5 times for 15 seconds). The lysate was centrifuged at 12000 rpm for 1 minute, and chitin binding domain protein in the supernatant was detected as follows.

（野生型と変異したキチンバインディングドメインの検出）
野生型と変異したキチンバインディングドメインが生成していることを、15%ゲル上でSDS-PAGEを行い、次いで抗キチンバインディングドメイン抗体（ニューイングランドバイオラブス）及びアルカリホスファターゼでラベルした抗ウサギIgG抗体を用いたウエスタンブロッティングを行うことにより確認した。一定量（1μL）のライゼートを9μLの水と2xSB（サンプルバッファー；100Mm Tris-HCl, pH6.8, 8%ドデシル硫酸ナトリウム、4% 2-メルカプトエタノール, 24% グリセロール, 0.01%ブロモフェノールブルー）と混合した。この溶液を95℃で５分間インキュベートし、その溶液の5μLをゲル電気泳動にかけ、PVDF膜上に電気ブロットした。膜をトリス緩衝生理食塩水（20 mM Tris/HCl, 150 mM NaClと0.1% Tween-20を含む）中で37℃１時間インキュベートすることにより3%BSAでブロッキングし、同じバッファーで1:5000に希釈した抗キチンバインディングドメイン抗体とインキュベートした。洗浄した後、1:5000で希釈したアルカリホスファターゼ結合抗ウサギIgG抗体とインキュベートし、ウエスタンブルーサブストレート（プロメガ、マジソン、WI）を用いてブロットを可視化した。 (Detection of wild-type and mutated chitin binding domains)
Performing SDS-PAGE on a 15% gel and confirming that the chitin binding domain mutated with the wild type is generated, and then anti-chitin binding domain antibody (New England Biolabs) and anti-rabbit IgG antibody labeled with alkaline phosphatase It was confirmed by performing the Western blotting used. A fixed amount (1 μL) of lysate with 9 μL of water and 2 × SB (sample buffer; 100 Mm Tris-HCl, pH 6.8, 8% sodium dodecyl sulfate, 4% 2-mercaptoethanol, 24% glycerol, 0.01% bromophenol blue) Mixed. This solution was incubated at 95 ° C. for 5 minutes and 5 μL of the solution was subjected to gel electrophoresis and electroblotted onto a PVDF membrane. The membrane was blocked with 3% BSA by incubating in Tris-buffered saline (containing 20 mM Tris / HCl, 150 mM NaCl and 0.1% Tween-20) for 1 hour at 37 ° C, and 1: 5000 with the same buffer. Incubated with diluted anti-chitin binding domain antibody. After washing, incubated with alkaline phosphatase conjugated anti-rabbit IgG antibody diluted 1: 5000 and blots visualized using Western Blue Substrate (Promega, Madison, WI).

図３はヒスチジン及びヒスチジンアナログのタンパク質への取り込みを示す、ブロッティングの写真である。各レーンにおいて、レーン１はIPTGを添加しなかったサンプルであり、レーン２はヒスチジンも本発明のアナログの添加しなかったサンプルの結果であり、レーン３はヒスチジンが添加されたサンプルの結果であり、レーン４、５、６、７は図１の化合物２、３、４、５について解析を行ったサンプルの結果である。図３の写真において、目的アミノ酸の導入は化合物２、３のみに認められた。すなわち、本発明のヒスチジンアナログ（化合物２）について、タンパク質中に効率良く導入されていることが確認された。   FIG. 3 is a blotting photograph showing the incorporation of histidine and histidine analogs into proteins. In each lane, lane 1 is a sample to which no IPTG was added, lane 2 was a result of a sample to which histidine was not added, and lane 3 was a result of a sample to which histidine was added. Lanes 4, 5, 6, and 7 are the results of samples analyzed for compounds 2, 3, 4, and 5 in FIG. In the photograph of FIG. 3, introduction of the target amino acid was observed only in compounds 2 and 3. That is, it was confirmed that the histidine analog (compound 2) of the present invention was efficiently introduced into the protein.

本発明のヒスチジンアナログは新規な非天然アミノ酸であるが、類似した他のヒスチジンアナログについて医薬として効果が報告されているために、本発明の非天然アミノ酸は医薬として有用である可能性がある。また、本発明の新規ヒスチジンアナログに含まれるトリアゾール環はイミダゾール環に比べて酸性が強く酸触媒としての機能が強いために、本発明のヒスチジンアナログをタンパク質に導入することにより、当該タンパク質の酸触媒活性を上げることができる。   Although the histidine analog of the present invention is a novel non-natural amino acid, the effect of the other similar histidine analogs as a pharmaceutical has been reported, and therefore the non-natural amino acid of the present invention may be useful as a pharmaceutical. Further, since the triazole ring contained in the novel histidine analog of the present invention has a higher acidity and a stronger function as an acid catalyst than the imidazole ring, the acid catalyst of the protein is introduced by introducing the histidine analog of the present invention into the protein. The activity can be increased.

図１は、本発明の実施例において使用したヒスチジン及びそのアナログの構造を示した図である。FIG. 1 is a diagram showing the structures of histidine and its analogs used in the examples of the present invention. 図２は、本発明の非天然ヒスチジンアナログの合成スキームを示した図である。FIG. 2 is a diagram showing a synthesis scheme of the non-natural histidine analog of the present invention. 図３はヒスチジン及びヒスチジンアナログのタンパク質への取り込みを示す、ブロッティングの写真であるFIG. 3 is a blotting photograph showing the incorporation of histidine and histidine analogs into proteins.

Claims (4)

下記の化学式
Figure 2005075765
からなる構造を有することを特徴とするアミノ酸。 The following chemical formula
Figure 2005075765
An amino acid having a structure consisting of 下記の化学式
Figure 2005075765
からなる構造を有するアミノ酸を製造する方法。 The following chemical formula
Figure 2005075765
A method for producing an amino acid having a structure consisting of: 下記の化学式
Figure 2005075765
からなる構造を有するアミノ酸が導入されたことを特徴とする蛋白質。 The following chemical formula
Figure 2005075765
A protein comprising an amino acid having a structure consisting of: 下記の化学式
Figure 2005075765
からなる構造を有するアミノ酸が導入された蛋白質を製造する方法。 The following chemical formula
Figure 2005075765
A method for producing a protein into which an amino acid having a structure is introduced.
JP2003307192A 2003-08-29 2003-08-29 New non-natural histidine analog amino acid Pending JP2005075765A (en)

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