通过工程化阿斯巴拉基尼尔结合酶的三级胺基键形成
Simon J de Veer1, Yan Zhou1, Thomas Durek1
1Institute for Molecular Bioscience, Australian Research Council Centre of Excellence for Innovations in Peptide and Protein Science, The University of Queensland Brisbane QLD 4072 Australia s.deveer@imb.uq.edu.au d.craik@imb.uq.edu.au fbhrehm@gmail.com.
Chemical science
|April 5, 2024
概括
经过工程设计的阿斯巴拉基尼尔结合酶产生三级胺基键,扩大了蛋白质修饰能力,超出了标准胺基键. 这种新的方法允许在温和条件下对先进的生物药物和治疗方法进行高效的,顺序的蛋白质标签.
科学领域:
- 生物化学 生物化学
- 化学生物学 化学生物学
- 蛋白质工程是指蛋白质的工程.
背景情况:
- 转酶对于特定位点的蛋白质修饰至关重要,使得定制生物制品的创建成为可能.
- 目前使用透酶的方法仅限于在蛋白质末端形成二次胺基键.
- 开发蛋白质修饰的新方法对于治疗和诊断的进步至关重要.
研究的目的:
- 研究一种工程化阿斯巴拉基尼尔结合酶形成三级胺基键的能力.
- 探索这种结合酶在序列和双蛋白标记方面的潜力.
- 为了扩大蛋白质修饰反应的化学多样性.
主要方法:
- 使用工程化阿斯巴拉基尼尔酶进行蛋白质结合反应.
- 采用多种二次氨基核来合成三级胺基键.
- 研究了酶的识别元素 (P1 Asn和P2' Leu) 和基质耐受性.
- 包含4-azidoproline用于单双标签.
主要成果:
- 在温和,近乎中性的pH条件下,工程化阿斯巴拉基尼尔结合酶有效地合成了三级胺基键.
- 在反应过程中,酶的最佳识别元素被保留了.
- 含有proline的产品对酶识别产生了抵抗力,从而促进了序列标记.
- 通过在结结处使用4-azidoproline实现了单双标签.
结论:
- 工程化阿斯帕拉基尼尔连接酶提供了一种新的合成三级胺基键的途径,克服了现有的转酶的局限性.
- 这种方法为简单,连续的蛋白质修饰提供了一个多功能平台.
- 这些发现扩大了创建定制生物,治疗和诊断的工具包.
相关概念视频
01:22Preparation of 1° Amines: Azide Synthesis
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Direct alkylation of ammonia produces polyalkylated amines, along with a quaternary ammonium salt. To exclusively prepare primary amines, the azide synthesis method can be used.
Azide ions act as good nucleophiles and react with unhindered alkyl halides to form alkyl azides. Alkyl azides do not participate in further nucleophilic substitution reactions, thereby eliminating the chances of polyalkylated products. Alkyl azides are reduced by hydride-based reducing agents, like lithium aluminum...
Azide ions act as good nucleophiles and react with unhindered alkyl halides to form alkyl azides. Alkyl azides do not participate in further nucleophilic substitution reactions, thereby eliminating the chances of polyalkylated products. Alkyl azides are reduced by hydride-based reducing agents, like lithium aluminum...
3.9K
01:28Preparation of 1° Amines: Gabriel Synthesis
3.5K
Direct alkylation is not a suitable method for synthesizing amines because it produces polyalkylated products. Gabriel synthesis is the most preferred method to exclusively make primary amines. The method uses phthalimide, which contains a protected form of nitrogen that participates in alkylation only once to predominantly give primary amines.
Strong bases like NaOH or KOH deprotonate the phthalimide to form the corresponding anion, which acts as a nucleophile. Further, the anion attacks an...
Strong bases like NaOH or KOH deprotonate the phthalimide to form the corresponding anion, which acts as a nucleophile. Further, the anion attacks an...
3.5K
01:07Acid Halides to Amides: Aminolysis
2.7K
Aminolysis is a nucleophilic acyl substitution reaction, where ammonia or amines act as nucleophiles to give the substitution product. Acid halides react with ammonia, primary amines, and secondary amines to yield primary, secondary, and tertiary amides, respectively.
In the first step of the aminolysis mechanism, the amine attacks the carbonyl carbon of the acyl chloride to form a tetrahedral intermediate. In the second step, the carbonyl group is re-formed with the elimination of a chloride...
In the first step of the aminolysis mechanism, the amine attacks the carbonyl carbon of the acyl chloride to form a tetrahedral intermediate. In the second step, the carbonyl group is re-formed with the elimination of a chloride...
2.7K
01:29Preparation of Amides
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Amides are synthesized by treating carboxylic acids with amines in the presence of dehydrating agents like dicyclohexylcarbodiimide (DCC).
The DCC-promoted synthesis of amides begins with the protonation of DCC by carboxylic acid. The protonation makes it a better acceptor. Next, the addition of carboxylate to the protonated carbodiimide gives a reactive acylating agent.
Subsequently, the amine acts as a nucleophile that attacks the acylating agent to form a tetrahedral intermediate. In the...
The DCC-promoted synthesis of amides begins with the protonation of DCC by carboxylic acid. The protonation makes it a better acceptor. Next, the addition of carboxylate to the protonated carbodiimide gives a reactive acylating agent.
Subsequently, the amine acts as a nucleophile that attacks the acylating agent to form a tetrahedral intermediate. In the...
3.0K
01:19Amines to Amides: Acylation of Amines
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Various carboxylic acid derivatives (such as acid chlorides, esters, and anhydrides) can be used for the acylation of amines to yield amides. The reaction requires two equivalents of amines. The first amine molecule functions as a nucleophile and attacks the carbonyl carbon to produce a tetrahedral intermediate. This is followed by the loss of the leaving group and restoration of the C=O bond.
Next, the second equivalent of amine serves as a Brønsted base and deprotonates the quaternary...
Next, the second equivalent of amine serves as a Brønsted base and deprotonates the quaternary...
2.4K
01:23Aldehydes and Ketones with Amines: Imine Formation Mechanism
5.5K
Imine formation involves the addition of carbonyl compounds to a primary amine. It begins with the generation of carbinolamine through a series of steps involving an initial nucleophilic attack and then several proton transfer reactions. The second part includes the elimination of water, as a leaving group, to give the imine.
Imines are formed under mildly acidic conditions. A pH of 4.5 is ideal for the reaction.
If the pH is low or the solution is too acidic, the reaction slows down in the...
Imines are formed under mildly acidic conditions. A pH of 4.5 is ideal for the reaction.
If the pH is low or the solution is too acidic, the reaction slows down in the...
5.5K