Short answer: Every common amino acid except glycine is chiral, so it exists as two mirror-image forms, the L- and D-enantiomers [2][6]. The L-forms are the ones used in ribosomal protein synthesis [1]. D-amino acids are their mirror images [5]. They turn up in many natural products, they can form by accident during peptide synthesis (racemization), and chemists sometimes put them into synthetic peptides on purpose [5][8][14]. Because the two enantiomers have the same mass, telling them apart takes chiral methods such as Marfey's reagent or ion mobility–mass spectrometry [5][10][12].
This article is educational chemistry for laboratory readers. It does not describe effects in people or animals and is not guidance for human or animal use.
What "D" and "L" mean
The α-carbon of most amino acids carries four different groups: the amino group, the carboxyl group, a hydrogen and the side chain. That makes it a stereocentre. IUPAC-IUB nomenclature designates its absolute configuration with a small-capital D or L prefix, which shows a formal relationship to D- or L-serine and, through serine, to D- or L-glyceraldehyde [2].
A few rules from the same recommendations [2]:
- Fischer projection: in the Fischer-Rosanoff convention the main chain is drawn vertically and the other two groups point toward the viewer; that is how L-α-amino acids are conventionally drawn.
- Omitting the prefix: the prefix may be dropped when an amino acid is obviously from a protein source and therefore assumed to be L.
- Racemic mixtures: an equimolar mix of D and L is called racemic and written DL- (for example DL-leucine), rac- or (±)-.
- Optical rotation is a separate label: (+) and (−) describe which way a compound rotates plane-polarized light, and IUPAC treats this as a separate designation from D/L, as in "(+)-L-glutamic acid".
D/L versus R/S
Chemists also use the more general Cahn-Ingold-Prelog R/S system [2]. The two systems are not interchangeable labels:
- The L-configuration of protein amino acids nearly always corresponds to S [2].
- The main exceptions are L-cysteine and L-cystine, which are R, because the sulfur on the side chain outranks the carboxylate oxygen in the priority rules [2]. (For more on cystine, see our disulfide bond guide.)
- Alanine shows the usual pattern. PubChem lists L-alanine as (2S)-2-aminopropanoic acid and D-alanine as (2R)-2-aminopropanoic acid. Both have the formula C3H7NO2 and a molecular weight of 89.09 g/mol [3][4].
- Threonine and isoleucine have two stereocentres. IUPAC prefers R/S for the second centre, e.g. (2S,3R)-threonine, and restricts the "allo" prefix to alloisoleucine and allothreonine [2].
Where D-amino acids are found
For a long time D-amino acids were thought to play only a minor role next to the L-enantiomers. Genchi's 2017 overview says many lines of evidence now show they are present in microorganisms, plants and mammals [7]. Some routes to D-residues:
- Non-ribosomal peptide synthesis (NRPS): Armstrong and Berthod describe NRPS as the main biosynthetic route to D-amino acid–containing natural products. In their example, valinomycin, an epimerization (E) domain in the NRPS converts L-valine into D-valine [5].
- Ribosomal peptides that are modified afterwards (RiPPs): Here the mRNA carries an ordinary L-amino acid codon at the position, and an enzyme converts the residue to the D-form after translation [5][6]. Ollivaux and colleagues call this step peptidyl aminoacyl L/D isomerisation [6].
- No direct ribosomal route: Armstrong and Berthod note there is so far no evidence that ribosomes can directly put a D-amino acid into a peptide chain [5].
- Chemical processing: food proteins can contain D-amino acids that arise during high-temperature, acid or alkali treatment and fermentation [7].
Natural products show how far the D-content can range. In Armstrong and Berthod's survey, daptomycin contains three D-residues among 14 amino acids (21%), and mattacin contains one among 10 (10%) [5]. Their table lists gramicidin from Bacillus brevis with 6 D-residues (D-Leu, D-Val) among 15 amino acids (40%), and cyclosporin A with one (D-Ala) among 11 (about 9%) [5]. For a wider look at what counts as a "natural" peptide, see are peptides natural?.
Racemization: unwanted D-residues in synthetic peptides
In a peptide made from L-building blocks, a D-residue can also appear by accident. During coupling, the activated amino acid can partly lose its configuration, which is called racemization [8]. The product then contains an epimer with the same mass as the target, so mass alone cannot reveal it [5].
Published examples from Fmoc solid-phase peptide synthesis (SPPS):
- Cysteine, histidine and aspartic acid were the residues susceptible to racemization during microwave SPPS of a model 20-mer that contained all 20 natural amino acids [8].
- Temperature: lowering the microwave coupling temperature from 80 °C to 50 °C limited racemization of histidine and cysteine [8].
- Base choice: using the hindered amine collidine in the coupling step minimized D-cysteine formation [8]. In a synthesis of the cystine-knot peptide protoxin II, using N-methylmorpholine as the base gave about 50% racemization during cysteine coupling. Switching to 2,4,6-collidine suppressed it [9].
- Aspartimide: aspartimide formation and the racemization of aspartic acid that follows were reduced by adding HOBt to the deprotection solution and/or using piperazine instead of piperidine [8].
How D- and L-residues are told apart
Enantiomers have identical physical and chemical properties in an achiral (isotropic) environment, except for how they interact with polarized light. That is why D-amino acids are easy to overlook [5]. A standard mass spectrometer cannot distinguish L- and D-enantiomers either, because they have the same exact mass [5]. The common approaches are listed below.
Marfey's method (hydrolysis + chiral derivatization)
- The peptide is hydrolysed to free amino acids. The hydrolysate is then reacted with Marfey's reagent, Nα-(2,4-dinitro-5-fluorophenyl)-L-alaninamide (FDAA), or with its D-enantiomer [5][11].
- This produces UV-absorbing diastereomers that separate on an ordinary C18 HPLC column. Peak positions are compared with FDAA derivatives of amino acid standards [5][11].
- A known caveat: strong-acid hydrolysis can partly racemize each chiral amino acid and converts asparagine and glutamine into aspartic and glutamic acid [5].
- Bhushan and Brückner's reviews cover the reagent's use for DL-amino acid mixtures, cysteine residues in peptides and the evaluation of racemization, as well as variants that swap L-Ala-NH2 for other chiral amino acid amides [10][11].
Chiral chromatography
Separating enantiomers requires a chiral selector, which forms transient diastereomeric complexes with each one [5]. Chiral stationary phases can now routinely separate amino acid enantiomers ahead of MS or other detectors [5].
Intact-peptide methods
Peptide epimers, which differ in the chirality of only one residue, are not enantiomers of each other [5]. Abdulbagi and colleagues explain that peptides containing D-amino acids have different ion mobility and collision cross-section values from their all-L counterparts. This allows baseline separation by ion mobility–mass spectrometry (IM-MS), and fragment-ion arrival times can show where the D-residue sits [12]. For the basics, see mass spectrometry peptide testing.
Why chemists use D-amino acids on purpose
In laboratory studies, D-residues are used to change how a peptide behaves toward protein-degrading enzymes (proteases):
- Substitution: Lu and colleagues substituted residues of the model peptide Pep05 with D-lysine and D-arginine, among other non-standard residues. In vitro, both the D- and the unnatural amino acids made the peptides more stable toward trypsin and plasma proteases. LC-MS was used to identify the degradation products [14].
- Mirror-image phage display: Schumacher and colleagues chemically synthesized a target protein domain in the D-configuration and screened it against a phage library of ordinary L-peptides. By symmetry, the mirror images of the hits (D-peptides) bind the natural L-form of the target. The authors note such D-peptide ligands should resist proteolytic degradation. Their example was a cyclic D-peptide that binds the SH3 domain of c-Src [13].
Frequently Asked Questions
What is the difference between D- and L-amino acids?
They are enantiomers, mirror images that cannot be superimposed, at the α-carbon. The D/L prefix gives the configuration by formal relationship to D- or L-glyceraldehyde [2][5].
Are amino acids in proteins L or D?
Ribosomal protein biosynthesis uses only the L-forms of the chiral amino acids [1]. D-residues in natural peptides come from non-ribosomal synthesis or from conversion of the residue after translation [5][6].
Is glycine D or L?
Neither. Glycine is achiral [2].
Is L the same as S?
Usually, but not always. L-cysteine and L-cystine are R [2].
What does "DL" mean on a label?
An equimolar (racemic) mixture of the two enantiomers [2].
Can mass spectrometry detect a D-amino acid?
Not by mass alone, because the enantiomers have the same exact mass [5]. Chiral derivatization, chiral chromatography or ion mobility separation is needed [5][11][12].
What causes racemization in peptide synthesis?
Loss of configuration during the activation and coupling of residues. In Fmoc SPPS this is best documented for cysteine and histidine, and coupling temperature and base choice both affect it [8][9].
Bottom line
D-amino acids are the mirror-image forms of the chiral protein amino acids [5]. IUPAC defines D/L by reference to glyceraldehyde, and L nearly always corresponds to S, with cysteine as the main exception [2]. D-residues occur in non-ribosomal and post-translationally modified natural peptides [5][6]. In synthetic peptides they can appear through racemization at sensitive residues such as cysteine and histidine [8][9], and chemists also add them deliberately, for example to study protease stability [13][14]. Because enantiomers share the same mass, confirming chirality takes chiral methods: Marfey's derivatization, chiral chromatography or ion mobility–MS [5][11][12]. For residue abbreviations, see our amino acid codes reference.
All products are for research use only — not for human or animal consumption.