Unusual Amino Acid Research Articles

Stalobacin: Discovery of Novel Lipopeptide Antibiotics with Potent Antibacterial Activity against Multidrug-Resistant Bacteria.

A novel lipopeptide antibiotic, stalobacin I (1), was discovered from a culture broth of an unidentified Gram-negative bacterium. Stalobacin I (1) had a unique chemical architecture composed of an upper and a lower half peptide sequence, which were linked via a hemiaminal methylene moiety. The sequence of 1 contained an unusual amino acid, carnosadine, 3,4-dihydroxyariginine, 3-hydroxyisoleucine, and 3-hydroxyaspartic acid, and a novel cyclopropyl fatty acid. The antibacterial activity of 1 against a broad range of drug-resistant Gram-positive bacteria was much stronger than those of "last resort" antibiotics such as vancomycin, linezolid, and telavancin (MIC 0.004-0.016 μg/mL). Furthermore, compound 1 induced a characteristic morphological change in Gram-positive and Gram-negative strains by inflating the bacterial cell body. The absolute configuration of a cyclopropyl amino acid, carnosadine, was determined by the synthetic study of its stereoisomers, which was an essential component for the strong activity of 1.

Microbes against microbial toxins

Biodegradation Algal blooms in polluted lakes are often associated with high levels of microbial toxins, including a class of cyclic peptides known as microcystins. Although these molecules are resistant to normal peptidases, some bacteria have developed specialized enzymes and pathways to metabolize them. Prospecting in the notoriously polluted and eutrophic Lake Taihu in China, Yang et al. isolated a bacterium capable of completely degrading microcystins. A multi-omics analysis confirmed many previously known steps of the pathway and suggested that genes involved in phenylacetic acid metabolism contribute to the breakdown of one of the unusual amino acid components. Water Res. 174 , 115638 (2020).

Effects of amino acid composition on the requirements for J‐proteins by artificial prions

Yeast prions are non‐Mendelian transmissible agents that are able to induce abnormal folding of specific proteins. The composition of yeast prions is usually in‐register, cross‐beta amyloid structures. Prion‐forming proteins have domains with unusual amino acid content that is typically rich in glutamine and/or asparagine. The chaperone protein network in yeast regulates the correct folding of proteins and is inexorably linked to the spreading of prions to daughter cells during division through a process called fragmentation. Specifically, the core “prion fragmentation machinery” at least minimally includes the proteins Hsp104, Hsp70 and one Hsp40, also known as a J‐protein. The relationship between the amino acid content of the prion‐forming domain (PrD) and chaperone proteins required for fragmentation, if any, is still unclear. Here we explored a system of engineered prions that resemble the Sup35 protein of S. cerevisiae, with single heterologous amino acids interspersed between polyQ sequences every fifth residue in the PrD of the construct. This domain determines the likelihood of a protein to form aggregates in vivo, and so by controlling the amino acid content in this region we can study the impact of specific amino acids and their interactions with chaperone proteins in the propagation of yeast prions. Specifically, requirements for the Hsp40 Sis1 is being investigated using multiple approaches, including plasmid‐shuffling experiments and repression of Sis1 levels using tetracycline. Strains have also been developed to test for requirements for additional J‐proteins for prion propagation. Overall, the long‐term goal for this research is to gain a deeper understanding of the relationship between amino acid content and chaperone protein interactions in prion propagation.Support or Funding InformationThis work was supported by the Lafayette College Chemistry Department, the EXCEL research scholarship program ( www.lafayette.edu), the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R15GM110606, and the Henry Dreyfus Teacher Scholar Award. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or the Dreyfus Foundation.

The Chemistry and Biology of Bactobolin: A 10-Year Collaboration with Natural Product Chemist Extraordinaire Jon Clardy.

Bactobolin is a hybrid natural product with potent cytotoxic activity. Its production from Burkholderia thailandensis was reported as part of a collaboration between the Greenberg and Clardy laboratories in 2010. The collaboration sparked a series of studies leading to the discovery of new analogues and associated structure-activity relationships, the identification of the bactobolin biosynthetic gene cluster and assembly of its unusual amino acid building block, the molecular target of and resistance to the antibiotic, and finally an X-ray crystal structure of the ribosome-bactobolin complex. Herein, we review the collaborations that led to our current understanding of the chemistry and biology of bactobolin.

Novel cilengitide-based cyclic RGD peptides as αvβ3 integrin inhibitors

In this letter, we report a series of five new RGD-containing cyclic peptides as potent inhibitors to αvβ3 integrin protein. We have incorporated various unnatural lipophilic amino acids into the cyclic RGD framework of cilengitide, which is selective for αvβ3 integrin. All the newly synthesized cyclic peptides were evaluated in vitrosolid phase binding assay and investigated for their bindingbehaviourtowards integrin subtypes. All the cyclic peptides were synthesized in excellent yield following solution-phase coupling strategy. The cyclic RGD peptides 1a-e exhibited IC50 of 9.9, 5.5, 72, 11 and 3.3 nM, respectively, towardsαvβ3 integrin protein. This finding offers further opportunities for the introduction unusual amino acids into the cyclic peptide framework of cilengitide.

The biology of ergothioneine, an antioxidant nutraceutical

Ergothioneine (ERG) is an unusual thio-histidine betaine amino acid that has potent antioxidant activities. It is synthesised by a variety of microbes, especially fungi (including in mushroom fruiting bodies) and actinobacteria, but is not synthesised by plants and animals who acquire it via the soil and their diet, respectively. Animals have evolved a highly selective transporter for it, known as solute carrier family 22, member 4 (SLC22A4) in humans, signifying its importance, and ERG may even have the status of a vitamin. ERG accumulates differentially in various tissues, according to their expression of SLC22A4, favouring those such as erythrocytes that may be subject to oxidative stress. Mushroom or ERG consumption seems to provide significant prevention against oxidative stress in a large variety of systems. ERG seems to have strong cytoprotective status, and its concentration is lowered in a number of chronic inflammatory diseases. It has been passed as safe by regulatory agencies, and may have value as a nutraceutical and antioxidant more generally.

Preparation of isodesmosine-KLH conjugate for ELISA system.

Chronic obstructive pulmonary disease (COPD) is a degenerative condition with limited diagnostic detection efficiency. Currently with no available cure, COPD is associated with irreversible elastic tissue degradation in lungs, which results in release of unusual amino acids, isodesmosine and desmosine. These biomarkers are potential key elements in enzyme-linked immunosorbent assay (ELISA), an analytical method, which can detect certain compounds including antigens and proteins in easy and affordable manner. In order to target a biomarker with ELISA, it is necessary to prepare its specific antibody, which can be achieved by immunization of host organism with appropriate antigen containing the biomarker. Although preparation of these types of conjugates has been published, desmosine and isodesmosine used by researchers are obtained from natural sources such as animal tissues. Here, we report the first synthetic preparation of isodesmosine and keyhole limpet hemocyanin (KLH) conjugate from commercially available chiral amino acids and carrier protein. Formation of the core pyridinium of isodesmosine was achieved through key reaction-Chichibabin pyridinium synthesis-to deliver a 1,2,3,5-tetrasubstituted pyridinium amino acid selectively. Further modifications involving KLH and maleimide linker provided the target conjugate, which could potentially invoke an immune response to produce anti-isodesmosine antibody for the ELISA system.

Quinoisobutyride A, an acyclic antibacterial tetrapeptide incorporating an unprecedented heterocyclic amino acid residue from the hypersaline lake-derived fungus Penicillium simplicissimum strain WSH17

Chromatographic refining of the EtOAc extract of a solid rice culture of the hypersaline lake-derived fungus Penicillium simplicissimum strain WSH17 afforded a new linear antibacterial peptide quinoisobutyride A (1) along with three known metabolites (2-4). The chemical structure of 1 was unambiguously determined based on extensive 1D and 2D NMR spectroscopic analyses together with HRESIMS. In addition, Marfey’s derivatization method along with biogenetic considerations were used to determine the absolute configuration of its amino acids’ entities. Interestingly, in this study we distinguished a new unusual heterocyclic amino acid which is comprised as a unit of compound 1. Among the isolated compounds, only 1 revealed a promising antibacterial activity against Staphylococcus aureus (ATCC700699) with a MIC value of 2.72 μM (2.0 μg/mL). Moreover, citrinamide A (3) was the only isolated compound that showed a cytotoxic activity against human epithelial colorectal adenocarcinoma (Caco-2) cell line with an IC50 value of 12.76 μM (5.5 μg/mL).

Isolation and structure determination of a new antibacterial peptide pentaminomycin C from Streptomyces cacaoi subsp. cacaoi.

A new antibacterial peptide named pentaminomycin C was isolated from an extract of Streptomyces cacaoi subsp. cacaoi NBRC 12748T, along with a known peptide BE-18257A. Pentaminomycin C was determined to be a cyclic pentapeptide containing an unusual amino acid, Nδ-hydroxyarginine (5-OHArg), by a combination of ESI-MS and NMR analyses. The structure of pentaminomycin C was determined to be cyclo(-L-Leu-D-Val-L-Trp-L-5-OHArg-D-Phe-). Pentaminomycin C exhibited antibacterial activities against Gram-positive bacteria including Micrococcus luteus, Bacillus subtilis, and Staphylococcus aureus. The biosynthetic gene cluster for pentaminomycin C and BE-18257A was identified from the genome sequence data of S. cacaoi subsp. cacaoi.

Design, Synthesis and Enzymatic Inhibition of Novel Unusual Amino Acids as a Transition State Analogue of Amyloid Precursor Protein Peptide

β-secretase 1 (BACE1) plays a pivotal role in the pathology of Alzheimer׳s disease via accumulation beta amyloid in the brain. In this context, identifying new scaffolds that block BACE1 is of great importance despite all pharmacokinetic drawbacks that peptide-like structures have. Here, we report a new core structure based on novel unusual amino acids by substituting phenyl amide group in the P1 position and small alkyl groups in the P1′ site that results in the formation of new biological active peptides in micromolar level. Three different scaffolds were designed based on docking studies to efficiently interact with critical Asp32 residue in the active site of BACE1 and incorporated in peptides synthesis by Fmoc solid-phase peptide synthesis (SPPS) methodology to achieve desired compounds in good yield. The inhibitory activity of all synthesized peptides was examined by FRET-based enzymatic assay. The peptide 7 showed the best inhibitory activity with IC50 = 98.14 µM. Results of this investigation revealed that utilizing unusual amino acids as building blocks for the synthesis of peptidomimetics would be an option for the development and optimization of pharmaceutical structures. The inhibition of β-secretase 1 (BACE1) is potentially important approach to treatment of Alzheimer disease (AD). Novel series of peptides coupled to the new unusual amino acids scaffold were investigated as BACE1 inhibitors in this study. The design of these peptides was mostly affected by OM00-3. Based on observations, in good agreement between BACE-1 inhibition assessment and docking methodology, peptide 7 was the most potent compound between all and could be the basis of future studies.

Synthesis of Novel Peptides Using Unusual Amino Acids.

Small peptides are valuable peptides due to their extended biological activities. Their activities could be categorized according to their low antigenicity, osmotic pressure, and also because of their astonishing bioactivities. For example, the aggression of Phe-Phe fibers via self-assembly and intermolecular hydrogen bonding is the main reason for the formation of Alzheimer’s β-amyloid fibrils. Hydrogen bonding is the main intramolecular interaction in peptides, while the presence of aromatic ring leads to the π-π stacking and affects the self-assembly and aggression. Thus, insertion of an unusual amino acid into peptide sequence facilitates the formation of intramolecular bonds, lipophilicity and its conformation. To design new small peptides with remarkable lipophilicity, it is an idea to employ γ-amino acid, such as gabapentin (H2N-Gpn-OMe) and baclofen (H2N-Baclofen-OMe), in the structure of small peptides to increase cell-penetrating properties and to prevent aggression of Phe-Phe fibrils in β-amyloids of Alzheimer’s disease. Some new tri- and tetrapeptides were synthesized through introducing biologically active gabapentin and baclofen to dipeptide of phenylalanine (Phe-Phe) through solution phase peptide synthesis strategy.

Effects of partial replacement of soybean meal in broiler diets with gelatin and mono-component protease on growth performance, carcass and blood biochemical characteristics, lipid oxidation of meat, and economics

Context Gelatin, presently a waste material from animal tissues and bones, may be suitable as a partial dietary replacement for soybean meal. In combination with protease, it may enhance growth performances of broiler chickens. However, limitations when introducing gelatin, are its unusual amino acid profile and digesta viscosity. Aim An experiment was conducted to elucidate effects on growth performance, blood biochemistry, carcass and meat characteristics, and feed costs of partial replacement of soybean meal with gelatin and protease in broilers diets. Methods The experimental design was a factorial arrangement of two levels of mono-component protease (0 and 200 g/t diet) and four levels of gelatin (0%, 2.82%, 4.23% and 5.64% of the diet) with six replicates and 15 male broiler chickens in each pen. Key results The higher levels of gelatin inclusion decreased bodyweight and increased feed conversion ratio (P < 0.001), whereas addition of enzyme improved both parameters (P < 0.001). Higher gelatin inclusion levels decreased abdominal fat, and enzyme addition resulted in greater carcass yield (P < 0.001). European performance efficiency factor reduced with incremental gelatin levels but increased with enzyme inclusion (P < 0.001). Feed cost per bird was reduced at higher gelatin inclusion levels, more so in the protease-treated diets up to 4.23% inclusion level. Lipid peroxidation values for breast and thigh meat decreased significantly with gelatin inclusion in the absence of protease but increased in the presence of protease. Breast and thigh lipid peroxidation was reduced (P < 0.001) by supplementation of protease in the control diet. Conclusions The results suggest that the performance-depressing effects of gelatin can be overcome to some extent by inclusion of a protease. Implications The use of gelatin may reduce dependence on soybean meal and reduce costs by using what is at present a waste material. Consideration is needed of the digestible amino acid profile of gelatin, and choice of more effective enzymes to reduce deleterious effects of gelatin.

L-(+)-Ergothioneine Significantly Improves the Clinical Characteristics of Preeclampsia in the Reduced Uterine Perfusion Pressure Rat Model

Preeclampsia is a multifactorial hypertensive disorder of pregnancy founded on abnormal placentation, and the resultant placental ischemic microenvironment is thought to play a crucial role in its pathophysiology. Placental ischemia because of fluctuations in the delivery of oxygen results in oxidative stress, and recent evidence suggests that mitochondrial dysfunction may be a prime mediator. However, large clinical trials of therapeutic antioxidants such as vitamins C and E for the treatment of preeclampsia have been disappointing. L-(+)-ergothioneine (ERG)-an unusual amino acid betaine derived from histidine-has important cytoprotective and antioxidant properties under conditions of high oxidative stress. In this study, we investigated the potential therapeutic effects of administration of ERG in the reduced uterine perfusion pressure (RUPP) rat model of preeclampsia. ERG (25 mg/kg per day) was administered to rats on gestational day 11. On gestational day 14, RUPP surgery was performed, and on gestational day 19, blood pressure (mean arterial pressure) and fetal growth were measured. Production of mitochondria-specific H2O2 was analyzed in vivo in kidney samples. ERG ameliorated the hypertension (129±3 versus 115±4 mm Hg; P=0.01; n=8) and significantly increased pup weight in RUPP rats. ERG also significantly decreased circulating levels of antiangiogenic sFlt-1 (soluble fms-like tyrosine kinase-1) in RUPP rats (1367±245 pg/mL; P=0.04). Mitochondria-specific H2O2 (0.022±0.003 versus 0.029±0.001; MitoP/B ratio, n=3; P=0.05) was also significantly decreased in kidney tissue in RUPP rats treated with ERG. These data support the potential use of ERG for the treatment of preeclampsia.

Early-earth nonprotein amino acid metabolites in modern cyanobacterial microbialites

Cyanobacteria are among the earth’s oldest known living groups of organisms and can form layered accretions called microbialites, found in both the fossil record and existing lakes. Studies of cyanobacterial biochemical processes help to understand the evolution of life on earth. The conserved metabolism of cyanobacterial species includes the biosynthesis of unusual nonprotein amino acids such as N-(2-aminoethyl)glycine, hypothesized to have constituted an early form of genetic information in cells. Pavilion Lake in British Columbia, Canada, hosts a population of unique, actively growing microbialites covered in biofilms dominated by cyanobacteria. We hypothesized that the living microbial communities produce dinitrogenous nonprotein amino acids, such as N-(2-aminoethyl)glycine and its structural isomers β-N-methylamino-l-alanine, 2,4-diaminobutyric acid and β-aminomethyl-l-alanine. We analyzed samples in sediment traps collected between 2007 and 2014 in depths ranging from 11 to 46 m. N-(2-aminoethyl)glycine, 2,4-diaminobutyric acid and β-aminomethyl-l-alanine were found in highest concentration in the shallowest microbialite biofilms with a maximum of 22 ng/g, 33 ng/g and 0.4 ng/g, respectively. In contrast, the concentration of β-N-methylamino-l-alanine was highest in collections between 18 and 26 m depths and only β-N-methylamino-l-alanine was found in the deepest water collections. These data provide evidence indicating that the production of these nonprotein amino acids is highly conserved through the evolution of cyanobacteria and suggest that the nitrogen-rich metabolites may have had both an important role in ancient and modern cyanobacterial metabolism. Further research will determine the role of N-(2-aminoethyl)glycine and its isomers in early life metabolism and their current function in photosynthetic cells.

Development of New Synthetic Strategies, Tactics and their Applications.

This account covers our work related to the development of various synthetic methodologies since 1994. We summarized our strategies and their application to design various functional molecules. In this regard, we also report the utility of our methodologies in others research work. These methods we report here are simple and efficient for the synthesis of a wide variety of intricate molecules such as heterocycles, polycycles, unusual α-amino acids, star-shaped molecules, and modified peptides. For this purpose, we used various transition metal-based reagents and catalysts. Various popular reactions such as metathesis, Suzuki coupling, [2+2+2] cyclotrimerization were used to assemble these targets. Moreover, rongalite has been used to expand the Diels-Alder chemistry.

Possibility of minimizing gluten intolerance by co-consumption of some fruits – A case for positive food synergy?

BackgroundStaple cereals and their products are important foods. Many cereals contain an important protein group, gluten, which provides structure to bakery products and pasta. Gluten proteins have an unusual amino acid composition, containing high proline and glutamine contents. These amino acids confer resistance to proteolysis by gastrointestinal tract enzymes, producing indigestible proline- and glutamine-rich peptides that can trigger immunogenic reactions responsible for gluten-related health disorders. These disorders represent a serious public health issue, affecting approximately 5% of the global population. The prescription of a gluten-free diet (GFD) is the only accepted therapy for gluten-related health disorders. Scope and approachAdherence to a GFD remains a difficult goal for many reasons. Alternative approaches have been proposed to supplement or substitute a GFD. Among them, the use of gluten-specific enzymes to degrade immunogenic peptides has shown promising results. Most of the enzymes used in this approach are of microbial origin. Identification of natural alternatives is a timely requirement. Key findingsSeveral enzymes of microbial origin have been identified that are claimed to digest gluten in the human gastrointestinal tract. However, only a few claim to be able to digest immunogenic gluten peptides. Most of these enzyme therapies are still being developed and are not yet viable practical solutions. Fruit-borne proteases are a possible alternative solution. Notably, green kiwifruit (Actinidia deliciosa), containing the cysteine protease actinidin, degraded gluten in animal trials and could be a natural way to manage gluten-related health disorders – an example of positive food synergy.

Cytotoxic Microcolin Lipopeptides from the Marine Cyanobacterium Moorea producens

Nine new linear lipopeptides, microcolins E-M (1-9), together with four known related compounds, microcolins A-D (10-13), were isolated from the marine cyanobacterium Moorea producens using bioassay-guided and LC-MS/MS molecular networking approaches. Catalytic hydrogenation of microcolins A-D (10-13) yielded two known compounds, 3,4-dihydromicrocolins A and B (14, 15), and two new derivatives, 3,4-dihydromicrocolins C and D (16, 17), respectively. The structures of these new compounds were determined by a combination of spectroscopic and advanced Marfey's analysis. Structurally unusual amino acid units, 4-methyl-2-(methylamino)pent-3-enoic (Mpe) acid and 2-amino-4-methylhexanoic acid (N-Me-homoisoleucine), in compounds 1-3 and 8, respectively, are rare residues in naturally occurring peptides. These metabolites showed significant cytotoxic activity against H-460 human lung cancer cells with IC50 values ranging from 6 nM to 5.0 μM. The variations in structure and attendant biological activities provided fresh insights concerning structure-activity relationships for the microcolin class of lipopeptides.

Characterization of glutamyl-tRNA-dependent dehydratases using nonreactive substrate mimics.

The peptide natural product nisin has been used as a food preservative for 6 decades with minimal development of resistance. Nisin contains the unusual amino acids dehydroalanine and dehydrobutyrine, which are posttranslationally installed by class I lanthipeptide dehydratases (LanBs) on a linear peptide substrate through an unusual glutamyl-tRNA-dependent dehydration of Ser and Thr. To date, little is known about how LanBs catalyze the transfer of glutamate from charged tRNAGlu to the peptide substrate, or how they carry out the subsequent elimination of the peptide-glutamyl adducts to afford dehydro amino acids. Here, we describe the synthesis of inert analogs that mimic substrate glutamyl-tRNAGlu and the glutamylated peptide intermediate, and determine the crystal structures of 2 LanBs in complex with each of these compounds. Mutational studies were used to characterize the function of the glutamylation and glutamate elimination active-site residues identified through the structural analysis. These combined studies provide insights into the mechanisms of substrate recognition, glutamylation, and glutamate elimination by LanBs to effect a net dehydration reaction of Ser and Thr.

The Amipurimycin and Miharamycin Biosynthetic Gene Clusters: Unraveling the Origins of 2-Aminopurinyl Peptidyl Nucleoside Antibiotics.

Peptidyl nucleoside antibiotics (PNAs) are a diverse class of natural products with promising biomedical activities. These compounds have tripartite structures composed of a core saccharide, a nucleobase, and one or more amino acids. In particular, amipurimycin and the miharamycins are novel 2-aminopurinyl PNAs with complex nine-carbon core saccharides and include the unusual amino acids (-)-cispentacin and N5-hydroxyarginine, respectively. Despite their interesting structures and properties, these PNAs have heretofore eluded biochemical scrutiny. Herein is reported the discovery and initial characterization of the miharamycin gene cluster in Streptomyces miharaensis (mhr) and the amipurimycin gene cluster (amc) in Streptomyces novoguineensis and Streptomyces sp. SN-C1. The gene clusters were identified using a comparative genomics approach, and heterologous expression of the amc cluster as well as gene interruption experiments in the mhr cluster support their role in the biosynthesis of amipurimycin and the miharamycins, respectively. The mhr and amc biosynthetic gene clusters characterized encode enzymes typical of polyketide biosynthesis instead of enzymes commonly associated with PNA biosynthesis, which, along with labeled precursor feeding studies, implies that the core saccharides found in the miharamycins and amipurimycin are partially assembled as polyketides rather than derived solely from carbohydrates. Furthermore, in vitro analysis of Mhr20 and Amc18 established their roles as ATP-grasp ligases involved in the attachment of the pendant amino acids found in these PNAs, and Mhr24 was found to be an unusual hydroxylase involved in the biosynthesis of N5-hydroxyarginine. Finally, analysis of the amc cluster and feeding studies also led to the proposal of a biosynthetic pathway for (-)-cispentacin.

Silk-Like Protein with Persistent Radicals Identified in Oyster Adhesive by Solid-State NMR.

The cement produced by the Eastern oyster, Crassostrea virginica, may provide blueprints for waterproof biocompatible adhesives synthesized under benign conditions. The composition of this organic-inorganic composite, and of an organic extract, was characterized by 13C and 1H solid-state NMR and also compared withC. virginica shell and its organic extract. Quantification of the organic fraction by 13C and 1H NMR spectroscopy consistently showed 3 wt % organics in cement, which was higher than the 1.2 wt % in the shell. According to 13C NMR with spectral editing, the organic fraction of cement consisted of 73% protein, 25% polysaccharide, and 2% lipid. The organic acid-insoluble extract from the cement was mostly made up of protein remarkably rich in alanine and glycine. The unusual amino acid content matched the composition of silk-like proteins in the C. virginica or C. gigas genomes, including spidron-1-like and shelk2 previously found to be upregulated at the mantle edge. The corresponding extract from the shell contained 32% glycine and was also enriched in serine but not alanine, which was consistent with a previous wet-chemistry study. The 13C and 1H NMR spin-lattice relaxation in the organic component of cement and the acid-insoluble extract was 4-40 times faster than in the shell and showedpronounced nonexponentiality, indicating a high concentration of persistent radicals in the organic components of cement, in agreement with a prior EPR study. The presence of radicals in the acid-insoluble cement fraction was confirmed by observation of a paramagnetic shift anisotropy. 13C NMR corroborated prior observations that the calcium carbonate in the shell and pseudonacre was mostly calcite, whereas cement had an enhanced aragonite fraction. Surprisingly, 1H-13C NMR indicated that aragonite in cement was more distant from the organic fraction than wascalcite. These results help advance our understanding of how oysters achieve adhesion within their wet environment.