pDobz/pDobb protected diaminodiacid as a novel building block for peptide disulfide-bond mimic synthesis

The diaminodiacid strategy has been widely studied in the chemical synthesis of peptide disulfide bond mimics. Diaminodiacid building blocks, which are key intermediates, are currently under the spotlight. However, one technical bottleneck inherent in existing building blocks is the contamination problem caused by the heavy metal reagents during the deprotection process, which makes the peptides less suitable for pharmaceutical use. Herein, we describe the successful development of a p-dihydroxyborylbenzyloxycarbonyl pinacol ester (pDobz)- and p-dihydroxyborylbenzyl pinacol ester (pDobb)-based novel diaminodiacid building block that can be easily deprotected via mild treatment with amine oxide. Its efficiency and practicability were also confirmed by the total synthesis of contryphan-Vn disulfide bond mimic. The results suggested that this novel diaminodiacid building block has satisfactory Fmoc SPPS compatibility, yet only required a facile, rapid, and metal-free deprotection process. We believe this novel diaminodiacid building block could promote further development of the diaminodiacid strategy.


Introduction
Disulde bonds play essential and indispensable roles in many peptides and proteins. They help proteins maintain their metabolic stability, biological activity, and target selectivity. [1][2][3][4][5] Studies have shown about one-fourth of the peptidic molecules in the protein data bank (PDB) contain at least one disulde bridge. 6 Many of them are under clinical or pre-clinical study for their potential therapeutic roles in pain disorders, cancer, coagulation disorders, and other conditions. 7-10 However, disulde bonds are unstable in vivo because of reduction, disulde isomerases, and enzymatic cleavage; the degradation then leads to structural distortion and activity loss. 11,12 To overcome these roadblocks, a number of approaches to synthetic disulde surrogates with improved redox stability and conformation rigidity have been developed. These include thioether, olen, diselenide, and triazole bridges. [13][14][15][16][17][18] The diaminodiacid strategy is one alternative to the postchain-strategy as a means of inserting these disulde replacements into the peptide backbone. This is because few types of disulde surrogates can be realized via the post-chain-strategy. 19 As shown in Fig. 1A, pre-prepared diaminodiacid building blocks can be readily installed through an amide coupling reaction rather than generation nonpeptidic bridge on solid support, which requires harsh reaction conditions and oen has unsatisfactory yield. [19][20][21] More importantly, when multiple disulde surrogates need to be introduced, the diaminodiacid strategy can overcome the regioselectivity misfolding problem associated with post-chain-assembly cyclization. 19,22 In previous studies, diaminodiacid building blocks were frequently orthogonally capped by allyloxycarbonyl (Alloc)/Allyl or p-nitrobenzyloxycarbonyl (pNZ)/p-nitrobenzyl (pNB) protective groups (Fig. 1B, 1 and 2). These two pairs of protecting groups can be removed utilizing Pd(PPh 3 ) 4 /PhSiH 23 and SnCl 2 / HCl, 24,25 respectively, on solid support. However, the use of heavy metal reagents may cause detrimental contamination during peptide and protein preparation, especially for pharmaceutical use. 22,26 Besides, the use of protective inert atmosphere and the time-consuming process hinder the convenient operation demanded by SPPS. To overcome this problem, Xu et al. 26 described the utility of 4-(N-[1-(4,4-dimethyl-2,6dioxocyclohexylidene)-3-methylbutyl]-amino)benzyl (Dmab)/ 1-(4,4-dimethyl-2,6-dioxocylcohex-1-ylidene)-3-meth-ylbutyl (ivDde) as diaminodiacid protection groups (Fig. 1B, 3) that can be removed by treatment with 2% hydrazinolysis in DMF solution. Nevertheless, some of degradation of Dmab/ivDde caused by repeated deprotection of the Fmoc group using 20% piperidine in DMF solution during SPPS can lead to undesirable byproducts, which may hinder the comprehensive application of this kind of diaminodiacid building block.
Although this strategy can be optimized to some extent by shortening the duration of each piperidine treatment or by using 2-methylpiperidine as a deprotection reagent, the discovery and development of a novel diaminodiacid building block, which is fully orthogonal with Fmoc SPPS and can be effectively deprotected under metal-free conditions, still remains a eld of intense research. We took inspiration from a series of arylboronate ester-based amino acid building blocks recently developed by our team. 27 The pDobz/pDobb protective groups were found to be stable with acid and base, 27,28 fully compatible with Fmoc SPPS and can be quickly and effectively removed in 30 minutes via treatment with amine oxide and the following low concentration of acid. 27 By taking these advantages into account, we here demonstrate the successful development of a pDobz/pDobb-protected diaminodiacid building block bearing a 1,4-disubstituted 1,2,3triazole bridge (Fig. 1B, 4) which was extensively applied in peptide chemistry because of its extraordinary thermal and metabolic stability. 17,29,30 Besides, we conrmed the efficiency and practicality of this novel diaminodiacid building block by preparing disulde bond mimetic of the model peptide contryphan-Vn, a disulde-constrained nonapeptide, which is a modulator of Ca 2+ -dependent K + channels. 31,32 This work makes it possible to provide a better key intermediate for use with the diaminodiacid strategy.
Further analysis and purication were carried out through reverse-phase high-performance liquid chromatography. As shown in Fig. 2, crude product contained only a single major component that can be easily puried in 98.6% purity, and the resulting total yield was 36% (according to initial resin load). Then, the molecular weight was conrmed using highresolution mass spectrometry (HRMS) and found to be identical to the theoretical molecular mass. Hence, these results suggested that the novel diaminodiacid building block was highly orthogonal with Fmoc SPPS and fairly efficient for synthesizing peptidomimetic components containing disulde bond surrogates.
It is important to emphasize that the installation of triazole surrogates in contryphan-Vn improved its stability. 19 To this end, we synthesized the native contryphan-Vn through Fmoc SPPS and dimethylsulfoxide-mediated solution-phase oxidation cyclization strategy (Scheme S1 †). Subsequently, we carried out the reduction stability experiment using dithiothreitol (DTT) as reducing agent. 33 Indeed, contryphan-Vn was completely reduced in eight hours in aqueous DTT solution, while peptidomimetics 14 remained intact aer eight hours' treatment ( Fig. 3A). Furthermore, the protease stability experiment was investigated. a-Chymotrypsin is a protease that preferentially cleaves peptide amide bonds where the side-chain of the amino acid N-terminal to the scissile amide bond is a large, hydrophobic amino acid such as Trp, Phe and Leu, and is used for peptide protease stability studies. 34 Contryphan-Vn and peptidomimetics 14 were subjected to the a-chymotrypsin mediated degradation test and monitored by HPLC. It was found that aer 32 hours' protease exposure, both of them were degraded completely. No signicant differences of protease stability were observed between peptidomimetics 14 and native model peptides (Fig. 3B). These results indicated that the introducing of triazole replacement could signicantly improve the reduction stability of peptide rather than the protease stability.

Conclusions
In summary, to our knowledge, this is the rst introduction of arylboronate ester protective groups to diaminodiacid building block construction, and the triazole bridge-based peptidomimetic of contryphan-Vn was also developed and studied for the rst time. More importantly, the low environmental cost, facile handling property, and the resulting high purity and good yield of the nal product suggest that this novel building block is a better key intermediate for the diaminodiacid strategy, so it may promote further development of disulde bond mimetic studies in the future.

General information
All reagents were purchased from Acros, Sigma-Aldrich, Alfa Aesar and Adamas. Amino acids were commercial available   from GL Biochem Shanghai Co. Ltd. All solvents used were bought from Sinopharm Chemical Reagent Co. Ltd. Dichloromethane (DCM) and N,N-dimethylformamide (DMF) were distilled over calcium hydride (CaH 2 ) under argon atmosphere and stored in ask containing 4Å molecular sieves. All reactions vessels were oven-dried before use. Reactions were monitored by thin-layer chromatography (TLC) and visualized by UV analyzer (254 nm), ninhydrin and/or phosphomolybdic acid. Peptides were analyzed and puried by reverse phase HPLC. A C18 analytic column (Shimazu Shim-pack VP-ODS, 4.6 Â 250 mm, 5 mm particle size, ow rate 1 mL min À1 ) was used for analytical RP-HPLC, and a C18 column (Shimazu Shim-pack PRC-ODS, 50 Â 250 mm, 15 mm particle size, ow rate 10 mL min À1 ) was used for preparative RP-HPLC. The solvents systems were buffer A (0.1% TFA in CH 3 CN) and buffer B (0.1% TFA in water). Data were recorded and analyzed using the soware system LC Solution. High resolution mass spectra (HR-MS) were measured on a Waters Xevo G2 QTOF mass spectrometer. 1 H-and 13 C-NMR spectrum was recorded on a Bruker Avance 300 MHz instrument. Chemical shis (d) were reported relative to TMS (0 ppm) for 1 H-and 13 C-NMR spectra. The coupling constants (J) were displayed in hertz (Hz) and the splitting patterns were dened as follows: singlet (s); broad singlet (s, br); doublet (d); doublet of doublet (dd); triplet (t); quartet (q); multiplet (m).

General procedures for the Fmoc solid phase peptide synthesis
The amino acid residues were attached to the resin with a single coupling procedure. All peptides were synthesized with a scale of 0.10 mmol.
(a) Standard pre-activation of resin protocol: the resin was swollen in DCM/DMF mixture solvent for 10 minutes.
(c) Standard coupling of natural amino acids protocol: aer pre-activation of 4.0 equivalents of Fmoc-protected amino acid in DMF for 5 minutes using 3.8 equivalents of HCTU and 8.0 equivalents of DIPEA, the solution was added to the resin. Aer 30 minutes, the resin was washed with DMF (Â5), DCM (Â5), and DMF (Â5). The coupling reaction was monitored with the ninhydrin test.
(d) Standard coupling of diaminodiacids protocol: aer preactivation of 1.5 equivalents of Fmoc-protected diaminodiacid in DMF for 1 minute using 2.0 equivalents of HATU and 8.0 equivalents of DIPEA, the solution was added to the resin. Aer 2 hours, the resin was washed with DMF (Â5), DCM (Â5), and DMF (Â5). The coupling reaction was monitored with the ninhydrin test. (g) Standard cleavage protocol: the cleavage cocktail (TFA : EDT : TIPS : water ¼ 95 : 2 : 2 : 1, v/v/v/v) was added to the resin. Aer stirring for 2 hours, the cleavage cocktail was collected. The solution was bubbled with argon for concentration and the chilled diethyl ether was added to precipitate the crude peptides. The peptide suspensions were centrifuged for 3 minutes at 3000 rpm and then the clear solution was decanted. The step of precipitation, centrifugation and decantation operations was repeated three times. The resulting white residues were dissolved in CH 3 CN/water, analyzed and puried by RP-HPLC.
(h) Standard oxidative folding protocol: peptide in the reduced form was dissolved in the oxidation buffer (0.5 mg mL À1 peptide in 6.0 M guanidine hydrochloride and 100.0 mM sodium dihydrogen phosphate PBS buffer, pH ¼ 7.4, with 10.0% DMSO). This mixture was allowed to stir for 24 h at room temperature. Then it was analyzed and puried by RP-HPLC.