Chemical Synthesis of Bioactive Proteins

Nature has developed a plethora of protein machinery to operate and maintain nearly every task of cellular life. These processes are tightly regulated via post-expression modifications—transformations that modulate intracellular protein synthesis, folding, and activation. Methods to prepare homogeneously and precisely modified proteins are essential to probe their function and design new bioactive modalities. Synthetic chemistry has contributed remarkably to protein science by allowing the preparation of novel biomacromolecules that are often challenging or impractical to prepare via common biological means. The ability to chemically build and precisely modify proteins has enabled the production of new molecules with novel physicochemical properties and programmed activity for biomedical research, diagnostic, and therapeutic applications. This minireview summarizes recent developments in chemical protein synthesis to produce bioactive proteins, with emphasis on novel analogs with promising in vitro and in vivo activity.     

Selective Peptide Cysteine Manipulation on Demand and Difficult Protein Chemical Synthesis Enabled by Controllable Acidolysis of N,S-Benzylidene Thioacetals

Although solid-phase peptide synthesis combining with chemical ligation provides a way to build up customized polypeptides in general, many targets are still presenting challenges for the conventional synthetic process, such as hydrophobic proteins. New methods and strategies are still required to overcome these obstacles. In this study, kinetic studies of Cys/Pen ligation and its acidolysis were performed, from which the fast acidolysis of substituted N,S-benzylidene thioacetals (NBTs) was discovered. The study demonstrates the potential of NBTs as a promising Cys switchable protection, facilitating the chemical synthesis of peptides and proteins by efficiently disrupting peptide aggregation. The compatibility of NBTs with other commonly adopted Cys protecting groups and their applications in sequential disulfide bond formation were also investigated. The first chemical synthesis of the native human programmed death ligand 1 immunoglobulin V-like (PD-L1 IgV) domain was achieved using the NBT strategy, showcasing its potential in difficult protein synthesis.     

Backbone-Installed Split Intein-Assisted Ligation for the Chemical Synthesis of Mirror-Image Proteins

Membrane-associated D-proteins are an important class of synthetic molecules needed for D-peptide drug discovery, but their chemical synthesis using canonical ligation methods such as native chemical ligation is often hampered by the poor solubility of their constituent peptide segments. Here, we describe a B ackbone- I nstalled S plit I ntein- A ssisted L igation (BISIAL) method for the synthesis of these proteins, wherein the native L-forms of the N- and C-intein fragments of the unique consensus-fast (Cfa) (i.e. L–Cfa^N and L–Cfa^C) are separately installed onto the two D-peptide segments to be ligated via a removable backbone modification. The ligation proceeds smoothly at micromolar (μM) concentrations under strongly chaotropic conditions (8.0 M urea), and the subsequent removal of the backbone modification groups affords the desired D-proteins without leaving any “ligation scar” on the products. The effectiveness and practicality of the BISIAL method are exemplified by the synthesis of the D-enantiomers of the extracellular domains of T cell immunoglobulin and ITIM domain (TIGIT) and tropomyosin receptor kinase C (TrkC). The BISIAL method further expands the chemical protein synthesis ligation toolkit and provides practical access to challenging D-protein targets.     

低浓度甲醛对多肽和蛋白化学修饰的质谱研究

采用基质辅助激光解析电离飞行时间质谱( MALDI-TOF MS)和纳升电喷雾四极杆飞行时间串联质谱( Nano-ESI -QTOF MS)技术,以标准肽段和流感病毒基质蛋白酶切肽段为模型,研究了甲醛对蛋白质和多肽主链的修饰作用。采用与实际病毒灭活过程一致的实验条件(4℃,0.025%(V/V)福尔马林(37%(w/w)甲醛溶液)处理72 h),进行甲醛与多肽的化学反应。结果表明,在实验条件下,甲醛能与标准肽段N端的氨基反应生成羟甲基加合物,再发生缩合反应生成亚胺,形成+12 Da的产物。此外,甲醛还能与标准肽段中的精氨酸、赖氨酸的侧链发生反应,生成+12 Da的反应产物。对流感病毒基质蛋白的酶切肽段与甲醛的反应的质谱分析结果显示,多数的肽段都生成了+24 Da的产物,质量的增加来源于肽段N端氨基(+12 Da)和C端精氨酸或赖氨酸的侧链(+12 Da)的贡献。此外,还观察到有一个漏切位点的肽段生成了+36 Da的产物。本研究结果表明,在实验条件下,低浓度甲醛主要与肽段和蛋白的N 端氨基,以及精氨酸和赖氨酸侧链发生反应。本研究为分析低浓度甲醛与蛋白质的反应产物提供了有效的质谱分析方法和解谱依据。     

N,O-Benzylidene Acetal Dipeptides (NBDs) Enable the Synthesis of Difficult Peptides via a Kinked Backbone Strategy

Proteins with highly hydrophobic regions or aggregation-prone sequences are typically difficult targets for chemical synthesis at the current stage, as obtaining such type of peptides via solid-phase peptide synthesis requires sophisticated operations. Herein, we report N,O-benzylidene acetal dipeptides (NBDs) as robust and effective building blocks to allow the direct synthesis of difficult peptides and proteins via a kinked backbone strategy. The effectiveness and easy accessibility of NBDs have been well demonstrated in our chemical syntheses of various challenging peptides and proteins, including chemokine, therapeutic hormones, histone, and glycosylated erythropoietin.     

Association of Radical Chemistry with LanD Flavoprotein Activity for C-Terminal Macrocyclization of a Ribosomal Peptide by Formation of an Unsaturated Thioether Residue

LanD flavoproteins catalyze oxidative decarboxylation of the C-terminal Cys residue of a peptide to produce an enethiol. This enethiol is highly reactive and can be coupled with an upstream dehydroamino acid through Michael addition to form S-[2-aminovinyl](3-methyl)cysteine, an unsaturated thioether residue known to be characteristic of an array of C-terminally macrocyclized, ribosomally synthesized and posttranslationally modified peptides (RiPPs). Based on a two-stage bioinformatics mining of posttranslational modifications (PTMs) related to C-terminal Cys processing, we report herein that LanD activity can couple with radical S-adenosylmethionine chemistry to provide a new unsaturated thioether residue, S-[2-aminovinyl]-3-carbamoylcysteine, by conjugating the resultant enethiol with Cβ of the Asn residue in the C-terminal NxxC motif of a peptide for macrocyclization. This study furthers our understanding of the variety of PTMs involved in creating the structure diversity of macrocyclic RiPPs.     

Synthesis of Derivatives of Chlorines Related to Chlorophyll‐a by Vilsmeier Reaction with Methyl Pyropheophorbide‐a

From methyl pyropheophorbide‐a (MPPa, 1 ), the vinyl group was converted into other functional groups including 2‐dimethoxylethyl, 1‐hydroxylalkyl, and alkylcarbonyl groups by addition and oxidization to form chlorin ( 2?5b ). The nickel complexes ( 6a?e ) were prepared by treatment with excess nickel acetate in MeOH by refluxing and were used directly for the next reaction without further separation. The Vilsmeier reactions of nickel chlorins with 3‐(dimethylamino)acrolein/phosphoryl chloride (3‐DMA/POCl₃) are carried out to give meso‐20‐fotmyl‐vinylpyropheophorbide‐a ( 7a?9b ).     

Modular Synthesis of Furans with up to Four Different Substituents by a trans-Carboboration Strategy

Propargyl alcohols, on treatment with MHMDS (M=Na, K), B₂(pin)₂, an acid chloride and a palladium/copper co-catalyst system, undergo a reaction cascade comprised of trans-diboration, regioselective acylation, cyclization and dehydration to give trisubstituted furylboronic acid pinacol ester derivatives in good yields; subsequent Suzuki coupling allows a fourth substituent of choice to be introduced and hence tetrasubstituted (arylated) furans to be formed. In terms of modularity, the method seems unrivaled, not least because each product can be attained by two orthogonal but convergent ways (“diagonal split”). This asset is illustrated by the “serial” formation of a “library” of all twelve possible furan isomers that result from systematic permutation of four different substituents about the heterocyclic core.     

Oxyma: An Efficient Additive for Peptide Synthesis to Replace the Benzotriazole‐Based HOBt and HOAt with a Lower Risk of Explosion[1]

Oxyma [ethyl 2‐cyano‐2‐(hydroxyimino)acetate] has been tested as an additive for use in the carbodiimide approach for formation of peptide bonds. Its performance in relation to those of HOBt and HOAt, which have recently been reported to exhibit explosive properties, is reported. Oxyma displayed a remarkable capacity to inhibit racemization, together with impressive coupling efficiency in both automated and manual synthesis, superior to those of HOBt and at least comparable to those of HOAt, and surpassing the latter coupling agent in the more demanding peptide models. Stability assays showed that there was no risk of capping the resin under standard coupling conditions. Finally, calorimetry assays (DSC and ARC) showed decomposition profiles for benzotriazole‐based additives that were consistent with their reported explosivities and suggested a lower risk of explosion in the case of Oxyma.     

A Regio‐ and Stereodivergent Synthesis of Homoallylic Amines by a One‐Pot Cooperative‐Catalysis‐Based Allylic Alkylation/Hofmann Rearrangement Strategy

Herein, we report a modular synthetic route to linear and branched homoallylic amines that operates through a sequential one‐pot Lewis base/transition‐metal catalyzed allylic alkylation/Hofmann rearrangement strategy. This protocol is operationally trivial, proceeds from simple and easily prepared substrates and catalysts, and enables all aspects of regio‐ and stereoselectivity to be controlled through a conserved experimental protocol. Overall, the high levels of enantio‐, regio‐, and diastereoselectivity obtained, in concert with the ability to access orthogonally protected or free amines, render this a straightforward and effective approach for the preparation of useful enantioenriched homoallylic amines. We have also demonstrated the utility of the products in the context of pharmaceutical synthesis.     

Synthesis and Photovoltaic Properties of the Copolymers Based on Carbazole with Tetrathiophene Porphyrin Side Chains Linked by a Flexible Alkyl‐interval

Four copolymers (XP10, XP11, XP12 and XP13) based on diketopyrrolopyrrole (DPP) and carbazole units with tetrathiophene porphyrin (TTP) side chains linked by a flexible alkyl‐interval were designed and synthesized. The effects of the introduction of TTP on the optical and electrochemical properties, the morphology, the mobility and the photovoltaic performance of copolymers were systematically studied. The results revealed that XP11 with a TTP/DPP ratio of 2/8 possessed the best performances, i.e., broad absorption spectra, balanced hole/electron mobility and suitable microphase separation. After optimizing via solution vapor annealing, the organic solar cell devices based on XP11 and PC₇₁BM showed the best power conversion efficiency of 5.11% with a short‐circuit current density (J_sc) of 10.36 mA·cm^–2, an open‐circuit voltage (V_oc) of 0.77 V, and a fill factor (FF) of 0.64.