Synthesis of lacto- N -tetraose

Craft, Kelly M.; Townsend, Steven D.

doi:10.1016/j.carres.2017.02.001

Cited by 44 publications

(26 citation statements)

References 54 publications

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“…LNT chemical and enzymatic syntheses can be grouped in two general strategies: the “linear approach” that mimics the biosynthetic pathway by sequential addition of two monosaccharide units on the non-reducing end of lactose, and the “convergent approach” through coupling of lacto- N -biose (LNB) and lactose as disaccharide building blocks. Recent chemical synthesis of LNT includes both strategies, the linear [ 11 ] and convergent [ 12 ] approaches, where the protecting-leaving group combinations were critical to achieve successful glycosylations. The low yields and multistep pathways currently limit the scalability of the chemical synthesis.…”

Section: Introductionmentioning

confidence: 99%

Transglycosylation Activity of Engineered Bifidobacterium Lacto-N-Biosidase Mutants at Donor Subsites for Lacto-N-Tetraose Synthesis

Castejón-Vilatersana

Faijes

Planas

2021

IJMS

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The health benefits of human milk oligosaccharides (HMOs) make them attractive targets as supplements for infant formula milks. However, HMO synthesis is still challenging and only two HMOs have been marketed. Engineering glycoside hydrolases into transglycosylases may provide biocatalytic routes to the synthesis of complex oligosaccharides. Lacto-N-biosidase from Bifidobacterium bifidum (LnbB) is a GH20 enzyme present in the gut microbiota of breast-fed infants that hydrolyzes lacto-N-tetraose (LNT), the core structure of the most abundant type I HMOs. Here we report a mutational study in the donor subsites of the substrate binding cleft with the aim of reducing hydrolytic activity and conferring transglycosylation activity for the synthesis of LNT from p-nitrophenyl β-lacto-N-bioside and lactose. As compared with the wt enzyme with negligible transglycosylation activity, mutants with residual hydrolase activity within 0.05% to 1.6% of the wild-type enzyme result in transglycosylating enzymes with LNT yields in the range of 10-30%. Mutations of Trp394, located in subsite -1 next to the catalytic residues, have a large impact on the transglycosylation/hydrolysis ratio, with W394F being the best mutant as a biocatalyst producing LNT at 32% yield. It is the first reported transglycosylating LnbB enzyme variant, amenable to further engineering for practical enzymatic synthesis of LNT.

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Section: Introductionmentioning

confidence: 99%

Transglycosylation Activity of Engineered Bifidobacterium Lacto-N-Biosidase Mutants at Donor Subsites for Lacto-N-Tetraose Synthesis

Castejón-Vilatersana

Faijes

Planas

2021

IJMS

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“…Indeed, we discovered that heterogeneous HMOs modulate growth and biofilm production for a number of bacterial pathogens (26,27). We also determined the identities of several single-entity HMOs with potent antimicrobial activity against GBS (28)(29)(30)(31). In addition to structure-activity relationship (SAR) studies, we found that HMOs potentiate the activity of select intracellular targeting antibiotics (32).…”

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confidence: 98%

A Solution to Antifolate Resistance in Group B Streptococcus : Untargeted Metabolomics Identifies Human Milk Oligosaccharide-Induced Perturbations That Result in Potentiation of Trimethoprim

Chambers

Moore

Craft

et al. 2020

mBio

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Adjuvants can be used to potentiate the function of antibiotics whose efficacy has been reduced by acquired or intrinsic resistance. In the present study, we discovered that human milk oligosaccharides (HMOs) sensitize strains of group B Streptococcus (GBS) to trimethoprim (TMP), an antibiotic to which GBS is intrinsically resistant. Reductions in the MIC of TMP reached as high as 512-fold across a diverse panel of isolates. To better understand HMOs’ mechanism of action, we characterized the metabolic response of GBS to HMO treatment using ultrahigh-performance liquid chromatography–high-resolution tandem mass spectrometry (UPLC-HRMS/MS) analysis. These data showed that when challenged by HMOs, GBS undergoes significant perturbations in metabolic pathways related to the biosynthesis and incorporation of macromolecules involved in membrane construction. This study represents reports the metabolic characterization of a cell that is perturbed by HMOs. IMPORTANCE Group B Streptococcus is an important human pathogen that causes serious infections during pregnancy which can lead to chorioamnionitis, funisitis, premature rupture of gestational membranes, preterm birth, neonatal sepsis, and death. GBS is evolving antimicrobial resistance mechanisms, and the work presented in this paper provides evidence that prebiotics such as human milk oligosaccharides can act as adjuvants to restore the utility of antibiotics.

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“…To achieve its regioselective synthesis, we investigated the effect of arming benzyl and disarming benzoyl groups at position 2 and 6 of the Gal acceptors in tuning the reactivity of the 3‐ and 4‐OH, respectively, in combination with various protecting and leaving groups in the glucosamine donors. Despite the expected higher reactivity of the equatorial 3‐OH versus the axial 4‐OH, regioselective glycosylation of position 3 has been shown not to be trivial . Accordingly, we synthesized a series of glucosamine thioglycoside and trichloroacetimidate donors with the amine protected by the participating phthalimido (Phth) or trichloroethyl carbamate (Troc) group (experimental procedures are provided as Supporting Information).…”

Section: Resultsmentioning

confidence: 99%

Regioselective Glycosylation Strategies for the Synthesis of Group Ia and Ib Streptococcus Related Glycans Enable Elucidating Unique Conformations of the Capsular Polysaccharides

Bino

Calloni

Oldrini

et al. 2019

Chemistry A European J

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Group B Streptococcus serotypes Ia and Ib capsular polysaccharides are key targets for vaccine development. In spite of their immunospecifity these polysaccharides share high structural similarity.B oth are composed of the same monosaccharide residues and differ only in the connection of the Neu5Aca2-3Gal side chain to the GlcNAc unit, which is a b1-4 linkage in serotype Ia and a b1-3 linkage in serotype Ib. The developmento fe fficient regioselective routes for GlcNAcb1-3[Glcb1-4]Gal synthons is described, which give access to different group B Streptococcus (GBS) Ia and Ib repeating unit frameshifts. These glycans were used to probe the conformation and molecular dynamics of the two polysaccharides, highlighting the different presentation of the protruding Neu5Aca2-3Gal moieties on the polysaccharide backbones and ah igherf lexibility of Ib polymer relative to Ia, which can impact epitope exposure.[b] Dr.I .C alloni, Dr.A .Ardµ,P rof. J.Supporting information (containingt he experimental procedures for the synthesis of oligosaccharides 1-5 and their characterization, molecular dynamicss imulations, and NMR spectra)and the ORCID identification number(s) for the author(s) of this article can be found under: https://doi.org/10.

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Synthesis of lacto- N -tetraose

Cited by 44 publications

References 54 publications

Transglycosylation Activity of Engineered Bifidobacterium Lacto-N-Biosidase Mutants at Donor Subsites for Lacto-N-Tetraose Synthesis

Transglycosylation Activity of Engineered Bifidobacterium Lacto-N-Biosidase Mutants at Donor Subsites for Lacto-N-Tetraose Synthesis

A Solution to Antifolate Resistance in Group B Streptococcus : Untargeted Metabolomics Identifies Human Milk Oligosaccharide-Induced Perturbations That Result in Potentiation of Trimethoprim

Regioselective Glycosylation Strategies for the Synthesis of Group Ia and Ib Streptococcus Related Glycans Enable Elucidating Unique Conformations of the Capsular Polysaccharides

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