1,2-Pyrrolidinedicarboxylic Acid, 4-Methylene-, 1-(1,1-Dimethylethyl) Ester, (2S)-

Within the scope of Boc/Benzyl solid-phase peptide synthesis strategies, (2S)-1-(tert-butoxycarbonyl)-4-methylenepyrrolidine-2-carboxylic acid is pre-activated as the 1-hydroxy-7-azabenzotriazole (HOAt) ester in anhydrous dimethylformamide. For a 0.25 mmol resin substitution on a hydroxymethylphenylacetamidomethyl (PAM) support, 1.0 mmol of the amino acid (4 equiv.) is combined with 0.95 mmol HATU and 2.0 mmol N,N-diisopropylethylamine (DIEA) at 0–5 °C for 3 minutes prior to transfer to the resin-filled vessel; the reaction is allowed to proceed for 45 minutes at 35 °C under gentle nitrogen agitation. Double coupling is mandated for sequences where the incoming residue bears a sterically hindered N-terminal valine or an α,α-disubstituted amino acid. Residual water content in the solvent is maintained below 50 ppm by Karl Fischer titration to suppress premature Boc deprotection during the activation step, and the entire process is documented under the batch record requirements of ICH Q7 Section 7.3 for yield-adjusted input of key starting materials. After final TFA-mediated cleavage, residual trifluoroacetic acid is reduced below 100 ppm per USP <467> Method IV by repeated diethyl ether trituration, and elemental impurities comply with ICH Q3D Option 1 limits as verified by inductively coupled plasma mass spectrometry (ICP-MS) on the lyophilized crude. The resulting peptide, typically incorporating the 4-methyleneproline residue as a turn-inducing element, is dissolved in pH 7.8 ammonium bicarbonate buffer, folded under oxidative conditions when cysteine residues are present, and lyophilized to an amorphous powder. Finished peptide formats include sterile-filtered lyophilisates filled in Type I borosilicate vials for preclinical in vivo pharmacokinetic studies, or spray-dried encapsulation-grade peptide–PLGA microparticles suitable for sustained-release injectable formulations.

Why Does Counterion Selection During the Post-Coupling Bicarbonate Wash Shift Epimerization Past 0.8%?

Extensive in-process differential scanning calorimetry (DSC) monitoring of 50 mmol-scale batch syntheses has revealed that the choice of counteranion during the post-coupling sodium bicarbonate wash exerts a measurable influence on the optical purity of the growing peptide chain. When 0.2 M sodium carbonate is substituted with potassium carbonate at pH 9.2, the D-enantiomer content of the released (2S)-4-methyleneproline residue increases from 0.3% to 0.8% as determined by Marfey’s test (LC-MS, C18 column, gradient acetonitrile/0.1% TFA, LOD 0.05%). Compliance with ICH Q6A decision tree #3 for chiral purity requires a validated acceptance limit of not more than 1.0% D-isomer in the final active pharmaceutical ingredient intermediate, directly linking this wash step to regulatory release. Optimal coupling performance is achieved with 3.5 equivalents of the Boc amino acid relative to resin loading, together with 3.3 equivalents of HBTU and 7.0 equivalents of DIEA, at a 0.2 M concentration in DMF; exceeding 4.0 equivalents leads to a nonlinear decrease in isolated yield due to formation of unreactive symmetric anhydride dimers that precipitate in the solvent delivery lines of automated synthesizers. Couplings are executed on a CEM Liberty Blue microwave synthesizer operating at 50 W power and 75 °C for 4 minutes, with a post-reaction hold time of 30 seconds to prevent thermal degradation of the acid-labile side-chain protecting groups. The final products are typically 18- to 24-mer peptides containing a single 4-methyleneproline incorporation, folded into single-conformation structures verified by 2D ¹H-¹⁵N HSQC, and used as biased ligands for the chemokine receptor CXCR4 in functional cAMP inhibition assays with an EC₅₀ shift relative to the native SDF-1α ligand.

Aqueous thiol-ene conjugation of the terminal vinyl group in (2S)-1-Boc-4-methyleneproline with thiolated polyethylene glycol (PEG-SH, 2 kDa) proceeds with quantitative conversion when a 1:1.05 molar ratio of amino acid to thiol is dissolved in degassed pH 7.4 phosphate-buffered saline containing 0.1 mol% lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) as photoinitiator and irradiated at 365 nm (10 mW/cm²) for 90 minutes. The conjugation is compliant with the biocompatibility assessment framework outlined in ISO 10993-5 when the product is intended for medical device diagnostic applications, and residual photoinitiator is removed by dialysis against a 1 kDa MWCO membrane until absorbance at 380 nm falls below 0.005 AU. The resulting PEGylated amino acid is purified by reversed-phase flash chromatography on a C18 cartridge using a step gradient of 5–95% acetonitrile in 0.1% aqueous formic acid, and the isolated conjugate retains the Boc protecting group, allowing direct incorporation into the N-terminus of a matrix metalloproteinase-2/9 (MMP-2/9) cleavable peptide sequence via standard HBTU/DIEA activation. Finished diagnostic probes are composed of the PEG-(4-methyleneproline)-peptide domain linked to a Förster resonance energy transfer (FRET) pair—typically 5-FAM as donor and QXL^™ 520 as quencher—and exhibit a 12-fold fluorescence activation upon 2 nM MMP-9 cleavage as determined by real-time fluorometry in a 96-well format per CLSI EP17-A2 guidelines for limit-of-blank establishment.

PROTAC Linker Rigidification Quantified via Cellular Thermal Shift Assay

Incorporation of the (2S)-4-methyleneproline scaffold into the alkyl–PEG linker region of cereblon-based PROTACs has been systematically correlated with ternary complex stabilization using cellular thermal shift assays (CETSA) in HEK293T cells expressing BRD4^BD1. When the Boc-protected amino acid is introduced as a central rigidifying element, the observed melting temperature shift (ΔT_m) for the target–ligase complex increases by 2.8–4.1 °C relative to a fully flexible hexaethylene glycol linker, indicating enhanced cooperative binding. The synthesis protocol for the linker module begins with coupling of 1.2 equivalents of (2S)-1-Boc-4-methyleneproline to the amine-terminated PEG chain using 1.1 equivalents of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and 1.5 equivalents of 1-hydroxy-7-azabenzotriazole (HOAt) in anhydrous dichloromethane at 0 °C to room temperature over 16 hours. Boc deblocking is subsequently performed with 4 M HCl in 1,4-dioxane (1 hour, 25 °C), and the resulting hydrochloride salt of the linker-amine is neutralized with 1.05 equivalents of DIEA prior to coupling with the VHL ligand VH032 acid in the presence of HATU. Residual palladium and heavy metal levels in the final PROTAC intermediate are controlled below 10 ppm as per the permitted daily exposure limits in ICH Q3D Table A.2.2, and the overall purity target is set at ≥98.5% by HPLC (220 nm) with a single impurity threshold of 0.15%. Preparative purification is achieved on a C18 column (250 × 50 mm, 10 µm) with a linear gradient of 30–70% acetonitrile in water over 45 minutes, and the isolated product is lyophilized to a white powder. Terminal PROTAC molecules containing this rigidified linker achieve a degradation efficiency DC₅₀ in the low nanomolar range (1.5–8.7 nM) against BRD4 after 12‑hour treatment, with no observable degradation of the BRD2 or BRD3 off-targets at 1 µM, as validated by quantitative Western blot and normalized to vinculin loading control.

Reported catalyst-loading optimizations for (S)-4-methyleneproline in asymmetric aldol additions between 4-nitrobenzaldehyde and acetone in DMSO at 25 °C identify 20 mol% as the minimum loading that secures both >85% conversion and 92% enantiomeric excess (ee) within 24 hours. The free amino acid catalyst is generated by treating the (2S)-1-Boc precursor with a mixture of trifluoroacetic acid and triisopropylsilane (95:5 v/v) for 2 hours at 25 °C, followed by precipitation from ice-cold diethyl ether and vacuum drying for 12 hours at 40 °C. For kilogram-scale catalyst preparation, residual solvent levels are required to fall below 600 ppm for methylene chloride and below 720 ppm for N,N-dimethylformamide per ICH Q3C options for Class 1 and Class 2 residual solvents, with headspace GC verification performed on each batch. The catalyst weight-% added to the reaction mixture is calculated on the basis of the aldehyde limiting reagent; increasing the loading to 30 mol% accelerates the reaction to 8‑hour completion but introduces detectable product racemization due to base-catalyzed retro-aldol pathways, while loadings below 10% result in incomplete conversion (<50%) even after 48 hours. Reaction progress is monitored by ¹H NMR using mesitylene as an internal standard, and the product is isolated by aqueous workup and silica plug filtration (ethyl acetate/hexane 1:4). Enantiomeric excess is determined by chiral stationary-phase HPLC on a Chiralpak AD‑H column (250 × 4.6 mm, 5 µm), mobile phase hexane/2‑propanol 90:10 at 1.0 mL/min, with UV detection at 254 nm; system suitability requires a resolution factor R_s > 2.0 between enantiomers per USP <621> guidelines. The resulting chiral β‑hydroxy ketone serves as a key building block for statin side-chain synthesis and has been employed as an intermediate in the preparation of atazanavir-analog aspartic protease inhibitors, where the stereochemical integrity of the hydroxy group directly determines the inhibitory K_i value.

When Orthogonal Protecting Group Patterns Are Required in the Synthesis of Aldehyde-Functionalized Peptide Macrocycles

Maintaining the acid-labile Boc group while performing palladium-catalyzed hydrogenolysis of a C-terminal benzyl ester is allowed by the steric and electronic environment of the tert-butyl carbamate in (2S)-1-Boc-4-methyleneproline, and this orthogonality has been exploited to prepare fully deprotected side-chain aldehyde handles without premature exposure of the N-terminal amine. In a typical procedure, 1.05 equivalents of the Boc-amino acid benzyl ester are dissolved in tetrahydrofuran, and 10% Pd/C (50% wet paste, 0.1 equiv. by weight) is added under nitrogen. Hydrogen gas is introduced at 1 atm pressure and the mixture is stirred vigorously for 3 hours; quantitative ester cleavage is confirmed by TLC (silca gel 60 F₂₅₄, ethyl acetate/hexane 1:1, visualised with ninhydrin). Process safety documentation aligns with ATEX 2014/34/EU for explosive atmospheres, and the hydrogenation equipment is purged with nitrogen to an oxygen level below 2% before hydrogen introduction. After filtration through celite and solvent evaporation, the free carboxylic acid is obtained in a purity suitable for direct coupling to an amino alcohol linker without further purification, eliminating the need for aqueous acid extraction that could cause premature Boc cleavage. The compound is subsequently utilized in the assembly of aldehyde-containing peptide macrocycles via a tandem oxime ligation and on-resin cyclization strategy, where the 4-methyleneproline residue acts as a helix-breaking kink that positions the reactive aldehyde within a 2.5 Å distance to an aminooxyacetyl side chain. Terminal macrocyclic products range from 14- to 19-membered rings and have been profiled as inhibitors of protein–protein interactions in the p53–MDM2 system, with SPR-derived K_D values in the low micromolar range; batch records for such research-grade intermediates comply with ISO 9001:2015 documentation standards, and each shipment is accompanied by a certificate of analysis reporting specific rotation [α]_D²⁰ at five concentrations and residual palladium analysis via graphite furnace atomic absorption spectroscopy with a detection limit of 2 ppm.