KG was supported by the R.A. Welch Foundation grant P-1760, TCU Andrews Institute of Mathematics &#38; Science Education (to KG), TCU Research and Creativity Activity Grant (to KG) and TCU SERC Grant (to JHS).

1. Synthesis of Biotin-Fc-cysteine (1)
<ol>
	<li>Solid phase methods to produce resin-bound 1.
	<ol>
		<li>Place biotin loaded resin (250 mg, 0.145 mmol) into a fritted syringe and swell the resin by drawing up dimethylformamide (5 ml) and shaking the syringe on a lab shaker for 20 min. Expel the solution and repeat dimethylformamide swelling one more time.</li>
		<li>Remove the Fmoc protecting group by adding 4-6 ml of 20% piperidine in dimethylformamide to the syringe followed by 10-15 min of shaking. Repeat the deprotection process with another 4-6 ml of piperidine. Wash the resin with a sequence of 3x dimethylformamide, 3x dimethylformamide:methanol (1:1), 3x methanol:dichloromethane (1:1), 3x dichloromethane, ~5 ml each. Do a ninhydrin test (+) on a small sampling (~10) of the beads to confirm successful deprotection by the presence of blue upon heating.</li>
		<li>Mix a solution containing 1&#8217;-Fmoc-amino-ferrocene-1-carboxylic acid (203.3 mg, 0.4350 mmol), 1-hydroxybenzotriazole hydrate (58.8 mg, 0.413 mmol), diisopropyl carbodiimide (0.0673 ml, 0.435 mmol), diisopropyl ethyl amine (0.0757 ml, 0.435 mmol), and a 4:1 mixture of dichloromethane and dimethylformamide. Draw this into the fritted syringe and gently shake on a lab shaker for 6 hr. Then expel the solution from the syringe and wash as previously described.</li>
		<li>Perform the ninhydrin test (-) as described above to confirm coupling. The ninhydrin test can still be useful in confirming coupling despite the orange color of the bead derived from the attachment of the iron containing moiety.</li>
		<li>Then remove the Fmoc group by the addition of 20% piperidine in dimethylformamide and washed as described above. The ninhydrin test (+) should be used to confirm Fmoc removal.</li>
		<li>Prepare a solution composed of Fmoc-Cys(Trt)-OH (254.8 mg, 0.4350 mmol), 1-hydroxybenzotriazole hydrate (58.8 mg, 0.4125 mmol), diisopropyl carbodiimide (0.0673 ml, 0.4350 mmol), diisopropyl ethyl amine (0.0757 ml, 0.4350 mmol), and a 4:1 mixture of dichloromethane and dimethylformamide. Add this cysteine coupling cocktail the fritted syringe and gently shake for 6 hr. Wash using the protocol described previously.</li>
		<li>Confirm coupling using the ninhydrin test (-), followed by removal of the Fmoc component with 20% piperidine and washing. Verify the free terminal amine using the ninhydrin test (+).</li>
	</ol>
	</li>
	<li>Cleavage of 1 from the resin.
	<ol>
		<li>Make a solution of TFA (9.45 ml), water (0.25 ml), 1,2-ethanedithiol (0.25 ml), and triisopropyl silane (0.1 ml), add it to the syringe and shake gently for 4 hr.</li>
		<li>Collect the resulting red-brown solution in an Eppendorf tube and evaporate the TFA slowly using a stream of air.</li>
		<li>Add cold diethyl ether (~15 ml) to the Eppendorf tube to precipitate 1, which will form gentle agitation. Isolate the product via centrifugation (1 g, 5 min). Then repeat cycles of diethyl ether washings (~60 ml total) and centrifuge to obtain 1 as a red/brown solid.</li>
	</ol>
	</li>
</ol>
2. Characterization and Analysis of 1
<ol>
	<li>Confirm that the identity matches the connectivity and composition shown in Figure 2 using 1H (16 scans)&#160;and 13C NMR (512 scans) in deuterated methanol (300 &#181;l) and ESI-MS analysis. 
	Expect the following results: 
	1H NMR spectrum (CD3OD) &#948;/ppm: 1.407-1.684 (m, 6H), 2.245 (t, 2H), 2.665-3.150 (m, 12H), 4.015 (t, 1H), 4.104 (d, 2H), 4.274 (q, 1H), 4.426 (d, 2H), 4.479 (q, 1H), 4.595 (t, 1H) and 13C NMR spectrum (CD3OD) &#948;/ppm: 24.644 (CH2), 25.472 (CH2), 28.051(CH2), 28.300 (CH2), 35.474 (CH2), 38.698 (CH2), 39.241 (CH2), 39.717 (CH2), 55.340/55.538 (Cp-ring), 60.286 (CH), 61.964 (CH), 62.521/62.821 (Cp-ring), 66.038/66.170 (Cp-ring), 69.153/69.328 (Cp-ring), 71.468/71.593 (Cp-ring), 76.466 (CH), 171.770 (C=O), 175.361 (C=O). 
	ESI-MS (m/z): Found: 639.00 [1+Na]+, Theoretical: 639.1 [1+Na]+ and HR-MS (m/z): Found: 617.2049 [1+H]+ , Theoretical: 617.1622 [1+H]+.</li>
	<li>Perform HPLC, elemental analysis to confirm the composition of isolated 1. 
	Carry out HPLC chromatograms using a C8 reversed phase column with 100% MeOH at a flow rate of 0.5 ml/min. Note: HPLC retention times were: 3.198-4.674 min.</li>
</ol>
3. Immobilization of 1 on a Gold Surface
<ol>
	<li>Cut polymer backed gold slides into squares of ~0.25 in2.</li>
	<li>Fill a 50 ml beaker with a DI water solution of 1 (~1 mM).</li>
	<li>Add the gold slide to the beaker and cover with a watch glass. Allow the glass slide to incubate O/N at RT with no agitation.</li>
	<li>Remove the gold slide from the solution and allow it dry in air.</li>
	<li>Obtain scanning electron microscopy images using a scanning electron microscope (or equivalent) to observe immobilized 1.</li>
</ol>

The authors have nothing to disclose.

The synthesis of asymmetric ferrocene derivatives is challenging in solution. For example, attempts to produce 1 in solution resulted in low yields of the desired product (less than 20%). Likewise, reactions utilizing 1&#8217;-amino-ferrocene carboxylic acid (sans Fmoc) and resin bound biotin resulted in insoluble product consistent with the polymerized product reported by Baristic et al. and minimal product.47 This is further complicated by ferrocene and its derivatives bein...

Biosensors are small devices that employ biomolecular recognition technology as the platform for selective analysis and are utilized for their specificity, speed, and low-cost. Electrochemical biosensors for the detection of biomolecules are at the forefront of this field due to their simplicity, cost effectiveness, and high sensitivity.1,3 The general anatomy of these sensors is an electrode equipped with a recognition molecule specific for the biological marker of interest. Binding of the biomarker by the recognition molecule results in a local change of potential or current that can be detected by simple measurement. To date the recognition moiety can range from enzymes,4-8 antibodies,9-12 whole cells,13-16 receptors,17-20 peptides21-23 and DNA24 and have largely focused on larger, biological molecules.25-28 Research efforts in this arena have concentrated mainly on immunosensors where an immunoglobulin is immobilized with a redox active core (such as ferrocene) and used to detect an antibody of interest. These studies have been excluded from clinical applications due to poor precision and time consumption stemming from the complications arising from use of antigen/antibodies.1,3 Growing attention has focused on the detection of small molecules (less than 1 kg/mol) of biomedical, food and environmental interest in addition to national security.29 The best known examples of biosensor devices are self-test glucose monitors, which have screen printed enzyme electrodes coupled to a pocket-size amperometric meter. These systems typically utilize a coulometric method where the total amount of charge generated by the glucose oxidation reaction is measured over a period of time. Marketable devices must be portable, robust and hand-held to make use facile for the population at large.
Redox tags such as ferrocene are necessary to provide the electrochemical detection of biomarkers or small molecules in solution as most biomarkers are not intrinsically electrochemically active.30-38 Ferrocene is an organometallic molecule that is a gold standard for electrochemistry, which makes it an excellent choice for integration into electrochemical biosensors. Ferrocene-based redox active species have already garnered considerable attention due to their small size, good stability, convenient synthetic access, easy chemical modification, relative lipophilicity, and ease of redox tuning.3,30-42 Small molecules based on the ferrocene core have been used extensively as detectors of metal ions and small molecules.32-38,43 Systems targeting larger species such as biomolecules have utilized the attachment of large antibodies or immunoglobulins to ferrocene derivatives that have been embedded onto an electrochemical surface.1,3,39,44 In each case, the potential and current intensity of the FeIII/FeII redox couple was altered upon molecular coupling, thus producing a new spectroscopic handle indicating the presence of the analyte molecule. This change arises from the extensive overlap that occurs between the pi-system of the cyclopentadienyl rings and the iron d-orbitals. If the pi-system is modified, i.e., derivatized or reacted, then the orbital interaction will, in turn, change. This will affect the Fe core and can be observed as a shift in the potential of the FeIII/FeII couple.40,45,46 These properties make such a system attractive for use as a quantifying agent in an electrochemical immunoassay or biosensor.
In order to produce ferrocene containing systems specific for biosensor capacities it is optimal to modify one Cp ring with the bio-receptor specific for a target molecule and utilize the other Cp ring as a molecular tether to the electrochemical readout or electrode (Figure 1). Synthesis of these asymmetric ferrocene derivatives is challenged by side reactions and the formation of dimeric and polymeric species formed upon intermolecular cross-linking.47 However, coupling chemistry producing an amide bond is the most direct route to provide simple derivatives of ferrocene involving biological components such as peptides and their metabolites. Therefore, solid phase techniques first developed in the 1950s by Merrifield for peptide synthesis can be applied to organometallic compounds containing ferrocene. Through the use of the orthogonally substituted 1&#8217;-Fmoc-amino-ferrocene-1-carboxylic acid molecule, a ferrocene system that can contain a receptor moiety (biotin), electrochemical readout (ferrocene), and immobilizing-linker component (cysteine) has been constructed and detailed herein. The synthesis of this bio-conjugate is discussed as well as evidence for immobilization on a gold surface. This work represents the first presentation of a system composed of biotin, ferrocene and an amino acid for immobilization on a gold surface.

<table><tbody><tr><td>Biotin Novatag Resin</td><td>NovaBiochem</td><td>8550510001</td><td></td></tr><tr><td>TORVIQ 10 ml Luer Lock Fritted Syringe</td><td>Fisher</td><td>NC9299151</td><td></td></tr><tr><td>piperidine</td><td>Acros</td><td>P/3520/PB05</td><td></td></tr><tr><td>ninhydrin test</td><td>Sigma-Aldrich</td><td>60017-1ea</td><td></td></tr><tr><td>1&amp;rsquo;-Fmoc-amino-ferrocene-1-carboxylic acid</td><td>Omm Scientific</td><td>Special Order</td><td></td></tr><tr><td>&lt;em&gt;N,N&amp;prime;&lt;/em&gt;-Diisopropylcarbodiimide</td><td>Sigma-Aldrich</td><td>D125407-5G</td><td></td></tr><tr><td>Fmoc-Cys(Trt)-OH</td><td>Novabiochem</td><td>8520080025</td><td></td></tr><tr><td>trifluoroacetic acid</td><td>Sigma-Aldrich</td><td>T5408</td><td></td></tr><tr><td>1,2-ethanedithiol</td><td>Sigma-Aldrich</td><td>2930</td><td></td></tr><tr><td>triisopropyl silane</td><td>Sigma-Aldrich</td><td>233781</td><td></td></tr><tr><td>Eppendorf tubes (20 ml)</td><td>any source</td><td></td><td></td></tr><tr><td>methanol</td><td>any source</td><td></td><td>dry with molecular sieves prior to use &amp;amp; store in 100 ml media bottle for easy usage</td></tr><tr><td>dichloromethane</td><td>any source</td><td></td><td>dry with molecular sieves prior to use &amp;amp; store in 100 ml media bottle for easy usage</td></tr><tr><td>dimethylformamide</td><td>any source</td><td></td><td>dry with molecular sieves prior to use &amp;amp; store in 100 ml media bottle for easy usage</td></tr><tr><td>centrifuge</td><td>any source</td><td></td><td></td></tr></tbody></table>

synthetic methodology for asymmetric ferrocene derived bio-conjugate systems via solid phase resin-based methodology

Early detection is a key to successful treatment of most diseases, and is particularly imperative for the diagnosis and treatment of many types of cancer. The most common techniques utilized are imaging modalities such as Magnetic Resonance Imaging (MRI), Positron Emission Topography (PET), and Computed Topography (CT) and are optimal for understanding the physical structure of the disease but can only be performed once every four to six weeks due to the use of imaging agents and overall cost. With this in mind, the development of &#8220;point of care&#8221; techniques, such as biosensors, which evaluate the stage of disease and/or efficacy of treatment in the clinician&#8217;s office and do so in a timely manner, would revolutionize treatment protocols.1 As a means to exploring ferrocene based biosensors for the detection of biologically relevant molecules2, methods were developed to produce ferrocene-biotin bio-conjugates described herein. This report will focus on a biotin-ferrocene-cysteine system that can be immobilized on a gold surface.

The resin bound form of 1 is shown in Figure 2. The covalent attachment of the ferrocene component gives rise to an orange tint to the resin beads that is persistent with continuous washing and indicative of an immobilized iron containing complex as opposed to iron absorption by the PEG component of the resin bead. The resin-free form of 1 is identical in color to the resin beads. Following removal of the compound from the resin-beads, the purity and yield (68%) resultin...

Watch this Scientific Journal Video about Synthetic Methodology for Asymmetric Ferrocene Derived Bio-conjugate Systems via Solid Phase Resin-based Methodology at JoVE.com

Synthetic Methodology for Asymmetric Ferrocene Derived Bio-conjugate Systems via Solid Phase Resin-based Methodology

The synthesis of asymmetric species of ferrocene is challenging using solution techniques. This report focuses on the methods carried out to produce a ferrocene-biotin bioconjugate using facile and clean reactions accomplished via solid-phase synthesis. Incorporation of a thiolate moiety is shown to impart the ability for immobilization on gold surfaces.

Early detection is a key to successful treatment of most diseases, and is particularly imperative for the diagnosis and treatment of many types of cancer. ...

synthetic-methodology-for-asymmetric-ferrocene-derived-bio-conjugate

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Chemistry

9.6K Views.  Texas Christian University. The synthesis of asymmetric species of ferrocene is challenging using solution techniques. This report focuses on the methods carried out to produce a ferrocene-biotin bioconjugate using facile and clean reactions accomplished via solid-phase synthesis. Incorporation of a thiolate moiety is shown to impart the ability for immobilization on gold surfaces.

Video: Synthetic Methodology for Asymmetric Ferrocene Derived Bio-conjugate Systems via Solid Phase Resin-based Methodology

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the development of antibody drug conjugates (ADCs) and nanomedicine, fluorescent microscopy and systems chemistry. For many of these applications, specificity of the bioconjugation method used is of prime concern. The Michael addition of maleimides with cysteine(s) on the target proteins is highly selective and proceeds rapidly under mild conditions, making it one of the most popular methods for protein bioconjugation.
We demonstrate here the modification of the only surface-accessible cysteine residue on yeast cytochrome c with a ruthenium(II) bisterpyridine maleimide. The protein bioconjugation is verified by gel electrophoresis and purified by aqueous-based fast protein liquid chromatography in 27% yield of isolated protein material. Structural characterization with MALDI-TOF MS and UV-Vis is then used to verify that the bioconjugation is successful. The protocol shown here is easily applicable to other cysteine - maleimide coupling of proteins to other proteins, dyes, drugs or polymers.

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

This protocol details the important steps required for the bioconjugation of a cysteine containing protein to a maleimide, including reagent purification, reaction conditions, bioconjugate purification and bioconjugate characterization.

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the ...

Synthesis of a Water-soluble Metal&#8211;Organic Complex Array

We demonstrate a method for the synthesis of a water-soluble multimetallic peptidic array containing a predetermined sequence of metal centers such as Ru(II), Pt(II), and Rh(III). The compound, named as a water-soluble metal-organic complex array (WSMOCA), is obtained through 1) the conventional solution-chemistry-based preparation of the corresponding metal complex monomers having a 9-fluorenylmethyloxycarbonyl (Fmoc)-protected amino acid moiety and 2) their sequential coupling together with other water-soluble organic building units on the surface-functionalized polymeric resin by following the procedures originally developed for the solid-phase synthesis of polypeptides, with proper modifications. Traces of reactions determined by mass spectrometric analysis at the representative coupling steps in stage 2 confirm the selective construction of a predetermined sequence of metal centers along with the peptide backbone. The WSMOCA cleaved from the resin at the end of stage 2 has a certain level of solubility in aqueous media dependent on the pH value and/or salt content, which is useful for the purification of the compound.

A potential general method for the synthesis of water-soluble multimetallic peptidic arrays containing a predetermined sequence of metal centers is presented.

We demonstrate a method for the synthesis of a water-soluble multimetallic peptidic array containing a predetermined sequence of metal centers such as ...

Constructing Thioether/Vinyl Sulfide-tethered Helical Peptides Via Photo-induced Thiol-ene/yne Hydrothiolation

Here, we describe a detailed protocol for the preparation of thioether-tethered peptides using on-resin intramolecular/intermolecular thiol-ene hydrothiolation. In addition, this protocol describes the preparation of vinyl-sulfide-tethered peptides using in-solution intramolecular thiol-yne hydrothiolation between amino acids that possess alkene/alkyne side chains and cysteine residues at i, i+4 positions. Linear peptides were synthesized using a standard Fmoc-based solid-phase peptide synthesis (SPPS). Thiol-ene hydrothiolation is carried out using either an intramolecular thio-ene reaction or an intermolecular thio-ene reaction, depending on the peptide length. In this research, an intramolecular thio-ene reaction is carried out in the case of shorter peptides using on-resin deprotection of the trityl groups of cysteine residues following the complete synthesis of the linear peptide. The resin is then set to UV irradiation using photoinitiator 4-methoxyacetophenone (MAP) and 2-hydroxy-1-[4-(2-hydroxyethoxy)-phenyl]-2-methyl-1-propanone (MMP). The intermolecular thiol-ene reaction is carried out by dissolving Fmoc-Cys-OH in an N,N-dimethylformamide (DMF) solvent. This is then reacted with the peptide using the alkene-bearing residue on resin. After that, the macrolactamization is carried out using benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBop), 1-hydroxybenzotriazole (HoBt), and 4-Methylmorpholine (NMM) as activation reagents on the resin. Following the macrolactamization, the peptide synthesis is continued using standard SPPS. In the case of the thio-yne hydrothiolation, the linear peptide is cleaved from the resin, dried, and subsequently dissolved in degassed DMF. This is then irradiated using UV light with photoinitiator 2,2-dimethoxy-2-phenylacetophenone (DMPA). Following the reaction, DMF is evaporated and the crude residue is precipitated and purified using high-performance liquid chromatography (HPLC). These methods could function to simplify the generation of thioether-tethered cyclic peptides due to the use of the thio-ene/yne click chemistry that possesses superior functional group tolerance and good yield. The introduction of thioether bonds into peptides takes advantage of the nucleophilic nature of cysteine residues and is redox-inert relative to disulfide bonds.

We present a protocol for the construction of thioether/vinyl sulfide-tethered helical peptides using photo-induced thiol-ene/thiol-yne hydrothiolation.

Here, we describe a detailed protocol for the preparation of thioether-tethered peptides using on-resin intramolecular/intermolecular thiol-ene ...

Solid-phase Synthesis of [4.4] Spirocyclic Oximes

A convenient synthetic route for spirocyclic heterocycles is well sought after due to the molecule&#39;s potential use in biological systems. By means of solid-phase synthesis, regenerating Michael (REM) linker strategies, and 1,3-dipolar cycloaddition, a library of structurally similar heterocycles, both with and without a spirocyclic center, can be constructed. The main advantages of the solid-support synthesis are as follows: first, each reaction step can be driven to completion using a large excess of reagents resulting in high yields; next, the use of commercially available starting materials and reagents keep the costs low; finally, the reaction steps are easy to purify via simple filtration. The REM linker strategy is attractive because of its recyclability and traceless nature. Once a reaction scheme is completed, the linker can be reused multiple times. In a typical solid-phase synthesis, the product contains either a part of or the whole linker, which can prove undesirable. The REM linker is &#34;traceless&#34; and the point of attachment between the product and the polymer is indistinguishable. The high diastereoselectivity of the intramolecular 1,3-dipolar cycloaddition is well documented. Limited by the insolubility of the solid support, the reaction progression can only be monitored by a change in the functional groups (if any) via infrared (IR) spectroscopy. Thus, the structural identification of intermediates cannot be characterized by conventional nuclear magnetic resonance (NMR) spectroscopy. Other limitations to this method stem from the compatibilities of the polymer/linker to the desired chemical reaction scheme. Herein we report a protocol that allows for the convenient production of spirocyclic heterocycles that, with simple modifications, can be automated with high-throughput techniques.

Here we present a protocol to demonstrate an efficient method for the synthesis of spirocyclic heterocycles. The five-step process utilizes solid-phase synthesis and regenerating Michael linker strategies. Generally difficult to synthesize, we present a customizable method for the synthesis of spirocyclic molecules otherwise inaccessible to other modern approaches.

A convenient synthetic route for spirocyclic heterocycles is well sought after due to the molecule&#39;s potential use in biological systems. By means of ...

Synthesis and Bioconjugation of Thiol-Reactive Reagents for the Creation of Site-Selectively Modified Immunoconjugates

Maleimide-bearing bifunctional probes have been employed for decades for the site-selective modification of thiols in biomolecules, especially antibodies. Yet maleimide-based conjugates display limited stability in vivo because the succinimidyl thioether linkage can undergo a retro-Michael reaction. This, of course, can lead to the release of the radioactive payload or its exchange with thiol-bearing biomolecules in circulation. Both of these processes can produce elevated activity concentrations in healthy organs as well as decreased activity concentrations in target tissues, resulting in reduced imaging contrast and lower therapeutic ratios. In 2018, we reported the creation of a modular, stable, and easily accessible phenyloxadiazolyl methyl sulfone reagent &#8212; dubbed &#8216;PODS&#8217; &#8212; as a platform for thiol-based bioconjugations. We have clearly demonstrated that PODS-based site-selective bioconjugations reproducibly and robustly create homogenous, well-defined, highly immunoreactive, and highly stable radioimmunoconjugates. Furthermore, preclinical experiments in murine models of colorectal cancer have shown that these site-selectively labeled radioimmunoconjugates exhibit far superior in vivo performance compared to radiolabeled antibodies synthesized via maleimide-based conjugations. In this protocol, we will describe the four-step synthesis of PODS, the creation of a bifunctional PODS-bearing variant of the ubiquitous chelator DOTA (PODS-DOTA), and the conjugation of PODS-DOTA to the HER2-targeting antibody trastuzumab.

In this protocol, we will describe the synthesis of PODS, a phenyoxadiazolyl methyl sulfone-based reagent for the site-selective attachment of cargos to the thiols of biomolecules, particularly antibodies. In addition, we will describe the synthesis and characterization of a PODS-bearing bifunctional chelator and its conjugation to a model antibody.

Maleimide-bearing bifunctional probes have been employed for decades for the site-selective modification of thiols in biomolecules, especially antibodies. ...

Preparation of Site-Specific Cytotoxic Protein Conjugates via Maleimide-thiol Chemistry and Sortase A-Mediated Ligation

Cancer is currently the second most common cause of death worldwide. The hallmark of cancer cells is the presence of specific marker proteins such as growth factor receptors on their surface. This feature enables development of highly selective therapeutics, the protein bioconjugates, composed of targeting proteins (antibodies or receptor ligands) connected to highly cytotoxic drugs by a specific linker. Due to very high affinity and selectivity of targeting proteins the bioconjugates recognize marker proteins on the cancer cells surface and utilize receptor-mediated endocytosis to reach the cell interior. Intracellular vesicular transport system ultimately delivers the bioconjugates to the lysosomes, where proteolysis separates free cytotoxic drugs from the proteinaceous core of the bioconjugates, triggering drug-dependent cancer cell death. Currently, there are several protein bioconjugates approved for cancer treatment and large number is under development or clinical trials.
One of the main challenges in the generation of the bioconjugates is a site-specific attachment of the cytotoxic drug to the targeting protein. Recent years have brought a tremendous progress in the development of chemical and enzymatic strategies for protein modification with cytotoxic drugs. Here we present the detailed protocols for the site-specific incorporation of cytotoxic warheads into targeting proteins using a chemical method employing maleimide-thiol chemistry and an enzymatic approach that relies on sortase A-mediated ligation. We use engineered variant of fibroblast growth factor 2 and fragment crystallizable region of human immunoglobulin G as an exemplary targeting proteins and monomethyl auristatin E and methotrexate as model cytotoxic drugs. All the described strategies allow for highly efficient generation of biologically active cytotoxic conjugates of defined molecular architecture with potential for selective treatment of diverse cancers.

Here we provide detailed protocols for a site-specific labeling of proteins with cytotoxic drugs using maleimide-thiol reaction and sortase A-mediated ligation.

Cancer is currently the second most common cause of death worldwide. The hallmark of cancer cells is the presence of specific marker proteins such as ...

Biochemistry

Construction of Cyclic Cell-Penetrating Peptides for Enhanced Penetration of Biological Barriers

Cancer has been a grand challenge in global health. However, the complex tumor microenvironment generally limits the access of therapeutics to deeper tumor cells, leading to tumor recurrence. To conquer the limited penetration of biological barriers, cell-penetrating peptides (CPPs) have been discovered with excellent membrane translocation ability and have emerged as useful molecular transporters for delivering various cargoes into cells. However, conventional linear CPPs generally show compromised proteolytic stability, which limits their permeability across biological barriers. Thus, the development of novel molecular transporters that can penetrate biological barriers and exhibit enhanced proteolytic stability is highly desired to promote drug delivery efficiency in biomedical applications. We have previously synthesized a panel of short cyclic CPPs with aromatic crosslinks, which exhibited superior permeability in cancer cells and tissues compared to their linear counterparts. Here, a concise protocol is described for the synthesis of the fluorescently labeled cyclic polyarginine R8 peptide and its linear counterpart, as well as key steps for investigating their cell permeability.

This protocol describes the synthesis of cyclic cell-penetrating peptides with aromatic cross-links and the evaluation of their permeability across biological barriers.

Cancer has been a grand challenge in global health. However, the complex tumor microenvironment generally limits the access of therapeutics to deeper ...

Constructing Cyclic Peptides Using an On-Tether Sulfonium Center

In recent years, cyclic peptides have attracted increasing attention in the field of drug discovery due to their excellent biological activities, and, as a consequence, they are now used clinically. It is, therefore, critical to seek effective strategies for synthesizing cyclic peptides to promote their application in the field of drug discovery. This paper reports a detailed protocol for the efficient synthesis of cyclic peptides using on-resin or intramolecular (intermolecular) bisalkylation. Using this protocol, linear peptides were synthesized by taking advantage of solid-phase peptide synthesis with cysteine (Cys) and methionine (Met) coupled simultaneously on the resin. Further, cyclic peptides were synthesized via bisalkylation between Met and Cys using a tunable tether and an on-tether sulfonium center. The whole synthetic route can be divided into three major processes: the deprotection of Cys on the resin, the coupling of the linker, and the cyclization between Cys and Met in a trifluoroacetic acid (TFA) cleavage solution. Furthermore, inspired by the reactivity of the sulfonium center, a propargyl group was attached to the Met to trigger thiol-yne addition and form a cyclic peptide. After that, the crude peptides were dried and dissolved in acetonitrile, separated, and then purified by high-performance liquid chromatography (HPLC). The molecular weight of the cyclic peptide was confirmed by liquid chromatography-mass spectrometry (LC-MS), and the stability of the cyclic peptide combination with the reductant was further confirmed using HPLC. In addition, the chemical shift in the cyclic peptide was analyzed by 1H nuclear magnetic resonance (1H NMR) spectra. Overall, this protocol aimed to establish an effective strategy for synthesizing cyclic peptides.

This protocol presents the synthesis of cyclic peptides via bisalkylation between cysteine and methionine and the facile thiol-yne reaction triggered by the propargyl sulfonium center.

In recent years, cyclic peptides have attracted increasing attention in the field of drug discovery due to their excellent biological activities, and, as ...

Solid Phase Synthesis of a Functionalized Bis-Peptide Using "Safety Catch" Methodology

In 1962, R.B. Merrifield published the first procedure using solid-phase peptide synthesis as a novel route to efficiently synthesize peptides. This technique quickly proved advantageous over its solution-phase predecessor in both time and labor. Improvements concerning the nature of solid support, the protecting groups employed and the coupling methods employed over the last five decades have only increased the usefulness of Merrifield's original system. Today, use of a Boc-based protection and base/nucleophile cleavable resin strategy or Fmoc-based protection and acidic cleavable resin strategy, pioneered by R.C. Sheppard, are most commonly used for the synthesis of peptides1.
Inspired by Merrifield's solid supported strategy, we have developed a Boc/tert-butyl solid-phase synthesis strategy for the assembly of functionalized bis-peptides2, which is described herein. The use of solid-phase synthesis compared to solution-phase methodology is not only advantageous in both time and labor as described by Merrifield1, but also allows greater ease in the synthesis of bis-peptide libraries. The synthesis that we demonstrate here incorporates a final cleavage stage that uses a two-step "safety catch" mechanism to release the functionalized bis-peptide from the resin by diketopiperazine formation.
Bis-peptides are rigid, spiro-ladder oligomers of bis-amino acids that are able to position functionality in a predictable and designable way, controlled by the type and stereochemistry of the monomeric units and the connectivity between each monomer. Each bis-amino acid is a stereochemically pure, cyclic scaffold that contains two amino acids (a carboxylic acid with an &alpha;-amine)3,4. Our laboratory is currently investigating the potential of functional bis-peptides across a wide variety of fields including catalysis, protein-protein interactions and nanomaterials.

The efficient solid-phase peptide synthesis of a functionalized bis-peptide trimer utilizing a "safety catch" cleavage procedure from HMBA resin is described.

In 1962, R.B. Merrifield published the first procedure using solid-phase peptide synthesis as a novel route to efficiently synthesize peptides. This ...

Biology

Solid Phase Synthesis

Source: Vy M. Dong and Diane Le, Department of Chemistry, University of California, Irvine, CA
Merrifield&#39;s solid-phase synthesis is a Nobel Prize winning invention where a reactant molecule is bound on a solid support and undergoes successive chemical reactions to form a desired compound. When the molecules are bound to a solid support, excess reagents and byproducts can be removed by washing away the impurities, while the target compound remains bound to the resin. Specifically, we will showcase an example of solid-phase peptide synthesis (SPPS) to demonstrate this concept.

Solid Phase Synthesis: Principles, Peptide Synthesis

Source: Vy M. Dong and Diane Le, Department of Chemistry, University of California, Irvine, CA
Merrifield&#39;s solid-phase synthesis is a Nobel Prize ...

Synthetic Methodology for Asymmetric Ferrocene Derived Bio-conjugate Systems via Solid Phase Resin-based Methodology

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Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

Synthesis of a Water-soluble Metal–Organic Complex Array

Constructing Thioether/Vinyl Sulfide-tethered Helical Peptides Via Photo-induced Thiol-ene/yne Hydrothiolation

Solid-phase Synthesis of [4.4] Spirocyclic Oximes

Synthesis and Bioconjugation of Thiol-Reactive Reagents for the Creation of Site-Selectively Modified Immunoconjugates

Preparation of Site-Specific Cytotoxic Protein Conjugates via Maleimide-thiol Chemistry and Sortase A-Mediated Ligation

Construction of Cyclic Cell-Penetrating Peptides for Enhanced Penetration of Biological Barriers

Constructing Cyclic Peptides Using an On-Tether Sulfonium Center

Solid Phase Synthesis of a Functionalized Bis-Peptide Using "Safety Catch" Methodology

Solid Phase Synthesis