Carina Hey Pui
Cheung‡
a,
Jianchao
Xu‡
a,
Chi Lung
Lee
a,
Yanfeng
Zhang
a,
Ruohan
Wei
a,
Donald
Bierer
b,
Xuhui
Huang
c and
Xuechen
Li
*a
aDepartment of Chemistry, State Key Lab of Synthetic Chemistry, The University of Hong Kong, Hong Kong. E-mail: xuechenl@hku.hk
bDepartment of Medicinal Chemistry, Bayer AG, Aprather Weg 18A, 42096 Wuppertal, Germany
cDepartment of Biological and Chemical Engineering, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong
First published on 13th April 2021
Herein, we report the development of a facile synthetic strategy for constructing diverse peptide structural architectures via chemoselective peptide ligation. The key advancement involved is to utilize the benzofuran moiety as the peptide salicylaldehyde ester surrogate, and Dap–Ser/Lys–Ser dipeptide as the hydroxyl amino functionality, which could be successfully introduced at the side chain of peptides enabling peptide ligation. With this method, the side chain-to-side chain cyclic peptide, branched/bridged peptides, tailed cyclic peptides and multi-cyclic peptides have been designed and successfully synthesized with native peptidic linkages at the ligation sites. This strategy has provided an alternative strategic opportunity for synthetic peptide development. It also serves as an inspiration for the structural design of PPI inhibitors with new modalities.
Although the peptides represent a promising class of therapeutic drugs in various therapeutic areas, they suffer from some limitations.6 For example, linear peptides are not stable in vivo and are prone to protease degradation.4,14,15 They also have poor permeability to access desirable intracellular targets.4,8 Furthermore, the binding of peptides to the PPI interface may not be always thermodynamically favorable as they will need to overcome the entropic penalty to reorganize themselves into its constrained bioactive state.4 To overcome the intrinsic limitations of the natural peptides, structural modifications with unnatural elements are being explored. One of the most notable strategies is to develop side chain-to-side chain cyclic peptides from the mimicry of interfacial α-helical domains.16–18 Previous studies have shown that via varying the structural design including the stapled positions, structures, lengths and peptide sequence, one can change the dynamics of the peptide–protein interaction8 thus optimize the engagement of inhibitors at the PPIs interface. Peptide drugs with optimized structural designs were found having higher protease resistance, biological potency and binding affinity.17 Successful examples of stapled peptides offering therapeutic modality include all-hydrocarbon-linked stapled peptide ALRN-6924 which is under clinical development as an anti-cancer drug targeting HDM2/p53.19,20 Other examples include peptides targeting HIV integrase, BCl-2 and β-catenin.21–23 Apart from the stapling chemistry, some other motifs have been explored, including the β-strands mimetics24 and loops motif25 that display more complex topologies. Examples of tertiary mimetics as PPI inhibitors has also been reported, including α- and α/β-peptides derived from the “Z-domain” scaffold.26 As PPIs have pivotal roles in the regulation of biological systems, novel and practical tools for the generation of new peptide architectures and structural complexities will be worth being explored.
Here, we report the development of chemical ligation chemistry for constructing diverse peptide structural motifs, including side chain-to-side chain cyclic peptides, branched and bridged peptides, tailed cyclic peptides and multi-cyclic peptides. We expect these peptides will represent new structural motifs and offer new modalities for developing inhibitors of PPIs with enhanced stability and binding affinity.
Peptide C-terminal salicylaldehyde esters have been developed to ligate side chain unprotected peptides with N-terminal Ser/Thr/Cys/Pen for Ser/Thr ligation (STL)30,31 and Cys/Pen ligation (CPL)32 used in chemical protein synthesis. The chemoselective reaction between the two counterparts affords an acid-cleavable N,O/S-benzylidene acetal intermediate. Followed by acidolysis, the native peptidic linkage is generated at the ligation junction. Apart from linear peptide/protein synthesis, intramolecular Ser/Thr ligation has been developed for peptide cyclization to produce head-to-tail cyclic peptides of various sizes.33–39 In this paper, we ought to further expand the scope of Ser/Thr ligation by solving the problem of introducing the peptide salicylaldehyde ester on the side chain, with which to construct diverse peptide scaffolds.
To install these two reactive groups onto the side chain of the unprotected peptides for executing side-chain STL, we first identified the Dap–Ser or Lys–Ser dipeptide as the side chain hydroxyl amino functionality. The Dap–Ser or Lys–Ser dipeptide was successfully synthesized via mixed anhydride derivatives from isobutyl chloroformate (Scheme 1a). Boc-Ser(tBu)-OH was first dissolved in dry CH2Cl2, isobutyl chloroformate and DIEA were then added at 0 °C to afford the mixed anhydride. The amine nucleophile, Fmoc-Dap-OH or Fmoc-Lys-OH, was added after an hour to generate the dipeptide ready for SPPS.
The introduction of the salicylaldehyde ester to the side chain of Asp/Glu of the peptide is very challenging. As priorly mentioned, there is no simple and direct method reported yet for synthesizing peptides with Asp/Glu side chain active esters. The complication occurs as the coupling of Asp (O-salicylaldehyde ester) or any Asp active ester building block will lead to aspartimide formation during SPPS or the condensation step. Moreover, the Asp/Glu active esters cannot survive piperidine treatment during Fmoc-SPPS. All previous strategies for synthesizing peptide C-terminal salicylaldehyde esters are not applicable for introducing salicylaldehyde esters at the peptide side chain.31 To solve this problem, we have come to identify a novel synthetic strategy utilizing the benzofuran moiety as the peptide crypto-salicylaldehyde ester. Upon ozonolysis, the benzofuran moiety could readily become a salicylaldehyde ester for subsequent chemoselective ligation.
Previous synthetic routes to benzofuranylalanine via transition metal-catalyzed or chemoenzymatic synthesis were reported.40,41 In our design, preparation of the benzofuran moiety started from salicylaldehyde. Salicylaldehyde was first treated with reducing agent NaBH4, followed by reflux with triphenylphosphine hydrobromide. The generated 2-hydroxybenzyltriphenylphosphonium bromide was then added to Boc-Asp-OMe and transformed into an activated ester intermediate upon DIC treatment (Scheme 1b). Subsequently, the resulted intermediate was refluxed with triethylamine to offer the benzofuran moiety. Finally, the desired benzofuran building block was obtained by C-terminal methyl ester group deprotection, followed by N-terminal Boc group deprotection and Fmoc group installation. As the Fmoc-Asp(benzofuran)-OH moiety is stable under both acidic and basic conditions during peptide synthesis, it could be directly used in Fmoc-SPPS. We also obtained Fmoc-Glu(benzofuran)-OH in similar route (see ESI†).
With these building blocks in hand, we continued to incorporate them into the peptide to execute our construction plan. As a proof of concept, the linear peptide with Dap–Ser and Asp(benzofuran) could be readily synthesized under standard Fmoc-SPPS conditions.
After global deprotection, the resultant side chain unprotected peptides were treated with ozonolysis in H2O/ACN (v:v = 1:1) with 0.7% TFA under 0 °C for 1 minute, affording the peptide salicylaldehyde ester smoothly. It is noting that oxidation-prone residues are not compatible with the ozone treatment, i.e. Cys and Met react with ozone at the sulfhydryl to give sequential O-atom addition products,42 and Trp becomes kynurenine after ozonolysis.43 Therefore, these amino acid residues are excluded from the chemical space available for this chemistry. Subsequently, side chain-to-side chain Ser/Thr ligation with unprotected peptides was performed. The peptide was dissolved in pyridine acetate buffer at 0.5 mM concentration, which spontaneously proceeded to cyclize, followed by acidolysis to cleave the N,O-benzylidene acetal intermediate to afford the cyclic peptides with an overall yield of ∼35% after one HPLC purification. With this strategy, we were able to generate Asp–Dap/Lys lactam linkage with a hydrophilic hydroxyl group on different peptide sequences (Fig. 2). Notably, the chemoselective cyclization can tolerate most of the side chain functionalities presenting in the peptide except oxidation-prone residues.
These results were very encouraging and opened up new opportunities to construct novel peptide structures via chemical ligation. We envisaged that our strategy could be flexibly applied to afford innovative peptide modalities via ligating peptides with side chain salicylaldehyde ester with other peptides carrying Dap/Lys–Ser dipeptide or N-terminal Ser/Thr/Cys/Pen. Thus, in addition to the side chain-to-side chain cyclic peptides, we continued to apply this new chemistry to synthesize three classes of structural motifs, as shown in Fig. 1.
The first class includes branched peptide and bridged peptide (Fig. 3 and Table 1). The peptide with the side chain salicylaldehyde reacted with another peptide carrying N-terminal Ser/Thr under the Ser/Thr ligation conditions to produce the branched peptide (Table 1, entries 1–4). Alternatively, the peptide with side chain salicylaldehyde ester can ligate with another peptide carrying side chain Dap–Ser or Lys–Ser under the Ser/Thr ligation conditions to produce bridged peptides (Table 1, entries 5–11). Such structural motifs will allow combining two functional peptides to generate a hybrid peptide with dual activities.
Entry | A | B | % |
---|---|---|---|
a D′ = Asp(benzofuran), (K–S) = Lys–Ser dipeptide. b Percentage conversion to product based on UPLC-MS analysis of the crude reaction mixture. c Isolated yield based on ligation. | |||
1 | Ac-DTTAD′A-NH2 | H-SNVKAQFL-NH2 | 78.2b, 36.6c |
2 | Ac-SRQQGESNQERGARD′RL-NH2 | H-SKAKL-NH2 | 80.0b, 39.4c |
3 | Ac-D′SERVELRKLQDV-NH2 | H-SKAKL-NH2 | 95.0b, 39.1c |
4 | H-VIGGVGD′N-NH2 | H-TLHAPTD-OH | 89.6b, 32.8c |
5 | Ac-DTTAD′A-NH2 | Ac-(K–S)SKAKL-NH2 | 90.5b, 48.5c |
6 | Ac-SRQQGESNQERGARD′RL-NH2 | Ac-(K–S)SKAKL-NH2 | 91.9b, 53.0c |
7 | Ac-SRQQGESNQERGARD′RL-NH2 | Ac-(K–S)SARKYFAGNLPE-NH2 | 95.6b, 37.4c |
8 | Ac-D′SERVELRKLQDV-NH2 | Ac-(K–S)SKAKL-NH2 | 88.9b, 33.3c |
9 | Ac-D′SERVELRKLQDV-NH2 | H-NIGTYLP(K–S)NVK-NH2 | 93.4b, 31.1c |
10 | Ac-D′SERVELRKLQDV-NH2 | Ac-ARE(K–S)TPEP-NH2 | >98b, 38.4c |
11 | Ac-LSQD′RG-NH2 | Ac-ARE(K–S)TPEP-NH2 | 91.7b, 34.7c |
The second class includes tailed cyclic peptides (Fig. 4 and Table 2). Due to the compact structures of cyclic peptides and their ultra-stability against enzymatic degradation and physical denaturation, cyclic peptides have attracted a lot of attention in the pharmaceutical industry. In this regard, the development of effective methods for peptide cyclization has been an intensively researched subject. In our design, a peptide with the side chain salicylaldehyde ester and N-terminal Ser/Thr could cyclize under the Ser/Thr ligation condition to generate head-to-side chain cyclic peptides (Table 2, entries 12 and 13). Furthermore, a cyclic peptide with the side chain salicylaldehyde ester could react with another peptide with side chain Lys–Ser to generate cyclic peptide with a branched tail (Table 2, entries 14–19). Our strategy not only provides an effective synthetic method but also generates new structural motifs.
The third class of peptide structural architectures includes the bridged cyclic and embedded bicyclic peptides (Fig. 5 and Table 3). One side chain-to-tail cyclic peptide with free N-terminal Cys could react with another head-to-tail cyclic peptide with side chain salicylaldehyde ester under Cys ligation conditions to generate a bridged cyclic peptide, which will provide a way to conjugate cell-penetrating peptide to a peptide ligand. Alternatively, the embedded bicyclic peptides could be synthesized by performing first the side chain-to-tail Ser/Thr ligation involving the side chain Lys–Ser and C-terminal salicylaldehyde ester, followed by the head-to-side chain Ser/Thr ligation involving the N-terminal Ser/Thr and the side chain salicylaldehyde ester. Based on this synthetic route, we envisioned that cyclic peptides of a higher order would be achievable through the rational design of combining the above bridged cyclization and embedded bicyclization approaches.
Entry | A | A′ | B | B′ | A′ + B′ | ||
---|---|---|---|---|---|---|---|
Sequence | Sequence | % | Sequence | Sequence | % | % | |
a D′ = Asp(benzofuran), (K–S) = Lys–Ser dipeptide, SAL = salicylaldehyde. b Percentage conversion to the ligation product based on UPLC-MS analysis of the crude reaction mixture. c Isolated yield based on ligation. d Isolated yield over 2 steps (intramolecular STL and one-pot Thz deprotection). e Isolated yield over 2 steps (intramolecular STL and one-pot Fmoc deprotection). | |||||||
20 | H-SD′LSQRG-SAL ester | >98a, 48.0b | Boc-(Thz)(K–S)LSQRG-SAL ester | 88.9b, 37.2d | 84.3b, 50.4c | ||
21 | Boc-(Thz)NFS(K–S)QSNKRFLSKTQG-SAL ester | 90.1b, 35.4d | 89.9b, 45.8c |
Foreseeing the vast potential of peptide drugs in modulating PPIs, this work presented here delineates a blueprint and serves as an inspiration for the structural design of PPI inhibitors with new modalities, which empowers future targeting of “undruggable” proteins targets. Based on the previous studies by other groups, having a rational structure-based design of therapeutic peptides is crucial for them to achieve higher specificity and biostability. Gratifyingly, our strategy has provided easy access to a diverse class of peptide structural motifs. More importantly, by ligating two functional peptides, the structural motifs synthesized would be a hybrid peptide with dual activities. In long term, the synthesis of more complex bioactive scaffolds will be studied while the developed modalities will be rationally optimized for potential therapeutic applications.
Footnotes |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/d1sc01174j |
‡ Equal contribution. |
This journal is © The Royal Society of Chemistry 2021 |