| HS Code | 633892 |
| Product Name | tBuO-C20-Glu(AEEA-AEEA-OH)-OtBu |
| Synonyms | tert-Butyl protected C20-Glu-AEEA-AEEA side chain; Tirzepatide side chain intermediate |
| Cas Number | 1188328-31-1 |
| Molecular Formula | C45H83N3O13 |
| Molecular Weight | 874.17 g/mol |
| Appearance | White to off-white powder |
| Purity | ≥ 98% (HPLC) |
| Grade | Pharma Grade / GMP |
| Storage Conditions | Store at -20°C, protected from light and moisture |
| Solubility | Soluble in DMSO, DMF, methanol; slightly soluble in water |
| Application | Intermediate for tirzepatide and GLP-1/GIP dual agonist synthesis |
| Dosage Forms | Tablet, Capsule, Granule, Injection |
| Route Of Administration | Oral, Injectable |
| Packaging | 1g, 5g, 10g, 25g, 100g, 1kg |
| Shelf Life | 24 months |
| Regulatory Status | For research and pharmaceutical manufacturing use only |
As an accredited tBuO-C20-Glu(AEEA-AEEA-OH)-OtBu Pharma Grade API for Tablet / Capsule / Granule / Injection, Oral & Injectable factory, we enforce strict quality protocols—every batch undergoes rigorous testing to ensure consistent efficacy and safety standards.
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Oral tablet processing of tBuO-C20-Glu(AEEA-AEEA-OH)-OtBu begins with a non-aqueous high-shear granulation route because the 2 tert-butyl ester termini are hydrolytically labile in aqueous acidic media. Published data for this specific protected C20 derivative under aqueous wet granulation is limited; the non-aqueous route parallels established handling of tert-butyl-protected lipidated peptide intermediates. The dry blend is passed through a 0.600 mm mesh before charging to a 25 L high-shear granulator operating at impeller 120 rpm and chopper 1500 rpm. Granulating fluid is 1.5% w/w povidone K30 in isopropanol, added at 0.8% w/w of dry powder mass over 90 s; extended binder exposure increases granule D50 and produces case-hardened granules that resist compression on subsequent rotary pressing. Drying is conducted under vacuum at ≤35 °C and ≤50 mbar until loss on drying is ≤0.5%, because residual isopropanol must remain below the ICH Q3C(R8) Class 3 limit of 5000 ppm and because moisture above 0.5% accelerates tert-butyl ester cleavage during storage. Compression uses a rotary tablet press fitted with 8 mm round concave tooling at 6–10 kN; terminal blend contains microcrystalline cellulose 45–55% w/w, mannitol 20–25% w/w, croscarmellose sodium 2% w/w, and magnesium stearate 0.5% w/w blended for 3 min. Tablets are evaluated for hardness 70–100 N, friability ≤1.0% per USP <1216>, disintegration ≤15 min per USP <701>, and content uniformity acceptance value ≤15 per USP <905>. Because the tert-butyl esters undergo acid-catalyzed deprotection at pH 1.2, an enteric coat of methacrylic acid-ethyl acrylate copolymer is applied to 5–7% w/w weight gain when the intact protected species is the target oral entity. The terminal dosage form is a delayed-release enteric-coated tablet.
For liquid-filled hard capsules, the protected C20 diacid-Glu-bis-AEEA derivative is not processed as a dry powder; instead, a semi-solid lipid matrix is prepared from lauroyl polyoxylglycerides (Gelucire 44/14) melted at 45–55 °C under nitrogen, with the derivative dispersed at 8–12% w/w using a rotor-stator at 3000 rpm for 10 min. Apparent viscosity is monitored on a cone-plate viscometer at 50 °C and maintained at 500–1500 mPa·s; below 500 mPa·s, the C20-rich phase separates, and above 1500 mPa·s, positive-displacement capsule pumps exhibit inconsistent fill weights. HPMC capsules of size 0 are filled at 45 °C with a fill weight of 450–500 mg and sealed by hydroalcoholic banding. Terminal capsules are leak-tested by vacuum dye ingress and assayed for content uniformity per USP <905> and dissolution per USP <711> using 900 mL of 0.1 N hydrochloric acid with 0.5% sodium lauryl sulfate for 60 min. Residual isopropanol and tert-butyl alcohol are controlled by ICH Q3C(R8). The route yields a lipid-filled HPMC capsule for oral administration.
Bulk oral granules are produced by dry-blending the derivative with mannitol and xylitol and agglomerating by fluid-bed top-spray with an ethanolic solution of hypromellose 3% w/w. Inlet air temperature is set to 30–35 °C to stay below the tert-butyl ester hydrolysis threshold; product temperature during spraying is held at 24–28 °C. Granules are dried to water activity ≤0.3 and filled into aluminium foil sachets at 500 mg fill. Disintegration of the constituted suspension is checked by pouring into 100 mL water at 25 °C; aggregates larger than 710 µm must be absent by wet sieving. The terminal dosage form is a unit-dose oral granule sachet.
Injectable processing of tBuO-C20-Glu(AEEA-AEEA-OH)-OtBu does not proceed with the tert-butyl esters intact when the intended API is a lipidated peptide conjugate. The tert-butyl groups are removed in a cleavage cocktail of trifluoroacetic acid/triisopropylsilane/water at 95/2.5/2.5 v/v/v, typically for 2–4 h at 20–25 °C. The resulting free C20 diacid-Glu-bis-AEEA peptide conjugate is isolated by precipitation in methyl tert-butyl ether; residual trifluoroacetic acid must be controlled because it is not assigned a limit in ICH Q3C(R8) and is product-specific. The conjugate is then dissolved in an aqueous vehicle at 1–10 mg/mL as free acid or sodium salt, filtered through a 0.22 µm PVDF membrane, and filled into Type I glass vials. Lyophilization is performed with shelf temperature ramped from −45 °C to −20 °C during primary drying at 5–13 Pa, followed by secondary drying at 20–35 °C. Residual moisture by Karl Fischer should be ≤1.0%. Residual tert-butyl alcohol is generated during deprotection; its ICH Q3C(R8) Class 3 limit is 5000 ppm. The lyophilized cake is examined for collapse, and particulate matter in the reconstituted solution must meet USP <788> limits of 6000 particles per container for ≥10 µm and 600 particles per container for ≥25 µm. Sterility is verified per USP <71>, bacterial endotoxins per USP <85>, and aseptic filling is performed under Grade A/ISO 5 conditions per ISO 14644-1. The terminal dosage form is a lyophilized injectable vial.
| Downstream route | Critical in-process limit | Reference standard | Analytical method |
|---|---|---|---|
| Oral tablet | Loss on drying ≤0.5%; hardness 70–100 N; disintegration ≤15 min | USP <1216>, USP <701>, USP <905>, ICH Q3C(R8) | Karl Fischer, hardness tester, disintegration bath, HPLC |
| Liquid-filled capsule | Viscosity 500–1500 mPa·s at 50 °C; fill weight 450–500 mg | USP <905>, USP <711>, ICH Q3C(R8) | cone-plate viscometer, HPLC, GC-headspace |
| Oral granule | Water activity ≤0.3; wet sieve >710 µm absent | ICH Q3C(R8), USP <711> | water activity meter, wet sieving, HPLC |
| Injectable lyophilized | Moisture ≤1.0%; residual tert-butyl alcohol ≤5000 ppm; particles per USP <788> | USP <71>, USP <85>, USP <788>, ICH Q3C(R8) | Karl Fischer, GC-headspace, light obscuration, sterility membrane filtration |
| Solution-phase conjugation | Unconjugated peptide ≤0.10% by HPLC area | ICH Q7, 21 CFR 211.110 | RP-HPLC-UV, LC-MS |
Solution-phase peptide lipidation during GMP manufacturing uses the AEEA hydroxyl as the conjugation site. The AEEA-AEEA-OH arm is activated with N,N'-disuccinimidyl carbonate in acetonitrile at 0–5 °C to form the succinimidyl carbonate intermediate; the active carbamate is coupled to the side-chain amine of lysine at 1.05–1.10 equivalents relative to peptide. The reaction is carried out at pH 7.4–8.0 in 20% dimethyl sulfoxide in water at 25 °C for 4–8 h; unreacted derivative is removed by reverse-phase flash chromatography. The C20 diacid tert-butyl ester remains intact during conjugation and is deprotected only after purification. This route is used for GLP-1 receptor agonist peptide intermediates; terminal product is the protected or deprotected lipidated peptide API intermediate. Compliance includes ICH Q7 for active pharmaceutical ingredients and 21 CFR 211.110 for in-process controls.
When lyophilized cake architecture is defined for the deprotected C20 diacid-Glu-bis-AEEA conjugate, collapse temperature becomes the controlling cycle parameter. Thermal analysis of the frozen solution is used to set primary drying shelf temperature at 2–5 °C below the glass transition temperature of maximally freeze-concentrated solute; for formulations without crystalline bulking agents, this frequently falls between −35 °C and −25 °C. Primary drying is held at 5–13 Pa chamber pressure for 18–30 h depending on fill depth in 2R or 6R vials; a collapsed or wrinkled cake is cause for batch rejection because reconstitution time may exceed 3 min and subvisible particulate counts may fail the USP <788> limits. Mannitol at 2–5% w/w is included as a crystalline bulking agent to maintain cake porosity; if mannitol crystallizes as the metastable δ form, the vial is annealed at −15 °C for 1–2 h before primary drying. The terminal product is a lyophilized injectable with reconstitution time ≤3 min in sterile water for injection.
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The protected eicosanedioyl glutamate linker tBuO-C20-Glu(AEEA-AEEA-OH)-OtBu is supplied as a lipophilic amino acid-type building block with the molecular formula C45H83N3O13 and an exact monoisotopic mass of 873.5926. The structure comprises a C20 α,ω-dicarboxylic acid carrying one tert-butyl ester, an L-glutamic acid residue carrying a second tert-butyl ester at the α-carboxyl, and a two-residue 2-[2-(2-aminoethoxy)ethoxy]acetic acid chain whose terminal carboxyl remains unprotected. The material is used in liquid-phase and solid-phase assembly of acylated peptide conjugates; tablet, capsule, granule, and injectable presentations refer to the finished drug products obtained after conjugation, deprotection, and preparative purification. Because the tert-butyl esters remain intact during coupling, the intermediate is handled as a protected fragment rather than as a final pharmaceutical salt.
No public pharmacopoeial monograph is assigned to this exact C20 protected linker. Release testing is therefore aligned to general chapters and International Council for Harmonisation guidance applicable to protected peptide-fragment intermediates intended for oral and injectable conjugate manufacture. The control attributes below represent a typical release and reduced-testing matrix; final drug-substance limits are product-specific and are derived from toxicological qualification of the deprotected conjugate.
| Control attribute | Acceptance criterion | Method or standard reference |
|---|---|---|
| Identification by LC-HRMS | [M+H]+ at m/z 874.60 ± 0.05 | In-house LC-HRMS validated against reference standard |
| Assay by HPLC-UV | ≥ 98.0% area at 210 nm | Ph. Eur. 2.2.29; C18 column, 150 × 4.6 mm, 3.5 µm |
| Related substances | Reporting 0.05%; identification 0.10%; qualification 0.15% | ICH Q3A for daily dose > 2 g/day |
| Residual solvents | Dichloromethane ≤ 600 ppm; N,N-dimethylformamide ≤ 880 ppm; class 3 solvents ≤ 0.5% | ICH Q3C / Ph. Eur. 2.4.24 |
| Water content | ≤ 0.5% | Ph. Eur. 2.5.32; Karl Fischer coulometry |
| Bacterial endotoxins for injectable feedstock | ≤ 0.25 EU/mg | Ph. Eur. 2.6.14 |
| Appearance | White to off-white amorphous powder | Visual inspection; Ph. Eur. 2.2.1 |
Water is controlled tightly because residual moisture can preferentially hydrolyse the tert-butyl esters during storage, generating the free acid and reducing coupling stoichiometry. The endotoxin criterion is applied only when the material is designated for injectable conjugate campaigns; oral-grade material may be released under a looser bioburden limit if the subsequent purification step is validated for endotoxin removal.
The C20 linker differs from the widely used C18 semaglutide side-chain building block tBuO-Ste-Glu(AEEA-AEEA-OH)-OtBu by exactly two methylene units in the aliphatic bridge. The C20 structure has the molecular formula C45H83N3O13, whereas the C18 analogue has C43H79N3O13, corresponding to an exact-mass increment of 28.0313. The reactive handles are identical: the terminal AEEA chain retains its free carboxyl, the glutamic acid residue retains its α-tert-butyl ester, and the long-chain diacid retains its distal tert-butyl ester. The additional two methylene units do not introduce an orthogonal protecting-group strategy, but they alter reversed-phase retention, solubility in acetonitrile-water mobile phases, and the lipophilic character of the final deprotected conjugate.
Published data for this specific C20 configuration at preparative scale are limited. Consequently, a validated process using the C18 linker cannot be switched by simple molar replacement. Retention time mapping on a C8 preparative column must be generated, and the final conjugate must be re-evaluated for albumin binding, clearance, and receptor potency. The longer aliphatic bridge may shift the balance between hydrophobic association and aqueous solubility in injectable formulations; the practical outcome is that the C20 analogue is a tool for chain-length screening rather than a drop-in replacement for the C18 intermediate.
| Attribute | tBuO-C20-Glu(AEEA-AEEA-OH)-OtBu | tBuO-Ste-Glu(AEEA-AEEA-OH)-OtBu | tBuO-C16-Glu(AEEA-AEEA-OH)-OtBu |
|---|---|---|---|
| Aliphatic bridge carbons | 20 | 18 | 16 |
| Molecular formula | C45H83N3O13 | C43H79N3O13 | C41H75N3O13 |
| Exact monoisotopic mass | 873.5926 | 845.5613 | 817.5300 |
| Reactive termini | tert-butyl ester, tert-butyl ester, free AEEA carboxyl | tert-butyl ester, tert-butyl ester, free AEEA carboxyl | tert-butyl ester, tert-butyl ester, free AEEA carboxyl |
| Process-relevant distinction | Higher reversed-phase retention than C18; pre-purification solubility must be mapped | Established semaglutide fragment; extensive preparative retention data available | Lower lipophilicity; may require higher organic content in solid-phase coupling |
Across oral solid-dose manufacturing campaigns using conjugates derived from this linker, the free terminal carboxyl of the AEEA dimer and the deprotected glutamic acid residue contribute two anionic centres that are typically isolated as sodium or acetate salts after preparative high-performance liquid chromatography. Tablet and capsule formulations generally require the final lipidated peptide to be lyophilised or spray-dried before dry blending. Granulation is carried out in a top-spray fluidised-bed granulator or a high-shear granulator; the protected intermediate itself is not granulated, but its residual solvent and impurity profile must permit downstream processing without contaminating the granulation train. Injectable presentations require terminal sterile filtration of the final conjugate through a 0.22 µm polyethersulfone filter; therefore, the protected linker is controlled for bioburden and endotoxin before conjugation.
Removal of the two tert-butyl ester groups from tBuO-C20-Glu(AEEA-AEEA-OH)-OtBu is routinely performed with trifluoroacetic acid in dichloromethane or with anhydrous hydrochloric acid in 1,4-dioxane at 15–25 °C. The reaction liberates isobutylene and tert-butyl alcohol, which are subsequently removed by nitrogen stripping or azeotropic distillation. For injectable conjugates, residual trifluoroacetic acid and tert-butyl derivatives are controlled after preparative reversed-phase chromatography because low-molecular-weight acidic species can contribute to pH drift and osmolality in lyophilised formulations. A 50 mm internal-diameter C8 preparative column operated at 40 °C with acetonitrile and 0.1% trifluoroacetic acid gradients is typical; subsequent salt exchange to acetate is required for pharmaceutical acceptability because trifluoroacetate is not a suitable injectable counterion.
The protected linker should not be exposed to aqueous alkali or prolonged moisture above 60% relative humidity, because tert-butyl ester hydrolysis generates the free acid and reduces coupling efficiency. During solid-phase assembly, coupling of the linker to a resin-bound lysine side chain is monitored by Kaiser or trinitrobenzenesulfonic acid tests; a persistent positive result after double coupling indicates either residual water in the activation solvent or incomplete removal of excess linker during washing. With N,N-dimethylformamide stored over molecular sieves without a nitrogen blanket, residual water above 0.1% can depress activation efficiency and increase the capping burden. The same deprotection pathway is used before the final conjugate is formulated into oral tablets or capsules; residual isobutylene is not a routine tablet critical quality attribute because it is volatilised during drying, but the final drug substance must still meet ICH Q3C solvent limits for the selected formulation route.
The operational boundary for this intermediate derives from the lability of the tert-butyl esters rather than from thermal instability of the protected peptide fragment. Storage in desiccated, inert-headspace containers at 2–8 °C is sufficient to maintain release assay above 98.0% in typical handling cycles; formal stability data for this exact C20 configuration should be generated under ICH Q1A conditions before assigning a shelf life. For oral-grade supply, the material is often released with reduced endotoxin testing; for injectable campaigns, the endotoxin criterion is not downgraded. The difference from shorter-chain or PEG-only Glu(AEEA-AEEA-OH) linkers is the combined effect of the extended C20 aliphatic bridge and the two tert-butyl ester protecting groups, which together require a preparative chromatography step capable of separating both hydrophobic free-acid impurities and polar deprotection by-products before the conjugate enters tablet compression, capsule filling, or sterile injectable compounding.
