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

Characterization of a Three-Component Coupling Reaction on Proteins by Isotopic Labeling and Nuclear Magnetic Resonance Spectroscopy

A three-component Mannich-type electrophilic aromatic substitution reaction was previously developed to target the phenolic side chain of tyrosine residues on proteins. This reaction proceeds under mild conditions and provides a convenient alternative to lysine-targeting strategies. However, the use of reactive aldehydes, such as formaldehyde, warrants careful inspection of the reaction products to ensure that other modifications have not occurred. Through the use of isotopically enriched reagents, nuclear magnetic resonance (NMR)-based studies were used to obtain structural confirmation of the tyrosine-modification products. These experiments also revealed the formation of a reaction byproduct arising from the indole ring of tryptophan residues. Cysteine residues were shown to not participate in the reaction, except in the case of a reduced disulfide, which formed a dithioacetal. We anticipate that this analysis method will prove useful for the detailed study of a number of bioconjugation reactions

Chemoselective Tryptophan Labeling with Rhodium Carbenoids at Mild pH

Significant improvements have been made to a previously reported tryptophan modification method using rhodium carbenoids in aqueous solution, allowing the reaction to proceed at pH 6−7. This technique is based on the discovery that N-(tert-butyl)hydroxylamine promotes indole modification with rhodium carbenoids over a broad pH range (2−7). This methodology was demonstrated on peptide and protein substrates, generally yielding 40−60% conversion with excellent tryptophan chemoselectivity. The solvent accessibility of the indole side chains was found to be a key factor in successful carbenoid addition, as demonstrated by conducting the reaction at temperatures high enough to cause thermal denaturation of the protein substrate. Progress toward the expression of proteins bearing solvent accessible tryptophan residues as reactive handles for modification with rhodium carbenoids is also reported

A Modular Approach for Assembling Aldehyde-Tagged Proteins on DNA Scaffolds

Expansion of antibody scaffold diversity has the potential to expand the neutralizing capacity of the immune system and to generate enhanced therapeutics and probes. Systematic exploration of scaffold diversity could be facilitated with a modular and chemical scaffold for assembling proteins, such as DNA. However, such efforts require simple, modular, and site-specific methods for coupling antibody fragments or bioactive proteins to nucleic acids. To address this need, we report a modular approach for conjugating synthetic oligonucleotides to proteins with aldehyde tags at either terminus or internal loops. The resulting conjugates are assembled onto DNA-based scaffolds with low nanometer spatial resolution and can bind to live cells. Thus, this modular and site-specific conjugation strategy provides a new tool for exploring the potential of expanded scaffold diversity in immunoglobulin-based probes and therapeutics

Impact of Assembly State on the Defect Tolerance of TMV-Based Light Harvesting Arrays

Self-assembling, light harvesting arrays of organic chromophores can be templated using the tobacco mosaic virus coat protein (TMVP). The efficiency of energy transfer within systems containing a high ratio of donors to acceptors shows a strong dependence on the TMVP assembly state. Rod and disk assemblies derived from a single stock of chromophore-labeled protein exhibit drastically different levels of energy transfer, with rods significantly outperforming disks. The origin of the superior transfer efficiency was probed through the controlled introduction of photoinactive conjugates into the assemblies. The efficiency of the rods showed a linear dependence on the proportion of deactivated chromophores, suggesting the availability of redundant energy transfer pathways that can circumvent defect sites. Similar disk-based systems were markedly less efficient at all defect levels. To examine these differences further, the brightness of donor-only systems was measured as a function of defect incorporation. In rod assemblies, the photophysical properties of the donor chromophores showed a significant dependence on the number of defects. These differences can be partly attributed to vertical energy transfer events in rods that occur more rapidly than the horizontal transfers in disks. Using these geometries and the previously measured energy transfer rates, computational models were developed to understand this behavior in more detail and to guide the optimization of future systems. These simulations have revealed that significant differences in excited state dissipation rates likely also contribute to the greater efficiency of the rods and that statistical variations in the assembly process play a more minor role

Supplementary Figure Legends from CAT-02-106, a Site-Specifically Conjugated Anti-CD22 Antibody Bearing an MDR1-Resistant Maytansine Payload Yields Excellent Efficacy and Safety in Preclinical Models

Supplementary Figure Legends</p

Site-Specific Tandem Knoevenagel Condensation–Michael Addition To Generate Antibody–Drug Conjugates

Expanded ligation techniques are sorely needed to generate unique linkages for the growing field of functionally enhanced proteins. To address this need, we present a unique chemical ligation that involves the double addition of a pyrazolone moiety with an aldehyde-labeled protein. This ligation occurs via a tandem Knoevenagel condensation–Michael addition. A pyrazolone reacts with an aldehyde to generate an enone, which undergoes subsequent attack by a second pyrazolone to generate a bis-pyrazolone species. This rapid and facile ligation technique is performed under mild conditions in the absence of catalyst to generate new architectures that were previously inaccessible via conventional ligation reactions. Using this unique ligation, we generated three site-specifically labeled antibody–drug conjugates (ADCs) with an average of four drugs to one antibody. The in vitro and in vivo efficacies along with pharmacokinetic data of the site-specific ADCs are reported

Figures S1-S11, Table S1 from CAT-02-106, a Site-Specifically Conjugated Anti-CD22 Antibody Bearing an MDR1-Resistant Maytansine Payload Yields Excellent Efficacy and Safety in Preclinical Models

Fig S1. ELISA formats for detection of various analytes. Fig S2. Biophysical characterization of CAT-02-106. Fig S3. Competitive ELISA data showing CAT-02-106 binding to CD22. Fig S4. CAT-02-106 internalization on Ramos, Granta-519, and WSU-DLCL2 cells. Fig S5. CAT-02-106 in vitro cytotoxicity test on CD22-negative cells. Fig S6. The CAT-02-106-related ADC, anti-HER2 RED-106, does not induce bystander killing. Fig S7. Comparison of cell surface CD22 expression levels on Ramos and WSU-DLCL2 cells. Fig S8. Ramos and WSU-DLCL2 xenograft study data plotted as individual animals. Fig S9. Mouse body weights from Ramos and WSU-DLCL2 xenograft studies. Fig S10. CAT-02-106 binding to cynomolgus monkey B cells as assessed by flow cytometry. Fig S11. CAT-02-106 cross-reactivity to human and cynomolgus tissues. Table S1. Summary of pharmacokinetic findings in rats dosed at 3 mg/kg with CAT-02-106.</p

Supplementary Methods and Synthesis of RED-106 from CAT-02-106, a Site-Specifically Conjugated Anti-CD22 Antibody Bearing an MDR1-Resistant Maytansine Payload Yields Excellent Efficacy and Safety in Preclinical Models

Supplementary Methods and Synthesis of RED-106</p