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The caspase enzymes are integral to cellular inflammation and apoptotic cascades, and are commonly studied by cell biologists, medicinal chemists, and chemical biologists. In particular, the assessment of caspase enzymatic activity is a standard method to evaluate cell death pathways and novel apoptosis-modulating agents. Caspase enzymatic activity can be conveniently monitored with peptidic chromogenic or fluorogenic substrates, with certain peptide sequences imparting selectivity for certain caspases. The synthesis of these peptide substrates is typically performed via solid-phase synthesis, a method that is not ideal for production of the gram quantities needed for high-throughput screening. Described herein is a facile method for the synthesis of the Ac-DEVD-pNA caspase-3 substrate using solution-phase peptide synthesis. This protocol, involving iterative PyBOP-mediated couplings and Fmoc deprotections, is rapid, operationally simple, and can be used to generate over one gram of product.
Caspases are a group of cysteine proteases that play critical roles in the inflammatory response (caspases-1, -4, -5) and apoptotic cascade (caspases-2, -3, -6, -7, -8, -9, -10, -12).1 Caspases reside in the cell as low activity zymogens called procaspases.2 During apoptosis, a caspase activation cascade begins with a signal that activates an “initiator” procaspase to the active caspase enzyme, with procaspase-8 and -10 being activated through the dimerization of cellular death receptors,3 and procaspase-9 being activated through cytochrome c release from the mitochondria and formation of the apoptosome.4,5 Once activated, caspase-8 and caspase-9 catalyze the hydrolysis of procaspase-3 and procaspase-7, forming the “executioner” caspases-3 and -7.6–8 Caspase-3 (and to a lesser extent caspase-7) is responsible for the proteolytic cleavage of over 100 different protein substrates,9,10 resulting in the systematic dismantling of the cell and apoptosis (Figure 1).
Given their central role in apoptotic cell death, aberrant caspase activity can have disastrous consequences for an organism. For example, in many degenerative diseases an increase in caspase activity is observed, and this is believed to lead to the premature cell death. Evidence for elevated caspase activity has been seen in Alzheimer’s disease,11–13 Parkinson’s disease,12,14 Huntington’s disease,15 ALS16 and several others. In these cases, caspase inhibitors have been long sought as potential cytoprotective agents, and indeed the first caspase inhibitors are now in clinical trials for the treatment of liver disease, myocardial infarction, and rheumatoid arthritis.17 Conversely, resistance to apoptosis is a hallmark of cancer,18 and cancer cells can evade apoptosis through a modulation in caspase activity. Caspase-8,19–24 and -1025,26 are mutated in some cancers, and elevated levels of endogenous caspase inhibitor proteins (e.g., XIAP, survivin) are commonly observed in a variety of cancers.27–29 Interestingly, the levels of procaspase proteins are elevated in many tumor types,30–35 and this has led to the hypothesis that drugs that directly activate procaspase enzymes could selectively induce apoptotic death in cancer cells.35–37
Shortly after the discovery of the caspases a series of elegant studies defined the preferred cleavage site for each caspase isozyme.38,39 As indicated by their name (cysteine aspartic acid protease), caspases recognize short peptidic sequences and universally cleave on the C-terminal side of aspartic acid residues. The substrate recognition sequence for each caspase has been largely elucidated; for the most part, these enzymes recognize tetrapeptide sequences. The discovery of the preferred recognition sequence for each caspase led directly to the development of peptidic inhibitors and substrates for each isozyme. In the development of inhibitors,39,40 an electrophilic “warhead” (typically an aldehyde or α-fluoro ketone) is appended to the carboxy terminus of the scissile aspartic acid. Likewise, substrates can be created by appending the appropriate chromophore or fluorophore to this same position. The use of the para-nitro aniline (pNA) chromophore is especially popular, and as such the pNA tetrapeptide substrates for caspases-1, -3, -6, -8, and -9 are available from many commercial vendors.
Caspase-3, with its multiple cellular substrates, is one of the most studied and important caspases. Procaspase-3 is activated to caspase-3 through the action of both caspase-8 and caspase-9, meaning that procaspase-3 is a lynchpin upon which both the death receptor and mitochondrial apoptotic pathways converge (Figure 1).3 As such, there is much interest in and usage of the preferred peptidic substrates for caspase-3, Ac-DEVD-pNA (colorometric) and Ac-DEVD-AMC (fluorescent), in cell biology, chemical biology, and medicinal chemistry. For example, Ac-DEVD-pNA and Ac-DEVD-AMC are widely used to monitor caspase-3/-7 activity in cell culture, have been used for the in vitro identification of caspase-3/-7 inhibitors,40–50 and in the identification and assessment of small molecule activators of procaspase-3/-7.35–37 Caspase-3 or caspase-7 will catalyze the hydrolysis of Ac-DEVD-pNA, releasing the pNA chromophore which has a λmax of 405 nm (Figure 2).
While the average cost of Ac-DEVD-pNA from commercial vendors (5 mg for ~$60) is reasonable when small quantities of this reagent are needed, it is prohibitively expensive to purchase the gram quantities required to screen a small molecule library for caspase inhibition/activation. For example, to screen a 200,000 compound library for caspase-3 inhibition under standard 384-well plate assay conditions would require ~1.25 g of Ac-DEVD-pNA. Described herein is a protocol to synthesize 1 gram of this Ac-DEVD-pNA substrate. Through this method, Ac-DEVD-pNA can be produced for approximately 1/50th of the commercial cost using rapid, continuous solution-phase peptide synthesis.51,52 The solution-phase peptide synthesis method provides several advantages over solid-phase peptide synthesis for the generation of large quantities of short peptides. In solid-phase peptide synthesis, a large excess (as high as 5–40 equivalents) of amino acids are used to drive the coupling reactions to completion. In the solution-phase synthesis described herein, the amino acids are used in only slight excess (1.1 equivalents). In addition, peptides can easily be produced on the gram scale using solution-phase peptide synthesis. Ac-DEVD-pNA synthesized through this method has been used to generate substrate for the assessment of caspase inhibitors,40 for the high-throughput screening for procaspase-3 activators,35 and for mechanistic studies on procaspase-3 activating compounds,36,37 and cytoprotective compounds.53
As shown in Figure 3, the Ac-DEVD-pNA peptide is synthesized from C- to N-terminus. The synthetic route begins by coupling para-nitroaniline to Fmoc-Asp(Ot-Bu)-OH using POCl3.54 After workup, the resulting Fmoc-Asp(Ot-Bu)-pNA amide (1 in Figure 3) is purified by silica gel and isolated in 77% yield. This product is then carried through iterative Fmoc deprotection, amino acid coupling, and workup steps without any intermediate purification: protected valine is first coupled to provide 2, then Fmoc deprotection and PyBOP coupling with protected glutamic acid provides 3, and finally Fmoc deprotection and coupling with acylated aspartic acid provides the fully protected peptide 4. This protected peptide is difficult to isolate via extraction and column chromatography. Attempted extraction of the resulting product into ethyl acetate, chloroform, methylene chloride and hexane results in a persistent emulsion and insoluble precipitate. Efforts to purify the crude material using column chromatography resulted in an insoluble material that precipitates on the column. However, this peptide can be isolated by trituration in diethyl ether. Protected peptide 4 is then globally deprotected using trifluoroacetic acid (TFA) to provide the final product. The product is isolated by trituration in diethyl ether to give an overall yield of 40% for the 3 couplings and the global deprotection.
This procedure has been performed on a 1 to 10 mM scale with minimal differences in protocol. When working on smaller scales, reaction times may be decreased to 20 minutes per coupling/deprotection step. Conversely, larger scales may require longer reaction times and should be monitored to verify completion. Other substrates or inhibitors can likely be synthesized using different chromophores or fluorophores on the carboxy terminus. Individuals wishing to perform such modified protocols should ensure that there is a free amine on the chromophore or fluorophore of choice, and the initial coupling reaction to the protected aspartic acid residue may require optimization. After coupling to aspartic acid, an analogous protocol would be followed for the remaining coupling/deprotection steps.
Standard solid-phase peptide syntheses involve coupling to a resin, followed by C- to N-terminal peptide construction in a similar iterative sequence involving deprotection, purification, and coupling. Purification of products is achieved by filtration, where impurities are washed through a filter which retains the solid support and the growing peptide chain. Because the peptide is forming on a solid support and not in solution, coupling reactions are not as efficient and require large excess of each amino acid to overcome this limitation. Furthermore, due to low loading efficiency on the solid support, a large amount of resin is often required produce gram quantities of the final peptide. The solution-phase synthesis described herein is advantageous when large quantities of a relatively short peptide must be synthesized. In this example, gram quantities of the Ac-DEVD-pNA caspase-3/-7 substrate can be synthesized, in good overall yield, using nearly stoichometric quantities of reagents. This route also allows for the addition of the chromophore prior to couplings, enabling the purification of this product to be carried out early in the synthesis. In contrast, in solid-phase synthesis the protected peptide is typically cleaved from the resin before being coupled to the chromophore.
Although the synthesis reported herein is specific for the Ac-DEVD-pNA substrate, analogous strategies could likely be used to produce other caspase substrates, inhibitors, or reporters including, Ac-DEVD-AFC, Ac-DEVD-AMC, FAM-DEVD-fmk and others. In addition, the method outlined in Figure 3 represents an improvement over our previously reported synthesis,40 as the earlier protocol requires an extraction (after final coupling) that results in an emulsion that is difficult to resolve. The method reported herein takes advantage of the insolubility of the product in common solvents in utilization of precipitation-based purifications. We anticipate this method and those related to it will be useful for the preparation of large quantities of caspase substrates required for high-throughput screening.
Phosphate buffer is 90 g of NaHPO4·H2O (EMD cat. no. SX0710) and 37.5 g NaH2PO4 (Sigma-Aldrich cat. no. S9763) in 500 mL H2O pH 5.5. Hünig’s base was dried via distillation over CaH2 immediately before use. Into a distillation flask was placed an appropriate volume of Hunig’s base, CaH2 was added, the flask was heated, and the distillate was collected. Alternatively, a sure-seal bottle of Hünig’s base may be purchased and used.
Thoroughly clean and dry overnight (12 h) all glassware in an oven (100 °C) prior to use.
If the fully protected product is not pure after washing with diethyl ether, the product may be further washed with diethyl ether to remove further impurities. Alternatively, the product may be purified via reverse phase HPLC.
As described, the synthesis will produce 700–1000 mg of Ac-DEVD-pNA. The procedure can be scaled if desired.
We are grateful to the National Institutes of Health (R01-CA120439) for the support of this work. Q.P.P. was partially supported by a Chemistry–Biology Interface Training Grant from the National Institutes of Health (Ruth L. Kirschstein National Research Service Award 1 T32 GM070421 from the National Institute of General Medical Sciences) and by a predoctoral fellowship from the ACS Division of Medicinal Chemistry. D.C.W. was partially supported by Ruth L. Kirschstein National Research Service Award 3F31CA130138-01S1.
AUTHOR CONTRIBUTION STATEMENTAll authors contributed extensively to the work presented in this paper.
Competing Financial Interest
The authors declare that they have no competing financial interests.