Stereoselective α-Deuteration of Serine, Cysteine, Selenocysteine and 2,3-Diaminopropanoic Acid Derivatives

Stereoselective α-deuteration of serine, cysteine, selenocysteine and 2,3-diaminopropanoic acid derivatives Claudio D. Navo,§* Paula Oroz,# Nuria Mazo,#† Marina Blanco,# Jesús M. Peregrina# and Gonzalo Jiménez-Osés§,‡* § Center for Cooperative Research in Biosciences (CIC bioGUNE), Basque Research and Technology Alliance (BRTA), Bizkaia Technology Park, Building 801A, 48160 Derio, Spain. # Departamento de Química, Centro de Investigación en Síntesis Química, Universidad de La Rioja, 26006 Logroño, La Rioja, Spain. ‡ Ikerbasque, Basque Foundation for Science, 48013 Bilbao, Spain. Supporting Information Placeholder ABSTRACT: Efficient methodologies have been developed to synthesize enantiopure α-deuterated derivatives of serine, cysteine, selenocysteine and 2,3-diaminopropanoic acid. H/D exchange was achieved by deprotonation of a chiral bicyclic serine equivalent followed by selective deuteration. Additionally, diastereoselective additions of thiols, selenols and amines to a chiral bicyclic dehydroalanine in deuterated alcohols allowed site-selective deuteration at the Cα of cysteine, selenocysteine, and 2,3-diaminopropanoic acid derivatives. A deuterated analog of carbocysteine, a drug to treat bronchiectasis was synthetized. Introduction Deuterated compounds have been lately used for different applications in organic chemistry and biochemistry.1 Their chemophysical properties differ from those of the non-deuterated counterparts, being particularly useful for analyses by mass spectroscopy,2 NMR,3 or IR spectroscopy,4 among other techniques. The kinetic isotopic effect makes deuterated drugs more stable to degradation, thus lasting longer in the organism and allowing lower doses.5–7 Although several deuterated drugs are currently under clinical trials, the FDA has recently approved the first deuterated drug, deutetrabenazine, for the treatment of chorea associated to Huntington's disease and tardive dyskinesia.8 Deuterated amino acids have applications for neutron crystallography,9 imaging,10 or the study of biosynthetic and metabolic pathways.11 Particularly, α-deuterated amino acids can be more stable to metabolic degradation and epimerization, and have been used to assist the structural characterization of proteins12 and the study of their dynamics by NMR.13 Despite the increasing development of new synthetic methods, regioand stereoselective deuteration remains a challenge. The most common methodologies involve acid/base-catalyzed deuteration,14,15 metal catalysts,16,17 organocatalytic stereoselective deuteration,20 enzymatic deuteration,21,22 or organophotocatalytic decarboxylative deuteration.23,24 However, just a few methodologies have been reported for the selective synthesis of β-X-α-deuterated amino acids (X = N, O, S, Se). α-Deuterated serine has been produced by H/D exchange of cyclic chiral serine derivatives promoted by strong bases.25,26 Soda et al. described the αand β-deuteration of S-methylcysteine, catalyzed by L-methionine γ-lyase.27 More recently, Davis et al. reported a site-selective protein α-carbon deuteration through an engineered dehydroalanine (Dha) leading to α-deuterium cysteine (cysteine-2-d).28 To our knowledge, α-deuterium selenocysteine29 and 2,3-diaminopropanoic acid30,31 (selenocysteine-2-d and 2,3-diaminopropanoic-2-d acid, respectively) have been only reported in their racemic forms as mechanistic probes in enzymatic reactions. Bicyclic serine equivalent 1, has been widely used for the synthesis of enantiopure α-alkylserine derivatives by diastereoselective alkylation. The reaction involves the formation of a chiral, slightly pyramidalized enolate, which reacts diastereoselectively with different electrophiles such as alkyl halides32 or Michael acceptors.33 More recently, chiral bicyclic dehydroalanine equivalent 4 has been reported as a versatile Michael acceptor reacting with diverse heteronucleophiles under mild conditions to form derivatives of cysteine,34,35 selenocysteine36 and 2,3-diaminopropanoic acid.37 We envisioned using deuterium as an electrophile to trap enolates formed from 1 deprotonation in the presence of a base and from 4 upon nucleophile addition to selectively install a deuterium atom at the α position of amino acids. Herein, we report the diastereoselective basepromoted H/D exchange (HDE) of 1, together with diastereoselective X-Michael (X = S, Se, N) addition reactions to 4, and subsequent trapping of the corresponding enolates with deuterated alcohols as a deuterium source. This strategy was applied for the synthesis of enantiopure α-deuterated L/D-amino acid derivatives on a preparative scale and a deuterated analog of the mucolytic drug carbocysteine (Scheme S1). Results Synthesis of serine-2-d derivatives and peptides. The conditions for the HDE reaction on 1, readily synthesized from NBoc-L-serine methyl ester,32 were optimized evaluating the solvent, the base, the deuterium source, and the reaction time. The temperature (-78 ºC) and the initial amount of 1 (25 mg, 0.10 mmol) were fixed. The crude reaction mixture was analyzed by 1H NMR to determine the exchange ratio as well as the occurrence of undesired side reactions (Table S1). First, we evaluated conditions analogous to those for the diastereoselective alkylation of 1, i. e. 2.0 equivalents of base, excess of electrophile, and O-deuterated alcohols (methanol-d4 or isopropanol-OD, iPrOD) as both co-solvents and deuterium sources. Accordingly, compound 1 was reacted with lithium bis(trimethylsilyl)amide (LHMDS) as a base in a 1:1 mixture of tetrahydrofuran (THF) and O-deuterated alcohol as a solvent for a few seconds (< 60 s). The reaction was quenched by adding either a saturated aqueous solution of ammonium chloride or a saturated solution of ammonium-d4 chloride (ND4Cl) in deuterium oxide. Unfortunately, no exchange was observed in any case probably due to a quick deactivation of the base by the alcohol, precluding the formation of the enolate of 1. On the other hand, treatment of 1 with LHMDS in THF for a few seconds (< 60 s) followed by addition of a deuterated quencher (saturated ND4Cl in D2O or methanol-d4) led to high exchange ratios (85-90%), yet significant amounts of byproducts were observed. Using potassium bis(trimethylsilyl)amide (KHMDS) as a base or additives (i. e. hexamethyl phosphoramide, HMPA) resulted in complete decomposition of compound 1. Diluting the mixture, adding more equivalents of base, or stirring the mixture for longer times were also detrimental. The solvent also showed a significant influence on the deuteration process. For instance, performing the reaction in either dichloromethane or toluene produced low to moderate (30-60%) isotopic enrichments and partial decomposition of 1. However, quantitative deuteration (≥95%) and conversion were achieved using diethyl ether as a solvent. The latter conditions were selected as the optimum conditions (Scheme 1). A comparison of the 1H NMR spectra for compounds 1 and 2 showed that the signal corresponding to the exchanged hydrogen (H3, δ = 4.76 ppm) had disappeared, and those for the adjacent hydrogens (H2, δ = 4.14 and 4.29 ppm) had been transformed from two doublets of doublets (J = 8.9, 5.9 Hz; and 8.9, 8.9 Hz, respectively) to two doublets (J = 8.9 Hz). All other signals appeared at the same chemical shift, suggesting that a single diastereomer with the same absolute configuration was generated (Figure S1). Once the conditions for the selective deuteration were optimized, acidic hydrolysis of 2 by treatment with an aqueous 6 M HCl solution under reflux for 16 h quantitatively afforded L-serine-2-d (3) as a hydrochloride. Notably, the use of a non-deuterated solution for the hydrolysis did not affect the isotopic enrichment (≥95%). The stereochemical integrity of the α-carbon was maintained upon hydrolysis, as verified by its optical properties ([α]D20 +9.7 (c 1.0, 6 M HCl) for 3; [α]D20 +14.0 (c 1.0, 6 M HCl) for L-serine). Of note, the specific rotation of deuterated compounds may vary with respect to that of the unlabeled variant.38 Analogously, enantiomer D-serine-2-d (ent-3) was prepared from D-serine equivalent ent-1 using the same methodology (79% global yield, 91% isotopic enrichment, Scheme S2). Scheme 1. Diastereoselective hydrogen-deuterium exchange (HDE) reaction on bicyclic L-serine (1) equivalent followed by acidic hydrolysis. Yields obtained after purification of the products are shown in black. Isotopic enrichments are shown in green and in brackets. As an application of the synthetized α-deuterated amino acids, L-serine-2-d (3) was adequately protected for solid phase peptide synthesis (SPPS); several variants of proto-oncogene tyrosine-protein kinase c-Src myristoylated peptide (myr-Src) featuring single or triple Ser to Ser-2-d substitutions were prepared and characterized (Scheme S3). Synthesis of cysteine-2-d, selenocysteine-2-d, and 2,3-diaminopropanoic-2-d acid derivatives. Compound 1 was converted into chiral bicyclic dehydroalanine 4 following the previously reported methodology.34 The tandem Michael addition/α-deuteration reaction was assessed by reacting compound 4 with stoichiometric amounts of 7-mercapto-4-methylcoumarin (a) as a nucleophile, and triethylamine (TEA) as a base at room temperature using a iPrOD as a solvent and deuterium source. Dichloromethane (10% v/v) was also added to completely dissolve compound 4. The reaction showed complete conversion after 15 min and good deuterium incorporation (84%), as judged by 1H NMR. To maximize the deuteration ratio by reducing the presence of protium species (e. g. moisture), the reaction was carried out under an Ar atmosphere for 15 min. Under these conditions, adduct 5a was obtained almost quantitatively with excellent incorporation of deuterium (≥95%). The 1H NMR spectrum for deuterated adduct 5a was compared to that for the corresponding unlabeled analog 5Ha, which was synthesized under the same conditions but using non deuterated iPrOH instead. As with compounds 1 and 2, both spectra showed signals at the same chemical shift, with exception of that corresponding to the exchanged hydrogen (Hα, δ = 4.50 ppm), which had disappeared, and those for the adjacent hydrogens (Hβ, δ = 3.22 and 3.58 ppm), which were decoupled (Figure S2). We then generalized this methodology using representative thiols to form different cysteine derivatives (Scheme 2). The use of 7-mercapto-4-methylcoumarin (a) provides a fluorescently tagged cysteine derivative, whereas triphenylmethanethiol (b, TrtSH) and 4-methoxybenzyl mercaptan (c, PMBSH) may readily generate unprotected or S-protected cysteine. On the other hand, S-glycosylated cysteine derivatives have shown to be relevant for the activity of certain antimicrobial peptides, such as sublancin39 or glycocin F,40 hence, tetra-O-acetyl-1thio-β-D-glucose (d) was used as a nucleophile. Finally, reaction with N-Boc-D-cysteine methyl ester (e) leads to lanthionine, a bis-(α-amino acid) found in natural antimicrobial lanthipeptides.41 Remarkably, all reactions were completed in less than 45 min and the corresponding deuterated Michael adducts 5a-e were isolated in excellent yields (91-99%) and isotopic enrichments (≥93%), highlighting the general scope of this reaction. 2D NOESY NMR experiments confirmed the configuration of the generated stereocenter, which was identical for all the adducts, and coincided with that found for previously reported non-deuterated adducts.34 Deuterated adducts 5a-e were completely hydrolyzed by treatment with aqueous 6 M HCl at reflux for 16 h to give the corresponding D-cysteine-2-d hydrochlorides 6a-e in high to excellent yields (74–95%) and isotopic enrichment (≥90%). The absolute configuration at the Cα was maintained, as verified by their NMR and optical properties. Of note, unnatural D-cysteine derivatives were obtained from the compound 4 as observed in other S-Michael additions to this substrate.34,35 Hydrolysis of S-trityl-protected adduct 5b gave rise to unprotected D-cysteine-2-d, due to the concomitant deprotection of the trityl group, as a hydrochloride (6b). The same strategy was applied on enantiomer ent-4 using triphenylmethanethiol (TrtSH) as a nucleophile to obtain L-cysteine-2-d hydrochloride ent-6b in good global yield (70%, 2 steps) and excellent deuteration ratio (≥95%) (Scheme S4). Scheme 2. Stereoselective synthesis of α-deuterium D-cysteine derivatives (6a-e) by a tandem Michael addition-deuteration reaction followed by acidic hydrolysis. Isolated yields determined after purification are given in black. Isotopic enrichments are shown in green and in brackets. D-Cysteine-2-d (6b) was obtained upon hydrolysis of 5b. This methodology could also be applied for the use of amines as nucleophiles to produce α-deuterium 2,3-diaminopropanoic acid (DAP) derivatives (Scheme 3). These scaffolds are found in natural products and have been used for the synthesis of chiral ligands and therapeutic compounds.42 The tandem Michael addition/deuteration reaction worked well using primary (benzylamine, f), secondary acyclic (diethylamine, g) and cyclic (pyrrolidine, piperidine and azepane, h-j) amines as nucleophiles and chiral dehydroalanine 4, obtaining adducts 5f-j with high conversions (≥91%) and deuterium incorporations (≥90%). Those adducts were directly hydrolyzed without previous purification using an aqueous 6 M HCl solution at 60 ºC for 16 h to obtain α-deuterium DAP derivatives 6f-j in high global yields (82-97%, 2 steps) and isotopic enrichments (88-91%). The same procedure was performed to synthesize enantiomer ent-6f, using ent-4 as the Michael acceptor and benzylamine (BnNH2) as the nucleophile, in a high global yield (82%, 2 steps) and deuterium incorporation (≥92%) (Scheme S5). Scheme 3. Stereoselective synthesis of α-deuterium DAP derivatives (6f-j) by a tandem Michael addition-deuteration reaction followed by acidic hydrolysis. Isolated global yields (2 steps) determined after purification are given in black. Isotopic enrichments are shown in green and in brackets. We have recently reported a selective 1,4-conjugate addition of Se-nucleophiles to chiral dehydroalanine ent-4 as a methodology for the synthesis of selenocysteine (Sec) derivatives.36 Sec is the 21st proteinogenic amino acid and is often present in enzymes, being usually pivotal for their catalytic efficiencies.43 In an attempt to carry out the tandem Se-Michael addition/deuteration reaction, benzeneselenolate was generated in situ from diphenyl diselenide [(PhSe)2] using deuterated sodium borohydride and acetic acid, and reacted with dehydroalanine ent-4 in a 9:1 mixture of iPrOD and dichloromethane under Ar atmosphere. Complete conversion to adduct ent-5k was achieved in 5 min, with a high deuterium incorporation (85%). The use of fully deuterated acetic acid-d4 and sodium borodeuteride to minimize the presence of protium in the mixture afforded adduct ent-5k in good yield (74%) and with excellent isotopic enrichment (≥95%). This methodology was also applied using bis- (4-methoxyphenyl) diselenide [(PMBSe)2] as a nucleophile to form adduct ent-5l in a good yield after purification by column chromatography (70%) and excellent deuterium incorporation (≥95%). Enantiomerically pure Se-phenyl-L-selenocysteine-2d (ent-6k) and L-selenocystine-2,2'-d (ent-6l) hydrochlorides were obtained after acidic hydrolysis of the corresponding adducts by treatment with an aqueous 6 M HCl solution at 60 ºC for 16 h in high yields (84% and 98%, respectively) maintaining the deuteration ratio (Scheme 4). Scheme 4. Stereoselective synthesis of α-deuterium Sec derivatives (ent-6k and ent-6l) by a tandem Michael addition/deuteration reaction followed by acidic hydrolysis. Isolated yields determined after purification are given in black. Isotopic enrichments are shown in green and in brackets. Synthesis of carbocysteine-2-d. Having established the scope of the reaction and considering the increasing interest in developing drugs incorporating deuterium atoms, we envisioned applying this methodology to synthesize a cysteine- based drug. On this regard, carbocysteine (CC) is a mucolytic drug with antioxidant and anti-inflammatory properties that reduces the viscosity of sputum and is used to mitigate the symptoms of chronic obstructive pulmonary disease (COPD) and bronchiectasis.44 Carbocysteine could be easily accessed using methyl 2-mercaptoacetate as a nucleophile precursor using dehydroalanine ent-4 to obtain the carbocysteine with the natural absolute R-configuration. The Michael addition/deuteration reaction proceeded in an excellent yield (99%) with high deuterium incorporation (97%) giving rise to adduct ent-5m (Figure 1A). As with other sulfur-adducts, acidic hydrolysis of ent-5m in aqueous 6 M HCl at reflux for 16 h gave carbocysteine-2-d hydrochloride (ent-6m, CCD) in a high yield (91%) and isotopic enrichment (93%). We next evaluated the ability of CCD (ent-6m) to increase resistance to degradation in a physiological environment and compared it to that of the commercially available drug (SigmaAldrich, C7757). To this aim, both compounds were separately dissolved in a human serum solution (20% in phosphate buffer saline pH 7.4) and incubated at 37 ºC. Considering that the elimination half-life of carbocysteine is approximately 2 h,44 an aliquot of each sample was analyzed by UPLC-MS after 2.5 h of incubation. However, no significant change was observed, as none of them showed any degradation at that time. On the other hand, after incubation for 48 h, deuterated analog CCD (ent6m) showed slightly higher resistance to degradation, since a 98±4% of compound remained, whereas a 91±6% was observed for CC (Figure 1B). Although further studies are needed and kinetic isotopic effects are not necessarily the cause of this slightly improved stability to degradation, these results indicate a potential pharmacological use of CCD (ent-6m), which would last longer within the organism and, therefore, a lower dose might be needed, reducing costs and adverse effects. Figure 1. A) Stereoselective synthesis of deuterated analogue of carbocysteine, CCD (ent-6m). Isolated yields determined after purification are given in black. Isotopic enrichments are shown in green and in brackets. B) Stability of CCD and carbocysteine (CC) in human plasma at 37 ºC after 2.5 h (black) and 48 h (grey). Summary We have developed a general methodology for synthesizing enantioand diastereomerically pure α-deuterium serine, cysteine, selenocysteine and 2,3-diaminopropanoic acid derivatives. The reported strategies involve either a diastereoselective basepromoted H/D exchange on a chiral bicyclic serine derivative, or Michael additions of heteronucleophiles to a chiral bicyclic dehydroalanine using inexpensive deuterated alcohols as solvents and deuterium sources, both followed by acidic hydrolysis. The high stereoselection and deuterium incorporation obtained during H/D exchange and the hetero-Michael reactions is preserved in the final amino acids. The broad scope of these methodologies has been demonstrated at the 0.1-0.3 mmol scale, with examples of scaled-up syntheses at ca. 2 mmol scale (compounds 3 and ent-6m, see Supporting Information). A deuterated version of the mucolytic drug carbocysteine was synthesized using this methodology, which showed slightly higher stability in plasma. These findings suggest that carbocysteine-2-d might need lower administration dose, resulting in lower costs and adverse effects. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Experimental procedures, characterization data, and copies of the NMR spectra. (PDF). AUTHOR INFORMATION Corresponding Author Claudio D. Navo – Center for Cooperative Research in Biosciences (CIC bioGUNE), Basque Research and Technology Alliance (BRTA), Bizkaia Technology Park, Building 800, 48160 Derio, Spain. ORCID: 0000-0003-0161-412X; E-mail: [email protected]. Gonzalo Jiménez-Osés – Center for Cooperative Research in Biosciences (CIC bioGUNE), Basque Research and Technology Alliance (BRTA), Bizkaia Technology Park, Building 800, 48160 Derio, Spain – Ikerbasque, Basque Foundation for Science, 48013 Bilbao, Spain. ORCID: 0000-0003-0105-4337; E-mail: gjo- [email protected]. Authors Paula Oroz – Departamento de Química, Centro de Investigación en Síntesis Química, Universidad de La Rioja, 26006 Logroño, La Rioja, Spain. Nuria Mazo – Departamento de Química, Centro de Investigación en Síntesis Química, Universidad de La Rioja, 26006 Logroño, La Rioja, Spain. ORCID: 0000-0001-6049-0871. Marina Blanco – Departamento de Química, Centro de Investigación en Síntesis Química, Universidad de La Rioja, 26006 Logroño, La Rioja, Spain. Jesús M. Peregrina – Departamento de Química, Centro de Investigación en Síntesis Química, Universidad de La Rioja, 26006 Logroño, La Rioja, Spain. ORCID: 0000-0003-3778-7065. Present Addresses †Nuria Mazo – 3P Biopharmaceuticals, 31110 Noáin, Navarra, Spain. Author Contributions The manuscript was written through contributions of all authors. / All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT We thank Agencia Estatal Investigacion of Spain (AEI; grants RTI2018-099592-B-C21 and RTI2018-099592-B-C22). P.O. thanks Universidad de La Rioja for a predoctoral grant. REFERENCES (1) Atzrodt, J.; Derdau, V.; Kerr, W. J.; Reid, M. Deuteriumand Tritium-Labelled Compounds: Applications in the Life Sciences. Angew. Chem. Int. Ed. 2018, 57, 1758. (2) Hoofnagle, A. N.; Resing, K. A.; Ahn, N. G. Protein Analysis by Hydrogen Exchange Mass Spectrometry. Annu. Rev. Biophys. Biomol. Struct. 2003, 32, 1. (3) Smith, A. E.; Zhang, Z.; Pielak, G. J.; Li, C. NMR Studies of Protein Folding and Binding in Cells and Cell-like Environments. Curr. Opin. Struct. Biol. 2015, 30, 7. (4) Adhikary, R.; Zimmermann, J.; Dawson, P. E.; Romesberg, F. E. IR Probes of Protein Microenvironments: Utility and Potential for Perturbation. ChemPhysChem 2014, 15, 849. (5) Harbeson, S. L.; Tung, R. D. Deuterium in Drug Discovery and Development. Annu. Rep. Med. Chem. 2011, 46, 403. (6) Katsnelson, A. Heavy Drugs Draw Heavy Interest from Pharma Backers. Nat. Med. 2013, 19, 656. (7) Russak, E. M.; Bednarczyk, E. M. Impact of Deuterium Substitution on the Pharmacokinetics of Pharmaceuticals. Ann. Pharmacother. 2019, 53, 211. (8) Schmidt, C. First Deuterated Drug Approved. Nat. Biotechnol. 2017, 35, 493. (9) Koruza, K.; Lafumat, B.; Végvári; Knecht, W.; Fisher, S. Z. Deuteration of Human Carbonic Anhydrase for Neutron Crystallography: Cell Culture Media, Protein Thermostability, and Crystallization Behavior. Arch. Biochem. Biophys. 2018, 645, 26. (10) Wei, L.; Shen, Y.; Xu, F.; Hu, F.; Harrington, J. K.; Targoff, K. L.; Min, W. Imaging Complex Protein Metabolism in Live Organisms by Stimulated Raman Scattering Microscopy with Isotope Labeling. ACS Chem. Biol. 2015, 10, 901. (11) Bornø, A.; Van Hall, G. Quantitative Amino Acid Profiling and Stable Isotopically Labeled Amino Acid Tracer Enrichment Used for in Vivo Human Systemic and Tissue Kinetics Measurements. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 2014, 951–952, 69. (12) Movellan, K. T.; Najbauer, E. E.; Pratihar, S.; Salvi, M.; Giller, K.; Becker, S.; Andreas, L. B. Alpha Protons as NMR Probes in Deuterated Proteins. J. Biomol. NMR 2019, 73, 81. (13) Sheppard, D.; Li, D. W.; Brüschweiler, R.; Tugarinov, V. Deuterium Spin Probes of Backbone Order in Proteins: 2H NMR Relaxation Study of Deuterated Carbon α Sites. J. Am. Chem. Soc. 2009, 131, 15853. (14) Mitulovi, G.; Lämmerhofer, M.; Maier, N. M.; Lindner, W. Simple and Efficient Preparation of R and S‐enantiomers of Alfa‐carbon Deuterium‐labelled Alfa‐amino Acids. J. Label. Compd. Radiopharm. 2000, 43, 449. (15) Lygo, B.; Humphreys, L. D. Enantioselective Synthesis of αCarbon Deuterium-Labelled L-α-Amino Acids. Tetrahedron Lett. 2002, 43, 6677. (16) Takeda, R.; Abe, H.; Shibata, N.; Moriwaki, H.; Izawa, K.; Soloshonok, V. A. Asymmetric Synthesis of α-Deuterated α-Amino Acids. Org. Biomol. Chem. 2017, 15, 6978. (17) Chatterjee, B.; Krishnakumar, V.; Gunanathan, C. Selective αDeuteration of Amines and Amino Acids Using D2O. Org. Lett. 2016, 18, 5892. (20) Moozeh, K.; So, S. M.; Chin, J. Catalytic Stereoinversion of LAlanine to Deuterated D-Alanine. Angew. Chem. Int. Ed. 2015, 54, 9381. (21) Chun, S. W.; Narayan, A. R. H. Biocatalytic, Stereoselective Deuteration of α-Amino Acids and Methyl Esters. ACS Catal. 2020, 10, 7413. (22) Doyon, T. J.; Buller, A. R. Site-Selective Deuteration of Amino Acids through Dual-Protein Catalysis. J. Am. Chem. Soc. 2022, 144, 7327. (23) Ji, P.; Zhang, Y.; Dong, Y.; Huang, H.; Wei, Y.; Wang, W. Synthesis of Enantioenriched α-Deuterated α-Amino Acids Enabled by an Organophotocatalytic Radical Approach. Org. Lett. 2020, 22, 1557. (24) Liu, L.; Deng, Z.; Xu, K.; Jiang, P.; Du, H.; Tan, J. Access to Deuterated Unnatural α-Amino Acids and Peptides by Photochemical Acyl Radical Addition. Org. Lett. 2021, 23, 5299. (25) Reider, P. J.; Eichen Conn, R. S.; Davis, P.; Grenda, V. J.; Zambito, A. J.; Grabowski, E. J. J. Synthesis of (R)-Serine-2-d and Its Conversion to the Broad Spectrum Antibiotic Fludalanine. J. Org. Chem. 1987, 52, 3326. (26) Rose, J. E.; Leeson, P. D.; Gani, D. Regiospecific Deuteriation of Chiral 3-Isopropyl-2,5-Dimethoxy-3,6-Dihydropyrazines in the Stereospecific Synthesis of α-Deuteriated α-Amino Acids. J. Chem. Soc. Perkin Trans. 1 1992, 1563. (27) Esaki, N.; Nakayama, T.; Sawada, S.; Tanaka, H.; Soda, K. 1H NMR Studies of Substrate Hydrogen Exchange Reactions Catalyzed by L-Methionine γ-Lyase. Biochemistry 1985, 24, 3857. (28) Galan, S. R. G.; Wickens, J. R.; Dadova, J.; Ng, W. L.; Zhang, X.; Simion, R. A.; Quinlan, R.; Pires, E.; Paton, R. S.; Caddick, S.; Chudasama, V.; Davis, B. G. Post-Translational Site-Selective Protein Backbone α-Deuteration. Nat. Chem. Biol. 2018, 14, 955. (29) Esaki, N.; Karai, N.; Nakamura, T.; Tanaka, H.; Soda, K. Mechanism of Reactions Catalyzed by Selenocysteine β-Lyase. Arch. Biochem. Biophys. 1985, 238, 418. (30) Wang, M.; Gould, S. J. Biosynthesis of Capreomycin. 2. Incorporation of L-Serine, L-Alanine, and L-2,3-Diaminopropionic Acid. J. Org. Chem. 1993, 58, 5176. (31) Van Der Baan, J. L.; Barnick, J. W. F. K.; Bickelhaupt, F. Biosynthesis of L-2,3-Diaminopropanoic Acid. J. Chem. Soc. Perkin Trans. 1 1984, 2809. (32) Aydillo, C.; Jiménez-Osés, G.; Busto, J. H.; Peregrina, J. M.; Zurbano, M. M.; Avenoza, A. Theoretical Evidence for Pyramidalized Bicyclic Serine Enolates in Highly Diastereoselective Alkylations. Chem. Eur. J. 2007, 13, 4840. (33) Aydillo, C.; Navo, C. D.; Busto, J. H.; Corzana, F.; Zurbano, M. M.; Avenoza, A.; Peregrina, J. M. A Double Diastereoselective Michael-Type Addition as an Entry to Conformationally Restricted Tn Antigen Mimics. J. Org. Chem. 2013, 78, 10968. (34) Gutiérrez-Jiménez, M. I.; Aydillo, C.; Navo, C. D.; Avenoza, A.; Corzana, F.; Jiménez-Osés, G.; Zurbano, M. M.; Busto, J. H.; Peregrina, J. M. Bifunctional Chiral Dehydroalanines for Peptide Coupling and Stereoselective S-Michael Addition. Org. Lett. 2016, 18, 2796. (35) Navo, C. D.; Asín, A.; Gómez-Orte, E.; Gutiérrez-Jiménez, M. I.; Compañón, I.; Ezcurra, B.; Avenoza, A.; Busto, J. H.; Corzana, F.; Zurbano, M. M.; Jiménez-Osés, G.; Cabello, J.; Peregrina, J. M. CellPenetrating Peptides Containing Fluorescent D-Cysteines. Chem. Eur. J. 2018, 24, 7991. (36) Oroz, P.; Navo, C. D.; Avenoza, A.; Busto, J. H.; Corzana, F.; Jiménez-Osés, G.; Peregrina, J. M. Toward Enantiomerically Pure βSeleno-α-Amino Acids via Stereoselective Se-Michael Additions to Chiral Dehydroalanines. Org. Lett. 2021, 23, 1955. (37) Navo, C. D.; Mazo, N.; Oroz, P.; Gutiérrez-Jiménez, M. I.; Marín, J.; Asenjo, J.; Avenoza, A.; Busto, J. H.; Corzana, F.; Zurbano, M. M.; Jiménez-Osés, G.; Peregrina, J. M. Synthesis of Nβ-Substituted α,βDiamino Acids via Stereoselective N-Michael Additions to a Chiral Bicyclic Dehydroalanine. J. Org. Chem. 2020, 85, 3134. (38) Coumbarides, G. S.; Dingjan, M.; Eames, J.; Suggate, M. J. On the Specific Rotation of Deuterium-Containing Isotopomers of 2-Dimethylaminocyclohexyl-1-Trimethylammonium Iodide. J. Label. Compd. Radiopharm. 2006, 49, 153. (39) Oman, T. J.; Boettcher, J. M.; Wang, H.; Okalibe, X. N.; Van Der Donk, W. A. Sublancin Is Not a Lantibiotic but an S-Linked Glycopeptide. Nat. Chem. Biol. 2011, 7, 78. (40) Amso, Z.; Bisset, S. W.; Yang, S. H.; Harris, P. W. R.; Wright, T. H.; Navo, C. D.; Patchett, M. L.; Norris, G. E.; Brimble, M. A. Total Chemical Synthesis of Glycocin F and Analogues: S-Glycosylation Confers Improved Antimicrobial Activity. Chem. Sci. 2018, 9 (6), 1686. (41) Yu, Y.; Zhang, Q.; Van Der Donk, W. A. Insights into the Evolution of Lanthipeptide Biosynthesis. Protein Sci. 2013, 22, 1478. (42) Viso, A.; Fernández de la Pradilla, R.; García, A.; Flores, A. α,β-Diamino Acids: Biological Significance and Synthetic Approaches. Chem. Rev. 2005, 105, 3167. (43) Johansson, L.; Gafvelin, G.; Arnér, E. S. J. Selenocysteine in Proteins - Properties and Biotechnological Use. Biochimica et Biophysica Acta - General Subjects. Elsevier October 2005, pp 1–13. (44) Mitchell, S. C.; Steventon, G. B. S-Carboxymethyl-L-Cysteine. Drug Metab. Rev. 2012, 44, 129.