Hydrazino-Pictet-Spengler Ligation as a Biocompatible Method for the Generation of Stable Protein Conjugates Paresh Agarwal, Romas Kudirka, Aaron E. Albers, Robyn M. Barfield, Gregory W. de Hart, Penelope M. Drake, Lesley C. Jones, and David Rabuka* Redwood Bioscience, 5703 Hollis Street, Emeryville, California 94608, United States * S Supporting Information ABSTRACT: Aldehyde- and ketone-functionalized biomole- cules have found widespread use in biochemical and biotechnological fields. They are typically conjugated with hydrazide or aminooxy nucleophiles under acidic conditions to yield hydrazone or oxime products that are relatively stable, but susceptible to hydrolysis over time. We introduce a new reaction, the hydrazino-Pictet-Spengler (HIPS) ligation, which has two distinct advantages over hydrazone and oxime ligations. First, the HIPS ligation proceeds quickly near neutral pH, allowing for one-step labeling of aldehyde-functionalized proteins under mild conditions. Second, the HIPS ligation product is very stable (>5 days) in human plasma relative to an oxime-linked conjugate (∼1 day), as demonstrated by monitoring protein-fluorophore conjugates by ELISA. Thus, the HIPS ligation exhibits a combination of product stability and speed near neutral pH that is unparalleled by current carbonyl bioconjugation chemistries. T he introduction of aldehydes and ketones into proteins has emerged as a powerful strategy for conjugation methods. The reactivity of these moieties as electrophilic functional groups renders them bioorthogonal, allowing for selective reactivity with nucleophiles in the presence of native amino acids and a variety of post-translational modifications (PTMs). As a result, the number of tools available to incorporate aldehydes and ketones into proteins, particularly using site-selective approaches, has grown dramatically in the past decade. There are now many ways to incorporate these functional groups into proteins using chemical, 1-6 enzy- matic, 7-10 and chemoenzymatic 11-14 methods. The introduc- tion of aldehydes and ketones into proteins has facilitated glycoproteomic studies, 15-18 protein imaging in live cells, 11,19 single-molecule imaging studies, 20 and protein purification, 21 as well as preparation of chemically modified therapeutic and heterobifunctional proteins, 22-24 functional protein-based materials, 25,26 and protein nucleic acid conjugates and glycoconjugates. 27,28 In most of these applications, a protein aldehyde or ketone is treated with a molecule of interest bearing an aminooxy nucleophile to generate an oxime-linked conjugate that is used in a downstream application. One major drawback to oxime conjugation chemistry is the requirement for acidic conditions to enable the reaction to proceed at an appreciable rate. While many biomolecules can tolerate extended incubation under typical oxime conjugation conditions (pH 4.5), some types of proteins and PTMs are susceptible to conformational changes or degradation under acidic conditions. For example, histidine phosphorylation, a PTM increasingly appreciated for its biological relevance, is unstable to mildly acidic conditions; 29 large protein assemblies such as viral capsids are prone to acid-induced disruption of quaternary structure; 30 proteins mediating viral infection such as influenza hemagglutinin undergo irreversible acid-triggered conformational changes; 31 and a variety of proteins implicated in amyloid diseases undergo acid-induced aggregation. 32,33 To prevent loss of function, the labeling of these types of proteins through oxime-based chemistry would need to be carried out close to neutral pH, where oxime formation is very slow. The use of aniline as a nucleophilic catalyst for oxime conjugations has obviated this problem to a degree, 34 but its efficiency varies according to the system. For some conjugations aniline catalysis works well, while for others it has a neutral or detrimental impact on reaction kinetics and conjugation efficiencies. 17,22 Furthermore, for sensitive biological applications it may prove problematic to use millimolar quantities of aniline as a catalyst due to concerns about its toxicity. 35 As an alternative to aminooxy nucleophiles, hydrazines are attractive functional groups for bioconjugation reactions with aldehydes and ketones because of their nucleophilicity near neutral pH. 36 However, reactions of aliphatic hydrazines to form hydrazones suffer from low equilibrium constants in water, requiring a large excess of hydrazine reagent to achieve good conversions in conjugation reactions. 37 Furthermore, alkylhydrazones are readily hydrolyzed under aqueous con- ditions, severely limiting the utility of these conjugates due to Received: January 27, 2013 Revised: May 19, 2013 Communication pubs.acs.org/bc © XXXX American Chemical Society A dx.doi.org/10.1021/bc400042a | Bioconjugate Chem. XXXX, XXX, XXX-XXX