Hydrophobic Tag Research Articles

Silicon-based hydrophobic tags applied in liquid-phase peptide synthesis: protected DRGN-1 and poly alanine chain synthesis.

Two new recyclable silicon-based hydrophobic tags were developed for installing them at the C-terminal of peptides to increase the solubility in organic solvents and the reactivity of the peptides during liquid-phase peptide synthesis (LPPS). They comprise a siloxy group containing tag and an arylsilyl group containing tag. The hydrophobicity of these tags is much greater than those of previously reported examples. The siloxy group containing tag is compatible with Fmoc-deprotection conditions (Fmoc-chemistry) and hydrogenation conditions (Cbz-chemistry), while the arylsilyl group containing one is resistant to Fmoc-deprotection conditions (Fmoc-chemistry) and Boc-deprotection conditions (Boc-chemistry). Using the siloxy group containing tag, protected DRGN-1, a peptide containing 14 amino acid residues was prepared successfully by using linear synthesis combined with one convergent synthesis step. Using the arylsilyl group containing tag, a poly alanine chain containing 7 alanine residues was synthesized. The arylsilyl group containing tag can also be installed at the N-terminal to elongate the peptides from the N-terminal to the C-terminal. The yields and the solubility of the products in organic solvents in each step were good to excellent with the assistance of these silicon-based tags.

Carborane as an Alternative Efficient Hydrophobic Tag for Protein Degradation.

Carboranes 1 and 2 were designed and synthesized for hydrophobic tag (HyT)-induced degradation of HaloTag fusion proteins. The levels of the hemagglutinin (HA)-HaloTag2-green fluorescent protein (EGFP) stably expressed in Flp-In 293 cells were significantly reduced by HyT13, HyT55, and carboranes 1 and 2, with expression levels of 49, 79, 43, and 65%, respectively, indicating that carborane is an alternative novel hydrophobic tag (HyT) for protein degradation under an intracellular environment. To clarify the mechanism of HyT-induced proteolysis, bovine serum albumin (BSA) was chosen as an extracellular protein and modified with maleimide-conjugated m-carborane (MIC). The measurement of the ζ-potentials and the lysine residue modification with fluorescein isothiocyanate (FITC) of BSA-MIC conjugates suggested that the conjugation of carborane induced the exposure of lysine residues on BSA, resulting in the degradation via ubiquitin E3 ligase-related proteasome pathways in the cell.

Enhanced protein degradation by intracellular delivery of pre-fused PROTACs using lipid-like nanoparticles

Proteolysis-targeting chimaera (PROTAC) technology is an emerging approach for achieving targeted degradation of a protein of interest (POI) intracellularly. However, the cell permeability of PROTACs is limited by their high molecular weight and total polar surface area. Moreover, the activation of the proteasome-mediated degradation by PROTAC requires the formation of a ternary (three-component) complex, composed of the PROTAC, the POIs, and E3-ligases related proteins (E3Ps). Simplifying the three-component system to two-component system could theoretically increase the efficiency of the formation of ternary complex and enhance the protein degradation efficiency. Herein, we demonstrate that pre-fusion of PROTACs with E3Ps (called “pre-fused PROTACs”) before administration could transform the original PROTAC system to two-component system. After delivery by lipid nanoparticles, the degradation of POI by pre-fused PROTACs was dramatically increased and accelerated compared with standard PROTACs. Moreover, we demonstrated that this approach could be generalized to another hydrophobic tag (HyT) degrader by demonstrating the improved targeted protein degradation after pre-fusion the HyT degrader with heat shock protein 70 (HSP70).

Reversible Oligomerization and Reverse Hydrophobic Effect Induced by Isoleucine Tags Attached at the C-Terminus of a Simplified BPTI Variant.

Protein amorphous aggregation has become the focus of great attention, as it can impair the ability of cells to function properly. Here, we evaluated the effects of three peptide tags, consisting of one, three, and five consecutive isoleucines attached at the C-terminus end of a simplified bovine pancreatic trypsin inhibitor (BPTI) variant, BPTI-19A, on the thermal stability and oligomerization by circular dichroism spectrometry and differential scanning calorimetry in detail. All of the BPTI-19A variants exhibited a reversible and apparently two-state thermal transition like BPTI-19A at pH 4.7. The thermal transition of the five-isoleucine-tagged variant showed clear protein-concentration dependence, where the apparent denaturation temperature decreased as the protein concentration increased. Quantitative analysis indicated that this phenomenon originated from the presence of reversibly oligomerized (RO) states at high temperatures. The results also illustrated that the thermodynamic stability difference between the native and the monomeric denatured state in all the proteins was destabilized by the hydrophobic tags and was well explained by the reverse hydrophobic effect due to the tags. The existence of the RO states was confirmed by both analytical ultracentrifugation and dynamic light scattering. This indicated that the five-isoleucine hydrophobic tag is strong enough to induce intermolecular hydrophobic contact among the denatured molecules leading to oligomerization, and even one- or three-isoleucine tags are effective enough to generate intramolecular hydrophobic contact, thus provoking denaturation through the reverse hydrophobic effect.

Design and Assembly of Membrane-Spanning DNA Nanopores.

Versatile lipid membrane-inserting nanopores have been made by functionalizing DNA nanostructures with hydrophobic tags. Here, we outline design and considerations to obtain DNA nanopores with the desired dimensions and conductance properties. We further provide guidance on their reconstitution into lipid membranes.

Targeted Protein Degradation: An Emerging Therapeutic Strategy in Cancer.

Drug discovery in the scope of cancer therapy has been focused on conventional agents that nonselectively induce DNA damage or selectively inhibit the activity of key oncogenic molecules without affecting their protein levels. An emerging therapeutic strategy that garnered attention in recent years is the induction of Targeted Protein Degradation (TPD) of cellular targets by hijacking the intracellular proteolysis machinery. This novel approach offers several advantages over conventional inhibitors and introduces a paradigm shift in several pharmacological aspects of drug therapy. While TPD has been found to be the major mode of action of clinically approved anticancer agents such as fulvestrant and thalidomide, recent years have witnessed systematic endeavors to expand the repertoire of proteins amenable to therapeutic ablation by TPD. Such endeavors have led to three major classes of agents that induce protein degradation, including molecular glues, Proteolysis Targeting Chimeras (PROTACs) and Hydrophobic Tag (HyT)-based degraders. Here, we briefly highlight agents in these classes and key advances made in the field with a focus on clinical translation in cancer therapy.

Improved Methods for Oxytocin Analysis from Human Serum and Urine by Orbitrap ESI‐LCMS after SPE Purification

IntroductionNative oxytocin (OT) levels in non‐pregnant/non‐lactating/non‐medicated (NPLM) humans are very low. The limit of detection (LOD, 10–25 pg/mL) of our previous liquid chromatography mass spectrometry (LCMS) method precluded their quantification in serum and urine. Thus, we sought to improve the LCMS sensitivity of OT measurements in these matrices by hydrophobic tagging or optimizing solid phase extraction (SPE) procedures to selectively concentrate native OT.MethodsOT was reduced then alkylated with N‐alkyl acetamide (C12, C14, C16, C18) tags or directly tagged with sulfonyl chloride reagents. Native serum OT was crashed with acetonitrile (ACN), extracted using a Sep‐Pak C18 cartridge washed 4× with increasing ACN concentrations to remove interferences, eluted with ACN, then concentrated by a flow of nitrogen gas. LCMS was performed using a Kinetex C18 column (150 × 3 mm, 2.6 μm) and Q‐Exactive orbitrap MS. Native urinary OT was crashed with ACN then extracted using a Strata‐X SPE cartridge washed 5× by gravity with increasing ACN concentrations. OT was eluted with ACN then concentrated online using a Strata‐X SPE (20 × 2.0 mm, 25 μm) coupled to a Kinetex C18 column followed by LCMS analysis using a Q‐Exactive orbitrap MS operated in ESI+ mode with targeted SIM scan. Quantification of the analytes was set within 5 ppm of the calculated exact isotope masses ([M+2H]2+: OT: 504.22551; OT‐d5: 506.74120).ResultsAlkylation with C12 and C14 yielded a di‐ and mono‐capped product, respectively, while C16 and C18 failed to react. Reactions of native OT with biphenyl sulfonyl chloride resulted in mono‐tagged adducts whereas reaction with 1‐methylimidazole sulfonyl chloride lead to mono‐ and bi‐tagged adducts in a 1:1 ratio. None of these products had better sensitivity than native OT. Thus, we decided to focus on native OT. The LOD of spiked serum using our SPE protocol was 2.5 pg/mL. The LOD in neat OT standards and spiked urine using our manual SPE and online concentrating protocol was 0.25 pg/mL and 1–5 pg/mL, respectively, depending on background noise. Applying this modified SPE method, we could detect OT consistently in one (1.6 pg/mL) and inconsistently in two lactating women’s urine.ConclusionWe were unable to improve the sensitivity of OT measurements using hydrophobic tags as previously described1. Our findings were confirmed by the Max Planck Institute for Psychiatry, (Munich, Germany), that performed the reaction on aq. OT and [Arg8]‐VP with the C12 and C14 reagents we synthesized followed by TOF based LCMS which revealed a 10‐fold lower sensitivity compared to native OT. Therefore, we chose to measure native OT after SPE purification to lower the LOD in serum and urine to allow measurement of the very low native OT (1–2 pg/mL) levels found in NPLM humans. We found that manual SPE was mandatory to improve sensitivity. Using online SPE alone, the LOD in spiked urine was > 20 pg/mL and resulted in more interferences. Modifying the previous SPE protocol for serum and urine extraction lowered the LOD to 2.5 pg/mL and 1–5 pg/mL, respectively. The increased sensitivity for urinary OT allowed OT detection in some but not all lactating women’s urine at levels lower than reported by ELISA.Support or Funding InformationP30 CA71789

Controlling aggregation of cholesterol-modified DNA nanostructures.

DNA nanotechnology allows for the design of programmable DNA-built nanodevices which controllably interact with biological membranes and even mimic the function of natural membrane proteins. Hydrophobic modifications, covalently linked to the DNA, are essential for targeted interfacing of DNA nanostructures with lipid membranes. However, these hydrophobic tags typically induce undesired aggregation eliminating structural control, the primary advantage of DNA nanotechnology. Here, we study the aggregation of cholesterol-modified DNA nanostructures using a combined approach of non-denaturing polyacrylamide gel electrophoresis, dynamic light scattering, confocal microscopy and atomistic molecular dynamics simulations. We show that the aggregation of cholesterol-tagged ssDNA is sequence-dependent, while for assembled DNA constructs, the number and position of the cholesterol tags are the dominating factors. Molecular dynamics simulations of cholesterol-modified ssDNA reveal that the nucleotides wrap around the hydrophobic moiety, shielding it from the environment. Utilizing this behavior, we demonstrate experimentally that the aggregation of cholesterol-modified DNA nanostructures can be controlled by the length of ssDNA overhangs positioned adjacent to the cholesterol. Our easy-to-implement method for tuning cholesterol-mediated aggregation allows for increased control and a closer structure–function relationship of membrane-interfacing DNA constructs — a fundamental prerequisite for employing DNA nanodevices in research and biomedicine.

Hydrophobic Tags for Highly Efficient Light-Activated Protein Release.

We have previously described the photoactivated depot (PAD) approach for the light-stimulated release of therapeutic proteins such as insulin. The aim of this method is to release insulin from a shallow dermal depot in response to blood glucose information, using transcutaneous irradiation. Our first approach utilized a photocleavable group that linked insulin to an insoluble but injectable polymer bead. The bead conferred insolubility, ensuring that the injected material stayed at the site of injection, until light cleaved the link, and allowed insulin to be absorbed systemically. While this proved to be effective, the use of a polymer to ensure insolubility introduces two major design problems: (1) low concentration of insulin, as a majority of the material is composed of polymer, and (2) upon release of the insulin, the polymer has to be cleared from the system. To address these two problems, in this work, we have pursued "hydrophobic tags", photocleavable small nonpolar molecules that confer insolubility to the modified insulin prior to irradiation without the bulk or need for biodegradation required of polymers. We developed a combined solid- and solution-phase synthetic approach that allowed us to incorporate a range of small nonpolar moieties, including peptides, into the final depot materials. The resulting materials are >90% w/w insulin and have sharply decreased solubilities relative to unmodified insulin (≤1000 × lower). We demonstrated that they can be milled into low micron-sized particles that can be readily injected through a 31G needle. These suspensions can be prepared at an effective concentration of 20 mM insulin, a concentration at which 120 μL contains 7 days of insulin for a typical adult. Finally, upon photolysis, the insoluble particles release soluble, native insulin in a predictable fashion. These combined properties make these new modified insulins nearly ideal as candidates for PAD materials.

A substrate tagging and two-step enzymatic reaction strategy for large-scale synthesis of 2,7-anhydro-sialic acid

A sialyltransferase acceptor tagging and two-step enzymatic reaction strategy has been developed for multigram-scale chemoenzymatic synthesis of 2,7-anhydro-N-acetylneuraminic acid (2,7-anhydro-Neu5Ac), a compound that can serve as a sole carbon source for the growth of Ruminococcus gnavus, a common human gut commensal. Different approaches of introducing hydrophobic UV-active tags to lactose as well-suited sialyltransferase acceptors have been explored and a simple two-step high-yield chemical synthetic procedure has been identified. The UV-active hydrophobic tag facilitates monitoring reaction progress and allows facile product purification by C18-cartridges. A two-step enzyme-catalyzed reaction procedure has been established to combine with C18 cartridge-based purification process for high-yield production of the desired product in multigram scales with the recycled use of chromophore-tagged lactoside starting material and sialoside intermediate. This study demonstrated an environmentally friendly highly-efficient synthetic and purification strategy for the production of 2,7-anhydro-Neu5Ac to explore its potential functions.

A MedChem toolbox for cereblon-directed PROTACs.

A modular chemistry toolbox was developed for cereblon-directed PROTACs. A variety of linkers was attached to a CRBN ligand via the 4-amino position of pomalidomide. We used linkers of different constitution to modulate physicochemical properties. We equipped one terminus of the linker with a set of functional groups, e.g. protected amines, protected carboxylic acids, alkynes, chloroalkanes, and protected alcohols, all of which are considered to be attractive for PROTAC design. We also highlight different opportunities for the expansion of the medicinal chemists' PROTAC toolbox towards heterobifunctional molecules, e.g. with biotin, fluorescent, hydrophobic and peptide tags.

Poloxin-2HT+: changing the hydrophobic tag of Poloxin-2HT increases Plk1 degradation and apoptosis induction in tumor cells.

We report the hydrophobically-tagged Plk1 PBD inhibitor Poloxin-2HT+, which selectively degrades the tumor target Plk1 and induces apoptosis in human tumor cells with higher potency than the hydrophobically-tagged inhibitor Poloxin-2HT. Our data provide further evidence that hydrophobically tagged inhibitors of protein-protein interactions can target and destroy disease-relevant proteins.

Selective Degradation of Polo-like Kinase 1 by a Hydrophobically Tagged Inhibitor of the Polo-Box Domain.

Hydrophobic tagging (HT) of bioactive compounds can induce target degradation via the proteasomal pathway. The first application of hydrophobic tagging to an existing inhibitor of protein-protein interactions is now presented. We developed Poloxin-2HT by fusing an adamantyl tag to Poloxin-2, an inhibitor of the polo-box domain of the protein kinase Plk1, which is a target for tumor therapy. Poloxin-2HT selectively reduced the protein levels of Plk1 in HeLa cells and had a significantly stronger effect on cell viability and the induction of apoptosis than the untagged PBD inhibitor Poloxin-2. The change in cellular phenotype associated with the addition of the hydrophobic tag to Poloxin-2 demonstrated that Poloxin-2HT targets Plk1 in living cells. Our data validate hydrophobic tagging of selective inhibitors of protein-protein interactions as a novel strategy to target and destroy disease-relevant proteins.

Selective Degradation of Polo‐like Kinase 1 by a Hydrophobically Tagged Inhibitor of the Polo‐Box Domain

AbstractHydrophobic tagging (HT) of bioactive compounds can induce target degradation via the proteasomal pathway. The first application of hydrophobic tagging to an existing inhibitor of protein–protein interactions is now presented. We developed Poloxin‐2HT by fusing an adamantyl tag to Poloxin‐2, an inhibitor of the polo‐box domain of the protein kinase Plk1, which is a target for tumor therapy. Poloxin‐2HT selectively reduced the protein levels of Plk1 in HeLa cells and had a significantly stronger effect on cell viability and the induction of apoptosis than the untagged PBD inhibitor Poloxin‐2. The change in cellular phenotype associated with the addition of the hydrophobic tag to Poloxin‐2 demonstrated that Poloxin‐2HT targets Plk1 in living cells. Our data validate hydrophobic tagging of selective inhibitors of protein–protein interactions as a novel strategy to target and destroy disease‐relevant proteins.

TDP-43 specific reduction induced by Di-hydrophobic tags conjugated peptides

TAR DNA binding protein 43 (TDP-43) is a key target in amyotrophic lateral sclerosis (ALS) treatment. Here, based on hydrophobic tagging strategy, we designed and synthesized a series of single or double hydrophobic tags conjugated peptides D1-D8. Among them, it was found that D4 displayed strongest ability to induce TDP-43 degradation in cells. D4 could reduce TDP-43 induced cytotoxicity. Besides, D4 could reduce TDP-43 levels in a transgenic drosophila model.

Water‐Soluble Sulfo‐Fluorous Affinity (SOFA) Tag‐Assisted Enzymatic Synthesis of Oligosaccharides

AbstractHerein, we report a bifunctional sulfo‐fluorous affinity (SOFA) tag‐assisted enzymatic synthesis and purification strategy for the facile preparation of bioactive glycans using fluorous solid‐phase extraction (FSPE). The incorporation of a sulfonate moiety onto the heavy fluorous tag significantly increases its water solubility, which allows the broad use of the inherently hydrophobic fluorous tag in aqueous buffers. In addition, the SOFA tag contains a photocleavable linker, enabling the easy release of amino‐functionalized oligosaccharides by UV irradiation. The SOFA tag was used in the synthesis of both negatively charged and neutral glycans to demonstrate its broad utility as an acceptor toward six different glycosyltransferases, significantly improving the feasibility of the preparation of complex glycans using FSPE. All the reactions were performed in an aqueous buffer, a minimum amount of methanol was used to purify the products, and the SOFA tag was easily recovered after photo‐irradiation. Thus, the entire synthetic process is environmentally benign.magnified image

BODIPY dyes in bio environment: Spectral characteristics and possibilities for practical application

Abstract Boron dipyrrin (BODIPY) dyes serve as perspective fluorescent sensors and probes in a vast range of biochemical and medical applications. Thus, mechanisms of interaction with biomolecules and accompanying spectral behavior of BODIPY are of major interest for fundamental science and practical applications. 8-aryl BODIPY derivatives were found to have high fluorescent respond upon the protein addition. Investigation of the interaction between the series of BODIPY complexes and Bovine Serum Albumin (BSA) using ultraviolet-visible (UV-vis) absorption, fluorescence spectroscopy under simulated physiological conditions (pH 7.4) and molecular docking methods was performed. The results of fluorescence spectroscopy indicated that the fluorescence of hydrophobic BODIPY dyes substantially increases in the presence of BSA. The prospected reason is binding of the dye to the BSA hydrophobic cavities in the subdomain IIA. Aromatic moiety size increase causes shifting of the interaction driving force from mostly non-specific to the π-stacking. Among all probes, the 8-phenyl substituted dye showed the highest sensitivity and the quantitative fluorescent response for BSA in aqueous solution. BODIPY with 8-phenyl substituent or analogs therefore could be used as protein surface polarity sensors, at the same time all of the investigated dyes with bulky aromatic groups could be used as hydrophobic molecule tags and markers for bioimaging tasks.

Reversible association of proteins into sub-visible amorphous aggregates using short solubility controlling peptide tags

Careful analysis of sub-visible amorphous aggregates, where proteins associate non-covalently in either native or denatured states without forming a specific quaternary structure, may shed insight into the mechanisms of protein aggregation and solubility. Here we report a biophysical and biochemical analysis of our model protein, a bovine pancreatic trypsin inhibitor variant (BPTI-19A), whose oligomerization were controlled by attaching solubility controlling peptide tags (SCP tags) to its C terminus, which are short peptides composed of a single type of amino acid that modulate protein solubility. The dynamic light scattering and static light scattering at 25°C indicated that 11 out of 15 SCP tags merely affected the hydrodynamic radius and light scattering intensity of our reference variants BPTI-19A and BPTI-C2G. On the other hand, hydrophobic SCP tags composed of 5 Ile (C5I) or 5 Leu (C5L) were associated into sub-visible aggregates. Circular dichroism indicated that all tagged BPTI variants had the same secondary structure contents as the reference BPTI-19A at 25°C, suggesting that BPTI-C5I and C5L kept their native structure upon association. Furthermore, the thermal denaturation of all of the BPTI variants was fully reversible and typical of natively folded small globular proteins, as monitored by CD at 222 nm. However, the thermal stability of BPTI-19A tagged with hydrophobic residues decreased with increasing protein concentration and tag's hydrophobicity, and BPTI-C5I and C5L were partially denatured at 37°C. Biochemical stability assessed by limited proteolysis with pepsin correlated with the extent of the variants' aggregation, and the large sub-visible aggregates formed by BPTI-C5I and C5L significantly increased their resistance to pepsin proteolysis. Altogether, these observations indicated that hydrophobic SCP tags led to the reversible association of native-like proteins into sub-visible soluble amorphous aggregates resistant to pepsin digestion.

De novo sequencing of highly modified therapeutic oligonucleotides by hydrophobic tag sequencing coupled with LC-MS.

Correct sequences are prerequisite for quality control of therapeutic oligonucleotides. However, there is no definitive method available for determining sequences of highly modified therapeutic RNAs, and thereby, most of the oligonucleotides have been used clinically without direct sequence determination. In this study, we developed a novel sequencing method called 'hydrophobic tag sequencing'. Highly modified oligonucleotides are sequenced by partially digesting oligonucleotides conjugated with a 5'-hydrophobic tag, followed by liquid chromatography-mass spectrometry analysis. 5'-Hydrophobic tag-printed fragments (5'-tag degradates) can be separated in order of their molecular masses from tag-free oligonucleotides by reversed-phase liquid chromatography. As models for the sequencing, the anti-VEGF aptamer (Macugen) and the highly modified 38-mer RNA sequences were analyzed under blind conditions. Most nucleotides were identified from the molecular weight of hydrophobic 5'-tag degradates calculated from monoisotopic mass in simple full mass data. When monoisotopic mass could not be assigned, the nucleotide was estimated using the molecular weight of the most abundant mass. The sequences of Macugen and 38-mer RNA perfectly matched the theoretical sequences. The hydrophobic tag sequencing worked well to obtain simple full mass data, resulting in accurate and clear sequencing. The present study provides for the first time a de novo sequencing technology for highly modified RNAs and contributes to quality control of therapeutic oligonucleotides. Copyright © 2016 John Wiley & Sons, Ltd.

Immuno-affinity Capture Followed by TMPP N-Terminus Tagging to Study Catabolism of Therapeutic Proteins.

Characterization of in vitro and in vivo catabolism of therapeutic proteins has increasingly become an integral part of discovery and development process for novel proteins. Unambiguous and efficient identification of catabolites can not only facilitate accurate understanding of pharmacokinetic profiles of drug candidates, but also enables follow up protein engineering to generate more catabolically stable molecules with improved properties (pharmacokinetics and pharmacodynamics). Immunoaffinity capture (IC) followed by top-down intact protein analysis using either matrix-assisted laser desorption/ionization or electrospray ionization mass spectrometry analysis have been the primary methods of choice for catabolite identification. However, the sensitivity and efficiency of these methods is not always sufficient for characterization of novel proteins from complex biomatrices such as plasma or serum. In this study a novel bottom-up targeted protein workflow was optimized for analysis of proteolytic degradation of therapeutic proteins. Selective and sensitive tagging of the alpha-amine at the N-terminus of proteins of interest was performed by immunoaffinity capture of therapeutic protein and its catabolites followed by on-bead succinimidyloxycarbonylmethyl tri-(2,4,6-trimethoxyphenyl N-terminus (TMPP-NTT) tagging. The positively charged hydrophobic TMPP tag facilitates unambiguous sequence identification of all N-terminus peptides from complex tryptic digestion samples via data dependent liquid chromatgraphy-tandem mass spectroscopy. Utility of the workflow was illustrated by definitive analysis of in vitro catabolic profile of neurotensin human Fc (NTs-huFc) protein in mouse serum. The results from this study demonstrated that the IC-TMPP-NTT workflow is a simple and efficient method for catabolite formation in therapeutic proteins.