Improvement of ACE inhibitory activity of chitooligosaccharides (COS) by carboxyl modification

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Abstract

In the present research, chitooligosaccharides (COS) were carboxylated with –COCH2CH2COO groups to obtain specific structural features similar to Captopril®. Angiotensin I converting enzyme (ACE) inhibitory activity of carboxylated COS was studied and observed to enhance its activity with increased substitution degree. Further, Lineweaver–Burk plot analysis revealed that inhibition was competitive via obligatory binding site of the enzyme. This was accompanied with substitution of positively charged quarternized amino groups to COS with different substitution degrees, in which negative impact on ACE inhibition was observed.

Graphical abstract

COCH2CH2COO group, which is similar to Captopril® in structure, was introduced to chitooligosaccharides (COS). Angiotensin I converting enzyme (ACE) inhibitory activity of COS was successfully improved by the introduced group. ACE inhibitory mechanism of COS derivative was competitive via obligatory binding site of the enzyme.

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Introduction

Hypertension is a major risk factor for the development of cardiovascular diseases and mortality in Western countries.1 Angiotensin I converting enzyme (ACE; EC 3.4.15.1), is responsible for hypertension by producing vasoconstrictor angiotensin II and degrading vasodilator, bradykinin. Therefore, inhibition of ACE is considered to be an important therapeutic approach for controlling hypertension. Initial interest in ACE inhibitors began with the discovery of snake venom peptides that could compete well with angiotensin I, the natural substrate of ACE.2, 3 With the model development of catalytic structure of ACE, specific inhibitors that bind more precisely to the enzyme active site were developed.4 Even though the synthetic inhibitors are remarkably effective as antihypertensive drugs, they often result adverse side effects. Certain functional foods containing ACE inhibitory compounds have exhibited to act as alternative treatment for hypertension.5 Therefore, during the past decades fundamental studies have opened a new field of study searching for ACE inhibitors from natural bioresources.6, 7

Chitosan is a biodegradable, non-allergenic deacetylated derivative of chitin, a polysaccharides abundantly found in nature. Numerous studies have demonstrated that chitosan has various biological activities such as antimicrobial, antitumor, and immune enhancing effects.8 Chitooligosaccharides (COS), partially hydrolyzed products of chitosan are of great interest in pharmaceutical and medicinal applications due to high solubility and non-toxicity. Moreover, recent advances in understanding the structure and properties of chitosan and its derivatives have opened new avenues for its applications.9 Improvement of structural properties of chitosan for a particular application can be easily brought about by chemical modifications. However, researches on synthesis of COS derivatives and identification of their biological activities have not been reported so often. Therefore, studies aimed for developing new COS derivatives and to test their bioactivities are of interest.10, 11

Recently, ACE inhibitory activity of COS was identified and brought about a new non-peptidic group of competitive inhibitors.12 However, properties of COS that would be beneficial to interact with the active site of ACE, are still remain to be elucidated and further studies on this would contribute to the knowledge for the development of safe and novel ACE inhibitors. According to already confirmed interactions of ACE inhibitors such as Captopril® and Lisinopril® with ACE, it can be presumed that specific structural characteristics along with higher negative charge density contribute for a greater affinity to the enzyme.13 Therefore, in the present research we introduced negatively charged carboxyl group to COS with structural similarities to Captopril® (see CCOS-1–3 in Scheme 1) and hypothesized that it would increase ACE inhibitory activity. In addition, positively charged quaternized amino groups were also separately introduced to COS and studied their influence on ACE inhibition (see ECOS-1–3 in Scheme 1).

Section snippets

Structural confirmation of new COS derivatives

Substitution of carboxyl or quaternized amino groups was clearly confirmed by 13C NMR, 1H NMR, and FT-IR spectra of COS derivatives. In comparison to the FT-IR spectrum of COS (Fig. 1A), both symmetric and asymmetric stretch absorptions of carboxyl groups (1560 cm−1 and 1410 cm−1, respectively, as in Fig. 1B),14, 15, 16 and the bend absorption of N(CH3)3+ (1480 cm−1 as in Fig. 1C)17, 18 confirmed the successful introduction of new groups. In addition, different substitution degrees of carboxyl and

Conclusions

In this study we found a facile way to modify the structure of COS and thereby to improve its ACE inhibitory activity. For this purpose, synthesis of carboxylated COS was carried out under mild conditions in which no possibilities for adverse influences on structural changes occurred. Therefore, substitution of OOC–CH2CH2–CO–Ndouble bond was predominantly under control. Carboxyl groups were identified to be beneficial for ACE inhibition by enhancing the binding ability of COS to the obligatory active

Materials and instruments

COS was kindly donated by Kitto. Life Co. (Seoul, Korea) with an acetylation degree of 27.92% (measured by elemental analysis) and molecular weight range 6.0–7.0 × 103 (measured by MALDI-TOF mass spectrometry).21 2,3-Epoxypropyl trimethylammonium chloride was prepared from 2,3-epoxypropyl chloride and trimethyl amine aqueous solution.19 All commercial reagents including succinic anhydride, ACE from rabbit lung and its substrate (hippuryl-histidyl-leucine) were obtained from Sigma Chemical Co.

Acknowledgements

This work was supported by the Brain Korea 21 project. The author also got support from APEC Postdoc Program funded by Korean Science and Engineering Foundation.

References and notes (23)

  • A. Rousseau-Plasse et al.

    Bioorg. Med. Chem.

    (1996)
  • M. Bala et al.

    Bioorg. Med. Chem.

    (2002)
  • H. Ronghua et al.

    Carbohydr. Polym.

    (2003)
  • R.A.A. Muzzarelli

    Carbohydr. Res.

    (1982)
  • H. Ronghua et al.

    Carbohydr. Polym.

    (2003)
  • D.W. Cushman et al.

    Biochem. Pharmacol.

    (1971)
  • D. Duprez et al.

    J. Hum. Hypertens.

    (2002)
  • M.A. Ondetti et al.

    Biochemistry

    (1971)
  • D.W. Cushman et al.

    Hypertension

    (1991)
  • L.A. Goretta et al.

    FEBS Lett.

    (2003)
  • D.H. Lee et al.

    Peptides

    (2004)
  • Cited by (0)

    View full text