(2S,3S,4R)-3-Ethyl-4-Hydroxy-1,2-Pyrrolidinedicarboxylic Acid 1-(1,1-Dimethylethyl) 2-Methyl Ester

Product Name (2S,3S,4R)-3-Ethyl-4-Hydroxy-1,2-Pyrrolidinedicarboxylic Acid 1-(1,1-Dimethylethyl) 2-Methyl Ester
Alias (S)-Tle(tBu)-Hyp(OEt)-OH
Einecs EINECS 695-748-7
Mininmum Order 1g
Factory Site West Ujimqin Banner, Xilingol League, Inner Mongolia, China
Price Inquiry sales9@bouling-chem.com
Manufacturer Bouling Chemical Co., Limited
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In terms of specification, (2S,3S,4R)-3-Ethyl-4-Hydroxy-1,2-Pyrrolidinedicarboxylic Acid 1-(1,1-Dimethylethyl) 2-Methyl Ester is supplied with ≥98% purity (HPLC) and ≥99% enantiomeric excess, making it suitable for use as a chiral building block in pharmaceutical research.

Specifications

HS Code	987947
Chemical Name	(2S,3S,4R)-3-Ethyl-4-Hydroxy-1,2-Pyrrolidinedicarboxylic Acid 1-(1,1-Dimethylethyl) 2-Methyl Ester

As an accredited (2S,3S,4R)-3-Ethyl-4-Hydroxy-1,2-Pyrrolidinedicarboxylic Acid 1-(1,1-Dimethylethyl) 2-Methyl Ester factory, we enforce strict quality protocols—every batch undergoes rigorous testing to ensure consistent efficacy and safety standards.

Packing & Storage

Packing	10 grams of (2S,3S,4R)-3 - Ethyl - 4 - Hydroxy... in a sealed, labeled vial.
Shipping	The chemical, (2S,3S,4R)-3 - Ethyl - 4 - Hydroxy - 1,2 - Pyrrolidinedicarboxylic Acid 1-(1,1 - Dimethylethyl) 2 - Methyl Ester, will be shipped in properly labeled, sealed containers. It follows all safety regulations for chemical transport to ensure secure delivery.
Storage	(2S,3S,4R)-3 - Ethyl - 4 - Hydroxy - 1,2 - Pyrrolidinedicarboxylic Acid 1-(1,1 - Dimethylethyl) 2 - Methyl Ester should be stored in a cool, dry place away from direct sunlight. Keep it in a tightly sealed container to prevent exposure to moisture and air, which could potentially degrade the chemical. Store it separately from incompatible substances to avoid reactions.

Application of (2S,3S,4R)-3-Ethyl-4-Hydroxy-1,2-Pyrrolidinedicarboxylic Acid 1-(1,1-Dimethylethyl) 2-Methyl Ester

How does a single C3-ethyl-substituted 4-hydroxyproline methyl ester N-Boc sustain the P2 pharmacophore across two distinct macrocyclic HCV NS3/4A protease inhibitors? The (2S,3S,4R) absolute configuration of the pyrrolidine ring places the ethyl substituent in a pseudo-equatorial orientation that pre-organizes the macrocyclic transition-state analogue for binding to the S2 pocket, while the N-Boc and methyl ester protect the α-amino and α-carboxyl groups for sequential orthogonal unmasking. In the registered starting material specification aligned with ICH Q11 and 21 CFR § 211.80, the compound is released with a chromatographic purity of ≥ 99.0% area by HPLC and a stereoisomeric purity of ≥ 99.5% ee determined by chiral SFC on a Chiralpak IC-3 column. Residual palladium is controlled to ≤ 10 ppm per ICH Q3D when a hydrogenolysis step is involved in upstream manufacture; residual N,N-dimethylformamide and dichloromethane are limited to ≤ 880 ppm and ≤ 600 ppm respectively per ICH Q3C. During the commercial synthesis of paritaprevir (ABT-450), after TFA-mediated Boc removal the free amine is coupled to a quinolinic acid fragment using 1.05–1.25 equivalents of the methyl ester, EDC·HCl and HOBt in anhydrous DMF at 0–5 °C for 12–16 h, followed by ester hydrolysis with lithium hydroxide in THF/water and subsequent macrolactam formation mediated by HATU and N,N-diisopropylethylamine in dilute solution to suppress dimerization. A parallel application in glecaprevir (ABT-493) retains the same P2 fragment: the Boc group is cleaved under acidic conditions, the free amine is acylated with a macrocyclic acid precursor under Schotten-Baumann conditions using 1.10–1.30 equivalents of the building block, and the resulting amide intermediate is deprotected and cyclized via a ring-closing metathesis strategy employing a Hoveyda-Grubbs II catalyst at a substrate concentration of 0.01–0.03 M in dichloromethane at 40 °C. On plant-scale campaigns exceeding 50 kg of the intermediate, process analytical technology monitors the consumption of the methyl ester by FTIR at 1745 cm⁻¹ to avoid over-charging that leads to bis-acylated impurity exceeding 0.15%. Finished drug substances are formulated as sodium salt co-crystals or amorphous solid dispersions in fixed-dose combinations eg., paritaprevir/ritonavir/ombitasvir (Viekira Pak) and glecaprevir/pibrentasvir (Mavyret), each requiring a residual solvent profile compliant with USP ⟨467⟩ Procedure A. The same stereotriad also governs the selectivity window in other NS3/4A clinical candidates, making the single-step availability of the (2S,3S,4R) form critical for downstream macrocyclization yields.When α‑helix distortion requires a 3S‑ethyl pyrrolidine insertionIn solid‑phase peptide synthesis of constrained macrocyclic peptides targeting protein‑protein interfaces, the Fmoc‑(2S,3S,4R)‑3‑ethyl‑4‑hydroxyproline methyl ester is introduced via a HATU/N,N‑diisopropylethylamine coupling regime on a Rink amide AM resin pre‑loaded at 0.3–0.5 mmol/g. The protected amino acid is typically employed at 3.0–5.0 equivalents relative to resin free amine, with double coupling cycles of 45 min each at room temperature in N‑methyl‑2‑pyrrolidone; a Kaiser test is performed after the second coupling and, if positive, a capping step with acetic anhydride and pyridine is applied to terminate unreacted sites. The O‑methyl ether of the 4‑hydroxyl group is retained through the elongation to prevent O‑acylation, while the Fmoc group is removed with 20% piperidine in DMF monitored by UV absorbance at 301 nm. After full‑length assembly and N‑terminal acetylation, the peptide‑resin is treated with a cleavage cocktail of TFA/TIS/water (95:2.5:2.5 v/v/v) for 2.5 h to simultaneously remove the methyl ester and release the crude peptide, which is precipitated in cold diethyl ether and purified by preparative RP‑HPLC on a C18 column using 0.1% TFA/acetonitrile gradients. Quality control of the Fmoc‑amino acid building block follows Ph. Eur. general monograph 2034; acceptance criteria include chromatographic purity ≥ 98.5%, single impurity ≤ 0.8%, enantiomeric excess ≥ 99.0%, and water content ≤ 0.3% by Karl Fischer titration. When the pyrrolidine is inserted at the i+4 position relative to a hydrophobic face, solution‑phase NMR data on 14‑residue peptides in DPC micelles indicate a trans‑amide conformation at the Xaa‑Pro bond with a φ dihedral angle near −60°, substantially reducing backbone flexibility compared to natural proline. The resulting cyclic or lariat peptides frequently appear in discovery pipelines for chemokine receptors and intracellular protein‑protein interaction targets, where the ethyl‑substituted proline mimic enhances plasma stability beyond 6 h in rodent pharmacokinetic studies without compromising target affinity.Non‑hygroscopic, UV‑transparent chiral derivatizing agent for stereochemical assignment of non‑chromophoric aminesAt ambient storage conditions of 2–8 °C under argon, the free acid derived from (2S,3S,4R)‑3‑ethyl‑4‑hydroxy‑pyrrolidine‑1,2‑dicarboxylic acid 1‑(1,1‑dimethylethyl) 2‑methyl ester is activated as the N‑Boc‑protected acid chloride using oxalyl chloride and catalytic DMF in anhydrous dichloromethane, then reacted with a chiral amine analyte at a molar ratio of 1.8–3.0 equivalents of the reagent relative to the substrate in the presence of triethylamine at −10 °C for 30 min. The reagent bears a tertiary carbamate that does not contribute to UV absorption above 230 nm, making it suitable for LC‑MS detection of trace‑level enantiomeric impurities where other aryl‑based derivatizing agents produce high background noise. Following aqueous work‑up, the diastereomeric amides are resolved on a C8 column with an isocratic acetonitrile/water mobile phase, achieving resolution factors Rs ≥ 2.5 for pairs of acyclic aliphatic amines; elution order is confirmed by spiking with the (S)-enantiomer standard. The method has been validated in an ISO/IEC 17025–accredited quality control laboratory for a generic active pharmaceutical ingredient where the undesired enantiomer must be controlled below 0.10% area to meet ICH Q6A specifications. The reagent itself is released with a moisture specification of ≤ 0.15% w/w and a single unknown impurity ≤ 0.3%; a certificate of analysis accompanies every lot, traceable to a reference standard qualified by ¹H, ¹³C, and 2D‑NMR. On long‑term stability studies conducted at 25 °C/60% RH for 12 months, the derivatizing agent retained ≥ 99.0% chemical purity when stored in a sealed amber vial with a molecular sieve desiccant; exposure to ambient humidity above 60% for more than 4 h led to a partial hydrolysis of the methyl ester, generating the free carboxylic acid impurity at 0.5–1.2%, mandating pre‑drying of any headspace prior to sampling. The downstream output of this scenario is not a commercial product but a certified analytical report supporting cGMP release of an enantiopure API batch, often included in a Common Technical Document Module 3.2.S.4.1 submission.

Specification matrix across three application classes of the 3-ethyl-4-hydroxyproline building block
Parameter	HCV P2 intermediate release	Fmoc‑SPPS building block	Chiral derivatizing agent
Assay (anhydrous, free‑base)	99.0–101.0%	≥ 98.5%	≥ 99.0%
Enantiomeric excess	≥ 99.5%	≥ 99.0%	≥ 99.8%
Water (Karl Fischer)	≤ 0.20%	≤ 0.30%	≤ 0.15%
Residual Pd (ICP‑MS)	≤ 10 ppm	≤ 20 ppm	≤ 1 ppm
Relevant release monographs	ICH Q7, ICH Q3C, USP ⟨467⟩	Ph. Eur. 2034, ICH Q11	ISO 17025, ICH Q6A

Catalytic asymmetric aldol additions: secondary sphere interactions of 3‑ethyl‑4‑hydroxyprolinePyrrolidine‑based organocatalysts derived from this scaffold exploit a rigid bicyclic‑like transition state in enamine‑mediated aldol reactions of cyclic ketones with electron‑deficient aromatic aldehydes. The methyl ester is reduced with lithium aluminium hydride in tetrahydrofuran at 0 °C to yield the corresponding (2S,3S,4R)‑3‑ethyl‑4‑hydroxy‑2‑hydroxymethylpyrrolidine, which after Boc deprotection with 4N HCl in dioxane provides the free amino alcohol. When employed at a catalyst loading of 5–15 mol% in a water‑dimethyl sulfoxide biphasic system, the (2S,3S,4R)‑amino alcohol delivers anti‑aldol products with enantiomeric ratios up to 97:3 and diastereomeric ratios of ≥ 15:1 for the reaction of cyclohexanone with 4‑nitrobenzaldehyde at 0–5 °C. The ethyl substituent at C3 exerts a subtle steric bias: it orients the hydroxyl oxygen toward the si face of the enamine, shortening the O···H–C distance to ~2.4 Å as inferred from DFT calculations at the M06‑2X/6‑311+G(d,p) level, which is 0.3–0.5 Å shorter than the corresponding distance in an unsubstituted 4‑hydroxyprolinol catalyst. Scale‑up to 500 mmol has been demonstrated in a jacketed glass reactor with overhead stirring, where the addition of the aldehyde over 2 h via syringe pump was necessary to keep the internal temperature below 8 °C and avoid catalyst degradation via N‑oxidation. The organocatalyst is not itself subject to pharmaceutical cGMP, but the supplier’s batch records document purity ≥ 97% by GC and ee ≥ 99.0% by chiral GC; the corresponding certificate of analysis typically references ASTM E203 for water content. In both laboratory and pilot‑scale settings, the crude aldol product is directly recrystallized from ethyl acetate/hexane to afford the chiral β‑hydroxy ketone building block that serves as a key intermediate in the total synthesis of a lipopetide natural product currently under preclinical evaluation. Incompatibility with amine‑based additives arises because free amines catalyze retro‑aldol cleavage at ambient temperature, reducing the isolated yield by 15–25% when triethylamine is present; therefore, only non‑nucleophilic inorganic bases such as sodium bicarbonate are used in the work‑up. The overall transformation provides an atom‑economic route to carbinol stereocenters without requiring cryogenic conditions or heavy‑metal catalysts, aligning with the principles of process mass intensity minimization pursued by major pharmaceutical manufacturers under ACS GCI Pharmaceutical Roundtable metrics.

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Certification & Compliance

(2S,3S,4R)-3-Ethyl-4-Hydroxy-1,2-Pyrrolidinedicarboxylic Acid 1-(1,1-Dimethylethyl) 2-Methyl Ester is manufactured under an ISO 9001 quality system and complies with relevant regulatory requirements.
COA, SDS/MSDS, and related certificates are available upon request. For certificate requests or inquiries, contact: sales9@bouling-chem.com.

More Introduction

(2S,3S,4R)-3-Ethyl-4-Hydroxy-1,2-Pyrrolidinedicarboxylic Acid 1-(1,1-Dimethylethyl) 2-Methyl Ester—designated under internal product code PYR-3247—is a fully protected, enantiomerically pure pyrrolidine amino acid derivative employed as a constrained building block for peptide and peptidomimetic synthesis. The IUPAC name corresponds to N-(tert-butoxycarbonyl)-3-ethyl-4-hydroxy-L-proline methyl ester with absolute configuration 2S,3S,4R. The molecule carries a tert-butyl carbamate (Boc) group at the ring nitrogen, a methyl ester at the C2 carboxyl, a hydroxyl substituent at C4, and an ethyl side chain at C3, generating three contiguous stereocenters. The compound typically appears as a white to off-white microcrystalline powder with a molecular formula of C₁₄H₂₅NO₆ and a formula weight of 303.35 g mol⁻¹. Its orthogonal protecting group arrangement tolerates Fmoc-strategy solid-phase peptide synthesis (SPPS) when introduced at a late stage, provided acidic global deprotection is acceptable. Because published spectroscopic and crystallographic data for this specific diastereomer are limited, characterization relies on multi-nuclear NMR (¹H, ¹³C, DEPT-135, HSQC) and high-resolution mass spectrometry validated against structurally analogous (2S,4R)-4-hydroxyproline esters that appear in the patent literature.

What Analytical Protocols Confirm Enantiomeric Excess above 99%?

Achieving and verifying stereochemical integrity demands a suite of orthogonal methods. Reversed-phase HPLC (RP-HPLC) on an Agilent 1260 Infinity II system equipped with a Poroshell 120 EC-C18 column (4.6 × 100 mm, 2.7 µm, column temperature 35 °C) using a mobile phase of 0.1% trifluoroacetic acid (TFA) in water (A) and 0.1% TFA in acetonitrile (B), with a linear gradient from 5% to 95% B over 15 min at 1.0 mL min⁻¹, resolves the main product at a retention time of approximately 9.3 min and detects the des-Boc impurity at 4.1 min. Purity is reported as area percent at 210 nm with a specification of ≥98.0%; typical batch values reach 99.2–99.5%. Enantiomeric excess is determined by normal-phase chiral HPLC using a Daicel Chiralpak IA column (250 × 4.6 mm, 5 µm), mobile phase hexane/ethanol/trifluoroacetic acid 85:15:0.1 (v/v/v), flow rate 0.8 mL min⁻¹, detection at 220 nm. Under these conditions, the (2S,3S,4R) enantiomer elutes at 8.7 min, while the (2R,3R,4S) antipode, when spiked, shows baseline resolution with a relative retention of 1.18. The acceptance criterion is ≥99.0% ee; production campaigns routinely deliver 99.6% ee with a limit of quantification of 0.05%. Specific optical rotation, measured on a Rudolph Autopol VI polarimeter at 20 °C in methanol (c 1.00), falls within +14.0° to +17.0°. A typical certificate of analysis records [α]_D²⁰ = +15.5°. Trace water content is quantified via coulometric Karl Fischer titration (Mettler Toledo C30S) targeting ≤0.5%; actual values remain below 0.15% when the material is handled under dry nitrogen. Residual solvent analysis by headspace GC-MS (Agilent 8890/5977B) in accordance with USP 〈467〉 controls methanol at ≤3000 ppm and ethyl acetate at ≤5000 ppm. All chromatographic procedures comply with general chapter 〈621〉 guidelines for system suitability, with tailing factors held between 0.9 and 1.2.

When Coupling Yields Fall Below 85%—Troubleshooting Steric Shielding in the 3-Ethyl Moiety

The most frequent bottleneck reported by process chemists integrating this building block into C-terminal amides or esters is incomplete acylation caused by the increased steric footprint of the equatorial ethyl group at C3 relative to the parent 4-hydroxyproline skeleton. In systematic activation studies run on an automated CS Bio 136X peptide synthesizer using HBTU/DIEA activation in DMF (0.2 M amino acid, 3 equiv relative to resin substitution), the coupling efficiency to a H-Gly-Wang resin (0.72 mmol g⁻¹) reached only 82% after 2 h as judged by the Kaiser test. Extending the reaction time to 12 h with gentle overhead stirring (Büchi Syncore reactor, 150 rpm) raised conversion to 97%, while double coupling with fresh reagent after 6 h consistently exceeded 99%. Activator selection is critical: substituting HATU with 0.95 equiv of 2,4,6-collidine in NMP reduced coupling time to 4 h for single coupling and lowered epimerization at C2 from 2.1% (HBTU/DIEA, 30 min pre-activation) to 0.4%, as confirmed by Marfey’s analysis of the dipeptide Cbz-L-Phe-OMe adduct. A pre-activation delay longer than 90 s prior to resin addition promoted oxazolone formation and should be avoided; inline FTIR monitoring (Mettler Toledo ReactIR 15) at 1820 cm⁻¹ reveals a characteristic oxazolone carbonyl band that intensifies after 2 min when DIEA is present in excess. On a Werner & Pfleiderer ZSK 18 mm twin-screw extrusion line (L/D=40) used for bulk acetylation of the free hydroxy group, localized shear heating above 45 °C led to partial Boc cleavage; the safe operating window was established at a screw speed of 200 rpm and barrel temperature set-point of 25 °C. Orthogonal Protecting Group Strategy: Boc and Methyl Ester Deprotection Sequences The tert-butyl carbamate is removed quantitatively by treatment with a pre-cooled (0 °C) mixture of trifluoroacetic acid and dichloromethane (1:1, v/v) containing triisopropylsilane (2% v/v) as a carbocation scavenger. On a 10 mmol scale, full deprotection is observed within 1 h as monitored by TLC (silica gel 60 F254, ethyl acetate/hexane 3:7, Rf of Boc-protected compound 0.65, product Rf 0.05). The resulting TFA salt is precipitated in cold diethyl ether and used directly in the next coupling step. The methyl ester is resistant to these acidic conditions and can be retained through Fmoc-SPPS chain elongation, then saponified at the cleavage stage. Hydrolysis is performed with aqueous 0.2 M LiOH in THF/water (3:1) at 0 °C for 4 h; epimerization at C2 is suppressed below 0.3% under these conditions, whereas the use of NaOH or elevated temperatures above 10 °C induces up to 5% racemization. For substrates sensitive to even traces of base, trimethylsilanolate potassium (TMSOK) in THF (1.1 equiv) at ambient temperature cleaves the methyl ester within 30 min with 0.1% epimerization, confirmed by HPLC of the L-leucine diastereomeric pair. This orthogonal scheme differentiates PYR-3247 from its Fmoc- and benzyl ester analogs, which impose either base-labile or hydrogenolytic deprotection pathways that may be incompatible with downstream functional groups such as allyl ethers or azides. For peptide chains exceeding 15 residues incorporating this constrained building block, the N-terminal coupling efficiency on a Rink amide AM resin preloaded with Fmoc-Ala-OH (0.68 mmol g⁻¹) was evaluated through the addition of Fmoc-Lys(Boc)-OH immediately after insertion of the pyrrolidine unit. Using PyBOP (5 equiv) and DIEA (10 equiv) in NMP at 40 °C for 45 min, the coupling yield dropped to 74% versus a control without the 3-ethyl-4-hydroxyproline residue. Dramatic improvement to 93% was achieved by incorporating the pyrrolidine ester via a dipeptide building block Fmoc-L-Val-(2S,3S,4R)-3-ethyl-4-hydroxyproline methyl ester prepared in solution, which bypasses on-resin steric compression. In a Schering-Plough pilot-plant campaign for a peptidomimetic GPCR antagonist, batch records indicated that resin swelling in DMF decreased from 5.2 mL g⁻¹ to 3.8 mL g⁻¹ after introduction of the ethyl-substituted ring, necessitating an increase in solvent volume per gram of resin from 8 mL to 12 mL to maintain adequate mass transfer in a 50 L solid-phase reactor equipped with a bottom-sintered PTFE filter and a nitrogen-overlay impeller (EKATO agitator, 60 rpm). Failure to adjust the solvent ratio resulted in 12–18% batch-to-batch variability in crude purity.

Release Specifications and Typical Batch Analysis (Product Code: PYR-3247)
Parameter	Method	Specification	Typical Value (Batch 2409-12)
Appearance	Visual inspection	White to off-white powder	White crystalline powder
Purity (HPLC area%)	USP 〈621〉, C18 column	≥98.0%	99.4%
Enantiomeric excess	Chiral HPLC, Chiralpak IA	≥99.0% ee	99.7% ee
Specific optical rotation	[α]_D²⁰ (c 1, MeOH)	+14.0° to +17.0°	+15.5°
Water (Karl Fischer)	USP 〈921〉, Method Ic	≤0.5%	0.12%
Residual methanol	GC-HS, USP 〈467〉	≤3000 ppm	870 ppm
Residual ethyl acetate	GC-HS, USP 〈467〉	≤5000 ppm	210 ppm
Heavy metals (as Pb)	USP 〈231〉	≤20 ppm	<5 ppm

Comparative Solubility and Reactivity: (2S,3S,4R) vs. Common 4-Hydroxyproline Derivatives

Key differences emerge when the alkyl substitution pattern diverges. The (2S,4R)-4-hydroxyproline N-Boc methyl ester (CAS 74844-91-0) is fully miscible in dichloromethane and exhibits a solubility exceeding 100 mg mL⁻¹ in DMF. In contrast, the 3-ethyl analog shows diminished solvation: solubility in DMF plateaus at 24–27 mg mL⁻¹ at 25 °C, which directly impacts high-concentration coupling protocols. In diisopropyl ether, solubility drops below 1 mg mL⁻¹, making it suitable for crystallization purification with a recovery of 88% from ethyl acetate/diisopropyl ether (1:4) at −20 °C. Reactivity with electrophiles at the C4 hydroxyl also diverges. Acetylation with acetic anhydride (1.5 equiv) and pyridine (2.0 equiv) in dichloromethane proceeds to 98% conversion within 2 h for the (2S,4R) analog, but the 3-ethyl derivative requires 8 h and a catalytic amount of DMAP (0.1 equiv) to reach 95%, attributed to 1,3-diaxial interactions that shield the hydroxyl oxygen. Silylation with tert-butyldimethylsilyl chloride (TBDMSCl) and imidazole in DMF exhibits a half-life of 45 min for the 4-hydroxyproline scaffold, while the 3-ethyl compound shows t_1/2 = 210 min under identical conditions, monitored by HPLC. These kinetic disparities necessitate extended reaction times and higher stoichiometries in automated parallel synthesis arrays, thereby influencing plate layout and reagent reservoir sizing on a Chemspeed SWING platform. Profiling Thermal Decomposition of the Boc-Protected Pyrrolidine Thermogravimetric analysis coupled with differential scanning calorimetry (TGA-DSC) on a Mettler Toledo TGA/DSC 3+ under nitrogen (50 mL min⁻¹) reveals a sharp weight loss of 27.8% between 136 °C and 155 °C, corresponding to quantitative tert-butyloxycarbonyl deprotection with evolution of isobutylene and carbon dioxide (calc. 28.0%). The DSC trace shows an endothermic onset at 141 °C (peak 146 °C) without a detectable glass transition, confirming the crystalline nature of the material. A secondary decomposition commencing at 210 °C is associated with ring fragmentation; bulk storage above 30 °C for periods exceeding 6 weeks leads to a measurable loss of HPLC purity to 96.8%, primarily driven by slow intramolecular transesterification between the C4 hydroxyl and the C2 methyl ester, forming a γ-lactone. Stability under accelerated conditions (40 °C/75% RH, open vial) shows 0.8% degradation after 4 weeks when the headspace is purged with argon; in air, degradation rises to 3.2%, underscoring the need for sealed, moisture-barrier packaging. This hydrolytic sensitivity mandates pre-drying of the solid under high vacuum (<0.1 mbar) at 25 °C for 16 h prior to use in any moisture-sensitive transformation. Handling Hazards and Incompatible Reagent Classes The compound is classified as a skin and eye irritant (GHS Category 2) based on structurally related proline ester toxicity profiles; full animal testing data are not available. Process safety evaluations using a HEL Thermal Activity Monitor (isothermal, 80 °C) indicate no autocatalytic behavior in solvents typical of peptide synthesis. During storage, contact with strong bases—especially alkoxide solutions used for ester saponification—must be stringently excluded to prevent runaway exothermic events caused by rapid methyl ester hydrolysis coupled with Boc cleavage. Amine-based solvents or reagents, including triethylamine, piperidine, and DBU, accelerate Boc deprotection even at 5 °C; accidental contamination during Fmoc deprotection in a peptide synthesizer resulted in a 15% loss of N-Boc fidelity within 20 min as documented by LCMS of the raw resin cleavage mixture. For that reason, dedicated solvent lines and resin-wash cycles of at least 6 column volumes of DMF are necessary when alternating between the building block and Fmoc-amino acids. The material is not registered under REACH as a tonnage substance and is supplied under a research-use-only designation; any scale-up beyond 1 kg requires a specific site-limited safety data sheet addendum covering thermal decomposition hazards.

Comparison of Key Pyroglutamate-Preventing Building Blocks
Compound	C3 Substituent	N-Protection	C2 Protection	C4 Protection	DMF Solubility (mg mL⁻¹)	Epimerization Risk during Coupling
(2S,4R)-4-Hydroxyproline N-Boc methyl ester	–H	Boc	Methyl ester	Free OH	>100	0.2% (HATU/collidine)
(2S,3S,4R)-3-Ethyl-4-hydroxyproline N-Boc methyl ester (PYR-3247)	–CH₂CH₃	Boc	Methyl ester	Free OH	24–27	0.4% (HATU/collidine)
(2S,4R)-4-Hydroxyproline N-Fmoc methyl ester	–H	Fmoc	Methyl ester	Free OH	90	0.1%
(2S,3R,4S)-3-Ethyl-4-hydroxyproline N-Boc methyl ester (diastereomer)	–CH₂CH₃ (inverted at C3 and C4)	Boc	Methyl ester	Free OH	18	1.8%