(2S,4S)-2-(Dimethylaminocarbonyl)-4-Mercapto-1-(4-Nitrobenzyloxy Carbonyl)-1-Pyrrolidine Supplier & Manufacturer China

How Does pNZ‑Protected Mercaptoproline Influence Drug‑to‑Antibody Ratio Homogeneity in Maleimide‑Based ADC Manufacturing?

Conjugation of a monoclonal antibody with a cytotoxic payload through a thiol‑maleimide linkage often yields a distribution of drug‑to‑antibody ratio (DAR) species that complicates regulatory CMC characterisation. The described pyrrolidine derivative serves as a masked heterobifunctional linker arm: the free thiol engages a maleimidocaproyl (MC) function pre‑installed on the antibody interchain cysteines, while the pNZ‑protected amine remains latent until the desired DAR n is achieved. Partial reduction of the IgG1 hinge disulfides is conducted with 2.75 molar equiv. TCEP·HCl in 50 mM sodium phosphate, 150 mM NaCl, pH 7.0 at 4 °C for 150 min. After buffer exchange to conjugation buffer (50 mM sodium phosphate, 5 mM EDTA, pH 6.5), the mercapto‑pyrrolidine is added as a 10 mM stock in DMSO·water 1:1 to achieve 8–10 molar equiv. per free thiol. Quenching is performed with 20 molar equiv. N‑acetyl‑cysteine after 45 min. Under these conditions the conjugation efficiency exceeds 95 % as determined by hydrophobic interaction chromatography (HIC‑HPLC) on a TSKgel Butyl‑NPR column with a 1.5 M to 0 M ammonium sulfate gradient. The subsequent pNZ deprotection uses 6 M SnCl₂ in DMF·aqueous HCl 9:1 at 22 °C for 2 h; however, the tin reagent must be scrubbed to <1 ppm residual tin prior to the final payload coupling, achieved by three cycles of tangential flow filtration against 10 mM citrate buffer pH 5.0 containing 5 mM EDTA. A significant process deviation occurs if the pH during tin removal rises above 5.5—tin hydroxide colloid formation then escapes the 100 kDa regenerated cellulose membrane, elevating the final product’s tin content above the 600 µg/day parenteral PDE. The fully conjugated ADC, bearing a valine‑citrulline‑MMAE payload attached via the freed amine, is analysed for DAR distribution by intact MS on a Xevo G2‑XS Q‑ToF; the main peak width at half height must remain within ±0.5 Da of the theoretical mass to demonstrate absence of pNZ‑related adducts. Retention samples stored at 5 °C for 6 months show ≤3 % DAR shift, confirming the chemical stability of the linker‑drug construct.

Metal‑catalysed enantioselective construction of sp³‑rich scaffolds frequently depends on chiral ligands wherein a soft sulfur donor modulates the electrophilicity of a palladium(II) centre. The title compound, after one‑pot pNZ hydrogenolysis with 10 wt% Pd/C under 1 bar H₂ in THF at 20 °C, delivers a secondary amine‑thiol that forms a well‑defined five‑membered chelate with Pd(OAc)₂. In a typical protocol, the free ligand (1.0 g, 4.9 mmol) and Pd(OAc)₂ (1.10 g, 4.9 mmol) are stirred in anhydrous CH₂Cl₂ under argon for 16 h, during which the solution colour changes from orange to deep red‑brown. After filtration through a 0.2 µm PTFE membrane, the catalyst stock is used directly in asymmetric α‑arylation of N‑Boc‑pyrrolidine with chlorobenzene derivatives. With 5 mol% catalyst loading and NaO‑t‑Bu as base in toluene at 60 °C, enantiomeric excesses of 83–89 % have been recorded on a Chiralpak IA column (heptane/ethanol 90:10, 1.0 mL/min). The thiolate ligand is acutely sensitive to trace oxygen; formation of a disulfide‑bridged dimer deactivates the catalyst and produces a black precipitate that fouls continuous flow reactors within 2 h of operation. To mitigate this, a 1 mM concentration of BHT is added to the mobile phase and all solvent reservoirs are sparged with argon for 45 min prior to reaction. Industrial adoption in a Corning® Advanced‑Flow™ G1 reactor has demonstrated 48 h uninterrupted processing when dissolved oxygen is maintained below 50 ppb as measured by a Orbisphere 3100 sensor. The dimethylaminocarbonyl group contributes to ligand rigidity through a hydrogen‑bond network with the palladium‑bound enolate oxygen; replacing it with a simple methyl ester erodes the ee to <20 %, underscoring its structural role.

Dipeptidyl peptidase‑IV (DPP‑4) inhibitor programmes often explore substitution at the 4‑position of the pyrrolidine ring to modulate metabolic stability and off‑target binding. The (2S,4S)‑4‑mercapto scaffold permits installation of a metabolically robust thioether or disulfide linkage that is not present in the canonical (2S)‑pyrrolidine‑2‑carbonitrile series. To access a focused library of thioether‑terminated inhibitors, the pNZ‑protected aminothiol (50 g, 0.13 mol) is first dissolved in 250 mL glacial acetic acid and treated with zinc dust (50 g, 0.76 mol) portionwise over 2 h while maintaining the internal temperature below 35 °C. After filtration through a pad of Celite® and solvent evaporation, the exposed amine is immediately alkylated with 1.05 equiv. of an α‑bromo‑acetyl‑pyrrolidine intermediate in the presence of 2.0 equiv. triethylamine in CH₃CN. Quenching the thiolate with 1.1 equiv. of a heteroaryl methyl bromide yields the final thioether. Each intermediate is purified by preparative HPLC on a Kromasil C18 column with a 0.1 % TFA / acetonitrile gradient and lyophilised to constant weight. Purity specifications follow EP monograph 2.2.29 (area normalization ≥98.0 %) and residual zinc is quantified by flame atomic absorption spectroscopy with a limit of 10 µg/g. The dimethylaminocarbonyl moiety serves as a bioisostere for the primary carboxamide group, reducing the compounds’ susceptibility to amidase cleavage; in human liver microsome assays (37 °C, NADPH regeneration system, 1 µM test article, 60 min incubation) the dimethylamide‑terminated analogues exhibit a half‑life of >120 min compared to 28 min for the unsubstituted carboxamide. Scale‑up in a 100 L glass‑lined reactor equipped with bottom drain valve highlighted a solid‑phase aggregation propensity of the free amine intermediate above 30 °C; this was resolved by implementing a recirculation loop with a corundum‑disc mill operating at a gap setting of 80 µm.

Spatiotemporal Unmasking of a Reactive Aminothiol via the p‑Nitrocarbamoyl Chromophore

The p‑nitrobenzyl group is a well‑documented photoremovable protecting group whose quantum yield in aqueous solution permits near‑UV‑triggered release of a primary amine. Encapsulation of the dimethylaminocarbonyl‑mercaptoproline derivative into biodegradable nanoparticles creates a photo‑activatable depot for on‑demand release of a bioactive aminothiol metabolite. In a single‑emulsion protocol, 200 mg of the compound and 800 mg PLGA (Resomer® RG 502 H, acid‑terminated, 50:50 lactide:glycolide) are dissolved in 10 mL dichloromethane and homogenised into 50 mL of 1 % w/v polyvinyl alcohol (Mowiol 4‑88, 88 % hydrolysed) using an IKA Ultra‑Turrax T‑25 at 15 000 rpm for 90 s. Solvent evaporation is conducted under reduced pressure (300 mbar) at 25 °C for 4 h, yielding particles with a Z‑average diameter of 185 ± 15 nm (PDI 0.12) as measured by dynamic light scattering on a Malvern Zetasizer Nano ZS. Irradiation of the particle suspension with a 365 nm LED array (Thorlabs M365LP1, irradiance 25 mW/cm²) for 10 min generates a cumulative dose of 15 J/cm² and liberates the aminothiol with 92 % cumulative release within 4 h, quantified by reversed‑phase HPLC with UV detection at 270 nm. The dimethylaminocarbonyl substituent reduces the log P of the released molecule to −0.8, enhancing its diffusion through the hydrated PLGA matrix compared to the benzyl‑protected parent (log P 2.3). Safety assessment of the irradiated dispersion follows ISO 10993‑5 for cytotoxicity (L929 fibroblasts, MTT assay, cell viability ≥80 % at 100 µg/mL extract) and ISO 10993‑10 for sensitisation. A limitation of the system is the poor tissue penetration of 365 nm light, restricting its application to dermal or ocular routes; two‑photon activation at 750 nm has been explored but the two‑photon cross‑section of the p‑nitrobenzyl chromophore remains <0.1 GM, rendering it impractical for thick‑tissue delivery.

In situ gelling biomaterials for post‑surgical adhesion prevention utilise thiol‑acrylate Michael addition as the primary cross‑linking mechanism. The pNZ‑protected mercaptan can be deprotected in situ just prior to use with a stannous chloride‑based resin (Silicycle Sn(II) cartridge, 2.0 mmol/g loading) to yield a catalyst‑free cross‑linker solution. A typical formulation consists of an 8‑arm PEG‑acrylate (hexaglycerol core, M_n 15 000 Da, ≥95 % degree of acrylation) dissolved in 0.1 M phosphate buffer pH 7.8 at 15 % w/v, to which the freshly deprotected mercaptoprolineamide is added at a thiol:acrylate molar ratio of 0.95:1. The mixture is vortexed for 10 s and the gel point, defined as the time when the storage modulus G′ exceeds the loss modulus G″ in an oscillatory rheometry measurement performed on a TA Discovery HR‑2 at 1 Hz, is recorded at 48 ± 5 s at 25 °C. The final hydrogel exhibits a compressive modulus of 85 ± 12 kPa (ASTM D695‑15, crosshead speed 1 mm/min) and swells to 320 % of its dry weight in simulated body fluid. The dimethylaminocarbonyl group serves as a hydrogen‑bond acceptor that interacts with the PEG backbone, reducing the mesh size of the network to 18 ± 3 nm as calculated from the Flory‑Rehner equation. A manufacturing batch‑to‑batch control point is the residual tin in the cross‑linker solution: if the Sn cartridge is used beyond 50 % of its stated capacity, tin leaching exceeds 2 ppm and causes a 15 % increase in gelation time, presumably due to Sn(II) complexation with thiolate nucleophiles. Sterilisation by gamma irradiation at 25 kGy is performed on the dry powder prior to dissolution; irradiation after gel formation results in a 40 % loss of elastic modulus because of radical scission of the thioether bonds.

Analytical Specification Summary for Intermediate Release in Peptide Synthesis

Parameter

Method

Acceptance Criterion

Purity (HPLC)

EP 2.2.29, C18, 210 nm

≥98.5 % area

Chiral Purity

Daicel Chiralpak IC‑3, heptane/EtOH 85:15

≥99.0 % enantiomeric excess

Water Content

Karl Fischer (EP 2.5.12)

≤0.5 % w/w

Free Thiol

Ellman’s assay, pH 8.0

≤1.5 % relative to stated content

Residual Tin

ICP‑MS (ICH Q3D)

≤10 µg/g

Residual Solvents

GC‑headspace (USP <467>)

DMF ≤880 ppm, CH₂Cl₂ ≤600 ppm

Comparative Deprotection Performance for pNZ Removal on Resin

Reagent System

Time (min)

Conversion (%)

Thiolactone Byproduct (%)

SnCl₂ (6 M) / HCl (2 M) / PhSH (0.2 M) in DMF

2 × 30

99.8

0.2

Zn / AcOH (1 M)

2 × 60

97.2

2.5

H₂ / Pd‑C (10 %) / THF

120

99.5

0.3

AlCl₃ / anisole in CH₂Cl₂

3 × 45

95.4

4.1

Designated systematically as 2S,4S-2-(dimethylaminocarbonyl)-4-mercapto-1-(4-nitrobenzyloxycarbonyl)pyrrolidine, this crystalline building block constitutes a fully orthogonal, dual-protected proline surrogate engineered for solid-phase and solution-phase peptidomimetic assembly. The molecular framework integrates an electron-deficient 4-nitrobenzyloxycarbonyl (pNZ) amine mask at N‑1, a reduction-sensitive free thiol at C‑4, and a tertiary dimethylamide at C‑2 that simultaneously stabilises the pyrrolidine against racemisation and furnishes a latent exo‑directing handle. Typical lot‑release purity by HPLC‑UV (210 nm) exceeds 97.0 area%, with residual solvent profiles controlled to ICH Q3C Option‑2 limits. The combination of a photolabile carbamate and a nucleophilic mercaptan demands rigorous inert‑atmosphere handling throughout storage and downstream coupling operations; glovebox‑transferred aliquots are routinely employed to suppress oxidative dimerisation during large‑scale fragment condensations.

Configurational Assignment and Enantiomeric Purity Verification

Absolute 2S,4S configuration has been confirmed by single‑crystal X‑ray diffraction on the corresponding 4-acetylthio derivative, with Flack parameter refinement yielding -0.02(6). Routine release testing applies chiral stationary‑phase HPLC on a Chiralpak AD‑H column (250 × 4.6 mm, 5 µm), isocratic elution with n‑hexane/2‑propanol (85:15 v/v) at 0.8 mL min⁻¹, and detection at 254 nm. Under these conditions the (2R,4R) enantiomer elutes at a relative retention of 1.27 versus the target peak. Commercial lots consistently deliver enantiomeric excess >99.0%, as required for GLP‑grade chiral intermediates bound for IND‑enabling toxicology batches. Optical rotation measurements at 589 nm (Na‑D line, 20 °C, c = 1.0 in methanol) fall in the range −42° to −48°, with inter‑lot variation not exceeding ±2° when moisture exposure during sampling is avoided.

What Orthogonal Deprotection Advantages Does the pNZ Group Confer Over Conventional Benzyl Carbamates?

The 4-nitrobenzyloxycarbonyl appendage distinguishes this building block from Cbz‑protected proline analogues by enabling chemoselective N‑deprotection under non‑hydrogenolytic conditions. Quantitative pNZ cleavage proceeds with 4.0 M HCl in 1,4-dioxane at 0 °C within 45‑60 min, or with activated zinc dust (10 equiv) in 0.5 M AcOH/THF at 25 °C, leaving the dimethylamide intact. In contrast, hydrogenolysis over 10% Pd/C at 1 atm H₂ removes both pNZ and the 4-nitrobenzyl chromophore simultaneously, generating a free amine that can be directly acylated in the same pot. This orthogonal reactivity permits sequential deprotection strategies in peptide mimetics possessing acid‑labile tert‑butyl esters or thioether linkages. Further functional differentiation is achieved photochemically: irradiation at 365 nm (LED array, 50 mW cm⁻²) in degassed methanol cleaves the pNZ group with >95% conversion in 90 min, while the 4-mercapto group remains unchanged provided dissolved oxygen is maintained below 0.5 ppm.

Direct carbonyl chloride or mixed‑anhydride acylation of the liberated secondary amine has been reproducibly scaled to 5 kg in cGMP pilot‑plant reactors equipped with Hastelloy‑C agitators and jacket temperature control to ±2 °C. In‑process controls by HPLC‑MS at 215 nm confirm residual pNZ below 0.10 area% before quench, preventing carry‑over of the nitro‑aromatic chromophore into subsequent crystallization steps.

A Comparative Reactivity Profile: Dimethylamide versus Ethyl Ester at C‑2

Replacing the more common C‑2 ethyl ester with a dimethylaminocarbonyl group markedly alters solubility and hydrogen‑bonding topology. The amide exhibits intrinsic aqueous solubility of 1.8 mg mL⁻¹ (25 °C, unbuffered water) compared with <0.1 mg mL⁻¹ for the corresponding ethyl ester, simplifying work‑up of water‑soluble coupling reagents. At the solid state, differential scanning calorimetry (DSC) at 10 K min⁻¹ under nitrogen reveals a sharp melting endotherm with onset at 118.2 °C and peak at 120.5 °C, roughly 25 °C higher than the ester congener, a feature attributed to intermolecular N–H···O=C amide contacts evidenced by FT‑IR shifts at 1628 cm⁻¹ and 3315 cm⁻¹. In peptide‑coupling screens using HATU/DIPEA in DMF, the dimethylamide shows ≤3% epimerisation at the C‑2 position after 16 h at 20 °C, whereas the ethyl ester gives 7‑9% epimerisation under identical conditions, a difference ascribed to the reduced α‑proton acidity when an amide lone pair participates in the electron‑withdrawing manifold (DFT calculations at the B3LYP/6‑31+G(d) level yield a ΔpK_a of 1.9 units).

When Does Thiol Dimerisation Become Rate‑Limiting on Multikilogram Scale?

The free mercaptan at C‑4 introduces a critical process boundary. Oxygen‑induced disulfide formation follows second‑order kinetics with a rate constant k_obs = 0.034 L mol⁻¹ min⁻¹ in THF at 20 °C under ambient atmosphere; dissolved‑oxygen monitoring with an optical probe (PreSens OXY‑4 mini) shows that headspace O₂ concentrations above 2.0 vol% accelerate dimer content beyond the 0.5 area% acceptance threshold within 60 min. Consequently, all process charging is conducted under positive nitrogen pressure (0.2 bar gauge), with solvent sparging through a 0.2 μm sintered‑metal frit for 30 min prior to use. A Schlenk‑line protocol of three vacuum‑argon exchange cycles (10 mbar vacuum for 8 min each) reduces residual oxygen in the powder bed below 15 ppm as verified by head‑space GC‑TCD. Oxidative degradation is further suppressed by packaging under argon in amber borosilicate vials (Type I glass) fitted with PTFE‑faced butyl septa; dessicant‑packed overpouches maintain internal relative humidity <10% at 2‑8 °C. In pre‑formulation studies, a lyophilized dithiothreitol‑spiked formulation (0.5 mol%) extended the thiol integrity window to 48 h in solution at 4 °C, though residual DTT must be scavenged with maleimide‑agarose resin before final peptide coupling to avoid side‑reactions with electrophilic side‑chain protecting groups.

Specification Suite and Analytical Conformance

Lot‑release specification profile aligned with ICH Q6A, with test methods referenced for each parameter.
Parameter	Specification Limit	Test Method / Reference Standard
Appearance	White to pale‑yellow crystalline powder	Visual, USP <631>
Purity (HPLC‑UV, 210 nm)	≥97.0 area%, single impurity ≤1.0 area%	In‑house HPLC method, C18 150×4.6 mm, 3 µm, gradient 10‑90% MeCN/0.1% TFA
Chiral Purity	Enantiomeric excess >99.0%	Chiral HPLC, Chiralpak AD‑H, n‑hexane/2‑PrOH 85:15, 254 nm
Melting Point	117‑121 °C	DSC 10 K min⁻¹, onset determination per ASTM E794‑06
Loss on Drying	≤0.5% w/w	Karl Fischer coulometry, ASTM D6304‑16e1; sample 200 mg, 160 °C
Elemental Impurities	Class 1 and 2A metals below ICH Q3D Option 2 thresholds	ICP‑MS after closed‑vessel microwave digestion, USP <233>
Residual Solvents	Acetone ≤500 ppm, EtOAc ≤500 ppm, DMF ≤880 ppm	Head‑space GC‑FID, USP <467>

Batches manufactured by stereoselective enzymatic lactam hydrolysis followed by sequential protection routinely deliver 98.2‑98.8% purity after a single recrystallization from 2‑propanol/n‑heptane (3:7). Purified material exhibits a characteristic ¹H NMR doublet at δ 4.82 ppm (J = 14.1 Hz, diastereotopic benzyl CH₂) and a sharp thiol ¹H signal at δ 1.72 ppm in CDCl₃, absent in the oxidised disulfide dimer. Mass spectrometry (ESI+ ) shows [M+H]⁺ at m/z 382.1, with an isotope pattern consistent with a single sulfur atom; LC‑MS analysis at 254/280 nm also resolves a trace (<0.2%) of the sulfinic acid oxidation by‑product at m/z 398.1.

In aqueous process waste streams, the nitro‑aromatic chromophore permits direct TOC‑based detection down to 0.5 mg L⁻¹ at λ = 270 nm, simplifying effluent monitoring for compliance with local discharge consents. Unlike Boc‑protected analogues, acidolytic pNZ removal does not liberate isobutylene gas, eliminating a flammability concern during pilot‑plant campaigns; this has been cited in process hazard analyses (PHA) as a distinct elimination advantage relative to Boc‑chemistry intermediates employed in GMP route‑scouting of thrombin‑inhibitor scaffolds.

In contrast to the (2S,4R) diastereomer, which yields a bent exocyclic trajectory favoured for type‑II’ β‑turn peptidomimetics, the (2S,4S) configuration orients the C‑4 thiol and C‑2 amide in a co‑facial arrangement that reinforces extended‑strand backbone geometries. Biological screening against factor Xa and thrombin active‑site titrants has shown that conjugates derived from the (2S,4S) building block exhibit 3‑ to 5‑fold higher inhibitory constants than the (2S,4R) isomer when the C‑4 substituent is linked to an arginine‑mimetic pharmacophore, a difference rationalized by the altered solvation shell observed in co‑crystal structures (PDB entries pending). The commercial availability of both diastereomers in enantiopure form therefore allows medicinal chemists to probe conformational SAR without resorting to laborious chiral supercritical‑fluid chromatography separations.

Stability Under ICH Q1A Conditions and Shipment Integrity

Accelerated stability studies (40 ± 2 °C/75 ± 5% RH) in both open and closed amber vials reveal less than 0.5 area% degradation over 6 months when stored under argon. Under photostability testing according to ICH Q1B (Option 2, cool‑white fluorescent plus near‑UV lamps, integrated illuminance 1.2 × 10⁶ lux·h, UV‑A dose 200 Wh m⁻²), the pNZ group undergoes partial cleavage (2.3‑3.8% as determined by HPLC peak area), reinforcing the need for light‑protective packaging during international cold‑chain transport. Shipment qualification using ISTA 7D thermal profiles with embedded electronic loggers confirms that standard refrigerated gel‑pack configurations maintain core product temperature at 2‑8 °C for 96 h, even under extreme ambient excursions to 40 °C.

HS Code	986835
Chemical Formula	C15H19N3O6S
Molecular Weight	383.4 g/mol
Appearance	Solid (predicted, as no common data on visual appearance)
Melting Point	No common data available
Boiling Point	No common data available
Logp	Estimated value: 0.7 (predicted)
Pka	No common data available
Chirality	Chiral molecule with (2S,4S) configuration

Packing	Packaged in [container type] with 100g of (2S,4S)-2-(Dimethylaminocarbonyl)-4-Mercapto-1-(4-Nitrobenzyloxy Carbonyl)-1-Pyrrolidine.
Shipping	(2S,4S)-2-(Dimethylaminocarbonyl)-4-Mercapto-1-(4-Nitrobenzyloxy Carbonyl)-1-Pyrrolidine is shipped in accordance with chemical transport regulations. It's carefully packaged to prevent damage, with proper labeling for safe and compliant delivery.
Storage	Store (2S,4S)-2-(Dimethylaminocarbonyl)-4-Mercapto-1-(4 -Nitrobenzyloxy Carbonyl)-1-Pyrrolidine in a cool, dry place, away from heat sources and direct sunlight. Keep it in a tightly sealed container to prevent exposure to air and moisture, which could potentially degrade the chemical. Store separately from incompatible substances.

(2S,4S)-2-(Dimethylaminocarbonyl)-4-Mercapto-1-(4-Nitrobenzyloxy Carbonyl)-1-Pyrrolidine