Fluorine Label Research Articles

What does fluorine do to a protein? Thermodynamic, and highly-resolved structural insights into fluorine-labelled variants of the cold shock protein

Fluorine labelling represents one promising approach to study proteins in their native environment due to efficient suppressing of background signals. Here, we systematically probe inherent thermodynamic and structural characteristics of the Cold shock protein B from Bacillus subtilis (BsCspB) upon fluorine labelling. A sophisticated combination of fluorescence and NMR experiments has been applied to elucidate potential perturbations due to insertion of fluorine into the protein. We show that single fluorine labelling of phenylalanine or tryptophan residues has neither significant impact on thermodynamic stability nor on folding kinetics compared to wild type BsCspB. Structure determination of fluorinated phenylalanine and tryptophan labelled BsCspB using X-ray crystallography reveals no displacements even for the orientation of fluorinated aromatic side chains in comparison to wild type BsCspB. Hence we propose that single fluorinated phenylalanine and tryptophan residues used for protein labelling may serve as ideal probes to reliably characterize inherent features of proteins that are present in a highly biological context like the cell.

19F NMR Studies of Cyclophilin a and its Interaction with HIV-1 Capsid

Human cyclophilin A (CypA) directly interacts with the HIV-1 capsid, the conical core within the virion that encloses the RNA genome, and modulates viral infectivity through ill-understood mechanism(s). To study the binding between CypA and capsid, we employed 19F NMR spectroscopy. The 100% naturally abundant spin-1/2 19F nucleus with high gyromagnetic ratio is absent from most naturally occurring biological molecules, and 19F chemical shifts are highly sensitive to the local conformation and chemical environment. As a result, 19F serves as a valuable NMR probe in biological systems. Using 4F-, 5F-, 6F-, or 7F-substituted indole as a precursor, single 19F labels were introduced into CypA at different positions of the sole tryptophan ring in the protein, which is conveniently located in the capsid binding site. X-ray crystallography and 1H-1H NOEs confirmed that CypA retains the same structure after fluorine labelling and only small chemical shift changes were present in the 1H-15N HSQC spectra of the F-CypA samples. Interestingly, all amides that exhibit chemical shift differences are connected through an extended hydrogen bonding network, and the topology of the networks differ between the differently labeled fluorinated F-CypA proteins. We hypothesize that the fluorine atom interacts with nearby polar groups and this electronic perturbation is passed on through the hydrogen bonding network to generate a unique, allosteric long-range effect. The solvent accessibility of the capsid binding site for the different positions on the 6-membered indole ring was probed by recording the effects of D2O content in D2O/H2O solutions on the 19F chemical shift. We also investigated the binding between capsid protein and CypA by 19F NMR titration experiment. Analysis of the titration shifts permitted the determination of individual binding constants for different fluorinated F-CypAs.

Correlative in vivo Fluorescence Imaging and 19F-MRI of Zebrafish Embryos

Monitoring the pathway of a drug in the body and confirming delivery to the desired target is important in drug development. To this aim, we are developing a correlative fluorescence imaging approach in combination with 19F-MRI. Our first attempts to develop a suitable 19F-labeling strategy will be monitored using well-established fluorescence imaging in transparent zebrafish embryos. Later on, 19F-MRI could then be applied to non-transparent organisms. Zero background and high NMR sensitivity render fluorine a promising label to be traced in vivo. Although 19F possesses almost the same magnetic resonance sensitivity as 1H, a mono-atomic fluorine label is far too insensitive for MRI detection in vivo. To enhance the sensitivity, we therefore explored labels possessing many 19F nuclei with, if possible, identical 19F chemical shift. As a first step, we constructed nanoparticles with encapsulated fluorescence dye and perfluorocarbons, using an emulsion diffusion method. Characterization by solid-state NMR and dynamic light scattering demonstrated a successful simultaneous encapsulation of the fluorocarbon and fluorescence dye. In our first confocal microscopy and MRI experiments in zebrafish embryos, we were able to characterize the distribution of the nanoparticles in the organism. Based on these first results, we are currently expanding our correlative 19F-MRI and fluorescence imaging approach to peptide-based drugs. To this aim, we are synthesized artificial amino acids bearing up to 27 equivalent 19F-nuclei, which can then be combined with fluorescence labels to allow tracing peptides in vivo.

Optimizing fluorine labelling for 19F solid-state NMR in oriented biological systems.

When planning a fluorine labeling strategy for 19F solid state NMR (ssNMR) studies of the structure and/or mobility of fluorine labeled compounds in situ in an oriented biological system, it is important to characterize the NMR properties of the label. This manuscript focuses on the characterization of a selection of aromatic fluorine compounds in dimyristoylphosphatidylcholine bilayers using 19F ssNMR from the standpoint of determiningthe optimum arrangement of fluorine nuclei on a pendant aromatic ring before incorporation into more complex biological systems.

2'-O-Trifluoromethylated RNA - a powerful modification for RNA chemistry and NMR spectroscopy.

New RNA modifications are needed to advance our toolbox for targeted manipulation of RNA. In particular, the development of high-performance reporter groups facilitating spectroscopic analysis of RNA structure and dynamics, and of RNA–ligand interactions has attracted considerable interest. To this end, fluorine labeling in conjunction with 19F-NMR spectroscopy has emerged as a powerful strategy. Appropriate probes for RNA previously focused on single fluorine atoms attached to the 5-position of pyrimidine nucleobases or at the ribose 2′-position. To increase NMR sensitivity, trifluoromethyl labeling approaches have been developed, with the ribose 2′-SCF3 modification being the most prominent one. A major drawback of the 2′-SCF3 group, however, is its strong impact on RNA base pairing stability. Interestingly, RNA containing the structurally related 2′-OCF3 modification has not yet been reported. Therefore, we set out to overcome the synthetic challenges toward 2′-OCF3 labeled RNA and to investigate the impact of this modification. We present the syntheses of 2′-OCF3 adenosine and cytidine phosphoramidites and their incorporation into oligoribonucleotides by solid-phase synthesis. Importantly, it turns out that the 2′-OCF3 group has only a slight destabilizing effect when located in double helical regions which is consistent with the preferential C3′-endo conformation of the 2′-OCF3 ribose as reflected in the 3J (H1′–H2′) coupling constants. Furthermore, we demonstrate the exceptionally high sensitivity of the new label in 19F-NMR analysis of RNA structure equilibria and of RNA–small molecule interactions. The study is complemented by a crystal structure at 0.9 Å resolution of a 27 nt hairpin RNA containing a single 2′-OCF3 group that well integrates into the minor groove. The new label carries high potential to outcompete currently applied fluorine labels for nucleic acid NMR spectroscopy because of its significantly advanced performance.

Surface Modification of Poly(ether ether ketone) through Friedel-Crafts Reaction for High Adhesion Strength.

Poly(ether ether ketone) (PEEK) possesses attractive mechanical and thermal properties but demonstrates poor adhesion. To overcome this disadvantage, in this study, the surface modification of PEEK or PEEK-based carbon-fiber-reinforced thermoplastics (CFRTP) was performed through the Friedel-Crafts reaction and successive epoxidation. Under optimized reaction conditions, surface modification was achieved without surface deterioration, and epoxy groups were introduced. The progress of the Friedel-Crafts reaction and epoxidation was demonstrated by X-ray photoelectron spectroscopy measurements after fluorine labeling through thiol-en reaction and amine addition, respectively. The adhesive strength between CFRTP and epoxy adhesives was increased to 23.5 MPa, and cohesive fracture of epoxy adhesives, rather than interfacial peeling, occurred. In addition, compared with conventional plasma treatment, the durability of the modified surface and thickness of the modified surface layer increased. Therefore, we succeeded in modifying the surface properties through the epoxidation of the PEEK surface.

Targeted expression and purification of fluorine labelled cold shock protein B by using an auxotrophic strategy

High resolution NMR spectroscopy is a seminal method in modern structural biology to obtain insights into proteins' structure, dynamics and function at dilute condition as well as in a cell-like environment or even intracellularly. Usually, 1H, 15N or 13C nuclei are predominantly used for the characterization of the protein of interest. These measurements are limited due to the wealth of chemical shifts and background signals arising from all molecules present in the NMR test tube. On top of that, the protein under study has to be isotopically enriched in nitrogen and/or carbon nuclei enabling to overcome the inherently low natural abundance of 13C and 15N NMR active isotopes. In this way switching to 19F NMR spectroscopy strongly reduces the total amount of signals seen in an NMR spectrum as it turns off background signals and is for this reason extremely attractive for highly-resolved investigations of proteins performance measured directly in cells or in a cell-like environment.Here we show the effective expression and purification of cold shock protein B from Bacillus subtilis (BsCspB) using fluorine labelled phenylalanine or fluorine labelled tryptophan residues. We reveal that fluorine labelled BsCspB represents the same fold on a secondary as tertiary level as seen for the wild type protein independent of the labelling position illuminating the soft character of fluorine insertion. This experimental setup of targeted fluorine labelling sets a profound ground for a broad range of highly-resolved 19F NMR applications to be performed in a complex cellular environment.

Synthesis and Postpolymerization Modification of Fluorine-End-Labeled Poly(Pentafluorophenyl Methacrylate) Obtained via RAFT Polymerization.

Chain-end-labeled polymers are interesting for a range of applications. In polymer nanomedicine, chain-end-labeled polymers are useful to study and help understand cellular internalization and intracellular trafficking processes. The recent advent of fluorescent label-free techniques, such as nanoscale secondary ion mass spectrometry (NanoSIMS), provides access to high-resolution intracellular mapping that can complement information obtained using fluorescent-labeled materials and confocal microscopy and flow cytometry. Using poly(N-(2-hydroxypropyl)methacrylamide) (PHPMA) as a prototypical polymer nanomedicine, this paper presents a synthetic strategy to polymers that contain trace element labels, such as fluorine, which can be used for NanoSIMS analysis. The strategy presented in this paper is based on reversible addition fragmentation chain transfer (RAFT) polymerization of pentafluorophenyl methacrylate (PFMA) mediated by two novel chain-transfer agents (CTAs), which contain either one (α) or two (α,ω) fluorine labels. In the first part of this study, via a number of polymerization experiments, the polymerization properties of the fluorinated RAFT CTAs were established. 19F NMR spectroscopy revealed that these fluorinated RAFT agents possess unique spectral signatures, which allow to directly monitor RAFT agent conversion and measure end-group fidelity. Comparison with 4-cyanopentanoic acid dithiobenzoate, which is a standard CTA for the RAFT polymerization of PFMA, revealed that the introduction of one or two fluorine labels does not significantly affect the polymerization properties of the CTA. In the last part of this paper, a proof-of-concept study is presented that demonstrates the feasibility of the fluorine-labeled poly(pentafluorophenyl methacrylate) polymers as platforms for the postpolymerization modification to generate PHPMA-based polymer nanomedicines.

Use of 19F NMR to probe conformational changes of arrestin

Signal specificity is achieved through specific and highly‐regulated molecular recognition. Arrestins, a small family of multi‐functional adaptor proteins, mediate surprisingly complex cellular signaling events. To elicit the appropriate cellular response, arrestins have to ensure that the correct components are linked together to prevent the formation of mismatched protein complexes. It is now clear that arrestins undergo significant conformational movements in many regulating processes; however, previous studies of arrestin conformational changes are almost completely focused on receptor‐induced conformational changes, and little is known about binding‐induced conformations that allow arrestins to accomplish their diverse signaling functions. We hypothesize that binding induced conformational changes play decisive roles in regulating the recruitment of binding partners. 19F NMR has proven to be a powerful technique in the study of protein structure and dynamics because the 19F nucleus is easily incorporated into protein through specific labeling. The chemical shift of 19F is extremely sensitive to the changes of local micro‐environment caused by the conformational movements of protein, making it possible to analyze the minor movements within a protein molecule. 19F NMR is performed by incorporating a fluorine label into the protein structure Fluorine labeling can be accomplished in a variety of ways. Here, we developed a 19F NMR‐based method to detect the conformational movements of arrestins by employing a thio‐based method to incorporate 19F to the specific residues on arrestin. We have observed the release of C‐tail in arrestin‐3 upon binding of IP‐6 (inositol hexaphosphate). The methods developed here can be employed to detect the conformational changes of arrestin with many other binding partners.Support or Funding InformationThis project is supported by NSF MRI project (1531870), TTU faculty research grant (XZ) and Student Research Grant (RS)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Glycation of human serum albumin impairs binding to the glucagon-like peptide-1 analogue liraglutide

The long-acting glucagon-like peptide-1 analogue liraglutide has proven efficiency in the management of type 2 diabetes and also has beneficial effects on cardiovascular diseases. Liraglutide's protracted action highly depends on its capacity to bind to albumin via its palmitic acid part. However, in diabetes, albumin can undergo glycation, resulting in impaired drug binding. Our objective in this study was to assess the impact of human serum albumin (HSA) glycation on liraglutide affinity. Using fluorine labeling of the drug and 19F NMR, we determined HSA affinity for liraglutide in two glycated albumin models. We either glycated HSA in vitro by incubation with glucose (G25- or G100-HSA) or methylglyoxal (MGO-HSA) or purified in vivo glycated HSA from the plasma of diabetic patients with poor glycemic control. Nonglycated commercial HSA (G0-HSA) and HSA purified from plasma of healthy individuals served as controls. We found that glycation decreases affinity for liraglutide by 7-fold for G100-HSA and by 5-fold for MGO-HSA compared with G0-HSA. A similarly reduced affinity was observed for HSA purified from diabetic individuals compared with HSA from healthy individuals. Our results reveal that glycation significantly impairs HSA affinity to liraglutide and confirm that glycation contributes to liraglutide's variable therapeutic efficiency, depending on diabetes stage. Because diabetes is a progressive disease, the effect of glycated albumin on liraglutide affinity found here is important to consider when diabetes is managed with this drug.

The Application of REDOR NMR to Understand the Conformation of Epothilone B.

The structural information of small therapeutic compounds complexed in biological matrices is important for drug developments. However, structural studies on ligands bound to such a large and dynamic system as microtubules are still challenging. This article reports an application of the solid-state NMR technique to investigating the bioactive conformation of epothilone B, a microtubule stabilizing agent, whose analog ixabepilone was approved by the U.S. Food and Drug Administration (FDA) as an anticancer drug. First, an analog of epothilone B was designed and successfully synthesized with deuterium and fluorine labels while keeping the high potency of the drug; Second, a lyophilization protocol was developed to enhance the low sensitivity of solid-state NMR; Third, molecular dynamics information of microtubule-bound epothilone B was revealed by high-resolution NMR spectra in comparison to the non-bound epothilone B; Last, information for the macrolide conformation of microtubule-bound epothilone B was obtained from rotational-echo double-resonance (REDOR) NMR data, suggesting the X-ray crystal structure of the ligand in the P450epoK complex as a possible candidate for the conformation. Our results are important as the first demonstration of using REDOR for studying epothilones.

Bafilomycin analogue site-specifically fluorinated at the pharmacophore macrolactone ring has potent vacuolar-type ATPase inhibitory activity

Based on previously reported structure–activity relationships, an analogue of bafilomycin A1 with a site-specific fluorine label at the C2 position was designed and efficiently synthesized. The fluorinated compound exhibited potent vacuolar-type ATPase (V-ATPase) inhibitory activity, comparable to that of the natural product, representing the first example of highly bioactive analogues with a modified macrolactone core from the plecomacrolide family compounds.

The Development of a Versatile Trifunctional Scaffold for Biological Applications

AbstractWe describe the synthesis of a trifunctional scaffold constructed from a planar core of trimesic acid derivatized with three propargylamine moieties. The scaffold was attached to a solid‐phase resin through the carboxylic group of a fluorinated alkyl spacer arm. The orthogonal protection of two of the alkyne groups with triethylsilyl and triisopropylsilyl moieties enabled modular and efficient derivatization of the scaffold with three different azides by using solid‐phase synthesis on amphiphilic ChemMatrix resin. We showed that a fluorine label can be used to quantify the content of fluorine‐containing compounds by 19F NMR spectroscopic analysis after cleavage from the resin. We have thus designed a versatile and convenient tool that could be useful for simple and rapid solid‐phase syntheses of combinatorial libraries of the scaffold‐based compounds, for example as new protein binders.

Fluorinated Fe(III) salophene complexes: optimization of tumor cell specific activity and utilization of fluorine labeling for in vitro analysis.

Fluorine-substituted iron(III) salophene complexes (salophene = N,N'-bis(salicylidene)-1,2-phenylenediamine) were synthesized and evaluated for biological activity. All complexes showed growth inhibitory effects with IC50 values ranging from 0.05 to 2.45 μM against HT-29 colon carcinoma as well as MCF-7 and MDA-MB-231 mammary carcinoma cells (cisplatin: 5.75, 12.72, 5.81 μM, respectively). HR-CS MAS investigations revealed that the complexes were highly protein-bound already after an incubation period of 10 min and accumulated more effectively in tumor cells than cisplatin. Interestingly, the ligands were enriched in the cells too, indicating that the salophene moiety acts as a carrier ligand and mediates the uptake of the complexes. Furthermore, induction of apoptosis proved to be dependent on the substitution pattern as well as on the tumor cell line, as evidenced from the annexin V-FITC/PI assay. Most of the complexes, especially the highly active 5-Fe, showed tumor cell specific effects and no/less influence on the proliferation of T-cells generated from the peripheral blood of healthy individuals.

Fluorinated aromatic amino acids are sensitive 19F NMR probes for bromodomain-ligand interactions.

We describe a 19F NMR method for detecting bromodomain–ligand interactions using fluorine-labeled aromatic amino acids due to the conservation of aromatic residues in the bromodomain binding site. We test the sensitivity, accuracy, and speed of this method with small molecule ligands (+)-JQ1, BI2536, Dinaciclib, TG101348, and acetaminophen using three bromodomains Brd4, BrdT, and BPTF. Simplified 19F NMR spectra allowed for simultaneous testing of multiple bromodomains to assess selectivity and identification of a new BPTF ligand. Fluorine labeling only modestly affected the Brd4 structure and function assessed by isothermal titration calorimetry, circular dichroism, and X-ray crystallography. The speed, ease of interpretation, and low concentration of protein needed for binding experiments affords a new method to discover and characterize both native and new ligands.

Specific fluorine labeling of the HyHEL10 antibody affects antigen binding and dynamics.

To more fully understand the molecular mechanisms responsible for variations in binding affinity with antibody maturation, we explored the use of site specific fluorine labeling and (19)F nuclear magnetic resonance (NMR). Several single-chain (scFv) antibodies, derived from an affinity-matured series of anti-hen egg white lysozyme (HEL) mouse IgG1, were constructed with either complete or individual replacement of tryptophan residues with 5-fluorotryptophan ((5F)W). An array of biophysical techniques was used to gain insight into the impact of fluorine substitution on the overall protein structure and antigen binding. SPR measurements indicated that (5F)W incorporation lowered binding affinity for the HEL antigen. The degree of analogue impact was residue-dependent, and the greatest decrease in affinity was observed when (5F)W was substituted for residues near the binding interface. In contrast, corresponding crystal structures in complex with HEL were essentially indistinguishable from the unsubstituted antibody. (19)F NMR analysis showed severe overlap of signals in the free fluorinated protein that was resolved upon binding to antigen, suggesting very distinct chemical environments for each (5F)W in the complex. Preliminary relaxation analysis suggested the presence of chemical exchange in the antibody-antigen complex that could not be observed by X-ray crystallography. These data demonstrate that fluorine NMR can be an extremely useful tool for discerning structural changes in scFv antibody-antigen complexes with altered function that may not be discernible by other biophysical techniques.

Direct Observation of Phenylalanine Orientations in Statherin Bound to Hydroxyapatite Surfaces

Extracellular biomineralization proteins such as salivary statherin control the growth of hydroxyapatite (HAP), the principal component of teeth and bones. Despite the important role that statherin plays in the regulation of hard tissue formation in humans, the surface recognition mechanisms involved are poorly understood. The protein-surface interaction likely involves very specific contacts between the surface atoms and the key protein side chains. This study demonstrates for the first time the power of combining near-edge X-ray absorption fine structure (NEXAFS) spectroscopy with element labeling to quantify the orientation of individual side chains. In this work, the 15 amino acid N-terminal binding domain of statherin has been adsorbed onto HAP surfaces, and the orientations of phenylalanine rings F7 and F14 have been determined using NEXAFS analysis and fluorine labels at individual phenylalanine sites. The NEXAFS-derived phenylalanine tilt angles have been verified with sum frequency generation spectroscopy.

Pentafluorophenylhalocarbenes

Pentafluorophenylchlorocarbene and pentafluorophenylfluorocarbene are highly reactive species and effective carriers of fluorine labels via addition to alkenes and insertion into C-H bonds.

ChemInform Abstract: Fluoroglycoproteins: Ready Chemical Site‐Selective Incorporation of Fluorosugars into Proteins.

AbstractA tag‐and‐modify strategy for the synthesis of homogenous fluorinated glyco‐amino acids, peptides, and proteins carrying fluorine labels in the sugar moiety is described, allowing, for example, access to directly radiolabelled 18F‐glycoproteins.

Fluorinated Retinoids and Carotenoids

Methods of preparing fluorinated retinoids with labels located on odd-numbered carbons as well on even-numbered carbons and those containing trifluoromethyl groups are reviewed. The use of such retinoids in studies of protein-bound species is summarized, including the application of (19)F NMR spectroscopy for elucidating the mechanism of cis/trans isomerization, restricted rotation within the protein binding pocket, and identification of specific protein-substrate interactions. The fluorine label was also useful for wavelength attenuation of protein-bound species (including formation of NIR absorbing pigments) and for other unique applications. The more limited studies available on fluorinated carotenoids are also reviewed.