Practical Synthetic Route to Functionalized Rhodamine Dyes

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

A Designed <i>A. vinelandii</i>–<i>S. elongatus</i> Coculture for Chemical Photoproduction from Air, Water, Phosphate, and Trace Metals

Microbial mutualisms play critical roles in a diverse number of ecosystems and have the potential to improve the efficiency of bioproduction for desirable chemicals. We investigate the growth of a photosynthetic cyanobacterium, <i>Synechococcus elongatus</i> PCC 7942, and a diazotroph, <i>Azotobacter vinelandii</i>, in coculture. From initial studies of the coculture grown in media with glutamate, we proposed a model of cross-feeding between these organisms. We then engineer a new microbial mutualism between <i>Azotobacter vinelandii</i> AV3 and <i>cscB Synechococcus elongatus</i> that grows in the absence of fixed carbon or nitrogen. The coculture cannot grow in the absence of a sucrose-exporting <i>S. elongatus,</i> and neither organism can grow alone without fixed carbon or nitrogen. This new system has the potential to produce industrially relevant products, such as polyhydroxybutyrate (PHB) and alginate, from air, water, phosphate, trace metals, and sunlight. We demonstrate the ability of the coculture to produce PHB in this work

Tyrosine-Selective Protein Alkylation Using π-Allylpalladium Complexes

A new protein modification reaction has been developed based on a palladium-catalyzed allylic alkylation of tyrosine residues. This technique employs electrophilic π-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 C17 chains on chymotrypsinogen A in water using little or no cosolvent. Following this, the C17 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

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 μM still led to appreciable levels of modification. Reconstitution experiments confirmed that myoglobin retained its ability to bind heme following modification

Attachment of Peptide Building Blocks to Proteins Through Tyrosine Bioconjugation

Recent efforts have yielded a number of short peptide sequences with useful binding, sensing, and cellular uptake properties. In order to attach these sequences to tyrosine residues on intact proteins, a three-component Mannich-type strategy is reported. Two solid phase synthetic routes were developed to access peptides up to 20 residues in length with anilines at either the N- or C-termini. In the presence of 20 mM formaldehyde, these functional groups were coupled to tyrosine residues on proteins under mild reaction conditions. The identities of the resulting bioconjugates were confirmed using mass spectrometry and immunoblot analysis. Screening experiments have demonstrated that the method is compatible with substrates containing all of the amino acids, including lysine and cysteine residues. Importantly, tyrosine residues on proteins exhibit much faster reaction rates, allowing short peptides containing this residue to be coupled without cross reactions

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 °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

Regioselective Labeling of Antibodies through N-Terminal Transamination

A convenient new method is described for the introduction of ketone groups at the N-termini of antibodies. The reaction occurs in the presence of pyridoxal-5′-phosphate under conditions mild enough to maintain antigen binding function, as confirmed by enzyme-linked immunosorbent assay. Further derivatization of these functional sites was accomplished through oxime formation, yielding well-defined antibody conjugates for a wide range of applications. The ability of the modified antibodies to bind their targets was confirmed via immunodot blot analysis. The generality of this method has been demonstrated on a number of monoclonal and polyclonal antibodies, all with different binding specificities

Recyclable Thermoresponsive Polymer–Cellulase Bioconjugates for Biomass Depolymerization

Here we report the construction and characterization of a recoverable, thermoresponsive polymer–endoglucanase bioconjugate that matches the activity of unmodified enzymes on insoluble cellulose substrates. Two copolymers exhibiting a thermoresponsive lower critical solution temperature (LCST) were created through the copolymerization of an aminooxy-bearing methacrylamide with <i>N</i>-isopropylacrylamide (NIPAm) or <i>N</i>-isopropylmethacrylamide (NIPMa). The aminooxy group provided a handle through which the LCST was adjusted through small-molecule quenching. This allowed materials with LCSTs ranging from 20.9 to 60.5 °C to be readily obtained after polymerization. The thermostable endoglucanase EGPh from the hypothermophilic <i>Pyrococcus horikoshii</i> was transaminated with pyridoxal-5′-phosphate to produce a ketone-bearing protein, which was then site-selectively modified through oxime linkage with benzylalkoxyamine or 5 kDa-poly­(ethylene glycol)-alkoxyamine. These modified proteins showed activity comparable to the controls when assayed on an insoluble cellulosic substrate. Two polymer bioconjugates were then constructed using transaminated EGPh and the aminooxy-bearing copolymers. After 12 h, both bioconjugates produced an equivalent amount of free reducing sugars as the unmodified control using insoluble cellulose as a substrate. The recycling ability of the NIPAm copolymer–EGPh conjugate was determined through three rounds of activity, maintaining over 60% activity after two cycles of reuse and affording significantly more soluble carbohydrates than unmodified enzyme alone. When assayed on acid-pretreated Miscanthus, this bioconjugate increased the amount of reducing sugars by 2.8-fold over three rounds of activity. The synthetic strategy of this bioconjugate allows the LCST of the material to be changed readily from a common stock of copolymer and the method of attachment is applicable to a variety of proteins, enabling the same approach to be amenable to thermophile-derived cellulases or to the separation of multiple species using polymers with different recovery temperatures

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 μM. 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