Broadening the Utility of Farnesyltransferase-Catalyzed Protein Labeling Using Norbornene–Tetrazine Click Chemistry

Bioorthogonal
chemistry has gained widespread use in the study
of many biological systems of interest, including protein prenylation.
Prenylation is a post-translational modification, in which one or
two 15- or 20-carbon isoprenoid chains are transferred onto cysteine
residues near the C-terminus of a target protein. The three main enzymesprotein
farnesyltransferase (FTase), geranylgeranyl transferase I (GGTase
I), and geranylgeranyl transferase II (GGTase II)that catalyze
this process have been shown to tolerate numerous structural modifications
in the isoprenoid substrate. This feature has previously been exploited
to transfer an array of farnesyl diphosphate analogues with a range
of functionalities, including an alkyne-containing analogue for copper-catalyzed
bioconjugation reactions. Reported here is the synthesis of an analogue
of the isoprenoid substrate embedded with norbornene functionality
(C10NorOPP) that can be used for an array of applications, ranging
from metabolic labeling to selective protein modification. The probe
was synthesized in seven steps with an overall yield of 7% and underwent
an inverse electron demand Diels–Alder (IEDDA) reaction with
tetrazine-containing tags, allowing for copper-free labeling of proteins.
The use of C10NorOPP for the study of prenylation was explored in
the metabolic labeling of prenylated proteins in HeLa, COS-7, and
astrocyte cells. Furthermore, in HeLa cells, these modified prenylated
proteins were identified and quantified using label-free quantification
(LFQ) proteomics with 25 enriched prenylated proteins. Additionally,
the unique chemistry of C10NorOPP was utilized for the construction
of a multiprotein–polymer conjugate for the targeted labeling
of cancer cells. That construct was prepared using a combination of
norbornene–tetrazine conjugation and azide–alkyne cycloaddition,
highlighting the utility of the additional degree of orthogonality
for the facile assembly of new protein conjugates with novel structures
and functions.

生物正交化学（Bioorthogonal chemistry）已在诸多受关注的生物系统研究中得到广泛应用，其中便涵盖蛋白质异戊二烯化（protein prenylation）相关研究。异戊二烯化是一类翻译后修饰（post-translational modification）过程，具体为将一条或两条15碳或20碳的类异戊二烯链转移至靶蛋白C端附近的半胱氨酸残基上。催化该反应的三种主要酶——法尼基转移酶（protein farnesyltransferase, FTase）、牻牛儿基牻牛儿基转移酶I（geranylgeranyl transferase I, GGTase I）以及牻牛儿基牻牛儿基转移酶II（geranylgeranyl transferase II, GGTase II）——已被证实可耐受类异戊二烯底物上的多种结构修饰。此前研究已利用这一特性，开发出一系列带有不同官能团的法尼基二磷酸类似物，其中便包括可用于铜催化生物偶联反应的含炔基类似物。本文报道了一种带有降冰片烯官能团的类异戊二烯底物类似物（C10NorOPP）的合成方法，该类似物可应用于从代谢标记到选择性蛋白质修饰等诸多场景。该探针通过7步反应合成，总产率达7%，可与含四嗪标签的分子发生反电子需求Diels-Alder（inverse electron demand Diels–Alder, IEDDA）反应，从而实现蛋白质的无铜标记。研究人员探索了C10NorOPP在异戊二烯化研究中的应用，将其用于HeLa细胞、COS-7细胞以及星形胶质细胞中异戊二烯化蛋白质的代谢标记。此外，在HeLa细胞中，研究人员通过无标记定量（label-free quantification, LFQ）蛋白质组学方法对这些修饰后的异戊二烯化蛋白质进行了鉴定与定量，共富集得到25种异戊二烯化蛋白质。除此之外，研究人员还利用C10NorOPP独特的化学反应特性，构建了一种多蛋白-聚合物偶联物用于癌细胞的靶向标记。该构建体通过结合降冰片烯-四嗪偶联与叠氮-炔环加成反应制备而成，凸显了额外的正交反应位点对于便捷组装具有新颖结构与功能的新型蛋白质偶联物的应用价值。