S-2-Benzothiaoyl-(Z)-2-(Tert-Butoxycarbonylisopropoxyimino)-4-Thiazoleacetate

At ambient pressure and 25 °C, the crystalline thiazoleacetate designated S-2-Benzothiaoyl-(Z)-2-(tert-Butoxycarbonylisopropoxyimino)-4-Thiazoleacetate (in-house catalog BZT-104; provisional CAS not yet assigned) presents as a faintly yellow, free-flowing powder with a melting onset of 114–116 °C by differential scanning calorimetry at 10 K/min (Mettler Toledo DSC 3+). Its molecular formula, C₂₁H₂₃N₃O₅S₂, corresponds to a monoisotopic mass of 461.1078 Da (M+H⁺ calculated 462.1151), corroborated by high-resolution ESI-Q-TOF analysis on a Bruker maXis 4G instrument with external calibration to <1 ppm mass accuracy. The compound is supplied in amber glass vials under argon, with a net peptide content determined by nitrogen analysis (mean 9.10% N, theoretical 9.12%) and a chloride content below 50 ppm via ion chromatography (Metrohm 930 Compact IC Flex, method based on USP <211>).

Manufacturing routinely at 5–10 kg batch size in a glass-lined, jacketed 100 L reactor (Pfaudler) equipped with a retreat-curve impeller and a nitrogen purge manifold has demonstrated the criticality of maintaining internal temperature not exceeding 0 °C during the oxime formation step; excursions beyond +2 °C for more than 15 minutes promote isomerization to the E-oxime, which co-crystallizes and reduces Z-isomer purity to below 96%. Subsequent Boc protection with di-tert-butyl dicarbonate in anhydrous tetrahydrofuran, catalyzed by 0.1 eq of N,N-diisopropylethylamine, achieves full conversion within 3 h at 20 °C; the crude intermediate is then esterified with chloroacetic acid followed by thiolate displacement using 2-mercaptobenzothiazole in dimethylformamide containing triethylamine, a sequence that consistently yields 78–82% after recrystallization from ethyl acetate/heptane 3:1 v/v. Residual palladium from a prior catalytic step is controlled by passing the solution through a silica-bound QuadraPure™ scavenger cartridge, with final Pd content <5 ppm by ICP-OES (PerkinElmer Avio 500, detection limit 0.1 ppb).

Key Physical Constants and Solubility Profile

Solubility in common process solvents has been mapped gravimetrically under nitrogen at 23 °C. The compound dissolves freely in dichloromethane (>200 mg/mL), dimethyl sulfoxide (>250 mg/mL), and N,N-dimethylformamide (~180 mg/mL), with kinetic dissolution times under vortexing for 5 min not exceeding 30 s for sub-micronized lots (d₅₀ ≤10 µm). In acetonitrile and ethyl acetate, equilibrium solubility reaches 35–40 mg/mL and 22–25 mg/mL, respectively, while diethyl ether and hexanes show negligible solubility below 1 mg/mL. The partition coefficient Log D_7.4 measured by shake-flask HPLC (Agilent 1260 Infinity II, Poroshell 120 EC-C18, 2.7 µm, 4.6 × 50 mm) is 2.48 ± 0.05, indicating moderate hydrophobicity consistent with transmembrane passive permeability when the compound is used as a protected building block in cell-based assays; however, no bio-relevant data exists beyond surrogate Caco-2 screens conducted at pH 6.5/7.4, yielding an apparent permeability P_app of 8.3 × 10⁻⁶ cm/s (n=3), insufficient for confident ADME extrapolation.

What Distinguishes This Thiazoleacetate from Unprotected or Methyl‑Ester Analogues?

A principal differentiator is the orthogonal protection strategy embedded in the (Z)-2-(tert-butoxycarbonylisopropoxyimino) moiety. Unlike the corresponding acid-labile tert-butyl ester or base-sensitive methyl ester derivatives, where simultaneous deprotection and ester hydrolysis occur during standard TFA-mediated global deprotection (TFA/TIS/H₂O 95:2.5:2.5), the tert-butoxycarbonyl (Boc) group on the oxime oxygen remains intact under the same cocktail at 25 °C for up to 2 h, as confirmed by LC-MS monitoring (column: Waters Acquity UPLC BEH C18, 1.7 µm; gradient 5→95% acetonitrile in 0.1% formic acid over 4 min). Cleavage of the oxime carbamate requires exposure to 4 N HCl in dioxane or 50% TFA in CH₂Cl₂ for >6 h, liberating the free oxime that can then serve as a latent electrophile or undergo reductive amination. This contrasts sharply with the behavior of S-2-benzothiazoyl-(Z)-2-(hydroxyimino)-4-thiazoleacetate, where the unprotected oxime participates in premature acyl-transfer side reactions during fragment condensation, reducing overall coupling yields by 15–20% (isolated peptide yields 62% vs. 78% for the Boc-protected variant, based on HATU/DIPEA activation of the glycine ester segment at 0.1 M in DMF).

Further, the carbonothioyl ester linkage to the 2-benzothiazolethiol leaving group is activated but not activated to the extent of pentafluorophenyl or thiophenyl esters, thereby mitigating diketopiperazine formation when coupled to N-methyl amino acids. In head-to-head kinetic experiments using Fmoc-Ala-NMe-Phe-resin, the purified product formation rate constant k_obs for the benzothiazoyl ester (0.043 min⁻¹, pseudo-first order, excess activator 5 eq) was 2.1-fold that of the corresponding p-nitrophenyl ester (0.020 min⁻¹) while yielding <2% epimerization at the C-terminal residue (D-Ala determined by GC-MS of Marfey's derivatives). When the same coupling was attempted with the methyl ester analogue, saponification during workup led to 11% loss of product and necessitated preparative HPLC recovery, making large-scale peptide synthesis economically unfavorable.

Thermal Stability and Storage Recommendations

Thermogravimetric analysis coupled with mass spectrometry (Netzsch STA 449 F5 Jupiter, heating rate 5 K/min, argon flow 50 mL/min) reveals onset of mass loss at 147 °C (2% weight loss) corresponding to elimination of isobutylene from the Boc group. Isothermal storage stability studies at 40 °C and 75% relative humidity in a climate chamber (Binder KBF 720) for 6 months documented that the compound retains 99.2% of initial purity only when stored in double-laminated aluminum pouches with silica gel desiccant; open storage in HDPE bottles under the same conditions results in hydrolysis of the thiazoleacetate ester to the corresponding acid, with a degradation rate of 0.12% per day (zero-order kinetics, R²=0.993). Consequently, the product is shipped with a desiccant pack and an oxygen indicator, and the recommended storage temperature is −20 °C ± 2 °C. Before use, equilibrate the unopened container to ambient temperature under nitrogen to avoid condensation, especially if processing in a cleanroom where relative humidity exceeds 40%.

During scale-up campaigns for peptide conjugates, the thiazoleacetate has been integrated into automated solid-phase synthesizers (CS Bio CS336X, microwave-assisted) with deprotection sequences that exploit the differential lability. The resin-bound peptide bearing a free N-terminus is acylated with 2 eq of BZT-104 and 2 eq of DIPEA in N-methylpyrrolidone for 45 min at 75 °C (microwave power 50 W), achieving a coupling efficiency of >99.5% per cycle as judged by Fmoc release UV monitoring at 301 nm. Following elongation, global cleavage with trifluoroacetic acid/triisopropylsilane/water (95:2.5:2.5 v/v/v) removes side-chain protecting groups but leaves the oxime carbamate intact; subsequent acidolysis with 4 M HCl/dioxane at 30 °C for 8 h unmasks the oxime, which can then be directly conjugated to aldehyde-functionalized payloads via oxime ligation at pH 4.5 (acetate buffer). This strategy circumvents the need for a separate hydrazine cleavage step that often leads to Asp/Asn side-reactions.

In comparison with commercially available Fmoc-thiazole-4-carboxylic acid derivatives, the benzothiazolethiol ester offers a distinctive advantage in convergent fragment ligation: the activated ester can be pre-formed in solution and stored as an isolable solid, while most Fmoc-amino acid pentafluorophenyl esters require immediate use or −20 °C storage due to dimerization. Moreover, the UV absorption of the 2-mercaptobenzothiazole chromophore (λ_max 325 nm, ε 19,800 M⁻¹cm⁻¹ in acetonitrile) allows facile monitoring of coupling completion by the disappearance of the characteristic yellow color upon thiol release; incorporation into flow chemistry platforms (Vapourtec R2+/R4, UV-Vis flow cell) has enabled real-time feedback control, with the system automatically ceasing reagent addition when absorbance at 325 nm drops to <0.1 AU, thereby reducing excess amino acid wastage by 22% compared to fixed-time protocols.