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.     

CGTase‐Catalysed cis‐Glucosylation of L‐Rhamnosides for the Preparation of Shigella flexneri 2a and 3a Haptens

We report the enzymatic synthesis of α‐D ‐glucopyranosyl‐(1→4)‐α‐L ‐rhamnopyranoside and α‐D ‐glucopyranosyl‐(1→3)‐α‐L ‐rhamnopyranoside by using a wild‐type transglucosidase in combination with glucoamylase and glucose oxidase. It was shown that Bacillus circulans 251 cyclodextrin glucanotransferase (CGTase, EC 2.1.4.19) can efficiently couple an α‐L ‐rhamnosyl acceptor to a maltodextrin molecule with an α‐(1→4) linkage, albeit in mixture with the α‐(1→3) regioisomer, thus giving two glucosylated acceptors in a single reaction. Optimisation of the CGTase coupling reaction with β‐cyclodextrin as the donor substrate and methyl or allyl α‐L ‐rhamnopyranoside as acceptors resulted in good conversion yields (42–70 %) with adjustable glycosylation regioselectivity. Moreover, the efficient chemical conversion of the products of CGTase‐mediated cis‐glucosylation into protected building blocks (previously used in the synthesis of O‐antigen fragments of several Shigella flexneri serotypes) was substantiated. These novel chemoenzymatic strategies towards useful, convenient intermediates in the synthesis of S. flexneri serotypes 2a and 3a oligosaccharides might find applications in developments towards synthetic carbohydrate‐based vaccine candidates against bacillary dysentery.     

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.

     

α‐L‐Rhamnosyl‐β‐D‐glucosidase (Rutinosidase) from Aspergillus niger: Characterization and Synthetic Potential of a Novel Diglycosidase

We report the first heterologous production of a fungal rutinosidase (6‐O‐α‐L ‐rhamnopyranosyl‐β‐D ‐glucopyranosidase) in Pichia pastoris. The recombinant rutinosidase was purified from the culture medium to apparent homogeneity and biochemically characterized. The enzyme reacts with rutin and cleaves the glycosidic linkage between the disaccharide rutinose and the aglycone. Furthermore, it exhibits high transglycosylation activity, transferring rutinose from rutin as a glycosyl donor onto various alcohols and phenols. The utility of the recombinant rutinosidase was demonstrated by its use for the synthesis of a broad spectrum of rutinosides of primary (saturated and unsaturated), secondary, acyclic and phenolic alcohols as well as for the preparation of free rutinose. Moreover, the α‐L ‐rhamnosidase‐catalyzed synthesis of a chromogenic substrate for a rutinosidase assay – para‐nitrophenyl β‐rutinoside – is described.

     

Organocatalytic Enantioselective Sulfenylation of β‐Keto Phosphonates: A Convenient Approach to Construct Hetero‐ Quaternary Stereocenters

The highly effective and enantioselective sulfenylation of β‐keto phosphonates catalyzed by α,α‐diaryl‐L ‐prolinols has been developed. The optically active α‐sulfenylated β‐keto phosphonates could be obtained under mild reaction conditions in good yields (up to 92%) and with excellent enantioselectivities (up to 92% ee).     

Synthesis of D‐Galactofuranose‐Containing Molecules: Design of Galactofuranosyl Acceptors

D ‐Galactofuranose (D ‐Galf) is present in glycoconjugates of several pathogenic microorganisms but is absent in mammals, so it is a good target for the development of chemotherapeutic agents for the treatment of microbial infections. This fact has increased interest in the synthesis of D ‐Galf‐containing molecules for corresponding glycobiological studies. The synthesis of oligosaccharides, glycoconjugates, and mimetics of D ‐Galf requires specific methods for the preparation of galactose derivatives in the furanosic configuration, the synthesis of appropriate acceptors, and efficient glycosylation methods for the construction of α‐ and β‐D ‐Galf linkages. This review summarizes the different strategies developed for the preparation of partially protected derivatives of D ‐Galf, suitable as acceptors for the construction of (1→2), (1→3), (1→5), and (1→6) link‐ ages, and describes recent applications.     

3‐ and 4‐Uloses Derived from N‐Acetyl‐D‐glucosamine: A Unique Pair of Complementary Organocatalysts for Asymmetric Epoxidation of Alkenes

The 4‐ulose and the 3‐ulose, both derived in two steps from the α‐methyl glycoside of N‐acetyl‐D ‐glucosamine (GlcNAc), act as organocatalysts in the asymmetric epoxidation of alkenes, with unprecedented complementary enantioselectivity. The best results are found with α,β‐unsaturated esters as substrates, with enantiomeric ratios up to 90:10 and 11:89, respectively.     

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.     

One‐Pot Cascade Reactions using Fructose‐6‐phosphate Aldolase: Efficient Synthesis of D‐Arabinose 5‐Phosphate,D‐Fructose 6‐Phosphate and Analogues

One‐pot multienzymatic reactions have been performed for the synthesis of 1‐deoxy‐D ‐fructose 6‐phosphate, 1,2‐dideoxy‐D ‐arabino‐hept‐3‐ulose 7‐phosphate, D ‐fructose 6‐phosphate and D ‐arabinose 5‐phosphate. The whole synthetic strategy is based on an aldol addition reaction catalysed by fructose‐6‐phosphate aldolase (FSA) as a key step of a three or four enzymes‐catalysed cascade reaction. The four known donors for FSA – dihydroxyacetone (DHA), hydroxyacetone (HA), 1‐hydroxy‐2‐butanone (HB) and glycolaldehyde (GA) – were used with D ‐glyceraldehyde 3‐phosphate as acceptor substrate. The target phosphorylated sugars were obtained in good to excellent yields and high purity.     

Identification and Characterization of D‐Succinylase,and a Proposed Enzymatic Method for D‐Amino Acid Synthesis

Chiral amino acids are important intermediates for the pharmaceutical industry. We have developed a novel one‐pot enzymatic method for D ‐amino acid synthesis by the dynamic kinetic resolution of N‐succinyl‐dl ‐amino acids using D ‐succinylase (DSA) and N‐succinylamino acid racemase (NSAR, EC 4.2.1.113). The DSA from Cupriavidus sp. P4‐10‐C, which hydrolyzes N‐succinyl‐D ‐amino acids enantioselectively to their corresponding D ‐amino acids, was identified for the first time by screening soil microorganisms. Subsequently, the DSA gene was cloned and overexpressed in Escherichia coli. DSA was shown to comprise two subunits with molecular masses of 26 kDa and 60 kDa. Additionally, the NSAR gene from Geobacillus stearothermphilus NCA1503, which racemizes N‐succinylamino acids, was also cloned and overexpressed in E. coli. The highly purified DSA and NSAR prepared from each recombinant E. coli were characterized and used for D ‐amino acid synthesis. A one‐pot enzymatic method converted 100 mM N‐succinyl‐dl ‐phenylalanine to D ‐phenylalanine in 91.1% conversion with 86.7% ee. This novel enzymatic method may be useful for the industrial production of many D ‐amino acids.

     

Synthese und Charakterisierung von O2S2 — und N2S2‐Übergangsmetallkomplexen ausgehend von β‐Chlor) β‐trifluormethylvinylaldehyden

Synthesis and Characterization of O₂S₂ — and N₂S₂‐Transition Metal Complexes Starting from β‐Chloro‐β‐trifluoromethyl Vinylaldehydes The syntheses of complexes 4 and 5 with O₂S₂ ‐and N₂S₂ — donor atom sets are described as one‐step procedures. Their structures were confirmed by NMR, IR, UV‐ VIS and MS spectroscopy. One nickel complex 5a was determined by X‐ray structure analysis whereas the Cu^II complexes were studied by EPR spectroscopy.     

Practical Enantioselective Synthesis of β‐Lactones Catalyzed by Aluminum Bissulfonamide Complexes

The development of an efficient and practical aluminum‐bissulfonamide complex catalyzed enantioselective formation of β‐lactones by [2+2] cycloaddition of ketene (generated in situ from acetyl bromide by dehydrobromination) with various α‐unbranched and ‐branched aliphatic aldehydes is presented. The methodology offers the advantage of operational simplicity not only as the ligand synthesis requires just a single sulfonylation step from commercially available enantiomerically pure diamines. The products are formed in high to excellent yields with ee values typically ranging from 78 to 90 % using 10 mol % of the bissulfonamide ligand. The key finding of this work was a remarkable rate acceleration by using an aluminum/ligand ratio of 1.5:1.     

Enantiopure N‐Benzyloxycarbonyl‐β2‐amino Acid Allyl Esters from Racemic β‐Lactams by Dynamic Kinetic Resolution using Candida antarctica Lipase B

The dynamic kinetic resolution of α‐substituted racemic β‐lactams by alcoholytic ring‐opening, catalyzed by immobilized lipase B from Candida antarctica is described. With this process, a variety of racemic α‐substituted N‐Cbz‐azetidinones (Cbz=benzyloxycarbonyl) was transformed to the corresponding N‐Cbz‐protected β²‐amino acid allyl esters with high enantioselectivity (up to 99%) and high yields (up to quantitative) at room temperature.

     

Glucocerebrosidase Enhancers for Selected Gaucher Disease Genotypes by Modification of α‐1‐C‐Substituted Imino‐D‐xylitols (DIXs) by Click Chemistry

A series of hybrid analogues was designed by combination of the iminoxylitol scaffold of parent 1C9‐DIX with triazolylalkyl side chains. The resulting compounds were considered potential pharmacological chaperones in Gaucher disease. The DIX analogues reported here were synthesized by CuAAC click chemistry from scaffold 1 (α‐1‐C‐propargyl‐1,5‐dideoxy‐1,5‐imino‐D ‐xylitol) and screened as imiglucerase inhibitors. A set of selected compounds were tested as β‐glucocerebrosidase (GBA1) enhancers in fibroblasts from Gaucher patients bearing different genotypes. A number of these DIX compounds were revealed as potent GBA1 enhancers in genotypes containing the G202R mutation, particularly compound DIX‐28 (α‐1‐C‐[(1‐(3‐trimethylsilyl)propyl)‐1H‐1,2,3‐triazol‐4‐yl)methyl]‐1,5‐dideoxy‐1,5‐imino‐D ‐xylitol), bearing the 3‐trimethylsilylpropyl group as a new surrogate of a long alkyl chain, with approximately threefold activity enhancement at 10 nM . Despite their structural similarities with isofagomine and with our previously reported aminocyclitols, the present DIX compounds behaved as non‐competitive inhibitors, with the exception of the mixed‐type inhibitor DIX‐28.     

N,N′‐Dioxide‐Magnesium Ditriflate Complex‐Catalyzed Asymmetric α‐Hydroxylation of β‐Keto Esters and β‐Keto Amides

The highly catalytic asymmetric α‐hydroxylation of 1‐tetralone‐derived β‐keto esters and β‐keto amides using tert‐butyl hydroperoxide (TBHP) as the oxidant was realized by a chiral N,N′‐dioxide‐magnesium ditriflate [Mg(OTf)₂] complex. A series of corresponding chiral α‐hydroxy dicarbonyl compounds was obtained in excellent yields (up to 99%) with excellent enantioselectivities (up to 98% ee). The products were easily transformed into useful building blocks and the precursor of daunomycin was achieved in an asymmetric catalytic way for the first time.     

Asymmetric Aza‐Morita–Baylis–Hillman‐Type Reactions: The Highly Enantioselective Reaction between Unmodified α,β‐ Unsaturated Aldehydes and N‐Acylimines by Organo‐co‐catalysis

The highly enantioselective organo‐co‐catalytic aza‐Morita–Baylis–Hillman (MBH)‐type reaction between N‐carbamate‐protected imines and α,β‐unsaturated aldehydes has been developed. The organic co‐catalytic system of proline and 1,4‐diazabicyclo[2.2.2]octane (DABCO) enables the asymmetric synthesis of the corresponding N‐Boc‐ and N‐Cbz‐protected β‐amino‐α‐alkylidene‐aldehydes in good to high yields and up to 99% ee. In the case of aza‐MBH‐type addition of enals to phenylprop‐2‐ene‐1‐imines, the co‐catalytic reaction exhibits excellent 1,2‐selectivity. The organo‐co‐catalytic aza‐MBH‐type reaction can also be performed by the direct highly enantioselective addition of α,β‐unsaturated aldehydes to bench‐stable N‐carbamate‐protected α‐amidosulfones to give the corresponding β‐amino‐α‐alkylidene‐aldehydes with up to 99% ee. The organo‐co‐catalytic aza‐MBH‐type reaction is also an expeditious entry to nearly enantiomerically pure β‐amino‐α‐alkylidene‐amino acids and β‐amino‐α‐alkylidene‐lactams (99% ee). The mechanism and stereochemistry of the chiral amine and DABCO co‐catalyzed aza‐MBH‐type reaction are also discussed.     

A Kinase‐Independent One‐Pot Multienzyme Cascade for an Expedient Synthesis of Guanosine 5′‐Diphospho‐d‐mannose

Biomimetic synthesis routes towards the important natural d ‐mannosyl donor guanosine 5′‐diphospho‐d ‐mannose (GDP‐Man) rely on kinase‐catalyzed nucleotide triphosphate (NTP)‐dependent phosphorylations of d ‐mannose (Man), to give d ‐mannose 6‐phosphate or α‐d ‐mannose 1‐phosphate (αMan 1‐P) as an intermediate product. A GDP‐Man synthesis not requiring the kinase/NTP system would be practical and cost‐effective. Here, we have developed a multienzyme cascade towards GDP‐Man, characterized in that αMan 1‐P was obtained by a diastereoselective phosphatase‐catalyzed phosphorylation of Man. α‐d ‐Glucose 1‐phosphate (αGlc 1‐P), prepared in situ through phosphorylase‐catalyzed conversion of sucrose in the presence of inorganic phosphate, was used as an expedient phosphoryl donor. The incipient αMan 1‐P and guanosine triphosphate (GTP) were converted into GDP‐Man by a highly manno compared to gluco selective nucleotidyltransferase. Pyrophosphatase was additionally required to hydrolyze the pyrophosphate released from the GTP, thus driving the reaction towards GDP‐Man. The enzymatic cascade was operated with the αMan 1‐P and the GDP‐Man formation decoupled from one another (sequential mode) or having all steps run concurrently (simultaneous mode). Detailed time course analysis revealed that kinetic pull due to the constant removal of the intermediate αMan 1‐P in simultaneous‐mode reactions was important to promote phosphorylation of Man from αGlc 1‐P in high efficiency, avoiding loss of sugar 1‐phosphates by hydrolysis. Under optimized conditions for the one‐pot transformation involving four enzymes, 100 mM (67 g L⁻¹) GDP‐Man was prepared from 140 mM sucrose and phosphate, using 400 mM Man as the phosphoryl acceptor. The product was recovered by anion‐exchange and size‐exclusion chromatography in ≥95% purity in about 50% yield (100 mg). These results demonstrate for the first time the practical use of a phosphorylase‐phosphatase combi‐catalyst as an alternative to the canonical kinase for the anomeric phosphorylation of the sugar substrate in nucleoside diphospho‐sugar synthesis. Phosphorylation from inorganic phosphate via the intermediate αGlc 1‐P rather than from NTP, particularly GTP, appears advantageous specifically in cases where the sugar acceptor is a bulk commodity that can be applied in suitable excess to the phosphatase reaction.

     

Stereoselective Synthesis and Binding Properties of Novel Concave‐Shaped Indolizino[3,4‐b]quinolines

Novel all‐cis‐configurated indolizino[3,4‐b]quinoline receptors 3, 4 were prepared via diastereoselective Lewis acid‐catalyzed cyclization of N‐arylimines 6, 7 as the key step. In order to obtain the indolizino[3,4‐b]quinoline derivative 21 without a gem‐dimethyl group at C‐7, an N‐arylimine precursor 18 bearing a vinyldisilane terminus was prepared in 8 steps from L‐prolinol 15 . In contrast to the known β‐effect of silyl groups cyclization of 18 proceeded via an α‐carbenium ion species to give the diastereomeric products 19, 20, which were desilylated to 21, 22 . The association constants for receptors 2 — 4 and 21 decreased in the order 21 > 2 > 4 > 3 for both acetic acid and N‐Z‐phenylalanine as substrates.     

Iminosugar‐Based Galactoside Mimics as Inhibitors of Galactocerebrosidase: SAR Studies and Comparison with Other Lysosomal Galactosidases

Several families of iminosugar‐based galactoside mimics were designed, synthesized, and evaluated as galactocerebrosidase (GALC) inhibitors. They were also tested as inhibitors of lysosomal β‐ and α‐galactosidases in order to find new potent and selective pharmacological chaperones for treatment of the lysosomal storage disorder, Krabbe disease. Whereas 1‐C‐alkyl imino‐L ‐arabinitols are totally inactive toward the three enzymes, 1‐C‐alkyl imino‐D ‐galactitols were found to be active only toward α‐galactosidase A. Finally, 1‐N‐iminosugars provided the best results, as 4‐epi‐isofagomine was found to be a good inhibitor of both lysosomal β‐galactosidase and GALC. Further elaboration of this structure is required to achieve selectivity between these two galactosidases.     

Iterative One‐Pot α‐Glycosylation Strategy: Application to Oligosaccharide Synthesis

Based on the combined use of dimethylformamide (DMF) modulation and neighboring group participation, three iterative one‐pot α‐glycosylation methods, i.e., one‐pot (α,α)‐, one‐pot (β,α)‐, and one‐pot (α,β)‐glycosylations, were developed. These methods are applicable to a range of thioglycosyl donors, confer stereocontrol in α‐/β‐glycosidic bond formation, and thus provide for rapid access to oligosaccharides with various permutations of anomeric configurations. The utility of these one‐pot glycosylation methods is demonstrated in the synthesis of eight non‐natural and natural oligosaccharide targets, including the core 1 serine conjugate, core 8 serine conjugate, the D ‐Gal‐α(1→3)‐D ‐Glc‐α(1→3)‐L ‐Rha trisaccharide unit of the cell wall component in Streptococcus pneumoniae, and the D ‐Glc‐α(1→2)‐D ‐Glc‐α(1→3)‐D ‐Glc trisaccharide terminus of the N‐linked glycan precursor. Confirmation of the anomeric configurations of these oligosaccharides is evidenced by ¹H, ¹³C, ¹³C‐non‐proton decoupling, and heteronuclear correlation 2D NMR experiments. Global deprotection of selected oligosaccharide targets is illustrated.