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Angiotensin II Proc. Nati Acad. Sci. USA Vol. 78, No. 2, pp. 757-760, February 1981 Biochemistry Synthesis of [a-methyltyrosine-4]angiotensin II: Studies of its conformation, pressor activity, and mode of enzymatic degradation (angiotensin II analog/resistance to a-chymotrypsin degradation/full pressor activity/NMR and circular dichroism spectra similar to those of angiotensin II) M. C. KHOSLA*, K. STACHOWIAK, R. R. SMEBY*, F. M. BUMPUS*, F. PIRIOUt, K. LINTNERt, AND S. FERMANDJIANt *Research Division, The Cleveland Clinic Foundation, Cleveland, Ohio 44106; and tService de Biochimie, Department de Biologie, Centre d'Etudes Nucleaires de Saclay, P. B. No. 2, F-91190 Gif-sur-Yvette, France Communicated by Irvine H. Page, October 15, 1980 ABSTRACT Modifications in angiotensin II and its antago- full biological activity (agonist or antagonist), the backbone and nistic peptides that should have increased in vivo half-lives but not side-chain structure of the analog should resemble that of the reduced biological activity were studied by determining the effect hormone, All (2, 4). These observations may be helpful in the of a-methylation of the tyrosine in position 4. [a-Methyltyrosine- 4]angiotensin II, synthesized by the solid-phase procedure, design of new potent analogs. showed 92.6 ± 5.3% pressor activity of angiotensin H. Incubation with a-chymotrypsin for 1 hr indicated absence of degradation EXPERIMENTAL although, under the same conditions, angiotensin H was com- O-Benzyl-a-Methyl-L-Tyrosine. The procedure used for the pletely degraded to two components. Comparison of the 'H NMR synthesis ofthis compound is a modification ofthe one used by spectra in aqueous solution and the circular dichroism spectra in trifluoroethanol of angiotensin II and [a-methyltyrosine- Khosla et aL (2) for the synthesis of 0-(2,6-dichlorobenzyl)-L- 4]angiotensin II suggested that a methylation of the tyrosine res- tyrosine. idue in angiotensin HI does not lead to major changes in the overall a-Methyl-L-tyrosine (3.904 g; 20 mmol) and 1.6 g of NaOH solution conformation. These results are in contrast to those ob- (20 mmol) in 10 ml of H20 were treated with 2.5 g of tained with N-methylation in position 4, which drastically reduced CuSO4-5H20 (10 mmol) in 10 ml of H20. The mixture was the biological activity and produced remarkable changes in the shaken on a wrist shaker for 10 min, and 60 ml of MeOH was peptide backbone and a severe limitation in rotational freedom of the side chains in tyrosine. Thus, it may be possible to synthesize added, followed by 2.38 ml of C6H5CH2Br (20 mmol). The flask potent angiotensin II analogs that have greater resistance to en- was stoppered, and the mixture was shaken for 20 hr at room zymatic degradation by a-methylation in position 4 (or 5) and si- temperature. The copper complex was removed by filtration, multaneous suitable modification at the NH2 and COOH termini. washed with three 10-ml portions of H20, two 10-ml portions of MeOH, and two 10-ml portions of Et2O. The semi-air-dried The antagonists for the pressor hormone angiotensin II (Asp- product (6.2 g) was suspended in 40 ml of 1 M HCl, pulverized, Arg-Val-Tyr-Ile-His-Pro-Phe; All) have proved useful in the and filtered out. The precipitate was repeatedly macerated with study ofexperimental hypertension and as possible new clinical 1 M HC1 and filtered out until the filtrate was almost colorless. diagnostic tools (1). However, their long-term application has The residue was then stirred with 20 ml of 16.4% AcONa, fil- been limited because they have a short in vivo half-life and, due tered out, washed with hot water, and dried at reduced pressure to degradation bypeptidases, are not orally active. We therefore over P205. O-Benzyl-a-methyl-L-tyrosine thus obtained was attempted to make these peptides resistant to this enzymatic homogeneous: thin layer chromatography (TLC) on silica gel, degradation by replacing the natural amino acid residues with RF 0.43 in n-BuOH/AcOH/H20 (4:1:5); RF 0.57 in n-PrOH/ N-methylamino (2) or /3homoamino (3) acid residues. These H20 (1:1). modifications drastically reduced the biological activity ofthese tert-Butyloxycarbonyl-O-Benzyl-a-Methyl-L-Tyrosine. A analogs, and conformation studies suggest that N-methylation suspension of 1.42 g ofO-benzyl-a-methyl-L-tyrosine (5 mmol) in positions 4 or 5 results in remarkable changes in the peptide in 30 ml of Me2SO was treated with 0.72 ml of NEt3 (5 mmol) backbone and a severe limitation in the rotational freedom of and 0.3 g oftert-butyloxycarbonyl azide (0.3 g), and the mixture the side chains in tyrosine, isoleucine, and histidine residues was stirred at 40°C under anhydrous conditions for 24 hr. The (4). The replacement ofan a proton by an a methyl group, how- addition ofBoc-N3 (0.3 g) and NEt3 (0.72 ml) was repeated every ever, is thought to produce minimal changes in backbone and 24 hr until the solid went into solution (1 week or more). The side-chain structures. The analogs thus obtained might mimic solution was diluted with 40 ml of H20, treated with NaOH to the parent hormone in recognizing and binding with the re- pH 9, and extracted with three 25-ml portions of Et2O. The ceptor on the cell membrane and, in addition, be stable to en- aqueous layer was evaporated to dryness at 20°C at reduced zymatic degradation (4, 5). Based on this hypothesis, we have pressure on a rotary evaporator. The residue was dissolved in synthesized [a-methyltyrosine4]angiotensin II ([a-MeTyr4]AII). 7 ml ofH20, and the solution was cooled to 0°C and treated with And, indeed, this peptide is resistant to chymotrypsin degra- ice-cold citric acid solution to pH 4.5. The solution was then dation and yet retains almost the full pressor activity of All. saturated with solid NaCl and extracted with AcOEt; the organic Conformation studies suggest minimum changes in backbone phase was washed with H20 and then with saturated NaCl so- and side-chain structures. These results again suggest that, for lution, dried with Na2SO4, and evaporated to yield 2.36 g of The publication costs ofthis article were defrayed in part by page charge Abbreviations: AII, angiotensin II; [a-MeTyr4]AII, [a-methyltyrosine- payment. This article must therefore be hereby marked "advertise- 4]angiotensin II; TLC, thin layer chromatography; CD, circular ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. dichroism. 757 Downloaded by guest on September 29, 2021 758 Biochemistry: Khosla et aL Proc. Natt Acad. Sci. USA 78 (1981) product: TLC on silica gel,RF0.67 in EtOH; RF0.81 in CHC13/ RESULTS AcOH (95:5); RF0.73 in CHClJMeOH/AcOH (85:10:5). [a-MeTyr4]AII. COOH-Terminal tert-butyloxycarbonyl-L- 'H NMR. In addition to the conformational information that phenylalanine was attached to 2% crosslinked chloromethyl can be extracted from the 'H NMR spectrum, one can ascertain polymer as described, and chain elongation was performed on the correct chemical nature of the analog studied. Comparison a manual nitrogen-stirred apparatus (6) by using the protocol of the spectrum of[a-MeTyr4]AII with that of All shows (i) the described (7). In general, the coupling reaction was carried out appearance of a methyl group single peak at 1.31 ppm; (ii) the twice for 6-12 hr each time. 1-Hydroxybenzotriazole (2 mmol disappearance of the tyrosine a-proton octuplet (4.52 ppm in per mmol of tert-butyloxycarbonylamino acid) was used as an All), (iii) the transformation of the amide proton doublet of ty- additive during coupling with N,N-dicyclohexylcarbodiimide. rosine (8.07 ppm in All) into a single peak at 7.47 ppm; (iv) an Apart from facilitating the coupling reaction, this procedure also upfield shift of the aromaticmeta protons oftyrosine (0.26 ppm avoids racemization of the histidine residue during coupling with respect to All); and (v) that all the othera-protons exhibit with tert-butyloxycarbonyl-Nim-benzylhistidine (8). Coupling the same chemical shifts and patterns as in the native peptide with tert-butyloxycarbonylvaline was difficult and was repeated (Fig. 1). These data leave no doubt as to the correct chemical three times by using a 3-fold excess of this derivative and a 6- structure of the peptide [a-MeTyr4]AII. fold excess of 1-hydroxybenzotriazole each time. At the end of The majority ofthe proton resonances are identical in All and the synthesis, the peptide was removed from the polymer with la-MeTyr4]AII, but differences in the chemical shifts are de- HBr/CF3COOH and hydrogenated over 5% palladium/BaSO4 tected on the NH and sidechain protons of the tyrosine-neigh- in MeOH/AcOH/H20 (5:1:1). The crude product was purified bor residues valine-3 and isoleucine-5. It is reasonable to at- on a column of Bio-Rad anion exchange resin (AG-1 X 2, tribute these differences to the inductive effect ofthe a-methyl 200-400 mesh, acetate form) by eluting with ammonium acetate group and to a slight reorientation and decrease of rotational buffer (pH 8.5). Fractions containing the major component properties of the tyrosine side chain (ring-current effects). were pooled, lyophilized, and rechromatographed on succes- Two types of conformationally related vicinal coupling con- sive columns of Sephadex G-25 by using n-BuOH/pyridine/ stants were measured in [a-MeTyr4]AII and compared with H20 (65:35:35, upper phase) and n-BuOH/AcOH/H20 (4:1:5, those of All: the JNH-CHa value, which is tied to the dihedral upper phase) as the solvent systems.
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