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Matthew B Francis

Publications and source records attributed to Matthew B Francis.

15 recordsLinked to original sources
PubMed2022

Tyrosinase-Mediated Synthesis of Nanobody-Cell Conjugates.

A convenient enzymatic strategy is reported for the modification of cell surfaces. Using a tyrosinase enzyme isolated from Agaricus bisporus, unique tyrosine residues introduced at the C-termini of nanobodies can be site-selectively oxidized to reactive o-quinones. These reactive intermediates undergo rapid modification with nucleophilic thiol, amine, and imidazole residues present on cell surfaces, producing novel nanobody-cell conjugates that display targeted antigen binding. We extend this approach toward the synthesis of nanobody-NK cell conjugates for targeted immunotherapy applications. The resulting NK cell conjugates exhibit targeted cell binding and elicit targeted cell death.

Journal Article↗
PubMed2007

Solubilization and stabilization of bacteriophage MS2 in organic solvents.

Several techniques were examined for the solubilization of bacteriophage MS2 in organic solvents. Direct extraction of the MS2 from an aqueous phase into isooctane containing 2 mM AOT, a proven approach for the organic solubilization of many proteins, was not successful. However, predried samples of MS2 were solubilized through the direct addition of organic solvents containing 500 mM AOT. As an alternative procedure, reverse micelles containing aqueous solutions of MS2 were prepared in isooctane using AOT, dehydrated through solvent evaporation and azeotropic drying, and resolubilized in a solvent of choice. The structure and microenvironment of organic-solubilized MS2 were investigated by UV absorbance, the fluorescence emission of an attached solvatochromatic dye, tryptophan fluorescence, and atomic force microscopy, all of which contributed evidence for a fully assembled capsid in the organic solvent. The solubilized MS2 was derivatized with stearic acid in chloroform, illustrating that bioconjugation reactions can be performed on organic-solubilized capsids using reagents that are completely insoluble in water. Furthermore, the organic-solubilized phage remained infectious after heating at 90 degrees C for 20 min, whereas phage in aqueous buffer or dried with nitrogen were nonviable following the heat treatment protocol. The extended range of available chemical modifications and the enhanced thermal stability of the organic-solubilized capsids bodes well for the formulation of storage-stable vaccines predicated on reactions in or exposure to organic media.

Biotechnology↗
PubMed2006

Modification of aniline containing proteins using an oxidative coupling strategy.

A new bioconjugation reaction has been developed based on the chemoselective modification of anilines through an oxidative coupling pathway. Aryl amines were installed on the surface of protein substrates through lysine acylation reactions or through the use of native chemical ligation techniques. Upon exposure to NaIO4 in aqueous buffer, the anilines coupled rapidly to the aromatic rings of N,N-dialkyl-N'-acyl-p-phenylenediamines. The identities of the reaction products were confirmed using ESI-MS and through comparison to small molecule analogs. Control experiments indicated that none of the native amino acids participated in the reaction. The resulting bioconjugates were found to be stable toward hydrolysis from pH 4 to pH 11 and in the presence of many commonly used oxidants, reductants, and nucleophiles. A fluorescent phenylenediamine reagent was synthesized for the selective detection of aniline labeled proteins in mixtures, and the reaction was used to append the C-terminus of the green fluorescent protein with a single PEG chain. When combined with techniques for the incorporation of unnatural amino acids into proteins, this bioorthogonal coupling method should prove useful for a number of applications requiring a high degree of labeling specificity.

Aniline Compounds↗
PubMed2006

Transition metal catalyzed methods for site-selective protein modification.

The broad utility of protein bioconjugates has created a need for new and diverse strategies for site-selective protein modification. In particular, chemical reactions that target alternative amino acid side chains or unnatural functional groups are emerging as a valuable complement to more commonly used lysine- and cysteine-based strategies. Considering their widespread use in organic synthesis, reactions catalyzed by transition metals could provide a particularly powerful set of transformations for the continued expansion of the bioconjugation toolkit. Recent efforts to apply transition metal catalysis to protein modification have resulted in new methods for protein cross-linking, tryptophan modification, tyrosine modification, reductive amination of protein amines, and unnatural amino acid labeling. These strategies have substantially expanded the synthetic flexibility of protein modification, and thus the range of applications for which bioconjugates can be used in chemical biology and materials science.

Amino Acids↗
PubMed2006

Tyrosine-selective protein alkylation using pi-allylpalladium complexes.

A new protein modification reaction has been developed based on a palladium-catalyzed allylic alkylation of tyrosine residues. This technique employs electrophilic pi-allyl intermediates derived from allylic acetate and carbamate precursors and can be used to modify proteins in aqueous solution at room temperature. To facilitate the detection of modified proteins using SDS-PAGE analysis, a fluorescent allyl acetate was synthesized and coupled to chymotrypsinogen A and bacteriophage MS2. The tyrosine selectivity of the reaction was confirmed through trypsin digest analysis. The utility of the reaction was demonstrated by using taurine-derived carbamates as water solubilizing groups that are cleaved upon protein functionalization. This solubility switching technique was used to install hydrophobic farnesyl and C(17) chains on chymotrypsinogen A in water using little or no cosolvent. Following this, the C(17) alkylated proteins were found to associate with lipid vesicles. In addition to providing a new protein modification strategy targeting an under-utilized amino acid side chain, this method provides convenient access to synthetic lipoproteins.

Alkylation↗
PubMed2005

Reductive alkylation of proteins using iridium catalyzed transfer hydrogenation.

An efficient transition metal catalyzed procedure for the reductive alkylation of proteins has been developed. Imines formed from the condensation of aldehydes (1 mM) with lysine residues and the N-terminus can be reduced efficiently by a [Cp*Ir(4,4'-dimethoxy-2,2'-bipyridine)(H2O)]SO4 catalyst in the presence of formate ions. The reaction proceeds readily at pH 7.4 in aqueous phosphate buffer at temperatures ranging from 22 to 37 degrees C, and reaches high levels of conversion for a number of aromatic aldehydes. UV experiments have confirmed that the catalyst does not bind to protein substrates. The utility of the reaction has been demonstrated through an efficient two-step procedure for the attachment of unfunctionalized poly(ethylene glycol) to protein targets.

Alkylation↗
PubMed2005

Dual-surface modification of the tobacco mosaic virus.

The protein shell of the tobacco mosaic virus (TMV) provides a robust and practical tubelike scaffold for the preparation of nanoscale materials. To expand the range of applications for which the capsid can be used, two synthetic strategies have been developed for the attachment of new functionality to either the exterior or the interior surface of the virus. The first of these is accomplished using a highly efficient diazonium coupling/oxime formation sequence, which installs >2000 copies of a material component on the capsid exterior. Alternatively, the inner cavity of the tube can be modified by attaching amines to glutamic acid side chains through a carbodiimide coupling reaction. Both of these reactions have been demonstrated for a series of substrates, including biotin, chromophores, and crown ethers. Through the attachment of PEG polymers to the capsid exterior, organic-soluble TMV rods have been prepared. Finally, the orthogonality of these reactions has been demonstrated by installing different functional groups on the exterior and interior surfaces of the same capsid assemblies.

Capsid↗
PubMed2004

A three-component Mannich-type reaction for selective tyrosine bioconjugation.

A new selective bioconjugation reaction is described for the modification of tyrosine residues on protein substrates. The reaction uses imines formed in situ from aldehydes and electron-rich anilines to modify phenolic side chains through a Mannich-type electrophilic aromatic substitution pathway. The reaction takes place under mild pH and temperature conditions and can modify protein substrates at concentrations as low as 20 muM. Using an efficient fluorescence-based assay, we demonstrated the reaction using a number of aldehydes and protein targets. Importantly, proteins lacking surface-accessible tyrosines remained unmodified. It was also demonstrated that enzymatic activity is preserved under the mild reaction conditions. This strategy represents one of the first carbon-carbon bond-forming reactions for protein modification and provides an important complement to more commonly used lysine- and cysteine-based methods.

Aldehydes↗
PubMed2004

Selective tryptophan modification with rhodium carbenoids in aqueous solution.

A new transition metal-based reaction has been developed for the selective modification of tryptophan residues on protein substrates. After activation of vinyl-substituted diazo compounds by Rh2(OAc)4, the resulting metallocarbenoid intermediates were found to modify indoles in aqueous media despite competing reactions with water. Both N- and 2-substituted indole products were observed in the reaction. Following initial small-molecule studies, the reaction was performed on two protein substrates. Both myoglobin and subtilisin Carlsberg were modified readily in aqueous solution, and the tryptophan selectivity of the reactions was confirmed through MS analyses of trypsin digest fragments. It was also demonstrated that myoblobin concentrations as low as 10 muM still led to appreciable levels of modification. Reconstitution experiments confirmed that myoglobin retained its ability to bind heme following modification.

Amino Acid Sequence↗
PubMed2004

Interior surface modification of bacteriophage MS2.

An efficient strategy for the interior surface functionalization of MS2 viral capsids is reported, featuring a new hetero-Diels-Alder bioconjugation reaction. After virus isolation, the RNA genome was removed from the spherical particles by exposure to pH 11.8 conditions for a period of 4 h. Following this, 180 tyrosine residues on the interior surface of each "empty" capsid shell were modified by using a site-selective diazonium-coupling reaction. To attach additional functionality, the azo conjugate was reduced with Na2S2O4 to afford an ortho-amino tyrosine derivative. Oxidation of this moiety with NaIO4 produced an o-iminoquinone on the protein surface, which was found to undergo an efficient hetero-Diels-Alder reaction with N-(4-aminophenyl)acrylamide. This four-step procedure can be carried out in under 4 h, reaches high levels of conversion, and yields the desired conjugates in >60% overall yield.

Bioreactors↗
PubMed2003

Practical synthetic route to functionalized rhodamine dyes.

[reaction: see text] An efficient method for the synthesis of functionalized rhodamine derivatives has been developed. Multigram quantities of these water-soluble fluorophores can be prepared from inexpensive precursors and purified without the use of chromatography. A series of protein-reactive functional groups has been installed through subsequent reactions, providing materials for biomolecule modification. For multicolor applications, a solid-phase purification strategy has been developed to afford rhodamine derivatives possessing a wide range of spectral properties.

Journal Article↗
PubMed2002

Self-assembled oligonucleotide-polyester dendrimers.

A new approach to the controlled synthesis of multicomponent dendrimers is presented, in which three oligonucleotide-dendron conjugates were synthesized using solid phase techniques and hybridized to create a second generation polyester dendrimer with DNA as a core and bearing two types of peripheral functional groups.

Chromatography, High Pressure Liquid↗
PubMedDate not supplied

An affinity-based method for the purification of fluorescently-labeled biomolecules.

Due to the difficulty of separating mixtures of labeled and unlabeled biomolecules, a general new method for the affinity purification of modified proteins has been developed. A Sepharose-based solid support bearing beta-cyclodextrin groups was used to capture chromophore-modified proteins selectively, while unmodified proteins remained in solution. After isolation of the resin, the modified proteins were released by treating the sample with a competitive cyclodextrin binder, such as adamantane carboxylic acid. This procedure was demonstrated for several dyes displaying a wide range of spectral characteristics and diverse chemical structures. Preliminary studies have shown that this method can also be used to enrich modified peptide fragments present in proteolytic digests. This technique is anticipated to accelerate the development of new protein modification reactions and could provide a useful tool for proteomics applications.

Affinity Labels↗