Asymmetric biomimetic transamination of α-keto amides to peptides

Peptides are important compounds with broad applications in many areas. Asymmetric transamination of α-keto amides can provide an efficient strategy to synthesize peptides, however, the process has not been well developed yet and still remains a great challenge in both enzymatic and catalytic chemistry. For biological transamination, the high activity is attributed to manifold structural and electronic factors of transaminases. Based on the concept of multiple imitation of transaminases, here we report N-quaternized axially chiral pyridoxamines 1 for enantioselective transamination of α-keto amides, to produce various peptides in good yields with excellent enantio- and diastereoselectivities. The reaction is especially attractive for the synthesis of peptides made of unnatural amino acids since it doesn’t need great efforts to make chiral unnatural amino acids before amide bond formation.

P eptides are one type of the most important compounds with high biological activities, which are widely present in many natural products, pharmaceutically relevant molecules, and biological systems [1][2][3] . Especially in recent years, there appears a growing interest in therapeutic peptides [1][2][3] and more and more peptide drugs have been developed (Fig. 1a) [4][5][6] . Development of alternative new methods for the synthesis of peptides is always highly desirable and potentially useful 7,8 .
Previous studies have suggested that asymmetric 1,3-proton shift between the ketimine intermediate and the aldimine is likely a key step for biological transamination (Fig. 1d) [11][12][13]46,47 . In order to accelerate this step, evolution has elegantly optimized transaminases by incorporating a Lys residue at an appropriate position [48][49][50] . The ε-NH 2 group of the Lys residue serves as an intramolecular base to deprotonate the benzylic C-H of the ketimine (Fig. 1d) [48][49][50] . In addition, the pK a values of the pyridine N in the coenzyme pyridoxal 5′-phosphate (PLP) and pyridoxamine 5′-phosphate (PMP) are around 8.5 [51][52][53] , thus the pyridine N is predominantly protonated in biological systems (near pH 7) [53][54][55][56] . The strong electron-withdrawing property of the protonated pyridine ring helps to increase the acidity of the benzylic C-H bond (Fig. 1d) [54][55][56][57][58] . The two effects work together to promote the transformation from the ketimine to the aldimine via 1,3-proton shift, magically accelerating transamination process. Inspired by the controlled protonation of the pyridine N of PLP in biological systems, Rapoport has developed N-methylpyridinium-4-carboxaldehyde benzenesulfonate (Rapoport's salt) as an effective transamination reagent for conversion of amines to carbonyl compounds 59 . Francis have found that Rapoport's salt displays significantly improved efficiency in the transamination of proteins as compared to pyridinium-4-carboxaldehyde, converting the N-termini into the corresponding carbonyl groups 44,45 . Recently, we have proved that introducing an amine side arm to a chiral pyridoxamine can remarkably increase its activity and enantioselectivity for asymmetric transamination of α-keto acids 26 and also have observed that quaternization of the pyridine N of a chiral pyridoxal leads to dramatical improvement of catalytic activity for asymmetric biomimetic Mannich reaction 60 and aldol reaction 61 of glycinate. On the basis of the structural characteristics of transaminases [46][47][48][49][50][53][54][55][56] as well as the previously reported studies 26,44,45,[59][60][61][62] , we has designed N-quaternized biaryl axially chiral pyridoxamines 1 bearing an amine side arm, mimicking transaminases in multiple aspects for catalytic asymmetric transamination of α-keto amides to peptides (Fig. 1c). The quaternization always keeps the pyridine ring with strong electron-withdrawing ability to improve the benzylic C-H acidity of the ketimine intermediate during transamination, no matter under acidic or basic conditions 60,61 . Like the Lys residue does in biological transamination, the amine side arm can serve as an intramolecular base to facilitate 1,3-proton shift.
Here we show that asymmetric biomimetic transamination of α-keto amides 2 can be achieved by using chiral pyridoxamines 1 as the catalyst, to produce various peptides 3 with excellent enantiopurities (Fig. 1e).

Results
Catalyst synthesis. The synthesis of chiral pyridoxamines 1 started with reductive amination of compound 5 26 to introduce the amine side chain. Protecting the two amine groups with ditert-butyl dicarbonate gave intermediates 6 ( Fig. 2). Treatment of 6 with methyl iodide and subsequent deprotection with hydrochloric acid afforded N-methyl pyridoxamines 1a-e in good yields.
In order to investigate the impacts of catalyst and substrates on diastereomeric induction, several representative α-keto amides (for 3m-n, 3r, 3v-y, and 3aa-ab) were examined respectively using (S)-  1b (5 mol%), (R)-1b (5 mol%), and achiral pyridoxamine 7 (20 mol %) as the catalyst. The corresponding peptides were formed with S configurations of the newly generated chiral centers from catalyst (R)-1b and R configurations from (S)-1b. The chiral pyridoxamine catalyst dominated the stereoselectivity, while the chiral groups on the amino acid residues of α-keto amides throwed little influence on the diastereomeric induction probably due to being far away from the reaction centers as well as the flexibility of the skeletons of the αketo amides. No matter which configuration of the catalyst 1b was applied, excellent diastereoselectivities were always obtained, even for α-keto amides (for 3n and 3aa) with a nearby bulky chiral amino acid residue and for those that displayed obvious substrateinduction on diastereoselectivity in 7-catalyzed non-asymmetric transamination (5:95 dr for 3r and 19:81 dr for 3x). Synthetic applications. Divergent extending an additional amino acid unit from a central peptide is of great interest for peptide drug screening and bioactivity studies. The synthesis would be difficult when the extended unit is a commercially unavailable unnatural amino acid. The transamination process provides an efficient strategy for the amino acid extending. For example, starting from the benzyl ester of protease inhibitor Ubenimex (8) 65 , condensation with α-keto acids and subsequent asymmetric transamination afforded a variety of enantiopure peptides 3ae-ai with one more amino acid residue extended (Fig. 5a)  unprotected OH group remained untouched during the condensation and transamination. Based on the "condensation-transamination" process, a new strategy for the synthesis of peptides also can be developed. As illustrated in Fig. 5b, the methyl ester of DPP-IV inhibitor Diprotin A (10) 66 underwent condensation with α-keto acid 9a and subsequent asymmetric transamination, forming tetrapeptide 3ad with excellent diastereoselectivity. Repeating the reaction sequence two more times afforded hexapeptide 3ak with high enantiopurity. The chirality of the extended amino acid residues was established along with the transamination process. The protocol is especially attractive for the synthesis of peptides made of unnatural amino acids, since it doesn't need great efforts to make NH 2 -protected chiral unnatural amino acids before the amide bond formation.
Reaction mechanism. A plausible mechanism was proposed for the transamination (Fig. 6a) [11][12][13]25,26 . Pyridoxamine 1b condenses with α-keto amide 2 to form ketimine 11, which undergoes asymmetric 1,3-proton shift to aldimine 13 under the assistance of the amine side arm [46][47][48][49][50] . Hydrolysis of aldimine 13 releases peptide 3 and generates the pydridoxal, which is in situ converted into iminium 14 via intramolecular condensation. The iminium 14 then undergoes decarboxylative transamination with the amine source diphenylglycine (4) back to pyridoxamine catalyst 1b 25,26,63 , completing a catalytic cycle. As expected, N-quaternization of the pyridine ring of the chiral pyridoxamines resulted in higher catalytic activity and better enantioselectivity for the asymmetric transamination (Fig. 6b,   1b vs 1g). The stronger electron-withdrawing property makes the benzylic C-H of ketimine 11 more acidic and also stabilizes the corresponding delocalized carbanion 12 better 54-58,67-69 , thus favoring the 1,3-proton shift and accelerating the transamination process. The control experiment confirmed the amazing effect of the amine side arm again (Fig. 6b, 1b vs 1f). Introducing an acetyl group onto the nitrogen to eliminate the basicity of the amine on the side arm led to marked decreases in activity and enantioselectivity. The amine side arm not only promotes the 1,3-proton shift by acting as an intramolecular base to deprotonate the benzylic C-H of ketimine 11 (Fig. 6a) but also helps to orient the α-keto amide by hydrogen bonding with the carbonyl oxygen of the amide group (Fig. 6c), resulting in improved activity and stereoselectivity [48][49][50] . Protonation of the delocalized carbanion 12 occurs at α−position of the amide group from the up side of the pyridine ring away from the amine side arm 26,46,47 , to form the newly generated chiral center with S configuration from catalyst (R)-1b.

Discussion
In summary, based on the concept of multiple imitation of transaminases, we have developed N-quaternized axially chiral pyridoxamines 1 containing an amine side arm. With pyridoxamine 1b as the catalyst, challenging substrates α-keto amides were successfully transaminated to peptides in good yields with excellent enantio-and diastereoselectivities. The catalyst dominated the diastereoselective control for the transamination of chiral α-keto amides. Thus, a pair of diastereomeric peptides could be respectively obtained with high enantiopurities by switching the configuration of the pyridoxamine catalyst. The strong electron-withdrawing property of the N-quaternized pyridine ring together with the cooperative catalysis of the amine side arm account for the increased catalytic activity and selectivity of the pyridoxamine 1b in the transamination. The reaction can provide an efficient strategy for divergent and successive extension of peptides via condensation-transamination reaction sequence, which is especially attractive for the synthesis of peptides made of unnatural amino acids.

Methods
General procedure for the asymmetric biomimetic transamination Reaction (Fig. 4). A mixture of α-keto amide 2 (0.10 mmol), chiral pyridoxamine 1b (0.0050 mmol), 2,2-diphenylglycine (4) (0.11 mmol), HOAc (0.40 mmol), Na 2 HPO 4 or KOAc (0.20 mmol), CF 3 CH 2 OH or MeOH (0.48 mL), and H 2 O (0.12 mL) was stirred at 16-25°C for the specified time. For glycinyl α-keto amides (for 3a-l) and α-keto phenylbutanamides of amino acid esters (for 3m-y), the crude reaction mixtures were treated with di-tert-butyl dicarbonate (0.3 mmol) at room temperature for 3 h after the transamination was completed, then concentrated via rotary evaporator to remove most of the solvent and isolated by column chromatography on silica gel with a mixed solvent ethyl acetate and petroleum ether as the eluant to give the products N-Boc-protected dipeptides 3a-y. For Peptidyl αketo amides (for 3z-ad), the reaction mixtures were submitted to concentration via rotary evaporator to remove most of the solvent and then isolated by column chromatography on silica gel with a mixed solvent of dichloromethane, methanol and ammonia solution in ethanol (2.9 M) as the eluant to give the transamination products tripeptides 3z-aa and tetrapeptides 3ab-ad without NH 2 -protection. The ee and dr values of 3a-ac were determined by HPLC analysis.