HSQC NMR Studies Research Articles

Novel betaine-amino acid based natural deep eutectic solvents for enhancing the enzymatic hydrolysis of corncob

A novel natural deep eutectic solvent (NDES) with water content ranging from 65 to 93 wt%, in which betaine (Bet) acts as the cation and amino acids (AAs) as the anions, was prepared by a simple and green chemical route. [Bet][AA] NDES showed excellent xylan and lignin solubility, however, scare cellulose solubility. A mild and facile pretreatment process with [Bet][AA] NDES was carried out at 60 °C for 5 h. The enzymatic hydrolysis efficiency of cellulose and corncob was significantly improved. Detailed characterization showed that the enhancement of cellulose digestibility derived mainly from xylan and lignin removal. Xylan and lignin removal for [Bet][Lys]-W87 was 47.68 and 49.06%, while it was 42.20% and 57.01% for [Bet][Arg]-W82, respectively. FT-IR, SEM, XRD, and HSQC NMR studies confirmed the effectiveness and mechanism of [Bet][Lys]-W87 and [Bet][Arg]-W82 on biomass pretreatment.

Galectin-3 binds selectively to the terminal, non-reducing end of β(1→4)-galactans, with overall affinity increasing with chain length.

Galactans are linear polysaccharides of β(1→4)-linked galactose residues. Although they can antagonize galectin function, the nature of their binding to galectins needs to be better defined to develop them as drugs. Here, we investigated interactions between galectin-3 (Gal-3) and a series of galactans ranging in weight average molecular weight from 670 to 7550 Da. 15N-1H HSQC NMR studies with 15N-labeled Gal-3 carbohydrate recognition domain (CRD) indicate that each of these galactans interacts primarily with residues in β-strands 4, 5 and 6 on the canonical, β-galactoside sugar binding S-face. Although these galactans also bind to full length Gal-3 (CRD plus N-terminal tail) to the same extent, it appears that binding to the S-face attenuates interactions between the CRD F-face and N-terminal tail, making interpretation of site-specific binding unclear. Following assignment of galactan 13C and 1H resonances using HSQC, HMBC and TOCSY experiments, we used 13C-1H HSQC data to demonstrate that the Gal-3 CRD binds to the terminal, non-reducing end of these galactans, regardless of their size, but with binding affinity increasing as the galactan chain length increases. Overall, our findings increase understanding as to how galactans interact with Gal-3 at the non-reducing, terminal end of galactose-containing polysaccharides as found on the cell surface.

Transglutaminase Factor XIII Cross-Links Reactive Glutamines in Disordered Regions of Fibrinogen αC

Fibrinogen is the most abundant protein involved in blood coagulation and has been implicated in cardiovascular disease. During the formation of a blood clot, thrombin cleaves fibrinopeptides A and B from fibrinogen (AαBβγ)2 to form fibrin monomers (αβγ)2. Fibrin polymerization proceeds with the formation of protofibrils and a hard clot is only generated when the transglutaminase factor XIII (FXIII) introduces covalent γ-glutamyl-e-lysyl bonds between specific reactive glutamines and lysines in the fibrin clot network. The αC region (Aα221-610) of fibrinogen contains numerous reactive glutamines in the flexible αC-connector (221-392) and a FXIII binding site within αC (371-425). MALDI-TOF mass spectrometry assays and 2D 15N-1H HSQC NMR studies have been carried out on fibrinogen αC (233-425), a segment containing three known reactive glutamines (Q237, Q328, and Q366). FXIIIa crosslinks these three glutamines to the lysine mimic glycine ethyl ester (GEE) in the order Q237 >> Q366 > Q328. To better characterize this system, single site mutants have been generated in which the reactive glutamines (Q) are individually replaced with inactive asparagines (N). Work with these mutants suggests that FXIIIa does not require crosslinking to occur in a sequential manner. Each glutamine can be cross-linked independently of the other. Due to its highly flexible nature, the fibrinogen αC region has not been observed by X-ray crystallography. 2D HSQC NMR studies on 15N-labeled αC (233-425) suggest that this protein is intrinsically disordered. FXIIIa substrate specificity for specific reactive glutamines is predicted to be controlled by a combination of neighboring residues and the presence of regions of disorder. The knowledge gained from these studies can be used for subsequent assay and drug designs.

Abstract 3658: Structure-guided de novo design of selective Mcl-1 Inhibitors: Synthesis, structural and biochemical characterization

Abstract Myeloid cell leukemia-1 (Mcl-1) is a potent anti-apoptotic protein, member of the prosurvival Bcl-2 family. Amplification of the gene encoding the Mcl-1 protein is a common genetic aberration in human cancer. Overexpression of Mcl-1 is associated with high tumor grade, resistance to chemotherapy and poor prognosis in many types of cancers. Thus Mcl-1 is emerging as a critical survival factor in a broad range of human cancers and represents an attractive molecular target for development of a new class of cancer therapy. There is still need for developing BH3 mimetics that can efficiently and selectively target Mcl-1 protein. Employing de-novo structure-based drug-design, we have designed a new class of potent small-molecule Mcl-1 inhibitors based on the initial lead compound discovered by high-throughput screening. Analyzing the binding model of this compound with Mcl-1 through computational docking supported by HSQC NMR studies, and using the structural information, we have designed and optimized a class of small-molecule inhibitors of Mcl-1 using a 2-(phenylsulfonamido) benzoic acid as a scaffold to reproduce the key interactions with Mcl-1 such as the conserved hydrogen bond between the aspartate in pro-apoptotic proteins and Arg263 in Mcl-1. Several co-crystal structures of this class inhibitors in complex with Mcl-1 have provided a basis for their further optimization which ultimately led to the discovery of ligands with low-nanomolar potency (Ki = 8 nM) and selectivity for Mcl-1. These compounds bind in the canonical BH3 binding groove. Interestingly, depending on the particular substitution pattern in the core scaffold, a “flip’ binding mode was observed where the substituents bind in a reverse orientation. These findings were confirmed by X-ray crystallography. The most potent compounds were further biologically characterized with a series of complementary biochemical and functional assays. Citation Format: Ahmed Mady, Lei Miao, Andrej Perdih, Chenzhong Liao, Naval Bajwa, Jeanne Stuckey, Zaneta Nikolovska-Coleska. Structure-guided de novo design of selective Mcl-1 Inhibitors: Synthesis, structural and biochemical characterization. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3658. doi:10.1158/1538-7445.AM2015-3658

Fractionation of bamboo hemicelluloses by graded saturated ammonium sulphate

The hemicelluloses were isolated with 10% KOH at 25°C from dewaxed and delignified bamboo powder. The alkali-soluble hemicelluloses from Sinocalamus affinis were fractionated by ammonium sulphate precipitation method. The bamboo alkali-soluble hemicelluloses yielded seven hemicellulosic fractions obtained at 0, 5, 15, 25, 40, 55, and 70% saturation with ammonium sulphate. It was found that the more branched hemicelluloses were precipitated at higher ammonium sulphate concentrations (55 and 70%), the more linear hemicelluloses were precipitated at lower ammonium sulphate concentrations (0, 5, 15, 25, and 40%). The molecular weights of hemicellulosic fractions become lower from 35,270 (H0) to 18,680 (H70)gmol−1 with the increasing concentrations of saturated ammonium sulphate from 0 to 70%. Based on the FT-IR, 1H, 13C and 2D HSQC NMR studies, the alkali-soluble hemicelluloses were 4-O-methyl-glucuronoarabinoxylans composed of the (1→4)-linked β-d-xylopyranosyl backbone with branches at O-3 of α-L-arabinofuranosyl or at O-2 of 4-O-methyl-α-d-glucuronic acid.

The trans influence in the modulation of platinum anticancer agent biology: the effect of nitrite leaving group on aquation, reactions with S-nucleophiles and DNA binding of dinuclear and trinuclear compounds.

To examine the effect of leaving group and trans influence on the general reactivity of polynuclear platinum antitumor agents we investigated substitution of the chloride leaving groups with nitrite ion, which forms strong bonds to Pt. It was of interest to explore whether nitrite could be used to modulate biological properties of these agents, in particular the deactivating reactions that occur on reaction with S-nucleophiles, involving loss of the linking diamine under the trans influence of sulfur. Reported herein is a study of the synthesis, aquation, DNA binding and reactions with glutathione (GSH), methionine (Met) and acetylmethione (AcMet) of nitrito derivatives of di- and trinuclear platinum antitumor compounds: [{trans-PtNO(2)(NH(3))(2)}(2)(mu-NH(2)(CH(2))(6)NH(2))](NO(3))(2) (1-NO(2)) and [{trans-PtNO(2)(NH(3))(2)}(2)(mu-trans-Pt(NH(3))(2){NH(2)(CH(2))(6)NH(2)}(2))](NO(3))(4) (1'-NO(2)). {(1)H,(15)N}-HSQC NMR studies revealed that 1-NO(2) is inert to aquation reactions, even after prolonged incubation at physiological pH. Monitoring of the interaction of 1-NO(2) with the duplex 5'-d(ATATGTACATAT)(2) (I) showed only unreacted complex, consistent with activation by aquation being a requirement for covalent DNA binding. The reaction of 1-NO(2) with GSH was studied by (1)H, (195)Pt, (15)N and {(1)H,(15)N}-HSQC NMR spectroscopy. For the parent dichlorido compounds (1 and 1') substitution of chloride by GS(-) leads to drug degradation involving liberation of the diamine linker. While the same final products trans-[Pt(SG)(2)(NH(3))(2)] (5) and trans-[{Pt(SG)(NH(3))(2)}(2)-mu-SG] (6) are formed, different mechanisms are involved, consistent with the trans influence NO(2)(-) > Cl(-); the half-life is slightly longer for 1-NO(2) (1.8 h) compared with 1 (1.3 h). Identification of the intermediate trans-[Pt(NH(3))(2)(NO(2))(SG)] (4) shows that the nitrito group remains coordinated while the linker amine is substituted by coordination of GS(-), and then trans labilization of the nitrito group occurs leading to 5 and 6. Reaction of the trinuclear 1'-NO(2) with GSH follows essentially the same reaction pathway. Reaction of 1-NO(2) with Met and AcMet is much slower and only 20 % liberated amine was observed after reaction with Met for 24 h at 37 degrees C. The final product from reaction with AcMet is trans-[Pt(NH(3))(2)(NO(2))(AcMet)], as in this case coordination of the S-nucleophile does not lead to trans labilization of the nitrito group.

Binding Properties of the C‐terminal Domain of VIAF

The protein X-linked inhibitor of apoptosis (XIAP) plays an important role in caspase inactivation and as a consequence in the inhibition of apoptosis. It is known that IAP antagonists are able to specifically bind XIAP and reactivate caspase activity stimulating apoptosis. The viral IAP-associated factor (VIAF) protein is a novel IAP-interacting factor able to modulate caspase activation during apoptosis. We show that the C-terminal domain of VIAF (c-VIAF) is not able in vitro to behave as a direct IAP antagonist. By [15N-1H] HSQC NMR studies we revealed that c-VIAF binds to the RING domain of XIAP and characterized the important residues involved in the binding. Through 1D 1H-NMR screening of 1000 compounds from an in-house collection, we found that compound BI-86-E10 is able to bind to c-VIAF in a region adjacent to that interacting with the RING domain of XIAP, as supported by homology modeling and computational docking studies. After an initial round of SAR, the compound analog BI-86-E6 was found to bind with a K(d) value of 16.5 microM. These initial compounds may serve as chemical probes for further functional studies, possibly aimed at validating c-VIAF as a novel target for antiviral drug development.

Acetylation of Human Hemoglobin by Methyl Acetylphosphate: EVIDENCE OF BROAD REGIO-SELECTIVITY REVEALED BY NMR STUDIES

The development of chemical modification agents that reduce the tendency of sickle hemoglobin (HbS) to aggregate represents an important chemotherapeutic goal. Methyl acetylphosphate (MAP) has been reported to bind to the 2,3-diphosphoglycerate (2,3-DPG) binding site of hemoglobin, where it selectively acetylates residues, resulting in increased solubility of HbS. We have prepared [1-(13)C]MAP and evaluated the adduct formation with hemoglobin using (1)H-(13)C HMQC and HSQC NMR studies. These spectra of the acetylated hemoglobin adducts showed 10-11 well resolved adduct peaks, indicating that the acetylation was not highly residue specific. The chemical shift pattern observed is in general similar to that obtained recently using [1'-(13)C]aspirin as the acetylating agent (Xu, A. S. L., Macdonald, J. M., Labotka, R. J., and London, R. E. (1999) Biochim. Biophys. Acta 1432, 333-349). Blocking the 2, 3-DPG binding site with inositol hexaphosphate (IHP) resulted in a selective reduction in intensity of adduct resonances, presumably corresponding to residues located in the 2,3-DPG binding cleft. The pattern of residue protection appeared to be identical to that observed in our previous study using IHP and labeled aspirin. Pre-acetylation of hemoglobin using unlabeled MAP, followed by acetylation with [1'-(13)C]aspirin indicated a general protective effect, with the greatest reduction of intensity for resonances corresponding to acetylated residues in the 2,3-DPG binding site. These studies indicated that both MAP and aspirin exhibit similar, although not identical, acetylation profiles and target primarily the betaLys-82 residue in the 2,3-DPG binding site, as well as sites such as betaLys-59 and alphaLys-90, which are not located in the beta-cleft of hemoglobin.

1H/15N HSQC NMR studies of ligand carboxylate group interactions with arginine residues in complexes of brodimoprim analogues and Lactobacillus casei dihydrofolate reductase.

1H and 15N NMR studies have been undertaken on complexes of Lactobacillus casei dihydrofolate reductase (DHFR) formed with analogues of the antibacterial drug brodimoprim (2,4-diamino-5-(3', 5'-dimethoxy-4'-bromobenzyl)pyrimidine) in order to monitor interactions between carboxylate groups on the ligands and basic residues in the protein. These analogues had been designed by computer modeling with carboxylated alkyl chains introduced at the 3'-O position in order to improve their binding properties by making additional interactions with basic groups in the protein. Specific interactions between ligand carboxylate groups and the conserved Arg57 residue have been detected in studies of 1H/15N HSQC spectra of complexes of DHFR with both the 4-carboxylate and the 4, 6-dicarboxylate brodimoprim analogues. The spectra from both complexes showed four resolved signals for the four NHeta protons of the guanidino group of Arg57, and this is consistent with hindered rotation in the guanidino group resulting from interactions with the 4-carboxylate group in each analogue. In the spectra of each complex, one of the protons from each of the two NH2 groups and both nitrogens are considerably deshielded compared to the shielding values normally observed for such nuclei. This pattern of deshielding is that expected for a symmetrical end-on interaction of the carboxylate oxygens with the NHeta12 and NHeta22 guanidino protons. The differences in the degree of deshielding between the complexes of the two structurally similar brodimoprim analogues and the methotrexate indicates that the shielding is very sensitive to geometry, most probably to hydrogen bond lengths. The 1H/15N HSQC spectrum of the DHFR complex with the brodimoprim-6-carboxylate analogue does not feature any deshielded Arg NHeta protons and this argues against a similar interaction with the Arg57 in this case. It has not proved possible to determine whether the 6-carboxylate in this analogue is interacting directly with any residue in the protein. 1H/15N HSQC spectra have been fully assigned for the complexes with the three brodimoprim analogues and chemical shift mapping used to explore interactions in the binding site. The 1H signals of the bound ligands for all three brodimoprim analogues have been assigned. Their 1H chemical shifts were found to be fairly similar in the different complexes indicating that the 2, 4-diaminopyrimidine and the benzyl ring are binding in essentially the same binding sites and with the same overall conformation in the different complexes. The rotation rate about the NepsilonCzeta bond in the brodimoprim-4,6-dicarboxylate complex with DHFR has been determined from a zz-HSQC exchange experiment, and its value is quite similar to that observed in the DHFR.methotrexate complex (24 +/- 10 s-1 at 8 degrees C and 50 +/- 10 s-1 at 15 degrees C, respectively). The 1H and 15N chemical shift differences of selected amide and guanidino NH groups, measured between the DHFR complexes, provided further evidence about the interactions involving Arg57 with the 4-carboxylate and 4,6-dicarboxylate brodimoprim analogues.

Multidimensional 109Ag, 31P, and 1H HMQC and HSQC NMR studies on a model homogeneous catalyst. Reactions of a chiral ferrocenylphosphine with Ag(CF3SO3)

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTMultidimensional 109Ag, 31P, and 1H HMQC and HSQC NMR studies on a model homogeneous catalyst. Reactions of a chiral ferrocenylphosphine with Ag(CF3SO3)Francesca Lianza, Alceo Macchioni, Paul Pregosin, and Heinz RueggerCite this: Inorg. Chem. 1994, 33, 22, 4999–5002Publication Date (Print):October 1, 1994Publication History Published online1 May 2002Published inissue 1 October 1994https://doi.org/10.1021/ic00100a025RIGHTS & PERMISSIONSArticle Views249Altmetric-Citations18LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (443 KB) Get e-Alerts Get e-Alerts