凝血酶适配体的功能基修饰及生物活性

doi:10.7503/cjcu20180168

摘要/Abstract

摘要：

在2'-脱氧胸苷(dT)的5位引入2-胺乙基胺酰基, 获得了化合物5-[(2-胺乙基)胺酰基甲基]-2'-脱氧胸苷(1), 将其用于对凝血酶适配体(TBA)的2个单链结构域(T3T4和T12T13)进行功能基修饰. 化合物1的引入对TBA的分子内折叠产生了一定的影响. TBA的结构单元T4和T13分别被化合物1取代后, 5位的2-胺乙基胺酰基降低了分子的稳定性及抗凝血作用, 表明T4和T13是高度保守的碱基; T3和T12分别被化合物1取代后, 5位上的2-胺乙基胺酰基可能处于适配体-凝血酶相互作用界面, 加强了与凝血酶的相互作用, 从而提高了抗凝血作用.

关键词: 凝血酶适配体, 热稳定性, 圆二色光谱, 凝血时间

Abstract:

Thrombin binding aptamer(TBA) is a small aptamer for human α-thrombin. Detailed structural analysis has demonstrated that the two-layer G-quartet acts as the structural backbone and three loops have different contributions for the intramolecular stability and the interaction with thrombin. Here, the chemical modification with 5-[(2-aminoethyl)amino-2-oxoethyl]-2'-deoxythymidine(1) on the two loops of TBA(T3T12 and T4T13) were reported. The 5-substituent induced different effect for each residue in the loops. The intramolecular stacking of T4 and T13 were negatively affected for the thermal stability and the inhibitory activity. In the case of the loop T3 and T12 extruding outward, the extra functional groups of the compound 1 were helpful for the interaction with thrombin, the 5-positioned extra amino and amido groups were probably located at the right positions for the hydrogen bonding interaction with the residues from thrombin. These results further confirmed the loop residues for their folding positions, and their contributions could be enhanced by 5-substituted functional groups of 2'-deoxythymidine at specific positions.

Key words: Thrombin binding aptamer(TBA), Thermal stability, Circular dichroism, Clotting time

中图分类号:

O625

TrendMD:

杜闪闪, 李阳, 郭磊, 李朋羽, 柴智龙, 王涛, 全东琴, 何军林. 凝血酶适配体的功能基修饰及生物活性. 高等学校化学学报, 2018, 39(11): 2445.

DU Shanshan, LI Yang, GUO Lei, LI Pengyu, CHAI Zhilong, WANG Tao, QUAN Dongqin, HE Junlin. Modification of Aptamer TBA with Extra Functional Groups and the Biological Activities^†. Chem. J. Chinese Universities, 2018, 39(11): 2445.

图/表 5

Scheme 1 TBA and 2'-deoxythymidine(dT) analogue 1

Table 1 TBA and its analogues modified with the monomer 1 and the mass spectra data

Name	Sequences d(5'-3')	MS, m/z(calcd. )
TBA	GGT TGG TGT GGT TGG	4727.4(4726.0)
TBA-T3-1	GG1 TGG TGT GGT TGG	4813.8(4812.1)
TBA-T4-1	GGT1GG TGT GGT TGG	4814.0(4812.1)
TBA-T12-1	GGT TGG TGT GG1 TGG	4814.3(4812.1)
TBA-T13-1	GGT TGG TGT GGT1GG	4815.4(4812.1)
TBA-T3-T12-1	GG1 TGG TGT GG1 TGG	4899.1(4898.2)
TBA-T4-T13-1	GGT1GG TGT GGT 1GG	4898.9(4898.2)

Table 2 Thermal stability of TBA and its analogues*

Name	T_m/℃	Name	T_m/℃
TBA	35.0	TBA-T13-1	30.6
TBA-T3-1	36.1	TBA-T3-T12-1	33.7
TBA-T4-1	30.5	TBA-T4-T13-1	27.4
TBA-T12-1	34.6

Fig. 1 CD spectra of TBA and its analogue(A), (B) in PBS(138 mmol/L NaCl+27 mmol/L KCl+10 mmol/L Na2HPO4+1.76 mmol/L KH2PO4, pH=7.4); (C)—(I) CD spectra of TBA and its analogue in the presence of thrombin(6 U, with incubation time of 5 min(labeled with a) and overnight(labeled with b) in PBS. (A) a. TBA; b. TBA-T3-1; c. TBA-T12-1; d. TBA-T3-T12-1; (B) a. TBA; b. TBA-T4-1; c. TBA-T13-1; d. TBA-T4-T13-1; (C) a. TBA; b. TBA-a; c. TBA-b; (D) a. TBA-T3-1; b. TBA-T3-1-a; c. TBA-T3-1-b; (E) a. TBA-T4-1; b. TBA-T4-1-a; c. TBA-T4-1-b; (F) a. TBA-T12-1; b. TBA-T12-1-a; c. TBA-T12-1-b; (G) a. TBA-T13-1; b. TBA-T13-1-a; c. TBA-T13-1-b; (H) a. TBA-T3-T12-1; b. TBA-T3-T12-1-a; c. TBA-T3-T12-1-b; (I) a. TBA-T4-T13-1; b. TBA-T4-T13-1-a; c. TBA-T4-T13-1-b.

Table 3 Anti-anticoagulant effect of TBA and its analogues

Name	Clotting time/s	Anticoagulant effect/s	Name	Clotting time/s	Anticoagulant effect/s
Control	20.1±0.2		TBA-T12-1	31.2±0.6	11.1
TBA	28.4±0.1	8.3	TBA-T13-1	24.4±0.1	4.3
TBA-T3-1	30.6±0.4	10.5	TBA-T3-T12-1	27.6±0.6	7.5
TBA-T4-1	23.6±0.5	3.5	TBA-T4-T13-1	24.3±0.2	4.2

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