The Chemical Synthesis of Pepstatin A

Morishima, Hazime; Takita, Tomohisa; Umezawa, Hamao

doi:10.7164/antibiotics.25.551

Cited by 16 publications

(6 citation statements)

References 4 publications

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“…Pepstatin is a naturally occurring inhibitor of aspartyl proteinases (1); it has the amino acid composition isovaleryl (Iva)-ValVal-Sta-Ala-StaOH, in which Sta is the residue of statine [(4S,3S)-4-amino-3-hydroxyl-6-methylheptanoic acid] (2). The interaction ofpepstatin with aspartyl proteases is characterized by small dissociation constants [i.e., 4.6 x 10-11 M with porcine pepsin (3) and 1.5 x 10-10 M with penicillopepsin (unpublished results)].…”

mentioning

confidence: 99%

Conformational flexibility in the active sites of aspartyl proteinases revealed by a pepstatin fragment binding to penicillopepsin.

James¹,

Sielecki²,

Salituro³

et al. 1982

Proc. Natl. Acad. Sci. U.S.A.

158

123

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BiochemistryConformational flexibility in the active sites of aspartyl proteinases revealed by a pepstatin fragment binding to penicillopepsin ( Communicated by Joseph S. Fruton, June 21, 1982 ABSTRACT Crystals of the molecular complex between the esterified tripeptide fragment of pepstatin and the aspartyl proteinase penicillopepsin are isomorphous with crystals of native penicillopepsin. The difference electron-density map at 1.8-A resolution, computed by using the amplitude differences and refined phases of reflections from the crystal of native penicillopepsin, unambiguously showed the binding mode of isovaleryl Pepstatin is a naturally occurring inhibitor of aspartyl proteinases (1); it has the amino acid composition isovaleryl (Iva)-ValVal-Sta-Ala-StaOH, in which Sta is the residue of statine [(4S,3S)-4-amino-3-hydroxyl-6-methylheptanoic acid] (2). The interaction ofpepstatin with aspartyl proteases is characterized by small dissociation constants [i.e., 4.6 x 10-11 M with porcine pepsin (3) and 1.5 x 10-10 M with penicillopepsin (unpublished results)]. It Structure I Penicillopepsin is an aspartyl proteinase isolated from the mold Penicillium janthinellum. Its crystal structure (6, 7) has been refined at 1.8-A resolution (8) to a conventional R-factor § of 0.136 for those reflections with I, the intensity of Bragg reflections, equal to or greater than the estimated standard deviation of the intensity measurement derived from counting statistics [I 2 o-(I)]. The aspartyl proteinases are characterized by a long binding cleft that can accommodate 7 or 8 amino acid residues of an oligopeptide substrate in an extended conformation. The two catalytically important aspartyl residues, Asp&jand Asp-213, are centrally located in this binding cleft (Asp32and Asp-215 in the porcine pepsin sequence numbering).Fluorescence measurements on the aspartyl proteinases with specific substrates have indicated that conformational mobility of groups in the active site may play an important role in the mechanism of these enzymes (9). MATERIALS AND METHODSThe enzyme, penicillopepsin, was prepared, purified, and crystallized as described (6,7,10). Crystals of the native enzyme The pepstatin derivative Iva-Val-Val-StaOEt was synthesized by the following procedure. t-Butoxycarbonyl (Boc)-StaOEt (12) was deprotected with 4 M HCl in dioxane and sequentially coupled with Boc-L-valine, Boc-L-valine, and isovaleryl anhydride by using procedures previously described for-the synthesis of pepstatin analogues (13) 6137The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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mentioning

confidence: 99%

Conformational flexibility in the active sites of aspartyl proteinases revealed by a pepstatin fragment binding to penicillopepsin.

James¹,

Sielecki²,

Salituro³

et al. 1982

Proc. Natl. Acad. Sci. U.S.A.

158

123

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“…Most of the other p-nitrophenyl esters were prepared as described previously.3 p-Nitrophenyl cyanoacetate was prepared by condensation of cyanoacetic acid (Aldrich) and p-nitrophenol in the presence of dicyclohexylcarbodiimide following the general procedure of Holmquist and Bruice. 5 The ester was purified by flash chromatography ( (Aldrich). The acyl chloride (10 g, 0.09 mol) was added to 250 mL of ice-cold ethyl acetate containing p-nitrophenol (10 g, 0.07 mol).…”

Section: Methodsmentioning

confidence: 99%

“…84 (3 , H16), 2.16 (3 H, Ac), 2.22 (H0, J4,4 = 15.0 Hz, J3,4Í = II. 2 Hz), 2.35 (H4a, t/3l4a = 4.4 Hz), 3.14 (H13), 3.18 (H13, c/x313 = 4.2 Hz), 3.85 (H), 3.93 (H2, J23 = 4.3 Hz), 4.29 (H16), 4.58 (H1S, J1516 = 12.0 Hz), 4.84 (Hu, J1011 = 5.7 Hz), 4.85 (H7), 5.25 (H3), 6.52 ( ), 6.61 (H10), 7.10-7.34 (5 H). C^H^NOg requires: C, 63.0; H, 5.95; N, 3.1. Found: C, 63.1; , 6.0; N, 3.0. Acetylation of 3-O-Acetyl-DON.…”

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confidence: 99%

Acyl substituent effects on ester aminolysis

Knowlton¹,

Byers²

1988

J. Org. Chem.

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“…Ackerman and Douglas have recently reported that a purified microbial peptide is a potent chemotactic agent for human leukocytes (1). This agent, pepstatin, is an N-blocked hydrophobic pentapeptide isolated from culture filtrates of actinomycetes (15) which has the amino acid sequence: N-isovaleryl-L-valyl-Lvalyl-AHMHA-L-alanyl-AHMHA (9), where AHMHA denotes the unusual amino acid 4amino-3-hydroxy-6-methyl heptanoic acid. To determine the relationship between the chemotactic properties of pepstatin and the f-methionyl peptides, we have considered the possibility that these cytotaxins share a common receptor site on the neutrophil membrane.…”

mentioning

confidence: 99%

Cytotaxin receptors of neutrophils: evidence that F-methionyl peptides and pepstatin share a common receptor

Nelson¹,

Ackerman²,

Fiegel³

et al. 1979

Infect Immun

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Pepstatin, a chemotactic microbial pentapeptide, competes with f-Met-Leu-[3H]Phe for binding to human neutrophils. Furthermore, porcine neutrophils, which neither specifically bind nor respond chemotactically to the synthetic fmethionyl peptides, also fail to respond chemotactically to pepstatin. These results suggest that pepstatin shares a receptor on the neutrophil with f-methionyl peptides, despite their completely different amino acid compositions. The specificity of this cytotaxin receptor may therefore be broader than expected and depend on ligand characteristics distinct from primary structure.

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The Chemical Synthesis of Pepstatin A

Cited by 16 publications

References 4 publications

Conformational flexibility in the active sites of aspartyl proteinases revealed by a pepstatin fragment binding to penicillopepsin.

Conformational flexibility in the active sites of aspartyl proteinases revealed by a pepstatin fragment binding to penicillopepsin.

Acyl substituent effects on ester aminolysis

Cytotaxin receptors of neutrophils: evidence that F-methionyl peptides and pepstatin share a common receptor

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