Efficient Tandem Biocatalytic Process for the Kinetic Resolution of Aromatic β‐Amino Acids

We describe a simple and efficient enzymatic tandem reaction for the preparation of enantiomerically pure β‐phenylalanine and its analogues from the corresponding racemates. In this process, phenylalanine aminomutase (PAM) catalyzes the stereoselective isomerization of (R)‐β‐phenylalanines to (S)‐α‐phenylalanines, which are in situ transformed to cinnamic acids by phenylalanine ammonia lyase (PAL). Preparative scale conversions are done with a mutated PAM with enhanced catalytic activity.     

Chemoenzymatic Preparation of Enantiopure Homoadamantyl β‐Amino Acid and β‐Lactam Derivatives

Racemic cis‐10‐azatetracyclo[7.2.0.1^2,6.1^4,8]tridecan‐11‐one was prepared from homoadamant‐4‐ene by chlorosulfonyl isocyanate addition. The transformation of the β‐lactam to the corresponding β‐amino ester followed by Candida antarctica lipase A‐catalyzed enantioselective (E>>200) N‐acylation with 2,2,2‐trifluoroethyl butanoate afforded methyl (1R,4R,5S,8S)‐5‐aminotricyclo[4.3.1.1^3,8]undecane‐4‐carboxylate and the (1S,4S,5R,8R)‐butanamide with>99% ee at 50% conversion. Alternatively, transformation of the β‐lactam to the corresponding N‐hydroxymethyl‐β‐lactam and the following Pseudomonas cepacia (currently Burkholderia cepacia) lipase‐catalyzed enantioseletive O‐acylation provided the (1S,4S,6R,9R)‐alcohol (ee=87%) and the corresponding (1R,4R,6S,9S)‐butanoate (ee>99%). In the latter method, competition for the enzyme between the (1R,4R,6S,9S)‐butanoate, 2,2,2‐trifluoroethyl butanoate and the hydrolysis product, butanoic acid, tended to stop the reaction at about 45% conversion and finally gave racemization in the (1S,4S,6R,9R)‐alcohol with time.     

Multi‐Enzymatic Synthesis of Optically Pure β‐Hydroxy α‐Amino Acids

A novel enzymatic production system of optically pure β‐hydroxy α‐amino acids was developed. Two enzymes were used for the system: an N‐succinyl L ‐amino acid β‐hydroxylase (SadA) belonging to the iron(II)/α‐ketoglutarate‐dependent dioxygenase superfamily and an N‐succinyl L ‐amino acid desuccinylase (LasA). The genes encoding the two enzymes are part of a gene set responsible for the biosynthesis of peptidyl compounds found in the Burkholderia ambifaria AMMD genome. SadA stereoselectively hydroxylated several N‐succinyl aliphatic L ‐amino acids and produced N‐succinyl β‐hydroxy L ‐amino acids, such as N‐succinyl‐L ‐β‐hydroxyvaline, N‐succinyl‐L ‐threonine, (2S,3R)‐N‐succinyl‐L ‐β‐hydroxyisoleucine, and N‐succinyl‐L ‐threo‐β‐hydroxyleucine. LasA catalyzed the desuccinylation of various N‐succinyl‐L ‐amino acids. Surprisingly, LasA is the first amide bond‐forming enzyme belonging to the amidohydrolase superfamily, and has succinylation activity towards the amino group of L ‐leucine. By combining SadA and LasA in a preparative scale production using N‐succinyl‐L ‐leucine as substrate, 2.3 mmol of L ‐threo‐β‐hydroxyleucine were successfully produced with 93% conversion and over 99% of diastereomeric excess. Consequently, the new production system described in this study has advantages in optical purity and reaction efficiency for application in the mass production of several β‐hydroxy α‐amino acids.

     

Phenylalanine Ammonia‐Lyase‐Catalyzed Deamination of an Acyclic Amino Acid: Enzyme Mechanistic Studies Aided by a Novel Microreactor Filled with Magnetic Nanoparticles

Phenylalanine ammonia‐lyase (PAL), found in many organisms, catalyzes the deamination of l ‐phenylalanine (Phe) to (E)‐cinnamate by the aid of its MIO prosthetic group. By using PAL immobilized on magnetic nanoparticles and fixed in a microfluidic reactor with an in‐line UV detector, we demonstrated that PAL can catalyze ammonia elimination from the acyclic propargylglycine (PG) to yield (E)‐pent‐2‐ene‐4‐ynoate. This highlights new opportunities to extend MIO enzymes towards acyclic substrates. As PG is acyclic, its deamination cannot involve a Friedel–Crafts‐type attack at an aromatic ring. The reversibility of the PAL reaction, demonstrated by the ammonia addition to (E)‐pent‐2‐ene‐4‐ynoate yielding enantiopure l ‐PG, contradicts the proposed highly exothermic single‐step mechanism. Computations with the QM/MM models of the N‐MIO intermediates from l ‐PG and l ‐Phe in PAL show similar arrangements within the active site, thus supporting a mechanism via the N‐MIO intermediate.     

Discovery of Novel Tyrosine Ammonia Lyases for the Enzymatic Synthesis of p-Coumaric Acid

p-Coumaric acid (p-CA) is a key precursor for the biosynthesis of flavonoids. Tyrosine ammonia lyases (TALs) specifically catalyze the synthesis of p-CA from l -tyrosine, which is a convenient enzymatic pathway. To explore novel and highly active TALs, a phylogenetic tree-building approach was conducted including 875 putative TALs and 46 putative phenylalanine/tyrosine ammonia lyases (PTALs). Among them, 5 TALs and 3 PTALs were successfully characterized and found to exhibit the proposed enzymatic activity. The TAL from Chryseobacterium luteum sp. nov (TAL_clu) has the highest affinity (K_m =0.019 mm ) and conversion efficiency (k_cat/K_m= 1631 s⁻¹ ⋅ mm ⁻¹) towards l -tyrosine. The reaction conditions for two purified enzymes and their E. coli recombinant cells were optimized and p-CA yields of 2.03 g/L after 8 hours by TAL_clu and 2.35 g/L after 24 h by TAL from Rivularia sp. PCC 7116 (TAL_rpc) in whole cells were achieved. These TALs are thus candidates for the construction of whole-cell systems to produce the flavonoid precursor p-CA.     

Catalytic Asymmetric Synthesis of Functionalized α,α‐Disubstituted α‐Amino Acid Derivatives from Racemic Unprotected α‐Amino Acids via in‐situ Generated Azlactones

Masked and activated highly enantioenriched α,α‐disubstituted α‐amino acids with an additional adjacent stereocenter were formed by a tandem reaction involving five steps using racemic unprotected amino acid substrates. Key step is the 1,4‐addition of in‐situ generated azlactones to a broad number of enones. The products of this step‐economic route can, e.g., be useful for a divergent and rapid access to biologically interesting unnatural glutamic acid derivatives.     

Efficient Synthesis of β,γ‐Alkynyl α‐Amino Acid Derivatives by Ag(I)‐Catalyzed Alkynylation of α‐Imino Esters

The first catalytic synthesis of β,γ‐alkynyl α‐amino acid derivatives was achieved by direct addition of terminal alkynes to α‐imino esters in the presence of an Ag(I) salt under mild reaction conditions.     

Ene‐Carbonyl Reductive Coupling Mediated by Zinc and Ammonia for the Synthesis of γ‐Hydroxybutyric Acid Derivatives

The reductive coupling of an aryl carbonyl group with an activated alkene to give the corresponding γ‐hydroxybutyric acid (GHBA) derivative was achieved in the presence of zinc and ammonia. A board scope of GHBA derivatives could be synthesized under ambient conditions by this method. A mechanism involving a Zn‐ketyl radical as the key intermediate is proposed.     

Organocatalytic Enantioselective Strecker Synthesis of α‐Quaternary α‐Trifluoromethyl Amino Acids

The first organocatalytic enantioselective Strecker synthesis of α‐quaternary α‐trifluoromethylated amino acids has been developed. Employing Takemoto’s thiourea catalyst the nucleophilic addition of trimethylsilyl cyanide to trifluoromethyl ketimines affords α‐amino nitriles in good to excellent yields (50–99%) and very good enantioselectivities (ee=83–95%). The enantiopure amino nitriles can be obtained by recrystallization. Deprotection and hydrolysis leads to the title amino acids.     

In vitro Synthesis of New Cyclodepsipeptides of the PF1022‐Type: Probing the α‐D‐Hydroxy Acid Tolerance of PF1022 Synthetase

The nonribosomal peptide synthetase PF1022‐synthetase (PFSYN) synthesises the cyclooctadepsipeptide PF1022 from the building blocks D ‐lactate, D ‐phenyllactate and N‐methylleucine. The substrate tolerance of PFSYN for hydroxy acids was probed by in vitro screening of a set of aliphatic and aromatic α‐D ‐hydroxy acids with various structural modifications in the side chain. Thus, new PF1022 derivatives for example, propargyl‐D ‐lactyl‐PF1022 and β‐thienyl‐D ‐lactyl‐PF1022 were generated. The promiscuous behaviour of PFSYN towards aliphatic and aromatic α‐D ‐hydroxy acids is considerably larger than that of related enniatin synthetase (ESYN) and thus gives rise to the enzymatic generation of various new PF1022 derivatives.     

重组大肠杆菌苯丙氨酸解氨酶稳定性和反式肉桂酸的转化

为了提高反式肉桂酸合成L-苯丙氨酸的产率,研究了效应物、还原剂和金属离子对重组大肠杆菌苯丙氨酸解氨酶(PAL)稳定性的影响,并利用响应面法优化了反式肉桂酸的转化条件.结果表明,体积浓度为10%的甘油水溶液可以显著提高酶稳定性,巯基乙醇、蔗糖、Mg2 能提高比酶活,但Cu2 和 Co2 使PAL失活.响应面分析结果表明,当pH为9.85,肉桂酸浓度为6.058 g/L, 底物与酶量的体积比率为3.3时, 反式肉桂酸转化率为80.5 %,比不优化条件下的转化率提高了15.7%.     

A General Approach to the Synthesis of β2‐Amino Acid Derivatives via Highly Efficient Catalytic Asymmetric Hydrogenation of α‐Aminomethylacrylates

A new strategy was developed for the synthesis of a valuable class of α‐aminomethylacrylates via the Baylis–Hillman reaction of different aldehydes with methyl acrylate followed by acetylation of the resulting allylic alcohols and S_N2′‐type amination of the allylic acetates. Asymmetric hydrogenation of these diverse olefinic precursors using rhodium(Et‐Duphos) catalysts provided the corresponding β²‐amino acid derivatives with excellent enantioselectivities and exceedingly high reactivities (up to >99.5% ee and S/C=10,000). The first hydrogenation of (Z)‐configurated substrates was studied for the synthesis of β²‐amino acid derivatives. The high influence of the substrate geometry and steric hindrance on the reactivity and enantioselectivity was also disclosed for this reaction. This protocol provides a highly practical, facile and scalable method for the preparation of optically pure β²‐amino acids and their derivatives under mild reaction conditions.     

Asymmetric Synthesis of α‐Amino Acids under Operationally Convenient Conditions

A new multipurpose glycine equivalent for the general asymmetric synthesis of α‐amino acids is introduced. The chiral reagent can be transformed to various amino acids by alkylations with alkyl halides as well as aldol and Michael addition reactions under operationally convenient reaction conditions at room temperature with virtually complete stereochemical control.

     

Ene Reductase‐Catalysed Synthesis of (R)‐Profen Derivatives

Enantiomerically pure (R)‐profen derivatives and intermediates are synthesised utilising the enzyme YqjM, an ene reductase from Bacillus subtilis. After optimisation of the reaction conditions, the chemoenzymatic approach was applied for the first time in the synthesis of (R)‐flurbiprofen methyl ester.     

Production of Dehydrogingerdione Derivatives in Escherichia coli by Exploiting a Curcuminoid Synthase from Oryza sativa and a β‐Oxidation Pathway from Saccharomyces cerevisiae

Gingerol derivatives are bioactive compounds isolated from the rhizome of ginger. They possess various beneficial activities, such as anticancer and hepatoprotective activities, and are therefore attractive targets of bioengineering. However, the microbial production of gingerol derivatives has not yet been established, primarily because the biosynthetic pathway of gingerol is unknown. Here, we report the production of several dehydrogingerdione (a gingerol derivative) analogues from a recombinant Escherichia coli strain that has an “artificial” biosynthesis pathway for dehydrogingerdione that was not based on the original biosynthesis pathway of gingerol derivatives in plants. The system consists of a 4‐coumarate:CoA ligase from Lithospermum erythrorhizon, a fatty acid CoA ligase from Oryza sativa, a β‐oxidation system from Saccharomyces cerevisiae, and a curcuminoid synthase from O. sativa. To our knowledge, this is the first report of the microbial production of a plant metabolite the biosynthetic pathway of which has not yet been identified.     

Different Enzymatic Processing of γ‐Phosphoramidate and γ‐Phosphoester‐Modified ATP Analogues

Monitoring the activity of ATP‐consuming enzymes provides the basis for elucidating their modes of action and regulation. Although a number of ATP analogues have been developed for this, their scope is restricted because of the limited acceptance by respective enzymes. In order to clarify which kind of phosphate‐modified ATP analogues are accepted by the α‐β‐phosphoanhydride‐cleaving ubiquitin‐activating enzyme 1 (UBA1) and the β‐γ‐phosphoanhydride‐cleaving focal adhesion kinase (FAK), we tested phosphoramidate‐ and phosphoester‐modified ATP analogues. UBA1 and FAK were able to convert phosphoramidate‐modified ATP analogues, even with a bulky modification like biotin. In contrast, a phosphoester‐modified analogue was poorly accepted. These results demonstrate that minor variations in the design of ATP analogues for monitoring ATP utilization have a significant impact on enzymatic acceptance.     

Kinetic Resolution of Aliphatic β‐Amino Acid Amides by β‐Aminopeptidases

Access to enantiopure β‐amino acids : β‐Aminopeptidases are hydrolases that possess the unique ability to cleave N‐terminal β‐amino acids from peptides and amides. Hydrolysis of racemic β‐amino acid amides catalyzed by these enzymes displays enantioselectivity with strong preference for substrates with the L ‐configuration, and gives access to various aliphatic β‐amino acids of high enantiopurity.

     

Asymmetric Synthesis of 3,4‐Dihydrocoumarins Bearing an α,α‐Disubstituted Amino Acid Moiety

An organocatalytic approach for the stereoselective synthesis of 3,4‐dihydrocoumarins with an α,α‐disubstituted amino acid moiety incorporated is presented. The developed methodology is based on the cascade reaction between α‐substituted azlactones and 2‐hydroxychalcones. It is initiated by a chiral Brønsted base‐catalyzed enantio‐ and diastereoselective Michael reaction followed by the azlactone ring opening to construct a 3,4‐dihydrocoumarin framework. Products bearing two adjacent stereogenic centers, one being quaternary, were formed with high enantioselectivities and excellent diastereoselectivities. Furthermore, the complete regioselectivity of the new cascade reactivity is worthy of notice.