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EDEdX 115 (l) ii SS 121 ^y* (2)...... 126 Vol. 196, No. 3, 1993 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS November 15, 1993 Pages 1490-1495 STABILIZATION OF a HELIX IN C-TERMINAL FRAGMENTS OF NEUROPEPTIDE Y Noboru Yumoto, Sachiko Murase, Takashi Hatton, Hitoshi Yamamoto, Toshiro Tatsu, and Susumu Yoshikawal Government Industrial Research Institute, Osaka, Ikeda, Osaka 563, Japan Received September 30, 1993 To elucidate the a-helix-stabilizing effect of amino acids at the helical ends, we prepared analogs of C-terminal fragments of neuropeptide Y (NPY) containing an a-helical part. The helix-stabilizing tendency of W-terminal amino acid in NPY(12-36) was found to be as follows: Thr > Ser > Gly > Gin > Cys > Asn > Asp > Val > Phe > Glu > Lys > Tyr > Ala = Trp > His > Arg, suggesting the importance of end capping. The capping effect was not evident when A-termini in NPY(ll-36) and NPY(13-36) were replaced. Under the same conditions as those for the receptor binding, [Thr12]NPY(12-36) had about 4-fold higher a- helix content than [Arg 12]NPY(12-36). However, there was no apparent relationship between the helix content and binding affinity to the Yz receptor. © 1993 Academic Press, Inc. Neuropeptide Y (NPY) is an amidated 36 amino acid peptide, and has been proposed to have a compact globular structure named the PP-fold as shown in Fig. 1 based on the X-ray analysis of the homologous avian pancreatic polypeptide (1). However, recent NMR studies indicated that the a-helical part extends over residues 11- 36 and the jV-terminal part (residues 1-9) is an unstructured mobile segment in aqueous solution (2, 3). NPY is widely distributed in the central and peripheral nervous systems, and thought to be involved in the sympathetic vascular control, the central regulation of endocrine and autonomic function, food intake, circadian rhythm, etc. (4). Two subtypes of receptors for NPY have been well characterized (5): Yi receptors are activated poorly by fragments of NPY, whereas Yz receptors can bind C-terminal fragments of NPY such iTo whom correspondence should be addressed at Government Industrial Research Institute, Osaka, 1-8-31 Midorigaoka, Ikeda, Osaka 563, Japan. Fax: (+81)-727-51-9628. Abbreviation used: NPY. neuropeptide Y. 0006-29 1X/93 $4.00 Copyright © 1993 by Academic Press, Inc. All rights of reproduction in any form reserved. 1 — Vol. 196, No. 3, 1993 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 36 Fig. 1. Schematic drawing of porcine neuropeptide Y. The PP-fold comprises two antiparallel helices, an ^-terminal polyproline helix (residues 1-8) and an amphipathic a- helix (residues 14-32), connected by a type I (3-turn. as NPY(13-36) effectively. A single substitution of the middle residue in the a-helical region with a helix-breaking proline residue caused decrease in the binding affinity to both types of receptors as well as in the a-helix content (6), suggesting that the formation of a-helical structure is very important for the receptor binding. To elucidate the a-helix- stabilizing effect of amino acids at the helical end in NPY, we have prepared analogs of C- terminal fragments of NPY, in which AMerminal residues were substituted. Two major interactions that stabilize a-helix at its ends are known; charged side- chain/helix dipole interactions (7) and side-chain/main-chain hydrogen bonding (8). a- Helices have a large macrodipole with a positive pole near the iV-terminus and a negative pole near the C-terminus, because individual peptide dipoles are aligned almost parallel to the helix axis. Therefore, electrostatic interactions between the macrodipole and charged side-chains at the ends of helix are important for a-helix stability (7, 9). An alternative determinant is the presence of residues at the helix ends whose side chains can form hydrogen bonds with the initial four NH groups and final four CO groups of the main chain, because these groups lack intrahelical hydrogen-bond partners. Such a type of side- chain/main-chain hydrogen bonding is called end capping or capping interaction (8, 10). In the PP-fold family, the charged side-chain/helix dipole interactions are reported to be important for the stabilization for the a-helical part (11, 12). In this study, however, we found that end capping at the AMerminus of the a-helical part in NPY is more important for the stabilization. MATERIALS AND METHODS Materials. NPY and Fmoc-L-amino acids were purchased from Peptide Institute, Inc. (Osaka, Japan), reagents for peptide synthesis and TentaGel TG-RAM resin from — 2 — Vol. [0] (deg • cm2/dmol) -20000 -15000 -10000 196, the Figure content spectra method by binding [Thr assay holder. temperature spectropolarimeter, solid-phase analytical and from assisted time-of-flight reagent Shimadzu CD, No. same aminomethanesulfonate 12 The purified CD Fig. [Thr Amersham were ]NPY(12-36) Wavelength The Receptor CD Peptide and indicating grade. of 3, 3 spectra laser to by 12 2. pH peptides shows ]NPY(12-36) HPLC, Shigeri Corp. Y 1993 secondary Measurements. typical prepared using CD of peptide 2 (pH desorption by receptors mass were peptide spectra Synthesis. reversed (Kyoto, International 7.5) the Binding and et the a examples measured synthesizer spectrometer had (nm) al. mixture were Model according a-helix the structure and solutions mean (18.0 (13). of ionization. about on Japan), molecular BIOCHEMICAL buffer phase RESULTS NPY(12-36), temperature Assay. dissolved ^M); the at J-500A, NPY are residue of (Amersham, contents 10°C 4-fold CD of was hippocampal a-helix PSSM-8, shown to curve (pH HPLC. and (Shimadzu/Kratos analogs analogs the (A), Porcine measurements controlled weights 7.5). ellipticity 125 higher equipped 3, Wavelength method AND in (37°C) 25°C in [Thr [Arg of — I-NPY and AND The Fig. cleaved of 5 Curve were 3 analogs U.K.). 12 hippocampal 12 (B) — a-helix were C-terminal mM random ]NPY(12-36) DISCUSSION ]NPY(12-36) 2. membranes purity by as BIOPHYSICAL of labeled with and synthesized at The 1, those using from confirmed 7, Shigeri All 222 NPY(12-36) of (nm) 37°C content Kompact A were a of analogs coil. f NPY(ll-36), the -[tris(hydroxymethyl)methyl]-2- data with the a fragments for nm, each (C) thermostatically membranes (14.3 other was and et resin the carried than Calculation Bolton processor with by -[<9)222 on al. showed peptide RESEARCH MALDIII) receptor nM). determined [Arg mass chemicals a (30.8 by [Arg (13). a of Shimadzu 0.2 trifluoroacetic (14) and 12 NPY out spectrometry 12 NPY(12-36) ]NPY(12-36). characteristic ^M); Wavelength cm used was ]NPY(12-36) DP-501. Hunter binding The with of revealed controlled light was COMMUNICATIONS used with curve checked for also the automated 125 path. analyzed a binding were matrix- a-helix reagent by I-NPY assay. 2, Jasco acid, on The that and (nm) cell CD the by of at a Vol. 196, No. 3, 1993 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 0 5 10 15 0 5 10 15 20 25 30 0 5 10 15 20 25 a-Helix Content (%) Fig. 3. a-Helix contents of analogs of NPY(ll-36) (A), NPY(12-36) (B) and NPY(13-36) (C). In all analogs, TV-terminal amino acids were replaced with the indicated amino acids. a-Helix contents were calculated by using [0)222 values at 37°C according to the method of Chen et al. (14). Arrowheads indicate the wild-type jV-terminal amino acids. *, not determined. NPY(13-36) at pH 7.5 and 37°C. When the CD spectra of analogs of NPY(12-36) were monitored as a function of temperature, the midpoint temperatures of thermal unfolding curves decreased in order of a-helical content shown in Fig. 3B. In NPY(12-36), TV- terminal residues whose side chains can accept a main chain NH proton (Thr, Ser, Gin, Cys, and Asn) are more effective for the helix-stabilization than negatively charged residues (Asp and Glu). Although the replacement of TV-terminal Pro with Thr or Ser in NPY(13-36) also stabilized the a-helix to some extent, other substitutions in NPY(13-36) and any substitution in NPY(ll-36) did not significantly affect the stability (Fig. 3AC). These results indicate that Ala-12 in NPY is located at the TV-cap position which demarcates the helix TV-terminus, and that TV-terminal capping is more important for helix stabilization than negatively charged side-chain/helix dipole interactions in NPY. The helix-stabilizing tendency at this position is consistent with the preferences observed in X- ray elucidated proteins (10,15) and short model peptides (16-18). The presence of Pro at 13th residue in NPY also coincides well with the preference at TV-cap + 1 (10). The helix-stabilizing effect of Gly at 12th residue cannot be attributed to capping or charge- dipole interactions. A similar result obtained in barnase was explained in terms of the least interference of Gly with solvation of the NH groups at the helix terminus (19). To reveal the relationship between formation of the a-helical part and receptor binding activity, we examined the inhibitory effect of above peptides on 125I-NPY binding — 4 — Vol. 196, No. 3, 1993 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Thr Ser Gly Gin Cys Asn Asp Val Phe Glu Lys Tyr Ala Trp His Arg 10'S 10'6 1Q-7 10-8 10-9 ICso (M) Fig. 4. Inhibition of 125I-NPY binding to Y% receptor by analogs of NPY(12-36). IC50 values were calculated from inhibition curves which were obtained by displacement of specific 125I-NPY binding to porcine hippocampal membranes by the analogs. to porcine hippocampal membranes, which were often used as a homogeneous Y2 receptor preparation (6, 13). IC50 values of the analogs of NPY(12-36) significantly differed as shown in Fig. 4, but there was no apparent relationship between the helix content and IC50 value. Recent studies on the arginine-rich region (17 amino acid peptide) of the HIV Rev protein revealed that a-helix content of its analogs correlated well with specific binding to Rev response element RNA (20). In this case, formation of a rigid secondary structure is a prerequisite for specific binding to its target. In contrast, it seems unnecessary for NPY to form a rigid a-helical structure before binding to the Y2 receptor. REFERENCES 1. Schwartz, T. W., Fuhlendorff, 1, Kjems, L. L., Kristensen, M. S., Vervelde, M., O'Hare, M., Krstenansky, J. L., and Bjpmholm, B. (1990)A/m. NY Acad. Sci. 611, 35-47. 2. Cowley, D. J., Hoflack, J. M., Pelton, J. T., and Saudek, V. (1992) Ear. J. Biochem. 205, 1099-1106. 3. Mierke, D. F., Durr, H., Kessler, H., and Jung, G. (1992) Eur. J. Biochem. 206, 39- 48. 4. Wahlestedt, C. and Reis, D. J. (1993 )Annu. Rev. Pharm. Toxicol. 33, 309-352. 5. Wahlestedt, C., Grundemar, L., Hakanson, R., Heilig, M., Shen, G. H., Zukowska- Grojec, Z., and Reis, D. J. (1990) .4/m. NY Acad. Sci. 611, 7-26. 6. Fuhlendorff, J., Johansen, N. L., Melberg, S. G., Thpgersen, H., and Schwartz, T. W. (1990)7. Biol. Chem. 265,11706-11712. 7. Shoemaker, K. R., Kim, P. S., York, E. J., Stewart, J. M., and Baldwin, R. L. (1987) Nature 326, 563-567. 8. Presta, L. G. and Rose, G. D. (1988) Science 240, 1632-1641. 9. Scholtz, J. M. and Baldwin, R. L. (1992) Anna. Rev. Biophys. Biomol. Struct. 21, 95-118. 10. Richardson, J. S. and Richardson, D. C. (1988) Science 240, 1648-1652. 11. Tonan, K., Kawata, Y., and Hamaguchi, K. (1990) Biochemistry 29, 4424-4429. — 5 — Vol. 196, No. 3, 1993 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 12. Bj0mholm, B., J0rgensen, F. S., and Schwartz, T. W. (1993) Biochemistry 32, 2954- 2959. 13. Shigeri, Y., Mihara, S., and Fujimoto, M. (1991)7. Neurochem. 56, 852-859. 14. Chen, Y.-H., Yang, J. T., and Chau, K. H. (1972) Biochemistry 13, 3350-3359. 15. Stickle, D. F., Presta, L. G., Dill, K. A., and Rose, G. D. (1992)7. Mol. Biol. 226, 1143-1159. 16. Lyu, P. C., Zhou, H. X., Jelveh, N., Wemmer, D. E., and Kallenbach, N. R. (1992) J. Am. Chem. Soc. 114, 6560-6562. 17. Lyu, P. C., Wemmer, D. E.,Zhou, H. X., Pinker, R. J., and Kallenbach, N. R. (1993) Biochemistry 32, 421-425. 18. Forood, B., Feliciano, E. J., and Nambiar, K. P. (1993) Proc. Natl. Acad. Sci. USA 90, 838-842. 19. Serrano, L. and Fersht, A. R. (1989) Nature 342, 296-299. 20. Tan, R., Chen, L., Buettner, J. A., Hudson, D., and Frankel, A. D. (1993) Cell 73, 1031-1040. — 6 — hit. J. Peptide Protein Res. 42. 1993, 216-226 Copyright © Munksgaard 1993 Printed in Belgium - all rights reserved INTERNATIONAL JOURNAL OF PEPTIDE & PROTEIN RESEARCH ISSN 0367-8377 Quantitative structure-activity relationship (QSAR) study of elastase substrates and inhibitors MOTOYOSHI NOMIZU1, TAKANORI IWAKIl, TAKEYOSHI YAMASHITA1, YOSHIMASA INAGAKI', KATSUHIKO ASANO1, MIKI AKAMATSU2 and TOSHIO FUJITA2 Pharmaceutical Laboratory, Kirin Brewery Co. Ltd., Maebashi Gunma, Japan and 2 Department of Agricultural Chemistry, Faculty of Agriculture, Kyoto University, Kyoto, Japan Received 27 August 1992, accepted for publication 17 January 1993 One hundred Suc-X-Y-Ala-pNA peptides (Sue: succinyl, pNA: /?-nitroanilide, X, Y: Gly, Ala, Val, Leu, lie, Phe, Pro, a-aminobutyric acid, norvaline, norleucine) were synthesized and their reaction constants with porcine pancreatic elastase (Km, kcat and kC!lJKm) were determined. These reaction constants were quanti tatively analyzed using the Free-Wilson/Fujita-Ban method. The contribution of amino acid side chains to the reaction constants Km, kcat and kcat/Km, expressed logarithmically, was found to be additive. On the other hand, 19 elastase inhibitors of the general formula CF3CO-X-Y-Ala-pNA (X,Y: ten amino acids) were synthesized, and their inhibition constants were compared with the Michaelis constant for the correspond ing substrates and analyzed using free-energy-related substituent constants. In the analysis of amino acid side chains in the Y position, the K\ value of the inhibitor was generally correlated to the Km value of the sub strate, which corresponded to the inhibitor, thus confirming the validity of the equation log(l/X;) = 1.271 log(l/XJ + 4.831 This study may serve as a prototypical approach to unraveling structure-activity relationships of peptide substrates and inhibitors of medicinal or agricultural importance. © Munksgaard 1993. Key words: elastase; Free-Wilson/Fujita-Ban analysis; inhibitor; peptide; QSAR analysis; substrate Elastase is a major serine proteases which is considered Recently, the quantitative structure-activity relation to participate in the pathogenesis of some diseases, ship (QSAR) analyses of biologically active peptides, particularly of emphysema (1-3). Porcine pancreatic oxytocin (11), enkephalin (12), renin inhibitors (13), elastase (PPE) has been the subject of many studies and bitter thresholds of peptides (14), have been stud using synthetic substrates and inhibitors (4-10). In par ied. The hydrophobicity of oligopeptides has also been ticular, the substrate specificities of PPE have been analyzed using QSAR techniques, and their structural determined and discussed with peptides /?-nitroanilides descriptors were discussed (15). (6), methyl esters (7), chloromethyl ketone inhibitors (8) In this paper we describe the QSAR analyses of the and natural peptides (10). PPE substrates of the general formula Suc-X-Y-Ala- pNA [Sue: succinyl, pNA: /Miitroanilide, X,Y; Gly, Ala, Val, Leu, lie, Pro, Phe, Aba (a-amino butyric acid), Nva (norvaline), Nle (norleucine)]. The QSAR analy Abbreviations used: Sue, succinyl; pNA, p-nitroanilide; Aba, sis of the effects of the amino acid side chain on the a-aminobutyric acid; Nva, norvaline; Nle, norleucine; PPE, porcine substrates was useful for designing inhibitors. One hun pancreatic elastase; QSAR, quantitative structure-activity relation dred substrates have been synthesized and their cleav ship; Boc, rm-butyloxycarbonyl; TFA, trifluoroacetic acid; DCC, N,N' -dicyclohexylcarbodiimide; Su, A-hydroxysuccinimidyl; Np, age by PPE was determined (Km, kcat and kcat/Km). p-nitrophenyl; TEC, thin-layer chromatography; HPLC, high- The reaction constants were quantitatively analyzed performance liquid chromatography; DMF, dimethylformamide; using the Free-Wilson/Fujita-Ban method (16), and THF, tetrahydrofuran; DCHA, dicyclohexylamine; DMSO, di the effects of amino acid side chains on the reaction methyl sulfoxide. constants were elucidated in detail. In studies of en- 7 QSAR of elastase substrate/inhibitor zyme and ligand, the specificity of the Pi and Pi sites uncleaved by PPE, and Suc-Gly-Gly-Ala-pNA showed (17) is the most important determinant, but in design extremely low activity. Therefore these compounds ing specific ligands or drugs, the specificities of the were not used in these analyses. The log(l/X m), log kcat other sites are also important factors. The quantitative and log^cat/Xm) values of the 89 compounds are shown analyses of the subtle effects of amino acid side chains in Table 1. In each position the mathematical contri at the Pi and P3 sites should provide useful information butions of amino acid side-chains to the reaction con for designing new inhibitors of elastase. stants were analyzed quantitatively using the Free- Nineteen trifluoroacetylated inhibitors (18, 19) with Wilson/Fujita-Ban method (16), using the regression the general formula CFsCO-X-Y-Ala-pNA were syn equations shown below." This analytical method was thesized, and their kinetic constants (K\) were deter based on the effect of a residue in each position rela mined. The effects of amino acid residues in the X and tive to that of alanine, using the indicator variables for Y position on inhibitory activity were also analyzed by each amino acid residue except alanine. In these equa QSAR techniques, providing an interesting correlation tions the variable takes a value of 0 or 1 [e.g. Gly(X) between the inhibitors and the corresponding sub is the variable which takes a value of 1 only when the strates. amino acid at position X is glycine in Suc-X-Y-Ala- pNA.]. RESULTS log(l/AT m) = 0.127Nva(X) - 0.519Gly(Y) + 0.166Phe(Y) + Synthesis of substrates and inhibitors (0.119) (0.134) (0.128) The synthesis of substrates and inhibitors is outlined 0.524Nle(Y) + 0.206Aba(Y) - 0.171 (1) as follows. Suc-tripeptide-pNA or CFsCO-tripeptide- (0.128) (0.128) (0.050) pNA was prepared by acylation with succinic anhy dride or trifluoro ac ety limid azole of the corresponding n = 89, s - 0.178, r = 0.815 trifluoroacetic acid (TFA) treated terr-butyloxycar bonyl (Boc)-tripeptide-pNA. Boc-tripeptide-pNA com log kcat = - 1.052Gly(X) - 0.309Phe(X) - 0.557Gly(Y) pounds were synthesized by two routes. One route uti (0.116) (0.110) (0.125) lized the N,N' -dicyclohexylcarbodiimide (DCC) (20) - 0.498Leu(Y) - 0.871Phe(Y) + 0.389Pro(Y) condensation reagent for coupling of Boc-X-Y-OH with (0.119) (0.119) (0.119) H-Ala-pNA. Boc-X-Y-OH was prepared using the A-hydroxylsuccinimidyl (Su) (21) or p-nitrophenyl (Np) -0.213Nle(Y) +0.939 (2) (22) active ester procedure. The other route utilized the (0.119) (0.052) DCC condensation through coupling of Box-X-OH n - 89, s - 0.163, r = 0.948 with HCl-dioxane-treated Boc-Y-Ala-pNA. Boc-Y- Ala-pNA was prepared by condensation of Boc-Y-OH log (kcaJKm) = - 1.158Gly(X) - 0.439Phe(X) - 0.147Leu(X) and H-Ala-pNA using the DCC method. (0.089) (0.085) (0.085) Enzyme assays and kinetic analysis - 1.050Gly(Y) - 0.527Leu(Y) - 0.674Phe(Y) Amidolytic activities of the above synthetic substrates (0.100) (0.095) (0.095) were assayed using PPE and spectrophotometric + 0.457Pro(Y) + 0.347Nle(Y) + 0.199Nva(Y) monitoring at 410 nm of the rate of production of (0.095) (0.095) (0.095) /7-nitroaniline. Kinetic studies were carried out to de termine Km, kcat and kcat/Km. The Michaelis constant, + 0.242Aba(Y) + 3.793 (3) Km, was calculated from the Lineweaver-Burk plot (0.095) (0.051) obtained with substrate concentrations in the range n = 89, j = 0.125, r= 0.979 0.01-2.5 ium; (fccat = v /[E]). The inhibitory activities of the above trifluoroacetyl In these and the following equations, n is the number tripeptide derivatives toward the enzyme were tested, of compounds, s is the standard deviation, r is the and kinetic studies were carried out in order to deter correlation coefficient, and the values in parentheses mine the mode and potency {K\) of the inhibition. are the 95% confidence intervals. Enzymatic activity was assayed by the use of the syn The constant values have been calculated for Suc- thetic substrate Suc-(Ala)]-pNA (compound 11) (9) Ala-Ala-Ala-pNA. and monitoring the rate of production of o-nitroaniline at 410 nm. Analysis of inhibitors Next, the inhibitory potencies of the synthetic inhibi Analysis of substrates tors, general formula CF3CO-X-Y-Ala-pNA, were The reaction constants which were observed for the quantitatively analyzed. The mathematical contribu synthetic substrates were analyzed quantitatively. Suc- tions of amino acid residues in the X and Y positions Pro-Y-Ala-pNAs (Y: ten amino acids) were essentially to the inhibitory potency were calculated. — 8 — M. Nomizu et al. TABLE 1 Synthetic substrates and their reaction constants No. Suc-X-Y-Ala-pNA !og(l/Xm) log k cal log (kcal/Km) X Y Obsd. Calcd. Obsd. Calcd. Obsd. Calcd. 1 Gly Ala -0.354 -0.170 0.049 -0.113 2.695 2.636 2 Gly Val -0.231 -0.171 -0.105 -0.113 2.703 2.636 3 Gly Leu 0.125 -0.171 -0.796 -0.611 2.328 2.109 4 Gly He 0.036 -0.171 -0.337 -0.113 2.704 2.636 5 Gly Pro -0.322 -0.171 0.068 0.276 2.746 3.092 6 Gly Phe - 0.260 - 0.006 -0.854 - 0.984 1.881 1.961 7 Gly Aba 0.059 0.034 - 1.121 -0.113 2.832 2.877 8 Gly Nva -0.220 -0.171 0.041 -0.113 2.882 2.834 9 Gly Nle 0.149 0.353 -0.155 -0.326 2.994 2.983 10 Ala Gly -0.686 - 0.690 0.107 0.383 2.422 2.743 11 Ala Ala -0.225 -0.171 0.936 0.939 3.711 3.793 12 Ala Val - 0.049 -0.171 0.979 0.939 3.930 3.793 13 Ala Leu -0.193 -0.171 0.515 0.441 3.322 3.267 14 Ala He -0.083 -0.171 0.951 0.939 3.869 3.793 15 Ala Pro -0.207 -0.171 1.436 1.328 4.230 4.250 16 Ala Phe - 0.072 - 0.006 0.375 0.068 3.303 3.119 17 Ala Aba -0.114 0.034 1.033 0.939 3.920 4.035 18 Ala Nva -0.061 -0.171 1.045 0.939 3.985 3.992 19 Ala Nle 0.638 0.353 0.622 0.726 4.265 4.141 20 Val Gly -0.493 - 0.690 0.467 0.383 2.974 2.743 21 Val Ala -0.215 -0.171 1.185 0.939 3.970 3.793 22 Val Val 0.041 -0.171 0.876 0.939 3.920 3.793 23 Val Leu - 0.243 -0.171 0.561 0.441 3.318 3.267 24 Val He -0.170 -0.171 0.880 0.939 3.710 3.793 25 Val Pro 0.033 -0.171 1.476 1.328 4.509 4.250 26 Val Phe 0.018 - 0.006 0.210 0.068 3.225 3.119 27 Val Aba 0.276 0.034 0.866 0.939 4.140 4.035 28 Val Nva 0.060 -0.171 0.965 0.939 4.025 3.992 29 Val Nle 0.228 0.353 0.864 0.726 4.090 4.141 30 Leu Gly -0.559 - 0.690 0.188 0.383 2.628 2.596 31 Leu Ala -0.326 -0.171 1.107 0.939 3.781 3.646 32 Leu Val -0.185 -0.171 0.780 0.939 3.595 3.646 33 Leu Leu -0.340 -0.171 0.412 0.441 3.072 3.119 34 Leu He - 0.452 -0.171 0.991 0.939 3.539 3.646 35 Leu Pro 0.142 -0.171 0.996 1.328 4.140 4.103 36 Leu Phe -0.152 - 0.006 0.033 0.068 2.881 2.971 37 Leu Aba -0.158 0.034 1.053 0.939 3.895 3.888 38 Leu Nva - 0.045 -0.171 0.925 0.939 3.880 3.844 39 Leu Nle 0.071 0.353 0.956 0.726 4.041 3.993 40 lie Gly - 0.708 - 0.690 0.607 0.383 2.900 2.743 41 lie Ala -0.371 -0.171 1.210 0.939 3.838 3.793 42 lie Val -0.252 -0.171 0.927 0.939 3.674 3.793 43 lie Leu -0.312 -0.171 0.358 0.441 3.045 3.267 44 He He -0.152 -0.171 0.738 0.939 3.585 3.793 45 He Pro 0.071 -0.171 1.274 1.328 4.346 4.250 46 He Phe -0.188 - 0.006 0.158 0.068 2.971 3.119 47 He Aba - 0.086 0.034 1.068 0.939 3.982 4.035 48 He Nva - 0.228 -0.171 1.061 0.939 3.833 3.992 49 He Nle 0.585 0.353 0.407 0.726 3.992 4.141 50 Phe Gly -0.812 -0.690 0.037 0.073 2.225 2.305 51 Phe Ala - 0.425 -0.171 0.890 0.630 3.465 3.355 52 Phe Val -0.207 -0.171 0.511 0.630 3.303 3.355 53 Phe Leu -0.344 -0.171 0.310 0.132 2.965 2.828 54 Phe He -0.255 -0.171 0.490 0.630 3.236 3.355 55 Phe Pro -0.107 -0.171 1.000 1.019 3.893 3.811 56 Phe Phe 0.013 - 0.006 - 0.409 -0.241 2.605 2.680 Continued — 9 — QSAR of elastase substrate/inhibitor TABLE 1 (continued) No. Suc-X-Y-Ala-pNA log (1/Xm) log fccat lOg (fcea,/Xm) X Y Obsd. Calcd. Obsd. Calcd. Obsd. Calcd. 57 Phe Aba 0.053 0.034 0.556 0.630 3.610 3.596 58 Phe Nva -0.033 -0.171 0.612 0.630 3.579 3.553 59 Phe Nle 0.108 0.353 0.553 0.417 3.660 3.702 60 Aba Gly - 0.906 - 0.690 0.565 0.383 2.658 2.743 61 Aba Ala -0.155 -0.171 1.086 0.939 3.931 3.793 62 Aba Val 0.215 -0.171 0.619 0.939 3.834 3.793 63 Aba Leu -0.037 -0.171 0.471 0.441 3.435 3.267 64 Aba lie 0.222 -0.171 0.653 0.939 3.876 3.793 65 Aba Pro -0.362 -0.171 1.581 1.328 4.220 4.250 66 Aba Phe 0.252 - 0.006 - 0.076 0.068 3.179 3.119 67 Aba Aba 0.060 0.034 1.045 0.939 4.107 4.035 68 Aba Nva - 0.244 -0.171 0.787 0.939 4.029 3.992 69 Aba Nle 0.538 0.353 0.589 0.726 4.124 4.141 70 Nva Gly -0.377 -0.563 0.199 0.383 2.822 2.743 71 Nva Ala -0.362 - 0.044 1.060 0.939 3.699 3.793 72 Nva Val 0.051 - 0.044 0.814 0.939 3.863 3.793 73 Nva Leu - 0.246 - 0.044 0.471 0.441 3.226 3.267 74 Nva lie - 0.228 - 0.044 0.860 0.939 3.631 3.793 75 Nva Pro -0.143 - 0.044 1.380 1.328 4.238 4.250 76 Nva Phe 0.222 0.121 -0.102 0.068 3.124 3.119 77 Nva Aba 0.131 0.161 0.925 0.939 4.061 4.035 78 Nva Nva 0.316 - 0.044 0.741 0.939 4.057 3.992 79 Nva Nle 0.569 0.480 0.604 0.726 4.057 3.992 80 Nle Gly -0.851 -0.690 0.581 0.383 2.730 2.743 81 Nle Ala -0.328 -0.171 1.143 0.939 3.814 3.793 82 Nle Val -0.188 -0.171 0.840 0.939 3.653 3.793 83 Nle Leu -0.371 -0.171 0.303 0.441 2.947 3.267 84 Nle He - 0.068 -0.171 0.730 0.939 3.662 3.793 85 Nle Pro -0.196 -0.171 1.380 1.328 4.185 4.250 86 Nle Phe 0.244 - 0.006 -0.086 0.068 3.158 3.119 87 Nle Aba 0.215 0.034 0.812 0.939 4.025 4.035 88 Nle Nva - 0.262 -0.171 1.176 0.939 3.914 3.992 89 Nle Nle 0.420 0.353 0.736 0.726 4.173 4.141 In the analysis of the X position, the log(l/Xi) val TABLE 2 ues of the seven CFsCO-X-Ala-Ala-pNAs are shown Inhibition of PPE by CFjCO-X-Ala-Ala-pNA in Table 2. %-Values for amino acid side chains were proposed on the basis of the experimentally measured No. CF3CO-X-AIa-Ala-pNA log(l/Xj) 7Ta Bb hydrophobicity (15,23). Equation (4) was derived using them. Obsd. Calcd. log(l/Xi) = 0.515%-0.3294 + 4.471 (4) 101 Ala 4.545 4.631 0.31 0 (0.193) (0.209) (0.249) 102 Val 4.773 4.770 1.22 1 103 Leu 5.062 5.018 1.70 1 n = l, s = 0.089, r = 0.967 104 He 5.022 5.069 1.80 1 107 Aba 4.988 4.909 0.85 0 In eqn. (4), B is an indicator variable: if the amino acid 108 Nva 5.274 5.187 1.39 0 X has a branched side chain, B = 1; if it has a linear side 109 Nle 5.384 5.415 1.93 0 chain, a = 0. a %-values from ref. (23). In the analysis of the Y position, the log(l/Xj) values b B = 0 if the amino acid has linear side chain, or B = 1 if the amino of the nine inhibitors are listed in Table 3. The corre- acid side chain is branched. 10 M. Nomizu et al. TABLE 3 DISCUSSION Inhibition of PPE by CFjCO-Ala-Y-Ala-pNA In eqns. (l)-(3) each slope value of the indicator vari No. CF3CO-Ala-Y-Ala-pNA log(l/ATj) log( 1 j )a able terms shows the degree of the variation of activ ity caused by replacing the alanine residue with the Obsd. Calcd. corresponding amino acid into this position. The mathe matical contributions of the amino acid side chains to 110 Gly 3.935 3.959 -0.354 101 Ala 4.545 4.545 -0.225 the reaction constants in the X and Y positions are 111 Val 4.847 4.769 -0.049 summarized in Table 4. 112 Leu 4.701 4.586 -0.193 The Free-Wilson/Fujita-Ban method assumes that 113 lie 4.569 4.726 -0.083 the mathematical contributions of each partial structure 114 Pro 4.582 4.568 -0.209 to the activity are additive. The contributions of amino 115 Phe 4.635 4.740 -0.072 acid side chains to log(l/AT m) and log kcat were found 116 Aba 4.756 4.686 -0.114 to be additive to some degree; each value is found 117 Nva 4.764 4.754 -0.061 within a small range. a log(l/Am) value corresponding to Suc-Ala-Y-Ala-pNA substrate Equation (3) for \og(k cat/Km) is a very satisfactory listed in Table 1. equation, as is shown by the standard deviation and the correlation coefficient. It suggests that effects of variation of the amino acids side chain in each posi lation between log( 1/ATi) for CFgCO-Ala-Y-Ala-pNA tion are almost completely explained by the additive and log(l/X m) for the corresponding succinyl tripeptide model. derivative was found, and eqn. (5) was derived. As shown in Table 4, an increase or decrease of the volume of amino acid side chain in the X position fails log(l/Xi)= 1.27Ilog(l/Xm) +4.831 (5) to increase log(l/X m) and log kca.t relative to substrates (0.392) (0.104) having alanine in this position. In the Y position, the n = 9, s = 0.093, r = 0.945 values of 1 /Km were found to show a tendency to in crease with the length of the amino acid side chains, In eqn. (5) log(l/X m) is the logarithmic reaction con and the presence of amino acids with branched side stant of the corresponding succinyl tripeptide deriva chains did not affect \/Km relative to the values with an tives. alanine residue in the same position. The £cat-values TABLE 4 Effect of amino acid residues at X and Y positions on the substrate activity, the reference being taken as alanine in each position X Y Side chain Alog(l/.W Alog kcat Alog (kcat/Km) Alog(l/X m) Alog(tcat) Alog(/ccat /Xm) Gly 0 - 1.05 - 1.16 -0.52 -0.56 - 1.05 -H Ala 0a 0a 0a 0a 0a 0a -ch 3 Aba 0 0 0 0.20 0 0.25 -ch 2-ch 3 Nva -0.13 0 0 0 0 0.20 -ch 2-ch 2-ch 3 Nle 0 0 0 0.52 -0.21 0.35 -ch 2-ch 2-ch 2-ch 3 ch 3 Val 0 0 0 0 0 0 -CH ch 3 ch 3 Leu 0 0 -0.15 0 -0.50 -0.52 -ch 2-ch ch 3 ch 3 lie 0 0 0 0 0 0 -CH ch 2-ch 3 Pro (negative) 0 0.39 0.46 -CH2-CH2-CH2-(N) Phe 0 -0.31 -0.44 0.17 -0.87 -0.67 -ch 2- a Based on the effect of an alanine residue in each position. — 11 — QSAR of elastase substrate/inhibitor relative to that of alamine were increased more by pro EXPERIMENTAL PROCEDURES line residues but were decreased by glycine, norleucine, leucine and phenylalanine. Amino acid derivatives and peptide reagents were ob These analyses suggest that the reaction constants tained from Kokusan Chemical Works, Ltd. (Tokyo, in each position were related to hydrophobicity and Japan). Melting points were determined on a Yanagim- steric effects of the amino acid side chains. Km relates oto melting point apparatus, model MP-J3. Optical ro to the binding of the substrate to the enzyme, and kCSLt tations were measured with a JASCO automatic pola- to the preparation of acylated enzyme and the release rimeter, model DIP-140. The hydrolyses of peptide of />-nitroaniline from the enzyme, which is related derivatives for amino acid analyses were carried out in to the conformational variation of the enzyme. Thus 6 N HC1 at 110 °C for 24 h. Amino acid compositions this seemed to require a more detailed investigation of acid hydrolysates were determined with a Hitachi by comparison with the effect of inhibition using inhibi amino acid analyzer, model L-8500. Thin-layer chro tors. matography (TLC) was performed on silica-gel plate The effect of the amino acid residue in the X posi (Kieselgel G60, Merck). HPLC was conducted with a tion is given in eqn. (4), which shows that 1 fK\ is in Waters compact model 204, using a Nucleosil 5Cig creased by hydrophobic amino acid side chains, and column (7.5 x 250 mm). decreased by branched side chains. For the set of 7 residues in the X position, a correlation of the inhibi General procedure for synthesis of Boc-Y-Ala-pNA tors and the substrates was not found. Thus the results (Y = Gly, Ala, Val, Leu, lie, Pro, Phe, Aba, Nva, Nle). suggested that in the case of a Suc-type substrate and DCC (5.92 g, 28.68 mmol) was added to a mixture of a CFgCO-type inhibitor, the amino acid in the X po Boc-Y-OH (26.68 mmol) and H-Ala-pNA (5.00 g, sition binds to a different site on the enzyme. 23.90 mmol) in dimethylformamide (DMF) (80 mL) at From eqn. (5), obtained by analysis of the effect of 0 0 C and the mixture, after being stirred at room tem the amino acid residue in the Y position, an interesting perature overnight, was filtered. The solvent was re phenomenon is observed: the 1/ATi and Km values are moved by evaporation and the residue was extracted neutrally correlated (for details see Table 3). This phe with AcOEt (200 mL). The organic phase was washed nomenon suggested that in the Y position the same with 5% citric acid, 5% NaHCOs and H2O, dried over amino acid is favored both in the substrate and in the NazSCL and concentrated. The residue was crystal inhibitor. lized from ether or isopropylether followed by recrys Many studies of the X-ray crystal structures of PPE tallization from MeOH with ether or isopropylether. with or without ligands have appeared (24), and the General procedure for synthesis of Boc-X-Y-OH X-ray structure of trifluoroacetyl-dipeptide-pNA with (X,Y= Gly, Ala, Val, Leu, lie, Pro, Phe). Boc-X-OSu or PPE has been determined by Hughes et al. (25). This ONp (30.0 mmol) in tetrahydrofuran (THF) (100 mL) X-ray study showed that the CF3CO group is at the S1 was mixed with a solution or suspension of H-Y-OH subsite in the active site of PPE, and dipeptide-pNA is (90.0 mmol) and Et2N (12.5 mL, 90.0 mmol) in H20 bound at sites close to the SI' to S3' subsite. Com (50 mL) in an ice-bath. After 6 h the solvent was re parison between the X-ray studies and our QSAR re moved by evaporation and the residue was dissolved in sults suggests that the Suc-type substrate and CF3CO- 5% NaHCCL (200 mL). The aqueous phase was type inhibitor are bound in different sites. Nle was most washed with ether and acidified with citric acid. The suitable in the X position of CFsCO-tripeptide-pNA, resulting precipitate was extracted with AcOEt and the Lys residue in CF3CO-dipeptide-pNA is bound (200 mL) and the extract was washed with 5 % citric at the SI' site of PPE. Nle and Lys have similar shapes acid and aqueous saturated NaCl solution, then dried but have differing charge distributions. Thus the two over Na2S04 and concentrated. The residue was pu residues seem to bind close together in an area which rified by recrystallization from appropriate solvents is narrow and hydrophobic. However, in our QSAR (MeOH-Et20, MeOH-isopropyl ether, AcOEt-n- study, a correlation of the amino acid effects in the Y hexane). If an oily precipate was obtained, it was con position of the substrates (Km) and the inhibitors (X;) verted into the dicyclohexylamine (DOHA) salt and the was observed. From this observation we postulate that salt was recrystallized or precipitated from MeOH- the Y residue of the Suc-type substrate and the CF3CO- ether or AcOEt-n-hexane. Boc-Pro-Pro-OH and its type inhibitor seem to bind to close or similar sites of DOHA salt were oily products, and could not purified PPE. by the above procedures. They were purified by column These quantitative analyses of the effects of the amino chromatography on silica gel using CHCL-MeOH acid composition on substrate and inhibitory activity (10:0.5) as an eluent. are very interesting enzymatically and lead to a useful index for the design of new elastase inhibitors and the General procedure for synthesis of Boc-X- Y-Ala-pNA from development of peptidic drugs. Boc-X-Y-OH and H-Ala-pNA (X,Y= Gly, Ala, Val, Leu, He, Pro, Phe). DCC (592 mg, 2.87 mmol) was added to a mixture of Boc-X-Y-OH (2.87 mmol) and H-Ala- — 12 — M. Nomizu et al. TABLE 5 Purification procedure, m.p., optical rotation, Rf values and HPLC retention time of Suc-X-Y-Ala-pNA No. Suc-X-Y-Ala-pNA Purification M.p. (°C) [oc Jd in DMSO Rft HPLC system 11 (cone.) retention X Y time (min)c 0 Gly Gly C 200-202 -29.2(0.3) 0.11 26.22 1 Gly Ala C 125-127 -28.7(0.1) 0.23 27.86* 2 Gly Val c 185-188 -27.6(0.7) 0.26 32.16 3 Gly Leu A 148-150 - 23.4 (0.6) 0.38 36.40 4 Gly He A 120-123 -35.2(0.2) 0.40 35.56* 5 Gly Pro A 216-218 - 57.8 (0.4) 0.16 29.40 6 Gly Phe A 102-105 -30.9(0.1) 0.40 38.22 7 Gly Aba A 151-153 -20.0 (0.3) 0.16 31.16 8 Gly Nva A 108-110 - 13.8 (0.5) 0.16 35.24 9 Gly Nle A 97-99 - 11.8(0.5) 0.16 37.46 10 Ala Gly A 130-132 -1.4 (0.2) 0.24 28.82 11 Ala Ala B 122-124 - 26.2 (0.5) 0.16 30.64 12 Ala Val B 122-124 - 39.8 (0.5) 0.29 34.04* 13 Ala Leu B 175-177 -35.4 (0.3) 0.45 38.90 14 Ala He B 225-227 -40.8(0.6) 0.37 38.60 15 Ala Pro B 130-132 -57.2 (0.4) 0.37 30.46 16 Ala Phe B 136-138 - 20.2 (0.3) 0.54 41.38 17 Ala Aba B 214-216 - 30.3 (0.5) 0.18 34.04* 18 Ala Nva B 190-192 -24.5(0.3) 0.27 36.90 19 Ala Nle B 142-144 -23.7 (0.5) 0.30 40.42 20 Val Gly A 190-194 - 11.2 (0.3) 0.38 34.12 21 Val Ala B 145-148 -21.5(0.4) 0.32 36.02 22 Val Val B 221-224 -29.8(0.3) 0.39 38.90 23 Val Leu B 122-125 -30.4 (0.3) 0.51 44.42 24 Val He B 182-184 - 27.2 (0.3) 0.59 41.80 25 Val Pro B 115-117 -39.8(0.3) 0.45 35.54* 26 Val Phe B 229-233 - 20.4 (0.4) 0.49 49.82 27 Val Aba B 237-239 - 26.7 (0.3) 0.26 38.80* 28 Val Nva B 223-227 -26.5(0.3) 0.26 41.96* 29 Val Nle B 196-198 - 17.5 (0.4) 0.30 45.42 30 Leu Gly A 192-195 -28.7 (0.5) 0.28 39.16* 31 Leu Ala B 170-173 -22.9(0.7) 0.36 40.80* 32 Leu Val B 222-225 - 29.9 (0.7) 0.40 42.74 33 Leu Leu B 120-122 - 30.6 (0.6) 0.68 48.60 34 Leu He B 214-217 -31.1(0.8) 0.56 45.80* 35 Leu Pro B 104-108 -46.8 (0.5) 0.39 39.84 36 Leu Phe B 198-200 - 17.4 (0.3) 0.64 49.60 37 Leu Aba B 212-214 -29.1 (0.4) 0.28 43.56 38 Leu Nva B 211-213 -30.5(0.5) 0.36 45.96* 39 Leu Nle B 178-180 -31.1 (0.3) 0.39 49.84 40 lie Gly C 113-115 - 9.2 (0.2) 0.32 37.56 41 lie Ala B 225-228 - 16.9(0.3) 0.47 40.20 42 He Val B 227-230 - 19.2 (0.3) 0.47 42.08 43 He Leu B 185-187 - 24.3 (0.3) 0.52 47.78 44 He He B 245-247 -26.1(0.3) 0.69 45.32 45 He Pro B 135-140 -49.2 (0.4) 0.40 38.56 46 He Phe B 115-118 - 13.2(0.3) 0.68 49.86 47 He Aba B 239-241 -21.6 (0.4) 0.27 43.16* 48 He Nva B 238-240 - 26.6 (0.4) 0.37 47.54* 49 He Nle B 201-203 -24.1 (0.3) 0.42 49.64* 50 Phe Gly A 105-107 - 14.6(0.3) 0.42 38.86 51 Phe Ala A 195-197 - 12.8 (0.2) 0.40 41.80* 52 Phe Val B 120-124 - 10.4 (0.4) 0.68 39.20 53 Phe Leu B 206-208 - 14.7 (0.4) 0.65 48.76 54 Phe He B 192-195 - 12.7(0.4) 0.68 46.66 55 Phe Pro A 100-102 -21.3(0.2) 0.46 41.46* Continued 13 QSAR of elastase substrate/inhibitor TABLE 5 (continued) No. Suc-X-Y-Ala-pNA Purification M.p. (°C) [(*]□ in DMSO Rfh HPLC system 3 (cone.) retention X Y time (min)" 56 Phe Phe B 210-223 -38.1 (0.3) 0.52 49.40 57 Phe Aba B 210-212 - 11.3(0.3) 0.56 43.76 58 Phe Nva B 212-215 - 19.2 (0.7) 0.54 46.62* 59 Phe Nle B 225-228 - 14.4 (0.5) 0.55 50.16* 60 Aba Gly C 215-217 - 13.8 (0.6) 0.15 31.42 61 Aba Ala A 219-221 - 13.6 (0.3) 0.23 34.96 62 Aba Val B 200-203 - 37.3 (0.4) 0.37 42.56 63 Aba Leu B 230-233 - 29.7 (0.4) 0.27 37.42* 64 Aba lie B 225-230 -40.1 (0.5) 0.30 39.80 65 Aba Pro C 92-95 -41.7 (0.4) 0.20 34.24 66 Aba Phe B 225-228 -64.4 (0.5) 0.42 44.40 67 Aba Aba B 249-253 -21.5(0.3) 0.21 36.98 68 Aba Nva B 224-227 - 33.3 (0.6) 0.24 39.42 69 Aba Nle B 192-194 - 30.0 (0.3) 0.26 42.62 70 Nva Gly C 209-212 - 12.2 (0.3) 0.20 35.58 71 Nva Ala B 199-202 -22.5 (0.3) 0.28 37.96 72 Nva Val B 228-230 -27.2(0.4) 0.62 46.86 73 Nva Leu B 221-223 - 32.3 (0.4) 0.28 40.16 74 Nva • lie B 234-237 -41.8(0.4) 0.31 44.02 75 Nva Pro A 96-99 - 69.5 (0.4) 0.24 37.44 76 Nva Phe B 216-219 -26.5(0.5) 0.34 47.20* 77 Nva Aba B 239-243 - 32.4 (0.5) 0.24 40.78* 78 Nva Nva B 228-231 - 26.6 (0.7) 0.32 43.18 79 Nva Nle B 198-200 -25.0 (0.7) 0.39 46.38 80 Nle Gly A 122-124 -10.4(0.3) 0.24 38.84 81 Nle Ala B 182-184 - 30.6 (0.5) 0.39 43.40 82 Nle Val B 172-174 - 19.1 (0.4) 0.40 49.98* 83 Nle Leu B 221-223 -25.6(0.5) 0.37 44.16 84 Nle He B 228-230 - 39.2 (0.6) 0.24 44.60 85 Nle Pro C 90-92 -57.1 (0.4) 0.25 40.88* 86 Nle Phe B 213-215 - 14.7 (0.3) 0.45 51.58 87 Nle Aba B 226-228 -31.2 (0.4) 0.31 47.16 88 Nle Nva B 216-218 -21.5(0.3) 0.27 47.02 89 Nle Nle B 174-177 - 20.5 (0.4) 0.39 49.56 90 Pro Gly C 92-94 - 19.3 (0.2) 0.40 33.10 91 Pro Ala C 102-104 - 26.9 (0.2) 0.51 35.22 92 Pro Val C 102-105 -31.5(0.3) 0.43 36.80 93 Pro Leu A 109-111 -39.4 (0.3) 0.69 42.34 94 Pro He C 116-117 -31.3(0.3) 0.51 39.94 95 Pro Pro C 118-120 -46.8 (0.4) 0.45 35.98 96 Pro Phe A 166-170 -28.3(0.3) 0.60 43.66 97 Pro Aba C 96-98 -50.7(0.3) 0.35 36.54* 98 Pro Nva C 94-98 -42.3(0.5) 0.38 39.60* 99 Pro Nle C 84-86 -40.3(0.5) 0.43 43.20 a Compounds were purified following procedures A, B and C. (A) recrystallized from MeOH with n-hexane; (B) recrystallized from DMF with ether; (C) purified by HPLC, followed by precipitation from MeOH with n-hexane. b Rf values were determined in the following solvent system: CH3Cl-MeOH-AcOH (9:1:0.5, v/v). c HPLC on Nucleosil 5Ci8 column (7.5 x 250 mm) with gradient of acetonitrile (10-60%, 60 min) in 0.1% aq. TEA at a flow rate of 2.0 mL/min. * Compounds were analyzed for C, H, and N within ±0.4% of the theoretical values. pNA (500 mg, 2.39 mmol) in THF (20 mL), and the AcOEt (50 mL). The organic phase was washed with mixture was stirred at room temperature overnight, then 5% citric acid, 5% NaHCO] and H20, dried over filtered and concentrated. The residue was dissolved in NazSOa and concentrated. The residue was crystal 14 M. Nomizu et al. lized or precipitated from appropriate solvents (DMF- with TFA (5 mL) in the presence of anisole (0.5 mL) at EtiO, MeOH-EtiO, MeOH-isopropyl ether, AcOEt- ice-bath temperatures for 60 min. After evaporation of n-hexane). If the residue did not dissolve in AcOEt, the TFA in vacuo at 30 °C or less, the residue was washed crude product was triturated with EtzO and 5% citric with n-hexane. The resulting oily precipitate was dis acid, and the resulting powder was washed with 5% solved in 4 N HCl-dioxane (10 mL) at ice-bath tem citric acid, 5% NaHCOg and H2O and crystallized or peratures, and after 10 min, n-hexane was added and precipitated from appropriate solvents (DMF-AcOEt, washed. The residue was dried over KOH pellets in- DMF—EtzO, MeOH—Et20). vacuo for 3 h and dissolved in DMF (20 mL), together with Et]N (348 pL, 2.50 mmol) and Boc-X-OH General procedure for synthesis of Boc-X- Y-Ala-pNA from (3.75 mmol). After addition of DCC (774 mg, Boc-X-OHandBoc- Y-Ala-pNA (X, Y = Gly, Ala, Val, Leu, 3.75 mmol), the solution was stirred overnight, then lie, Pro, Phe, Aba, Nva, Nle; at least one ofXorYis Aba, filtered and concentrated. The crude sample was puri Nva or Nle). Boc-Y-Ala-pNA (2.50 mmol) was treated fied by the above same procedures. TABLE 6 M.p., optical rotation, Rf values and C, H, N analysis data of CF3CO-X-Y-Ala-pNA No. CF3CO-X-Y-Ala-pNA M.p. (°C) [a]“in DMSO Rf- Formula Calcd. (Found) (cone.) X Y C H N 100 Gly Ala 220-223 -32.3 0.20 CieHigNsO^F; 44.35 4.19 16.16 (0.46) (44.63 4.21 16.13) 101 Ala Ala 245-247 -40.3 0.38 C17H20N5O6F3 45.64 4.51 15.66 (0.45) (45.60 4.48 15.72) 102 Val Ala 272-274 -48.7 0.36 C19H24N5O6F3 48.00 5.09 14.73 (0.48) (47.81 4.99 14.57) 103 Leu Ala 243-245 -43.0 0.41 C20H26N5O6F3 49.08 5.35 14.31 (0.45) (48.83 5.26 14.23) 104 lie Ala 239-243 -49.8 0.51 C20H26N5O6F3 49.08 5.35 14.31 (0.35) (48.88 5.38 14.26) 105 Phe Ala 255-257 -9.3 0.54 C19H22N5O6F3 48.21 4.68 14.80 (0.49) (48.51 4.70 14.67) 106 Pro Ala 252-256 -64.9 0.35 C23H24N5O6F3 52.77 4.62 13.38 (0.66) (52.63 4.64 13.44) 107 Abu Ala 249-253 -39.1 0.43 C18H22N5O6F3 46.86 4.81 15.18 (0.48) (46.80 4.81 15.18) 108 Nva Ala 259-263 -42.1 0.50 C19H24N5O6F3 48.00 5.09 14.73 (0.42) (48.04 5.02 14.62) 109 Nle Ala 254-258 -36.5 0.44 C20H26N5O6F3 49.08 5.35 14.31 (0.50) (48.90 5.36 14.23) 110 Ala Gly 222-224 -55.8 0.28 CieHigNsOeFs 44.35 4.19 16.16 (0.49) (44.30 4.20 16.02) 111 Ala Val 274-277 -40.3 0.34 C19H24N5O6F3 48.00 5.09 14.73 (0.45) (47.81 5.07 14.47) 112 Ala Leu 199-203 -32.4 0.48 C20H26N5O6F3 49.08 5.35 14.31 (0.50) (48.81 5.33 14.18) 113 Ala lie 207-210 -36.3 0.49 C20H26N5O6F3 49.08 5.35 14.31 (0.39) (48.79 5.22 14.15) 114 Ala Phe 240-245 - 19.2 0.45 G19H22N5O6F 3* 47.31 4.81 14.45 (0.52) 1/2H20 (47.60 4.76 13.91) 115 Ala Pro 105-108 - 115.1 0.39 C23H24N5O6F3 52.77 4.62 13.38 (0.31) (52.55 4.60 13.25) 116 Ala Abu 269-272 -40.4 0.36 C18H22N5O6F3 46.86 4.81 15.18 (0.44) (46.92 4.80 15.14) 117 Ala Nva 256-258 -30.0 0.38 C19H24N5O6F3 48.00 5.09 14.73 (0.45) (47.86 5.03 14.60) 118 Ala Nle 271-273 -30.4 0.33 C20H26N5O6F3 49.08 5.35 14.31 (0.45) (48.90 5.36 14.27) a Rf values were determined in the following solvent system: CH3Cl-MeOH-AcOH (9:1:0.5, v/v). 15 QSAR of elastase substrate/inhibitor General procedure for synthesis of Suc-X-Y-Ala-pNA Enzyme inhibition studies. In a typical experiment, 250 pL (X,Y= Gly, Ala, Val, Leu, lie, Pro, Phe, Aba, Nva and of the inhibitor (40-800 pM in 50 mM Tris buffer Nle). Boc-tripeptide-pNA (1.0 mmol) was cleaved by containing 10% DMSO) and 250 pL of Suc-(Ala)]- TFA (ca. 10 mL per g of peptide) in the presence of pNA (4 mM in 50 mM Tris buffer containing 2% anisole (0.5 mL) at 0 °C for 60 min. After evaporation DMSO) were added to 450 pL of 50 mM Tris buffer of TFA in vacuo at 30 ° C or less, the residue was treated in a quartz cuvette and thermally equilibrated in the with dry ether. If a powder was obtained, it was col spectrophotometer for 10 min, and the absorbance was lected by filtration, dried over KOH pellets in vacuo for balanced at 410 nm. The enzyme (50 pL of 656 mM in 3 h and then used for the coupling reaction. If an oily 50 mM Tris buffer) was added to the sample cuvette, precipitate was obtained, it was washed with n-hexane, and the increase in absorbance was monitored for dried over KOH pellets in vacuo for 3 h and then used 10 min. Ki-values were determined by plotting the for the coupling reaction. The TFA-treated samples slopes from Lineweaver-Burk curves vs. inhibitor con were dissolved in DMF (20 mL) together with EtgN centration. (418 pL, 3 mmol) and succinic anhydride (368 mg, 2 mmol). After stirring for 4 h, the solvent was evapo rated and the residue was redissolved in n-butanol ACKNOWLEDGMENT (100 mL). The solution was washed with 5% citric acid We thank Drs. G.W. Milne, T.R. Burke and P.P. Roller, Laboratory and H2O, and n-butanol was evaporated in vacuo. Prod of Medicinal Chemistry, NCI, NIH for encouragement and critical ucts were purified by crystallization and precipitation reading of the manuscript. We thank M. Mayuzumi and A. Ohkubo from MeOH with n-hexane or DMF with EtiO. If the for their skilful technical assistance in this work. obtained products had not been completely purified, these samples were purified by HPLC on a Nucleosil 5Cis column (7.5 x 250 mm), which was eluted with a REFERENCES linear gradient of acetonitrile in 0.1% TFA. Then the 1. Robert, L. & Hornebeck, W. (1989 ) Elastin and Elastases, vol. 1, sample was precipitated from MeOH with n-hexane. CRC Press, Boca Raton The purity and identity of the sample were confirmed 2. Mittman, C. & Taylor, J.C. (eds.) (1988) Pulmonary Emphysema by TLC, HPLC and amino acid analysis. The final and Proteolysis, vol. 2, Academic Press, New York purification procedure, m.p., optical rotation, Rf values 3. Morrison, H.M. (1987) Clin. Sci. 72, 151-158. and retention time of analytical HPLC are shown in 4. Powers, J.C. & Zimmerman, M. (1989) in Design of Enzyme Table 5. Inhibitors as Drugs (Sandler, M. & Smith, H.J., eds.), pp. 596- 619, Oxford University Press, New York General procedure for synthesis of CF3 CO-X- Y-A la-pNA 5. Del Mar, E G., Largman, C., Brodrick, J.W., Fassett, M., & (X,Y= Gly, Ala, Val, Leu, lie, Pro, Phe, Aba, Nva, Nle, Geokas, M.C. (1980) Biochemistry 19, 468-472 where either X or Y was an alanine residue). TFA-treated 6. Kasafirek, E., Eric, P., Slaby, J. & Mails, F. (1976) Eur. J. samples of Boc-tripeptide-pNA (1.0 mmol) were dis Biochem. 69, 1-13 solved in DMF (20 mL) together with Et]N (418 p.L, 7. Thomson, A. & Kapadia, S B. (1979) Eur. J. Biochem. 102, 3 mmol) and trifluoroacetylimidazole (630 mg, 3 mmol). 111-116 The solution, after being stirred for 24 h, was concen 8. Powers, J.C., Gupton, B E., Hartley, A.D., Nishino, N. & Whit trated and the residue was triturated with ether and 5 % ley, R.J. (1977) Biochim. Biophys. Acta 485, 156-166 9. Bieth, J., Spiess, B. & Wermuth, C.G. (1974) Biochem. Med. 11, citric acid. The resulting powder was washed with 5% 350-357 citric acid, 5% NaHCOs and H2O and precipitated 10. Narayanan, A.S. & Anwar, R.A. (1969) Biochem. J. 114, 11- from DMF with Et20 or AcOEt. Their physical con 17. tents and analytical data are shown in Table 6. 11. Pliska, V. & Heiniger, J. (1988) Int. J. Peptide Protein Res. 31, 520-536 Enzyme assay. PPE was purchased from Sigma Co. All 12. Fauchere, J.-L. (1984) QSAR in Design of Bioactive Compounds: enzyme assays were performed spectrophotometrically Proc. 1st Telesymposium on Medicinal Chemistry, pp. 135-144, at 40 °C on a Shimadzu UV-3000 spectrophotometer. J.R. Prous, Barcelona In a typical experiment the substrate [500 pL, 2.0- 13. Nisato, D., Wagnon, J., Callet, G., Mettefeu, D., Assens, J.-L., 0.25 mM in 50 mM Tris buffer containing 2% dimethyl Plouzane, C., Tonnerre, B., Pliska, V. & Fauchere, J.-L. (1987) sulfoxide (DMSO)] was added to 450 juL of a 50 mM J. Med. Chem. 30, 2287-2291 Tris buffer solution (pH 8.0) in a quartz cuvette and 14. Asao, M., Iwamura, H., Akamatsu, M. & Fujita, T. (1987) J. thermally equilibrated in the spectrophotometer for Med. Chem. 30, 1873-1879 2 min, and the absorbance was balanced at 410 nm. 15. Akamatsu, M., Yoshida, Y., Nakamura, H., Asao, M., Iwamura, H. & Fujita, T. (1989) Quant. Struct.-Act. Relat. 8, 195-203, The enzyme (50 pL, 656 pu in 50 mM buffer) was added to the sample cuvette, and the increase in ab Akamatsu, M. & Fujita, T. (1992) J. Pharm. Sci., 81, 164-174 16. Fujita, T. & Ban, T. (1971) J. Med. Chem. 14, 148-152 sorbance was monitored for 10 min. The Michaelis 17. Schechter, I. & Berger, A. (1967) Biochem. Biophys. Res. Com- constant Km was calculated from the Lineweaver—Burk mun. 27, 157-162 plot obtained with substrate concentrations in the range 18. Dimicoli, J.-L., Renaud, A., Lestienne, P. & Bieth, J.G. (1979) 2.0-0.25 mM; kcat = V/[E]. J. Biol. Chem. 254, 5208-5218 16 — M. Nomizu et al. 19. Renaud, A., Lestienne, P., Hughes, D.L., Bieth, J.G. & Dimicoli, 25. Hughes, D.L., Sieker, L.C., Bieth, J. & Dimicoli, J.-L. (1982)/. J.-L. (1983)7. Biol. Chem. 258, 8312-8316 Mol. Biol. 162, 645-658 20. Sheehan, J.C. & Hess, G.P., (1955)/. Am. Chem. Soc. 77, 1067— 1068 21. Anderson, G.W., Zimmerman, J.E. & Callahan, F. (1964)/. Am. Address: Sbc. 86, 1839-1842 Motoyoshi Nomizu, Ph D. 22. Bodanszky, M. & du Vigneaud, V. (1959)/. Am. Chem. Soc. 81, Laboratory of Developmental Biology 5688-5691 NIDR, NIH, Bldg 30, Rm 427 23. Fauchere, J.-L. & Pliska, V. (1983) Eur. J. Med. Chem. Chim. Bethesda, MD 20892 Ther. 18, 369-375 USA 24. Bode, W., Meyer, E., Jr. & Powers, J.C. (1989) Biochemistry 28, Tel: (301 >496-9855 1951-1963 Fax: (301)-402-0897 — 17 — K CD fflz6.1t — a^^f K©^< &SfMW*St£:£aS &£a LT#(*©fi'&&©#|}#CSgtitSSI£jg/.: LT va, # sms® a warn LTvagi£> K'lC-KSSnapSBttB^^f'JfflL/cVfc^a" 4t*EE" A< medicinal chemist cmWWcflTUao KCfllii^Sic* 6tt> J: D f#ffl#M© A t Wb^# (pep t idomimei i cs) ©f'-T-f >. iBfifc li^<©W5S#Kit^Tffl*S Peptide A V Constrained Peptidomimetics A V Peptoid — 18 — Outline of the General Strategy in the Design of Peptides Primary structure of peptide of interest f Structure-activity relationship and elucidation of the importance of individual residues for biological action Biological testing ^ > Secondary structure search o , . , , , „ , and peptide design Synthesis of globally^y(structural refinement and optimization) constrained peptide Statistical and Biophysical methods model-building 1. X-ray methods 2. N.m.r. 3. Computer-assisted drug design 4. Other spectroscopic methods — 19 — (3-Pleated Sheet Peptidomimetics linked pyrrolinone compound mimics shape of J3-pleated sheet Pyrrolinone-based peptidomimetic : l i ! i Ar H Angiotensinogen fragment Ar = Aromatic group Pyrrolinone-based peptidomimetic simu lates p-pleated sheet conformation of tet- rapeptidyl angiotensinogen fragment. Dashed lines show alignment of carbonyl groups in the two structures. Side chains are also aligned ■Mi nh =V° ' 20 — v vj >mmx iz «t 5 1H-NMR Analyses of cls-trans Isomerization — Imidapril and Imidaprilat — RO=f )~N- COjH frans-form (co - 180°) (Bloactlve form) Compound Solvent cis / frans ratio Imidapril hydrochloride D20 >99% trans Imidapril (free) D20 >99% trans imidaprilat D20 +TFA >99% trans 1H-NMR Analyses of cis-trans Isomerization — Enalapril and Enalaprilat — Oh « COjH c/s-form (co » 0°) frans-form (co - 180°) (Bloactlve form) Compound Solvent cis / trans ratio Enalapril maleate D20 44 / 56 Enalapril (free) d2o 52 / 48 Enalaprilat d2o 20 / 80 D20 + TFA 27 / 73 The conformation of the amide bond of Imidaprilat is restricted to the bioactive trans geometry. — 21 — References 1. G. Holzemann, Kontakte(Darmstadt) 1, 3(1991). 2. V. J. Hruby et. al., Biochem. J. 268 . 249(1990). 3. A. F. Spatola, Chemistry and Biochemistry of Amino Acids, Peptides and Proteins(Edited by B. Weinstein), 7, 267(1983). 4. C. Toniolo, Int. J. Peptide Protein Res., 35, 287(1990). 5. A. C. Bach et. al., Int. J. Peptide Protein Res., 38, 314(1991). 6. M. J. Genin et, al., J. Org. Chem., 58, 860(1993). 7. P. Juwadi et al., Int. J. Peptide Protein, 40 163(1992). 8. M. J. Genin et. al., J. Amer. Chem. Soc..114. 8778(1992). 9. D. J. Kyle et. al., Peptide Res., 5, 206(1992). 10. J. W. Bean et. al., J. Amer. Chem. Soc., 114. 5328(1992). 11. J. E. Baldwin et. al., Tetrahedron Lett., 34, 1665(1993). 12. R. W. Hoffmann, Angew. Chem. Int. Ed. Engl., 31, 1124(1992). 13. A. B. Smith ffi et. al., J. Amer. Chem Soc., 1_14, 10672(1992). 14. A. B. Smith H et. al., Tetrahedron Lett., 34, 63(1993). 15. T. D. Gordon et. al., 34,1901 (1993). 16. R. Hirschmann et. al., J. Amer. Chem. Soc., 114, 9217(1992). 17. K. Nunami et. al., Peptide Chemistry., in press. — 22 — 3004 (c) 1993 The Chemical Society of Japan Bull. Chem. Soc. Jpn.,66 , 3004—3008(1993) [Vol. 66, No. 10 Synthesis of Barnase Site-Specifically Labelled with Two 13C Atoms Using Partially Protected Peptide Thioester Building Blocks Hironobu Hojo and Saburo Aimoto* Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565 (Received May 6, 1993) Barnase, site-specifically labelled with two 13C atoms, was synthesized using partially protected peptide thioesters as building blocks. Four partially protected peptide segments (Boc-[Lys(Boc) 19,27 ]-barnase(l—34)— SC(CH3)2CH2CO-Nle-NH2, zNoc-[Lys(Boc) 39 ’49 ]-barnase(35—52)-SC(CH3)2CH2CO-Nle-NH2, %Noc-[Lys- (Boc)62,66]-barnase(53—81)-SC(CH 3)2CH2CO-Nle-NH2, [Lys(Boc) 98 ’108 ]-barnase(82—110)) were successively condensed in the presence of silver ions and iV-hydroxysuccinimide (HONSu). Finally, barnase with full ribonu- clease activity was obtained in a yield of 11% based on the carboxyl terminal peptide segment. Most protein production is not performed by a chemi was purified by reversed-phase HPLC (RPHPLC). Boc cal method, but by recombinant DNA technology. This groups were introduced to the side-chain amino groups is because neither the solid-phase nor solution methods of an HPLC-purified peptide thioester in order to real of peptide synthesis are appropriate for the rapid prepa ize selective removal of the amino protecting groups af ration of highly pure proteins. In order to overcome ter segment condensation. The partially protected pep the problems involved in both methods, we developed tide segments were prepared in good yields without any a procedure in which partially protected peptide thio problems, and were used for the barnase synthesis. esters prepared via a solid-phase method are used as All of the partially protected peptide segments used building blocks for protein synthesis in a solution. This for barnase synthesis are listed in Table 1. The yields procedure 1) was applied to the syntheses of c-Myb Pro of the peptide segments were calculated based upon the tein (142—193) amide,2) HU-type DNA-binding protein amino groups in the MBHA resin. The linker contain (HBs) of 90 amino acids3) and its stable-isotope-labelled ing Nle and S-1-alkyl thioester moieties, where (-alkyl derivative.4) means 1,1-disubstituted alkyl, gave satisfactory yields To demonstrate the usefulness of this method for en in the preparation of peptide segments as in a previous zyme synthesis, we synthesized barnase, a protein com synthesis. 4) prising 110 amino acids with RNase activity (Fig. 1). Synthesis of Barnase by Segment Coupling: In this study, barnase site-specifically labelled with two Segment condensation was performed according to the 13C atoms was synthesized for a structural study of bar scheme shown in Fig. 2. The typical coupling conditions nase in the future. This paper describes the results of were as follows: Peptides 3 (120 mg, 17 pmol) and 4 the synthesis and the use of this method for protein (100 mg, 13 pmol) were dissolved in dimethyl sulfoxide synthesis. (DMSO) (2.3 ml). HONSu (30 mg, 260 pmol), AgN0 3 (13 mg, 77 pmol), and 4-methylmorpholine (NMM) (9 Results and Discussion pi, 82 pmol) were then added in succession. The solu Preparation of Peptide Segments: For syn tion was stirred overnight at room temperature in the thetic purposes, the barnase sequence was divided into dark. The peptide was precipitated with distilled water four peptide segments, as shown in Fig. 1. Gly 52 and and washed twice. After the precipitate was dissolved Ala74 were labelled with 13C as (2-13C) Gly and (1-13C) in 70% aqueous acetic acid, 800 mg of zinc dust was Ala, respectively. added. The solution was sonicated for 7 h under a nitro A partially protected peptide thioester was pre gen stream. After removing the zinc dust by centrifu pared according to the procedure described in a pre gation, the supernatant was dialyzed against distilled vious paper. 4) To a 4-methylbenzhydrylamine resin water using a Spectrapor membrane 6 and freeze-dried (MBHA resin or NHg-resin), t- butoxy car bony 1- L- nor- to give a mixture containing peptide 5 (170 mg). Ac leucine (Boc-Nle) and Boc-Gly-SC(CH 3)2CH2COOH cording to a similar procedure, peptides 2 and 5, then were successively introduced using dicyclohexylcarbodi- peptides 1 and 6, were successively condensed. The imide (DCC) in the presence of 1-hydroxybenzotri- condensation reactions were monitored by HPLC using azole (HOBt) to obtain Boc-Gly-SC(CH 3)2CH2CO- a C4 column. The HPLC elution profiles of the reaction Nle-NH-resin.4) On this resin, Boc-amino acids were mixtures are shown in Fig. 3. The segment coupling successively condensed. After peptide chain assem of peptide 5 and 2 and that of peptide 6 and 1 were bly, the terminal amino group was protected with a 4- almost complete within 6 h. During peptide chain elon pyridylmethoxycarbonyl (zNoc) group. The protected gation by segment coupling no HPLC purification was peptide resin was treated with anhydrous hydrogen fluo performed. ride5) to give a crude fiMoc-peptide thioester, which After segment condensation of peptides 1 and 6, dis- — 23 — October, 1993] Synthesis of Barnase 1 10 Ala-Qln-Val-Ile-Aan-Thr-Phe-Aap-Gly-Val-Xla-Aap-Tyr-Leu-Gln- 20 30 Thr-Tyr-Hla-Lya-Leu-Pro-Aap-Aan-Tyr-lie-Tbr-Lya-Ser-Glu-Ala- t 40 Gln-Ala-Leu-Gly-Trp-Val-Ala-Ser-Lya-Qly-Aan-Leu-Ala-Aap-Val- 50 * 1 60 Ala-Pro-Gly-Lya-Ser-lle-Gly-Gly-Aap-lle-Phe-Ser-Aan-Arg-Glu- 70 * Gly-Lya-Leu-Pro-Gly-Lya-Ser-Gly-Arg-Thr-Trp-Arg-Glu-Ala-Asp- 80 t 90 Ile-Aan-Tyr-Thr-Ser-Gly-Phe-Arg-Aan-Ser-Aap-Arg-Ile-Leu-Tyr- 100 Ser-Ser-Aap-Trp-Leu-lie-Tyr-Lya-Thr-Thr-Aap-Hla-Tyr-Gln-Thr- 110 Phe-Thr-Lya-Ile-Arg Fig. 1. Amino acid sequence of barnase. The arrows indicate the sites of segment coupling; * indicates amino acids labelled with 13C; (2-13C)Gly 52 and (l-13C)Ala74. Table 1. Partially Protected Peptide Segments Prepared for Segment Coupling Peptide segments Yield/% Boc-[Lys(Boc) 19 ’27]-Barnase(l—34)-SC(CH3)2CH2CO-Nl^NH2 (1) 12 Boc-Ala-Gln-Val-Ile-Asn-Thr-Phe-Asp-Gly-Val-Ala-Asp-Tyr-Leu-Gln-Thr-Tyr-His — Lys(Boc)-Leu-Pro-Asp-Asn-Tyr-Ile-Thr-Lys(Boc)-Ser-Glu-Ala-Gln-Ala-Leu-Gly- SC(CH3)2CH2C0-N1^NH2 iNoc-[Lys(Boc) 39 ’49 ]-Barnase(35—52)-SC(CH3)2CH2CO-Nl^NH2 (2) 32 *Noc-Trp-Val-Ala-Ser-Lys(Boc)-Gly-Asn-Leu-Ala-Asp-Val-Ala-Pro-Gly-Lys(Boc)-Ser- Ile-Gly-SC(CH 3)2CH2CO-Nle-NH2 iNoc-[Lys(Boc) 62,66]-Barnase(53—81)-SC(CH 3)2CH2CO-Nle-NH2 (3) 12 zNoc-Gly-Asp-Ile-Phe-Ser-Asn-Arg-Glu-Gly-Lys(Boc)-Leu-Pro-Gly-Lys(Boc)-Ser-Gly- Arg-Thr-Trp-Arg-Glu-Ala-Asp-Ile-Asn-Tyr-Thr-Ser-Gly-SC(CH 3) 2 CH2 CO-Nle-NH2 [Lys(Boc) 98,108 ]-Barnase(82—110) (4) 12 Phe-Arg-Asn-Ser-Asp-Arg-Ile-Leu-Tyr-Ser-Ser-Asp-Trp-Leu-Ile-Tyr-Lys(Boc)-Thr-Thr- Asp-His-Tyr-Gln-Thr-Phe-Thr-Lys(Boc)-Ile-Arg (1) (2) (3) (4) lowed by Pharmacia HiLoad S-Sepharose HP (Fig. 4) (Boc )2 (Boch (Boch (Bock to give the final product, barnase (1—110) (7) in 11% Boc~ { 1-34 }-SRfloc- ( 35-52 )~SR — 24 — Hironobu HOJO and Saburo AlMOTO [Vol. 66, No. 10 Bamase[1-110] (7) 0.2 co Retention time / min 45 2 Fig. 4. Ion-exchange chromatography of the HPLC- purified barnase(l—110) (7) by Pharmacia HiLoad S-Sepharose HP (16x100 mm). The broken line indi cates the NaCl concentration in 0.05 M sodium phos phate buffer (pH 6.0). Arg(Tos)-OCH 2-C6H4CH2CONHCH2-C6H4-resin (Boc- Arg(Tos)-OCH 2~PAM-resin) was purchased from Applied Biosystems Inc. (Foster City, CA.). (l-13C)Ala and (2-13C) Gly were purchased from Isotec Inc. (Miamisburg, OH.). The solvents and reagents used for solid- phase peptide synthesis were purchased from Watanabe Chemical Ind. Ltd. (Hiroshima). Analytical RPHPLC was performed on YMC-Pack ODS-AM or C4 (4.6x250 mm) and prepara tive RPHPLC was on YMC-Pack ODS-AM or PROTEIN- RP (20x250 mm) (YMC, Kyoto). The amino acids were analyzed on an L-8500 amino acid analyzer (Hitachi Ltd., Tokyo) after hydrolysis with 4 M* methanesulfonic acid at Retention time / min 110 °C for 24 h in an evacuated sealed tube. The peptide Fig. 3. HPLC elution profiles of the reaction mix mass number was determined by fast atom bombardment tures of segment couplings after an overnight re mass spectrometry using a JMS-HX100 (JEOL Ltd., Tokyo) action. Arrows in panel A,B,C indicate iNoc- equipped with a JMA-3100 mass data system. Although the [Lys(Boc) 62’66,98 ’108 ]-barnase(53—110), iNoc-[Lys- peptide weight was an observed value, the yield was calcu (Boc)39 ’49 ’62’66’98 ,108 ]-barnase(35—110), and Boc- lated based upon the amino acid analysis data. Sonication [Lys(Boc) 19 '27-39 * 49 '62-66'98 ' 108 ]-barnase( 1—110), re was performed using a Branson Model B-220. Dialysis was spectively. Column: YMC-Pack C4 (4.6x250 mm). carried out using a Spectrapor 6 membrane (M. W. cut off The broken line indicates the acetonitrile concentra 1000). Native barnase was a gift from Dr. H. Yanagawa of tion in 0.1% TFA. Mistubishi-Kasei Institute of Life Sciences. Yeast RNA was purchased from Kohjin Co., Ltd. (Tokyo). protecting groups was well solvated by DMSO, kept good flexibility around the reaction sites and, hence, Synthesis retained high reactivity. Thus, the thioester building Peptide Chain Elongation on a Solid Support. block method, which uses a minimal protection strat Solid-phase syntheses of peptide segments were performed egy, is suitable for protein synthesis, not only because on a peptide synthesizer 430A (Applied Biosystems Inc.) of the ease of segment preparation, but also because of using the 0.5 mmol scale, double-coupling protocol of the the high reactivity during segment condensation. The benzotriazole-active ester method of the system software points which must be overcome in this method are to version 1.40 NMP/HOBt t-Boc. Unreacted amino groups find an easily-removable protecting group instead of 2,2, were capped by acetic anhydride after each amino acid in 2-trichloroethoxycarbonyl (Troc) or iNoc for the termi troduction. The side-chain-protecting groups of Boc-amino nal amino group, and to establish a strategy with which acids were ochlorobenzyloxycarbonyl (Cl-Z) for the A6 of Lys, benzyl (Bzl) for the alcoholic OH of Thr and Ser, cyclo to synthesize cysteine-containing proteins. 7) hexyl ester (OcHex) for the /3-carboxyl group of Asp, benzyl Materials and Methods ester for the 7-carboxyl group of Glu, tosyl (Tos) for the N9 of Arg, benzyloxymethyl for the AT of His, 2-bromobenzyl- Boc-amino acid derivatives and MBHA resin were pur chased from the Peptide Institute Inc. (Osaka). Boc- #1M = 1 mol dm-3. — 25 — October, 1993] Synthesis of Barnase oxycarbonyl for the phenolic OH of Tyr and formyl (For) for was prepared by the procedure described for the synthesis the iV* of Trp. Boc-Gly-SC(CH 3)2CH2COOH was prepared of peptide 2. Ala74 was incorporated manually, by mix according to the method described in a preceding paper.4) ing with Boc-(l-13C)Ala (0.75 mmol), 1 M HOBt in NMP tNoc-[Lys(Boc) 39,49 , (2-13C)Gly 52]-barnase(35— (0.75 ml) and 1 M DCC in NMP (0.75 ml) for 4 h. Yield of 52)-SC(CH3)2CH2CO-Nle-NH2 (2). Boc-Nle (170 peptide 3: 12% based on the amino groups in the MBHA mg, 0.75 mmol) was mixed with 1 M HOBt in 1-methyl- resin. Found: m/z 3775.5. Calcd for (M + H)+ : 3775.9. 2-pyrrolidinone (NMP) (0.75 ml) and 1 M DCC in NMP Amino acid analysis of peptide 3: Asp4.29 Thr2.03Ser3.00 (0.75 ml). After 30 min, this solution was mixed with neu Glu2.i6Proi.o3Gly5.o5AlaiIlei.9oLeui.oo(Tyr+Nle)i.98Pheo.97 tralized MBHA-resin (850 mg, 0.54 mequiv of NH2 group) Lys 2.01Trpo.63Arg 2.98 - and shaken for 4 h. After the Boc group was removed [Lys(Boc) 98,108 ]—barnase(82—110) (4). Starting with 55% TFA in dichloromethane (DCM) for 5 and 15 from Boc-Arg(Tos)-OCH 2-PAM-resin (0.83 g, 0.5 mmol), min followed by neutralization with 5% N, N- diisopropyl- zNoc-[Lys(Boc) 98,108 ]-barnase(82—110) (440 mg, 62 pmol) ethylamine (DIEA) in A,iV-dimethylformamide (DMF) for was prepared by the procedure described for peptide 2. 5 min (x2), Boc-(2- 13C)Gly-SC(CH 3)2CH2COOH (220 This peptide (130 mg, 18 pmol) was sonicated with zinc mg, 0.75 mmol), prepared as the same procedure described dust (200 mg) in 75% aqueous acetic acid (4 ml) under previously, 4) was introduced to the Nle-NH-resin using nitrogen for 2 h. After removing zinc dust, the solution 1 M HOBt in NMP (0.75 ml) and 1 M DCC in NMP was dialyzed against distilled water (1 dm3x3) and freeze- (0.75 ml) in a similar manner, to give Boc-(2-13C)Gly-SC- dried to give peptide 4 (120 mg, 17 pmol, 12% based on (CH3)2CH2CO-Nle-NH-resin. Using this resin, a protected Arg in the starting resin). Found: m/z 3868.3. Calcd peptide resin corresponding to the sequence of barnase for (M + H)+ : 3868.0. Amino acid analysis of peptide 4: (35—52), Boc-Trp (For )-Val-Ala-Ser (Bzl)-Lys(Cl-Z)-Gly- Asp4.19Thr4.13Ser3.02Glu1.nIle2.73Leu2Tyr3.05Phe1.79Lys2.10 Asn- Leu- Ala- Asp (OcHex)- Val- Ala- P ro- G ly- Lys(Cl-Z)- His1.03Trp0.40Arg2.77. Ser(Bzl)-Ile-(2-13C)Gly-SC(CH 3)2CH2CO-Nle-NH-resin Synthesis of [(2-13C)Gly 52, (l-13C)Ala74]-barnase was prepared on a synthesizer by means of double cou (1—110) (7). Peptides 3 (120 mg, 17 pmol), 4 (100 pling. After this resin was treated with 55% TFA in mg, 13 pmol) and HONSu (30 mg, 260 pmol) were dis DCM for 5 and 15 min, followed by neutralization with solved in DMSO (2.3 ml) containing NMM (9.0 pi, 82 pmol). 5% DIEA in DMF for 5 min (x2), 4-pyridylmethyl p-ai- AgNOs (13 mg, 77 pmol) was then added and the mixture trophenyl carbonate (zNoc-ONp) (410 mg, 1.5 mmol) was was stirred for 5 h at room temperature in the dark. The allowed to react with the terminal amino group in 80% solution was stirred overnight after adding more NMM (4.0 DMSO-NMP overnight, to give a protected peptide resin pi, 36 pmol). A precipitate obtained by adding distilled wa (1.9 g). An aliquot of the resin (510 mg) was treated ter to the solution, was freeze-dried to give a powder (220 with HF (8 ml), p-cresol (0.5 ml), and 1,4-butanedithio- mg). This peptide was sonicated with zinc dust (800 mg) 1 (1.5 ml) at 0 °C for 90 min to give 250 mg of a crude in 70% acetic acid (25 ml) under nitrogen for 7 h at room product. This product was purified on RPHPLC to obtain temperature. The solution was dialyzed against distilled iNoc-[(2-13C)Gly 52]-barnase (35—52)-SC(CH3)2CH2CO- water (1 dm3x3) and freeze-dried to give a mixture (170 Nle-NH2 (150 mg, 50 pmol, 35% based on the amino mg) containing peptide 5. Following the same procedure, groups in the MBHA resin). Found: m/z 2134.5 (M + peptides 2 (81 mg, 20 pmol) and 1 (93 mg, 14 pmol) were H)+. Calcd for (M + H)+ : 2134.1. Amino acid composition: successively condensed. The crude peptide obtained (320 Asp2.i6Ser2.o3Proi.o3Gly 3 Ala3.27Val2.o4 Ileo. 98 Leu i .os Nleo. 99 mg) was treated with TFA (2.6 ml) containing 5% 1,4-bu- Lys 2.osTrpo.54. tanedithiol (v/v) at room temperature for 15 min. TFA To the iNoc-[(2-13C)Gly 52]-barnase (35—52)-SC(CH3)2- was removed under a nitrogen stream and the peptide was CH2CO-Nle-NH2 (150 mg, 50 pmol) dissolved in DMSO precipitated with ether. This peptide was purified on RPH (2.6 ml), N-(Z-butoxycarbonyloxy)succinimide (Boc-ONSu) PLC using PROTEIN-RP to give powdered peptide 7 (81 (56 mg, 260 pmol) and triethylamine (36 pi, 260 pmol) were mg, 3.7 pmol) after freeze-drying. This peptide was further added; the resulting solution was stirred for 5 h. A mixed purified by ion-exchange chromatography using Pharmacia solvent of ether and ethyl acetate was added to the reaction HiLoad S-Sepharose HP (16x100 mm), which was equili mixture. The formed precipitate was collected by centrifu brated with 0.05 M sodium phosphate (pH 6.0) and eluted gation and freeze-dried from a dioxane suspension to give with a 0 to 0.3 M NaCl gradient in the buffer over 30 min 180 mg of peptide 2 (46 pmol, 32% based on the amino at a flow rate of 2.5 ml min-1. The elution of the pep groups in the MBHA resin). Found: m/z 2335.0 (M + H)+, tide was monitored by absorbance at 220 nm. The main Calcd for (M + H)+ : 2334.2. Amino acid analysis of pep- fraction was collected and desalted by RPHPLC to give [(2- tide 2: Asp2.08 Ser1.8 sPro1.07Gly 3.05Ala3Val1.93 Ile0.98 Leu1.02 13C)Gly 52, (l-13C)Ala74]-barnase(l—110) (22 mg, 1.4 pmol, Nleo.99Lys2.02Trpo.50- 11% based on peptide 4). Amino acid analysis of peptide Boc- [Lys(Boc) 19,27 ]— barnase(l— 34)- SC(CH3)2 7: Asp14.94 Thr8 .50Ser8 .39 Glu7.25Pro2.96 Gly 10.21 AlasVal3.54 CH2CO—Nle— NH2 (1). This peptide was pre Ile6.87Leu6.77Tyr6.80Phe3.8iLyS8.13His2.12Trp1.79Arg5.82- pared following the procedure described for the synthe Measurement of RNase Activity. Yeast RNA (1.6 sis of peptide 2. Yield of peptide 1: 12% based on the mg) was dissolved in 0.125 M Tris-HCl pH 8.5 (0.8 ml) and amino groups in the MBHA resin. Amino acid analy 0.2 ml of appropriately diluted enzyme was added. The mix sis of peptide 1: Asp5.06Thr2.82Ser0.89Glu4.0sPro1.02Gly2.01 ture was incubated at 37 °C for 15 min. The reaction was Ala4Vali.69 Ilei.61Leu2.99(Tyr-t-Nle)4.o2Pheo.97Lys2.ooHisi.oo- stopped by adding a solution containing 6% HCIO4 and 1% iNoc-[Lys(Boc) 62,66, (l-13C)Ala74]-barnase(53— lanthanum acetate (1 ml). The mixture was kept at 0 °C for 81) —SC(CH3)2CH2CO—Nle—NH2 (3). This peptide 15 min and the precipitate was removed by centrifugation. — 26 — 3008 Hironobu HOJO and Saburo AlMOTO [Vol. 66, No. 10 The supernatant (0.5 ml) was diluted with 4.5 ml of water, cording to the process shown below: and the absorbance at 260 nm (A260) was measured. An R-CO-SR' + Ag + + HONSu ^ [R-CO-ONSu + Ag - SR'l increase in A26O of 1.0 under these conditions was defined as nh2-r" 100 units of enzyme activity. ------►R-CO-NH-R', where R-CO-SR is a partially pro tected peptide thioester, -SR' is -SC(CH3)2CH2CO-Nle- We express our thanks to Professor Mitiko Go of Fac NH2 and NH2-R" is an amino component peptide. ulty of Science, Nagoya University, for her kind ad 2) H. Ho jo and S. Aimoto, Bull. Chem. Soc. Jpn., 64, 111 (1991). vice throughout this study, Dr. Hiroshi Yanagawa of 3) H. Ho jo and S. Aimoto, Bull. Chem. Soc. Jpn., 65, Mitsubishi-Kasei Institute of Life Sciences for the gift of 3055 (1992). native barnase and Professor Fumio Sakiyama and Mr. 4) H. Hojo, Y. D. Kwon, Y. Kakuta, S. Tsuda, I. Tanaka, Satoshi Kuroda of the Institute for Protein Research, H. Hikichi, and S. Aimoto, Bull. Chem. Soc. Jpn., in press. Osaka University, for measuring the enzymatic activity. 5) S. Sakakibara, Y. Shimonishi, Y. Kishida, M. Okada, We also thank Professor Yasutsugu Shimonishi of the and H. Sugihara, Bull. Chem. Soc. Jpn., 40, 2164 (1967). Institute for Protein Research, Osaka University, for use 6) G. W. Rushizky, A. E. Greco, R. W. Hartley, and H. of the mass spectrometer. This study was partly sup A. Sober, Biochemistry, 2, 787 (1963). ported by a Grant-in-Aid for Scientific Research No. 7) Y. D. Kwon, R. H. Zhang, M. P. Bemquerer, M. 02263101 from the Ministry of Education, Science and Tominaga, H. Hojo, and S. Aimoto, Chem. Lett., 1993, 881. Culture. References 1) In this method, segment condensation proceeds ac 27 — Kzff- to)$6RE3£$IB£ 2. 1 K*K# SS IE KAhStfil 9 0 7¥E. Fischer Ic K©6hg^fTt>ti.f;©lc(i Ci !) "< M. Bergmann „ L. Zervas K J: 5> y ;h^"4L •> A /b** —-'blEOSFA21 lc J: -o T MSWlc^ggffi^'ia^Lfco $ blc^+y d" >x ') >*~ c> tEi'©^fiSicfi£S) L* Kt>^fi£°rt8attofc„ ^©IWESBtEx «SX> ®5«Si$IF©gfl|gfcitt © 1 9 6 3^icMerrifieldii^Sttffiflg±T^y ,f' KB*@fiLTV< BfflSlc J:5 mmMO:AhK&="&M%L^©#Kyf Kl)fl®lcirBgT8 5»'©J: ^icSbn/co ic 6 *<&#-< yf KizZ(6aay%* 2. 2 #K#©m%w aSO'C/f- KirfiSlcMt-5-ffc¥69E%(iKlcfi< E95dli. -eoA/SaLlifflSfiKSti Ac i> 2.2.1 %m@lcZ6AGEM RNase © A6%" Ifc^Afig LfcSSH^Sffi£ LrSSIt L Fig. 1. Structure of Bovine Pancreatic Ribonuclease A — 28 — 20 64 | 65 69 ItO j(^(H^)(Ar|)(Glu)(w)(L*^^Af^(lT^^e))(Lys)(Sej)(ser)^ @P rS5jQ©©®©©©)<3)©©©(3tg)-( Fig. 1. Primary structure of angiogenin 2.2.2 J: & ati HIV yDfT--t?'OM,7) (H)-P1 QIT LWQRP L10VT I [W] I GGQL E21 A L L D TGAD D3°T VLEEMNL Pg" Q4Vv K P K M I G G l50G G F I K V R Q Y rf50 Q61 I E l(Ab§G Hlf A IGTVL VGpf p81 V N I I G R N L L®°T Q I G |Aba|T L N l^-(OH) h'7-1'^7 v -18> ? >yAic#< KS-—$ics?eKLx -eci**' k$■ &6J5&o Coupling Step I < vl v-e trendomize. **l) I (9 Apeptldes) AA -# GA-0 va -e AG-# GG-0 VG -# Av-e GV-# VV -# irendomue, *o*0 I 1. Stained bead removed and washed (27 tripeptides) 2. Peptide mlcrosequenced trom a single bead AAA -0 GAA -# VAA-0 AAG-# GAG "0 VAG-0 AAV-e GAV -0 VAV-0 AGA-0 GGA -0 VGA-0 AGG-S GGG -0 VGG-0 AGV-# GGV -0 VGV-0 AVA -0 GVA -0 VVA-0 AVG -0 GVG -0 VVG-0 AVV -0 GW -0 vvv-0 -29 — •? )\z^~ JUS'S.'7 f- K ABE 191 I'Jif- V >©»!,'t°>©S SXIiTea Bag ©«fc 9 «gCAtlfcSlli±lc^X'f- K£ ABEL. ffliJE©«S$£B£L*:gK t" > X fi mi± T-Stt £ MS ih 6 ;5 j$0 fftfft ©$£ o £->-:? x>x£ABEo <7f K*Sffl«t01KBILr#lli-r 5cifcBJ„ polyethylene bottle cleavage of the Fmoc protecting O) group ______tea bag --0 piperidine/dime Ihylformomide CH,-O-CO-NH- 1CH5),-CO-NM-(CH, j4-nh -CO-tCH t washing steps Fmoc-ff-alanin* polyethylene heBSmeihyienediamioe lunclionollzed with acrylic acid solvent : dimethyltormomide methanol coupling Fmoc-amino acids TBTU/1-hydroxybenzolriazole 10-50 nmol peplide diisopr opylelhylomlne covalently bound in dimethyllormomide washing steps onliserum in microtiler plate solvent: dimethyltormomide methanol 2.2.3 mts. D N A^^S6(HBs) j; Ltz7 7 / > h O f* # x. X x ;U £ cT fi£lh 6 o 1 10 i Me t-Asn-Lys-Thr-Glu-Leu-Ile-Asn-Ala-Val-Ala-Glu-Thr-Ser-Gly- 20 30 Leu-Ser-Lys- Lys-Asp-Ala-Thr-Lys -Ala-Val-Asp-Ala-Val -Phe - Asp - I 40 Ser-Ile - Thr-Glu-Ala - Leu-Arg-Lys - Gly-Asp - Lyg-Val - Gin - Leu -1 le - 50 601 Gly-Phe -Gly-Asn - Phe-Glu-val -Arg-Glu-Arg-Ala-Ala-Arg- Lys -Gly- * 70 Arg-Asn-Pro-Gln-Thr-Gly-Glu-Glu-Met-Glu-Ile-Pro-Ala-Ser-Lys - 80 90 Val-Pro-Ala-Phe-Lya-Pro-Gly-Lyg-Ala-Leu-Lya-Aap-Ala-Val-Lyg Fig. 1. Amino acid sequence of MBs. The arrows indicate the sites of segment coupling. Met* indicates methionine-me/Av/- 30 — 0) (2) (3) (4) (Boc), (Boc), (Boc), (Boc), Boc- ( 1-15 )-SR *toc- fl6-39} -Sfl fftoc -(40-60)-SR H-(61-90 }-OH 0 , II). Ul). Iv). v) (Boc), ------H-( 40-90 )-OH (S-A) o . II). HI). Iv). v) (<5%) (Boc),, ------H-( 16-90 )-OH (7-A) 041). HI) (19%l H-{ {-90 }-OH (l-A) (1.2%) I) AgNO, ♦ HONSo, 1) TFA ,IR) HPLC, Iv) Boc-ONSu, v) Zrv'AeOH •SR: -SCHjCHjCONHj Fig. 4. Synthetic route of HBs(l—90) by segment coupling. 2. 3 9rH«B8©E55 (*7 V h-yv -> K©S6g) K • * 7 V h yv -> 7 V 4^-7 A ;M<- RSXlcffl vr*toATS:^6gL/c<, HgNC HCO-Pro-Leu-Phe-Gly- 10 15 20 lle-Ala-Gly-Glu-Asp-Gly-Pro-Thr-Gly-Pro-Ser-Gly-lle-Val-Gly-GIn-OH Proposed Structure of Nephritogenoside 2. 4 1) E. Fischer, Chem. Ber., 40, 1754 (1907). 2) M. Bergmann, and L. Zervas, Chem. Ber., 65B, 1192 (1932). — 31 — 3) V. du Vigneaud, C. Kessler, J. M. Swan, C. W. Roberts, P. G. Katsoyannis, J. Am. Chem. Soc., 76, 3115, (1954). 4) J. Meienhofer, E. Schnabel, H. Bremer, 0. Brinkhoff, R. Zabel, W. Sroka, H. Klostermeyer, D. Brandenburg, T. Okuda, H. Zahn, Z. Naturforsch. , 18b, 1120 (1963). 5) P. G. Katsoyannis, K. Fukuda, A. Thometsko, K. Suzuki, M. Tilak, J. Am. Chem. Soc., 86, 932 (1964). 6) Y. T. Rung, Y. G. Du, W. T. Huang, C. C. Chen, L. T. Ke, S. C. Hu, R. G. Jiang, S. Q. Chu, C. I. Niu, J. Z. Hsu, W. C. Chang, L. L. Chen, H. S. Li, Y. Wang, T. P. Loh, A. H. Chi, C. H. Li, P. T. Shi, Y. H. Yien, K. L. Tong, C. Y. Hsing, Sienta Sinica, 15, 544 (1966). 7) H. Yajiraa, N. Fujii, Chem. Pharm. Bull., 29, 600 (1981) 8) T. Kimura, N. Chino, S. Kumagae, H. Kuroda, J. Emura, and S. Sakakibara, Biochem. Soc. Transactions, 18, 1297 (1990). 9) R. B. Merrifield, J. Am. Chem. Soc., 85, 2149 (1963). 10) 21#, p.122 (1957) 11) f 1#, P.361 (1969) 12) , A# (1975 ) 13) mmfM, (1985) 14) f v m A, P.207 (1977) 15) iv " , BAAYk#^^, m Mlk^|5)A, P.401 (1977) 16) Atm#, (T) (#&%# ^0#m2) " , p. 641 (1987) 17) A. flodawer, M. Miller, M. Jaskolski, B. K. Sathyanarayana, E. Baldwin, I. T. Weber, L. M. Selk, L. Clawson, J. Schneider, S. B. H. Kent, Science, 245, 616 (1989) 18) a)K. S. Lam, S. E. Salmon, E. M. Hersh, V. J. Hruby, W. M. Kazmierski, R. J. Knapp, Nature, 354, 82 (1991) b)R. A. Houghten, C. Pinella, S. E. Blondel le, J. R. Appel, C. T. Dooley, J. H. Cuervo, ibid, 354, 84 (1991) 19) G. Jung, and A. G. Beck-Sickinger, Angew. Chem. Int. Ed., 31, 367 (1992) 20) H. Ho jo, and S. Aimoto, Bull. Chem. Soc. Jpn., 65, 3055 (1992) 21) T. Teshima, K. Nakajima, M. Takahashi, and T. Shiba, Tetrahedron Lett., 33, 363 (1992) — 32 mmiM) nmm i. (1) (L5V75 /6$) X&Z. (2) (3) mm&mzFgv&z (5) n. 7a-r7--¥<0£imfi0 Enzyme Preferred cleavage site Enzyme Preferred cleavage site N-terminal C-terminal N-terminal C-terminal Serine proteases Thiol proteases 1 1 Trypsin -Arg (or lys)-Yaa- Papain, Streptococcus protease -Phe (or Val, Leu)-Xaa-Yaa- i Achromobacter protease -lys-Yaa- Clostripain, cathepsin B —Arg — Yaa — i Chymotrypsin, subtilisin -Trp (or Tyr, Phe, Leu)-Yaa- Cathepsin C H-X-Phe (or Tyr, Arg)-Yaa- • Metal proteases Elastase, a-lytic protease -Ala (or Ser)-Yaa- 1 i Thermolysin -Xaa-leu (or Phe)- Proline-specific protease — Pro — Ya a ~* i i • Myxobacter protease II — Xaa — Lys- Staphylococcus V8 protease -Asp (or Glu)-Yaa- Aspartic proteases i -Xaa-Yaa- i Carboxypeptidase Y Pepsin -Phe (orTyr, Leu)-Trp (or Phe, Tyr)- m. XMi 2) 3;> (1) 5FS?I^6977'n-7 - T-^TOT’a-f7—f^MX^ 6 R-COOH h 2n -r - R-COO" + H3N+-R' ^ R-COOH + H2N-R' ^ R-CO-NH-R' + H20 (1) R-CO-NH-R1 . H20 Y_____ [R-CO-NH-R'|0h (2) ^ ~ [R-C00Hjo b [R'-NH2J0b R-COOH K (3) Kob — (1 + lOpH-pk ‘) (1 + 10pN'-pH ) R-CO-NH-R' rig. i — Table 2------Effect of blocking residues on (a) product solubility Soluble reactant 1 Enzyme^ QjSS0|vec j product Solubility3/ 4- Solid product (precipitate) Product pM Soluble reactant 2 + HjO Ac-Phe-Leu-NH2 104 (in water) (b) Z-Phe-Leu-NH2 27 Z-Phe-Leu-OEt 17 Z-Phe-OH + Leu-NH2 ______0.2 mw g-chymotrypsin______Z-Phe-Leu-NH2 Z-Phe-leu-OBu* 1.7 (0.1m) (0.1 m) pH 7.0.25'C. 16 h (Yield. 70-80%) Z-Phe-Leu-NHC6H5 0.8 Z-Phe-Leu-ODPM 0.03 Equilibrium controlled synthesis (1) - Precipitation. 'In pH 7.0 buffer containing 10% dimethylformamide. — 33 — b V y •>y 21> £ij •>x22)(c j:6 Z-Phe-Leu-NHz 'ZnZftfct 80 100 pH (mM) (A) pH Equilibrium controlled synthesis (2) - Biphasic system. Fig. 3------ (a) (Solvent phase) Ii (Water phase) I Reactants Reactants I N 1I Enzyme I I N I Product Product + H20 I I (b) Ac-Trp-OH + Leu-NHj 5 pM g-chymotrypsin, pH 7.0______Ac-Trp-Leu-NH2 (0.4 him ) (0.4 mw) 98% ethyl acetate (Yield. 44%) Equilibrium controlled synthesis (3) - Dissolved state system. r-Fig.4 ------Reactant 1 Enzyme, organic cosolvent (a) + ------—------x Product + H20 Reactant 2 (excess) 0.2 mM trypsin (b) Z-Arg-Leu-NH 2 Z-Arg-OH + Leu-NH2 37‘C. 20 h (0.05 m) (0.5 m)------:------► (Yield. 11%) (0.05 m) (1.0 m)------► (Yield. 40%) 50% dimethylformamide (0.05 m) (0.5 m)------► (Yield. 46%) (c) Reactant 1 Enzvme molecular trap + Product + H20 ------^ :----- ^ Product—molecular trap Reactant 2 — 34 — (2) immmvn-T- - v LmmvsfAt* rFig. s------b) R-CO-X ♦ H2N-R- _ Ac-Phe-OH + E /h 2o R-CO-NH-R" . HX Ac-Phe-X + E Ac-Phe-X • E Ac-Phe-E k_, + \ Nu X Ac-Phe-Nu + E R-COOH Kinetically controlled synthesis. The catalytic mechanism of a-chymotrypsin: the covalent-linked enzyme- R-CO-NH-R- peptide intermediate (Ac-Phe-E) is subjected to nucleophilic attack from H20 or other nucleophiles (Nu): the rates of the reactions (k3 and kJ dictate its R-CO-X outcome. iv. -try>7 ,n-r7—fff)T5 s) s) 7J^ - E7->;l'7ii$f$3i®PS'C. p HKiiWt-E) Organic cosolvent ellect on porcine trypsin 120 - 100 iL esterase activity cosolvent modified enzyme amidase activity no organic cosolvent • 60 100 • % organic cosolvent Time (min.) ■ Figure 6- Effect of water-miscible organic solvents on the activities of Figure 7. Comparison of cosolvent-modified enzyme and all-aqueous porcine trypsin. The esterase activity toward TV-benzoylargininc methyl system for the synthesis of Bzl-Arg-Gly-NH 2, as monitored by HPLC ester (40 mM) was determined in 0.02 M CaCl2 (2 mL) containing with a C-18 column. Conditions: 0.2 M Bzl-Arg-OEt, 0.2 M Gly-NH 2, organic solvent. A 50-jiL enzyme solution (4.17 X 10~s M) in 0.004 M 0.25 M Tris, 0.02 M CaCl2, 0.1 mM TPCK-trcated bovine trypsin, pH Tris, pH 7, was added to the substrate solution, and the solution was held 10; cosolvent-mod ified system is 60% by volume dioxane, and approxi at pH 8 by automatic addition of 0.025 M NaOH through a Radiometer mately 30% of the initial esterase activity is retained after 2.5 h. No pH-stat. The activity was determined on the basis of consumption of peptide formation was observed in the absence of enzymes. The product base. (□) dioxane; (▲) DMF. To determine the amidase activity, the is soluble in both systems. enzyme was added to a solution (1 mL) containing jV-benzoyl-DL-arginine p-nitroanilide, Tris (0.1 M), CaCl3 (0.02 M), pH 8.0. The absorbance change at 410 nm was monitored. (O) acetonitrile; (a ) DMF; (□) dioxane. — 35 — 10) II) |1) v. lyv* j: umi&cT; / ssfomx crude papain (150mg) Cbz-Ata-OMe (0.1 M) + Gly-CW (0.2 M) + D-Lcu-OMe (0.2 M) ______Cbz-Ala-Gly-D-Lcu-OMe (69%) (4) 60% MeOH, pH 9.5. 10 mL, 12 h subtilisin, 0.1 g Ac-D-Phc-OEt + Phe-NH2 Ac-D-Phe-Phe-NH2 (67%) /ert-Amylalcohol, 31 mL. 45°C. 3 days 3.1 mmol 3.1 mmol Table3. Aminolysis of alanine methyl ester with 3-aminopentane catalyzed by D- A(a Achromobacier D-alanine aminopeptidase [a]. Acyl donor Solvent Yield [%] [b] AA D-alanine methyl ester HCI butyl acetate 98 [|imol] D, L-alanine methyl ester HCI butyl acetate 48 L-alanine methyl ester HCI butyl acetate 0 D-alanine methyl ester HCI benzene 98 D-alaninc methyl ester HCI trichlorocthanc 91 D-alanine methyl ester HCI toluene 67 [a] A reaction mixture containing PU-6-immobilized D-alanine aminopepti dase (7.5 units of partially purified enzyme of 210 units mg" '), 0.1 mmol of alanine methyl ester hydrochloride, and 0.5 mmol of 3-ami nopen tanc in water- saturated organic solvent (1 mL) was incubated at 30 °C for 1 h. [b] The yield t (min was calibrated with chemically synthesized authentic products with ninhydrin. Fig. 8. Kinetic resolution of d, L-alanine amide by Achromobacier D-alanine aminopeptidase. vn. 7nT7--b'C>$E6<7)rSLkl4; Aqueous solution Table4. Kinetic Constants for Subtilisin BPN' and Mutants' BPN' 8399 Km. Km. 20 40 60 substrate S"1 n M M'1 s* 1 S'* iiM M-1 s-> Time, hr Suc-AAPF-pNA* 47 172 2.7 X 105 76 112 6.8 X 10J NTCF 0.2 76 2.2 X I01 0.2 33 6.0 X I0J Figure Y Stability of subtilisin BPN' and mutants in aqueous solution Z-Lys-SBzl 46 531 8.7 X 104 32 948 3.4 X 104 (0.05 M Tris-HCl, pH 8.4) and in DMF at 25 °C: D, wild-type BPN'; Bz-Tyr-OEt 70 1700 4.1 X I04 73 2358 3.1 X I05 ■, 8350; e. 8397; ▲, 8399. The rate of inactivation was measured on the basis of the remaining activity as described previously. 4 The inac tivation is not simple first order. 8350: Met 50 Phc (hydrophobic). Gly 169 Ala (hydrophobic and configurational entropy), Asn 76 Asp (Ca2+ binding and H bonding). Gin 206 Cys (oxidized to Cys-SH. van dcr Waals), Tyr 217 Lys (H bonding), Asn 218 Scr (H bonding). 8397: The same as 8350 except no change for Tyr 217. 8399: The same as 8350 except no changes for Gly 169 and Tyr 217. — 36 — isjiQiT; VUI , Protease from S. cellulosae Table S Relative Activity of the Enzyme for Various Synthetic Peptides The final concentration of the substrate was 10 nut, except for those given in parentheses. The rate of u- Phe-L-Leu-NH2 was taken as 100. Relative Relative Peptide Peptide Peptide Relative activity activity activity Gly-He 21.8 Leu-Val* 5.4 Phe-Leu-NH2 100 Gly-Phe 12.3 Leu-Tyr* 2.2 Pro-Phe-NH : 48.1 Gly-Leu 5.2 Leu-Pro 0 Pro-Leu-Gly-NH 2 37.7 Gly-Lys 0 D-Leu-Tyr 0 Gly-Leu-NHj 10.3 Gly-Glu 0 Tyr-Leu* 3.2 Asp-Phe-OMe 0 Gly-Ala 0 Ser-Leu 3.8 Cbz-Ser 0 Gly-Asn 0 His-Leu* 0 Cbz-Phe 0 Gly-Gly 0 Pro-Leu 0 Cbz-GIy-Pro 0 Gly-Asp 0 Val-Phe 6.3 Bz-Gly-Lys 0 Gly-Pro 0 Lys-Glu N Cbz-Ile-Pro 0 Gly-D-Leu 0 Glu-Val N Cbz-Phe-Leu 4.7 Gly-D-Phe N Leu-Leu-Leu 2.4 Cbz-Leu-Tyr* 6.3 SO.H S0_H s°3h SO.H i 3 l 3 1 J Cly-Ile-•Val-Clu-Gln-Cye-Cys-Ala- Ser-Val-Cye-Ser- Leu-Tyr-Cln-Leu-Clu-Asn-Tyr-Cya- t S. cellulosae t T protease ——— 18) t T i Chymotrypsin A ! t t t T t r t Qiyoocrypiin r 1 t i r t r rep.ia20) P«p*in21) r r r r r r r t A»p. oryzee22^ r i • r i r r r r proteinase Asp. ochraceua^^^ r r r r r r r proteinase Scop, brevlcaulie*^ T T • i r r r t • proteinase Fig . IQ Cleavage Site of Oxidized A-Chain of Insulin by the Protease from 5. cellulosae and by Other Enzymes. The arrows indicate a major site of action. The broken-line arrows indicate other bond split less readily. Temperature (°C) 0 2 4 6 8 Fig. 11. The temperature dependences of the initial velocities of formation of (u-Leu-Gly) 2, (u-Leu-Gly) 3, Reaction Time (h) and u-Leu (or Gly) from L-Leu-Gly. A, vt (initial Fig. 12. An example of the time courses of formation velocity of (u-Leu-Gly) 2 formation); #, v2 (initial of (L-Leu-Gly),, (u-Leu-Gly) 3, u-Leu, and Gly. A, velocity of (L-Leu-Gly) 3 formation); O, vlx (the sum of (n-Leu-Gly) 3; e, (u-Leu-Gly) 3; O, sum of (u-Leu-Gly) 2 v2 and v2); □, vH (initial velocity of u-Leu (or Gly) and (u-Leu-Gly) 3; ■, u-Leu; □, Gly. Enzyme con centration: 1.4 x 10~s m. Substrate concentration : 1.3 x formation). In condensation and hydrolytic reactions, 10-i M_ the relative velocities of 100% correspond to v;T and vH at 65°C, respectively. Enzyme concentration: 3.0x 10-s m. Substrate concentration: 1.3 x 10-1 m. — 37 — x . 1 Morihara, K. & Oka, T. (1982) Kagaku no Ryouiki 36, 444-455 2 Jakubke, H-D., Kuhl, P., & Konnecke, A. (1985) Angew. Chem. 97, 79-87 3 Kasche, V. (1986) Enzyme Microb. Technol. 8, 4-16 4 Barbas, C. F. Ill, Matos, J. R. West, J. B. Wong, C-H. (1988) J. Ame. Chem. Soc. 110, 5162-5166 5 Barbas, C. F. Ill, & Wong, C-H. (1987) J. Chem. Soc. Chem. Commun. (1987) 532-534 6 Coletti-Previero, M.A., Previero.M. & Zuckerkandl, E., (1969) J. Mol. Biol. 39, 493-501 7 Wong, C.H. Chen, S.T. Hennen, W.J. Bibbs, J.A. Wang, Y. F., Liu, J.L.C., Pantoliano, M.W., Whitlow,M., & Bryan, P.N. (1990)112, 945-953 8 West, J.B., Hennen, W.J., Lalonde, J.L., Bibbs, J.A., Zong, Z., Meyer, E.F., & Wong, C.H. (1990) 112, 5314-5320 9 Widmer, F., Breddam, K., & Johanson, J.T. (1980) Carlsberg Res. Commun. 45, 453-463 1 0 Petkov, D.D., & Stoineva, LB. (1984) Tetrahedron Lett. 25, 3751-3754 1 1 West, J.B., & Wong, C.H. J. Org. Chem. (1986)51, 2728-2735 1 2 Margolin, A.L., Tai, D.F., & KEbanov, A.M. (1987) J. Ame. Chem. Soc. 109, 7885-7887 1 3 Asano, Y., Nakazawa, A., Kato, Y., &Kondo, K. (1989) Angew. Chem. Int. Ed. Engl. 28, 450-451 1 4 Zhong, Z., Jennifer, L.C.L., Dinterman, L.M., Finkelman, M.A., Mueller.T., Rollence.M.L., Whitlow,M., & Wong, C.H. (1991) 113, 683-684 1 5 Muro.T., Tominaga.Y. , & Okada, S. (1984) Agric. Biol. Chem. 48, 1223-1230 1 6 Muro, T., Tominaga, Y., & Okada, S. (1986) J. Biochem. 99, 12625-1630 1 7 Muro, T., Murakami, T., Tominaga, Y., & Okada, S. (1987) Agric. Biol. Chem. 51, 607-609 — 38 — K J: L v> X * 5 xETX^x (-©HIS (It) ^y h V -&%#####%# *46 $& • 6* l. iscfttc L-X;i/7 5 > EliHI?L1W<04=«#EmI: ti it -6Settejl®gt LT±g&?S:*£ffit 1111:, S £ftSi:&9 IBISES ^ fc^Si$5tLttg **K>tv ^0 Hoi 7 K# #ict)^6&.a##(o#ai:uyf yx 5 >Ei/t x? - ixtX7-tX;i/f 5 ymifrfr*>Zfr IPmWi, co2h co2h L-Glu (extended form) L-Glu (folded form) COjH L-CCG-I (NC) L-CCG-II (CC) L-CCG-III (NC) L-CCG-IV (CC) mGluR agonist uptake inhibitor uptake Inhibitor NMDA agonist — 39 — 2. L-2-fl->1/0 7*0 MU'?') ~>7 (L-CCG-riV) tQlggf fc>ft.;blUi;7;U7 5 y^O 377*7-73 7 S-extendedSifcSWi foldedSt: il tttffft L-C4lnL-2-#;v#+'>->^ d-/d •>> (l-ccg -hv ) 6-Eat, (hi ) o cccEii^x5> a#ibf siljRto^rf-x b-e*9 ##&#«##%tcSffl&yn #1 Neuropharmacological Profile of CCG Isomers (relative potency ratios; L-Glu = 1) L-CCG-I L-CCG-II L-CCG-III L-CCG-IV Depolarizing Binding IPs formation f CAMP \ Receptor CCG activity* affinity * rat* mGluRt d mGluR2 * subtype Remarks extended Selective metabotropic agonist CCG-I 6 0.006 5 1 70 metabotropic [QA > CCG-I > t-ACPD > L-Glu) Depression of monosynaptic reflex CCG-II 0.3 ~0 <1 0.1 0.07 L-Glu uptake inhibitor folded L-Glu uptake inhibitor (potentiation) CCG-III 0.5 ~0 - - - Potent NMDA aaonist CCG-IV 100 17 NMDA [CCG-IV > NMDA > L-Glu] NMDA 40 0.2 - - - NMDA KA 100 ~0 . - KA 0.01 AMPA & mixed agonist QA 300 0.15 350 45 metabotropic (1S,3R)- metabotropic ACPD 2 not tested 1 0.2 1 * Depolarizing action on the rat spinal cord. (Shinozaki etaL.Br. J. Pharmacol.. 1989.) b Binding affinity to NMDA receptor in the rat cerebral cortex ([3H]CPP). (Kawal el at, Eur. J. Pharmacol.. 1992.) c Enhancement of IP3 metabolism in the rat hippocampal synaptoneurosomes. (Nakagawa el al., Eur. J. Pharmacol., 1990.) ^Stimulation of PI hydrolysis In mGluRt-expressing Chinese hamster ovary (CHO)cell. (Hayashieta!., Br. J. Pharmacol., 1992.) "Inhibition of forskolin-stimulates cAMP formation In mGluR2-expressing CHO cell. (Hayashiet al., Br. J. Pharmacol, 1992.) NHCHj HO,C,V^cO,H xj —/>< 5'smi'-ty?-vzma&mntlx, mmn (2) @8Ey-b7'5'-liextendedSO^lV ^ 5 7@EO 3 7 7 * itz (1) <9 7 *>NMDA$ ■ffi'-i 7,l±foldedS<0^;v^ 5 7®f<9377 Ztib i±^;v7 5 >E •b t' 7 - <9 tT&lBESIS $• $ e> KfflS-fbt 4 £ i: **-e § s „ -fi-e (1) ccoS 40 — 3. CC CCGEOa-7 < y ^ #/J^t: f 6W * >&#Wl < 1~ 6 #^<7) 2il0 z.btl&o NMDAE Wr 7 X - CD #7) &77-X YXhZ> L-CCG-IV 1-- 02. L-CCG-IV^) 03. CCGmOpH^J# L-CCG-I L-CCG-II L-CCG-IV NC-rotamer CC-rotamer (ci-cr 4. MCG#(0^^ CCG#OgB^##^6, CCGE<7)a-T < y «9 igLtv>Si t # Zc0 LT, C3' f&K###&#A LZ:cis-MCG-I, cis-j3 Z tf trans-MCG-III t IV <7)^$ £ £ ^.ta^tz (cisE ti C34£ <9 E$& a — 41 — D-t v±A#(onmm#& tmhtifzE m& cls-MCG-l cls-MCG-111 (R|=H, Ffe-CH^DMe) cis-MCG-IV (Ri=H, R2=CH2OMe) trans-MCG-lll (Rj =CH20Me, Rg^H) trans-MCG-IV (R^ zGHgOMe, F^=H) cls-MCG-l : (2S,1'S,2,R,3,R)-2-(2-carboxy-3-methoxymethylcyclopropyl)glyclne cis-MCG-lll : (2S, 1 ,S,2'S,3'R)-2-(2-carboxy-3-methoxymethylcyclopropyl)glyclne trans-MCG-lll : (2S,1 'S,2'S,3'S)-2-(2-carboxy-3-methoxymethylcyclopropyl)glyclne cis-MCG-IV : (2S,1'R,2,R,3'S)-2-(2-carboxy-3-methoxymethylcyclopropyl)glyclne trans-MCG-IV : (2S,1 'R,2,R,3'R)-2-(2-carboxy-3-methoxymethylcyclopropyl)glyclne E-ether trans-MCG-lll OTBS OMe OCH 1.F 2. Mel 3. AcOH H NHo 4. Ba(OH)2 NHBoc 5. BtxyO cis-MCG-lll (45%) 1. H3cr LOH/MeOH (65%) 2. Jones Ox. OTBS 3. CH jN 2 1. CSA/MeOH OTBS HOgC COgH. - 2. CSA/MejC(OMe)2 4. LiOH/MeOH 1. DIBAL H 5. INNaOH 2. FSQ3Me OTBS -y- T5TBS 6. 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DCG-IV l±L-CCG-I h & ;&,, 14- 15) 9. 4ffiB%y;v? ? : mtmmffiMr*-* h ?> HS y 7 * X - -y 3 >1:5.1? K > --> 3 y £-0H£cdB Bffii; LT •) ttii&Ly ny-OV 4^;vy V -> y ES#ro »;i/ ** - Mb Ir SI® P8 £ UTt^VX-f >>#& L£„ ^ ursa^-ffc-S-ti^BIi OOi ? tc-S-Si-eS^o 16> 010. 4i6®^yJi-5' 5 >®<0£j$ ho2c nh2 ho2c nh2 J 4 f I f ^^^OOgH Xy^^^CQgH 0 L-Asp i \> S^Av0 88% (R'=protecting group) A: Pd(OAc) 2, P(Oi-Pr)3,Et2MeNHI, morpholine, CO, 25 atm, 120 °C, 20 h, Ste7-y 3@&ty^^ — 47 — £X± ^(iAMPAEi/-try^ -(Dismtwm^a, ^ 9 Iffl^ftKifimLcm References 1. Monaghan, D. T.; Bridges, R. J.; Cotman, C. W. Ann. Rev. Pharmacol. Toxicol. 1989 , 29, 365. 2. (a) Curtis, D. R.; Phillis, J. W.; Watkins, J. C. Brit J. Pharmacol. 1961 ,16, 262. (b) Davies, J.; Evans, R. H.; Francis, A. A.; Jones, D.; Smith, A. S.; Watkins, J. C. Neurochem Res. 1982,7, 1119. 3. (a) Yamanoi, K.; Ohfune, Y.; Watanabe, K.; Li. P. N.; Takeuchi, H. Tetrahedron Lett. 1988, 29, 1181. (b) Shimamoto, K.; Ishida, M.; Shinozaki, H.; Ohfune, Y. J. Org. Chem. 1991 ,56,4167. 4. (a) Shinozaki, H.; Ishida, M.; Shimamoto, K.; Ohfune, Y. Brain Res 1989 ,480, 355. (b) Shinozaki, H.; Ishida, M.; Shimamoto, K.; Ohfune, Y. Br. J. Pharmac. 1989 ,98,1213. 5. (a) Nakagawa, Y.; Saitoh, K.; Ishihara, T.; Ishida, M.; Shinozaki, H. Eur. J. Pharmacol. 1990 , 184, 205. (b) Ishida, M.; Akagi, H.; Shimamoto, K.; Ohfune, Y.; Shinozaki, H. Brain Res. 1990 , 537, 311. (c) Hayashi, Y.; Tanabe, Y.; Aramori, I.; Masu, M.; Shimamoto, K.; Ohfune, Y.; Nakanishi, S. Br. J. Pharmacol. 1992 , 107,539. 6. (a) Kudo, Y.; Akita, K.; Ishida, M.; Shinozaki, H. Brain Res. 1991 ,567, 342. (b) Kawai, M.; Horikawa, Y.; Ishihara, T.; Shimamoto, K.; Ohfune, Y. Eur. J. Pharmacol. 1992, 211, 195. 7. We thank Dr. N. Hamanaka, Ono Pharmaceutical Co. Ltd., for X-ray crystallography of CCGs. 8. (a) Shimamoto, K.; Ohfune, Y. Tetrahedron Lett 1990, 31,4049. (b) Ishida, M.; Ohfune, Y.; Shimada, Y.; Shimamoto, K.; Shinozaki, H. Brain Res. 1991, 550,152. 9. (a) Lehmann, M. S.; Koetzle, T. F.; Hamilton, W. C. /. Cryst. Mol. Struct. 1972, 2, 225. (b) Lehmann, M. S.; Nunes, A. C. Acta Cryst. 1980 , B36, 1621. 10. Hashimoto, K.; Shirahama, H. J. Syn. Org. Chem. Japan. 1989 ,47,212. 11. (a) Slevin, J. T.; Collins, J. F.; Coyle, J. T. Brain Res. 1983, 265,169. (b) Conway, G. A.; Park, J. S.; Maggiora, L.; Mertes, M. P. J. Med. Chem. 1984, 27,52. 12. Hashimoto, K.; Shirahama, H. In: Amino Acids: Chemistry, Biology and Medicine; Lubec, G.; Rosenthal, G. A. Eds.; ESCOM: Leiden, 1990; p572. 13. Raghavan, S.; Ishida, M.; Shinozaki, H.; Nakanishi, K.; Ohfune, Y. Tetrahedron Lett in press. 14. Ohfune, Y.; Shimamoto, K.; Ishida, M.; Shinozaki, H. Biorg. Med. Chem. Lett. 1993, 3,15. 15. Ishida, M.; Saitoh, T.; Shimamoto, K.; Ohfune, Y.; Shinozaki, H. Br. J. Pharmacol, in press. 16. Ouerfelli, O.; Ishida, M.; Shinozaki, H.; Nakanishi, K.; Ohfune, Y. Synlett. 1993 , 409. — 48 — Volume 335, number 1, 47-50 FEES 13289 November 1993 © 1993 Federation of European Biochemical Societies 00145793/93/S6.00 Adaptability of nonnatural aromatic amino acids to the active center of the E. coli ribosomal A site Takahiro Hohsakaa, Ken Sato3, Masahiko Sisido3 *, Kazuyuki Takaib, Shigeyuki Yokoyama b aResearch Laboratory of Resources Utilization, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 227, Japan b Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan Received 9 September 1993; revised version received 7 October 1993 3,-Ar-Aminoacyl analogs of puromycin with nonnatural aromatic amino acids were synthesized and their inhibitory activity in E. coli in vitro protein synthesizing system was evaluated. The analogs with L-2-naphthylalanine, L-p-biphenylalanine, L-2-anthrylalanine and /razu-L-p-phenylazophenyla- lanine were found to inhibit the protein synthesis with high efficiency. The inhibition suggests that these nonnatural amino acids are accepted by the active center of the E. coli ribosomal A site. A model for the adaptability of nonnatural aromatic amino acids to the active center is proposed. Nonnatural amino acid; Puromycin; Protein biosynthesis; Ribosome; A site 1. INTRODUCTION were used. Puromycin is an inhibitor of protein syn thesis and known to bind to the ribosomal A site and Until recently, the amino acid replacements in pro teins are limited to 20 natural amino acids in the ordi nary protein engineering. Recently, however, several groups reported techniques for the incorporation of nonnatural amino acids into proteins [1-3] by the use of chemically misacylated tRNAs [4,5]. By introducing nonnatural amino acids into biosynthetic proteins, the NHOH scope of protein engineering will be extended to include CO a wide variety of artificial functions. In protein biosyn CH-R thesis, the selection of amino acids takes place essen nh2 tially at the aminoacylation of tRNA under the control (I) of aminoacyl tRNA synthetase. Therefore, once a block the entry of aminoacyl tRNA. The amino group tRNA is misacylated with a noncognate amino acid, the of bound puromycin forms a peptide bond with the latter amino acid will be directly incorporated into pro carboxyl group of the peptidyl tRNA on the P site. The teins. This was actually shown by Noren et al. [2] and resulting peptide with a terminal puromycin moiety Bain et al. [3] However, they reported that the incorpo leaves the ribosome. The inhibitory activity of puromy ration efficiency depends sharply on the type of amino cin analogs has been examined as a measure for the acids. For instance, D-phenylalanine was exclusively re adaptability of amino acids to the active center of ribo jected in their biosynthesizing system. This must be be somal A site. The analogs of aromatic amino acids, cause other steps such as the peptide bond formation in L-phenylalanine and L-tyrosine were most effective ribosome, discriminate nonnatural amino acids from [6-8], and those with O-benzyl-L-serine, S-benzyl-L- natural ones. cysteine [7,8], L-homocitrulline [9] and L-lysine [10] were In this study, we evaluated the adaptability of non moderately active, whereas the analogs of other amino natural amino acids carrying a variety of aromatic acids were almost inactive. The optical configuration of groups, to the A site of E. coli ribosomal peptidyl trans the amino acids was also important since the D-phenyla- ferase center. For this purpose, 3'-./V-aminoacyl analogs lanyl analog was far less active than the L-isomer [6]. of puromycin with nonnatural amino acids (Compound I) We examined the inhibitory activity of puromycin analogs with nonnatural amino acids carrying large aro matic groups (Fig. 1), and predicted whether or not * Corresponding author. Present address: Department of Bioengineer ing Science, Okayama University, 3-1-1 Tsushima-Naka, Okayama these nonnatural amino acids can be incorporated into 700, Japan. proteins. Published by Elsevier Science Publishers B. V. — 49 — Volume 335, number 1 FEES LETTERS November 1993 2. MATERIALS AND METHODS 3. RESULTS Puromycin, puromycin aminonucleoside, L-l-naphthylalanine (2) The results of the inhibition experiments are summa and L-2-naphthylalanine (3) were purchased from Sigma. L-l-pyren- rized in Table I. Puromycin and L-phenylalanyl analogs ylalanine (4), L-2-pyrenylalanine (5), L-p-biphenylalanine (6), L-2-an- showed strong inhibitory activity as reported previously thrylalanine (7), L-2-anthraquinonylalanine (8), L-9-carbazolylalanine [6]. Similarly, the analogs of L-2-naphthylalanine, l-p- (9), L-9-ethyl-3-carbazolylalanine (10), L-9-phenanthrylalanine (11) and L-p-phenylazophenylalanine (12) were synthesized in our labora biphenylalanine, L-2-anthrylalanine, and L-p-phenyl- tory [11]. L-AzOCn (13-16) and L-CarCn (17,18) were gifts from Profes azophenylalanine were found to inhibit with good effi sor Nishino of Kyushu Institute of Technology. ciency. On the other hand, the analog of L-l-naphthyl The puromycin analogs were synthesized as follows: puromycin alanine showed negligible inhibitory activity, in contrast aminonucleoside (0.01 mmol), /erz-butyloxycarbonyl-protected amino acid (0.01 mmol) and 1-hydroxybenzotriazole hydrate (0.011 mmol) to that of L-2-naphthylalanine. The analogs of L-l-py- were dissolved in 0.1 ml of anhydrous dimethylformamide. To this renylalanine, L-2-pyrenylalanine, L-9-phenanthrylalan- solution, l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (0.011 ine, L-2-anthraquinonylalanine, L-9-carbazolylalanine mmol) was added at 0°C and the mixture was stirred for 2 h at 0°C and L-9-ethyl-3-carbazolylalanine were also inactive. and 22 h at room temperature. The mixture was diluted with 2 ml of The inhibitory activity was markedly decreased when ethyl acetate and washed twice with 4% NaHC03, and twice with saturated NaCl. The organic phase was dried over MgS0 4 and the the fra/M-azobenzene group of the p-phenylazophenyl- solvent was evaporated. The resulting solid was recrystallized from alanyl analog was photoisomerized to the ds-form, as ethylacetate and /i-hexane. For deprotection, the product was dis shown in Fig. 2. Irradiation with UV light at 350 nm solved in 0.1 ml of trifluoroacetic acid at 0°C. After 30 min at 0°C, resulted in conversion of about 80% of azobenzene the solvent was flushed off by N2 gas, then the deprotected product was washed with ether for several times and dried in vacuo. The purity group to the dj-form under photostationary state. The was confirmed by reverse-phase HPLC and ’H-NMR. inhibitory activity of the puromycin analog was low In the inhibition experiment, E. coli S-30 extract [12], mRNA encod under this condition. The extrapolation to 100% cis- ing bacteriophage T7 gene 10, and l4C-labeled leucine were used. Pro form showed that the analog with ds-p-phenylazophen- tein synthesis was carried out at 37°C for 30 min in the presence of ylalanine was virtually inactive. various concentrations of puromycin analogs. The amount of the protein synthesized was measured by filter paper disk technique [13]. The effect of spacer lengths between the C“ atom and The inhibitory activity of the analogs of puromycin was expressed aromatic groups was also examined. In Table IB, the relative to a control experiment without inhibitor. results of azobenzene derivatives and carbazole deriva- Table I Inhibitory activity of 3'-A-aminoacyl analog of puromycin with a variety of aromatic natural and nonnatural amino acids in E.coli in vitro protein synthesizing system Inhibition (%) Concentration (M) X Amino acid o 1 x 10"3 3 x 10"4 1 x 10~4 3 x 10"5 1 x 10 A. O-Methyltyrosine _* 98.8 99.0 99.0 98.5 80.6 Phenylalanine 97.3 98.9 98.3 94.4 96.2 70.1 1-Naphthylalanine 65.6 29.2 0 2-Naphthylalanine 99.2 90.0 92.3 93.9 74.6 28.4 1-Pyrenylalanine _** 29.0 4.5 0 2-Pyrenylalanine _** _** 0 p-Biphenylalanine 97.8 90.4 71.6 64.5 55.4 56.3 9-Phenanthrylalanine 83.4 62.1 28.6 0 2-Anthrylalanine 97.8 98.5 98.9 95.5 81.2 73.0 2-Anthraquinonylalanine 58.1 33.0 34.6 0 9-Carbazolylalanine 25.5 5.7 7.1 0 9-Ethyl-3-carbazolylalanine 0.8 11.0 4.4 0 p-Phenylazophenylalanine 96.8 99.0 91.3 67.3 59.4 32.7 B. AzOC3 _* 99.9 89.6 37.8 14.1 8.1 AzOC4 97.8 96.5 79.7 64.5 33.8 0 AzOC5 _** 15.3 8.7 0 AzOC6 _** 16.2 13.7 0 CarC3 34.6 11.5 0 CarC5 1.9 0 *Not tested. **Insoluble. — 50 Volume 335, number 1 FEES LETTERS November 1993 h 2n ~^cooh Fig. 2. Inhibitory activity of the puromycin analogs with L-p-phenyla- h ^v ch 2 zophenylalanine: (□), trans-form (before irradiation); (a ), 80% c/5-form (after irradiation at 350 nm for 5 min); (•), 100% m-form (extrapolated values); (o), puromycin as a reference. react with peptidyl tRNA. If the latter is the case, how ever, a constant inhibitory efficiency that is lower than 9 100% will be observed after all the binding sites were occupied by the addition of an excess amount of the H2N>f,COOH H,N^COOH puromycin analog. The results in Table I exclude this H^fCH2)n H'"(GU2)n possibility, because the inhibitory activity of each ana log is not constant over the concentration range of KT4-1(T3 M and is increasing with the concentration. The concentration dependence in Table I shows that the low activities are due to low affinity for the active cen ter. Therefore, it is concluded that L-2-naphthylalanine, 13-16 (n=3-6) L-p-biphenylalanine, L-2-anthrylalanine, trans-L-p- phenylazophenylalanine, AzOC3 and AzOC4 adapt to Fig. 1. Structure of aromatic natural and nonnatural amino acids used the active center of ribosomal A site, but l-1-naphthyl- in this study. alanine, l- 1 -pyrenylalanine, L-2-pyrenylalanine, L-2-an- thraquinonylalanine, L-9-carbazolylalanine, L-9-ethyl- 3-carbazolylalanine, L-9-phenanthrylalanine, cis-L-p- lives are shown. The azobenzene derivatives having short spacers were active but those having long spacers were inactive. The carbazole derivatives were found to aal aalaalaa be inactive in every case. CHR CH-CH- 4. DISCUSSION The results in Table I show that the inhibitory activ ities of the puromycin analogs are widely different. The analogs are clearly divided into two groups depending on the inhibitory activity, those that show high activity at 10~5 M and those that show low activity even at 10"3 M. The analogs of high inhibitory activity may bind to the active center and form peptidyl puromycin, as has P-SITE A SITE been demonstrated previously for the phenylalanine an alogs, tyrosine, O-benzylserine, and S-benzylcysteine. Fig. 3. A model for the adaptability of nonnatural aromatic amino The puromycin analogs of those amino acids inhibited acids to the active center of E. coli ribosomal A site. The 3'-Ar-ami- the protein synthesis and formed peptidyl puromycin in noacyl analog of puromycin and peptidyl tRNA exist in ribosomal A the ribosome [7,8]. On the other hand, those of low site and P site, respectively. Nonnatural amino acids carrying benzene rings in regions B, E, G, H and I can adapt to the active center of A activity may either have low affinity for the active cen site, whereas those carrying benzene rings in other regions do not ter, or effectively bind to the active center but do not adapt. — 51 Volume 335, number 1 FEES LETTERS November 1993 phenylazophenylalanine, AzOC5, AzOC6, CarC3 and The results in Table IB suggest that spacer length is CarC5 do not adapt. also an important factor in determining the adaptabil The next question was why some nonnatural amino ity. In the case of the azobenzene derivatives, spacers acids adapt to the active center but others do not. In with n> 5 are clearly unfavorable, suggesting that previous studies, the analogs with aromatic amino acids azobenzene groups very remote from C“ atom do not containing one benzene ring have been found to adapt adapt to the ribosomal A site. In the case of carbazole to the active center [6-8]. However, the adaptability derivatives, however, no proper spacer length was does not depend only on the size of aromatic group, found for the incorporation of 9-substituted carbazole because 2-naphthylalanine adapts but 1-naphthylalan- group. ine does not. Therefore, the active center may discrimi The incorporation of the photofunctional nonnatural nate nonnatural amino acids not only by their size but amino acids into proteins is now in progress, and will their geometry. From a consideration of the results in appear in future reports. Table IA, a simple model for the adaptability of aro matic nonnatural amino acids to the active center can Acknowledgements: The authors wish to thank professor Norikazu be proposed as shown in Fig. 3. The benzene rings of Nishino of Kyushu Institute of Technology for the gift of nonnatural amino acids carrying carbazole or azobenzene group (13-18). arylalanine-type amino acids denoted as A, B, C, etc., can be divided into allowed regions (B, E, G, H, I) and disallowed regions (A, C, D, F). The amino acids that REFERENCES adapt to the active center contain benzene rings only in [1] Baldini, G., Martoglio, B., Schachenmann, A., Zugliani, C. and the allowed regions. On the other hand, those that do Brunner, J. (1988) Biochemistry 27, 7951-7959. not adapt contain benzene rings in the disallowed re [2] Noren, C.J., Anthony-Cahill, S.J., Griffith, M.C. and Schultz, P.G. (1989) Science 244, 182-188. gions. [3] Bain, J.D., Glabe, C.G., Dix, T.A., Chamberlin, A.R. and Diala, Based on this model, nonnatural amino acids that E.S. (1989) J. Am. Chem. Soc. Ill, 8013-8014. adapt to the active center can be predicted. For exam [4] Heckler, T.G., Zama, Y., Naka, T. and Hecht, S.M. (1983) J. ple, L-2-phenanthrylalanine that occupies regions B, E Biol. Chem. 258, 4492-4495. [5] Heckler, T.G., Chang, L.-H., Zama, Y„ Naka, T., Chorghade, and H may adapt, whereas l- 1 -phenanthrylalanine that M.S. and Hecht, S.M. (1984) Biochemistry 23, 1468-1473. occupies regions A, B and D will not. This model [6] Nathans, D. and Niedle, A. (1963) Nature 197, 1076-1077. explains that the analogs with benzyl derivatives of [7] Symons, R.H., Harris, R.J., Clarke, L.R, Wheldrake, J.F. and L-serine and L-cysteine are active [7,8], because the ben Elliott, W.H. (1969) Biochim. Biophys. Acta 179, 248-250. zene rings of these derivatives are located in region E. [8] Harris, R.J., Hanlon, J.E. and Symons, R.H. (1971) Biochim. Biophys. Acta 240, 244-262. The present results predict what type of nonnatural [9] Guarino, A.J., Ibershof, M.L. and Swain, R. (1963) Biochim. amino acids can be incorporated into proteins when Biophys. Acta 72, 62-68. they are attached to tRNA and added to the in vitro [10] Lichtenthaler, F.W., Cuny, E., Morino, T. and Rychlik, I. (1979) protein biosynthesizing system [1-3]. In the case of Chem. Ber. 112, 2588-2601. naphthylalanine, for instance, L-2-naphthylalanine may [11] Sisido, M. (1992) Progr. Poly. Sci. 17, 699-764. [12] Pratt, J.M. (1984) in: Transcription and Translation (Hames, be incorporated into proteins but l-1-naphthylalanine B.D. and Higgins, S.D., Eds.) pp. 179-209, IRL Press, Oxford. may not. Similarly, L-2-phenanthrylalanine is a proper [13] Mans, R.J. and Novell!, G.D. (1961) Arch. Biochem. Biophys. 94, amino acid for the incorporation of phenanthryl group. 48-53. — 52 W$T < 7° A (It) tty h V jz <& ^ A 1. g#r $ ymtii 3 2 OEE<07 5 y# 6SS7 ?y® (2 OEE) 7 5 y#— #@8#®75y (7 0 0 EEM±) (ABA*) #7 5 yE- #A#7 5 ym (f^v\ 4-##m6:^3) (AI7 5 yf) ###i±A#m*<07 5 ymm — 53 (2) (3) (4) kj:a, 5 y ®aWtK It () # tt/cATto5:MS7 5 y^Eth £-J§fctt (4) KM'Zfrvmu kcoo, t>tit>ti 2. ##7 < y#(Q^-M li (2) icMotltM ^zlIS A V — F ., 3. 7 < y##^)— 7 5 y@£ST>'i§-j&<7)3=A&tl:i#x.;£7;i±5ii3i L#31LA#&(0 $ W:###&?);m ^ A^ *6. Sto^t^.7 5 yE^SSKSCT^fifc^fFlfe^StUf — 54 — ft (1) (2) Gabriel'n'jS ( 3 ) Curtins 6H5 ( 4 ) Hoffmann Is {i (5) Schmidt|s{5 ( 6 ) Bucherer-Berg^JS (t: ^ > b d" >&) (7) Strecker^Si (8) UgiE95%a#-&& (9) (1) r,r2nch 2co 2r3 —— r1r2nchco 2r3 HgNCHCOgH r4 R4 (2) BrCHR1C02R2 — PhtNCHR1C02R; h 2nchr 1co 2h (3) RCHC02Et ----- RCHC02Et — rchco 2h co 2h con 3 nh 2 (4) RCHC02Et —►- RCHC02Et RCHCOgH co 2h conh 2 NH2 (5) RCHC02Et —- RCHCOgEt rchco 2h COMe NHCOMe nh 2 HN~Vi iki R \P)(fl\ RCHO ». rchco 2h o^ N>o nh 2 H (7) RCHO + HCN + NH3 -—— RCHCN - rchco 2h NH2 nh 2 (8) RCHO + R’NH2 + PhCOgH + t-BuNC —► RCH0O2H nh 2 t < r*=nch 2co 2r (R* = chiral template) — 55 (2) 9 - h cogm^tor 5 Jit OLi O O O Ri^n Aq BocN=NBoc ri V^a° Rix ^/C02H ,NBoc ^—( nh 2 % BocHN c (3) Bu 2x B o' o O O NBS R1V^ANA0 R'sAn\ vj N, v_/ r 2 R, (4) h l/'y RCHO + (5) ftKnr E%<7)65fcigT** 3^ HS-S-fiS:t|b]®KS®£1:©%©%^ ^ IcjEffl T* § & v^A (6) 75/iSyRoiBS-chS; c: £5titv^0 4. ##7 < iit^77 FSt^tlZK^V » y-7'yS(7)-§-S;5r^lk LT @1 Aculeatine A y (2) R1 =R2=Me, R3=X Echinocandin Pneumocandin A0 (L-671,329) (3a) B(1a), R1=R2=OH R1=Me, FfLcHzCONHg, F?=Y C (1b), F?=OH, R2=H Pneumocandin B0 (L-688,786) (3b) D (1c), R1=R2=H R1=H, FfczCHgCONHg, R3=Y Mulundocandin (4) R1=Me, R2=H, R3=Z 56 — gitr 5 oh l x pe^ye: k*ex £ i> os-s- ##v'o WS&yytKSJi: LT, x + y^yy^SttiwiTir? yESc9S-§-&<7) 4 y &$it£ & T fc 9 8> (01) v #j&7 5 yE 02 Synthesis of 1,2-amino hydroxyl system. NHBoc NHBoc NHC02TBS NHC02TBS II 1 I OMs R = ChiOCHgPh, R’ = Cl or OCOPh, Boc = CQ-f-Bu, TBS = SiMg-t-Bu Synthesis of 1,3-amino hydroxy! system. nhco 2tbs I NHBoc (f) ------► H02C (a), (b) epoxidation with MCPBA; (c) S%2 type cyclic carbamate formation; (d), (e) ScN - type cyclic carbamate formation; (f) halolactoniozation. 5. Aij£et7 < yE@<7)#^mm Man/woman-made amino acid 1475E^"C § fz — 57 — (1) mmtm&vmmfi (2) (3) a-75 k^-s-o r^r^j t 6. ##!%iSfc (1) Amino acids, peptides and proteins', The Chemical Society: 1968-1992, vol 1-23. (2) Hunt, S. Chemistry and Biochemistry of the Amino Acids ; Barrett, G. C. Ed.; Chapman and Hall: London, 1985; pp 55. (3) Ohfune, Y.; Shimamoto, K. J. Synth. Org. Chem. Jpn. 1991, 49, 302-313. (5) Coppola, G. M.; Shuster, H. F. Asymmetric Synthesis, Construction of Chiral Molecules Using Amino Acids ; John Wiley & Sons: New York, 1987. (6) Williams, R. M. Synthesis of Optically Active a-Amino Acids ; Pergamon Press: London, 1989. (7) a-Amino Acid Synthesis', Tetrahedron Symposium-in-Print; O'Donnell, M. J. Ed.; Tetrahedron 1988, 44, 5253. (8) (a) Benz, F.; Kniisel, F.; Nuesch, J.; Treichler, H.; Voser, W.; Nyfeler, R.; Keller- Schierlein, W. Helv. Chim. Acta 1974,57, 2459. (b) Keller-Juslen, C.; Kuhn, M.; Loosli, H.-R.; Petcher, T. J.; Weber, H. P.; von Wartburg, A. Tetrahedron Lett. 1976, 4147. (c) For a review, see: Inoue, S.; Sezaki, M. J. Synth. Org. Chem. Jpn 1993,57,73. (9) (a) Ohfune, Y.; Kurokawa, N. J. Synth. Org. Chem. Jpn. 1986, 44, 647-659. (b) Ohfune, Y. Acc. Chem. Res. 1992,25, 360. (10) (a) Kurokawa, N.; Ohfune, Y. J. Am. Chem. Soc. 1986, 108, 6041. (b) Kurokawa, N.; Ohfune, Y. J. Am. Chem. Soc. 1986, 108, 6043. (c) Evans, D. A.; Weber, A. E. J. Am. Chem. Soc. 1987, 109, 7151. (d) Kurokawa, N.; Ohfune, Y. Tetrahedron in press. — 58 i j 7° f- k (M)$6sromi@£ EF«S 3fl 22 B ^3- K'C/f- FfciO'T ; ym 30 mmarID K©Wa h&Cfvo y V X h 1. str30n,reps 7 U t°- 1. str30n,seq30n 2. seq30n,repq 2. str30ref,seq30ref 2.#o-K 1 s g#^l:6>l63 “fcffl” ©£EJ£SjSi:ii|sgBejt»'ijE.6ft3. f©#Tj#0^B:^f jr^x.-5= “t>©” ©tsssii^ofimrtet 3. awbia^f rx. 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F©^#, -@ij:labaditin,phakellistatin ^IJ:ristocetin,echinocandine,malonichrome,astin,microcystin f FT'ifcSW^Sft-a'So 3^iJTIi€©a®T £>5„ #lj :microcolin KU'C/f- K©tSiS^#ia6T©5ife5:r-fc, x t'5„ lot, Ufc*li$-C(iff|g©J£i9 4't'fc©fe*-Bo 4-@©P$T-tt^3- F 1. TlVAod- Fto 2.TX^J-XF 3. 4. 5. &%© g# e. 7.#mm# 8. 9 . YkA%aui%^©## 10. n.stt 12. FiiFJ-$rePs®*-7- F^liJfflUT&srCiSSo ASSS£©$fgi:M;i L/c^rf- Ftt+-7- F “bind” -CWM-&. mAm-f, ii F i 37A, a Giu(Cys) f»<*5„ F^'*-5o t#0AKPa#8©igBFL f&ffctLTOitk ##, me. m#©^&l:MaL-C#4 FI:3B 9l:*b, V * x> / 5-y^ii — 61 — 3 7 ~ / @§ 30 K:seq30n ;:©5d,7'9 i;-l;tiilK?0t!fgl;:$-dt,'T-£i$$4l.£$65l©7t>£6XlSE30HT©t> ©S-E^iJt-7^-7. (SEQDB)^bS©/cfc©T*5» 58#©EMK#&, 6 6€©9#m±li#m©E9ij76%6t,7TL'5. imefr&5#m©E9ijl:*7^T ^$75^660^7, AT©m8#0E^^#m©E^J7 6*6 67.7^6 6^7 7 0-Cii4'l'„ #l:@^58#7&5^7f KICK L7Hx tiL5*A%75aWYI:#0% 7$l#$fk -£©7 5 7 MEW**^nsto^l'. E^r-ene.!* GenBank,EMBL ©#MEW-7<-XI=&#&:h.7('%(,'. 65�<7f F©7; 7ME^A j: 7 c /:<77 F76£^%##L,7:&#^#%5 6Em — 62 angiotensin oxytocin CYIQNCPLG ox DRVYVHPFHL vasopressm rat mesotocin ------j_ chicken ------S- isotocin snake ------Y- glumitocin ---- S----- Q- turtle valitocin ------V- alligator ------A- aspargtocin bullfrog ------N- vasotocin ------R- goosefish N------pheny press in -FF------R- salmon N------N- conopressin -F-R------K- eel N------G- cephalotocin —FR------I- dogfish N-P-I---- Q- annetocin -FVR------T- ±©*©£ A:\agrep -2 "DRVYVHPFHL" seq30n > out E^IJX-^©(il 'o Tl '-S 7 7 'f vl/ seqSOn A'b 2 $$$©j$l'£Ff UTE£lJ “DRVYVHPFHL” liSfSS-II© “x-i'OflJffl” 1. *>*>8 2. ss 3. mm# 4. ##m 5. z®m 6. 7. $ a* s. am# 9 . K-f=f- k Slffl ti#$ repq ICE® £ ft*a ' -5 „ tt$®*-7- F£*Uffl UT#j}-SaB©^7°^ K @l^7°f F©#< 7# 1 ©&©-r&a. 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ACE, acyltransferase, alkaline phosphatase, APase, apase M, ATPase, carboxyl protease, enkephalinase, farnesyl transferase, Cys protease, HIV protease, phosphatase, phosphodiesterase, phosphoolipase, protease, Pro endopeptidase, thiol protease, thrombin, trypsin, tyrosinase 8. inhibit growth; cell growth, plant growth, antineoplastic, antitumor, promote Serratia growth 9. inhibit glycan syn, melanin syn, superoxide prodn 10. inhibit; K channel, ion channel, Ca blocker, ion transporter act, increase membrane permeability 11. toxin; insect toxin, phytotoxic, phytotoxin, my cotoxin, cytostatic, cytotoxic, toxin prodn, hepatotoxic, hepatotoxin, leaf spot disease pathogen, Cd resistance 12. posttranslational modification repq iSSjK:^ K©»» hor, tox, inh, fuc, mic, not, pro, orf, pep 1. horrhormonal; adipokinetic hormone, adrenorphin, AKH, allatostatin, allatotropin, alpha bag cell peptide, alpha factor, alytesin, amidorphin, ANF, angiotensin, ANP, ant ho RFamide, antho RIamide, antho RNamide, antho RWamide, APGWamide, apidaecin aspartocin, atria peptide, atriopeptin, BNP, bombesin, bombinin, bombolitin, bombyxin, bradykinin, buccalin, bursin, caerulein, cailiFMRFamide, calliMIRFamide CCK-PZ, chromogranin A-derived peptide, color change hormone, conantokin, conopressin, corticotropin like interm lobe peptide, deltorphin, dermenkephalin, dermorphin, diapuase hormone, diuretic hormone, dynorphin, egg assocd peptide, egg laying peptide, eledoisin, endorphin, endothelin, enkephalin, F sex factor, FMRFamide, galanin, gastric proventricular peptide, gastrin, gastrin releasing peptide, germ specificity regulating peptide, glucagon, glucagon like peptide, glumitocin, glycentin related peptide, gonadotropin releasing peptide, granuliberin, head activator peptide, hypertrehalosemic peptide, hypothalamic peptide, hypotrehalosemic hormone, insulin, isotocin, kallidin, kassinin, kinin, kyotorphin, leucokinin, leucosulfakinin, leukopyrokinin, litorin, locustakinin, locustapyrokinin, locustatachykinin, luliberin, mating factor, melanin concentrating hormone, melanostatin, — 65 melanotropin, mesotocin, mosact, motilin, MSH, neo-endorphin, neurokinin, neuromedin, neuropeptide, neurotensin, opioid peptide, ornitho kinin, oxyntomodulin, oxytocin, pancreatic polypeptide, peptide BAM12P, peptide HI, peptide PHI, perisulfakinin, phenypressin, pheromonotropic neuropeptide, phyllocaerulein, phyllokinin, phyllolitorin, phyllomedusin, physalaemin, pigment dispersing hormone, Pol-RFamide, polisteskinin, polyphemusin, proctolin ranakinin, ranatachykinin, ranatensin, red pigment-concentrating hormone, relaxin, resact, rhodotorucine, rimorphin, RPCH neuropeptide, SADPNFLRFamide, SALMFamide, secretin, sex pheromone, somatoliberin, somatostatin, speract, sperm activating peptide, substance P, tachykinin, tachyplesin, thymic factor, thymic humoral factor, thymosin, thyroliberin, thyroliberin-like peptide, tremerogen, urokinase, urotensin, valitocin, valosin, vasoactive intestinal peptide, vasoactive peptide, vasopressin, vasotocin, vespakinin, vespulakinin, VIP, waspkinin, xenopsin, 2. tox:toxin; conotoxin, conus toxin, crotoxin, delta-like toxin, enterotoxin, halo toxin, lethal toxin, omega agatoxin, sarafotoxin, toxic peptide, toxin 3. inhrinhibitor; ACE, alphal protease, galactose oxidase, PI, protease, trypsin 4. fuc:functional; anorexigenic peptide, anti arrhythmic peptide, antigonadotropic peptide, antimicrobial peptide, ATPase accelerating peptide, bioact hydrophobic peptide, bradykinin potentiating peptide, carbohydrate carrying peptide, cardio-excitatory peptide, cardioactive peptide, cardioactive peptide, cardioexcitatory peptide, catch relaxing peptide, chemotactic peptide, cholinergic neurostimulating peptide, conditioned avoidance response peptide contraceptive peptide, corticostatic peptide, corticotropin releasing peptide CRF active peptide, delicious taste peptide, delta sleep inducing peptide, DFT stimulating peptide, diuretic peptide, encephalitogenic peptide, enkephalin releasing peptide, growth blocking peptide, growth modulating peptide, growth/mitosis inhibitory peptide, histamine releasing peptide, inhibitory peptide, light adapting hormone, locustamyoinhibiting peptide, locustamyotropin, Lom-AG-myotropin, lymphocyte stimulating peptide, macrophage chemotactic factor, mast cell degranulating peptide, mitosis inhibiting peptide, morphogenetic peptide, mytilus inhibitory peptide, paragonial peptide paralytic peptide, pedal peptide, proliferation inhibiting peptide, redox active peptide, sleeper peptide, somatotropin releasing 10-peptide, sperm activating peptide, steroidogenesis activator, thymocyte growth peptide, trpEG attenuator peptide, trypsinogen activation peptide, tumor invasion inhibiting factor, uremic peptide 5. mic:miscellaneous; adrenal medullary peptide, brain peptide, chromaffin granule peptide, ganglion peptide, gastric peptide, glutathione, internal peptide, intestinal peptide, learning induced brain peptide, locusta. peptide, myelin peptide, neutrophil granule peptide, pancreatic islet peptide, posterior pituitary peptide, salivary His rich peptide, salivary peptide, seminal peptide, spermatozoal antigen, spinal cord 3-peptide, synaptosomal peptide 6. not: achatin, acrosin, ameletin, anantin, apamine, bactenecin, califin, carassin, cationic Cys-rich peptide, cephalotocin, cerebellin, cinnamycin, cionin, clupeine, corazonin, crabrolin, cycloleonurinin, defensin, duramycin, ferritin, fulicin, gallidermin, gelsolin, — 66 — guanylin, histatin, hydrin, hylambatin, indolicidin, kinetensin, marinostatin, mastoparan, melittin, metallothionein, metorphamide, microcin, myomodulin, myotropin, nephritogenoside , periplanetin, pneumadin, Pro rich peptide, protaminerigin, Pyr-Glu-Pro-NH2, scyliorhinin, secapin, sillucin, stellin, sturine, systemin, tertiapin, tryptophyllin, tuftsin, uperolein, vitellogenin, 7. pro:protein; agglutinin, albumin prepiece, albuminamide, alpha2HS glycoprotein B, amylase alpha, antifreeze glycoprotein, antioncogene protein pt27, ATPase 9, FO ATPase beta, capsid protein, casomorphin beta, collagen signal peptide, complement C3f, creatine kinase B, dopa decarboxylase, early leader protein, fibrinogen alpha, hbrinopeptide, fibroin peptide, hbronectin Cys region, flavoprotein, G protein, genome linked protein VPg, gliadin, globin, hemolysin, histone HI, histone H3, Ig, immunostimulating casein 6-peptide, maltodextrin phosphorylase, MHC, myeloma protein, pepsinogen major glycopeptide, phosphoglycerate kinase, phosphoribosyl transferase, phycobiliprotein , phycocyanin, placental lactogen, polysialo glycoprotein, protein C, pyruvate carboxylase biotin peptide, reaction center protein, regulatory protein araE, replication initiation protein, ribosomal protein, RNA polymerase, terminal protein, tropomyosin, Zn finger protein 8. orf: gene; leader peptide ermG, shl leader peptide, trp operon leader peptide, trpGDC operon leader peptide 9. pep: peptide; peptide 2, peptide A, peptide A12d, peptide A17c, peptide aPY, peptide B, peptide B12, peptide HR, peptide I, peptide II, peptide OA24b, peptide POL236, peptide PYF, peptide SCP, — 67 — J. Biochem. 113, 55-60 (1993) Molecular Conformation of Porcine Amelogenin in Solution: Three Folding Units at the N-Terminal, Central, and C-Terminal Regions 1 Yuji Goto,* Eiichi Kogure,** Toshio Takagi,** Saburo Aimoto,** and Takaaki Aoba***- 2 * Department of Biology, Faculty of Science, Osaka University, Toyonaka, Osaka 530; ** Institute for Protein Research, Osaka University, Suita, Osaka 565; and *** Forsyth Dental Center, 140 Fenway, Boston, MA 02115, U.S.A. Received for publication, June 29, 1992 Circular dichroism (CD) studies were conducted to gain a better insight into the conforma tion of amelogenins, which were isolated from developing enamel of piglets. The intact porcine amelogenin and its degraded products were purified chromatographically. The 25-residue peptide corresponding to the segment at the C-terminus was synthesized. CD spectra of these samples were measured at pH 5.0-5.3 in the temperature range between 4 and 90°C. The most remarkable finding was that the CD spectrum of the intact amelogenin was accounted for by the sum of the spectra of the three fragments at the N-terminal, central, and C-terminal regions, supporting the hypothesis that the structure of the whole protein consists of discrete folding units. Furthermore, low-angle laser light scattering analysis provided evidence that the 20 kDa amelogenin, the most abundant extracellular matrix protein in forming enamel tissue, exists in a monomeric form at pH 5.3 and 25 C. It was tentatively concluded that the N-terminal region contains £-sheet structures, while the spectral characteristics of the C-terminal region are similar to those of a random coil conformation. The conformation of the central region was characterized by a strong negative ellipticity at 203 nm, although its nature remains to be defined. Tooth enamel is the hardest tissue in mammals and is apatite crystals and that adsorbed amelogenin inhibited the composed of the largest calcium apatite crystals in the crystal growth of hydroxyapatite in media, as found in vivo. body. It is believed that enamel matrix proteins, secreted More importantly, the high adsorption affinity and inhibi by the ameloblasts, play a regulatory role in the process of tory activity of the intact amelogenin were weakened by enamel crystal formation, but the mechanism has not yet enzymatic cleavage of the hydrophilic segment at the been defined. In developing enamel, the secreted matrix C-terminus and almost completely lost with the subsequent proteins have been classified into two groups, amelogenins cleavage at the N-terminus (4). These observations support and enamelins (2). The amelogenins are major matrix the contention that the expression and modulation of constituents, corresponding to 90% or more of the total functions of the amelogenins are intimately related to their proteins secreted during developmental stages. The pri molecular structures. However, there is a paucity of infor mary structure of the intact (i.e., secreted) amelogenin has mation about the molecular structures of the amelogenins already been determined in human, porcine, murine, and in solution and in the solid state. bovine (see below). The intact amelogenin is degraded In the present study, circular dichroism (CD) and low- shortly after secretion, decreasing in quantity dramatically angle laser light scattering (LALLS) were utilized to gain a with developmental advancement, finally leaving the hard better insight into the molecular conformation of porcine est tissue, composed almost exclusively (95 wt%) of in amelogenin in solution. The intact porcine amelogenin organic crystals. consists of 173 amino acid residues (5) and has an apparent Concerning the functional roles of the amelogenins in molecular mass of 25 kDa by SDS - polyacrylamide gel enamel mineralization, it has been postulated that the electrophoresis (SDS-PAGE). It was demonstrated that, amelogenins, which are hydrophobic in nature, may con following the cleavages of segments, the 25 kDa porcine tribute passively to the growth of enamel crystals by filling amelogenin gives rise to products having molecular masses the intercrystallite space and providing a space for further of 20,13, and 5 kDa (6). As mentioned above, the degrada growth upon their removal (2). However, recent work (3) tion of porcine amelogenin occurring in a programmed showed that intact amelogenin is adsorbed selectively onto manner appears to modulate the functions of amelogenin prior to massive loss from the formed enamel. An intrigu 1 This study was supported in part by Grants-in-Aid for Scientific ing hypothesis is that the intact amelogenin may consist of Research from the Ministry of Education, Science and Culture of independent folding domains having unique properties and Japan (YG, TT, and SA) and USPHS Research Grant DE07623 from that the cleavage sites may correspond to sites linking the the National Institute of Dental Research (TA). 2 To whom correspondence should be addressed. independent folding domains. To test this hypothesis, we Abbreviations: CD, circular dichroism; LALLS, low-angle laser light analyzed CD spectra of intact amelogenin and its frag scattering; PAGE, polyacrylamide gel electrophoresis; SDS, sodium ments. The results were consistent with the presence of dodecyl sulfate. three independent folding units at the N-terminal, central, Vol. 113, No. 1, 1993 — 68 — Y. Goto et al and C-terminal regions. acid residues at the C-terminus, the 13 kDa fragment corresponding to the central region, and the N-terminal 5 MATERIALS AND METHODS kDa fragment. Following the degradation of the intact 25 kDa amelogenin shortly after its secretion, the 20 kDa Preparation of Porcine Enamel Samples—Enamel polypeptide exists as a major matrix constituent in secre matrix proteins were isolated from the secretory enamel of tory porcine enamel. Those porcine amelogenins of 25, 20, permanent teeth of a 6-month-old freshly slaughtered 13, and 5 kDa were purified by chromatography as de piglet. Tooth germs were dissected out of the mandible. scribed in the following section. After removal of the covering soft tissues, the exposed Chromatography —Gel filtration chromatography of the enamel surface was wiped gently with a paper tissue dipped matrix protein extracted from the secretory enamel was in cold saline containing protease and phosphatase in conducted at 4°C on a Sephadex G-100 (Pharmacia-LKB) hibitors. The secretory enamel was dissected with fine equilibrated with 50 mM carbonate buffer (pH 10.8). Each spatulas and then dissolved in 0.5 M acetic acid containing protein fraction was eluted with the same alkaline buffer, protease and phosphatase inhibitors. All procedures for and concentrated using the YM-5 membrane. Further extraction of proteins from the dissected enamel samples purification of each of the 25, 20, and 13 kDa amelogenins were conducted at 0°C; details of the procedures were was carried out by seven cycles of chromatography under reported previously (3). The proteins extracted in the acid the same conditions. Following the final chromatography, solution were desalted through a YM-5 ultrafiltration the fractionated sample was titrated with 0.5 M acetic acid membrane (Mr cut-off 5,000, Amicon) and then freeze- to a pH of around 4, desalted by ultrafiltration with the dried. The obtained proteins were stored at — 30°C until YM-5 membrane and then freeze-dried. The 5 kDa amelo used for analyses. genin corresponding to the N-terminal 45-residue fragment Figure 1 shows the primary sequences of human (7), was most sparing soluble and tended to associate with other porcine (5), murine (8), and bovine (9) amelogenins. As minor fragments having similar molecular masses. Thus, indicated by asterisks in the figure, the amelogenin se following the Sephadex G-100 chromatography , the frac quences are highly conserved among the mammalian tion corresponding to 5 kDa fragment was further separat species, particularly at the N- and C-termini. The primary ed on a DEAE-cellulose column using 10 mM Tris buffer structures in the central region of the proteins are species- (pH 8.3) in the presence of 6 M urea. The proteins were dependent but are commonly characterized by the presence eluted with a 0.05 to 0.15 M NaCl gradient in the buffer. of repeated sequences of -Pro-X-Pro- or -Pro-X-X-Pro-. Finally, the 5 kDa polypeptide fraction was purified on an Table I shows the amino acid sequences of intact porcine SP-Sephadex C-25 column with a pH gradient from 3.6 to amelogenin and its degraded products. Enzymatic cleav 6.5; the buffer used was 1 M citrate in the presence of 6 M ages of porcine amelogenins occur at the sites between urea. Details of the experimental procedures and typical Trp45 and Leu46, and Serl48 and Metl49 (6), yielding the chromatograms obtained for porcine amelogenins were protein of 20 kDa (by SDS-PAGE) lacking the last 25 amino reported previously (10). The purity of the separated sample was determined by amino acid analysis, electro phoresis (15% polyacrylamide gels containing SDS for the 1 10 20 30 40 50 25, 20, and 13 kDa proteins and 5% gel in the presence of 6 Human MPLPPHPGHPGYINFSYEVLTPLKWYQS-IRPPYPSYGYEPMGGWLHHQI M urea for the 5 kDa fragment), and partial sequencing at Porcine MPLPPHPGHPGYINFSYEVLTPLKWYQNMIRHPYTSYGYEPMGGWLHHQI the N- and C-termini. Murine MPLPPHPGHPGYINFSYEVLTPLKWYQSMIRQPYPSYGYEPMGGWLHHQI Since the fragment corresponding to the C-terminal 25 Bovine MPLPPHPGHPGYINFSYEVLTPLKWYQSMIRHPYPSYGYEPMGGWLHHQI amino acid residues has not been identified in vivo (the reason is unknown), the corresponding 25-residue peptide 60 70 80 90 100 (C25) was synthesized by a standard solid-phase method. IPVLSQQHPPTHTLQPHHHIPWPAQQPVIPQQPMMPVPGQHSMTPIQHH The crude product was purified by reversed-phase HPLC. IPWSQQTPQSHALQPHHHIPMVPAQQPGIPQQPMMPLPGQHSMTPTQHH The purity of the final product was determined by amino IPVLSQQHPPSHTLQPHHHLPWPAQQPVAPQQPMMPVPGHHSMTPTQHH IPWSQQTPQNHALQPHHHIPMVPAQQPWPQQPMMPVPGQHSMTPTQHH acid analysis and Fab mass spectrometry (JEOL, Tokyo). Circular Dichroism Measurements —CD spectra of the purified protein and peptide samples were recorded using a 110 120 130 140 150 JASCO spectropolarimeter (model J-500A) equipped with QPNLPPPAQQPY--- QPQPVQPQPHQPMQPQPP------a thermostatically controlled cell holder. Prior to measure QPNLPLPAQQPF--- QPQPVQPQPHQPLQPQSP------QPNIPPSAQQPFQQPFQPQAIPPQSHQPMQPQSP------ments, a weighed amount of the sample was dissolved in QPNLPLPAQQPF--- QPQSIQPQPHQPLQPHQPLQPMQPMQPLQPLQPL deionized water to yield concentrations of 0.1-0.3 mg protein (or peptide)/ml. Since some acetate (used in the solvent for protein/fragment purification) still remained in 160 170 180 190 200 ---- VHPMQPLPPQPPLPPMFPMQPLPPMLPDLTLEAWPSTDKTKREEWSIF --- PMHPIQPLLPQPPLPPMFSMQSL-- L PDL PLEAWPATDKTKREEVD ---- LHPMQPLAPQPPLPPLFSMQPLSPILPELPLEAWPATDKTKREEVD Q PQ P PVH PIQPLPPQPPLPPIFPMQPLP PML PDL PLEAWPATDKTKREEVD TABLE I. Structure of porcine amelogenin and its fragments. Species Structure Fig. 1. Amino acid sequence of human, porcine, murine, and 25-kDa 1 173 bovine amelogenins. (*) indicates homology in these four se 5-kDa 1 45 quences. The sequences of human, porcine, murine, and bovine 20-kDa 1 148 amelogenins are taken from Shimokawa et al. (7), Yamakoshi et al. 13-kDa 46 148 (5), Snead et al. (8), and Shimokawa et al. (9), respectively. C25 149 173 J. Biochem. — 69 — CD Studies on Folding Units of Porcine Amelogenin the freeze dried samples, the resulting experimental solu amino acid composition is not markedly biased. The tions were about 5 mM with respect to acetate (the concen amelogenin fragments and the proteins used as molecular trations were determined by Dionex ion chromatography) . weight standards were judged to be such proteins. The It was demonstrated (6) that the amelogenin proteins are value of K-(dn/dc)-1, unique to the experimental set-up, sparing soluble at neutral pH and room temperature but was determined from the slope of a plot between the values become soluble on cooling or by decreasing the pH below 6. of (output) LS (output)tu" 1 obtained for standard proteins Thus, the present CD measurements were always conduct and their molecular weights. The standard proteins used ed at pH 5.0 to 5.3. Weighed samples of 25, 20, and 13 kDa were: bovine serum albumin (66,000), ovalbumin amelogenins and synthetic peptide were solubilized without (45,000), carbonic anhydrase (29,000), a -chymotrypsin- any visible clouding in the solution, whereas the solution of ogen (25,000), and RNase A (13,700). the 5 kDa fragment was slightly turbid. In order to examine In practice, an aliquot (100 /H) of the sample solution, thermal transitions of the molecular structures of the adjusted to a concentration of 1 to 5 mg/ml, was injected amelogenins, CD spectra were recorded at various temper into a column of either TSK-GEL G3000-XL (Tosoh) atures. In addition, the change in ellipticity at 230 nm with packed with modified silica gel particles or Superdex 75 increase in temperature was followed at a rate of l°C/min. packed with dextran particles (Pharmacia-LKB). The Temperature was monitored with a Sensortek Model column was equilibrated with 20 mM sodium acetate buffer BAT-12 thermometer. The reversibility of the thermal at pH 5.3. The protein was eluted with the same buffer transition was checked by measuring the ellipticity after containing various concentrations of NaCl (50,200, and 500 lowering the temperature to 20°C. mM), at a flow rate of 0.2 ml/min at 25°C. The elution All CD spectra were recorded with a 1 mm cell in the far profile was monitored with a LALLS photometer (model UV range of 250 to 190 nm. The instrument was calibrated LS-8000, Tosoh), which was connected serially with a with ammonium d- 10-camphorsulfonate. At the end of precision differential refractometer (model RI-8000). each experiment, the actual concentration of the sample used was determined by amino acid analysis. The CD data RESULTS AND DISCUSSION obtained were expressed as mean residue ellipticity [0], which is defined as [ 0] = 100 X dohsA /(lc), where 0obsd is the Circular Dichroism Spectra of Porcine Amelogenins — observed ellipticity in degrees, c and l are the concentration Figure 2A shows CD spectra of porcine amelogenins and in residue moles per liter and the length of the light path in synthetic peptide at pH 5.3 and 20°C. The CD spectrum of centimeters, respectively. the intact 25 kDa amelogenin had a minimum at 203 nm Low-Angle Laser Light Scattering Analysis —The sys with an ellipticity of — 22,500, suggesting the presence of tem used was reported previously (11, 12). According to some conformational structures. Similar characteristic this technique, the molecular weight (Mr) of a protein can features, showing the minimum ellipticity at 203 nm, were be determined by the equation, also recognized in CD spectra of the 20 and 13 kDa ame logenins. As compared to the value of minimum ellipticity M _ jf (output) LS(output) Rr1 m Mt~K (dn/dc) Uj obtained for the 25 kDa intact protein, the 20 kDa ame logenin showed a slightly smaller value ( — 20,000) but, where K is a constant determined by the instrumental and interestingly, the 13 kDa fragment showed a significantly experimental conditions, dn/dc is the specific refractive greater value ( — 30,000). Those spectra of the porcine index increment, and (output) LS (output) R r1 is the ratio of amelogenins were similar in shape to that of bovine the outputs of the detectors of the scattering photometer amelogenin reported by Renugopalakrishnan et al. (13), and differential refractometer. The value of dn/dc can be who proposed that the CD spectrum of bovine amelogenin assumed to be virtually constant for proteins of which the in acidic solutions resembled the CD spectra of “unorder- ■'o -20 240 250 Wavelength (nm Wavelength (nm) Wavelength (nm) Fig. 2. Far-UV CD spectra of the porcine amelogenins in 5 mM acetate buffer at pH 5.3 and 20°C. Panel A: the 25 kDa (1), 20 kDa (2), 13 kDa (3), and 5 kDa (4) amelogenins and the C-terminal synthetic peptide (5). Panel B: Measured (solid line) and calculated (broken line) spectra of the 25 kDa amelogenin. Panel C: Measured (solid line) and calculated (broken line) spectra of the 20 kDa amelogenin. Vol. 113, No. 1, 1993 — 70 Y. Goto et al. Fig. 3. Far-UV CD spectra of the 25 kDa (A) and 13 kDa (B) amelogenins in 5 mM acetate buffer at pH 5.3 at various tem peratures. Temperatures were 6 (1), 20 (2), 40 (3), 60 (4), and 79 (5) "C. Wavelength (nm) Wavelength (nm) Temperature (°C) Fig. 4. Dependence on temperature of the ellipticity at 230 nm (LS)/(RI) of the 25 kDa (solid line) and 13 kDa (broken line) amelogenins Fig. 6. Determination of the molecular mass of the 20 kDa in 5 mM acetate buffer at pH 5.3. The circle and square show the amelogenin (square) by the low-angle laser light scattering signal obtained after heating the 25 kDa and 13 kDa amelogenins, respectively, to 90°C. method. The calibration line was obtained by a plot of (output) Ls/ (output) ri against the molecular weights of standard proteins (cir cles). Standard proteins were bovine serum albumin (66,000), ovalbumin (45,000), carbonic anhydrase (29,000), a -chymotrypsin- ogen (25,000), and RNase A (13,700). The values of (output)us/ (output) R, were obtained on the basis of the peak area. C-terminus, was distinct from those of the porcine ame logenins of 25, 20, and 13 kDa. The spectrum of the N-terminal fragment showed a weak minimum ([9] = — 3,000) at around 215 nm, typical of a >9 -sheet conforma tion. This finding is in good agreement with the observa tions by Fourier-transform infrared and Raman spectros copy (14), suggesting that bovine amelogenin has a low a -helical content, and high /?-tum and/9-sheet composition. On the other hand, the CD spectrum of the 25-residue peptide corresponding to the segment at the C-terminus showed a sharp minimum at 200 nm ([9] = — 35,000). The Elution Volume ( ml ) overall spectral features of the peptide fragment resem Fig. 5. Elution pattern from Superdex 75 of the 20 kDa amelo- bled those of a fully unfolded protein observed at pH 2 in genin (1), carbonic anhydrase (2), a -chymotrypsinogen (3), and the absence of salt (15). Thus it is likely that the C- RNase A (4) monitored by measuring the absorption at 280 nm. terminal fragment itself has a random coil conformation in solution. Next, we examined whether the CD spectra of the 25 and ed” structures. 20 kDa porcine amelogenins could be predicted from those The CD spectrum of either the 45-amino acid residue of the degraded products. Assuming that the CD spectra fragment at the N-terminus or the synthetic peptide originating from the fragments contribute addictively to corresponding to the last 25 amino acid residues at the the CD spectrum of the whole molecule, the ellipticities of J. Biochem. 71 — CD Studies on Folding Units of Porcine Amelogenin the 20 and 25 kDa proteins, [0*] 2OkDa and [0*] 25kDa respec at 230 nm as a function of temperature. A plot of the tively, are calculated on the basis of the experimental [0] experimental data for the 25 kDa intact protein yielded a values of the 5 and 13 kDa fragments and the synthetic continuously descending line, indicating that the thermal peptide C25 according to the following equations: transition taking place in the structure of the 25 kDa intact amelogenin was a gradual process without a cooperative 45 103 transition. Again, the data points obtained for the 13 kDa [e*l. [*]. i + [0] 13 (2) 148 148 fragment yielded a line almost parallel to that of the 25 kDa protein. It is also notable that the thermal transition of the [0*]25kDa — ^yg[^]skDa+ [ $] 13kDa + ^yg[^]c25- amelogenins was fully reversible, as supported by the reproducible value of [0] (indicated by the circle and Figure 2B shows the results of [0*] calculation with square), which was re-measured at 20°C after heating the respect to the 25 kDa intact amelogenins. Interestingly sample to 90°C. enough, the calculated spectrum (shown by the dotted line) Putative Oligomeric State of Porcine Amelogenin —Since was very close to the experimental spectrum (shown by a porcine (as well as other mammalian) amelogenins tend to solid line). The excellent agreement lends support to our form intermolecular association in solution (6), we at hypothesis that the intact amelogenin consists of three tempted to determine monomeric or oligomeric state of independent folding units at the N-terminal, central, and porcine amelogenin in solution by LALLS coupled with C-terminal regions. It should be pointed out that the high-performance gel chromatography . A specific point to putative folding units appear to work in a cooperative be tested was whether CD spectral features of porcine manner to realize the postulated function of the protein in amelogenins common with those of collagens originate from enamel mineralization. Indeed, previous studies (4) de the presence of collagen-like triple helical structure in the monstrated that the whole molecular structure, involving former. both the N- and C-termini, was required to maximize the In pilot experiments, it was found that neither the 25 nor adsorption of porcine amelogenin onto apatite crystals. 20 kDa protein was eluted from the TSK-G3000SW-XL In the case of the 20 kDa porcine amelogenin shown in column (silica gel) with 20 mM acetate buffer (pH 5.3) Fig. 2C, there was a small discrepancy between the containing various concentrations of NaCl from 0.05 to 0.5 experimental and calculated spectra. A plausible explana M. When the dextran-base Superdex 75 column was used, tion for the obtained discrepancy is that, following the the 25 kDa protein was still adsorbed strongly to the cleavage of the hydrophilic fragment at the C-terminus, the column but the 20 kDa protein was eluted with the same folding structures of the N-terminal and central regions acetate buffer. The remarkable difference in adsorption (now the latter is the C-terminal region of the 20 kDa property between the 25 and 20 kDa amelogenins is consis degraded product) might be modulated slightly. Some tent with the finding (3) that the 25 kDa amelogenin is evidence for this was obtained in previous ^H-NMR studies adsorbed selectively onto apatite crystals, whereas its using photo-CIDNP (chemically induced dynamic nuclear degraded product of 20 kDa, lacking the C-terminal seg polarization), showing that accessibility of flavin probes to ment, has lost the adsorption affinity for apatite surfaces. the His residues (11 out of the 14 residues are localized in Figure 5 shows typical elution patterns of the 20 kDa the central region, see Fig. 1) differed significantly between amelogenin and the standard proteins monitored by the 25 and 20 kDa proteins (16). measuring the absorption at 280 nm, obtained at pH 5.3 in Thermal Transitions —Figure 3A shows typical CD the presence of 0.2 M NaCl. The 20 kDa amelogenin spectra of the intact amelogenin measured at various comprises 148 amino acid residues and its theoretical temperatures. With increasing temperature from 4 to molecular mass, calculated on the basis of its amino acid 90°C, the negative band at 203 nm decreased in magnitude composition, is 17,065 Da; it was also determined (19) that and, concomitantly, the ellipticity at 220 nm increased. The the 20 kDa porcine amelogenin is not glycosylated. The increase in [0] at 220nm suggests an increase in the elution profile of the 20 kDa amelogenin was broader than or-helix content. As can be seen in the figure, careful those of the standard proteins. We also found that elution examination of the spectra measured at different tempera profiles of the 20 kDa amelogenin became much broader in tures disclosed the presence of an isodichroic point at 214 the presence of NaCl at 50 and 500 mM. A comparison of nm, supporting a two-state transition between the low- and the chromatograms showed that this amelogenin was eluted high-temperature forms. As shown in Fig. 2B, similar slightly earlier than chymotrypsinogen (25 kDa). This results including the isodichroic point at 214 nm were observation does not imply that the real molecular mass of obtained using the 13 kDa fragment. The finding that both the 20 kDa amelogenin is larger than that of chymotryp the 25 and 13 kDa amelogenin samples displayed similar sinogen because it is known (20) that amelogenins do not thermal transitions, further demonstrates that the central behave in solution like a globular protein. region of the whole amelogenin molecule constitutes a Figure 6 shows plots of (output) LS/(output) RI versus relatively isolated domain having little interaction with the molecular weight, Mr, according to Eq. 1. The values of folding units at the N- and C-termini. Considering the high (output) LS/ (output) Ri were obtained on the basis of the content of proline residues or the presence of Pro-x-Pro or peak area. The data points obtained with the standard Pro-x-x-Pro sequences in the central region of porcine proteins yielded a line having the slope value of 53,300, amelogenin (see Fig. 1), it is interesting to note that the which corresponds to K- (dn/dc)_1. Based on this calibra overall characteristics of the thermal transitions of the tion line, it was estimated that the molecular mass of the amelogenin protein resemble those of collagen and related 148 -residue amelogenin was 20.9 kDa. Since the peak proteins (17, 18). profile of this porcine amelogenin was broadened, the Figure 4 shows changes of the ellipticity [0] determined values of (output) Ls/(output) R, were also obtained from Vol. 113, No. 1, 1993 — 72 — Y. Goto et al. multiple points around the peak on the basis of the signal Birmingham, AL intensities. By using the calibration line of Fig. 6, the 3. Aoba, T., Fukae, M., Tanabe, T., Shimizu, M., & Moreno, E.C. molecular mass was estimated to be 19.8 ±2.1 kDa. The (1987) Calcif. Tissue Int. 41, 281-289 4. Aoba, T., Moreno, E.C., Kresak, M., & Tanabe, T. (1989) J. Dent. good agreement between the weight-average molecular Res. 68, 1331-1336 mass determined for the 20 kDa amelogenin and the 5. Yamakoshi, Y., Tanabe, T., Fukae, M., & Shimizu, M. (1989) in molecular mass of its monomeric unit indicates that the Tooth Enamel V (Feamhead, R.W., ed.) pp. 314-318, Florence amelogenin exists as a monomer and not as an oligomer. Publ., Yokohama The agreement between the above weight-average value 6. Shimizu, M. & Fukae, M. (1983) in Mechanisms of Tooth Enamel and the molecular weights obtained one by one around the Formation (Suga, S., ed.) pp. 125-141, Quintessence Publ., Tokyo peak maximum also supports the above conclusion. 7. Shimokawa, H., Tamura, M., Ibaraki, K., & Sasaki, S. (1989) in In conclusion, the present CD studies support the hypo Tooth Enamel V (Feamhead, R.W., ed.) pp. 301-305, Florence thesis that the intact porcine amelogenin consists of three Publ., Yokohama independent folding units, i.e., the N-terminal, central, and 8. Snead, M.L., Lau, E.C., Zeichner-David, M., Fincham, A.G., C-terminal regions. Possible secondary structures existing Woo, S.L.C., & Slavkin, H.C. (1985) Biochem. Biophys. Res. in the central region of the intact porcine amelogenin or in Commun. 129, 812-818 the 13 kDa fragment remain to be determined. It is most 9. Shimokawa, H., Sobel, M.E., Sasaki, S., Termine, J.D., & Young, M.F. (1987) J. Biol. Chem. 262, 4042-4047 likely that the hydrophilic segment at the C-terminus has a 10. Tanabe, T., Aoba, T., Moreno, E.C., Fukae, M., & Shimizu, M. random coil structure. An important observation, relevant (1990) Calcif. Tissue Int. 46, 205-215 to the functional significance of amelogenin, is that the 11. Maezawa, S. & Takagi, T. (1983) J. Chromatogr. 280, 124-130 corresponding region affects markedly the adsorption 12. Takagi, T. (1990) J. Chromatogr. 506, 409-416 properties of the whole protein. On the other hand, the 13. Renugopalakrishnan, V., Strawich, E.S., Horowitz, P.M., & assumed /?- sheet conformation in the N-terminal region Glimcher, M.J. (1986) Biochemistry 25, 4879-4887 14. Zheng, S., Tu, A.T., Renugopalakrishnan, V., Strawich, E.S., & may mediate the intermolecular association of the cleaved Glimcher, M.J. (1987) Biopolymers 26, 1809-1813 fragments or the amelogenin proteins as commonly found 15. Goto, Y., Calciano, L.J., & Fink, A.L. (1990) Proc. Natl. Acad. at neutral pH. Further investigation using LALLS is Sci. USA 87, 573-577 needed to elucidate the aggregation process of the ameloge- 16. Aoba, T., Kawano, K., & Moreno, E.C. (1990) J. Biol. Buccale nins in physiological media. 18, 189-194 17. Brodsky-Doyle, B., Leonard, K.R., & Reid, K.M.B. (1976) Biochem. J. 159, 279-286 REFERENCES 18. Hayashi, T., Curran-Patel, S., &Prockop, D.J. (1979) Biochemis try 18, 4182-4187 1. Termine, J.D., Belcourt, A.B., Christner, P.J., Conn, K.M., & 19. Akita, H., Fukae, M., Shimoda, S., & Aoba, T. (1992) Arch. Oral Nylen, M.V. (1980) J. Biol. Chem. 255, 9760-9768 Biol. 37, 953-962 2. Warshawsky, H. (1984) in The Chemistry and Biology of 20. Strawich, E.S., Poon, P.H., Renugopalakrishnan, V., & Glimcher, Mineralized Tissues (Butler, W.T., ed.) pp. 33-45, Ebsco Media, M.J. (1985) FEBS Lett. 184, 188-192 — 73 — sjh m 1. emmom# 1)2)W#ES 3 . K <%)#%= ^ W&&T i / m 2) $g#B^%T ;yl NH2-CH-C00H NH2-CH CH-NH2 , H2N-C-COOH 3) HelixB^7 ^VE | KcHtA R V R R:bipy ■ y’-f- K^oa&A »cflKt©Mh K^xdSA 2) ^|$S & A) ^ V 7 ^ ' B) 4-1—'>* 'sm C) 5StSz£ . D) j<'s K V v KCDjlM ODistance Geometry (Cj; O<2-helix content©Wiffi V nmmmyjzm —74 6-2 -3V7 * a 5 7 $ y® » ---- e-0 Fig. 19 J. Ramachandran plots illustrating the sterically allowed regions for 1231 Ramachanndran map1* 1 (o) (t) (Zt*) (2) m2 (zft) (1) (2) 6-2-1 succinyl-Tyr-Ser-Glu 4-Lys <-(Xaa,)-Glu4-Lys 4-NIIi KOXaa,^(C##g#%T < 'J -y ? * Ala>Leu>lle>Yal K o tz" 0 £S>{CN yfi$CD^AK ^2 J: 9 Nle.Nva, Abu i; ,y 7 XZ> <0 (c£J L C £ & 3 &®TleT liGlyi^Alc a ^ v 7 y SISic -5 C -S 0 o ' — 75 — SflLl Peptide “(0)222 /.% A AG, keal/mol Ala Abu Nva Nle Ala, 27,300 ± 800 85 ± 3 -0.74 ± 0.07 1 1 Abu, 27,000 ± 900 84 ± 4 -0.70 ± 0.08 ch2 ch2 ch2 ch3 Nvaj 27,520 ± 800 86 ± 3 -0.76 ± 0.07 ch ch3 2 ch2 NIc, 27,370 ± 600 85 ± 2 -0.74 ± 0.06 ch3 ?h2 Leuj 24,100 ± 700 75 ± 3 -0.55 ± 0.04 ch3 He, 17,800 * 500 56 ± 2 -0.32 ± 0.02 Val, 16,000 ± 450 50 ± 2 -0.27 ± 0.02 SfiLll Tic, 4,800 ± 300 15 ± 4 0.22 ± 0.04 Ala Abu Val Tie -[5)222. Mean residue cllipticity (dcgrec-cm 2/dmol) of peptides at 1 I 222 nm./ » [6]obl - [0)o/[5) m»x - [5)0 - the fraction of helix. [6]oh ,, ch3 ch2 ch3-ch ch3-c-ch3 [0] observed from a previous column; [6]0 m 0 ± 500 degree-cm 2/ ch3 ch3 ch3 dmol, obtained by titrating a scries of peptides with the denaturing solvent guanidine hydrochloride (23); [6]mix — (/1 - 4/n)[6)« - the * maximal mean residue cllipticity value for chain length where n = the SaLlil number of residues and [6]„ ** —40,000 degree cmVdmol (24). AAG NIq Leu lie Tie ■ free energy for helix formation of each guest amino acid related to 1 1 glycine. AAGX ■ AG, - AGciy, where AGciy “ 0.31 keal/mol (12). ch2 ch2 ch3-ch ch3-c-ch3 ch2 ch3-ch ch2 ch3 ch3 ch2 ch3 o K 6 — 2 — 2 ax>9 0*0^ A Ta, yS^^fDT $ ttc. histidine a mmonia lyase tZ £ © 7 y 7 %K fcgSa6 £, tlX V Z 0 C tl b {i5fcl&7 y'< 7 ® OgjEjfclftggffclc •U 5 0 £. ^Et3&©Z${cfiAPhe,ALeu, AAla, APro©4 o»bftT&9 s C P Og£gli 7 $ /I tefcfLT cis {&&#&„ 1 7^7*2© ^ - y#%=# (i+1) (i+2)&g0]-±-(c < «, A Phe^W* V =f"<7'f KTtis £.* - y ©NII(i) t C0(i + 3) t *$[□]&$&!.' C t&t> *> L *> t&%£' 9/M < x ftS&©APhe££frJ£$tt&U-*7p^ Kt V y ^ {is 7yffiiI£JR9^'C — 76 6-2-3 a , a-2E&T 5 /MO^A D-a-T5/66® (<6X A 1 KT(i^##g%ty(±^E#^0 a 60 . • 120 ^ v -y / c 6^tobnrvs0 E) 5 pBrBz-(Aib-L-Ala) 8«r «-0Me-4. r2H20O5}ir[*l 7K^fa a 6 ^ ORamachandran Plot111 77 Scheme I Table I. [0]222nm for Peptides 2-5 (15-30 mM) in Water (0.1% TFA) at 0 and 60 °Ce 0 e *-[t?]222, mean residue cllipticity (dcgrcc-cnV/dmol) of peptides at 222 nm. / = [fl]^ - [5]0/([5]„ux “ [fllo) = the fraction of helix. [0]oW, [0] observed from a previous column. (0]o = 0 ± 500 deg- cm2/dmol.: [0]m„ = ((/i - 4)/n)[0]. - the maximal mean residue cl- liplicity" value for chain length where n = the number of residues and [0]„ = -40000 dcg-cm 2/dmol.17 me i/i+7 TbIO S-S gsigicza .a helix Schultz l+7#gOT C f- KTteD, LON7Fmoc-S-(acetamidomethyl)-2-amino-6-mercaptohexanoic acid (El 6 4*1) V) El 6 *2 ~ 5 (Aon) ffr-y'cDffltitfck K) vJX;l/7 j K7*V v tl/cQ .Ctlb0^7*f KliV'-fftks 6 0° Clc&VT&lSV-'x V % =) y T y b^fjk LtZo 5W$VlfTlcx L.DftRtfL, LftThlA £/£$ -fro ' 78 Table Helix content (pH 7.0, 1*C) degreeW-dmol- 1 Helix dipole Peptide 0.01 M NaCI .. 1.0 M NaCI interaction 0>4)E,K 29,000 24,800 + (z+4)K,E 25,300 25,700 — (i+3)E,K 17,600 • 17,400 • + 0>3)K,E 8,500 12,000 — 220 . Fig. CD spectra of peptide (/+4)E,K (17 phi) at four temper atures in 0.01 M KF (pH 7.0). deg, Degree. V>4) E.K: Ac A E A A A K E A A A K E A A A K A HHj (1+4) K,E: AcAKAAAEKAAAEKAAAEA NH, + ■ - + * + - * (1+3) E,K: AcAEAAKAEAAKAEAAK anh 2 (1+3) K,E: AcAKAAEAKAAEAKAAE ANH, +■ * + • + - ^ {Upper) Sequences of the four peptides designed. Ac, acetyl; A, .alanine; E, glutamic acid; K, lysine. {Lower) Diagram illustrates the potential helix-anchoring effect caused by salt bridges or ion pairs for the z+4 peptides. Note that the charged groups spiral around the surface of the helix. • m7 GUi-LyswcDmnwmz&z«^ v * ? — 79 — AcWH Table Summary of Changes in -(0)an on Addition of Metal Ions to Peptides la-d, 2a-d, 3, and 4c change in -[#]322 with additive,1 % -iV ■ peptide deg cmydmol Co'+ Ni,+ Cuu ZnJ* Cd1+ la 20 000 . -45 -57 -17 -41 -26 lb 14 200 -5 -14. 50 -1 4 AcNII Ic 15 500 31 -5 •0 3 -22 £ Id , 13 800 26 -43 ■ 48 -8 -16 2a 18600 -30 -48 -34 -34 50 Eq.2 2b 18 300 50 35 32 28 II 2c 18400 26 -7 56 22 43 sp- 2d 17800 33 23 18 23 24 3 17 500 -2 -5 0 -3 -8 HjNOC,rr-'y ' 4c 19300 -10 -10 -20 -15 -13 'Peptide, 6-14 pM, pH 7.9 (200 mM aqueous sodium borate), 25 L » Llpnd *C. ‘Metal ion, 200>tM. M » Mclil Ion Peptides 1 and 2 were designed to probe the structural optimum for helix stabilization by simultaneous chelation of two amino- diacetic acid bearing side chains. • eo- 1 a-d: Ac-XAIa 2X(Ala4GluLys)3-NH2 2a-d: Ac-XAlajXfAla^GIuLysJj-NHj 3: Ac-Ala 3(AlauGluLys)3-NH2 4c: Ac-AlaLtX(AJa4GluLys)3-NH2 5a: Ac-XAIaaXAIa^GluLys-NHz H /(CH2)nN(CH2C02H)2 X - 1' Wavelength (nm) Figure CD spectra of (A) peptide 2* (12 yM) in the absence of metal ions (II) and in the presence of CdJf (III) or NiJ+ (I) (200 yiM in metals) a "n- 1 c n-3 at 25 *C and (B) peptide 5a (36 #iM) in the absence of metals at 4 and 25 eC (I) and in the presence of Cd1* (200 mM) at 25 (II) and 4 °C b n-2 d n-4 (HI) El9 y ? K lc £ £ a ^ V v ^ o 'sYiW.r '6 N-backbone to aide chain a N to N-backbone i C to C-backbona t C-backbona to aid* chain £ N-backbona to end r\ * -R-S-S-R- , -R-CO-NH-R- , -R-CHj-S-R- , etc ^ * ‘R-S-S-R* 9 -R-CO-HH-R- , *R-CHj*S-R* » ®tc Figure The concept of N-backbone cyclization. Figure The concept of C-backbone cyclization. ■.Ell 0 N a -backbone cycl izationi C a-backbone cyclization Id £ * a «) — 80 — ill* C cmm#] 1 ) lils&iz ^7°f K\ LTHfcHSSWfc&©fiN X WttBiMWfe t AUtE—fet£ 7^7 h ^ (CD) t^5)o Ctlb©^&7 %'V%(:£l"<^ @fz#7y4; v hK%o^#^v\_ h\ y 7 y j^Tfc N M R a „ 2) NMRgg#g#tlT& (NMR) &3WLf:g8mo#ie#)e®mE(i, 1 9 6 0^ft(C%(c%a6>,tT,TU NMR^mem9f^oi5^Rf^%^o^LTi^o NMR X 1 (ca t&tZo m-tpo&jg 1?® finite & t D £ < ttdFicJ: Do < £ ua6, X b-y^A^C = J (# ) =1. 9-1. 4cos (#-60) + 6. 4cos (#-60) NO E#x ^fO&6 7'D h 7 ^0^0 h y 57"D h yo#tP0%jR^^^3^-e^ f (T) NO E = K ------ — 81 mi NMR A' 6 ®X - f k »\«* & # ^2 \ b m m m # \ (r?;s, xf K) ae #^B'l'S8*®pK® it ;gdr«i3t **«£, s-s*g£®#as # »**8IHI®ffl*N®» '> S^-X tf>$® 7 s£^#BMK:.fc5irsgs®s5 b SpKffi ('N'Jyn-a-f (7 *islk EEElig) ft @^f®m^a;ekf®#E®#m (s*. Saift-f^X SS5J?*;i/) XX6 tftfiac @^f#m @$Lfc»^±S®IS^A v' < i Sft©«£*l o ft $6A^'ilSl®8IS®^* a ft *$ - S*SE#i®il$^E^ ft ffiSLfc@$Sffl®ffl58-i$E (NOEZO) STi ME®$HtBie ^f±*®#m. 0®si£Sc (S#6SS£tlCt)-t$f) S^-®^®SS@@ (*M, 'f3h» WfT,/) 5 S^gESSi ®E« iTv_^ iD x’n bXb %##» JtXtff— — 82 — K tenure* tK f (r ) r 0^0^^ r (i7°P h >H»3©ffl>tfffi8li’C h K> s h y*f ($b5 AJ2AM) i^o^tii^ti^o E2 2^^NMR#m&0^#6^Oj5m^^L/Co C$X« o.( '-1 Pseudo atom g^k-BA©® 2 %%^NMR©^#^f©j&@ ^ # WWW IS M # & & 2 D J(-resolved) J<9E>£ H-C 2DJ 2D dipole ffl M & 'H$/7 bttM COSY DQF-COSY (as) lK-'9 CS/y bttM H-C COSY HMBC • Xt^ CHE^) HMQC MSOT'>7 bffiM OHE £) NOEffiK NOES Y OT&St #Xtf>3c& V RELAY bfflgg HOHAHA h7-£<9S 2 83 — 3) N M R 0 #1# ## m2 K N M R (C j; a ]%#:#)### 2 ^NMR©CO SYiNOESY OES Yii9l^0^DX b°- ? (Dfcm £ D 7XS^F b1 © EEUflfg^li £ ft 3 o (1213 N 4) ! ai, itr «u>$ 6 ecias w I rgTB NMRf“9 pns| &*»NKR/DGE (ffiSjsa) :&5cNMR(DaO) rAicNMR(HaO) DQF-C0SY, RELAY, NOESY DQl'-C0SY(7fy»"-7'!l>hW (thf W#W*&@ mBSEoxry&e . n/mmmutt'ym 7'mcsnEe @«(7»AAE.S«E) M&mr- mm) f-hf &m) mmnkmm '&*mx~mvriz. NOESY(da'N,dNN,da #) XY'y^aS^V-Yayi - N0ESY(N0E)t"->6 m&mmr-tn ■ ttE XGASVC------INLKH~ 1 AAQ/O'-vOW mmmmwmmm -?y'f (MolSkopS'YYA) I mm»%m rmnvim- 10h~20h/S,$zL-2'3Z OW-W) DADASE 7-n"-3>fz->$:EM DISHAN.FEDER.tzYMd (a"7?@S) (10~100aY5zk->3» / t FimHv't' i-l x ^fisas @2 NMR$EStfrEK*5g6SSOTI®iBtff#l« — 84 — NMR/D Gmvm&kftgilliX RD;Restrained Molecular Dynamics * nmr /d g (rmn-/am-) 1976 2-Dimension NMR(COSY) 1977 Crippen J.Comp.Phys. 24, 96 Embedding 2DJ(-resolved) 1978 Crippen, Havel Acta Crystallogr. A34, 282 Metric matrix H-C COSY 1979 SECSY 1980 NOESY 1981 Crippen et al. Int.J.Pep.Pro.Res. 17, 156 Bleomycin (7+2) Braun, Go, & B.B.A. 667, 377 Glucagon (29) Wlithrich et al. 1982 BPTI sequential assignment 1983 Braun et al. J.Mol.Biol. 169, 921 Glucagon refinement Pure phase spectra Wlithrich, J.Mol.Biol. 169, 949 Pseudo atom DQF-COSY Billeter & Braun TOCSY Noguchi, Go J.Phy.Soc.Japan 52, 3685 Derivative in dihearal angle (D.A.) 1984 Havel, Wlithrich Bull.Math.Biol. 46, 673 DISGEO (Pascal language) ROESY Abe, Braun, Go Comp.Chem. 8, 239 ’DISMAN’ algorithm Secondary structure et al. from J & NOE 1985 Kaptein, van J.Mol.Biol. 182,179 Model + RD, Lac repressor HOHAHA Gunsteren et al. head (51) E-COSY Havel, Wlithrich DISGEO, BPTI(58) simulation Williamson, Havel, J.Mol.Biol. 182,295 DISGEO, BUSI HA (57) & Wlithrich Braun, Go J.Mol.Biol. 186,611 DISGEO, BPTI simulation 1986 Kline, Braun J.Mol.Biol. 189,377 DISMAN, Tendamistat (74) & Wlithrich C=t>X-ray diffractioin «=>R. Huber, et al., J.Mol.Biol. 189,377) method Brunger, Karplus, P.N.A.S. 83, 3801 RD, Crambin simulation (46) & Gronenborn Clore, Karplus, EMBO J. 5, 2729 DISGEO + RD, Purothionin & Gronenborn, et al. (45) 1987 Wagner, Braun, J.M.B. 196, 611 DISGEO/DISMAN, BPTI(58) PE-COSY Havel, Go, & Wlithrich et al. Kraulis, Jones Proteins 2, 188 NOEDIA (Ada), Crambin simulation Wako, Go J.Comp.Chem. 8, 625 Vectorized derivative in D.A. 1988 Kline, Braun, J.Mol.Biol. 204, 675 DISMAN, Tendamistat 3-Dimension NMR & Wlithrich refinement Nilges, Gronen- Protein Engineer. 2, 27 Simulated Annealing, born et al. Crambin etc. Moore, Havel, Science 240, 314 DISGEO + RD, Plastocyanin & Wright et al. (97) 1989 Wlithrich Science 243, 45 Protein Structure Determi- nation by NMR/DG — 85 — : CH3 CH3; \ // P C' H; 0 NOESY <02 (ppm) NC m 3 BPTI#NOESYf-f (500MHz) t? nx fcT-* £ 9 El 4- BPTI(Bovine Pancreatic Trypsin Inhibitor > TVl&^ljSl&SS) Wc NMR/DG&CD'>Ul/-'>3 — 86 — ItJKffVx 3r*#$^g6S, #m6tT,6 2K7cNMR?'-f ®no e s y »> 5, ©#$©7j<$e^ isigufiii ©COS Y»>6©X h-yS6rSI6»>6»f#6.tv3-ffie©lBHfllB ® H - D 3£&iS® »> £> f# £> ft -5 *$*S£tl8 W !.**> 3#bft5S-S #Am# ti Eifihho NM RSgliHlfff'ffiCW^i Lt, ©Jt«W®SraK*tt«®'»$*s'5Jfg ®#s©as^toiweT^Rj|g ibiSIf 6 ft 60 1 9 8 6 ^ K < #3: CXSISSIBtfrSjrNMR i£$tMVr&T- H—$ 6 K X a-7 i 5--fc?-f y t fyyu?, F (Tendamistat) ©SC^flliSPtfrAsisieSliCffbfts &$£ ft-ftHSliB 6/La-mLfCo (05) (a)NMRJ: 0$Ateig$-t>O)*iS [A.D. Kline et al„ ]. Mol. Biol., 189, 380, Fig. 3 (1986)] (blxig-t ') [J.W.Pflugrath et al.J.Mol. BioL.W, 385, Fig.l (1986)] 05 NMRJlE)gtff$kXE^SSyf$ NMRiggHWtfrffi ©H@;5.t It, ®*3ftftTT©aj3«t£«jgtt*©bftsa>\ *ft;6S£"ftli;fjEE6^©*iJ$X»6iaS©@r«SLT VD ^)NO (D53‘:7'S^zk§ ( 5 {c-oti fcf — y 7 ©5}pEJW$T^ffll|{£(^"Fll l TjUll! ;£ t) — 87 — CHEMISTRY LETTERS, pp. 1873 1876, 1993. © 1993 The Chemical Society of Japan Total Synthesis of Aureobasidin A, an Antifungal Cyclic Depsipeptide Toru KUROME, Kaoru INAMT,f Tetsuya INOUE, Katsushige IKAI, Kazutoh TAKES AKO,* Ikunoshin KATO, and Tetsuo SHIBA*t t Pep tide Institute, Protein Research Foundation, 4-1-2 Ina, Minoh, Osaka 562 Biotechnology Research Laboratories, Takara Shuzo Co., Ltd., 3-4-1 Seta, Otsu, Shiga 520-21 A total synthesis of an antifungal cyclic depsipeptide aureobasidin A was first achieved mainly using PyBroP 1) as a coupling reagent. The synthetic cyclized product was completely identical with the natural antibiotic in all respects. Some unexpected reactions due to A-methylamino acid were also described. A new cyclic depsipeptide aureobasidin A (1), isolated as a major component from the culture medium of the black yeast Aureobasidium pullulans R106, exhibits a strong antifungal activity against pathogenic fungi such as Candida albicans, Cryptococcus neoformans, and some species of Aspergillus with a low toxicity. 2) A whole structure of the peptide was determined mainly by the HMBC technique and chemical degradation. 3) The structures of more than twenty congeners of aureobasidin were determined in comparison with aureobasidin A.4) MePhe MeVal D-Hmp' HOMeVal MeVal a lie Aureobasidin A (1) For purpose of investigation of a structure-activity relationship of the aureobasidin family antibiotic, we attempted a total synthesis of aureobasidin A aiming an establishment of a synthetic technique of the cyclic depsipeptide containing A-methyl amino acids, which is known to be difficult to condense by usual peptide coupling methods. The linkage between alle1 and Pro 9 was chosen as a site of the final cyclization to avoid the coupling at an A-methyl amino acid as an amine component, which otherwise may diminish a yield of the cyclization reaction 88 — Chemistry Letters, 19 9 3 due to its steric characters. A linear nonapeptide(l-9) was synthesized according to the Boc strategy. Thus, a coupling between Leu3-HOMeVal4-Hmp 5 (Segment B) and MeVal6-Phe7-MePhe8 -Pro 9 (Segment C) was first attempted, followed by condensation of the coupling product (3-9) with alle^MeVal2 (Segment A) by the fragment condensation as shown below. Segment C was prepared by a stepwise elongation method starting from Boc-L-Pro-OPac (11) as a carboxyl terminus by the successive condensation using PyBroP 5) and DIEA in dichloromethane.6) Segment A Boc-L-allc-OH PyBroP, DIEA Zn HC1»H- L-MeVal-OPac Boc-L-alle-L-MeVal-OPac Boc-L-dzIle-L-MeVal-OH AcOH 2 (41%) 3 4 (67%) Segment B 1) BSTFA 1) HC1 (96%) H-DL-HOMeVal-OH ~~—Boc-DL-HOMeVal-OBzl ------—Boc-L-Leu-DL-HOMeVal-OBzl 2) B0C2O 2)Boc-L-Lcu-OH 3) BzlBr, Cs2C03 PyBroP, DIEA (94%) (65%) H? / Pd H-D-Hmp-OPac ^ —------Boc-L-Leu-DL-HOMeVal-OH------— —-► Boc-L-Leu-DL-HOMeVal-D-Hmp-OPac (100%) DCC, <>n2] (79%) CP Boc-L-Leu-L-HOMeVal-D-Hmp-OPac --- ——► Boc-L-Leu-L-HOMeVal-D-Hmp-OH on silica gel AcOH 9L (74%) 10 Segment C 1) HCl (100%) 1) HC1 (100%) Boc-L-Pro-OPac ------► Boc-L-MePhe-L-Pro-OPac ------2) Boc-L-McPhe-OH \ 2 2)Boc-L-Phe-OH 11 PyBroP, DIEA (83%) PyBroP, DIEA (86 %) 1) HCl (92%) Boc-L-Phe-L-MePhe-L-Pro-OPac ------► B oc- L-MeVal-L-Phe-L-MePhe-L-Pro-OPac 2)Boc-L-McVal-OH 13 PyBroP, DIEA 14 (86 %) HCl HCl *H-L-MeVal-L-Phe- L-MePhe-L-Pro-OPac (93%) 15 DL-(3-Hydroxymethylvaline (HOMeVal) in Segment B was prepared according to the method by Izumiya et al7) Although HOMeVal could not be protected with the Boc group by the usual manner, the protection could be achieved after activation and solubilization by trimethylsilylation with BSTFA. The introduction of the benzyl group to its carboxyl function and the removal of the Boc group were carried out, followed by coupling with Boc-L-Leu-OH using PyBroP without protection of (3-OH group in HOMeVal to afford 7 in a moderate yield. After deprotection of Bzl group, a mixture of the diastereomers of the product 8 was coupled with H-D-Hmp- OPac with DCC and 4-pyrrolidinopyridine as an acylation catalyst. In this condensation reaction, DCC was — 89 Chemistry Letters, 19 9 3 superior to PyBroP in the yield. Both coupling reagents without the base catalyst did not afford the desired product owing to the formation of the oxazolinium compound of Boc-Leu-HOMeVal as a main intermediate.8 ) The diastereomers of the product 9 were separated each other by silica gel column chromatography, 9 ) and the L-L- D compound (9L) was deprotected with zinc/acetic acid to give, 10 (Segment B). The coupling of 10, whose C- terminus is a hydroxy acid, Hmp, with 15 was carried out using PyBroP to avoid a racemization. After removal of the Boc group in the peptide 16 with TFA , a fragment condensation with 4 (Segment A) by the WSCD- HOObt method was carried out to give the protected linear nonapeptide 17 with a slight racemization as shown in the scheme, although the same condensation by the PyBroP method gave a 1:1 mixture of the completely racemized diastereomers. COOH PyBroP DIEA COOPac Segment C COOPac Segment A Segment B HOOC 1) TFA 2) HCl/dioxane WSCD-HOObt COOPac 17 1) Zn / AcOH PyBroP ------► Aureobasidin A (1) 2) TFA DIEA 3) HCl/dioxane in CH2C12 (91%) (45%) 18 The protecting groups of both carboxyl and amino groups in the linear nonapeptide 17, which was purified diastereomerically, were removed successively, and then the free peptide 18 was cyclized with PyBroP in CH2CI2 under a high-dilution condition (10-3 M) to afford predominantly the cyclic monomeric peptide (PD- MS: Found, M+ 1100; Calcd for cyclic monomer, 1100). The cyclization of AZ-hydroxysuccinimide ester of the nonapeptide at the same concentration in CH2CI2 as in that of PyBroP reaction gave a cyclic dimer as the major product. In case of PyBroP, the reaction seemed to proceed promptly compared to that in the active ester. There- — 90 Chemistry Letters, 19 9 3 Table 1. Minimum Inhibitory Concentrations (pg/ml) of Natural and Synthetic Aureobasidin A Natural Synthetic Candida albicans TIMM 0136 <0.0125 <0.0125 Candida albicans TIMM 0171 <0.0125 <0.0125 Candida kefyr TIMM 0301 0.4 0.4 Candida glabrata TIMM 1062 0.1 0.2 Cryptococcus neoformans TIMM 0354 0.8 0.8 Saccharomyces cerevisiae ATCC 9763 0.2 0.2 Aspergillus fumigatus F48 >25 >25 fore, a condensation in a high-dilution condition was actually achieved preventing the dimerization in that case. On the other hand, the cyclization of the active ester in DMF proceeded very slowly even though the cyclic monomer was obtained as a major product besides the dimer. Such difference depending on the solvent seemed to arise from the solvation feature of the peptide molecule in DMF which may be advantageous to the monomeric cyclization. The synthetic cyclic monomer thus obtained is completely identical with the natural antibiotic in all respects (TLC, HPLC, ^H-NMR, and antifungal activities). Thus we now achieved the first total synthesis of aureobasidin A with the unique pattern of the antifungal activity. References 1) Abbreviations: PyBroP: bromotris(pyrrolidino)phosphonium hexafluorophosphate; HMBC: heteronuclear multiple-bond connectivity; Boc: r-butoxycarbonyl; Pac: phenacyl; DIE A: diisopropylethylamine; BSTFA: N, O-bistrimethylsilyltrifluoroacetamide; DCC: dicyclohexylcarbodiimide; Hmp: (2R)-hydroxy-(3R)~ methylpentanoic acid; WSCD: water-soluble carbodiimide [A-ethyl-/V'-(dimethylaminopropyl)carbodiimide (EDC)]; HOObt: 3-hydroxy-4-oxo-3,4-dihydro- 1,2,3-benzotriazine; PD-MS: plasma-desorption mass spectroscopy; HOBt: 1-hydroxybenzotriazole; lM=lmol dm* 3. 2) K. Takesako, K. Ikai, F. Haruna, M. Endo, K. Shimanaka, E. Sono, T. Nakamura, I. Kato, and H. Yamaguchi, J. Antibiot., 44, 919 (1991). 3) K. Ikai, K. Takesako, K. Shiomi, M. Moriguchi, Y. Umeda, J. Yamamoto, I. Kato, and H. Naganawa, J. Antibiot., 44, 925 (1991). 4) K. Ikai, K. Shiomi, K. Takesako, S. Mizutani, J. Yamamoto, Y. Ogawa, and I. Kato, J. Antibiot., 44, 1187 (1991); K. Ikai, K. Shiomi, K. Takesako, and I. Kato, ibid., 44, 1199 (1991). 5) J. Coste, E. Frerot, P. Jouin, and B. Castro, Tetrahedron Lett., 32, 1967 (1991). 6) The coupling reaction of Boc-L-MePhe-OH with HCl*H-L-Pro-OPac by the WSCD-HOBt method gave the product in a yield of 87%, accompanying L-D isomer (A ratio of L-L and L-D isomers was 10 : 3). This racemization was due to the intramolecular formation of the charged Schiff base in Pro residue between the imino group and the carbonyl function of the Pac ester. (H. Kuroda, S. Kubo, N. Chino, T. Kimura, and S. Sakakibara, Int. J. Peptide Protein Res., 40, 114 (1992). The reaction with PyBroP afforded the desired product without racemization. 7) N. Izumiya and A. Nagamatsu, J. Chem. Soc. Jpn., 72, 336 (1951). 8) N. L. Benoiton and F. M. F. Chen, Can. J. Chem., 59, 384 (1981). This oxazolinium-forming phenomenon was recently reported in case of A-MeVal and some N-methylamino acids by B. Castro et al. independently. (E. Frerot, P. Jouin, J. Coste, and B. Castro, Tetrahedron Lett., 33, 2815 (1992). 9) These diastereomeric peptides (L-L-D or L-D-D) after separation were hydrolyzed with 6N HC1 for 19 h to give respectively L-HOMeVal and D-HOMeVal, which were assigned in comparison with the authentic amino acids on Daicel Chiralpak WH. 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MLTTttjBS^L, 7)cE±ktiM^oA: IIE£$xA:i#£-c7Kx9 .y^x W^ttl-WLTIi. ^7"^- FSrT/i^A'E Tt£tf|itSW6\ ^eoEfttcioT.-AdllS A:, - LtlfhET^Tfci), mjxli\ t17h 1\'/ — 98 — CHEMISTRY LETTERS, pp. 1305 1308, 1993. © 1993 The Chemical Society of Japan Synthesis of 2'(3')-0-Aminoacyl-pdCpA Carrying a Photofunctional Nonnatural Amino Acid Takaaki NIINOMI and Masahiko SISIDO* + Research Laboratory of Resources Utilization, Tokyo Institute of Technology 4259 Nagatsuta, Midori-ku, Yokohama 227 A mixed dinucleotide pdCpA, was synthesized and characterized by spectroscopic as well as enzymic analysis. The dinucleotide was acylated with an A-protected nonnatural amino acid (Boc-azoAla). The acylation was found to occur at the 2'(3')-0 position exclusively. Transfer RNA misacylated with a noncognate amino acid is known to work in protein biosynthetic system and insert the amino acid into a specific site of a protein. 0 Therefore, if one can acylate a tRNA with a nonnatural amino acid, one will obtain an unusual protein in which the nonnatural amino acid is incorporated at the site that should have been occupied by the amino acid cognate to the tRNA. Since direct ami noacylation of tRNA at the 3 '-O position of the terminal A unit seems intractable, an alternative pathway using enzymic ligation of 3'-0- aminoacylated pCpA with a truncated tRNA missing a terminal pCpA unit, has been proposed. 2-7) The semisynthetic tRNA was actually shown to work for the site-specific incorporation of nonnatural amino acids, by Brunner,5) Schultz,6) and Chamberlin.^) Chamberlin and coworkers^) showed that the C unit of pCpA can be replaced by a deoxy C (dC) unit. Although this markedly simplified the synthesis of the dinucleotide-amino acid conjugate, synthesis of the 2'(3')-0-aminoacyl-pdCpA is still a key step in the entire process. In this communication we describe a synthetic route that is much simpler than earlier,3-7) to an aminoacyl-pdCpA carrying a nonnatural amino acid, L-p-phenylazophenylalanine, i.e., 5'-0-phosphoryl-2'-deoxycytidylyl(3'-5')-[2 ,(3')-0-L-phenylazophenylalanyladenosine] = pdCpA-azoAla. The azobenzene units in proteins will photocontrol protein functions. The synthetic route for pdCpA-azoAla is shown in Scheme 1. As starting materials, a commercially available deoxycytidine derivative, 4-A-benzoyl-5'-<9-(4,4'-dimethoxytrityl)-2'- deoxycytidylyl-3'[(2-cyanoethyl)-(A,A-diisopropyl)]phosphoramidite 1 (American Bionetics, Inc.) was used as a dC component and 6-N,T-0, 3'-0-tribenzoyladenosine^) 2 was employed as an A component. The coupling of the dC component (480 mg) with the A component (277 mg) was carried out in a usual manner.^) The product was fractionated with a silica gel column [MeOHZdichloromethane(DM)] in 61% yield. The S-0 protecting group of the dinucleotide was removed with 5% trichloroacetic acid in DM. The crude product was ^ Present addess: Department of Bioengineering Science, Faculty of Engineering, Okayama University, 3-1-1 Tsushima-Naka, Okayama 700. 99 — 1306 Chemistry Letters, 19 9 3 o O — P—o—ch2 C ii h 3c^n ^ch 3 O—P—O—GHg “ kjy OH kp 1 o "o-p=o 1) Thiophenol ? Boc-NHCH(CHqR)COOCH2CN r ^_CH A 2) NH3 '0-P=0 , & o—ch2 .66, I H HO OH c=o I I hc-ch2r r=-CH- q HC —CH2R NHg NHI Boc Scheme 1. Synthetic route to pdCpA-azoAla-Boc (6) and structure of puromycin analog (7). fractionated with a silica gel column (MeOH/DM) in 86% yield. Phosphorylation of the free 5'-0 position was achieved with cyanoethyl(diisopropylamino)methoxyphosphi ne.9, 10) The crude product was fractionated with a silica gel column (MeOH/DM) in 80% yield. 31p NMR showed 4 large peaks that are interpreted by a predominance of two diastereomers among RR, RS, SR, and SS isomers of the two chiral centers on the phosphorus atoms. The fully protected dinucleotide was deprotected in two steps. The methyl ester at the 5' terminal was removed with thiophenol and the benzoyl group at the nucleic bases and the cyanoethyl esters were deprotected with aqueous ammonia. The product was fractionated with reversed phase HPLC (ODS column, eluent = acetonitrile/0.1 M ammonium acetate, pH 7.0, linear gradient with increasing acetonitrile from 0% to 50% in 50 min). The chromatogram showed three peaks. The first fraction (15%) showed the same NMR spectrum as pdCpA reported by Schultz and coworkers.6) The third fraction (70%) was assigned to pdCpA with cyanoethyl ester remained at the 5' phosphate. When this fraction was further treated with aqueous ammonia for 24 h, the product was identical to the first fraction. The 31p NMR spectrum of the first fraction showed two equivalent peaks, indicating the absence of chirality on the phosphorus atoms. The assignment of the first fraction to free pdCpA was further supported by enzymic digestion using snake venom phosphodiesterase or nuclease PI. An HPLC analysis of the products of the enzymic reaction indicated an equimolar amount of — 100 Chemistry Letters, 19 9 3 in trifluoroacetic acid at 0 °C for 30 min, and the acid was purged off by a stream of nitrogen. The residue was washed with ether and dried under vacuum. HPLC analysis showed complete removal of the Boc group. The pdCpA-azoAla could be stored for several weeks at -20 °C. In conclusion, pdCpA aminoacylated with a nonnatural amino acid at the 2'(3')-0 position was synthesized from commercially available intermediates. The pdCpA-amino acid will be used to incorporate the nonnatural amino acid into proteins at any specific sites. The authors appreciate professors T. Hata and M. Sekine of Tokyo Institute of Techno logy for their suggestions on the synthesis and enzymic analysis of pdCpA. We also wish to thank professor S. Yokoyama of The University of Tokyo for suggesting puromycin analogs. References 1) F. Chapeville, F. Lipmann, G. von Ehrenstein, B. Weisblum, W.J. Ray, and S. Benzer, Proc. Natl. Acad. Sci. U.S.A., 48, 1086 (1962). 2) S.S. Jones, B. Rayner, C.B. Reese, A. Ubasawa, and M. Ubasawa, Tetrahedron, 36, 3075 (1980). 3) T.G. Heckler, L.H. Chang, Y.Zama, T. Naka, and S.M. Hecht,Tetrahedron, 40, 87 (1984); T.G. Heckler, Y. Zama, T. Naka, and S.M. Hecht, J. Biol. Chem., 258, 4492 (1983). 4) H. Hotoda, R. Saito, M. Sekine, and T. Hata, Tetrahedron, 46, 1181 (1990);. Y. Ueno, T. Mishima, H. Hotoda, and T. Hata, Chem. Lett., 1992, 595. 5) G. Baldini, B. Martoglio, A. Schachenmann, C. Zugliani, and J. Brunner, Biochemistry, 27, 7951 (1988). 6) CJ. Noren, S.J. Anthony-Cahill, M.C. Griffith, and P.G. Schultz, Science, 244, 182 (1989); S.A. Robertson, CJ. Noren, S.J. Anthony-Cahill, M.C. Griffith, and P.G. Schultz, J. Am. Chem. Soc., 113, 2722 (1991); D. Mendel, J.A. Ellmann, and P.G.Schultz, ibid. , 113, 2758 (1991); J.A. Ellman, D. Mendel, and P.G.Schultz, Science, 255, 197 (1992); D. Mendel, J.A. Ellman, Z. Chang, D.L. Veenstra, P.A. Kollman, and P.G. Schultz, ibid., 2 5 6, 1798(1992). 7) J.D. Bain, E.S. Diala, C.G. Glabe, T.A. Dix, and A.R. Chamberlin, J. Am. Chem. Soc., Ill, 8013 (1989); J.D. Bain, E.S. Diala, C.G. Glabe, D A. Wacher, M.H. Lyttle, T.A. Dix, and A.R. Chamberlin, Biochemistry, 30, 5411 (1991); J. D. Bain, D A. Wacker,E.E. Kuo, and A.R. Chamberlin, Tetrahedron, 47, 2389 (1991); J.D. Bain, D A. Wacker, E.E. Kuo, M.H. Lyttle, and A.R. Chamberlin, J. Org. Chem., 56, 4625 (1991). 8) M. Ikehara, F. Harada, and E. Ohtsuka, Chem. Pharm. Bull., 14, 1338 (1966). 9) M.J. Gait, "Oligonucleotide Synthesis, A Practical Approach, "IRE Press, Oxford, Washington, D.C., 1984. 10) (3-Cyanoethanol (2 g) was dissolved in DM (50 mL) at 0 °C. Diisopropylaminomethoxy- chlorophosphine (5 g) and N, Wdiisopropylethylamine (26.5 g) were mixed and stirred at 25 °C for 30 min and the precipitate was removed. The filtrate was diluted with ethyl acetate, washed with water, dried over Na2S04, and evaporated. Yield 3.9 g. 11) M. Taiji, S. Yokoyama, and T. Miyazawa, J. Biochem ., 98, 1447 (1985). 12) T. Hohsaka, M. Sisido, T. Takai, and S. Yokoyama, unpublished work. (Received May 6, 1993) — 101 — Chemistry Letters, 19 9 3 5-dCMP and 5’-AMP, indicating a 1:1 presence of dC and A unit. The total yield of the deprotection was 84%. pdCpA-azoAla-Boc Aminoacylationof pdCpA was carried out using an A-protected amino acid activated ester in DMF, according to Schultz et al.6) Free pdCpA (3x10-3 mmol) was azoAla-PAN converted to a pyridinium form using a Dowex 50 W column (pyridinium form, 3 mL, eluted with pyridine/water = 1/1,12 mL). The pyridinium salt was then converted to tetrabutylammonium form using a Dowex 50 W (tetrabutylammonium form, 3 mL, eluted with pyridine/water = 1/1, 12 mL). The eluted solution was evaporated and dried under vacuum. The latter was dissolved in anhydrous DMF (75 pL) containing triethylamine (6 pL) and Wavelength /nm mixed with 5 equivalents of r-butyloxycar bonyl L-/7-phenylazophenylalanine cyanome- Fig.l. CD spectra of 6 and 7 in 0.1 M thyl ester (Boc-azoAla-OCM) under argon ammonium acetate (pH 7). atmosphere. The mixture was stored at room temperature for 4 days and diluted with 50 mM ammonium acetate (pH 4.5)./acetonitrile=l/l (600 pL). The resultant mixture was subjected to preparative HPLC under the same condition as described above. The HPLC chart showed 6 large peaks. Reasonable absorption spectra were obtained only for two fractions that appeared at 22 min (fraction 1) and at 25 min (fraction 2). The two fractions may be assigned to pdCpA-azoAla-Boc's aminoacylated at 2'-0 and 3 '-0 position of the adenosine unit. This was confirmed by the interconversion of the two peaks. When fraction 1 was lyophilized and analyzed again with HPLC, the chromatogram showed two peaks with the same intensity ratio (1:4) as before. The same phenomenon was observed for the fraction 2. Similar interconversion has been reported on 2'(3')-0-phenylalanyladenosine with a preference of the 3'-0 derivative than the T-0 derivative. 11) Therefore, the fraction 1 may be the 2'-0 derivative and the fraction 2 may be the 3'-0 isomer. The combined yield of the two isomers was 69%. Both fractions showed trans/cis photoisomerization when they were irradiated with a 350 nm light (trans to cis) or with a 450 nm light (cis to trans), indicating the presence of azobenzene unit. pdCpA-azoAla-Boc showed a characteristic CD pattern at the azobenzene absorption band (Fig.l). The CD pattern is similar to that of a puromycin analog A-acylated with azoAla (7).12) The resemblance strongly supports that the ami noacylation is occurring at the 3-0 position. The A-protecting group (Boc) of pdCpA- azoAla-Boc could be removed by an acid treatment. The lyophilized substance was dissolved — 102 Reprinted from the Journal of the American Chemical Society, 1994, 116. Copyright © 1994 by the American Chemical Society and reprinted by permission of the copyright owner. Photoswitching of NAD+-Mediated Enzyme Reaction through Photoreversible Antigen-Antibody Reaction dark/Ab Takahiro Hohsaka, Kazuo Kawashima, and Masahiko Sisido* Research Laboratory of Resources Utilization uv/Ab Tokyo Institute of Technology 4259 Nagatsuta, Midori-ku, Yokohama 227, Japan Revised Manuscript ReceivedReceived SeptemberNovember 15,17, 1993 Switching of biological reactions by external signals, such as Time (min) light irradiation, has been attracting the interest of a number of researchers.1-5 On/off switching of enzymic reactions will have a large effect on the yield of final products because of the catalytic reactions of the enzymes. For this idea to be realized, however, it is crucial to attain absolute zero reactivity in the “off” state, since even minor reactivity in the “off” state will be amplified to cause significant total reactivity. In the past, chemical modification of inhibitors, cofactors, or I 0.95 enzymes to attach photochromic groups, such as azobenzene or spiropyran, has been reported.1-8 In these examples, one of the photoisomers shows higher activity than the other, resulting in the photocontrol of biological reactions. In most cases, however, the difference of the activities of the two photochromic states is not large enough, except for a few cases.8 To achieve on/off switching, one should design a system in which a large effect is Time (min) induced from a relatively small change in the molecular shape Figure 1. (a) Reduction of DCIP by the coupled enzyme system mediated of photochromic groups. Another factor that may suppress the with azoAla-NAD +: (—) in the dark without antibody; (—) after UV activity ratio in a system with azobenzene is that a pure cis state irradiation without antibody; (------) in the dark with antibody; (-~) cannot be attained under UV irradiation. Therefore, the system after UV irradiation with antibody, (b) A profile of on/off switching of should be “on” in the cis form and “off” in the trans form to the coupled enzymic reaction. achieve zero activity in the “off” state. In this communication, we report a general approach to important features of the azobenzene/antibody system is that switching biological reactions using an azobenzene group com the antibody binds and inactivates the trans form and can provide bined with an antibody against a tra/w-azobenzene group. A the “off” state. monoclonal antibody against a nonnatural amino acid carrying We have chosen NAD+ as the key compound and modified it an azobenzene group, L-p-(phenylazo)phenylalanine, 9 has been with an azobenzene group. NAD+ acts as a common coenzyme prepared by our group. 10 The antibody binds an azobenzene for a large number of oxidoreductases in the respiration system. group when it is in the trans form, but releases it when the latter The control of NAD+ activity by the binding of antibody has is photoisomerized to the cis form. The binding and release could been reported by Carrico et al.12 They showed that DNP-bound be repeated many times. If one can attach an azobenzene group NAD+ was inactivated by the addition of anti-DNP antibody. to some key substances, such as enzymes, substrates, ligands, As an azobenzene-bound NAD+ derivative, a conjugate of receptors, etc., the biological activity of these substances can be L-p-(phenylazo)phenylalanine (azoAla) methyl ester and N6- photocontrolled in the presence of the antibody. 11 One of the carboxymethyl-substituted NAD+ 13 (azoAla-NAD+, 1) was designed. 14 * To whom correspondence should be addressed. Present address: De partment of Bioengineering Science, Faculty of Engineering, Okayama X-ray crystallographic analysis of several enzyme-NAD + University, 3-1-1 Tsushimanaka, Okayama 700, Japan. complexes revealed that the amino group of the adenine ring is (1) Erlanger, B. F. Annu. Rev. Biochem. 1976, 45, 267-283. exposed to the solvent,15 suggesting that substitution of this amino (2) Martinek, K.; Berezin, I. V. Photochem. Photobiol. 1979, 29, 637- group will not severely reduce its activity. In fact, ^-substituted 649. (3) (a) Namba, K.; Suzuki, S. Chem. Lett. 1975, 947-970. (b) Aizawa, NAD+ has been shown to retain high activity. 12-13-16-17 The M.; Namba, K.; Suzuki, S. Arch. Biochem. Biophys. 1977, 182, 305-310. azobenzene-bound NAD+ was added to a coupled enzyme system (4) For recent examples of irreversible control of enzyme activities, see: (a) Turner, A. D.; Pizzo, S. V.; Rozakis, G. W.; Porter, N. A. J. Am. Chem. (12) (a) Carrico, R. J.; Christner, J. E.; Boguslaski, R. C.; Yeung, K. K. Soc. 1987, 109, 1274-1275. (b) Turner, A. D.; Pizzo, S. V.; Rozakies, G. W.; Anal. Biochem. 1976, 72, 271-282. (b) Schredcr, H. R.; Carrico, R. J.; Porter, N. A. J. Am. Chem. Soc. 1988, 110,244-250. (c) Mendel, D.; Ellman, Boguslaski, R. C.; Christner, J. E. Anal. Biochem. 1976, 72, 283-292. J. A.; Schultz, P. G. J. Am. Chem. Soc. 1991, 113, 2758-2760. (13) Lindberg, M.; Larsson, P. O.; Bosbach, K. Ear. J. Biochem. 1973, 40, (5) (a) Willner, I.; Rubin, S.; Riklin, A. J. Am. Chem. Soc. 1991, 113, 187-193. 3321-3325. (b) Willner, I.; Rubin, S.; Cohen, Y. J. J. Am. Chem. Soc. 1993, (14) The azoAla-NAD + was synthesized from azoAla methyl ester and 115, 4937-4938. A^-carboxymethyl-substituted NAD"*1 ' 2by 3 using4 5 6 water7 8 9 10 soluble 11 carbodiimide in (6) Westmark, P. R.; Kelly, J. P.; Smith, B. D. J. Am. Chem. Soc. 1993, 50% aqueous dioxane. The crude product was purified to a single peak in 115, 3416-3419. reversed-phase HPLC. The product showed 1:1 incorporation of the azobenzene (7) Karube, I.; Nakamoto, Y.; Suzuki, S. Biochim. Biophys. Acta 1976, group and N AD* in the absorption spectrum and reasonable enzymatic activity. 445, 774-779. (15) Rossmann, M. G.; Liljas, A.; Brandcn, C.-I.; Banaszak, L. J. In The (8) Willner, I.; Rubin, S.; Zor, T. J. Am. Chem. Soc. 1991, 113, 4013- Enzymes, 3rd ed.; Boyer, P. D., Ed.; Academic Press: New York, 1975; Vol. 4014. 11, Chapter 2. (9) Goodman, M.; Kossoy, A. J. Am. Chem. Soc. 1966, 88, 5010-5015. (16) Windmueller, H. G.; Kaplan, N. O. J. Biol. Chem. 1961, 236,2716- (10) Harada, M.; Sisido, M.; Hirose, J.; Nakanishi, M. FEBS Lett. 1991, 2726. 286, 6-8. (17) As PEG-NAD+ conjugates, for example, see: (a) Furukawa, S.; (11) Similar experiments have been reported as a method for enzyme Katayama, N.; Iizuka, T.; Urabe, I.; Okada, H. FEBS Lett. 1980, 121,239- immunoassay: Rubenstcin, K. E.; Schneider, R. S.; Ullman, E. F. Biochem. 242. (b) Buckmann, A. F.; Kula, M.-R.; Wichmann, R.; Wandrey, C. J. Appl. Biophys. Res. Commurt. 1972, 47, 846-851. Biochem. 1981, 3, 301-315. 0002-7863/94/1516-0413$04.50/0 © 1994 American Chemical Society — 103 — J. Am. Chem. Soc., Vol. 116, No. 1, 1994 Communications to the Editor .CONH, Scheme 1. Schematic Illustration of Photoreversible NAD+-Mediated Coupled Enzyme Reaction. ADH = HO-P-O- alcohol dehydrogenase; DI = diaphorase Ab-NAD Ab-NADH inactive ? OHOH NHCH£ONHCHCOOCH3 „i .06 k uv ^ ^ vis Red OH OH Ox system in the presence of the antibody in the dark also recovered the mediation activity. This is interpreted in terms of the inhibitory effect of the azoAla methyl ester that competes with consisting of alcohol dehydrogenase, which reduces NAD+ to the azoAla-NAD+ for the binding sites of the antibody. NADH, and diaphorase, which oxidizes NADH to NAD+ with Visible light irradiation of the UV-irradiated mixture again an appropriate electron acceptor. As the latter acceptor, we have suppressed the mediation effect, and the photocontrol could be chosen 2,6-dichlorophenol-indophenol sodium salt (DCIP), be repeated many times under alternating photoirradiation with UV cause the reduction process can be followed by the fading of blue (on) and visible (off) light. This is expected since the azobenzene- color. antibody binding has been shown to be photoreversible. 10 As shown in Figure 1, the reduction of DCIP in the coupled To conclude, the azoAla-N AD+-mediated enzyme reaction enzyme system 18 is mediated by the azoAla-NAD+ either in the could be photoswitched reversibly in the presence of the antibody trans state or in the cis state, with a little smaller rate in the latter against the trans -azobenzene group. The mechanism of the state.19 When the antibody was added to the enzyme system in photoswitching may be illustrated as depicted in Scheme 1. There the dark, the reduction of DCIP was completely stopped. The are several advantages in the present system. First, a complete mediation activity recovered to about 72% of the inherent activity “off” state was attained by the blocking of the trans -azobenzene of azoAla-NAD+ by the UV irradiation.20 The incomplete group with the antibody. Second, since NAD+ is a commonly recovery may be due to incomplete photoisomerization to the cis occurring mediator in a variety of oxidoreductases, this principle state. Addition of a large amount of azoAla methyl ester to the can be easily applied to other systems. Furthermore, since the antibody against the azobenzene group may undergo little cross (18) The reaction mixture contained 50 mM Tris-HCl (pH 7.5), 100 mM ethanol, 0.02 mM DCIP sodium salt, 1.25 units/mL yeast alcohol dehydro reactions toward other proteins, the azobenzene/antibody couple genase, and 2.5 units/mL Clostridium kluyveri diaphorase. The reaction was can be utilized in a wide variety of biological systems, if one can initiated by adding azoAla-N AD+ (total 0.5 >iM) with or without the antibody attach an azobenzene group to some key substances, such as (total 0.25 /im). The reaction was carried out at 25 °C and followed by the absorbance of DCIP at 600 nm. enzymes, coenzymes, inhibitors, etc. (19) A Hg-Xe lamp with a bandpass filter (350 ± 20 nm) was used for UV irradiation of azoAla-NAD +. A cutoff filter (>420 nm) was used for visible light irradiation. Acknowledgment. The authors wish to thank Professor K. (20) NADH is also photoexcited by the UV irradiation, and the excited Ichimura of the Tokyo Institute of Technology for helpful NADH would reduce DCIP directly. The possible nonenzymatic reduction of DCIP, however, could not be detected in a control experiment in the absence discussion. They also appreciate Mr. M. Harada for preparing of diaphorase and the antibody. the antibody against tro/LS-azobenzene. — 104 — f m $ # (i) m^±9y u 4'%#? UTKx > >^9-9 6 f 7/i7 £*T*A-i: Lfc, ^7'f Kfflfl-f liglKigt:: ov'T & IR-g-t & o 1.^7"f- Klcti>9 y^yjmmn IJ sesbmsm ^ e-e- i;@igffiti * es-t a ^ 7> k c H t £ fflftffl-&%, < S h-Tv> S „ a -* c>^7> K r [^J 1 ;thrombin(3«t Sfibinogen^M. Potencies of synthetic peptides as inhibitors of thrombin's clotting activity Peptide IC50 (/vH) Hirudin(54-65)S04 0.17 Hirudin(54-65) 1.3 thrombin HCII(49-75) 28 HCII(54-75) 38 ------> yB-chain(410-427) 130 fibrinogen fibrin Tm(426-444) 140 AP-19 >500* CCK.(1-8)S04 >100* hirudin inhibitor heparin cofactor II *Highest concentration tested . Fibrin Clot Inhibition no. compound IC«4,° 1 hirudin54^5 Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-Gln-OH 3.7s 2 hirudin PAM^ Gly-Asp-Phe-Glu-Pro-Ile-Pro-Glu-Asp-Ala-Tyr-Asp-Glu-OH 2.78 3 Suc-Tyr-Glu-Pro-Ile-Pro-Glu-Glu-Ala-Cha-D-Glu-OH 0.15 4 Suc-Tyr(OS0 3H)-Glu-Pro-I!e-Pro-Glu-Glu-Ala-Cha-D-Glu-OH 1.7 5 Suc-Phe(p-N02)-Glu-Pro-Ile-Pro-Glu-Glu-Ala-Cha-D-Glu-OH 2.8 6 Suc-Phe(p-NH2)-GIu-Pro-IIe-Pro-Glu-Glu-Ala-Cha-D-Glu-OH 1.3 7 Suc-Phe(p-NHS03H)-Glu-Pro-Ile-Pro-Glu-Glu-Alfl-Cha-D-Glu-OH 13.1 8 Suc-Phe-Glu-Pro-Ile-Pro-Glu-Glu-Tyr-Leu-o-Glu-OH 1.6 9 Suc-Phe-Glu-Pro-Ile-Pro-Glu-Glu-Tyr(OS0 3H)-Leu-D-Glu-OH 0.087 10 Suc-Tyr-Glu-Pro-Ile-Pro-Glu-G!u-Phe(p-N0 2)-Leu-D-Glu-OH 2.8 11 Suc-Tyr-Glu-Pro-Ile-Pro-Glu-Glu-Phe(p-NH 2)-Leu-D-Glu-OH 0.38 12 Suc-Tyr-Glu-Pro-Ile-Pro-Glu-Glu-Phe(p-NHS0 3H)-Leu-D-Glu-OH 0.12 13 Suc-Phe-Glu-Pro-Ile-Pro-Glu-Glu-Ala-Tyr-D-Glu-OH 1.2 14 Suc-Phe^Glu-Pro-Ile-Pro-Glu-Glu-Ala-Tyr(OS0 3H)-D-Glu-OH 0.30 15 Suc-Tyr-.Glu-Pro-Ile-Pro-Glu-Glu-Ala-Phe(p-N0 2)-D-Glu-OH 0.71 16 Suc-Tyr-Glu-Pro-Ile-Pro-Glu-Glu-Ala-Phe(p-NH 2)-D-Glu-OH 2.9 17 Suc-Tyr-Glu-Pro-IIe-Pro-Glu-Glu-Ala-Phe(p-NHS0 3H)-D-Glu-OH 2.5 18 Suc-Tyr-Ser-Pro-Ile-Pro-Ser-Ser-Ala-Cha-Ser-OH >76 19 Suc-Tyr-Ser(0S0 3H)-Prti-Ile-Pr0-Ser(0S03H)-Ser(0S03H)-Ala-Cha-Ser(0S03H)-0H 0.21 0ICM = molar dose of peptide that results in 50% inhibition of fibrin clot formation relative to a blank control after thrombin added to plasma. 2. KK X £ U~te -f 9 9 yfibronectin h. 7 7 ■?> CL t. ip £> N fibronectin (D n [553^ 7° 7 K •£■ ftmL, ^#4,5)o Williams^^ZO Reo3 virus t Reo3R Kh 7 7 9 n - -f JUffLificD 7 < / h ^ Reo3R tc — 105 a Concentration for Code Sequence 50% cell attachment Fibroncctin 0.10 nmol ml-* IV YAVTGRGOSPASSKP I S INYRTE I 0 K P S 0 M (C) 0.25 IVA VTGRGDSPASSKP 1(C) 1.6 IVB S I NYRTE I OKPSQM(C) >50.0 IVA1 V T G R G D S P A(C) 2.5 IVA2 SPASSKP I S(C) >50.0 IVA la V T G R G 0(C) 10.0 IVA 1b G R G 0 S (C) 3.0 IVA 1c R G D 5 P A(C) 6.0 RVDS R V D S P A(C) >50.0 Attachment of NRK cells to immobilized fibroncctin and synthetic fibroncctin peptides, a. Peptides were synthesized by Peninsula Laboratories (Belmont, California) according to our specifications, to include a COOH-terminal cysteine. (C). The IV A 1b activity of each peptide is indicated on the right of the figure by the concentration necessary to achieve 50% maximal attachment in the assay described below, b, The synthetic peptides were assayed for their ability to promote the attachment of NRK cells by first attaching the peptides via the heterobifunctional cross-linker SPDP (;V-succinimidyl-3-(2-pyridyldithio) propionate; Sigma) to rabbit IgG which had been immobilized on polystyrene 3. By using radio- 8 50 labelled peptides or antibodies to the larger peptides, we have determined that coupling of the peptides using this method is more than 85% complete (data not shown). Peptides not containing a cysteine are not included in the figure as the methods used to immobilize them appeared to be less efficient (see text). The attachment assay was carried out as described33 using freshly lrypsinized NRK cells. After incubation for 1 h at 37eC, those cells which had attached were fixed, stained and quantitated using cither an Artek cell counter or a vertical pathway spec IVA2.IVB, trophotometer. In all cases, maximum attachment was~ 80-90% RVDSPAC 01 'he cells plated. The titration curves for whole fibroncctin (FN) 1.0 and peptide IV (ref. 3) are shown here (or comparison. (Peptide) (nmol ml"') ANTKIOA/O ANTBOOY ANThdOTYPIC/ANTRCCEPTOR AMT IB 00 Y 4 V oncanwc amwo ac © 5 DEVELOP PEPTBES scoucncc ACOT*a *( ------^ WITH PROPER IT Its TO L10 AMO SC0VCNCC 0PTHEII0AN0 A 4 ANTRECEPTOR ANTIBODY ^ General strategy for the development of receptor acid sequence of the variable regions of the antireceptor binding peptides based on antibody structure. A ligand- antibody arc determined. These sequences are compared to receptor interaction is depicted. In (1), the ligand is utilized as the sequence of the ligand to establish areas of sequence an immunogen to develop anti-ligand antibodies. In (2), these similarity. In (5), peptides are developed based on these anti-ligand antibodies are utilized as an immunogen to devel •sequences, and their activities tested. These peptides can be op anti-idiotypic/antireccptor antibodies, which mimic the further modified, to investigate specific structural features ligand functionally. In (3), the receptor is utilized directly as involved in receptor binding, and tnc information gained can the immunogen for the development of antireceptor antibod be utilized to develop novel biologically active compounds that ies, some of which also mimic the ligand. In (4), the amino interact with the receptor. — 106 — ° Sequence similarities between amino acids 317-332 of the HA3 and a combined determinant composed of the second complementarity determining regions (CDR I Is) of both VH (residues 43-56) and Vl (residues 39-55) of 87.92.6. These sequences are shown here, termed Reo for the HA3 determinant, Vl for the 87.92.7 VL CDR II, and VH for the 87.92.6 V% CDR II. Identities are shown with an (o) while amino acids of the same class are shown with a (+). VH: Gin Gly Leu Glu Trp lie GlyArglle Asp Pro Ala Ash Gly o + + o o o . + ' Reo: Gin Ser Met — Trp lie Gly He Val Ser TyrScr Gly Ser Gly Leu Asn + .+ o o o o o + VL: Lys Pro Gly Lys Thr AsnLys Leu Leu He TyrScr Gly ,Ser ThrLeu Gin 3. ^7"7- KC X Z> fcfoV'/rt-miiS f o 1.5 — — % £|1.0 — — & 0.5 — ~ 11111111111 m ii 11f111111111 f 11 u 6 mer 7 mer i jmtmiif 111111 111 II g|4 K<7>7 5 = * ?'o t' y ^>-ytiS$i££ {>tlz4, 5, 6fcJ:[f77; / *£ i> 7 •/ 7*^7* -f- K L X, 7 v -^;i/rr V 6 6 G^#(%AChR^L#:)^T ^ h <1 t K <£ 6 ^ (OT\ S5 half-life time (hours) 5 10 15 control anti-(Torpedo a 183-200) anti-(Torpedo — 107 model peptide, ol07-116-#6776-#107-116 Torpedo #183 -200 model peptide, CKGGLR- #67-76-KC human #183-200 IE 6 AChR a -y-yzL- .y h 4 K OottAlttT'S/8$ae*IJ*iI£lf2"E K) computer-graphic (NMR) T0.005M $ ;m XOf-f m >502’® P is>502«d> BffcTfl B 10-502® d-' a 10-502® d-' m ±m a ±102 a^TTH m >102*® tw aSH t 12 7 iTii,i:m«»i,E22wi' fc-17 4(mumitT) — 108 — 4. 1) Hortin, L. et al.:Inhibition of thrombin's clotting activity by synthetic peptide segments of its inhibitors and substrates. Biochem. Biophys. Res. Commun. 169: 437,1990 2) Krstenansky, J. L. et al.: Design, Synthesis and antithrombin activity for conformationally restricted analogs of peptide anticoagulants based on the C-termnal region of the leech peptide, hirudin. Biochim. Biophys. Acta, 957:53, 1988 3) Payne, M. H. et al.: Positional effects of sulfation in hirudin and hirudin PA related anticoagulant peptides. J. Med. Chem. , 34: 1184, 1991 4) Pierschbacher, M. D. et al.: Cell attachment activity of fibronectin can be duplicated by small synthetic fragments of the molecule. Nature, 309(3): 30, 1984 io= i8 ,1992 6) Williams, W. V. et al.: Development and use of receptor binding peptides derived from antireceptor antibodies. BIO/TECHNOLOGY, 7:471, 1989 7) b b 10: 2117, 1992 8) Geysen, H. M. et al.Strategies for epitope analysis using peptide synthesis. J. Immunol. Methods, 102 : 259, 1987 9) Takamori, M. et al.: A synthetic peptide, torpedo califomica a 183-200, of the acetylcholine receptor as a tool for immunoadsorption via plasma perfusion in myasthenia gravis. Artif. Organs Today, 1:53, 1991 10) Takamori, M. et al. : Myasthenogenic significance of synthetic a -subunit peptide 183-200 of Torpedo califomica and human acetylcholine receptor. J. Neurol. Sci., 85: 121, 1988 11) 7: 503, 1990 12) TtTVl/nV AZm#f,22:194,1993 13) t(§##&#&, 165: 102, 1993 14) M: Z 6 ^ <[#, 43: 202, 1988 15) Luckas, T. J. et al.: Calmodulin binding domains: characterization of a phosphorylation and calmodulin binding site from myosin light chain kinase. Biochemistry, 25: 1458, 1986 109 — xmxwmffiu&pji #7C # immmi 10"9 MBIT) b*i £0 ^ft^£i;fti;mw©7 < y #^^^6 7 k£3 m%ijL% Ztz&iz. it, ^0#%T ; y BgE^jT, £'©fl$©:E£ ^llti:©-?# hofri comm Il^LTEo T^D-f ^$R# $ nr V 6 : (D x y 7" A • 7f k • 7^7 y — © x 7 y - - y 7" (D*05ftffi 7 y yx° 7 H© 7 7 7' y y Hk ^)D N K& (D^W FnA"->- (i^7Xll7j ^ C T\ J^T(z:^n^n©7 7°n -^©9f%0tJ£^ L/Co [9f%W (D x y f A • ^ 7°y K • H7*7 y - © x 7 y - - y 7' 7Sy® 5#Ai3%a^7'f ^tT,^^^^©20@#©7 ; y # & 3 © & 5% & ©^#% It k^K^-e H- 1 Ltz X o (^7°f K • 70'?y - Zlofe ^ & 6o j; (El-1) T#X7 y--y/(cj;r)T#^^y:^ yf ^^m7;y@gL^m^]iA$!)^uc6^, m# ©7y/< 7#©^%(c-;yfK&a!l!&&#a7:aX " 7 4 7* 7 y - t? id; {@7 © ^ 7° y Kl^'i^til!c^^/;i6x7 y - - y 7'©g^^@^ — no — - 1 iooo k • 7^7'J -©SS& 7?-/'& 7'77 ^&©m# GMy/AitET© (D77/AmE?© 0967x7* ©k'-/***'j7*n ®i# 7^®* #A(NNK)„ #A(nnk )„ 7 bey^fflS t* wn**/T < / ^SI^oa ' 7/ ^ ©#E 'J7* W--7*7;UK A* 7* f K IrJ—JSF'g-e^j® iVc (Fmoc££) (Fmoc, Boc^) V7*fK ‘&fiR 0x7 y - ~ y t* 0x 7 1; - - y 7* )X 7 y — — y 7* ix 7 V — — y 7' 0x7 y — - y 7* @DNA$ttEfllft^ (DDNAi&SE^J&S iffSRJtg $ /# ##^7 $ y% e?'J&^ (DWQ&&Rjt^ ©moa&'maB ©^DiA^-RJIb m PJr V7*fKgtePB£ y 7* UVy - * 7* 77< K k*y±©^^»^ M&mm JfcJW] ConAM^A'7'fK £1-7 7 V Tin:£i7' K £iiyKJl^yK&dD ©## M^E^'J©^ ©*££E?iJ©&S |§£E?iJ©£to ( Y P Y) (RQFKVV) ( E T T E T) (RRWWCR) (YGAFM) (12) (6) (14) (8) (9) — 111 — random peptide M13 PL-6 JacO lac' Peptidase cleavage site Signal sequence Kpnl BstXl S • * amber ▼ 3 • ” A G TG G T A C CTTTCTA GTCCCACTCCGCTCTTG X6 G G 0 C T T T C T A T T C C C A C T C C (NN§ ^CCCGCCGCC T C C G |CATGG AAACATAACAGTC A(T(^ [cGGCGGCGcj Xg LIBRARY C-X6-C LIBRARY El — 1 . m fe ~P X ^ K J: a 7 y ^ 7° -f- K • y 4 7* y 'J — © |jf |j£ a ) 7 7 - b ) 7° 7 X * K '/£ 65 y yx° 7 g © 7 7 7* 7 y h it & & K L T##8 t(c|S^f a 7 y 7 g© 7 ; 7 #E^ja^Hg ^ ^ (c % r) T ail£s 20BSSE J: 7 ^ 4%a## #?&aa7'y#a h 4- y y © IS £ 7 y 7 g © SR# ^ 7°^ k ^)D N Kj& & a ^ 7° 4 K & 7 - FtaDNAiffllW^DNAiaotn - K $ tl 6 ^ 7° 4 K (fg K^nf(f^a) awgaft tvaQ A C T H IC)[f L t 0, 3 n M © ft$fQ f£ *C IS £ * a ^7°f K^jl. V/c 5 *1 T V a Q (D y W K n /n° y — (#7k#7j<^) tg*$& ^ 7°f K © #] # © /\ 4 K d yx° y-as-^g^^Hi:^ >B ck 7 (c ##©^7°f a^i& ?&ao fRjihL #7K%j^a^<^4 KD/^y-#^-4.5^#t)#U77i/f-^(c^LT (tyx 4 KoA»y-f ©fluffy o 4 y y (+4.5) n y y (+4.2) > □ 4 y y ( + 3.8) ^r^Ta6^7j:7%^m?&ao m#^^B?lc^LT10nM©^#fn %Tis^f a yf K (i4^m©^yf K© g ##) a^ >, ti-c^a (# - 2) o — 112— -2. a y' y ft & F 4- y y (74) (20) 2 X 1 0 ™6 (16) ) (116) a f 7' 7° f K (26) aE (17) 0 -1 0 y (ACTH) (3 9) DNAtiJifi^ 7° 4 K (24) 3 X 1 (19) 7 X y K ju 7 a v (17) DNAfB###^ 7°f K (17) 2 X 1 0 "B (19) K (20) DNA%##m^7°4 K (20) 1 X 1 0 "6 (19) f 7* x ^ y x p (11) DNAffltfift^ 7°4 K (9) 6 X 1 0 "8 (19) 4 y y a v y (21+30) DNA####^7°4 K (6) 3 X 1 0 "9 (21) m##fE0?(TNP) (157) my m\- r fy (14x8) 1 X 1 0 ~8 (22) c-raf^iafe-FSi^Jal^V 7 '?r (20) (20) 8 X 1 0 ~8 (23) l . ? y ? a . ^y°f- k . y 'j y* 7 y - (D x 7 V - - y y 1 ) $o 1^ 1. G. Jung & A.G. Beck-Sickinger (1992) Multiple peptide synthesis methods and their applications., Angew. Chem. Int. Ed. Engl. , 31, 367-383. 2) 7 4 7* 7 2. R.N. Zuckermann et al. (1992) Identification of highest-affinity ligands by affinity selection from equimolar peptide mixtures generated by robotic synthesis., Proc. Natl. Acad. Sci. USA, 89, 4505-4509. 3. R. Frank (1992) Spot-synthesis: An easy technique for the positionally addressable, parallel chemical synthesis on a membrane support. ,Tetrahedron, 48, 9217-9232. 4. R.B. Christian et al. (1992) Simplified methods for construction, assessment and rapid screening of peptide libraries in bacteriophage. , J. Mol. Biol. , 227, 711-718. 5. K.T. O’Neil et al. (1992) Identification of novel peptide antagonists for GPIIb/IIIa from a conformationally constrained phage peptide library., Proteins: Structure, Function, and Genetics, 14, 509-515. 6. M.G. Cull et al. (1992) Screening for receptor ligands using large libraries of peptides linked to the C terminus of the lac repressor. 7. K.S. Lam et al. (1991) A new type of synthetic peptide library for — 113 — identifying ligand-binding activity. , Nature, 354, 82-84. 8. R.A. Houghten et al. (1991) Generation and use of synthetic peptide combinatorial libraries for basic research and drug discovery., Nature, 354, 84-86. 9. S.P.A. Fodor et al. (1991) Light-directed, spatially addressable parallel chemical synthesis. , Science, 251, 767-773. 3) % ? V - - y 7oH 10. Z. Songyang et al. (1993) SH2 domains recognize specific phosphopeptide sequences., Cell, 72, 767-778. 11. Y.-H. Chu et al. (1993) Using affinity capillary electrophoresis to identify the peptide in a peptide library that binds most tightly to vancomycin. , J. Org. Chem., 58, 648-652. 12. K.R. Oldenburg et al. (1992) Peptide ligands for a sugar-binding protein isolated from a random peptide library., Proc. Natl. Acad. Sci. USA, 89, 5393-5397. 13. J.K. Scott et al. (1992) A family of concanavalin A-binding peptides from a hexapaptide epitope library., Proc. Natl. Acad. Sci. USA, 89, 5398-5402. 14. A.M. Lew et al. (1989) A protective monoclonal antibody recognizes a linear epitope in the precursor to the major merozoite antigens of Plasmodium chabaudi adami., Proc. Natl. Acad. Sci. USA, 86, 3768-3772. 2. ? 'S ? W® 7 y ? * > b it 15. H.M. Rarick et al. (1992) A site on rod G protein a subunit that mediates effector activation., Science, 256, 1031-1033. 16. K.E. McLane et al. (1991) Identification of sequence segments forming the a -bungarotoxin binding sites on two nicotinic acetylcholine receptor a subunits from the avian brain., J. Biol. Chem., 266, 15230-15239. 17. S.R. Krystek, Jr. et al. (1991) Identification of subunit contact sites on the a -subunit of 1utropin., Biochemistry, 30, 1858-1864. 18. T.A. Santa-Coloma & L.E. Reichert, Jr. (1991) Determination of a -subunit contact regions of human follicle-stimulating hormone J3 -subunit using synthetic peptides., J. Biol. Chem., 266, 2759-2762. 3 . D N A m ^ 7° T K & 19. K.L. Bost & J.E. Balalock (1989) Preparation and use of complementary peptides., Methods Enzymol ., 168, 16-28. 20. J.E. Balalock et al. (1989) Use of peptides encoded by complementary RNA for generating anti-idiotypic antibodies of predefined specificity., Methods Enzymol. , 178, 63-74. 21. V.P. Knutson (1988) Insulin-binding peptide., J. Biol. Chem., 263, 14146— 14151. 4. H 7k g;) 22. G. Fassina et al. (1992) Binding of human tumor necrosis factor a to multimeric complementary peptides., Arch. Biochem. Biophys. , 296, 137-143. 23. G. Fassina et al. (1989) Recognition properties of peptides hydropathically complementary to residues 356-375 of the c-raf protein., J. Biol. Chem.,264, 11252-11257. — 114 — § iwnm [SilHS] <5^ H T (D ^ u 6o tM'ft £ o C xV^ ft £ <1 /< A. fc ti ft 6 ij isovalerate D-Hydroxy- Zo & / 4- ( (A23187) ( & 7 / F + ^ i; -f ± C >J X / y T ^ C m ^ "7 L-c, y f - P yf ) ^ y # *4 ^ ) O' y H # C-N 0 L H (12) CH T Yil L & | 0 C-X — 6 it H Clj CH, 115 ¥■ 0 C-X H Unj | Al« — L ^ CH zm* 0 C-N t$ dH, u A Val y Lei D CH I C-N £ CH, 0 14 h B Prol3 All CH ' | L #n fln t s u s & z n r * ' * ? ? f * . 0 C-N • dH, H X- /°L CH Yd D | Glu C-N 0 CH, /c y Aib F H r ^7 Yd L VH CH 17 -? | y 7 12 C-N ( ( 0 ( y CH, H ^ 7° ; CH Yd D | ± r 7 y ; CH, C-N 0 4 ~ 4 y -Y t y H Try L Leu y CH CHi | (Dm$£^ > 7 * K 0 % C-N v ; • y Gln dfSau ^o- ; 11 H.|. ^ e CH y Lei D y -r CH, t° ? y y C-N 0 18 tg y f V cm>x A f- H Try L A CH Oil | ) y y © (is) 0 y C-N K#*n dfuS:H, H ) • ffl CH Lee D tmzfr> (15) CH. I y y C-N 0 b H >, Trp L CH CHi | ) 0 C-N ut Aib4 dH, JK H CH Lei D CH, & I C-N 0 CH, Val8 3 U < Tip L CH CHe | C-NH 0 9* CH, OH | 1 a-a-ac vk i 3K+-s^k-a-#(#;n%m: -^jr« V V3 'xStiSiSTS^i. Si%(a')T- £$£80. p. 186 (1978) £0). o — —O M g=o ---- =3o JS«-^JS« ' O— —■o (a) (b) yjxW's A2^frci5^ +v^/u (Sung, S.S. & Jordan, P.C. :/. Theor. Biol., 140, 369 (1989) Z 9). 1UUMLI #—ft^/i-3 vjft* yx?LMi. RHtfcfflSToHKaFBKoH 4 y-y yA^rSa^-CS^Lfc t> T J A VC. ZSSKlHtiC (Muller, P. & Rudin, D.O. : Nature, 217, 713 (1968) Z 9). 1*1 I3X!10-1 A 1 r T y A f-•> y *■ + y* TvM--/ o (c)^+ '✓*>£&*. + + jS-Hit/Z < , 3 yj}t y ^ A‘y*Ci? 4 /£ £ (Fox, R.O., Jr. & Richards, F.M. : Nature, 300, 325 '>307 ?/ f-y ySST-col» 6 r. KC1 iftff 2 m, (1982) *&). HE 210 my %-jhx. "C — 116 — U V ft 4 4 W | 0 3 D 3 0 o' 3 o' 8 fc §. „» " g 236 237 -5 F t/i ^ i !C-EK PI : • : ^0 ? Z - PA- >0 ? • . ? • . 00b|> Units) ; 2 M ! • 2 W > OCCURRENCE (Arbitrary FREQUENCY i U O Q (rn) @ * ) 3 d 33 " > 3 C D ) ) 3 3 > > 3 T O 3 CD T > 33 < O CD (/)<£/)> > > 33 > O y b o t t o m o f t h es t r u c t u r e . IA ^ p| 5 |»VtViV o o o > ( 2 ) m P )fffl f ° 7 ^ i i e g i * * Z 3 s V 1 N> > n rr S< -X O I + + + a # Sr H 3* * A V 1 # H M, < o > 1 v> St $F 1 1 V CD 1 m 1 fp a m o r CL CL X CD CD St 1 © i + + + t3 •a 6 -t 1 v= Relative frequency Relative frequency Relative frequency Relative frequency I # V 1 fDpF -V- 1 V it Or V 1 X o 0 1 1 H y- £ Si e R S V ^ O V i 1 K + + n 5 1 + ^ H © X " CD ^ ^ H VI x a o z :o VI N -r *7* ^ ^: x ~7* AmpKtude (mv) >= + ^ o: - O --ih CD x> tn V K' to V ^ * 2- » s V to V o y ° z O V + + I + + + I? o X °* ~ Vt II o — K " . l 3 J H CJ o O) <3 z VI o O £ /'"’N X to V-Z *1% O 3 5* ( ^ / 7 ^ 7 7" f K ) ffiHS -b y * - ' (1 9 9 0) K. S. Akerf eldt, et a 1. ( 1 9 9 3 ) "Synthetic peptides as models for ion channel proteins" Acc.Chem.Res. 26:191-197 ( T -fe f a V S.Oiki, et al. (1988) "M2 5 , a candidate for the structure lining the ionic channel of the nicotinic cholinergic receptor” Proc.Natl.Acad.Sci.USA 85:8703-8707 S.Oiki, et al. (1990) "Bundles of amphipathic transmembrane a -helices as a structural motif for ion-conducting channel protein" Protein: Structure,Function,andGenetics 8:226-236 J.-L.Galzi, et al. (1992) "Mutations in the channel domain of a neuronal nicotinic receptor convert ion selectivity from cationic to anionic” Nature, 359:500-505 (Na f ^ y $ ) S. Oiki, et al. (1988) "Channel protein engineering: Synthetic 22-mer peptide from the voltage-sensitive sodium channel forms ionic channels in lipid bilayers” Proc.Natl.Acad.Sci. USA. 85:2392-2397 M. T. Tosteson, et al. ( 1 9 8 9 ) "Voltage-gated channels formed in lipid bilayers by a positively charged segment of the Na- channel polypeptide ” Proc.Natl.Acad.Sci.USA, 86:707-710 ( M C D ^ 7° f K ) E.Cherubini, et al. (1 98 7) "Long-term potentiat ion of synaptic transmission in the hipocampus induced by a bee venom peptide” Nature, 328:70-73 T. Ide, et al. ( 1 9 8 9 ) "Mast cell degranulating peptide forms voltage gated and cation selective channels in lipid bilayers” Biochem.Biophys. Res. Comm. 163:155-160 T. Kondo, et al. ( 1 9 9 2 ) "K channel involvement in induction of synaptic enhancement by mast cell degranulating peptide” Neurosci.Res. 13:207-216 (1990 ) 30 : 22-2 7 ( 7* v K n h * y ) H. Rehm, et al. (1 98 9 ) "Dendrotoxin-binding brain membrane protein displays a K channel activity that is stimulated by both cAMP- dependent and endogenous phosphorylations ” Biochemistry 28: 6455-6460 D.N.Parcel and J.0.Dolly (1989) "Dendrotoxin acceptor from bovine synaptic plasma membrane" Biochem.J. 257:899-903 ( D-enantiomer) D.Wade, et al. ( 1 9 9 0 ) ”A11 — D amino acid-containing channel-forming antibiotic peptides” Proc.Natl.Acad.Sci.USA 87:4761-4765 120 a* (i) v ^ © w m ^ t a H M- m & HI off l 0 SB 0 e- sft -ft ff VJ vji V. 'S 0 0 & 3ft S M ^ -ft SB m A s< ^ + H m s\ -7% £ % 0 3500 2 0 0 0 □ □ a □ □ IT CZI -jE: 50 150 250 350 550 650 450 750 850 950 1150 1250 1350 1450 1550 1650 1750 1850 1950 ^1050 : I I I X Si 0 >o Vj- H -ft S^ V Q 0 3ft & # <1 V -ft vji V. 0 ~r 3 o o CD O X CL g CD ■O COOH VJ,U —Lys-Qiy-COOH Leu 41 / Pro 40 Cys39 \ 3.1 + S'yT?Oft-f* ’/J5ao^^?- KUO ‘/C6H5-CH20CNHCHC0NHCHC0NHCH-C02CH3\ 12- \ o ch2 CH ch3 I s CH2XCH3 ch3 L (Fe/4S4) ® 3.2 LT<0- 3E 3.2 %Fe4s4 »£© 1/K y * Attffc (DMF 4>, 7^='xT)4; V K -y ^ (V us. SCE) It # 2-/1- 3-/2- [Fe4S4(Z-Cys-Ile-Ala-OMe) 4]2- + 0.12 -1.00 [Fe4S4(Z-Cys-Pro-Leu-OMe) 4]2~ —0.02a) -1.19 [Fe4S4(Z-Cys-Pro-Val-OMe) 4j2- -1.18 [F e4S4 (Z-Cys-Pro-Gly-OMe) 4]2- n.o.* > -1.07 [Fe4S4(S-2,4,6- F !> y +vi" OH (Fe.SOffffc Ph-C-CH-Ph + Ph-C-C-Ph + ii i 25'C 0 OH THF/DMF 66 OH J. Am. Chem. Soc. 1989, Ilf 380-381 Helichrome: Synthesis and Enzymatic Activity of a Designed Hemeproteih* Tomikazu Sasaki* and Emil T. Kaiser* Laboratory of Bioorganic Chemistry and Biochemistry The Rockefeller University New York, New York 10021 amphiphilic a.helix 1 ■ 5 10 15 . Ala-Glu-GIn-Leu-Leu-GIn-Glu-Ala-Glu-GIn-Leu-Leu-GIn-Glu-Leu-amide . :.\Cn\ • substrate Ea?r E.: o-lK::' :p i vE: A: Ala, E: Glu, Q: Gin, L: Leu , . .. L(axial ligand) ,1! a Figure 2. Amino acid sequence of peptide: (a) helix wheel and (b) helix •:* ’ Figure 1. Proposed structure of helichrome 1 after folding of the peptide, i chains. • ai/i diagram, in which the circle represents hydrophobic amino acids. < . i.‘! * " ■ •; r.-t ' i* -.- Biopolymers, Vol. 29, 79-88 (1990) Synthesis and Structural Stability of Helichrome as an Artificial Hemeproteins TOMIKAZU SASAKI** and EMIL THOMAS KAISER Laboratory of Bioorganic Chemistry and Biochemistry, The Rockefeller University, New York, New York 10021 SYNOPSIS A detailed procedure is described for the synthesis of helichrome, which is the first successful example of polypeptide-based artificial hemeprotein. The segment synthesis- condensation approach used for the assembly of small proteins has proven to be extremely useful for protein mimetics as well. The final deprotection was performed using the TMSOTf-thioanisole method instead of the less-convenient hydrogen fluoride method. The unfolding transition of the a-helical conformation of helichrome induced by guanidine hydrochloride was studied to understand the stability and dynamics of the folded structure. The resulting parameters (Q,5 = 5.2Af and AGH2° = —4.4 kcal mol-1) characterizing helichrome denaturation were comparable to that of native globular proteins. 123 — Soi&uca. MS") , I'4 Synthesis of a Sequence-Specific DNA-Cleaving peptide UMES P , Sluka, Suzanna J. Horvath, Michael F. Bruist, I. Simon, Peter B. Dervan Melvin i ««nthctic 52-rcsiduc peptide based on the sequence-specific DNA-binding domain of rt^ccombinasc (139 —190) has been equipped with ethylcncdiaminctctraacctic acid icnTA) at the amino terminus. In the presence ofFc(H), this synthetic EDTA-pcptidc ^yes DNA at Hin recombination sites. The cleavage data reveal that the amino jetnljnus of Hin(139 —190) is bound in the minor groove of DNA near the symmetry jjjs of Hin recombination sites. This work demonstrates the construction of a hybrid peptide combining two functional domains: sequence-specific DNA binding and DNA cleavage. Minor Groove Major Groove 1 10 20 GRPRAINKHEQEQISRLLEKG 30 50 HPRQQLAIIFGIGVSTLYRYFPASSIKKRMN Oj Ctj B HO. °o ^ °o ° " Hln(139-190) 5" N N N N HH N N N N 3‘ S'KKNMNHNNHNJ 3'NNNNNNNNNNS' J'NNNNNHNHHNr Fig. 1. Cleavage patterns produced by a diffusible Fig. 2. (A) The 52-residue peptide is the COOH- oxidant generated by EDTA ■ Fc localized in the terminus of Hin, amino acids 139 to 190. Tenta minor and major grooves ot right-handed DNA. tive a helix assignments arc underlined; helices a2 The edges of the bases arc shown as open and and a3 arc from alignment with CAP and X-cro, crosshatchcd bars for the minor and major a, was assigned using the Gamier algorithm (21). grooves, respectively. (B) Synthetic peptide 1, EDTA-Hin(139 —190). Hlxi. Secondary Hin Start CfTT CTTCAAAACCAACCTTTTTCATAAPCCAATCCTCCATCACAAAACCCACTAAA 1/T TCTTCCTTATCTCATCTA A AC C ACA Ait ATCffll ctAACAACTTTTGCTTCCAAAAACTATTfrCOTTAOCACOTACTCTTTTCCCTqATTTnAACAACGAATACACTACATTTCCTCTTTrTAMrK; 10 pM Hln(139 -190 ) -i MPE-Fe Fig. 4. The sequence left to right represents the rAjTTCCTICTTCAAAUCCAACCTTfFTTe cleavage data from the bottom to the middle of A TlA ACCAACAACTT ITT CCTTCCAaIaAAC the gel in Fig. 3. (A) Boxes arc the 26-bp hixL binding site, secondary Hin-binding site, and start of the hin gene. (B) Bars represent extent of 10 pM FE.EDTA-Hln(139 -190 )| protection from MPE • Fc cleavage in the pres «h ence of Hin(139-190) (Fig. 3, lanes 5 and 14). (C) Arrows represent extent of cleavage for ♦ttl ttl» t Fc • EDTA-Hin(139 —190) at 10 pm (Fig. 3, lanes 9 and 10). (D) Cleavage patterns for 1-5 MM I I , Y.Sjf Fc • EDTA-Hin(139-190) at 1.5 pM (Fig. 3, F..EDTA.HH„«»,l|,l |||, . ,|ll -J lanes 7 and 12). Extent of cleavage was deter i'TTTATT6CTTCTTCAAAACCAACCTTTTTCATAAACCAATCCTCCATCACAAAACCCACTAAAATTCTTCCTTATCTCATCTAAAC6ACAAtATCtlf mined by dcnsitomctric analysis of the gel autora lAAATAACCAAGAACTTTTCCTTCCAAAAACTATTTCCTTACCACCTACTCTTTTCCCTCATTTTAACAACCAATACACTACATTTCCTCTTTTACUC tin tttjt tin diogram. — 124 — 1987, (3), p. 345-350) © 1987 The Chemical Society of Japan FtJ:5 y7 /1 F V (1986 *p 8 n 13 a g S) -t|S7; K L-a-i/y f-^7 ; 7-e-* 7 ^ ^ Lt, 7yi^-rk K-\<0-7 7 y-fb2KS<0 ##cZ6-7 7y t K V yo^M^7^ft Jx7co tfc, tn LT, 7^7k Ki7-t F y -y 7 y kH y 1987, (3), p. 378-385) © 1987 The Chemical Society of Japan — 5 -feJl.3?x7.7L5—tf^E-rJU-Ol'T— (1986 ^ 8 >120 H St 5E) ± pa an ^ m £%**- & * i% f 's^tr-y^ h !) y f-/u7y- ;&-7A:7 p ^ K (CTAC1) ^JHWc; t’4'S!fLv£$'C, g^ ^^txyu^ U""C/13U '7c 77—< y >//u41 -y 7? yt' — yt^-t- "7 s - yu7 7 - yu —l- ti x i/ yu —l- p 'f y y ^^#^-7" y^/h* 7- — 3S^ (77- Kt * y -7 yu-D-Jo JiO'-L-y i-^7y- v-p- — h p 7 = r/o ojjdtk^jw^joV'-c, t-^< * - -kyy^mo 2#-c, (2) y- 7-^-7 KifCiEftTio £7c, (3) CTAC1 i -t/ui ^^7^^ f ^7y^ - >) p y K (2C,,N 2C.C1) ^ t £ 6, 23mol% CTAC1/77 mol% 2CI6N2 C,C1 Uftlt X'fr:?%£te £ — 125 — K jiiee »s A:til #s 1 . # U # K - ^(C#SL, LT#%f6o yoftRUt, ZOfrji'ty 50BS® V -fe 7° y - (c* 7 £ ctfc j; z ^yy n* N i u- a^eH© ,1/t y ©J#^©y 9 (c , ^#Uc#y © y * y n y= ^ x h it s /Mifilti a -?yy Kic. ^SE^ifi: v N $ ^jc ity ammm^m-^L. #Lu##e##^#mya c 6^gm^yao u-c LT©m^fpm^&a©Tx ^[EKi^yy K©m^#M#ijm©^^ l^ootist^ C 6 (C L /: o ^©#fb/c##(c j:(9it<]:#^. x 7o /:/:##%(;:&%,& (i&ao ##%##??&% , T^a/c^)^^#(c^(/ a^6 x #^%E#© ^ ©Ja^^i%#"e& aAT ^,60 ymbL/cma-r&a^yy r ^^t;#^St4© AIil0f?i^O^^^4 t X'iZ. b'rtfrtix £ tz o $7° a v= i 7 h ©>tf ^#E#" 2 . 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U 4 + A ^ M^*Z$:Z'&&m&2-fiZ.Q><}G>3rA ±A>' (6) °2/i ya* a&a ^'lax/c^ ^$^514 aa ^^v ^9^$1U2IP^y6 fi/z^^igiol^ n n^/C $ Z^^## v?5 28 t, 0 s 5 c 6 M##c: 6 ^ ^ -Y-y 6(i^8S"C&&6o &##"& s a - V v ^ K;PO#^(c J; ^ - V K y ^ #U^x.^ur^^m^Tyo-f 0^0^^6/La-e&r)/:o 6 C 6^199 3^ (c , yy KO# AA t it± < £5£#rtfJfc&ftj (pepzyme) #£$*1 tZo <1 CD&Z&JI* > ^ y y F © PS D & V qJ|£f££A Lfc^0T\ A^g, 6|sj#(c#rL^m#m^^^c Ay D ^ h (i^yf KK#rLi\##^#ijaif 6 C6^BM(CLTU6^, * &(c:# K(i^AT<^,5^0-c^6o xm (1) ATgmrn Bf^#A (aA%) it^mn NO.89 PAGE.71-84 1981 (2) $T '/§ EE 7n (^ A$ A%) VOL. 33, NO. 10 PAGE. 887-892 1982 mf^#A (aA%) taDDIl VOL. 27, NO. 18 PAGE. 32-38 1 984 (4) 'M t ^ t -r A y $ 6 (^aZAZ) — 132 — y r 4 y >r i ft ) v VOL.13, NO.18 PAGE.3-14 1984 (5) Synthesis and evaluation of a miniature organic model of chymotrypsin. D’SOUZA V T, HANABUSA K, O'LEARY T, GADWOOD R C, BENDER M L(Northwestern Univ., IL) Biochem Biophys Res Commun VOL.129, NO. 3 PAGE. 727-732 1985 (6) #A#A (ft. AX) VOL.39, NO.2 PAGE.102-105 1986 #X#A (ftAX) VOL. 4, NO. 2 PAGE. 114-1 23 1 986 (8) Artifiicial enzymes and enzyme models. BRESLOW R(Columbia Univ., New York) Adv Enzymol VOL. 58 PAGE. 1-60 1986 (9) Reduction of ninhydrin with Cyclodextrin-1, 4- dihydronicot inam ides as NADH models. YOON C-J, IKEDA H, KOJIN R, IKEDA T, TODA F(Tokyo Inst. Technology) J Chem Soc Chem Commun NO.14 PAGE.1080-1081 1986 Axgm XSXS OKA X) VOL. 35, NO. 10 PAGE. 930-933 1986 (11) ; ; 't^mAX0^®#XE8::t ALiftl—S|5 (AESA X) — 133 — VOL.35, NO.10 PAGE. 938-941 1986 (12)Thermal and pH stability of " yS-benzyme" D*SOUZA V T. LU X L, GINGER R D, BENDER M [(Northwestern Univ., IL, USA) Proc Natl Acad Sci USA VOL.84, NO.3 PAGE.673-674 1987 dm x) VOL.49 PAGE.123-129 1986 (14) axbs £ mmm (bA X) $ii,mm VOL.32, NO.9 PAGE.1102-1112 1987 (15) $e@^>f y y -y k b#x Ax#mo^:t^MA- (aA x) Bio Ind VOL. 5, NO. 7 PAGE. 53 5-541 1 988 (16) AXBS©9F3mc <1 tt'Ztz x) 0 L VOL. 26, NO. 11 PAGE. 30-35 1988 (17) Emb# s y #X#A. (AA X) x#xm VOL. 39, NO. 11 PAGE. 948-954 1988 (18) ^ n -r> X h v tJIPI (m#A %mx#m) ## VOL. 59, NO. 2 PAGE. 105-112 198 9 (19) '> ? O 7 T y AX Hz y* - • AlSii L T©#XBI$t£ 134 — W-L^A n (AA X) VOL.47, NO.6 PAGE.484-492 1989 #X#A (AA X) VOL.31, NO.6 PAGE.322-323 1989 (21) Artificial enzymes:Synthesis of imidazole substituted at c(2) of fi -cyclodextrin as an efficient enzyme model of chymotrypsin. RAO K R, SRINIVASAN T N, BHANUMATHIN, SATTUR P BOndian Inst. Chemical Technology, Hyderabad, IND) J Chem Soc Chem Commun NO.1 PAGE.10-11 1990 (22) tfom MStes csbxsa x) VOL.55 PAGE. 197-203 1989 (23) b'f ; >Bi2AX@^0^^ #X$A (AA) imtxm VOL. 43, NO. 5 PAGE. 786-789 1990 (24) %D7K##A##^t f'vP Amm-aiK (AR5$A x) mm VOL. 39, NO. 10 PAGE. 744-752 1990 (25) m##m NO.119 PAGE.133-144 1991 (26) ## cc^-e^AAXg# — 135 — #I#A (AA) VOL.45, NO.7 PAGE.442-445 1990 (27) X^X#^AX##0^^ #_k#A. (AA X) AJtlHHKE NO.13 PAGE.3-10 1991 (28) ## m&vmmmumft f v IHiliS (SA X) x#xm VOL.42, NO.8 PAGE.615-619 1991 (29) ## r4=#GM#*8J : y B izAX^mo^Xm# #X$A. (AA X) VOL.32, NO.5 PAGE.287-293 1990 (30) k' f ; y B (,fccLO' B 12^MU b oX^XJlMAXH# #X$A. ARM# (AA X) VOL.17, NO.8 PAGE.298-303 1991 (3D@^x ib#^X# VOL.65, NO.6 PAGE.250-264 1991 (32) X#X#m!k'? ; yB6AX##(Cj:6T ; $^fX#A (AA X) ; (feMA II) Bio Ind VOL. 8, NO. 9 PAGE. 63 7-642 1 991 (33) Catalytic Functions of Artificial Enzyme Composed of Simple Vitamin BiModel3 and Synthetic Bilayer Membrance. MURAKAMI Y. HISAEDA Y, SONG X-M, FAN S-D, OHNO T(Kyushu Univ.. Fukuoka) — 136 — Bull chem Soc Jpn VOL.64, NO.9 PAGE.2744-2750 1991 VOL.46, NO.3 PAGE.208-213 1993 — 137 — 1 No. 1 (%) No. ma# () *-f h;L ilo'tc /0 /SttSfltit Cys-25 IcStfblTc 212 (1<7)7 i yEA><7>7j:5A^W7Cirg& Howard L. Levin XCct-5yt Kn-3f-> §B&(:7- a ramu-iQ-twmm^A m&AT#A L 7 j* w / 1 E. T. Kaiser 7$ KcQSHb Mit qAh ^qA,h’ 7a L L7:, yt KOX3777; K£±JE<7)7 5A'< {'>***& m) /w J. Am. Chem. Soc. , 100(24)7670-7677(1978) R R /0 Cys-25 lc&fbI5c N-7Un.yhFo*:f>m' ( SH )-~f2#{t# ^#.©£Stok#A¥S£R' x-, w >St4S|5{5 James T. Slama ^uM6ESt4^WL/cS4M #tty<77hV<77 K 2$Wl 5|#;v CH3(CHz),C00Ph(N0z) -*CHa(CHz)aCOOH bX7y>^St;^^77<77 tXfy K eyelo ( D-Leu-L-Glu-L-His )z f Ph(NOz) VT, g-fIC0777L+>>^p-X hD7i-it -7; CH, (CHz). oCOOPh(NOz)—CH] (CHz). oCOOH X X 77KT)Jin7k^¥(cfc(t 6^514^^ 0 + Ph(NOz) 7a l 17 L tz0 £E5SM.t:>$enf4(7)l§S±ie<7‘7 Kii Val-OPh(NOz) - HCl - D-Val + L-Val y i A7-7UCj:b^T3 ~20E Barry Robson &##%=@i4%f v Koisat AimR. 6 >©m g mtthm&m mt'mik, muco (9im^m% (7/W?- ) CRC Grit.Rev.Biochem. . 127 m 'C/f- KtOtogfEffltc I2& D &"£ A Tld: 14 (4)273-296(1983) t'^Oo ( ## >® @1495(5 Cys-25 ic$(fcil* 0 CHa 0 CHa @1495(5 E. T. Kaiser 577h'>C7i-D Cys-25KK{taK®E©ffiB*-C* 675 8^ * / 1 \ R;C2-m *#@147 i m. AAms& *#@147 ; 7RA#©A:*©%&K yX/fF % : R-NHCHCOO "X^-NOa 0 V/t, S vawmoz M Boc-Lys-A-Lys- 1 ys-lys-NH-PEGM M@ p-;FD7xUt7tf-K M@) E7kl4?*^ 5 PI. 9 imi. K5maS0SK( ?lffl*![ 39 (4) OWfFtt KE K L-Leu-L-His (Dy^fF C#&4c4:'jif Mi's ’ 75->xx f-;u—•€-©JjQ7k#f!?#j ?IW1. (Y.Kimura b Chem Lett. 1984 429 ) 1 0 tivkfim KL li4Ojj07k5>S?i$JtJ:bk(L)/kTCF3.6 9IM2. ( liiBA immb^) $\m. 91 M2. (R. Ueno i Chem. Lett. 1984 1807 ) , 35(10)486-492(1986) L-Phe-L-His-L-Leu (%) No. (murnm ) ^7 hvl/ #[/(: fctiA mm t77Z>^^t'y'i/:ll h V^77 K^r. V toi hU 7/7 F % N-acetylphenylalanine p-nitrophenyl N, N-yf Hfy/t N, N-LW/KtATov-f F x%77i/#A#*777W:&cf< s$l 16( ^7K )!5!t ( 7'x#> ) Science 243,622-628(1989) 5Zf*EEr^7y^77 K*< 2 oSALA tgA^> 4 ot ) m&7MM Wt57;/ Ep-7lD7xmf/t tZ.f-iS'simftlZ&ZT i 7|iXf;t *X8ffik 5EB & ?5JEES txfy «t i' tz cDiz:(*litR6^[]7k^?Er^Wt L 7c0 JIOT1S « =HL m&m=. Z-L-Leu-L-His 1 7 (Kit m*y %#SK) 7; y^xxf-;i/03H*ii{Rfi^ja7k^? L Z-L-Leu-L-His Ov^f mtilMb L7: Z-L-Leu-L-His-L-Leu Cio-Phe-PNP + L-Tyr-OEt it. Cio-Phe-PNP OjD7k^?!CfeI^5 m&u&f- (mtir-LHm-x) L/D = 23 £f#7s0 ( „ „ ) - Z-Phe-L-Tyr-OEt + H0-C.H4-N0* v-A‘y Lima's m 13-16(1990 ) mssma b t f v yyy b 1 8 y.9iY &— (Qlxlt smmm so # 1(4)63-70(1990) ffl* ®lbi59WlT De Novo fittSftfc V Hy y XfijSfEffl£Wlh3Zn( H )®^ Ala-GIu-Gln-Leu-Gln-Glu-Ala-GIu-GIn- V yoA/g-A^L7:o 1 9 'xAy >/’y Efbil^ 7a L SKemmi6t%7k&(Df y ? -Leu-Leu-Gln-Glu-Leu-C0NH2 U y oAi-f#7:o Proc. Ann. Int. Conf. IEEE Eng. Med. Bio. Sci coiuOli/f : yy'-7L N-7yA7;:A77:7 p-:Fo7i:Wf& N-7y4-7xl«-75x> p-l)07iziUm Wb zjiy •> ?7-% S6H KHf£ 1 Op-c-O™" m&=Pitz^7 #m m S(i'^7P-yftH Ifctt h KBEaf&&^[Fe4S4] ##* [Fe4S4(Z-CysS-Pro-Va 1-OMe).]2 ~ 2 3 X lIjo W -Bp-^ > v> y x=«k6 ®Hk OH 0 tel 'K* 1 m&2£ tXfy>tfy- itii h V^Xf- K N-dodecanoyl L(D)-phenylalanine XXT7—f-4- ±@S1- p-nitrophenyl ester Z-L-His-L-Leu Z;N-BOC C,, CONHCHC00 ^NO, R;CH2Ph SETi y$exxf-yu C,2-L(D)-Phe-PNP 2 4 uu7kftm tel (DAo;K4#&fr6tlr7:. Z-L-Phe-L-His (lXf7-f) 1)000^ R rgftMCBzAC) (Di^K^zFn-^ (D^l Z-L-Phe-L-His-L-Leu Es^i-byUiimXhT L/D=38 i (#x± ) It+Xm&ZM 17 (3)518-523(1991) -* a&^%7k^A$% + HO—C«H i —NO 2 n/c0 /N4Dry)K>y)l-y) Hfl'jizSjL(CBzAC) ;U % {RJtf jk Ws -n xu V -hS^SStf-R Z A AgB-J4; mm %gS#g V it-SiK^E'-S'SSSt .r,?K £A^»ES ^HSM I H®-> 8 ^ A i*l($Qy^> % i ISH-k^t1^ S4J A^g^lSg ^K-Mi A^i^Sg: I i>)b».MJi't'fl -frtl^-k A^togtits hA& , -M^thlK „p iK ^ ^ rif h ijg u tjgfl A u -(% liv-fUO —O U —C/3 143 — i c m~t~ -s. —m. No. 7 (%) No. #2# ( ) 94b)\* 360'ir ttito om-m muf raw V-mmi(D£{k¥£f&; D EPS N-benzoyl-L-trypsine ethyl ester a- ( aCT) ©St£ffif4£jll& L M. Zouhair Atassi <77 K#a"Pepzyme" ISft ChPepz ;aWJ7yy gf£®$ tI®cU29 (15 BTEE ) 7cChPepz£ h ') 7v > (Trypsin) ■S-fltRL <77 k —i*^toK$M¥B!( mum ChPepz ) fcTrPepz££®UtoteM¥«tt6$ 3 3 Taghi Manshouri a@y ;2P->3>CO£fi£<77 tak$M¥ Kl mh!,29tf) -WFT, S-S&STWfgiiT'ii , TrPeps ;HJ/yy SI4B4 tllttl/:29fi -tffF aCTOSStfM BTEE^A®P*<7)ChPepz? N-Tosy 1-L-arginine-methyl ester Jn*5M¥ttTiaCTi fslflS® * ( BaylorE#±# ) Proc. Natl. Acad. Sci. USA (■5 TAME ) Lto Trypsin <7)g$m5 TAME^TrPepzTtD 90 8282-8286(1993) —m#07K#am( mum TrPepz ) 7Kfffitit JLWm Trypsin(D$%0X O# [email protected] 3 4 3 5 {K-Jf • WMffl88i)j (S52S : IsS • St ESS A A Rxf4 :t- % % A y 9 — f 550 AEm@EBAPJ 1-8-4 TEL (06)443-5321 FAX (06)443-5319