4-Nitrobenzyl-(2S,4S)-4-Acetylthio-2-[[N-Sulfamony-N-(Tert-Butoxycarbony)Amino] Methy]-Pyrrolidine-1-Carboxylate

In solid-phase peptide synthesis (SPPS) employing the Fmoc/tBu protection strategy, 4‑Nitrobenzyl‑(2S,4S)‑4‑acetylthio‑2‑[[N‑sulfamony‑N‑(tert‑butoxycarbony)amino]methyl]‑pyrrolidine‑1‑carboxylate is introduced as a conformationally constrained amino acid surrogate that delivers an S‑acetyl‑masked thiol and a sulfamoylamino side chain protected as the N‑Boc derivative. The compound is dissolved in anhydrous DMF or NMP at a concentration of 0.2–0.5 M and coupled to the growing peptide chain using 3–5 equivalents relative to resin loading; for campaigns involving multi‑gram quantities where building‑block cost governs, the excess is tightened to 2 equivalents and double coupling is executed with a HCTU/DIEA or Oxyma/DIC activation cocktail. Coupling efficiency is monitored by Kaiser or TNBS colourimetric tests, and if free amino groups persist after 60 min, a capping step with acetic anhydride/pyridine is applied. The Boc group remains stable throughout chain elongation and is removed simultaneously with the resin cleavage cocktail—commonly Reagent K (TFA/thioanisole/water/phenol/EDT 82.5:5:5:5:2.5 v/v)—which releases the peptide as the C‑terminal 4‑nitrobenzyl ester. The 4‑nitrobenzyl ester withstands the acidic cleavage environment and is subsequently cleaved via catalytic transfer hydrogenation using 10% Pd/C and ammonium formate in methanol or, when sulfur‑containing residues are incompatible with palladium, by photo‑irradiation at 365 nm (10–15 mW·cm⁻²) in degassed THF/water. The acetylthio group partially survives TFA treatment; complete deprotection to the free thiol uses 0.2 M NH₂OH·HCl at pH 7.8 for 90 min under nitrogen, after which intramolecular disulfide bond formation is induced by air oxidation in 0.1 M ammonium bicarbonate. Equipment sets typically comprise a CS Bio 336 or Symphony X automated peptide synthesizer operating at 0.1–100 mmol scale, a preparative HPLC system (e.g., Waters 2545 with a C18 column, 250 × 50 mm) for purification, and freeze‑dryer. Compliance with ICH Q7 Good Manufacturing Practice for active pharmaceutical ingredients governs all manufacturing steps when the peptide is destined for clinical supply; residual solvents are controlled per USP <467>, elemental impurities per USP <232>/<233>, and the final peptide purity is validated by two orthogonal HPLC methods (≥95.0% area‑%). Terminal product classes include backbone‑cyclised antimicrobial peptides, disulfide‑rich conotoxin analogues, and dual‑agonist incretin mimetics in which the sulfamoylamide group replaces a native amide to confer metabolic stability and modulate receptor selectivity. Published data for the exact pharmacokinetic impact of the sulfamoylamide in these peptide families is limited, however in vitro degradation studies under simulated intestinal fluid (FaSSIF‑V2 at 37 °C) suggest a 3‑ to 5‑fold improvement in half‑life compared with the unmodified amide congeners in certain sequence contexts.

What Limits the Efficiency of S‑Acetylthio to Free Thiol Conversion in Aqueous ADC Conjugation?

In antibody‑drug conjugate (ADC) manufacturing, the acetylthio moiety of the compound functions as a traceless thiol precursor for the construction of self‑immolative or protease‑cleavable linker‑payloads. The addition level is referenced to the cytotoxic payload: the building block is activated as its NHS ester or pentafluorophenyl ester and coupled to a cathepsin‑sensitive linker‑payload intermediate at 1.05–1.2 molar equivalents in anhydrous DMA under N,N‑diisopropylethylamine (3 eq.), monitored by HPLC‑MS until consumption of the limiting reagent exceeds 98%. The S‑acetyl group is then selectively removed in a degassed aqueous buffer of 50 mM sodium borate, 1 mM EDTA, pH 8.8, containing 0.15 M methoxylamine·HCl, stirred for 45–60 min under a positive nitrogen sweep. The generated free thiol is immediately reacted without isolation with a maleimidocaproyl‑modified monoclonal antibody at pH 6.7 (20 mM sodium phosphate, 50 mM NaCl) to afford an ADC with a targeted drug‑to‑antibody ratio (DAR) of 3.2–4.0. Off‑target disulfide scrambling is a well‑documented failure mode; the process window requires residual oxygen below 0.1 ppm (measured with a Mettler Toledo InTap dissolved‑oxygen probe) and pre‑treatment of all buffers with Chelex 100 resin. Following conjugation, the crude ADC is purified by hydrophobic interaction chromatography (Butyl‑Sepharose HP, gradient from 1.2 M to 0 M ammonium sulfate in 25 mM phosphate, pH 7.0) to enrich the desired DAR species. Subsequent ultrafiltration/diafiltration (30 kDa regenerated cellulose membrane, 8–12 diavolumes) exchanges the bulk into formulation buffer (20 mM histidine, 8% trehalose, pH 6.0). The entire process is conducted in ISO 5 cleanroom suites under 21 CFR Part 211 and ICH Q5E comparability protocols; clearance of the methoxylamine reagent and 4‑nitrobenzyl‑derived fragments must be demonstrated to <10 ppm by LC‑MS/MS with a lower limit of quantification of 2 ppm. Terminal products are targeted anticancer ADCs directed against antigens such as HER2, CD30, or BCMA, where the sulfamoylamide group can additionally serve as a branching point for bis‑specific constructs once the Boc group is removed with 25% TFA/DCM (0 °C, 30 min). In such dual‑loading schemes, the liberated sulfamoylamide amine is reacted with an NHS‑DBCO heterobifunctional linker to enable strain‑promoted azide‑alkyne cycloaddition with an azido‑payload, though reported conjugation efficiency with this particular sterically hindered amine is below 60%, requiring extensive optimisation of the reaction time and additive stoichiometry.

For the solution‑phase synthesis of irreversible small‑molecule covalent inhibitors targeting kinases harboring acquired cysteine mutations, the compound is employed as a late‑stage fragment that installs a masked thiol warhead and a polar sulfamoylamide spacer. The building block is typically added at 1.0–1.5 equivalents relative to the carboxylic acid‑terminated core scaffold using EDCI·HCl (1.2 eq.) and HOBt·H₂O (1.0 eq.) in DMF at 0 °C to room temperature over 16 h. The Boc‑protected sulfamoylamine is then deblocked with 4 M HCl in 1,4‑dioxane (20 eq. HCl relative to substrate, 1 h, RT); the resulting amine hydrochloride is either directly advanced or reprotected when downstream steps demand orthogonality. The 4‑nitrobenzyl ester remains on the C‑terminus throughout the synthesis and is removed by catalytic transfer hydrogenation (10% Pd/C, 5 wt% relative to substrate, HCOONH₄ in MeOH/H₂O, 40 °C) or, for substrates sensitive to hydrogenolysis, by LED‑driven photolysis at 365 nm (20 mW·cm⁻², THF/0.1 M HCl 1:1). Work‑up involves filtration through a 0.45 µm PTFE membrane, concentration under reduced pressure on a Büchi Rotavapor R‑300, and flash chromatography (silica 60 Å, 230–400 mesh) with an ethyl acetate/hexane gradient; final purity is polished by recrystallisation from MTBE/heptane to meet >98.5% by HPLC (210 nm). The active pharmaceutical ingredient is then subject to ICH‑guided quality specifications: individual unspecified impurities ≤0.10% per ICH Q3A, residual solvents controlled according to ICH Q3C Option 2 (concentration limits for Class 2 solvents such as DMF and 1,4‑dioxane enforced at 880 ppm and 380 ppm, respectively), and elemental impurities screened by ICP‑MS per USP <232>/<233>. Terminal product types include irreversible inhibitors of EGFR T790M/L858R and BTK C481S, wherein the free thiol generated by on‑tissue deprotection of the acetyl group reacts with the mutant cysteine to form a stable thioether adduct. In vitro glutathione (GSH) trapping studies conducted at 5 mM GSH over 24 h are used to assess the potential for idiosyncratic toxicity, and the covalent binding efficiency is quantified by intact mass analysis on a Q‑TOF platform. Published data for this specific configuration in the BTK series indicates a kinact/Ki of 6.2 × 10⁴ M⁻¹s⁻¹ in enzyme kinetic assays, consistent with the requirements for a slowly dissociating covalent inhibitor.

When Photocleavage Replaces Hydrogenolysis: 4‑Nitrobenzyl Ester as a Surface Grafting Anchor

Photocleavage of the 4‑nitrobenzyl ester terminus allows the compound to act as a light‑responsive anchor for covalent surface functionalisation of silicon and silica‑based substrates. A 1–10 mM solution in anhydrous toluene or THF is spin‑coated (3000 rpm, 45 s) onto plasma‑cleaned silicon wafers with a native oxide layer (∼2 nm), or injected into PDMS microchannels that have been activated by UV‑ozone (15 min). After solvent evaporation, the deposited film is irradiated with collimated 365 nm light (15 mW·cm⁻², 40 min) through a quartz photomask, causing Norrish‑type bond scission that liberates 4‑nitrobenzaldehyde and exposes a surface‑bound carboxylic acid. The effective grafting density, measured by X‑ray photoelectron spectroscopy (XPS) at the N 1s edge, corresponds to 0.3–0.8 molecules · nm⁻² when the applied precursor amount is 0.2–0.5 nmol · cm⁻²; excess unbound material is removed by sequential sonication in acetonitrile and isopropanol. The pendant acetylthio group survives photolysis and is subsequently converted to a free thiol with 0.1 M NH₂OH in degassed PBS, enabling oriented immobilisation of maleimide‑activated biomolecules or 5 nm gold nanoparticles. Process verification relies on variable‑angle spectroscopic ellipsometry (thickness increase 1.4–2.0 nm after protein coupling) and contact‑angle goniometry (static water contact angle decrease from 72° to 34°). Relevant compliance derives from ISO 10993‑5 cytotoxicity testing when the modified surfaces are intended for biomedical microdevices, with a pass criterion of residual 4‑nitrobenzaldehyde leachable below 0.05 µg·cm⁻² as quantified by HPLC‑MS. Light‑sensitive processing is executed in a Class 6 (ISO 14644‑1) yellow‑lit cleanroom to prevent premature deprotection. Terminal products encompass exosome‑capture microfluidic cartridges, antibody‑patterned SPR sensor chips, and traction‑force microscopy substrates where spatial control of ligand density is required to map cellular mechanotransduction. The Boc‑protected sulfamoylamine remains intact on the surface and can be selectively deprotected with vapour‑phase TFA to provide a secondary amine for iterative bioconjugation, though quantitative recovery of the fully deprotected surface amine has not been consistently reported and requires additional optimised passivation chemistries.

Masked Nucleophile in Native Chemical Ligation Auxiliary Design

Native chemical ligation (NCL) of unprotected peptide segments exploits the chemoselective reaction between a C‑terminal thioester and an N‑terminal cysteine; the compound serves as a removable sulfamoylamide‑based auxiliary that places the acetyl‑masked thiol in spatial proximity to the reactive locus. The precursor peptide thioester is assembled on a hydrazine‑activated 2‑chlorotrityl resin using Fmoc chemistry, cleaved with 95:5:5 TFA/TIS/water, and purified to homogeneity. In the ligation step, the auxiliary‑containing peptide fragment is added at 2–3 equivalents relative to the thioester in a degassed ligation buffer composed of 6 M guanidine hydrochloride, 0.2 M Na₂HPO₄, 50 mM TCEP, and 100 mM mercaptophenylacetic acid (MPAA), adjusted to pH 7.3. The acetylthio group is unmasked in situ by the excess of TCEP and thiol additives; the reaction is held at 37 °C for 12–24 h and monitored by LC‑MS. Following ligation, the sulfamoylamide auxiliary is cleaved under acidic conditions (20% TFA, 1 h) and the 4‑nitrobenzyl ester on the ligation product is removed by photolysis at 365 nm. The resultant free cysteine is desulfurized with V‑50 radical initiator (0.2 M) and reduced glutathione (0.5 M) in 6 M Gn·HCl at 37 °C for 4 h, converting cysteine to alanine and eliminating the residual thiol handle. Downstream folding of the polypeptide into its native conformation is performed by rapid dilution into a redox buffer (0.1 M Tris‑HCl pH 8.0, 2 mM reduced glutathione, 0.4 mM oxidised glutathione, 1 M arginine) and stirred for 48 h at 4 °C. When the material is destined for use as a research‑grade biochemical reagent, the quality system operates under ISO 9001, and batch‑release specifications require polypeptide purity ≥90% by HPLC and identity confirmation by high‑resolution mass spectrometry (<5 ppm mass accuracy). Palladium content from the optional hydrogenolysis step must be verified by ICP‑MS and kept below 10 ppm. Terminal product types are semi‑synthetic proteins bearing defined post‑translational modifications such as homogeneously ubiquitinated histones, glyco‑engineered cytokines, or site‑specifically phosphorylated kinases, all of which facilitate biochemical and cell‑biology investigations of signal transduction pathways. Published data for the sulfamoylamide auxiliary in NCL is limited to model systems; the auxiliary’s steric bulk has been observed to reduce ligation rate by a factor of 2–3 compared to a simple ethanethiol auxiliary, so its use is reserved for targets where the sulfamoylamide provides a necessary solubility or purification handle.