4-(Fmoc-Amino)-1-Methyl-1H-Pyrrole-2-Carboxlyic Acid

In automated Fmoc-strategy solid-phase peptide synthesis (SPPS) executed on a Prelude X bench-scale synthesizer (Gyros Protein Technologies), the incorporation of 4-(Fmoc-amino)-1-methyl-1H-pyrrole-2-carboxylic acid as a heterocyclic building block demands a deliberate departure from canonical amino acid coupling protocols. Departure is most pronounced during activation of the sterically hindered carboxylic acid at the pyrrole 2-position, where competitive racemization risk is negligible but acylation kinetics are retarded relative to proteinogenic Fmoc-amino acids. Production-scale campaigns targeting peptide APIs for Phase I clinical supply typically enforce a starting material specification under ICH Q7A Section 7.3, requiring individual organic impurities ≤1.0% (area % by RP-HPLC at 220 nm, C18 column, 1.0 mL/min linear gradient of 0.1% TFA in acetonitrile/water) and residual piperidine below 50 ppm as determined by headspace GC-FID per ICH Q3C(R8) Class 3 solvent limits. The compound is routinely coupled to Rink amide AM resin (loading 0.48 mmol/g) at a 5.0 molar equivalent excess with activation by HCTU (4.9 equiv) and N,N-diisopropylethylamine (10.0 equiv) in dry DMF at a reagent concentration of 0.25 M. Double-coupling cycles of 45 minutes each are employed when the subsequent residue is a β-branched amino acid or when real-time UV monitoring at 304 nm indicates a residual free amine peak area exceeding 4% of the preceding deprotection trace, a batch-specific failure mode traced to inadequate resin bed swelling. Downstream manufacturing proceeds on scale-adjusted resin quantities—typically 15 mmol per batch for early toxicology material—with resin-bound peptide chains cleaved using a mixture of TFA/TIS/H₂O (95:2.5:2.5, v/v/v) for 2.5 h under nitrogen, precipitated in cold diethyl ether, and lyophilized. The final crude peptide, bearing an N-methylpyrrole-2-carboxyamide cap at the designated position, is purified by preparative RP-HPLC on a 50 mm ID C18 column with a 1.0%/min acetonitrile gradient and converted to acetate salt form. Table 1 lists the Class 2 residual solvent compliance thresholds most frequently monitored, as the compound’s final crystallization from ethyl acetate/hexane demands rigorous drying at 40 °C under 5 mbar for 18 h to avoid dimethylformamide carryover into the synthesizer fluidic path.

*Class 3 solvent; limit set at 50 ppm per internal specification justified by ICH Q3A risk assessment for parenteral peptide APIs.

When Does N-Methylpyrrole-2-Carboxylate Replace Proline in β-Turn Mimetics?

Replacement of L-proline with 4-(Fmoc-amino)-1-methyl-1H-pyrrole-2-carboxylic acid in linear peptide sequences is exploited during the design of conformationally constrained turn mimetics targeting protein–protein interfaces, particularly where a cis amide geometry must be artificially stabilized without elevating the sequence’s tendency toward diketopiperazine formation. The pyrrole ring imposes a torsional restraint distinct from the pyrrolidine of proline, as its planar aromatic system flattens the ψ angle and the N-methyl substituent forces a trans amide conformation across the preceding residue. Synthesis proceeds on 2-chlorotrityl chloride resin (substitution 1.2 mmol/g) at 0.25 mmol scale to minimize interchain aggregation during coupling of this hydrophobic building block. Activation with HATU (4.5 equiv) in the presence of 2,4,6-collidine (8.0 equiv) in NMP at 50 °C for 20 min has been verified to suppress lactam formation between the free pyrrolic amino group (post-deprotection) and the nascent C-terminal activated ester of the growing chain—a side reaction observed when HBTU is used at ambient temperature. Table 2 documents the coupling efficiencies recorded under three activation regimens, determined by post-deprotection Fmoc quantification at 304 nm using a 1 cm path-length flow cell on the synthesizer. Industry compliance is aligned with ICH Q6A decision tree #3 for synthetic peptides intended as reference standards, requiring peptide identity confirmation by amino acid analysis following 24 h hydrolysis in 6N HCl at 110 °C and mass accuracy within 3 ppm by Q-TOF. The purified mimetic, typically a 12–20 residue monocyclic peptide constrained via side-chain lactam bridges, is lyophilized as a TFA salt and subsequently converted to an acetate salt by ion-exchange chromatography on Dowex® 1×8 resin. Terminal products include intravenously administered antagonists of the Bcl-2 family or MDM2/p53 disruptors requiring serum half-lives exceeding 6 h.

Chelation-Functionalized Oligopeptide Precursors and Their Process-Scale Purification

A 1.0 mmol-scale synthesis of a hexapeptide containing a single 4-(Fmoc-amino)-1-methyl-1H-pyrrole-2-carboxylate residue at the N-terminus was executed on an AAPPTec Focus XC automated synthesizer using a PL-Rink resin (loading 0.39 mmol/g) with a 3.0 equiv symmetric anhydride pre-formed from the Fmoc-pyrrole building block and DIC in DCM/DMF (1:1) at 0.1 M for 15 min at 4 °C. The symmetric anhydride method was elected to circumvent premature deprotonation of the pyrrolic NH by excess tertiary amine, a phenomenon that accelerates an undesired N-acyl urea shift when the carboxylic acid is activated as a uronium salt. Following chain assembly, global deprotection and cleavage released a crude peptide that was precipitated in methyl tert-butyl ether and dissolved in 20 mM ammonium bicarbonate (pH 8.0) prior to incubation with ZnCl₂ (1.5 equiv) for 3 h at 25 °C to generate the zinc-coordinated peptide complex. Process-scale purification employed a 100 mm ID C18 column on a Novasep Hipersep® SYSTEM operating at 400 mL/min with 0.1% TFA–acetonitrile gradient; the metalated fraction eluted at 28.5% acetonitrile and was distinguished from the free peptide by a bathochromic shift of the absorption maximum from 272 nm to 287 nm. Compliance with ISO 17025:2017 for analytical testing of metal content is verified by ICP-MS (Agilent 7900) following microwave digestion in concentrated HNO₃, with a specification of Zn content 1.00 ± 0.05 mol per mol peptide. The terminal product is a white to off-white lyophilized powder intended as an antimicrobial peptide–metal complex active against metallo-β-lactamase-expressing Gram-negative pathogens, stored under argon at −20 °C to prevent metal dissociation.

For the construction of peptide-based hydrogels that rely on π-π stacking interactions to achieve shear-thinning rheological profiles suitable for injectable cell delivery, the incorporation of a 4-(Fmoc-amino)-1-methyl-1H-pyrrole-2-carboxylic acid unit at the hydrophobic terminus of an amphiphilic β-sheet-forming sequence has been adopted to raise the storage modulus (G’) above 800 Pa at 1 rad/s and 1% strain on a TA Instruments Discovery HR-2 rheometer with a 40 mm parallel plate geometry. The peptide is assembled on a 0.1 mmol scale using Fmoc-Gly-Wang resin (loading 0.73 mmol/g), and the heterocyclic building block is coupled as a 4.0 equiv pre-activated HOBt ester (prepared offline with DIC, HOBt, 3.8 equiv each, in DMF at 0 °C for 10 min) to minimize resin esterification by-product. Because the terminal product is destined for cell culture and in vivo biocompatibility screening, the bulk lyophilized peptide is subjected to endotoxin reduction via an anion-exchange chromatography step (CaptoTM Q ImpRes, 5 mL column) that achieves <0.5 EU/mg per USP <85> kinetic chromogenic LAL assay, and residual TFA is exchanged against acetate to a final TFA content below 50 ppm by ion chromatography. The freeze-dried hydrogel precursor peptide is stored in sealed glass vials under vacuum and is reconstituted at 10 mg/mL in PBS (pH 7.4) to form a self-supporting gel within 15 min at 37 °C for 3D encapsulation of mesenchymal stem cells.

If Conformational Locking of Linear Epitopes Is Required for Vaccine Conjugate Design

Antigenic determinant peptides incorporating 4-(Fmoc-amino)-1-methyl-1H-pyrrole-2-carboxylic acid as a rigidifying spacer between the B-cell epitope and the T-helper epitope are manufactured under controlled conditions compatible with EMA/CHMP/VWP/164653/05 guidance on the development of peptide-based vaccines. The synthesis is executed on a 2 mmol scale using a ChemMatrix® PEG resin (loading 0.45 mmol/g) to accommodate the poorly solvated β-sheet aggregation that accompanies the all-hydrocarbon linker sequence. Coupling of the pyrrole monomer is performed with PyBOP (5.0 equiv) and DIEA (12 equiv) in NMP at 45 °C for 30 min, repeated once; incomplete acylation is detected by a residual bromophenol blue indicator spot test on resin beads. The carboxylic acid handle of the fully protected peptide segment is subsequently derivatized on-resin with 6-aminohexanoic acid followed by N-hydroxysuccinimide/DIC to generate a C-terminal NHS ester for conjugation to lysine ε-amino groups of CRM197 carrier protein under 100 mM HEPES buffer (pH 7.2) with 10% v/v DMSO as cosolvent. The unconjugated peptide is removed by diafiltration against 10 mM PBS using a 10 kDa MWCO tangential flow filtration membrane (Pellicon® 3 Cassette). Lot release testing includes identity confirmation by MALDI-TOF MS of the conjugate (mass shift corresponding to 8–12 peptide haptens per carrier), free peptide content <2% by RP-HPLC, and sterility per EP 2.6.1. The final conjugate is presented as a sterile-filtered liquid formulation containing 50 µg/mL of peptide-CRM197 conjugate, 0.5 mg/mL aluminum phosphate adjuvant, and 0.9% sodium chloride, filled in 2 mL Type I glass vials.

Monitoring the Conversion of Resin-Bound Carboxylic Acid to NHS Ester by Colorimetric Assay

Site-specific biotinylation of a diagnostic peptide probe via the C-terminal carboxylate of an inserted 4-(Fmoc-amino)-1-methyl-1H-pyrrole-2-carboxylic acid residue is performed while the N-terminus remains Fmoc-protected to ensure monofunctionalization. The peptide is synthesized on HMPA-PEGA resin (loading 0.2 mmol/g) at 0.05 mmol scale with the pyrrole building block coupled as the penultimate residue using DIC/OxymaPure (6.0 equiv each) in DMF at 25 °C for 45 min. After removal of the 2-chlorotrityl-protecting group on the amino side chain (if present), the C-terminal carboxylic acid is activated to the NHS ester by treatment with N,N’-disuccinimidyl carbonate (10 equiv) and DIEA (20 equiv) in dry acetonitrile for 16 h under nitrogen; conversion is assessed qualitatively by the Kaiser test negative response and quantitatively by cleavage of a small resin aliquot and RP-HPLC analysis showing complete consumption of the acid-bearing peptide peak. The manufacturing line operates under a quality management system certified to ISO 13485:2016, with documented risk management per ISO 14971:2019 for design control of peptide reagents used in in vitro diagnostic immunoassays. The resin-bound NHS ester is reacted with biotinamidohexanoic acid hydrazide (3.0 equiv) in DMSO:DMF (1:4) containing 0.5% triethylamine for 12 h at 25 °C, followed by deprotection and cleavage. The crude biotinylated peptide is purified by semi-preparative HPLC on a 10 mm ID C4 column, and the product fraction is identified by streptavidin-agarose pull-down assay and LC-MS (expected [M+2H]²⁺ mass increment of 226.1 Da per biotin moiety). Terminal products are supplied as lyophilized aliquots of 1.0 mg per vial with a peptide content ≥95% by elemental nitrogen analysis, intended for conjugation to streptavidin-coated magnetic beads in chemiluminescent ELISA platforms.

Resin-bound oligopeptide chains undergo on-bead modification with 4-(Fmoc-amino)-1-methyl-1H-pyrrole-2-carboxylic acid at the focal point of a second-generation Lys₃ dendron scaffold, creating a trivalent presentation platform for receptor clustering studies. The synthesis starts from a Fmoc-β-Ala-Wang resin (loading 0.8 mmol/g) onto which three copies of a tetrapeptide spacer arm terminated in Fmoc-Lys(Fmoc)-OH are assembled divergently. After removal of the N-terminal Fmoc groups, the heterocyclic building block is introduced as a 3.5 equiv excess per amine site using COMU (3.3 equiv) and 2,4,6-collidine (7.0 equiv) in DMSO for 2 h at 30 °C to achieve capping of the three exposed amino termini. Because the intended final product is a ⁶⁴Cu-labeled multimer for PET imaging, the downstream production environment complies with 21 CFR Part 212 current good manufacturing practice for PET drugs, particularly regarding sterile filtration and bacterial endotoxins testing. After cleavage and HPLC purification to >99% purity, the trimeric peptide is conjugated to a DOTA chelator via the pyrrole amino groups following Fmoc removal and subsequent acylation with DOTA-NHS ester in 0.1 M NaHCO₃ buffer (pH 8.5) for 4 h. The metal-free purified conjugate is aliquoted into sterile vials and shipped under a dry ice temperature chain to radiopharmacies where labeling with ⁶⁴CuCl₂ (37 MBq/nmol specific activity) in 0.25 M ammonium acetate (pH 5.5) at 45 °C for 20 min yields the terminal diagnostic radiopharmaceutical. Residual organic solvent levels are controlled within ICH Q3C limits for Class 2 solvents acetonitrile (≤410 ppm) and DMSO (≤5000 ppm) as validated by gas chromatography on the final drug product formulation.