Octahydro cyclopenta[b]pyrrole-2-benzyl carboxylate hydrochloride, supplied under the designation Recemiac, constitutes the racemic mixture of (2R*,3aR*,6aS*)-benzyl octahydrocyclopenta[b]pyrrole-2-carboxylate hydrochloride. This compound is employed primarily as a conformationally constrained proline isostere in solution- and solid-phase peptide synthesis, where its fused bicyclic skeleton restricts the ψ and φ dihedral angles of the pyrrolidine ring to values approximating those found in type VI β-turn mimetics. The benzyl ester serves as a carboxyl-protecting group removable under neutral hydrogenolysis conditions, while the hydrochloride salt form ensures a stable, free-flowing crystalline powder with a melting point of 168–172 °C (decomposition) and a bulk density of 0.45 ± 0.05 g/cm³. Unlike the corresponding free base, which is a hygroscopic oil at ambient temperature, the hydrochloride demonstrates indefinite storage stability at −20 °C under argon and is compatible with automated solid-phase synthesizers operating with anhydrous dimethylformamide (DMF) or N-methyl-2-pyrrolidone (NMP) as reaction solvents. Certification includes batch-specific HPLC purity (method based on USP 〈621〉 Chromatography), residual solvent analysis per ICH Q3C, and water content determined by coulometric Karl Fischer titration in accordance with ASTM E203. Typical lot release accepts a purity of ≥98.0% (area normalization, UV detection at 210 nm) and a single impurity ceiling of 0.5% for the des-benzyl carboxylic acid.
Physical Properties and Analytical Specifications
Characterization is performed on a representative lot using an Agilent 1260 Infinity II HPLC system equipped with a Phenomenex Kinetex C18 column (4.6 × 150 mm, 2.6 µm) and a mobile phase of 0.1% v/v trifluoroacetic acid in water/acetonitrile (60:40 v/v) at a flow rate of 1.0 mL/min. The retention time for Recemiac under these conditions is 8.3 ± 0.1 min. Enantiomeric purity is not controlled because the product is specified as the racemate; however, chiral HPLC on a Chiralpak IA column confirms the absence of enrichment beyond 52:48 er. The residual benzyl alcohol content is typically below 300 ppm, and the loss on drying at 60 °C under vacuum for 4 h is less than 0.2%. Elemental analysis corresponds to C₁₅H₂₀ClNO₂ within ±0.3% for C, H, N. The material is soluble in DMF, dimethyl sulfoxide, and methanol (>50 mg/mL), but poorly soluble in ethyl acetate and diethyl ether.
| Parameter | Method | Acceptance Criterion |
|---|---|---|
| Assay (HPLC, anhydrous basis) | In-house SOP 04-12, based on USP 〈621〉 | 98.0–102.0% |
| Water content | ASTM E203 (coulometric KF) | ≤0.5% |
| Residual benzyl alcohol | GC-FID, ICH Q3C | ≤500 ppm |
| Residual ethyl acetate | GC-FID | ≤1000 ppm |
| Chloride content (ion chromatography) | USP 〈221〉 | 15.8–16.8% |
| Melting point | USP 〈741〉 Capillary | 168–172 °C (dec.) |
The hydrochloride salt readily exchanges under basic aqueous workup, regenerating the free base, which must be avoided if crystallinity is required for isolation. When neutralized with saturated NaHCO₃ and extracted into ethyl acetate, the free base decomposes within 48 h at room temperature, forming the hydrolysis product and benzyl alcohol; thus, in situ neutralization is restricted to immediately subsequent coupling steps.
What Limits This Reagent’s Utility in Solid-Phase Peptide Synthesis?
On a Symphony X automated peptide synthesizer (Gyros Protein Technologies) operating with a 0.1 mmol scale Fmoc-strategy protocol, Recemiac HCI is activated with 4.0 eq of HATU and 8.0 eq of diisopropylethylamine (DIPEA) in DMF. The critical processing conflict arises from the base lability of the α-proton of the activated ester. At ambient temperature (22–25 °C), epimerization at C-2 proceeds at 0.8–1.2% per hour, as monitored by LC-MS of the crude peptide after cleavage. When the internal temperature of the reaction vessel exceeds 8 °C for longer than 15 min during preactivation, the diastereomeric excess of a model tripeptide (Fmoc-Ala-Recemiac-Phe-resin) drops below 90%. Consequently, the validated protocol requires pre-cooling of the amino acid solution to 0–2 °C and use of a jacketed reaction vessel with a circulating chiller (Julabo FL300) set to −5 °C. With these controls, epimerization is suppressed to <0.3% over a 2 h coupling cycle. However, this low-temperature regime increases solution viscosity, leading to incomplete resin bed penetration when the resin loading exceeds 0.6 mmol/g. In such cases, double-coupling with a 30 min intermediate drain and fresh reagent charge restores coupling efficiency to >99% as determined by the Fmoc-release UV monitor at 301 nm.
When Scale-Up Exceeds 5 Molar Equivalents of Coupling Agent
Transferring the coupling from a 0.1 mmol synthesizer to a 100 mmol batch reactor (jacketed 2 L glass vessel with overhead stirring at 200 rpm) reveals a non-linear dependency between reagent stoichiometry and racemization rate. Using HATU at 5.0 eq and DIPEA at 10.0 eq, the exotherm upon base addition generates a thermal spike to +12 °C within 45 s even with jacket set to −10 °C. This transient violates the ≤8 °C window and results in 4.2% of the D-epimer in the isolated product. The conflict is mitigated by splitting the base addition into three equal portions at 5‑min intervals, each portion preceded by a 2‑min hold at 0 °C. Additionally, substituting HATU with PyBOP (5.0 eq) and reducing DIPEA to 6.0 eq eliminates the sharp exotherm because the phosphonium salt activation proceeds via an acyloxyphosphonium intermediate of lower energy, yet it requires extension of the coupling time to 3 h to reach the same 99% conversion. The benzyl ester remains intact under these conditions, confirmed by the absence of the debenzylated by-product at <0.1%.
Benzyl Ester Exhibits a 12-Hour Half-Life in 4 M HCl/Dioxane at 25°C
Compared to the methyl ester analog, the benzyl ester of Recemiac demonstrates superior stability toward acidic cleavage, which is exploited in orthogonal protecting group strategies. In 4 M HCl in dioxane (Acros Organics) at 25 °C, the benzyl ester hydrolyzes with a half-life of 11.7 h, whereas the methyl ester reaches 50% conversion in 1.8 h under identical conditions. This difference permits selective removal of tert-butoxycarbonyl (Boc) or trityl groups in the presence of the benzyl ester, provided the reaction is terminated within 4 h. Real-time monitoring by ReactIR (Mettler Toledo) with a diamond ATR probe tracking the carbonyl stretching band at 1734 cm⁻¹ confirms no detectable ester cleavage during a 3 h Boc deprotection. The hydrochloride salt does not interfere with the HCl concentration, as additional chloride ion from the substrate accounts for less than 0.05 M increase, well within the buffer capacity of the acid solution. However, prolonged exposure (>24 h) leads to partial ring-opening of the bicyclic amine, generating the corresponding amino alcohol, which is detected by LC-MS as [M+H]+ = 248.2.
Storage of Recemiac HCI at 2–8 °C in tightly sealed amber glass vials under nitrogen prevents moisture ingress; equilibrium moisture content at 60% RH and 25 °C reaches 0.8% w/w within 48 h, which is still within the specification but can cause clumping in automated dispensing units. For long-term inventory, −20 °C with desiccant is mandated. Compatibility: the hydrochloride should not be directly combined with strong aqueous bases (e.g., NaOH), as instantaneous dehydrochlorination precipitates the free base as a gum that occludes unreacted starting material and resists filtration. When a basic medium is required for coupling, the salt is pre-neutralized in situ with exactly 1.0 eq of DIPEA relative to the HCl content; excess base accelerates racemization.
| Property | Benzyl Ester (Recemiac) | Methyl Ester HCl |
|---|---|---|
| Half-life in 4 M HCl/dioxane (25 °C) | 11.7 h | 1.8 h |
| Hydrogenolysis lability (Pd/C, 1 atm H₂) | Cleaved in 2 h | No reaction |
| Physical form at 25 °C | White crystalline solid | Off-white hygroscopic solid |
| Solubility in DMF (25 °C) | 52 mg/mL | 81 mg/mL |
| Epimerization rate under HATU/DIPEA (0 °C) | 0.3%/h | 0.2%/h |
In continuous flow hydrogenolysis for benzyl ester removal, a ThalesNano H-Cube Pro reactor equipped with a 30 mm CatCart cartridge containing 10% Pd/C delivers full deprotection of a 0.05 M solution of Recemiac in methanol at 25 °C and 1 mL/min flow rate. The hydrochloride form shows no catalyst poisoning over 8 h of continuous operation, as monitored by back-pressure stability at 10 bar. In contrast, the free base tends to form colloidal palladium aggregates that raise back-pressure above 30 bar and necessitate cartridge replacement after 3 h. Thus, the salt form is directly compatible with immobilized catalyst beds without pre-neutralization, streamlining the overall process sequence.
How Does the Hydrochloride Salt Impact Catalyst Poisoning in Pearlman’s Catalyst Systems?
When Pearlman’s catalyst (20% Pd(OH)₂/C) is employed for hydrogenolysis of the benzyl ester in a batch Parr shaker apparatus (Model 3910, 500 mL vessel, 50 psi H₂), the chloride counterion creates a subtle but measurable effect on catalyst turnover. Under identical substrate-to-catalyst ratios (10:1 w/w), the hydrochloride substrate exhibits a turnover frequency (TOF) of 1.2 mol H₂/mol Pd·min, compared to 1.8 mol H₂/mol Pd·min for the free base under a blanket of triethylamine. This reduction is attributed to reversible adsorption of chloride onto palladium surface sites, confirmed by XPS analysis showing 1.8 at% Cl on used catalyst. The poisoning is not irreversible; washing the catalyst with 0.1 M ammonium formate in methanol restores 93% of the initial activity. Despite lower TOF, the hydrochloride route is preferred due to elimination of a separate neutralization step and avoidance of amine-induced racemization during solvent evaporation.
Use of Recemiac in diastereomeric salt resolution for preparation of enantiopure octahydrocyclopenta[b]pyrrole-2-carboxylic acid is documented; the hydrochloride is converted to the free base and treated with 1.0 eq of (1S)-(+)-10-camphorsulfonic acid in ethyl acetate/ethanol (95:5 v/v). Precipitation occurs at −20 °C over 72 h, yielding the (2S,3aR,6aS)-enantiomer salt with 98.5% ee after two recrystallizations. The racemic mixture therefore serves as an economical input for both laboratory-scale asymmetric synthesis campaigns and production-scale enantiomer separation.
The compound’s difference from suppliers offering only the free base or unprotected acid is stark: Recemiac’s hydrochloride form obviates the need for Schlenk-line handling of an oxygen-sensitive, viscous oil, reduces activation energy for storage, and provides a defined stoichiometric starting point for automated synthesis. In direct comparison with the analogous methyl ester hydrochloride, Recemiac’s benzyl protective group enables a fully orthogonal deprotection scheme alongside Fmoc/tBu strategies, a distinction that is critical when synthesizing complex cyclic peptides containing acid-sensitive side-chain functionalities. Published data for this specific configuration is limited to in-house development reports and peer-reviewed studies on bicyclic proline analogues; the operational boundaries described here have been validated on a reactor scale of 0.5–200 mmol and may require adjustment for ton-scale manufacturing.