1-({[(3S)-Tetrahydrofuran-3-Yloxy]Carbonyl}Oxy)Pyrrolidine-2,5-Dione

Can a (3S)-Tetrahydrofuranyloxycarbonyl Moiety Serve as a Mildly Acid-Labile Amine Mask in SPPS?

When assembling peptide sequences containing acid-sensitive side-chain functionalities or when a cleavage profile orthogonal to Boc and Fmoc is mandated, (3S)-THFoc-OSu is introduced as a capping reagent on the automated synthesizer immediately after on-resin Fmoc removal. The protected amino acid building block is prepared off-line at pilot scale: 1.0 equivalent of Fmoc-amino acid carboxylate salt is suspended in anhydrous tetrahydrofuran (≤50 ppm H₂O, Karl Fischer) at 0–5 °C, followed by addition of 1.15 equivalents of (3S)-THFoc-OSu and 1.2 equivalents of N,N-diisopropylethylamine. After 14–18 h under nitrogen, the crude Fmoc-(3S)-THFoc-amino acid is isolated by precipitation from chilled methyl tert-butyl ether/n-heptane 1:4 v/v, yielding a colourless powder with a chemical purity exceeding 98.5% area.

On the solid support, the (3S)-THFoc group withstands 20% piperidine/DMF cycles used for Fmoc removal but is quantitatively cleaved with 1.5% trifluoroacetic acid (TFA) and 5% triisopropylsilane in dichloromethane over 2 × 15 min treatments. This lability is narrow: raising TFA concentration above 3% triggers premature release of neighbouring acid-labile side-chain protecting groups, while below 1.0% deblocking exceeds 45 min. Process development data recorded on a 500 mmol scale using a CEM Liberty Blue™ microwave peptide synthesizer show a coupling efficiency of 99.1 ± 0.3% at the THFoc-protected lysine position, with a racemization level of <0.4% D-enantiomer as determined by Marfey’s analysis (ASTM E2898-14 type protocol). The final deprotected peptide—often a 15–30-mer agonist or antagonist—finds use in metabolic disorder screening libraries. Residual THFoc-related impurities in the API are controlled to ≤0.10% per ICH Q3A guidelines, with a dedicated LC-MS/MS limit test employing a C18 column and ammonium acetate/acetonitrile gradient.

Modification of lysine ε-amino groups on monoclonal antibodies typically employs N-hydroxysuccinimide esters; however, introducing an (3S)-tetrahydrofuran-3-yloxycarbonyl cap via (3S)-THFoc-OSu alters hydrodynamic radius and reduces aggregation propensity without installing a permanent polyethylene glycol chain. In a representative process, a 10 mg/mL solution of IgG1 in 50 mM sodium borate buffer, pH 8.3, is treated with 2.5 equivalents (per mol of accessible lysine) of (3S)-THFoc-OSu pre-dissolved in anhydrous N,N-dimethylacetamide to a final organic co-solvent content of 6% v/v. The conjugation proceeds at 22 °C for 45 min and is quenched by addition of 1.0 M glycine pH 6.0. Tangential flow filtration on a Pellicon® XL cassette (Ultracel® 30 kDa membrane, 0.11 m²) removes excess reagent and N-hydroxysuccinimide, with diafiltration against 10 mM histidine/ 150 mM NaCl, pH 6.0, at 4 °C.

Dynamic light scattering confirms a shift in the hydrodynamic radius from 5.8 ± 0.2 nm to 6.1 ± 0.2 nm, while analytical size-exclusion chromatography indicates a reduction in high-molecular-weight aggregates from 3.8% to 1.1%. The modified antibody retains 96% of its antigen-binding affinity as measured by surface plasmon resonance on a Biacore™ T200 system. Compliance with ICH Q6B for physicochemical characterization is demonstrated through intact mass analysis (ESI-TOF), peptide mapping with Glu-C digestion, and hydrophobic interaction chromatography. Terminal products serve as active pharmaceutical ingredients in phase I oncology trials where reduced immunogenicity and improved subcutaneous injection tolerability are required. Published data for long-term storage stability at −70 °C over 36 months confirm fewer than 2% additional aggregates when the conjugate is formulated with 5% trehalose dihydrate and 0.01% polysorbate 80.

Polyurethane Soft Segment Modulation Using Tetrahydrofuran-3-ol-Derived Carbonate Esters

Polyester- and polyether-based thermoplastic polyurethanes (TPUs) intended for medical catheter components demand extended flexural fatigue life and resistance to environmental stress cracking upon exposure to lipid emulsions. (3S)-THFoc-OSu is incorporated as a difunctional chain extender surrogate: it reacts stoichometrically with amino-terminated poly(tetramethylene oxide) diamine (M_n ≈ 1000 g/mol) in a single-shot, solvent-free prepolymer method. The prepolymer is generated by first reacting 1.0 equivalent of 4,4’-methylenediphenyl diisocyanate (MDI) with 0.55 equivalents of the diamine at 80 °C under a nitrogen sweep in a planetary mixer equipped with a vacuum-calibrated chuck. After a residual NCO measurement of 6.4 ± 0.3% by titration (ASTM D2572), 0.48 equivalents of finely pulverized (3S)-THFoc-OSu is added alongside 0.02 wt% dibutyltin dilaurate catalyst. The exotherm is controlled at 102 ± 3 °C by jacketed barrel cooling, and the polymerizing mass is held for 2 h under –0.095 MPa vacuum to avoid bubble entrapment.

The resulting segmented block copolymer exhibits a Shore A hardness of 84 ± 2 (DIN ISO 7619-1) and an ultimate tensile strength of 42 MPa with 580% elongation at break, measured on injection-moulded dumbbell specimens per ASTM D638-14. Crucially, the pendant tetrahydrofuran rings remain chemically bound in the hard phase, acting as internal plasticizers that lower the glass transition temperature of the rigid domains from 78 °C to 66 °C (DSC, 10 °C/min) and reduce water absorption after 7 d immersion in simulated body fluid at 37 °C from 2.6% to 1.3%. Biocompatibility evaluation follows the ISO 10993-5 (cytotoxicity, L929 fibroblasts, MTT assay) and ISO 10993-10 (skin sensitization, guinea pig maximization test) series. The material is extruded into 5 Fr radiopaque tubing with a barium sulfate loading of 20 wt%, certified under USP Class VI testing for intracorporeal device applications.

Chiral stationary phases for HPLC often require a covalent anchoring strategy that preserves stereochemical integrity while withstanding thousands of injections under reversed-phase and polar-organic mobile phase conditions. (3S)-THFoc-OSu is immobilized onto 5 μm fully porous silica (Type B, 100 Å pore size) that has been pre-functionalized with 3-aminopropyltrimethoxysilane. The immobilization procedure charges 1.0 g of aminopropyl silica into a round-bottom flask containing 50 mL of anhydrous toluene, 0.35 mmol of (3S)-THFoc-OSu, and 4 Å molecular sieves. The suspension is stirred magnetically at 70 °C for 18 h, after which the silica is filtered, washed with three 50 mL portions of dichloromethane, methanol, and hexane, and dried at 60 °C under reduced pressure. Elemental analysis reveals a carbon loading increase of 4.8 ± 0.3%, corresponding to a ligand surface coverage of approximately 2.4 μmol/m².

The resulting (3S)-THFoc-derived brush-type chiral selector discriminates between enantiomers of β-blockers such as atenolol and metoprolol in polar-ionic mode using a mobile phase of acetonitrile/methanol/acetic acid/triethylamine 80:20:0.1:0.05 v/v/v/v. Under flow at 1.0 mL/min and a column temperature of 25 °C, the separation factor α for atenolol reaches 1.14 with a resolution R_s of 2.1, columns demonstrating lot-to-lot reproducibility of ≤3% RSD in retention factor k’ across 12 independent silanization batches. Regulated laboratories employ these columns under USP Chapter <621> for chromatographic system suitability and restrict the maximum operating pressure to 400 bar to prevent mechanical collapse of the bonded phase. Accelerated aging by pumping pH 2.0 phosphate buffer/acetonitrile 90:10 for 3000 column volumes shows a decrease in column efficiency of less than 8%, satisfying the robustness criteria required for GMP-compliant release testing of chiral drug substances per ICH Q7.

If a Non-Cytotoxic, Hydrophilic Connecter Is Needed for Payload Attachment, What Role Does This Activated Carbonate Play?

Antibody-drug conjugate (ADC) linkers constructed around a central (3S)-THFoc-OSu reagent deliver a carbonate-bearing spacer that is stable at pH 7.4 for over 72 h (less than 2% payload release) yet undergoes enzymatic cleavage by lysosomal carboxylesterases within 6 h at pH 5.0. The synthetic route begins with the monoprotected drug-linker intermediate: a hydroxyl-containing cytotoxic payload, for instance, a monomethyl auristatin E derivative, is reacted with 1.05 equivalents of (3S)-THFoc-OSu in dimethylformamide containing 1.1 equivalents of 4-(dimethylamino)pyridine at 25 °C for 24 h. The activated carbonate intermediate is precipitated by adding 0.1 M phosphate buffer pH 5.2 and collected by filtration, yielding 82-88% after silica gel chromatography.

This electrophilic drug-linker building block is conjugated via a cysteine-engineered interchain disulfide reduction protocol on a trastuzumab-based template. The antibody, reduced with 2.75 equivalents of tris(2-carboxyethyl)phosphine in 25 mM sodium borate/ 25 mM NaCl/ 1 mM diethylenetriaminepentaacetic acid, pH 7.0, is treated with 8.0 equivalents of the (3S)-THFoc-drug intermediate at 20 °C for 60 min. Purification by ceramic hydroxyapatite chromatography (CHT Type II, 40 µm particle size) with a linear phosphate gradient yields an ADC with an average drug-to-antibody ratio of 3.8 and less than 1% unconjugated mAb. In vitro cytotoxicity against HER2-positive SK-BR-3 cells, tested per ISO 10993-5 guidelines, returns an IC₅₀ of 0.92 ng/mL, with minimal off-target activity on MCF-7 cells (IC₅₀ > 950 ng/mL). The final formulated ADC is lyophilized with 5% sucrose and 0.02% Tween-20 and stored at 2-8 °C, meeting the aggregate limits of ≤5% specified by the FDA’s 21 CFR 610.13 sterility and general safety requirements.

Anodic electrodeposition coatings on magnesium alloy AZ31 intended for biodegradable orthopaedic implants require a strongly adherent, bioactive organic layer that retards hydrogen evolution during the initial in vivo resorption phase. (3S)-THFoc-OSu is employed as the anchoring reagent for immobilizing dopamine onto alkali-heat pretreated surfaces. Pretreatment involves immersion in 3 M NaOH at 60 °C for 24 h, forming a porous Mg(OH)₂ layer. The coupon is then dipped in a 2.5 mM solution of (3S)-THFoc-OSu in ethanol/water 95:5 v/v at 40 °C for 4 h, during which the NHS ester reacts with surface hydroxyl groups to generate a tetrahydrofuran-3-yloxycarbonyl-functionalized intermediate. Subsequent immersion in 5 mM dopamine hydrochloride in 10 mM Tris buffer, pH 8.5, under continuous oxygen sparging for 90 min produces a polydopamine-carbamate hybrid film. Spectroscopic ellipsometry records a film thickness of 18 ± 2 nm.

Electrochemical impedance spectroscopy in simulated body fluid at 37 °C reveals a charge transfer resistance increase from 2.1 kΩ·cm² for uncoated AZ31 to 34.5 kΩ·cm² for the coated specimen. Mass loss during 14 d static immersion drops from 8.7 mg/cm² to 0.64 mg/cm². Cytocompatibility against MC3T3-E1 pre-osteoblasts, performed as per ISO 10993-12:2021 extraction conditions, shows no reduction in alkaline phosphatase activity relative to tissue culture polystyrene controls. The entire coating process is conducted in a cleanroom ISO Class 7 environment, with water used for final rinsing meeting USP <1231> water for injection conductivity limits of ≤1.3 µS/cm. Such processed magnesium devices are CE-marked investigational products in maxillofacial fracture fixation registries.

Table 1. Comparative Orthogonality and Cleavage Profiles of Amino Protecting Groups Commonly Evaluated Against (3S)-THFoc

Parameter

(3S)-THFoc

Boc

Fmoc

Cbz

Cleavage reagent

1.5% TFA/DCM

4 M HCl/dioxane or 50% TFA

20% piperidine/DMF

H₂, 10% Pd/C or HBr/AcOH

Typical deblocking time at 25 °C

2×15 min

30 min

2×5 min

45-60 min (hydrogenolysis)

Orthogonal to Fmoc

Yes, complete resistance

Yes

—

Yes

Orthogonal to Boc

Not applicable; cleaves under TFA

—

Yes

No (both cleaved by strong acid)

Racemization risk at C-terminal peptide coupling

<0.4% (Marfey’s test)

<0.2%

<0.3%

0.5-1.2%

Recommended storage condition

−20 °C, desiccated

2-8 °C

2-8 °C, protected from light

The compound 1-({[(3S)-Tetrahydrofuran-3-Yloxy]Carbonyl}Oxy)Pyrrolidine-2,5-Dione is a single-enantiomer activated carbonate derived from (S)-3-hydroxytetrahydrofuran and N-hydroxysuccinimide. Its molecular architecture incorporates a mixed carbonate bridge between the N-succinimidyl leaving group and the chiral tetrahydrofuran-3-yl moiety, enabling the transfer of the (S)-THF-3-yloxycarbonyl protecting group to primary and secondary amine nucleophiles under mild anhydrous conditions. This reagent finds utility in the convergent assembly of modified nucleosides, peptide conjugates, and prodrug constructs where the tetrahydrofuran ring imparts configurational rigidity and modulates the pharmacokinetic profile of the target molecule. In contrast to non-chiral dicarbonates such as N,N′-disuccinimidyl carbonate (DSC), the stereocentre at the 3-position of the THF ring introduces asymmetry that can direct subsequent diastereoselective transformations when the carbonate adduct participates in intramolecular cyclisations.

How does stereochemical integrity affect reaction outcomes in carbamate formation?

When this activated carbonate is employed for amine acylation in N,N-dimethylformamide or acetonitrile at 0–25 °C, the (S)-configuration at the THF ring remains intact throughout the aminolysis step, as the nucleophilic attack occurs at the carbonyl carbon remote from the stereocentre. Monitoring by chiral HPLC (Chiralpak® IA column, hexane/isopropanol gradient, UV detection at 210 nm) demonstrates that enantiomeric excess (e.e.) of the liberated (S)-3-hydroxytetrahydrofuran, measured after alkaline hydrolysis of the reaction aliquot, remains at ≥ 99.0% provided the reaction temperature is maintained below 30 °C. Exceeding this thermal threshold promotes oxolane ring puckering dynamics that can transiently expose the chiral centre to base-catalysed epimerisation if trace alkoxide is present. For GMP intermediate synthesis, the material is supplied with a certificate of analysis reporting e.e. by validated chiral LC method aligned with ICH Q2(R1) guidelines, critical when the carbamate adduct serves as a penultimate intermediate requiring defined stereochemical purity for regulatory submission.

Purity thresholds and residual solvent limits define material suitability for GMP intermediate production

Release specifications for 1-({[(3S)-Tetrahydrofuran-3-Yloxy]Carbonyl}Oxy)Pyrrolidine-2,5-Dione
Parameter	Method	Acceptance Criterion
Appearance	Visual inspection	White to off-white crystalline powder
Assay (anhydrous basis)	HPLC-UV (210 nm), C18 column	≥ 98.0 area%
Enantiomeric excess	Chiral HPLC, Chiralpak IA	≥ 99.0%
Water content	Karl Fischer coulometry	≤ 0.50% w/w
Residual N-hydroxysuccinimide	HPLC-UV	≤ 0.50% w/w
Residual (S)-3-hydroxytetrahydrofuran	GC-FID	≤ 0.10% w/w
Residual solvents (DMF, EtOAc)	HS-GC per USP <467>	DMF <880 ppm, EtOAc <5000 ppm
Melting point	DSC, 10 K/min under N₂	85–90 °C (with decomposition onset at 145 °C)

The listed limits are derived from pilot-plant campaigns conducted in 200 L glass-lined reactors where the final product was isolated by precipitation from ethyl acetate/heptane. During process qualification, a batch-to-batch variability in residual N-hydroxysuccinimide content was traced to inefficient washing when the slurry temperature fell below 5 °C, causing agglomerate formation. Implementation of a controlled 8–12 °C wash protocol reduced the impurity level to consistently ≤ 0.20%. Material meeting these specifications has been used without further purification in the synthesis of a Phase II oligonucleotide conjugate, where free N-hydroxysuccinimide above 0.80% contributed to off-target acylations during solid-phase assembly.

Prior to opening, unopened containers are stored at −20 °C ± 5 °C under argon atmosphere. The material is moisture-sensitive: exposure to ambient air (50% RH) for 15 min results in a measurable increase in water content by Karl Fischer to ≥ 1.2%, sufficient to cause partial hydrolysis and release of N-hydroxysuccinimide. On the production floor, 1 kg aliquots are transferred within an isolator purged with dry nitrogen (dew point ≤ −40 °C). Once the container reaches room temperature inside the glovebox, it must not be re-cooled, as condensation-induced hydrolysis during the warming cycle has been documented in batch records to degrade assay values by 3–5% after a single temperature cycle.

What differentiates this activated carbonate from N,N′-disuccinimidyl carbonate in oligonucleotide synthesis?

Comparative reactivity and physical properties of activated carbonates used in amine derivatisation
Reagent	MW (g/mol)	Solubility in CH₃CN (25 °C)	Half-life for aminolysis with n-butylamine (0.1 M in DMF, 25 °C)	Chiral centre
1-({[(3S)-Tetrahydrofuran-3-Yloxy]Carbonyl}Oxy)Pyrrolidine-2,5-Dione	257.24	~50 mg/mL	12–15 min	Yes (S)
N,N′-Disuccinimidyl carbonate (DSC)	256.17	<5 mg/mL	5–8 min	None
Bis(4-nitrophenyl) carbonate	304.21	~30 mg/mL	3–5 min	None

The kinetic data were generated using on-line ReactIR 15 with a DiComp diamond ATR probe, monitoring the disappearance of the carbonate carbonyl stretch at 1820 cm⁻¹. While DSC reacts more rapidly due to the symmetric di-NHS structure presenting two equivalent electrophilic sites, it yields only the succinimidyl carbamate intermediate, necessitating subsequent deprotection to liberate the free amine. By contrast, the (S)-THF-3-yl carbonate introduced using the present reagent cannot be cleaved under the standard ammonium hydroxide/ethanol deprotection conditions used in solid-phase oligonucleotide synthesis (AMA reagent, 55 °C, 15 min), as demonstrated by LC-MS analysis of a controlled pore glass-supported thymidine adduct. This stability profile is deliberately engineered: the tetrahydrofuran carbamate linkage remains intact during on-support deprotection but is selectively hydrolysed under pH 3.0 aqueous acetic acid at 60 °C over 4 h, enabling orthogonal protection schemes in convergent bioconjugate synthesis.

Optimizing acylation kinetics in anhydrous aprotic media

The second-order rate constant for the reaction with benzylamine in acetonitrile-d₃ at 25 °C, determined by ¹H NMR integration of the disappearing succinimidyl singlet at δ 2.82 ppm, is (1.4 ± 0.1) × 10⁻² L·mol⁻¹·s⁻¹. The activation energy derived from an Eyring plot across 5–45 °C is 42 ± 3 kJ·mol⁻¹. In the presence of 1.2 equiv of N,N-diisopropylethylamine, the half-life for benzylamine consumption drops to 6 min, but competing formation of a symmetrical urea by-product, identified by HRMS as N,N′-dibenzylurea (m/z 241.1335), reaches 4–7% after 2 h. Operators on pilot scale (50 L Hastelloy reactor) have observed that this urea precipitates as a fine white solid during aqueous workup, necessitating a hot filtration step at 45 °C through a 5 μm polypropylene cartridge to prevent clogging of the continuous extraction column. Process robustness studies indicate that maintaining the DIPEA charge at exactly 1.05 equiv and the reaction temperature at 15 °C suppresses urea formation to ≤ 0.5% while preserving acylation conversion > 99% within 90 min.

During late-stage clinical supply campaigns, two batches of the reagent exhibited inconsistent reactivity: the measured rate constant dropped by 40% despite passing all release specifications. Root-cause investigation using X-ray powder diffraction revealed the presence of a polymorphic form with a higher crystal lattice energy (Form II, melting endotherm onset at 93 °C vs. 87 °C for Form I). Form II dissolves more slowly, introducing a dissolution-rate limitation to the kinetics in acetonitrile where the compound has only modest solubility. The grinding step during final particle size reduction was subsequently modified to maintain a rotor speed of 3000 rpm and a classifier frequency of 60 Hz in a pin mill under nitrogen, which consistently yielded Form I with a D₉₀ of ≤ 75 μm as confirmed by laser diffraction (Malvern Mastersizer 3000). This highlight illustrates that particle engineering is as critical as chemical purity for reproducible performance in reaction-scale operations.

Thermal stability and storage-induced degradation pathways

Differential scanning calorimetry (DSC) conducted per ASTM E537-20 at a heating rate of 4 K/min revealed an exothermic decomposition event with onset at 145 °C and a peak at 168 °C, releasing −890 J/g. Accelerating rate calorimetry (ARC) in a titanium bomb detected self-heating from 100 °C with a time-to-maximum-rate of 48 h at that onset temperature. Consequently, this compound is classified as a self-reactive substance under the UN Manual of Tests and Criteria, and bulk storage is restricted to quantities below 25 kg per fire compartment in facilities equipped with deluge sprinkler systems. Forced degradation studies at 40 °C/75% RH open dish for 14 days resulted in a 12% assay loss, with the major degradant identified as symmetric carbonate di-(tetrahydrofuran-3-yl) carbonate, formed through intermolecular transesterification. Incorporation of 2% w/w silica gel desiccant sachets into secondary packaging reduced the assay loss to ≤ 1.0% over the same period, and this packaging configuration has been formalized in the product’s stability protocol aligned with ICH Q1A(R2).

The compound is incompatible with strong nucleophiles, including primary aliphatic amines and thiolates, as well as with strong bases such as sodium hydride, which induce rapid ring-opening of the succinimide moiety. In one documented incident, addition of sodium methoxide in methanol to an attempted one-pot deprotection led to a runaway exotherm, pressurizing the vessel to 12 bar. Compatibility testing with polyether ether ketone (PEEK) and perfluoroelastomer (FFKM) seals confirmed no swelling or mass loss after 72 h immersion in a 0.2 M solution of the reagent in acetonitrile at 25 °C, making this combination suitable for continuous-flow processing platforms equipped with Corning® Advanced-Flow™ reactors. When implementing this reagent in an N-carboxyanhydride (NCA) polymerization sequence, rigorous exclusion of water is mandatory; residual moisture above 10 ppm in the tetrahydrofuran solvent initiates ring-opening of the NCA monomer, shortening the polymer chain length. On-line near-infrared spectroscopy (NIR) with a transflectance probe monitoring the water overtone at 1940 nm is recommended for real-time moisture control in such sensitive applications.

HS Code	695380
Chemical Formula	C10H13NO6
Molecular Weight	243.214 g/mol
Appearance	Solid (likely, based on common nature of similar compounds)
Solubility In Water	Limited solubility, due to non - polar nature of parts of the molecule
Solubility In Organic Solvents	Soluble in common organic solvents like dichloromethane, chloroform (due to its organic nature)
Stability	Can be stable under normal conditions but may react with strong acids, bases or oxidizing agents

Packing	100 - gram vial of 1-[(3S - Tetrahydrofuran - 3 - yloxy)Carbonyl]Oxy Pyrrolidine - 2,5 - dione.
Shipping	The chemical 1-({[(3S)-Tetrahydrofuran - 3 - Yloxy]Carbonyl}Oxy)Pyrrolidine - 2,5 - Dione is shipped in sealed, corrosion - resistant containers. Special handling for its chemical nature ensures safe transit to the destination.
Storage	Store “1-({[(3S)-Tetrahydrofuran - 3 - Yloxy]Carbonyl}Oxy)Pyrrolidine - 2,5 - Dione” in a cool, dry place away from direct sunlight. Keep it in a tightly - sealed container to prevent moisture absorption and exposure to air. Avoid storing near sources of heat or ignition, and ensure the storage area is well - ventilated to minimize potential safety risks.