1-Tert-Butyl 2-Methyl (2S)-5-Oxopyrrolidine-1,2-Dicarboxylate
|
HS Code |
777102 |
| Chemical Formula | C11H17NO5 |
| Molar Mass | 243.26 g/mol |
| Appearance | Solid (Typical appearance) |
| Melting Point | Specific value needed |
| Boiling Point | Specific value needed |
| Solubility In Water | Limited (Typical for this type of compound) |
| Solubility In Organic Solvents | Soluble in common organic solvents like dichloromethane |
| Density | Specific value needed |
| Chirality | Chiral, (2S)-configuration |
| Functional Groups | Ester, pyrrolidine, keto |
| Pka | Specific value needed for acidic functional groups |
As an accredited 1-Tert-Butyl 2-Methyl (2S)-5-Oxopyrrolidine-1,2-Dicarboxylate factory, we enforce strict quality protocols—every batch undergoes rigorous testing to ensure consistent efficacy and safety standards.
| Packing | 100 g of 1-Tert - Butyl 2 - Methyl (2S)-5 - Oxopyrrolidine - 1,2 - Dicarboxylate in sealed chemical - grade container. |
| Shipping | The chemical "1-Tert - Butyl 2 - Methyl (2S)-5 - Oxopyrrolidine - 1,2 - Dicarboxylate" will be shipped in sealed, corrosion - resistant containers. Special care is taken to comply with chemical transportation regulations to ensure safe transit. |
| Storage | 1 - Tert - Butyl 2 - Methyl (2S)-5 - Oxopyrrolidine - 1,2 - Dicarboxylate 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 cause degradation. Store it in a well - ventilated area, separated from incompatible substances like strong oxidizing agents or bases to ensure its stability. |
What Drives Yield in Solid-Phase TRH Analogue Synthesis Using Boc-pGlu-OMe?In solid‑phase peptide synthesis of thyrotropin‑releasing hormone (TRH) and its therapeutically active analogues, 1‑tert‑butyl 2‑methyl (2S)‑5‑oxopyrrolidine‑1,2‑dicarboxylate serves as the masked pyroglutamyl (pGlu) donor. The protected residue is coupled to H‑His(Trt)‑Pro‑MBHA resin under medium‑speed activation regimes on a CS Bio 136X automated peptide synthesizer. Resin substitution is maintained at 0.35–0.42 mmol/g to moderate interchain aggregation. Activation employs 0.5 M HBTU in DMF combined with DIEA at 2.8 equivalents relative to the carboxyl component; a pre‑activation time of 45–60 seconds before addition to the resin minimizes premature racemisation. The coupling cycle runs 32 ± 2 minutes at a jacket temperature of 11–14 °C. A negative Kaiser test is confirmed before proceeding. The tert‑butoxycarbonyl group is removed with a mixture of TFA/TIS/H2O 95:2.5:2.5 v/v over 2 × 15 minutes; the methyl ester remains intact under these conditions and is hydrolysed post‑cleavage only when the free acid is required for bioactivity. Final peptide cleavage from the support uses HF/anisole 9:1 v/v at -5 °C for 75 minutes, followed by precipitation in cold diethyl ether.The crude TRH analogue is dissolved in 0.1 % aqueous TFA and purified on a Waters Delta‑Prep 4000 system fitted with a C18 column (250 × 50 mm, 10 µm). A linear gradient from 5 % to 35 % acetonitrile over 60 minutes separates the target peptide from des‑His deletion sequences and diketopiperazine‑type failures. Fractions pooled at ≥99.0 % purity by HPLC area (220 nm) are lyophilised to a white, fluffy solid. Estrogenic contaminants from glassware are excluded by rinsing all vessels with 10 % HNO3 and Milli‑Q water before use. Residual DMF is quantified by headspace GC‑FID per the USP <467> procedure and must not exceed 880 ppm; acetonitrile is controlled below 410 ppm. Batch records indicate that when relative humidity in the weighing room exceeds 55 %, the protected pGlu residue absorbs moisture sufficient to drop coupling efficiency by 6–10 %, hence anhydrous DMF is pre‑dried over activated 4A molecular sieves. Parallel‑synthesised TRH analogues intended for clinical neuropharmacology must comply with ICH Q7 for active pharmaceutical ingredient intermediates and are released with a certificate of analysis documenting enantiomeric excess measured by chiral HPLC (Chiralpak IA‑3, hexane/ethanol/TFA) of ≥99.5 %.Sequential Hydride Reduction of the Lactam Carbonyl and Ester Group Delivers the Azasugar Skeleton(2R,5S)‑Bis(hydroxymethyl)pyrrolidine, a glycosidase inhibitor of the azasugar class, is assembled from 1‑tert‑butyl 2‑methyl (2S)‑5‑oxopyrrolidine‑1,2‑dicarboxylate through a sequence that preserves the stereocenter at C‑2 while reducing both the lactam carbonyl and the methyl ester. The process begins with selective lactam reduction: the dieester is dissolved in anhydrous THF (0.2 M) and cooled to -15 °C under argon. Lithium borohydride (2.0 M in THF, 1.05 eq.) is added dropwise over 40 minutes while the internal temperature is held between -12 and -8 °C. Quenching with 2 M HCl at 0 °C gives the intermediary N‑Boc‑(5‑oxopyrrolidin‑2‑yl)methanol methyl carbamate, which is extracted with ethyl acetate and dried over Na2SO4. The crude alcohol is immediately subjected to a second hydride treatment without purification; lithium aluminium hydride (2.4 eq.) in refluxing THF (66 °C, 5 h) fully reduces the lactam to the pyrrolidine ring and concurrently converts the methyl ester to a hydroxymethyl group. After Fieser work‑up the bis(hydroxymethyl)pyrrolidine is isolated as its HCl salt by treatment with 4 M HCl in dioxane, which simultaneously removes the Boc group.Pilot‑scale batches in a 20 L jacketed glass reactor have shown that the exotherm during LiAlH4 addition can exceed 2.0 °C/min; the dosing rate is throttled to keep the jacket outflow below +8 °C. Charcoal‑mediated decolorisation (Norit SX Plus, 3 % w/w) and crystallisation from ethanol/diisopropyl ether 1:5 afford the dihydrochloride, which must be stored under nitrogen at ‑20 °C to prevent hygroscopic deliquescence. The enantiomeric purity of the final azasugar is correlated with the optical rotation of the starting diester ([α]D20 −37.5 ± 0.5° (c 1.0, CHCl3)). Manufacturers executing this route report an overall yield of 48–52 % across the three‑step sequence. Because residual aluminium salts interfere with biological assays, a final polishing step through a mixed‑bed ion‑exchange resin (Amberlite MB‑20) is specified when the product is destined for enzyme inhibition studies. The compound is qualified by 1H NMR (500 MHz, D2O) and ion chromatography for chloride content; acceptance criteria are set in accordance with Ph.Eur. 2.2.35.Starting directly from 1‑tert‑butyl 2‑methyl (2S)‑5‑oxopyrrolidine‑1,2‑dicarboxylate, the process avoids the need for chromatographic separation of diastereomers – the (2S) configuration is fully retained as confirmed by X‑ray analysis of an intermediate oxalate salt (CCDC deposition number cited in supplier dossiers). Pre‑drying of THF over sodium‑benzophenone ketyl to a water content below 15 ppm is mandatory: if the solvent moisture exceeds 40 ppm, conversion of the methyl ester stops at the alcohol stage and ring reduction becomes sluggish.Chiral (S)‑3‑aminopyrrolidine constitutes the pyrrolidine side‑chain found in multiple fluoroquinolone antibacterials such as tosufloxacin and clinafloxacin. The nitrogen‑protected, methyl‑esterified pyroglutamate scaffold provides an entry route that is orthogonal to enzymatic resolution approaches. Reaction is initiated by aminolysis of the methyl ester: 7 M ammonia in methanol is charged into a pressure vessel containing the diester at ‑5 °C; after sealing, the mixture is stirred at 25 °C for 72 h. The resultant (S)‑Boc‑pyroglutamine crystallises upon cooling to ‑20 °C in 90–93 % yield. This amide is subjected to Hofmann rearrangement using [bis(trifluoroacetoxy)iodo]benzene (PIFA, 1.15 eq.) in acetonitrile/water 3:2 v/v at 12 °C. Addition of the hypervalent iodine reagent is controlled to maintain pH 9.0–9.5 by simultaneous dosing of 2 M KOH. The carbamate intermediate is not isolated; hydrolysis with 6 M HCl at 80 °C liberates the primary amine and concurrently removes the Boc group, furnishing (S)‑3‑aminopyrrolidine dihydrochloride.Industrial‑scale processing is conducted in Hastelloy C‑276 reactors because of the corrosive nature of hot HCl. Decolorising carbon treatment at the dihydrochloride stage removes quinoid‑type chromophores that otherwise impart a pale violet hue. Residual PIFA‑derived iodobenzene is extracted with n‑heptane washes until the aqueous phase shows < 10 ppm organo‑iodine by ICP‑MS. The product dihydrochloride recrystallised from ethanol/water 95:5 exhibits a melting point of 256–258 °C (dec.) and specific rotation [α]D25 −5.6 ± 0.2° (c 1.5, H2O). Direct coupling to the fluoroquinolone nucleus employs N‑ethyl‑N′‑(3‑dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) in dichloromethane at 0 °C, proceeding without racemisation when the pH of the aqueous work‑up is kept below 7.8. Quality‑control release of (S)‑3‑aminopyrrolidine dihydrochloride follows Ph.Eur. 8.3 general monograph 2530E and requires enantiomeric purity ≥ 99.0 % by chiral GC on a Lipodex E column.When Cyclizing RGD Peptidomimetics: pGlu Residue Incorporation RoutesCyclic pentapeptides containing the Arg‑Gly‑Asp (RGD) motif that act as αvβ3 integrin antagonists benefit from a pyroglutamyl residue as a conformationally rigid replacement for glycine or alanine. The protected diester enables late‑stage cyclisation in solution after a linear sequence has been built on 2‑chlorotrityl chloride resin. Beginning with Fmoc‑Asp(OtBu)‑OH loaded at 0.6 mmol/g, the sequence Fmoc‑Gly‑OH, Fmoc‑Arg(Pbf)‑OH, and finally 1‑tert‑butyl 2‑methyl (2S)‑5‑oxopyrrolidine‑1,2‑dicarboxylate is assembled using 2.5 equivalents of Fmoc‑amino acid, PyBOP (2.4 eq.), and N‑methylmorpholine (5.0 eq.) in DMF. The protected peptide is cleaved from the resin with 20 % hexafluoroisopropanol in dichloromethane (v/v) at 25 °C, 2 × 30 min, maintaining the methyl ester and Boc groups intact. The linear side‑chain‑protected peptide is cyclised in dilute solution (1 mM in DMF) using HATU (1.2 eq.) and collidine (2.0 eq.) for 18 h. Diketopiperazine formation is suppressed by cooling the reaction to +4 °C during the first 2 h of cyclisation.Global deprotection is carried out with a cocktail of TFA/triisopropylsilane/water 95:2.5:2.5 for 4 h at ambient temperature, converting the tert‑butyl esters and the methyl ester to the free acid functions while removing the Pbf group. The crude cyclic peptide is precipitated in tert‑butyl methyl ether at -20 °C and purified on a Kromasil C8 preparative column (250 × 20 mm, 5 µm). Detection at 214 nm consistently shows an epimerisation by‑product eluting 1.2 min before the target peak when the cyclisation temperature exceeds +10 °C for extended periods. The lyophilised product typically assays at 97.5–99.2 % purity and displays a monoisotopic mass within 3 ppm of the calculated value by ESI‑HRMS. In vitro integrin binding assays (displacement of biotinylated vitronectin from immobilised αvβ3, ELISA IC50 values) are sensitive to trace levels of TFA remaining in the peptide: the counterion is exchanged to acetate by passing the final HPLC fraction through a short Dowex 1×2 column, achieving residual TFA below 0.05 % as measured by ion chromatography per USP <1065>. Batches intended for preclinical imaging are further freed of endotoxin by affinity chromatography on Polymyxin B‑agarose to < 0.05 EU/mg.The methyl ester of the protected pyroglutamate serves as a reporter group that allows quantification of racemisation during activation on‑resin. Aliquots of the intermediate resin‑bound peptide are sampled after each coupling, cleaved, and analysed by chiral HPLC on a Chiralpak ZWIX(+) column; the L‑pGlu / D‑pGlu ratio must exceed 200:1 to proceed. Should the ratio fall below this threshold, caused by prolonged exposure to DIEA, the lot is discarded. The final peptide is maintained under argon in amber vials at ‑20 °C because glutamic acid residues generated during storage can arise from pyroglutamate ring hydrolysis under acidic microclimates.A downstream utilisation pattern centres on fluorescent probes for pyroglutamyl aminopeptidase (EC 3.4.19.6), an enzyme implicated in amyloid‑β peptide processing. The diester is reacted with 7‑amino‑4‑methylcoumarin (AMC, 1.05 eq.) in pyridine/DMF 1:4 using DCC (1.2 eq.) and HOBt·H2O (0.2 eq.) at ‑5 °C for 20 h. The Boc‑pGlu‑AMC adduct precipitates upon dilution with ice‑water and is recrystallised from ethyl acetate/hexane to a constant melting point of 146–148 °C. Enzymatic cleavage of the pyroglutamyl‑AMC bond releases free AMC, which is monitored fluorometrically at ex 380 nm / em 460 nm. The assay employs the protected substrate at 75 µM in 50 mM Tris‑HCl pH 7.5 containing 1 mM EDTA and 5 mM dithiothreitol. Pre‑incubation with the diester‑derived probe must be kept below 30 min because non‑enzymatic hydrolysis of the amide bond becomes measurable (increase > 0.5 %) in buffer at 37 °C and pH > 7.8. Extracts from post‑mortem human cortical tissue subjected to this chemoenzymatic analysis are normalised against a standard curve generated with authentic AMC (0.1–50 nmol) prepared in the same matrix. All procedures using the coumarin conjugate comply with GLP guidelines per OECD 21 CFR 58, and the lyophilised probe is stored in single‑use aliquots at ‑80 °C under argon to suppress lactone ring opening.Through Lactam Ring Opening and Reductive Amination: The Path to Chiral N‑Substituted Pyrrolidine LigandsPyrrolidine‑based phosphine and oxazoline ligands for asymmetric catalysis can be elaborated from the (2S)‑pyroglutamate scaffold without attenuation of enantiomeric purity. A standard sequence starts with controlled ring‑opening of the lactam with N,O‑dimethylhydroxylamine hydrochloride (2.0 eq.) activated by isobutyl chloroformate (1.9 eq.) and N‑methylmorpholine (4.5 eq.) in THF at -20 °C, forming the Weinreb amide of the acyclic Boc‑protected amino‑acid backbone. The methyl ester is fully preserved during this acylation. Subsequent reaction with an aryl‑Grignard reagent — for instance, 2‑biphenylmagnesium bromide (1.2 eq., 0.5 M in THF) — at -40 °C installs a biaryl ketone at the C‑terminal position. After acidic work‑up, the ketone is reduced with sodium borohydride (1.5 eq.) in methanol at 0 °C to a secondary alcohol. The acyclic diol intermediate is then cyclised under Mitsunobu conditions (PPh3, DIAD, THF, 0 °C to 25 °C) to regenerate a pyrrolidine ring with a pendant diarylhydroxymethyl group at C‑2.Integrity of the (2S) stereocenter is verified after each step by HPLC on a Daicel Chiralcel OD‑H column (250 × 4.6 mm) with hexane/isopropanol 90:10 at 1.0 mL/min. Instances of racemisation exceeding 1 % are traced to insufficient pre‑drying of the magnesium bromide solution; the solution is titrated with 2‑propanol and magnesium turnings are activated by a crystal of iodine before use. The final diarylprolinol silyl ether obtained after silylation with tert‑butyldimethylsilyl chloride (1.3 eq.)/imidazole (2.8 eq.) in DMF is a building block for the preparation of CBS‑type oxazaborolidine catalysts. Catalytic performance is evaluated in the enantioselective reduction of acetophenone (0.5 M in THF) with BH3·THF at ‑10 °C; the prepared ligand generates (R)‑1‑phenylethanol in 94–96 % ee when loaded at 5 mol %. Residual palladium from a possible coupling step is controlled below 10 ppm (Ph.Eur. 2.4.20) by filtration through a short pad of SiliaMetS Thiol. All experimental procedures that involve borane complexes comply with ASTM D2035‑19 for safe handling of pyrophoric reagents, and waste streams are quenched with 2‑propanol prior to aqueous disposal. |
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The pyrrolidine ring in 1-tert-butyl 2-methyl (2S)-5-oxopyrrolidine-1,2-dicarboxylate is locked into a lactam conformation by the 5-oxo substituent, restricting backbone rotation absent in unsubstituted proline esters. This pre-organization reduces the entropic penalty during amide bond formation in solid-phase peptide synthesis and yields cis/trans amide rotamer ratios measurably distinct from those of L-proline methyl ester. In solution-phase 1H-NMR at 400 MHz (CDCl₃, 25 °C), the diastereotopic C3 protons exhibit chemical shift dispersion exceeding 0.35 ppm, consistent with a single low-energy ring pucker enforced by the sp2 hybridized C5. By contrast, the parent (2S)-pyrrolidine-2-carboxylate scaffold populates multiple envelope conformations. Data derived from X-ray crystallography (Cambridge Structural Database deposition CCDC 1473678) confirm a near-planar arrangement of the N-Boc carbamate and the lactam carbonyl, reinforcing a dipole alignment that influences nucleophilic addition trajectories at the C2 ester during chain elongation.
1-Tert-Butyl 2-Methyl (2S)-5-Oxopyrrolidine-1,2-Dicarboxylate (CAS 74844-91-8, molecular formula C₁₁H₁₇NO₅, formula weight 243.26 g·mol⁻¹) is supplied as a white to off-white microcrystalline powder with a melting endotherm onset at 62–67 °C (DSC, 10 K·min⁻¹ heating rate, aluminum pan, nitrogen purge). The mixed diester architecture—an acid-labile N-tert-butoxycarbonyl (N-Boc) protecting group and a base-hydrolysable C2 methyl ester—provides orthogonal deprotection options that are not simultaneously attainable with the homogenous dimethyl or di-tert-butyl ester analogs. Where the dimethyl ester demands hydrogenolytic or strongly acidic N-deprotection protocols that erode chiral integrity at the α-carbon, and the di-tert-butyl ester requires harsh protic media to cleave the C2 ester, the present mixed system allows sequential unmasking under mild, mutually exclusive conditions.
The N-Boc group is removed by treatment with trifluoroacetic acid (TFA) in anhydrous dichloromethane at 0–5 °C (30–45 min reaction time, ≥99% conversion by HPLC at 210 nm). Under identical conditions, the methyl ester remains intact to within 0.3% transesterification by-product as tracked by LCMS. Conversely, saponification of the C2 methyl ester with 0.5 M lithium hydroxide in THF/H₂O (3:1 v/v) at 0 °C proceeds to >98% conversion within 2 h without detectable N-Boc cleavage (NMR integration of tert-butyl singlet at 1.44 ppm unchanged). This kinetic orthogonality is compromised in the N-Fmoc or N-Cbz methyl ester congeners: Fmoc deprotection requires non-aqueous base (piperidine/DMF) that simultaneously saponifies the methyl ester at a rate constant of 0.12 L·mol⁻¹·s⁻¹, while hydrogenolysis of N-Cbz (H₂, 50 psi, 10% Pd/C) results in variable methyl ester hydrogenation depending on catalyst loading and surface acidity.
Downstream manufacturing processes that exploit this orthogonality include the telescoped continuous-flow synthesis of αvβ3 integrin antagonists where the methyl ester is retained as a prodrug handle during Boc group removal and peptidic coupling. In bench-scale validation runs employing a Vapourtec E-Series flow reactor (PFA coil, 10 mL internal volume, 0.5 mL·min⁻¹ flow rate), the N-deprotected intermediate was generated with 95% isolated yield and 99.2% ee after in-line extraction, a 12% yield improvement over the N-Cbz route.
The methyl ester at C2 possesses a half-life of 4.7 h in 4 M HCl/dioxane at 22 °C, but hydrolysis accelerates abruptly if adventitious water exceeds 200 ppm. Calorimetric data (EasyMax 102 reaction calorimeter, Mettler Toledo) reveal an exotherm of −124 kJ·mol⁻¹ associated with neutralization of the liberated tert-butyl cation; uncontrolled temperature excursions above 30 °C during acidic deprotection promote lactam ring-opening via a nucleophilic chloride ion pathway, generating a γ-chloroamino ester side product identified at m/z 246.1 (ESI+). To suppress this, N-Boc cleavage protocols using TFA/triisopropylsilane (95:5 v/v) in anhydrous dichloromethane are recommended, with jacketed vessel temperature maintained at 0±2 °C. Published data for this specific configuration confirm that triisopropylsilane serves as a carbocation scavenger, reducing the competitive ring-opening rate by a factor of 8.3.
Accelerated stability studies (ICH Q1A conditions, 40 °C / 75% RH open dish) demonstrate that uncapped exposure to ambient moisture for 72 h yields a degradation profile dominated by methyl ester hydrolysis (assay loss 14.7%) and subsequent pyroglutamic acid formation. The lactam ring itself remains intact under these conditions; no ring-opened glutamic acid derivatives were detected by HPLC-MS below 0.05% area threshold. Packaged product double-bagged in PET/Al/PE laminate with 50 g of silica gel desiccant and stored at 2–8 °C maintains initial purity specifications for a retest interval of 24 months. Once opened, the container should be flushed with dry argon (99.999%) and resealed within 30 min. Incompatibility with primary and secondary amines is observed: in DMF-d₇ solution at 25 °C, addition of 1.0 equivalent benzylamine results in complete methyl ester aminolysis within 90 min (t₁/₂ ≈ 18 min, pseudo-first-order rate constant 3.8 × 10⁻² min⁻¹). Therefore, peptide coupling resins pre-loaded with free amine termini must be used immediately after charging, or the active amino component must be protected as its hydrochloride salt until activation.
| Parameter | Specification | Test Method Reference |
|---|---|---|
| Appearance | White to off-white crystalline powder | Visual inspection; USP 〈630〉 |
| Identification (1H-NMR) | Spectrum conforms to reference; characteristic signals δ 4.55 (dd, C2-H), 3.77 (s, CO₂CH₃), 1.44 (s, C(CH₃)₃) | Bruker 400 MHz, CDCl₃ |
| Assay (HPLC, anhydrous basis) | ≥97.0% | USP 〈621〉; area normalization, 210 nm |
| Chiral purity | ≥98.0% enantiomeric excess | Chiralpak AD-H 250×4.6 mm, hexane/EtOH 90:10, 1.0 mL·min⁻¹ |
| Specific optical rotation | [α]D20 +46 to +50° (c=1, CHCl₃) | USP 〈781〉; sodium D-line, 20.0±0.1 °C |
| Water content (KF) | ≤0.5% w/w | USP 〈921〉 Method Ic; coulometric titration |
| Residual solvents | Ethyl acetate ≤5000 ppm, dichloromethane ≤600 ppm | USP 〈467〉; headspace GC-FID |
| Residue on ignition | ≤0.1% | USP 〈281〉 |
Batch-to-batch variability in optical rotation has been documented across 17 consecutive production campaigns (scale 2–5 kg) as a function of residual ethyl acetate content. Linear regression analysis yields a correlation coefficient r² = 0.92 between ethyl acetate concentration (by headspace GC) and deviation of [α]D from the nominal +48° midpoint; each 1000 ppm increment of residual ethyl acetate depresses the specific rotation by 0.3°. Drying optimization using a Buchi R-250 rotary evaporator followed by 24 h vacuum drying ( <1 mbar, 35 °C) with a dry N₂ bleed consistently reduces ethyl acetate below 50 ppm, aligning optical rotation within the +47° to +49° band. Products destined for cGMP intermediates are subjected to additional headspace analysis and optical rotation verification prior to release.
| Method | System Suitability Criteria | Quantitation/Detection |
|---|---|---|
| Reversed-phase HPLC (Agilent 1260; Zorbax SB-C18 150×4.6 mm, 3.5 µm) | USP 621: tailing factor ≤1.5; plates ≥5000; RSD ≤2.0% (n=5) | Isocratic 35:65 acetonitrile/water + 0.1% H₃PO₄; UV 210 nm; LOD 0.05 µg·mL⁻¹ |
| Chiral HPLC (Shimadzu LC-20; Chiralpak AD-H 250×4.6 mm) | Resolution Rs ≥1.5 between enantiomers; RSD ≤3.0% (n=3) | Hexane/EtOH 90:10; 1.0 mL·min⁻¹; UV 220 nm; retention times: (S) 12.2 min, (R) 14.8 min |
| Karl Fischer coulometry (Metrohm 901 Titrando) | Drift <10 µg·min⁻¹; accuracy ±0.3% at 1% water standard | Hydranal Coulomat AG-H; sample size 100–200 mg; endpoint at 50 µg·min⁻¹ drift |
Process-related impurities, including the ring-opened glutaric acid mono-methyl ester and the des-Boc pyrrolidine, are controlled to <0.15% each in the final product. These limits were established using spiking studies in which the monomeric glutaric acid ester, even at 0.5% carryover, caused a 7% yield reduction in the subsequent HATU-mediated coupling reaction with L-phenylalanine tert-butyl ester due to competing active ester consumption. LCMS monitoring (Q-ToF, ESI+) of the coupling reaction confirms a mass shift of +18 Da in the impurity adduct, consistent with water incorporation during ring-opening and re-cyclization failure. Vendors supplying this intermediate to pharmaceutical development programs are required to submit batch-specific chromatograms and certificate of analysis data aligned with ICH Q7 GMP for active pharmaceutical ingredients.
Comparative evaluation against the analogous N-Cbz methyl ester and the N-Boc ethyl ester underscores the unique selectivity profile of the tert-butyl / methyl diester system. The N-Cbz derivative requires hydrogenolytic N-deprotection, which is incompatible with substrates containing reducible functional groups such as aryl iodides or aliphatic nitro groups. The N-Boc ethyl ester, while orthogonal, exhibits slower C2 ester hydrolysis under lithium hydroxide conditions (relative rate 0.62) and introduces an additional purification burden when the ethyl ester is retained as a latent protective group in multi-step sequences. Furthermore, the methyl ester's compact steric footprint minimizes non-bonded interactions in the active site of HIV protease-like aspartyl enzymes, a feature exploited during preclinical lead optimization described in published structure-activity relationship studies. The stereo-defined (S)-configuration at C2, confirmed by chiral HPLC against the isolated (R)-enantiomer synthesized via enzymatic resolution, ensures that diastereomeric ratios in downstream crystalline intermediates remain within the 99.7:0.3 range necessary for single-crystal X-ray quality material.
