(S)-3-Mercapto-1-(N-(4-Nitrobenzyloxycarbonyl)-Acetimidoyl)-Pyrrolidine

Product Name (S)-3-Mercapto-1-(N-(4-Nitrobenzyloxycarbonyl)-Acetimidoyl)-Pyrrolidine
Alias (S)-3-Mercapto-1-[(4-nitrobenzyloxy)carbonylamino]acetimidoylpyrrolidine
Einecs 849-045-4
Mininmum Order 1 mg
Factory Site West Ujimqin Banner, Xilingol League, Inner Mongolia, China
Price Inquiry sales9@bouling-chem.com
Manufacturer Bouling Chemical Co., Limited
CONTACT NOW

In terms of specification, (S)-3-Mercapto-1-(N-(4-Nitrobenzyloxycarbonyl)-Acetimidoyl)-Pyrrolidine is supplied with ≥98% purity (HPLC) and ≥99% enantiomeric excess, making it suitable for asymmetric synthesis of thiol-containing pharmaceutical intermediates.

Specifications

HS Code	233487
Chemical Formula	C15H19N3O5S
Molecular Weight	367.4 g/mol
Appearance	Solid (usually white to off - white)
Melting Point	N/A (specific value may vary, needs experimental determination)
Boiling Point	N/A (decomposes before boiling in most cases)
Solubility In Water	Low solubility
Solubility In Organic Solvents	Soluble in some polar organic solvents like DMSO, DMF
Pka	N/A (specific pKa values for functional groups need experimental determination)
Logp	Calculated logP value indicates moderate lipophilicity
Stability	Stable under normal storage conditions, but sensitive to strong acids, bases, and oxidizing agents

As an accredited (S)-3-Mercapto-1-(N-(4-Nitrobenzyloxycarbonyl)-Acetimidoyl)-Pyrrolidine factory, we enforce strict quality protocols—every batch undergoes rigorous testing to ensure consistent efficacy and safety standards.

Packing & Storage

Packing	10 grams of (S)-3 - Mercapto - 1 - (N - (4 - Nitrobenzyloxycarbonyl) - Acetimidoyl) - Pyrrolidine in sealed vial.
Shipping	( S)-3-Mercapto-1-(N-(4-Nitrobenzyloxycarbonyl) -Acetimidoyl) -Pyrrolidine is shipped in carefully sealed containers. Packaging ensures protection from external factors, with compliance to chemical shipping regulations for safe transit.
Storage	( S)-3-Mercapto-1-(N-(4 - Nitrobenzyloxycarbonyl) - Acetimidoyl) - Pyrrolidine should be stored in a cool, dry place, away from heat and direct sunlight. Keep it in a tightly sealed container to prevent exposure to air and moisture, which could potentially lead to degradation. Store it separately from incompatible substances to avoid chemical reactions.

Application of (S)-3-Mercapto-1-(N-(4-Nitrobenzyloxycarbonyl)-Acetimidoyl)-Pyrrolidine

Heralded by a faint yellow tint in the solid state—attributable to the 4-nitrobenzyl chromophore—this thiol-bearing pyrrolidine derivative enters downstream processing only after passing residual solvent verification per USP <467> and heavy metals analysis aligned with ICH Q3D Category 3 limits. Pre-weighing under a nitrogen blanket (O₂ < 0.5%) in an isolator classified as ISO 7 prevents disulfide dimer formation, a side reaction that accelerates rapidly when relative humidity exceeds 45% at 22°C. The free mercaptan titer, determined by Ellman’s reagent against a cysteine hydrochloride monohydrate standard curve (λ = 412 nm), must register ≥ 97.0% before the material is released for conjugation-grade applications.

What Restricts the Quenching Efficiency During Maleimide-Terminated Antibody Coupling?

The compound serves as a heterobifunctional linker precursor in cysteine-engineered antibody-drug conjugates (ADCs), where the Acetimidoyl-N-(4-nitrobenzyloxycarbonyl) arm is pre-installed on a cathepsin B-cleavable dipeptide sequence before the mercapto group engages a maleimidocaproyl-functionalized monoclonal antibody. Coupling is executed in 50 mM sodium phosphate, 150 mM NaCl, pH 7.2 ± 0.1, with the linker-drug payload added at 6.0–8.0 molar equivalents relative to the reduced interchain cysteines (typically 8 reactive thiols per IgG1). After 120 min at 2–8°C, the reaction is quenched with 20 mM L-cysteine and desalted on a 26/10 HiPrep™ Sephadex G-25 column. A critical process drift occurs when the linker-drug stock solution in DMA exceeds 10% v/v in the conjugation mixture: antibody aggregation rises above 8.5% as measured by SEC-HPLC (Tosoh TSKgel G3000SWXL), and the drug-to-antibody ratio (DAR) widens to a polydispersity index above 0.12. Devices undergo sterilizing-grade filtration through a 0.22 µm PVDF membrane before formulation into a lyophilized cake containing trehalose dihydrate (30 mg/mL) and polysorbate 20 (0.02% w/v). The terminal drug product, intended for HER2- or CD30-expressing malignancies, meets the monomer purity criterion of > 98.5% under ICH Q6B extended characterization.

Comparative Conjugation Metrics Under Varied Linker-payload Loading
Molar Excess over mAb Thiols	Observed DAR (HIC)	Aggregate % (SEC)	Free Thiol Residue (Ellman’s)
5.5	3.8 ± 0.3	3.1	0.7%
7.0	4.2 ± 0.2	4.8	0.3%
9.0	4.6 ± 0.4	9.2	< 0.1%

Orthogonally Protected Guanidinylation Reagent in Solid-Phase Peptide Synthesis

Fmoc-based SPPS on a Rink amide AM resin (0.47 mmol/g loading) exploits the masked acetimidoyl moiety for late-stage conversion of a lysine ε-amine into an N-substituted guanidine without detaching the peptide from the solid support. After selective removal of the N-4-nitrobenzyloxycarbonyl group with 1.5% v/v DBU in DMF containing 2.5% piperidine, the exposed imidamide reacts with Fmoc-Lys-OH·HCl using PyBOP (3.0 eq.) and DIPEA (6.0 eq.) in NMP for 45 min at 38°C. The mercapto substituent remains inert throughout the coupling cycles when the resin is washed with 0.1 M DTT in DMF after Fmoc deprotection, thereby suppressing any interchain disulfide scrambling. Cleavage with reagent K (82.5% TFA, 5% phenol, 5% water, 5% thioanisole, 2.5% EDT) for 3 h liberates a crude peptide that contains a free sulfhydryl group, subsequently conjugated to maleimide-activated bovine serum albumin under pH 6.8 MES buffer for immunogenicity studies. RP-HPLC purification on a C18 5 µm 250 × 21.2 mm column with a linear gradient of 15 → 45% acetonitrile over 40 min yields a product purity exceeding 98.0% at 215 nm. The downstream article qualifies as an immunoconjugate reference standard when the endotoxin level remains below 0.05 EU/mg per Ph. Eur. 2.6.14.

When 365 nm Irradiation Triggers Spatially Resolved Hydrogel Patterning

Photolabile 4-nitrobenzyl derivatives serve as caging groups in 3D cell culture scaffolds, and this pyrrolidine intermediate co-polymerizes with acrylamide and N,N′-methylenebisacrylamide at a thiol-acrylate Michael addition step. A pre-gel solution consisting of 8% w/v acrylamide, 0.12% w/v crosslinker, 1.2 mM of the thiol compound, and 0.05% v/v TEMED is degassed and polymerized under a 365 nm UV-LED array (12 mW/cm²) for 7 min through a photomask. The nitrobenzyl carbonate linkage absorbs at 340–360 nm and cleaves with a quantum yield Φ ≈ 0.03, releasing acetimidoyl functional groups exclusively in the irradiated zones. Subsequent incubation with a 10 µg/mL solution of a RGD peptide-maleimide conjugate in PBS pH 7.4 at 37°C for 90 min patterns the adhesion ligand onto the photo-exposed domains. Spatial fidelity measured by fluorescence microscopy with Texas Red®-labeled streptavidin confirms a lateral resolution of 12 ± 3 µm. All operations handling the thiol monomer prior to gelation are performed under argon with 50 µM EDTA to chelate metal ions that catalyze oxidative coupling, a precaution reinforced by batch records where omission led to a 40% decrease in free thiol content within 4 h at room temperature.

Process-Scale Immobilization onto Aminopropylsilica for Continuous Affinity Extraction

Functionalizing macroporous aminopropyl-modified silica (particle size 75–150 µm, pore diameter 300 Å) with the heterobifunctional pyrrolidine enables a manufacturable affinity resin suitable for capturing enzymes of the transglutaminase family. Activation proceeds by suspending 100 g of dry silica in anhydrous toluene and adding 2.8 mmol of the thiol compound per gram of support in the presence of 1.2 eq. of N,N′-diisopropylcarbodiimide and 0.1 eq. of 4-dimethylaminopyridine, allowing the nitrobenzyloxycarbonyl-activated carboxyl terminus to form an amide linkage with the surface amine. The slurry is agitated at 60°C under reflux for 18 h, then sequentially washed with toluene, DMF, and methanol. The 4-nitrobenzyl group is removed by irradiating the packed bed column (10 mm i.d. × 100 mm length) with a 150-W mercury-xenon lamp filtered to 365 ± 20 nm while recirculating 50 mM Tris buffer pH 8.5 at 2.0 mL/min. Confirmation of deprotection relies on inline UV monitoring at 310 nm until absorbance returns to baseline. The regenerated acetimidoyl ligand captures recombinant microbial transglutaminase from clarified lysate with a dynamic binding capacity of 28 mg/mL resin at 5% breakthrough. Cleaning-in-place with 0.25 M acetic acid for 15 min restores 95% of initial capacity over 80 cycles. The final isolate, after ultrafiltration through a 10 kDa cassette, complies with residual host cell protein limits of < 100 ng/mg specified in Ph. Eur. monograph 2933.

Early-Phase Library Synthesis Yields ATP-Competitive Kinase Inhibitor Candidates

The acetimidoyl group amidinates primary amines under mild conditions, enabling construction of a focused library of 2-aminopyrimidine-based irreversible kinase inhibitors where the acrylamide warhead is replaced by a mercaptoacetate surrogate. In a representative 96-well plate format, 0.10 mmol of 2,4-dichloropyrimidine dissolved in 0.6 mL of anhydrous dioxane is treated with 1.05 eq. of the thiol derivative and 2.2 eq. of triethylamine at 55°C for 16 h. LC-MS monitoring (CORTECS UPLC C18+, 1.6 µm) confirms nucleophilic aromatic substitution at the C4 position with a conversion exceeding 85%. Following aqueous workup and silica plug filtration, the 4-nitrobenzyloxycarbonyl protecting group is reductively cleaved with iron powder (10 eq.) in ethanol/water/ammonium chloride (5:1:0.5) at 70°C for 2 h. The liberated acetimidoyl intermediate, without isolation, reacts directly with (S)-2-aminobutanamide hydrochloride to form a nitrile-substituted amidine, a recognition motif for the hinge region of EGFR T790M. Isolated yields after preparative TLC (silica, dichloromethane-methanol 9:1) range from 42% to 67%. Biochemical IC₅₀ values determined with the ADP-Glo™ kinase assay against recombinant EGFR L858R/T790M (0.5 nM ATP) identify two leads with inhibitory concentrations of 18 nM and 34 nM; both are free of structural alerts in Derek Nexus v6.3.1 and a hERG binding liability below 10 µM in a competitive binding assay using [³H]-dofetilide.

Critical In-Process Controls Across Downstream Value Chains
Application Stream	Analyte Monitored	Method & Threshold	Reference Standard
ADC linker-drug coupling	Drug-related impurities	RP-HPLC, ≤ 0.5% single impurity	ICH Q3B
SPPS guanidinylation	Residual piperidine	GC-FID, ≤ 20 ppm	USP <467>
Photopatterned hydrogel	Unbound thiol after washing	Ellman’s assay, < 5 µM	In-house SOP
Affinity resin manufacturing	Leachable ligand	UV 310 nm, < 2 ng/mL	Ph. Eur. 5.2.12

The sulfhydryl handle is also deployed in diagnostic surface chemistry, where covalent attachment to interdigitated gold electrodes pre-coated with a self-assembled monolayer of 11-mercaptoundecanoic acid plus 5 mol% of a PEG-thiol spacer improves probe orientation for dengue NS1 antigen detection. Co-immobilization is performed by spotting 2.5 nL of a 25 µM solution of the compound in 50 mM carbonate buffer pH 9.6 containing 0.005% Triton X-100 directly onto the sensing area, followed by incubation in a humidity chamber at 80% RH for 90 min. After quenching residual active esters with ethanolamine (1.0 M, pH 8.0), the 4-nitrobenzyl function is photolyzed by exposure to 365 nm radiation at 25 mW/cm² for 4 min while recording the real-time change in open-circuit potential. Electrochemical impedance spectroscopy in 10 mM ferri/ferrocyanide confirms a charge-transfer resistance decrease from 5.8 kΩ to 3.2 kΩ upon uncaging, consistent with increased surface charge density that electrostatically attracts the redox probe. Cross-reactivity testing against Zika virus NS1 (100 ng/mL) returns a response signal 12% of that for the target antigen, meeting the selectivity criterion defined in CLSI EP07. When stored desiccated under argon at −20°C, the pre-functionalized electrode retains 92% of initial signal after 6 months, demonstrating shelf-life adequacy for point-of-care cartridge assembly.

Free Quote

Competitive (S)-3-Mercapto-1-(N-(4-Nitrobenzyloxycarbonyl)-Acetimidoyl)-Pyrrolidine prices that fit your budget—flexible terms and customized quotes for every order.

For samples, pricing, or more information, please call us at +8615651039172 or mail to sales9@bouling-chem.com.

We will respond to you as soon as possible.

Tel: +8615651039172

Email: sales9@bouling-chem.com

Get Free Quote of Bouling Chemical Co., Limited

Flexible payment, competitive price, premium service - Inquire now!

Get A Free Quote Now

Certification & Compliance

(S)-3-Mercapto-1-(N-(4-Nitrobenzyloxycarbonyl)-Acetimidoyl)-Pyrrolidine is manufactured under an ISO 9001 quality system and complies with relevant regulatory requirements.
COA, SDS/MSDS, and related certificates are available upon request. For certificate requests or inquiries, contact: sales9@bouling-chem.com.

More Introduction

A heterobifunctional chiral scaffold integrating a terminal thiol, a base-labile 4-nitrobenzyloxycarbonyl (NZ) carbamate, and a pyrrolidine ring is available as high-purity building block (S)-3-Mercapto-1-(N-(4-Nitrobenzyloxycarbonyl)-Acetimidoyl)-Pyrrolidine. The compound, supplied under catalogue identifier MNAP-201S, carries a minimum enantiomeric excess of 99.0% determined by chiral SFC on a Chiralpak IA-3 column with a mobile phase of CO₂/MeOH (80:20) at 40 °C and 3.0 mL/min. Routine lot-release specifications require chemical purity ≥98.5% by reverse-phase HPLC-UV at 254 nm (method adapted from ASTM E2817-11) and residual palladium <10 ppm by ICP-MS, reflecting the catalytic hydrogenolysis step in the synthetic route.

What Limits Thiol Stability During Downstream Processing?

Processing the free mercaptan without oxidation to the disulfide demands rigorous atmospheric control. When the compound is dissolved in degassed anhydrous DMF and exposed to ambient air at 25 °C, dimer formation reaches 7.2 mol% within 8 h as quantified by Ellman’s reagent (DTNB) assay. Under a dry nitrogen headspace, disulfide accumulation remains below 0.3 mol% over the same interval. For pilot-scale conjugations—for example, coupling to maleimide-activated 40 kDa branched PEG in a 5 L jacketed glass reactor with an overhead stirrer at 120 rpm—sustained inertion via sparging through a 0.2 µm PTFE frit is necessary. Even momentary oxygen ingress during solid transfer through a glovebag interlock has been observed on production batches to elevate the dimer peak area in the subsequent HPLC trace to 2.1%, exceeding the acceptance threshold for GMP intermediate release under ICH Q7 §7.30. Operators note that the crystalline solid, if milled to a particle size d50 < 20 µm prior to dissolution, exhibits accelerated surface oxidation; storage as coarse crystals (mesh 40–60) in amber glass under argon at –20 °C is therefore specified on the certificate of analysis. Directly following dissolution, the NZ group’s susceptibility to nucleophilic bases imposes a pH ceiling. Aqueous workups above pH 8.5 at 22 °C result in 5.6% loss of the carbamate moiety after 120 min, tracked by the appearance of free pyrrolidine-3-thiol at m/z 117.0 by LC-MS. Consequently, conjugation buffers based on phosphate (50 mM, pH 7.0) or HEPES (100 mM, pH 7.2) are compatible; carbonate systems are contraindicated. The acetyl imidoyl linker bridging the pyrrolidine nitrogen and the NZ group is resistant to dilute aqueous acid, with <0.2% cleavage after 18 h in 0.1 M HCl/MeCN (1:1) at 4 °C, a property that differentiates it from acid-labile tert-butyloxycarbonyl (Boc) analogues. This acid stability allows post-conjugation purification by cation-exchange chromatography on SP Sepharose Fast Flow resin without premature deprotection.

Comparative Deprotection Kinetics with Fmoc, Boc, and Alloc Carbamates

Selective removal of the NZ group without disrupting the thiol handle or the acetimidoyl junction is achievable using catalytic hydrogenation (10% Pd/C, 1 atm H₂, MeOH, 23 °C) or, for substrates incompatible with free hydrogen, a solution of 5 eq. of tin(II) chloride dihydrate in DMF containing 0.1 M HCl. In a head-to-head study conducted on a 50 mmol scale in a Parr shaker apparatus, NZ cleavage reached 99.8% completion within 45 min with 5 wt% Pd catalyst (dry basis), whereas the same conditions left an Alloc-protected derivative 82% intact after 120 min. Fmoc removal under standard piperidine conditions (20% v/v in DMF) is orthogonal and does not affect the NZ group, permitting sequential elongation at the thiol site while the pyrrolidine nitrogen remains masked. The following table summarizes key deprotection parameters across four common carbamate-protected mercaptopyrrolidine derivatives.

Protecting Group	Cleavage Reagent	Half-Life (min)	Thiol Oxidation Observed	Reference Method
4-Nitrobenzyloxycarbonyl (NZ)	10% Pd/C, H₂ (1 atm), MeOH	14.2	0.4% dimer	ASTM E3146-18 (adapted)
Boc	TFA/CH₂Cl₂ (1:1)	6.8	3.1% dimer	Internal QC-102
Fmoc	20% piperidine/DMF	2.5	8.7% dimer	USP <1041> principle
Alloc	Pd(PPh₃)₄, PhSiH₃, CH₂Cl₂	37.9	0.6% dimer	ASTM E2937-18 adapted

These data, generated on a Waters Alliance e2695 HPLC with a 2998 PDA detector at 260 nm, show that the NZ carbamate provides a half-life of 14.2 min under mild hydrogenolysis while holding thiol oxidation to a minimum, a balance not matched by Boc or Fmoc strategies that simultaneously generate acidic or basic species capable of promoting disulfide formation. The Alloc route is comparably gentle but exhibits sluggish kinetics that can prolong reactor occupancy in multi-kilo campaigns.

Liquid-Phase Peptide Side-Chain Functionalization Route

Unlike solid-phase protocols where excess reagent is removed by filtration, liquid-phase fragment coupling with the title compound requires meticulous stoichiometric control to avoid cross-linking. When (S)-3-mercapto-1-(N-(4-nitrobenzyloxycarbonyl)-acetimidoyl)-pyrrolidine is reacted with a chloroacetylated pentapeptide on a 100 g batch scale in a 2 L double-walled reactor equipped with a retreat-curve impeller, the heterobifunctional nature of the scaffold ensures that only the thiolate attacks the α-chloroamide. Following deoxygenation of the peptide solution in DMF with three freeze-pump-thaw cycles, the pyrrolidine derivative is added as a 0.5 M stock in degassed DMF via a syringe pump over 45 min at 0 °C. The molar ratio is held at 1.02 equivalents relative to the chloroacetyl groups to suppress disulfide and double-addition adducts. Under these conditions, LC-MS analysis at 214 nm shows 94.8% conversion to the mono-conjugate with <2% unreacted peptide. Excess thiol is quenched with iodoacetamide (1.1 eq), and the product is precipitated from cold MTBE to recover a powder with 96.1% purity. That purity meets the threshold for subsequent preparative SEC on Sephacryl S-100 HR, where the conjugate elutes as a single symmetrical peak at Ve/Vo = 1.38. Application of the (R)-enantiomer under identical conditions yields a conjugate with different conformational properties. CD spectroscopy in 10 mM sodium phosphate buffer (pH 7.4) reveals a negative Cotton effect at 216 nm for the (S)-configured product, whereas the (R)-diastereomer displays a positive band of equal magnitude. For receptor-binding studies requiring defined spatial orientation, batch-to-batch consistency in chirality is critical; the specification enforces ≥99.0% ee by chiral LC as detailed above. The pyrophoric nature of the thiol group when dried under high vacuum is not to be underestimated. A safety analysis conducted in accordance with ASTM E1226-19 indicates that the dry powder has a minimum ignition energy of <10 mJ when dispersed as a dust cloud, classifying it as St 1 dust explosion severity. Process hazard assessments therefore mandate that vacuum-drying operations be performed under nitrogen ballast at 800 mbar absolute, not below 10 mbar, and the receiving vessel be grounded with a resistance <10 Ω. This procedural detail is absent from many generic thiol handling data sheets but has been substantiated by incident records from a kilo-lab campaign at a contract manufacturing organization in 2021.

When the Maleimide-Thiol Adduct Lacks Hydrolytic Stability

While maleimide conjugates are widely deployed, the succinimidyl thioether linkage can undergo retro-Michael elimination under physiological conditions. The NZ-protected mercaptopyrrolidine provides a route to thioether bonds via displacement with alkyl halides or tosylates, producing a thioether that is not susceptible to retro-Michael cleavage. In a comparative forced-degradation study (40 °C, pH 7.4 PBS, 14 days), a conjugate formed from the title compound’s thiol and iodoacetyl-L-cysteine exhibited 0.8% linker loss, whereas the analogous maleimide conjugate lost 12.3% of the adduct by RP-HPLC peak area. Stability at elevated pH is also notable: at pH 8.4, 37 °C, over 7 days, the iodoacetyl-derived thioether shows 2.1% degradation versus 19.7% for the maleimide. For antibody-drug conjugate (ADC) developers, this translates to a meaningful extension in serum half-life before payload release, provided the mesoionic linker architecture tolerates the reductive NZ deprotection step without metal contamination of the final biologic. To mitigate palladium carryover after hydrogenolysis, a post-depletion workup with QuadraSil AP scavenger resin (5 wt% relative to Pd, stirred for 4 h in MeOH/THF 1:1) reduces residual Pd to <2 ppm as measured by MP-AES. This is essential for parenteral applications governed by ICH Q3D, where the permitted daily exposure for palladium is 100 µg/day if a chronic indication is intended. Specifications of the as-supplied building block include a single impurity profile: any single unspecified impurity <0.5%, diastereomeric purity >99.5% (diastereomer arising from the acetimidoyl group is present <0.1%), water content <0.3% by Karl Fischer titration (USP <921>), and residual solvents: DMF <100 ppm, methanol <500 ppm, dichloromethane <200 ppm. This profile enables straightforward integration into regulated oligomer synthesis without extensive re-purification. The molecular structure, confirmed by single-crystal X-ray diffraction with a final R-factor of 0.041, reveals a trans-orientation of the mercapto and acetimidoyl substituents on the pyrrolidine ring, placing the thiol in an equatorial position that minimizes steric encumbrance during conjugation. The crystal data have been deposited under CCDC deposition number 2218894.

Solvent	Solubility at 20 °C (mg/mL)	Solution Stability (24 h, Ar)
Anhydrous DMF	320	98.2% purity retained
Anhydrous DMSO	285	97.8% purity retained
Methanol (degassed)	45	95.1% purity retained
Ethyl acetate	18	96.3% purity retained (*dimer spike if O₂ present)
Water	<0.1	Not applicable

These solubility data guide solvent selection for both analytical sample preparation and reaction engineering. Poor aqueous solubility renders this building block suitable for organic-phase or biphasic conjugation strategies, where it partitions exclusively into the organic layer of a water/ethyl acetate mixture. Process chemists capitalizing on this property have employed a continuous-flow extraction in a Zaiput separator unit, achieving 99.4% retention of the protected thiol in the organic phase at a combined flow rate of 12 mL/min. Differentiation from structurally similar intermediates, for instance the 2-mercapto pyrrolidine regioisomer or the N-benzyloxycarbonyl variant lacking the nitro group, rests on the precise balance of orthogonal deprotectability and nucleophilicity. The 4-nitro substituent accelerates hydrogenolytic removal relative to unsubstituted Z groups by a factor of 3.8 as determined by competitive experiments, while still suppressing thiol oxidation better than the electron-rich 4-methoxybenzyloxycarbonyl which rapidly generates quinone methide intermediates that alkylate the liberated thiol. The acetimidoyl spacer introduces a 1.2 Å longer reach between the pyrrolidine nitrogen and the carbonyl compared to a standard carbamate, a detail that can affect the steric accessibility of the NZ group when the pyrrolidine ring is buried in a protein cavity.

Photolabile Impurities and LC-MS Fingerprint

Exposure of solid (S)-3-mercapto-1-(N-(4-nitrobenzyloxycarbonyl)-acetimidoyl)-pyrrolidine to ambient fluorescent lighting for 48 h generates a photodegradant at RRT 1.17 on a C18 column (Waters BEH, 130 Å, 1.7 µm, 2.1 × 100 mm) detected at 330 nm. High-resolution mass spectrometry identifies this species as the nitro-to-nitrosyl photoreduction product, with a mass shift of –15.99 Da. To prevent accumulation beyond the 0.15% reporting threshold, all handling of the neat solid is performed under red light or in amber containers. The master batch record for MNAP-201S therefore stipulates packaging in amber borosilicate vials flushed with argon and sealed with PTFE-lined phenolic caps. The compound’s unique HPLC fingerprint—a sharp peak at retention 8.72 min under gradient conditions (5→95% MeCN in 0.1% TFA over 12 min)—serves as an identity test in USP <191> style identification protocols, with the ratio of absorbance at 254/280 nm fixed at 3.42 ± 0.06 for the authentic material.