Synthesis of Peptide Disulfide-Bond Mimics by Using Fully

Letter Cite This: Org. Lett. 2018, 20, 6074−6078

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Synthesis of Peptide Disulfide-Bond Mimics by Using Fully Orthogonally Protected Diaminodiacids Tao Wang,†,§,# Jian Fan,‡,# Xiao-Xu Chen,† Rui Zhao,‡ Yang Xu,‡ Donald Bierer,⊥ Lei Liu,∥ Yi-Ming Li,§ Jing Shi,*,‡ and Ge-Min Fang*,† †

School of Life Science, Institute of Physical Science and Information Technology, Anhui University, Hefei 230601, P. R. China Department of Chemistry, University of Science and Technology of China, Hefei 230026, P. R. China § School of Biological and Medical Engineering, Hefei University of Technology, Hefei 230009, P. R. China ∥ Tsinghua University, Beijing 100084, P. R. China ⊥ Department of Medicinal Chemistry, Bayer AG, Aprather Weg 18A, 42096 Wuppertal, Germany

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‡

S Supporting Information *

ABSTRACT: A new strategy was developed for the synthesis of peptide disulfide-bond mimics using fully orthogonally protected diaminodiacids. This method overcomes the previous problems of heavy-metal contamination and poor compatibility with Fmoc chemistry and provides a practical avenue for the efficient preparation of peptide disulfide-bond mimics.

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isulfide-rich polypeptide scaffolds, which are frequently present in the structure of FDA-approved peptide drugs such as insulin, linaclotide, and plecanatide, have attracted a great deal of attention from peptide-based drug discovery.1 Although disulfide-rich scaffolds confer peptides with enhanced enzymatic stability and structural diversity, the disulfide bridge can be unstable toward thiol and disulfide isomerases in vivo.2 A promising and effective strategy to improve the metabolic integrity of disulfide-containing polypeptides is to replace reducible disulfide bridges with nonreducible bonds such as thioether, selenoether, triazole, or hydrocarbon bridges.3 This strategy, proposed as early as the 1960s, has been widely used by many groups to engineer a variety of bioactive peptides4 and displayed an attractive prospect in peptide drug discovery, as showcased by an uterotonic and antihemorrhagic drug, carbetocin, a recently approved oxytocin analogue containing a thioether bond.5 A number of methods have been developed for the preparation of disulfide peptide mimics. Among them, one efficient and versatile method is diaminodiacid-based solidphase peptide synthesis (SPPS) (Figure 1).6,7 This approach avoids the use of intermolecular reactions such as thiol alkylation, azide−alkyne cycloaddition, and olefin metathesis that are commonly used to construct disulfide-bonded mimic scaffolds on peptide substrates, thereby reducing the number of synthetic steps and increasing the overall yield of the final product. More importantly, many different diaminodiacids can be readily incorporated into the peptides using the same synthetic strategy, thus enabling the synthesis of diverse © 2018 American Chemical Society

Figure 1. Orthogonally protected diaminodiacids used for the preparation of disulfide-bond surrogates. Fmoc, Tbe, and Mtt are fully orthogonal to each other.

molecular structures of disulfide mimics through the same synthetic chemistry strategy.3a The diaminodiacids used in this strategy contain two amino groups and one carboxyl group that needs to be temporarily protected. More specifically, one amino protecting group is Fmoc, and the remaining protecting groups can be Alloc, pNz, Dmab, or ivDde groups, which are Received: August 2, 2018 Published: September 14, 2018 6074

DOI: 10.1021/acs.orglett.8b02459 Org. Lett. 2018, 20, 6074−6078

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Organic Letters orthogonal to Fmoc.7,9 When using Alloc- and pNz-protected diaminodiacids, we encountered an annoying issue, namely, metal contamination of target peptides,8 probably as a result of the high affinity of the remaining palladium to thiol-rich peptides. Recently developed Dmab/ivDde-protected diaminodiacids avoid the use of heavy-metal reagents; however, neither Dmab nor ivDde is fully stable toward piperidine,9 rendering them unsuitable for Fmoc-based peptide synthesis of long sequences and for use in an automatic synthesizer. In addition, removing the side-chain carboxyl protecting group of the diaminodiacid also liberates its side-chain amino functional group, which during the subsequent cyclization reaction tends to react with the coupling reagent, thereby lowering the yield of the peptide synthesis.10 Therefore, there is a strong need to develop new diaminodiacids whose protecting groups are fully compatible with Fmoc SPPS and can be removed under metal-free conditions. Herein we report a new, practical strategy using thioethercontaining diaminodiacids bearing three completely orthogonal protecting groups (Fmoc, Mtt, and Tbe) as the key building blocks to synthesize cysteine-rich disulfide-bond mimics. This method becomes possible on the foundation of the following findings: (1) hexafluoroisopropanol (HFIP) can chemoselectively release the Mtt protecting group in the presence of multiple Trt protecting groups of cysteine residues (of note, 1% TFA in DCM, which is widely used for Mtt deprotection, can result in partial removal of Trt from the Cys residue) and (2) the thiol-sensitive carboxyl protecting group, Tbe, is fully compatible with Fmoc chemistry. The efficiency and practicality of this method are illustrated by the successful synthesis of thioether-containing oxytocin and hepcidin analogues. The user-friendly diaminodiacid bearing three fully orthogonal protecting groups makes solid-phase synthesis of Cys-rich disulfide-bond mimics much simpler and more efficient. The acid-sensitive Mtt protecting group was used to protect the side-chain amino group of the diaminodiacid, but very few high-quality protecting groups match our requirement for the side-chain carboxyl group.11 The widely used allyl-based protecting groups are unacceptable because of the use of palladium-based reagents.12 We turned our attention to our recently developed 2-(tert-butyldisulfanyl)ethyloxycarbonyl (Tbeoc) group for N-terminal cysteine protection in chemical protein synthesis.13 The metal-free conditions for Tbeoc deprotection may become completely orthogonal to both Fmoc and Mtt, so we envisioned the possibility of developing a practically useful, Fmoc-chemistry-compatible carboxylic protecting group by coupling 2-(tert-butyldisulfanyl)ethanol (Tbe) to the side-chain carboxylic acid of the diaminodiacid. Our interest in the Tbe ester was further stimulated by the recent finding from Kent’s group that the peptide α-ester having 2-mercaptoethanol at the C-terminus can be converted quantitatively into the peptide α-carboxylate via an intramolecular displacement mechanism under weakly alkaline conditions.14 In order to study the compatibility of the Tbe protecting group with Fmoc-based SPPS, the following two issues needed to be addressed: (1) during peptide chain elongation, the Tbe ester should be completely stable toward 25% piperidine in DMF, and (2) the Tbe protecting group must be able to be efficiently removed while the other side-chain protecting groups of the polypeptide are preserved. The stability of the

Tbe ester toward piperidine was confirmed by the high stability of Boc-Phe-Tbe in 25% piperidine over 24 h (Figure S2). Then we investigated the conditions for Tbe deprotection (Table S2). Boc-Phe-Tbe was nearly quantitatively converted to BocPhe-OH after reduction by 2-mercaptoethanol in the presence of an organic base (Et3N, DIPEA, or DBU). We used 2mercaptoethanol/DIPEA solution as the deprotection conditions for the subsequent peptide synthesis. In view of the widespread use of the thioether linkage in the synthesis of disulfide-bond mimics,15 the Fmoc/Mtt/Tbe-protected C−Sbridged or S−C-bridged diaminodiacids were synthesized according to the previously reported procedure, as shown in Scheme 1 (also see Schemes S1 and S3). These two Scheme 1. Chemical Synthesis of Fmoc/Mtt/Tbe-Protected Diaminodiacids

diaminodiacids can provide two peptide disulfide mimics with subtle differences in the topological structure (∼CH2−S∼ vs ∼S−CH2∼), which could enable in-depth studies of the disulfide-bond function of bioactive peptides. With thioether-containing diaminodiacids in hand, we next tested their compatibility with Fmoc-based SPPS by synthesizing a model peptide, namely, a cystathionine analogue of oxytocin. The purity of crude oxytocin prepared from previous Dmab/ivDde-protected diaminodiacids was less than 50%.9 Therefore, it was tested here whether Mtt/Tbe is more compatible with Fmoc chemistry than Dmab/ivDde. The synthesis of the oxytocin analogue started with the Rink amide AM resin, as shown in Figure 2. After assembly of the tripeptide, Pro-Leu-Gly, on the resin, the C−S-bridged diaminodiacid was coupled to the α-N-terminal amino group of Pro via a double coupling method. Subsequently, the linear precursor of the oxytocin mimic was finished by assembly of the tetrapeptide Tyr-Ile-Gln-Asn onto the N-terminal amino group of the diaminodiacid. After removal of the Tbe protecting group by 2-mercaptoethanol in DIPEA-containing NMP, the side-chain carboxyl group of the diaminodiacid was efficiently cyclized with the N-terminal amino group of Tyr using PyAOP/NMM. It is noteworthy that although the sidechain protecting group Mtt is very bulky, it does not hinder the intramolecular cyclization between the side-chain carboxylic acid group of the diaminodiacid and the N-terminal amino group of the peptide. Finally, the intact oxytocin analogue was cleaved from the resin in TFA. The purity of the crude oxytocin analogue, as shown in Figure 2, reached 70%, which is significantly higher than that synthesized using the previous 6075

DOI: 10.1021/acs.orglett.8b02459 Org. Lett. 2018, 20, 6074−6078

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Figure 2. Synthetic route for the preparation of oxytocin and hepcidin disulfide mimics and analytical RP-HPLC traces of the crude synthetic peptide intermediates (methionine was replaced by norleucine to avoid side reactions). Conditions for Tbe removal: 2-mercaptoethanol (5 M), DIEA (1.25 M) in NMP. Conditions for Mtt removal: HOBt (5.0 equiv), HFIP (50 vol %) in 1,2-dichloroethane (DCE). Conditions for hepcidin folding: GSSG (5.0 equiv), GSH (5.0 equiv), 2:1 water/acetonitrile, pH 7.5, rt, 14 h.

Dmab/ivDde-based diaminodiacid,7 demonstrating the high compatibility of the Tbe/Mtt-protected diaminodiacid with Fmoc-based peptide synthesis. We further tested the practical applicability of the method by synthesizing the mimics of a more challenging but pharmaceutically interesting peptide, hepcidin, containing four pairs of disulfide bonds. This 25 amino acid peptide hormone has been used as a key element in regulating iron entry into the circulation, and diseases associated with hepcidin dysregulation can be treated by intravenous injection of synthetic hepcidin.16 The folding yield of synthetic linear hepcidin is less than 12% because of misfolding of the disulfide bonds.17 Replacement of one disulfide bond with a C−S bridge significantly increases the yield of hepcidin folding by up to 60%, and therefore, a diaminodiacid-based approach appears to be a promising hepcidin engineering method.5b However, the previous method for synthesizing thioether-containing hepcidin encountered the issue of palladium contamination. In this context, we attempted to establish a practical, metal-free

strategy to access thioether-containing hepcidin by using the Mtt/Tbe-based diaminodiacid. The S−C-bridged diaminodiacid (Figure 1) was selected to synthesize a hepcidin derivative in which the Cys11−Cys19 disulfide bond was replaced by a thioether bond. After the S− C diaminodiacid coupling, we analyzed the purity of the peptide intermediate by HPLC. As expected, the predominant peak in the HPLC corresponded to the desired peptide. After assembly of the tetrapeptide and the heptapeptide onto the Nterminus of the diaminodiacid, we confirmed the high efficiency of peptide synthesis and the high stability of the Tbe group toward piperidine by HPLC. Prior to the intramolecular cyclization reaction, the Tbe group was quantitatively removed with high chemoselectivity by 2mercaptoethanol/DIPEA/NMP, as shown in Figure 2. The following steps were intramolecular cyclization, Mtt deprotection, and assembly of the remaining amino acid residues. Unexpectedly, when 1% TFA was used to remove Mtt, we encountered the problem of Trt deprotection of the Cys side 6076

DOI: 10.1021/acs.orglett.8b02459 Org. Lett. 2018, 20, 6074−6078

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chain, which resulted in multiple side products after the subsequent amino acid coupling (Figure S16). To solve this problem, we used HFIP/HOBt/DCE to deprotect the Mtt group.18 To our delight, the HFIP conditions could selectively remove Mtt without interfering with the Trt protecting group of Cys residues. Of note, HFIP is not the first choice for Mtt deprotection because of its potential toxicity. After completing the linear thioethercontaining hepcidin, we obtained a crude product with an HPLC purity of over 80%. The subsequent peptide folding was carried out under common neutral conditions: 33% acetonitrile, 1:1 GSSG/GSH, pH 7.5. As analyzed by HPLC, the folding yield of the hepcidin derivative was 70%, which is significantly higher than that of wild-type hepcidin. The IC50 value for the hepcidin disulfide thioether mimic was measured to be 241 nM, which is similar to that of disulfide-linked hepcidin (224 nM). Taken together, these results show that the described Mtt/Tbe-protected diaminodiacid is fully compatible with Fmoc SPPS and avoids the use of heavy metals, thus providing a user-friendly and practically useful method of synthesizing bioactive cysteine-rich disulfide-bond mimics. To conclude, we have developed user-friendly diaminodiacids for Fmoc-based SPPS of cysteine-rich disulfide-bond mimics. The diaminodiacids reported here contain three orthogonal protecting groups (Fmoc, Mtt, and Tbe). Compared with the previously developed pNz/pNb, Alloc/ Allyl, and Dmab/ivDde-protected diaminodiacids, the present Mtt/Tbe type avoids the use of cytotoxic heavy-metal reagents and is fully compatible with Fmoc SPPS. The practicability of this method has been exemplified by the efficient synthesis of oxytocin and hepcidin analogues. We anticipate that this work will provide a highly efficient and practically useful strategy for the efficient preparation and optimization of cysteine-rich disulfide-bond mimics for diagnostic and therapeutic applications.

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Key R&D Program of China (2017YFA0505200), the National Natural Science Foundation of China (21572214, 21532004, 21807001, and 91753205), and the Fundamental Research Funds for the Central Universities (PA2017GDQT0021).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.8b02459. General information, chemical synthesis of Fmoc/Mtt/ Tbe-protected diaminodiacids, stability of Boc-PheOTbe toward 25% piperidine in DMF, optimization of the Tbe ester deprotection conditions, SPPS of disulfide peptide mimics, and NMR and MS data for Fmoc/Mtt/ Tbe-protected diaminodiacids (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected] ORCID

Lei Liu: 0000-0001-6290-8602 Yi-Ming Li: 0000-0001-6716-8199 Jing Shi: 0000-0003-0180-4265 Ge-Min Fang: 0000-0003-4149-4062 Author Contributions #

T.W. and J. F. contributed equally. 6077

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