Chemical Deuteration of α-Amino Acids and Optical Resolution: Overview of Research Developments

Abstract

Deuterium-labelled amino acids have found extensive applications in such research areas as pharmaceutical, bioanalytical, neutron diffraction, inelastic neutron scattering, in analysis of drug metabolism using mass spectrometry (MS), and, structuring of biomolecules by NMR. For these reasons, interest in new methodologies for the deuterium labelling of amino acids and the extent of their applications are equally rising. The ideal method will be able to label target compounds rapidly and cost-effectively by the direct exchange of a hydrogen atom by a deuterium atom. Most of these exchange reactions can often be carried out directly on the final target compound or a late intermediate in the synthesis, and often D₂O can be used as the deuterium source. This review aims to provide a high-level overview of the chemical deuteration of amino acids in various groups (aromatic, heterocyclic, and non-aromatic α-amino acids). It primarily focuses on metal-catalyzed H/D exchange under hydrothermal conditions, with some attention given to studies on stereoselectivity and chemically synthesized perdeuteration and selective deuteration. In addition, we present different methods tested, manipulated, and developed for versatile new scalable protocols for preparation of selective and perdeuterated biologically important amino acids and their enzymatic and kinetic resolution to give pure enantiomers. Different methods for the synthesis of stereocontrolled selective and perdeuterated amino acids, including synthetic, and methods for preparing optically pure amino acids are presented.

1. Introduction

Since the discovery of deuterium [1], there has been considerable interest in the preparation of deuterated compounds. The characteristic properties of deuterium compared with hydrogen generate a vast demand in numerous fields. Although deuterium (²H) is a stable isotope and not detectable by radioactive tracer techniques, it is readily monitored by other commonly available analytical methods, thus making deuterium a useful labelling agent. By analyzing the characteristic behavior of a deuterium-substituted compound using infrared spectroscopy [2,3], proton magnetic resonance (PMR) [4,5,6,7], mass spectrometry [8,9,10], and neutron scattering techniques [11,12], it is possible to obtain a broad range of information that is not easily accessible through conventional radioactive tracers. Some of the research has targeted the synthesis of deuterated compounds for biological investigations. The breakdown of drugs by Cytochrome P450 (CYP450) enzymes can be slowed by strategically replacing vulnerable carbon–hydrogen (C-H) bonds with more stable carbon–deuterium (C-D) bonds. This technique enhances a drug’s pharmacokinetics, improving how it functions in the body. The success of this method was validated in 2017 with the FDA approval of deutetrabenazine, a pioneering deuterated drug used to treat Huntington’s disease (Figure 1) [7]. Since amino acids are fundamental chemical building blocks for many biological components and serve as key components of peptide-based therapeutics, studies related to biomaterials might be facilitated using deuterated amino acids [5,7,10,13,14,15]. Deuterium-labelled amino acid-based compounds have found extensive applications in such research areas as pharmaceutical (Figure 1) [16,17,18,19], bioanalytical [19,20], biological, (Figure 1) [5,13,14,21] neutron diffraction [11,12,22], inelastic neutron scattering [12], Raman scattering [12], and in analysis of drug metabolism mechanisms [19,23] using mass spectrometry (MS) [8,18] and the structure of biomolecules by NMR [24,25]. Deuterium-labeled amino acids and their derivatives have proven to be invaluable tools for studying biosynthetic pathways, enzyme mechanisms, and the structures of peptides and proteins. Additionally, they play a significant role in improving absorption, distribution, metabolism, and excretion (ADME) profiling, as well as enhancing drug efficacy [26]. Furthermore, the pharmacokinetics and predicted metabolic sites of amino acid drug candidates deuterated at the α-position adjacent to nitrogen have been extensively examined (Figure 1). While the current review focuses on the deuteration of non-exchangeable protons, it is worth noting that deuteration of exchangeable protons has also been shown to affect protein–ligand binding affinities [27,28].

While improving metabolic stability is a well-known advantage of deuteration, its effect on protein–ligand binding affinity is a crucial and often overlooked aspect. This phenomenon can significantly influence the behavior of deuterated molecules in biological systems. Instead of synthesizing deuterated molecules, ligand binding to the histamine H₂ receptor was studied by performing the experiments in heavy water (D₂O) [28]. This method replaces all exchangeable hydrogen atoms in the protein with deuterium, allowing for the study of how hydrogen bonding affects the interaction. Using a range of computational techniques, the authors showed that deuteration increases histamine’s binding affinity for its H₂ receptor. They concluded that this increased affinity is due to changes in hydrogen bonding, both at the receptor site and within the surrounding aqueous environment [27,28]. The results of the study highlight the importance of deuteration for the development of new drugs, as the selective replacement of exchangeable hydrogen atoms with deuterium can increase the duration of action due to their slower decomposition. This growing interest highlights the demand for these compounds and drives researchers to develop more efficient methods for their synthesis and direct deuteration methods for their production have appeared in the literature.

Neutron scattering offers a compelling complementary analytical approach. Its strength comes from its high sensitivity to deuterium. This unique property allows for researchers to exploit the differences in neutron scattering cross sections by substituting hydrogen atoms with deuterium in a sample [29]. The result is a significant reduction in background noise and enhanced contrast in specific areas [30]. Furthermore, the ability to perform site-selective deuteration provides an unparalleled means to obtain high-resolution information regarding conformational dynamics and structural alterations within targeted domains.

NMR spectroscopy is an invaluable technique for understanding protein structure and dynamics in solution, offering a robust method for determining 3D structures of proteins that resist crystallization [31]. However, its utility diminishes with increasing molecular mass due to spectral crowding and line broadening from fast transverse relaxation. While deuteration has long been used to simplify NMR spectra, achieving this requires stereospecific and regiospecific deuterated amino acids. Perdeuteration, while simplifying spectra, leads to a significant loss of crucial NOE information by removing all carbon-bound protons [31].

Deuteration of amino acids is achieved by either by direct deuteration or using deuterated precursors to synthesize final amino acids. Deuterium-labelled precursors are often prepared by direct exchange using transition metal-catalyzed hydrothermal or acid- or base-mediated reactions. These deuterated precursors are used in a multi-step reaction pathway to the final compound. The target compound is labelled rapidly and cost effectively by the direct exchange of a hydrogen atom by a deuterium atom [32]. Most of these exchange reactions can often be carried out directly on the final target compound or a late intermediate in the synthesis, and often D₂O can be used as the deuterium source [33]. Direct deuteration on amino acids often resulted in partially deuterated compounds and the accompaniment of racemization. Direct deuteration is also sometimes used to achieve selective or site-specific H/D exchange. Direct deuteration, particularly using D₂O as the deuterium source, offers several compelling advantages: D₂O is generally the most affordable and readily available deuterium source. This significantly reduces the overall cost of deuteration, making it a highly economical option, especially for large-scale production. The exchange reaction can often be performed directly on the final target compound or a late-stage intermediate. This eliminates the need for lengthy, multi-step syntheses using expensive deuterated precursors, which can be time-consuming and resource-intensive. As a result, direct deuteration simplifies the experimental setup and execution, streamlining the entire process. However, its major limitations lie in achieving precise site-specificity and maintaining stereochemical integrity, which may necessitate alternative, more expensive, and complex methods for specific research or development goals. The commercial interest in these methods is evidenced from appearance of series of patents [34,35,36,37,38,39].

This review aims to provide a high-level overview of the chemical deuteration of amino acids in various groups (aromatic, heterocyclic, and non-aromatic α-amino acids). Methods for metal-catalyzed or chemically catalyzed selective deuteration and perdeuteration will be discussed. Most of the previous studies related to deuteration of amino acids are confined to specific deuteration [40,41,42,43,44]. In addition, methods related to deuteration through chemical synthesis and deuteration through stereoselective chemical synthesis also presented. Investigations related to different methods of transition metal- or chemical-catalyzed deuteration of biologically significant amino acids and their optical resolution methods from our laboratory will be discussed. A comprehensive summary of different selective and perdeuteration methods added at the end of this paper as a Table 1. It is our hope that this paper serve as useful guidance for scientists involved in the chemical deuteration of amino acids. Metal-catalyzed hydrothermal H/D exchange reactions in D₂O are generally used to produce completely or partially deuterated organic compounds [45,46]. Direct deuteration of amino acids using metal-catalyzed hydrothermal H/D exchange reactions (typically >200 °C and >20 bar) would cause several types of undesirable side reactions, such as epimerization or the cleavage of amine or acid groups, and decomposition.

2. Methods

A direct and non-destructive perdeuteration method would be a valuable method for the commonly available amino acids so that they may be obtained in high quantity, in a more routine-based manner, high in isotopic (above 90%D) content, without loss of chirality, and at low cost. The reactivity of the C-H bonds of amino acids in supercritical-temperature deuteroxide solutions was tested to examine the acid or base reactivity of C-H bonds. Deuteration of aromatic and non-aromatic compounds at the supercritical temperature in D₂O is a known technique [47,48,49]. Ying Yang and Evilia attempted direct deuteration on commonly available amino acids at 400 °C in basic conditions (Scheme 1) with heating time varied between 5 to 10 min [50]. H/D exchange is found to be rapid at α carbon in all examples and lower deuteration also observed at alkyl chains and aromatic rings. Decomposition occurred in cases when having OH, SH, SS, or C=N groups on alkyl chains except for methionine 5 (Scheme 1). However, if any deuteration was observed at the chiral center, it was often accompanied by racemisation [50]. Extended heating significantly enhanced deuteration in various amino acids. For alanine, complete deuteration at both the α-position and methyl groups was achieved within 40 min. Methionine’s S-CH₃ group also reached full deuteration with a shorter reaction time of 10 min. However, for isoleucine, leucine, and valine, longer reaction times (30 to 40 min) resulted in only partial deuteration: 27% for isoleucine, 70% for leucine, and 55% for valine’s methyl groups. Rapid and complete deuteration was observed at methylene -CH₂CO₂H in glutamic acid within 5 min, even before α-deuteration. The mechanism for this exchange seems to go through an increase in C-H activation (acidity) under basic supercritical temperature conditions, leading to carbanion formation. The reason for the rapid exchange at the α-position is lower stability of the tertiary carbanion. Loss of chirality at chiral centers might have resulted due to the incoming deuterium attacking either side of the carbanion ion.

The group further explored direct deuteration of amino acids at 400 °C in strong acid [50], with α-carbon exchange observed in all cases [50]. Racemization occurred at the chiral centers, as was previously observed for the reaction under basic conditions.

In case of glutamic acid 9 and histidine 11, it seems supercritical temperature is not required for deuteration (Scheme 2) [50]. Phenylalanine 12 was found to be deuterated at α-carbon, -CH₂-, and ring deuteration was observed at ortho to -CH₂- group at 400 °C; similarly, ring deuteration on tyrosine 13 was observed at ortho to OH at the same temperature. Deuteration pattern in tryptophan 14 was completely deuterated at α-β-carbon, 2, 4, and 6 positions on hetero cyclic ring and partial deuteration occurred at 5 and 7 positions. Regarding deuteration under supercritical strong acidic (8% DCl) conditions, all the amino acids studies were found to be deuterated at α-carbon (Scheme 2) with loss of chirality except for cysteine, methionine, serine, and threonine, which decomposed rapidly [50]. Acidic conditions are less suitable for production of deuterated amino acids because of the greater degree of decomposition and racemization than basic conditions. All others that survived under these conditions with the deuteration sites are shown in Scheme 2. The mechanism seems to be slightly different compared to basic conditions. There are two possible mechanisms proposed in the paper [50] (Scheme 3A,B), the first is the loss of chirality and deuteration at α-carbon center through the formation of super acid, and the second is through formation of carbocation, as shown in Scheme 3A,B [50].

2.1. Specific Deuteration

Selectively deuterium-labelled amino acids are incorporated biosynthetically into fully assembled biological units and subsequently use the label to monitor the site of interest on the macromolecule [51]. Selective deuteration of amino acids is often useful for the study of biological macromolecules by NMR and other biophysical techniques. Proton NMR investigations of proteins have provided a great deal of information about conformation, dynamics, and microscopic states of amino acid residues. For example, the C2-proton of histidine, which resonates downfield from the rest of the aromatic resonance envelope, has been studied extensively in a number of proteins [51]. Regio- and stereoselective deuterium-labelled amino acids also have an important application as probes for the elucidation of biosynthetic pathways or reaction mechanisms by NMR and MS either as a substrate or added into peptides or proteins. One hurdle for such studies has been the limited availability and/or high cost of selectively deuterated amino acids. α-Deuterated amino acids play an indispensable role in the emerging area of clinical functional metabolomics, providing various essential data on in vivo amino acid metabolism and protein turnover [32]. The goal of clinical metabolomics is to identify metabolic signatures in bodily fluids. These signatures can then be used to assess a person’s current health and predict their risk of developing diseases [52]. α-Deuterated L-valine is incorporated biosynthetically into L. casei dihydrofolate reductase (DHFR); this allows comparison of the α-CH-NH fingerprint regions of COSY spectra of deuterated and normal DHFR (Dihydrofolate Reductase) complexes to identify cross-peaks from 15 of the 16 valine residues [5]. Over recent years, the asymmetric synthesis of α-deuterated α-amino acids has received due attention; however, the issues of selectivity, level of deuteration, and enantiomeric purity of the target products still leave room for improvement [32]. Therefore, the development of new methods for the synthesis and resolution of α-deuterated amino acids is highly desirable.

Through a simple method, racemic α-deuterated amino acids were prepared by heating corresponding amino acids with benzaldehyde in acetic acid-d₄ [18,44]. The isotopic purity of the products was more than 99.5% and α-deuterated amino acids were racemized. Authors used the racemization method (via Schiff base intermediate) to exchange the deuterium at α-position of amino acids in deuterated acid (Scheme 4) [44]. These DL-α-deuterated amino acids were converted to methyl esters subsequently resolved by the enzyme alcalase via selective ester hydrolysis to give highly enantiomerically pure amino acids [53].

A general rapid, inexpensive, and generally applicable method for preparation of α-deuterated α-amino acids starting from commercially available amino acids was described by Tikhonov et al. [42]. DL-α-Amino acids were deuterated at the α-position using D₂O and phenol-crosslinked salicylaldehyde–formaldehyde polymer catalyst (Scheme 5). This process involves replacing hydrogen atoms with deuterium atoms at the α-carbon of the amino acid. The use of a polymer catalyst suggests a heterogeneous reaction, where the catalyst is a solid material, and the reaction takes place on its surface. The deuteration is likely achieved through an exchange reaction facilitated by the catalyst. The preparation of the catalyst is also described in the paper [42]. The method of separation of two isomers seems achievable on N-boc-protected amino acids using a chiral HPLC technique [42].

Selective α-deuteration also occurred through the racemization of amino acids, which employs refluxing acetic acid-d₁ and acetic anhydride to give α-deuterated racemic N-acetylated amino acids [41]. A mixture of large excess acetic anhydride with D₂O is used to give a solution of Ac₂O in AcOD. Treatment of amino acids with this solution at reflux temperature for a few minutes leads to acylation, racemization, and H/D exchange at the α-carbon. One possible mechanism for the reaction is through the formation of the oxazolidinone intermediate as shown in Scheme 6 [41]. Several other amino acids were also deuterated at the α-position; the first cycle gave 70–80% exchange and a second cycle gave 90–100% exchange. This method can also be used for the exchange of tritium onto the α-position of amino acids [41]. This process directly yields acetylated products, which are the starting material for enzymatic resolution. This method also proved to preserve stereocenters other than at the α-carbon. Mitulovi et al. also reported that the above racemic α-deuterated N-acetylated amino acids separated into (S) and (R) by HPLC liquid chromatography on preparative scale using a chiral stationary phase based on quinine carbamate [54]. Enantiomerically pure (R) and (S) amino acids can be obtained in good yields by hydrolyzing the N-protecting group. Usually, an enzyme is required for the preparation of stereoselectively labelled amino acids. This method is notable for the absence of any enzyme involvement. The authors also demonstrated that this can be extended to secondary amino acids like proline [54].

Novel radiosyntheses of [¹⁸F]fluorinated aromatic amino acids and their α-deuterated forms were developedand utilized compounds in preclinical positron emission tomography (PET) studies in order to test the hypothesis that α-deuteration should afford superior tracers for PET studies of dopamine and serotonin synthesis in living brain. Part of the synthesis of [¹⁸F]DOPA image tracer and its α-deuterated isotopologue can be achieved through synthesis of the corresponding bromo analogues followed by nucleophilic [¹⁸F]-fluorine labelling. The commercially available L-DOPA (Scheme 7) was brominated at the 6 position by using molecular bromine in acetic acid to give bromo compound 15 in good yield [55]. Pre-exchanged compound 15 was heated in acetic anhydride and monodeuterated acetic acid to give a racemic mixture of α-deuterated N-acetylated 16 with 92%D level [56]. The racemic mixture 16 was esterified and followed by an efficient alcalase enzymatic selective hydrolysis of the L-isomer only to give 21 and 22 unhydrolyzed as a Br-D-DOPA-d₁ acetyl ester (Scheme 7) [57]. The enantiomeric purity of 21 is confirmed by comparing optical rotation of the protonated version of 17 [57], which was synthesized according to a procedure from the literature (Supplementary Information) [58]. N-acetyl-L-DOPA-d₁ was hydrolyzed by refluxing in 6 M DCL for 5 h to give a white solid 6-Br-L-DOPA-d₁ 23 with 92% deuteration level. This compound can be used for ¹⁸F labelling though a nucleophilic substitution. The method can be extended to all other aliphatic and aromatic amino acids to give α-deuterated L-amino acids.

The selectively α-deuterated L- and D-tyrosine were used as analytes to test against a chiral metal-organic framework (CMOF) chemical sensor. The chiral analyte coordinates with the open metal sites of the CMOF [59]. The dynamics of this guest are taken up into the host and can be measured using ²H Solid-State NMR. ²H Solid-State NMR has been demonstrated as an effective tool for determining the molecular dynamics of guests within short-range ordered MOFs [60]. The aim of the ²H solid-state NMR experiment is to observe molecular motional differences between enantiomers of deuterated tyrosine within a CMOF. The above α-deuteration through acetylation method extends to D- and L-α-deuterated tyrosine derivatives (Scheme 8). The commercially available L-tyrosine (Scheme 8)-exchangeable protons exchanged with D₂O a couple of times by evaporation. Pre-exchanged compound 24 was heated in large excess of acetic anhydride and a small amount of monodeuterated acetic acid to give a racemic mixture of α-deuterated N-acetylated 25 [56]. The racemic mixture was esterified to give 26, followed by an efficient alcalase enzymatic-selective hydrolysis to ester-hydrolyzed N-acetylated L-isomer 27 and unhydrolyzed D-isomer acetyl ester 29 (Scheme 8) (Supplementary Information) [57]. Acid hydrolysis of 27 gave α-deuterated L-tyrosine-d₁ as a white solid. Unhydrolyzed D-isomer acetyl ester 29 recrystallized in ethyl acetate followed by acid hydrolysis gave α-deuterated D-tyrosine-d₁ 30. The enantiomeric purity of 28 and 30 is confirmed by comparing optical rotation of the protonated version of 28 and 30, which was reported in the literature (Supplementary Information) [61].

A general procedure for the preparation of α,β-deuterium-labelled amino acids is reported by LeMaster and team [43,62]. Exchange of α,β-protons is catalyzed by AlSO₄ and pyridoxal hydrochloride via Schiff base tautomerization (Scheme 9i–iii). This method is applied to ten commonly available amino acids successfully with high deuteration levels (Scheme 9) [42]. The amino acids are known to be reversibly transaminated and racemized by pyridoxal and Al ions. Details about the preparation of amino acids α- or β-deuterated selectevly or combined using pyridoxal and Al(III) ions and without using enzymes have been reported [42]. They clearly illustrated that selectivity between α or α,β combined is pH-dependent; therefore, the method represents a convenient and inexpensive technique for preparing selectively deuterated amino acids (Scheme 9). Further, selective β-deuteration was achieved via back-exchange of the α-position from α,β-deuterated amino acid reflux in water the above same reaction at pH 10. A couple of other papers also reported preparation of α-deuterated valine, isoleucine, and phenylalanine by treating with pyridoxal hydrochloride and NaOD [5,63]. This procedure was unsuccessful for the deuteration of cysteine, serine, threonine, histidine, and tryptophan. Functional groups interfere with Schiff’s base formation, which is essential for α,β-deuteration. A comprehensive summary of different selective and perdeuteration methods added at the end of this paper as a Table 1.

For our in-house deuterated protein biosynthesis efforts using Pichia pastoris, L-alanine-d₇ was required as a carbon source. We successfully prepared perdeuterated DL-alanine-d₇ 31 on a 100 g scale by treating commercially available L-alanine with AlSO₄ and pyridoxal hydrochloride in D₂O at reflux temperature for 24 h. After a single cycle, the overall deuteration was found to be 90% ± 1, with 97% deuteration at the α-position and 86% at the β-position (Scheme 10). Gram-scale kinetic resolution of 31 was achieved using the N-acetyl-alanine-d₄ salt with L-leucinamide as a simple base. L-leucinamide 35 was prepared by treating L-ethyl leucinate 34 with an excess of aqueous NH₃ (Supplementary Information) (Scheme 10). DL-Alanine-d₇ 31 was acetylated with excess acetic anhydride in D₂O at 70 °C. Mole equivalents of N-Acetyl-DL-alanine-d₄ (32) and L-leucinamide were dissolved in ethanol at 50 °C. After being kept overnight at room temperature, separated needle crystals were filtered, washed with a small amount of ethanol and dried in vacuo to give salt of L-leucinamide and N-acetyl-L-alanine-d₄ 33 in good yield [64]. The salt was showing an optical rotation of $[{\alpha ]}_D^{25}$ −8.96° (C = 2, water) reference $[{\alpha ]}_D^{25}$ −10.5° (C = 2, water). N-acetyl-L-alanine-d₄ was separated from its salt by passing through an Amberlite-IR−120 column to give a white solid; N-acetyl-L-alanine-d₄ showed an optical rotation of $[{\alpha ]}_D^{25}$ 60.7° (C = 2, water) reference $[{\alpha ]}_D^{25}$ 63.6° (C = 2, water) (Scheme 10). N-acetyl-L-alanine-d₄ was hydrolyzed in 6 M DCl (deuterium chloric acid) reflux for 5 h to give a white solid 37. Optical rotation for the resolved L-alanine-d₇ 37 $[{\alpha ]}_D^{25}$ 12.2° (C = 1, 5 M HCl) reference $[{\alpha ]}_D^{25}$ 14.2° (C = 1, 5 M HCl) (Scheme 10).

As part of an in-house project to biosynthesize and characterize mutant GPCR (G-protein-coupled receptor) suitable for X-ray crystallography and NMR studies, deuterated L-methionine and L-homocysteine were required. Initially, exchange of α,β-protons of L-methionine was achieved with a 91% D level using AlSO₄ and pyridoxal HCl, yielding racemized DL-methionine-d₃ 38. To achieve perdeuteration across the entire methionine molecule, the sulfur in compound 38 was oxidized with hydrogen peroxide in acetic acid to give dioxidized sulfone 43. This sulfone moiety activated the adjacent methyl and methylene groups, facilitating perdeuteration to yield product 44 with a 90% D level (Scheme 11) (Supplementary Information). Unfortunately, attempts to reduce the sulfone back to give perdeuterated DL-methionine-d₈ were unsuccessful. Therefore, compound 38 was used to proceed further to obtain L-methionine-d₃ and L-homocysteine-d₃. The racemic mixture 38 was esterified and subsequently subjected to an efficient alcalase enzymatic selective hydrolysis of only the L-isomer, which gave 41, leaving 40 unhydrolyzed as an ester (Scheme 11). The optical rotation for the resolved L-methionine-d₃ 41 was measured as 17.09° (C = 1, 1 M HCl), compared to a reference of 23.1° (C = 1, 1 M HCl). L-methionine-d₃ 41 was then demethylated with sulfuric acid to yield L-homocysteine-d₃ 42 (Scheme 11) without loss of its stereochemistry. The optical rotation for the resolved L-homocysteine-d3 42 was $[{\alpha ]}_D^{25}$ 20.06° (C = 1, 1 M HCl) reference $[{\alpha ]}_D^{25}$ 25.0° (C = 1, 1 M HCl) (Supplementary Information).

2.2. Selective and Perdeuteration of Aromatic or Heterocyclic Amino Acids

A straightforward, inexpensive, and large-scale procedure for the selective deuteration of phenylalanine, tyrosine, tryptophan, and histidine has been reported, making them suitable for incorporation into bacterial proteins (Scheme 12) [21]. This method greatly facilitates the use of selective deuteration in detailed ¹H NMR studies for elucidating protein structure and function. In this procedure, amino acids were treated with deuterated sulfuric acid to yield selectively deuterated racemic mixtures. To achieve high deuteration levels, the exchangeable N-H and O-H protons in the four amino acids were first exchanged with deuterium oxide prior to the main deuteration. Tyrosine was heated at 180–190 °C in 50% D₂SO₄ in D₂O for 48 h. Two cycles of this treatment yielded α,2,3,5,6-pentadeuterated tyrosine 45 with 99% deuterium levels and a 50% yield. Tri-α,2,6-deuterated tyrosine 46 was subsequently prepared from α,2,3,5,6-pentadeuterated tyrosine 45 by heating at 150 °C for 3 days in water with sufficient sulfuric acid to dissolve the tyrosine (Scheme 12). It was observed that tyrosine tended to decompose at higher temperatures, with 190 °C being determined as the optimum temperature for the exchange. When phenylalanine was heated under the same conditions for two cycles, the reaction yielded α,2,3,4,5,6-hexadeuterated phenylalanine 47 with 99% deuterium levels and yields around 60% (Scheme 12) [21]. The use of D₂SO₄, due to its toxicity and corrosive nature, must be carried out under strict safety precautions.

In tryptophan, a complete exchange of all aromatic protons for deuterium was achieved by maintaining a solution of tryptophan in approximately 9% D₂SO₄ in D₂O at 60–70 °C for two weeks. Conversely, α,4,5,6,7-pentadeuterated tryptophan was prepared via back exchange at the C2 indole position from α,2,4,5,6,7-hexadeuterated tryptophan 49 by heating in water at pH 7 and 145 °C for 3 days. The acid labile nature of tryptophan limits the concentrations of D₂SO₄ that can be employed. For histidine, the trideutero compound 50 can be obtained by heating at 140 °C in D₂O with sufficient D₂SO₄ to dissolve the histidine for 20 h. After two cycles, this process yielded racemic histidine with 99%D levels and a 96% yield (Scheme 12).

As part of an internal project to synthesize ampicillin with a selectively deuterated phenylglycine moiety from 6-aminopenicillanic acid (6-APA), we needed perdeuterated D-phenylglycine-d₆. We started with commercially available D-phenylglycine, which was heated to reflux in D₂O with reduced PtO₂ (Adam’s catalyst) (pre-treated with NaBH₄). Reduced PtO₂ refers to the process of converting platinum dioxide (PtO₂) to metallic platinum (Pt). This transformation is crucial because PtO₂ itself is not catalytically active, but it readily reduces to highly dispersed, catalytically active platinum metal under conditions like exposure to hydrogen or certain reducing agents (caution must be exercised when using PtO₂, as it is a strong oxidizing agent). This yielded a racemic mixture of DL-phenylglycine-d₆ 51 in high yield with 96% deuteration (Scheme 13) (Supplementary Information). The racemic mixture 51 was then esterified in acidic ethanol and subjected to an efficient kinetic resolution with (+)-tartaric acid, yielding the tartrate salt of D-ester-d₆ 53. Specifically, the racemic ethyl ester 52 was resolved with 1 molar equivalent of (+)-tartaric acid in aqueous alcohol, providing the D-ester hydrogen (+)-tartrate salts 53 in 41% yield (or 82% based on the D-component) (Scheme 13) (Supplementary Information). Finally, enantiomerically pure D-phenylglycine-d₆ 54 was obtained upon DCl hydrolysis of 53. Its optical rotation was confirmed by comparing it to commercially available D-phenylglycine ($[{\alpha ]}_D^{25}$ −151.90° (C = 1, 6 M HCl) reference $[{\alpha ]}_D^{20}$ −155.0° (C = 1, 1 M HCl) Sigma Aldrich Australia).

Perdeuterated ¹⁰B-boronophenylalanine-d₇ (¹⁰BPA) is being developed for Neutron Capture Enhanced Particle Therapy (NCEPT), with the expectation that its perdeuteration could significantly enhance this treatment. This is due to the vastly different neutron scattering properties of hydrogen (¹H) and deuterium (²H or D), allowing for substantial enhancement of a material’s neutron contrast through molecular deuteration. This can increase neutron contrast in tissues, which is helpful in therapies and imaging involving neutron capture. If hydrogens replace in a molecule with deuteriums, the way neutrons interact with that molecule changes a lot. As part of this effort, perdeuterated tyrosine-d₇ and phenylalanine-d₈ are crucial requirements. To prepare these, commercially available L-tyrosine and L-phenylalanine were separately refluxed in D₂O with reduced PtO₂ (Adam catalyst) (caution must be exercised when using PtO₂, as it is a strong oxidizing agent) (pre-treated with NaBH₄). This process yielded a racemic mixture of DL-tyrosine-d₇ 58 in moderate yields (Scheme 14). However, phenylalanine degraded under these conditions, resulting in a very low yield of DL-phenylalanine-d₈ 56 (approximately 10%). The racemic mixture 58 was then esterified in acidic methanol, followed by efficient enzymatic resolution with alcalase at pH 8, to successfully obtain L-tyrosine-d₇ 60. Acid hydrolysis of the unhydrolyzed D-tyrosine-d₇ ester 61 yielded enantiomerically pure D-tyrosine-d₇ 62. Phenylalanine was perdeuterated using two different methods. Initially, the exchange of α,β-protons of L-phenylalanine was achieved using AlSO₄ and pyridoxal HCl, which produced racemized DL-phenylalanine-d₃ 55. The aromatic ring was then deuterated with a high yield and 90%D level by refluxing in D₂O with 10% w/w Pd/C/Pt/C (1:1) (Scheme 14). After bubbling with H₂ gas, the heterogeneous mixture was refluxed at 140 °C for 8 h. The resulting racemic mixture DL-phenylalanine-d₈ 56 was also esterified in acidic methanol, followed by enzymatic resolution with alcalase at pH 8, to give L-phenylalanine-d₈ 57 (Scheme 14), (Supplementary Information). Prior to acid hydrolysis, both the unhydrolyzed D-phenylalanine methyl ester and D-tyrosine methyl ester were recrystallized from ethyl acetate (Supplementary Information). All the D and L enantiomers were confirmed by comparing their respective optical rotations with commercially available protonated versions. The mechanism of aromatic H/D exchange proceeds via heterogeneous catalysis. Garnett and colleagues proposed that a π-complex mechanism is involved in the heterogeneous catalysis of H/D exchange [65] (Scheme 15). Kinetic studies revealed that, alongside the associative mechanism A, a competing dissociative π-complex mechanism B also played a role. The key distinction between these two reaction pathways lies in the substitution process: in the associative mechanism A, a hydrogen atom is directly replaced by a deuterium atom coordinated to the metal center (Scheme 15). In contrast, the dissociative mechanism B involves substitution of a proton from the initially formed π-complex by the metal atom [18,65]. The intermediate phenyl radical d is subsequently formed. It is only in the second step that the metal atom is replaced by a deuterium atom, leading to the formation of the final product c. For platinum, the dissociative mechanism is believed to play a more prominent role.

2.3. Specific Deuteration with Retention of Stereochemistry

Many existing methods for α-, β-, or α,β-deuteration of amino acids often lead to a loss of optical activity at the chiral α-carbon [66]. To avoid this, deuteration typically needs to be performed either on the intact molecule or by synthesizing the molecule from deuterated precursors. Celine Taglang and colleagues, however, reported a method for the selective and enantiospecific α-deuteration of several common amino acids and their derivatives [67]. This was achieved through enantiospecific C-H activation using RuNP@PVP nanoparticles as a catalyst and D₂ gas at 55 °C (Figure 2). Their mechanistic studies suggest that the selectivity for the α-position of the directing heteroatom stems from a four-membered dimetallacycle, which acts as a key intermediate. For serine and threonine, additional C-H activation occurred at the β-carbon, with both cases retaining stereoselectivity. Notably, in threonine, α,β-deuteration proceeded with full retention of configuration for both chiral centers (Figure 2). This method demonstrated high regioselectivity and full enantiospecificity across various solvents, including D₂O, THF, and DMF. This enantiospecific C-H activation was also successfully extended to biologically active peptides, where similar deuterium incorporation was observed. More recently, a significant phytic acid-modulated Ru (Ru/NPC-600) catalyst was developed for the regioselective deuteration of amines, diamines, and amino acids [68]. While Ru/NPC-600 still achieves over 95% perdeuteration on alanine, proline, glycine, and lysine, the authors focused on the regioselective deuteration of these compounds and did not explicitly mention the stereoselectivity at the α-carbon [68].

A new method has been reported for the stereoselective α-deuteration of L-alanine to yield deuterated D-alanine with an inversion of stereochemistry, all under milder reaction conditions (25 °C) [69]. This catalytic deuteration utilizes a combination of an achiral dichloropyridoxal analogue and a chiral base, notably without requiring the protection of amine and acid groups (Scheme 16). The versatility of this method also allows for the catalytic deuteration of D-alanine with retention of stereochemistry, resulting in deuterated D-alanine. Furthermore, the researchers demonstrated that a racemic mixture of alanine can be catalytically deuterated to produce an enantiomeric excess of deuterated D-alanine [69].

Alessia Michelotti and colleagues have reported a scalable method for the stereoselective deuteration of amino acids using 5% Ru/C under basic conditions in D₂O [70]. Under optimal conditions—10% catalyst loading, 3 mol equivalents of NaOH in D₂O under an H₂ atmosphere at 70 °C for 12 h—they achieved 99%D levels on L-alanine (Scheme 17). High α-deuteration was also achieved for other unfunctionalized amino acids such as glycine, leucine, and valine. For proline and lysine, deuteration was observed at all positions adjacent to the amine group. Notably, platinum and palladium on carbon catalysts were found to be ineffective for this H/D exchange on L-alanine, a finding consistent with other reported literature methods. The optimized reaction conditions involved using 1 mol of substrate with 3 mol of NaOH and 10% w/w Ru/C (5% Ru on carbon) in D₂O, heated at 90 °C under 1 atm of H₂. These conditions resulted in approximately 95% deuterium incorporation at the α-carbon without any loss of stereochemical configuration.

Azetidine-2-carboxylic acid (AZE) is a non-protein amino acid (NPAA) and a toxic metabolite found in several plant species, including members of the Beta vulgaris group like sugar beet and garden beet. As an analog of proline, AZE can be mistakenly incorporated into proteins during synthesis. Fodder beets present a potential pathway for AZE to enter the human food chain. To investigate whether the fodder beet cultivar group of Beta vulgaris produces AZE, we used deuterated AZE-d₅ in a mass spectrometry experiment. In our lab, the four-membered cyclic AZE was deuterated under Ru/C/H₂-mediated hydrothermal conditions. While this method yielded AZE-d₅, the yield was low (50%), and the deuteration level was only 60%D after two cycles. For purification, the final product was converted into its N-Boc derivative, which then underwent acid hydrolysis to give pure AZE-d₅ as an HCl salt (Scheme 18) (Supplementary Information). Site-specific deuteration levels were determined using a previously reported formula [71], and overall deuteration levels were calculated with DGet! Software [72].

Robert C. Woodworth and colleagues developed a catalytic method for the selective deuteration of aromatic amino acids under mild conditions. Their approach utilized Raney nickel as a catalyst, employed under either acidic or basic conditions at room temperature [66]. They successfully used this catalyst to prepare selectively deuterated derivatives of phenylalanine 63, tyrosine 64, and tryptophan 65, suggesting the potential to extend this method to other amino acids. Deuteration was observed not only on the aromatic rings but also on the aliphatic chains of all three amino acids. Interestingly, while other positions on the rings of phenylalanine and tyrosine showed significant deuteration, there was no substantial deuteration at the ortho-to-methylene positions. In the heterocyclic ring of tryptophan, only the 7′ and 2′ positions were significantly deuterated 65 (Scheme 19). The authors also noted that the overall optical activity remained high for all three amino acids. However, exchange at the α-proton was slow, requiring 500 h and accompanied by racemization.

The heterocyclic ring in tryptophan has been successfully deuterated with retention of stereochemistry at the α-carbon using four distinct sets of conditions (Scheme 19). One method involved using a homogenous activated Adam’s catalyst (PtO₂) for hydrothermal reflux in D₂O for 24 h, yielding tryptophan (2′,5′,6′,7′-d₄) 66 [4]. Further deuteration to obtain tryptophan (2′,4′,5′,6′,7′-d₅) 67 was achieved by subjecting 66 to a second 24 h exchange round (Scheme 20).

Both R- and S-tryptophan were also deuterated to give R- and S-tryptophan (2′,4′,5′,6′,7′-d₅) by heating them in Raney/nickel/D₂O at 100 °C for 10 days [34]. Winnicka and colleagues also reported an acidic condition for L-tryptophan indole ring deuteration, involving stirring at room temperature in CF₃COOD/D₂O for 3 days to produce S-tryptophan (2′,4′,5′,6′,7′-d₅) 67 (Scheme 20) [51]. After three cycles of exchange, 93% deuterium incorporation was reported on the tryptophan ring. Randall and Mathew and their team also reported the deuteration of all protons of the heterocyclic ring of L-tryptophan 67 using trifluoroacetic acid in 25% D₂O, crucially without losing optical activity at the α-carbon [51]. Two rounds of treatment, each lasting three days, resulted in L-tryptophan-d₅ with 100% recovery and 93% deuteration. Selective deuteration at the 4′-position of L-tryptophan was achieved to give 68 by subjecting it to UV light for 72 h. When L-tryptophan was treated with a mixture of D₂O and ³H₂O under irradiation conditions, it resulted in doubly 4′-labeled deuterium and tritium L-tryptophan (Scheme 20) [73].

Activated Adam’s catalyst plays a role in preparing L-phenylalanine-d₅ 69 and tyrosine-d₄ 70 under basic conditions (Scheme 21) [4]. This L-tyrosine-d₄ was then converted to selectively protonated L-phenylalanine-d₄ 72 by treating it with 1-phenyl-5-chlorotetrazole, followed by heating with 5% Pd/C [4]. The aromatic ring of L-phenylalanine was deuterated to yield L-phenylalanine-d₅ using 85% D₂SO₄ heated at 50 °C for 2.5 days [51]. This process achieved up to 90%D ring deuteration without any loss of stereochemistry. L-Tyrosine-d₂ (3′,5′) 71 was also prepared from L-tyrosine by heating it in 20% D₂SO₄ in D₂O for over 2 days (Scheme 21). For L-histidine, ring deuteration occurred at 190 °C in NaOD/D₂O (pH 6), resulting in DL-histidine-d₂ 73. Similarly, when compound 73 was heated at 80 °C in NaOH/H₂O (pH 8), it produced DL-histidine-d₁ (2′) 74. L-Histidine-d₁ 75 can be prepared from L-histidine by heating it in basic D₂O at 80 °C for 2 days. In all three amino acids, acid- or base-mediated deuteration is achieved through an equilibration mechanism. To reach the desired deuteration level without losing stereochemistry at the α-carbon, the reaction was repeated three times for each case [51].

Lei Wang and colleagues reported a straightforward method for the aromatic ring deuteration of L-tyrosine and L-tryptophan without affecting the stereochemistry at the α-carbon [10]. The exchange was carried out at room temperature using deuterated triflic acid (Scheme 22). The researchers also attempted the exchange on L-tyrosine and N-acetylated L-tyrosine. They observed that a 90% H/D exchange occurred at the 2′ and 3′ positions of L-tyrosine 70 within 24 h at room temperature (Scheme 22). Crucially, the exchange rate significantly improved when using N-acetylated L-tyrosine. Over 90% exchange was achieved within 6 h at the 3- and 2-positions, respectively, for compound 76 (Scheme 22). Similar trends were observed for L-tryptophan derivatives 67 and 77 [10]. The H/D exchange for the aromatic protons of L-tryptophan reached up to 80% at room temperature. While unprotected compound 70 took over 8 h, N-Ac-L-tryptophan 77 completed the exchange in less than 3 h (Scheme 22) [10]. These results clearly indicate that the exchange rate for N-acetyl-protected aromatic α-amino acids is faster than that for unprotected compounds. Importantly, the exchange occurred without any racemization at the α-carbon.

L-Tyrosine, a precursor of L-DOPA, plays an essential role in a variety of metabolic processes. The enzyme tyrosinase, classified as an oxidoreductase, catalyzes two sequential oxidation reactions: first, the conversion of L-tyrosine to L-DOPA, and then the oxidation of L-DOPA to dopaquinone. Kanska et al. investigated the deuterium kinetic isotope effect (KIE) in the tyrosinase-catalyzed hydroxylation of L-tyrosine-d₂ (3′,5′-positions) to produce L-DOPA. For these kinetic studies, selective ring deuteration of L-tyrosine at the 3′ and 5′ positions was successfully achieved with nearly 100% deuterium incorporation (Scheme 23) [74].

2.4. Metal Mediated Heterogenous Conditions Selective Direct Deuteration

Kokel and colleagues have developed an environmentally benign method for the selective deuteration of amino acids and synthetic building blocks using a Pd/C-Al-D₂O catalytic system. This approach is particularly valuable given the growing importance of isotope-labeled compounds in medicinal chemistry and structural biology, where they serve as enhanced drug candidates and crucial biological probes. The core of their method lies in selective H-D exchange reactions that utilize simple D₂O as the deuterium source. D₂ gas is generated in situ from the reaction of aluminum and D₂O, while a commercially available palladium catalyst facilitates the H-D exchange. The procedure’s high selectivity and efficiency, coupled with its simplicity and safety, establish it as an environmentally friendly alternative to existing methods (Scheme 24) [33]. Sajiki and coworkers also reported an efficient and selective method for the α,β-deuteration of phenylalanine derivatives [75]. They observed that at 110 °C, Pd/C-mediated exchange in D₂O exclusively occurred at the β-position with retention of stereochemistry, yielding compound 79 (Scheme 24). However, increasing the temperature to 160 °C extended the exchange to both α- and β-position, albeit with a loss of stereochemistry, resulting in racemic product 80 (Scheme 24). The authors also noted the formation of a small amount of ring-saturated product under these conditions.

Fei-Fei Sheng and colleagues have developed a highly efficient method for synthesizing β-deuterated amino acids. Their protocol utilizes palladium diacetate-catalyzed H/D exchange from N-protected aminoamides. A key advantage of this approach is its simplicity: the β-deuterated amino acid can be obtained simply by removing the protecting groups (Scheme 24) [76]. This versatile method is not limited to a narrow scope; it can be readily extended to produce a variety of other natural and unnatural α-amino amides. The authors proposed a mechanism in which palladium acetate first reacts with the amino amide substrate, replacing the acetate group to form intermediate A. This is followed by C–H bond activation, resulting in the formation of cyclopalladium complex B with the release of a second acetic acid molecule. The liberated acetic acid undergoes H/D exchange with heavy water, generating deuterated acetic acid. This deuterated acid then assists in the cleavage of the C–Pd bond, leading to the incorporation of a deuterium atom at the β-position of the amino amide. Finally, ligand exchange regenerates palladium acetate and produces the deuterated amino amide.

Aibo Li and his team have developed a highly efficient protocol for the enantioselective deuteration of amidoacrylates. This method, catalyzed by cobalt, yields α,β-dideuterio-α-amino esters with excellent enantiomeric ratios and almost complete deuteration (99%). A significant advantage of this approach is its use of deuterated methanol as a cost-effective deuterium source (Scheme 25) [77]. This new protocol has already been successfully applied to prepare dideuterated amino acid fragments found in various drugs. Furthermore, the stereoselective deuteration has been directly utilized in the synthesis of L-DOPA-d₂.

While efforts to deuterate L-tyrosine showed lower deuterium efficiency at the β-position, a minor amount of deuterium incorporation (17%D) was observed at the ortho-positions of the phenolic hydroxyl group. In related work, Basujit Chatterjee and colleagues developed an easy and stereoselective method for α-deuteration of amines and amino acids. Their technique utilizes a monohydrido-bridged dinuclear Ru complex as a catalyst and deuterium oxide (D₂O) as the deuterium source (Scheme 26) [78].

For an in-house project focused on preparing deuterated polymyxin analogues to study their structure–activity relationship on Gram-negative bacterial membranes, L-2,4-diaminobutyric acid was needed. Initially, attempts to deuterate commercially available L-2,4-diaminobutyric acid using Ru/C in D₂O under reflux only achieved an overall deuteration level of 65%D. To improve this, L-2,4-diaminobutyric acid was then refluxed in D₂O with reduced PtO₂ (Adam’s catalyst) (which was pre-treated with NaBH₄). This approach successfully yielded a racemic mixture of DL-2,4-diaminobutyric acid-d₅ 81 with approximately 94%D level in quantitative yield (Scheme 27) (Supplementary Information). The racemic mixture 81 was subsequently diacetylated by heating it in acetic anhydride and D₂O to produce 82. This diacetylated product was then purified using flash column chromatography. The racemic diacetylated mixture 82 was esterified in acidic ethanol, followed by an efficient enzymatic resolution with alcalase at pH 8 to give the L-diacylated product. The unhydrolyzed product was extracted using ethyl acetate. Finally, enantiomerically pure L-2,4-diaminobutyric acid-d₅ 81a was obtained after hydrolysis of 82 with 6 M DCl (deuterium HCl).

3. Deuteration by Chemical Synthesis

In the early 1960s, researchers successfully synthesized fully deuterated L-phenylalanine 57, glutamic acid, aspartic acid, and asparagine. These were initially obtained as their DL N-acetylated mixtures, with the L-amino acids then resolved using the acylase-1 enzyme (Scheme 28) [79,80]. Phenylalanine, for instance, was synthesized from benzaldehyde-d₆ 85 and N-acetyl-glycine 86 (Scheme 28) [79]. This particular approach was not a convergent method, meaning each amino acid was synthesized individually. For example, glutamic acid-d₅ was synthesized from acylamido malonic ester and acetylene dicarboxylic acid. Similarly, asparagine-d₃ was prepared from phthalidomide and malonic ester by treating them with an appropriate alkyl halide (Scheme 29). In all these cases, the resulting DL-amino acids were resolved into their L-amino acid forms using the acylase-1 enzyme.

One of the most straightforward and convergent laboratory routes for synthesizing DL-amino acids involves starting from commercially available acylamino malonic ester 108 (Scheme 30) [40,80]. This method begins by generating a carbanion from the acylamino malonic ester in a basic medium, such as ethanolic sodium ethoxide [80]. This carbanion then acts as a nucleophile, displacing the halide from any given alkyl halide. The resulting intermediate, 109, is typically hydrolyzed, deacylated, and decarboxylated in a single step by boiling it in either deuterated or protonated acid, which produces the desired DL-amino acid [40,80]. If the goal is specifically to create α-deuterated amino acids, the final step would involve boiling the intermediate in a deuterated acid environment. Furthermore, if RX (the alkyl halide) is a fully deuterated precursor, the resultant product will be a fully deuterated DL-amino acid.

Deuteration by Stereoselective Chemical Synthesis

A novel approach for the stereoselective synthesis of α-deuterated amino acids starts with the chiral auxiliary diketopiperazine 110. Diketopiperazine templates like 110 are effective building blocks for chiral amino acid synthesis because they are easily monofunctionalized and can be hydrolyzed to their corresponding amino acids (Scheme 31) [81]. Efficient mono-alkylation of this chiral template using the desired electrophile successfully yields the (S)-substituted adducts (Scheme 31). While initially reported for α-deuterated amino acids, this method can be extended to produce fully deuterated amino acids while maintaining stereoselectivity. Introducing two deuterium atoms on each side can be achieved by generating a carbanion at the active methylene group on the chiral auxiliary 110 (Scheme 31), which is then quenched with D₂O to form the central intermediate 111 [82]. Subsequently, introducing an R alkyl group (RX), including different deuterated alkyl halides, yields the desired fully deuterated or selective α-L-deuterated amino acids. This method has been applied to produce L-phenylalanine, L-tyrosine, L-alanine, glycine, L-leucine, L-isoleucine, L-valine, and L-tryptophan [81]. The authors note that the nature of the electrophile plays a significant role in the observed high stereochemical excess. Specifically, the bulkiness of the incoming electrophile is crucial. For instance, no selectivity was observed when methyl iodide was used as the electrophile; however, the resulting 50:50 mixture of isomers was easily separated by column chromatography.

Rose and colleagues developed another valuable chiral auxiliary: (3S)-isopropyl-2,5-dimethoxy-dihydropyrazine 112. When subjected to base-catalyzed deuteration in MeOD/D₂O, this compound yields 113 in excellent yield, crucially without altering the stereocenter at C3 (Scheme 32) [83,84]. This auxiliary allows for the selective alkylation at C3 via a butyllithium-generated carbanion, providing a diverse range of enantiomerically pure (S) and (R) amino acids (Scheme 32). Here, RX represents various protonated or deuterated alkyl halides. A significant advantage of (3S)-isopropyl-2,5-dimethoxy-dihydropyrazine is its commercial availability at a low cost, and it can also be readily synthesized from glycine and (2R)-valine. This method’s versatility extends to the preparation of tritiated L or D-amino acids. It is worth noting that longer straight-chain alkyl halides require extended reaction times compared to benzyl chloromethyl ether (Scheme 32). Finally, acid hydrolysis of the alkylated dihydropyrazine produces a mixture of the corresponding amino acid and valine methyl ester.

The chiral template diacetone-D-glucos-3-ulose 114 has proven highly effective in the stereoselective synthesis of a variety of chiral molecules, including chiral acetic acid, chiral glycine, D- or L-alanine, 3-alkylmalic acids, and chirally monodeuterated glycerol [85]. A key advantage of this carbohydrate template is its inherent double steric constraint, which directs the stereochemical outcome of reactions. The addition of acetylene to 114 was achieved using either lithium acetylide or an ethynyl magnesium bromide solution, yielding compounds 115 or 116. Compound 117, a starting material for L-alanine, was prepared in a three-step sequence involving silylation of the hydroxyl group, methylation of the acetylene, and desilylation, with an overall yield of 70% [86]. Reduction of 115, 116, or 117 using reagents like LiAlH₄, LiAlD4, or Pd/C/H₂CaCO₃ provided stereospecifically Z or E olefin isomers 118, 119, and 120 (Scheme 33) [17,86]. Compound 120 was subsequently transformed into D- or L-alanine through a three-step process: protecting the hydroxyl group with trichloroacetamide, followed by a sigmatropic rearrangement in heated xylene, and finally periodate cleavage, as depicted in Scheme 33.

Due to the bulky α-oriented isopropylidene group at C4, only the β-face of the olefinic bond in intermediates 118 and 119 is accessible to incoming reactants. This steric hindrance ensures that electrophiles preferentially attack the less hindered side, leading to highly predictable stereochemistry. Indeed, epoxidation of 118 and 119 with m-chloroperbenzoic acid proceeded stereoselectively, yielding a diastereoisomeric mixture of 121, 122, and 122, 123, respectively, in an 87% yield and a 5:1 ratio [17]. This mixture was then separated using medium pressure column chromatography. Nitrogen functionality was introduced stereospecifically at the C-2′ position with an inversion of configuration by treating 121 and 122 with potassium phthalimide, which gave 125 and 126 [17]. In parallel, the methyl derivative 120 was transformed into L-alanine-d₃ 133 through a sequence involving ring opening with an amine and oxidative periodate cleavage. Periodate cleavage of 125 and 126 successfully yielded phthalimide glycinals 127 and 128 in good yields. Further oxidation of 127 and 128 with KMnO4 and H₂SO₄ provided the R and S phthaloyl glycines 129 and 130. Finally, hydrolysis of 129 and 130 with Pb(OAc)₄ in acetic acid and methanol effectively cleaved the phthaloyl group, affording the R and S glycines 131 and 132 (Scheme 33) [17].

Maeda and colleagues developed a highly enantioselective method for synthesizing chirally pure 3R and 3S L-serines, utilizing intermediates 118 and 119 [85]. This approach relies on a sugar template, which ensures predictable stereochemistry in subsequent reactions. Intermolecular halogen addition to compounds 134 and 135, both bearing an N-benzyl-methylthioformimidoyl group on the C-3α-hydroxyl group, proceeded diastereoselectively. This yielded 136 and 137 as the major products, rather than 138 and 139. Consequently, the resulting stereochemistry at C-1′ and C2 could be precisely predicted to give L-amino acids. The halomethyl groups of 136 and 137 are versatile and can be transformed into various substituents and functional groups. This characteristic allows for the expansion of this methodology to prepare a range of other chirally pure β-labelled L-amino acids (Scheme 34).

A general method has been developed for the enantioselective synthesis of isotopically labeled L-leucine or isoleucine. This approach involves preparing 2-oxo-4-methylpentanoic acid 146 from Evans chiral auxiliary 148, which is selectively labelled with either 13C or deuterium in the R or S configured methyl group. The process then continues with a reductive amination of ketone 147, catalyzed by leucine dehydrogenase (Scheme 35) [87]. This strategy has been successfully applied to the total synthesis of (2S,4R)-leucine-d₃ using CD₃I as the deuterium source. If the R group is ethylbromide-d₅, the resulting compound will be (2S,4R)-isoleucine-d₅.

Christopher J. Easton and his team have reported a novel method for the stereoselective synthesis of α-deuterated and (R), (S) α,β-dideuterated phenylalanine derivatives from (S)-phenylalanine. Their work also extends to preparing α-deuterated (R) alanine, (R) valine, and (R) leucine from their respective S-isomers. This new synthetic approach for chiral (R), (S) α-deuterated and (2S,3R) and (2R,3S) α,β-dideuterated phenylalanine derivatives is particularly significant because it bypasses the need for enzyme-catalyzed resolution or any other enantiomer separation techniques. This simplification streamlines the production of these valuable labelled compounds [88].

As previously discussed (referencing Scheme 7 and Scheme 8), racemic α-deuterated amino acids can be prepared using acetic anhydride and D₂O, followed by enzymatic resolution of the esterified α-deuterated amino acids to yield optically pure L-α-deuterated amino acids. However, achieving high enantiomeric purity for the (D) R-isomer can sometimes be challenging. To address this, the authors developed a method for preparing both the (L) S- and (D) R-α-deuterated phenylalanines, L-153 and D-156, directly from L-phenylalanine. This approach leverages the concept of self-regeneration of chirality through manipulation of an N-protecting group (Scheme 36) [88]. The diastereoisomers shown in Scheme 36 were successfully separated by silica gel chromatography. Reduced amides 150 and 151 were then treated with NaOMe in MeOD at reflux for 4 h, resulting in mixtures of the monodeuterides 152 and 155, and 154 and 157, respectively. Individual compounds were separated from these mixtures via silica chromatography, and subsequent acid hydrolysis yielded 153 and 156 from 152 and 154, and 155 and 157, respectively. Ultimately, the monodeuterated phenylalanines 153 and 156 were obtained as highly enantiomerically pure compounds (greater than 95% ee).

A similar methodology has been successfully applied to prepare four distinct α,β-deuterated phenylalanine isomers (165, 168, 173, and 176) in a stereocontrolled manner. This synthesis begins with the phthaloyl derivative of phenylalanine 149. Halogenation of 149 with N-bromosuccinimide (NBS) yielded bromides 158 and 159, crucially without any loss of stereochemistry at the α-carbon. These bromides then underwent β-deuteration with D₂/Pd/C to give 160 and 161, respectively. As a result, all four α,β-deuterated isomers 165, 168, 173, and 176 were obtained as single enantiomers, as detailed in Scheme 37 and Scheme 38 [88]. All the diastereomers produced were pure and thoroughly characterized using NMR spectroscopy. Their mass spectra confirmed a high deuteration level, indicating they were 95% deuterated.

The same methodology extended to the conversion of (L) S-isomers of alanine 178a, valine 178b, and leucine 178c to the corresponding α-deuterated R-isomers 184a, 184b, and 184c as shown in Scheme 39.

Chirally deuterium-labelled proline is highly valuable for probing peptide residue conformations and conducting detailed stereochemical investigations [89]. Makoto Oba’s group reported a method for the stereoselective deuteration of L-proline [89]. They achieved this by first performing a catalytic deuteration of protected 3,4-dehydro-L-proline 186 using RuCl₂(PPh₃)₃. This was followed by RuO₄-oxidation, which yielded a 3,4-dideuterated L-pyroglutamic acid 189 derivative (Scheme 40). This derivative is a promising precursor for various other deuterated amino acids. Furthermore, a stereoselective reduction of the amide carbonyl group led to L-proline-3,4,5-d₃ 191, where all the ring methylenes are stereoselectively labelled with deuterium. Additionally, Proline-d₂ 188 was prepared through acid hydrolysis of 187.

More recently, a new enantioselective deuteration method at the α-position of amino acids like proline, serine, cystine, phenylalanine, and lysine has been reported, notably without the need for external chiral sources (Scheme 41) [90]. When derivative A was treated with NaOEt in EtOD at room temperature, the α-deuterated product formed with retention of the C-α configuration. Interestingly, using dimethyl formamide significantly reduced the reaction’s efficiency and selectivity, and adding a nonpolar solvent also led to a considerable decrease in both yield and enantioselectivity. A supporting mechanism for enantioselective deuteration has also been proposed, in which deuteration likely occurs via direct transfer from the EtOD solvent to the chiral enolate.

Deuterated L-methionine tracers, like L-methionine-d₇, are important tools for studying methionine metabolism, particularly in investigations such as the rat methionine loading test. Hiroshi and colleagues developed a straightforward method for synthesizing DL-methionine-d₇ from DL-methionine-d₄ (Scheme 42) [91]. This involved converting the S-CH₃ group to S-CD₃ using Birch reduction conditions. The resulting racemic methionine-d₇ 194 was then resolved to give the desired 195 through selective hydrolysis of its N-acylated derivative using kidney acylase [91].

Both protonated and deuterated DL-methionine derivatives can be prepared from DL-homocysteine thiolactone hydrochloride (HCTL) (Scheme 43) [92]. In this process, DL-homocysteine thiolactone hydrochloride is dissolved in NaOMe in methanol. After 30 min, an alkyl halide is added to yield the DL-S-alkyl homocysteine ester. This ester is subsequently converted to its free acid form via saponification with either LiOH or NaOH.

Stereoselective deuteration of amino acids is crucial for determining the 3D solution structure of peptides and proteins using NMR spectroscopy. Makoto Oba and colleagues developed a method for the stereoselective β-deuteration of serine and cysteine, synthesizing them from deuterated aldehyde 198 without the need for biotransformations [25]. As shown in Scheme 44, protected serine 196 was first reacted with dimethyl pyrazole to afford compound 197, which was subsequently reduced to yield the aldehyde intermediate 198. Asymmetric reduction of 198 using S-alpine borane resulted in the chirally labelled alcohol 199. Compound 199 underwent acetylation and debenzylation, followed by RuO₄ oxidation of its hydroxymethyl group, to give protected serine 202. Acid hydrolysis of 202 then successfully yielded the target compound, L-serine-d₁ 203, in good yield. L-cysteine-d₁ 208 was also synthesized from protected serine 202 in four steps (Scheme 44). This involved removing the acetate group, introducing a tosyl leaving group, and then treating the intermediate with KSAc to obtain protected L-cysteine-d₁-thioacetate 207. Finally, acid hydrolysis of 207 provided L-cysteine-d₁ 208.

Table 1. Comparative Analysis of Deuteration Methods.

Method	Cost	Scalability	Stereochemistry/Site Specific	Applicability
Direct Deuteration (Acid/Base Catalyzed)	Low	High (with D2O)	Typically loses stereochemistry; challenges in site-selectivity without directing groups.	Broad applicability for many functional groups; effective for introducing multiple deuterium atoms.
Specific Deuteration (Acetic Anhydride/Acid/Aldehyde)	Low	High (with D2O)	Loss of stereochemistry but high regiospecificity.	Broad applicability for many functional groups.
Specific Deuteration (Pyridoxal/AlSO4)	Low	High (with D2O)	Loss of stereochemistry but high regiospecificity.	Not applicable to cysteine, serine, threonine, histidine, and tryptophan.
Metal-Mediated Hydrothermal Deuteration (Pt/C, Pd/C, PtO2)	Low	High (with D2O)	Loss of stereochemistry and less regiospecific.	Applicable to a limited number of amino acids like phenylalanine, tyrosine, and phenylglycine.
Ruthenium-Catalyzed Deuteration	High	Low to Medium	May or may not be stereoselective; high regiospecificity.	Broad applicability for many amino acids; not suitable for perdeuteration.
Metal or nonmetal ligand-Based Deuteration	High	Low	Highly stereoselective and highly regiospecific.	Broad applicability for many amino acids; not suitable for perdeuteration.
Chemical Synthesis	High	Low	May be stereoselective.	Broad applicability for many functional groups; good for introducing multiple deuterium atoms.
Stereoselective Chemical Synthesis (Chiral Auxiliary)	High	Low	Highly stereoselective and highly regiospecific.	Broad applicability for many amino acids, enabling both perdeuteration and site-specific labelling.

Table 1. Comparative Analysis of Deuteration Methods.

Method	Cost	Scalability	Stereochemistry/Site Specific	Applicability
Direct Deuteration (Acid/Base Catalyzed)	Low	High (with D2O)	Typically loses stereochemistry; challenges in site-selectivity without directing groups.	Broad applicability for many functional groups; effective for introducing multiple deuterium atoms.
Specific Deuteration (Acetic Anhydride/Acid/Aldehyde)	Low	High (with D2O)	Loss of stereochemistry but high regiospecificity.	Broad applicability for many functional groups.
Specific Deuteration (Pyridoxal/AlSO4)	Low	High (with D2O)	Loss of stereochemistry but high regiospecificity.	Not applicable to cysteine, serine, threonine, histidine, and tryptophan.
Metal-Mediated Hydrothermal Deuteration (Pt/C, Pd/C, PtO2)	Low	High (with D2O)	Loss of stereochemistry and less regiospecific.	Applicable to a limited number of amino acids like phenylalanine, tyrosine, and phenylglycine.
Ruthenium-Catalyzed Deuteration	High	Low to Medium	May or may not be stereoselective; high regiospecificity.	Broad applicability for many amino acids; not suitable for perdeuteration.
Metal or nonmetal ligand-Based Deuteration	High	Low	Highly stereoselective and highly regiospecific.	Broad applicability for many amino acids; not suitable for perdeuteration.
Chemical Synthesis	High	Low	May be stereoselective.	Broad applicability for many functional groups; good for introducing multiple deuterium atoms.
Stereoselective Chemical Synthesis (Chiral Auxiliary)	High	Low	Highly stereoselective and highly regiospecific.	Broad applicability for many amino acids, enabling both perdeuteration and site-specific labelling.

4. Conclusions

Chemical deuteration of α-amino acids and their optical resolution is a rapidly evolving field with significant implications for various research areas. The presented manuscript provides overview of the different methods of metal-catalyzed and chemical-mediated selective perdeuteration and their optical resolution of amino acids. Papers related to deuteration by chemical synthesis using deuterated precursors followed enzymatic resolution to give enantio-pure amino acids, and deuteration by stereoselective chemical synthesis using chiral auxiliary regents were also covered in this paper. Papers related to synthesis of both (S or L)- and (R or D)-α-deuterated amino acids from L-amino acids through manipulation of an N-protecting group, based on the concept of self-regeneration of chirality, were also presented. Investigations from our labs provided different methods of metal-catalyzed or chemical-mediated selective perdeuteration, and their enzymatic and kinetic resolution of biologically important amino acids such as L-DOPA, L-alanine, D-phenylglycine, L-methionine, L-cysteine, L-phenylalanine, L-tyrosine, and diaminobutyric acid were presented. These days, deuterated biologically important amino acids play crucial role in neutron scattering, neutron reflectometry, and NMR, and as internal standard in quantitative analysis by the LC-MS technique. Some perdeuterated or specific deutersted amino acids are commercially available at exuberant cost. Although deuteration has become a well-established methodology in chemistry, this field still offers interesting potential for innovation (e.g., with respect to heterogenous and homogenous catalysts). Most heterogeneous catalysis that support Pd, Pt, and Ru materials are commonly used to catalyze multi-H/D exchange of the arene part of amino acids. Unfortunately, the tolerance of these catalysts toward easily susceptible functional groups in amino acids as well as the selectivity is still challenging. Therefore, a strong need for the development of new methods for the selective and perdeuteration of amino acids and their optical resolution.