Peptide Bioconjugates Research Articles

Effective Access to Multivalent Inhibitors of Carbonic Anhydrases Promoted by Peptide Bioconjugation.

Multivalency has impressive effects on (bio)molecular recognition, through the simultaneous presentation of multiple copies of a ligand, which can change a weak millimolar binder into a potent nanomolar one. The implementation of multivalency in enzyme inhibition is rather recent, being exemplified by few serendipitous discoveries, and hitherto relying on the random exploration of new multivalent structures as potential enzyme inhibitors. Here, a straightforward and versatile method is reported that enables the construction of multivalent systems for the inhibition of carbonic anhydrases (CA), widespread enzymes that catalyze a fundamental biochemical reaction. Oxime and hydrazone click-type bioconjugation techniques were successfully used for the preparation of tetravalent peptide conjugates tethered with sulfonamide CA inhibitors. The enzyme inhibition assays show that multivalent effects were present with these novel compounds, but also reveal various structural effects provided by the scaffolds. The versatility of this approach may facilitate the exploration of structure-activity relationships for other types of enzyme inhibitors.

Thiazolidine-Masked α-Oxo Aldehyde Functionality for Peptide and Protein Modification.

α-Oxo aldehyde-based bioconjugation chemistry has been widely explored in peptide and protein modifications for various applications in biomedical research during the past decades. The generation of α-oxo aldehyde via sodium periodate oxidation is usually limited to the N-terminus of a target protein. Internal-site functionalization of proteins with the α-oxo aldehyde handle has not been achieved yet. Herein we report a novel method for site-specific peptide and protein modification using synthetically or genetically incorporated thiazolidine-protected α-oxo aldehyde. Efficient unmasking of the aldehyde was achieved by silver ion-mediated hydrolysis of thiazolidine under mild conditions for the first time. A model peptide and a recombinant protein were used to demonstrate the utility of this new method, which were site-specifically modified by oxime ligation with an oxyamine-functionalized peptide labeling reagent. Therefore, our current method has enriched the α-oxo aldehyde synthetic tool box in peptide and protein bioconjugation chemistry and holds great potential to be explored in novel applications in the future.

Propargyl-Assisted Selective Amidation Applied in C-terminal Glycine Peptide Conjugation.

Alkyl esters, such as propargyl esters, typically lack the electron-withdrawing inductive effects needed to participate in nucleophilic acyl substitution reactions. Herein, we report an unusual observation in which glycine propargyl ester derivatives displayed selective, base-independent reactivity towards linear alkylamines under mild, metal-free conditions. Through global reaction route mapping (GRRM) modeling calculations, it is predicted that these observations may be governed by factors related to hydrogen-bonding and intermolecular interactions, rather than electron-withdrawing inductive effects. Based on this concept of propargyl-assisted selective amidation, a direct application was made to develop a novel site-specific C-terminal glycine peptide bioconjugation technique as a proof-of-concept, which relies upon the selective reactivity of glycine propargyl esters over that of aspartate and glutamate side-chain-linked propargyl esters.

An Overview of the Synthesis of Highly Versatile N-Hydroxysuccinimide Esters

N-Hydroxysuccinimide esters (NHS-esters) are important and widely used tools in various areas of chemistry including peptide synthesis, bioconjugate chemistry, functionalized materials and polymers. The usual strategy employed to prepare these active esters generally relies on the coupling reaction with a carboxylic acid and N-hydroxysuccinimide in the presence of a coupling agent. However, more recently, many other efficient strategies have emerged in the literature. This short review covers the literature devoted to the preparation of these valuable N-hydroxysuccinimide esters. 1 Introduction 2 N-Hydroxysuccinimide Coupling Reaction with Activated Carboxylic Acids 3 Carboxylic Acid Coupling Reaction with Activated N-Hydroxysuccinimide 4 Alcohol and Aldehyde Coupling Reaction with N-Hydroxysuccinimide under Oxidizing Conditions 5 Carbonylative Cross-Coupling Reaction 6 Conclusion

Extent of the Oxidative Side Reactions to Peptides and Proteins During the CuAAC Reaction.

The copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction is a powerful tool for bioconjugation of biomolecules, particularly proteins and peptides. The major drawback limiting the use of the CuAAC reaction in biological systems is the copper-mediated formation of reactive oxygen species (ROS), leading to the oxidative degradation of proteins or peptides. From the studies on a limited number of proteins and peptides, it is known that, in general, the copper mediated oxidative damage is associated with the copper coordination environment and solvent accessibility. However, there is a lack of data to help estimate the extent of copper-mediated oxidation on a wide range of proteins and peptides. To begin to address this need, we quantitatively measured the degree of copper-mediated oxidation on libraries of 1200 tetrapeptides and a model protein (bovine serum albumin, BSA) using liquid chromatography mass spectrometry (LC-MS). The collected data will be useful to researchers planning to use the CuAAC reaction for bioconjugaton on peptides or proteins.

RGDS- and TAT-Conjugated Upconversion of NaYF4:Yb(3+)/Er(3+)&SiO2 Nanoparticles: In Vitro Human Epithelioid Cervix Carcinoma Cellular Uptake, Imaging, and Targeting.

Starting NaYF4:Yb(3+)/Er(3+) nanoparticles with size tuned from 24 to 33 nm were prepared by high-temperature coprecipitation of lanthanide chlorides in high-boiling organic solvents. To enhance colloidal stability in aqueous medium, an aminosilica shell was introduced on the surface by hydrolysis and condensation of tetramethyl orthosilicate and (3-aminopropyl)trimethoxysilane using a reverse microemulsion technique; to form alkyne groups, reaction with 4-pentynoic acid followed. Finally, the cell adhesive and cell penetrating azidopentanoyl-GGGRGDSGGGY-NH2 (RGDS) and azidopentanoyl-GGGRKKRRQRRR-NH2 (TAT) peptides were conjugated to the upconversion particles via Cu(I)-catalyzed alkyne-azide cycloaddition. The concentrations of the peptides bound to the nanoparticle surfaces and amount of adsorbed residual Cu(I) catalyst were determined using an (125)I-radiolabeled RGDS peptide and a (64)Cu(I)-doped catalyst, respectively. Targeting and uptake of the RGDS- and TAT-conjugated NaYF4:Yb(3+)/Er(3+)&SiO2 nanoparticles by human cervix carcinoma HeLa cells were monitored by confocal microscopy. RGDS-conjugated nanoparticle probes were mainly localized on the cell plasma membrane due to specific binding of the peptide to the corresponding integrins. In contrast, the TAT-conjugated nanoparticles were able to cross the cell membrane and accumulate in the cell cytoplasm. Thus, this new peptide bioconjugation approach supported both extra- and intracellular nanoparticle uptake, enabling targeting and imaging of the specific tumor phenotypes.

Abstract 1341: PRINT: A protein bioconjugation method with exquisite N-terminal specificity

Abstract The use of proteins and peptides for therapeutic applications are often compromised by low biological stability, high renal clearance, and non-optimal biodistribution. Chemical attachment of poly-(ethylene glycol) (PEGylation) is often considered the most effective way to improve these pharmacologic properties by increasing circulation half-life as well as reduced renal clearance, immunogenicity and protease mediated degradation. However, random conjugation results in heterogeneous derivatives with undefined composition and can substantially lower the bioactivity of the modified protein, leading to unpredictable in vivo behavior. Site-specific modification of proteins is therefore an attractive approach to circumvent the non-specificity resulting from random conjugation. We have developed a novel technique named PRINT (PRotect, INcise Tag) for N-terminal specific bioconjugation of proteins and peptides. Conceptually, PRINT can be performed on any protein that has any N-terminal tag for purification and a protease cleavage site following the tag. The recombinant protein is first treated with an excess of citraconic anhydride to reversibly block all reactive primary amine sites. Proteolytic cleavage then exposes only a single amine (the primary amine at the N-terminus) for desired bioconjugation by amine-reactive NHS ester chemistry. Lowering of reaction pH results in removal of the citraconates, leaving N-terminal specific mono PEGylated protein molecules. We used Tumor Necrosis Factor-α (TNF-α) as a model protein as it suffers from inherent instability and short biological half-life, and exhibits toxic side effects at therapeutic concentrations in both small animals and human patients. We demonstrate that PRINT results in a single product with exquisite selectivity and specificity in contrast to conventional reaction using the same NHS reagent, which was further confirmed by mass spectrometric analyses. Subsequent de-blocking generated an N-terminal protected TNF-α molecule with enhanced serum stability, superior pharmacokinetic properties, and reduced systemic toxicity. Importantly, N-terminal protection by PRINT did not affect the bioactivity of TNF-α. Existing site-selective bioconjugation approaches are either specific to amino acid tags or involve substantial non-trivial chemical or biotechnological manipulations to synthesize a desired bioconjugate. In contrast, PRINT employs ubiquitously used recombinant DNA techniques and easily acquired commercial reagents to generate exquisite N-terminal selective protection. We show that PRINT is a robust, reproducible and mild strategy which is able to target the α -amine and provide N-terminal specific protection to proteins or peptides that suffer from similar issues. We believe that this approach is strongly orthogonal to current methods and will be applicable to many biotherapeutics and bioprobes that are currently being designed to treat cancer. Citation Format: Surojit Sur, Yuan Qiao, Anja C. Fries, Robert N. O’Meally, Robert N. Cole, Kenneth W. Kinzler, Bert Vogelstein, Shibin Zhou. PRINT: A protein bioconjugation method with exquisite N-terminal specificity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1341.

Hyaluronic Acid Hydrogels Formed in Situ by Transglutaminase-Catalyzed Reaction.

Enzymatically cross-linked hydrogels can be formed in situ and permit highly versatile and selective tethering of bioactive molecules, thereby allowing for a wealth of applications in cell biology and tissue engineering. While a number of studies have reported the bioconjugation of extracellular matrix (ECM) proteins and peptides into such matrices, the site-specific incorporation of biologically highly relevant polysaccharides such as hyaluronic acid (HA) has thus far not been reported, limiting our ability to reconstruct this key feature of the in vivo ECM. Here we demonstrate a novel strategy for transglutaminase-mediated covalent linking of HA moieties to a synthetic poly(ethylene glycol) (PEG) macromer resulting in the formation of hybrid HA-PEG hydrogels. We characterize the ensuing matrix properties and demonstrate how these cytocompatible gels can serve to modulate the cellular phenotype of human mammary cancer epithelial cells as well as mouse myoblasts. The use of HA as a novel building block in the increasingly varied library of synthetic PEG-based artificial ECMs should have applications as a structural as well as a signaling component and offers significant potential as an injectable matrix for regenerative medicine.

Oxime conjugation in protein chemistry: from carbonyl incorporation to nucleophilic catalysis.

Use of oxime forming reactions has become a widely applied strategy for peptide and protein bioconjugation. The efficiency of the reaction and robust stability of the oxime product has led to the development of a growing list of methods to introduce the required ketone or aldehyde functionality site specifically into proteins. Early methods focused on site-specific oxidation of an N-terminal serine or threonine and more recently transamination methods have been developed to convert a broader set of N-terminal amino acids into a ketone or aldehyde. More recently, site-specific modification of protein has been attained through engineering enzymes involved in posttranslational modifications in order to accommodate aldehyde-containing substrates. Similarly, a growing list of unnatural amino acids can be introduced through development of selective amino-acyl tRNA synthetase/tRNA pairs combined with codon reassignment. In the case of glycoproteins, glycans can be selectively modified chemically or enzymatically to introduce aldehyde functional groups. Finally, the total chemical synthesis of proteins complements these biological and chemoenzymatic approaches. Once introduced, the oxime ligation of these aldehyde and ketone groups can be catalyzed by aniline or a variety of aniline derivatives to tune the activity, pH preference, stability and solubility of the catalyst. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Organic chemistry. Strain-release amination.

To optimize drug candidates, modern medicinal chemists are increasingly turning to an unconventional structural motif: small, strained ring systems. However, the difficulty of introducing substituents such as bicyclo[1.1.1]pentanes, azetidines, or cyclobutanes often outweighs the challenge of synthesizing the parent scaffold itself. Thus, there is an urgent need for general methods to rapidly and directly append such groups onto core scaffolds. Here we report a general strategy to harness the embedded potential energy of effectively spring-loaded C-C and C-N bonds with the most oft-encountered nucleophiles in pharmaceutical chemistry, amines. Strain-release amination can diversify a range of substrates with a multitude of desirable bioisosteres at both the early and late stages of a synthesis. The technique has also been applied to peptide labeling and bioconjugation.

Bioconjugation of peptides using advanced nanomaterials to examine their interactions in 3D printed flow-through device.

Peptide-peptide interactions are crucial in the living cell as they lead to the formation of the numerous types of complexes. In this study, synthetic peptides containing 11 of cysteines (α-domain of metallothionein (MT)) and sialic acid binding region (130-loop of hemagglutinin (HA)) were employed. The aim of the experiment was studying the interactions between MT and HA-derived peptides. For this purpose, fragments were tagged with cysteines at C-terminal part to serve as ligand sites for PbS and CuS quantum dots (QDs), and therefore these conjugates can be traced and quantified during wide spectrum of methods. As a platform for interaction, γ-Fe2O3 paramagnetic particles modified with tetraethyl orthosilicate and (3-aminopropyl)triethoxysilane (hydrodynamic diameter 30-40 nm) were utilized and MT/HA interactions were examined using multi-instrumental approach including electrochemistry, electrophoretic methods, and MALDI-TOF/TOF mass spectrometry. It was found that peptides enter mutual creation of complexes, which are based on some of nonbonded interactions. The higher willingness to interact was observed in MT-derived peptides toward immobilized HA. Finally, we designed and manufactured flow-through electrochemical 3D printed device (reservoir volume 150 μL) and utilized it for automated analysis of the HA/MT metal labels. Under the optimal conditions, (deposition time and flow rate 80 s and 1.6 mL/min for CuS and 120 s and 1.6 mL/min PbS, respectively), the results of peptide-conjugated QDs were comparable with atomic absorption spectrometry.

Noncovalent Modulation of the Inverse Temperature Transition and Self-Assembly of Elastin-b-Collagen-like Peptide Bioconjugates.

Stimuli-responsive nanostructures produced with peptide domains from the extracellular matrix offer great opportunities for imaging and drug delivery. Although the individual utility of elastin-like (poly)peptides and collagen-like peptides in such applications has been demonstrated, the synergistic advantages of combining these motifs in short peptide conjugates have surprisingly not been reported. Here, we introduce the conjugation of a thermoresponsive elastin-like peptide (ELP) with a triple-helix-forming collagen-like peptide (CLP) to yield ELP-CLP conjugates that show a remarkable reduction in the inverse transition temperature of the ELP domain upon formation of the CLP triple helix. The lower transition temperature of the conjugate enables the facile formation of well-defined vesicles at physiological temperature and the unexpected resolubilization of the vesicles at elevated temperatures upon unfolding of the CLP domain. Given the demonstrated ability of CLPs to modify collagens, our results not only provide a simple and versatile avenue for controlling the inverse transition behavior of ELPs, but also suggest future opportunities for these thermoresponsive nanostructures in biologically relevant environments.

Ultrafiltration membrane-based purification of bioconjugated gold nanoparticle dispersions

Abstract Functionalization of nanoparticles (NP) with biomolecules to form bioconjugated systems has received large attention in biomedical applications. However, purification of these nanoparticle bioconjugates from unbound free biofunctional ligands (e.g., peptides) remains a significant challenge in the production of well-defined materials. The conventional separation methods often compromise the product’s properties and recovery. In this work, removal of excess of unbound peptides after the bioconjugation step to yield functionalized gold nanoparticles (AuNP) was achieved by exploiting the sieving properties of commercial regenerated cellulose (RC) ultrafiltration (UF) membranes. The RC membrane with nominal molecular weight cut-off (NMWCO) of 30 kDa precisely fractionated the mixtures and purified gold nanoparticle–peptide bioconjugates in a pressure driven semi-continuous diafiltration process. The RC 30 kDa membrane showed absolute rejection of the bioconjugated AuNP and the recovery of AuNP–peptide bioconjugate in the retentate was >87% relative to the initial amount in the mixture. In addition, the separation efficiency and throughput results were much better compared to the centrifugal membrane filtration method using an analogous membrane. All results indicate that by choice of an appropriate membrane type and barrier pore size, and with optimized solution chemistry and filtration parameters, ultrafiltration membranes, and in particular RC membranes, can be very well suited for the purification of bioconjugated nanoparticle dispersions, and the diafiltration mode is very well suited for upscaling.

Solid-Phase-Based Synthesis of Ureidopyrimidinone–Peptide Conjugates­ for Supramolecular Biomaterials

Supramolecular polymers have shown to be powerful scaffolds for tissue engineering applications. Supramolecular biomaterials functionalized with ureidopyrimidinone (UPy) moieties, which dimerize via quadruple hydrogen-bond formation, are eminently suitable for this purpose. The conjugation of the UPy moiety to biologically active peptides ensures adequate integration into the supramolecular UPy polymer matrix. The structural complexity of UPy–peptide conjugates makes their synthesis challenging and until recently low yielding, thus restricted the access to structurally diverse derivatives. Here we report optimization studies of a convergent solid-phase based synthesis of UPy-modified peptides. The peptide moiety is synthesized using standard Fmoc solid-phase synthesis and the UPy fragment is introduced on the solid-phase simplifying the synthesis and purification of the final UPy–peptide conjugate. The convergent nature of the synthesis reduces the number of synthetic steps in the longest linear sequence compared to other synthetic approaches. We demonstrate the utility of the optimized route by synthesizing a diverse range of biologically active UPy–peptide bioconjugates in multimilligram scale for diverse biomaterial applications. 1 Introduction 2 Divergent Synthesis 3 Convergent Synthesis 4 UPy–Amine Strategy 5 UPy–Carboxylic Acid Strategy 6 Conclusion

Peptide Bioconjugates of Electron‐Poor Metallocenes: Synthesis, Characterization, and Anti‐Proliferative Activity

We report the synthesis of metallocene compounds Cp2 M with two different electron-withdrawing substituents on both cyclopentadienyl rings (hexafluoroacetone (HFA) and chlorobenzoyl (1-5); HFA and COOH (6 and 7), M=Fe or Ru). The COOH-containing derivatives were used to synthesize peptide bioconjugates with enkephalin (8 and 9) and neurotensin (10 and 11) as well as fluorescein-labeled neurotensin (12). All the molecules were fully characterized, including X-ray structures for 6 and 7. The physicochemical properties (lipophilicity and electrochemistry) and cytotoxicity on MCF-7, HT-29, and PT-45 cancer cells were evaluated for selected compounds. Electrochemical investigation by cyclic voltammetry revealed that all bis-substituted metallocenes are up to 300 mV harder to oxidize compared to the monosubstituted 2-ferrocenylhexafluoropropan-2-ol (FcHFA: Δ${E{{{\rm f}\hfill \atop 0\hfill}}}$=214 mV; disubstituted derivatives: up to Δ${E{{{\rm f}\hfill \atop 0\hfill}}}$=512 mV; both vs. FcH(0/+) ). For the bis-substituted compounds, log P determinations by RP-HPLC showed increased lipophilicity in comparison to the monosubstituted FcHFA and RcHFA. Cellular uptake was investigated by fluorescence microcopy, and this revealed endosomal entrapment for 12.

A cleavable cytolysin–neuropeptide Y bioconjugate enables specific drug delivery and demonstrates intracellular mode of action

Myxobacterial tubulysins are promising chemotherapeutics inhibiting microtubule polymerization, however, high unspecific toxicity so far prevents their application in therapy. For selective cancer cell targeting, here the coupling of a synthetic cytolysin to the hY1-receptor preferring peptide [F7,P34]-neuropeptide Y (NPY) using a labile disulfide linker is described. Since hY1-receptors are overexpressed in breast tumors and internalize rapidly, this system has high potential as peptide–drug shuttle system. Molecular characterization of the cytolysin–[F7,P34]-NPY bioconjugate revealed potent receptor activation and receptor-selective internalization, while viability studies verified toxicity. Triple SILAC studies comparing free cytolysin with the bioconjugate demonstrated an intracellular mechanism of action regardless of the delivery pathway. Treatments resulted in a regulation of proteins implemented in cell cycle arrest confirming the tubulysin-like effect of the cytolysin. Thus, the cytolysin–peptide bioconjugate fused by a cleavable linker enables a receptor-specific delivery as well as a potent intracellular drug-release with high cytotoxic activity.

Delivery and tracking of quantum dot peptide bioconjugates in an intact developing avian brain.

Luminescent semiconductor ∼9.5 nm nanoparticles (quantum dots: QDs) have intrinsic physiochemical and optical properties which enable us to begin to understand the mechanisms of nanoparticle mediated chemical/drug delivery. Here, we demonstrate the ability of CdSe/ZnS core/shell QDs surface functionalized with a zwitterionic compact ligand to deliver a cell-penetrating lipopeptide to the developing chick embryo brain without any apparent toxicity. Functionalized QDs were conjugated to the palmitoylated peptide WGDap(Palmitoyl)VKIKKP9GGH6, previously shown to uniquely facilitate endosomal escape, and microinjected into the embryonic chick spinal cord canal at embryo day 4 (E4). We were subsequently able to follow the labeling of spinal cord extension into the ventricles, migratory neuroblasts, maturing brain cells, and complex structures such as the choroid plexus. QD intensity extended throughout the brain, and peaked between E8 and E11 when fluorescence was concentrated in the choroid plexus before declining to hatching (E21/P0). We observed no abnormalities in embryonic patterning or embryo survival, and mRNA in situ hybridization confirmed that, at key developmental stages, the expression pattern of genes associated with different brain cell types (brain lipid binding protein, Sox-2, proteolipid protein and Class III-β-Tubulin) all showed a normal labeling pattern and intensity. Our findings suggest that we can use chemically modified QDs to identify and track neural stem cells as they migrate, that the choroid plexus clears these injected QDs/nanoparticles from the brain after E15, and that they can deliver drugs and peptides to the developing brain.

ChemInform Abstract: Phenylthiocarbamate or N‐Carbothiophenyl Group Chemistry in Peptide Synthesis and Bioconjugation

AbstractReview: 104 refs.

Synthesis, Characterization, and Cytotoxic Activity of AuI N,S‐Heterocyclic Carbenes Derived from Peptides Containing L‐Thiazolylalanine

AbstractThiazolium salts 2a–b derived from a dipeptide Boc‐Gly‐(Thz‐Ala)‐OMe (1b) containing the nonproteinogenic amino acid L‐thiazolylalanine (Thz‐Ala) can be used to generate the corresponding N,S‐heterocyclic carbene (NSHC) gold(I) (3a–b) and silver(I) (4) complexes. Reaction of the NSHC‐gold(I) iodide 3b with Boc‐Cys‐Gly‐OMe gives access to the peptide bioconjugate 5, which contains a NSHC‐Au‐(S‐Cys) unit. Compounds 3–5 constitute the first coin metal NSHC‐peptide complexes. All new compounds were comprehensively characterized by 1H, 13C and 2D‐NMR spectroscopy, IR spectroscopy, and mass spectrometry. Their cytotoxicity was studied in vitro against three different tumor cell lines (A549, Jurkat T and MiaPaca2) and IC50 values in the low micromolar range (< 25 μM), and as low as 0.4 μM in the best case were observed. All new Au complexes show good stability and promising properties and, as a result, this novel type of gold(I) carbene complex opens possibilities for the design of new metal‐based drugs with promising antitumor characteristics.

Phenylthiocarbamate orN-Carbothiophenyl Group Chemistry in Peptide Synthesis and Bioconjugation

The design of novel chemoselective and site-specific ligation methods provides new tools for obtaining complex scaffolds, peptidomimetics, and peptide conjugates. The chemistry of the N-phenylthiocarbonyl group has led to several developments in peptide ligation chemistry and peptide bioconjugation during the last 10 years. The aim of this review is to provide an overview of this emerging field.