1Department of Chemistry and Biotechnology, School of Engineering, University of Tokyo, 7-3-1 Hongo, Tokyo 113-8656, 2Gene Function Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Tukuba Science City 305-8562 and 3Department of Material and Biological Chemistry, Faculty of Science, Yamagata University, Yamagata 990-8560, Japan
4 To whom correspondence should be addressed, at the Tokyo address. e-mail: taira{at}chembio.t.u-tokyo.ac.jp
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Abstract |
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Keywords: amino acid analogue/histidine/incorporation/translation/1,2,3-triazole
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Introduction |
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For the introduction of unnatural amino acids into proteins, several methods are available (Kochendoerfer and Kent, 1999; Liu and Schultz, 1999
; Sisido and Hohsaka, 1999
). Some amino acid analogues, in addition to the 20 natural amino acids, can be introduced into proteins (Koide et al., 1988, 1994
; van Hest et al., 2000
). Translational studies showed that all the introduced unnatural amino acids have structural similarity with a natural amino acid (van Hest et al., 2000
). Thus, the amino acid analogue to be incorporated in vivo in protein synthesis has structural similarity with a natural amino acid.
In this study we prepared several histidine analogues including a novel histidine analogue, ß-(1,2,3-triazol-4-yl)-DL-alanine (2), and their incorporations into a protein in vivo were investigated and compared with each other.
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Materials and methods |
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All solvents and reagents were commercially available and used without further purification. 1H NMR spectra (400 MHz) and 13C NMR spectra (100 MHz) were recorded on a Varian AS400 spectrometer. Column chromatography was performed with Wako Gel-200 silica gel; Wako Silicagel 70F was used for thin-layer chromatography. All assays were repeated at least three times. The experimental errors represent the standard deviations of these independent experiments.
Synthesis of ß-(1,2,3-triazol-4-yl)-DL-alanine
2-L-tert-Butoxycarbonylamino-3-cyanopropionic acid methyl ester (7). To a solution of N--Boc-L-asparagine (6) (1.54 g, 6.65 mmol) in dry CH2Cl2 (15 ml) was added DCC (3.02 g, 14.6 mmol). After stirring at room temperature for 6 h, methanol (400 µl, 9.98 mmol) was added and stirred for another 6 h. The precipitated dicyclohexylurea was removed by filtration and the filtrate was washed with 5% NaHCO3 twice, then washed with brine and dried. After evaporation, the residue was purified by column chromatography, yielding 1.06 g (70%) of 7. 1H NMR (CDCl3):
(ppm) 1.41 [s, 9H, C(CH3)3]; 2.92 (dd, J = 16.8, 5.3 Hz, 2H, CH2CN); 3.79 (s, 3H, CH3); 4.49 (m, 1H, CH2CH); 5.55 (m, 1H, NH). 13C NMR (CDCl3):
(ppm) 21.9, 28.4, 50.4, 53.5, 81.1, 116.5, 155.1, 169.7. IR (KBr) (cm1): 3350 br (NH); 2253 (CN); 1739 (C=O, ester); 1678 (C=O, carbamate). HRMS: found 229.1179. Calcd for (MH+), C10H17N2O4: 229.1187.
2-L-tert-Butoxycarbonylamino-3-cyanopropanol (8). To a solution of 7 (1.48 g, 6.51 mmol) in dry THF (6.8 ml) was added LiBH4 (70.8 mg, 3.23 mmol) under nitrogen and the mixture was refluxed for 3 h. The resulting mixture was cooled and acidified to pH 4 with 2 M KHSO4. After filtration, the filtrate was evaporated. The residue was extracted with CHCl3 and washed with water and dried. After evaporation, the residue was purified by column chromatography, yielding 1.17 g (90%) of 8. 1H NMR (CDCl3): (ppm) 1.44 [s, 9H, C(CH3)3]; 2.22 (br s, 1H, CH2OH); 2.71 (m, 2H, CH2CN); 3.77 (dd, J = 10.9, 4.1 Hz, 2H, CH2OH); 3.93 (m, 1H, CH2CH); 5.12 (m, 1H, NH). 13C NMR (CDCl3):
(ppm) 20.3, 28.5, 49.0, 62.9, 80.6, 117.9, 155.7. IR (KBr) (cm1): 3339 br (NH); 2253 (CN); 1695 (C=O). HRMS: found MH+ 201.1264. C9H17N2O3 requires 201.1238.
2-L-tert-Butoxycarbonylamino-3-cyanopropyl tert-butyldimethylsilyl ether (9). To a solution of 8 (1.17g, 5.86 mmol) in dry DMF were added tert-butyldimethylchlorosilane (1.32 g, 8.79 mmol) and imidazole (798 mg, 11.72 mmol) and the mixture was stirred for 1 day. After removing the solvent in vacuo, the residue was dissolved in CHCl3, washed with water and dried. After evaporation, the residue was purified by column chromatography, yielding 1.44 g (78%) of 9. 1H NMR (CDCl3): (ppm) 0.06 [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 Hz, 2H, CH2CN); 3.68 (dd, J = 10.1, 5.0 Hz, 2H, CH2OSi); 3.92 (m, 1H, CH2CH); 4.83 (m, 1H, NH). 13C NMR (CDCl3):
(ppm) 5.7, 18.0, 19.9, 25.7, 28.2, 48.6, 63.1, 79.6, 117.2, 154.8. IR (KBr) (cm1): 3358 br (NH); 2251 (CN); 1718 (C=O). HRMS: found 314.2034. Calcd for (MH+), C15H30N2O3Si1: 314.2024.
2-DL-tert-Butoxycarbonylamino(4-trimethylsilyl-1,2,3-triazol-5-yl)propyl tert-butyldimethylsilyl ether (10). A 2.73 ml (4.34 mmol) amount of n-butyllithium (1.59 M hexane solution) was added dropwise to a solution of trimethylsilyldiazomethane (2.0 M hexane solution, 2.74 ml, 5.48 mmol) in diethyl ether (28 ml) at 0°C and the mixture was stirred at 0°C for 20 min. To the resulting solution was added dropwise a solution of 9 (1.44 g, 4.57 mmol) in diethyl ether (14 ml) at 0°C, then the mixture was stirred at 0°C for 3 h. The mixture was treated with saturated aqueous ammonium chloride and extracted with diethyl ether. The ethereal extract was washed with water and dried. After evaporation, the residue was purified by column chromatography, yielding 1.47 g (75%) of 10. Considering the basic conditions of this 1,3-dipolar addition reaction, racemization occurred in this step. 1H NMR (CDCl3): (ppm) 0.02 [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 Hz, 2H, CH2C=C); 3.62 (dd, J = 9.9, 6.0 Hz, 2H, CH2OSi); 3.95 (m, 1H, CH2CH); 5.34 (m, 1H, NH). 13C NMR (CDCl3):
(ppm) 5.5, 1.0, 18.2, 25.9, 27.5, 28.3, 52.2, 58.1, 64.3, 79.0, 155.6. IR (KBr) (cm1): 3134 br (NH); 1716 (C=O); 1687 (C=N). HRMS: found MH+ 429.2678. Calcd for C19H41N4O3Si2: 429.2715.
2-DL-tert-Butoxycarbonylamino(1,2,3-triazol-4-yl)propanol (11). To a solution of 10 (1.46 g, 3.23 mmol) in THF (7.1 ml) was added TBAF (1.0 M THF solution, 7.1 ml, 7.1 mmol) and the mixture was stirred for 2 h. After evaporation, the ammonium salt was removed by ion-exchange chromatography using Dowex 50WX-200 ion-exchange resin, then purified by silica column chromatography, yielding 634 mg (81%) of 11. 1H NMR (CDCl3): (ppm) 1.32 [s, 9H, C(CH3)3]; 2.98 (d, J = 6.6 Hz, 2H, CH2C=C); 3.57 (d, J = 3.9 Hz, 2H, CH2OH); 3.88 (m, 1H, CH2CH); 5.48 (m, 1H, NH); 7.55 (s, 1H, C=CH).13C NMR (CDCl3):
(ppm) 26.8, 28.5, 52.2, 63.4, 80.1, 110.0, 156.4. IR (KBr) (cm1): 3312 br (NH/OH); 1685 (C=O/C=N). HRMS: found MH+ 243.1455. C10H19N4O3 requires 243.1453.
N--Boc-ß-(1,2,3-triazol-4-yl)-DL-alanine (12). To a solution of 11 (634 mg, 2.62 mmol) in DMF (25 ml) were added PDC (3.44 g, 9.17 mmol) and Celite (5.1 g) and the mixture was stirred for 1 day. After removing the insoluble materials through a pad of Celite, the residue was washed with diethyl ether. The solvent was evaporated and the product (12) was obtained (658 mg, 98%). HRMS: found M+ 256.1164. C10H16N4O4 requires 256.1123.
ß-(1,2,3-Triazol-4-yl)-DL-alanine (2). To a solution of 12 (658 mg, 2.57 mmol) in CH2Cl2 was added TFA and anisole and the mixture was stirred for 12 h. After removing the solvent in vacuo, the residue was triturated with diethyl ether, yielding the TFA salt. This was purified by ion-exchange chromatography using Dowex 50WX-200 ion-exchange resin. The crude TFA salts were loaded in aqueous solution and eluted using 2 M aqueous ammonia solution. The product (2) was obtained (612 mg, 98%). We used a mixture of D- and L-amino acid analogues. However, only the L-isomer should be incorporated into the protein (Noren et al., 1989). M.p. 244245°C. 1H NMR (D2O):
(ppm) 3.25 (m, 2H, CH2); 3.95 (dd, J = 6.8, 5.2 Hz, 1H, CH); 7.69 (s, 1H, C=CH). IR (KBr) (cm1): 31002000 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%.
ß-1,2,3,4-Tetrazol-5-yl)-DL-alanine (4). A 1 equiv. amount of highly toxic nBu3SnN3 was used in the previously reported synthetic route to 4 (Ornstein et al., 1993). We prepared 4 by the procedure shown in Figure 2 (Duncia et al., 1991
; Adlington et al., 1999
). Compound 13 was prepared from L-aspartic acid by following a literature procedure (Ornstein et al., 1993
).
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2-L-tert-Butoxycarbonylamino-N-(2-cyanoethyl)succinamic acid tert-butyl ester (14). To a solution of 13 (860 mg, 2.96 mmol) in DMF (8 ml) was added N-hydroxybenzotriazole (401 mg, 2.96 mmol), (2-cyanoethyl)amine (219 µl, 2.96 mmol) and dicyclohexylcarbodiimide (614 mg, 2.96 mmol) and the mixture was stirred at 0°C. After 48 h, the DMF was removed in vacuo and the residue was purified by column chromatography, yielding 900 mg (89%) of 14. 1H NMR (CDCl3): (ppm) 1.44 [s, 18H, 2xC(CH3)3]; 2.60 (dd, J = 6.6, 6.2 Hz, CH2CN); 2.92 (dd, J = 15.6, 4.2 Hz, 2H, CH2CH); 3.50 (dd,, J = 6.6, 6.2 Hz, CH2NH); 4.38 (m, 1H, CH2CH); 5.58 (m, 1H, CHNH); 6.36 (m, 1H, CH2NH).
2-L-tert-Butoxycarbonylamino-3-[1-(2-cyanoethyl)-1H-tetrazol-5-yl]propionic acid tert-butyl ester (15). Compound 14 (900 mg, 2.63 mmol), triphenylphosphine (690 mg, 2.63 mmol), diethylazodicarboxylate (40% in toluene, 1.14 ml, 2.63 mmol), trimethylsilylazide (349 µl, 2.63 mmol) and THF (7 ml) were mixed and stirred at room temperature under nitrogen. After 24 h, a further 1 equiv. of each of the above reagents was added and the mixture was stirred for 24 h. The mixture was cooled to 0°C and an excess of a 5% aqueous solution of ammonium cerium(IV) nitrile (230 ml) was slowly added. THF (200 ml) was added to dissolve the precipitated organic matter and the solution was stirred for 1 h. The aqueous mixture was extracted with CHCl3 (3x200 ml). The organic layers were combined and dried (MgSO4). After evaporation, the residue was purified by column chromatography, yielding 800 mg (83%) of 15. 1H NMR (CDCl3): (ppm) 1.26 [s, 18H, 2xC(CH3)3]; 3.10 (dt, J = 6.9, 1.0 Hz, CH2CN); 3.45 (m, 2H, CH2CH); 4.46 (m, 1H, CH2CH); 4.62 (dt, J = 6.9, 2.0 Hz, CH2N=N); 5.58 (m, 1H, CHNH); 6.43 (m, 1H, CH2NH). 13C NMR (CDCl3):
(ppm) 18.7, 26.2, 28.0, 28.4, 42.8, 52.4, 80.7, 83.6, 116.2, 152.7, 155.6, 169.2. IR (KBr) (cm1): 3389 br (NH); 2270 (CN); 1732 (C=O); 1693 (C=O). HRMS: found MH+ 367.2063. C16H27N6O4 requires 367.2082.
ß-1,2,3,4-Tetrazol-5-yl)-DL-alanine (4). To 15 (800 mg, 2.18 mmol) in THF (16 ml) was added 1 N NaOH (2.18 ml, 2.18 mmol) and the mixture was stirred at room temperature for 8 h. The THF was removed in vacuo. The residue was dissolved in CH2Cl2 (50 ml) and TFA (12.5 ml) and anisole (1.25 ml) were added. After stirring for 12 h, the solvent was removed in vacuo. The residue was triturated with diethyl ether, yielding the TFA salt. This was purified by ion-exchange chromatography using Dowex 50WX-200 ion-exchange resin. The crude TFA salts were loaded in aqueous solution and eluted using 2 M aqueous ammonia solution to afford 277 mg of 4 (81%). This product is a racemic form. 1H NMR (D2O): (ppm) 3.45 (m, 2H, CH2CH); 4.14 (t, J = 6.2 Hz, 1H, CH2CH). 13C NMR (CDCl3):
(ppm) 26.8, 52.3, 167.0. HRMS: found M+ 157.0576. C4H7N2O5 requires 157.0594.
Plasmid construction of the chitin-binding domain (CBD)
An expression vector fragment was generated by a standard inverse polymerase chain reaction (PCR) method as follows. A 1 µmol amount of each synthetic DNA primer was mixed with the commercial expression vector pGEX-4T-3 (Pharmacia, Uppsala, Sweden), which contains a tac promoter for high-level expression, then PCR reaction was carried out with Ex. Taq DNA polymerase (Takara, Shiga, Japan). The sequences of the sense and antisense primers were 5'-GCCAAAGC ATATGGGATCCCCGAATTCCCG-3' and 5'-CCGGGAT CCCTCTTCATATTTTTCTTCAAGA-3', respectively. The sense primer contains EcoRI sites and antisense primer contains BamHI sites (underlined). The PCR product (4 kbp) was isolated by 1% agarose gel electrophoresis (AGE) and digested with BamHI and EcoRI. The digested product was purified by phenolchloroform extraction and precipitated with ethanol.
The plasmid vector pHEX-CBD, which express the chitin-binding domain (CBD) chimeric proteins with a short GST fragment tag at the N-terminus, was constructed as follows. A 1 µmol amount of each synthetic DNA primer was mixed with the CBD gene cloned in the commercial CBD expression vector pTYB1 (New England Biolabs, Beverly, MA), then PCR reaction was carried out with Ex. Taq DNA polymerase (Takara). The sequences of the sense and antisense primers were 5'-GCGGGATCCACGACAAATCCTGGTGTATC-3' and 5'-CGGAATTCTCATTGAAGCTGCCACAAGG-3', respectively. The PCR product was isolated by 4% polyacrylamide gel electrophoresis (PAGE) and digested with EcoRI and BamHI. The digested product was purified by the phenolchloroform extraction and precipitated with ethanol. The precipitated DNA fragment was ligated into EcoRI and BamHI sites of the expression vector fragment described above.
We used Escherichia coli strain JM109 (Toyobo, Osaka, Japan) for multiplication of the plasmids, pHEX and pHEX-CBD, described above. We transformed the E.coli. strain with each plasmid and plated the bacteria on LB containing ampicillin. The single colony was cultured in 3 ml of YT medium and the plasmids were isolated with a plasmid miniprep kit (Qiagen, Hilden, Germany). The sequences of the plasmids were confirmed by DNA sequencing.
Expression of the chitin-binding domain (CBD)
Histidine-auxotrophic bacterial expression host UTH780 competent cells were transformed with the expression vector pHEX-CBD. Then the transformant was grown in 5 ml of minimal medium [10 mg of Fe(NH4)2(SO4)3, 55 mg of MgSO4·6H2O, 4.4 g of KH2PO4, 6.0 g of K2HPO4·3H2O, 1.0 g of NH4Cl, 6 g of glucose, 10 mg of thiamine and 20 mg of histidine per litre, pH 7] at 37°C with 20 µg/ml ampicillin until the turbidity at 600 nm (A600) reached 0.6. The bacteria were harvested by centrifugation at 3000 g for 10 min, washed twice with M9 medium and resuspended in 5 ml of a minimal medium lacking histidine. The cells were shaken for 20 min at 37°C to eliminate substantial amounts of histidine inside the cells. Histidine or histidine isostere was then added to the medium. Induction was performed by adding isopropyl ß-D-thiogalactoside (IPTG, final concentration 0.4 mM) and the bacteria were cultured for an additional 4 h at 37°C, then harvested by centrifugation at 3000 g for 10 min. 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 for 15 s, five times). The lysate was centrifuged at 12 000 g for 1 min and the CBD protein in the supernatant was then analyzed as described below.
Detection of wild-type and mutant CBD
The syntheses of the wild-type and mutant CBD were confirmed on a 15% gel by SDSPAGE followed by western blotting using an anti-CBD antibody (New England Biolabs) and alkaline phosphatase-labeled anti-rabbit IgG (Promega, Madison, WI). An aliquot (1 µl) of the lysate was mixed with 9 µl of water and 2x SB [sample buffer; 100 mM TrisHCl (pH 6.8), 8% sodium dodecyl sulfate, 4% 2-mercaptoethanol, 24% glycerol and 0.01% bromophenol blue]. This solution was incubated at 95°C for 5 min, then 5 µl of the solution were subjected to gel electrophoresis. It was electrotransblotted on to poly(vinylidene difluoride) (PVDF) membranes. The membranes were blocked for 1 h at 37°C with 3% BSA in Tris-buffered saline containing 20 mM TrisHCl, 150 mM NaCl and 0.1% Tween-20 and incubated with a 1:5000 dilution of the anti-CBD antibody in the same buffer. After washing, the blots were incubated for 1 h with a 1:5000 dilution of the alkaline phosphatase-conjugated anti-rabbit IgG and visualized using Western Blue substrates (Promega).
Computation
Theoretical calculations were carried out using Spartan (Wavefunction, Irvine, CA). Molecular orbital calculations were carried out using the AM1 Hamiltonian. The electrostatic potential was mapped on the total electron density surface. The structure optimization and energy estimation of each regio isomer were considered in AM1.
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Results |
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For the histidine analogue, we considered the replacement of the imidazole moiety of histidine with a different five-membered heterocyclic moiety. Among the five-membered heterocyclic compounds, a pKa value of the 1,2,3-triazole conjugate acid is 5 units lower than that of imidazole (Katritzky and Rees, 1984
). Because of its structual similarity with histidine, 2 is expected to be incorporated effectively into proteins. Another histidine analogue, ß-(1,2,4-triazol-3-yl)-DL-alanine (Figure 1) (3), is now commercially available (Sigma); however, there is no report on the synthesis of a histidine analogue that contains 1,2,3-triazole in its side chain.
Figure 3 shows the route for the synthesis of 2. Commercially available N--Boc-L-asparagine (6) was used as a starting material. The first step in the synthesis of 2 is the conversion of the amide group to nitrile. Commonly, a primary amide is converted to nitrile under drastic conditions, e.g. by reaction with POCl3 or butyllithium (Smith, 1994
). However, Ressler and Ratzkin reported convenient and mild conditions to convert the primary amide of L-asparagine and L-glutamine to nitrile by the use of N,N'-dicyclohexylcarbodiimide (DCC) (Ressler and Ratzkin, 1961
). We used this procedure and carboxylic acid was also converted to methyl ester under these conditions. Considering the nucleophilic reagent (lithiotrimethylsilyldiazomethane) used for forming the 1,2,3-triazole moiety, the carbonyl group should be reduced to alcohol. There are two other moieties which are sensitive to reduction, so a milder reducing reagent, LiBH4, was used here (Brown et al., 1982
). After protection of the hydroxy group with tert-butyldimethylsilyl chloride (TBDMSCl), 9 was reacted with lithiotrimethylsilyldiazomethane (Seyferth et al., 1972
), affording protected 1,2,3-triazol-4-yl-alaninol (10) (Aoyama et al., 1982
). This reaction was carried out in diethyl ether. When THF was used as the solvent, the yield was low (
20%). After removing the TBDMS and trimethylsilyl (TMS) groups with tetrabutylammonium fluoride (TBAF), 11 was oxidized with pyridinium dichromate (PDC) (Corey and Schmidt, 1979
) in dimethylformamide. Finally, the removal of the Boc group of 12 with trifluoroacetic acid (TFA) gave the TFA salt of the amino acid. This salt was purified by ion-exchange chromatography using Dowex 50WX-200 ion-exchange resin and yielded 2.
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For the introduction of the various analogues, we used an in vivo auxotrophic strain method because of its simplicity. Producing a large amount of a mutant protein is another advantage of this method. However, this in vivo protein-synthesizing system is known to be limited both by the toxicity of unnatural amino acids to the host cell and by strict discrimination of aminoacyl-tRNA synthetase (ARS) and/or ribosomes. Only a study of the in vivo incorporation for some methionine analogues has been reported (van Hest et al., 2000) and no systematic evaluation for various kinds of histidine analogues is available.
Of several histidine-auxotrophic E.coli strains that were tested, we found that the UTH780 strain had the best properties for the expression of our histidine analogue-containing proteins. Hence this E.coli strain was first transformed with a plasmid vector encoding a target protein. We chose the chitin-binding domain (CBD) as the model target protein because it contains only one histidine residue. Wild-type or mutant CBD was expressed by the transformant in a minimal medium containing histidine or the histidine analogue, respectively. Synthesis of the wild-type or mutant CBD was confirmed by SDSPAGE followed by western blotting analysis using anti-CBD antibody and alkaline phosphatase-labeled anti-rabbit IgG.
As shown in Figure 4, our novel histidine analogue 2 was efficiently introduced into the protein, as was 3 in accordance with previous reports (Beiboer et al., 1996; Soumillion and Fastrez, 1998
). To our surprise, the required molar concentration of these analogues in the present system was as low as that of pristine histidine (0.16 mM). The yield of the mutant CBD was 85% compared with the wild-type CBD. Hence both 2 and 3 are efficiently recognized by the his-tRNA synthetase and the resulting misaminoacylated tRNAHis was easily accepted by E.coli ribosome during in vivo translation. In contrast, 4 and 5 were not incorporated into the protein at all.
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Discussion |
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Table I shows the relative energies of the regioisomers (1a5). [There are several regioisomers which originated from the position of protonation on the heterocycle moiety. The stability of each regioisomer was considered in AM1. The electron density maps of the each regioisomer (15a) are shown in Figure 5.] Figure 6 shows the electron density maps of the most stable regioisomer of each amino acid. The computational study shows that the location of a hydrogen atom on the heterocyclic moiety is different between the incorporated compounds 13 and the rejected compound 4 in their most stable regioisomers.
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In conclusion, we have synthesized a novel histidine analogue 2 and its incorporation efficiency was systematically compared with that of several other histidine analogues. Importantly, the analogues that contain 1,2,3-triazole and 1,2,4-triazole could be introduced efficiently into a protein in vivo. Calculated results suggest that the hydrogen atom located at the position might be essential for recognition by his-tRNA synthetase. To our knowledge, this incorporation study is the first to indicate that the hydrogen atom at this position seriously affects this incorporation. Moreover, these analogues with similar steric factors should be useful for the future analysis of enzyme reaction mechanisms by means of, for example, Brönsted plots (Brönsted and Pedersen, 1923
) within proteinous enzymes that have never been reported in the past.
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Acknowledgements |
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Received April 10, 2003; revised June 20, 2003; accepted July 22, 2003.