| HS Code | 819167 |
| Product Name | Fmoc-D-serine |
| Synonyms | N-alpha-Fmoc-D-serine; (R)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-hydroxypropanoic acid |
| Cas Number | 116861-26-8 |
| Molecular Formula | C18H17NO5 |
| Molecular Weight | 327.33 g/mol |
| Purity | ≥98% (HPLC) |
| Appearance | White to off-white powder |
| Solubility | Soluble in DMF, DMSO, and methanol; sparingly soluble in water |
| Storage Conditions | Store at 2-8°C, protected from light and moisture |
| Iupac Name | (2R)-2-({[(9H-fluoren-9-ylmethoxy)carbonyl]amino})-3-hydroxypropanoic acid |
| Smiles | C([C@H](C(=O)O)NC(=O)OCC1c2ccccc2-c3ccccc13)O |
| Inchi | InChI=1S/C18H17NO5/c20-10-15(16(21)22)19-18(23)24-9-17-13-7-3-1-5-11(13)12-6-2-4-8-14(12)17/h1-8,15,17,20H,9-10H2,(H,19,23)(H,21,22)/t15-/m1/s1 |
As an accredited Fmoc-D-serine factory, we enforce strict quality protocols—every batch undergoes rigorous testing to ensure consistent efficacy and safety standards.
| Packing | Fmoc-D-serine, 5 g, packaged in an amber glass vial with a polypropylene cap and desiccant, stored under nitrogen. |
| Container Loading (20′ FCL) | Container Loading (20′ FCL): Fmoc-D-serine packed in sealed drums on pallets, secured properly, labeled, and containerized for safe, efficient transport. |
| Shipping | Fmoc-D-serine is shipped at ambient temperature in a sealed, light-protected container with desiccant. It is not classified as dangerous goods for transport. Avoid prolonged exposure to heat, moisture, and UV light. Upon receipt, store at 2–8°C in a dry, well-ventilated area. |
| Storage | Store Fmoc-D-serine in a tightly sealed container at -20°C, protected from light and moisture. Keep desiccant present and allow the vial to reach room temperature before opening to prevent condensation. Under these dry, cool, dark conditions, the compound remains stable for long-term peptide synthesis use. |
| Shelf Life | Shelf life: typically 2 years when stored at -20°C, dry, and protected from light. |
In peptide API manufacturing, Fmoc-D-serine is handled as an unprotected hydroxyl acid in Fmoc-strategy solid-phase peptide synthesis for sequences in which a D-serine residue is required at an internal position and the adjacent amino acid is not a sterically hindered N-methylated residue. The building block is coupled onto 0.40 mmol/g Fmoc-Rink Amide AM resin after deprotection with 20% v/v piperidine in DMF for 5 min and 10 min at 20–25 °C. A typical coupling mixture consists of 3.0 equiv Fmoc-D-serine, 2.85 equiv HCTU, and 6.0 equiv N,N-diisopropylethylamine in DMF at 0.30 M, preactivated at 0–5 °C for 2–3 min, then delivered to the deblocked resin for 45 min at 20–25 °C. A positive Kaiser test triggers a second coupling with fresh 1.5 equiv Fmoc-D-serine, 1.43 equiv HCTU, and 3.0 equiv DIPEA for 30 min. The unprotected β-hydroxyl is not a passive spectator: when preactivation exceeds 5 min at room temperature or the uronium reagent is charged above 3.0 equiv, activated carboxyl species can acylate the β-hydroxyl of an adjacent Fmoc-D-serine molecule and create a resin-bound ester branch that survives Fmoc deprotection and yields truncated/deletion impurities. For sequences longer than 8 residues or where multiple acylating cycles follow the D-serine position, manufacturers ordinarily replace Fmoc-D-serine with Fmoc-D-Ser(tBu)-OH under the same activation protocol; the free hydroxyl form is retained for short-chain peptides or C-terminal positions near cleavage. Cleavage uses TFA/H₂O/TIS 95:2.5:2.5 v/v/v for 2.5 h at 20–25 °C, followed by cold methyl tert-butyl ether precipitation, preparative C18 reversed-phase HPLC with 0.1% TFA/acetonitrile gradients, and lyophilization as acetate salts. The terminal products are linear or cyclic peptide APIs and reference standards containing D-serine at specified positions; batch release includes LC-MS purity at 214 nm, HPLC system suitability under USP 621, and residual DMF/dichloromethane control under ICH Q3C.
Solution-phase coupling of Fmoc-D-serine to amino acid esters is constrained by competition between the carboxyl group and the β-hydroxyl for the O-acylisourea intermediate. A representative dipeptide synthesis charges 1.0 equiv Fmoc-D-serine, 1.0 equiv glycine methyl ester hydrochloride, 1.1 equiv hydroxybenzotriazole monohydrate, 1.1 equiv EDC·HCl, and 2.0 equiv N-methylmorpholine in DMF at 0–5 °C, then warms to 20 °C for 4 h. Under these conditions the desired amide bond forms, but the unprotected β-hydroxyl can attack an activated Fmoc-D-serine molecule to produce Fmoc-D-Ser-O-Fmoc-D-Ser as an organic-soluble impurity that co-elutes with the target dipeptide during ethyl acetate extraction and requires flash chromatography for removal. Published data for this specific side reaction as a function of Fmoc-D-serine concentration is limited; process-development batches indicate that preactivation beyond 10 min at 20 °C increases the O-acylated impurity and depresses isolated yield below 85%. The process window is therefore established by spiking studies with the suspected dimer and by monitoring the reaction with LC-MS at 220 nm. For longer solution-phase fragment couplings, the free hydroxyl is masked as a tert-butyl ether before carbodiimide activation; Fmoc-D-Ser(tBu)-OH tolerates longer preactivation and higher activation temperatures. The product class from this route comprises Fmoc-D-Ser-Gly ester fragments, Fmoc-D-Ser-amino acid ester fragments, and D-serine-containing intermediates for subsequent fragment condensation. Compliance for enantiomeric purity relies on chiral HPLC without derivatization on a Crownpak CR-I(+) column eluted with pH 2.0 perchloric acid at 25 °C; hydrolysis followed by Marfey’s reagent provides an orthogonal check of D-serine configuration. Residual solvents are controlled under ICH Q3C; HOBt and EDC residues are confined to the mother liquor and monitored by LC-MS.
Fmoc-D-serine is used as a glycosyl acceptor in solution-phase synthesis of Fmoc-protected glycopeptide building blocks because the β-hydroxyl is free while the carboxyl is protected as an allyl ester. The first process step converts Fmoc-D-serine to Fmoc-D-Ser-OAll by alkylation with 1.5 equiv allyl bromide and 1.5 equiv potassium carbonate in DMF at 20 °C for 6 h; the allyl ester is isolated by ethyl acetate extraction and used without silica-gel chromatography in some pilot batches. Glycosylation then combines 1.0 equiv Fmoc-D-Ser-OAll with 1.5 equiv 2,3,4,6-tetra-O-acetyl-α-D-galactopyranosyl trichloroacetimidate and 0.2 equiv trimethylsilyl trifluoromethanesulfonate in dichloromethane at −20 °C for 2 h, followed by gradual warming to 0 °C. The acetyl protecting groups direct the reaction toward the β-configuration; anomeric purity is confirmed by 1H NMR coupling constants and reversed-phase HPLC. Allyl ester removal uses 0.02 equiv tetrakis(triphenylphosphine)palladium(0) and 2.0 equiv phenylsilane in THF under inert atmosphere, producing Fmoc-D-Ser(β-D-Gal(Ac)₄)-OH as a protected glycopeptide building block. The terminal products are mucin-type D-serine glycopeptide vaccine candidates, microbial adhesion antagonists, and antifreeze glycopeptide analogues. Residual palladium is controlled by ICP-MS at less than 10 ppm; residual dichloromethane and THF are controlled under ICH Q3C; the final building block is released by HPLC purity at 214 nm and mass spectrometry.
Phosphoserine isostere synthesis from Fmoc-D-serine requires a protected carboxyl and an unprotected β-hydroxyl; direct phosphitylation of the free acid is not performed because the carboxyl group competes with the hydroxyl for the phosphoramidite under tetrazole activation. The carboxyl is first protected as the allyl ester, then reacted with 1.5 equiv N,N-diisopropyl dibenzylphosphoramidite and 3.0 equiv 1H-tetrazole as a 0.45 M solution in acetonitrile in dichloromethane at −20 °C for 30 min; oxidation uses 2.0 equiv of 5.5 M tert-butyl hydroperoxide in decane at 0 °C for 1 h. The D configuration of the α-carbon is retained during phosphite transfer and oxidation; batch release includes 31P NMR and chiral HPLC after acidic hydrolysis. The terminal products are Fmoc-D-Ser(PO(OBzl)OH)-OH building blocks and D-phosphoserine-containing peptides used as stereochemical probes for protein phosphatase substrate recognition and 14-3-3 binding studies. Because Fmoc-D-serine is a D-amino acid derivative, the resulting phosphoserine analogue is not a direct mimic of endogenous L-phosphoserine; its application is confined to mechanism-of-action studies and co-crystallization campaigns where the unnatural configuration is intentionally selected. Residual allyl alcohol and palladium are controlled under ICH Q3C and by ICP-MS; purity is quantified by reversed-phase HPLC at 214 nm.
When Fmoc-D-serine replaces Fmoc-L-serine in a peptide sequence, the change in α-carbon stereochemistry alters on-resin aggregation, Fmoc deprotection rates, and final peptide proteolytic stability. In difficult sequences containing polyalanine or isoleucine-rich stretches, insertion of a single D-serine residue has been observed to reduce resin-bound NH-Fmoc aggregation and improve subsequent coupling yields; however, the effect is sequence-dependent, and no universal threshold exists for predicting the benefit. Published data for specific D-serine-containing peptide sequences is limited, so manufacturing campaigns rely on resin substitution checks, Kaiser test results, and UPLC-MS profiles rather than general rules. The same coupling protocol used for L-serine is applied: 2.5 equiv Fmoc-D-serine, 2.4 equiv HATU, 5.0 equiv DIEA, 0.15 M in NMP, with double coupling for 20 min at 40 °C when the Kaiser test remains positive. Cleavage uses TFA/TIS/H₂O 95:2.5:2.5 v/v/v for 2.5 h at 20–25 °C; thioanisole is added at 5% v/v only when methionine or cystine is present in the same sequence. The terminal products include D-amino acid-containing antimicrobial peptides and receptor ligand libraries where the D-serine residue is selected to restrict proteolytic degradation. Chiral amino acid analysis of acid hydrolysate with Marfey’s reagent or chiral GC confirms D-serine recovery and racemization below 0.5%. Residual piperidine, NMP, and TFA are controlled under ICH Q3C and by ion chromatography; peptide purity is assessed by UPLC-MS at 214 nm.
Direct loading of Fmoc-D-serine onto 2-chlorotrityl chloride resin is used to generate C-terminal D-serine peptide acids in a single resin-capture step. The resin, with initial substitution 1.0–1.5 mmol/g, is swollen in dichloromethane and treated with 1.0–1.2 equiv Fmoc-D-serine relative to trityl chloride sites and 4.0 equiv DIEA in DCM/DMF 9:1 v/v for 60 min at 20–25 °C; unreacted trityl chloride is end-capped with methanol/DIEA. The unprotected β-hydroxyl does not normally compete with the carboxyl for trityl chloride during the first 60 min; if loading extends beyond 90 min, hydroxyl attachment increases and produces resin-bound Fmoc-D-serine with a lower final acid cleavage yield. This threshold is monitored by Fmoc loading assay after piperidine deprotection; typical loadings are 0.60–0.90 mmol/g. The loaded resin is then elongated by standard Fmoc SPPS cycles and cleaved with TFA/TIS/H₂O 95:2.5:2.5 v/v/v to release C-terminal D-serine peptide acids. The terminal products are used in structure-activity relationship studies and as intermediates for side-chain-specific conjugation. Residual DCM is controlled under ICH Q3C; Fmoc loading is quantified by UV absorbance at 290 nm using the dibenzofulvene-piperidine adduct.
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N-alpha-(9-fluorenylmethoxycarbonyl)-D-serine, designated Fmoc-D-serine or Fmoc-D-Ser-OH, is supplied as a white to off-white powder with CAS registry number 116861-26-8, molecular formula C18H17NO5, molecular weight 327.33 g/mol, and monoisotopic mass 327.1106 Da. The molecule consists of the D-serine skeleton protected at the alpha-amino position by the base-labile 9-fluorenylmethoxycarbonyl group; the primary alcohol remains free. This unprotected side chain distinguishes Fmoc-D-serine from Fmoc-D-Ser(tBu)-OH, the tert-butyl ether-protected derivative, and confers both synthetic utility and process risk.
In Fmoc-based solid-phase peptide synthesis and solution-phase fragment condensation, Fmoc-D-serine introduces a D-serine residue whose hydroxyl can remain free during chain assembly or can be functionalized orthogonally. The D configuration allows synthesis of mirror-image peptides, D-peptide antagonists, and racemic protein-crystallography constructs; configurational integrity is critical, and standard chiral purity release limits for the monomer are ≥99.0%. Fmoc-D-serine is distinct from the L enantiomer by opposite optical rotation and chiral HPLC retention; in achiral reversed-phase HPLC, the two enantiomers co-elute.
Fmoc removal proceeds through amine-mediated beta-elimination of the fluorenylmethoxycarbonyl group, releasing dibenzofulvene, which is trapped by the secondary amine, and carbamic acid, which decarboxylates. The dibenzofulvene chromophore absorbs at 301 nm; this provides a UV-visible signal for deprotection monitoring in flow-through synthesizers such as the CEM Liberty Blue or the Biotage Syro I. Standard deprotection uses 20% piperidine in DMF or NMP. In solution, cleavage is very fast; in resin-bound systems, contact times of 3–15 min per cycle are common because diffusion into the polymer matrix governs overall rate rather than intrinsic cleavage kinetics. The shortest reproducible deprotection interval should be selected, since prolonged piperidine contact can increase side reactions and may degrade the unprotected serine hydroxyl at elevated temperature.
Fmoc-D-serine does not require acid-labile hydroxyl deprotection, but the unprotected primary alcohol makes the dry powder moisture-sensitive. Storage at 2–8°C in tightly sealed containers under dry inert gas is standard. Material removed from cold storage should be equilibrated to ambient temperature inside a desiccator before weighing; condensation on cold powder raises water content and can alter the mass balance of kilogram-scale coupling solutions.
The analytical profile is designed to detect the two most likely failure modes: enantiomeric contamination from L-serine and moisture-induced mass errors. Although the molecule has no official pharmacopoeial monograph in the major compendia, supplier certificates of analysis are generally harmonized with ICH Q3C for residual solvents and ICH Q3D for elemental impurities if the material is intended for peptide active pharmaceutical ingredients.
| Parameter | Specification | Reference method |
|---|---|---|
| Appearance | White to off-white powder | Visual and photometric |
| Identity | IR spectrum corresponds to reference | FTIR |
| Purity | ≥98.0% | HPLC at 220 nm, C18 column, 0.1% TFA system |
| Enantiomeric purity | ≥99.0% D-serine | Chiral HPLC |
| Water content | ≤0.5% | Karl Fischer titration |
| Residual solvents | DMF ≤0.1%, dichloromethane ≤0.05% | Headspace GC according to ICH Q3C |
| Specific rotation | +10.0° to +12.0° (c=1, DMF) | Polarimetry at 20°C |
Purity by HPLC is generally performed with a C18 stationary phase and a mobile phase of 0.1% trifluoroacetic acid in water and acetonitrile; detection at 220 nm is selected because the Fmoc chromophore absorbs strongly. The method resolves Fmoc-D-serine from the dibenzofulvene cleavage product and from small-molecule protecting-group fragments. Chiral HPLC uses a weak cation-exchange chiral stationary phase or a polysaccharide-based chiral column; resolution between Fmoc-D-serine and Fmoc-L-serine should be at least 2.0. For identity confirmation, LC-MS electrospray ionization typically detects the [M+H]+ ion at m/z 328.1 and the [M+Na]+ adduct at m/z 350.1.
For coupling, Fmoc-D-serine is dissolved in DMF or NMP at 0.10–0.30 mol/L. At 0.20 mol/L, the required mass is 65.47 g/L for the free hydroxyl derivative and 76.69 g/L for Fmoc-D-Ser(tBu)-OH; substitution without mass correction produces a 14.6% reduction in molarity. Dissolution under anhydrous conditions is generally rapid; if turbidity persists after 5 min, the solution should be filtered through a polypropylene frit rather than a hydrophobic polytetrafluoroethylene membrane. The free hydroxyl increases polarity relative to the tert-butyl ether, reducing distribution into ethyl acetate or methyl tert-butyl ether during aqueous workup and shortening reversed-phase HPLC retention.
In stepwise SPPS, the unprotected hydroxyl can be acylated by activated carboxyl components, particularly when uranium salts or carbodiimides generate high local concentrations of active ester. The resulting O-acyl ester is base-labile and may be removed during the next piperidine treatment, causing mass loss rather than chain termination. Under basic conditions, O→N acyl migration is possible, a property that is exploited in O-acyl peptide ligation methods but can create process-related impurities in resin-bound synthesis. Published data for this specific configuration is limited at process scale; the extent of O-acylation is strongly sequence-dependent and cannot be predicted from monomer data alone.
Manufacturers therefore reserve Fmoc-D-serine with free hydroxyl for solution-phase segment synthesis, selective O-functionalization, or short sequences in which coupling conditions do not expose the hydroxyl to a large molar excess of active ester. For routine stepwise SPPS, Fmoc-D-Ser(tBu)-OH is preferred because the tert-butyl ether blocks this pathway and is removed only during final acid cleavage. When the free hydroxyl is retained deliberately, preactivation with Oxyma/DIC at 0.10 M and lower reaction temperatures can reduce unwanted O-acylation relative to prolonged carbodiimide exposure; however, multifactorial optimization is required and no single set of conditions applies to all sequences.
Microwave-assisted SPPS using Fmoc-D-serine at elevated temperatures can shorten coupling times to 2–5 min on some commercial synthesizers, but thermal acceleration also increases the risk of side reactions at the free hydroxyl. Published data for this specific configuration is limited at process scale; process development should compare crude purity and isolated yield against the corresponding Fmoc-D-Ser(tBu)-OH under identical resin loading and sequence conditions.
Because the hydroxyl is unprotected, Fmoc-D-serine can be selectively O-acylated, O-glycosylated, or O-phosphorylated before peptide assembly; the Fmoc group remains intact under many neutral and mildly acidic functionalization conditions. The free carboxylic acid must be masked as an ester or anchored to the resin to avoid competing carboxylate acylation. This selectivity is not available with Fmoc-D-Ser(tBu)-OH unless the tert-butyl ether is first removed, which is impractical under Fmoc-compatible conditions.
Fmoc-D-serine and Fmoc-L-serine share molecular formula, molecular weight, and reversed-phase HPLC retention; chiral HPLC is required to resolve them. The D-enantiomer exhibits positive specific rotation in DMF, while the L-enantiomer gives negative rotation; certificate-of-analysis values are reported at 20°C with c=1 in DMF. A chiral purity limit of ≥99.0% for the D-isomer is common. Incorporation of the wrong enantiomer at a single residue can alter peptide folding, receptor binding, and proteolytic stability because most proteases recognize L-amino acid side chains. Racemization of D-serine during Fmoc SPPS is generally low under standard carbodiimide or uranium activation; configurational inversion should be monitored in the final peptide by acid hydrolysis followed by chiral HPLC or chiral GC. Acceptance limits for L-serine content in finished peptides are sequence-specific and are derived from the biological target; for peptide APIs, the allowable L-serine content is typically controlled to ≤0.5% or lower if the D configuration is critical for pharmacological activity.
Replacement of L-serine by D-serine changes backbone geometry and hydrogen-bonding patterns in peptide ligands. In medicinal chemistry, D-serine-containing peptides can display reduced susceptibility to serine proteases, altered receptor selectivity, and modified aggregation propensity. These biological effects are sequence-dependent and are not directly predicted from monomer properties; they must be evaluated in the target peptide using relevant biochemical assays.
Fmoc-D-Ser(tBu)-OH differs from Fmoc-D-serine by a molecular weight increase of 56.11 g/mol and by the presence of a tert-butyl ether on the serine oxygen. The protected derivative is stable to piperidine-mediated Fmoc removal and is the preferred monomer for most resin-bound synthesis, as it prevents O-acylation at the hydroxyl. The tert-butyl group is removed during final cleavage with trifluoroacetic acid containing scavengers such as triisopropylsilane and water; cleavage is typically conducted at room temperature for 1–3 h, but process-specific hold times require validation. Incomplete tert-butyl removal can produce a peptide with residual tert-butylated serine, a critical quality defect detectable by LC-MS as a mass shift of +56.11 relative to free serine.
Boc-D-serine carries an acid-labile N-terminal protection removed by 25–50% TFA in dichloromethane or 4 M HCl in dioxane, and is compatible with Boc SPPS but not with Fmoc-protected side chains. Cbz-D-serine is removed by hydrogenolysis over palladium or by transfer hydrogenation, making it orthogonal to Fmoc chemistry but less compatible with reducing conditions. Fmoc-D-serine therefore provides base-labile protection that is orthogonal to Boc, Cbz, and tert-butyl groups, while its free hydroxyl requires selective handling during activation.
| Derivative | N-terminal removal | Side chain | Main process risk |
|---|---|---|---|
| Fmoc-D-serine | 20% piperidine in DMF | Free hydroxyl | O-acylation during coupling; moisture uptake |
| Fmoc-D-Ser(tBu)-OH | 20% piperidine in DMF | tert-butyl ether | Incomplete tert-butyl removal during TFA cleavage |
| Boc-D-serine | 25–50% TFA or 4 M HCl in dioxane | Free hydroxyl | Acid lability limits orthogonal uses |
| Cbz-D-serine | Hydrogenolysis or transfer hydrogenation | Free hydroxyl | Catalyst contamination and reducing conditions |
Fmoc-D-serine for peptide API intermediates is typically controlled under current good manufacturing practice according to ICH Q7 when the amino acid is designated as a critical raw material. Residual solvents are assessed against ICH Q3C, and elemental impurity risk may be covered by supplier declarations under ICH Q3D. The product is not sterile; if used in downstream sterile peptide APIs, terminal sterilization or aseptic processing is required after drug substance manufacture.
Fmoc-D-serine is supplied in amber or fluoropolymer-lined containers to limit light exposure and moisture ingress. Static charge on Fmoc amino acids can cause powder dispersal during weighing; balances used for controlled substances should be operated with relative humidity below 40% and anti-static ionizers. The product should not be stored in solution for extended periods at room temperature unless stability data support longer hold times, because free-acid species in DMF can absorb atmospheric water and traces of secondary amines can initiate Fmoc removal. If storage in solution is unavoidable, headspace moisture should be excluded by nitrogen blanketing and the solution should be assayed by HPLC immediately before use.
