A 13C-NMR study of the inhibition of papain by a dipeptide-glyoxal inhibitor

Z-Phe-Ala-glyoxal (where Z is benzyloxycarbonyl) has been synthesized and shown to be a competitive inhibitor of papain with a Ki = 3.30±0.25nM. 13C-NMR has been used to show that in aqueous media, Z-Phe-[2-13C]Ala-glyoxal gives signals at 207.7p.p.m. and 96.3p.p.m. showing that both the α-keto carbon and its hydrate are present. When this inhibitor is bound to papain a single signal at 209.7p.p.m. is observed due to the 13C-enriched carbon. This demonstrates that the glyoxal α-keto carbon is not hydrated when it is bound to papain and that it does not form a thiohemiketal with the thiol group of Cys-25. Z-Phe-[1-13C]Ala-glyoxal has also been synthesized and its aldehyde carbon is fully hydrated in aqueous solution giving signals at 88.7p.p.m. and 90.2p.p.m. when the α-keto carbon and its hydrate are present respectively. When this inhibitor is bound to papain a single signal at 71.04p.p.m. was observed due to the 13C-enriched carbon showing that the 13C-enriched aldehyde carbon forms a thiohemiacetal with Cys-25.

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13C-NMR study of the inhibition of δ-chymotrypsin by a tripeptide-glyoxal inhibitor

Biochemical Journal ◽

10.1042/bj3620339 ◽

2002 ◽

Vol 362 (2) ◽

pp. 339-347 ◽

Cited By ~ 7

Author(s):

Aleksandra DJURDJEVIC-PAHL ◽

Chandralal HEWAGE ◽

J. Paul G. MALTHOUSE

Keyword(s):

13C Nmr ◽

Exchange Process ◽

Competitive Inhibitor ◽

Minor Role ◽

Oxyanion Hole ◽

Inhibitor Complex ◽

Rapid Exchange ◽

Nmr Study ◽

A Minor ◽

Hydroxy Groups

A new inhibitor, Z-Ala-Pro-Phe-glyoxal (where Z is benzyloxycarbonyl),has been synthesized and shown to be a competitive inhibitor of δ-chymotrypsin, with a Ki of 25±8nM at pH7.0 and 25°C. Z-Ala-Pro-[1-13C]Phe-glyoxal and Z-Ala-Pro-[2-13C]Phe-glyoxal have been synthesized, and 13C-NMR has been used to determine how they interact with δ-chymotrypsin. Using Z-Ala-Pro-[2-13C]Phe-glyoxal we have detected a signal at 100.7p.p.m. which we assign to the tetrahedral adduct formed between the hydroxy group of Ser-195 and the 13C-enriched keto-carbon of the inhibitor. This signal is in a pH-dependent slow exchange with a signal at 107.6p.p.m. which depends on a pKa of ∼ 4.5, which we assign to oxyanion formation. Thus we are the first to detect an oxyanion pKa in a reversible chymotrypsin—inhibitor complex. A smaller titration shift of 100.7p.p.m. to 103.9p.p.m. with a pKa of ∼ 5.3 is also detected due to a rapid exchange process. This pKa is also detected with the Z-Ala-Pro-[1-13C]Phe-glyoxal inhibitor and gives a larger titration shift of 91.4p.p.m. to 97.3p.p.m., which we assign to the ionization of the hydrated aldehyde hydroxy groups of the enzyme-bound inhibitor. Protonation of the oxyanion in the oxyanion hole decreases the binding efficiency of the inhibitor. From this decrease in binding efficiency we estimate that oxyanion binding in the oxyanion hole reduces the oxyanion pKa by 1.3 pKa units. We calculate that the pKas of the oxyanions of the hemiketal and hydrated aldehyde moieties of the glyoxal inhibitor are both lowered by 6.4–6.9 pKa units on binding to chymotrypsin. Therefore we conclude that oxyanion binding in the oxyanion hole has only a minor role in decreasing the oxyanion pKa. We also investigate how the inhibitor breaks down at alkaline pH, and how it breaks down at neutral pH in the presence of chymotrypsin.

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13C NMR Study of Poly(N-vinylpyrrolidone)–Cu(II) Complex in Aqueous Solution

Polymer Journal ◽

10.1295/polymj.26.479 ◽

1994 ◽

Vol 26 (4) ◽

pp. 479-483 ◽

Cited By ~ 2

Author(s):

Ken-ichi Tsuchiya ◽

K Takegoshi ◽

Kunio Hikichi

Keyword(s):

Aqueous Solution ◽

13C Nmr ◽

Nmr Study

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Room Temperature Suzuki Reactions in Aqueous Media under Air by Palladium(II) Complexes with Pyrazole Derived Ligands

Australian Journal of Chemistry ◽

10.1071/ch09383 ◽

2010 ◽

Vol 63 (2) ◽

pp. 315 ◽

Cited By ~ 6

Author(s):

Kai Xu ◽

Xin-Qi Hao ◽

Jun-Fang Gong ◽

Mao-Ping Song ◽

Yang-Jie Wu

Keyword(s):

Aqueous Solution ◽

13C Nmr ◽

Room Temperature ◽

Phenylboronic Acid ◽

Aqueous Media ◽

Palladium Complexes ◽

Coupling Reactions ◽

X Ray ◽

Suzuki Reactions ◽

Aryl Bromides

Two new palladium complexes with pyrazole derived ligands 2a–2b have been easily prepared and well characterized by elemental analysis, 1H NMR, 13C NMR, and IR spectra. Their detailed structures are determined by a single-crystal X-ray analysis of 2a. The two compounds were successfully applied to the Suzuki coupling reactions of aryl bromides with phenylboronic acid, in aqueous solution at room temperature under air, giving the desired coupled products in good to excellent yields with catalyst loadings as low as 0.01–0.05 mol-%.

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