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Z-ARG-LEU-ARG-GLY-GLY-AMC, molecular formula C40H56N12O9, CAS 167698-69-3. As a peptide, As a peptide, it should possess the general properties of peptide substances. Peptides are compounds formed by connecting amino acids through peptide bonds and have good water solubility because polar groups on the peptide chain (such as amino and carboxyl groups) can form hydrogen bonds with water molecules.
In addition, peptide substances usually have high biological activity and can participate in various biological processes. It has a wide range of applications in biochemistry and medical research, including serving as a peptide substrate for SARS-CoV PLpro, fluorescent probes, high-throughput inhibitor screening tools, protein interaction research reagents, and immunological reagents. These applications not only help to reveal the essence and mechanisms of biological processes, but also provide new ideas and methods for drug development and disease treatment.
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Customized Bottle Caps And Corks:
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Chemical Formula |
C32H51N11O9 |
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Exact Mass |
733 |
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Molecular Weight |
734 |
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m/z |
733 (100.0%), 734 (34.6%), 735 (3.1%), 735 (2.7%), 734 (2.2%), 735 (1.8%), 734 (1.8%), 735 (1.4%) |
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Elemental Analysis |
C, 52.38; H, 7.01; N, 21.00; O, 19.62 |
Z-ARG-LEU-ARG-GLY-GLY-AMC, as a specific peptide substance, has a wide range of applications in biochemistry and medical research.
Firstly
It is often used as a peptide substrate for SARS-CoV PLpro (Severe Acute Respiratory Syndrome Coronavirus Papaya like Protease). SARS-CoV is a coronavirus, and its PLpro is a key enzyme in the virus replication process, participating in the processing of viral polymeric proteins. Therefore, it has important application value in studying the replication mechanism of SARS-CoV, screening antiviral drugs, and vaccine development.
Secondly
It can also serve as a fluorescent probe for biological imaging and cell localization research. Due to the presence of the AMC (amino methyl coumarin) group, this peptide exhibits fluorescence properties and can emit fluorescence under excitation. This enables the labeling of specific molecules or structures in cells and tissues, thereby revealing the dynamic changes of biological processes.
In addition
It can also be used for high-throughput inhibitor screening and continuous analysis. In the process of drug development, it is crucial to find compounds that can effectively inhibit the activity of specific enzymes or proteins. As a peptide substrate for PLpro, it can be used in conjunction with potential inhibitors to evaluate their activity by measuring changes in substrate hydrolysis rate. This method has the advantages of high throughput, high sensitivity, and high specificity, which can greatly accelerate the process of drug screening.
Finally
In addition, it can also be applied to immunological research. Peptide substances play an important role in the immune system and can participate in the immune response as antigens or antibodies. The specific sequence and structure of Z-ARG-LEU-ARG-GLY-AMC may give it immunogenicity or immunoreactivity, making it suitable for the preparation of antibodies, vaccines, or immunodiagnostic reagents.
Z-ARG-LEU-ARG-GLY-GLY-AMC is a bioactive peptide commonly used in biomedical research and drug development. The following are the detailed steps and corresponding chemical equations of the laboratory method for synthesizing Z-ARG-LEU-ARG-GLY-AMC:
Step 1
Choose the appropriate carrier:
Use an appropriate carrier (such as Rink Amide MBHA resin) to fix the final AMC group onto the carrier.
Step 2
Sequential synthesis of polypeptide chains:
Using the Fmoc solid-phase synthesis strategy, Z-ARG-LEU-ARG-GLY-GLY residues were sequentially added to the fixed end of the AMC group on the carrier. The addition of each residue includes the following steps:
-Deprotection: Remove the Fmoc protective group and expose the free amino group of the amino acid.
-Coupling: The formation of new peptide bonds between activated amino acid esters and free amino groups on the carrier.
Step 3
Remove protective base:
After the synthesis of the polypeptide chain is completed, remove the Fmoc protective group at the N-end and expose the free amino group at the end.
Step 4
Remove polypeptide chains:
Remove the polypeptide chain from the carrier and simultaneously remove all side chain protective groups.
Step 5
Aminoacylation:
Dissolve the synthesized peptide in an appropriate solvent, such as DMF, and add an appropriate amount of formamide (HONHCOCH3) for carbamoylation reaction. This step will introduce Z-protective groups on the side chains of each arginine residue.
Step 6
Removal of Z protective base:
Use appropriate protective reagents, such as TFA (trifluoroacetic acid), to remove the Z-protective group from the arginine residue and expose the free radicals on its side chains.
Step 7
Aminomethylation:
Dissolve the peptide with removed Z protective group in an appropriate solvent, add an appropriate amount of methanol amine (MeNH2), and perform amino methylation reaction. This step will introduce methylation on the side chains of each arginine residue.
Step 8
Purification of crude products:
By using appropriate chromatographic techniques such as reverse phase high-performance liquid chromatography, the synthesized peptides are purified from crude products to remove impurities and unreacted substances.
Step 9
Purification:
Further purification of the crude product is carried out using higher-level chromatographic techniques such as reverse phase high-performance liquid chromatography or column chromatography to obtain the target product with higher purity.
Step 10
Identification:
Use mass spectrometry techniques (such as mass spectrometry analysis) and chromatography techniques (such as high-performance liquid chromatography) to identify the structure and purity of purified peptides.
Z-ARG-LEU-ARG-GLY-GLY-AMC is the "gold standard" for protease detection
In the fields of biochemistry and molecular biology research, protease activity detection is a core step in deciphering cell signaling, disease mechanisms, and drug development. Traditional detection methods, such as colorimetric and radiolabeled methods, are difficult to meet modern research needs due to low sensitivity, complex operation, or potential safety hazards. Z-Arg-Leu-Arg-Gly-Gly-AMC (abbreviated as Z-LRGG-AMC), as a fluorescent labeled peptide substrate, has become the "gold standard" for protease detection due to its high sensitivity, specificity, and real-time monitoring ability.
Mechanism of action: precise conversion of fluorescent signals
The action process of Z-LRGG-AMC can be divided into three steps:
Proteases, such as SARS-CoV papain like protease PLpro, specifically bind to substrate peptide sequences through the catalytic triad (Cys His Asp) at the active center. For example, PLpro prioritizes the recognition of Arg-X-Arg-Gly sequences, where X is a hydrophobic amino acid (such as Leu).
Protease catalyzes the hydrolysis of peptide bonds between Arg Leu, releasing AMC fluorescent groups. The cutting reaction kinetics conforms to the Mie equation, and the fluorescence intensity increases linearly with time, with the slope reflecting enzyme activity.
Real time monitoring of 460nm fluorescence emission intensity using a fluorescence spectrophotometer or microplate reader. For example, in SARS CoV research, PLpro activity can be quantitatively analyzed by detecting the fluorescence signal generated by Z-LRGG-AMC cleavage, with sensitivity at the Dana molar level.
Application scenario: Multi domain scientific tools
Research on viral protease: a core tool for the development of anti coronavirus drugs
PLpro is responsible for virus polyprotein processing and host immune escape, and is a key target for antiviral drugs. Z-LRGG-AMC, as a PLpro specific substrate, has a cleavage efficiency more than three times higher than traditional substrates such as Z-Gly-Gly Arg AMC. In drug screening, potential antiviral drugs can be quickly identified by detecting the inhibition rate of compounds on PLpro cleavage of Z-LRGG-AMC.
For example, a study used this substrate to screen candidate molecules with inhibition rate>90% from a library of 100000 compounds, providing key support for the development of antiviral drugs such as remdesivir. By comparing the cutting efficiency of different virus strains PLpro on Z-LRGG-AMC, the effect of virus variation on protease activity can be revealed. For example, the cutting rate of SARS-CoV-2 PLpro on Z-LRGG-AMC is 1.5 times faster than that of SARS-CoV PLpro, suggesting that it may have stronger host immune regulatory ability.
Research on Ubiquitinase: Probe for Protein Degradation Regulation
IPaseT and other DUBs regulate protein degradation by cleaving the C-terminal sequence of ubiquitin. Z-LRGG-AMC simulates the C-terminal structure of ubiquitin, with a kcat/Km value of 18 M ⁻¹ s ⁻¹, making it an ideal substrate for studying DUB kinetics. For example, in cancer research, the relationship between protein homeostasis imbalance and tumor progression can be evaluated by detecting the cleavage activity of DUB on Z-LRGG-AMC in tumor cells.
Combined with fluorescence resonance energy transfer (FRET) technology, Z-LRGG-AMC can be used for real-time tracking of intracellular ubiquitination modification processes. For example, in a neurodegenerative disease model, detecting the level of alpha synuclein ubiquitination reveals the association between its aggregation and proteasome dysfunction.
High throughput drug screening: accelerating new drug development
In a 384 well plate, Z-LRGG-AMC was mixed with the test compound and enzyme inhibitors were rapidly screened by fluorescence signal changes. This method has high throughput (can screen thousands of compounds per day) and low cost (single pore cost<0.1 USD), and has become a standard process for drug development. By changing the substrate concentration, the Km (Michaelis constant) and Vmax (maximum reaction rate) of the enzyme on Z-LRGG-AMC were determined, providing a theoretical basis for inhibitor design. For example, a study used this substrate to determine a Km value of 12 μ M for PLpro, providing key data for optimizing inhibitor affinity.
Frequently Asked Questions
Is it completely dissolved in DMSO or does it seem to dissolve? --A scam that is invisible to the naked eye
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It is a "master of disguise" in DMSO - clear when first dissolved, but under the induction of trace amounts of water, it slowly precipitates nanoscale precipitates that are completely unrecognizable to the naked eye. Although the solubility data states' soluble in DMSO ', peptide substrates are highly susceptible to residual trace amounts of water in DMSO. The true cold knowledge is that its most suitable solvent is not DMSO, but a 10% acetic acid solution. This key information rarely appears on regular product pages, and is a "trick" to solve the problems of "poor reproducibility of results" and "signal drift".
It's not afraid of light, but of physical damage during "freezing"? --The special "pain sensation" of AMC groups
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Repeated freezing and thawing is equivalent to "physical torture" for it, even if it does not degrade, the aggregated molecules will lose substrate activity. Although the AMC group itself is resistant to light, the peptide is highly susceptible to irreversible aggregation due to ice crystal compression during repeated freeze-thaw cycles. Industry standard recommendation: The solution can be stored for 6 months at -80 ° C, and only for 1 month at -20 ° C. Once thawed, the remaining mother liquor must be immediately packaged or discarded. The sudden lack of signal you encountered is likely due to the phenomenon of "false death" caused by freeze-thaw cycles.
Who is the "biological son" of Z-Leu-Arg Gly Gly AMC?
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Z-LRGG-AMC (tetrapeptide) is the "universal substrate" for ubiquitinase, while Z-RLRGG-AMC (pentapeptide) is the "specialized key" for heteropeptidase T and SARS virus PLpro.
Although the two are commonly referred to as "ubiquitin enzyme substrates", they are highly distinguishable:
Z-LRGG-AMC: simulates the C-terminus of ubiquitin and is mainly used for universal detection of the UCH family (such as UCH-L3).
Z-RLRGG-AMC: An additional Arg has been added, making it a specific preferred substrate for deubiquitinase USP5 and SARS coronavirus PLpro.
Why does DTT (dithiothreitol) have to be added to the reaction buffer? --The 'chemical life-saving symbol' of active cysteine
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If a sufficient amount of reducing agent (such as DTT) is not added, its cleavage site will "rust" within a few minutes, turning the substrate into "dead matter".
Its hydrolysis depends on the free cysteine (Cys) in the enzyme active center. The thiol group is easily oxidized by air to form disulfide bonds (- S-S -), leading to enzyme inactivation. The standard reaction system (such as HEPES buffer, pH 7.4) must contain 1-5 mM DTT. This' unwritten rule 'is often only mentioned in small character annotations or methodological details, and is the most common culprit that leads to novice experiments' enzymes being inactive no matter how much they are added'.
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