Synthesis of Ub-ACA, a Fluorogenic DUB Substrate via ACPL Method

Abstract

This chapter details the step-by-step synthesis ubiquitin–7-amino-4-coumarinyl-acetic acid (Ub-ACA) using the activated cysteine-directed protein ligation (ACPL) technique. Ub-ACA is a fluorogenic substrate for deubiquitinases (DUBs). It can be readily synthesized via ACPL to a high quantity by exchanging a C-terminal cysteine residue in a recombinantly expressed Ub-G76C mutant with Gly-ACA, a process mediated simply by 2-nitro-5-thiocyanatobenzoic acid (NTCB). This chapter provides protocols for protein expression, thiol activation, fluorophore conjugation, and product purification, yielding Ub-ACA with high purity suitable for enzymatic assays.

1. Introduction

Ubiquitin is a small, evolutionarily conserved protein present in nearly all eukaryotic organisms.(1) It regulates diverse cellular processes by covalently attaching to target proteins(2), thereby modulating their activity, localization, or stability. This post-translational modification, known as ubiquitination.(3) The post-translational modification of proteins with ubiquitin plays a pivotal role in regulating proteostasis, signal transduction, cell cycle progression, and immune responses.(4, 5) As such this is a target for therapeutic intervention.(6-8) This ubiquitination modification is dynamically controlled by a cascade of enzymes that includes ubiquitin-activating (E1), conjugating (E2), and ligating (E3) enzymes, along with deubiquitinases (DUBs) that reverse ubiquitination. Many of these enzymes, particularly within the DUB superfamilies (USP, UCH, OTU, and MJD)(9), utilize an active-site cysteine for catalysis. The ability to monitor DUB activity with molecular precision is essential for understanding their biological roles and for developing therapeutic inhibitors.

Fluorogenic substrates such as ubiquitin-7-amino-4-methylcouramin (Ub-AMC) have historically enabled real-time DUB activity assays by releasing a fluorescent AMC moiety upon enzymatic cleavage.(10) The synthesis of Ub-AMC is typically achieved by reacting Gly-AMC with a recombinantly expressed Ub^1-75-intein that naturally forms a thioester between Ub^1-75 and its intein fusion partner. This process is called expressed protein ligation. There are two limitations related to this existing approach. The first is complication related to handling purification of Ub^1-75-intein that undergoes hydrolysis at the thioester site and the second is the low solubility of Gly-AMC which limits the yield of the overall synthesis. To overcome these limitations, we replaced the expressed protein ligation process with the activated cysteine-based protein ligation (ACPL, shown in Figure 1) technique(11, 12) that involves only a small molecule-triggered exchange of a C-terminal cysteine followed by a 6×His tag with an amine-containing compound and therefore is easy to handle and replaced Gly-AMC with Gly-7-amino-4-coumarinyl-acetic acid (Gly-ACA as shown in Figure 2) that retains similar excitation/emission characteristics of AMC while offering superior water solubility, chemical stability, and signal-to-noise ratio in enzymatic assays.(13)

Figure 1:

The ACPL Reaction Mechanism

Figure 2:

The Synthesis of Ub-ACA via ACPL.

In this chapter, we describe a detailed and reproducible protocol for the synthesis of Ub-ACA via the ACPL approach (Figure 3). We include the design and expression of the Ub-G76C mutant, its activation with 2-nitro-5-thiocyanatobenzoic acid (NTCB) at the introduced cysteine residue, and subsequent ligation with Gly-ACA under mild conditions. The resulting Ub-ACA probe is suitable for quantitative fluorogenic DUB assays in recombinant systems, cell lysates, and tissue extracts. This methodology not only enables the preparation of Ub-ACA but is also generalizable to other ubiquitin and ubiquitin-like (Ubl) probes modified with amine-containing nucleophiles, facilitating broader applications in enzymology and chemical biology.

Figure 3:

The Synthesis of Gly-ACA

2. Materials

2.1. Reagents

Prepare all solutions using either ultrapure water (prepared by purifying deionized water, to attain a sensitivity of 18 MΩ-cm at 25 °C) or analytical grade reagents as specified. Store all reagents at designated temperatures. Diligently follow all waste disposal regulations when disposing waste materials.

1. pET duet vector harboring Ub-G76C-6×His expression plasmid in BL21 (DE3) E.coli cells.

2. 2xYT medium: Ready-to-use. Use 1L medium for expressing the protein.

3. Ampicillin: Prepare 100 μg/mL aliquots in water, store at −20 °C.

4. Isopropyl Thio-β-Galactoside (IPTG): Dissolve 2.38g of IPTG in 10mL of Milli-Q water to prepare 1M stock. Store at −20 °C.

5. Disodium hydrogen Phosphate (Na₂HPO₄): Prepare freshly before use. Store at room temperature.

6. Imidazole: Prepare freshly before use. Store at room temperature.

7. Sodium Chloride (NaCl): Prepare freshly before use. Store at room temperature.

8. Lysis Buffer: Dissolve 7.1g Na₂HPO₄, 29.22g NaCl, 340.4mg imidazole, 276mg TCEP into 800mL of Milli-Q water, adjust the pH till 7.8, make up the volume till 1L, filter and store at room temperature (Final concentration: 50 mM Na₂HPO₄, 500 mM NaCl, 5 mM imidazole, 1 mM TCEP).

9. Wash Buffer: Dissolve 7.1g Na₂HPO₄, 29.22g NaCl, 2.04g imidazole, 276mg TCEP into 800mL of Milli-Q water, adjust the pH till 7.8, make up the volume till 1L, filter and store at room temperature (Final concentration: 50 mM Na₂HPO₄, 500 mM NaCl, 30 mM imidazole, 1 mM TCEP).

10. Elution Buffer: Dissolve 7.1g Na₂HPO₄, 14.6g NaCl, 20.4g imidazole, 276mg TCEP into 800mL of Milli-Q water, adjust the pH till 7.8, make up the volume till 1L, filter and store at room temperature (Final concentration: 50 mM Na₂HPO₄, 250 mM NaCl, 300 mM imidazole, 1 mM TCEP).

11. NTCB: Prepare 500 mM stock solution of NTCB in DMSO. Store at −20 °C.

12. Gly-ACA: Weigh directly before use at required concentration.

13. Phosphate Buffer Saline (PBS, 20×): 202.8mM Na₂HPO₄, 35.2mM KH₂PO₄, 54mM KCl, 2.74M NaCl, pH -7.4: Store at 4 °C. Dissolve 50mL of 20×PBS to 950mL of Milli-Q water to prepare 1×PBS solution.

14. TCEP: Prepare 500 mM stock solution of TCEP in Milli-Q water. Store at −20°C.

15. 50 mM ammonium bicarbonate buffer: Dissolve 4 g of ammonium bicarbonate into 1 L of Milli-Q water. Filter and store at 4 °C.

16. ACA Assay Buffer: Dissolve 605.7 mg of Tris, 584.5 mg NaCl, 14.6 mg EDTA, 30.8 mg DTT, 10 mg BSA (Final concentration: 50 mM Tris, 100 mM NaCl, 0.5 mM EDTA, 2mM DTT, 0.1% BSA (v/v)) in 70 mL of Milli-Q water. Adjust the pH to 7.6, make up the volume till 100 mL, filter and store at 4 °C.

17. Hi-trap and Hi-prep26/10 desalting columns: Use for buffer exchange purpose, store at 4 °C.

18. Deubiquitinases: UCHL1, UCHL5, USP25. All three DUBs were acquired commercially.

19. 3KDa MWCO amicon ultracentrifugation filter.

20. Pierce 660 protein assay kit (Thermo Scientific, Cat: 22662): Use for measuring protein concentration at 660nm.

21. Microsyringe filters.

22. Tris: Prepare freshly before use. Store at room temperature.

23. Sodium Chloride (NaCl): Prepare freshly before use. Store at room temperature.

24. Disodium hydrogen Phosphate (Na₂HPO₄): Prepare freshly before use. Store at room temperature.

25. Imidazole: Prepare freshly before use. Store at room temperature.

26. Dithiothreitol (DTT): Prepare freshly before use. Store at −20 °C.

27. Ethylenediaminetetraacetate (EDTA): Prepare freshly before use. Store at room temperature.

28. Bovine Serum Albumin (BSA): Prepare freshly before use. Store at 4 °C.

29. Ni-NTA Agarose: Store at 4 °C.

30. Tetrahydrofuran (THF): Store at room temperature.

31. Methanol: Store at room temperature

32. Dichloromethane (DCM) anhydrous: Store at room temperature

33. Trifluoroacetic acid (TFA): Store at room temperature

34. Hydrogen balloon

35. Celite pad: Used for filtration purpose.

36. Palladium (Pd) catalysts

3. Methods

The ACPL method is a chemo selective strategy for C-terminal protein modification that enables the installation of diverse nucleophiles—such as small molecules, fluorophores, or peptides—onto a recombinant protein bearing a C-terminal cysteine residue. Unlike traditional expressed protein ligation (14), which requires intein fusion constructs or enzymatic catalysis, ACPL is a purely synthetic methodology, does not involve enzymes, and can be carried out under mild aqueous conditions. This technique features a nitrile donor molecule, e.g., NTCB, to activate a protein cysteine as shown in Figure 1 to form a 1-acyl-2-iminothiazolidine intermediate that subsequently undergoes a nucleophilic acyl substitution reaction with a small molecule amine. The overall reaction leads to replacing cysteine with the amine at the C-terminus of the target protein.

3.1. Expression and Purification of Ub-G76C-6×His

1. An overnight culture of E. coli BL21(DE3) cells harboring a pETDuet expression vector coding Ub-G76C-6×His was inoculated (1:100 dilution) into a 2xYT medium containing 100 μg/mL ampicillin.

2. Cells were let grow at 37 °C until OD600 reached 0.6-0.9 and 1mL IPTG (From 1M stock, Final concentration: 1 mM) was added to induce protein expression.

3. The cells were let grow in a shaker at 37 °C for 8 h or 18 °C for 16 h and collected by centrifugation (10000×g, 30 min, 4 °C).

4. The cell pellets were resuspended in a 100 mL lysis buffer and lysed by sonication.

5. The cell lysates were clarified by centrifugation (20000 ×g, 30 min, 4 °C) and the supernatant was decanted.

6. 6 M HCl solution was then added dropwise into the supernatant to adjust pH to 2 to drive the formation of white precipitate inside the solution. The precipitate was pelleted by centrifugation (20000 ×g, 30 min, 4 °C) and removed.

7. The pH value of the supernatant was then adjusted back to 7.8 by adding 6 M NaOH. This supernatant was loaded onto 4mL high-affinity Ni²⁺-NTA resins (pre-equilibrated with 50mL lysis buffer) and incubated with shaking for 1 h at room temperature and then washed with 10mLof wash buffer five times to remove other unbound/unwanted proteins.

8. The bound Ub-G76C-6×His was eluted by 7mL of elution buffer and then desalted into a 50 mM ammonium bicarbonate (ABC) buffer using HiPrep 26/10 desalting column.

9. All proteins were concentrated using 3 kDa MWCO Amicon ultracentrifugation filter devices and concentrations were measured using pierce 660 protein assay kit.

10. Eventually, all proteins were aliquoted at 500μM concentration (100μL each), lyophilized, and stored at −80 °C.

3.2. Synthesis of Gly-ACA

1. Using an established synthesis method (15), benzyl 2-(7-(2-((tert-butoxycarbonyl)amino)acetamido)-2-oxo-2H-chromen- 4-yl)acetate (1) was synthesized first.

2. 2-(7-(2-((tert-butoxycarbonyl)amino)acetamido)-2-oxo-2H-chromen-4-yl)acetic acid (2): To a stirred solution of 1 (2.0 g, 3.43 mmol) in THF: Methanol (1:1, 20 mL) was added 10% Pd/C (160 mg). The reaction flask was sealed, and the air inside was removed under the Schlenk line. A hydrogen balloon was then attached to the reaction flask and the reaction mixture was stirred at room temperature for 2 h. After the completion of deprotection, the reaction mixture was filtered with a pad of Celite. The filtrate was concentrated in vacuo and the residue was used for the next step without further purification.

3. 2-(7-(2-aminoacetamido)-2-oxo-2H-chromen-4-yl)acetic acid or Gly-ACA (3): To a stirred solution of 2 (1.50 g, 3.98 mmol) in anhydrous DCM (10 mL) at 0 °C was added TFA (3 mL). The reaction mixture was stirred at room temperature for 3 h. After completion of the reaction, remove the solvent in vacuo and wash with hexane to afford Gly-ACA as a white solid.

3.3. Ub-ACA synthesis using ACPL

1. Dissolve 68.75 mg of Gly-ACA (500 mM) in 250 μL of 1×PBS buffer, pH-7.4 (see Note 1). Dropwise add 6 M NaOH to adjust the pH to 9.5 (see Note 2) and mix very well.

2. At this stage, add 100 μL of 500 μM of Ub-G76C-6×His, 5 μL of NTCB (from 500 mM stock) and, 1μL of TCEP (from 500 mM stock) sequentially and mix very well (see Note 3). Make up the volume to 500 μL using PBS buffer (final concentrations: 500 μM Ub-G76C-6×His, 500 mM Gly-ACA, 5 mM NTCB, 1 mM TCEP). Incubate the reaction mixture at 37 °C overnight.

3. Equilibrate a hi-trap desalting column in 50 mM ammonium bicarbonate buffer. Inject the incubated reaction mixture into the column and buffer exchange using FPLC (see Note 4). Collect and combine the fractions containing the purified, ligated product.

4. Equilibrate a 2 mL nickel NTA agarose resin slurry in 20 mL of 50 mM ammonium bicarbonate buffer. Add 100 μL of the resin slurry into the combined fractions and incubate at 4 °C for 30 min to capture any unreacted Ub-G76C-6×His. Store the remaining resin at 4 °C and reuse later.

5. Centrifuge the sample at 16000×g for 5 min to precipitate the resin and collect the supernatant containing Ub-ACA (see Note 5), filter through micro syringe filters.

6. Concentrate using 3KDa MWCO amicon ultracentrifugation filter (centrifuge at 3000×gfor 20 mins), collect the top fraction containing the probe.

7. Measure the concentration by pierce 660 (absorbance at 660nm) assay reagents (follow manufacturer’s protocol) and store aliquots at 50 uM concentration at −80 °C.

3.4. Applications of Ub-ACA

Ub-ACA is a versatile fluorogenic probe designed for activity assays of deubiquitinases (DUBs). Upon cleavage of the ACA moiety, it emits a fluorescent signal (Ex: 380 nm, Em: 460 nm), enabling sensitive, real-time monitoring of enzymatic activity.

In cell or tissue lysates, Ub-ACA enables detection of endogenous DUB activity with minimal background. It can reveal tissue-specific activity patterns and is useful for evaluating pharmacological inhibition or genetic perturbation. Ub-ACA is well-suited for high-throughput screening (HTS) platforms due to its homogeneous assay format, rapid kinetics, and broad linear range, supporting inhibitor discovery and screening campaigns.

1. In a 96 well plate, add 1 μL (from a 5 μM stock prepared in PBS, pH-7.4) of three cysteine-based DUBs such as UCHL1, UCHL5 and USP25 to a final concentration of 50 nM in 100 μL of ACA assay buffer (see Note 6). Incubate the mixture at 30 °C.

2. Add 800 nL of Ub-ACA (from a 50 μM stock, final concentration 400 nM) and take fluorescence measurements (Ex: 380 nm, Em: 460 nm) for 1.5 h at every 2 min interval (Figure 4).

Figure 4:

DUB-catalyzed ACA Release from Ub-ACA Indicated by Fluorescence Increase.

4. Notes

1. Set a designated volume for the reaction (e.g., 500 μL). Do not add excess buffer.

2. Adjust the pH slowly. Avoid adding higher volume of 6 M NaOH, otherwise, sudden increase in pH may hamper the chemical ligation process.

3. After adding TCEP and NTCB, it is possible that the pH may decrease. Adjust the pH back, if that happens.

4. It is recommended to do the buffer exchange process at lower temperature such as 4 °C. This prevents the protein/probe from degradation and maintain its stability.

5. While collecting the supernatant, do not disturb the nickel resin, use a needle syringe to aspirate out the supernatant.

6. Always use BSA in fluorescence assay buffer. By preventing non-specific interactions, BSA helps to lower background fluorescence and improve signal-to-noise ratios in real-time fluorescence assays.

7. Generate a curve by plotting the fluorescence intensity at Y-axis and run time at X-axis.