Amine of Proteins Via Biomimetic Ortho-Quinone-Mediated Oxidation
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ARTICLE https://doi.org/10.1038/s41467-021-22654-7 OPEN Modification of N-terminal α-amine of proteins via biomimetic ortho-quinone-mediated oxidation Siyao Wang1,6, Qingqing Zhou1,6, Xiaoping Chen2,6, Rong-Hua Luo3,4,6, Yunxue Li1, Xinliang Liu1, ✉ ✉ Liu-Meng Yang3,4, Yong-Tang Zheng 3,4,7 & Ping Wang 1,5,7 Naturally abundant quinones are important molecules, which play essential roles in various biological processes due to their reduction potential. In contrast to their universality, the 1234567890():,; investigation of reactions between quinones and proteins remains sparse. Herein, we report the development of a convenient strategy to protein modification via a biomimetic quinone- mediated oxidation at the N-terminus. By exploiting unique reactivity of an ortho-quinone reagent, the α-amine of protein N-terminus is oxidized to generate aldo or keto handle for orthogonal conjugation. The applications have been demonstrated using a range of proteins, including myoglobin, ubiquitin and small ubiquitin-related modifier 2 (SUMO2). The effect of this method is further highlighted via the preparation of a series of 17 macrophage inflam- matory protein 1β (MIP-1β) analogs, followed by preliminary anti-HIV activity and cell viability assays, respectively. This method offers an efficient and complementary approach to existing strategies for N-terminal modification of proteins. 1 Shanghai Key Laboratory for Molecular Engineering of Chiral Drugs, School of Chemistry and Chemical Engineering, Frontiers Science Center for Transformative Molecules, Shanghai Jiao Tong University, Shanghai, China. 2 Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province College of Physics and Optoelectronic Engineering, Shenzhen University, Shenzhen, China. 3 Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan, China. 4 Center for Biosafety Mega-Science, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan, China. 5 Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Center for Systems, Shanghai Jiao Tong University, Shanghai, China. 6These authors contributed equally: Siyao ✉ Wang, Qingqing Zhou, Xiaoping Chen, Rong-Hua Luo. 7These authors jointly supervised this work: Yong-Tang Zheng, Ping Wang. email: [email protected]. ac.cn; [email protected] NATURE COMMUNICATIONS | (2021) 12:2257 | https://doi.org/10.1038/s41467-021-22654-7 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-22654-7 xidation reactions play important roles in organic oxidants, which can convert the N-termini of proteins into aldo chemistry and are widely involved in crucial biological or keto functionalities for oxime formation or other bio- O 1,2 47–49 fi transformations . In line with the broad interest in the conjugation reactions . However, the rate or ef ciency of development of selective and mild oxidation reactions, ortho- transamination/oxime formation may vary at different N-termini quinone cofactors of copper amine oxidases (CuAOs) have been or even different amino acid combinations50,51. Room for extensively studied due to their high catalytic efficiency. In the improvements is still remained in the field of N-terminal mod- 52 catalytic cycle, the Tyr side chain is converted into lysyl tyr- ification strategy . Marginally, the salt nature of NaIO4, PLP or osylquinone, which can readily oxidize primary amines into RS salt makes them difficult to be removed from aqueous med- aldehydes via a quinone-mediated transamination pathway using ium after transamination of peptide or small protein targets, 3,4 53 O2 as a co-oxidant to complete the catalytic cycle (Fig. 1a) . which may potentially interfere the subsequent conjugations . Recently, significant progress has been achieved toward the Thus, given the generality of quinone and its derivatives in design of quinone-based catalysts with major contributions from the effective oxidation of amines, we seek to investigate whether Corey5, Fleury6, Kobayashi7, Stahl8–10 and Luo (Fig. 1b)11–13. or not the quinones may be suitable reagents for the selective and These biomimetic quinone-based catalysts show specific che- efficient oxidation of the N-terminal α-amines of proteins to moselectivity toward the dehydrogenation of primary, secondary, aldehyde or ketone with fast kinetics as well as a wide scope of tertiary amines or other reactions14–16. The applications of qui- amino acids. none oxidation have been largely limited to small molecule In this work, we show a selective and rapid method for mod- transformations despite the significant research progress in this ifying the N-termini of proteins via a quinone-mediated oxidation area17,18. On the other side, in nature, the reactions between o- of N-terminal α-amine of complex peptides and proteins under quinones such as DOPA19 and protein side chains are important physiological conditions (Fig. 1c). Several examples have been but complex in fundamental process of life20. Although the early demonstrated using a range of peptides and proteins, including work provided by Mason in 1955 showed that o-quinones could ubiquitin, myoglobin, E3 ubiquitin-protein ligase RNF4 (32-133) react with proteins via the N-terminal residue21, the reaction fragment and small ubiquitin-related modifier 2 (SUMO2). between o-quinone motifs and proteins have not been fully elu- Moreover, we prepared a library of macrophage inflammatory cidated on a molecular level22. Nevertheless, the intrinsic design protein 1β (MIP-1β)54 analogs using a combination of native of quinones by nature may provide an effective opportunity to chemical ligation and quinone-mediated transamination, where modify proteins under physiological conditions. the late-stage modification of the N-terminus of MIP-1β can lead Existing methods used to modify proteins are mainly confined to a 20-fold increase in its anti-HIV-1 activity. to nucleophilic amino acids, such as Cys and Lys, and potentially result in heterogeneous conjugates due to the high frequency of Results and discussion these amino acids23–25. In this context, selective modification of Development of ortho-quinone-mediated oxidation. To begin the N-terminus of proteins has led to the single site functionali- – our journey, a range of quinone derivatives Ox1–Ox8 were pre- zation of proteins26 30. Although significant progress has been pared containing different functionalities with an aim toward made, the majority of these methods required specific N-terminal fine-tuning their reactivity to be selective for the α-amine rather residue, owing to their assistance of the side-chain functionality, – than the ε-amine of Lys, as well as other functionalities7,11,12.We such as β-nucleophilically-functionalized Cys31 35, Ser/Thr36,37. tested the transamination reaction of model peptide 1 (1 mM) Transamination reactions under physiological conditions have with the sequence of GFHAKGY in an aqueous solution buffered been elegantly developed by Francis and co-workers utilizing – at pH 6.5 using 10 mM (saturated) of the quinones (Ox1–Ox8)as pyridoxal-5-phosphate (PLP)38 45 or N-methylpyridinium-4- the oxidants (Fig. 2a). The oxidized product was subsequently carboxaldehyde benzenesulfonate salt (Rapoport’s salt)46 as reacted with EtONH2 to form its corresponding oxime 1a.4-tert- Fig. 1 Development of site-specific N-terminal protein modification via quinone-mediated transamination. a Natural catalytic cycle of CuAOs; b Oxidation of primary amines with ortho-quinone to form imines; c Biomimetic quinone-mediated transamination inspired by CuAOs. the quinones may be suitable reagents for the selective and efficient oxidation of the N-terminal α-amines of proteins to aldehyde or ketone with fast kinetics as well as a wide scope of amino acids. 2 NATURE COMMUNICATIONS | (2021) 12:2257 | https://doi.org/10.1038/s41467-021-22654-7 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-22654-7 ARTICLE Fig. 2 Optimization of the reaction conditions used in the biomimetic transamination reaction. a Screening of oxidants Ox1-Ox8 used for the transamination reaction with model peptide 1; b LC trace obtained for the transamination reaction using Ox1 in yellow or Ox2 in blue; c pH Optimization of the transamination reaction using Ox1 in yellow or Ox2 in blue (the rate of conversion was calculated by integrating the related peaks of the LC trace measured at 280 nm. Centers represent means, error bars represent standard deviation, n = 3 independent replicates). Butyl-5-methoxy-4-tert-butyl-o-benzylquinone Ox1 achieved observation of the expected side-products. Asp peptide 7 near quantitative conversion (>90%) to generate product 1a in underwent oxidative decarboxylation during the oxidation and 3 h with <2% Lys oxidation observed via LCMS due to the therefore formed the methylated product 11a. Ser peptide 8, Thr extraordinary low pKa of the N-terminal amine compared to that peptide 9 and Trp peptide 10 were oxidized, but all formed the of the Lys side chain amine26.4-tert-Butyl-o-benzylquinone Ox2 side-chain cleaved oxime product 1a cleanly. Peptides 11−15 could also achieve 55% conversion at pH 6.5; the major by- (containing Ala, Phe, Ile, Tyr and Val as N-termini, respectively) product 1b was attributed to the undesired Lys side chain oxi- could also be converted into their desired oxime products, but dation (>40%) (Fig. 2b). Further optimization of the pH of the with lower conversions. Asn peptide 16 was oxidized in the first transamination reactions with either quinone Ox1 or Ox2 step, but the resulting ketone product could not be converted to showed that Ox1 had an optimal pH range of 6.0–6.5, while the oxime effectively in 3 h. Further extension of oxime reaction time side reaction during using quinone Ox2 could be minimized by to 5 h led to the formation of product 16a with 88% conversion. lowering reaction pH to 4.0 (90% conversion to give target pro- Although Gln peptide 17 could be transaminated, it tended to duct 1a) (Fig.