Automated solid‐phase synthesis of metabolically stabilized triazolo‐peptidomimetics

The use of 1,4‐disubstituted 1,2,3‐triazoles as trans‐amide bond surrogates has become an important tool for the synthesis of metabolically stabilized peptidomimetics. These heterocyclic bioisosters are generally incorporated into the peptide backbone by applying a diazo‐transfer reaction followed by CuAAC (click chemistry) with an α‐amino alkyne. Even though the manual synthesis of backbone‐modified triazolo‐peptidomimetics has been reported by us and others, no procedure has yet been described for an automated synthesis using peptide synthesizers. In order to efficiently adapt these reactions to an automated setup, different conditions were explored, putting special emphasis on the required long‐term stability of both the diazo‐transfer reagent and the Cu(I) catalyst in solution. ISA·HCl is the reagent of choice to accomplish the diazo‐transfer reaction; however, it was found instable in DMF, the most commonly used solvent for SPPS. Thus, an aqueous solution of ISA·HCl was used to prevent its degradation over time, and the composition in the final diazo‐transfer reaction was adjusted to preserve suitable swelling conditions of the resins applied. The CuAAC reaction was performed without difficulties using [Cu (CH3CN)4]PF6 as a catalyst and TBTA as a stabilizer to prevent oxidation to Cu(II). The optimized automated two‐step procedure was applied to the synthesis of structurally diverse triazolo‐peptidomimetics to demonstrate the versatility of the developed methodology. Under the optimized conditions, five triazolo‐peptidomimetics (8–5 amino acid residues) were synthesized efficiently using two different resins. Analysis of the crude products by HPLC‐MS revealed moderate to good purities of the desired triazolo‐peptidomimetics (70–85%). The synthesis time ranged between 9 and 12.5 h.


| SCOPE AND COMMENTS
The importance of peptides in medicine has notably grown. 1 Their ability to interact with clinically relevant proteins with exquisite affinity and specificity make them ideal drug candidates. Peptides are also excellent vehicles (vectors) to transport selectively imaging probes and cytotoxic payloads to the site of diseases due to their suitable pharmacokinetic profiles and tissue penetration properties, as well as convenient and economic availability. 2 However, the use of peptides in the clinic is hampered by their metabolic lability, which can limit their accumulation at the biological target(s). 3 As a result, many research groups have reported different methods for the stabilization of peptides, for example, by structural modifications (e.g., N-methylation 4 or reduction of amide bonds, employment of amide bond surrogates, or incorporation of unnatural amino acids 5 ) and/or conformational restraints (e.g., head-to-tail cyclization and stapling 6 ). The use of metabolically stable amide bond bioisosters represents an attractive approach to enhance the metabolic stability of a peptide while preserving its biological function(s). Among the examples reported (e.g., sulfonamides and semicarbazides 7 ), 1,4-disubstituted 1,2,3-triazoles (Tz) have been shown to be suitable trans-amide bond surrogates. 8 On the other hand, 1,5-disubstituted 1,2,3-trizoles can serve as analogs of cis-amine bonds. 9 These metabolically stable Tz-based bioisosters of amide bonds share similarities with the amido functional group in terms of size, planarity, H-bonding properties, and polarity ( Figure 1).
The incorporation of triazoles into the peptide's backbone can be accomplished in different ways. An elegant approach relies on the use of solid-phase chemistry for both peptide elongation and triazole incorporation. This way, the peptide is elongated following standard Fmoc/ t Bu chemistry until the position of Tz-insertion, and this is accomplished on solid support in two sequential synthetic steps. The first reaction makes use of a diazo-transfer reagent (e.g., imidazole-1-sulfonyl azide 10 ) for the amide-to-azide conversion at the N-terminus of the peptide. The second reaction consists of the [2+3] dipolar cycloaddition between the introduced azide functionality and an appropriate alkyne derivatives of amino acids. 11 The in solution synthesis of α-amino alkynes starting from commercial amino acid derivatives is described elsewhere. 12 Employment of specific metal catalysts yields different regioisomers of the triazole heterocycle. While Copper(I) 13 is used for the synthesis of 1,4-disubstituted 1,2,3-triazoles, Ruthenium(II) 14 has proven useful to synthesize the 1,5-disubstituted regioisomers. Albeit reported, 15 the introduction of 1,5-disubstituted 1,2,3-triazoles into peptides on solid-phase is challenging, at least in our hands. 16 We therefore focus here on the application of 1,4-disubstituted 1,2,3-triazoles as trans-amide bond surrogates.
The emergence and widespread availability of automated peptide synthesizers offers an opportunity for the rapid synthesis of not only peptides but also peptidomimetics, provided that the chemistry used is compatible with SPPS. Even though the diazo-transfer 17 reaction and CuAAC 18  19 Unlike in manual synthesis, all solutions of reagents need to be prepared in advance, and the long-term stability of the reagents is essential for automated and programmed reaction sequences. The first attempts to perform the diazo-transfer reaction were conducted in DMF using ISAÁHCl  Table 1, approximately 99% conversion of the N-terminal amine of the peptide to an azido-functionality was achieved providing the azido-peptide in 90% purity ( Figure 2; Table 1 Automated addition of solutions of the catalyst, the α-amino alkyne (  Figure S2). Therefore, no special precautions had to be applied for the automated CuAAC on solid support.
The optimized two-step automated procedure was applied to the synthesis of different triazolo-peptidomimetics in order to demonstrate the general applicability of our new methodology. First, we looked at the application of different resins. Thus, the triazolecontaining minimal binding sequence of bombesin 19 was synthesized F I G U R E 1 Comparison between the trans-amide bond (left) and the 1,4-disubstituted 1,2,3-triazole (right). The orange arrows indicate H-bond acceptors, and the green arrows the H-bond donors. The length of the functional groups is specified in Angstroms (Å). R 1 and R 2 correspond to different (protected) side chains specific for an amino acid.
on MBHA and Wang resins (Table 3, entries 1 and 2). In both cases, the desired triazolo-peptidomimetic was obtained in a total synthesis time of 10-11 h with comparable purities. Thus, no preference for either resin became apparent. In the following, the procedure was also applied to the automated solid-phase synthesis of reported triazolecontaining analogs of Leu-enkephalin 22 and neurotensin 23 (Table 3, (Table 3, entry 5). 24,25 In all cases, the desired triazolopeptidomimetics were obtained within reasonable time (max. overnight), with good conversion for both standard SPPS (Fmoc/ t Bu chemistry) and the diazo-transfer/CuAAC steps (S3-S7).
In conclusion, we have developed a robust and versatile protocol for the automated synthesis of triazolo-peptidomimetics on solid support using a microwave-assisted peptide synthesizer. The T A B L E 1 Different reaction conditions examined for the diazo-transfer reaction.

| Amino alkynes
The amino alkynes were synthesized from the corresponding amino acids following a three-step synthetic route. These three steps include the formation of the Weinreb amide, the reduction to the corresponding amino aldehyde, and a Seyferth-Gilbert homologation using the Bestmann-Ohira reagent (Figure 3, blue route). In case of amino acids containing acidic side chain functionalities, an alternative route is recommended (Figure 3, green route). In this case, the carboxylic acid is reduced to the alcohol followed by Swern oxidation. Once the aldehyde is obtained, the homologation is performed with the Bestmann-Ohira reagent as described above. If deprotection of the Fmoc protecting group is observed as a result of the presence of base (K 2 CO 3 ) required for the Seyferth-Gilbert homologation in the last step, the amino group of the obtained crude product can be protected again using Fmoc-ONSu. These synthesis protocols can, at least in our hands, result in the partial racemization of the resulting amino alkynes Abbreviations: Gly, Fmoc-Gly-CCH; Phe, Fmoc-Phe-CCH; Arg, Fmoc-Arg (Boc) 2 -CCH; Glu, Fmoc-Glu ( t Bu)-CCH; Tyr, Fmoc-Tyr ( t Bu)-CCH. a Crude purity of the final triazolo-peptidomimetic after overall deprotection and cleavage from the resin was determined by HPLC-MS. b Given the limited stability of Fmoc-Arg (Boc) 2 -CCH in DMF, the amino alkyne solution was prepared shortly before the CuAAC reaction.
(likely at the stage of the aldehyde intermediate). 26 The presence of enantiomers is best analyzed by chiral-HPLC. 27 Alternatively, the amino alkynes can be coupled with an enantiomerically pure amino acid, followed by analysis of the diastereomeric purity of the resulting dipeptide by NMR or HPLC. 28 The incorporation of the partially racemized amino alkynes in the peptide backbone via the CuAAC reaction results in the formation of a mixture of diastereomers. The desired product can be readily identified by the comparison of the diastereomeric ratio of the peptide (determined by UV-HPLC) and the enantiomeric ration of the chiral amino alkyne. 25 Alternatively, the (partially) racemized amino alkyne can be subjected to (semi-)preparative chiral-HPLC purification prior to use. All diasteromeric mixtures of final triazolo-peptidomimetics studied by us so far could be separated efficiently by HPLC using a C18 column.
F I G U R E 3 Representation of the two general strategies to synthesize α-amino alkynes starting from amino acids.