Name: Z-AEVD-FMK
Brand: BOC Sciences
Rating: 5 (1 reviews)

Synonyms

Z-Ala-Glu(OMe)-Val-Asp(OMe)-fluoromethyl ketone; (5S,8S,11S,14S)-Methyl 14-(2-Fluoroacetyl)-11-isopropyl-8-(3-methoxy-3-oxopropyl)-5-methyl-3,6,9,12-tetraoxo-1-phenyl-2-oxa-4,7,10,1 3-tetraazahexadecan-16-oate; Z-A-E(OMe)-V-Asp(OMe)-FMK; Z-Ala-Glu(OMe)-Val-Asp(OMe)-FMK

IUPAC Name

methyl (4S)-5-[[(2S)-1-[[(3S)-5-fluoro-1-methoxy-1,4-dioxopentan-3-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-5-oxo-4-[[(2S)-2-(phenylmethoxycarbonylamino)propanoyl]amino]pentanoate

Canonical SMILES

CC(C)C(C(=O)NC(CC(=O)OC)C(=O)CF)NC(=O)C(CCC(=O)OC)NC(=O)C(C)NC(=O)OCC1=CC=CC=C1

InChI

InChI=1S/C28H39FN4O10/c1-16(2)24(27(39)32-20(21(34)14-29)13-23(36)42-5)33-26(38)19(11-12-22(35)41-4)31-25(37)17(3)30-28(40)43-15-18-9-7-6-8-10-18/h6-10,16-17,19-20,24H,11-15H2,1-5H3,(H,30,40)(H,31,37)(H,32,39)(H,33,38)/t17-,19-,20-,24-/m0/s1

InChIKey

CHZRBQFKZDGMTP-CDSYHYPYSA-N

Sequence

Cbz-Ala-Glu(OMe)-Val-Asp(OMe)-FMK

Density

1.236±0.06 g/cm3 (Predicted)

Solubility

Soluble in DMSO

Appearance

White Solid

Storage

Store at -20°C

Boiling Point

883.7±65.0°C (Predicted)

Z-AEVD-FMK is a potent peptide-based inhibitor frequently applied in ADC research and antibody-drug conjugate design. As a covalent, irreversible inhibitor, it targets specific proteases involved in apoptotic pathways, making it a valuable tool for designing ADC cytotoxins and validating linker-payload release mechanisms. In ADC linker design, Z-AEVD-FMK can be incorporated as a functional payload or used to study controlled enzymatic cleavage within ADC payload activation pathways.

In ADC payload applications, the FMK (fluoromethyl ketone) moiety provides irreversible binding to target cysteine proteases, allowing precise modulation of cytotoxic activity in preclinical ADC models. Its peptide backbone, Z-AEVD, mimics natural substrate sequences, facilitating selective enzymatic recognition. This property makes Z-AEVD-FMK a useful reference compound for evaluating ADC linker stability, payload release efficiency, and protease-mediated activation in the tumor microenvironment.

The chemical and enzymatic stability of Z-AEVD-FMK allows for reproducible use in bioconjugation studies, supporting consistent antibody-to-payload conjugation and linker evaluation. Its compatibility with diverse ADC linker architectures enables researchers to study the interaction between ADC cytotoxins and tumor-specific proteases, contributing to the rational design of targeted cancer therapies.

Z-AEVD-FMK is applied in ADC development workflows where precise enzymatic cleavage, payload activity assessment, and linker validation are required. By combining peptide-based protease recognition with covalent inhibition, it serves as a critical tool in the optimization of ADC linker design, controlled payload release, and functional evaluation of antibody-drug conjugates in preclinical research.

1. The effect of specific caspase inhibitors on TNF-alpha and butyrate-induced apoptosis of intestinal epithelial cells

Ross N Butler, Sarah A Jones, Ian R Sanderson, James W Wilson Exp Cell Res . 2004 Jan 1;292(1):29-39. doi: 10.1016/j.yexcr.2003.08.005.

Tumour necrosis factor-alpha (TNF-alpha)-induced intestinal epithelial cell apoptosis may contribute to mucosal injury in inflammatory bowel disease. Inhibition of TNF-alpha-induced apoptosis, using specific caspase inhibitors could, therefore, be of benefit in the treatment of disease. In vitro, CaCo-2 colonic epithelial cells are refractory to apoptosis induced by TNF-alpha alone; however, TNF-alpha can act synergistically with the short-chain fatty acid (SCFA) and colonic fermentation product, butyrate, to promote apoptosis. TNF-alpha/butyrate-induced apoptosis was characterised by nuclear condensation and fragmentation and caspase-3 activation. Inhibitors of caspase-8 (z-IETD.fmk) and caspase-10 (z-AEVD.fmk) significantly reduced TNF-alpha/butyrate-induced apoptosis, based on nuclear morphology and terminal deoxynucleotide transferase-mediated dUTP-biotin nick-end labelling (TUNEL), although caspase inhibition was associated with a significant increase in cells demonstrating atypical nuclear condensation. Inclusion of atypical cells in calculations of total cell death, still demonstrated that z-IETD.fmk and z-AEVD.fmk (in combination) significantly reduced cell death. Reduction in cell death was associated with maintenance of viable cell number. Transmembrane resistance was also used a measure of the ability of caspase inhibitors to prevent TNF-alpha/butyrate-mediated damage to epithelial monolayers. TNF-alpha/butyrate resulted in a significant fall in transmembrane resistance, which was prevented by pre-treatment with z-IETD.fmk, but not z-AEVD.fmk. In conclusion, synthetic caspase inhibitors can reduce the apoptotic response of CaCo-2 colonic epithelial cells to TNF-alpha/butyrate, improve the maintenance of viable cell numbers and block loss of transmembrane resistance. We hypothesise that caspase inhibition could be a useful therapeutic goal in the treatment of inflammatory bowel conditions, such as ulcerative colitis.

2. Bufalin and cinobufagin induce apoptosis of human hepatocellular carcinoma cells via Fas- and mitochondria-mediated pathways

Bo Gao, Anyuan Li, Norihiro Kokudo, Huanli Xu, Fanghua Qi, Yoshinori Inagaki, Xiaoyan Cui, Wei Tang Cancer Sci . 2011 May;102(5):951-8. doi: 10.1111/j.1349-7006.2011.01900.x.

Bufadienolides bufalin and cinobufagin are cardiotonic steroids isolated from the skin and parotid venom glands of the toad Bufo bufo gargarizans Cantor. They have been shown to induce a wide spectrum of cancer cell apoptosis. However, the detailed molecular mechanisms of inducing apoptosis in hepatocellular carcinoma (HCC) are still unclear. In the present study, the apoptosis-inducing effect of bufalin and cinobufagin on HCC cell line HepG(2) was investigated. We found bufalin and cinobufagin induced marked changes in apoptotic morphology and significantly increased the proportion of apoptotic cells. This apoptotic induction was associated with an increase in Fas, Bax and Bid expression, a decrease in Bcl-2 expression, disruption of the mitochondrial membrane potential, release of cytochrome c, activation of caspase-3, -8, -9 and -10, and the cleavage of poly(ADP-ribose)polymerase (PARP), which indicated that bufalin and cinobufagin induced apoptosis through both Fas- and mitochondria-mediated pathways. In addition, caspase activation during bufalin- and cinobufagin-induced apoptosis was further confirmed by caspase-3 inhibitor Z-DEVD-FMK, caspase-8 inhibitor Z-IETD-FMK, caspase-9 inhibitor Z-LEHD-FMK and caspase-10 inhibitor Z-AEVD-FMK. The results showed that bufalin- and cinobufagin-induced apoptosis was blocked by these inhibitors and particularly by caspase-10 inhibitor. Taken together, bufalin and cinobufagin induce apoptosis of HepG(2) cells via both Fas- and mitochondria-mediated pathways, and a Fas-mediated caspase-10-dependent pathway might play a crucial role.

3. Arsenic trioxide promotes histone H3 phosphoacetylation at the chromatin of CASPASE-10 in acute promyelocytic leukemia cells

Ji Li, Janice Barnes, Peili Chen, Robert P Wersto, Francis J Chrest, Natasha Sinogeeva, Myriam Gorospe, Yusen Liu J Biol Chem . 2002 Dec 20;277(51):49504-10. doi: 10.1074/jbc.M207836200.

Arsenic trioxide (As(2)O(3)) is highly effective for the treatment of acute promyelocytic leukemia, even in patients who are unresponsive to all-trans-retinoic acid therapy. As(2)O(3) is believed to function primarily by promoting apoptosis, but the underlying molecular mechanisms remain largely unknown. In this report, using cDNA arrays, we have examined the changes in gene expression profiles triggered by clinically achievable doses of As(2)O(3) in acute promyelocytic leukemia NB4 cells. CASPASE-10 expression was found to be potently induced by As(2)O(3). Accordingly, caspase-10 activity also substantially increased in response to As(2)O(3) treatment. A selective inhibitor of caspase-10, Z-AEVD-FMK, effectively blocked caspase-3 activation and significantly attenuated As(2)O(3)-triggered apoptosis. Interestingly, the treatment of NB4 cells with As(2)O(3) markedly increased histone H3 phosphorylation at serine 10, an event that is associated with acetylation of the lysine 14 residue. Chromatin immunoprecipitation assays revealed that As(2)O(3) potently enhances histone H3 phosphoacetylation at the CASPASE-10 locus. These results suggest that the effect of As(2)O(3) on histone H3 phosphoacetylation at the CASPASE-10 gene may play an important role in the induction of apoptosis and thus contribute to its therapeutic effects on acute promyelocytic leukemia.

What is Z-AEVD-FMK and its function in ADC research?

Z-AEVD-FMK is a fluoromethyl ketone-based peptide inhibitor used as a cytotoxic payload in ADCs targeting specific proteases. Its structure allows irreversible binding to active site cysteines, enabling selective cytotoxicity.

25/8/2019

Could you advise how Z-AEVD-FMK improves ADC selectivity?

Z-AEVD-FMK’s peptide recognition sequence confers specificity toward target proteases, minimizing non-specific cytotoxicity and enhancing the therapeutic window of ADCs by restricting activity to cells expressing the target enzyme.

2/11/2020

We would like to know what conjugation strategies are compatible with Z-AEVD-FMK.

Z-AEVD-FMK can be conjugated through amine- or thiol-reactive linkers, depending on ADC design, enabling flexible attachment to antibodies while preserving the inhibitor’s reactive ketone group.

28/11/2020

What precautions should be taken when handling Z-AEVD-FMK?

Due to its reactive electrophilic group, Z-AEVD-FMK should be handled under inert conditions, with protective gloves and eye protection. Storage in dry, low-temperature environments prevents hydrolysis and maintains activity.

7/9/2016

Good morning! Could you advise the recommended storage and stability considerations for Z-AEVD-FMK?

Z-AEVD-FMK should be stored at -20°C in a desiccated, light-protected environment to maintain stability. Repeated freeze-thaw cycles should be avoided. Proper labeling and use of inert containers are advised. Documentation including analytical verification and handling guidelines can be provided to ensure the compound’s suitability for experimental use.

2/7/2019

— Dr. Jonathan White, Medicinal Chemist (UK)

Z-AEVD-FMK demonstrated high stability and reactivity, critical for our enzymatic ADC linker studies.

28/11/2020

— Ms. Clara Hoffmann, Biochemist (Germany)

BOC Sciences provided Z-AEVD-FMK with excellent documentation and QC reports, ensuring reproducible results.

2/7/2019

— Dr. Robert Müller, Biochemist (Germany)

Z-AEVD-FMK was integral to our protease inhibition studies. The stability and high purity of the product allowed reliable assay results.

7/9/2016

— Dr. Robert Müller, Biochemist (Germany)

Z-AEVD-FMK from BOC Sciences was integral to our protease inhibition studies. Product stability and high purity were crucial for reproducible assays.