Prevotella copri and microbiota members mediate the beneficial effects of a therapeutic food for malnutrition.

Microbiota-directed complementary food (MDCF) formulations have been designed to repair the gut communities of malnourished children. A randomized controlled trial demonstrated that one formulation, MDCF-2, improved weight gain in malnourished Bangladeshi children compared to a more calorically dense standard nutritional intervention. Metagenome-assembled genomes from study participants revealed a correlation between ponderal growth and expression of MDCF-2 glycan utilization pathways by Prevotella copri strains. To test this correlation, here we use gnotobiotic mice colonized with defined consortia of age- and ponderal growth-associated gut bacterial strains, with or without P. copri isolates closely matching the metagenome-assembled genomes. Combining gut metagenomics and metatranscriptomics with host single-nucleus RNA sequencing and gut metabolomic analyses, we identify a key role of P. copri in metabolizing MDCF-2 glycans and uncover its interactions with other microbes including Bifidobacterium infantis. P. copri-containing consortia mediated weight gain and modulated energy metabolism within intestinal epithelial cells. Our results reveal structure-function relationships between MDCF-2 and members of the gut microbiota of malnourished children with potential implications for future therapies.

Quantifying Gut Microbial Short-Chain Fatty Acids and Their Isotopomers in Mechanistic Studies Using a Rapid, Readily Expandable LC-MS Platform.

Short-chain fatty acids (SCFAs) comprise the largest group of gut microbial fermentation products. While absorption of most nutrients occurs in the small intestine, indigestible dietary components, such as fiber, reach the colon and are processed by the gut microbiome to produce a wide array of metabolites that influence host physiology. Numerous studies have implicated SCFAs as key modulators of host health, such as in regulating irritable bowel syndrome (IBS). However, robust methods are still required for their detection and quantitation to meet the demands of biological studies probing the complex interplay of the gut-host-health paradigm. In this study, a sensitive, rapid-throughput, and readily expandible UHPLC-QqQ-MS platform using 2-PA derivatization was developed for the quantitation of gut-microbially derived SCFAs, related metabolites, and isotopically labeled homologues. The utility of this platform was then demonstrated by investigating the production of SCFAs in cecal contents from mice feeding studies, human fecal bioreactors, and fecal/bacterial fermentations of isotopically labeled dietary carbohydrates. Overall, the workflow proposed in this study serves as an invaluable tool for the rapidly expanding gut-microbiome and precision nutrition research field.

Bioactive glycans in a microbiome-directed food for children with malnutrition.

Evidence is accumulating that perturbed postnatal development of the gut microbiome contributes to childhood malnutrition1-4. Here we analyse biospecimens from a randomized, controlled trial of a microbiome-directed complementary food (MDCF-2) that produced superior rates of weight gain compared with a calorically more dense conventional ready-to-use supplementary food in 12-18-month-old Bangladeshi children with moderate acute malnutrition4. We reconstructed 1,000 bacterial genomes (metagenome-assembled genomes (MAGs)) from the faecal microbiomes of trial participants, identified 75 MAGs of which the abundances were positively associated with ponderal growth (change in weight-for-length Z score (WLZ)), characterized changes in MAG gene expression as a function of treatment type and WLZ response, and quantified carbohydrate structures in MDCF-2 and faeces. The results reveal that two Prevotella copri MAGs that are positively associated with WLZ are the principal contributors to MDCF-2-induced expression of metabolic pathways involved in utilizing the component glycans of MDCF-2. The predicted specificities of carbohydrate-active enzymes expressed by their polysaccharide-utilization loci are correlated with (1) the in vitro growth of Bangladeshi P. copri strains, possessing varying degrees of polysaccharide-utilization loci and genomic conservation with these MAGs, in defined medium containing different purified glycans representative of those in MDCF-2, and (2) the levels of faecal carbohydrate structures in the trial participants. These associations suggest that identifying bioactive glycan structures in MDCFs metabolized by growth-associated bacterial taxa will help to guide recommendations about their use in children with acute malnutrition and enable the development of additional formulations.

Eat your beets: Conversion of polysaccharides into oligosaccharides for enhanced bioactivity.

Bioactive oligosaccharides with the potential to improve human health, especially in modulating gut microbiota via prebiotic activity, are available from few natural sources. This work uses polysaccharide oxidative cleavage to generate oligosaccharides from beet pulp, an agroindustry by-product. A scalable membrane filtration approach was applied to purify the oligosaccharides for subsequent in vitro functional testing. The combined use of nano-LC/Chip Q-TOF MS and UHPLC/QqQ MS allowed the evaluation of the oligosaccharide profile and their monosaccharide complexity. A final product containing roughly 40 g of oligosaccharide was obtained from 475 g of carbohydrates. Microbiological bioactivity assays indicated that the product obtained herein stimulated desirable commensal gut bacteria. This rapid, reproducible, and scalable method represents a breakthrough in the food industry for generating potential prebiotic ingredients from common plant by-products at scale. INDUSTRIAL RELEVANCE: This work proposes an innovative technology based on polysaccharide oxidative cleavage and multi-stage membrane purification to produce potential prebiotic oligosaccharides from renewable sources. It also provides critical information to evidence the prebiotic potential of the newly generated oligosaccharides on the growth promotion ability of representative probiotic strains of bifidobacteria and lactobacilli.

Plant-based production of diverse human milk oligosaccharides.

Human milk oligosaccharides (HMOs) are a diverse class of carbohydrates that aid in the health and development of infants. The vast health benefits of HMOs have made them a commercial target for microbial production; however, producing the ∼130 structurally diverse HMOs at scale has proven difficult. Here, we produce a vast diversity of HMOs by leveraging the robust carbohydrate anabolism of plants. This diversity includes high value HMOs, such as lacto-N-fucopentaose I, that have not yet been commercially produced using state-of-the-art microbial fermentative processes. HMOs produced in transgenic plants provided strong bifidogenic properties, indicating their ability to serve as a prebiotic supplement. Technoeconomic analyses demonstrate that producing HMOs in plants provides a path to the large-scale production of specific HMOs at lower prices than microbial production platforms. Our work demonstrates the promise in leveraging plants for the cheap and sustainable production of HMOs.

Inducible CRISPR-targeted "knockdown" of human gut Bacteroides in gnotobiotic mice discloses glycan utilization strategies.

Understanding how members of the human gut microbiota prioritize nutrient resources is one component of a larger effort to decipher the mechanisms defining microbial community robustness and resiliency in health and disease. This knowledge is foundational for development of microbiota-directed therapeutics. To model how bacteria prioritize glycans in the gut, germfree mice were colonized with 13 human gut bacterial strains, including seven saccharolytic Bacteroidaceae species. Animals were fed a Western diet supplemented with pea fiber. After community assembly, an inducible CRISPR-based system was used to selectively and temporarily reduce the absolute abundance of Bacteroides thetaiotaomicron or B. cellulosilyticus by 10- to 60-fold. Each knockdown resulted in specific, reproducible increases in the abundances of other Bacteroidaceae and dynamic alterations in their expression of genes involved in glycan utilization. Emergence of these "alternate consumers" was associated with preservation of community saccharolytic activity. Using an inducible system for CRISPR base editing in vitro, we disrupted translation of transporters critical for utilizing dietary polysaccharides in Phocaeicola vulgatus, a B. cellulosilyticus knockdown-responsive taxon. In vitro and in vivo tests of the resulting P. vulgatus mutants allowed us to further characterize mechanisms associated with its increased fitness after knockdown. In principle, the approach described can be applied to study utilization of a range of nutrients and to preclinical efforts designed to develop therapeutic strategies for precision manipulation of microbial communities.

Prevotella copri-related effects of a therapeutic food for malnutrition.

Preclinical and clinical studies are providing evidence that the healthy growth of infants and children reflects, in part, healthy development of their gut microbiomes1-5. This process of microbial community assembly and functional maturation is perturbed in children with acute malnutrition. Gnotobiotic animals, colonized with microbial communities from children with severe and moderate acute malnutrition, have been used to develop microbiome-directed complementary food (MDCF) formulations for repairing the microbiomes of these children during the weaning period5. Bangladeshi children with moderate acute malnutrition (MAM) participating in a previously reported 3-month-long randomized controlled clinical study of one such formulation, MDCF-2, exhibited significantly improved weight gain compared to a commonly used nutritional intervention despite the lower caloric density of the MDCF6. Characterizing the 'metagenome assembled genomes' (MAGs) of bacterial strains present in the microbiomes of study participants revealed a significant correlation between accelerated ponderal growth and the expression by two Prevotella copri MAGs of metabolic pathways involved in processing of MDCF-2 glycans1. To provide a direct test of these relationships, we have now performed 'reverse translation' experiments using a gnotobiotic mouse model of mother-to-offspring microbiome transmission. Mice were colonized with defined consortia of age- and ponderal growth-associated gut bacterial strains cultured from Bangladeshi infants/children in the study population, with or without P. copri isolates resembling the MAGs. By combining analyses of microbial community assembly, gene expression and processing of glycan constituents of MDCF-2 with single nucleus RNA-Seq and mass spectrometric analyses of the intestine, we establish a principal role for P. copri in mediating metabolism of MDCF-2 glycans, characterize its interactions with other consortium members including Bifidobacterium longum subsp. infantis, and demonstrate the effects of P. copri-containing consortia in mediating weight gain and modulating the activities of metabolic pathways involved in lipid, amino acid, carbohydrate plus other facets of energy metabolism within epithelial cells positioned at different locations in intestinal crypts and villi. Together, the results provide insights into structure/function relationships between MDCF-2 and members of the gut communities of malnourished children; they also have implications for developing future prebiotic, probiotic and/or synbiotic therapeutics for microbiome restoration in children with already manifest malnutrition, or who are at risk for this pervasive health challenge.

Bioactive glycans in a microbiome-directed food for malnourished children.

Evidence is accumulating that perturbed postnatal development of the gut microbiome contributes to childhood malnutrition1-4. Designing effective microbiome-directed therapeutic foods to repair these perturbations requires knowledge about how food components interact with the microbiome to alter its expressed functions. Here we use biospecimens from a randomized, controlled trial of a microbiome-directed complementary food prototype (MDCF-2) that produced superior rates of weight gain compared to a conventional ready-to-use supplementary food (RUSF) in 12-18-month-old Bangladeshi children with moderate acute malnutrition (MAM)4. We reconstructed 1000 bacterial genomes (metagenome-assembled genomes, MAGs) present in their fecal microbiomes, identified 75 whose abundances were positively associated with weight gain (change in weight-for-length Z score, WLZ), characterized gene expression changes in these MAGs as a function of treatment type and WLZ response, and used mass spectrometry to quantify carbohydrate structures in MDCF-2 and feces. The results reveal treatment-induced changes in expression of carbohydrate metabolic pathways in WLZ-associated MAGs. Comparing participants consuming MDCF-2 versus RUSF, and MDCF-2-treated children in the upper versus lower quartiles of WLZ responses revealed that two Prevotella copri MAGs positively associated with WLZ were principal contributors to MDCF-2-induced expression of metabolic pathways involved in utilization of its component glycans. Moreover, the predicted specificities of carbohydrate active enzymes expressed by polysaccharide utilization loci (PULs) in these two MAGs correlate with the (i) in vitro growth of Bangladeshi P. copri strains, possessing differing degrees of PUL and overall genomic content similarity to these MAGs, cultured in defined medium containing different purified glycans representative of those in MDCF-2, and (ii) levels of carbohydrate structures identified in feces from clinical trial participants. In the accompanying paper5, we use a gnotobiotic mouse model colonized with age- and WLZ-associated bacterial taxa cultured from this study population, and fed diets resembling those consumed by study participants, to directly test the relationship between P. copri, MDCF-2 glycan metabolism, host ponderal growth responses, and intestinal gene expression and metabolism. The ability to identify bioactive glycan structures in MDCFs that are metabolized by growth-associated bacterial taxa will help guide recommendations about use of this MDCF for children with acute malnutrition representing different geographic locales and ages, as well as enable development of bioequivalent, or more efficacious, formulations composed of culturally acceptable and affordable ingredients.

Dietary Intake of Monosaccharides from Foods is Associated with Characteristics of the Gut Microbiota and Gastrointestinal Inflammation in Healthy US Adults.

BACKGROUND: Current assessment of dietary carbohydrates does not adequately reflect the nutritional properties and effects on gut microbial structure and function. Deeper characterization of food carbohydrate composition can serve to strengthen the link between diet and gastrointestinal health outcomes. OBJECTIVES: The present study aims to characterize the monosaccharide composition of diets in a healthy US adult cohort and use these features to assess the relationship between monosaccharide intake, diet quality, characteristics of the gut microbiota, and gastrointestinal inflammation. METHODS: This observational, cross-sectional study enrolled males and females across age (18-33 y, 34-49 y, and 50-65 y) and body mass index (normal, 18.5-24.99 kg/m2; overweight, 25-29.99 kg/m2; and obese, 30-44 kg/m2) categories. Recent dietary intake was assessed by the automated self-administered 24-h dietary recall system, and gut microbiota were assessed with shotgun metagenome sequencing. Dietary recalls were mapped to the Davis Food Glycopedia to estimate monosaccharide intake. Participants with >75% of carbohydrate intake mappable to the glycopedia were included (N = 180). RESULTS: Diversity of monosaccharide intake was positively associated with the total Healthy Eating Index score (Pearson's r = 0.520, P = 1.2 × 10-13) and negatively associated with fecal neopterin (Pearson's r = -0.247, P = 3.0 × 10-3). Comparing high with low intake of specific monosaccharides revealed differentially abundant taxa (Wald test, P < 0.05), which was associated with the functional capacity to break down these monomers (Wilcoxon rank-sum test, P < 0.05). CONCLUSIONS: Monosaccharide intake was associated with diet quality, gut microbial diversity, microbial metabolism, and gastrointestinal inflammation in healthy adults. As specific food sources were rich in particular monosaccharides, it may be possible in the future to tailor diets to fine-tune the gut microbiota and gastrointestinal function. This trial is registered at www. CLINICALTRIALS: gov as NCT02367287.

Quantitative Bottom-Up Glycomic Analysis of Polysaccharides in Food Matrices Using Liquid Chromatography-Tandem Mass Spectrometry.

Carbohydrates are the most abundant biomolecules in nature, and specifically, polysaccharides are present in almost all plants and fungi. Due to their compositional diversity, polysaccharide analysis remains challenging. Compared to other biomolecules, high-throughput analysis for carbohydrates has yet to be developed. To address this gap in analytical science, we have developed a multiplexed, high-throughput, and quantitative approach for polysaccharide analysis in foods. Specifically, polysaccharides were depolymerized using a nonenzymatic chemical digestion process followed by oligosaccharide fingerprinting using high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (HPLC-QTOF-MS). Both label-free relative quantitation and absolute quantitation were done based on the abundances of oligosaccharides produced. Method validation included evaluating recovery for a range of polysaccharide standards and a breakfast cereal standard reference material. Nine polysaccharides (starch, cellulose, ß-glucan, mannan, galactan, arabinan, xylan, xyloglucan, chitin) were successfully quantitated with sufficient accuracy (5-25% bias) and high reproducibility (2-15% CV). Additionally, the method was used to identify and quantitate polysaccharides from a diverse sample set of food samples. Absolute concentrations of nine polysaccharides from apples and onions were obtained using an external calibration curve, where varietal differences were observed in some of the samples. The methodology developed in this study will provide complementary polysaccharide-level information to deepen our understanding of the interactions of dietary polysaccharides, gut microbial community, and human health.

Multi-Glycomic Characterization of Fiber from AOAC Methods Defines the Carbohydrate Structures.

Dietary fiber has long been known to be an essential component of a healthy diet, and recent investigations into the gut microbiome-health paradigm have identified fiber as a prime determinant in this interaction. Further, fiber is now known to impact the gut microbiome in a structure-specific manner, conferring differential bioactivities to these specific structures. However, current analytical methods for food carbohydrate analysis do not capture this important structural information. To address this need, we utilized rapid-throughput LC-MS methods to develop a novel analytical pipeline to determine the structural composition of soluble and insoluble fiber fractions from two AOAC methods (991.43 and 2017.16) at the total monosaccharide, glycosidic linkage, and free saccharide level. Two foods were chosen for this proof-of-concept study: oats and potato starch. For oats, both AOAC methods gave similar results. Insoluble fiber was found to be comprised of linkages corresponding to ß-glucan, arabinoxylan, xyloglucan, and mannan, while soluble fiber was found to be mostly ß-glucan, with small amounts of arabinogalactan. For raw potato starch, each AOAC method gave markedly different results in the soluble fiber fractions. These observed differences are attributable to the resistant starch content of potato starch and the different starch digestion conditions used in each method. Together, these tools are a means to obtain the complex structures present within dietary fiber while retaining "classical" determinations such as soluble and insoluble fiber. These efforts will provide an analytical framework to connect gravimetric fiber determinations with their constituent structures to better inform gut microbiome and clinical nutrition studies.

An approach for evaluating the effects of dietary fiber polysaccharides on the human gut microbiome and plasma proteome.

Increases in snack consumption associated with Westernized lifestyles provide an opportunity to introduce nutritious foods into poor diets. We describe two 10-wk-long open label, single group assignment human studies that measured the effects of two snack prototypes containing fiber preparations from two sustainable and scalable sources; the byproducts remaining after isolation of protein from the endosperm of peas and the vesicular pulp remaining after processing oranges for the manufacture of juices. The normal diets of study participants were supplemented with either a pea- or orange fiber-containing snack. We focused our analysis on quantifying the abundances of genes encoding carbohydrate-active enzymes (CAZymes) (glycoside hydrolases and polysaccharide lyases) in the fecal microbiome, mass spectrometric measurements of glycan structures (glycosidic linkages) in feces, plus aptamer-based assessment of levels of 1,300 plasma proteins reflecting a broad range of physiological functions. Computational methods for feature selection identified treatment-discriminatory changes in CAZyme genes that correlated with alterations in levels of fiber-associated glycosidic linkages; these changes in turn correlated with levels of plasma proteins representing diverse biological functions, including transforming growth factor type ß/bone morphogenetic protein-mediated fibrosis, vascular endothelial growth factor-related angiogenesis, P38/MAPK-associated immune cell signaling, and obesity-associated hormonal regulators. The approach used represents a way to connect changes in consumer microbiomes produced by specific fiber types with host responses in the context of varying background diets.

The Development of the Davis Food Glycopedia-A Glycan Encyclopedia of Food.

The molecular complexity of the carbohydrates consumed by humans has been deceptively oversimplified due to a lack of analytical methods that possess the throughput, sensitivity, and resolution required to provide quantitative structural information. However, such information is becoming an integral part of understanding how specific glycan structures impact health through their interaction with the gut microbiome and host physiology. This work presents a detailed catalogue of the glycans present in complementary foods commonly consumed by toddlers during weaning and foods commonly consumed by American adults. The monosaccharide compositions of over 800 foods from diverse food groups including Fruits, Vegetables, Grain Products, Beans, Peas, Other Legumes, Nuts, Seeds; Sugars, Sweets and Beverages; Animal Products, and more were obtained and used to construct the "Davis Food Glycopedia" (DFG), an open-access database that provides quantitative structural information on the carbohydrates in food. While many foods within the same group possessed similar compositions, hierarchical clustering analysis revealed similarities between different groups as well. Such a Glycopedia can be used to formulate diets rich in specific monosaccharide residues to provide a more targeted modulation of the gut microbiome, thereby opening the door for a new class of prophylactic or therapeutic diets.

A Multidimensional Mass Spectrometry-Based Workflow for De Novo Structural Elucidation of Oligosaccharides from Polysaccharides.

Carbohydrates play essential roles in a variety of biological processes that are dictated by their structures. However, characterization of carbohydrate structures remains extremely difficult and generally unsolved. In this work, a de novo mass spectrometry-based workflow was developed to isolate and structurally elucidate oligosaccharides to provide sequence, monosaccharide compositions, and glycosidic linkage positions. The approach employs liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based methods in a 3-dimensional concept: one high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (HPLC-QTOF MS) analysis for oligosaccharide sequencing and two ultra high performance liquid chromatography-triple quadrupole mass spectrometry (UHPLC-QqQ MS) analyses on fractionated oligosaccharides to determine their monosaccharides and linkages compositions. The workflow was validated by applying the procedure to maltooligosaccharide standards. The approach was then used to determine the structures of oligosaccharides derived from polysaccharide standards and whole food products. The integrated LC-MS workflow will reveal the in-depth structures of oligosaccharides.

Dietary fiber, starch, and sugars in bananas at different stages of ripeness in the retail market.

The goal of this work was to evaluate changes in dietary fiber measured by the traditional enzymatic-gravimetric method (AOAC 991.43) and the more recently accepted modified enzymatic-gravimetric method (AOAC 2011.25), mono- and disaccharides, and starch as a function of assessed ripeness in a controlled study of a single lot of bananas and in bananas at the same assessed stages of ripeness from bananas purchased in retail stores, from different suppliers. Sugars, starch, and dietary fiber were analyzed in bananas from a single lot, at different stages of ripeness, and in retail samples at the same assessed stages of ripeness. Mean fiber measured by the traditional enzymatic-gravimetric method (EG) was ~2 g/100g and not affected by ripeness. Mean fiber assessed with the recently modified method (mEG) was ~18 g/100g in unripe fruit and decreased to 4-5 g/100g in ripe and ~2 g/100g in overripe bananas. Slightly ripe and ripe bananas differed by ~1.1 g/100g in the controlled single-lot study but not among retail samples. There was a large increase in fructose, glucose and total sugar going from unripe to ripe with no differences between ripe and overripe. Aside from stage of ripeness, the carbohydrate composition in retail bananas is likely affected by differences in cultivar and post-harvest handling. Results from this study demonstrate the importance of measuring dietary fiber using the mEG approach, developing more comprehensive and sensitive carbohydrate analytical protocols and food composition data, and recognizing the impact of different stages of maturity and ripeness on carbohydrate intake estimated from food composition data.

Evaluating microbiome-directed fibre snacks in gnotobiotic mice and humans.

Changing food preferences brought about by westernization that have deleterious health effects1,2-combined with myriad forces that are contributing to increased food insecurity-are catalysing efforts to identify more nutritious and affordable foods3. Consumption of dietary fibre can help to prevent cardiovascular disease, type 2 diabetes and obesity4-6. A substantial number of reports have explored the effects of dietary fibre on the gut microbial community7-9. However, the microbiome is complex, dynamic and exhibits considerable intra- and interpersonal variation in its composition and functions. The large number of potential interactions between the components of the microbiome makes it challenging to define the mechanisms by which food ingredients affect community properties. Here we address the question of how foods containing different fibre preparations can be designed to alter functions associated with specific components of the microbiome. Because a marked increase in snack consumption is associated with westernization, we formulated snack prototypes using plant fibres from different sustainable sources that targeted distinct features of the gut microbiomes of individuals with obesity when transplanted into gnotobiotic mice. We used these snacks to supplement controlled diets that were consumed by adult individuals with obesity or who were overweight. Fibre-specific changes in their microbiomes were linked to changes in their plasma proteomes indicative of an altered physiological state.

Strain-level functional variation in the human gut microbiota based on bacterial binding to artificial food particles.

Greater understanding of the spatial relationships between members of the human gut microbiota and available nutrients is needed to gain deeper insights about community dynamics and expressed functions. Therefore, we generated a panel of artificial food particles with each type composed of microscopic paramagnetic beads coated with a fluorescent barcode and one of 60 different dietary or host glycan preparations. Analysis of 160 Bacteroides and Parabacteroides strains disclosed diverse strain-specific and glycan-specific binding phenotypes. We identified carbohydrate structures that correlated with binding by specific bacterial strains in vitro and noted strain-specific differences in the catabolism of glycans that mediate adhesion. Mixed in vitro cultures revealed that these adhesion phenotypes are maintained in more complex communities. Additionally, orally administering glycan beads to gnotobiotic mice confirmed specificity in glycan binding. This approach should facilitate analyses of how strains occupying the same physical niche interact, and it should advance the development of synbiotics, more nutritious foods, and microbiota-based diagnostics.

Evaluation of dicopper azacryptand complexes in aqueous CuAAC reactions and their tolerance toward biological thiols.

A series of dicopper azacryptand complexes was evaluated in copper-catalysed azide-alkyne cycloaddition (CuAAC) in water at 37 °C. It was found that they showed high activity at concentrations as low as 5 µM. These dinuclear catalysts were more susceptible toward inhibition by cysteine rather than glutathione under the conditions tested. Interestingly, the mononuclear copper complexes that are ligated by tripodal amine ligands, also exhibited good catalytic activity in aqueous media and showed similar sensitivity toward biological thiols as that of the dinuclear complexes. Our results suggest that the azacryptand catalysts are too structurally flexible to adequately prevent interactions of their metal centres with external nucleophiles. However, further steric tuning of the ligand structure might enable more effective substrate gating in future studies.