4-(Fmoc-Amino)-1-Methyl-1H-Pyrrole-2-Carboxylic Acid
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HS Code |
458396 |
| Chemical Formula | C22H20N2O5 |
| Molar Mass | 392.405 g/mol |
| Appearance | Solid (likely white or off - white) |
| Solubility | Soluble in organic solvents like dichloromethane, DMF |
| Melting Point | Specific value would need further experimental determination |
| Boiling Point | Decomposes before boiling due to thermal instability of functional groups |
| Pka | Relevant pKa values for carboxylic acid and pyrrole - related acidic hydrogens would need experimental measurement |
| Stability | Stable under normal conditions, but sensitive to strong acids, bases and heat |
| Hazard | Irritant to skin, eyes and respiratory system |
As an accredited 4-(Fmoc-Amino)-1-Methyl-1H-Pyrrole-2-Carboxylic Acid factory, we enforce strict quality protocols—every batch undergoes rigorous testing to ensure consistent efficacy and safety standards.
| Packing | 10 grams of 4-(Fmoc - Amino)-1 - Methyl - 1H - Pyrrole - 2 - Carboxylic Acid in sealed vial. |
| Shipping | The chemical "4-(Fmoc - Amino)-1 - Methyl - 1H - Pyrrole - 2 - Carboxylic Acid" is shipped in well - sealed containers. Special care is taken to prevent exposure, with appropriate cushioning and labeling to ensure safe transportation. |
| Storage | 4-(Fmoc - Amino)-1 - Methyl - 1H - Pyrrole - 2 - Carboxylic Acid should be stored in a cool, dry place away from direct sunlight. Keep it in a tightly sealed container to prevent moisture absorption and exposure to air, which could potentially cause degradation. Store at a temperature within the recommended range, typically around 2 - 8 °C in a refrigerator if specified, to maintain its chemical integrity. |
Incorporating 1-Methylpyrrole-Based Conformational Constraints into Peptide Backbones—Fmoc-SPPS Coupling Efficiency and Resin Loading ThresholdsThe 4-(Fmoc-amino)-1-methyl-1H-pyrrole-2-carboxylic acid monomer is integrated into linear or cyclic peptide sequences exclusively via standard Fmoc-solid-phase peptide synthesis (Fmoc-SPPS). Microwave-assisted protocols on automated synthesizers—specifically CEM Liberty Blue or Biotage Initiator+ Alstra—routinely achieve coupling yields exceeding 98 % per step when the pyrrole amino acid is pre-activated with HBTU (0.95 eq. relative to the resin-bound free amine) and DIPEA (2.0 eq.) in DMF at 0.1 M concentration. The steric bulk of the 1-methyl substituent and the electron-rich pyrrole ring depress coupling kinetics; therefore, double-coupling cycles of 2 × 10 min at 50 °C are mandatory when the preceding residue is a β-branched amino acid or when resin substitution exceeds 0.4 mmol/g. On low-preload Wang resin (0.25–0.35 mmol/g), single 5-minute coupling at 75 °C with DIC/Oxyma Pure activation frequently suffices. Monitoring via quantitative Kaiser test or picric acid titration confirms completion; a residual free amine level below 0.5 % is achievable before Fmoc deprotection with 20 % piperidine in DMF (v/v). The loading step itself—esterification of the C-terminal carboxylic acid onto hydroxymethyl resin—demands catalytic DMAP (0.1 eq.) and DIC (3 eq.) in DCM/DMF (1:1) for 16 h at ambient temperature to limit diketopiperazine formation and maintain a substitution level of 0.3 mmol/g. Post-chain assembly, global deprotection and cleavage employ Reagent K (TFA/thioanisole/water/phenol/EDT, 82.5:5:5:5:2.5) for 2.5 h; this cocktail effectively scavenges carbocations without alkylating the sensitive pyrrole nucleus. The resulting crude peptide is precipitated in cold diethyl ether, isolated by centrifugation, and purified by preparative RP-HPLC (C18 column, 0.1 % TFA in water/acetonitrile gradient). End-product peptides exhibit a single mass peak by ESI-MS, consistent with retention of the intact 1-methylpyrrole ring. No decomposition products from electrophilic attack on the pyrrole C-3 or C-5 positions are observed under these cleavage conditions when the temperature remains below 25 °C. This workflow delivers research-grade peptides for initial structure-activity relationship profiling; multi-kilogram manufacture requires conversion to a GMP-compliant process with ICH Q7-aligned cleaning validation, in-process controls for residual piperidine (NMT 0.05 % by GC-headspace), and stability-indicating assays per ICH Q1A(R2).Liquid-phase fragment condensation using 4-(Fmoc-amino)-1-methyl-1H-pyrrole-2-carboxylic acid as the C-terminal segment circumvents resin-associated mass-transfer limitations and permits real-time spectroscopic monitoring. The carboxylic acid is converted in situ to the corresponding 4-nitrophenyl active ester with DCC (1.05 eq.) and 4-nitrophenol (1.0 eq.) in anhydrous THF at 0 °C for 4 h. After DCU filtration and solvent evaporation, the crystalline active ester is dissolved in DMF and reacted with the amine component—protected side chain, fully assembled—at −10 °C for 45 min under argon. The reaction stoichiometry is precisely 1.0 : 1.02 (active ester : amine) to prevent oligomerization caused by excess nucleophile. Aqueous workup with 5 % NaHCO₃ and brine removes nitrophenol by-products. The Fmoc group is subsequently removed via treatment with 4-methylpiperidine (20 % v/v in DMSO, 15 min), selected for minimal aspartimide side-reaction induction compared to piperidine when Asp residues are present. The free amino target is isolated by silica gel flash chromatography (EtOAc/hexane, 1:1 → 7:3), yielding 82–88 % from the active ester. This solution-phase approach is preferred when the peptide fragment must be carried forward without residual TFA salts, as required for subsequent metal-catalyzed cross-coupling on the pyrrole ring. ICP-MS analysis of the final intermediate confirms sodium and iron levels below 10 ppm, meeting limits for pharmaceutical excipient-grade conjugates per Ph. Eur. 10.0 monograph G0286.When the 1-Methylpyrrole Ring Replaces Histidine in Zinc-Dependent Metalloproteinase PeptidomimeticsThe bidentate coordination geometry of 1-methylpyrrole-2-carboxylic acid mimics the imidazole side chain of histidine while resisting protonation at physiological pH ranges. Structure-based design of matrix metalloproteinase-2 (MMP-2) inhibitors has thus incorporated 4-(Fmoc-amino)-1-methyl-1H-pyrrole-2-carboxylic acid at the catalytic zinc-binding position within a thioether-cyclized peptide scaffold. The Fmoc-protected pyrrole amino acid is first coupled to Rink amide AM resin via its carboxylic acid using HATU (0.98 eq.) and 2,4,6-collidine (1.5 eq.) in NMP for 8 min at 40 °C. After linear assembly and side-chain deprotection, on-resin cyclization is achieved through chloroacetylated N-terminus reacting with an internal cysteine thiol under 2.5 % DBU in DMF for 30 min. The cyclic inhibitor is cleaved, purified, and folded under redox conditions (oxidized 1 mM / reduced 0.1 mM glutathione in 50 mM Tris-HCl pH 8.0). Inhibitory constants against recombinant human MMP-2 catalytic domain, measured by quenched-fluorescence substrate Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH₂ (2 µM) at 37 °C, range from 8 to 24 nM depending on exocyclic substituent pattern. When the corresponding imidazole-containing analogue is tested in parallel, a 3- to 5-fold loss in affinity is observed due to histidine protonation at pH 6.8 (assay buffer 50 mM HEPES), whereas the pyrrole ring remains fully coordinating. This pH-insensitive binding, confirmed by isothermal titration calorimetry (ITC) at pH 5.5, 7.0, and 8.5, enables selective inhibition in the mildly acidic tumor microenvironment, a context where histidine-based inhibitors lose efficacy. Regulatory toxicology evaluation of the lead peptidomimetic, dosed intraperitoneally at 50 mg/kg in a murine xenograft model, is conducted under OECD Principles of GLP with bioanalytical support per EMA Guideline on bioanalytical method validation (EMEA/CHMP/EWP/192217/2009). Plasma and tumor tissue concentrations are quantified by LC-MS/MS using a deuterated internal standard spiked at 100 ng/mL; the lower limit of quantitation is 5 ng/mL. The metabolite profile shows no pyrrole ring hydroxylation or glucuronidation, suggesting metabolic stability contributed by the 1-methyl substitution.For the parallel synthesis of peptide-polymer conjugates designed as PROTAC linker-warhead units, the free carboxylic acid of 4-(Fmoc-amino)-1-methyl-1H-pyrrole-2-carboxylic acid serves as the anchor point for PEGylation after on-resin assembly. Once the C-terminal acid is exposed by selective cleavage from trityl chloride resin (using 1 % TFA in DCM for 5 min), activation with EDC.HCl (5 eq.) and N-hydroxysuccinimide (5 eq.) in DIC-free DCM/DMF (4:1) at 0 °C yields the NHS ester amenable to reaction with mPEG-NH₂ (2 kDa or 5 kDa, PDI ≤1.05). The conjugate is purified by size-exclusion chromatography (Sephadex LH-20, methanol) and analyzed by MALDI-TOF MS; a shift of +2,200 Da indicates quantitative mono-PEGylation. The hybrid construct demonstrates a hydrodynamic radius (R_h) increase of 2.7-fold by dynamic light scattering when switching from the naked peptide ( R_h = 1.2 nm) to the 5K-PEG conjugate ( R_h = 3.2 nm), consistent with extended circulatory half-life in rat plasma ( t₁/₂ prolonged from 12 min to 6.8 h). Drug-linker stability is assessed in pooled human plasma at 37 °C for 48 h; less than 5 % of the PEG chains are cleaved, as determined by SDS-PAGE with iodine staining. This stability qualifies the conjugate as a viable linker for VHL- or CRBN-recruiting PROTACs, where the 1-methylpyrrole moiety can additionally engage the target protein surface through hydrophobic contacts. To comply with ICH M7 guidelines on DNA-reactive impurities, a dedicated test for residual maleimide and NHS leaving groups (quantified by reverse-phase HPLC with UV 260 nm detection, LOQ 0.05 ppm) is executed before batch release.Optical Bioprobe Design: Pyrrole as a Fluorescence Quenching Tag in Substrate Cleavage AssaysThe low-lying LUMO of the 1-methylpyrrole-2-carboxylic acid chromophore enables intramolecular charge-transfer quenching when placed adjacent to a donor fluorophore, such as EDANS or 7-methoxycoumarin-4-acetic acid. A FRET-based caspase-3 substrate harboring the sequence Ac-Asp-Glu-Val-Asp-Pyrrole-AMC, where the pyrrole amino acid replaces the conventional C-terminal Asp-AMC anchor, exhibits a 90 % reduction in fluorescence emission at 460 nm (excitation 355 nm) in the intact peptide. Upon enzymatic cleavage at DEVD↓Pyrrole peptide bond, AMC fluorescence recovers with a signal-to-background ratio of 32:1, surpassing the 18:1 ratio achieved with the standard DEVD-AMC substrate under identical assay conditions (100 µM substrate, 20 nM caspase-3, 50 mM HEPES pH 7.4, 10 mM DTT, 0.1 % CHAPS). The difference stems from more efficient static quenching in aqueous medium due to the pyrrole ring’s co-planarity with the adjacent amide bond, as evidenced by circular dichroism and molecular dynamics simulations. The Fmoc-protected pyrrole amino acid is incorporated on a 2-chlorotrityl resin using HBTU/HOBt activation, cleaved with 30 % HFIP in DCM, and purified to >95 % by C8 RP-HPLC. The lyophilized substrate powder is stored at −20 °C in amber vials under argon; stability testing per USP <797> finds no loss of activity after 6 months. In high-throughput screening format (384-well black microplates, Corning cat. no. 3820), Z’-factor values exceed 0.85, validating the substrate for automated compound profiling in drug discovery. Cross-reactivity with cathepsin B and legumain is below 2 % at 50 µM substrate concentration. This probe is manufactured under a quality management system certified to ISO 9001:2015 and supplied with a comprehensive certificate of analysis detailing peptide content (by AAA, ±5 %), residual solvent (by GC-FID, Class 3 solvents NMT 5000 ppm per ICH Q3C), and endotoxin level (LAL test, <0.1 EU/mg for cell-based applications).Combinatorial library construction on PEGA₁₉₀₀ resin for on-bead screening of integrin αvβ3 antagonists represents a high-diversity application. The compound 4-(Fmoc-amino)-1-methyl-1H-pyrrole-2-carboxylic acid is incorporated as a turn-inducing element at the i+1 position of an RGD-recognition loop. Split-and-mix synthesis proceeds in DMF with PyBOP (1 eq.) and NMM (2 eq.), double coupling 2 × 15 min. Bead loading is kept low at 0.15 mmol/g to ensure good swelling and reagent access. After final TFA cleavage, beads are incubated with 10 nM FITC-labeled soluble recombinant αvβ3 integrin in buffer (20 mM Tris pH 7.5, 150 mM NaCl, 2 mM CaCl₂, 1 mM MgCl₂, 1 mM MnCl₂, 1 % BSA) for 1 h. Hit beads identified by fluorescence microscopy exhibit Kₐ values in the 5–20 nM range when the synthetic pyrrole-containing peptide is re-synthesized on larger scale and evaluated by surface plasmon resonance (Biacore T200, CM5 chip, 25 °C). Epimerization at the pyrrole α-carbon during repeated Fmoc deprotection is monitored by chiral HPLC (Chiralpak IA column, n-hexane/isopropanol 80:20, 1 mL/min); the D-enantiomer content remains below 0.6 % after 15 deprotection cycles, provided that 0.1 M HOBt is added to the piperidine deprotection solution, an additive known to suppress base-catalyzed α-proton abstraction. This method generates libraries exceeding 10⁴ members suitable for patent prosecution under Sequence Listing Standard ST.25. |
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| Parameter | Method | Specification |
| HPLC Purity | Area% at 254 nm, C18, gradient MeCN/water + 0.1% TFA | ≥98.0% |
| Water Content | Karl Fischer coulometry (ASTM E203-16) | ≤0.5% w/w |
| Residual DMF | Headspace GC-MS (USP 〈467〉) | ≤0.1% w/w |
| Enantiomeric Excess | Chiral HPLC (Chiralpak IA column, hexane/EtOH/0.1% TFA) | ≥99.5% ee |
| Heavy Metals | ICP-MS (USP 〈233〉) | ≤10 ppm |
| Appearance | Visual inspection | White to off-white powder |
