| HS Code | 836317 |
| Product Name | Fmoc-D-asparagine |
| Synonyms | Fmoc-D-Asn-OH; N-[(9H-Fluoren-9-ylmethoxy)carbonyl]-D-asparagine |
| Cas Number | 108334-68-5 |
| Molecular Formula | C19H18N2O5 |
| Molecular Weight | 354.36 g/mol |
| Appearance | White to off-white powder |
| Purity | ≥98% (HPLC) |
| Melting Point | 180-182 °C (dec.) |
| Optical Rotation | [α]20/D = +7.5° (c=1 in DMF) |
| Storage Conditions | Store at -20 °C, protected from light and moisture |
| Solubility | Soluble in DMF and DMSO |
| Applications | Used in solid-phase peptide synthesis for introducing D-asparagine, and in preparation of D-amino acid-containing peptides |
As an accredited Fmoc-D-asparagine factory, we enforce strict quality protocols—every batch undergoes rigorous testing to ensure consistent efficacy and safety standards.
| Packing | Fmoc-D-asparagine is supplied as a white crystalline powder in a 5 g amber glass bottle, sealed under argon with desiccant. |
| Container Loading (20′ FCL) | 20′ FCL container loading of Fmoc-D-asparagine: sealed drums, palletized and secured, ensuring safe, compliant transport for this fine chemical. |
| Shipping | Fmoc-D-asparagine ships at ambient temperature in a sealed, desiccated container. Keep away from moisture, heat, and direct light. Standard laboratory courier with tamper-evident packaging is used; no special hazardous transport classification applies. Verify customs documentation for international orders. |
| Storage | Store Fmoc-D-asparagine in a tightly sealed container, protected from light and moisture. For long-term stability, keep it at -20°C in a desiccator or with desiccant. Allow the vial to reach room temperature before opening to prevent condensation, and minimize exposure to air to avoid degradation. |
| Shelf Life | Shelf life: 2–3 years when stored at -20°C, desiccated, and protected from light. |
In GMP peptide synthesis trains that produce D-Asn-containing peptide APIs, Fmoc-D-asparagine is fed as a pre-weighed solid from isolator dispensing booths held at 20–24 °C and 35–45 % relative humidity. The building block is released against a certificate of analysis that includes identity, specific rotation, water content by Karl Fischer titration, and residual solvent testing aligned with Ph. Eur. 2.4.24 and USP 467. Coupling solutions are prepared at 0.20 M in anhydrous DMF with 4 eq Fmoc-D-asparagine, 4 eq HBTU, 4 eq HOBt, and 8 eq DIPEA. Discovery-scale runs on automated synthesizers with 5–100 mL reaction vessels apply the activated monomer for 45 min at 25 °C; pilot-scale campaigns in 50 L jacketed reactors running 0.5–10 mol batches extend coupling to 90 min at 15–20 °C to maintain acylation control. Fmoc removal is restricted to two cycles of 20 % piperidine in DMF for 5 min and 10 min, because the unprotected carboxamide side chain of Fmoc-D-Asn is vulnerable to aspartimide pathway activation under excessive base exposure. Cleavage from Rink amide AM resin uses TFA/TIS/H2O 95:2.5:2.5 v/v/v for 2.5 h; peptide acids from Wang resin use TFA/thioanisole/water/phenol 90:5:3:2 v/v/v/v for 3 h. Crude D-Asn-containing peptide is precipitated in cold MTBE, dissolved in 10 % acetonitrile/water, and purified on preparative C18 columns with 0.1 % TFA/acetonitrile gradients. Coupling completeness is monitored by Kaiser test or chloranil test, and residual Fmoc-D-Asn is tracked by HPLC against a qualified reference standard. ICH Q7 requires process controls and retained samples when the resulting peptide is intended for clinical supply.
| Scale | Resin/equipment | Coupling conditions | Deprotection | Analytical control |
| 0.05 mmol | Rink amide PEG-polystyrene, 0.4 mmol/g, CEM Liberty Blue HT | 4 eq Fmoc-D-Asn, HBTU/HOBt/DIPEA in DMF, 45 min at 25 °C | 20 % piperidine/DMF, 5 + 10 min | Kaiser test, LC-MS |
| 10 mmol | 2-chlorotrityl chloride resin, 250 mL reactor | 4 eq Fmoc-D-Asn, HATU/DIPEA in NMP, 60 min at 20 °C | 20 % piperidine/NMP, 5 + 10 min | HPLC-UV |
| 100 mmol | 50 L jacketed reactor, Wang resin | 4 eq Fmoc-D-Asn, DIC/HOBt in DMF, 90 min at 15 °C | 20 % piperidine/DMF, 5 + 10 min | UPLC-MS, chiral amino acid analysis |
During Fmoc deprotection of peptides containing Fmoc-D-Asn, the side-chain primary amide can participate in intramolecular cyclization with the C-terminal ester of the same residue, yielding a five-membered aspartimide. The pathway is initiated by base-mediated deprotonation of the backbone amide, not by direct side-chain attack, which explains why sequence context adjacent to D-Asn influences impurity levels more than the D-configuration itself. Under standard 20 % piperidine in DMF at 25 °C, aspartimide formation may remain below the detection limit when the following residue is sterically hindered, such as Val or Ile. When the adjacent residue is Gly or Gln, the same deprotection can generate 1–5 % aspartimide-containing sequences after two 10-min cycles, resolved by LC-MS as a −18 Da mass shift from the target peptide. Microwave-assisted SPPS at 70–80 °C reduces coupling time but aggravates this side reaction; process operators therefore use lower-temperature coupling for D-Asn residues or select Fmoc-D-Asn(Trt)-OH for sequences longer than 15 amino acids. Addition of 0.1 M HOBt to the deprotection solution has been reported in the literature to open the intermediate and reduce piperidide adducts, but the reproducibility of this approach across resin types is limited. On-line UV monitoring at 300 nm is not sufficient to detect aspartimide because the released dibenzofulvene chromophore is generated in all deprotection events. Release testing therefore uses UPLC-MS/MS with a C18 column of 1.7 µm particle size and extracted-ion chromatograms for target, −18 Da, and +67 Da piperidide adducts. The operational boundary for Fmoc-D-Asn without side-chain protection is set at 2 × 5 min piperidine contact and ≤40 °C, with immediate neutralization by washing with DMF containing 0.5 M HOBt after deprotection.
Antimicrobial peptide discovery programs use Fmoc-D-asparagine to introduce D-Asn at positions that would otherwise be cleaved by serum proteases. Parallel synthesis on a 96-well Intavis MultiPep RSi automated synthesizer at 5–10 µmol scale on ChemMatrix Rink amide resin applies 5 eq Fmoc-D-Asn, 5 eq HATU, and 10 eq DIPEA in DMF for 30 min at 20 °C. Double coupling is programmed when the preceding residue is a β-branched amino acid such as Thr or Ile, because the D-configuration slows acylation at sterically constrained positions. Deprotection uses 20 % piperidine in DMF with a single 5-min cycle to avoid aspartimide ring closure; the resin is washed with 0.5 M HOBt in DMF after each deprotection step. Cleavage with TFA/thioanisole/EDT/anisole 90:5:3:2 v/v/v/v for 2 h yields crude D-Asn-containing AMPs that are precipitated in cold diethyl ether and purified on C18 flash cartridges. Purity is assessed by UPLC at 214 nm and identity by MALDI-TOF MS using α-cyano-4-hydroxycinnamic acid. Minimum inhibitory concentration testing follows CLSI M07-A10 broth microdilution against Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922; results are interpreted only against quality-control ranges defined in the standard. D-Asn substitutions at hydrophilic positions are screened for effect on cationicity and not for direct antimicrobial activity claims. The building block is certified for research use and is released by identity, chiral purity, and water content.
Retro-inverso peptide analogues that replace native L-amino acid backbones with reversed amide bonds and D-amino acid side chains are assembled using Fmoc-D-asparagine for the D-Asn residue. The synthesis requires repetitive amide bond formation at sterically hindered amines; coupling in NMP with 5 eq Fmoc-D-Asn pentafluorophenyl ester and 10 eq DIPEA at 30 °C for 60 min is used when activating agents like HATU produce incomplete acylation. Deprotection with 20 % piperidine in NMP is limited to 2 × 5 min because the reversed backbone positions the Asn amide side chain near the C-terminal ester of the preceding residue, increasing aspartimide susceptibility. To suppress deamidation, the peptide-resin is never exposed to aqueous base above pH 9; post-cleavage workup avoids aqueous ammonia and uses dilute acetic acid for clarification. Cleavage from MBHA resin with TFA/trifluoromethanesulfonic acid/thioanisole/EDT 90:5:3:2 v/v/v/v at 0 °C for 2 h releases the retro-inverso construct; for constructs with a free C-terminal carboxyl group, the cleavage uses 95 % TFA with 2.5 % TIS and 2.5 % water. Purification on a C4 reversed-phase column with 0.1 % TFA in acetonitrile/water removes truncated sequences, while chiral amino acid analysis after hydrolysis in 6 M HCl at 110 °C for 24 h confirms D-Asn configuration. Plasma stability is tested at 37 °C in human plasma over 0–24 h with sampling points analyzed by LC-MS/MS, but published data for specific D-Asn-containing retro-inverso clinical candidates remains limited. End products are investigational peptide drugs and tool compounds for target validation.
C-terminal loading of Fmoc-D-asparagine onto 2-chlorotrityl chloride resin is governed by the steric bulk of the Fmoc group and the hydrogen-bonding capacity of the unprotected carboxamide side chain. Loading is performed by dissolving Fmoc-D-Asn in anhydrous DCM/DMF 1:1 v/v with 2.0 eq DIPEA and adding to resin at 0.5–1.0 mmol/g target capacity. The mixture is agitated for 45–60 min at 15–20 °C; residual trityl chloride sites are capped with methanol/DIPEA for 30 min. Actual loading is determined by Fmoc UV assay at 290 nm after deprotection of a dried resin aliquot with 20 % piperidine in DMF. Values outside 0.2–1.2 mmol/g trigger rework or rejection; high loadings above 1.3 mmol/g can reduce coupling efficiency in subsequent residues due to electrostatic repulsion and resin volume constraints. The free carboxamide of Fmoc-D-Asn can form interchain hydrogen bonds that reduce swelling in DCM, but DMF swells 2-chlorotrityl resin sufficiently for uniform substitution. Cleavage from 2-chlorotrityl chloride using 1 % TFA in DCM yields a protected peptide fragment with Fmoc-D-Asn at the C-terminus, which is used in fragment condensation or cyclization. Equipment for this loading step includes jacketed glass synthesizers with 50–250 mL volume and sintered frit dispersion plates. The released D-Asn-containing peptide is analyzed by LC-MS and chiral TLC using a CHIRALPLATE system; no GMP release is performed unless the fragment enters an API synthesis chain.
For asparagine-specific protease probe libraries, Fmoc-D-asparagine occupies non-cleavable positions in short 6–12 mer substrates assembled on PAM resin using HBTU/DIPEA coupling in NMP. The peptide is capped with an N-terminal fluorescent donor such as 5-carboxyfluorescein and a C-terminal quencher, or with the donor/quencher pair reversed. Because D-Asn is not recognized by asparagine endopeptidase or legumain, the probe remains intact in negative-control wells, while the L-asparagine counterpart is hydrolyzed. Cleavage and deprotection use TFA/TIS/H2O 95:2.5:2.5 for 2 h; purification is performed by reverse-phase HPLC on C18 with 0.1 M triethylammonium acetate/acetonitrile. Enzymatic assays follow published conditions at pH 5.0–7.4 and 37 °C, with fluorescence readout every 5 min for 120 min on a microplate reader. The material is not GMP-controlled and requires no pharmacopeial monograph; identity and chiral purity are confirmed by LC-MS.
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Fmoc-D-asparagine (CAS 108321-39-7), also listed as Fmoc-D-Asn-OH and Nα-(9-fluorenylmethoxycarbonyl)-D-asparagine, is a base-labile protected D-amino acid used in Fmoc/tBu solid-phase peptide synthesis. The empirical formula is C19H18N2O5, corresponding to a molecular weight of 354.36 g/mol. The stereocenter carries the (R) descriptor under Cahn–Ingold–Prelog rules. Product catalog numbers are supplier-specific; the compound is indexed under the CAS registry number rather than a unified model designation. Solid-phase synthesis grade material is commonly supplied as a white to off-white powder and controlled at ≥98.5% HPLC purity at 220 nm, with the L enantiomer held below 0.5% area by chiral HPLC. Fmoc-D-asparagine contains an unprotected side-chain carboxamide, unlike the trityl-protected variant Fmoc-D-Asn(Trt)-OH. This unprotected amide defines much of the coupling strategy and creates a process boundary because carbodiimide activation can dehydrate the side chain to a β-cyanoalanine derivative. The compound dissolves in DMF, NMP, and DMSO, is sparingly soluble in dichloromethane, and is essentially insoluble in water.
At production scale, Fmoc-D-asparagine is typically dissolved in DMF at 0.2–0.5 M and activated with HATU/DIEA or DIC/Oxyma. In automated peptide synthesizers using single-use reactors of 10–100 mL and resin substitution of 0.1–0.5 mmol/g, standard coupling times range from 30–60 min at ambient temperature. Batch records show that prolonged pre-activation above 5 min with excess carbodiimide raises the β-cyanoalanine side product. For sequences longer than 20 residues, or when coupling above 50 °C is required, the trityl-protected derivative is generally substituted. Fmoc removal is monitored by UV absorbance at 290–301 nm through the dibenzofulvene-piperidine adduct. Coupling to Wang, Rink amide, and 2-chlorotrityl resins is feasible because the unprotected side-chain amide does not require resin-specific scavengers under standard Fmoc/tBu cleavage conditions.
The unprotected primary carboxamide of D-asparagine is the main process constraint. Under carbodiimide activation with DIC or DCC, the side-chain amide can be dehydrated to a β-cyanoalanine derivative. The reaction is promoted by excess carbodiimide relative to the carboxylic acid, elevated temperature, and extended pre-activation. A controlled process window therefore uses 1.0–1.2 equivalents of DIC relative to Fmoc-D-asparagine, activation at 0–4 °C for 2–5 min, and immediate addition to the resin-bound amine. When Oxyma is included at 1.0 equivalent, the nitrile impurity measured by RP-HPLC at 220 nm is maintained below 1.0% area in routine protocols; published data for this specific configuration is limited to vendor optimization studies. Racemization at the α-carbon is controlled by limiting DIEA to 2.0 equivalents and avoiding pre-activation above 5 min. The D enantiomer can convert to the L form through an oxazolone intermediate if the base is added before the activated ester is fully formed. The preferred addition sequence is therefore Fmoc-D-Asn-OH plus activator, followed by DIEA, followed by transfer to the resin.
In process development, Fmoc-D-asparagine is often introduced into β-turn or D-peptide sequences where the side-chain carboxamide provides hydrogen-bonding capacity but the backbone stereochemistry is altered. The unprotected amide can compete with the resin-bound N-terminal amine for activated carboxylate when resin loading is high. Coupling protocols derived from vendor optimization studies often use 1.5–2.0 equivalents of Fmoc-D-Asn-OH and 1.5–2.0 equivalents of activator relative to resin loading, with a single 30 min recoupling if a qualitative Kaiser test remains positive. Residual water above the Karl Fischer limit reduces first-pass coupling efficiency because water competes for the activated carboxylate. Material with water content above 0.5% is therefore dried under vacuum at ambient temperature before use in moisture-sensitive activation.
Batch release documentation for solid-phase synthesis grade Fmoc-D-asparagine follows the methods summarized in Table 1. Manufacturer limits vary, but the following acceptance profile is representative of material used in cGMP peptide manufacturing.
| Parameter | Method | Acceptance limit |
|---|---|---|
| Appearance | Visual inspection | White to off-white powder |
| Identification | FTIR vs reference standard | Matches reference spectrum |
| Assay | USP 621 RP-HPLC, C18, 5 μm, 250 × 4.6 mm, 220 nm | ≥98.5% area |
| Single impurity | USP 621 RP-HPLC | ≤0.5% area |
| Total impurities | USP 621 RP-HPLC | ≤1.5% area |
| Enantiomeric purity | Chiral HPLC | L enantiomer ≤0.5% area |
| Specific rotation | USP 781, c=1, DMF, 20 °C | +11.0° to +12.5° |
| Water content | USP 921 Karl Fischer | ≤0.5% |
| Residual solvents | USP 467 GC headspace | DCM ≤600 ppm, DMF ≤880 ppm |
| Loss on drying | USP 731 | ≤0.5% |
System suitability for the assay is run according to USP 621, with tailing factor ≤2.0 and injection precision RSD ≤2.0% for the main peak. Residual solvent limits follow ICH Q3C Option 1 Class 2 exposure limits for dichloromethane and DMF. Certificate of analysis documentation should include batch-specific chromatograms and the methods listed above. If optical rotation falls outside the specified range, enantiomeric contamination or residual solvent can be the cause; the batch should not be released without chiral HPLC confirmation.
Storage at −20 ± 5 °C in a sealed, desiccated container is required to maintain the release specification. Before opening, the container should be equilibrated to ambient temperature for at least 30 min to avoid moisture condensation when relative humidity exceeds 60%. Fmoc-D-asparagine is base-labile; contact with primary or secondary amines, including ammonia and piperidine vapours, must be excluded. Strong aqueous alkali above pH 10 removes the Fmoc group, and strong acid can hydrolyse the side-chain amide after prolonged exposure. Repeated freeze-thaw cycles should be minimized because absorbed water accelerates Fmoc cleavage and reduces coupling yield. Contact with strong oxidizing agents should also be avoided due to the fluorenylmethoxycarbonyl moiety.
Fmoc-D-asparagine and Fmoc-L-asparagine share the empirical formula C19H18N2O5 and molecular weight 354.36 g/mol. Conventional C18 reversed-phase HPLC does not resolve the enantiomers. Differentiation requires a chiral stationary phase, polarimetric detection, or enzymatic assay. The D derivative exhibits positive specific rotation under the conditions in Table 1, while the L derivative shows the corresponding negative value. In preparative peptide synthesis, the choice between D and L asparagine alters backbone stereochemistry and can affect proteolytic stability, receptor binding, and chromatographic retention of the final peptide. The D isomer is not a drop-in replacement for the L isomer in biological assays; potency and selectivity must be re-established using the target assay or a qualified surrogate.
| Derivative | Side-chain protection | Main deprotection | Molecular difference | Typical use |
|---|---|---|---|---|
| Fmoc-D-asparagine | Unprotected carboxamide | Piperidine 20% in DMF | MW 354.36 g/mol; positive rotation | Direct SPPS; short-to-medium sequences; D-peptide synthesis |
| Fmoc-L-asparagine | Unprotected carboxamide | Piperidine 20% in DMF | MW 354.36 g/mol; negative rotation | Natural peptide sequences; identical coupling chemistry |
| Fmoc-D-Asn(Trt)-OH | Trityl on carboxamide | Piperidine 20%, then TFA cleavage of trityl | Higher molecular weight; no nitrile side product | Long, hydrophobic, or heated sequences |
| Boc-D-asparagine | Unprotected carboxamide | TFA or 4 M HCl/dioxane | Lower molecular weight; no strong UV chromophore | Boc/Bzl SPPS or solution-phase orthogonal schemes |
Because the unprotected carboxamide in Fmoc-D-asparagine can undergo dehydration, the trityl-protected derivative is preferred when coupling at elevated temperature or when repeated coupling cycles are required. The trade-off is an additional acid-labile protecting group that must be removed with TFA in the presence of scavengers. Fmoc-D-asparagine and Fmoc-D-aspartic acid are also not interchangeable. Fmoc-D-aspartic acid is typically supplied as the β-tert-butyl ester to avoid aspartimide formation, while Fmoc-D-asparagine supplies a neutral carboxamide without side-chain protection. The difference changes side-chain ionization, pI, metal-binding, and receptor interaction; capillary electrophoresis or ion-exchange HPLC is required to confirm the intended charge state of the final peptide.
Fmoc-D-asparagine and Boc-D-asparagine are not interchangeable without adjusting the deprotection strategy. Fmoc removal uses a secondary amine, typically 20% piperidine in DMF, while Boc removal uses trifluoroacetic acid. In a hybrid synthesis that requires both acid-labile and base-labile protection, the two can be used orthogonally: a Boc-D-asparagine can remain intact during Fmoc deprotection, and an Fmoc-D-asparagine can remain intact during mild acid treatment if the side-chain protecting groups and resin linker are selected accordingly. The Fmoc derivative provides a strong UV chromophore for automated deprotection monitoring at 290–301 nm; the Boc derivative lacks this chromophore and requires alternative endpoint detection such as ninhydrin or chloranil tests. The larger Fmoc group also reduces volatility and increases molecular weight relative to the Boc analogue, which shifts loading calculations and may alter resin swelling in DMF versus DCM.
In automated synthesizer protocols, Fmoc-D-asparagine is delivered as a DMF solution through preconditioned lines with dead volumes below 0.5 mL to prevent cross-contamination. The delivery line is rinsed with DMF before and after addition. Coupling efficiency is assessed by Kaiser test or quantitative Fmoc UV release at 290–301 nm. On preparative columns packed with 10–20 μm C18 silica, peptides containing D-asparagine may show retention-time shifts relative to the L analogue; method development should not assume identical retention. Mass confirmation accounts for the formal incorporation of D-asparagine with a monoisotopic residue mass of 114.0429 Da. Peptide sequences containing D-Asn can display altered hydrogen-bonding in turn regions; crystallography or NMR analysis is required to confirm the structural consequence. The unprotected side-chain carboxamide cannot be selectively modified on the solid phase; if further side-chain diversification is planned, an orthogonally protected derivative or post-synthetic conjugation should be considered.
