酪氨酸衍生物调控酶催化路径可控合成功能黑色素★

doi:10.6023/A23050240

化学学报 ›› 2023, Vol. 81 ›› Issue (11): 1486-1492.DOI: 10.6023/A23050240 上一篇下一篇

所属专题：庆祝《化学学报》创刊90周年合辑

研究论文

酪氨酸衍生物调控酶催化路径可控合成功能黑色素^★

苏东芮^a^,^b, 任小康^b, 于沄淏^b, 赵鲁阳^b^,^*(), 王天宇^a^,^*(), 闫学海^b^,^c^,^d^,^*()

a 北京科技大学化学与生物工程学院功能分子与晶态材料科学与应用北京市重点实验室北京 100083
b 中国科学院过程工程研究所生化工程国家重点实验室北京 100083
c 中国科学院大学化学工程学院北京 100049
d 中国科学院过程工程研究所介尺度科学研究中心北京 100083

投稿日期:2023-05-19 发布日期:2023-07-28
作者简介:
^★庆祝《化学学报》创刊90周年.
基金资助:
国家自然科学基金(22025207); 国家自然科学基金(22232006); 国家自然科学基金(22077122); 国家自然科学基金(22072004)

Tyrosine Derivative Regulated Enzyme Catalytic Pathway for Controllable Synthesis of Functional Melanin^★

Dongrui Su^a^,^b, Xiaokang Ren^b, Yunhao Yu^b, Luyang Zhao^b(), Tianyu Wang^a(), Xuehai Yan^b^,^c^,^d()

a Beijing Key Laboratory for Science and Application of Functional Molecular and Crystalline Materials, School of Chemistry and Chemical Engineering, University of Science and Technology Beijing, Beijing 100083
b State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100083
c School of Chemical Engineering, University of Chinese Academy of Sciences, Beijing 100049
d Center for Mesoscience, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100083

Received:2023-05-19 Published:2023-07-28
Contact: *E-mail: yanxh@ipe.ac.cn (X. Y.); zhaoly@ipe.ac.cn (Y. Z.); twang@ustb.edu.cn (T. W.)
About author:
^★Dedicated to the 90th anniversary of Acta Chimica Sinica.
Supported by:
National Natural Science Foundation of China(22025207); National Natural Science Foundation of China(22232006); National Natural Science Foundation of China(22077122); National Natural Science Foundation of China(22072004)

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摘要/Abstract

利用酶催化自组装将生物小分子构筑成具有独特功能的生物大分子聚合物是制备功能生物材料极具前景的新策略, 然而其挑战在于如何在底物层面调控生物大分子的结构和功能. 以从酪氨酸构筑黑色素为例, 通过底物结构的简单衍生化, 实现了对酶催化自组装过程中关键聚合位点的控制, 得到一系列尺寸、形貌各异的黑色素产物. 进一步表征了各黑色素产物的光热转换性能, 在细胞层次验证了结构修饰的黑色素用于光热材料的潜力. 揭示了通过改变底物核心基团周边化学结构调控酶催化路径, 进一步调控黑色素产物性质及功能的可行性, 为构筑新型功能黑色素材料提供了新思路, 同时对揭示生物大分子结构与生物功能的关系提供了有益启示.

关键词: 黑色素, 酪氨酸酶, 酶催化自组装, 酪氨酸衍生物, 光热材料

Construction of biomacromolecules via enzyme-mediated catalytic assembly from small biomolecules is fascinating for preparing functional biological materials. The challenge remains on how to control the structure and functions of biomacromolecules through substrate regulation. A sequence of crucial biomacromolecules, melanin, were prepared via simple substrate derivation, which controls the key polymerization sites in enzyme catalyzed self-assembly process. In detail, we designed three tyrosine derivatives, namely, 3-fluorotyrosine [Tyr(F)], N-acetyltyrosine [Tyr(N-Ac)], and tyrosine ethyl ester [Tyr(OEt)]. The three substrates corresponded to the blockage of different tyrosinase-mediated polymerization active site, and tyrosine was used as the reference. The above small molecules as substrates (1.0 mmol/L) and tyrosinase (2 U) were mixed in phosphate buffer (pH=8.5, 0.10 mol/L, 2.0 mL), which was stirred in an air environment of 25 ℃. After 24 h, the reaction was quenched and the mixture was centrifuged to obtain different melanin nanoparticle products (MNPs). Characterizations from transmission electron microscopy (TEM), infrared spectroscopy (IR), and X-ray photoelectron spectroscopy (XPS) showed that all the melanin products had eumelanin-like skeleton, but were different in the degree of polymerization and microscopic chemical structures. These structure-modified MNPs showed overall absorption in the ultraviolet-visible-near infrared (UV-vis-NIR) region, thus enabling photothermal conversion in the NIR-I region. The photothermal conversion efficiency of MNP, MNP(F), and MNP(OEt) (3 mg•mL^-1) was measured to be 46.6%, 37.0%, and 25.8% [laser 808 nm, 1.5 W, where MNP(N-Ac) was not available due to rather low temperature increase]. Interestingly, in vitro experiment showed that MNP(OEt) exhibited better photothermal cytotoxicity than MNP, and this was probably because MNP(OEt) had a smaller particle size and less negative ζ-potential, which could ease cell endocytosis. This work demonstrated the feasibility to regu-late enzyme-mediated catalytic pathway via simple substrate derivation. It provides insights for the construction of new functional melanin materials and for revealing the relationship between biological macromolecular structure and function.

Key words: melanin, tyrosinase, enzyme-mediated catalyztic assembly, tyrosine derivative, photothermal material

引用此文

苏东芮, 任小康, 于沄淏, 赵鲁阳, 王天宇, 闫学海. 酪氨酸衍生物调控酶催化路径可控合成功能黑色素^★[J]. 化学学报, 2023, 81(11): 1486-1492.

Dongrui Su, Xiaokang Ren, Yunhao Yu, Luyang Zhao, Tianyu Wang, Xuehai Yan. Tyrosine Derivative Regulated Enzyme Catalytic Pathway for Controllable Synthesis of Functional Melanin^★[J]. Acta Chimica Sinica, 2023, 81(11): 1486-1492.

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图/表 6

图1. (a)酪氨酸酶催化酪氨酸(Tyr)底物生成黑色素机理、(b)酪氨酸反应聚合活性位点及本文采用的酪氨酸衍生物结构(依次为Tyr(OEt)、Tyr(F)和Tyr(N-Ac))、(c)通过酪氨酸底物衍生化进行活性位点调控, 经酪氨酸酶催化制备黑色素策略示意图以及(d)由不同底物制得黑色素溶液样品图像[从左到右依次为MNP、MNP(OEt)、MNP(F)和MNP(N-Ac), 分别对应于由底物Tyr、Tyr(OEt)、Tyr(F)和Tyr(N-Ac)所得产物]

Figure 1. (a) Mechanism of tyrosinase catalysis from tyrosine (Tyr) substrate to melanin product, (b) active site of tyrosine reaction polymerization and the structure of tyrosine derivatives used in this work, (c) schematic diagram of tyrosinase-mediated melanin production by regulating the active polymerization site through the derivatization of tyrosine substrates, and (d) images of melanin solution samples prepared from different substrates [from left to right: MNP, MNP(OEt), MNP(F), and MNP(N-Ac) corresponding to Tyr, Tyr(OEt), Tyr(F), and Tyr(N-Ac)]

图2. 黑色素的形貌及结构表征 (a~d)原黑色素、氟代黑色素、N-乙酰黑色素和黑色素乙酯的TEM图像; (e)四种黑色素的红外光谱, 其中特征信号(1)指示醌和邻苯二酚结构; (f)四种黑色素的XPS全谱

Figure 2. Morphology and structural characterizations of melanin products (a~d) TEM images of MNP, MNP(F), MNP(N-Ac) and MNP(OEt), respectively; (e) infrared spectra of four types of melanin, where the feature peaks (1) indicated catechol and quinone groups, respectively; (f) XPS spectra of four types of melanin

图3. 分子对接模拟不同酪氨酸底物在酪氨酸酶活性口袋中具有能量极小值的结构示意图. (a~d)底物依次为酪氨酸、3-氟-酪氨酸、N-乙酰酪氨酸和酪氨酸乙酯. 其中底物(省略非活性氢原子)以球棍模型表示, 与底物相距3 ?以内的氨基酸残基以彩色棍模型表示, 双铜氧催化核心以球模型表示.

Figure 3. Structural diagram of molecular docking simulation of different tyrosine substrates with energy minima in the tyrosinase activity pocket. The substrates (a~d) are Tyr, Tyr(F), Tyr(N-Ac), and Tyr(OEt), respectively. The substrate (omitting nonactive hydrogen atoms) is represented by a ball stick model, the amino acid residues within 3 ? of the substrate are represented by a colored stick model, and the oxo-dicopper catalytic core is represented by a ball model

图4. 四种黑色素的光热转换效果表征 (a)四种黑色素的紫外-可见-近红外吸收光谱; (b)四种黑色素在激光照射下的升降温循环曲线(其中黑色素样品浓度均为3 mg?mL–1, 激光为808 nm, 功率为1.5 W, 取黄色区域中的曲线段用于光热效率计算)

Figure 4. Characterization of the photothermal conversion effect of four types of melanins (a) UV-Vis-NIR absorption spectra of four types of melanins; (b) temperature rise-fall cycles of four types of melanin under laser irradiation (the concentration of melanin sample is 3.0 mg?mL–1, the laser is 808 nm, and the laser power density is 1.5 W?cm–2. The curve segments in the yellow area are taken for photothermal efficiency calculation)

	[(Q_NP+Q_dis)/ξ+T₀]^a/K	C₀^a/K	(–ξ/C_p)^a/s^–1	A^b	ξ/(W•K^–1)	Q_NP/W	η	R²^c
MNP	49.5	–19.8	–0.00751	1.073	0.031	0.650	0.473	1.000
MNP(F)	50.7	–18.2	–0.00432	1.959	0.018	0.386	0.260	1.000
MNP(OEt)	51.4	–18.4	–0.00610	1.431	0.026	0.572	0.396	0.999

表1. 黑色素、氟代黑色素和黑色素乙酯光热效率计算参数

Table 1. Photothermal conversion efficiency calculation parameters of MNP, MNP(F), and MNP(OEt)

图5. 4T1细胞分别与(a)原黑色素、(b)氟代黑色素、(c) N-乙酰黑色素和(d)黑色素乙酯共孵育24 h, 然后用808 nm激光(1.5 W?cm–2)照射3 min的存活率[误差棒代表标准偏差(n=6)]

Figure 5. MTT assay of 4T1 cell vialibity after exposure to different concentrations of (a) MNP, (b) MNP(F), (c) MNP(N-Ac), and (d) MNP(OEt) with or without 808 nm laser irradiation (1.5 W?cm–2) for 3 min [error bars represent the standard deviation (n=6)]

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酪氨酸衍生物调控酶催化路径可控合成功能黑色素^★

Tyrosine Derivative Regulated Enzyme Catalytic Pathway for Controllable Synthesis of Functional Melanin^★

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酪氨酸衍生物调控酶催化路径可控合成功能黑色素★

Tyrosine Derivative Regulated Enzyme Catalytic Pathway for Controllable Synthesis of Functional Melanin★

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酪氨酸衍生物调控酶催化路径可控合成功能黑色素^★

Tyrosine Derivative Regulated Enzyme Catalytic Pathway for Controllable Synthesis of Functional Melanin^★