Frustrated Al/M Heterobimetallic Complexes (M = Cr, Mo, W) That Exhibit Both Lewis and Radical Pair Behavior.

Exploration of new heterobinuclear Al/M combinations is relevant to contemporary strategies for cooperative bond activation. Here, we report the synthesis and characterization of six new Al/M heterobimetallic complexes (M = Cr, Mo, W) that exhibit end-on "isocarbonyl"-type Al─O═C═M bridges with metalloketene character rather than featuring Al─M─C≡O motifs with metal-metal bonding. The new compounds were characterized experimentally by nuclear magnetic resonance and infrared spectroscopies and theoretically using density functional theory, natural bond orbital, and quantum theory of atoms in molecules calculations. Factors influencing Al─O═C═M vs Al─M─C≡O isomerism were probed both experimentally and computationally. Crossover experiments between different group VI Al/M derivatives and regioselective epoxide ring opening indicate that the Al/M complexes act as masked frustrated Lewis pairs in solution under certain conditions. However, crossover experiments between group VI Al/M complexes and a previously studied Al-Fe complex, as well as computational modeling, imply that the same complexes can also reasonably act as masked frustrated radical pairs (FRPs). FRP reactivity with the group VI Al/M complexes was achieved under photochemical conditions, producing unsaturated metal-carbonyl dimers [(CpCr)2(CO)3]2- and [Mn2(CO)8]2-, which would otherwise be unstable under standard conditions but that are isolable here due to Al(III) coordination. The metal-metal bonding in these unsaturated metal-carbonyl dimers was also analyzed theoretically.

A γδ T cell-IL-3 axis controls allergic responses through sensory neurons.

In naive individuals, sensory neurons directly detect and respond to allergens, leading to both the sensation of itch and the activation of local innate immune cells, which initiate the allergic immune response1,2. In the setting of chronic allergic inflammation, immune factors prime sensory neurons, causing pathologic itch3-7. Although these bidirectional neuroimmune circuits drive responses to allergens, whether immune cells regulate the set-point for neuronal activation by allergens in the naive state is unknown. Here we describe a γδ T cell-IL-3 signalling axis that controls the allergen responsiveness of cutaneous sensory neurons. We define a poorly characterized epidermal γδ T cell subset8, termed GD3 cells, that produces its hallmark cytokine IL-3 to promote allergic itch and the initiation of the allergic immune response. Mechanistically, IL-3 acts on Il3ra-expressing sensory neurons in a JAK2-dependent manner to lower their threshold for allergen activation without independently eliciting itch. This γδ T cell-IL-3 signalling axis further acts by means of STAT5 to promote neuropeptide production and the initiation of allergic immunity. These results reveal an endogenous immune rheostat that sits upstream of and governs sensory neuronal responses to allergens on first exposure. This pathway may explain individual differences in allergic susceptibility and opens new therapeutic avenues for treating allergic diseases.

Quantifying effects of second-sphere cationic groups on redox properties of dimolybdenum quadruple bonds.

A series of four dimolybdenum paddlewheel complexes supported by anionic N,N-dimethylglycinate (DMG) or zwitterionic N,N,N-trimethylglycine (TMG) ligands was synthesised to examine the effects of charged groups in the second coordination sphere on redox properties of MoMo bonds. An average shift in reduction potential of +35 mV per cationically charged group was measured, which is approximately half of what would be expected for an analogous mononuclear complex.

Cu site differentiation in tetracopper(i) sulfide clusters enables biomimetic N2O reduction.

Copper clusters feature prominently in both metalloenzymes and synthetic nanoclusters that mediate catalytic redox transformations of gaseous small molecules. Such reactions are critical to biological energy conversion and are expected to be crucial parts of renewable energy economies. However, the precise roles of individual metal atoms within clusters are difficult to elucidate, particularly for cluster systems that are dynamic under operating conditions. Here, we present a metal site-specific analysis of synthetic Cu4(µ4-S) clusters that mimic the Cu Z active site of the nitrous oxide reductase enzyme. Leveraging the ability to obtain structural snapshots of both inactive and active forms of the synthetic model system, we analyzed both states using resonant X-ray diffraction anomalous fine structure (DAFS), a technique that enables X-ray absorption profiles of individual metal sites within a cluster to be extracted independently. Using DAFS, we found that a change in cluster geometry between the inactive and active states is correlated to Cu site differentiation that is presumably required for efficient activation of N2O gas. More precisely, we hypothesize that the Cu δ+⋯Cu δ- pairs produced upon site differentiation are poised for N2O activation, as supported by computational modeling. These results provide an unprecedented level of detail on the roles of individual metal sites within the synthetic cluster system and how those roles interplay with cluster geometry to impact the reactivity function. We expect this fundamental knowledge to inform understanding of metal clusters in settings ranging from (bio)molecular to nanocluster to extended solid systems involved in energy conversion.

Lessons from recent theoretical treatments of Al-M bonds (M = Fe, Cu, Ag, Au) that capture CO2.

Complexes with Al-M bonds (M = transition metal) have emerged as platforms for discovering new reaction chemistry either through cooperative bond activation behaviour of the heterobinuclear unit or by modifying the properties of the M site through its interaction with the Al centre. Therefore, elucidating the nature of Al-M bonding is critical to advancing this research area and typically involves careful theoretical modelling. This Frontier article reviews selected recent case studies that included theoretical treatments of Al-M bonds, specifically highlighting complexes capable of cooperative CO2 activation and focusing on extracting lessons particular to the Al-M sub-field that will inform future studies with theoretical/computational components.

IL-2 family cytokines IL-9 and IL-21 differentially regulate innate and adaptive type 2 immunity in asthma.

BACKGROUND: Asthma is often accompanied by type 2 immunity rich in IL-4, IL-5, and IL-13 cytokines produced by TH2 lymphocytes or type 2 innate lymphoid cells (ILC2s). IL-2 family cytokines play a key role in the differentiation, homeostasis, and effector function of innate and adaptive lymphocytes. OBJECTIVE: IL-9 and IL-21 boost activation and proliferation of TH2 and ILC2s, but the relative importance and potential synergism between these γ common chain cytokines are currently unknown. METHODS: Using newly generated antibodies, we inhibited IL-9 and IL-21 alone or in combination in various murine models of asthma. In a translational approach using segmental allergen challenge, we recently described elevated IL-9 levels in human subjects with allergic asthma compared with nonasthmatic controls. Here, we also measured IL-21 in both groups. RESULTS: IL-9 played a central role in controlling innate IL-33-induced lung inflammation by promoting proliferation and activation of ILC2s in an IL-21-independent manner. Conversely, chronic house dust mite-induced airway inflammation, mainly driven by adaptive immunity, was solely dependent on IL-21, which controlled TH2 activation, eosinophilia, total serum IgE, and formation of tertiary lymphoid structures. In a model of innate on adaptive immunity driven by papain allergen, a clear synergy was found between both pathways, as combined anti-IL-9 or anti-IL-21 blockade was superior in reducing key asthma features. In human bronchoalveolar lavage samples we measured elevated IL-21 protein within the allergic asthmatic group compared with the allergic control group. We also found increased IL21R transcripts and predicted IL-21 ligand activity in various disease-associated cell subsets. CONCLUSIONS: IL-9 and IL-21 play important and nonredundant roles in allergic asthma by boosting ILC2s and TH2 cells, revealing a dual IL-9 and IL-21 targeting strategy as a new and testable approach.

Airway basal stem cells are necessary for the maintenance of functional intraepithelial airway macrophages.

Adult stem cells play a crucial role in tissue homeostasis and repair through multiple mechanisms. In addition to being able to replace aged or damaged cells, stem cells provide signals that contribute to the maintenance and function of neighboring cells. In the lung, airway basal stem cells also produce cytokines and chemokines in response to inhaled irritants, allergens, and pathogens, which affect specific immune cell populations and shape the nature of the immune response. However, direct cell-to-cell signaling through contact between airway basal stem cells and immune cells has not been demonstrated. Recently, a unique population of intraepithelial airway macrophages (IAMs) has been identified in the murine trachea. Here, we demonstrate that IAMs require Notch signaling from airway basal stem cells for maintenance of their differentiated state and function. Furthermore, we demonstrate that Notch signaling between airway basal stem cells and IAMs is required for antigen-induced allergic inflammation only in the trachea where the basal stem cells are located whereas allergic responses in distal lung tissues are preserved consistent with a local circuit linking stem cells to proximate immune cells. Finally, we demonstrate that IAM-like cells are present in human conducting airways and that these cells display Notch activation, mirroring their murine counterparts. Since diverse lung stem cells have recently been identified and localized to specific anatomic niches along the proximodistal axis of the respiratory tree, we hypothesize that the direct functional coupling of local stem cell-mediated regeneration and immune responses permits a compartmentalized inflammatory response.

Metabolomics of IgE-Mediated Food Allergy and Oral Immunotherapy Outcomes based on Metabolomic Profiling.

Background: The immunometabolic mechanisms underlying variable responses to oral immunotherapy (OIT) in patients with IgE-mediated food allergy are unknown. Objective: To identify novel pathways associated with tolerance in food allergy, we used metabolomic profiling to find pathways important for food allergy in multi-ethnic cohorts and responses to OIT. Methods: Untargeted plasma metabolomics data were generated from the VDAART healthy infant cohort (N=384), a Costa Rican cohort of children with asthma (N=1040), and a peanut OIT trial (N=20) evaluating sustained unresponsiveness (SU, protection that lasts after therapy) versus transient desensitization (TD, protection that ends immediately afterwards). Generalized linear regression modeling and pathway enrichment analysis identified metabolites associated with food allergy and OIT outcomes. Results: Compared with unaffected children, those with food allergy were more likely to have metabolomic profiles with altered histidines and increased bile acids. Eicosanoids (e.g., arachidonic acid derivatives) (q=2.4×10 -20 ) and linoleic acid derivatives (q=3.8×10 -5 ) pathways decreased over time on OIT. Comparing SU versus TD revealed differing concentrations of bile acids (q=4.1×10 -8 ), eicosanoids (q=7.9×10 -7 ), and histidine pathways (q=0.015). In particular, the bile acid lithocholate (4.97[1.93,16.14], p=0.0027), the eicosanoid leukotriene B4 (3.21[1.38,8.38], p=0.01), and the histidine metabolite urocanic acid (22.13[3.98,194.67], p=0.0015) were higher in SU. Conclusions: We observed distinct profiles of bile acids, histidines, and eicosanoids that vary among patients with food allergy, over time on OIT and between SU and TD. Participants with SU had higher levels of metabolites such as lithocholate and urocanic acid, which have immunomodulatory roles in key T-cell subsets, suggesting potential mechanisms of tolerance in immunotherapy. Key Messages: - Compared with unaffected controls, children with food allergy demonstrated higher levels of bile acids and distinct histidine/urocanic acid profiles, suggesting a potential role of these metabolites in food allergy. - In participants receiving oral immunotherapy for food allergy, those who were able to maintain tolerance-even after stopping therapyhad lower overall levels of bile acid and histidine metabolites, with the exception of lithocholic acid and urocanic acid, two metabolites that have roles in T cell differentiation that may increase the likelihood of remission in immunotherapy. Capsule summary: This is the first study of plasma metabolomic profiles of responses to OIT in individuals with IgE-mediated food allergy. Identification of immunomodulatory metabolites in allergic tolerance may help identify mechanisms of tolerance and guide future therapeutic development.

Single-cell transcriptomic analyses reveal distinct immune cell contributions to epithelial barrier dysfunction in checkpoint inhibitor colitis.

Immune checkpoint inhibitor (ICI) therapy has revolutionized oncology, but treatments are limited by immune-related adverse events, including checkpoint inhibitor colitis (irColitis). Little is understood about the pathogenic mechanisms driving irColitis, which does not readily occur in model organisms, such as mice. To define molecular drivers of irColitis, we used single-cell multi-omics to profile approximately 300,000 cells from the colon mucosa and blood of 13 patients with cancer who developed irColitis (nine on anti-PD-1 or anti-CTLA-4 monotherapy and four on dual ICI therapy; most patients had skin or lung cancer), eight controls on ICI therapy and eight healthy controls. Patients with irColitis showed expanded mucosal Tregs, ITGAEHi CD8 tissue-resident memory T cells expressing CXCL13 and Th17 gene programs and recirculating ITGB2Hi CD8 T cells. Cytotoxic GNLYHi CD4 T cells, recirculating ITGB2Hi CD8 T cells and endothelial cells expressing hypoxia gene programs were further expanded in colitis associated with anti-PD-1/CTLA-4 therapy compared to anti-PD-1 therapy. Luminal epithelial cells in patients with irColitis expressed PCSK9, PD-L1 and interferon-induced signatures associated with apoptosis, increased cell turnover and malabsorption. Together, these data suggest roles for circulating T cells and epithelial-immune crosstalk critical to PD-1/CTLA-4-dependent tolerance and barrier function and identify potential therapeutic targets for irColitis.

Photochemical Synthesis of Acyl Fluorides Using Copper-Catalyzed Fluorocarbonylation of Alkyl Iodides.

Acyl fluorides are important reagents due to their unique balance between reactivity and stability. Here, we report a copper-catalyzed carbonylative coupling strategy for synthesizing acyl fluorides under photoirradiation. Alkyl iodides were transformed in high yields into acyl fluorides by using a commercially available copper precatalyst (CuBr·SMe2) and a readily available fluoride salt (KF) at ambient temperature and mild CO pressure (6 atm) under blue light irradiation.

Coordination-induced O-H/N-H bond weakening by a redox non-innocent, aluminum-containing radical.

Several renewable energy schemes aim to use the chemical bonds in abundant molecules like water and ammonia as energy reservoirs. Because the O-H and N-H bonds are quite strong (>100 kcal/mol), it is necessary to identify substances that dramatically weaken these bonds to facilitate proton-coupled electron transfer processes required for energy conversion. Usually this is accomplished through coordination-induced bond weakening by redox-active metals. However, coordination-induced bond weakening is difficult with earth's most abundant metal, aluminum, because of its redox inertness under mild conditions. Here, we report a system that uses aluminum with a redox non-innocent ligand to achieve significant levels of coordination-induced bond weakening of O-H and N-H bonds. The multisite proton-coupled electron transfer manifold described here points to redox non-innocent ligands as a design element to open coordination-induced bond weakening chemistry to more elements in the periodic table.

Triazenide-supported [Cu4S] structural mimics of CuZ that mediate N2O disproportionation rather than reduction.

As part of the nitrogen cycle, environmental nitrous oxide (N2O) undergoes the N2O reduction reaction (N2ORR) catalyzed by nitrous oxide reductase, a metalloenzyme whose catalytic active site is a tetranuclear copper-sulfide cluster (CuZ). On the other hand, heterogeneous Cu catalysts on oxide supports are known to mediate decomposition of N2O (deN2O) by disproportionation. In this study, a CuZ model system supported by triazenide ligands is characterized by X-ray crystallography, NMR and EPR spectroscopies, and electronic structure calculations. Although the triazenide-ligated Cu4(µ4-S) clusters are closely related to previous formamidinate derivatives, which differ only in replacement of a remote N atom for a CH group, divergent reactivity with N2O is observed. Whereas the formamidinate-ligated clusters were previously shown to mediate single-turnover N2ORR, the triazenide-ligated clusters are found to mediate deN2O, behavior that was previously unknown to natural or synthetic copper-sulfide clusters. The reaction pathway for deN2O by this model system, including previously unidentified transition state models for N2O activation in N-O cleavage and O-O coupling steps, are included. The divergent reactivity of these two related but subtly different systems point to key factors influencing behavior of Cu-based catalysts for N2ORR (i.e., CuZ) and deN2O (e.g., CuO/CeO2).

Gene-based association study of rare variants in children of diverse ancestries implicates TNFRSF21 in the development of allergic asthma.

BACKGROUND: Most genetic studies of asthma and allergy have focused on common variation in individuals primarily of European ancestry. Studying the role of rare variation in quantitative phenotypes and in asthma phenotypes in populations of diverse ancestries can provide additional, important insights into the development of these traits. OBJECTIVE: We sought to examine the contribution of rare variants to different asthma- or allergy-associated quantitative traits in children with diverse ancestries and explore their role in asthma phenotypes. METHODS: We examined whole-genome sequencing data from children participants in longitudinal studies of asthma (n = 1035; parent-identified as 67% Black and 25% Hispanic) to identify rare variants (minor allele frequency < 0.01). We assigned variants to genes and tested for associations using an omnibus variant-set test between each of 24,902 genes and 8 asthma-associated quantitative traits. On combining our results with external data on predicted gene expression in humans and mouse knockout studies, we identified 3 candidate genes. A burden of rare variants in each gene and in a combined 3-gene score was tested for its associations with clinical phenotypes of asthma. Finally, published single-cell gene expression data in lower airway mucosal cells after allergen challenge were used to assess transcriptional responses to allergen. RESULTS: Rare variants in USF1 were significantly associated with blood neutrophil count (P = 2.18 × 10-7); rare variants in TNFRSF21 with total IgE (P = 6.47 × 10-6) and PIK3R6 with eosinophil count (P = 4.10 × 10-5) reached suggestive significance. These 3 findings were supported by independent data from human and mouse studies. A burden of rare variants in TNFRSF21 and in a 3-gene score was associated with allergy-related phenotypes in cohorts of children with mild and severe asthma. Furthermore, TNFRSF21 was significantly upregulated in bronchial basal epithelial cells from adults with allergic asthma but not in adults with allergies (but not asthma) after allergen challenge. CONCLUSIONS: We report novel associations between rare variants in genes and allergic and inflammatory phenotypes in children with diverse ancestries, highlighting TNFRSF21 as contributing to the development of allergic asthma.

Single-cell RNA sequencing of murine ankle joints over time reveals distinct transcriptional changes following Borrelia burgdorferi infection.

Lyme disease is caused by the bacterial pathogen Borrelia burgdorferi, which can be readily modeled in laboratory mice. In order to understand the cellular and transcriptional changes that occur during B. burgdorferi infection, we conducted single-cell RNA sequencing (scRNA-seq) of ankle joints of infected C57BL/6 mice over time. We found that macrophages/monocytes, T cells, synoviocytes and fibroblasts all showed significant differences in gene expression of both inflammatory and non-inflammatory genes that peaked early and returned to baseline before the typical resolution of arthritis. Predictions of cellular interactions showed that macrophages appear to communicate extensively between different clusters of macrophages as well as with fibroblasts and synoviocytes. Our data give unique insights into the interactions between B. burgdorferi and the murine immune system over time and allow for a better understanding of mechanisms by which the dysregulation of the immune response may lead to prolonged symptoms in some patients.

Uncovering a CF3 Effect on X-ray Absorption Energies of [Cu(CF3 )4 ]- and Related Copper Compounds by Using Resonant Diffraction Anomalous Fine Structure (DAFS) Measurements.

Understanding the electronic structures of high-valent metal complexes aids the advancement of metal-catalyzed cross coupling methodologies. A prototypical complex with formally high valency is [Cu(CF3 )4 ]- (1), which has a formal Cu(III) oxidation state but whose physical analysis has led some to a Cu(I) assignment in an inverted ligand field model. Recent examinations of 1 by X-ray spectroscopies have led previous authors to contradictory conclusions, motivating the re-examination of its X-ray absorption profile here by a complementary method, resonant diffraction anomalous fine structure (DAFS). From analysis of DAFS measurements for a series of seven mononuclear Cu complexes including 1, here it is shown that there is a systematic trifluoromethyl effect on X-ray absorption that blue shifts the resonant Cu K-edge energy by 2-3 eV per CF3 , completely accounting for observed changes in DAFS profiles between formally Cu(III) complexes like 1 and formally Cu(I) complexes like (Ph3 P)3 CuCF3 (3). Thus, in agreement with the inverted ligand field model, the data presented herein imply that 1 is best described as containing a Cu(I) ion with dn count approaching 10.

Immune Responses in Checkpoint Myocarditis Across Heart, Blood, and Tumor.

Immune checkpoint inhibitors (ICIs) are widely used anti-cancer therapies that can cause morbid and potentially fatal immune-related adverse events (irAEs). ICI-related myocarditis (irMyocarditis) is uncommon but has the highest mortality of any irAE. The pathogenesis of irMyocarditis and its relationship to anti-tumor immunity remain poorly understood. We sought to define immune responses in heart, tumor, and blood during irMyocarditis and identify biomarkers of clinical severity by leveraging single-cell (sc)RNA-seq coupled with T cell receptor (TCR) sequencing, microscopy, and proteomics analysis of 28 irMyocarditis patients and 23 controls. Our analysis of 284,360 cells from heart and blood specimens identified cytotoxic T cells, inflammatory macrophages, conventional dendritic cells (cDCs), and fibroblasts enriched in irMyocarditis heart tissue. Additionally, potentially targetable, pro-inflammatory transcriptional programs were upregulated across multiple cell types. TCR clones enriched in heart and paired tumor tissue were largely non-overlapping, suggesting distinct T cell responses within these tissues. We also identify the presence of cardiac-expanded TCRs in a circulating, cycling CD8 T cell population as a novel peripheral biomarker of fatality. Collectively, these findings highlight critical biology driving irMyocarditis and putative biomarkers for therapeutic intervention.

Interstitial lung diseases in inborn errors of immunity.

PURPOSE OF REVIEW: Our goal is to review current understanding of interstitial lung disease (ILD) affecting patients with inborn errors of immunity (IEI). This includes understanding how IEI might predispose to and promote development or progression of ILD as well as how our growing understanding of IEI can help shape treatment of ILD in these patients. Additionally, by examining current knowledge of ILD in IEI, we hope to identify key knowledge gaps that can become focus of future investigative efforts. RECENT FINDINGS: Recent identification of novel IEI associated with ILD and the latest reports examining treatment of ILD in IEI are included. Of noted interest, are recent clinical studies of immunomodulatory therapy for ILD in common variable immunodeficiency. SUMMARY: ILD is a frequent complication found in many IEI. This article provides a guide to identifying manifestations of ILD in IEI. We review a broad spectrum of IEI that develop ILD, including antibody deficiency and immune dysregulation disorders that promote autoimmunity and autoinflammation. This work integrates clinical information with molecular mechanisms of disease and diagnostic assessments to provide an expedient overview of a clinically relevant and expanding topic.

Resident memory T cell development is associated with AP-1 transcription factor upregulation across anatomical niches.

Tissue-resident memory T (T RM ) cells play a central role in immune responses to pathogens across all barrier tissues after infection. However, the underlying mechanisms that drive T RM differentiation and priming for their recall effector function remains unclear. In this study, we leveraged both newly generated and publicly available single-cell RNA-sequencing (scRNAseq) data generated across 10 developmental time points to define features of CD8 T RM across both skin and small-intestine intraepithelial lymphocytes (siIEL). We employed linear modeling to capture temporally-associated gene programs that increase their expression levels in T cell subsets transitioning from an effector to a memory T cell state. In addition to capturing tissue-specific gene programs, we defined a consensus T RM signature of 60 genes across skin and siIEL that can effectively distinguish T RM from circulating T cell populations, providing a more specific T RM signature than what was previously generated by comparing bulk T RM to naïve or non-tissue resident memory populations. This updated T RM signature included the AP-1 transcription factor family members Fos, Fosb and Fosl2 . Moreover, ATACseq analysis detected an enrichment of AP-1-specific motifs at open chromatin sites in mature T RM . CyCIF tissue imaging detected nuclear co-localization of AP-1 members Fosb and Junb in resting CD8 T RM >100 days post-infection. Taken together, these results reveal a critical role of AP-1 transcription factor members in T RM biology and suggests a novel mechanism for rapid reactivation of resting T RM in tissue upon antigen encounter.

Activation of robust bonds by carbonyl complexes of Mn, Fe and Co.

Metal carbonyl complexes possess among the most storied histories of any compound class in organometallic chemistry. Nonetheless, these old dogs continue to be taught new tricks. In this Feature, we review the historic discoveries and recent advances in cleaving robust bonds (e.g., C-H, C-O, C-F) using carbonyl complexes of three metals: Mn, Fe, and Co. The use of Mn, Fe, and Co carbonyl catalysts in controlling selectivity during hydrofunctionalization reactions is also discussed. The chemistry of these earth-abundant metals in the field of robust bond functionalization is particularly relevant in the context of sustainability. We expect that an up-to-date perspective on these seemingly simple organometallic species will emphasize the wellspring of reactivity that continues to be available for discovery.

Metal Site-Specific Electrostatic Field Effects on a Tricopper(I) Cluster Probed by Resonant Diffraction Anomalous Fine Structure (DAFS).

Studies of multinuclear metal complexes are greatly enhanced by resonant diffraction measurements, which probe X-ray absorption profiles of crystallographically independent metal sites within a cluster. In particular, X-ray diffraction anomalous fine structure (DAFS) analysis provides data that can be interpreted akin to site-specific XANES, allowing for differences in metal K-edge resonances to be deconvoluted even for different metal sites within a homometallic system. Despite the prevalence of Cu-containing clusters in biology and energy science, DAFS has yet to be used to analyze multicopper complexes of any type until now. Here, we report an evaluation of trends using a series of strategically chosen Cu(I) and Cu(II) complexes to determine how energy dependencies of anomalous scattering factors are impacted by coordination geometry, ligand shell, cluster nuclearity, and oxidation state. This calibration data is used to analyze a formally tricopper(I) complex that was found by DAFS to be site-differentiated due to the unsymmetrical influence on different Cu sites of the electrostatic field from a proximal K+ cation.