This compound functions strictly as a linker scaffold and does not directly bind any therapeutic target; its role is to connect an E3 ubiquitin ligase ligand to a target protein-binding warhead in PROTAC design. The pent-4-ynoate chain (C4) provides a very short spacer (approximately 5-6 Angstrom extended) with a terminal alkyne for copper-catalyzed azide-alkyne cycloaddition (CuAAC). The tert-butyl ester can be hydrolyzed under acidic conditions to reveal a free carboxylic acid for amide coupling with amine-containing ligands. The tert-butyl ester is stable under CuAAC conditions (which typically use copper and reducing agents in aqueous mixtures), allowing orthogonal stepwise conjugation: the alkyne can be reacted with an azide-functionalized ligand first, then the tert-butyl ester can be hydrolyzed and coupled to the second ligand. Alternatively, the order can be reversed: hydrolyze the tert-butyl ester to the carboxylic acid, couple to one ligand via amide bond formation, then perform CuAAC with the alkyne to attach the second ligand. The short C4 spacer is among the shortest PROTAC linkers available and is suitable only for target-E3 ligase pairs where the distance between binding pockets is extremely short. For most target-ligase pairs, longer linkers (C6-C12) are required to span the distance for productive ternary complex formation.

PROTAC contains two distinct ligands linked by a single linker: one is the ligand for the E1088 ubiquitin ligase, and the other is the ligand for the target protein. PROTAC utilizes the intracellular ubiquitin-proteasome system to selectively degrade the target protein.

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In vitro, tert-Butyl pent-4-ynoate has no independent biological activity; its function is realized only when incorporated into a complete PROTAC molecule. Characterization of PROTACs containing this linker involves treating target-expressing cell lines with the PROTAC and quantifying target protein degradation by Western blot. The short C4 chain length may be suboptimal for many PROTAC applications; if the linker is too short to span the distance between the two binding pockets on the target protein and E3 ligase, no ternary complex forms and no degradation occurs. However, for certain target-ligase pairs with closely apposed binding sites, very short linkers can be effective. The CuAAC reaction produces a 1,4-disubstituted 1,2,3-triazole ring, which adds an aromatic heterocycle and approximately 2-3 Angstrom of additional length to the linker. The triazole ring is metabolically stable and can participate in pi-pi stacking interactions. The tert-butyl ester is stable under basic and neutral conditions but is cleaved under acidic conditions (e.g., TFA/DCM, 4M HCl in dioxane) or under basic hydrolysis (LiOH in THF/MeOH/H2O) if milder conditions are desired. The tert-butyl ester provides orthogonal protection to the alkyne, allowing stepwise conjugation without protecting group interference.

In vivo, the linker alone has no pharmacological activity; all in vivo effects derive from intact PROTACs incorporating this linker. Complete PROTACs are evaluated in xenograft mouse models for target knockdown and tumor growth inhibition. The very short C4 linker may limit in vivo efficacy if the ternary complex geometry is unfavorable. For PROTACs where this linker is used, the short length may require that the two ligands be positioned very close to each other, which may be achievable only for a small subset of target-ligase pairs. The triazole ring formed during CuAAC is highly stable and resistant to metabolism. The tert-butyl ester is hydrolyzed during PROTAC synthesis (to the carboxylic acid) and then converted to a stable amide bond, so the ester is not present in the final PROTAC; therefore, no concerns about esterase-mediated hydrolysis in vivo. The amide bond formed is resistant to hydrolysis under physiological conditions. For the final PROTAC, the short, rigid linker (due to the triazole ring) may have reduced flexibility compared to a pure alkyl chain of similar length, which could enhance degradation potency if the geometry is correct, or ablate it if the geometry is mismatched.

Standard linker characterization involves orthogonal conjugation strategies. Option 1 (CuAAC first, then ester hydrolysis and amide coupling): Step A (CuAAC): Dissolve tert-Butyl pent-4-ynoate and an azide-functionalized ligand (1.0-1.2 equivalents) in a mixture of t-BuOH/H2O or DMF/H2O. Add CuSO4·5H2O (0.1 equivalents), sodium ascorbate (0.2 equivalents), and a stabilizing copper ligand such as TBTA or THPTA (0.1-0.2 equivalents). Stir at room temperature or 40degC for 2-12 hours. The reaction produces a 1,4-disubstituted 1,2,3-triazole ring linking the alkyne to the azide. Step B (Ester hydrolysis): Dissolve the product from step A in THF/MeOH/H2O (3:1:1) with 5-10 equivalents of LiOH or NaOH. Stir at room temperature for 2-6 hours. Acidify with 1M HCl to pH 2-3, extract with DCM or ethyl acetate, dry over Na2SO4, concentrate to yield the free carboxylic acid. Step C (Amide coupling): Dissolve the free carboxylic acid and an amine-containing ligand (1.0-1.2 equivalents) in DMF. Add HATU (1.2-1.5 equivalents) or EDCI/HOBt, and DIEA (2-3 equivalents). Stir at room temperature for 2-12 hours. Purify by flash column chromatography (silica gel, DCM/MeOH gradient) or preparative reversed-phase HPLC (C18 column, acetonitrile/water with 0.1% TFA). Option 2 (Ester hydrolysis and amide coupling first, then CuAAC): Step A: Hydrolyze the tert-butyl ester as described above to yield pent-4-ynoic acid. Step B: Amide coupling of pent-4-ynoic acid with an amine-containing ligand (1.0-1.2 equivalents) using HATU/DIEA or EDCI/HOBt in DMF at room temperature for 2-12 hours. The product is an amide with a terminal alkyne. Step C: CuAAC of the terminal alkyne with an azide-functionalized ligand as described above. Purify after each step. Characterization by 1H NMR: tert-butyl ester delta 1.45 ppm (singlet, 9H), CH2 groups delta 2.2-2.5 ppm (multiplet, CH2C≡CH and CH2CO), terminal alkyne CH delta 1.95-2.05 ppm (singlet). After CuAAC, terminal alkyne proton disappears and triazole proton appears at delta 7.8-8.0 ppm (singlet). After ester hydrolysis, tert-butyl peak disappears and carboxylic acid proton appears (broad, delta 11-12 ppm). After amide coupling, amide NH appears (broad, delta 7.5-8.5 ppm). ESI-MS: parent [M+H]+ m/z 155.2 (C9H14O2, MW 154.21); after hydrolysis (pent-4-ynoic acid), [M-H]- m/z 97.1; after CuAAC, molecular weight increases by the mass of the azide ligand minus N2, plus the triazole ring.

Cell-based evaluation of PROTACs built from tert-Butyl pent-4-ynoate follows standard protocols. Target-expressing cells (e.g., HEK293T, HeLa, HCT116, MCF-7, or disease-relevant lines) are cultured in DMEM or RPMI1640 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37degC in a 5% CO2 humidified incubator. Cells are seeded in 6-well plates at a density of 2-5 × 10^5 cells per well and allowed to adhere overnight. PROTAC stock solutions in DMSO (10 mM) are serially diluted in culture medium to final concentrations ranging from 0.01 nM to 10 microM, with final DMSO concentration maintained at ≤0.1%. PROTAC-containing medium is added to the cells and the cells are incubated for 4, 8, 16, or 24 hours depending on the target protein degradation kinetics. For cell harvesting, medium is aspirated, and cells are washed twice with ice-cold phosphate-buffered saline (PBS). Cells are lysed in RIPA lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) freshly supplemented with a protease inhibitor cocktail (e.g., 1 mM PMSF, 1 ug/mL leupeptin, 1 ug/mL aprotinin) and phosphatase inhibitors (1 mM sodium orthovanadate, 10 mM sodium fluoride, 10 mM beta-glycerophosphate). Lysis is performed on ice for 20-30 minutes with periodic vortexing. Lysates are clarified by centrifugation at 14,000 × g for 15 minutes at 4degC. Protein concentration is quantified using the bicinchoninic acid (BCA) assay with bovine serum albumin (BSA) as standard. For Western blot analysis, equal amounts of protein (20-50 ug per lane) are mixed with Laemmli sample buffer containing beta-mercaptoethanol or dithiothreitol (DTT), heated at 95degC for 5-10 minutes, and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 4-12% gradient or 10% polyacrylamide gels. Separated proteins are transferred electrophoretically onto polyvinylidene difluoride (PVDF) or nitrocellulose membranes (0.45 um pore) using wet or semi-dry transfer apparatus. Membranes are blocked with 5% nonfat dry milk or 3% bovine serum albumin (BSA) in TBST (Tris-buffered saline with 0.1% Tween-20) for 1 hour at room temperature. Primary antibodies specific to the target protein (diluted 1:500 to 1:2000 in blocking buffer) are incubated with the membranes overnight at 4degC with gentle shaking. Membranes are washed three times with TBST (10 minutes each), then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (diluted 1:2000 to 1:5000 in blocking buffer) for 1 hour at room temperature. Following three additional TBST washes, protein bands are visualized using enhanced chemiluminescence (ECL) substrate and detected with a chemiluminescence imaging system. Densitometric analysis is performed using ImageJ or similar software, and DC50 values are calculated by nonlinear regression analysis using the four-parameter logistic equation in GraphPad Prism. Negative controls: vehicle (DMSO only) and a linker-only control where the linker is conjugated to only one ligand (either the E3 ligase ligand or the target ligand) but not both. To confirm proteasome-dependent degradation, co-treatment with 10 uM MG132 for 4-6 hours should block PROTAC-induced degradation.

Animal studies are conducted with the final PROTAC constructed from tert-Butyl pent-4-ynoate. Female athymic nude or NSG mice (6-8 weeks old, body weight 18-22 g) bearing subcutaneous xenografts (established by injecting 5 × 10^6 cells in 100 uL of PBS/Matrigel 1:1 into the right flank) are used. When tumor volumes reach 100-200 mm3 (typically 7-14 days post-inoculation), animals are randomized into treatment groups (n=6-10 per group). PROTAC is formulated fresh daily: typical vehicles include (1) 10% DMSO, 40% PEG400, 50% saline; (2) 5% DMSO, 40% 10% hydroxypropyl-beta-cyclodextrin (HP-beta-CD), 55% water; (3) 10% ethanol, 30% PEG300, 60% water. Dosing routes: intraperitoneal (IP) injection (most common), intravenous (tail vein, IV), or oral gavage (PO). Dosing schedules: once daily (QD) or every other day (Q2D) for 14-28 days. Typical dose range: 1-30 mg/kg body weight, adjusted based on preliminary tolerability studies. A vehicle control group receives formulation only without PROTAC. Tumor volume is measured by digital caliper every 2-3 days: volume = (length × width2)/2. Body weight is recorded daily; animals experiencing >20% weight loss from baseline are euthanized. At the study endpoint (either when control tumors reach ~1500 mm3 or after completing the dosing period), tumors are excised, weighed, and divided: one portion snap-frozen in liquid nitrogen for immunoblot analysis; one portion fixed in 10% neutral buffered formalin for IHC. Blood is collected via cardiac puncture or retro-orbital bleeding into EDTA-coated tubes, centrifuged at 1,000 × g for 10 minutes at 4degC, and plasma is stored at -80degC for PK LC-MS/MS analysis. Tumor lysates are immunoblotted for target protein; IHC on formalin-fixed paraffin-embedded sections (4-5 um) involves deparaffinization, antigen retrieval, blocking, primary antibody incubation, HRP-secondary detection, DAB development, and hematoxylin counterstain.

No pharmacokinetic data are available for tert-Butyl pent-4-ynoate as an isolated compound; all PK properties are determined by the final PROTAC. For PROTACs containing the short C4 linker with a triazole ring (from CuAAC), the following generalizations apply. The short alkyl portion (only three methylene groups between the triazole ring and the amide bond) is too short to undergo significant CYP-mediated omega-hydroxylation because the terminal methyl group is not exposed; instead, the triazole ring and amide bond dominate the linker structure. The triazole ring is highly stable and resistant to metabolism. The amide bond (from the carboxylic acid after ester hydrolysis, conjugated to a ligand) is resistant to hydrolysis under physiological conditions. The overall linker length (C4 plus triazole ring) is very short, approximately 7-9 Angstrom. For PROTACs where this linker successfully induces degradation, the short length likely forces the two ligands into very close proximity, which may be optimal for only a small number of target-ligase pairs. For the final PROTAC, typical PK parameters in mice after IV administration at 1 mg/kg: half-life (t1/2) 2-6 hours, clearance (CL) 10-30 mL/min/kg, volume of distribution (Vd) 1-2 L/kg. After PO administration at 10 mg/kg: Cmax 0.1-0.5 uM, Tmax 1-2 hours, oral bioavailability 5-25%. Plasma protein binding typically >95% for high-molecular-weight PROTACs. Metabolic stability in human liver microsomes: t1/2 30-60 minutes, CLint 10-50 uL/min/mg protein. CYP profiling identifies CYP3A4 as the primary isoform responsible for metabolism of attached ligands, but the short linker itself is not a significant site of metabolism.

No formal toxicity data exist for tert-Butyl pent-4-ynoate as an isolated compound. Standard chemical safety information from suppliers indicates the compound is a colorless to pale yellow liquid, purity typically 95-98%. GHS hazard statements: H302 (harmful if swallowed), H315 (causes skin irritation), H319 (causes serious eye irritation), H335 (may cause respiratory irritation). Precautionary statements: P261 (avoid breathing dust/fume/gas/mist/vapors/spray), P280 (wear protective gloves/protective clothing/eye protection/face protection), P301+P310 (IF SWALLOWED: immediately call a POISON CENTER or doctor), P305+P351+P338 (IF IN EYES: rinse cautiously with water for several minutes; remove contact lenses if present and easy to do; continue rinsing). Precautionary statements: wear protective gloves, protective clothing, and eye/face protection; avoid breathing dust/fume/gas/mist/vapors/spray; use only outdoors or in a well-ventilated area; wash hands thoroughly after handling; store in a tightly sealed container in a cool, dry place away from light and moisture. In case of skin contact, wash with plenty of soap and water; in case of eye contact, rinse cautiously with water for several minutes and remove contact lenses if present; if swallowed, rinse mouth and seek medical attention; if inhaled, remove person to fresh air. For the final PROTAC molecule containing this linker, toxicity assessment in animal models includes daily monitoring of clinical signs (lethargy, hunched posture, piloerection, diarrhea, respiratory distress) and body weight changes. Serum chemistry analysis: alanine aminotransferase (ALT) and aspartate aminotransferase (AST) for hepatotoxicity; blood urea nitrogen (BUN) and creatinine for nephrotoxicity; creatine phosphokinase (CPK) for muscle damage. Hematology: complete blood count (CBC) with white blood cell differential, platelet count, hemoglobin, and hematocrit. Histopathology: hematoxylin and eosin (H&E) staining of major organs including liver, kidney, heart, lung, spleen, and intestines. The maximal tolerated dose (MTD) is determined through dose escalation studies. Off-target degradation of essential proteins is a class-specific toxicity risk for PROTACs; this is assessed by mass spectrometry-based proteomics to identify unintended degradation targets. This compound has not entered clinical trials nor received regulatory approval for any therapeutic indication; all uses are strictly for research purposes in PROTAC development and drug discovery.

C9H14O2

154.21

154.099

185986-76-9

10877341

Colorless to light yellow liquid

0

4

11

177

0

CC(C)(C)OC(=O)CCC#C

GRWJMJVACAWLAO-UHFFFAOYSA-N

InChI=1S/C9H14O2/c1-5-6-7-8(10)11-9(2,3)4/h1H,6-7H2,2-4H3

tert-butyl pent-4-ynoate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)