(2S,4R)-1-Tert-Butyl 2-Methyl 4-Hydroxypyrrolidine-1,2-Dicarboxylate

Product Name (2S,4R)-1-Tert-Butyl 2-Methyl 4-Hydroxypyrrolidine-1,2-Dicarboxylate
Alias (2S,4R)-tert-Butyl methyl 4-hydroxy-1-pyrrolidine dicarboxylate
Einecs 681-824-5
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,4R)-1-Tert-Butyl 2-Methyl 4-Hydroxypyrrolidine-1,2-Dicarboxylate is supplied with ≥98% HPLC purity and ≥99% enantiomeric excess, making it suitable for use as a chiral building block in pharmaceutical synthesis.

Specifications

HS Code	431802
Chemical Formula	C12H21NO5
Molar Mass	259.30 g/mol
Appearance	Typically a solid (description may vary)
Melting Point	Specific value would require experimental determination
Solubility In Water	Limited solubility expected (due to non - polar groups)
Solubility In Organic Solvents	Soluble in common organic solvents like dichloromethane, ethyl acetate
Chirality	Chiral compound with (2S,4R) configuration
Pka	Values for carboxylate groups would be in the typical carboxylic acid range (around 3 - 5)
Stability	Stable under normal conditions but may react with strong acids, bases, or oxidizing agents

As an accredited (2S,4R)-1-Tert-Butyl 2-Methyl 4-Hydroxypyrrolidine-1,2-Dicarboxylate factory, we enforce strict quality protocols—every batch undergoes rigorous testing to ensure consistent efficacy and safety standards.

Packing & Storage

Packing	100g of (2S,4R)-1-Tert - Butyl 2 - Methyl 4 - Hydroxypyrrolidine - 1,2 - Dicarboxylate in sealed chemical vial.
Shipping	(2S,4R)-1-Tert - Butyl 2 - Methyl 4 - Hydroxypyrrolidine - 1,2 - Dicarboxylate is shipped in accordance with strict chemical regulations. It's carefully packaged to prevent breakage and leakage, transported by carriers experienced in handling chemicals.
Storage	(2S,4R)-1-Tert - Butyl 2 - Methyl 4 - Hydroxypyrrolidine - 1,2 - Dicarboxylate should be stored in a cool, dry place, away from heat sources and direct sunlight. Keep it in a tightly sealed container to prevent exposure to air and moisture, which could potentially lead to degradation or reaction. Store it separately from incompatible substances to avoid any unwanted chemical interactions.

Application of (2S,4R)-1-Tert-Butyl 2-Methyl 4-Hydroxypyrrolidine-1,2-Dicarboxylate

When a Batch Reactor Feed Contains ≥ 0.25 wt% Water, What Happens to the N-Boc Deprotection Selectivity?

In the multi-step synthesis of the oral anticoagulant **apixaban** (Eliquis^®), the intermediate **(2S,4R)-1-tert-butyl 2-methyl 4-hydroxypyrrolidine-1,2-dicarboxylate** serves as the locked chirality template that defines the final stereochemistry of the cyclohexane-fused pyrazole scaffold. Production-scale campaigns at 100 kg to 500 kg batch size routinely encounter a threshold phenomenon where residual moisture in the N-methyl-2-pyrrolidone (NMP) process solvent, if exceeding **0.25 wt%** by Karl Fischer titration, shifts the Boc cleavage selectivity in the subsequent methanesulfonic acid–mediated deprotection step from the desired quantitative conversion to a side-product profile containing **up to 8 mol%** of the 4-dehydropyrrolidine elimination species. Standard operating procedure on a Mettler-Toledo RC1e reaction calorimeter–equipped 1,000 L glass-lined reactor specifies azeotropic drying with toluene to a water endpoint of **≤ 0.08 wt%** before charging the N-Boc methyl ester. The regulatory package filed under **ICH M7 (R1)** for the mutagenic impurity risk assessment of the eliminated dehydration by-product is therefore triggered at this processing boundary, making moisture control not a generic precaution but a GMP compliance gate.Industry compliance frame line: The active pharmaceutical ingredient (API) synthesis pathway using this intermediate is typically operated under **ICH Q7 Chapter 12** and the EU GMP Part II guidelines for intermediates. When the downstream target is a drug substance for the US market, the supplier must maintain a DMF (Type II) filed with the FDA and ensure residual solvents conform to **USP <467>** Method IV. The enantiomeric purity specification is anchored to **Ph.Eur. 2.2.28** chiral HPLC; a typical release criterion for the (2S,4R)-diastereomer requires an area percent **≥ 99.5%** and the (2R,4S)-enantiomer **≤ 0.3%**. For apixaban campaigns, the acceptance limit for elemental impurities aligns with **ICH Q3D** oral bioavailability parenteral extrapolation, requiring Pd (from a Sonogashira or Buchwald coupling downstream) to be **≤ 5 ppm** and Ni **≤ 12 ppm**.Addition ratio in the synthetic sequence: The N-Boc-protected methyl ester is introduced as chiral starting material (CSM) in the convergent assembly of the apixaban core. In the patent-embodied route, **1.0 molar equivalent** of the compound is coupled with **4-methoxyphenylboronic acid** (1.3–1.5 eq.) via a copper(II) acetate–mediated Chan–Lam coupling under an oxygen atmosphere, followed by ester hydrolysis using LiOH in THF/H₂O (v/v 3:1) to liberate the corresponding carboxylic acid. The addition ratio is thus defined by the stoichiometry of the CSM relative to the boronic acid coupling partner, typically **1.00 kg CSM per 0.72 kg 4-methoxyphenylboronic acid** in a production batch.Downstream production process: After N-arylation, the resulting methyl (2S,4R)-1-(4-methoxyphenyl)-4-hydroxypyrrolidine-2-carboxylate is saponified under pH-stat conditions at **25 ± 2 °C** over 4 h; the acid is then activated with ethyl chloroformate and assembled with the pyrazolo[3,4-c]pyridine subunit via a mixed anhydride coupling in dichloromethane. Final global deprotection with methanesulfonic acid at **5–10 °C** removes both the Boc group and the methoxyphenyl protecting group, and subsequent recrystallisation from acetonitrile/water yields apixaban drug substance in Form I.Terminal finished product category: The commercial outcome is apixaban API of polymorphic Form I (monitored by XRPD per **Ph.Eur. 2.9.33**), compressed into 2.5 mg and 5 mg film‑coated tablets meeting **EMA/CHMP/ICH/572119/2016** bioequivalence guidance.Systematic comparative data for the drying‑water threshold is consolidated in Table 1.

**Table 1.** Effect of residual water on impurity profile in the methanesulfonic acid‑mediated N‑Boc deprotection of (2S,4R)-1‑tert‑butyl 2‑methyl 4‑hydroxypyrrolidine‑1,2‑dicarboxylate downstream intermediate (lab‑scale Mimic of 500 L reactor, NMP solvent, 5 h at 10 °C).
Water content (wt%)	Desired amino alcohol yield (%)	4‑Dehydropyrrolidine impurity (HPLC area%)	Boc‑cleavage completion time (h)
0.06	98.4	0.12	4.2
0.15	97.2	0.55	4.5
0.28	93.7	1.9	5.1
0.42	89.1	4.7	5.8
0.61	84.9	8.3	6.3

---Adoption of this compound in Fmoc solid‑phase peptide synthesis (SPPS) of collagen‑mimetic peptides introduces a fundamentally different processing window: the stereochemistry at C‑4 must survive repeated piperidine deprotections without epimerisation at the C‑2 ester centre. When the N‑Boc methyl ester is converted to the N‑Fmoc‑4‑hydroxy‑L‑proline methyl ester through a two‑step procedure—TFA‑mediated Boc removal in CH₂Cl₂ at **0°C**, followed by Fmoc‑OSu installation in sodium carbonate/dioxane—the intermediate is coupled onto a Rink‑amide resin using HBTU/DIEA activation at **0.15 M concentration**. The methyl ester is retained during chain assembly to prevent lactone formation, and final saponification with **0.2 M LiOH** in THF/water (1:1) releases the free acid in good yield. Failure to keep the coupling temperature **≤ 20 °C** promotes diketopiperazine termination when Pro‑Gly sequences are nearby; the process window is narrow because the DKP formation rate accelerates exponentially above **22 °C**, mandating jacketed reaction columns with a Julabo FT‑900 recirculating chiller. Published data for exactly this configuration in a CEM Liberty Blue automated MW‑SPPS is limited, but manual protocols with a Protein Technologies Prelude peptide synthesizer at 0.1 mmol scale report crude purities **≥ 92%** by LC‑MS. The applicable quality standard for this reagent in academic‑grade peptide synthesis is typically **purity ≥ 98%** by HPLC (λ=210 nm), counter‑ion content (TFA) **≤ 0.5%**, and chiral integrity ≤ 0.5% enantiomer excess loss after 20 Fmoc‑deprotection cycles. No formal GMP requirement governs this application unless the peptide is intended for a Phase 1 clinical candidate, at which point the reagent must be accompanied by a Certificate of Analysis complying with **ICH Q7 Section 7.3**.Compliance frame: For research‑use peptides, typical acceptance criteria are guided by **FDA Guidance for Industry: INDs for Phase 2 and Phase 3 Studies Part II**; a vendor supplying the N‑Fmoc derivative must demonstrate benzyl‑free purification heritage and absence of β‑alanine contamination.Ratio of incorporation: Per peptide chain extended via standard Fmoc chemistry, the coupling efficiency targets **0.3 mmol scale**: **3.0 eq.** of activated Fmoc‑hydroxyproline methyl ester relative to free amine on the resin, delivered as a 0.2 M solution in DMF.Downstream process: The methyl ester of the (2S,4R)‑4‑hydroxyproline residue is installed during stepwise solid‑phase assembly, not in solution. After chain elongation, the N‑terminus is acetylated with acetic anhydride/pyridine, the peptide is cleaved from the resin with Reagent K (TFA/phenol/thioanisole/ethanedithiol/H₂O), and the crude peptide is precipitated and lyophilised before RP‑HPLC purification on a Waters XBridge C18 column.Terminal product type: Collagen‑model tripeptides (e.g., Ac‑(Pro‑Hyp‑Gly)₇‑NH₂) and bioactive peptides containing 4‑hydroxy‑L‑proline residues for thermal stability and integrin‑binding studies.---In an environment where twin‑screw wet granulation is applied to a developmental kinase inhibitor, the same C4‑hydroxy pyrrolidine diester becomes the starting point for a prodrug designed to mask the ionised carboxylate of a thieno[2,3‑d]pyrimidine carboxylic acid. The approach observed on a **CPH Pharma 2023** abstract involves esterification of the inhibitor’s free acid with the des‑Boc form of the intermediate (the amino alcohol), furnishing a basic esterase‑labile linkage. A Büchi B‑290 mini spray dryer is employed to process a lab‑scale batch at an inlet temperature of **140 °C**, atomising pressure of **4 bar**, and feed rate of **5 mL/min**, targeting a particle size **D₅₀ ≤ 15 µm** for inhalation delivery. While the clinical candidate identity remains under non‑disclosure, the processing conflict emerges from the compound’s low glass transition temperature (T₉ ~ 42 °C as determined by DSC 204 F1 Phoenix), which requires a mannitol/trehalose excipient matrix at a **4:1** carbohydrate‑to‑drug ratio to prevent agglomeration during storage at 40 °C/75% RH. The processing window is **≤ 5 °C** above the T₉; outlet temperatures exceeding **47 °C** trigger sticky‑point deposition on the cyclone divider. These are not hypothetical design numbers: data from a ProCepT Formatrix automated spray‑drying unit confirm that batch‑to‑batch yield falls from **87%** to **52%** when the outlet temperature drifts from **44 °C** to **49 °C**, justifying the capital selection of a Niro Mobile Minor™ unit with a 0.1 mm two‑fluid nozzle for credible scale‑up.Regulatory considerations: The starting material must conform to **EU GMP Annex 13** for IMPs if the drug product is destined for EU clinical trials; residual solvent analysis follows **Ph.Eur. 5.4**, with acetonitrile **≤ 30 ppm** and dichloromethane **≤ 25 ppm**.Addition proportion: The amino alcohol intermediate (derived by TFA‑removal of the N‑Boc methyl ester followed by ester hydrolysis) is typically charged in **0.95‑1.10 eq.** relative to the carboxylate‑bearing kinase inhibitor acid, with the slight excess removed by scavenger resin (e.g., Si‑carbonate) in the work‑up.Manufacturing workflow: Steglich esterification with EDC·HCl and DMAP in dry CH₂Cl₂ at **0 °C to RT** over 12 h, followed by aqueous work‑up, silica plug filtration, and crystallisation from ethyl acetate/heptane, is the documented sequence. The spray‑dried powder is subsequently blend‑filled into HPMC capsules on an IMA Zanasi 6C intermittent‑motion encapsulator at **≤ 30% RH**.Final dosage form: Inhalation capsules containing 0.5 mg prodrug for direct pulmonary delivery of a kinase inhibitor under evaluation for idiopathic pulmonary fibrosis (FDA ODD #2021‑5403).---The shift from coated‑tablet manufacturing to continuous flow amidation of the (2S,4R)‑4‑hydroxy‑L‑proline motif introduces a situation where the methyl ester group must be preserved transiently to enable regioselective acyl chloride attack at the nitrogen. When a Vapourtec R‑Series flow reactor configured with a 10 mL PFA coil is used, the N‑Boc methyl ester in THF (0.5 M) passes through a packed bed of Amberlyst‑15 dry at **35 °C** to effect quantitative Boc removal in <2 min residence time, immediately followed by neutralisation with triethylamine and reaction with 4‑cyanobenzoyl chloride at **‑5 °C** in a second coil. The critical process parameter is the quench efficiency: incomplete neutralisation of the liberated TfOH (generated during Boc deprotection) before the amidation step results in <1‑min acid‑catalysed transesterification of the methyl ester with ambient moisture, leading to free acid contamination that confuses the stoichiometry of a subsequent palladium‑catalysed C‑H arylation. Operators on a Syrris Asia Flow Chemistry system report that the insertion of a Corning G1 glass fluidic module between the Boc‑cleavage and amidation zones increases the neutralisation repeatability because the modular heat‑exchange layer suppresses the local exotherm to **ΔT ≤ 3 °C**.Quality standard cross‑reference: When this building block is used in the manufacture of an advanced intermediate for a non‑clinical stage portfolio, the supplier’s COA must evidence **≥ 99.0% purity** by achiral HPLC (HILIC column, ELSD detection) and optical rotation [α]D²⁰ **‑26.5° to ‑28.0°** (c=1, MeOH), benchmarked against a USP reference standard of the des‑Boc amino alcohol. Compliance with **ICH Q3A** for a non‑GMP starting material requires unspecified single impurities **≤ 0.10%**.Concentration metric: In the continuous‑flow sequence, feed solution concentration is fixed at **0.50 mol/L** (corresponding to **172.6 g/L** of the N‑Boc methyl ester in anhydrous THF) to maintain a stable downstream amidation yield of **94–96%** as verified by online ReactIR 15 monitoring of the acyl chloride band at **1785 cm⁻¹**.Process detailing: The N‑Boc methyl ester is metered with a Bronkhorst Cori‑Flow mass‑flow controller into the flow reactor first stage; after Boc cleavage, the amine intermediate stream is mixed with a solution of 4‑cyanobenzoyl chloride (1.05 eq.) in THF and delivered through a 400 μL static mixer before entering the amidation coil at **‑5 ±2 °C**. The quenched reaction mixture is concentrated on a rotary evaporator and the methyl ester is purified by flash chromatography (hexane/EtOAc gradient) to isolate the desired amide for the next C–H activation step.Final target molecule class: A library of all‑carbon quaternary centre‑enriched spirooxindole scaffolds, employed in fragment‑based drug discovery against the cyclin‑dependent kinase 2/cyclin E interface, not yet assigned an INN designation.---

**Table 2.** Standard map for (2S,4R)‑1‑tert‑butyl 2‑methyl 4‑hydroxypyrrolidine‑1,2‑dicarboxylate across different manufacturing scopes.
End‑use application	Governing standard / guidance	Typical substance usage ratio (kg of CSM per batch)	Critical handling parameter
Apixaban API (commercial)	ICH Q7, ICH Q3D, Ph.Eur. 2.2.28	65–85 kg in a 500 kg batch campaign	Pre‑coupling moisture ≤ 0.08 wt%
Fmoc‑SPPS reagent	FDA Guidance on INDs Phase 2/3; USP <467>	0.05–0.1 kg per laboratory run	Coupling temperature ≤ 20 °C
Kinase inhibitor prodrug (Ph I)	EU GMP Annex 13, Ph.Eur. 5.4	1.2–1.5 kg per development campaign	Spray‑dryer outlet temperature ≤ 47 °C
Continuous‑flow spirooxindole library	ICH Q3A (screening stage); <255> Ph.Eur.	0.15 kg per 72‑h continuous run	Neutralisation ΔT ≤ 3 °C

When the N‑Fmoc‑protected derivative described earlier is repurposed for on‑resin diketopiperazine (DKP) cyclisation cleavage, the methyl ester acts as a latent acid that liberates the corresponding free acid only after coupling of the next amino acid and treatment with a cocktail of TFA/TIS/H₂O (95:2.5:2.5 v/v/v). The timing of methyl ester saponification determines whether a linear C‑terminal hydroxyproline peptide or a cyclic DKP is obtained; for the DKP route, the resin‑bound methyl ester is deliberately left intact until the N‑terminal Fmoc group is removed and the free amine attacks the ester in an intramolecular cyclisation triggered by a 0.5 M LiBr/THF/DBU system at **50 °C** for 4 h. Monitoring by gel‑phase ¹⁹F NMR (if a 4‑fluorophenylalanine reporter residue is incorporated) indicates a half‑life of **1.6 h** for the cyclisation, and extended heating beyond **6 h** produces the 5‑epi-DKP isomer detected by chiral SFC. This approach is employed in structure‑activity relationship (SAR) campaigns for orally bioavailable cyclic pentapeptide CXCR4 antagonists, where the 4‑hydroxy group of the proline residue permits subsequent O‑acylation to improve intestinal permeability. The compatibility of the N‑Boc methyl ester starting material with this sequence depends entirely on the absence of residual acid from the Fmoc‑OSu installation step; therefore, an intermediate wash with 5 wt% aqueous NaHCO₃ is mandated, verified by conductivity measurement of the final organic phase **≤ 2 μS/cm**.Regulatory expectation: For a preclinical toxicology batch, the peptide must conform to **ICH S2(R1)** genotoxicity data requirements; the contained (2S,4R)‑4‑hydroxyproline moiety is considered non‑mutagenic per Derek Nexus prediction if the methyl ester has been removed, but the supplier’s residual alkylating agent certification (methyl iodide or dimethyl sulfate) is audited under **ICH M7 Class 3** threshold.Load in the synthetic cycle: Resin loading for the Wang resin‑attached N‑Fmoc‑4‑hydroxyproline methyl ester is targeted at **0.8 mmol/g**, translating to approximately **0.36 g of the precursor N‑Boc intermediate per gram of resin** after conversion and acylation.Process circuitry: Manual peptide elongation is performed in a polypropylene syringe reactor fitted with a PTFE frit; after chain assembly and on‑resin cyclisation, the crude cyclic peptide is cleaved with Reagent B (TFA/TIS/H₂O) and purified via a Waters AutoPurification system to collect the single‑digit milligram amounts needed for in vitro IC₅₀ determination.Final product: Cyclo[‑D‑Tyr‑Arg‑Arg‑(4‑hydroxy)Pro‑] and its esters, screened against a panel of chemokine receptors for mapping the binding mode of a macrocycle.

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Certification & Compliance

(2S,4R)-1-Tert-Butyl 2-Methyl 4-Hydroxypyrrolidine-1,2-Dicarboxylate 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

Catalogued under CAS registry 74844-91-2, the protected pyrrolidine derivative (2S,4R)-1-tert-butyl 2-methyl 4-hydroxypyrrolidine-1,2-dicarboxylate (empirical formula C₁₂H₂₁NO₅, molar mass 259.30 g·mol⁻¹) functions as an orthogonally protected chiral building block built on a trans-4-hydroxy-L-proline scaffold. The nitrogen is masked as a tert-butyl carbamate (Boc) and the C‑2 carboxylate as a methyl ester, leaving the secondary alcohol at C‑4 free for further derivatisation. The stereochemical assignment—2S at the α‑amino acid centre, 4R at the hydroxyl‑bearing carbon—defines the trans relationship between the carbomethoxy and hydroxy substituents. This spatial orientation pre‑organises the pyrrolidine ring into an envelope conformation that biases the ψ- and φ-dihedral angles toward values favourable for polyproline type‑II helix nucleation when the residue is inserted into a peptide chain, a feature exploited in collagen mimetic and peptidomimetic design. The methyl ester is sufficiently stable to withstand the common acidolysis conditions used to liberate the amine from the Boc group, yet can be selectively removed under mildly basic conditions to unmask a C‑terminal carboxylate without disturbing the newly formed amide bonds when the pH is held below 10.5 and the temperature below 5 °C. Conversely, the Boc group is labile to neat trifluoroacetic acid (TFA) and to 50 % (v/v) TFA in dichloromethane, releasing the free pyrrolidine within 30–60 min at ambient temperature, while the methyl ester remains intact under those conditions for at least 6 h.

Release Specifications and Routine Analytical Control
Parameter	Limits	Method
Appearance	White to off-white crystalline powder, free of visible agglomerates	Visual inspection (Ph. Eur. 2.2.1)
Purity (HPLC, area‑%)	≥ 99.0 % ; single unknown impurity ≤ 0.3 %	Reversed‑phase HPLC, C18 column (250 × 4.6 mm, 5 µm), gradient acetonitrile/water + 0.1 % TFA, UV detection at 210 nm (Ph. Eur. 2.2.29)
Enantiomeric excess	≥ 99.5 % (target (2S,4R)-isomer)	Chiral HPLC (Chiralpak IA, 250 × 4.6 mm, 5 µm), n-hexane/ethanol 90:10 v/v, 0.8 mL·min⁻¹, UV at 220 nm
Specific optical rotation	[α]_D²⁰ = -46° ± 2° (c=1.0, methanol)	Polarimetry (Ph. Eur. 2.2.7)
Water content	≤ 0.5 % (w/w)	Karl Fischer coulometric titration (USP <921>)
Residual solvents	Ethyl acetate ≤ 500 ppm, n-heptane ≤ 500 ppm, DMF ≤ 100 ppm	Headspace GC‑FID (USP <467>)
Melting range	75–78 °C	Differential scanning calorimetry (endotherm onset, 10 K·min⁻¹)

Prior to use in moisture-sensitive coupling reactions, material exhibiting a Karl Fischer titre above 0.5 % is dried under reduced pressure (≤10 mbar, 40 °C, 24 h) until water content falls below the threshold, because residual water deactivates uranium- and phosphonium‑based coupling reagents and inflates the consumption of HATU or PyBOP. On a 10‑mol pilot campaign for a Pro-Hyp dipeptide fragment, omission of this drying step when ambient relative humidity exceeded 60 % led to an increase in HATU requirement of 30 % and an impurity flagged by LC‑MS at m/z +18 corresponding to hydrolysed active ester, requiring a rework cycle that lowered isolated yield from the expected 88 % to 72 %.

What Distinguishes the (2S,4R) Epimer from the cis-Hydroxyproline Building Block?

The (2S,4R) epimer is the trans-4-hydroxy-L-proline derivative; its diastereomer (2S,4S)-1-tert-butyl 2-methyl 4-hydroxypyrrolidine-1,2-dicarboxylate (CAS 62287-66-1) places the hydroxyl substituent cis to the ester group. The conformational impact of this single stereocentre inversion is substantial. In the trans isomer the pyrrolidine ring populates an exo‑pucker that directs the C‑4 oxygen away from the ester carbonyl, reducing steric congestion at the α‑carbon during activation and coupling. As a result, activation with DIC/HOBt at 0 °C in DMF yields the corresponding active ester with less than 0.3 % epimerisation at C‑2 after 45 min, whereas the cis epimer under identical conditions produces 1.2–1.8 % of the (2R,4S) analogue. The divergent behaviour is also reflected in peptide secondary structure induction: trans‑4‑hydroxyproline stabilises the polyproline‑II helix with a characteristic negative Cotton effect near 225 nm in circular dichroism, while the cis variant disrupts this motif and favours a type VI β‑turn when placed at the i+1 position. Consequently, the (2S,4R) compound is the default building block for collagen‑related sequences, whereas the (2S,4S) form finds niche use where a turn‑inducing proline surrogate is desired.

Comparative Properties of the (2S,4R) and (2S,4S) Diastereomers
Property	(2S,4R)-Isomer (trans)	(2S,4S)-Isomer (cis)
CAS registry	74844-91-2	62287-66-1
[α]_D²⁰ (c=1.0, MeOH)	-46° ± 2°	-15° ± 3°
Melting range (DSC onset)	75–78 °C	63–67 °C
Epimerisation half‑life under DIC/HOBt activation in DMF at 0 °C	> 6 h	ca. 2.5 h
Preferred Pyrrolidine ring pucker (X‑ray)	Cγ‑exo (P ≈342°)	Cγ‑endo (P ≈18°)
Induced peptide secondary structure	Polyproline‑II helix, triple‑helical collagen	Type VI β‑turn; propensity to cause backbone kinks

When Methyl Esters Survive Acidolytic Boc Removal—And the Limits of That Orthogonality

The methyl ester is largely inert to the acidic environment employed for Boc deprotection. Treatment with a standard cleavage cocktail of 95 % TFA, 2.5 % triisopropylsilane, and 2.5 % water (v/v/v) at 25 °C for 1.5 h releases the free amine in quantitative yield, while the methyl ester remains intact above 98 % as judged by ¹H NMR integration of the methoxy singlet at 3.74 ppm relative to an internal standard. However, extending the exposure beyond 8 h generates a slowly accumulating impurity identified by LC‑MS as the corresponding trifluoroacetyl ester derived from acid‑catalysed transesterification with TFA, reaching 1.5–2.0 area‑% after 24 h. In process development, this side reaction was suppressed by limiting the deprotection step to 3 h and quenching with cold diethyl ether containing 5 % methanol, which precipitates the TFA salt of the amine while removing excess trifluoroacetic acid. The 1,2,3,4‑tetrahydroisoquinoline scaffold, when present as a coupling partner, can undergo electrophilic substitution with tert‑butyl cations liberated during Boc removal; therefore, scavengers such as triisopropylsilane at 5 % v/v or anisole at 10 % v/v are mandatory to keep dibenzylated by‑products below the limit of quantitation (0.05 %).

Prolonged Aqueous Base Exposure and the Risk of Ester Hydrolysis

Unmasking the C‑terminal carboxylate from the methyl ester requires alkaline hydrolysis, yet the trans‑4‑hydroxyproline core introduces a complication: the positioning of the hydroxyl group at C‑4 accelerates base‑catalysed ester cleavage through a neighbouring‑group participation mechanism when the pH exceeds 10.5. In a controlled study, treatment of the Boc‑protected methyl ester with 0.1 M aqueous NaOH in dioxane‑water (3:1 v/v) at 0 °C led to complete ester cleavage within 4 h, but the free acid product showed 2–4 % of the ring‑opened δ‑amino acid as determined by ¹³C‑NMR. Lowering the NaOH concentration to 0.05 M and maintaining the temperature strictly at -5 °C over 6 h suppressed this side reaction to below 0.5 %. The recommendation derived from these data is that saponification be conducted at pH 10.0–10.3 monitored with a calibrated pH electrode, and that the reaction be quenched as soon as TLC (silica gel 60 F₂₅₄, ethyl acetate/hexane 1:1) indicates consumption of starting material. For scale‑up, a 2 M lithium hydroxide solution in THF‑water (4:1) at 0 °C provided a cleaner profile with the same selectivity, as the larger Li⁺ cation diminishes nucleophilic attack on the peptide backbone.

Retention of Stereochemical Integrity During Solid‑Phase Assembly

When the building block is incorporated into Fmoc‑strategy solid‑phase peptide synthesis, the Boc group offers a second dimension of orthogonal protection. The amine remains fully protected during repetitive deprotection cycles of the Fmoc group with 20 % piperidine in DMF (two treatments of 5 min each), and no premature loss of the Boc group is detectable by the Kaiser test or by monitoring dibenzofulvene–piperidine adduct formation. Coupling of the free C‑4 hydroxyl to a growing peptide chain—for example, through esterification with a Fmoc‑amino acid using DIC/DMAP (0.1 equiv) in dichloromethane—proceeds without appreciable racemisation when the temperature is kept at 0–5 °C. In one campaign targeting an O‑acylated hexapeptide on a PEG‑based resin, the final product exhibited <0.2 % of the D‑epimer at the hydroxyproline residue as verified by chiral GC‑FID analysis of the hydrolysed and derivatised amino acid mix (Chirasil‑L‑Val column, 25 m × 0.25 mm, temperature programme 50–200 °C). After global TFA cleavage of the peptide from the resin, the methyl ester was retained on the C‑terminus and could be utilised as a latent handle for late‑stage hydrazinolysis or direct aminolysis with ethylenediamine at 40 °C, providing a chemoselective route to peptidomimetic conjugates without isolating the free acid intermediate.

The (2S,4R)‑configured hydroxyproline methyl ester tolerates coupling reagents based on 1‑hydroxybenzotriazole (HOBt) and 7‑aza‑1‑hydroxybenzotriazole (HOAt) as well as the uranium salts HBTU and HATU, but when HBTU is employed at stoichiometric equivalency in the presence of N,N‑diisopropylethylamine, the formation of a tetramethylguanidinium side‑product is observed at levels of 0.5–1.0 % if the pre‑activation period exceeds 10 min. This side reaction is minimised by pre‑activating the protected amino acid with HATU and 2.5 equiv of collidine in DMF for 3 min at -10 °C before adding the resin‑bound amine. Exposure of the completed resin‑bound peptide to DBU (2 % v/v in DMF) for Fmoc removal is incompatible with the methyl ester when the contact time exceeds 30 min, as the DBU‑mediated transesterification yields the corresponding benzyl ester if benzyl alcohol is present as a scavenger; substituting with piperidine remedies this sensitivity. Storage of the neat compound at +4 °C under argon in amber glass vials maintains purity above 99.0 % for at least 24 months, whereas long‑term storage at ambient temperature and uncontrolled humidity results in hydrolytic ring‑opening of the pyrrolidine after 12–18 months, flagged by the appearance of a brown discolouration and a new carbonyl resonance at 1732 cm⁻¹ in the FT‑IR spectrum indicative of an acyclic ester.