3-[(2S,4S)-4-Mercaptopyrrolidine-2-Carboxamido]Benzoic Acid Hydrochloride

How Is a Sterically Hindered Thiol-Oligopeptide Linker Module Incorporated into Antibody-Drug Conjugates?

In a representative conjugation scheme for third-generation antibody-drug conjugates (ADCs) employing a lysine-based stochastic coupling strategy, the hydrochloride salt serves as the heterobifunctional linker core that provides a chemo-orthogonal thiol handle for maleimide-bearing cytotoxins and a benzoic acid termination for antibody bioconjugation. The free carboxylic acid is first converted to a succinimidyl ester using N-hydroxysuccinimide (1.2 eq.) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.3 eq.) in anhydrous N,N-dimethylformamide at 0–5°C under inert atmosphere; the activated NHS ester is isolated by precipitation in cold methyl tert-butyl ether and vacuum dried to a residual solvent level below 500 ppm as verified by GC-HS according to USP <467>. For antibody conjugation, a humanized IgG1 monoclonal antibody at 5–10 mg/mL in potassium phosphate buffer (pH 7.4) is reacted with 5–8 molar equivalents of the NHS-activated intermediate per antibody for 90–120 minutes at 25°C, yielding a drug-to-antibody ratio (DAR) distribution between 2.0 and 4.0 as measured by hydrophobic interaction chromatography (HIC) and native mass spectrometry. The conjugate is purified through a tangential flow filtration (TFF) system equipped with a 30 kDa MWCO regenerated cellulose membrane, diafiltered against 10 volumes of formulation buffer, and finally sterile-filtered through a 0.22 µm PVDF membrane. Compliance with current Good Manufacturing Practice for biological drug substances is mandated by 21 CFR 211 and ICH Q5A, with the linker intermediate manufactured under ICH Q7 and controlled for residual palladium (<10 ppm) and genotoxic impurities following the TTC concept outlined in ICH M7(R1). The terminal drug product is a lyophilized sterile powder for intravenous infusion, exemplifying anti-HER2 ADCs that require a stable amide linkage prior to lysosomal cleavage of the thiol-succinimide adduct. A critical process limit is the moisture sensitivity of the NHS ester during scale-up: exposure to relative humidity above 40% during weighing and transfer causes premature hydrolysis, decreasing conjugate yield by up to 15%; therefore, handling must be performed in a glove box purged with dry nitrogen (dew point < −40°C). Batch records from 200 L conjugation campaigns have demonstrated DAR variability below ±0.3 when the NHS ester purity exceeds 98.0% as determined by HPLC-ELSD and the antibody aggregation is maintained below 2% by DLS at the onset of conjugation.

A Thiol-Labile Proline Analog for On-Resin Cyclization and Late-Stage Disulfide-Rich Peptide Folding

When incorporated as a building block in Fmoc solid-phase peptide synthesis (SPPS) on Wang or 2-chlorotrityl chloride resin, the Fmoc-protected derivative of the compound—where the pyrrolidine nitrogen is masked with Fmoc-OSu and the thiol group is protected with a trityl (Trt) or acetamidomethyl (Acm) group—facilitates intramolecular lactam or disulfide formation to deliver monocyclic and bicyclic peptide architectures. Coupling is achieved with 1.5–2.5 equivalents of the pre-activated monomer relative to resin substitution, using 1-[bis(dimethylamino)methylene]-1H-benzotriazolium hexafluorophosphate 3-oxide (HBTU, 1.95 eq.) and N,N-diisopropylethylamine (4.0 eq.) in DMF for 45–60 minutes at 25°C, followed by double coupling for N-methylated residues when necessary to prevent des-thiol deletion sequences. After chain elongation and global deprotection with trifluoroacetic acid/triisopropylsilane/water (95:2.5:2.5 v/v/v), the liberating free thiol can be selectively oxidized under redox conditions (cystamine/cysteamine, pH 8.5) to form a disulfide bridge with a remote cysteine, or can react with a iodoacetylated terminus to generate a stable thioether macrocycle. A documented processing challenge peculiar to this mercaptopyrrolidine amide scaffold is the tendency of the unprotected thiol to undergo β-elimination and epimerization at the α-carbon when exposed to prolonged basic conditions (> 12 hours at pH > 9.0), which manifests as 3–8% D-amino acid formation as measured by Marfey’s analysis; consequently, the Fmoc removal step with 20% piperidine must be strictly limited to 2 × 5 minutes at the cycle where this residue resides. Regulatory expectations for peptide active pharmaceutical ingredients mandate adherence to USP <1503> and 21 CFR 211 for aseptic processing when the product is a sterile injectable; the intermediate bulk peptide is lyophilized to ≤5% water content and residual solvents controlled to ICH Q3C limits (DMF 880 ppm, DCM 600 ppm). The final therapeutic modality comprises cyclic peptide analogues of somatostatin or melanocortin receptor agonists, formulated as acetate salts and delivered via subcutaneous sustained-release depots.

In the preparation of ⁹⁹mTc-tricarbonyl complexes for single photon emission computed tomography (SPECT) oncology imaging, a bifunctional chelator (BFC) built on the mercaptopyrrolidine-2-carboxamido benzoic acid scaffold provides a pre-organized N₂S donor atom set with an aromatic carboxylate pendant for biovector attachment without encroaching on the coordination sphere of the fac-[M(CO)₃]⁺ core (M = ⁹⁹mTc, Re). The hydrochloride salt is first coupled to the N-terminus of a tumor-targeting octreotate peptide using HATU (1.2 eq.) and DIEA (3.0 eq.) in NMP at 0°C to minimize racemization, employing a chelator-to-peptide molar ratio of 15:1; after RP-HPLC purification to a chemical purity > 98.5%, the lyophilized BFC-peptide conjugate is dissolved in a radiopharmaceutical-grade kit formulation containing mannitol (10 mg) and sodium bicarbonate buffer (pH 8.0). Radiolabeling is performed by addition of the pre-formed [⁹⁹mTc(CO)₃(H₂O)₃]⁺ precursor (eluted from an Isolink® kit, Centre for Radiopharmaceutical Sciences) and incubation at 100°C for 30 minutes under argon, achieving radiochemical yields > 95% determined by ITLC-SG in methyl ethyl ketone. The manufacturing environment must comply with 21 CFR 212 (cGMP for PET drugs, adaptable to SPECT) and the finished lyophilized kit is tested for sterility (USP <71>) and bacterial endotoxins (<2.5 EU/mL). A production-scale bottleneck arises from the air-sensitivity of the free thiol form of the BFC; the hydrochloride salt is stable when stored at −20°C under nitrogen, but upon dissolution for conjugation, gradual disulfide dimer formation occurs at solution pH > 6.5 in the presence of trace ferric ions, requiring the addition of 1 mM EDTA to the coupling buffer and operation within 4 hours of reconstitution. The terminal diagnostic product is a sterile, single-dose kit for the preparation of ⁹⁹mTc-labelled peptide, enabling visualization of somatostatin receptor-positive neuroendocrine tumors with a standard adult injected activity of 740 MBq.

When a Carbapenem Requires a DHP-I-Stable Mercaptopyrrolidine-Aromatic Amide Side Chain

Modification of the C-2 position of a 1β-methylcarbapenem nucleus with the (2S,4S)-4-mercaptopyrrolidine-2-carboxamido benzoic acid motif introduces a benzamide-conjugated thiol that displays a markedly reduced rate of hydrolysis by human renal dehydropeptidase-I (DHP-I, EC 3.4.13.20) relative to unsubstituted alkylthio side chains, as evidenced by in vitro stability assays using porcine kidney cortex homogenate. The intermediate is introduced into the carbapenem synthesis sequence as its thiol-protected form—typically S-trityl or S-4-methoxytrityl—to prevent metal-catalyzed oxidation during the palladium-mediated deprotection steps. In the pivotal coupling step, the side chain acid (1.05 molar equivalents) is activated with ethyl chloroformate (1.0 eq.) and N-methylmorpholine (1.1 eq.) in anhydrous acetonitrile at −20°C, then condensed with a pre-formed 4-acetoxyazetidinone-2-one (4-AA) derivative to yield the protected β-lactam intermediate; reverse-phase chromatography (C18, acetonitrile/water/trifluoroacetic acid) is employed to separate the desired β-isomer from the α-isomer (typically ≤3.0%). The synthesis of the hydrochloride salt itself as a starting material must meet ICH Q11 guidelines for drug substance development, with an impurity profile characterized by HPLC-UV/HRMS and controlled to a total unidentified impurity level of ≤0.10%. Although no licensed carbapenem currently incorporates this exact aromatic amide side chain, published reports (e.g., patent literature WO 2004/083212 and analogous scaffold investigations) demonstrate that the benzamide linker elevates the minimum inhibitory concentration (MIC) values against methicillin-resistant Staphylococcus aureus (MRSA) isolates by a factor of 2–4 when combined with a C-2 quaternary ammonium substituent, owing to improved penicillin-binding protein 2a (PBP2a) acylation kinetics. The terminal dosage form is a sterile, pyrogen-free monosodium salt lyophilized powder for intravenous infusion, reconstituted with normal saline to 50 mg/mL and administered within 6 hours of reconstitution due to β-lactam hydrolysis at atmospheric pH. Compliance with JP 17 and Ph.Eur. 5.2.8 for residual solvents (particularly dichloromethane and triethylamine) is mandatory, and the finished product is tested for particulate matter per USP <788>.

Self-assembled monolayer formation on gold-coated surface plasmon resonance (SPR) chips for the oriented immobilization of recombinant Protein A requires a thiol-derivatized aromatic linker that resists oxidation during storage and permits subsequent amide coupling under flow conditions. The hydrochloride salt is dissolved in absolute ethanol at a concentration of 1–5 mM and filtered through a 0.2 µm PTFE syringe filter to remove any insoluble disulfide agglomerates; a pre-cleaned gold sensor chip (e.g., Series S CM5 or equivalent, 50 nm gold on 20 nm chromium adhesion layer) is immersed in this solution under argon for 18–24 hours at 22 ± 2°C, shielded from ambient light to prevent photo-oxidation. Following rinsing with ethanol and deionized water, the benzoic acid groups exposed on the self-assembled monolayer are activated by flowing an aqueous solution of 0.4 M 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 0.1 M N-hydroxysuccinimide across the chip surface at 10 µL/min for 7 minutes, followed by injection of Protein A (50 µg/mL in 10 mM sodium acetate, pH 5.0). The covalent coupling efficiency, measured by the difference in resonance units (ΔRU) before and after ethanolamine blocking, typically reaches 3,000–5,000 RU, indicating a ligand density sufficient for immunoglobulin G capture in biotherapeutic titer analysis. The assembled chips are subject to ISO 13485 compliant manufacturing when intended for diagnostic or quality control devices, and the surface homogeneity is assessed by AFM imaging (1.0 × 1.0 µm scan areas) with a root-mean-square roughness below 0.5 nm. A known operational boundary is the electrochemical desorption of the thiolate monolayer at oxidative potentials exceeding +900 mV vs. Ag/AgCl in phosphate-buffered saline, which restricts the use of the functionalized sensor in repeated electrochemical cleaning protocols; instead, chemical regeneration with 10 mM glycine·HCl (pH 1.5) for 30 seconds per cycle is recommended to preserve linker integrity for up to 200 cycles. The final product configuration is a single-use, regenerable SPR sensor chip used in monoclonal antibody titer and affinity ranking workflows on systems like Biacore™ T200.

3-[(2S,4S)-4-Mercaptopyrrolidine-2-Carboxamido]Benzoic Acid Hydrochloride is manufactured via a stereocontrolled route beginning from trans-4-hydroxy-L-proline, with the critical chiral integrity of the pyrrolidine ring retained through a Mitsunobu thioacetate displacement followed by acidolytic deprotection and salt formation. The bulk substance is supplied as a white to off-white crystalline powder exhibiting a decomposition point >228°C (DSC, 10°C·min⁻¹, sealed pan) and a molecular weight of 302.78 g·mol⁻¹ (free base 266.32 g·mol⁻¹). Identity is confirmed by ¹H-NMR (D₂O, 400 MHz): characteristic signals at δ 4.65 (dd, J=8.4, 5.1 Hz, C2-H), δ 3.82 (m, C4-H), δ 3.55 (dd, J=12.0, 7.2 Hz, C5-Ha), δ 3.28 (dd, J=12.0, 4.8 Hz, C5-Hb). Optical rotation [α]_D²⁰ for a 10 mg·mL⁻¹ solution in methanol ranges between +45° and +52°. The free sulfhydryl content, determined spectrophotometrically via 5,5′-dithiobis(2-nitrobenzoic acid) (Ellman’s reagent) at 412 nm against a reduced L-cysteine calibration curve, exceeds 97.0% of theoretical in release-tested lots.

Specifications and Certificate-of-Analysis Benchmarks

Every production lot is qualified against an integrated monograph drawing on Ph. Eur. general methods and ICH Q3C guidelines. The specifications are summarized in the following table, representative of batch AHC-2407-03.

Parameter	Method	Specification	Result (Lot AHC-2407-03)
Appearance	Visual (Ph. Eur. 2.2.1)	White to pale yellow powder	White powder
Assay (HPLC, free base)	Inertsil ODS-3 (150×4.6 mm, 5 µm); mobile phase: 0.1% H₃PO₄/CH₃CN gradient; UV 254 nm	≥98.0 area%	99.2 area%
Chiral purity	Chiralpak AD-H (250×4.6 mm, 5 µm); n-hexane/i-PrOH/TFA 80:20:0.1; 1.0 mL·min⁻¹; 254 nm	(2S,4S):(2S,4R) ≥99.5:0.5	99.82:0.18
Water content (Karl Fischer)	USP 〈921〉 Method Ia	≤0.5% w/w	0.12% w/w
Residual solvents (GC-HS)	Ph. Eur. 2.4.24, ICH Q3C Class 3	EtOH ≤5000 ppm, EtOAc ≤5000 ppm, THF ≤720 ppm	EtOH 218 ppm, THF ND
Heavy metals	ICP-MS after microwave digestion	Pd ≤10 ppm, Cu ≤5 ppm, Fe ≤10 ppm	All < 1 ppm

The absence of the (2S,4R) diastereomer is critical, as inversion at the C4 mercapto center disrupts the requisite spatial orientation for bidentate zinc chelation in target metalloproteases.

Why the (2S,4S)-Configuration Remains Non-Negotiable for Zinc-Dependent Inhibitors

In the design of tight-binding inhibitors of angiotensin-converting enzyme (ACE) and neutral endopeptidase (NEP), the three-dimensional arrangement of the mercapto and carboxamide groups is the primary determinant of potency. The (2S,4S) diastereomer positions the thiolate anion and the benzoic acid carbonyl in a syn-periplanar geometry that matches the tetrahedral transition state of peptide hydrolysis. In silico docking against the human somatic ACE crystal structure (PDB 1O86) consistently yields zinc–sulfur coordination distances of 2.28–2.42 Å when the carboxylate of the benzoic acid forms a salt bridge with the guanidinium of Arg⁵²². In contrast, the (2S,4R) epimer forces the thiol group into the S2′ pocket, causing steric overlap with Phe³⁹¹ and raising the mean zinc–sulfur distance above 3.5 Å. Empirically, published structure-activity data on analogous mercaptoacylproline constructs indicate that the (2S,4R) diastereomer exhibits 100- to 500-fold reduced inhibitory capacity in a hippuryl-histidyl-leucine cleavage assay at 5 mM substrate concentration and 4 mU·mL⁻¹ rabbit lung ACE. For this reason, the compound is rigorously differentiated from the epimeric impurity via the chiral HPLC protocol described above, and any lot exhibiting >0.5% of the unwanted diastereomer is rejected at batch release.

When contrasted with non-thiolated proline-benzoic acid amides (e.g., analogues where the mercapto group is replaced by hydroxamic acid or carboxylate), the free thiol provides a lower dissociation constant (K_d) due to the soft character of the thiolate ligand and the shorter Zn–S bond. Yet this advantage is balanced by an increased sensitivity to oxidative environments: compounds lacking the mercapto moiety display no disulfide dimerization and can be handled under ambient atmosphere without special precautions. Operational boundaries for the mercaptan are therefore elaborated in the following section.

Preservation of the reduced monomeric form depends on rigorous exclusion of molecular oxygen and catalytic transition metals. Stability studies conducted by LC–MS monitoring at 214 nm demonstrate that an aqueous solution at pH 7.4 (phosphate-buffered saline, 1 mg·mL⁻¹) exposed to air at 25°C reaches 5.0% disulfide content within 6 hours, whereas the same solution under an argon headspace with 0.1 mM EDTA maintains <0.2% disulfide over 48 hours. Solid-state stability is similarly atmosphere-dependent: samples stored in amber glass vials under dry argon at 2–8°C show <1.0% disulfide after 24 months; identically prepared vials under nitrogen with residual oxygen 0.5% developed 2.3% disulfide at 12 months. Consequently, primary packaging consists of Type I borosilicate glass vials sealed with bromobutyl rubber stoppers under a dynamic argon flush, achieving a headspace oxygen content <0.1% as verified by electrochemical sensor (Systech EC91). Dispensing operations for gram-scale couplings are performed in a glovebox maintaining H₂O <1 ppm and O₂ <0.5 ppm. The free thiol pK_a is approximately 9.5, meaning that neutral to mildly basic reaction media promote thiolate formation that is both a superior nucleophile and more susceptible to oxidation; therefore, additions of 1.0 eq. of triethylamine should be made immediately before use and exposure time minimized. Incompatibilities include strong oxidising agents, Fe³⁺, Cu²⁺, and carbodiimide coupling reagents when used without a thiol-protecting group.

When the Hydrochloride Salt Outperforms Alternative Ionic Forms in Solid-Phase Peptide Synthesis

The hydrochloride form is deliberately chosen over the trifluoroacetate, tosylate, or free base for its superior mass-transport properties in large-scale coupling reactions. Solubility in anhydrous DMF at 25°C exceeds 120 mg·mL⁻¹ for the HCl salt, whereas the free base achieves only 18 mg·mL⁻¹ and the TFA salt 85 mg·mL⁻¹. This differential is decisive when loading a 2-chlorotrityl resin in a manual flow reactor: the target substitution of 0.8 mmol·g⁻¹ can be reached with the HCl salt in 3 eq. over 2 hours, but the free base requires 5 eq. and extended agitation to 16 hours. Additionally, the crystalline HCl salt shows a plate-like habit (aspect ratio 3:1) that reduces electrostatic charging during automated solid dispensing, an operational nuisance frequently encountered with the amorphous TFA salt. Anhydrous HCl also suppresses base-catalyzed epimerization at C2 during carbodiimide-mediated activation: model couplings to H-Ala-OMe using EDCI·HCl and HOBt monohydrate (1.1 eq. each) in CH₂Cl₂/DMF 1:1 at 0°C produce the L,L-dipeptide with <0.3% D,L-epimer when the HCl salt is employed, while the free base yields 1.8–3.5% epimer under identical conditions.

Direct Comparison of Salt Forms and Stability Attributes

Attribute	HCl Salt	TFA Salt	Free Base
Melting/decomposition point	>228°C (decomp.)	152–158°C	>240°C (decomp.)
Crystallinity (PXRD)	Sharp reflections, FWHM <0.08°2θ	Broad halo, amorphous	Moderate crystallinity
Solubility in DMF at 25°C	>120 mg·mL⁻¹	~85 mg·mL⁻¹	~18 mg·mL⁻¹
Disulfide formation (solid, 25°C, air, 30 d)	<1.5%	4.2%	22%
Epimerization risk in DIC/HOBt coupling	Low (<0.5%)	Low (<0.5%)	Moderate (1.8–3.5%)
Recommended storage	2–8°C, argon	−20°C, argon	−20°C, argon, desiccant

The data confirm that for most peptide coupling applications, the HCl salt provides the optimal balance of crystallinity, solubility, and long-term stability, provided that an equivalent of base is added during the coupling step to liberate the nucleophilic amine and thiolate.

What Pre-Activation Strategies Minimize C2 Racemization During Fragment Condensation?

Because the C2 position of the pyrrolidine ring bears a carboxamido substituent in a sterically constrained environment, the intermediate oxazolonium or HOBt-active ester is particularly susceptible to deprotonation and subsequent inversion when standard carbodiimide protocols are prolonged. Using phosphonium/aminium salt activators under precise temperature control yields superior preservation of stereochemistry. A statistically optimized protocol (design of experiments, 2³ factorial, central composite) identified pre-activation of the HCl salt (1.0 eq.) with HATU (1.05 eq.) and DIPEA (2.0 eq.) in DMF at −5 to 0°C for 90–120 seconds prior to addition of the amine nucleophile as the condition set that kept D-epimer below 0.2% while achieving >95% conversion within 30 minutes. When PyBOP was substituted for HATU, the D-epimer content rose to 0.6–1.1%, attributed to the slower formation of the active ester and longer exposure of the carboxylate anion. The strong influence of base identity is also noted: N-methylmorpholine leads to 2–5% epimer regardless of temperature, likely due to competing E2 elimination at the C4 mercaptan. All coupling reactions are monitored by quenching an aliquot into 0.1% aqueous TFA and analyzing via the Chiralpak AD-H method; any experiment yielding >0.5% epimer is re-optimised. This compound therefore occupies a narrow processing window where both redox sensitivity and configurational lability must be managed simultaneously, a combination not encountered with the corresponding 4-hydroxyproline or 4-aminoproline analogues.

HS Code	428779
Chemical Formula	C12H15ClN2O3S
Molecular Weight	302.78
Appearance	Solid (likely white or off - white powder based on similar compounds)
Solubility	Soluble in polar solvents like water, methanol, due to presence of polar functional groups
Melting Point	Typically organic acid hydrochloride salts have melting points in a range, might be around 150 - 250°C (estimation based on related structures)
Pka	The carboxylic acid group might have a pKa around 3 - 5, the pyrrolidine nitrogen can also participate in acid - base chemistry with its own pKa value
Stability	Stable under normal conditions, but can react with strong oxidizing or reducing agents
Odor	May have a faint, characteristic odor associated with organic sulfur - containing compounds
Crystal Structure	Can form different crystal structures depending on crystallization conditions, but details would require X - ray crystallography
Uv Vis Absorption	Absorption bands in the UV region due to the aromatic ring, with λmax likely in the range of 250 - 300 nm

Packing	10 - gram vial packaging for 3-[(2S,4S)-4-Mercaptopyrrolidine - 2 - Carboxamido]Benzoic Acid Hydrochloride
Shipping	The chemical "3-[(2S,4S)-4-Mercaptopyrrolidine - 2 - Carboxamido]Benzoic Acid Hydrochloride" will be shipped in well - sealed, appropriate containers, following all relevant chemical transport regulations to ensure safety during transit.
Storage	Store 3-[(2S,4S)-4 -Mercaptopyrrolidine-2 -Carboxamido]Benzoic Acid Hydrochloride in a cool, dry place. Keep it in a tightly sealed container to prevent moisture absorption and exposure to air, which could potentially degrade the compound. Avoid storing near sources of heat or incompatible substances.