Process Biochemistry 43 (2008) 119123 www.elsevier.

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Aminoethyl chitooligosaccharides inhibit the activity of angiotensin converting enzyme

Dai-Nghiep Ngo a, Zhong-Ji Qian a, Jae-Young Je b, Moon-Moo Kim c, Se-Kwon Kim a,d,*
b a Department of Chemistry, Pukyong National University, Busan 608-737, Republic of Korea Division of Food Science and Aqualife Medicine, Chonnam National University, Yeosu 550-749, Republic of Korea c Department of Chemistry, Dong-Eui University, Busan 614-714, Republic of Korea d Marine Bioprocess Research Center, Pukyong National University, Busan 608-737, Republic of Korea

Received 21 June 2007; received in revised form 10 October 2007; accepted 30 October 2007

Abstract In the present research, chitooligosaccharides (COS) with molecular weight 8003000 Da and 90% of deacetylation were chemically modied by grafting 2-chloroethylamino hydrochloride at C-6 position to synthesize angiotensin I converting enzyme (ACE) inhibitory chitin derivatives based on the properties of ACE inhibitors. The synthetic product was designated as aminoethyl chitooligosaccharide (AE-COS) with molecular weight 800.794765 Da. Its IC50 value on ACE was 0.8017 mg/mL. Furthermore, Lineweaver-Burk plots revealed that the inhibition was competitive via obligatory binding site of ACE. Therefore, these results exhibited that substitution of the hydrogen atom at the C-6 position of pyranose residue by the aminoethyl group promotes ACE inhibitory effects of COS. # 2007 Elsevier Ltd. All rights reserved.
Keywords: Chitooligosaccharides (COS); Aminoethyl COS; Competitive inhibitor; Angiotensin I converting enzyme (ACE); Chitosan; Chitin

1. Introduction Hypertension is one of the major risk factor for the development of cardiovascular diseases, stroke and the end stage renal disease [1]. Angiostensin I converting enzyme (peptidylpeptide hydrolase, EC 3.4.15.1, ACE) belongs to the class of zinc proteases that need zinc and chloride for its enzymic activation. It plays an important physiological role in regulating blood pressure by converting an inactive form of decapeptide, angiotensin I, to a potent vasocontrictor, octapeptide, angiotensin II, and by inactivating catalytic function of bradykinin, which has depressor action. Therefore, inhibiton of ACE is considered to be an important therapeutic approach for controlling hypertension [2]. More recently, with the model development of catalytic structure of ACE, specic inhibitors that can bind to the enzyme active site have been developed. Although some synthetic inhibitors are remarkably effective as antihypertensive drugs [3], they often result in adverse side effects. Therefore, a number of natural ACE

* Corresponding author. Tel.: +82 51 6206375; fax: +82 51 6288147. E-mail address: sknkim@pknu.ac.kr (S.-K. Kim). 1359-5113/$ see front matter # 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2007.10.018

inhibitors have been isolated from functional food and natural bioresources [4]. At present, most of natural ACE inhibitors are peptides which have been isolated from various protein hydrolysates such as wakame, casein, fermented soymilk, cheese whey, soybean, corn gluten [5], yellown sole, bonito meat, Alaska pollack, short-necked clam and pearl oyster, and bovine plasma proteins [6], while only a few non-peptidic ACE inhibitors have been reported. Chitosan, a partially deacetylated polymer of Nacetyl glucosamine, is prepared with alkaline deacetylation of chitin, a polysaccharide abundantly found in nature. It has a number of biological functions such as antitumor effects [7], immuno-enhancing effects [8], antimicrobial activity [9], antioxidant effects, and antihypertensive effects. Chitooligosaccharides (COS), partially hydrolyzed products of chitosan are of great interest in pharmaceutical and medicinal applications due to its non-toxic and high solubility properties [8]. Moreover, the structure and properties of chitosan and its derivatives are being understood clearer and clearer. This point helped to improve the structural properties of chitosan for a particular application by chemical modications. However, researches on synthesis of COS derivatives and identication of their biological activities have been seldom reported. Therefore, our aim was to develop a new COS derivative with

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D.-N. Ngo et al. / Process Biochemistry 43 (2008) 119123 various incubation times following the above procedure. The Lineweaver-Burk plot was plotted as 1 V (min/OD at 228) against inhibitor concentration.

improved ACE inhibitory activity, and to study its inhibitory mechanism.

2. Materials and methods 2.1. Materials
Chitooligosaccharides (MW 8003000 Da, degree of deacetylation, DD, 90%) prepared from crab shells were donated by Kitto Life Co. (Seoul, Korea). ACE (from rabbit lung) and substrate peptide (hippurylhistidylleucine) of ACE, 2-chloroethylamino hydrochloride were purchased from Sigma Chemical Co. (St. Louis, MO). All other reagents were of the highest grade available commercially.

2.6. Statistics
All assays were carried out in triplicate, and results are reported as means standard deviation.

3. Results and discussion 3.1. Synthesis of AE-COS Chitooligosaccharides, hydrolytic products of chitosan, have amino groups or acetamide groups at C-2 position depending on their degree of acetylation. Partly based on these functional groups, they have exhibited a number of biological activities such as antibacterial activity, antifungal activity [8], antitumor activity, immunoenhancing effects [12], protective effects against infections and enzyme inhibition [2]. In this study, AE-COS were synthesized by grafting aminoethyl functionality to improve its ACE inhibitory potential. It was known that the hydroxyl groups of pyranose ring structure at different positions are different chemical attraction with the same one reagent in the one reaction. Herein, the hydroxyl group at C-6 was successfully replaced by aminoethyl group while the structure of COS was maintained because the C-6 hydroxyl groups had the highest reactivity for aminoethylation and the product was completely dissolved in water. The 1H NMR spectra were used to conrm the existence of substituted group. As shown in Fig. 1, a new chemical shift that appeared in the spectrum of AE-COS at 2.8 ppm was assigned to proton of CH2N. The peak at 2.0 ppm was residual acetyl peak, and protons of pyranose unit superimpose the NH2 of aminoethyl group (2.93.6 ppm). 3.2. ACE inhibitory activities of AE-COS Recently, a number of ACE inhibitory peptides, which were isolated from various food proteins, have been extensively studied, because food components are closely related to hypertension and these peptides may be associated with the presence of an antihypertensive peptide motif in controlling hypertension. In addition, following the discovery of captopril in 1977, a lot of potential ACE inhibitors have been synthesized and used extensively in the treatment of essential hypertension and heart failure in humans as enalapril, perindopril, lisinopril, fosinopril, etc [13]. Howerver, the carbohydrate-based ACE inhibitors have not been clearly reported until now. In this study, AE-COS with molecular mass ranging between 800.79 and 4765 Da was prepared from COS with molecular weight 8003000 Da by grafting aminoethyl groups at C-6 position of pyranose ring. Therefore, molarcular weight of AECOS increased and its effects on ACE inhibition were investigated (Fig. 2). AE-COS exhibited an 89.3% ACE inhibition at 2.5 mg/mL and its IC50 values was determined as 0.8017 mg/mL. The ACE inhibitory activities of COS and recognized that all COS exhibit ACE inhibitory activity.

2.2. Synthesis of aminoethyl chitooligosaccharides (AE-COS)

AE-COS were prepared by modifying our previous method [10] as shown in Scheme 1. Briey, aqueous 3.0 M (20 mL) 2-chlorethylamino hydrochloride was added to COS (0.40 g) while stirring at 40 8C. NaOH (3.0 M, 20 mL) was added to the reaction mixture dropwise, and continuously stirred for 48 h. After reaction, the solution was ltered using a lter paper. Subsequently, the reaction mixture was acidied with 0.1 N HCl, and dialyzed against water for 2 days. The product was freeze dried to give AE-COS (0.334 g).

2.3. Characterization of AE-COS

AE-COS were characterized by 1HNMR. 1HNMR measurements were performed on a JEOL JNM ECP-400 NMR spectrometer under a static magnetic eld of 400 MHz and chemical shift values are given in y (ppm). Molecular masses of AE-COS were determined by MALDI-TOF mass spectrometry on a Voyager mass spectrometer (Applied Biosystems, USA) using 2, 5dihydroxybenzoic acid as the matrix.

2.4. ACE inhibitory assay

ACE inhibitory activity was measured by the method of Cushman and Cheung [11] with slight modications. Briey, a sample solution (50 mL) was preincubated at 37 8C for 10 min with 50 mL of ACE solution (25 mU/mL), and the mixture was incubated with 150 mL of substrate (4.15 mM HipHisLeu in 50 mM sodium borate buffer containing 0.5 M NaCl at pH 8.3) for another 30 min at the same temperature. Finally, the reaction was terminated by the addition of 250 mL of 1.0 M HCl and the resulting hippuric acid was extracted with 0.5 mL of ethylacetate. After centrifugation (2000 g for 10 min), 200 mL of the upper layer was transferred into a test tube and evaporated at room temperature for 2 h in a vacuum. The hippuric acid was dissolved in 1.0 mL of distilled water, and the absorbance was measured at 228 nm using an UV-spectrophotometer (Tecan Austria GmbH, Austria). The IC50 value was dened as the concentration of inhibitor required to inhibit 50% of the ACE activity.

2.5. ACE inhibitory pattern of AE-COS

To clarify the inhibitory mechanism of AE-COS on ACE, Lineweaver-Burk plots were plotted with two different concentrations of the inhibitor and three different concentrations of ACE substrate (4.15, 2.075, and 1.0375 mM) with

Scheme 1. Synthesis of aminoethyl-chitooligosaccharides (AE-COS).

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Fig. 1. 1H NMR spectrum (400 MHz) of AE-COS in D2O.

Specically the trimer had a lower IC50 value (0.9 mM) than the other molecular weight COS [14]. Also Park et al. [2] investigated ACE inhibitory activities of different molecular weight COS those were produced using an ultraltration membrane bioreactor system. Their results showed that medium molecular weight COS (15 kDa) with 50% deacetylation possess the highest ACE inhibitory activity (1.22 mg/ mL) compared to high molecular weight COS (510 kDa) and low molecular weight COS (below 1 kDa). Moreover, they also compared ACE inhibitory effects of different COS from chitosan having 90%, 75% and 50% degree of deacetylation (DD) and concluded that DD of chitosans inuence their ACE inhibitory activity. These results suggested that the molecular weight and DD of COS are important factors of ACE inhibition.

Furthermore, Huang et al. [15] synthesized carboxyl COS having negative charge and similar structure to Captopril. In a previous study, our group synthesized aminoethyl chitin for ACE inhibition [16]. Most of the synthezied products increase ACE inhibitory activity higher than their original starting compounds. Also in this study, ACE inhibitory activity of AECOS has increased more than that of COS. At 2.5 mg/mL concentration, COS and AE-COS exhibited ACE inhibitory activities of 18.6% and 89.3%, respectively (Fig. 2). Furthermore, a marked dose-dependent inhibitory effect was observed in both COS and AE-COS treatment groups, which was consistent with previous reports [15,16].

Fig. 2. ACE inhibitory activity of AE-COS and COS at different concentrations. Experiment was carried out in the presence or absence (control) of AECOS or COS as described in the text using HHL as the enzyme substrate for 30 min at 37 8C.

Fig. 3. Lineweaver-Burk plot for the determination of inhibitory mode of ACE by AE-COS. ACE inhibitory activity was determined in the presence (0.75 and 1.50 mg/mL) or absence of AE-COS as described in the text using HHL as the enzyme substrate.

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Fig. 4. Hypothesized interaction between AE-COS and obiligatory site of ACE. S1 and S01 , S02 are subsite or pocket in the structure of ACE active site. Zn++ ion and X H are postulated hydrogen bond donors.

3.3. Determination of the inhibitory pattern on ACE ACE inhibitory pattern of the AE-COS was investigated by Lineweaver-Burk plots and was found to be competitive (Fig. 3). It means that AE-COS competes with the substrate to bind with the active site of ACE. Among the reported natural peptides for inhibition ACE activity, the most potent peptides possessed similar structures with the peptides isolated from the venom of pit viper. The interactional model between the substrate and active site of ACE was proposed by Ondetti and Cushman [17]. According to this model, the C-terminal tripeptide may interact with three subsites or pockets at active site of ACE. ACE appears to prefer substrates or competitive inhibitors which contain hydrophobic amino acids at three positions of the C terminal [18]. In general, ACE inhibiors contain one or more of the following functional groups such as a zinc binding ligand (normally a hydroxamic acids, thiols, phosphinyl and carboxyl group, ketones) [13], a hydrogen bond donor [3] and carboxyl terminal group [19]. Lisinopril, a synthetic ACE inhibitor, competes with the natural substrate of the enzyme for several binding sites in the catalytic domain and buried in the active site with interaction directly with the zinc ion by carboxyl group and hydrogen bonds by the central carbonyl and amino group [20]. In the case of COS, it can possess ion chelating ability and interact with the zinc ion of the active site easily. Furthermore, the OH and NH2 group may interact with the hydrogen of the enzyme binding site via hydrogen bonding [14]. In this study, AE-COS has pyranose residues which contain 90% amino groups and 10% acetyl groups; it may interact via hydrogen bonding and chelating zinc ion in ACE active site. Furthermore, AE-COS has an amino group in the aminoethyl group to substitute the hydrogen atom at C-6 position. This structure may interact with S01 porket of ACE active site (Fig. 4) and act as one of the major determinant of ACE inhibitory activity [13]. Therefore, it was presumed to be the higher ACE inhibitory effect observed for AE-COS than COS and its competitive structural interactions with ACE active site. This is further in agreement with previous similar reports [16].

4. Conclusion In this study, we found a facile way to modify the structure of COS and thereby to improve its ACE inhibitory activity. For this aim, the synthesis of AE-COS was carried out under mild conditions, and it exhibited a higher ACE inhibitory activity than COS. Moreover, ACE inhibitory pattern of AE-COS was found to be competitive via hydrogen bonding and chelating effects. Furthermore, we identied that amino group in aminoethyl groups at C6 position is important for ACE inhibition since it enhance the binding ability of COS to obligatory active site of the enzyme. Finally, these results illustrate the possibilities of improving ACE inhibitory activity of COS for their potential applications. Acknowledgement The authors acknowledge Marine Bioprocess Research Center of Marine Bio 21 Project (M2007-01), funded by the Ministry of Maritime Affairs and Fisheries, Republic of Korea. References
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