Available online at www.sciencedirect.com
ScienceDirect New chemistries for chemoselective peptide ligations and the total synthesis of proteins Thibault JR Harmand1, Claudia E Murar1 and Jeffrey W Bode1,2 The identification of fast, chemoselective bond-forming reactions is one of the major contemporary challenges in chemistry. The requirements of the native chemical ligation — an N-terminal cysteine and C-terminal thioesters — have encouraged a search for alternative amide-forming ligation reactions. Among successful alternatives to native chemical ligation, are the a-ketoacid–hydroxylamine ligation with 5oxaproline and, serine/threonine ligation, and potassium acyltrifluoroborate (KAT) ligation. In addition, the KAT ligation, along with the non-amide forming alkyne–azide ligation, is very useful for synthetic conjugations. All of these recent ligation methods were applied to synthesize different proteins, and have allowed chemists to incorporate unnatural amino acids, or to modify the peptide backbone. Addresses 1 Laboratory fu¨r Organische Chemie, Department of Chemistry and Applied Biosciences, ETH Zu¨rich, Zu¨rich 8093, Switzerland 2 Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Chikusa, Nagoya 464-8602, Japan Corresponding author: Bode, Jeffrey W ([email protected])
Current Opinion in Chemical Biology 2014, 22:115–121 This review comes from a themed issue on Synthetic biomolecules
homogeneous protein segments. Although some proteins have been made using exclusively SPPS [2–5] it is often difficult to synthesize protein fragments longer than 40– 50 amino acid residues. In order to access larger peptides and proteins it is necessary to employ chemoselective segment assembly reactions, commonly called chemical ligations. In these reactions two mutually reactive functional groups undergo a chemoselective reaction independent of all the unprotected functional groups borne by the amino acid side chains. Ideally, these reactions would proceed in aqueous buffers, at low concentrations and under mild conditions (Figure 1). The development of the native chemical ligation (NCL) reaction by Dawson et al. [6] in 1994 has been a remarkable breakthrough, allowing an unprotected segment bearing a thioester function at the C-terminus to ligate with another unprotected segment bearing an N-terminal cysteine residue, under mild aqueous conditions, and in a highly chemoselective way. The reaction typically takes only a few hours at room temperature. NCL has allowed the synthesis of a large number of important proteins [7– 10] and has been very well documented in recent years [11–14].
Edited by Paul F Alewood and Stephen BH Kent
http://dx.doi.org/10.1016/j.cbpa.2014.09.032 1367-5931/# 2014 Elsevier Ltd. All rights reserved.
Introduction The chemical synthesis of peptides and proteins is increasingly important for two major reasons. First, chemical protein synthesis allows the scientist to incorporate unnatural amino acids, posttranslational modifications, or labeling agents, thereby opening new opportunities for the understanding of protein molecules and controlling their biological mechanism of action. Second, the chemical synthesis of peptides and proteins can avoid contamination and other issues that can arise from their production from animal or recombinant sources. Solid phase peptide synthesis (SPPS) developed by Merrifield [1] is a powerful method for the synthesis of www.sciencedirect.com
The major limitations of NCL are the difficulty of generating the thioester moiety, especially with Fmoc SPPS, and the low abundance of cysteine residues in protein sequences. Although desulfurization approaches to overcome the second limitation are expanding the available ligation sites, their application in multi-segment ligations remains challenging. Furthermore, there is great desire to have multiple ligation reactions that can be performed orthogonally to one another for the rapid assembly of proteins. In this review we will focus on the recent developments that has have made in the chemical ligation of peptides using reactions that are mechanistically distinct from the native chemical ligation.
KAHA ligation with 5-oxaproline
In an effort to develop a general peptide forming ligation, the Bode group reported the a-ketoacid–hydroxylamine (KAHA) ligation as a chemoselective coupling of large, unprotected peptide segments [15]. The KAHA ligation can be classified into two mechanistically distinct reactions: the Type I ligation with O-unsubstituted hydoxylamines and the Type II ligations with O-substituted variants [16]. They have demonstrated that the KAHA Current Opinion in Chemical Biology 2014, 22:115–121
116 Synthetic biomolecules
Figure 1 OH NH2 H2N H3N
O
NH
O O
NH3
OH
Unprotected Peptide SH
HN
NH2
Unprotected Peptide NH3
OH
NH
• Aqueous Buffers • Low concentration • Mild temperature NH2 H2N H3N
COO
OH
O
NH
O O
NH3 O
Unprotected Peptide SH
HN NH
OH N H
NH2
Unprotected Peptide OH
COO
NH3
Current Opinion in Chemical Biology
General concept of peptide chemical ligation reactions.
ligation is compatible with the synthesis of small peptides such as GLP-1 using the type I ligation [17], however this variant is not well suited to reactions in aqueous media generally employed for peptide solubilization and handling. More recently, the Bode group designed 5-oxaproline as a monomer ideally suited for protein synthesis via type II KAHA ligations. The incorporation of 5-oxaproline on the N-terminus of one peptide segment gives clean ligation reactions with C-terminal peptide a-ketoacids under aqueous conditions [18,19]. 5-Oxaproline has proved to be a powerful N-hydroxyamino acid, sufficiently reactive in aqueous solvent, and at the same time stable to ligation conditions and solid phase peptide synthesis. After further investigations and preliminary mechanistic studies (TG Wucherpfennig et al. [45]), we have recently determined that the primary products of KAHA ligations with 5-oxaproline are esters, which readily rearrange to the amides in basic buffers, leading to a homoserine at the ligation site (Figure 2a,b). The ligation reaction proceeds cleanly for the assembly of peptide segments with acceptable rates. The low solubility of some peptide segments has created a particular interest in finding methods in which peptide solubility can be enhanced in order to facilitate the purification of peptide segments and chemical ligations. The utilization of depsipeptides during SPPS has shown great efficiency in overcoming this problem because of the high solubility compared to their amide counterparts [20]. Incorporation of isoacyl dipeptides is often used to improve the biophysical properties, preparation, and purification of hydrophobic peptides [21,22]. The isoacyl peptide esters can be converted to the native peptide Current Opinion in Chemical Biology 2014, 22:115–121
amides by an O-to-N acyl shift in basic aqueous buffers. Many syntheses of depsipeptides employ solid phase peptide synthesis by coupling a-hydroxy acids onto protected a-amino acids. KAHA ligation with 5-oxaproline may have similar advantages in altering the properties of ligation products by the generation of depsipeptides inaccessible by expression or other ligation methods. The versatility of the KAHA ligation was showcased by the chemical synthesis of Pup [19] (1 ligation), CspA [19] (1 ligation), UFM1 [18] (2 ligations), SUMO2 (3 ligations), and SUMO3 (2 ligations) proteins (TG Wucherpfennig et al. [45]) (Figure 2c); the preparation of several other proteins with >100 residues is currently in progress. For the synthesis of proteins by multiple KAHA 5-oxaproline ligations, we have shown that the 5-oxaproline moiety can be easily masked with an Na-Fmocprotecting group that is retained throughout resin cleavage, HPLC purification, and KAHA ligation(s). NaFmoc-protecting group can be removed by brief treatment with an amine base in DMSO, providing a facile approach to the synthesis of larger proteins by multiple segment ligations. In our work to date, the mutation of 2– 3 amino acids to a homoserine residue has not influenced the ability of the described proteins to form the expected tertiary structures, as assayed by CD spectroscopy and biochemical assays. At the moment we anticipate that the homoserine residue could serve as a surrogate for threonine, serine, methionine, aspartic acid, and asparagine residues. Unless these residues play a structural or functional role we assume to have multiple choices for the ligation sites. The KAHA ligation with 5-oxaproline provides direct access to depsipeptides and creates a new class of clean chemoselective, ester-forming ligation which may present possible advantages in protein total synthesis by increasing the solubility of the ligated products, glycoproteins, and therapeutic peptides [23,24]. Serine/threonine ligation (STL)
Tam et al. demonstrated in 1994 that an unprotected peptide segment bearing a glycolaldehyde ester at the carboxyl terminus was able to ligate to another unprotected segment if there was a serine, threonine or cysteine residue at the N-terminus. The ligation proceeds in pyridinium acetate buffer in a chemoselective manner and results in the formation of a pseudoproline at the site of ligation [25–27]. Unfortunately the reaction was rather slow and they did not find a way to convert this pseudoproline into a native peptide bond. In 2010 Li et al. reported a modified version of this aldehyde by using an O-salicylaldehyde ester (SAL) at the C-terminus. After ligation with another unprotected fragment bearing a terminal threonine or serine they were www.sciencedirect.com
New chemistry for chemoselective peptide ligations Harmand, Murar and Bode 117
Figure 2
(a)
H2N NH3
H2N
NH2 NH
H2N
OH
O
CO2H
O
CO2H
-ketoacid (KA)
NH3 O
Peptide segment 2
N H
O
NH3
NH
OH
O
H N
OH
Peptide segment 1
(b)
NH2
OH
CONH2
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5- oxaproline (Opr)
HO
NH3 H2N
NH
OH
Peptide segment 1 CO2H
NH2
O O
H N
CO2H
OH
CONH2
H2N
NH2 NH
H2N
OH H N
Peptide segment 1 CO2H
NH3
O
OH
O N H
R
H N O
O N H
N H
O
H N
R R
–H O
N H
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depsi
+H O
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H N
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R
N H
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R
O
H N
R
N H
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R
R
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+H O O O H
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HO H N
OH
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NH3 O
Peptide segment 2
O
NH3
R
–CO
Path A
O ketoacid
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R
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N H
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Ligation 20 mM, DMSO/H2O 0.1 M oxalic acid 50-60 °C, 8 h H2N
O
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–CO Path B
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+H O Path C
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CONH2
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(c)
SUMO 2/3 proteins 95/92 residues, 3/2 ligations
UFM1 protein 83 residues, 2 ligations
Pup protein 64 residues, 1 ligation
CspA protein 67 residues, 1 ligation Current Opinion in Chemical Biology
KAHA ligation with 5-oxaproline: (a) Formation of depsipeptides by KAHA ligation with 5-oxaproline and O-to-N acyl shift to form the homoserine and the amide at the ligation site. (b) Possible mechanisms for depsipeptide formation. (c) Proteins synthesized by KAHA ligation along with the number of ligations required for each of them (after each KAHA ligation, the rearrangement of the newly formed depsipeptide to the corresponding amide was performed to give amide bond at each ligation site).
able to generate a pseudoproline that could rearrange and then be converted to a natural peptide link under acidic condition [28] (Figure 3a). Like the C-terminal thioester moiety used in NCL, the phenyl ester moiety for Ser/Thr ligation is sensitive to piperidine treatment and must be introduced after Fmoc SPPS synthesis. For this purpose Li et al. used the Nbz resin method developed previously by Dawson for the generation of a peptide-thioester after Fmoc SPPS [29]. After the resin-bound protected peptide segment has www.sciencedirect.com
been assembled, the protected peptide-resin is treated with salicylaldehyde dimethyl acetal in CH2Cl2/THF, followed by TFA treatment to give the peptide salicylaldehyde ester. The Ser/Thr ligation reaction proceeds in a pyridinium acetate buffer at room temperature and the concentration of the peptide ester segment can be as low as 10 mM. Final treatment with acid generates the ligation product with a native amide bond at the Ser/Thr ligation site. They successfully applied this method for the synthesis of human acylphosphatase [30] and the human growth hormone-releasing hormone [31]. Current Opinion in Chemical Biology 2014, 22:115–121
118 Synthetic biomolecules
Figure 3
(a)
H2N
H2N
HO
O
VQGQ...ETRG
H
R
OH
CHO
N3
OH
NH2
O
HN
HN
OH
H MIPG...ETYG
OH
H
O
H2N
VQGQ...ETRG O
O
HO
N
O
H2N
NH2
H
KVIQ...HSKP
O
HN
O
OH
N N N
H MIPG...ETYG
R
H2N
N H
VQGQ...ETRG H2N
HN
NH2
OH
H MIPG...ETYG
N H
OH
HN
OH O
O
O
OH
NH2
O
NH3
H2N
NH3
PKSH...QIVK
N
O
57% Yield
OH
O
HO
H
3rd Fragment, CuI Phosphate buffer pH 8 15 min
NH3
NH3
NH2
O HN
H2N
KLEAV...VRAG
OH
HN NH3
N H
NH2
OH
KVIQ...HSKP
HO
HN
H
O
O
H2N
NH2
HN
OH
VQGQ...ETRG O
R
H2N
NH3
O
OH
OH
O
NH2
HN
O
Si(iPr)3
47% Yield over 2 steps
H2N
NH2
O HN
KLEAV...VRAG HO
Pyridine acetate buffer (1:1), 10h
H2N
NH2
1) CuI Phosphate buffer pH 8 15 min 2) TBAF/DMF
N H
H2N
H2N
NH3
OH O
PKSH...QIVK
H2N
O
OH
O
(b)
NH3
NH2
O HN
H2N
N N N
KLEAV...VRAG HO
DNKY..LTGF OH N N N OH H2N O
O
OH
TFA/H2O/iPr3SiH 10 min H2N H2N H
VQGQ...ETRG H2N
31% Yield
NH2
O HN
O
OH
O N H
NH3
OH
PKSH...QIVK HN
O
OH
NH2
N H
Current Opinion in Chemical Biology
Serine/threonine (STL) and azide–alkyne (PTL) ligations. (a) Human erythrocyte acylphosphatase first ligation by STL. (b) Analog cystatin A synthesis by PTL.
Alkyne–azide ligations (PTL)
The copper catalyzed alkyne–azide cycloaddition developed by Meldal [32] and Sharpless [33] has been used widely in chemical biology, particularly for bioconjugation because of its orthogonality (i.e. compatibility with functional groups found in native proteins) and fast reaction rate [34–37]. The triazole generated during the cycloaddition can be considered as a good mimic of the native amide bond because of its high dipole moment, its H-bond donor–acceptor character and its planar structure [38]. Delmas et al. reported in 2012 the utilization of CuImediated cycloaddition of azides and terminal alkynes (CuAAC) as a chemical ligation method with unprotected peptides for the synthesis of protein analogs [39]. They introduced a-azide acids and a-amino alkynes at the N and C termini respectively of the peptide segments to be reacted, by azidation of amino free protected amino acids Current Opinion in Chemical Biology 2014, 22:115–121
[40] and by functionalization of the resin by reductive amination of propargylamine [41,42]. By extending their previous work on multiple successive CuAAC [43] using triisopropylsilyl as a protecting group for the alkyne moiety that can be mildly removed with TBAF, they developed an iterative version of this peptidomimetic triazole ligation (PTL). The TIPS protecting group is easily introduced by changing the propargylamine to 3(triisopropyl)prop-2-yn-1-amine during the functionalization of the resin. The Cu-promoted ligation reaction proceeds under degassed phosphate buffer at pH 8 at concentration of 1 mM of the alkyne peptide and a slightly excess of the azide peptide in only 15 min, leading to a clean triazolopeptide that can be directly deprotected by treatment with TBAF for one hour. They applied the PTL method for the synthesis of an analog of Cystatin A, which they obtained in an overall yield of 27% over two ligation steps www.sciencedirect.com
New chemistry for chemoselective peptide ligations Harmand, Murar and Bode 119
Figure 4
H2N COOH
BF3 K H3N
O MeO
O
O
COOH
N
n
COOH
NH
Et
N
O
EFIAWLVRGRG
OH NH NH H2N
HN O
O
PEG5,000 , PEG20,000
N H
O CONH2
KAT
COOH
O
H N
HAEGTFTSDVSSYLEGQAA
NH2 NH
NH2
Et
GLP-1 analogue
KAT ligation
tBuOH/H2O 0.1 M oxalic acid 23 °C H2N
COOH
COOH H3N
H N
HAEGTFTSDVSSYLEGQAA
COOH
N NH
O CONH2
COOH
O N H
NH2 NH O
EFIAWLVRGRG
OH NH NH H2N
HN
NH2
O MeO
O
O n
Current Opinion in Chemical Biology
Chemoselective amide-formation with an equimolar ratio of reactants by KAT ligation.
and one deprotection (Figure 3b). The biological tests performed on the synthetic cystatin A showed that the presence of two peptidomimetic bonds did not disrupt the biological activity and that the potency of this cystatin A analog was not diminished. These data confirmed that the PTL can be used as a powerful, easy and fast method for the synthesis of biological active protein analogs.
In order to demonstrate the utility of this method, the Bode group used the KAT ligation for the synthesis of conjugates of unprotected peptides with large polymers. They chose a 31-amino acid residue analog of the antidiabetic peptide GLP-1 as an initial target. Using the KAT ligation, PEG, lipids, biotin, and dyes were introduced onto the unprotected peptide with excellent yields and small reaction times.
KAT ligation
As part of our search for amide-forming chemical ligation reactions, the Bode group recently reported the chemoselective amide-forming ligations of potassium acyltrifluoroborates (KATs) and O-carbamoylhydroxylamines [44]. KATs showed rapid amide formations along with excellent stability under mild aqueous conditions. Additionally, the KAT ligation allows the use of 1:1 stoichiometry of the two reagents, tolerates all unprotected functional groups, and operates without toxic reagents or byproducts (Figure 4). In other words, KAT ligation fulfills all of the criteria for an ideal chemoselective reaction, although further work on methods to prepare and introduce the KAT ligation reactive moieties to unprotected peptide segments at the C and Ntermini will be necessary. www.sciencedirect.com
Improved methods for conjugating molecules to unprotected peptides are in great demand. The high stability of both hydroxylamine and acyltrifluoroborate moieties to aqueous conditions, HPLC purification, and standard reagents used in peptide synthesis offers the possibility to imagine the KAT ligation as a powerful method for the future synthesis of large proteins.
Conclusions Numerous chemoselective ligations for the synthesis of proteins and peptides have been proposed in recent years. Here we have focused on several new ligation chemistries that promise to have practical utility. The KAHA ligation with 5-oxaproline holds great promise in the formation of depsipeptides, especially for hydrophobic proteins. Current Opinion in Chemical Biology 2014, 22:115–121
120 Synthetic biomolecules
Further design of alkoxyamine-containing monomers for the KAHA ligation will allow it to offer other unnatural as well as completely natural residues at the ligation sites. The introduction of methods for amide-forming ligation at N-terminal serine and threonine residues represents a versatile solution to generate natural Xaa–Ser/Thr linkages. The alkyne–azide ligation is an intriguing addition to the tools for chemical protein synthesis that allows for the formation of a peptidomimetic group directly into the protein backbone. Finally, the KAT ligation affords synthetic access to a great number of protein conjugates, and could provide an excellent method for preparing even larger proteins and protein-conjugates that are currently one of the frontiers of synthetic organic chemistry.
Acknowledgements This work was supported by ETH Zu¨rich and the Swiss National Science Foundation (200020_150073).
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest
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40. Goddard-Borger ED, Stick RV: An efficient, inexpensive, and shelf-stable diazotransfer reagent: imidazole-1-sulfonyl azide hydrochloride. Org Lett 2007, 9:3797-3800.
43. Valverde IE, Delmas AF, Aucagne V: Click a` la carte: robust semiorthogonal alkyne protecting groups for multiple successive azide/alkyne cycloadditions. Tetrahedron 2009, 65:7597-7602. 44. Noda H, Eros G, Bode JW: Rapid ligations with equimolar reactants in water with the potassium acyltrifluoroborate (KAT) amide formation. J Am Chem Soc 2014, 136:5611-5614. The KAT ligation offers a natural-bond forming ligation fast enough for synthetic approaches to protein engineering and biomolecule conjugates. 45. Wucherpfennig TG, Rohrbacher F, Pattabiraman VR, Bode JW: Formation and Rearrangement of Homoserine Depsipeptides and Depsiproteins in the a-Ketoacid–Hydroxylamine Ligation with 5-Oxaproline. Angew. Chem. Int. Ed. 2014 http://dx.doi.org/ 10.1002/anie.201406097. Early View.
Current Opinion in Chemical Biology 2014, 22:115–121
ScienceDirect New chemistries for chemoselective peptide ligations and the total synthesis of proteins Thibault JR Harmand1, Claudia E Murar1 and Jeffrey W Bode1,2 The identification of fast, chemoselective bond-forming reactions is one of the major contemporary challenges in chemistry. The requirements of the native chemical ligation — an N-terminal cysteine and C-terminal thioesters — have encouraged a search for alternative amide-forming ligation reactions. Among successful alternatives to native chemical ligation, are the a-ketoacid–hydroxylamine ligation with 5oxaproline and, serine/threonine ligation, and potassium acyltrifluoroborate (KAT) ligation. In addition, the KAT ligation, along with the non-amide forming alkyne–azide ligation, is very useful for synthetic conjugations. All of these recent ligation methods were applied to synthesize different proteins, and have allowed chemists to incorporate unnatural amino acids, or to modify the peptide backbone. Addresses 1 Laboratory fu¨r Organische Chemie, Department of Chemistry and Applied Biosciences, ETH Zu¨rich, Zu¨rich 8093, Switzerland 2 Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Chikusa, Nagoya 464-8602, Japan Corresponding author: Bode, Jeffrey W ([email protected])
Current Opinion in Chemical Biology 2014, 22:115–121 This review comes from a themed issue on Synthetic biomolecules
homogeneous protein segments. Although some proteins have been made using exclusively SPPS [2–5] it is often difficult to synthesize protein fragments longer than 40– 50 amino acid residues. In order to access larger peptides and proteins it is necessary to employ chemoselective segment assembly reactions, commonly called chemical ligations. In these reactions two mutually reactive functional groups undergo a chemoselective reaction independent of all the unprotected functional groups borne by the amino acid side chains. Ideally, these reactions would proceed in aqueous buffers, at low concentrations and under mild conditions (Figure 1). The development of the native chemical ligation (NCL) reaction by Dawson et al. [6] in 1994 has been a remarkable breakthrough, allowing an unprotected segment bearing a thioester function at the C-terminus to ligate with another unprotected segment bearing an N-terminal cysteine residue, under mild aqueous conditions, and in a highly chemoselective way. The reaction typically takes only a few hours at room temperature. NCL has allowed the synthesis of a large number of important proteins [7– 10] and has been very well documented in recent years [11–14].
Edited by Paul F Alewood and Stephen BH Kent
http://dx.doi.org/10.1016/j.cbpa.2014.09.032 1367-5931/# 2014 Elsevier Ltd. All rights reserved.
Introduction The chemical synthesis of peptides and proteins is increasingly important for two major reasons. First, chemical protein synthesis allows the scientist to incorporate unnatural amino acids, posttranslational modifications, or labeling agents, thereby opening new opportunities for the understanding of protein molecules and controlling their biological mechanism of action. Second, the chemical synthesis of peptides and proteins can avoid contamination and other issues that can arise from their production from animal or recombinant sources. Solid phase peptide synthesis (SPPS) developed by Merrifield [1] is a powerful method for the synthesis of www.sciencedirect.com
The major limitations of NCL are the difficulty of generating the thioester moiety, especially with Fmoc SPPS, and the low abundance of cysteine residues in protein sequences. Although desulfurization approaches to overcome the second limitation are expanding the available ligation sites, their application in multi-segment ligations remains challenging. Furthermore, there is great desire to have multiple ligation reactions that can be performed orthogonally to one another for the rapid assembly of proteins. In this review we will focus on the recent developments that has have made in the chemical ligation of peptides using reactions that are mechanistically distinct from the native chemical ligation.
KAHA ligation with 5-oxaproline
In an effort to develop a general peptide forming ligation, the Bode group reported the a-ketoacid–hydroxylamine (KAHA) ligation as a chemoselective coupling of large, unprotected peptide segments [15]. The KAHA ligation can be classified into two mechanistically distinct reactions: the Type I ligation with O-unsubstituted hydoxylamines and the Type II ligations with O-substituted variants [16]. They have demonstrated that the KAHA Current Opinion in Chemical Biology 2014, 22:115–121
116 Synthetic biomolecules
Figure 1 OH NH2 H2N H3N
O
NH
O O
NH3
OH
Unprotected Peptide SH
HN
NH2
Unprotected Peptide NH3
OH
NH
• Aqueous Buffers • Low concentration • Mild temperature NH2 H2N H3N
COO
OH
O
NH
O O
NH3 O
Unprotected Peptide SH
HN NH
OH N H
NH2
Unprotected Peptide OH
COO
NH3
Current Opinion in Chemical Biology
General concept of peptide chemical ligation reactions.
ligation is compatible with the synthesis of small peptides such as GLP-1 using the type I ligation [17], however this variant is not well suited to reactions in aqueous media generally employed for peptide solubilization and handling. More recently, the Bode group designed 5-oxaproline as a monomer ideally suited for protein synthesis via type II KAHA ligations. The incorporation of 5-oxaproline on the N-terminus of one peptide segment gives clean ligation reactions with C-terminal peptide a-ketoacids under aqueous conditions [18,19]. 5-Oxaproline has proved to be a powerful N-hydroxyamino acid, sufficiently reactive in aqueous solvent, and at the same time stable to ligation conditions and solid phase peptide synthesis. After further investigations and preliminary mechanistic studies (TG Wucherpfennig et al. [45]), we have recently determined that the primary products of KAHA ligations with 5-oxaproline are esters, which readily rearrange to the amides in basic buffers, leading to a homoserine at the ligation site (Figure 2a,b). The ligation reaction proceeds cleanly for the assembly of peptide segments with acceptable rates. The low solubility of some peptide segments has created a particular interest in finding methods in which peptide solubility can be enhanced in order to facilitate the purification of peptide segments and chemical ligations. The utilization of depsipeptides during SPPS has shown great efficiency in overcoming this problem because of the high solubility compared to their amide counterparts [20]. Incorporation of isoacyl dipeptides is often used to improve the biophysical properties, preparation, and purification of hydrophobic peptides [21,22]. The isoacyl peptide esters can be converted to the native peptide Current Opinion in Chemical Biology 2014, 22:115–121
amides by an O-to-N acyl shift in basic aqueous buffers. Many syntheses of depsipeptides employ solid phase peptide synthesis by coupling a-hydroxy acids onto protected a-amino acids. KAHA ligation with 5-oxaproline may have similar advantages in altering the properties of ligation products by the generation of depsipeptides inaccessible by expression or other ligation methods. The versatility of the KAHA ligation was showcased by the chemical synthesis of Pup [19] (1 ligation), CspA [19] (1 ligation), UFM1 [18] (2 ligations), SUMO2 (3 ligations), and SUMO3 (2 ligations) proteins (TG Wucherpfennig et al. [45]) (Figure 2c); the preparation of several other proteins with >100 residues is currently in progress. For the synthesis of proteins by multiple KAHA 5-oxaproline ligations, we have shown that the 5-oxaproline moiety can be easily masked with an Na-Fmocprotecting group that is retained throughout resin cleavage, HPLC purification, and KAHA ligation(s). NaFmoc-protecting group can be removed by brief treatment with an amine base in DMSO, providing a facile approach to the synthesis of larger proteins by multiple segment ligations. In our work to date, the mutation of 2– 3 amino acids to a homoserine residue has not influenced the ability of the described proteins to form the expected tertiary structures, as assayed by CD spectroscopy and biochemical assays. At the moment we anticipate that the homoserine residue could serve as a surrogate for threonine, serine, methionine, aspartic acid, and asparagine residues. Unless these residues play a structural or functional role we assume to have multiple choices for the ligation sites. The KAHA ligation with 5-oxaproline provides direct access to depsipeptides and creates a new class of clean chemoselective, ester-forming ligation which may present possible advantages in protein total synthesis by increasing the solubility of the ligated products, glycoproteins, and therapeutic peptides [23,24]. Serine/threonine ligation (STL)
Tam et al. demonstrated in 1994 that an unprotected peptide segment bearing a glycolaldehyde ester at the carboxyl terminus was able to ligate to another unprotected segment if there was a serine, threonine or cysteine residue at the N-terminus. The ligation proceeds in pyridinium acetate buffer in a chemoselective manner and results in the formation of a pseudoproline at the site of ligation [25–27]. Unfortunately the reaction was rather slow and they did not find a way to convert this pseudoproline into a native peptide bond. In 2010 Li et al. reported a modified version of this aldehyde by using an O-salicylaldehyde ester (SAL) at the C-terminus. After ligation with another unprotected fragment bearing a terminal threonine or serine they were www.sciencedirect.com
New chemistry for chemoselective peptide ligations Harmand, Murar and Bode 117
Figure 2
(a)
H2N NH3
H2N
NH2 NH
H2N
OH
O
CO2H
O
CO2H
-ketoacid (KA)
NH3 O
Peptide segment 2
N H
O
NH3
NH
OH
O
H N
OH
Peptide segment 1
(b)
NH2
OH
CONH2
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5- oxaproline (Opr)
HO
NH3 H2N
NH
OH
Peptide segment 1 CO2H
NH2
O O
H N
CO2H
OH
CONH2
H2N
NH2 NH
H2N
OH H N
Peptide segment 1 CO2H
NH3
O
OH
O N H
R
H N O
O N H
N H
O
H N
R R
–H O
N H
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R
depsi
+H O
N H
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H N
R
R
N H
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R
O
H N
R
N H
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R
R
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+H O O O H
O to N shift and deprotection pH 9.5 buffer, 23 °C H2N
CO H O H N
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HO H N
OH
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NH3 O
Peptide segment 2
O
NH3
R
–CO
Path A
O ketoacid
NH2 NH
OH
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N H
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Ligation 20 mM, DMSO/H2O 0.1 M oxalic acid 50-60 °C, 8 h H2N
O
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amide
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+H O Path C
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CONH2
OH peptide
(c)
SUMO 2/3 proteins 95/92 residues, 3/2 ligations
UFM1 protein 83 residues, 2 ligations
Pup protein 64 residues, 1 ligation
CspA protein 67 residues, 1 ligation Current Opinion in Chemical Biology
KAHA ligation with 5-oxaproline: (a) Formation of depsipeptides by KAHA ligation with 5-oxaproline and O-to-N acyl shift to form the homoserine and the amide at the ligation site. (b) Possible mechanisms for depsipeptide formation. (c) Proteins synthesized by KAHA ligation along with the number of ligations required for each of them (after each KAHA ligation, the rearrangement of the newly formed depsipeptide to the corresponding amide was performed to give amide bond at each ligation site).
able to generate a pseudoproline that could rearrange and then be converted to a natural peptide link under acidic condition [28] (Figure 3a). Like the C-terminal thioester moiety used in NCL, the phenyl ester moiety for Ser/Thr ligation is sensitive to piperidine treatment and must be introduced after Fmoc SPPS synthesis. For this purpose Li et al. used the Nbz resin method developed previously by Dawson for the generation of a peptide-thioester after Fmoc SPPS [29]. After the resin-bound protected peptide segment has www.sciencedirect.com
been assembled, the protected peptide-resin is treated with salicylaldehyde dimethyl acetal in CH2Cl2/THF, followed by TFA treatment to give the peptide salicylaldehyde ester. The Ser/Thr ligation reaction proceeds in a pyridinium acetate buffer at room temperature and the concentration of the peptide ester segment can be as low as 10 mM. Final treatment with acid generates the ligation product with a native amide bond at the Ser/Thr ligation site. They successfully applied this method for the synthesis of human acylphosphatase [30] and the human growth hormone-releasing hormone [31]. Current Opinion in Chemical Biology 2014, 22:115–121
118 Synthetic biomolecules
Figure 3
(a)
H2N
H2N
HO
O
VQGQ...ETRG
H
R
OH
CHO
N3
OH
NH2
O
HN
HN
OH
H MIPG...ETYG
OH
H
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H2N
VQGQ...ETRG O
O
HO
N
O
H2N
NH2
H
KVIQ...HSKP
O
HN
O
OH
N N N
H MIPG...ETYG
R
H2N
N H
VQGQ...ETRG H2N
HN
NH2
OH
H MIPG...ETYG
N H
OH
HN
OH O
O
O
OH
NH2
O
NH3
H2N
NH3
PKSH...QIVK
N
O
57% Yield
OH
O
HO
H
3rd Fragment, CuI Phosphate buffer pH 8 15 min
NH3
NH3
NH2
O HN
H2N
KLEAV...VRAG
OH
HN NH3
N H
NH2
OH
KVIQ...HSKP
HO
HN
H
O
O
H2N
NH2
HN
OH
VQGQ...ETRG O
R
H2N
NH3
O
OH
OH
O
NH2
HN
O
Si(iPr)3
47% Yield over 2 steps
H2N
NH2
O HN
KLEAV...VRAG HO
Pyridine acetate buffer (1:1), 10h
H2N
NH2
1) CuI Phosphate buffer pH 8 15 min 2) TBAF/DMF
N H
H2N
H2N
NH3
OH O
PKSH...QIVK
H2N
O
OH
O
(b)
NH3
NH2
O HN
H2N
N N N
KLEAV...VRAG HO
DNKY..LTGF OH N N N OH H2N O
O
OH
TFA/H2O/iPr3SiH 10 min H2N H2N H
VQGQ...ETRG H2N
31% Yield
NH2
O HN
O
OH
O N H
NH3
OH
PKSH...QIVK HN
O
OH
NH2
N H
Current Opinion in Chemical Biology
Serine/threonine (STL) and azide–alkyne (PTL) ligations. (a) Human erythrocyte acylphosphatase first ligation by STL. (b) Analog cystatin A synthesis by PTL.
Alkyne–azide ligations (PTL)
The copper catalyzed alkyne–azide cycloaddition developed by Meldal [32] and Sharpless [33] has been used widely in chemical biology, particularly for bioconjugation because of its orthogonality (i.e. compatibility with functional groups found in native proteins) and fast reaction rate [34–37]. The triazole generated during the cycloaddition can be considered as a good mimic of the native amide bond because of its high dipole moment, its H-bond donor–acceptor character and its planar structure [38]. Delmas et al. reported in 2012 the utilization of CuImediated cycloaddition of azides and terminal alkynes (CuAAC) as a chemical ligation method with unprotected peptides for the synthesis of protein analogs [39]. They introduced a-azide acids and a-amino alkynes at the N and C termini respectively of the peptide segments to be reacted, by azidation of amino free protected amino acids Current Opinion in Chemical Biology 2014, 22:115–121
[40] and by functionalization of the resin by reductive amination of propargylamine [41,42]. By extending their previous work on multiple successive CuAAC [43] using triisopropylsilyl as a protecting group for the alkyne moiety that can be mildly removed with TBAF, they developed an iterative version of this peptidomimetic triazole ligation (PTL). The TIPS protecting group is easily introduced by changing the propargylamine to 3(triisopropyl)prop-2-yn-1-amine during the functionalization of the resin. The Cu-promoted ligation reaction proceeds under degassed phosphate buffer at pH 8 at concentration of 1 mM of the alkyne peptide and a slightly excess of the azide peptide in only 15 min, leading to a clean triazolopeptide that can be directly deprotected by treatment with TBAF for one hour. They applied the PTL method for the synthesis of an analog of Cystatin A, which they obtained in an overall yield of 27% over two ligation steps www.sciencedirect.com
New chemistry for chemoselective peptide ligations Harmand, Murar and Bode 119
Figure 4
H2N COOH
BF3 K H3N
O MeO
O
O
COOH
N
n
COOH
NH
Et
N
O
EFIAWLVRGRG
OH NH NH H2N
HN O
O
PEG5,000 , PEG20,000
N H
O CONH2
KAT
COOH
O
H N
HAEGTFTSDVSSYLEGQAA
NH2 NH
NH2
Et
GLP-1 analogue
KAT ligation
tBuOH/H2O 0.1 M oxalic acid 23 °C H2N
COOH
COOH H3N
H N
HAEGTFTSDVSSYLEGQAA
COOH
N NH
O CONH2
COOH
O N H
NH2 NH O
EFIAWLVRGRG
OH NH NH H2N
HN
NH2
O MeO
O
O n
Current Opinion in Chemical Biology
Chemoselective amide-formation with an equimolar ratio of reactants by KAT ligation.
and one deprotection (Figure 3b). The biological tests performed on the synthetic cystatin A showed that the presence of two peptidomimetic bonds did not disrupt the biological activity and that the potency of this cystatin A analog was not diminished. These data confirmed that the PTL can be used as a powerful, easy and fast method for the synthesis of biological active protein analogs.
In order to demonstrate the utility of this method, the Bode group used the KAT ligation for the synthesis of conjugates of unprotected peptides with large polymers. They chose a 31-amino acid residue analog of the antidiabetic peptide GLP-1 as an initial target. Using the KAT ligation, PEG, lipids, biotin, and dyes were introduced onto the unprotected peptide with excellent yields and small reaction times.
KAT ligation
As part of our search for amide-forming chemical ligation reactions, the Bode group recently reported the chemoselective amide-forming ligations of potassium acyltrifluoroborates (KATs) and O-carbamoylhydroxylamines [44]. KATs showed rapid amide formations along with excellent stability under mild aqueous conditions. Additionally, the KAT ligation allows the use of 1:1 stoichiometry of the two reagents, tolerates all unprotected functional groups, and operates without toxic reagents or byproducts (Figure 4). In other words, KAT ligation fulfills all of the criteria for an ideal chemoselective reaction, although further work on methods to prepare and introduce the KAT ligation reactive moieties to unprotected peptide segments at the C and Ntermini will be necessary. www.sciencedirect.com
Improved methods for conjugating molecules to unprotected peptides are in great demand. The high stability of both hydroxylamine and acyltrifluoroborate moieties to aqueous conditions, HPLC purification, and standard reagents used in peptide synthesis offers the possibility to imagine the KAT ligation as a powerful method for the future synthesis of large proteins.
Conclusions Numerous chemoselective ligations for the synthesis of proteins and peptides have been proposed in recent years. Here we have focused on several new ligation chemistries that promise to have practical utility. The KAHA ligation with 5-oxaproline holds great promise in the formation of depsipeptides, especially for hydrophobic proteins. Current Opinion in Chemical Biology 2014, 22:115–121
120 Synthetic biomolecules
Further design of alkoxyamine-containing monomers for the KAHA ligation will allow it to offer other unnatural as well as completely natural residues at the ligation sites. The introduction of methods for amide-forming ligation at N-terminal serine and threonine residues represents a versatile solution to generate natural Xaa–Ser/Thr linkages. The alkyne–azide ligation is an intriguing addition to the tools for chemical protein synthesis that allows for the formation of a peptidomimetic group directly into the protein backbone. Finally, the KAT ligation affords synthetic access to a great number of protein conjugates, and could provide an excellent method for preparing even larger proteins and protein-conjugates that are currently one of the frontiers of synthetic organic chemistry.
Acknowledgements This work was supported by ETH Zu¨rich and the Swiss National Science Foundation (200020_150073).
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest
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Current Opinion in Chemical Biology 2014, 22:115–121
