Expedited synthesis of α-amino acids by single-step enantioselective α-amination of carboxylic acids

The conversion of C‒H bonds to C‒N bonds offers a sustainable and economical strategy for the synthesis of nitrogen-containing compounds. However, challenges regarding the control of regio- and stereoselectivity currently limit the broad applicability of intermolecular C(sp3)‒H amination reactions. We address these restrictions by directed nitrene-mediated C‒H insertion using a metal-coordinating functional group. We report a highly stereocontrolled, iron-catalysed direct α-amination of abundant carboxylic acid feedstock molecules. The method provides in a single step high-value N-Boc-protected α-monosubstituted and α,α-disubstituted α-amino acids, which can then be immediately used for applications including solution- and solid-phase peptide synthesis. This method fulfils important aspects of sustainability by being highly step efficient and utilizing non-toxic, Earth-abundant iron as the catalytic metal. The amination of C–H bonds is a sustainable approach to prepare important nitrogen-containing molecules; however, regio- and stereoselectivity is difficult to control. Now the synthesis of α-monosubstituted and α,α-disubstituted α-amino acids from abundant carboxylic acids has been achieved through Fe-catalysed asymmetric intermolecular C(sp3)–H amination by directed stereocontrolled nitrene insertion.

Amines are some of the most prevalent functional groups found in natural molecules and pharmaceuticals 1,2 . As such, the demand for economical and sustainable synthetic methods for the introduction of amino groups remains strong and growing. The direct replacement of a hydrogen by an amine functionality 3 through transition metalcatalysed, nitrene-mediated intermolecular C-H amination represents an attractive and highly step-efficient solution [4][5][6][7] . However, despite the considerable research efforts that have been focused on this area, the abundance of C-H bonds in organic molecules typically poses a formidable regioselectivity challenge for intermolecular C-H aminations. Furthermore, control of stereoselectivity is often difficult to achieve for the outer-sphere C-N bond formation step in which the substrate is not bound to the metal (Fig. 1a). These two aspects currently limit the broad applicability of stereocontrolled nitrene-mediated intermolecular C(sp 3 )-H aminations in synthetic chemistry.
Directed C-H activation by transition metal catalysis has proven to be a powerful tool for addressing the regio-and stereoselectivity issues confronted in C-H functionalizations such as the amination of nonactivated C(sp 3 )-H bonds 8 . Directed C-H amination exploits a metalcoordinating directing group for site-selective activation of a specific C-H bond, usually by the formation of a metallacycle intermediate, followed by reaction of the metal-carbon bond with an external or metal-bound nitrogen species. The sustainability of these methods, however, is often diminished by the reliance on noble metals, such as Pd, Ru, Rh and Ir, for inducing cyclometalation. In a different mechanistic manifold, directed C(sp 3 )-H amination could be achieved through nitrene insertion into C-H bonds without the preceding formation of a C-M bond, where M is a metal (Fig. 1b). Surprisingly, this mechanistic scheme is rarely explored and has only been realized non-racemically through hydrogen bond-mediated catalysis 9,10 and in racemic reactions through high-valence iridium 11 and rhodium 12 nitrene intermediates, and not in a catalytic asymmetric manner.
We intended to achieve asymmetric intermolecular C(sp 3 )-H amination through directed C(sp 3 )-H nitrene insertion. Specifically, we envisioned that carboxylic acids might be suitable directing groups for regioselective and stereocontrolled nitrene-mediated C-H aminations to directly convert abundant feedstock carboxylic acids into highly valuable non-racemic chiral α-amino acids bearing unnatural side chains, which are sought-after synthetic building blocks due to their modulated chemical, physical and pharmaceutical properties [13][14][15][16][17][18] (Fig. 1c). Direct α-amination of carboxylic acids has rare precedents and is highly challenging, in contrast with the well-established α-amination of aldehydes, Article https://doi.org/10.1038/s44160-023-00267-w applications, we elected to use the Boc group, one of the most commonly employed N-protecting groups in amino acid chemistry 26 .
We proceeded to explore the suitability of N-Boc-protected carbamates with various leaving groups at the nitrogen (BocNH-X; X = leaving group) as nitrene precursors [27][28][29][30][31][32][33] . While no conversion was observed with benzoate as the leaving group (entry 1), sulfonates provided more encouraging results. With toluenesulfonate as the leaving group, the desired N-Boc-protected phenylglycine was formed with 29% NMR spectroscopy yield and 91% e.e. after 20 h at -15 °C in an incomplete conversion (entry 2). An isopropyl sulfonate leaving group did not perform significantly better (entry 3). The best results were obtained with mesylate (Ms) as the leaving group. Subjecting PAA to (R,R)-FeBIP (8 mol%), BocNHOMs (2 equiv.) and piperidine (4 equiv.) in 1,2-dichlorobenzene (o-DCB) at −15 °C provided N-Boc-protected (S)-phenylglycine (1) in 78% NMR spectroscopy yield (77% isolated yield) with 93% e.e. (entry 4). Bases such as K 2 CO 3 (entry 5) or Et 3 N (entry 6) afforded inferior results. Other solvents, including dichloromethane or acetonitrile, led to decreased yields and enantioselectivities (entries 7 and 8). It is worth noting that higher temperatures also provided diminished yields (entries 9 and 10), presumably due to a Lossen-type rearrangement of BocN-HOMs induced by the base at elevated temperatures 29 . Less favourable results were also obtained when using an excess of aminating reagent to drive conversion to completion, which resulted in a decreased yield due to oxidative decomposition of the amino acid product (compare entries 4 and 11) (see also Supplementary Information section 'Oxidative decomposition of α-amino acids under reaction conditions' for details).
Finally, two popular related chiral iron catalysts featuring tetradentate N4-donor ligands were tested for comparison and displayed inferior results. The bis-pyridine complex (R,R)-Fe1 (ref. 34 ) provided R-configurated 1 in a significantly lower yield and with significantly lower enantioselectivity (20% yield with 48% e.e.; entry 12), while the bis-benzimidazole diacetonitrile iron complex (R,R)-Fe2 (ref. 35 ), which is prepared from (R,R)-FeBIP by silver-promoted ligand exchange, provided comparable enantioselectivity but a significantly decreased yield (entry 13). These results confirm the benefit of both the benzimidazole and chloride ligands of the FeBIP catalyst scaffold.

Substrate scope
With optimized reaction conditions in hand, we next investigated the substrate scope for this protocol (1-49) ( Table 2). The reaction ketones, 1,3-dicarbonyl compounds, esters and some carboxylic acid surrogates [19][20][21] . This can be attributed to both the acidity of the carboxylic acid moiety and the typically high pK a value of the α-hydrogen. In 1972, Yamada et al. 22 was the first to report the preparation of α-amino acids from carboxylic acids by a one-pot procedure through double deprotonation followed by reaction with O-methylhydroxylamine. However, this method requires a strong base and provides only racemic products. In 2012, Smith and colleagues 23 reported the enantioselective organocatalytic α-amination of carboxylic acids with N-aryl-N-aroyldiazenes in a one-pot, two-step procedure via in situ-generated acid anhydrides. This method is limited to the synthesis of N-aryl-α-glycine derivatives after SmI 2 -induced cleavage of the initially obtained hydrazides. In 2019, Shimizu and colleagues 24 introduced a boron-catalysed α-hydrazination of carboxylic acids with diisopropyl azodicarboxylate. In one example, the presence of a chiral ligand provided modest enantioselectivity (45% enantiomeric excess (e.e.)). Unfortunately, converting the α-hydrazino into an amino group required harsh reaction conditions.
Herein, we report a previously elusive stereocontrolled intermolecular one-step α-C(sp 3 )-H amination to afford high-value N-protected chiral α-amino acids. Our method refrains from multi-step synthetic sequences or inconvenient starting materials. Instead, it utilizes abundant carboxylic acid feedstock, sustainable and Earth-abundant iron catalysis and the popular tert-butyloxycarbonyl (Boc)-protecting group, which does not require a subsequent step-intensive exchange of the protection group. These characteristics render this method highly attractive for the expedited synthesis of α-monosubstituted and α,α-disubstituted α-amino acids.

Mechanistic investigations
Having established the broad scope and utility of our iron-catalysed α-amination reaction, we turned our attention to mechanistic considerations. Formation of transition metal nitrenes from N-protected carbamates has been well investigated by several research groups [30][31][32][33] and was used as the starting point for our new intermolecular α-C-H amination of carboxylic acids. Several control experiments were conducted to interrogate the intermediates involved in this reaction (Fig. 2a). Under the standard conditions, methyl phenylacetate failed to afford any α-amination product, which was in a stark contrast with the productive PAA substrate, supporting the notion that the carboxylic acid functional group is important for the mechanism. Moreover, when N-benzoate carbamate was utilized as the aminating reagent, no α-amino acid product was detected ( Table 1, entry 1), despite the fact that N-benzoate carbamate has proven very effective for metal nitrene formation 42 . However, the base-induced conversion of N-benzoate carbamate to a metal nitrene releases benzoate, which will compete with the carboxylate substrate for metal binding. Control experiments also rule out that the reaction occurs through the in situ generation

Fig. 3 | Proposed catalytic cycle.
Simplified mechanism for the catalytic asymmetric one-step α-amino acid synthesis. B, base.
Article https://doi.org/10.1038/s44160-023-00267-w of azanyl ester under standard conditions, which distinguishes this reaction mechanistically from our previous intramolecular 1,3-nitrene migratory insertion 25 . By comparing the reaction rate constants of nondeuterated PAA (k H ) versus deuterated PAA (PAA-d 2 ) (k D ) of two parallel reactions, a kinetic isotope effect of k H /k D = 2.0 was obtained, indicating that cleavage of the C-H occurs during the rate-determining step (Fig. 2b). When (Z)-4-phenylbut-3-enoic acid was subjected to standard reaction conditions, complete Z/E-isomerization of the C=C bond occurred to provide the corresponding (E)-α-amino acid in 23% NMR spectroscopy yield with an E/Z ratio of >100/1 (Fig. 2c). This implies a radical mechanism proceeding through the formation of an allylic radical intermediate. Furthermore, reactions starting from enantiopure (R)-or (S)-2-phenylpropanoic acid (2-PPA) confirmed that the ironcatalysed α-amination of α,α-disubstituted carboxylic acids proceeds in a stereoconvergent fashion. In both cases, S-configurated α-amino acid was predominantly formed and the reisolated intact carboxylic acid after reaction did not show any significant racemization (Fig. 2d). These results suggest that an irreversible stereoablative process occurred in the coordination sphere before the C-N bond formation. Moreover, Hammett studies also revealed that the intermediate formed during the rate-determining step has a strong radical character (see the Supplementary Information section 'Hammett plots' for details). Based on these experiments, a proposed simplified catalytic cycle for our developed intermolecular C(sp 3 )-H amination of carboxylic acids is shown in Fig. 3. Base-induced reaction of the iron catalyst with the amination reagent leads to the formation of the iron nitrene 43  We also perfomed density functional theory calculations to further reveal the mechanism of stereocontrol in the reaction. Propionic acid and a nitrene intermediate containing a methyl carbamate protection group were used as the model system for the computations. Our calculated free energy diagram for the C(sp 3 )-H abstraction and subsequent radical rebound steps is shown in Fig. 4a. The quintet spin state was found to be the most stable for I, the resting-state iron complex immediately before C(sp 3 )-H abstraction (the triplet state and open-shell singlet states were calculated to be ~6 and ~8 kcal mol −1 higher in free energy, respectively; the free energy barrier for the N-O cleavage step to generate the iron nitrene intermediate was calculated to be low at 7.9 kcal mol −1 ; see the Supplementary Information section 'Calculated free energy diagram' for details). Complex I undergoes 1,5-HAT via the quintet transition state TS-1 with a free energy barrier of 9.8 kcal mol −1 , while an alternative outer-sphere HAT mechanism was calculated to have a higher free energy barrier of 13.3 kcal mol −1 (see the Supplementary Information section 'Calculated free energy diagram' for details). The resulting diradical II was calculated to be lowest in energy in its triplet state. While radical rebound (recombination) is often fast or barrierless for C-H functionalizations catalysed by iron-containing enzymes such as P450 (ref. 44 ), the C-N bond-forming radical rebound step in our reaction had an unusually high barrier of 8.5 kcal mol −1 through the quintet transition state TS-2. This result suggested that the diradical intermediate II was relatively long lived, which might lead to stereoablation via bond rotation (such stereoablative bond rotation   Article https://doi.org/10.1038/s44160-023-00267-w may involve rotation of the more flexible Fe-O coordinative bond) and render the subsequent radical rebound step enantiodetermining 45 . Our calculations also showed that the 1,5-HAT step favoured the abstraction of the pro-R hydrogen by 2.1 kcal mol −1 ( 5 TS-1-R versus 5 TS-1-S), which would yield the R product enantiomer instead of the experimentally observed S enantiomer if it were stereodetermining. In contrast, stereoablation of II through facile conformational change 45 and subsequent stereodetermining radical rebound would be in agreement with the preferential formation of the S product. Our experimental examples of enantioconvergent C(sp 3 )-H amination of racemic α-branched carboxylic acids also indicate that the radical rebound step, not the C(sp 3 )-H abstraction, controls the stereochemical outcome. Examination of the radical rebound transition states (Fig. 4b) revealed that the lower free energy of 5 TS-2-S compared with 5 TS-2-R can be at least partially attributed to a more favourable conformation about the forming C-N bond, where the carbon substituents are staggered rather than near-eclipsed.

Conclusions
We developed an expedited catalytic asymmetric synthesis of α-amino acids by intermolecular α-C(sp 3 )-H amination of carboxylic acids. Our protocol employs abundant and readily available carboxylic acids as starting materials and converts them in a single step into highly valuable non-racemic N-protected α-amino acids suitable for direct use in peptide synthesis and for other synthetic applications. This method utilizes the convenient Boc-protecting group and sustainable iron catalysis without the need for more toxic or less abundant transition metals. This approach also merges nitrene-mediated C(sp 3 )-H functionalization with directed C(sp 3 )-H functionalization without the requirement for an intermediate cyclometalation. Upon deprotonation, the carboxylic acid substrate coordinates to the iron centre and serves as the directing group, which allows regioselective nitrene-mediated C(sp 3 )-H abstraction and ensures high stereocontrol for the subsequent C-N bond formation step. Mechanistic evidence supports a radical mechanism in which the radical rebound step, rather than the initial C(sp 3 )-H abstraction, is responsible for stereodiscrimination. Thus, in addition to providing a general method for the expedited synthesis of a wide range of chiral α-monosubstituted and α,α-disubstituted amino acids, including examples of late-stage amination of carboxylic acidcontaining pharmaceuticals and natural products, this study is also prototypical for the development of intermolecular asymmetric C-H aminations through directed C(sp 3 )-H nitrene insertion. Finally, it is noteworthy that this study discloses enantioselective intermolecular nitrene-mediated C(sp 3 )-H aminations catalysed by a synthetic chiral iron complex.

General procedure for the iron-catalysed α-amination of carboxylic acids
To a dry Schlenk tube was added the carboxylic acid (1 equiv.), BocN-HOMs (2-5 equiv.) and (R,R)-FeBIP (8-15 mol%). The tube was evacuated and backfilled with N 2 five times. The indicated solvent (0.1 M) and piperidine (4-7 equiv.) were added via syringe and the tube was sealed. The reaction mixture was further degassed via freeze-pump-thaw once. Then, the reaction mixture was stirred at -15 °C for the indicated time. After completion, the reaction mixture was diluted with Et 2 O and washed with aqueous NaHSO 4 (1 M). The aqueous layer was extracted with Et 2 O. The combined organic layer was dried over anhydrous sodium sulfate. After filtration, the solvent was evaporated under decreased pressure and the residue was purified by column chromatography on silica gel using a mixture of EtOAc and hexane (with 0.2% acetic acid as the additive) to obtain the non-racemic α-amino acids 1-49.