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One possibility for the non-invasive imaging of encapsulated cell grafts is to label the lumen of cell embedding capsules with a redox-responsive probe, as an increased extracellular reducing potential can be considered as a marker of hypoxia-induced necrosis. A Gd(III)-HPDO3A-like chelate has been conjugated to glycol-chitosan through a redox-responsive disulphide bond to obtain a contrast agent for Magnetic Resonance Imaging (MRI). Such a compound can be interspersed with fibroblasts within the lumen of alginate-chitosan capsules. Increasing reducing conditions within the extracellular microenvironment lead to the reductive cleavage of the disulphide bond and to the release of gadolinium in the form of a low molecular weight, non-ionic chelate. The efflux of such chelate from capsules is readily detected by a decrease of contrast enhancement in T 1 -weighted MR images.

[Image: see text] A recently developed synthetic protocol allowed for the functionalization of the active peptide A9 with a fluorogenic probe, which is useful for studying biomolecular interactions. Essentially, a nucleophilic attack on a halo-substituted benzofurazan is selectively performed by a cysteine sulfhydryl group. The process is assisted by the basic catalysis of activated zeolites (4 Å molecular sieves) and promoted by microwave irradiation. Fluorescence studies revealed that a donor–acceptor pair within the peptide sequence was introduced, thus allowing a deeper investigation on the interaction process between the peptide ligand and its receptor fragment. The obtained results allowed us to come full circle for all the currently understood structural determinants that were found to be involved in the binding process.

International audience; Dysregulated remodeling of the extracellular matrix (ECM) can lead to excessive accumulation of ECM proteins (primarily collagen, elastin/ tropoelastin, fibronectin and fibrin) resulting in tissue fibrosis. In many pathologies, changes in the molecular pattern of ECM components have been related to the progression and severity of fibrosis. Thus, magnetic resonance imaging (MRI) probes sensing specific ECM components hold promise for accurate staging of fibrotic diseases. This paper focuses on gadolinium-based contrast agents (GBCA) targeted to ECM components, including structural proteins and enzymes. According to available examples, they can be grouped into: 1) GBCA conjugated to targeting vectors that recognize and noncovalently bind to specific sites on the molecular target; 2) GBCA carrying a reactive chemical function able to bind covalently to the complementary chemical function of the molecular target; and 3) enzyme-responsive probes, whose relaxivity and pharmacokinetics change after enzymatic processing. Pros and cons of each approach are discussed.

International audience; Dysfunctional elastin turnover plays a major role in the progression of atherosclerotic plaques. Failure of tropoelastin cross-linking into mature elastin leads to the accumulation of tropoelastin within the growing plaque, increasing its instability. Here we present Gd4-TESMA, an MRI contrast agent specifically designed for molecular imaging of tropoelastin within plaques. Gd4-TESMA is a tetrameric probe composed of a tropoelastin-binding peptide (the VVGS-peptide) conjugated with four Gd(III)-DOTA-monoamide chelates. It shows a relaxivity per molecule of 34.0 ± 0.8 mM-1 s-1 (20 MHz, 298 K, pH 7.2), a good binding affinity to tropoelastin (KD = 41 ± 12 μM), and a serum half-life longer than 2 h. Gd4-TESMA accumulates specifically in atherosclerotic plaques in the ApoE-/- murine model of plaque progression, with 2 h persistence of contrast enhancement. As compared to the monomeric counterpart (Gd-TESMA), the tetrameric Gd4-TESMA probe shows a clear advantage regarding both sensitivity and imaging time window, allowing for a better characterization of atherosclerotic plaques.

Presentation at Open Repository 2021

An efficient synthesis of vinyl‐[1‐13C]pyruvate has been reported, from which 13C hyperpolarized (HP) ethyl‐[1‐13C]pyruvate has been obtained by means of ParaHydrogen Induced Polarization (PHIP). Due to the intrinsic lability of pyruvate, which leads quickly to degradation of the reaction mixture even under mild reaction conditions, the vinyl‐ester has been synthesized through the intermediacy of a more stable ketal derivative. 13C and 1H hyperpolarizations of ethyl‐[1‐13C]pyruvate, hydrogenated using ParaHydrogen, have been compared to those observed on the more widely used allyl‐derivative. It has been demonstrated that the spin order transfer from ParaHydrogen protons to 13C, is more efficient on the ethyl than on the allyl‐esterdue to the larger J‐couplings involved. The main requirements needed for the biological application of this HP product have been met, i. e. an aqueous solution of the product at high concentration (40 mM) with a good 13C polarization level (4.8 %) has been obtained. The in vitro metabolic transformation of the HP ethyl‐[1‐13C]pyruvate, catalyzed by an esterase, has been observed. This substrate appears to be a good candidate for in vivo metabolic investigations using PHIP hyperpolarized probes., 13C hyperpolarized ethyl‐[1‐13C]pyruvate by means of PHIP‐SAH: Vinyl‐[1‐13C]pyruvate has been prepared on laboratory scale through a new synthesis route. After its hydrogenation with p‐H2 and magnetization transfer (via MFC), polarized ethyl‐[1‐13C]pyruvate has been obtained in aqueous solution at high concentration (40 mM) and good polarization level (4.8 %). In the end, the in‐vitro conversion of HP ethyl‐[1‐13C]pyruvate into [1‐13C]pyruvate catalysed by an esterase, has been observed.

Aziridine derivatives involved in nucleophilic ring-opening reactions have attracted great interest, since they allow the preparation of biologically active molecules. A chemoselective and mild procedure to convert a peptide cysteine residue into lanthionine via S-alkylation on aziridine substrates is presented in this paper. The procedure relies on a post-synthetic protocol promoted by molecular sieves to prepare lanthionine-containing peptides and is assisted by microwave irradiation. In addition, it represents a valuable alternative to the stepwise approach, in which the lanthionine precursor is incorporated into peptides as a building block.

A green and efficient method for preparing lanthionine peptides by a highly chemoselective and stereochemically controlled procedure is presented. It involves anS-alkylation reaction, promoted by activated molecular sieves, on chiral cyclic sulfamidates, bothN-protected and unprotected. Of note, the reaction yield was high also for cyclic sulfamidates bearing a free amine group, while other strategies failed to achieve a ring-opening nucleophilic reaction withN-unprotected substrates. To prove the feasibility of the procedure, the synthesis of a thioether ring B mimetic of the natural lantibiotic haloduracin β was performed.

Cell scaffolds are often used in cell transplantation as they provide a solid structural support to implanted cells and can be bioengineered to mimic the native extracellular matrix. Gadolinium fluoride nanoparticles (Gd-NPs) as a contrast agent for Magnetic Resonance Imaging (MRI) were incorporated into poly(lactide-co-glycolide)/chitosan scaffolds to obtain Imaging Labelled Cell Scaffolds (ILCSs), having the shape of hollow spherical/ellipsoidal particles (200&ndash, 600 &mu, m diameter and 50&ndash, 80 &mu, m shell thickness). While Gd-NPs incorporated into microparticles do not provide any contrast enhancement in T1-weighted (T1w) MR images, ILCSs can release Gd-NPs in a controlled manner, thus activating MRI contrast. ILCSs seeded with human mesenchymal stromal cells (hMSCs) were xenografted subcutaneously into either immunocompromised and immunocompetent mice without any immunosuppressant treatments, and the transplants were followed-up in vivo by MRI for 18 days. Immunocompromised mice showed a progressive activation of MRI contrast within the implants due to the release of Gd-NPs in the extracellular matrix. Instead, immunocompetent mice showed poor activation of MRI contrast due to the encapsulation of ILCSs within fibrotic capsules and to the scavenging of released Gd-NPs by phagocytic cells. In conclusion, the MRI follow-up of cell xenografts can report the host cell response to the xenograft. However, it does not strictly report on the viability of transplanted hMSCs.

Numerous studies on molluscs have been carried out to clarify the physiological roles of haemolymph serum proteins and haemocytes. However, little is known about the presence and functional role of the serum metabolites. In this study, Nuclear Magnetic Resonance (NMR) was used to assess whether changes of the metabolic profile of Mytilus galloprovincialis haemolymph may reflect alterations of the physiological status of the organisms due to environmental stressors, namely copper and temperature. Mussel haemolymph was taken from the posterior adductor muscle after a 4-day exposure to ambient (16 °C) or high temperature (24 °C) and in the absence or presence (5 μg/L, 20 μg/L, or 40 μg/L) of sublethal copper (Cu2+). The total glutathione (GSH) concentration in the haemolymph of both control and treated mussels was minimal, indicating the absence of significant contaminations by muscle intracellular metabolites due to the sampling procedure. In the 1H-NMR spectrum of haemolymph, 27 metabolites were identified unambiguously. The separate and combined effects of exposure to copper and temperature on the haemolymph metabolic profile were assessed by Principal Component Analysis (PCA) and Ranking-PCA multivariate analysis. Changes of the metabolomic profile due to copper exposure at 16 °C became detectable at a dose of 20 μg/L copper. Alanine, lysine, serine, glutamine, glycogen, glucose and protein aliphatics played a major role in the classification of the metabolic changes according to the level of copper exposition. High temperature (24 °C) and high copper levels caused a coherent increase of a common set of metabolites (mostly glucose, serine, and lysine), indicating that the metabolic impairment due to high temperature is enforced by the presence of copper. Overall, the results demonstrate that, as for human blood plasma, the analysis of haemolymph metabolites represents a promising tool for the diagnosis of pollutant-induced stress syndrome in marine mussels.

The redox microenvironment within a cell graft can be considered as an indicator to assess whether the graft is metabolically active or hypoxic. We present a redox-responsive MRI probe based on porous silica microparticles whose surface has been decorated with a Gd-chelate through a disulphide bridge. Such microparticles are designed to be interspersed with therapeutic cells within a biocompatible hydrogel. The onset of reducing conditions within the hydrogel is paralleled by an increased clearance of Gd, that can be detected by MRI.

A one-pot, high-yield procedure for synthesizing lanthionine-containing peptides was developed. It relies on the S-alkylation of cysteine-containing peptides with chiral cyclic sulfamidates. The key feature of this approach is the use of mild reaction conditions (only activated molecular sieves are employed as the catalyst), leading to good chemoselectivity and excellent stereochemical control. The potential of the new methodology has been investigated by synthesizing the thioether ring of a natural lantibiotic, Haloduracin β.

An efficient and rapid procedure for synthesizing S-linked glycopeptides is reported. The approach uses activated molecular sieves as a base to promote the selective S-alkylation of readily prepared cysteine-containing peptides, upon reaction of appropriate glycosyl halides. Considering the very mild conditions employed, the chemoselective linkage of the electrophilic sugar with a peptide sulfhydryl group occurred in satisfactory yield, allowing the incorporation of mono and disaccharide moieties. The sugar-peptide conjugates obtained from α-d-glycosyl derivatives adopt a β-S-configuration, indicating the high stereoselectivity of the substitution reaction.

Molecular imaging requires the specific accumulation of contrast agents at the target. To exploit the superb resolution of MRI for applications in molecular imaging, gadolinium chelates, as the MRI contrast agents (CA), have to be conjugated to a specific vector able to recognize the epitope of interest. Several Gd(III)-chelates can be chemically linked to the same binding vector in order to deliver multiple copies of the CA (multimers) in a single targeting event thus increasing the sensitivity of the molecular probe. Herein three novel bifunctional agents, carrying one functional group for the bioconjugation to targeting vectors and four Gd(III)-AAZTA chelate functions for MRI contrast enhancement (AAZTA = 6-amino-6-methylperhydro-1,4-diazepinetetraacetic acid), are reported. The relaxivity in the tetrameric derivatives is 16.4 ± 0.2 mMGd–1 s–1 at 21.5 MHz and 25 °C, being 2.4-fold higher than that of parent, monomeric Gd(III)-AAZTA. These compounds can be used as versatile building blocks to insert pre...

Three different 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetamide (DOTAM) derivatives bearing as amide N-substituents phenyl, p-methoxyphenyl and p-ethylbenzoate groups were synthesized and the 1H and 17O NMR relaxometric behaviour of the Gd(III)-chelates and chemical exchange saturation transfer (CEST) effect of the Eu(III) complexes were evaluated. The electronic properties of the substituents were shown to strongly influence the coordinated water exchange rate (kex), resulting in five times faster kex for the electron donating phenylmethoxy group compared to the electron withdrawing ethylbenzoate group.

Commercial Gd-containing complexes are often used as MRI reporters in cellular labeling procedures as they are internalized into endosomes by pinocytosis. A methodology has been applied to assess the relative stability of three commercial Gd contrast agents following cellular uptake in fibroblasts and macrophages. It has been found that the acyclic series of Gd MRI contrast agents are degraded much more rapidly than their macrocyclic analogues, following endosomal internalization into living cells. This helps to explain their causal role in the development of nephrogenic systemic fibrosis in renally impaired patients. The methodology has also been applied to assess the fate of Gd-DTPA-BMA-loaded liposomes upon their endosomal internalization. Resistant liposomes prevent the degradation of the complex, whereas liposomes designed to release their payload in the acidic environments show a loss of integrity of Gd-DTPA-BMA analogous to the one observed upon internalization of the free complex.

The approach to molecular imaging of enzymes by MRI typically relies upon imaging probes composed of an enzymecleavable moiety conjugated with a paramagnetic imaging reporter, such as a GdIII chelate.[1] Upon enzymatic processing, the probe is transformed into a fragment with an altered relaxivity, leading to a different capability to enhance contrast in MR images with respect to the parent species. Ideally, the unprocessed (intact) form of the probe should be completely silent while the processed (cleaved) form should have a high relaxivity (that is, high contrast enhancement). In such a way the appearance of contrast within images can be unambiguously attributed to the result of enzymatic activity and not to dynamic changes of tissue probe concentration. However, gadolinium-based agents as enzyme responsive agents are never completely silent and both forms (unprocessed and processed) contribute to the overall contrast enhancement as a function of their respective relaxivities and tissue local concentrations.[2] Exact knowledge of the total concentration of Gd is essential to translate image contrast enhancement into the molar ratio of unprocessed versus processed forms, and thus into true enzyme activity maps. Aviable solution to the concentration problem can be provided by the R2p/R1p.

// Cecilia Roux 1, 2 , Chiara Riganti 3 , Sammy Ferri Borgogno 1, 2 , Roberta Curto 1, 2 , Claudia Curcio 1, 2 , Valeria Catanzaro 4 , Giuseppe Digilio 4 , Sergio Padovan 5 , Maria Paola Puccinelli 6 , Monica Isabello 6 , Silvio Aime 2, 7 , Paola Cappello 1, 2, 7, * and Francesco Novelli 1, 2, 7, * 1 Center for Experimental Research and Medical Studies, Citta della Salute e della Scienza di Torino, 10126 Turin, Italy 2 Department of Molecular Biotechnology and Health Sciences, University of Turin, 10126 Turin, Italy 3 Department of Oncology, University of Turin, 10126 Turin, Italy 4 Department of Science and Technologic Innovation, Universita del Piemonte Orientale “A. Avogadro”, 15121 Alessandria, Italy 5 Institute for Biostructures and Bioimages (CNR) c/o Molecular Biotechnology Center, 10126 Turin, Italy 6 Clinical Biochemistry Laboratory, Citta della Salute e della Scienza di Torino, 10126 Turin, Italy 7 Molecular Biotechnology Center, University of Turin, 10126 Turin, Italy * These authors have contributed equally to this work Correspondence to: Francesco Novelli, email: franco.novelli@unito.it Paola Cappello, email: paola.cappello@unito.it Keywords: PDAC; amino acid; circulating biomarkers; pancreatitis; diagnosis Received: March 24, 2017 Accepted: August 04, 2017 Published: August 24, 2017 ABSTRACT Pancreatic ductal adenocarcinoma (PDAC) is becoming the second leading cause of cancer-related death in the Western world. The mortality is very high, which emphasizes the need to identify biomarkers for early detection. As glutamine metabolism alteration is a feature of PDAC, its in vivo evaluation may provide a useful tool for biomarker identification. Our aim was to identify a handy method to evaluate blood glutamine consumption in mouse models of PDAC. We quantified the in vitro glutamine uptake by Mass Spectrometry (MS) in tumor cell supernatants and showed that it was higher in PDAC compared to non-PDAC tumor and pancreatic control human cells. The increased glutamine uptake was paralleled by higher activity of most glutamine pathway-related enzymes supporting nucleotide and ATP production. Free glutamine blood levels were evaluated in orthotopic and spontaneous mouse models of PDAC and other pancreatic-related disorders by High-Performance Liquid Chromatography (HPLC) and/or MS. Notably we observed a reduction of blood glutamine as much as the tumor progressed from pancreatic intraepithelial lesions to invasive PDAC, but was not related to chronic pancreatitis-associated inflammation or diabetes. In parallel the increased levels of branched-chain amino acids (BCAA) were observed. By contrast blood glutamine levels were stable in non-tumor bearing mice. These findings demonstrated that glutamine uptake is measurable both in vitro and in vivo . The higher in vitro avidity of PDAC cells corresponded to a lower blood glutamine level as soon as the tumor mass grew. The reduction in circulating glutamine represents a novel tool exploitable to implement other diagnostic or prognostic PDAC biomarkers.

Summary Protection against oxidative damage caused by excessive reactive oxygen species (ROS) by an antioxidant network is essential for the health of tissues, especially in the cardiovascular system. Here, we identified a gene with important antioxidant features by analyzing a null allele of zebrafish ubiad1, called barolo (bar). bar mutants show specific cardiovascular failure due to oxidative stress and ROS-mediated cellular damage. Human UBIAD1 is a nonmitochondrial prenyltransferase that synthesizes CoQ10 in the Golgi membrane compartment. Loss of UBIAD1 reduces the cytosolic pool of the antioxidant CoQ10 and leads to ROS-mediated lipid peroxidation in vascular cells. Surprisingly, inhibition of eNOS prevents Ubiad1-dependent cardiovascular oxidative damage, suggesting a crucial role for this enzyme and nonmitochondrial CoQ10 in NO signaling. These findings identify UBIAD1 as a nonmitochondrial CoQ10-forming enzyme with specific cardiovascular protective function via the modulation of eNOS activity., Graphical Abstract Highlights ► UBIAD1 is a Golgi prenyltransferase ► UBIAD1 contributes to the nonmitochondrial pool of CoQ10 ► UBIAD1 protects cardiovascular tissues from NOS-dependent oxidative damage ► UBIAD1 is a target for therapeutic strategies by limiting the side effects of statins, UBIAD1 is identified as a nonmitochondrial CoQ10 biosynthetic enzyme required for oxidative damage protection. Through CoQ10 synthesis, UBIAD1 modulates endothelial nitric oxide synthase activity and nitric oxide signaling necessary for cardiovascular development and homeostasis.

Inflammation is signaled by the overexpression of epitopes on the vascular endothelium that primarily aim at recruiting immune cells into the inflamed area. The intravascular localization of these biomarkers makes them suitable targets for the MRI visualization of inflammation. Phospholipid-based nanosystems appear excellent candidates in virtue of their good biocompatibility, ability to deliver a high number of imaging units at the target site, and for the easy functionalization with targeting vectors. In this work, phospholipid-based micelles (hydrodynamic diameter of 20 nm) loaded with the amphiphilic Gd(III)-complex Gd-DOTAMA(C18)2 were vectorized with a small peptide able to specifically bind VCAM-1 receptors. The micelles displayed a high longitudinal relaxivity (36.4 s-1mmolGd-1 at 25 °C and 0.7 T). A 1H- and 17O-water relaxometry study indicated that the paramagnetic complex embedded in the nanoparticles adopted two isomeric conformations, likely reflecting the well-known square antiprismatic (SAP) and twisted square antiprismatic (TSAP) configurations typically observed in DOTA-like lanthanide complexes. Interestingly, the TSAP structure, showing a much faster exchange rate for the water molecule coordinated to the metal ion, was the most abundant, thus explaining the high relaxivity of the micellar agent. The systemic administration of the micelles into a lipopolysaccharide-induced murine model of acute inflammation successfully demonstrated the ability of the targeting agents to detect the diseased area by T1 contrast enhanced MRI.

Two novel Gd-based contrast agents (CAs) for the molecular imaging of matrix metalloproteinases (MMPs) were synthetized and characterized in vitro and in vivo. These probes were based on the PLG*LWAR peptide sequence, known to be hydrolyzed between Gly and Leu by a broad panel of MMPs. A Gd–DOTA chelate was conjugated to the N-terminal position through an amide bond, either directly to proline (compd Gd–K11) or through a hydrophilic spacer (compd Gd–K11N). Both CA were made strongly amphiphilic by conjugating an alkyl chain at the C-terminus of the peptide sequence. Gd–K11 and Gd–K11N have a good affinity for β-cyclodextrins (KD 310 and 670 µ m respectively) and for serum albumin (KD 350 and 90 µ m respectively), and can be efficiently cleaved in vitro at the expected site by MMP-2 and MMP-12. Upon MMP-dependent cleavage, the CAs lose the C-terminal tetrapeptide and the alkyl chain, thus undergoing to an amphiphilic-to-hydrophilic transformation that is expected to alter tissue pharmacokinetics. To prove this, Gd–K11 was systemically administered to mice bearing a subcutaneous B16.F10 melanoma, either pre-treated or not with the broad spectrum MMP inhibitor GM6001 (Ilomastat). The washout of the Gd-contrast enhancement in MR images was significantly faster for untreated subjects (displaying MMP activity) with respect to treated ones (MMP activity inhibited). The washout kinetics of Gd-contrast enhancement from the tumor microenvironment could be then interpreted in terms of the local activity of MMPs. Copyright © 2012 John Wiley & Sons, Ltd.

Iopamidol (Isovue®-Bracco Diagnostic Inc.) is a clinically approved X-Ray contrast agent used in the last 30 years for a wide variety of diagnostic applications with a very good clinical acceptance. Iopamidol contains two types of amide functionalities that can be exploited for the generation of chemical exchange saturation transfer effect. The exchange rate of the two amide proton pools is markedly pH-dependent. Thus, a ratiometric method for pH assessment has been set-up based on the comparison of the saturation transfer effects induced by selective irradiation of the two resonances. This ratiometric approach allows to rule out the concentration effect of the contrast agent and provides accurate pH measurements in the 5.5-7.4 range. Upon injection of Iopamidol into healthy mice, it has been possible to acquire pH maps of kidney regions. Furthermore, it has been also shown that the proposed method is able to report about pH-changes induced in control mice fed with acidified or basified water for a period of a week before image acquisition.

The metal-thiolate connectivity of recombinant Cd(7)-MT10 metallothionein from the sea mussel Mytilus galloprovincialis has been investigated for the first time by means of multinuclear, multidimensional NMR spectroscopy. The internal backbone dynamics of the protein have been assessed by the analysis of (15)N T (1) and T (2) relaxation times and steady state {(1)H}-(15)N heteronuclear NOEs. The (113)Cd NMR spectrum of mussel MT10 shows unique features, with a remarkably wide dispersion (210 ppm) of (113)Cd NMR signals. The complete assignment of cysteine Halpha and Hbeta proton resonances and the analysis of 2D (113)Cd-(113)Cd COSY and (1)H-(113)Cd HMQC type spectra allowed us to identify a four metal-thiolate cluster (alpha-domain) and a three metal-thiolate cluster (beta-domain), located at the N-terminal and the C-terminal, respectively. With respect to vertebrate MTs, the mussel MT10 displays an inversion of the alpha and beta domains inside the chain, similar to what observed in the echinoderm MT-A. Moreover, unlike the MTs characterized so far, the alpha-domain of mussel Cd(7)-MT10 is of the form M(4)S(12) instead of M(4)S(11), and has a novel topology. The beta-domain has a metal-thiolate binding pattern similar to other vertebrate MTs, but it is conformationally more rigid. This feature is quite unusual for MTs, in which the beta-domain displays a more disordered conformation than the alpha-domain. It is concluded that in mussel Cd(7)-MT10, the spacing of cysteine residues and the plasticity of the protein backbone (due to the high number of glycine residues) increase the adaptability of the protein backbone towards enfolding around the metal-thiolate clusters, resulting in minimal alterations of the ideal tetrahedral geometry around the metal centres.

Under HR-MAS conditions, cells are subjected to high centrifugal forces that may cause irreversible cell damage. First, conditions have been defined to monitor and keep to a minimum unwanted effects in HR-MAS spectra arising from the loss of cell integrity. Then, the HR-MAS spectra of reasonably intact cells have been analyzed. Cell suspensions subjected to MAS rates as low as 1 kHz split into a two-compartment system that is composed of a cell-rich phase (H2Oi) and a cell-free phase (H2Oo). Each of these phases is characterized by its own water 1H-NMR signal. Transport of water molecules between the cell-rich and cell-free compartments is limited by the very low contact area between the two compartments, and water exchange dynamics consequently fall into the slow exchange limit on the NMR timescale. Since the exchange between the two water populations is “frozen,” the separation between the H2Oo and H2Oi water signals (Δνwater) detected in an HR-MAS experiment is not affected by chemical exchange but reflects only chemical differences in the two environments. Different cell lines show a different Δνwater, leading to the concept of “cellular water shift.” This shift roughly correlates with the cellular protein content, supporting the view that the most important determinant of the cellular water shift is the interaction between water and proteins in the intracellular compartment. Magn Reson Med, 2005. © 2005 Wiley-Liss, Inc.

HMGB1 (high mobility group B1) is a conserved chromosomal protein composed of two similar DNA binding domains (HMG box A and box B) linked by a short basic stretch to an acidic C-terminal tail of 30 residues. The acidic tail modulates the DNA binding properties of HMGB1, and its length differentiates the various HMGB family members. We synthesized a peptide that corresponds to the acidic tail in HMGB1 (T-peptide) and studied its binding to the single boxes and to the fragment corresponding to tailless HMGB1 (designated as AB(bt) fragment). CD spectroscopy showed that T-peptide stabilizes significantly the AB(bt) fragment and that the complex has an identical thermal stability as full-length HMGB1. Calorimetric and NMR data showed that T-peptide binds with a dissociation constant of 9 microM to box A and much more weakly to box B. (1)H-(15)N HSQC spectra of full-length HMGB1 and of the AB(bt) fragment are very similar; the small chemical shift differences that exist correspond to those residues of the AB(bt) fragment that were affected by the addition of the T-peptide. We conclude that the T-peptide mimics closely the acidic tail and that the basic stretch and the acidic tail form an extended and flexible segment. The tail interacts with specific residues in the boxes and shields them from other interactions.

Two novel mono-PEGylated derivatives of hGRF(1-29)-NH(2) [human growth hormone-releasing factor, fragment 1-29] have been synthesized by regio-specific conjugation of Lys(12) or Lys(21) to a monomethoxy-PEG(5000) chain (compounds Lys(12)PEG-GRF and Lys(21)PEG-GRF). The PEG moiety has been covalently linked at the amino group of a norleucine residue via a carbamate bond. The Lys(12)PEG-GRF regioisomer was found to be slightly less active in vitro than both the unmodified peptide and Lys(21)PEG-GRF. To assess whether the differences in the biological activity of the PEGylated analogues could be related to conformational rearrangements induced by the PEG moiety, the structure of these PEGylated derivatives has been worked out (TFE solution) by means of NMR spectroscopy and molecular dynamics. Secondary structure shifts, hydrogen/deuterium exchange kinetics, temperature coefficients of amide protons, and NOE-based molecular models point out that hGRF(1-29)-NH(2), Lys(21)PEG-GRF and Lys(12)PEG-GRF share a remarkably similar pattern of secondary structure. All three compounds adopt an alpha-helix conformation which spans the whole length of the molecule, and which becomes increasingly rigid on going from the N-terminus to the C-terminus. Residues Lys(12) and Lys(21) are enclosed in all the compounds considered into well-defined alpha-helical domains, indicating that PEGylation either at Lys(12) or Lys(21) does not alter the tendency of the peptide to adopt a stable alpha-helix conformation, nor does it induce appreciable conformational mobility in the proximity of the PEGylation sites. No significant variation of the amphiphilic organization of the alpha-helix is observed among the three peptides. Therefore, the different biological activities observed for the PEGylated analogues are not due to conformational effects, but are rather due to sterical hindrance effects. The relationship between the biological activitiy of the mono-PEGylated derivatives and sterical hindrance is discussed in terms of the topology of interaction between hGRF(1-29)-NH(2) and its receptor.

The segment 32-47 of the N-terminal extracellular domain of the type A cholecystokinin receptor, CCKA-R(32-47), was synthesized and structurally characterized in a membrane mimicking environment by CD, NMR and molecular dynamics calculations. The region of CCKA-R(32-47) encompassing residues 39-46 adopted a well-defined secondary structure in the presence of DPC micelles, whereas the conformation of the N-terminal region (segment 32-37) could not be uniquely defined by the NOE derived distance constraints because of local flexibility. The conformation of the binding domain of CCKA-R(32-47) was different from that found for the intact N-terminal receptor tail, CCKA-R(1-47). To assess whether CCKA-R(32-47) was still able to bind the nonsulfated cholecystokinin C-terminal octapeptide, CCK8, a series of titrations was carried out in SDS and DPC micelles, and the binding interaction was followed by fluorescence spectroscopy. These titrations gave no evidence for complex formation, whereas a high binding affinity was found between CCKA- R(1-47) and CCK8. The different affinities for the ligand shown by CCKA-R(32-47) and CCKA-R(1-47) were paralleled by different interaction modes between the receptor segments and the micelles. The interaction of CCKA-R(32-47) with DPC micelles was much weaker than that of CCKA-R(1-47), because the former receptor segment lacks proper stabilizing contacts with the micelle surface. In the case of SDS micelles CCKA-R(32-47) was found to form non-micellar adducts with the detergent that prevented the onset of a functionally significant interaction between the receptor segment and the micelle. It is concluded that tertiary structure interactions brought about by the 1-31 segment play a key role in the stabilization of the membrane bound, biologically active conformation of the N-terminal extracellular tail of the CCKA receptor. Copyright  2003 European Peptide Society and John Wiley & Sons, Ltd.

OBJECTIVES: The aim of this study was to evaluate 4 nonionic x-ray iodinated contrast agents (CAs), commonly used in radiographic procedures, as novel chemical exchange saturation transfer (CEST) magnetic resonance imaging (MRI) agents by assessing their in vitro exchange properties and preliminary in vivo use as tumor enhancing agents. MATERIALS AND METHODS: The CEST properties, as function of pH (range, 5.5-7.9) and of radio frequency conditions (irradiation field strength range of 1-9 ?T and time of 1-9 seconds), have been determined at 7 T and 310 K for 4 x-ray CAs commonly used in clinical settings, namely, iomeprol, iohexol, ioversol, and iodixanol. Their in vivo properties have been investigated upon intravenous injection in a murine HER2+ breast tumor model (n = 4 mice for each CA) using both computed tomography (CT) and MRI modalities. RESULTS: The prototropic exchange rates measured for the 4 investigated iodinated molecules showed strong pH dependence with base catalyzed exchange rate that was faster for monomeric compounds (20-4000 Hz in the pH range of 5.5-7.9). Computed tomography quantification showed marked (up to 2 mg I/mL concentration) and prolonged accumulation (up to 30 minutes postinjection) inside tumor regions. Among the 4 agents we tested, iohexol and ioversol display good CEST contrast properties at 7 T, and in vivo results confirmed strong and prolonged contrast enhancement of the tumors, with elevated extravasation fractions (74%-91%). A strong and significant correlation was found between CT and CEST-MRI tumor-enhanced images (R = 0.70, P < 0.01). CONCLUSIONS: The obtained results demonstrate that iohexol and ioversol, 2 commonly used radiographic compounds, can be used as MRI perfusion agents, particularly useful when serial images acquisitions are needed to complement CT information.

Antide is a decapeptide [(N-Ac-D-Nal(1)-D-Cpa(2)-D-Pal(3)-Ser(4)-Lys(Nic)(5)-D-Lys(Nic)(6)-Leu(7)-Ilys(8)-Pro(9)-D-Ala(10)-NH(2)] that acts in vivo as an antagonist of GnRH (gonadotropin-releasing hormone). The conformational behavior of antide has been studied in water, TFE, DMF, and DMSO solutions by means of 2D-NMR spectroscopy and molecular dynamics calculations. Antide adopts in aqueous solution a delta-shaped backbone conformation, which is characterized by an irregular turn around residues D-Pal(3)-Ser(4) and by the close spatial proximity of the side chains belonging to D-Nal(1) and Ilys(8) (as many as 17 NOE peaks were detected between these side chains). The side-chain protons of Ilys(8) (especially the H(gamma) ones) present remarkably upfield shifted resonances, because of ring current effects induced by the naphthyl moiety. The upfield shifted resonances of the Ilys(8) H(gamma) hydrogen atoms are strictly characteristic of the water delta-shaped conformation and can be considered as structure markers. The observation of ring current shifted Ilys(8) H(gamma) resonances under different conditions (temperature, pH, solvent) indicates a remarkable stability of the water delta-shaped conformation. Such a conformation is at least partially disrupted in solvent mixtures containing high percentages of organic solvents. TFE can induce a well-defined conformation, which is characterized by an S-shaped backbone conformation. In DMF and DMSO solution, the molecule is basically endowed with a random coil conformation and high fluxionality. Antide fulfills the conformational requirements that are known to play a crucial role in receptor recognition, namely (i) the presence of a turn in the backbone and (ii) the all-trans nature of peptide bonds. In addition, the structural rigidity of antide likely adds a further contribution to the receptor binding affinity.

Magnetic resonance imaging (MRI) relies on the topological distribution of the intense water NMR signal and may be used to report about changes in the internal structures of fruits associated to ripening, storing, pathogen infection. Herein the use of CEST–MRI (chemical exchange saturation transfer) is introduced to show that in addition to structural information, insights into the presence in the fruits of specific chemicals may be gained. Asparagine is present in plums at relatively high concentration (≈10–20 mM) and owns two amide protons (at 2.1 and 2.8 ppm down field from water) in slow exchange with water protons. By irradiating the amide resonances with a proper rf-field it is possible to transfer saturated magnetization to the “bulk” water signal. The attained change in signal intensity reflects the extent of prototropic exchange between amide and water protons that is modulated by the local pH.

The low temperature (–100 °C) X-ray structure of the complex K2[Yb(DTPA)(H2O)] has been determined. The metal ion is at the center of a tricapped trigonal prism and is nine-coordinate, binding to the three nitrogens and five oxygens of the ligand and one water molecule. From the structure obtained, three well-defined hydration shells can be observed consisting of: i) one coordinated water molecule; ii) several water molecules in the outer coordination sphere of the YbIII ion and iii) one water molecule surprisingly close to the metal center and hydrogen-bonded to proximate carboxylate groups. A variable-field and temperature NMRD study of the corresponding Gd complex has been performed and the data interpreted by taking into account the presence of second-sphere water molecule(s). The results of the analysis are in excellent agreement with the X-ray structural data.

A new assay for the measurement of the antioxidant capacity of biomolecules by high resolution 19F-NMR spectroscopy is presented here. This method is based on the use of trifluoroacetanilidic detectors, namely trifluoroacetanilide, N-(4-hydroxyphenyl)-trifluoroacetamide and 2-hydroxy-4-trifluoroacetamidobenzoic acid. Upon hydroxyl radical attack, such fluorinated detectors yield trifluoroacetamide and trifluoroacetic acid that can be quantitatively determined by 19F-NMR spectroscopy. Trifluoroacetamide was found to be a reliable reporter of hydroxyl radical attack on the fluorinated detectors, whereas N-(4-hydroxyphenyl)-trifluoroacetamide was found to be the most sensitive detector amongst the ones considered. Therefore, N-(4-hydroxyphenyl)-trifluoroacetamide has been used in competition experiments to assess the antioxidant capacity of a number of low and high molecular weight antioxidants. The antioxidant capacity of a given compound has been scaled in terms of an adimensional parameter, kF, that represents the ratio between the scavenger abilities of the fluorinated detector and the competitor. kF values obtained for low-molecular-mass compounds fall in the range 0.17 < kF < 1.5 and are in good agreement with second order rate constants (k2OH) for the reaction of the antioxidant with hydroxyl radicals. The kF value for serum albumin is much larger (46.9) than that predicted from the reported k2OH value. This finding supports the view that the protein can very effectively scavenge hydroxyl radicals as well as secondary radicals. Human blood serum showed that its antioxidant capacity is even higher than that shown by aqueous solutions of albumin at physiologic concentration suggesting a further contribution from other macromolecular serum components.

Complexes of the cyanomercury cation with various polypyrazolylborato ligands of the type HB(pz)3 · Hg-CN or pzB(pz)3 · Hg-CN (pz = pyrazolyl or substituted pyrazolyl) have been synthesised and characterised by IR, conductivity, 1H, 13C, and 199Hg NMR spectroscopy. The crystal structure of the cyanomercury hydridotris(1H-3,5-dimethylpyrazol-1-yl)borate has been resolved (space group Pī with a = 7.863(3), b = 11.157(5), c = 13.117(5)A; α = 89.32(3), β = 78.31(3), γ = 79.13(4)o, V = 1106.22A3, Z = 2) showing a distorted tetrahedral coordination around Hg. The tris(pyrazolyl)borato complexes contain four-coordinated Hg and are rigid in solution at r.t., while the tetrakis(pyrazolyl)borates are fluxional. The 15N-CPMAS-NMR spectrum of the pzTp · HgCN derivative suggests a tetracoordination around mercury in this complex in the solid state.

Fluorinated, arylsulfone-based inhibitors of Matrix Metalloproteinases (MMP) have been used, in the [ 18 F]-radiolabelled version, as radiotracers targeted to MMP-2/9 for Positron Emission Tomography (PET). Although they showed acceptable tumour uptake, specificity was rather low. To get further insights into the reason of low specificity, the binding interaction of these compounds with Human Serum Albumin (HSA) has been investigated. 19 F NMR spectroscopy showed that all compounds considered partition between multiple HSA binding sites, being characterized by either slow-exchange kinetics (with K a in the order of 10 5 M −1 ) and fast-exchange kinetics (with K a in the order of 10 4 M -1 ). For 2-(2-(4′-(2-fluoroethoxy)biphenyl-4-ylsulfonyl)phenyl)acetic acid ( 1a ) and 2-(2-(4′-(2-fluoroacetamido)biphenyl-4-ylsulfonyl)phenyl)acetic acid ( 1c ), these slow and fast-exchanging binding sites could be mapped to Sudlow's site I and II, respectively. It is shown that high affinity albumin binding constitutes a theoretical limitation for the specificity achievable by MMP-inhibitors as MMP-targeted PET tracers in cancer imaging, because albumin accumulating aspecifically in tumours lowers the binding potential of radiotracers.

Two novel Gd-based contrast agents (CAs) for the molecular imaging of matrix metalloproteinases (MMPs) were synthetized and characterized in vitro and in vivo. These probes were based on the PLG*LWAR peptide sequence, known to be hydrolyzed between Gly and Leu by a broad panel of MMPs. A Gd-DOTA chelate was conjugated to the N-terminal position through an amide bond, either directly to proline (compd Gd-K11) or through a hydrophilic spacer (compd Gd-K11N). Both CA were made strongly amphiphilic by conjugating an alkyl chain at the C-terminus of the peptide sequence. Gd-K11 and Gd-K11N have a good affinity for β-cyclodextrins (K(D) 310 and 670 µ m respectively) and for serum albumin (K(D) 350 and 90 µ m respectively), and can be efficiently cleaved in vitro at the expected site by MMP-2 and MMP-12. Upon MMP-dependent cleavage, the CAs lose the C-terminal tetrapeptide and the alkyl chain, thus undergoing to an amphiphilic-to-hydrophilic transformation that is expected to alter tissue pharmacokinetics. To prove this, Gd-K11 was systemically administered to mice bearing a subcutaneous B16.F10 melanoma, either pre-treated or not with the broad spectrum MMP inhibitor GM6001 (Ilomastat). The washout of the Gd-contrast enhancement in MR images was significantly faster for untreated subjects (displaying MMP activity) with respect to treated ones (MMP activity inhibited). The washout kinetics of Gd-contrast enhancement from the tumor microenvironment could be then interpreted in terms of the local activity of MMPs.

The solid state and solution NMR spectra of the tetrakis(pyrazol-l-yl) borate (L a ) and tris(3,5-dimethylpyrazol-l-yl) hydroborate (L b ) ligands and their complexes with MeHg II and RSHg II (R = Me, Et) are reported. The solid state 13 C and 15 N solid state NMR spectra of the MeHgL a complex are consistent with the reported X-ray structure. The close similarity of the spectral data shown by MeHgL a and MeSHgL a suggests that the two species have the same structure. In order to confirm this hypothesis, the structure of MeSHgL a has been determined by single crystal X-ray diffraction. The chemical shift anisotropy (CSA) of 199 Hg resonance is too large in these T-shaped complexes and does not allow the observation of the 199 Hg resonance under cross polarisation-magic angle spinning (CPMAS) experimental mode. On the other hand 199 Hg CPMAS NMR spectra were obtained for the complexes with the L b ligand. The reduced 199 Hg CSA value together with the information gained from the 13 C and 15 N CPMAS NMR spectra suggest a tetra coordination around mercury in these complexes. The tight co-ordination mode shown by complexes with the L b ligand is reflected in a decrease in the thiol intermolecular exchange rate in solution. This decreased ligand lability allows the detection of 3 J Hg-H couplings (inside HgSCH 3 and HgSCH 2 CH 3 moieties) in the low temperature NMR limiting spectra.

Murine melanoma B16 cells display on the extracellular side of the plasma membrane a large number of reactive protein thiols (exofacial protein thiols, EPTs). These EPTs can be chemically labeled with Gd-DO3A-PDP, a Gd(III)-based MRI contrast agent bearing a 2-pyridinedithio chemical function for the recognition of EPTs. Uptake of gadolinium up to 10(9) Gd atoms per cell can be achieved. The treatment of B16 cells ex vivo with a reducing agent such as tris(2-carboxyethyl)phosphine (TCEP) results in an increase by 850% of available EPTs and an increase by 45% of Gd uptake. Blocking EPTs with N-ethylmaleimide (NEM) caused a decrease by 84% of available EPTs and a decrease by 55% of Gd uptake. The amount of Gd taken up by B16 cells is therefore dependent upon the availability of EPTs, whose actual level in turn changes according to the extracellular redox microenvironment. Then Gd-DO3A-PDP has been assessed for the labeling of tumor cells in vivo on B16.F10 melanoma tumor-bearing mice. Gd-DO3A-PDP (or Gd-DO3A as the control) has been injected directly into the tumor region at a dose level of 0.1 μmol and the signal enhancement in MR images followed over time. The washout kinetics of Gd-DO3A-PDP from tumor is very slow if compared to that of control Gd-DO3A, and 48 h post injection, the gadolinium-enhancement is still clearly visible. Therefore, B16 cells can be labeled ex vivo as well as in vivo according to a common EPTs-dependent route, provided that high levels of the thiol reactive probe can be delivered to the tumor.

A novel class of paramagnetic liposome-based systems acting as dual T(1) and CEST (1)H-MRI contrast agents is described. The vesicles contain a shift reagent in the aqueous core and a Gd-complex on the external surface conjugated through a biodegradable linker. As such, the probe can generate T(1) contrast only, but after the cleavage and removal of the Gd-coating, the CEST contrast is switched on.

Four novel MRI Gd(III)-based probes have been synthesized and evaluated for their labeling properties on cultured cell lines K562, C6, and B16. The labeling strategy relies upon the fact that cells display a large number of reactive exofacial protein thiols (EPTs) that can be exploited as anchorage points for suitably activated MRI probes. The probes are composed of a Gd(III) chelate (based on either DO3A or DTPA) connected through a flexible linker to the 2-pyridyldithio chemical function for binding to EPTs. GdDO3A-based chelates could efficiently label cells (up to a level of 1.2 x 10(10) Gd(III) atoms/cell), whereas GdDTPA-based chelates showed poor or no cell labeling ability at all. Among the GdDO3A based compounds, that having the longest spacer (compound GdL1A) showed the best labeling efficacy. The mechanism of EPT mediated cell labeling by GdL1A involves probe internalization without sequestration of the Gd(III) chelate within subcellular structures such as endosomes.

Cells display on the outer surface of the plasma membrane a large number of protein thiols that can be reversibly labelled with suitably designed Gd(III)-based contrast agents for cell tracking by MRI.

Melanin granules (MGs) have been extracted from human Chinese black hairs by either acid hydrolysis (CH-type MGs) or enzymatic digestion (CP-type MGs), and their chemical structure investigated at the solid state by means of (13)C cross polarization magic angle spinning (CPMAS NMR) and EPR spectroscopy. Both types of MGs contain a large amount of protein that is tightly bound to the true melanin polymer, with CP-type MGs having a larger protein content than CH-type ones. Moreover, MGs may also contain variable amounts of lipid-like material. A high amount of paramagnetic metals is detected by EPR in CP-type MGs, in particular Fe(III). Iron can be bound in two chemical forms: as isolated high spin Fe(III) ions with rhombic symmetry and as small oxy-hydroxy Fe(III) aggregates. Iron is poorly available to chelators. CH-type MGs contain much fewer metals. CP-type MGs have then been subjected to partial bleaching by hydrogen peroxide in ammonia, yielding a residual solid, called residual oxidized melanin (ROM) and a soluble but still pigmented fraction called melanin free acid (MFA). MFA can be isolated by precipitation at acidic pH. The (13)C-CPMAS NMR and EPR spectra of these derivatives indicated that ROM has a structure very similar to that of parent MGs, whereas MFA shows a decrease of the protein content with respect to the melanin and a decreased amount of bound iron. Thus, the oxidative degradation of CP-type MGs is a process not involving the bulk of MGs, but rather it proceeds from the solvent-exposed outer parts to the interior.

A novel Gd(III) complex with a modified DO3A-like chelating cage has been synthesized and characterized as a candidate contrast agent responsive to the concentration of free thiols in tissues (essentially represented by reduced glutathione, GSH). The novel compound (called Gd-DO3AS-Act) bears a flexible linker ending with a 2-pyridyl-dithio group, that can promptly react with free thiols (XSH) to form mixed disulfides of the form Gd-DO3AS-SX. Compound Gd-DO3AS-Act is characterized by a millimolar relaxivity as high as 8.1 mM(-1) s(-1) (at 20 MHz, 25 degrees C and pH 7.4). Upon reaction with GSH, the Gd-DO3AS-SG covalent adduct is formed and the millimolar relaxivity drops to 4.1 mM(-1) s(-1). Such a decrease in relaxivity is explained on the basis of the formation of an intramolecular coordinative bond between one of the glutathionyl carboxyl groups and the Gd(III) centre, lowering the hydration state of the paramagnetic centre. (1)H-NMR dispersion profiles together with (17)O-NMR transverse relaxation time versus temperature profiles confirm that the hydration of the Gd(III) centre is strongly reduced ongoing from Gd-DO3AS-Act to the Gd-DO3AS-SG adduct. The relaxivity difference brought about by the reaction of Gd-DO3AS-Act with GSH can be enhanced up to 60% in the presence of poly-beta-cyclodextrin.

A library of 14 cyclic peptide analogues derived from the octapeptide C-terminal sequence of the human cholecystokinin hormone (CCK(26-33), or CCK8) was designed, synthesized, and characterized. The 14 peptide analogues were rationally designed to specifically interact with the CCK type B receptor (CCK(B)-R) on the basis of the structure of the bimolecular complex between CCK8 and the third extracellular loop of CCK(B)-R, namely CCK(B)-R(352-379). The rational design of new ligands for CCK(B)-R has relied on stabilization by cyclic constraints of the structural motifs that bring the key residues of the ligand (especially Trp 30, Met 31, and Phe 33) in the proper spatial orientation for optimal interaction with the receptor. The binding affinity of the new ligands for CCK(B)-R was assessed by displacement experiments of (111)In-radiolabeled CCK8 in cells that overexpress the CCK(B) receptor. The new ligands generally showed binding affinities lower than that of parent CCK8, with the best compounds having IC50 values around 10 microM. Structure-activity relationship data show that preservation of the Trp 30-Met 31 motif is essential and that the Phe 33 side chain must be present. NMR conformational studies of the compound with maximal binding affinity (cyclo-B11, IC50=11 microM) in DPC micelles shows that this compound presents a turn-like conformation centered at the Trp 30-Met 31 segment, as planned by rational design. Such a conformation is stabilized by its interaction with the micelle rather than by the cyclic constraint.

The interaction between different HO(z.rad;) radical scavengers in a three-component antioxidant system has been investigated by means of 19F-NMR spectroscopy. This system is composed of bovine serum albumin (BSA), trolox, and N-(4-hydroxyphenyl)-trifluoroacetamide (CF(3)PAF). The antioxidant capacity of BSA and trolox has been assessed by measuring the amount of trifluoroacetamide (TFAM) arising from the radical mediated decomposition of CF(3)PAF. When assayed separately, both trolox and BSA behaved as antioxidants, as they were effective to protect CF(3)PAF from HO* radical-mediated decomposition. By contrast, trolox enhanced the production of TFAM in the presence of BSA, thus behaving as a pro-oxidant. Urate, carnosine, glucose, and propylgallate showed antioxidant properties both with or without BSA. CF(3)PAF and trolox were found to bind to BSA with association constants in the order of 5 x 10(3)M(-1) and to compete for the same binding sites. These results have been discussed in terms of BSA-catalysed cross-reactions between trolox-derived secondary radicals and CF(3)PAF.

Zinc finger domains of the classical type represent the most abundant DNA binding domains in eukaryotic transcription factors. Plant proteins contain from one to four zinc finger domains, which are characterized by high conservation of the sequence QALGGH, shown to be critical for DNA-binding activity. The Arabidopsis thaliana SUPERMAN protein, which contains a single QALGGH zinc finger, is necessary for proper spatial development of reproductive floral tissues and has been shown to specifically bind to DNA. Here, we report the synthesis and UV and NMR spectroscopic structural characterization of a 37 amino acid SUPERMAN region complexed to a Zn(2+) ion (Zn-SUP37) and present the first high-resolution structure of a classical zinc finger domain from a plant protein. The NMR structure of the SUPERMAN zinc finger domain consists of a very well-defined betabetaalpha motif, typical of all other Cys(2)-His(2) zinc fingers structurally characterized. As a consequence, the highly conserved QALGGH sequence is located at the N terminus of the alpha helix. This region of the domain of animal zinc finger proteins consists of hypervariable residues that are responsible for recognizing the DNA bases. Therefore, we propose a peculiar DNA recognition code for the QALGGH zinc finger domain that includes all or some of the amino acid residues at positions -1, 2, and 3 (numbered relative to the N terminus of the helix) and possibly others at the C-terminal end of the recognition helix. This study further confirms that the zinc finger domain, though very simple, is an extremely versatile DNA binding motif.