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. 2024 Jan 18;35(2):245–253. doi: 10.1021/acs.bioconjchem.3c00541

N-tert-Butoxycarbonyl-N-(2-(tritylthio)ethoxy)glycine as a Building Block for Peptide Ubiquitination

Lingling Peng †, Elizabeth Helgason ‡, Rafael Miranda ‡, Jeffrey Tom †, Jennifer Zhang §, Erin C Dueber ‡, Aimin Song †,*
PMCID: PMC10885006  PMID: 38236171

Abstract

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N-Boc-N-(2-(tritylthio)ethoxy)glycine has been developed as a building block for peptide ubiquitination, which is fully compatible with solid-phase Fmoc chemistry and common peptide modifications including phosphorylation, methylation, acetylation, biotinylation, and fluorescence labeling. The optimal conditions for peptide cleavage and auxiliary removal were obtained. The utility of this building block in peptide ubiquitination was demonstrated by the synthesis of seven ubiquitinated histone and Tau peptides bearing various modifications. Cys residues were well tolerated and did not require orthogonal protection. The structural integrity and folding of the synthesized ubiquitinated peptides were confirmed by enzymatic deubiquitination of a fluorescently labeled ubiquitin conjugate. The synthetic strategy using this building block provides a practical approach for the preparation of ubiquitinated peptides with diverse modifications.

Introduction

Ubiquitin (Ub) is a small protein consisting of 76 amino acids, that is highly conserved throughout eukaryotes. The post-translational ligation of Ub via its C-terminus to the side chain of lysine (Lys) residues in a target protein is catalyzed by a cascade of enzymes including E1 activating enzyme, E2 conjugating enzyme, and E3 ubiquitin ligase and is reversed by deubiquitinases (DUBs).1,2 Ub can also be conjugated to itself to form polyubiquitin chains. Ubiquitination is a crucial post-translational modification (PTM) of eukaryotic proteins, which plays profound roles in a myriad of biological processes.1−3 The complexity of ubiquitination and deubiquitination processes is reflected by the fact that the human genome encodes 2 E1s, approximately 40 E2s, over 600 E3s, and nearly 100 DUBs.4−6 The preparation of Ub conjugates with a high homogeneity and sufficient quantities is of substantial interest for studying ubiquitination.

Nonenzymatic methodologies for the preparation of Ub conjugates have attracted increasing attention over the past decades. Chemical synthesis approaches offer attractive solutions to generate precise and homogeneous Ub conjugates, enabling facile and site-specific incorporation of noncanonical amino acids, probes, functional groups, and nonpeptide chemical bonds for various applications.7−15 The first synthesis of ubiquitinated peptides was achieved by Chatterjee et al. in 2007 via expressed protein ligation (EPL) using a photocleavable ligation auxiliary.16 Since then, several synthetic approaches for ubiquitinated peptides have been developed based on native chemical ligation (NCL)17 using a glycyl auxiliary18−22 or a mercaptolysine building block.23−27 In an alternative approach, chemical ubiquitination was accomplished by native chemical ligation of two Ub segments, one of which was preconstructed on the side chain of the designated Lys residue in the acceptor peptide during solid-phase peptide synthesis (SPPS).28 Furthermore, a chemical strategy for the synthesis of atypical Ub chains has been developed using an isopeptide-linked Ub isomer (isoUb) synthon.29

In 2014, Weller et al. reported a semisynthetic strategy for chemical ubiquitination and SUMOylation using a 2-(aminooxy)ethanethiol auxiliary (Scheme 1).20 In this strategy, the Lys residue for ubiquitination in the acceptor peptide was orthogonally protected during SPPS. Upon completion of the peptide assembly, the ε-amino group of the designated Lys residue was bromoacetylated, followed by the nucleophilic substitution of bromine with O-(2-(tritylthio)ethyl)hydroxylamine (1). After cleavage and purification, the resulting peptide bearing an N-(2-mercaptoethoxy)glycyl group was ligated with the recombinant Ub(1–75)-thioester. Removal of the 2-(aminooxy)ethanethiol auxiliary with zinc (Zn)20 or 4-mercaptophenylacetic acid (MPAA)21 afforded the desired ubiquitinated peptide. This strategy was first validated using model peptides20 and was then successfully applied to the preparation of full-length SUMOylated histones H2B and H4.21 The 2-(aminooxy)ethanethiol auxiliary strategy features compatibility with native cysteine (Cys) residues and mild conditions for auxiliary removal, providing a promising tool for preparing ubiquitinated and SUMOylated peptides.

Scheme 1. Peptide Ubiquitination Using a 2-(Aminooxy)ethanethiol Auxiliary20,21.

Scheme 1

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Adapted with permission from ref (20) Copyright 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

In an effort to develop reagents for detecting DUB activities, we applied the 2-(aminooxy)ethanethiol auxiliary strategy to the ubiquitination of biotinylated or fluorescently labeled peptides bearing PTMs and encountered a few difficulties. First, the efficiency of auxiliary installation varied greatly, depending on the peptide sequence, and low yields were obtained with long peptides. Second, a partial loss of phosphate groups from phosphopeptides was observed during the auxiliary installation due to the strongly nucleophilic conditions. Third, the auxiliary installation has to be the last step of the peptide synthesis because the formed N-(2-tritylthioethoxy)glycyl group is still reactive to acylation, which interferes with further modifications such as fluorescent labeling. We envisioned that these difficulties could be overcome by presynthesizing the glycyl auxiliary with appropriate protecting groups such as tert-butoxycarbonyl (Boc) and trityl (Trt) for SPPS. Herein, we report the development of N-Boc-N-(2-(tritylthio)ethoxy)glycine as a building block for peptide ubiquitination, which is fully compatible with solid-phase 9-fluorenylmethyloxycarbonyl (Fmoc) chemistry30 and common peptide modifications.

Results and Discussion

The synthesis of N-Boc-N-(2-(tritylthio)ethoxy)glycine (4) is illustrated in Scheme 2. O-(2-(tritylthio)ethyl)hydroxylamine (1) was synthesized using the literature method.20,31 The aminooxy group was then protected with Boc, followed by alkylation with methyl bromoacetate in the presence of sodium hydride. Saponification of the methyl ester with trimethyltin hydroxide32 afforded N-Boc-N-(2-(tritylthio)ethoxy)glycine (4) in an overall yield of 19% after three steps of reactions. The Boc and Trt protecting groups in building block 4 are stable to Fmoc SPPS and are readily removable by treatment with trifluoroacetic acid (TFA).

Scheme 2. Synthesis of N-Boc-N-(2-(tritylthio)ethoxy)glycine.

Scheme 2

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We used histone H2A(113–129) with a sequence of Ac-AVLLPKKTESHHKAKGK as a model peptide (Table 1, entry 1) to study the application of N-Boc-N-(2-(tritylthio)ethoxy)glycine (4) in peptide ubiquitination (Scheme 3A). Lysine 119 (K119) in H2A is known to undergo monoubiquitination in response to DNA damage.33 During the solid-phase synthesis of H2A(113–129), the ε-amino group of K119 was protected with 4-methyltrityl (Mtt). Upon completion of the peptide assembly, the N-terminus of the peptide was acetylated, followed by the removal of the Mtt-protecting group with a mixture of TFA/triisopropylsilane (TIS)/dichloromethane (DCM) (v/v/v 1:5:94). Compound 4 was readily coupled to the side chain of K119 using 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) and N,N-diisopropylethylamine (DIEA) as the activating system. The auxiliary-bearing peptide, H2A(113–129)-Aux, was then released from the resin by TFA treatment for analysis and purification by reversed-phase (RP) high-performance liquid chromatography (HPLC). Several cleavage cocktails were tested, and the optimal results were obtained with a mixture of TFA/DCM/water/TIS/phenol/3,6-dioxa-1,8-octanedithiol (DODT) (v/v/v/v/w/v 70:12.5:5:5:5:2.5).

Table 1. Design and Synthesis of Ubiquitinated Peptides Using N-Boc-N-(2-(tritylthio)ethoxy)glycine (Scheme 3).

entry peptide sequence modifications route
1 H2A(113–129)-K119Ub Ac-AVLLPK-K(Ub)-TESHHKAKGK N-Ac, K119Ub A
2 H2A(113–129)-K119Ub-pT120 Ac-AVLLPK-K(Ub)-(pT)-ESHHKAKGK N-Ac, K119Ub, pT120 A
3 FAM-H2A(113–129)-K119Ub (5-FAM)-AVLLPK-K(Ub)-TESHHKAKGK N-(5-FAM), K119Ub B
4 FAM-H2B(117–127)-K120Ub (5-FAM)-AVT-K(Ub)-YTSSNPR N-(5-FAM), K120Ub B
5 H3(1–21)-K4Me-K14Ub ART-K(Me)-QTARKSTGG-K(Ub)-APRKQLA K4Me, K14Ub B
6 biotin-H4(52–67)-K59Ub biotin-PEG4-EETRGVL-K(Ub)-VFLENVIR N-biotin-PEG4, K59Ub A
7 Tau(309–326)-K311Ac–K317Ub VY-K(Ac)-PVDLS-K(Ub)-VTSKCGSLG K311Ac, K317Ub B
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Scheme 3. Peptide Ubiquitination Using N-Boc-N-(2-(tritylthio)ethoxy)glycine.

Scheme 3

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Figure 1A,B shows the liquid chromatography (LC)–mass spectrometry (MS) analysis results of crude H2A(113–129)-Aux that was synthesized using the literature method (Scheme 1) and the N-Boc-N-(2-(tritylthio)ethoxy)glycine building block (4) (Scheme 3A), respectively. The desired peptide eluted at 2.37 min with an m/z of 1024.64 for [M+2H]2+. The literature method yielded only a small amount of H2A(113–129)-Aux with a crude purity of 17%. The major byproduct with a retention time of 2.12 min had a molecular mass of 1954.26 Da, which is 80 Da lower than that of the bromoacetylated H2A(113–129) precursor. Additionally, the isotopic pattern of bromine was not observed in the MS signals, indicating dehydrobromination during the auxiliary installation step. In contrast, H2A(113–129)-Aux was obtained as the major product with a crude purity of 51% using the N-Boc-N-(2-(tritylthio)ethoxy)glycine building block (4). The side reaction was completely suppressed.

Figure 1.

Figure 1

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LC–MS analysis results (UV detection at 220 nm) of crude H2A(113–129)-Aux (Ac-AVLLPK-K((HSEtO)G)-TESHHKAKGK) synthesized using (A) literature method and (B) N-Boc-N-(2-(tritylthio)ethoxy)glycine.

The purified H2A(113–129)-Aux was ligated with Ub(1–75)-hydrazide,34 which was synthesized using a published procedure.27 The in situ activation of Ub(1–75)-hydrazide with sodium nitrite (NaNO2) and MPAA, followed by ligation with H2A(113–129)-Aux proceeded smoothly to afford the ubiquitinated H2A(113–129)-Aux (H2A(113–129)-Aux-Ub) using a published method.35 A small amount of the heterodimer of H2A(113–129)-Aux-Ub and MPAA via a disulfide bond was observed as a byproduct, which was readily reduced with tris(2-carboxyethyl)phosphine (TCEP) to regenerate H2A(113–129)-Aux-Ub. The reaction solution was desalted by dialysis against water, followed by lyophilization. The obtained crude H2A(113–129)-Aux-Ub was used for subsequent auxiliary removal without further purification.

The attempt to remove the 2-mercaptoethoxy group in H2A(113–129)-Aux-Ub with MPAA was unsuccessful due to the formation of the heterodimer of H2A(113–129)-Aux-Ub and MPAA. The reductive cleavage of the N-O bond using Zn resulted in the desired product, K119-ubiquitinated H2A(113–129) (H2A(113–129)-K119Ub), along with a significant amount (∼65% by MS signals) of Ub (1–75) as the major byproduct from the hydrolysis of the alkoxyamide bond.20 Ub(1–75) coeluted with H2A(113–129)-K119Ub during HPLC analysis and purification, resulting in difficult separation (Figure 2A). After an extensive optimization of the reaction conditions, it was found that the addition of 0.1 M zinc chloride (ZnCl2) dramatically improved the efficiency (∼74% by MS signals) of auxiliary removal (Figure 2B). A possible explanation is that the chelation of the histidine (His) residues in H2A(113–129) by Zn2+ prevented their catalytic effect on the hydrolysis of the alkoxyamide bond. The resulting H2A(113–129)-K119Ub was purified with RP-HPLC (overall yield, 20%), characterized with LC–MS (Figure 2C) and high-resolution MS (HRMS) (Figure 2D), and refolded by dialysis against a refolding buffer (50 mM tris(hydroxymethyl)aminomethane (Tris), 150 mM sodium chloride (NaCl), pH 7.5). The secondary structure of H2A(113–129)-K119Ub was confirmed by circular dichroism (CD) spectroscopy (Figure 2E).

Figure 2.

Figure 2

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Characterization of H2A(113–129)-K119Ub (Ac-AVLLPK-K(Ub)-TESHHKAKGK). (A–C) Total ion chromatogram (TIC) of the crude product after auxiliary removal using Zn without (A) and with the addition of 0.1 M ZnCl2 (B), and the purified product (C); deconvoluted spectra are shown as insets. (D) HRMS spectrum of the [M + 14H]14+ signal. Calculated for C464H775N131O140S, 747.7735; found, 747.7732. (E) CD spectrum of the refolded Ub conjugate.

The N-Boc-N-(2-(tritylthio)ethoxy)glycine building block (4) is fully compatible with solid-phase Fmoc chemistry and can be incorporated in any step of peptide synthesis. During the synthesis of a fluorescently labeled K119 Ub conjugate of H2A(113–129) (FAM-H2A(113–129)-K119Ub, Table 1, entry 3), the N-terminal Fmoc was left on after the completion of the peptide assembly, while the Mtt-protecting group on the ε-amino group of K119 was removed (Scheme 3B). Building block 4 was coupled to the side chain of K119, followed by the removal of the N-terminal Fmoc with 20% 4-methylpiperidine in N,N-dimethylformamide (DMF). 5-Carboxyfluorescein (5-FAM) was then attached to the N-terminus of the peptide using N,N′-diisopropylcarbodiimide (DIC)/1-hydroxybenzotriazole (HOBt) as the activating system. After TFA cleavage and RP-HPLC purification, the obtained auxiliary-bearing N-(5-FAM)-H2A(113–129) (FAM-H2A(113–129)-Aux) was ligated with Ub(1–75)-hydrazide, followed by auxiliary removal with Zn/ZnCl2 to afford FAM-H2A(113–129)-K119Ub.

Based on the results, we designed and synthesized seven ubiquitinated histone36,37 and Tau38,39 peptides containing different modifications, including phosphorylation, methylation, acetylation, biotinylation, and fluorescent labeling (Table 1). All of the seven ubiquitinated peptides were successfully obtained, demonstrating the compatibility of the N-Boc-N-(2-(tritylthio)ethoxy)glycine building block with common peptide modifications. The presence of an unprotected Cys residue in Tau(309–326)-K311Ac–K317Ub (Table 1, entry 7) was well tolerated and did not require orthogonal protection.

Fluorescently labeled Ub conjugates have a broad range of applications in understanding ubiquitin signal transduction.10 The N-Boc-N-(2-(tritylthio)ethoxy)glycine building block is ideally suited for the chemical synthesis of fluorescently labeled ubiquitinated peptides. Using the established procedures, we prepared two 5-FAM-labeled ubiquitinated histone peptides, FAM-H2A(113–129)-K119Ub (Table 1, entry 3) and FAM-H2B(117–127)-K120Ub (Table 1, entry 4). The reactivity of the latter toward a DUB, namely ubiquitin C-terminal hydrolase L3 (UCH-L3), was examined by LC–MS, sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), and fluorescence polarization (FP) (Figure 3). LC–MS analysis showed full consumption of FAM-H2B(117–127)-K120Ub after incubation with UCH-L3 (0.5 μM) at room temperature for 30 min along with the formation of free Ub (Figure 3A). SDS-PAGE (Figure 3B) and FP (Figure 3C) analyses revealed that FAM-H2B(117–127)-K120Ub was hydrolyzed by UCH-L3 in a dose- and time-dependent manner. The results not only confirmed the structural integrity and folding of FAM-H2B(117–127)-K120Ub but also demonstrated its application as an FP reagent in detecting DUB activity.40−42 Fluorescein is one of the most widely used fluorophores in biological research. It was reported that fluorescein is incompatible with the desulfurization step in the chemical ubiquitination methods involving desulfurization.43 Our strategy provides a facile approach for the synthesis of fluorescein-labeled ubiquitinated peptides.

Figure 3.

Figure 3

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Enzymatic deubiquitination of FAM-H2B(117–127)-K120Ub by UCH-L3. (A) TIC of FAM-H2B(117–127)-K120Ub (2.5 μM) before (left) and after (right) incubation with UCH-L3 (0.5 μM) at room temperature for 30 min. (B) SDS-PAGE analysis results of UCH-L3-catalyzed deubiquitination of FAM-H2B(117–127)-K120Ub (2.5 μM) by fluorescence scanning (left) and Coomassie staining (right). Untreated FAM-H2B(117–127)-K120Ub (1 μM) was used as the control sample. (C) FP assay results of UCH-L3-catalyzed deubiquitination of FAM-H2B(117–127)-K120Ub (0.5 μM).

Conclusions

To summarize, we have developed N-Boc-N-(2-(tritylthio)ethoxy)glycine as a building block for peptide ubiquitination, which extends the applicability of the 2-(aminooxy)ethanethiol auxiliary strategy.20,21 The optimal conditions for peptide cleavage and auxiliary removal have been established. The building block is fully compatible with Fmoc SPPS and a variety of peptide modifications including phosphorylation, methylation, acetylation, biotinylation, and fluorescent labeling. Cys residues are well tolerated and do not require orthogonal protection. The usefulness of N-Boc-N-(2-(tritylthio)ethoxy)glycine in peptide ubiquitination has been demonstrated by the synthesis of ubiquitinated histone and Tau peptides bearing PTMs and fluorescent labeling. This building block is ideally suited for the preparation of ubiquitinated peptides with diverse modifications.

Experimental Procedures

Synthesis of tert-Butyl 2-(Tritylthio)ethoxycarbamate (2)

O-(2-(Tritylthio)ethyl)hydroxylamine (1) was prepared using a published procedure.20,31 Di-tert-butyl dicarbonate (Boc2O, 9.76 g, 44.7 mmol, 1.50 equiv) and sodium bicarbonate (NaHCO3, 5.01 g, 59.6 mmol) were added successively to a stirred solution of O-(2-(tritylthio)ethyl)hydroxylamine (1, 10.0 g, 29.8 mmol, 1.00 equiv) in tetrahydrofuran (THF, 25.0 mL) and water (25.0 mL) at 0 °C. The resulting mixture was stirred at room temperature for 8 h and then diluted with water (300 mL) and ethyl acetate (EtOAc, 500 mL). The organic phase was separated, and the aqueous phase was extracted with EtOAc (100 mL × 2). The combined organic phase was washed with brine (100 mL) and water (100 mL), dried over anhydrous sodium sulfate (Na2SO4), and concentrated under reduced pressure to dryness. The residue was purified by column chromatography (silica gel, 100–200 mesh, 0–30% EtOAc in petroleum ether) to afford tert-butyl 2-(tritylthio)ethoxycarbamate (2) as a white solid (6.00 g, yield 46.2%).

1H NMR (400 MHz, CDCl3) δ (ppm) 7.36–7.34 (m, 5H), 7.24–7.19 (m, 7H), 7.16–7.12 (m, 3H), 3.56 (t, J = 16 Hz, 2H), 2.42 (t, J = 16 Hz, 2H), 1.37 (s, 9H). 13C NMR (101 MHz, CDCl3) δ (ppm): 144.61, 129.56, 127.90, 126.69, 81.76, 74.51, 66.74, 30.05, 28.15. HRMS (ESI-TOF) m/z: [M + Na]+ calcd for C26H29NO3S 458.1760, found 458.1774.

Synthesis of Methyl N-(tert-Butoxycarbonyl)-N-(2-(tritylthio)ethoxy)glycinate (3)

A solution of 2 (1.50 g, 3.44 mmol, 1.00 equiv) in anhydrous DMF (10.0 mL) was added dropwise to a stirred suspension of sodium hydride (NaH, 248 mg, 10.33 mmol, 3.00 equiv) in anhydrous DMF (10.0 mL) at room temperature over 10 min under a nitrogen atmosphere. The resulting mixture was stirred at room temperature for 10 min and then chilled in an ice bath, followed by dropwise addition of methyl 2-bromoacetate (1.30 mL, 13.8 mmol). The reaction was stirred at room temperature for 8 h and was then quenched by the addition of saturated ammonium chloride (NH4Cl) solution (100 mL) and EtOAc (100 mL). The organic phase was separated, and the aqueous phase was extracted with EtOAc (50 mL × 2). The combined organic phase was washed with water (50 mL) and brine (50 mL), dried over anhydrous Na2SO4, and then concentrated under reduced pressure to dryness. The residue was purified by column chromatography (silica gel, 100–200 mesh, 0–25% EtOAc in petroleum ether) to afford N-(tert-butoxycarbonyl)-N-(2-(tritylthio)ethoxy)glycinate (3) as a yellowish solid (1.60 g, yield 91.5%).

1H NMR (400 MHz, methanol-d4) δ (ppm) 7.40 (d, J = 8.0 Hz, 6H), 7.31–7.27 (m, 6H), 7.24–7.21 (m, 3H), 4.07 (s, 2H), 3.70 (s, 3H), 3.55 (t, J = 16 Hz, 2H), 2.46 (t, J = 12 Hz, 2H). 13C NMR (101 MHz, methanol-d4) δ (ppm): 169.62, 145.25, 129.93, 128.12, 127.03, 82.68, 73.42, 51.77, 30.20, 27.52. HRMS (ESI-TOF) m/z: [M + Na]+ calcd for C29H33NO5S 530.1972, found 530.1982.

Synthesis of N-(tert-Butoxycarbonyl)-N-(2-(tritylthio)ethoxy)glycine (4)

Trimethyltin hydroxide (25.7 g, 141.8 mmol, 20.0 equiv) was added to a stirred solution of 3 (4.0 g, 7.88 mmol) in 1,2-dichloroethane (40 mL). The reaction was refluxed for 48 h and was then cooled to room temperature, followed by filtration. The filtrate was concentrated under reduced pressure to dryness. The residue was purified by RP-HPLC (C18 column, 30 mm × 150 mm, 5 μm, 100 Å, 40 mL/min, 25 min gradient from 50 to 80% aqueous acetonitrile containing 0.225% formic acid (FA)) to afford N-(tert-butoxycarbonyl)-N-(2-(tritylthio)ethoxy)glycine (4) as a white solid (1.90 g, yield 44.5%).

1H NMR (400 MHz, methanol-d4) δ (ppm) 7.41–7.38 (m, 6H), 7.30–7.26 (m, 6H), 7.23–7.19 (m, 3H), 3.99 (s, 2H), 3.54 (t, J = 12 Hz, 2H), 2.44 (t, J = 24 Hz, 2H), 1.43–1.42 (m, 9H). 13C NMR (101 MHz, methanol-d4) δ (ppm): 157.32, 144.72, 127.58, 129.39, 126.47, 81.61, 72.58, 67.47, 66.59, 29.70, 27.08, 25.11, 17.95. HRMS (ESI-TOF) m/z: [M–H]− calcd for C28H31NO5S 492.1850, found 492.1860.

Peptide Synthesis

Ub(1–75)-hydrazide was synthesized on hydrazine 2-chlorotrityl resin44 using a published procedure.27 The acceptor peptides were synthesized using microwave-assisted Fmoc SPPS.45 Commercially available preloaded Wang resin (loading 0.5–0.7 mmol/g) was used as the solid support, and Fmoc-Lys(Mtt)–OH was used as the building block for the Lys residue at the ubiquitination site. In each amino acid coupling cycle, the resin (200 mg) was first agitated with a solution of 4-methylpiperidine in DMF (v/v 20%, 4 mL) under microwave irradiation at 90 °C for 2 min twice for Fmoc removal. The supernatant was drained, and the resin was washed thoroughly with DMF (4 mL × 5). A solution of an Fmoc-protected amino acid in DMF (0.2 M, 5 mL) was then added to the resin, followed by the addition of ethyl (2E)-2-cyano-2-hydroxyiminoacetate solution (1 M, 1 mL) and DIC solution (1 M, 2 mL) in DMF. The resulting mixture was purged with nitrogen under microwave irradiation at 90 °C for 2 min, with the exceptions of Arg (75 °C, 2 min), Cys (50 °C, 4 min), and His (50 °C, 4 min). The supernatant was drained, and the resin was then washed thoroughly with DMF (6 mL × 5). The coupling cycle was repeated until the peptide assembly was complete. The resin-bound peptide was then subjected to Mtt removal and incorporation of the N-Boc-N-(2-(tritylthio)ethoxy)glycine building block (4) via either route A or B (Scheme 3).

In route A (Table 1, entries 1, 2, and 6), the N-terminal Fmoc was removed using the procedure described above. The N-terminus was capped with an acetyl group (Ac) or biotin-PEG4 by mixing the resin with a solution of acetic anhydride (113.2 μL, 1.20 mmol) and DIEA (62.7 μL, 0.36 mmol) in DCM (4 mL) or a solution of biotin-PEG4-acid (177 mg, 0.36 mmol), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU, 137 mg, 0.36 mmol), and DIEA (125.4 μL, 0.72 mmol) in a mixture of dimethyl sulfoxide (DMSO)/DMF/DCM (v/v/v 2:1:1, 4 mL), respectively, at room temperature for 2 h. The supernatant was drained, and the resin was washed thoroughly with DMF (4 mL × 3) and DCM (4 mL × 4). For Mtt removal, the resin was agitated with a mixture of TFA/TIS/DCM (v/v/v 1:5:94, 4 mL) at room temperature for 10 × 2 min, followed by thoroughly washing with DCM (4 mL × 3), 0.5% DIEA in DCM (4 mL × 2), and DMF (4 mL × 4). A solution of compound 4 (89 mg, 0.18 mmol), HATU (68 mg, 0.18 mmol), and DIEA (62.7 μL, 0.36 mmol) in DMF (3 mL) was then added to the resin. The resulting mixture was agitated at room temperature for 3 h. The supernatant was drained, and the resin was washed thoroughly with DMF (4 mL × 3) and DCM (4 mL × 4).

In route B (Table 1, entries 3–5 and 7), the Mtt-protecting group was removed, and building block 4 was coupled to the designated Lys residue, followed by the removal of the N-terminal Fmoc using the procedures described above. For the synthesis of 5-FAM-labeled peptides (Table 1, entries 3 and 4), the resin was agitated with a solution of 5-FAM (135 mg, 0.36 mmol), HOBt (49 mg, 0.36 mmol), and DIC (56.4 μL, 0.36 mmol) in DMF (3 mL) at room temperature for 2 h. The supernatant was drained, and the resin was washed with DMF (4 mL × 3). The resin was then mixed with a solution of 4-methylpiperidine in DMF (v/v 20%, 4 mL) at room temperature for 10 min twice. The supernatant was drained, and the resin was washed thoroughly with DMF (4 mL × 3) and DCM (4 mL × 4).

For peptide cleavage, a chilled mixture of TFA/DCM/water/TIS/phenol/DODT (v/v/v/w/v 70:12.5:5:5:5:2.5, 10 mL) was added to the dried peptide-bound resin. The resulting mixture was agitated at room temperature for 3 h. The supernatant was collected by filtration, and the resin was washed with the cleavage mixture (2 mL × 3). The combined filtrate was concentrated under reduced pressure to approximately one-fourth of the original volume, which was diluted with cold anhydrous diethyl ether (40 mL). The resulting mixture was chilled in an ice bath for 30 min and then centrifuged. The precipitate was washed with anhydrous diethyl ether (40 mL × 3). The obtained crude peptides were analyzed by LC–MS (C18 column, 4.6 × 50 mm, 5 μm, 100 Å, 1.0 mL/min, 2 min gradient from 5 to 95% aqueous acetonitrile containing 0.1% TFA, 220 nm) and purified by RP-HPLC (C18 column, 21.2 × 100 mm, 5 μm, 100 Å, 20 mL/min, 40 min gradient from 5 to 40% aqueous acetonitrile containing 0.05% TFA).

H2A(113–129)-Aux, HRMS (ESI-Orbitrap) m/z: [M+4H]4+ calcd for C90H155N27O25S 512.5425, found 512.5424.

H2A(113–129)-pT120-Aux, HRMS (ESI-Orbitrap) m/z: [M+4H]4+ calcd for C90H156N27O28PS 532.5341, found 532.5350.

FAM-H2A(113–129)-Aux, HRMS (ESI-Orbitrap) m/z: [M+5H]5+ calcd for C109H163N27O30S 473.4429, found 473.4443.

FAM-H2B(117–127)-Aux, HRMS (ESI-Orbitrap) m/z: [M+3H]3+ calcd for C77H103N17O26S 572.2400, found 572.2408.

H3(1–21)-K4Me-Aux, HRMS (ESI-Orbitrap) m/z: [M+6H]6+ calcd for C99H181N37O30S 401.0655, found 401.0656.

biotin-H4(52–67)-Aux, HRMS (ESI-Orbitrap) m/z: [M+3H]3+ calcd for C110H186N28O34S2 836.7782, found 836.7795.

Tau(309–326)-K311Ac-Aux, HRMS (ESI-Orbitrap) m/z: [M+3H]3+ calcd for C89H150N22O29S2 686.0200, found 686.0212.

Ligation of Ub(1–75) to Auxiliary-bearing Peptides35

A solution of Ub(1–75)-hydrazide (1 mM, 1 mL) in 0.2 M phosphate buffer containing 6.0 M guanidinium chloride (Gn·HCl) (pH 3.0) was stirred in an ice-salt bath at −10 °C, and an aqueous solution of NaNO2 (200 mM, 100 μL) was added. The resulting mixture was stirred at −10 °C for 20 min, followed by the addition of an aqueous solution of MPAA (200 mM, 0.9 mL). The pH of the resulting solution was adjusted to approximately 7 with a 2 M sodium hydroxide (NaOH) solution. The auxiliary-bearing peptide (1.5–5 μmol) was then added. The resulting mixture was stirred at room temperature under an inert gas atmosphere until the LC–MS analysis (PLRP-S column, 2.1 × 50 mm, 8 μm, 1000 Å, 0.8 mL/min, 8 or 15 min gradient from 15 to 60% aqueous acetonitrile containing 0.1% TFA) indicated a complete reaction (8–24 h). The solution was dialyzed (molecular weight cutoff (MWCO) 3,500 Da) against 100 mM Tris buffer containing 150 mM NaCl and 1 mM TCEP (pH 7.0), 1 mM aqueous solution of TCEP, and then water, followed by lyophilization. The crude products were used for subsequent auxiliary removal without further purification.

Auxiliary Removal

The crude ligation product was dissolved in a degassed 6 M Gn·HCl solution containing 0.1 M ZnCl2 (pH 3.0) at a concentration of 1 mg/mL, and freshly activated Zn powder46 (200 mg per 1 mg protein) was added. The reaction was incubated at 37 °C under an inert gas atmosphere until the LC–MS analysis (PLRP-S column, 2.1 × 50 mm, 8 μm, 1000 Å, 0.8 mL/min, 8- or 15 min gradient from 15 to 60% aqueous acetonitrile containing 0.1% TFA) indicated complete reaction (24–48 h), followed by centrifugation. The supernatant was collected, and the precipitated Zn was washed 5 times with 6 M Gn·HCl solution (pH 3.0, one-third of the reaction volume). The combined supernatant was dialyzed against 100 mM Tris buffer containing 150 mM NaCl, 5 mM ethylenediaminetetraacetic acid (EDTA) and 1 mM TCEP (pH 7.0), and then water, followed by lyophilization. The obtained crude ubiquitinated peptides were purified by RP-HPLC (C18 column, 21.2 × 100 mm, 5 μm, 100 Å, 20 mL/min, 30 min gradient from 5 to 40% aqueous acetonitrile containing 0.05% TFA).

H2A(113–129)-K119Ub (2.1 mg, yield 20%), HRMS (ESI-Orbitrap) m/z: [M+14H]14+ calcd for C464H775N131O140S 747.7735, found 747.7732.

H2A(113–129)-K119Ub-pT120 (1.7 mg, yield 16%), HRMS (ESI-Orbitrap) m/z: [M+15H]15+ calcd for C464H776N131O143PS 703.3202, found 703.3200.

FAM-H2A(113–129)-K119Ub, (1.3 mg, yield 12%), HRMS (ESI-Orbitrap) m/z: [M+14H]14+ calcd for C483H783N131O145S 770.3476, found 770.3483.

FAM-H2B(117–127)-K120Ub (1.0 mg, yield 10%), HRMS (ESI-Orbitrap) m/z: [M+12H]12+ calcd for C451H723N121O141S 844.5310, found 844.5345.

H3(1–21)-K4Me-K14Ub (1.4 mg, yield 13%), HRMS (ESI-Orbitrap) m/z: [M+14H]14+ calcd for C473H801N141O145S 773.0741, found 773.0728.

biotin-H4(52–67)-K59Ub (0.5 mg, yield 5%), HRMS (ESI-Orbitrap) m/z: [M+12H]12+ calcd for C484H806N132O149S2 910.6655, found 910.6643.

Tau(309–326)-K311Ac–K317Ub (1.9 mg, yield 18%), HRMS (ESI-Orbitrap) m/z: [M+12H]12+ calcd for C463H770N126O144S2 872.9760, found 872.9752.

Refolding of Ubiquitinated Peptides

A solution of the ubiquitinated peptide (1 mg/mL) in 100 mM phosphate buffer containing 6 M Gn·HCl (pH 7.5) was dialyzed (MWCO 3500 Da) against 100 mM Tris buffer containing 150 mM NaCl (pH 7.5) and then 50 mM Tris buffer containing 150 mM NaCl (pH 7.5).

CD Analysis

CD measurements were performed using a cuvette with a 1 mm cell path length at room temperature. The ubiquitinated peptide was dissolved in 20 mM potassium phosphate buffer (pH 7.4) at a 100 μM concentration. Three cumulative measurements were made, and the average was calculated and plotted.

Deubiquitination of FAM-H2B(117–127)-K120Ub by UCH-L3

In the LC–MS study, a solution of UCH-L3 (1 μM, 100 μL) in 50 mM Tris buffer containing 150 mM NaCl and 10 mM dithiothreitol (DTT) (pH 7.5) was preactivated at room temperature for 20 min and was then added to a solution of FAM-H2B(117–127)-Ub (5 μM, 100 μL) in 50 mM Tris buffer containing 150 mM NaCl (pH 7.5). The resulting mixture was incubated at room temperature for 30 min. A 50 μL sample was taken out, quenched with the addition of 10% FA (5 μL), and analyzed by LC–MS (PLRP-S column, 2.1 mm × 50 mm, 8 μm, 1000 Å, 0.8 mL/min, 8 min gradient from 15 to 60% aqueous acetonitrile containing 0.1% TFA).

For SDS-PAGE analysis, a solution of FAM-H2B(117–127)-Ub (5 μM, 50 μL) in 50 mM Tris buffer containing 150 mM NaCl (pH 7.5) was mixed with a solution of UCH-L3 (20, 100, and 500 nM, respectively, 50 μL) in 50 mM Tris buffer containing 150 mM NaCl and 10 mM DTT (pH 7.5). The resulting mixtures were incubated at room temperature. Aliquots (5 μL) were removed at different time points (0, 10, 30, and 60 min) and diluted with 2× SDS sample loading buffer (100 mM Tris hydrochloride buffer containing 4% SDS, 20% glycerol, 200 mM DTT, and 0.01% bromophenol blue, pH 6.8, 5 μL). The samples (5 μL) were loaded without boiling on a 4–12% Bis–Tris polyacrylamide gel, separated, and imaged by fluorescence scanning first (excitation wavelength of 480 nm and detection wavelength of 510–540 nm) and then with Coomassie staining. Untreated H2B(117–127)-K120Ub (1 μM, 2 μL) was used as the control sample.

In the FP assay, a solution of FAM-H2B(117–127)-Ub (1 μM, 5 μL) in 50 mM Tris buffer containing 150 mM NaCl (pH 7.5) was mixed with a solution of UCH-L3 (0, 100, 200, and 400 nM, respectively, 5 μL) in 50 mM Tris buffer containing 150 mM NaCl and 10 mM DTT (pH 7.5) in a 384-well costar black plate. FP was measured (excitation wavelength, 485 nm; detection wavelength, 525 nm) every 3 min for 1 h. Samples were analyzed in triplicate, and the average was calculated and plotted.

Acknowledgments

We thank Hao Wu for assistance with the LC–MS analysis.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.bioconjchem.3c00541.

  • 1H and 13C NMR spectra, HRMS, and LC–MS chromatograms (PDF)

The authors declare no competing financial interest.

Supplementary Material

bc3c00541_si_001.pdf (1.9MB, pdf)

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Supplementary Materials

bc3c00541_si_001.pdf (1.9MB, pdf)

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