Acid Pentafluorophenyl Esters Research Articles

A Platform for Site-Specific DNA-Antibody Bioconjugation by Using Benzoylacrylic-Labelled Oligonucleotides.

Many bioconjugation strategies for DNA oligonucleotides and antibodies suffer limitations, such as site‐specificity, stoichiometry and hydrolytic instability of the conjugates, which makes them unsuitable for biological applications. Here, we report a new platform for the preparation of DNA‐antibody bioconjugates with a simple benzoylacrylic acid pentafluorophenyl ester reagent. Benzoylacrylic‐labelled oligonucleotides prepared with this reagent can be site‐specifically conjugated to a range of proteins and antibodies through accessible cysteine residues. The homogeneity of the prepared DNA‐antibody bioconjugates was confirmed by a new LC‐MS protocol and the bioconjugate probes were used in fluorescence or super‐resolution microscopy cell imaging experiments. This work demonstrates the versatility and robustness of our bioconjugation protocol that gives site‐specific, well‐defined and plasma‐stable DNA‐antibody bioconjugates for biological applications.

A Platform for Site‐Specific DNA‐Antibody Bioconjugation by Using Benzoylacrylic‐Labelled Oligonucleotides

AbstractMany bioconjugation strategies for DNA oligonucleotides and antibodies suffer limitations, such as site‐specificity, stoichiometry and hydrolytic instability of the conjugates, which makes them unsuitable for biological applications. Here, we report a new platform for the preparation of DNA‐antibody bioconjugates with a simple benzoylacrylic acid pentafluorophenyl ester reagent. Benzoylacrylic‐labelled oligonucleotides prepared with this reagent can be site‐specifically conjugated to a range of proteins and antibodies through accessible cysteine residues. The homogeneity of the prepared DNA‐antibody bioconjugates was confirmed by a new LC‐MS protocol and the bioconjugate probes were used in fluorescence or super‐resolution microscopy cell imaging experiments. This work demonstrates the versatility and robustness of our bioconjugation protocol that gives site‐specific, well‐defined and plasma‐stable DNA‐antibody bioconjugates for biological applications.

Acoustic wave biosensor for the detection of the breast and prostate cancer metastasis biomarker protein PTHrP

There are currently no biosensors that are able to reliably detect the process of cancer metastasis. We describe the first label-free real-time ultra-high frequency acoustic wave biosensor prototype capable of detecting the breast and prostate cancer metastasis biomarker, parathyroid hormone-related peptide (PTHrP). Two different linkers – 11-trichlorosilyl-undecanoic acid pentafluorophenyl ester (PFP) and S-(11-trichlorosilyl-undecanyl)-benzothiosulfonate (TUBTS) – were used to immobilize whole anti-PTHrP antibodies and Fab’ fragments to surfaces as biorecognition elements. The biosensor surfaces were optimized using X-ray photoelectron spectroscopy (XPS) and the ultra-high frequency electromagnetic piezoelectric acoustic sensor (EMPAS). One optimized whole antibody-based surface (PFP/protein G'/whole antibodies/ethanolamine) and one optimized Fab' fragment-based surface (TUBTS/Fab' fragments) were tested as biosensors. It was determined that an in-line injection of bovine serum albumin prior to analyte injection yielded the most minimally fouling surfaces. Each surface was tested with no mass amplification and with sandwich-type secondary antibody mass amplification. The whole antibody-based mass-amplified biosensor yielded the lowest limit of detection (61ng/mL), highest sensitivity, and a linear range from 61ng/mL to 100μg/mL. However, the Fab' fragment-based biosensor displayed better regenerability as a loss of ~20% of the initial analyte signal intensity was observed with each subsequent injection. The whole antibody-based biosensor was only capable of producing an analyte signal in the first injection.

Diphenyl-benzo[1,3]dioxole-4-carboxylic acid pentafluorophenyl ester: a convenient catechol precursor in the synthesis of siderophore vectors suitable for antibiotic Trojan horse strategies

Catechols are components of many metal-chelating compounds, including siderophores that are naturally occurring iron(III) chelators excreted by microorganisms. Catechol derivatives are poorly soluble in organic media and the synthesis of catechol-containing molecules requires the use of protected catechol precursors with improved organic solubility. We therefore developed 2,2-diphenyl-benzo[1,3]dioxole-4-carboxylic acid pentafluorophenyl ester. This activated ester reacts with an amine functionalized scaffold to generate chelators in which the catechol functions are protected in the form of diphenyl-benzodioxole moieties. The catechol can subsequently be deprotected, at the end of the synthesis, with trifluoroacetic acid (TFA). This strategy was applied to the synthesis of two catechol compounds functionalized with a terminal propargyl extension. These two compounds were shown to promote iron uptake in Escherichia coli and Pseudomonas aeruginosa. These two compounds are suitable for use as vectors in antibiotic Trojan horse approaches, as they could be conjugated with azide-functionalized antibiotics using the Huisgen dipolar 1,3-cycloaddition.

Morphological Stabilization by Supramolecular Perfluorophenyl‐C60 Interactions Leading to Efficient and Thermally Stable Organic Photovoltaics

A new PC61BM‐based fullerene, [6,6]‐phenyl‐C61 butyric acid pentafluorophenyl ester (PC61BPF) is designed and synthesized. This new n‐type material can replace PC61BM to form a P3HT:PC61BPF binary blend or serve as an additive to form a P3HT:PC61BM:PC61BPF ternary blend. Supramolecular attraction between the pentafluorophenyl group of PC61BPF and the C60 cores of PC61BPF/PC61BM can effectively suppress the PC61BPF/PC61BM materials from severe aggregation. By doping only 8.3 wt% PC61BPF, device PC61BPF651 exhibits a PCE of 3.88% and decreases slightly to 3.68% after heating for 25 h, preserving 95% of its original value. When PC61BP with non‐fluorinated phenyl group is used to substitute PC61BPF, the stabilizing ability disappears completely. The efficiencies of PC61BP651 and PC61BP321 devices significantly decay to 0.44% and 0.11%, respectively, after 25 h isothermal heating. Most significantly, this strategy is demonstrated to be effective for a blend system incorporating a low band‐gap polymer. By adding only 10 wt% PC61BPF, the PDTBCDTB:PC71BM‐based device exhibits thermally stable morphology and device characteristics. These findings demonstrate that smart utilization of supramolecular interactions is an effective and practical strategy to control morphological evolution.

Parallel in Vivo and in Vitro Selection Using Phage Display Identifies Protease-dependent Tumor-targeting Peptides

We recently developed activatable cell-penetrating peptides (ACPPs) that target contrast agents to in vivo sites of matrix metalloproteinase activity, such as tumors. Here we use parallel in vivo and in vitro selection with phage display to identify novel tumor-homing ACPPs with no bias for primary sequence or target protease. Specifically, phage displaying a library of ACPPs were either injected into tumor-bearing mice, followed by isolation of cleaved phage from dissected tumor, or isolated based on selective cleavage by extracts of tumor versus normal tissue. Selected sequences were synthesized as fluorescently labeled peptides, and tumor-specific cleavage was confirmed by digestion with tissue extracts. The most efficiently cleaved peptide contained the substrate sequence RLQLKL and labeled tumors and metastases from several cancer models with up to 5-fold contrast. This uniquely identified ACPP was not cleaved by matrix metalloproteinases or various coagulation factors but was efficiently cleaved by plasmin and elastases, both of which have been shown to be aberrantly overexpressed in tumors. The identification of an ACPP that targets tumor expressed proteases without rational design highlights the value of unbiased selection schemes for the development of potential therapeutic agents.

Controlled Synthesis of Reactive Polymeric Architectures Using 5‐Norbornene‐2‐carboxylic Acid Pentafluorophenyl Ester

AbstractExo‐5‐norbornene‐2‐carboxylic acid pentafluorophenyl ester was synthesized from 5‐norbornene‐2‐carboxylic acid and polymerized by ring‐opening metathesis polymerization. The obtained polymers were soluble polymeric active esters that could be used for the preparation of multifunctional polymers. The molecular weight of the polymers was controlled by variation of the monomer to initiator ratio. The precursor polymers reacted quantitatively with primary and secondary amines. Time resolved FT‐IR studies at different temperatures of the polymer analogous reactions were performed and rate constants were determined.

Studies on Heterobifunctional Cross-Linking Reagents, 6-Maleimidohexanoic Acid Active Esters

6-maleimidohexanoic acid N-hydroxysuccinimide ester has been used widely for preparation of enzyme immunoconjugates as a unique heterobifunctional cross-linking reagent. Its heterobifunctional reactivity is good, but its ester portion hydrolyzes easily in the presence of water. Several 6-maleimidohexanoic acid active esters (6-maleimidohexanoic acid 4-nitrophenyl ester, 6-maleimidohexanoic acid N-hydroxy-5-norbornene-endo-2,3-dicarboximide ester, and 6-maleimidohexanoic acid pentafluorophenyl ester) were prepared and their reactivity and stability in an aqueous media were tested. Of the synthetic esters, the pentafluorophenyl ester exhibited the highest reactivity and stability in aqueous media.

A Practical One‐Pot Synthesis of New S‐Glycosyl Amino Acid Building Blocks for Combinatorial Neoglycopeptide Synthesis

S‐Glycosyl L‐aspartic acid building blocks were synthesized starting from 1‐thiosugars by reaction with 5‐aminopentanol and suitably protected L‐aspartic acid pentafluorophenyl ester in a one‐pot procedure under Mitsunobu conditions using 1,1′‐azodicarbonyl dipiperidine and trimethyl phosphine. The method allowed for the preparation of S‐glycosyl amino acid building blocks in one step without protection of the amino function for the Mitsunobu condensation. Alternatively, the title compounds were prepared by a stepwise approach via 5‐aminopentyl 1‐thioglycosides. This paper is dedicated to Joseph Lloyd Stiles.

Use of the excluded protecting group (EPG) method for peptide synthesis

The excluded protecting group (EPG) method has been used for the solution synthesis of several peptides including Merrifield's Model Tetrapeptide, linear antamanide and an analogue of magainin-1, [Ala(19), Asn(22)]magainin-1. In the approach reported, the C-terminal amino acid is esterified to the 2-position of cholestane as the [2s,3s]iodohydrin ester and the penultimate amino acid added to the aminoacyl-steroid as the Fmoc-pentafluorophenyl-ester. The Fmoc group is removed with Et(2)NH/DMF ( approximately 15% v/v) and, after evaporation to approximately 10 mL, the solution chromatographed on Sephadex LH-20 in DMF. The dipeptidyl-steroid elutes as the free amine well separated from other reaction mixture components. Fractions containing the dipeptide, as determined by counting and TLC, are pooled and reacted with the next Fmoc-amino acid-pentafluorophenyl ester in the sequence. Repetition of the deprotection/purification/reaction cycle yields the fully protected peptide. On completion of the synthesis, the cholestane iodohydrin ester is selectively removed by treatment with Zn degrees /AcOH to yield the peptide with intact alpha-amino and side chain protecting groups. Global deprotection is achieved with HF. All intermediates from the syntheses reported were characterized. The magainin analogue was shown to have full biologic activity. The Fmoc iodohydrin esters of 16 of the 20 proteogenic amino acids have been prepared and characterized for use as the C-terminal amino acids in other EPG syntheses.

A structure–activity study of nociceptin-(1–13)-peptide amide. Synthesis of analogues substituted in positions 0, 1, 3, 4 and 10

A series of analogues of nociceptin, Noc(1–13)NH 2 (an agonist at the ORL1 receptor) was synthesized with following modifications: (1) N-terminal extension with Arg 0; (2) replacement of Gly 3 by basic or polar amino acids—Arg, Asn, Lys(For) or deletion; (3) exchange of Phe 1 or Phe 4 by Phe(NO 2); (4) substitution of Ser 10 with d-Ser, Pro, d-Pro. The analogs were synthesized by solid-phase methodology using Fmoc-amino acid pentafluorophenyl esters. The affinity for the ORL1 and for the κ, μ and δ-opioid receptors was investigated by radioligand binding assay and bioactivity by a mouse vas deferens (MVD) assay. The addition of the amino acid residue Arg to the N-terminal enhances the opioid receptor affinity of Noc(1–13)NH 2 while retaining ORL1 receptor affinity at a moderate level. The replacement of Gly in position 3 by the basic or polar amino acids—Arg, Asn, Lys(For) or its deletion led to inactive analogues. The replacement of Ser in position 10 by its d-isomer, Pro and d-Pro resulted in a series of analogues with the following order of activity: Ser 10> d-Ser 10>Pro 10> d-Pro 10. In [ d-Ser 10]Noc(1–13)NH 2, introduction of an additional Phe(NO 2) 4 led to a >60-fold increase of ORL1 affinity, completely attenuating the loss of affinity brought about by Ser 10. In other analogues, introduction of Phe(NO 2) 4 did not change the magnitude of ORL1 binding significantly. Generally, while modifications in position 3 frequently led to a loss of most or all bioactivity, modifications in position 0 (Arg 0) or 4 (Phe(NO 2) 4) and 10 ( d-Ser 10, Pro 10) are tolerated.

A sequential high-yielding large-scale solution-method for synthesis of philanthotoxin analogues

A general, improved procedure for rapid synthesis of philanthotoxin analogues, a pharmacologically important class of polyamine conjugates, is described. The solution-phase procedure is illustrated by gram-scale synthesis of philanthotoxins PhTX-343 and PhTX-12. Selectively protected polyamines are coupled to N α-Fmoc-protected amino acid pentafluorophenyl esters. After removal of the N α-Fmoc group, the amine is coupled with carboxylic acid pentafluorophenyl esters. Deprotection followed by a rapid and efficient purification by vacuum liquid chromatography on octadecylsilyl silica (RP-18 phase) gave the philanthotoxin analogues in 74–78% overall yield.

Electrochemical polymerization and characterization of a functional dicarbazole conducting polymer

The electropolymerization of the unique dicarbazole monomer, 2,6-bis-carbazole-9-yl-hexanoic acid pentafluorophenyl ester, is reported. This chiral monomer possesses two carbazole units and a chiral grafting center, activated ester for covalent amine-coupling. Contrary to monocarbazole monomers unable of internal cross-linking, its specifically designed dicarbazole skeleton allowed us to obtain highly reticulated polydicarbazole films functionalized for probe coupling. Full analysis of its electrochemical polymerization revealed different oxidation potentials for the two structurally different carbazole units. The electrochemical activity of the polydicarbazole-coated electrode has been characterized in different redox electrolytes. Contacted by a model protein (BSA), this film has been easily passivated at the Rubpy 3(PF 6) electrochemical window.

Synthesis of mucin-type glycopeptide (β hCG 130–145) by on-resin fragment condensation of the glycopeptide segments carrying unmasked oligosaccharides

Use of amino acid pentafluorophenyl esters was found effective for chemoselective N-acylation in the peptide elongation. On-resin fragment condensation between 9 and 13 using DCC-PfpOH as an activator afforded coupling product 1 in good yield.

Carbopeptides: carbohydrates as potential templates for de novo design of protein models.

De novo design of proteins has evolved into a powerful approach for studying the factors governing protein folding and stability. Among the families of structures frequently studied is the 'four-helix bundle' in which four alpha-helical peptide strands, linked by loops, form a hydrophobic core. Assembly of protein models on a template has been suggested as a way to reduce the entropy of folding. Here we describe the potential use of a carbohydrate as such a template. The monosaccharide D-galactose was per-O-acylated with (Nbeta-Fmoc-betaAla)2O to give a penta-substituted derivative, which was converted to the corresponding glycosyl bromide and used for the glycosylation of 4-hydroxymethylbenzoic acid pentafluorophenyl ester (HMBA-OPfp). The beta-glycosidic carbohydrate template (Nbeta-Fmoc-3Ala)4-beta-D-Galp-(1-O)-MBA-OPfp thus obtained was coupled to a PAL-PEG-PS resin and simultaneously extended at the four arms to yield, after cleavage from the solid support, a carbopeptide with four identical peptide strands. Extension of this concept to, for example, synthesis of novel multiple antigenic peptides (MAPs) and synthesis of carbohydrate clusters can be easily envisioned. The ability to efficiently synthesize such structures sets the stage for further studies to test whether the carbohydrate templates do indeed nucleate folding.

Homologation of alpha-amino acids to beta-amino acids using Fmoc-amino acid pentafluorophenyl esters.

The homologation of alpha-amino acids to beta-amino acids by the two-step Arndt-Eister method is achieved by using Fmoc-alpha-amino acid pentafluorophenyl esters for the acylation of diazomethane, synthesizing the key intermediates Fmoc-aminoacyldiazomethanes as crystalline solids in good yields and purity.

Solid-phase synthesis of huwentoxin-I and its structure and bioactivity analysis

Huwentoxin-I, a neurotoxic peptide from the spiderSelenocosmia huwena, was synthesized by solid-phase method with Fluorenylmethoxycarbonyl amino acid pentafluorophenyl esters (Fmoc-AA-OPfp). The carboxyl and the hydroxy groups were protected by tBu; the side chains of Lys and His were protected by Boc; the guanidine group of Arg was protected by Mtr and the mercaptan group of Cys was protected by Trt. The solid-phase carrier was ethylene diamine-polyethylene-polystyrene (DEA-PEG-PS) resin. The synthetic peptide was cleaved from the resin and deprotected by a 90 % TFA solution containing 5 % thioanisole, 3% ethanedithiol and 2 % anisole. The product was reduced with DTT and then incubated with GSSG and GSH to form the correct disulfide bond linkages. The synthetic peptide was purified by HPLC and then characterized by amino acid composition and sequence analysis, peptide mapping and NMR. The biological activity of the synthetic product was tested by electrophysiological method using the isolated mouse phrenic nerve diaphragm preparation. The results indicated that the synthetic huwentoxin-I has the same chemical and conformational structure and biological activity as those of the native huwentoxin-I from the spider.

Preparation and properties of N alpha-Bpoc-amino acid pentafluorophenyl esters.

The preparation and properties are reported of several N alpha-Bpoc -amino acid pentafluorophenyl esters, including those bearing tert-butyl-, allyl- and trityl-based protecting groups. These derivatives have been used in the solid-phase peptide synthesis of several short peptides.

An approach to cyclic peptide libraries: Reducing epimerization in medium sized rings during solid phase synthesis

Using an amino acid linked by its side chain to a solid support, head-to-tail cyclic peptide libraries of varying ring size have been prepared via resin-bound cyclization. These mixtures complement the use of linear peptide libraries for drug lead discovery and extend molecular diversity to conformationally constrained systems. During our synthesis of cyclic peptide mixtures, C-terminal epimerization was identified as a problem during chain elongation. This was significantly overcome by coupling with preactivated amino acid pentafluorophenyl esters.

Synthesis of 2-Acetamido-2-deoxy-β-d-glucopyranoseO-Glycopeptides fromN-Dithiasuccinoyl-Protected Derivatives1-3

2-Acetamido-2-deoxy-β-d-glucopyranose (β-d-GlcpNAc), in O-glycosidic linkage to the side chain hydroxyls of serine (Ser) and threonine (Thr) residues, is often found in nuclear and cytoplasmic proteins. The “active ester” approach for solid phase glycopeptide synthesis calls for the direct glycosylation of Nα-(9-fluorenylmethyloxycarbonyl)amino acid pentafluorophenyl esters (Nα-Fmoc-AA-OPfp's). The synthesis of the required Ser(β-d-GlcpNAc) and Thr(β-d-GlcpNAc) building blocks poses special problems arising from the 2-amino substituent in the corresponding glycosyl donors. Activation of donors with a 2-N-acyl group provides relatively unreactive oxazoline intermediates, whereas the otherwise promising phthaloyl (Phth) group requires prolonged base treatment at elevated temperatures for its removal, and incomplete deprotection is often encountered. The dithiasuccinoyl (Dts) group provides bivalent protection in the same way as the Phth group, but has the advantage of being removed rapidly under mild condit...