Article Cite This: Acc. Chem. Res. 2018, 51, 1643−1655
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Serine/Threonine Ligation: Origin, Mechanistic Aspects, and Applications Han Liu and Xuechen Li*
Acc. Chem. Res. 2018.51:1643-1655. Downloaded from pubs.acs.org by IOWA STATE UNIV on 01/03/19. For personal use only.
Department of Chemistry, State Key Laboratory of Synthetic Chemistry, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, P. R. China
CONSPECTUS: Synthetic proteins are expected to go beyond the boundary of recombinant DNA expression systems by being flexibly installed with site-specific natural or unnatural modification structures during synthesis. To enable protein chemical synthesis, peptide ligations provide effective strategies to assemble short peptide fragments obtained from solid-phase peptide synthesis (SPPS) into long peptides and proteins. In this regard, chemoselective peptide ligation represents a simple but powerful transformation realizing selective amide formation between the C-terminus and N-terminus of two side-chainunprotected peptide fragments. These reactions are highly chemo- and regioselective to tolerate the side-chain functionalities present on the unprotected peptides, highly reactive to work with millmolar or submillimolar concentrations of the substrates, and operationally simple with mild conditions and accessible building blocks. This Account focuses on our work in the development of serine/threonine ligation (STL), which originates from a chemoselective reaction between an unprotected peptide with a C-terminal salicylaldehyde (SAL) ester and another unprotected peptide with an N-terminal serine or threonine residue. Mechanistically, STL involves imine capture, 5-endo-trig ring−chain tautomerization, O-to-N [1,5] acyl transfer to afford the N,O-benzylidene acetal-linked peptide, and acidolysis to regenerate the Xaa−Ser/Thr linkage (where Xaa is the amino acid) at the ligation site. The high abundance of serine and threonine residues (12.7%) in naturally occurring proteins and the good compatibility of STL with various C-terminal residues provide multiple choices for ligation sites. The requisite peptide C-terminal SAL esters can be prepared from the peptide fragments obtained from both Fmoc-SPPS and Boc-SPPS through four available methods (a safety-catch strategy based on phenolysis, direct coupling, ozonolysis, and the n + 1 strategy). In the synthesis of proteins (e.g., ACYP enzyme, MUC1 glycopeptide 40-mer to 80-mer, interleukin 25, and HMGA1a with variable post-translational modification patterns), both C-toN and N-to-C sequential STL strategies have been developed through selection of temporal N-terminal protecting groups and proper design of the switch-on/off C-terminal SAL ester surrogate, respectively. In the synthesis of cyclic peptide natural products (e.g., daptomycin, teixobactin, cyclomontanin B, yunnanin C) and their analogues, intramolecular head-to-tail STL has been implemented on linear peptide SAL ester precursors containing four to 10 amino acid residues with good efficiency and minimized oligomerization. As a thiol-independent chemoselective ligation complementary to native chemical ligation, STL provides an alternative tool for the chemical synthesis of homogeneous proteins with site-specific and structure-defined modifications and cyclic peptide natural products, which lays foundation for chemical biology and medicinal studies of those molecules with biological importance and therapeutic potential.
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INTRODUCTION In a letter to Adolf Baeyer in 1905, Emil Fischer stated that “my entire yearning is directed towards the first synthetic enzyme: if its preparation falls into my lap with the synthesis of a natural protein material, I will consider my mission fulfilled”.1 After Fischer reported the first synthesis of a dipeptide, GlyGly, in 1901,2 it took several decades to finally realize his © 2018 American Chemical Society
dream. Along this path, critical advances in peptide synthesis have been made, including the development of suitable protecting groups (Cbz, Fmoc, tBoc),3 coupling reagents,4 and solid-phase peptide synthesis (SPPS).5 Currently, by Received: April 4, 2018 Published: July 6, 2018 1643
DOI: 10.1021/acs.accounts.8b00151 Acc. Chem. Res. 2018, 51, 1643−1655
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Figure 1. Protein chemical synthesis via chemoselective peptide ligation with generation of the natural peptidic linkages.
the N-terminus of another peptide, such a method needed the side chain of lysine to be protected and caused epimerization at C-terminal non-Gly/Pro sites.10 In NCL, the peptide thioester undergoes reversible transthioesterification with the thiol group of the N-terminal cysteine, followed by intramolecular [1,4] S-to-N acyl transfer. Such a reaction pathway is highly chemoselective and allows for the use of unprotected peptides. Without further in situ activation of the thioester during NCL, epimerization at the C-terminal stereocenter is minimal. With NCL, chemical synthesis of sizable proteins has become a feasible task for many research laboratories. Over the past two decades since its inception, dramatic developments have been made to further advance NCL, including various ways to prepare the requisite peptide thioesters,11,12 expressed protein ligation (EPL)/intein-mediated ligation,13 NCL auxiliaries,14 NCL desulfurization15−17 to enable NCL at non-cysteine sites, and selenocysteine ligation (Figure 1).18,19 Alternatively, researchers have been attempting to develop other types of thiol-independent peptide ligations.20 Compared with amide formation using simple substrates, practical peptide ligation methods have more demanding requirements: high (or absolute) chemoselectivity, high reactivity with millimolar concentrations of the substrates, mild conditions (room temperature, no metal additives, etc.), and accessible building blocks. To this end, chemoselective peptide ligation has been enabled using paired unnatural amino acids at the reacting N/ C-terminus, such as α-keto acid−hydroxylamine (KAHA) ligation21,22 (Figure 1). All of these ligation methods have their own advantages and limitations, and thus, the development of novel and effective ligation methods based on different
means of these methods and strategies, peptides can be routinely synthesized in any organic synthesis lab with commercially available building blocks/reagents and apparatus by trained researchers. Indeed, even proteins had been synthesized either by direct SPPS (e.g., ribonuclease)6 or in combination with fragment coupling (e.g., green fluorescent protein).7 However, direct SPPS of long peptide sequences often gave very low overall yields because of the difficult product purification (therefore limiting SPPS mostly to 50 amino acids), and the fragment coupling approaches were often associated with difficult handling of side-chain-protected peptide fragments. It was the concept of regioselective peptide ligation coined by Kemp that made a paradigm shift in protein chemical synthesis. The key to peptide ligation is the chemoselectivity, which allows side-chain-unprotected peptide fragments to be used, thus overcoming the poor solubility and difficult handling issues met in the fragment coupling approach. In the 1980s, Kemp devised the “prior thiol capture” approach, in which an unprotected peptide with a C-terminal 4-hydroxy-6mercaptodibenzofuran ester was brought into close proximity of the N-terminal cysteine of the second peptide via reversible disulfide bond formation. Subsequent [1,11] O-to-N acyl transfer produced the amide bond to enable the selective reaction between the N- and C- termini.8 The breakthrough in peptide chemical ligation was native chemical ligation (NCL), reported in 1994, in which an unprotected peptide with a C-terminal thioester is able to ligate with a second unprotected peptide with an N-terminal cysteine.9 Although it had been shown that upon activation by silver ion a peptide thioester could undergo aminolysis with 1644
DOI: 10.1021/acs.accounts.8b00151 Acc. Chem. Res. 2018, 51, 1643−1655
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Figure 2. Serine/threonine ligation (STL) for peptide/protein synthesis.
manner. Next, the sp3-hybridized nitrogen formed in this ring− chain tautomerization underwent a [1,3] O-to-N acyl transfer with a putative fused [4,5] bicyclic transition state to generate the amide linkage, followed by fragmentation of the 2hydroxyloxazolidine. After next noting that Tam’s pseudoproline ligation involved a [1,4] O-to-N acyl transfer with a putative fused [5,5] bicyclic transition state (Scheme 1b),24,25 we were driven by curiosity to study [1,n] O-to-N acyl transfers with different-sized fused bicyclic transition states as a fundamental organic chemistry problem. The first case in our study was [1,5] O-to-N acyl transfer with a fused [6,5] bicyclic transition state (Scheme 1c). In our model reactions, the SAL esters of N-protected amino acids were treated with serine/threonine methyl esters.26 The SAL ester was chosen as the reacting group for several reasons: easy formation of the aromatic imine without the possibility of enamine formation, the good leaving property of the phenolic hydroxyl group, and preorganization of the ester and aldehyde functionalities, which could minimize the entropy penalty in the highly organized acyl transfer transition state. To our delight, this reaction proceeded smoothly and cleanly, even more rapidly than Tam’s pseudoproline ligation. Although the [1,4] acyl transfer as in the pseudoproline ligation via a fivemembered cyclic transition state is commonly believed to be more favorable than the [1,5] acyl transfer via a six-membered cyclic transition state, it is very likely that the priority is reversed when a fused bicyclic transition state and phenolic oxygen with its better leaving property are involved. This outcome provided the basis for realizing STL soon after we found that the resultant N,O-benzylidene acetal inter-
mechanisms and terminal functionalities is still a demanding and attractive research topic for synthetic protein chemistry.
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ORIGIN OF SERINE/THREONINE LIGATION The central element of NCL is the use of natural cysteine with unique reactivity (e.g., superior nucleophilicity, reversible transthioesterification, and rapid S-to-N acyl transfer) as the reacting N-terminus. To identify other N-terminal natural amino acids to mediate chemoselective peptide ligation, different strategies should be conceived. In our efforts, we developed an alternative and thiol-independent chemoselective peptide ligation directly using natural serine or threonine building blocks, termed Ser/Thr ligation (STL) (Figure 2a). We found that an N-terminal serine or threonine of one unprotected peptide carrying the 1,2-hydroxylamine bifunctional group can undergo chemoselective oxazolidine formation with the aldehyde group of a salicylaldehyde (SAL) ester installed at the C-terminus of a second unprotected peptide as the reversible prior capture, and subsequent O-to-N acyl transfer affords an N,O-benzylidene acetal-linked peptide that upon acidolysis generates the natural peptidic Xaa−Ser/ Thr linkage at the ligation site.26 Prior to the discovery of STL, one of the authors observed that when an N-protected glycine reacted with a serine-derived isonitrile, a dipeptide structure with formate modification of the serine side chain was formed in 25% yield at room temperature (Scheme 1a).23 This reaction likely proceeded through the formation of the imidate adduct between the carboxylic acid and isonitrile followed by attack of the sidechain hydroxyl group at the electrophilic carbon in a 5-endo-trig 1645
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Scheme 1. Reactions Involving [1,n] O-to-N Acyl Transfers with Putative Fused Bicyclic Transition States (X Indicates No Reaction)
peptides well, and 2,2,2-trifluoroethanol (TFE) or 2,2,2,2′,2′,2′-hexafluoroisopropanol (HFIP) could be added as a cosolvent to better solubilize the peptides if necessary. The internal side-chain functionalities in the peptides were welltolerated. It is worth noting that the side-chain amine of the internal lysine might compete to react with the aldehyde, but this reversible and unproductive imine formation could not interfere with the ligation pathway.29 To explore the scope and limitations of STL, the effect of the C-terminal residue on the ligation efficiency was investigated using model decapeptide SAL esters with 17 different C-terminal amino acids (the peptide SAL esters with C-terminal Lys/Asp/Glu could not be prepared as stable compounds).29 Under standard ligation conditions, seven peptides gave >65% conversion (FAST) at 2 h, while six and four peptides gave 33−45% (MEDIUM) and 150 amino acids). To maximize the efficiency, a convergent synthetic strategy had to be developed, for which N-to-C sequential STL was needed. In contrast to the C-to-N STL, the middle peptide fragment with a free N-terminal Ser/Thr and the protected C-terminal SAL ester played the central role. After the preceding ligation at the N-terminus, the aldehyde group of the peptide SAL ester should be regenerated under mild conditions to enable the next ligation. To achieve this goal, after extensive investigations, we found that the semicarbazone was a good surrogate of the aldehyde group for peptide SAL ester
protection. The peptide SAL ester protected as the semicarbazone ester (SALoff) could survive the TFA-mediated global deprotection of peptides and remain inert under the STL conditions, while the aldehyde could be regenerated (switched-on) using a TFA/pyruvic acid cocktail to generate the peptide SAL ester (SALon) for further ligation. The desired peptide SALoff esters could be readily prepared via method D in an “n + 1” manner.39 Interleukin 25 (IL25) is expressed by normal mammary epithelial cells and mediates the apoptosis of breast cancer cells through interaction with the IL25 receptor. IL25 is composed of 145 amino acid residues, with 10 Cys residues and one Nglycan at Asn104. Thus, this protein was a good platform to test the compatibility of our STL with widely used NCL. We finished the assembly of the full-length IL25 polypeptide from six fragments via two STL and three NCL reactions (Scheme 8).39 After the N-to-C STL between fragments (80−89) and (90−118), the C-terminal SALoff ester was switched on. Subsequently, the STL between fragments (80−118) and (119−123) and the following NCL with fragment (124−145) were implemented in one-pot manner without purification of the thioester, which showcased the excellent compatibility of STL with NCL. Finally, the purified fragment (80−145) was ligated with the peptide (1−79) thioester, which was prepared from fragments (1−41) and (42−79) via hydrazide-based NCL,42,43 to achieve the target polypeptide. This convergent synthesis of glycosylated IL25 illustrated well the great potential of STL in the synthesis of complex protein targets. PTMs finely regulate the function of proteins in a dynamic manner. Revealing the effect of PTMs on the protein−protein, protein−carbohydrate, and protein−nucleic acid interactions is of great importance for understanding cellular events and pathogenic processes. However, the difficulties in obtaining homogeneous proteins with designated PTMs at specific positions through isolation or expression methods have impeded PTM−function studies. In this regard, protein chemical synthesis is expected to generate proteins with site1650
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Accounts of Chemical Research Scheme 8. Synthesis of Glycosylated Interleukin 25 via Sequential N-to-C STL
worth pointing out that although all of the selected protein synthesis examples presented here involved Ser/Thr ligation at glycine sites, ligations at other C-terminal residues are also feasible (Figure 2b).
specific modifications installed at a stoichiometric level. The protein of interest to us was high-mobility group protein 1a (HMGA1a), which belongs to the family of non-histone nuclear proteins with diverse roles in transcription, replication, and DNA repair. HMGA1a is composed of 106 amino acid residues with multiple PTMs, including Ser/Thr phosphorylation, Arg methylation, and Lys acetylation. To investigate the effect of PTMs on the function of HMGA1a, we synthesized a small library of HMGA1a analogues with different PTM patterns (Scheme 9).44 The full-length protein was constructed from three fragments (1−37), (38−62), and (63−106) via sequential STL. Introduction of the Hmb group at Gly96 was found to be necessary for the synthesis, as it significantly improved the solubility of fragment (63−106) and the efficiency of STL. Armed with this facile approach, we synthesized eight full-length HMGA1a proteins carrying Ser/ Thr phosphorylation, Arg dimethylation, and N-terminal biotin modification with different patterns.44 We are currently implementing chemical biology research on HMGA1a using our synthetic protein library and related protein probes. It is
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APPLICATION OF SERINE/THREONINE LIGATION IN CYCLIC PEPTIDE SYNTHESIS Cyclic peptides play important roles in drug discovery because of their high target-binding affinity/specificity, benefiting from the conformation-confined peptide backbone, and their smallmolecule-like pharmacokinetic properties. Cyclic peptide natural products have provided countless lead structures for medicinal research and serve as promising targets for drug discovery and development. During the past eight years, intramolecular STL has been found to be effective in peptide cyclization (Figure 3).45 Cyclic peptides ranging from highly strained cyclic tetrapeptides38,46 to a flexible cyclic decapeptide could be synthesized efficiently via STL with minimal oligomerization of the linear precursors. The first natural product we pursued was daptomycin (6), a 1651
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Accounts of Chemical Research Scheme 9. Synthesis of HMGA1a Proteins with Site-Specific PTMs via STL
to generate proteins of reasonable sizes. In particular, the true value of protein chemical synthesis is the flexibility of installing site-specific natural (phosphorylation, glycosylation, methylation, etc.) or unnatural (D-amino acid, photoaffinity group, etc.) modifications during the synthesis. Protein chemical synthesis opens up new opportunities for chemists to answer biological questions that pose challenges for traditional biology and biochemistry. To this end, the development of creative, novel, and practical chemoselective peptide ligation methods should be continuously welcomed. In our own adventure over the past eight years (from 2009 to 2017), we have developed a novel peptide ligation based on the chemoselective reaction of peptide C-terminal salicylaldehyde esters and N-terminal Ser/Thr residues followed by mild acidolysis, termed serine/threonine ligation (STL). As an important complement to the widely used native chemical ligation (NCL), STL has several advantages, including the high abundance of Ser/Thr residues in native proteins (12.7%), no need for N-terminal modification, good compatibility with the C-terminal residues in the SAL esters (with Lys, Glu, and Asp as exceptions), traceless ligation delivering the native peptide linkage, simple reaction systems (pyridine/acetic solutions without additives), and operational simplicity. With efficient preparation methods for the peptide SAL esters established, STL has been applied to protein chemical synthesis. To achieve complex protein targets, we have developed C-to-N and N-to-C sequential STL strategies and investigated the combined application of STL with NCL in a
lipopeptide antibiotic that was approved by the U.S. Food and Drug Administration in 2003 for the treatment of infections caused by MRSA and VRE. Daptomycin consists of 13 amino acid residues, 10 of which cyclize to form a 31-membered ring. In 2013, we finished the first total synthesis of daptomycin via a combination of solution-phase and Fmoc-based solid-phase peptide synthesis.47 As the key step, serine ligation facilitated the macrocyclization at 5 mM in pyridine/acetic acid (1:1 mol/mol) solution and delivered the final product in 67% yield after HPLC purification. This synthetic route was highly efficient, as it could be used to generate one analogue within 1 week.48 In addition, STL was also used for the total synthesis of the bioactive cyclic heptapeptides cyclomontanin B (7)49 and yunnanin C (8).50 In 2016, we finished the total synthesis of teixobactin (9),51 a new antibiotic discovered in 2015 with high antibacterial activity and no observable resistance. In our convergent synthetic approach, the linear hexapeptide fragment was installed on the cyclic core via STL. By means of this strategy, more than 80 analogues were synthesized within 1 year for structure−activity relationship studies.52,53 Currently, the total synthesis of additional cyclic peptides with intriguing structures and potent bioactivities is ongoing in our lab.
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CONCLUSION Although proteins can be readily and cost-effectively obtained via recombinant DNA expression systems, protein chemical synthesis certainly provides an important and irreplaceable way 1652
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Figure 3. Total synthesis of cyclic peptide natural products via STL.
Notes
one-pot operation. With these developments, various cyclic peptides, long peptides/proteins with different functions and complexities, and peptoid−peptide/protein conjugates54−56 have been successfully synthesized with STL by others and us. In particular, we recently applied STL to the synthesis of more than 20 HMGA1a proteins with various site-specific PTMs, which expectedly laid a solid foundation to investigate how PTMs affect HMGA proteins in chromatin biology and chemical epigenetics. We expect that STL will become a useful tool for the synthesis of peptides/proteins and related constructs.
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The authors declare no competing financial interest. Biographies Han Liu got his B.Sc. in 2005 and Ph.D. in 2010 from Peking University. In 2010, he joined Professor Xuechen Li’s group at the University of Hong Kong and has worked as a postdoctoral fellow since then. His research interests include peptide chemistry and total synthesis of oligosaccharides. Xuechen Li got his B.Sc. from Nankai University in 1999 and his M.Sc. from The University of Alberta in 2003. After obtaining his Ph.D. from Harvard University in 2006, he did postdoctoral research at the Memorial Sloan-Kettering Cancer Center from 2007 to 2009. He joined the University of Hong Kong in 2009 as an assistant professor and was then promoted to associate professor in 2014 and full professor in 2018. His research interests include synthetic protein/peptide chemistry, carbohydrate chemistry, medicinal chemistry, and chemical biology.
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Tel: 852-22194992. Fax: 85228571586. ORCID
Xuechen Li: 0000-0001-5465-7727
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Funding
This work was supported by the Research Grants Council of Hong Kong (17309616, 17303617,C7038-15G, C6009-15G), the National Natural Science Foundation of China (21672180, 91753101), and the Area of Excellence Scheme of the University Grants Committee of Hong Kong (Grant AoE/P705/16).
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DOI: 10.1021/acs.accounts.8b00151 Acc. Chem. Res. 2018, 51, 1643−1655
