Design of Lanthanide Fingers: Compact Lanthanide‐Binding Metalloproteins

Lanthanides have interesting chemical properties; these include luminescent, magnetic, and catalytic functions. Toward the development of proteins incorporating novel functions, we have designed a new lanthanide‐binding motif, lanthanide fingers. These were designed based on the Zif268 zinc finger, which exhibits a ββα structural motif. Lanthanide fingers utilize an Asp₂Glu₂ metal‐coordination environment to bind lanthanides through a tetracarboxylate peptide ligand. The iterative design of a general lanthanide‐binding peptide incorporated the following key elements: 1) residues with high α‐helix and β‐sheet propensities in the respective secondary structures; 2) an optimized big box α‐helix N‐cap; 3) a Schellman α‐helix C‐cap motif; and 4) an optional D ‐Pro‐Ser type II’ β‐turn in the β‐hairpin. The peptides were characterized for lanthanide binding by circular dichroism (CD), NMR, and fluorescence spectroscopy. In all instances, stabilization of the peptide secondary structures resulted in an increase in metal affinity. The optimized protein design was a 25‐residue peptide that was a general lanthanide‐binding motif; this binds all lanthanides examined in a competitive aqueous environment, with a dissociation constant of 9.3 μM for binding Er³⁺. CD spectra of the peptide‐lanthanide complexes are similar to those of zinc fingers and other ββα proteins. Metal binding involves residues from the N‐terminal β‐hairpin and the C terminal α‐helical segments of the peptide. NMR data indicated that metal binding induced a global change in the peptide structure. The D ‐Pro‐Ser type II’ β‐turn motif could be replaced by Thr–Ile to generate genetically encodable lanthanide fingers. Replacement of the central Phe with Trp generated genetically encodable lanthanide fingers that exhibited terbium luminescence greater than that of an EF‐hand peptide.     

N‐Alkyl‐, 1‐C‐Alkyl‐, and 5‐C‐Alkyl‐1,5‐dideoxy‐1,5‐imino‐(l)‐ribitols as Galactosidase Inhibitors

A series of 1,5‐dideoxy‐1,5‐imino‐(l )‐ribitol (DIR) derivatives carrying alkyl or functionalized alkyl groups were prepared and investigated as glycosidase inhibitors. These compounds were designed as simplified 4‐epi‐isofagomine (4‐epi‐IFG) mimics and were expected to behave as selective inhibitors of β‐galactosidases. All compounds were indeed found to be highly selective for β‐galactosidases versus α‐glycosidases, as they generally did not inhibit coffee bean α‐galactosidase or other α‐glycosidases. Some compounds were also found to be inhibitors of almond β‐glucosidase. The N‐alkyl DIR derivatives were only modest inhibitors of bovine β‐galactosidase, with IC₅₀ values in the 30–700 μm range. Likewise, imino‐l ‐ribitol substituted at the C1 position was found to be a weak inhibitor of this enzyme. In contrast, alkyl substitution at C5 resulted in enhanced β‐galactosidase inhibitory activity by a factor of up to 1000, with at least six carbon atoms in the alkyl substituent. Remarkably, the ‘pseudo‐anomeric’ configuration in this series does not appear to play a role. Human lysosomal β‐galactosidase from leukocyte lysate was, however, poorly inhibited by all iminoribitol derivatives tested (IC₅₀ values in the 100 μm range), while 4‐epi‐IFG was a good inhibitor of this enzyme. Two compounds were evaluated as pharmacological chaperones for a GM1‐gangliosidosis cell line (R301Q mutation) and were found to enhance the mutant enzyme activity by factors up to 2.7‐fold.     

An Engineered Tryptophan Synthase Opens New Enzymatic Pathways to β‐Methyltryptophan and Derivatives

β‐Methyltryptophans (β‐mTrp) are precursors in the biosynthesis of bioactive natural products and are used in the synthesis of peptidomimetic‐based therapeutics. Currently β‐mTrp is produced by inefficient multistep synthetic methods. Here we demonstrate how an engineered variant of tryptophan synthase from Salmonella (StTrpS) can catalyse the efficient condensation of l ‐threonine and various indoles to generate β‐mTrp and derivatives in a single step. Although l ‐serine is the natural substrate for TrpS, targeted mutagenesis of the StTrpS active site provided a variant (βL166V) that can better accommodate l ‐Thr as a substrate. The condensation of l ‐Thr and indole proceeds with retention of configuration at both α‐ and β‐positions to give (2S,3S)‐β‐mTrp. The integration of StTrpS (βL166V) with l ‐amino acid oxidase, halogenase enzymes and palladium chemocatalysts provides access to further d ‐configured and regioselectively halogenated or arylated β‐mTrp derivatives.     

Efficient Biosynthesis of (R)‐ or (S)‐2‐Hydroxybutyrate from l‐Threonine through a Synthetic Biology Approach

An efficient multi‐enzyme cascade reaction for the synthesis of (R)‐ or (S)‐2‐hydroxybutyric acid [(R)‐ or (S)‐2‐HB] from l ‐threonine was developed by using recombinant Escherichia coli cells expressing separately or co‐expressing l ‐threonine deaminase from Escherichia coli K‐12 (ilvA), formate dehydrogenase (FDH) from Candida boidinii and l ‐lactate dehydrogenase (l ‐LDH) from Oryctolagus cuniculus or d ‐lactate dehydrogenase (d ‐LDH) from Staphylococcus epidermidis ATCC 12228. Up to 750 mM of l ‐threonine were completely transformed to (R)‐ or (S)‐2‐HB in optically pure form (>99% ee) with high isolated yields. This one‐pot multi‐enzyme transformation provides a new practical method for the synthesis of these important optically pure compounds.

     

Chiral Lewis Base‐Catalyzed,Enantioselective Reduction of Unprotected β‐Enamino Esters with Trichlorosilane

Catalytic asymmetric reduction of N‐unsubstituted β‐enamino esters represents a major challenge for asymmetric catalysis. In this paper, the first organocatalytic system that could be used for the asymmetric hydrosilylation of N‐unsubstituted β‐enamino esters has been developed. Using N‐tert‐butylsulfinyl‐L ‐proline‐derived amides and L ‐pipecolinic acid‐derived formamides as catalyst, a broad range of β‐aryl‐ and β‐alkyl‐substituted free β‐amino esters could be prepared with high yields and enantioselectivities. The practicality was illustrated by the gram‐scale asymmetric synthesis of ethyl (R)‐3‐amino‐3‐phenylpropanoate and isopropyl (S)‐3‐amino‐4‐(2,3,5‐trifluorophenyl)butanoate. The resulting product can be smoothly transformed to the FDA approved medicines dapoxetine and sitagliptin in a short synthetic route.

     

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.

     

Pipecolic Acid Hydroxylases: A Monophyletic Clade among cis‐Selective Bacterial Proline Hydroxylases that Discriminates l‐Proline

Proline hydroxylases are iron(II)/2‐oxoglutarate‐dependent enzymes that hydroxylate l ‐proline and derivatives, such as l pipecolic acid, which is the six‐membered‐ring homologue of l ‐proline. It has been established that there is a distinct group of conserved bacterial enzymes that hydroxylate l ‐pipecolic acid and trans‐3‐ and trans‐4‐methyl‐l ‐proline, but virtually no l ‐proline. This allows the organism to produce hydroxyproline congeners without hydroxylation of the physiologically omnipresent l ‐proline. In vitro conversions showed that the substrate spectrum of the pipecolic acid hydroxylases GetF (from a Streptomyces sp.; producer of the tetrapeptide antibiotic GE81112) and PiFa (from Frankia alni) overlaps that of proline hydroxylases, except for the nonacceptance of l ‐proline and smaller homologues. Distinct and conserved residues were determined for both types of enzymes. However, site‐directed mutagenesis in GetF did not yield variants that accepted l ‐proline; this suggested a complex interaction of several residues around the active site, which resulted in delicate changes in substrate specificity. This is supported by substrate docking in a homology model of GetF, which revealed an altered orientation for l ‐proline relative to that of preferred substrates.     

Two Opposing d‐Amino Acids Give Zigzag Hairpin Epitopes an Additional Kink to Create Antibody‐Selective Peptide Antigens

We have developed peptides that are able to distinguish between subgroups of polyclonal antibodies. These β‐hairpin peptides act as conformational epitopes with specific shape and flexibility; they have been analyzed by NMR and CD spectroscopy, and have been shown to identify known disease markers. As a standalone mini β‐sheet, a hairpin is stabilized by alternating pairs of hydrogen‐bonded and non‐bonded amino acids on its two opposing peptide strands. A single d mutation disrupts this secondary structure, the correlated double‐d mutation of two opposing amino acids compensates for this destabilizing effect. The designed kink was introduced into both hydrogen‐bonded and ‐non‐bonded positions of an all‐l hairpin that is a known conformational epitope in molecular recognition. Our peptides enabled the discrimination of different human rheumatoid arthritis autoantibodies in an ELISA assay.     

Fully bio‐based poly(ɛ‐capolactone)/poly(lactide) alternating multiblock supramolecular polymers: Synthesis,crystallization behavior,and properties

Supramolecular poly(?‐capolactone)/poly(lactide) alternating multiblock copolymers were prepared by UPy‐functionalized poly(lactide)‐b‐ poly(?‐capolactone)‐b‐ poly(lactide) copolymers. The prepared supramolecular polymers (SMPs) exhibit the characteristic properties of thermoplastic elastomers. The stereo multiblock SMPs (sc‐SMPs) were formed by blending UPy‐functionalized poly(l ‐lactide)‐b‐ PCL‐b‐ poly(l ‐lactide) (l ‐SMPs) and UPy‐functionalized poly(d ‐lactide)‐b‐ PCL‐b‐ poly(d ‐lactide) (d ‐SMPs) due to stereocomplexation of the PLLA and PDLA blocks. Sc‐SMPs with low content of d ‐SMPs (≤20%) are transparent, elastic solids, while those having high d ‐SMPs content are opaque, brittle solids. The effects of l ‐SMPs/d ‐SMPs mixing ratios on thermal, crystallization behaviors, crystal structure, mechanical and hydrophilic properties of sc‐SMPs were deeply investigated. The incorporation of UPy groups depresses the crystallization of polymer, and the stereocomplex formation accelerates the crystallization rate. The used initiator functionalized polyhedral oligomeric silsesquioxanes causes a different effect on the crystallization of PLA and PCL blocks. The tensile strength and elongation at break of l _d /d _d ‐SMPs (d represents the initiator diethylene glycol) are significantly larger than that of l _p /d _p ‐SMPs (p represents the initiator polyhedral oligomeric silsesquioxanes), and their heat resistance and hydrophilicity can be also modulated by the l ‐SMPs/d ‐SMPs mixing ratios and the different initiators. © 2017 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2017 , 134 , 45575.     

Reverse Micellar Encapsulation of d‐ and l‐Enantiomers of Some Aromatic α‐Amino Acids and Nucleobases by Glucose‐Derived Non‐ionic Gemini Surfactants in Neat n‐Hexane

Novel carbohydrate‐based non‐ionic gemini surfactants consisting of two sugar head groups, two hydrophobic tails having chain lengths of C12, C14, and C16 and a flexible –(CH₂)₆– spacer were synthesized and investigated for their reverse micellar encapsulation properties. The head groups of the geminis comprise glucose entities (with reducing function blocked in a cyclic acetal group) connected through C‐6 to tertiary amines. These surfactants were explored for reverse micellar encapsulation of d ‐ and l ‐enantiomers of aromatic α‐amino acids viz. histidine (His), phenylalanine (Phe), tyrosine (Tyr) and tryptophan (Trp) in neat n‐hexane. Similar studies were carried out for encapsulation of nucleobases viz. adenine (Ade), guanine (Gua), thymine (Thy), cytosine (Cyt) and Uracil (Ura). Reverse micellar studies revealed that aromatic α‐amino acids were encapsulated in the sequence His>Tyr>Phe>Trp. In most cases, a difference in the degree of encapsulation of d ‐ and l ‐enantiomers of aromatic amino acids in reverse micellar phases of gemini amphiphiles in neat n‐hexane, was revealed. For Tyr, l ‐enantiomer was better encapsulated than its antipode, i.e., d ‐enantiomer but for Trp, d ‐enantiomer was better encapsulated then l ‐enantiomer. In the case of nucleobases, Ura was found selectively encapsulated by reverse micelles formed by these new amphiphiles.     

Characterization of a Galactosynthase Derived from Bacillus circulans β‐Galactosidase: Facile Synthesis of D‐Lacto‐ and D‐Galacto‐N‐bioside

Glycosynthases—retaining glycosidases mutated at their catalytic nucleophile—catalyze the formation of glycosidic bonds from glycosyl fluorides as donor sugars and various glycosides as acceptor sugars. Here the first glycosynthase derived from a family 35 β‐galactosidase is described. The Glu→Gly mutant of BgaC from Bacillus circulans (BgaC‐E233G) catalyzed regioselective galactosylation at the 3‐position of the sugar acceptors with α‐galactosyl fluoride as the donor. Transfer to 4‐nitophenyl α‐D ‐N‐acetyl‐glucosaminide and α‐D ‐N‐acetylgalactosaminide yielded 4‐nitophenyl α‐lacto‐N‐biose and α‐galacto‐N‐biose, respectively, in high yields (up to 98 %). Kinetic analysis revealed that the high affinity of the acceptors contributed mostly to the BgaC‐E233G‐catalyzed transglycosylation. BgaC‐E233G showed no activity with β‐(1,3)‐linked disaccharides as acceptors, thus suggesting that this enzyme can be used in “one‐pot synthesis” of LNB‐ or GNB‐containing glycans.     

Chemoenzymatic Synthesis of GDP‐L‐Fucose Derivatives as Potent and Selective α‐1,3‐Fucosyltransferase Inhibitors

Fucosyltransferases (FucTs) usually catalyze the final step of glycosylation and are critical to many biological processes. High levels of specific FucT activities are often associated with various cancers. Here we report the development of a chemoenzymatic method for synthesizing a library of guanosine diphosphate β‐L ‐fucose (GDP‐Fuc) derivatives, followed by in situ screening for inhibitory activity against bacterial and human α‐1,3‐FucTs. Several compounds incorporating appropriate hydrophobic moieties were identified from the initial screening. These were individually synthesized, purified and characterized in detail for their inhibition kinetics. Compound 5 had a K_i of 29 nM for human FucT‐VI, and is 269 and 11 times more selective than for Helicobacter pylori FucT (K_i=7.8 μM) and for human FucT‐V (K_i=0.31 μM).     

Endo‐β‐Glucosidase Tag Allows Dual Detection of Fusion Proteins by Fluorescent Mechanism‐Based Probes and Activity Measurement

β‐Glucoside‐configured cyclophellitols are activity‐based probes (ABPs) that allow sensitive detection of β‐glucosidases. Their applicability to detect proteins fused with β‐glucosidase was investigated in the cellular context. The tag was Rhodococcus sp. M‐777 endoglycoceramidase II (EGCaseII), based on its lack of glycans and ability to hydrolyze fluorogenic 4‐methylumbelliferyl β‐d ‐lactoside (an activity absent in mammalian cells). Specific dual detection of fusion proteins was possible in vitro and in situ by using fluorescent ABPs and a fluorogenic substrate. Pre‐blocking with conduritol β‐epoxide (a poor inhibitor of EGCaseII) eliminated ABP labeling of endogenous β‐glucosidases. ABPs equipped with biotin allowed convenient purification of the fusion proteins. Diversification of ABPs (distinct fluorophores, fluorogenic high‐resolution detection moieties) should assist further research in living cells and organisms.     

First Examples of Proline‐Catalyzed Domino Knoevenagel/Hetero‐Diels–Alder/Elimination Reactions

A novel and efficient one‐pot synthesis of lactones (pyranones) has been achieved by domino Knoevenagel/hetero‐Diels–Alder/elimination reactions of O‐ and N‐prenyl aldehyde derivatives with Meldrum's acid in the presence of L ‐ or D ,L ‐proline. The reaction proceeds cleanly at room temperature to afford cis‐ or trans‐fused products in good yields with high diastereoselectivity.     

Biosynthetic Gene Cluster of a d‐Tryptophan‐Containing Lasso Peptide,MS‐271

MS‐271, produced by Streptomyces sp. M‐271, is a lasso peptide natural product comprising 21 amino acid residues with a d ‐tryptophan at its C terminus. Because lasso peptides are ribosomal peptides, the biosynthesis of MS‐271, especially the mechanism of d ‐Trp introduction, is of great interest. The MS‐271 biosynthetic gene cluster was identified by draft genome sequencing of the MS‐271 producer, and it was revealed that the precursor peptide contains all 21 amino acid residues including the C‐terminal tryptophan. This suggested that the d ‐Trp residue is introduced by epimerization. Genes for modification enzymes such as a macrolactam synthetase (mslC), precursor peptide recognition element (mslB1), cysteine protease (mslB2), disulfide oxidoreductases (mslE, mslF), and a protein of unknown function (mslH) were found in the flanking region of the precursor peptide gene. Although obvious epimerase genes were absent in the cluster, heterologous expression of the putative MS‐271 cluster in Streptomyces lividans showed that it contains all the necessary genes for MS‐271 production including a gene for a new peptide epimerase. Furthermore, a gene‐deletion experiment indicated that MslB1, ‐B2, ‐C and ‐H were indispensable for MS‐271 production and that some interactions of the biosynthetic enzymes were essential for the biosynthesis of MS‐271.     

Phage Display on the Anti‐infective Target 1‐Deoxy‐d‐xylulose‐5‐phosphate Synthase Leads to an Acceptor–Substrate Competitive Peptidic Inhibitor

Enzymes of the 2‐C‐methyl‐d ‐erythritol‐4‐phosphate pathway for the biosynthesis of isoprenoid precursors are validated drug targets. By performing phage display on 1‐deoxy‐d ‐xylulose‐5‐phosphate synthase (DXS), which catalyzes the first step of this pathway, we discovered several peptide hits and recognized false‐positive hits. The enriched peptide binder P12 emerged as a substrate (d ‐glyceraldehyde‐3‐phosphate)‐competitive inhibitor of Deinococcus radiodurans DXS. The results indicate possible overlap of the cofactor‐ and acceptor‐substrate‐binding pockets and provide inspiration for the design of inhibitors of DXS with a unique and novel mechanism of inhibition.     

Walking a Fine Line with Sucrose Phosphorylase: Efficient Single‐Step Biocatalytic Production of l‐Ascorbic Acid 2‐Glucoside from Sucrose

The 2‐O‐α‐d ‐glucoside of l ‐ascorbic acid (AA‐2G) is a highly stabilized form of vitamin C, with important industrial applications in cosmetics, food, and pharmaceuticals. AA‐2G is currently produced through biocatalytic glucosylation of l ‐ascorbic acid from starch‐derived oligosaccharides. Sucrose would be an ideal substrate for AA‐2G synthesis, but it lacks a suitable transglycosidase. We show here that in a narrow pH window (pH 4.8–6.0, with sharp optimum at pH 5.2), sucrose phosphorylases catalyzed the 2‐O‐α‐glucosylation of l ‐ascorbic acid from sucrose with high efficiency and perfect site‐selectivity. Optimized synthesis with the enzyme from Bifidobacterium longum at 40 °C gave a concentrated product (155 g L^?1; 460 mm ), from which pure AA‐2G was readily recovered in ～50 % overall yield, thus providing the basis for advanced production. The peculiar pH dependence is suggested to arise from a “reverse‐protonation” mechanism in which the catalytic base Glu232 on the glucosyl–enzyme intermediate must be protonated for attack on the anomeric carbon from the 2‐hydroxyl of the ionized l ‐ascorbate substrate.     

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.     

The Thioesterase Bhp is Involved in the Formation of β‐Hydroxytyrosine during Balhimycin Biosynthesis in Amycolatopsis balhimycina

The putative hydrolase gene bhp from the balhimycin biosynthetic gene cluster has been cloned and overexpressed in Escherichia coli. The corresponding enzyme Bhp was purified to homogeneity by nickel‐chelating chromatography and characterized. Although Bhp has sequence similarities to hydrolases with “haloperoxidase”/perhydrolase activity, it did not show any enzymatic activity with standard “haloperoxidase”/perhydrolase substrates (e.g., monochlorodimedone and phenol red), nonspecific esterase substrates (such as p‐nitrophenyl acetate, p‐nitrophenyl phosphate and S‐thiophenyl acetate) or the model lactonase substrate dihydrocoumarin. However, Bhp could be shown to catalyse the hydrolysis of S‐β‐hydroxytyrosyl‐N‐acetyl cysteamine thioester (β‐OH‐Tyr‐SNAC) with 15 times the efficiency of S‐L ‐tyrosyl‐N‐acetyl cysteamine thioester (L ‐Tyr‐SNAC). This is in agreement with the suggestion that Bhp is involved in balhimycin biosynthesis, during which it was supposed to catalyse the hydrolysis of β‐OH‐Tyr‐S‐PCP (PCP=peptidyl carrier protein) to free β‐hydroxytyrosine (β‐OH‐Tyr) and strongly suggests that Bhp is a thioesterase with high substrate specificity for PCP‐bound β‐OH‐Tyr and not a “haloperoxidase”/perhydrolase or nonspecific esterase.     

6‐Amino‐6‐deoxy‐5,6‐di‐N‐(N′‐octyliminomethylidene)nojirimycin: Synthesis,Biological Evaluation,and Crystal Structure in Complex with Acid β‐Glucosidase

6‐Amino‐6‐deoxy‐5,6‐di‐ N ‐( N ′‐octyliminomethylidene)nojirimycin , a reducing analogue of N‐nonyl‐1‐deoxynojirimycin, proved to be a potent and very selective inhibitor of β‐glucosidases, including human acid β‐glucosidase. Structural studies of the enzyme–inhibitor complex showed a binding mode in which the anomeric hydroxy group is accommodated in the “wrong” α configuration.