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ScienceDirect Serine/threonine ligation for the chemical synthesis of proteins Chi Lung Lee1 and Xuechen Li1,2 Advances in the development of efficient peptide ligation methods have enabled the total synthesis of complex proteins to be successfully undertaken. Recently, a Ser/Thr ligation has emerged as a new tool in synthetic protein chemistry. The chemoselective reaction between an N-terminal serine or threonine of an unprotected peptide segment and a C-terminal salicylaldehyde ester of another unprotected peptide segment gives rise to an N,O-benzylidene acetal linked product, which upon acidolysis produces a native peptide bond at the site of ligation. Ser/Thr ligation has been used for the synthesis of the human erythrocyte acylphosphatase protein and MUC1 glycopeptide segments, semisynthesis of peptoid/PEG-RNase S protein hybrids, and cyclic peptide synthesis including cyclic tetrapeptides, cyclomontanin B, yunnanin C, mahafacyclin B, and daptomycin. Addresses 1 Department of Chemistry, The University of Hong Kong, Hong Kong, China 2 The State Key Laboratory of Synthetic Chemistry, The University of Hong Kong, Hong Kong, China Corresponding author: Li, Xuechen ([email protected])
Current Opinion in Chemical Biology 2014, 22:108–114 This review comes from a themed issue on Synthetic biomolecules Edited by Paul F Alewood and Stephen BH Kent For a complete overview see the Issue and the Editorial Available online 6th October 2014 http://dx.doi.org/10.1016/j.cbpa.2014.09.023 1367-5931/# 2014 Elsevier Ltd. All rights reserved.
generation of a native peptide bond (Xaa-Cys) at the ligation site (Xaa represents any amino acids) [5]. It involves the chemoselective reaction between a peptide thioester and the N-terminal cysteine of a second peptide and tolerates all side chain functionalities normally found in proteins; it is the method of choice for protein chemical synthesis [3,6]. However, the low abundance of the cysteine residue content in proteins [7] may limit the widest application of NCL in synthetic protein chemistry, though the introduction of various b-thiolated residues has broadened its scope [8,9]. Nevertheless, the need for synthetic proteins has stimulated researchers to explore chemoselective peptide ligation at other amino acid sites [8,10]. It is challenging to develop chemoselective reactions in the presence of various nucleophilic groups including amino side chain of Lys, imidazole of His, carboxylic acid of Asp/Glu, hydroxyl of Ser/Thr/Tyr, thiol of Cys, and guanidine of Arg. More importantly, the generation of the natural peptidic linkage at the ligation site increases the challenge. Many chemoselective peptide ligation methods have been developed so far [8,10], including NCL-desulfurization [9,11,12], a-ketoacid-hydroxylamine ligation [13], and the Staudinger ligation [14]. Partially chemoselective ligations by direct aminolysis require the internal lysine residue to be protected and include thioester-mediated ligation [15,16], phenol estermediated ligation [17], and thioacid based-ligation [18]. Nevertheless, methods capable of delivering synthetic proteins remain limited, and in addition, most of the above mentioned methods require laborious additional steps to make them chemoselective and have found only limited practical application.
Development of serine/threonine ligation Introduction The advent of Merrifield’s solid phase peptide synthesis (SPPS) [1] opened up the possibility of synthesizing small proteins via chemical synthesis. Nevertheless, it was debated in the 60s, 70s and 80s whether the synthesis of proteins up to 100 amino acids and beyond could ever be achieved. This led to an increasing effort to optimize all aspects of SPPS including the fidelity of the amino acids, the power of current coupling agents, the choices of solvent and resins — all underpinned by better quality control [2–4]. However, it was not until the development of native chemical ligation (NCL) that chemical protein synthesis became a reality. NCL allows the chemoselective ligation of unprotected peptide segments with the Current Opinion in Chemical Biology 2014, 22:108–114
We wished to develop a novel chemical ligation strategy using natural amino acids other than cysteine at the Nterminus of the second ligating peptide. Towards this goal, we came to realize that the chemoselectivity of cysteine-based NCL lies in the bi-functionality of the N-terminal cysteine being a 1,2-mercapto amine. This was the key to differentiate itself from the internal nucleophilic mono-functional groups (e.g., thiol, amino) as the thiol group first undergoes reversible and chemoselective transthioesterification with the thioester at the C-terminus and subsequently undergoes rapid and irreversible 1,4 S to N acyl transfer to produce a more stable amide bond. Since only the N-terminal Cys can undergo this rearrangement, NCL is regioselective even in the www.sciencedirect.com
Serine/threonine ligation for synthetic biomolecules Lee and Li 109
presence of internal Cys residues in either reacting segment.
peptide segment could react selectively with the 1,amino2-hydroxyl functionalities on another unprotected peptide segment with N-terminal serine or threonine to produce an N,O-benzylidene ketal/or acetal linked product. The key issue would then become whether the resultant N,O-benzylidene ketal/or acetal intermediate, if generated, could be readily converted into the natural peptidic linkage under the peptide-compatible conditions (Scheme 1).
Thus, we considered using N-terminal serine-containing or threonine-containing peptides, which are also bi-functional amino acids, to mediate a chemoselective peptide ligation. Since serine and threonine residues are very abundant in natural proteins (12% content in protein) [7], enabling serine/threonine ligations should be attractive in protein chemical synthesis. Indeed, to realize a chemoselective serine/threonine ligation is apparently difficult if not impossible. Different from the ‘‘soft’’ thiol group of cysteine used in NCL, the hydroxyl and amino groups of the N-terminal serine or threonine are ‘‘hard’’ nucleophiles; neither of them can compete with other internal side chain nucleophilic ‘‘hard’’ functionalities (e.g., hydroxyl of Ser/Thr/Tyr, amino of Lys). Thus, the lack of superior nucleophilicity renders the N-terminal serine or threonine difficult to mediate a chemoselective reaction. As a result, we believe that the realization of chemoselectivity using N-terminal serine or threonine has to involve the synergetic action of the 1,amino-2hydroxyl functionalities.
Indeed, along the proposed reaction pathway, several issues concerned us. Firstly, since the first capture step relies on imine formation between the carbonyl group and the amino group of the N-terminal serine or threonine, would other internal amino groups of Lys also compete for imine formation, thereby affecting the desired pathway? Secondly, the oxazolidine formation via 5-endo-trig cyclization is an anti-Baldwin cyclization [21], whose effectiveness thus remains uncertain. Thirdly, the third step involves a 1,5 O to N acyl transfer, via a 6-membered ring transition state. In NCL, a 1,4 S to N acyl transfer via a 5membered ring transition state is very rapid, while the efficiency of 1,5 O to N acyl transfer of complex peptide moieties remains to be explored. Lastly, if the ligation were to occur, would the resultant N,O-benzylidene ketal/or acetal group be easily removed to reveal the natural peptidic linkage? To our best knowledge, N,O-benzylidene ketal/or acetal moieties have rarely been reported in the literature, thus no mild and peptide-compatible conditions to remove the N,O-benzylidene ketal/or acetal group had been described. Indeed, most reaction conditions that work for small molecules are not applicable for peptides,
Previously, Kemp [19] and Tam [20] pioneered the development of the imine-induced intramolecular acyl transfer strategy. Though these strategies have not been translated into practical chemoselective peptide ligations, or ones that deliver synthetic peptides or proteins with a native peptidic linkage at the ligation site, we were encouraged to investigate whether a phenol ester with a carbonyl group at the C-terminus of an unprotected
Scheme 1 O H2 N
O CO2H
NH 2
O OH
HN
H2N R O
HN
NH2
C O
CO2H
OH
O OH
HN
H 2N N
CO2H
NH2
HO H2N
R
NH2
R O O
HN
NH
CO2H
OH
R
H2 N
NH
R = H or Me N HO O
O R
R
NH
1,5 O → N acyl transfer
5-endo-trig cyclization
N
O H2 N
O H2 N NH2
CO2H
HN
NH2
H+
O OH
HN
NH2
N H HO
R
OH
CO2H
CO2H OH
HN HN
NH
N R
R
O H 2N
OH
CO2H
OH
H2 N N
NH2
O
N
NH
N,O-benzylidene acetal/ketal Current Opinion in Chemical Biology
Principle of Ser/Thr Ligation. Two unprotected peptides join together to produce an N,O-benzylidene acetal/ketal intermediate, which upon acidolysis results in a ligated product with a native Xaa-Ser/Thr at the conjunction site. www.sciencedirect.com
Current Opinion in Chemical Biology 2014, 22:108–114
110 Synthetic biomolecules
due to the complexity of the functional groups present and the fragility of the peptides themselves. In our model studies, the peptide salicylaldehyde (SAL) ester reacted with the N-terminal Ser/Thr peptide very smoothly to produce the proposed N,O-benzylidene acetal intermediate [22]. The proposed mechanism has been recently supported by a fluorogenic experiment [23]. As importantly, we found that the resultant N,Obenzylidene acetal is very acid-labile. Upon acidolysis with TFA, the natural Xaa-Ser/Thr linkage at the ligation site was obtained [22]. Among the carbonyl-containing phenol groups we had explored, the salicylaldehyde ester turned out to work the best. We have examined the epimerization issue at the Cterminus under the serine/threonine ligation conditions and epimerization was not observed [24]. In fact, the major pathway resulting in epimerization during peptide coupling is through the formation of a 5(4H)-oxazolone which racemizes via enolization [25] with subsequent reopening of the oxazolone by the amino component yielding epimeric products. If such reaction pathway were to occur during the serine/threonine ligation condition, this product would have a different molecular weight
from the N,O-benzylidene acetal linked product and be distinguishable by LC–MS analysis. We carefully analyzed the crude ligation mixture and did not observe the undesired product. Next, we studied the effect of an unprotected lysine on the serine/threonine ligation [26]. The first step of Ser/Thr ligation is the imine formation between the amino group of the N-terminal serine or threonine residue and the aldehyde group of the C-terminal SAL ester; thus it is likely that the internal Lys would compete for the imine formation, thereby slowing down or inhibiting the ligation, even though it could not form the productive oxazoline intermediate. To address this question, we have used a competitive experiment to study the effect of Lys at different positions of the N-terminal Ser/Thr peptide on the ligation, in which an N-terminal peptide without any Lys residue (K0) and another N-terminal peptide with Lys residue at varied positions (K2–K5) competitively react with the same C-terminal peptide. Our studies have revealed that an internal Lys did not adversely impact the efficiency of the ligation. Indeed, Lys placed at the adjacent site to the N-terminal Ser (i + 1) helped accelerate the ligation, possibly due to a proximity-induced imine exchange effect [27]. The systematic studies on the effect
Scheme 2
(a) Fmoc-SPPS HN H2N
(b)
O
H N
protected peptide
Fmoc-SPPS N H
H2N Unprotected peptide
N H
O H2N
O
O
protected peptide
O
O
O
N
(1) on-resin phenolysis
O
Crude N3-SQEPPISLSLTFHLLREVLEM-SAL-ester
O
N H
Crude NH2-YADAIFTNSYRKVLGQL-SAL-ester
salicylaldehyde
N H
H (2) global deprotection
(c) Time (min)
Boc-SPPS OH
O O
O
protected peptide
Boc-SPPS
O
(d) N H
N H
Crude NH2-SAAA-SAL-ester
HF
TMSOTf/TFA O H2N Unprotected peptide
O
O
O N H
H2N
O3 CH2CI2/TFA H2N Unprotected peptide
Unprotected peptide
O
O
Crude NH2-TINA-SAL-ester NH2
O3 MeCN/H2O
O O
O Time (min) H
Current Opinion in Chemical Biology
The synthesis of the peptide salicylaldehyde esters. (a) The approach via Fmoc chemistry SPPS, (b) Representative analytical HPLC-traces of the crude reaction mixture from Fmoc-SPPS of peptide SAL esters. (c) The approach via Boc chemistry SPPS. (d) Representative analytical HPLC-traces of the crude reaction mixture from Boc-SPPS of peptide SAL esters. Current Opinion in Chemical Biology 2014, 22:108–114
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Serine/threonine ligation for synthetic biomolecules Lee and Li 111
of the Lys residue at the different position of the peptide SAL ester on the ligation have not been conducted yet.
Scope and limitations of Ser/Thr ligation
have systematically evaluated the compatibility and reactivity of all 20 proteinogenic amino acids at the C-terminus of the peptide SAL ester under Ser/Thr ligation conditions.
The amino acid residues at the C-terminus of the peptide thioester reactant can dramatically affect the efficiency of a ligation reaction as observed in NCL studies [28]. In particular, b branched amino acids, i.e., Val, Ile, Thr retard the ligation significantly. In addition ligation with a C-terminal proline is very slow [28]. Analogously, we
We used NH2-AEGSQAKFGX-SAL ester and NH2SPKALTFG-CO2H (1 mM) to examine the scope and limitations of Ser/Thr ligation. Among the 20 naturally occurring amino acids, all except Asp, Glu and Lys were found to be suitable at the C-terminal of the peptide SAL
Scheme 3 HO
Me
FmocHN
47
(a)
HO
O O
70
71
H2N
CHO
98
OH
1. serine ligation 2. de-Fmoc with Et2NH/CH2CI2 HO
O 46
1
O CHO
Me 70
47
H2N
OH
O N H
71
98
OH
threonine ligation
O 1
46
HO
Me
N H
47
OH
O 70
N H
71
OH
98
folding
human erythrocyte acylphosphatase
(b)
S-Peptide CHO
Peptoid/PEG
O O
Subtilisin OH
O HO
RNase A
HN
Peptoid/PEG
NH2
O
S-Protein Current Opinion in Chemical Biology
Synthesis of human erythrocyte acylphosphatase via two Ser/Thr ligations. (a) Chemical synthesis of human erythrocyte acylphosphatase; (b) Semisynthesis of a peptoid/PEG-protein hybrids. www.sciencedirect.com
Current Opinion in Chemical Biology 2014, 22:108–114
112 Synthetic biomolecules
ester used for the ligation. Interestingly, the ligation at the bulky b branched amino acid Thr behaved similarly to that at the least hindered amino acid Gly. Accordingly, we have classified the C-terminal amino acids into three groups: fast reaction with Ala, Gly, Ser, Glu, Thr, Phe and Cys(StBu), modest reaction with Val, Leu, Ile, Asn Phe, Met and Tyr, and slow reaction with Arg, Trp and Pro [26]. The same trend was observed in serine/threonine ligation-mediated peptide cyclization. During the synthesis of yunnanin C analogues, it is found that the cyclization at Pro-Ser site was extremely slow, while the cyclization at Val-Ser proceeded similarly to that at Tyr-Ser [29]. An explanation for the similar anomalouslylow reactivity of Pro-thioesters has recently been proposed [30].
this end, we took advantage of the strategies developed for the synthesis of peptide thioesters by Fmoc chemistry SPPS [31]. In particular, Dawson and co-workers have been able to synthesize peptide thioesters via in-solution thiolysis of the peptide N-acyl-benzimidazolinone (Nbz) which is readily generated via Fmoc-SPPS [32]. We conducted on-resin phenolysis of the peptide Nbz with salicylaldehyde dimethyl acetal to release the peptide SAL ester from the resin in overall yields of 20–30% based on resin loading, without observing epimerization of the C-terminal residue [24]. This strategy provides a reliable approach to the Fmoc chemistry solid phase synthesis of peptide SAL esters (Scheme 2a,b). Alternatively, we have developed Boc chemistry SPPS methods to generate the peptide SAL ester (Scheme 2c,d). To this end, 20 -hydroxyl cinnamate was first linked to the AM resin. After completion of the peptide chain using Boc-SPPS and TMSOTf/TFA mediated global deprotection, the side chain unprotected peptide SAL ester was generated and then released from resin upon onresin ozonolysis [33]. Similarly, Liu et al. used 20 hydroxyl cinnamate-MBHA resin where the peptide
Synthesis of the requisite peptide SAL ester In applying Ser/Thr ligation to peptide/protein synthesis one urgent issue to address is the preparation of the requisite peptide SAL ester via SPPS. As the O-SAL ester is labile to piperidine-mediated Fmoc removal conditions, direct solid phase synthesis of the peptide SAL ester by way of Fmoc chemistry is not possible. Towards
Scheme 4
H 3N O NH CONH 2
O
NH
H N
N H
O
O N H
O
N H
NH
O
O
COOH O
O
H N
O HOOC HOOC HO H N
O
N H
O
O
N H HN
O O NH
N H
O
COOH
NH 2 Daptomycin (Cubicin)
Ser Ligation OH
O H 2N O N H O
HN
Ph O
O O
NH O
O
H N
N
O O
NH
O
NH
NH 2
OH Cyclomontanin B
Ph
HN
N H O
HO
H N
H N
O
O
NH O
Thr Ligation
N H
HN
Ph NH
Thr ligation
O
OO O
NH
Ph
HN
N
HN NH
O
O Thr Ligation Mahafacyclin B
OH
O
H N O Yunnanin C
Current Opinion in Chemical Biology
Natural cyclic peptides synthesized by way of Ser/Thr ligation-mediated peptide cyclization. Current Opinion in Chemical Biology 2014, 22:108–114
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Serine/threonine ligation for synthetic biomolecules Lee and Li 113
chain was first cleaved from the resin with either TFMSA/ TFA or HF, followed by in-solution ozonolysis to generate the peptide SAL esters [34]. It is noted that Cys, Met and Trp are incompatible with this method.
address explicit biological questions. In this regard, continuing efforts to further optimize existing methods and develop innovative and efficient methods and strategies to synthesize bio-macromolecules are urgently needed.
Applications of serine/threonine ligation in the synthesis of peptides/proteins and cyclic peptides
Acknowledgements
Serine/threonine ligation has recently been successfully applied to the synthesis of the human erythrocyte acylphosphatase protein (11 kDa) [24]. This protein molecule contains a number of well-distributed serine and threonine residues but no cysteine residues and thus is a good model to demonstrate the effectiveness of the serine/threonine ligation. Human erythrocyte acylphosphatase protein was assembled via two ligations at Ser71 and Thr47 respectively (Scheme 3a). The folded synthetic enzyme exhibited similar bioactivity to the natural protein [24]. Later, the Li group synthesized glycopeptide, MUC1 80-mer segments, via 4 iterative serine ligations [35]. Recently, Kirshenbaum and co-workers applied Ser/Thr ligation to the semi-synthesis of hybrid peptoid-protein conjugates and to protein modifications. The N-terminal portion of RNase A (residue 1–20) was cleaved by proteolysis to reveal a serine residue at the N-terminus, affording the S-protein (Scheme 3b). Next, the peptide salicylaldehyde ester was ligated with the S-protein to generate the desired peptoid-protein hybrid [36]. More recently, they have extended the same strategy for side-specific N-terminal protein PEGylation [37]. In another application, serine/threonine ligation has been applied intramolecularly to synthesize cyclic peptides that contain serine or threonine residues. Various cyclic peptides of sizes from 4 to 9 amino acids have been successfully prepared by way of serine/threonine ligation-mediated peptide cyclization [33,34]. As such, several natural bioactive cyclic peptides have been synthesized, including cyclomontanin B [38], yunnanin C [29], mahafacyclin B [34] and daptomycin [39] (Scheme 4). The cyclization proceeded smoothly, even in the presence of the unprotected lysine residue. In particular, highly constrained cyclic tetrapeptides that have been very difficult to obtain using other methods could be readily synthesized by serine/threonine ligationmediated peptide cyclization [33,34].
Conclusion Ser/Thr ligation has emerged as an alternative peptide ligation chemistry to synthesize peptides, proteins and cyclic peptides. Over the past decades, synthetic protein chemistry has provided a unique tool to generate proteins for the biological and medicinal studies of proteins, particularly those carrying post-translational modifications. As such, both chemists and biologists need to embrace and appreciate the power of chemical synthesis to synthesize and manipulate bio-macromolecules to www.sciencedirect.com
This work was supported by the Research Grants Council-General Research Fund of Hong Kong (HKU703811P, 707412P, 702813P), the National Basic Research Program of China (973 Program, 2013CB836900), the Peacock Program-Project Development Fund (KQC201109050074A) and the University of Hong Kong.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
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9. Wong CTT, Tung CL, Li X: Synthetic cysteine surrogates used in native chemical ligation. Mol Biosyst 2013, 9:826-833. A up-to-date review paper on native chemical ligation-desulfurization. 10. Pattabiraman VR, Bode JW: Rethinking amide bond synthesis. Nature 2011, 480:471-479. 11. Yan LZ, Dawson PE: Synthesis of peptides and proteins without cysteine residues by native chemical ligation combined with desulfurization. J Am Chem Soc 2001, 123:526-533. 12. Wan Q, Danishefsky SJ: Free-radical-based, specific desulfurization of cysteine: a powerful advance in the synthesis of polypeptides and glycopolypeptides. Angew Chem Int Ed Engl 2007, 46:9248-9252. 13. Pattabiraman VR, Ogunkoya AO, Bode JW: Chemical protein synthesis by chemoselective a-ketoacid-hydroxylamine (KAHA) ligations with 5-oxaproline. Angew Chem Int Ed Engl 2012, 51:5114-5118. 14. Nilsson BL, Kiessling LL, Raines RT: Staudinger ligation: a peptide from a thioester and azide. Org Lett 2000, 2:1939-1941. 15. Aimoto S: Contemporary methods for peptide and protein synthesis. Curr Org Chem 2001, 5:45-87. 16. Payne RJ, Ficht S, Greenberg WA, Wong CH: Cysteine-free peptide and glycopeptide ligation by direct aminolysis. Angew Chem Int Ed Engl 2008, 47:4411-4415. 17. Chen G, Wan Q, Tan Z, Kan C, Hua Z, Ranganathan K, Danishefsky SJ: Development of efficient methods for accomplishing cysteine-free peptide and glycopeptide coupling. Angew Chem Int Ed Engl 2007, 46:7383-7387. Current Opinion in Chemical Biology 2014, 22:108–114
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18. Crich D, Sharma I: Epimerization-free block synthesis of peptides from thioacids and amines with the Sanger and Mukaiyama reagents. Angew Chem Int Ed Engl 2009, 48: 2355-2358. 19. Kemp DS: The amine capture strategy for peptide-bond formation – an outline of progress. Biopolymers 1981, 20: 1793-1804. 20. Liu CF, Tam JP: Peptide segment ligation strategy without use of protecting groups. Proc Natl Acad Sci U S A 1994, 91: 6584-6588. 21. Gilmore K, Alabugin IV: Cyclizations of alkynes: revisiting Baldwin’s rules for ring closure. Chem Rev 2011, 111:6513-6556. 22. Li X, Lam HY, Zhang Y, Chan CK: Salicylaldehyde ester-induced chemoselective peptide ligations: enabling generation of natural peptidic linkages at the serine/threonine sites. Org Lett 2010, 12:1724-1727. 23. Tung CL, Lam HY, Xu J, Li X: A fluorogenic probe for recognizing 5-hydroxylysine inspired by serine/threonine ligation. Chem Commun 2014, 50:5298-5300. 24. Zhang Y, Xu C, Lam HY, Lee CL, Li X: Protein chemical synthesis by serine and threonine ligation. Proc Natl Acad Sci U S A 2013, 110:6657-6662. This paper demonstrated the application of Ser/Thr ligation for protein total chemical synthesis. In addition, a method to synthesize the requisite peptide salicylaldehyde ester has been developed via Fmoc-SPPS.
30. Pollock SB, Kent SBH: An investigation into the origin of the dramatically reduced reactivity of peptide-prolyl-thioesters in native chemical ligation. Chem Commun 2011, 47:2342-2344. 31. Mende F, Seitz O: 9-Fluorenylmethoxycarbonyl-based solidphase synthesis of peptide a-thioesters. Angew Chem Int Ed Engl 2011, 50:1232-1240. 32. Blanco-Canosa JB, Dawson PE: An efficient Fmoc-SPPS approach for the generation of thioester peptide precursors for use in native chemical ligation. Angew Chem Int Ed Engl 2008, 47:6851-6855. 33. Wong CTT, Lam HY, Song T, Chen G, Li X: Synthesis of constrained head-to-tail cyclic tetrapeptides by an imineinduced ring-closing/contraction strategy. Angew Chem Int Ed Engl 2013, 52:10212-10215. This paper reported a method to synthesize peptide salicylaldehyde ester by Boc chemistry SPPS. In addition, Ser/Thr ligation-mediated peptide cyclization was successfully used to synthesize cyclic tetrapeptides. Molecular modeling was used to explain the success of the tetrapeptide cyclization via Ser/Thr ligation. 34. Zhao JF, Zhang XH, Ding YJ, Yang YS, Bi XB, Liu CF: Facile synthesis of peptidyl salicylaldehyde esters and its use in cyclic peptide synthesis. Org Lett 2013, 15:5182-5185. This paper reported a method to synthesize peptide salicylaldehyde ester by Boc chemistry SPPS. In addition, Ser/Thr ligation-mediated peptide cyclization was used to synthesize cyclic peptide of various sizes and mahafacyclin B.
25. Bodanszky M, Bodanszky A: Racemization in peptide synthesis. Mechanism-specific models. Chem Commun 1967:591-593.
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Current Opinion in Chemical Biology 2014, 22:108–114
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ScienceDirect Serine/threonine ligation for the chemical synthesis of proteins Chi Lung Lee1 and Xuechen Li1,2 Advances in the development of efficient peptide ligation methods have enabled the total synthesis of complex proteins to be successfully undertaken. Recently, a Ser/Thr ligation has emerged as a new tool in synthetic protein chemistry. The chemoselective reaction between an N-terminal serine or threonine of an unprotected peptide segment and a C-terminal salicylaldehyde ester of another unprotected peptide segment gives rise to an N,O-benzylidene acetal linked product, which upon acidolysis produces a native peptide bond at the site of ligation. Ser/Thr ligation has been used for the synthesis of the human erythrocyte acylphosphatase protein and MUC1 glycopeptide segments, semisynthesis of peptoid/PEG-RNase S protein hybrids, and cyclic peptide synthesis including cyclic tetrapeptides, cyclomontanin B, yunnanin C, mahafacyclin B, and daptomycin. Addresses 1 Department of Chemistry, The University of Hong Kong, Hong Kong, China 2 The State Key Laboratory of Synthetic Chemistry, The University of Hong Kong, Hong Kong, China Corresponding author: Li, Xuechen ([email protected])
Current Opinion in Chemical Biology 2014, 22:108–114 This review comes from a themed issue on Synthetic biomolecules Edited by Paul F Alewood and Stephen BH Kent For a complete overview see the Issue and the Editorial Available online 6th October 2014 http://dx.doi.org/10.1016/j.cbpa.2014.09.023 1367-5931/# 2014 Elsevier Ltd. All rights reserved.
generation of a native peptide bond (Xaa-Cys) at the ligation site (Xaa represents any amino acids) [5]. It involves the chemoselective reaction between a peptide thioester and the N-terminal cysteine of a second peptide and tolerates all side chain functionalities normally found in proteins; it is the method of choice for protein chemical synthesis [3,6]. However, the low abundance of the cysteine residue content in proteins [7] may limit the widest application of NCL in synthetic protein chemistry, though the introduction of various b-thiolated residues has broadened its scope [8,9]. Nevertheless, the need for synthetic proteins has stimulated researchers to explore chemoselective peptide ligation at other amino acid sites [8,10]. It is challenging to develop chemoselective reactions in the presence of various nucleophilic groups including amino side chain of Lys, imidazole of His, carboxylic acid of Asp/Glu, hydroxyl of Ser/Thr/Tyr, thiol of Cys, and guanidine of Arg. More importantly, the generation of the natural peptidic linkage at the ligation site increases the challenge. Many chemoselective peptide ligation methods have been developed so far [8,10], including NCL-desulfurization [9,11,12], a-ketoacid-hydroxylamine ligation [13], and the Staudinger ligation [14]. Partially chemoselective ligations by direct aminolysis require the internal lysine residue to be protected and include thioester-mediated ligation [15,16], phenol estermediated ligation [17], and thioacid based-ligation [18]. Nevertheless, methods capable of delivering synthetic proteins remain limited, and in addition, most of the above mentioned methods require laborious additional steps to make them chemoselective and have found only limited practical application.
Development of serine/threonine ligation Introduction The advent of Merrifield’s solid phase peptide synthesis (SPPS) [1] opened up the possibility of synthesizing small proteins via chemical synthesis. Nevertheless, it was debated in the 60s, 70s and 80s whether the synthesis of proteins up to 100 amino acids and beyond could ever be achieved. This led to an increasing effort to optimize all aspects of SPPS including the fidelity of the amino acids, the power of current coupling agents, the choices of solvent and resins — all underpinned by better quality control [2–4]. However, it was not until the development of native chemical ligation (NCL) that chemical protein synthesis became a reality. NCL allows the chemoselective ligation of unprotected peptide segments with the Current Opinion in Chemical Biology 2014, 22:108–114
We wished to develop a novel chemical ligation strategy using natural amino acids other than cysteine at the Nterminus of the second ligating peptide. Towards this goal, we came to realize that the chemoselectivity of cysteine-based NCL lies in the bi-functionality of the N-terminal cysteine being a 1,2-mercapto amine. This was the key to differentiate itself from the internal nucleophilic mono-functional groups (e.g., thiol, amino) as the thiol group first undergoes reversible and chemoselective transthioesterification with the thioester at the C-terminus and subsequently undergoes rapid and irreversible 1,4 S to N acyl transfer to produce a more stable amide bond. Since only the N-terminal Cys can undergo this rearrangement, NCL is regioselective even in the www.sciencedirect.com
Serine/threonine ligation for synthetic biomolecules Lee and Li 109
presence of internal Cys residues in either reacting segment.
peptide segment could react selectively with the 1,amino2-hydroxyl functionalities on another unprotected peptide segment with N-terminal serine or threonine to produce an N,O-benzylidene ketal/or acetal linked product. The key issue would then become whether the resultant N,O-benzylidene ketal/or acetal intermediate, if generated, could be readily converted into the natural peptidic linkage under the peptide-compatible conditions (Scheme 1).
Thus, we considered using N-terminal serine-containing or threonine-containing peptides, which are also bi-functional amino acids, to mediate a chemoselective peptide ligation. Since serine and threonine residues are very abundant in natural proteins (12% content in protein) [7], enabling serine/threonine ligations should be attractive in protein chemical synthesis. Indeed, to realize a chemoselective serine/threonine ligation is apparently difficult if not impossible. Different from the ‘‘soft’’ thiol group of cysteine used in NCL, the hydroxyl and amino groups of the N-terminal serine or threonine are ‘‘hard’’ nucleophiles; neither of them can compete with other internal side chain nucleophilic ‘‘hard’’ functionalities (e.g., hydroxyl of Ser/Thr/Tyr, amino of Lys). Thus, the lack of superior nucleophilicity renders the N-terminal serine or threonine difficult to mediate a chemoselective reaction. As a result, we believe that the realization of chemoselectivity using N-terminal serine or threonine has to involve the synergetic action of the 1,amino-2hydroxyl functionalities.
Indeed, along the proposed reaction pathway, several issues concerned us. Firstly, since the first capture step relies on imine formation between the carbonyl group and the amino group of the N-terminal serine or threonine, would other internal amino groups of Lys also compete for imine formation, thereby affecting the desired pathway? Secondly, the oxazolidine formation via 5-endo-trig cyclization is an anti-Baldwin cyclization [21], whose effectiveness thus remains uncertain. Thirdly, the third step involves a 1,5 O to N acyl transfer, via a 6-membered ring transition state. In NCL, a 1,4 S to N acyl transfer via a 5membered ring transition state is very rapid, while the efficiency of 1,5 O to N acyl transfer of complex peptide moieties remains to be explored. Lastly, if the ligation were to occur, would the resultant N,O-benzylidene ketal/or acetal group be easily removed to reveal the natural peptidic linkage? To our best knowledge, N,O-benzylidene ketal/or acetal moieties have rarely been reported in the literature, thus no mild and peptide-compatible conditions to remove the N,O-benzylidene ketal/or acetal group had been described. Indeed, most reaction conditions that work for small molecules are not applicable for peptides,
Previously, Kemp [19] and Tam [20] pioneered the development of the imine-induced intramolecular acyl transfer strategy. Though these strategies have not been translated into practical chemoselective peptide ligations, or ones that deliver synthetic peptides or proteins with a native peptidic linkage at the ligation site, we were encouraged to investigate whether a phenol ester with a carbonyl group at the C-terminus of an unprotected
Scheme 1 O H2 N
O CO2H
NH 2
O OH
HN
H2N R O
HN
NH2
C O
CO2H
OH
O OH
HN
H 2N N
CO2H
NH2
HO H2N
R
NH2
R O O
HN
NH
CO2H
OH
R
H2 N
NH
R = H or Me N HO O
O R
R
NH
1,5 O → N acyl transfer
5-endo-trig cyclization
N
O H2 N
O H2 N NH2
CO2H
HN
NH2
H+
O OH
HN
NH2
N H HO
R
OH
CO2H
CO2H OH
HN HN
NH
N R
R
O H 2N
OH
CO2H
OH
H2 N N
NH2
O
N
NH
N,O-benzylidene acetal/ketal Current Opinion in Chemical Biology
Principle of Ser/Thr Ligation. Two unprotected peptides join together to produce an N,O-benzylidene acetal/ketal intermediate, which upon acidolysis results in a ligated product with a native Xaa-Ser/Thr at the conjunction site. www.sciencedirect.com
Current Opinion in Chemical Biology 2014, 22:108–114
110 Synthetic biomolecules
due to the complexity of the functional groups present and the fragility of the peptides themselves. In our model studies, the peptide salicylaldehyde (SAL) ester reacted with the N-terminal Ser/Thr peptide very smoothly to produce the proposed N,O-benzylidene acetal intermediate [22]. The proposed mechanism has been recently supported by a fluorogenic experiment [23]. As importantly, we found that the resultant N,Obenzylidene acetal is very acid-labile. Upon acidolysis with TFA, the natural Xaa-Ser/Thr linkage at the ligation site was obtained [22]. Among the carbonyl-containing phenol groups we had explored, the salicylaldehyde ester turned out to work the best. We have examined the epimerization issue at the Cterminus under the serine/threonine ligation conditions and epimerization was not observed [24]. In fact, the major pathway resulting in epimerization during peptide coupling is through the formation of a 5(4H)-oxazolone which racemizes via enolization [25] with subsequent reopening of the oxazolone by the amino component yielding epimeric products. If such reaction pathway were to occur during the serine/threonine ligation condition, this product would have a different molecular weight
from the N,O-benzylidene acetal linked product and be distinguishable by LC–MS analysis. We carefully analyzed the crude ligation mixture and did not observe the undesired product. Next, we studied the effect of an unprotected lysine on the serine/threonine ligation [26]. The first step of Ser/Thr ligation is the imine formation between the amino group of the N-terminal serine or threonine residue and the aldehyde group of the C-terminal SAL ester; thus it is likely that the internal Lys would compete for the imine formation, thereby slowing down or inhibiting the ligation, even though it could not form the productive oxazoline intermediate. To address this question, we have used a competitive experiment to study the effect of Lys at different positions of the N-terminal Ser/Thr peptide on the ligation, in which an N-terminal peptide without any Lys residue (K0) and another N-terminal peptide with Lys residue at varied positions (K2–K5) competitively react with the same C-terminal peptide. Our studies have revealed that an internal Lys did not adversely impact the efficiency of the ligation. Indeed, Lys placed at the adjacent site to the N-terminal Ser (i + 1) helped accelerate the ligation, possibly due to a proximity-induced imine exchange effect [27]. The systematic studies on the effect
Scheme 2
(a) Fmoc-SPPS HN H2N
(b)
O
H N
protected peptide
Fmoc-SPPS N H
H2N Unprotected peptide
N H
O H2N
O
O
protected peptide
O
O
O
N
(1) on-resin phenolysis
O
Crude N3-SQEPPISLSLTFHLLREVLEM-SAL-ester
O
N H
Crude NH2-YADAIFTNSYRKVLGQL-SAL-ester
salicylaldehyde
N H
H (2) global deprotection
(c) Time (min)
Boc-SPPS OH
O O
O
protected peptide
Boc-SPPS
O
(d) N H
N H
Crude NH2-SAAA-SAL-ester
HF
TMSOTf/TFA O H2N Unprotected peptide
O
O
O N H
H2N
O3 CH2CI2/TFA H2N Unprotected peptide
Unprotected peptide
O
O
Crude NH2-TINA-SAL-ester NH2
O3 MeCN/H2O
O O
O Time (min) H
Current Opinion in Chemical Biology
The synthesis of the peptide salicylaldehyde esters. (a) The approach via Fmoc chemistry SPPS, (b) Representative analytical HPLC-traces of the crude reaction mixture from Fmoc-SPPS of peptide SAL esters. (c) The approach via Boc chemistry SPPS. (d) Representative analytical HPLC-traces of the crude reaction mixture from Boc-SPPS of peptide SAL esters. Current Opinion in Chemical Biology 2014, 22:108–114
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Serine/threonine ligation for synthetic biomolecules Lee and Li 111
of the Lys residue at the different position of the peptide SAL ester on the ligation have not been conducted yet.
Scope and limitations of Ser/Thr ligation
have systematically evaluated the compatibility and reactivity of all 20 proteinogenic amino acids at the C-terminus of the peptide SAL ester under Ser/Thr ligation conditions.
The amino acid residues at the C-terminus of the peptide thioester reactant can dramatically affect the efficiency of a ligation reaction as observed in NCL studies [28]. In particular, b branched amino acids, i.e., Val, Ile, Thr retard the ligation significantly. In addition ligation with a C-terminal proline is very slow [28]. Analogously, we
We used NH2-AEGSQAKFGX-SAL ester and NH2SPKALTFG-CO2H (1 mM) to examine the scope and limitations of Ser/Thr ligation. Among the 20 naturally occurring amino acids, all except Asp, Glu and Lys were found to be suitable at the C-terminal of the peptide SAL
Scheme 3 HO
Me
FmocHN
47
(a)
HO
O O
70
71
H2N
CHO
98
OH
1. serine ligation 2. de-Fmoc with Et2NH/CH2CI2 HO
O 46
1
O CHO
Me 70
47
H2N
OH
O N H
71
98
OH
threonine ligation
O 1
46
HO
Me
N H
47
OH
O 70
N H
71
OH
98
folding
human erythrocyte acylphosphatase
(b)
S-Peptide CHO
Peptoid/PEG
O O
Subtilisin OH
O HO
RNase A
HN
Peptoid/PEG
NH2
O
S-Protein Current Opinion in Chemical Biology
Synthesis of human erythrocyte acylphosphatase via two Ser/Thr ligations. (a) Chemical synthesis of human erythrocyte acylphosphatase; (b) Semisynthesis of a peptoid/PEG-protein hybrids. www.sciencedirect.com
Current Opinion in Chemical Biology 2014, 22:108–114
112 Synthetic biomolecules
ester used for the ligation. Interestingly, the ligation at the bulky b branched amino acid Thr behaved similarly to that at the least hindered amino acid Gly. Accordingly, we have classified the C-terminal amino acids into three groups: fast reaction with Ala, Gly, Ser, Glu, Thr, Phe and Cys(StBu), modest reaction with Val, Leu, Ile, Asn Phe, Met and Tyr, and slow reaction with Arg, Trp and Pro [26]. The same trend was observed in serine/threonine ligation-mediated peptide cyclization. During the synthesis of yunnanin C analogues, it is found that the cyclization at Pro-Ser site was extremely slow, while the cyclization at Val-Ser proceeded similarly to that at Tyr-Ser [29]. An explanation for the similar anomalouslylow reactivity of Pro-thioesters has recently been proposed [30].
this end, we took advantage of the strategies developed for the synthesis of peptide thioesters by Fmoc chemistry SPPS [31]. In particular, Dawson and co-workers have been able to synthesize peptide thioesters via in-solution thiolysis of the peptide N-acyl-benzimidazolinone (Nbz) which is readily generated via Fmoc-SPPS [32]. We conducted on-resin phenolysis of the peptide Nbz with salicylaldehyde dimethyl acetal to release the peptide SAL ester from the resin in overall yields of 20–30% based on resin loading, without observing epimerization of the C-terminal residue [24]. This strategy provides a reliable approach to the Fmoc chemistry solid phase synthesis of peptide SAL esters (Scheme 2a,b). Alternatively, we have developed Boc chemistry SPPS methods to generate the peptide SAL ester (Scheme 2c,d). To this end, 20 -hydroxyl cinnamate was first linked to the AM resin. After completion of the peptide chain using Boc-SPPS and TMSOTf/TFA mediated global deprotection, the side chain unprotected peptide SAL ester was generated and then released from resin upon onresin ozonolysis [33]. Similarly, Liu et al. used 20 hydroxyl cinnamate-MBHA resin where the peptide
Synthesis of the requisite peptide SAL ester In applying Ser/Thr ligation to peptide/protein synthesis one urgent issue to address is the preparation of the requisite peptide SAL ester via SPPS. As the O-SAL ester is labile to piperidine-mediated Fmoc removal conditions, direct solid phase synthesis of the peptide SAL ester by way of Fmoc chemistry is not possible. Towards
Scheme 4
H 3N O NH CONH 2
O
NH
H N
N H
O
O N H
O
N H
NH
O
O
COOH O
O
H N
O HOOC HOOC HO H N
O
N H
O
O
N H HN
O O NH
N H
O
COOH
NH 2 Daptomycin (Cubicin)
Ser Ligation OH
O H 2N O N H O
HN
Ph O
O O
NH O
O
H N
N
O O
NH
O
NH
NH 2
OH Cyclomontanin B
Ph
HN
N H O
HO
H N
H N
O
O
NH O
Thr Ligation
N H
HN
Ph NH
Thr ligation
O
OO O
NH
Ph
HN
N
HN NH
O
O Thr Ligation Mahafacyclin B
OH
O
H N O Yunnanin C
Current Opinion in Chemical Biology
Natural cyclic peptides synthesized by way of Ser/Thr ligation-mediated peptide cyclization. Current Opinion in Chemical Biology 2014, 22:108–114
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Serine/threonine ligation for synthetic biomolecules Lee and Li 113
chain was first cleaved from the resin with either TFMSA/ TFA or HF, followed by in-solution ozonolysis to generate the peptide SAL esters [34]. It is noted that Cys, Met and Trp are incompatible with this method.
address explicit biological questions. In this regard, continuing efforts to further optimize existing methods and develop innovative and efficient methods and strategies to synthesize bio-macromolecules are urgently needed.
Applications of serine/threonine ligation in the synthesis of peptides/proteins and cyclic peptides
Acknowledgements
Serine/threonine ligation has recently been successfully applied to the synthesis of the human erythrocyte acylphosphatase protein (11 kDa) [24]. This protein molecule contains a number of well-distributed serine and threonine residues but no cysteine residues and thus is a good model to demonstrate the effectiveness of the serine/threonine ligation. Human erythrocyte acylphosphatase protein was assembled via two ligations at Ser71 and Thr47 respectively (Scheme 3a). The folded synthetic enzyme exhibited similar bioactivity to the natural protein [24]. Later, the Li group synthesized glycopeptide, MUC1 80-mer segments, via 4 iterative serine ligations [35]. Recently, Kirshenbaum and co-workers applied Ser/Thr ligation to the semi-synthesis of hybrid peptoid-protein conjugates and to protein modifications. The N-terminal portion of RNase A (residue 1–20) was cleaved by proteolysis to reveal a serine residue at the N-terminus, affording the S-protein (Scheme 3b). Next, the peptide salicylaldehyde ester was ligated with the S-protein to generate the desired peptoid-protein hybrid [36]. More recently, they have extended the same strategy for side-specific N-terminal protein PEGylation [37]. In another application, serine/threonine ligation has been applied intramolecularly to synthesize cyclic peptides that contain serine or threonine residues. Various cyclic peptides of sizes from 4 to 9 amino acids have been successfully prepared by way of serine/threonine ligation-mediated peptide cyclization [33,34]. As such, several natural bioactive cyclic peptides have been synthesized, including cyclomontanin B [38], yunnanin C [29], mahafacyclin B [34] and daptomycin [39] (Scheme 4). The cyclization proceeded smoothly, even in the presence of the unprotected lysine residue. In particular, highly constrained cyclic tetrapeptides that have been very difficult to obtain using other methods could be readily synthesized by serine/threonine ligationmediated peptide cyclization [33,34].
Conclusion Ser/Thr ligation has emerged as an alternative peptide ligation chemistry to synthesize peptides, proteins and cyclic peptides. Over the past decades, synthetic protein chemistry has provided a unique tool to generate proteins for the biological and medicinal studies of proteins, particularly those carrying post-translational modifications. As such, both chemists and biologists need to embrace and appreciate the power of chemical synthesis to synthesize and manipulate bio-macromolecules to www.sciencedirect.com
This work was supported by the Research Grants Council-General Research Fund of Hong Kong (HKU703811P, 707412P, 702813P), the National Basic Research Program of China (973 Program, 2013CB836900), the Peacock Program-Project Development Fund (KQC201109050074A) and the University of Hong Kong.
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18. Crich D, Sharma I: Epimerization-free block synthesis of peptides from thioacids and amines with the Sanger and Mukaiyama reagents. Angew Chem Int Ed Engl 2009, 48: 2355-2358. 19. Kemp DS: The amine capture strategy for peptide-bond formation – an outline of progress. Biopolymers 1981, 20: 1793-1804. 20. Liu CF, Tam JP: Peptide segment ligation strategy without use of protecting groups. Proc Natl Acad Sci U S A 1994, 91: 6584-6588. 21. Gilmore K, Alabugin IV: Cyclizations of alkynes: revisiting Baldwin’s rules for ring closure. Chem Rev 2011, 111:6513-6556. 22. Li X, Lam HY, Zhang Y, Chan CK: Salicylaldehyde ester-induced chemoselective peptide ligations: enabling generation of natural peptidic linkages at the serine/threonine sites. Org Lett 2010, 12:1724-1727. 23. Tung CL, Lam HY, Xu J, Li X: A fluorogenic probe for recognizing 5-hydroxylysine inspired by serine/threonine ligation. Chem Commun 2014, 50:5298-5300. 24. Zhang Y, Xu C, Lam HY, Lee CL, Li X: Protein chemical synthesis by serine and threonine ligation. Proc Natl Acad Sci U S A 2013, 110:6657-6662. This paper demonstrated the application of Ser/Thr ligation for protein total chemical synthesis. In addition, a method to synthesize the requisite peptide salicylaldehyde ester has been developed via Fmoc-SPPS.
30. Pollock SB, Kent SBH: An investigation into the origin of the dramatically reduced reactivity of peptide-prolyl-thioesters in native chemical ligation. Chem Commun 2011, 47:2342-2344. 31. Mende F, Seitz O: 9-Fluorenylmethoxycarbonyl-based solidphase synthesis of peptide a-thioesters. Angew Chem Int Ed Engl 2011, 50:1232-1240. 32. Blanco-Canosa JB, Dawson PE: An efficient Fmoc-SPPS approach for the generation of thioester peptide precursors for use in native chemical ligation. Angew Chem Int Ed Engl 2008, 47:6851-6855. 33. Wong CTT, Lam HY, Song T, Chen G, Li X: Synthesis of constrained head-to-tail cyclic tetrapeptides by an imineinduced ring-closing/contraction strategy. Angew Chem Int Ed Engl 2013, 52:10212-10215. This paper reported a method to synthesize peptide salicylaldehyde ester by Boc chemistry SPPS. In addition, Ser/Thr ligation-mediated peptide cyclization was successfully used to synthesize cyclic tetrapeptides. Molecular modeling was used to explain the success of the tetrapeptide cyclization via Ser/Thr ligation. 34. Zhao JF, Zhang XH, Ding YJ, Yang YS, Bi XB, Liu CF: Facile synthesis of peptidyl salicylaldehyde esters and its use in cyclic peptide synthesis. Org Lett 2013, 15:5182-5185. This paper reported a method to synthesize peptide salicylaldehyde ester by Boc chemistry SPPS. In addition, Ser/Thr ligation-mediated peptide cyclization was used to synthesize cyclic peptide of various sizes and mahafacyclin B.
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