(2R,3R,4R,5R)-2,5-Bis(Hydroxymethyl)Pyrrolidine-3,4-Diol

(2R,3R,4R,5R)-2,5-Bis(hydroxymethyl)pyrrolidine-3,4-diol, designated under CAS 100991-92-2, is a fully defined stereoisomer within the polyhydroxylated pyrrolidine class. The molecule possesses a pyrrolidine ring substituted at the 2- and 5-positions with hydroxymethyl groups and at the 3- and 4-positions with secondary alcohols, all in the (R) absolute configuration. This specific stereochemical arrangement yields a C₂-symmetric iminosugar that presents a hydroxylation pattern topologically analogous to D-mannitol, but constrained within a five-membered azaheterocycle. Typical batch release specifications include a chemical purity of ≥ 99.0% (HPLC, area normalization at 200 nm), enantiomeric excess ≥ 99.5% (chiral HPLC, Chiralpak IA column, n-hexane:ethanol 80:20), water content ≤ 0.5% (Karl Fischer titration, according to Ph. Eur. 2.5.12), and residue on ignition ≤ 0.1%. The compound is supplied as a white to off-white crystalline powder with a melting range of 162–165 °C (decomposition) determined by differential scanning calorimetry at a heating rate of 10 K/min under nitrogen purge.

What Differentiates This Iminosugar from D-galacto- and D-gluco-configured Analogues?

The biological target profile of polyhydroxylated pyrrolidines is exquisitely sensitive to the relative and absolute stereochemistry of the ring substituents. The (2R,3R,4R,5R) configuration orients all four substituents in a steric arrangement that mimics D-mannopyranose in its ⁴C₁ chair conformation, an unusual feature among commercially available iminosugars which more commonly duplicate D-glucose or D-galactose topologies. A direct structural comparator is (2R,3R,4R,5S)-2,5-bis(hydroxymethyl)pyrrolidine-3,4-diol (the D-glucitol analogue), in which the C-5 epimerization inverts the relative orientation of the 5-hydroxymethyl group, shifting glycosidase inhibition selectivity from α-mannosidases toward α-glucosidases. The (2R,3R,4R,5R)-isomer exhibits a K_i value in the low micromolar to nanomolar range against jack bean α-mannosidase (measured spectrophotometrically with 4-nitrophenyl-α-D-mannopyranoside substrate at pH 4.5, 37 °C), whereas the 5S epimer typically yields K_i values one to two orders of magnitude higher against the same enzyme. This orthogonal inhibition fingerprint is exploited in glycoprocessing enzyme research and in the design of pharmacological chaperones for lysosomal storage disorders such as α-mannosidosis, where mutant α-mannosidase ERT requires small-molecule stabilization without inhibiting endogenous GH38 mannosidases systemically.

Synthetic access to the all-R isomer is more constrained than to epimeric mixtures. The chiral pool synthesis from D-mannitol proceeds via a double nucleophilic displacement sequence that preserves configuration at C-3 and C-4, but requires rigorous control of Mitsunobu conditions or triflate displacement steps to avoid competing elimination at the 2,5-ditosylate intermediate. An alternative enzymatic aldolase route using dihydroxyacetone phosphate and D-glyceraldehyde-3-phosphate catalyzed by a stereoselective aldolase has been published, but published data for this specific configuration at preparative scale (>100 g) is limited. Consequently, the (2R,3R,4R,5R)-isomer commands a significantly higher cost-per-gram than the mixed epimers, and commercial availability is restricted to specialized chiral building block suppliers.

Pharmacopoeial and Analytical Benchmarking

No dedicated monograph exists for this compound in the major pharmacopoeias; however, analytical method validation protocols applied in batch release adhere to ICH Q2(R2) guidelines. Identity is confirmed by ¹H NMR (D₂O, 400 MHz) with characteristic signals: δ 3.95 (ddd, J = 8.2, 5.6, 2.1 Hz, 2H, H-3/H-4), δ 3.87 (dd, J = 11.8, 3.3 Hz, 2H, H-1a/H-6a), δ 3.70 (dd, J = 11.8, 5.9 Hz, 2H, H-1b/H-6b), δ 3.28 (dt, J = 8.2, 3.0 Hz, 2H, H-2/H-5). ¹³C NMR exhibits signals at δ 65.3 (C-2, C-5), δ 75.8 (C-3, C-4), and δ 59.9 (CH₂OH). High-resolution mass spectrometry (ESI-TOF) yields [M+H]⁺ = 180.0866, matching the theoretical mass for C₆H₁₄NO₄ within 3 ppm. Residual solvent analysis per USP <467> residual solvents procedure (headspace GC-FID) confirms levels of methanol ≤ 300 ppm and dichloromethane ≤ 60 ppm, consistent with ICH Q3C limits for Class 2 solvents. Heavy metals by USP <231> method II are controlled to ≤ 10 ppm.

For enantiomeric purity determination, baseline resolution of the (2R,3R,4R,5R) peak from the (2S,3S,4S,5S) enantiomer is achieved with a retention time difference of 2.8 min using a Chiralpak IA-3 column (250 × 4.6 mm, 3 µm) at 25 °C with a mobile phase flow rate of 0.8 mL/min. The detection limit for the undesired enantiomer is established at 0.05% (S/N ≥ 3). In-process controls during manufacturing monitor the optical rotation at the final recrystallization step; the specific rotation [α]_D²⁰ in water (c = 1.0) is consistently recorded between +48° and +52°, any deviation beyond this window triggering re-analysis by chiral HPLC.

Storage, Handling, and Incompatibility Boundaries

The compound is hygroscopic. Dynamic vapor sorption analysis at 25 °C reveals a mass gain of 2.8% when relative humidity increases from 0% to 60% RH, and a sharp mass increase to 8.5% at 80% RH. Long-term storage under argon or nitrogen in sealed amber glass vials with desiccant and at a temperature of −20 ± 5 °C is recommended. For processing in aqueous media, freshly prepared solutions should be used within 24 hours if held at 2–8 °C, as the free amine functionality undergoes slow oxidative degradation catalyzed by dissolved metal ions; treatment of water with Chelex 100 resin prior to dissolution extends solution stability to approximately 48 hours. Incompatibility is observed with strong oxidizing agents, acid chlorides, and aldehydes — the latter condition conducive to imine formation and subsequent Amadori rearrangement products when the compound is exposed to reducing sugars at elevated temperatures (>50 °C). During lyophilization, cryoprotectant addition is unnecessary; primary drying at −40 °C and 0.1 mbar for 24 hours yields a free-flowing powder with residual water < 0.2%.

When Is It Used as a Chiral Scaffold Rather Than an Inhibitor?

Beyond glycosidase inhibition, the C₂-symmetry and dense functionality of (2R,3R,4R,5R)-2,5-bis(hydroxymethyl)pyrrolidine-3,4-diol render it a valuable chiral ligand precursor and organocatalyst scaffold. The secondary amine can be selectively N-alkylated or N-acylated without perturbing the four hydroxyls when using temporary silyl protection (TBSCl, imidazole in DMF at 0 °C to room temperature, 18 hours). The resulting N-functionalized derivatives have been employed in enantioselective aldol reactions: N-proline-derived amides of this pyrrolidine catalyze the reaction between acetone and 4-nitrobenzaldehyde with enantiomeric excesses reported up to 89% (determined by chiral HPLC of the aldol product). In asymmetric epoxidation, titanium(IV) complexes of the diprotected tetraol catalyze epoxidation of allylic alcohols with tert-butyl hydroperoxide, achieving conversion rates exceeding 85% and enantioselectivities in the range of 70–80% ee. The relative rigidity imparted by the contiguous all-R array of substituents restricts the conformational space accessible to metal chelates, thereby enhancing enantiofacial discrimination compared to analogues with mixed stereochemistry. When used as a ligand for Cu(II) in Henry reactions, the complex formed in situ in methanol/water (9:1) delivers 2.5-fold higher asymmetric induction than the ligand derived from the corresponding (2R,3R,4R,5S)-diastereomer under identical conditions.

Industrial application profiles, however, are shaped by cost and supply chain reliability. The necessity for multistep chiral pool synthesis or enzymatic routes constrains catalog prices to approximately €1,500–3,000/g for research-grade material (≥98% purity), orders of magnitude above the cost of racemic or mixed-epimer iminosugars. For medicinal chemistry programs targeting oral administration, the hydrochloride salt of the parent amine is often prepared in situ (treatment with 1.05 eq. HCl in dioxane) to enhance aqueous solubility and reduce hygroscopic variability; the salt form exhibits a solubility of >150 mg/mL in phosphate-buffered saline (pH 7.4) compared to ~40 mg/mL for the free base.

Comparative Table of Polyhydroxylated Pyrrolidine Isomers

The following table enumerates key stereoisomers in the 2,5-bis(hydroxymethyl)pyrrolidine-3,4-diol family, their primary glycosidase inhibition targets, and selected physical constants where available.

Values derived from suppliers' certificates of analysis and peer-reviewed enzyme inhibition assays; “DOL” indicates dissemination on lab scale without a published CAS index number.

Processing Parameters in Solid Oral Dosage Forms

While formulated primarily as a research tool and not registered as an active pharmaceutical ingredient, the compound’s behavior in solid blends is relevant to medicinal chemistry laboratories scaling tablet formulations for preclinical toxicology studies. Direct compression with microcrystalline cellulose (Avicel PH-102) and 1% w/w magnesium stearate in a tablet press operating at 15 kN compression force yields compacts with a tensile strength exceeding 2.2 MPa and friability below 0.4% (USP <1216>). However, blend homogeneity when the active load is below 2% w/w requires geometric dilution and end-over-end blending at 25 rpm for 20 minutes to achieve an acceptance value of ≤15 per USP <905> content uniformity testing. Wet granulation with aqueous binder solutions is not recommended due to the hygroscopic response and amine-water interactions generating sticky granules; ethanol-based granulation with 5% PVP K-30 binder yields acceptable granules with Hausner ratio 1.12 and angle of repose 32°.

Chromatographic Retention in Hydrophilic Interaction Liquid Chromatography (HILIC)

The high polarity of this tetraol requires separation modes diverging from reversed-phase chromatography. A ZIC-HILIC column (150 × 4.6 mm, 3.5 µm) with acetonitrile/50 mM ammonium formate buffer pH 4.5 (80:20 v/v) at a flow rate of 0.5 mL/min resolves the parent peak from process-related impurities with a retention time of 9.4 min. Under these conditions, the tailing factor at 10% peak height ranges from 1.0 to 1.3. Detection is performed by evaporative light scattering (ELSD) with nitrogen nebulizer gas at 3.5 bar, drift tube temperature 45 °C, and gain 8. The limit of quantitation for potential N-methyl impurity arising from reductive amination side-reactions is established at 0.03% w/w relative to the main analyte.

Batch-to-Batch Variability and Root-Cause Investigations

Manufacturing campaigns tracked over 18 months across 12 batches (lot sizes ranging from 50 g to 500 g) have identified two primary sources of deviation: residual palladium from hydrogenolysis of N-benzyl or N-Cbz protecting groups, and color body formation during recrystallization. Palladium content is quantified by ICP-MS (method adapted from USP <232>) and typically maintained below 5 ppm; one campaign exhibited an excursion to 12 ppm linked to inadequate charcoal filtration post-hydrogenation, corrected by the insertion of a 0.2 µm polypropylene depth filter inline prior to concentration under reduced pressure. Color body formation (yellow to light brown discoloration) correlates with exposure to trace oxygen during hot filtration of the recrystallization solution. Implementation of a nitrogen-blanketed filtration skid with 0.5 psi positive pressure reduced the incidence of out-of-specification appearance from 8% to <1% of batches.

Thermal stability assessed by thermogravimetric analysis (TGA) indicates the onset of decomposition at 185 °C, with 2% mass loss by 200 °C. Differential scanning calorimetry reveals an endothermic event associated with melting immediately followed by an exothermic decomposition peak, rendering melt-processing above 170 °C inadvisable for any downstream application. Storage at accelerated stability conditions (40 °C/75% RH) for 6 months resulted in a purity decrease of 0.4% and a total impurity profile that remained within specification, though a slight increase in the epimer content was noted (from 0.15% to 0.32%), attributed to the base-catalyzed epimerization at C-2 mediated by trace ammonium present in the lyophilizate.

Are There Any Regulatory-Specific Considerations?

The compound is not listed in the European Inventory of Existing Commercial Chemical Substances (EINECS) and is supplied solely for research and development purposes under a TSCA R&D exemption. For laboratories operating under ISO/IEC 17025 accreditation, certificates of analysis include measurement uncertainty budgets for chiral purity (expanded uncertainty ±0.2% with k=2) and water content. No REACH registration dossier has been submitted; import quantities into the EU as a substance on its own must remain below 1 tonne per annum to maintain compliance with the registration exemption threshold. For shipments to Japan, compliance with the Chemical Substances Control Law (CSCL) is verified through the supplier’s notification as a General Chemical Substance under the designation of “pyrrolidine derivative.” The absence of a harmonized classification under CLP (Regulation (EC) No 1272/2008) necessitates a self-classification based on available toxicological data; acute oral toxicity studies in Sprague-Dawley rats (OECD 423) indicate an LD₅₀ > 2000 mg/kg, supporting no acute toxicity classification, but skin sensitization data (OECD 406 guinea pig maximization) are not available, and conservative classification as Skin Sensitizer Category 1B is applied in the provisional safety data sheet.