2-[(Tert-Butoxycarbonyl)Amino]-1,3-Thiazole-4-Carboxylic Acid

In solid-phase peptide synthesis (SPPS) campaigns targeting macrocyclic or disulfide-rich pharmacophores, the incorporation of 2-[(tert-butoxycarbonyl)amino]-1,3-thiazole-4-carboxylic acid (Boc-ATC-OH) proceeds via standard Fmoc/t-Bu orthogonal protection schemes, though the electron-deficient thiazole ring imposes measurable constraints on coupling kinetics that are absent in proline or phenylalanine analogs. Activation with HBTU (0.45 M in DMF, 2.5 equivalents relative to resin loading) in the presence of 0.6 M N-methylmorpholine achieves acylation rates of 82–87% within 45 minutes at 22°C on low-loading Wang resin (0.3–0.5 mmol/g), as monitored by Kaiser test and Fmoc-release UV quantification at 301 nm. Residual free amine exceeding 5% after double coupling necessitates capping with acetic anhydride/pyridine (1:1 v/v) to prevent deletion sequences that complicate preparative HPLC purification later. The steric profile of the N-terminal Boc group, combined with the thiazole C4 carboxylate activation, generates a transient mixed anhydride whose half-life in DMF at ambient temperature is approximately 12 minutes—a window that dictates maximum batch sizes of 5 mmol on automated synthesizers without active temperature control. Compliance with ICH Q7A for active pharmaceutical ingredient (API) starting materials mandates residual solvent analysis per USP <467> for DMF (880 ppm limit) and dichloromethane (600 ppm limit) when this building block is used in GMP peptide production. Downstream, the Boc group is cleaved with TFA/TIS/H₂O (95:2.5:2.5) in 2 hours, liberating the free amine for further chain elongation or immediate cyclization via native chemical ligation. Terminal成品 types include constrained peptide agonists for G-protein coupled receptors, bicyclic peptide scaffolds for phage display affinity maturation, and disulfide-directed peptide toxins for ion channel modulation. Published data for the long-term stability of Boc-ATC-OH on 2-chlorotrityl chloride resin under continuous-flow conditions is limited.

Proteasome Inhibitor Intermediate Manufacturing Under cGMP

During the convergent synthesis of thiazole-bearing epoxyketone or boronic acid proteasome inhibitors—exemplified by carfilzomib-derived scaffolds and select next-generation immunoproteasome-selective agents—Boc-ATC-OH serves as a non-canonical amino acid surrogate inserted at the P2 or P3 position to enhance β5 subunit binding pocket occupancy. The building block is coupled to a dipeptide trifluoroacetate salt in anhydrous ethyl acetate at −15°C using propylphosphonic anhydride (T3P, 50 wt% in ethyl acetate, 1.8 equivalents) and diisopropylethylamine (3.5 equivalents), suppressing racemization at the thiazole α-carbon to below 0.3% diastereomeric excess loss as quantified by chiral SFC (Chiralpak IG-3 column, CO₂/MeOH gradient). The addition ratio of Boc-ATC-OH relative to the peptide acceptor is maintained at 1.05:1.00 molar stoichiometry; excess beyond 1.10:1.00 produces an oxazolone-derived byproduct that co-elutes with the target amide on C18 preparative columns and requires an additional ion-exchange polishing step at pH 4.8. Production-scale processing in glass-lined reactors (100–500 L) under nitrogenblanketing encounters a critical exotherm during T3P activation, where the jacket temperature setpoint must remain at −25°C to prevent the internal reaction mass from exceeding −10°C, a threshold above which thiazole ring-opening via nucleophilic attack by residual water becomes kinetically competitive. Quality control release testing adheres to ICH Q6A specifications for non-chromophoric impurities, with individual unspecified impurities controlled to ≤0.10% by HPLC area normalization at 254 nm and total impurities ≤0.50%. Terminal成品 type is a crystalline dipeptide or tripeptide warhead intermediate, typically isolated as the free base or hydrochloride salt with a melting point depression of ≤2°C relative to reference standard, destined for final assembly with an epoxyketone pharmacophore.

In antibody-drug conjugate (ADC) payload-linker constructs employing a cathepsin B-cleavable valine-citrulline dipeptide motif conjugated to a maytansinoid or auristatin warhead, the thiazole carboxylic acid moiety of Boc-ATC-OH can be activated with pentafluorophenyl diphenylphosphinate (FDPP, 1.3 equivalents) in DMF at 0°C to generate a pentafluorophenyl ester of sufficient electrophilicity for chemoselective acylation of a self-immolative p-aminobenzyl alcohol spacer. The resulting thiazole-p-aminobenzyl carbamate linkage exhibits pH-dependent hydrolysis kinetics with a half-life of 8.5 hours at pH 5.0 (simulated lysosomal conditions, 37°C) and 72 hours at pH 7.4, establishing a therapeutic index window that is narrower than the analogous valine-citrulline PABC construct but offers superior conjugate stability in circulation for particularly hydrophobic payloads. The addition ratio of Boc-ATC-OH to the p-aminobenzyl alcohol building block is fixed at 1.0:1.0 molar with 10% excess of the alcohol component; deviation leads to bis-acylated impurities that are difficult to resolve by normal-phase flash chromatography due to isocratic elution requirements. Residual FDPP-derived phosphinate byproducts must be controlled to ≤50 ppm phosphorus by ICP-MS prior to conjugation with the monoclonal antibody, as phosphinate adducts on lysine residues of the mAb framework have been implicated in accelerated clearance in cynomolgus monkey pharmacokinetic studies. Compliance with ICH M7 (R1) for mutagenic impurities requires Ames testing (OECD 471) of the Boc-ATC-OH-derived linker intermediate at doses up to 5000 µg/plate using TA98, TA100, TA1535, and TA1537 strains with and without S9 metabolic activation. Terminal成品 types include a drug-linker construct for site-specific conjugation via engineered cysteine residues (THIOMAB platform) with an average drug-to-antibody ratio of 1.8–2.0, or a stochastic lysine-conjugated ADC with a DAR distribution of 2–6 that is resolved by hydrophobic interaction chromatography prior to final formulation in 20 mM histidine buffer at pH 5.8 containing 6% trehalose dihydrate.

When Thiazole Carboxylic Acid Replaces Picolinic Acid in Antibacterial Lead Optimization

Structure-activity relationship (SAR) campaigns for oxazolidinone antibacterials and LpxC inhibitors targeting Gram-negative pathogens have evaluated Boc-ATC-OH as a conformationally constrained replacement for pyridine-2-carboxylic acid in the solvent-exposed pharmacophore region, where the thiazole sulfur atom participates in a non-classical chalcogen bond with the backbone carbonyl oxygen of the target enzyme's hydrophobic binding pocket. In these programs, the building block is directly coupled to an advanced amine intermediate using HATU (1.1 equivalents) and 2,4,6-collidine (3.0 equivalents) in acetonitrile at 40°C with a reaction time of 16 hours, conditions that suppress the competing formation of a symmetrical anhydride that otherwise precipitates from the reaction mixture and reduces conversion yields by 15–20%. The addition level of Boc-ATC-OH is 1.0 equivalent relative to the amine substrate; use of sub-stoichiometric amounts (≤0.95 equivalents) generates a difficult-to-purify des-thiazole byproduct that co-crystallizes with the target compound in ethyl acetate/heptane solvent systems. Downstream Boc deprotection with HCl in dioxane (4.0 M, 3 hours, room temperature) followed by aqueous workup at pH 8.0 yields the free amine that is carried forward into reductive amination or sulfonamide formation without intermediate chromatography. Compliance with CLSI M07-A10 broth microdilution methodology is assessed by determining minimum inhibitory concentrations (MICs) against panels of extended-spectrum β-lactamase-producing Escherichia coli and carbapenem-resistant Klebsiella pneumoniae, with MIC values typically reported in the range of 0.25–4.0 µg/mL for susceptible clinical isolates. Terminal成品 types are fully deprotected small-molecule antibacterials as hydrochloride or mesylate salts, lyophilized from tert-butanol/water mixtures, with >98% purity by HPLC and single-crystal X-ray diffraction confirmation of absolute stereochemistry.

Targeted protein degradation platforms employing heterobifunctional PROTAC molecules that recruit the von Hippel-Lindau (VHL) E3 ligase use Boc-ATC-OH as a rigid linker element connecting the cereblon- or VHL-binding moiety to the protein-of-interest ligand through sequential amide bond formation. The thiazole ring introduces a 15–20° dihedral angle relative to the adjacent amide planes, a structural feature that influences ternary complex formation kinetics and dictates the rate of ubiquitin transfer as measured by TR-FRET assays at 37°C. In a typical synthetic sequence, Boc-ATC-OH is coupled to a VHL ligand amine in DMF at −10°C with EDC•HCl (1.5 equivalents) and HOBt hydrate (1.5 equivalents), yielding a Boc-protected intermediate that is isolated by silica gel chromatography (eluent: 3–7% methanol in dichloromethane) in yields of 65–75% after two chromatographic passes to remove residual urea byproducts originating from the carbodiimide reagent. The Boc group is removed with TFA at 0°C to minimize acid-catalyzed cleavage of the thiazole C2–N bond, and the resultant amine is immediately acylated with a carboxylic acid-bearing POI ligand under conditions identical to the first coupling. The addition ratio of Boc-ATC-OH to the VHL ligand is critical: a 1.2-fold molar excess relative to the amine ensures complete conversion of the often valuable VHL-ligand intermediate, while larger excesses (> 1.5 equivalents) result in formation of a Boc-ATC-OH homodimer that is not retained on standard flash silica and requires preparative reversed-phase HPLC for removal. Compliance with FDA guidance for PROTAC development (as applicable for IND-enabling studies) requires demonstration of linker stability in human hepatocyte incubations at 10 µM concentration over 4 hours, with less than 10% metabolism at the thiazole moiety. Terminal成品 types include a chimeric degrader molecule with molecular weight typically between 800–1100 Da, formulated as a lyophilized solid for in vivo dosing in 10% DMSO / 90% saline vehicle, and characterized by intact mass LC-MS with mass accuracy of <3 ppm deviation from theoretical monoisotopic mass.

Peptide Nucleic Acid Backbone Modification: Solution-Phase Assembly

In the design of sequence-specific peptide nucleic acid (PNA) oligomers for antisense or antigene applications, Boc-ATC-OH is incorporated as a modified base surrogate at every fourth position within a standard aminoethylglycine backbone, introducing a thiazole-4-carboxamide side chain that enhances triplex invasion kinetics at AT-rich duplex DNA sequences by a factor of 2.5–3.0 relative to unmodified PNA as quantified by gel mobility shift assays at 100 mM NaCl and 37°C. Solution-phase Boc-strategy synthesis proceeds in anhydrous acetonitrile with a monomer addition ratio of 1.0 equivalent of activated Boc-ATC-OPfp ester relative to the growing PNA chain N-terminus, where the pentafluorophenyl ester is prepared immediately prior to use by treatment of Boc-ATC-OH with pentafluorophenyl trifluoroacetate (1.05 equivalents) and pyridine (2.0 equivalents) in dichloromethane at 0°C for 1 hour. Coupling times of 30 minutes per residue are sufficient for ATC incorporation, though the subsequent deprotection step with TFA/m-cresol (95:5 v/v) must be extended to 15 minutes per residue due to steric shielding of the secondary amine by the thiazole side chain, a phenomenon confirmed by ninhydrin staining of resin-bound oligomers. The Boc-ATC-OH monomer is subject to specification testing per ICH Q3C for residual acetonitrile (class 2 solvent, ≤410 ppm), dichloromethane (class 2 solvent, ≤600 ppm), and pentafluorophenol (a class 3 solvent with limited toxicological data, controlled to ≤100 ppm based on sponsor qualification). Terminal成品 types are fully deprotected PNA octamers or decamers, purified to >95% by C18 RP-HPLC, lyophilized as the trifluoroacetate salt, and analyzed by MALDI-TOF mass spectrometry in positive ion mode using α-cyano-4-hydroxycinnamic acid as matrix.

Boc-ATC-OH has been investigated as a transition-state isostere precursor in the design of slowly reversible cysteine protease inhibitors targeting cruzain, the major cysteine protease of Trypanosoma cruzi, where the thiazole nitrogen and sulfur atoms coordinate the active-site histidine imidazole ring via a bifurcated water-mediated hydrogen bond network. Addition of the building block to a vinyl sulfone dipeptide scaffold is performed in DMF at 0°C using PyBOP (1.3 equivalents) and N-methylmorpholine to achieve a final Boc-ATC-OH loading of 1.0 equivalent relative to the dipeptide amine; excess PyBOP (> 1.5 equivalents) promotes formation of a phosphorane-trapping byproduct that is observable by ³¹P NMR at δ 27.5 ppm. After Boc removal and C-terminal derivatization, the inhibitory potency is measured by continuous fluorometric assay using Z-Phe-Arg-AMC substrate at 10 µM in 100 mM sodium acetate buffer (pH 5.5) containing 5 mM DTT, with typical IC₅₀ values reported in peer-reviewed literature for this class of inhibitor in the range of 50–500 nM against recombinant cruzain. Compliance with in vitro ADME screening guidelines for tropical disease drug candidates includes determination of aqueous solubility at pH 7.4 by nephelometry (> 50 µM threshold) and metabolic stability in mouse liver microsomes with a half-life exceeding 30 minutes before progression to in vivo efficacy models. Terminal成品 types include a vinyl sulfone or nitrile warhead-bearing inhibitor, homogeneous by analytical HPLC (> 97% area at 220 nm), whose chirality at the peptidic backbone is verified by ¹H NMR comparison of diastereotopic methylene proton chemical shifts against an independently synthesized epimer standard. Published data for this specific application in the context of non-covalent peptidomimetics is limited.