Article pubs.acs.org/bc
General Dialdehyde Click Chemistry for Amine Bioconjugation Sina Elahipanah,† Paul J. O’Brien,† Dmitry Rogozhnikov,† and Muhammad N. Yousaf*,†,‡ †
Department of Chemistry and Biology, Laboratory for Biomolecular Interactions, York University, Toronto, Ontario, Canada M3J 1P3 ‡ OrganoLinX Inc., Toronto, Ontario, Canada M3J 1P3 S Supporting Information *
ABSTRACT: The development of methods for conjugating a range of molecules to primary amine functional groups has revolutionized the fields of chemistry, biology, and material science. The primary amine is a key functional group and one of the most important nucleophiles and bases used in all of synthetic chemistry. Therefore, tremendous interest in the synthesis of molecules containing primary amines and strategies to devise chemical reactions to react with primary amines has been at the core of chemical research. In particular, primary amines are a ubiquitous functional group found in biological systems as free amino acids, as key side chain lysines in proteins, and in signaling molecules and metabolites and are also present in many natural product classes. Due to its abundance, the primary amine is the most convenient functional group handle in molecules for ligation to other molecules for a broad range of applications that impact all scientific fields. Because of the primary amine’s central importance in synthetic chemistry, acid−base chemistry, redox chemistry, and biology, many methods have been developed to efficiently react with primary amines, including activated carboxylic acids, isothiocyanates, Michael addition type systems, and reaction with ketones or aldehydes followed by in situ reductive amination. Herein, we introduce a new traceless, high-yield, fast click-chemistry method based on the rapid and efficient trapping of amine groups via a functionalized dialdehyde group. The click reaction occurs in mild conditions in organic solvents or aqueous media and proceeds in high yield, and the starting dialdehyde reagent and resulting dialdehyde click conjugates are stable. Moreover, no catalyst or dialdehyde-activating group is required, and the only byproduct is water. The initial dialdehyde and the resulting conjugate are both straightforward to characterize, and the reaction proceeds with high atom economy. To demonstrate the broad scope of this new click-conjugation strategy, we designed a straightforward scheme to synthesize a suite of dialdehyde reagents. The dialdehyde molecules were used for applications in cell-surface engineering and for tailoring surfaces for material science applications. We anticipate the broad utility of the general dialdehyde click chemistry to primary amines in all areas of chemical research, ranging from polymers and bioconjugation to material science and nanoscience.
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amines are of intense interest that impacts many fields of research. In particular, there is tremendous growing interest in the field of bioconjugation and, therefore, the development of reagents that react with primary amines in biological systems.18−21 From recent history, each time a new strategy to react with amines has been developed, it has led to new breakthrough research and new innovations that impact many research fields including polymer chemistry, biology, and material and nanoscience. To conjugate amines to a broad range of substrates, several ligation strategies have been developed that span activated acids, isothiocyanates, and the reductive amination of imine groups.22−28 At present, the most popular ligation strategy relies on the activation of carboxylic acids to react with primary amines to form a stable amide bond. Although there are several conjugation reactions available for various functional groups, with bio-orthogonal chemistry gaining importance, the overwhelming strategy for bioconjugation is methods of reacting to primary amines.29−36 In fact, the largest commercial industry
INTRODUCTION The ability to generate a diverse suite of molecules that react with primary amine groups has revolutionized chemical, material, and biological research.1−3 Primary-amine-containing molecules are abundant, a key functional group in biological systems, and found in many important raw materials in nature and in starting materials for diverse chemical processes and syntheses.2−5 Primary amines are the most important base and nucleophile used in acid base chemistry and as a molecular handle for ligation chemistry.6−8 In biological systems, primary amines are found in every amino acid. Free amine groups are found in proteins via the lysine amino acid and are critical for a variety of diverse functions including hydrogen bonding, as a base for buffering aqueous environments, and as a nucleophile for a broad range of enzymatic processes.9−12 The primary amine is ubiquitous in chemistry and used to synthesize polymers and covalent and metal organic frameworks (COFs and MOFs), as metal-chelating groups, and as a molecular handle to immobilize a broad range of molecules to all types of materials and nanoparticles.13−17 Due to the fact that the primary amine is a key functional group in all fields of chemistry, methods to generate molecules that contain primary amine groups and strategies to react molecules to primary © XXXX American Chemical Society
Received: February 24, 2017 Revised: April 6, 2017
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DOI: 10.1021/acs.bioconjchem.7b00106 Bioconjugate Chem. XXXX, XXX, XXX−XXX
Article
Bioconjugate Chemistry for conjugation is dedicated to providing a broad suite of ligands and molecules with activated acids. The majority of these carboxylic acids are activated by the classic N-hydroxy succinimide−dicyclocarbodiimide (NHS−DCC) system and variations. Although molecules with activated acids are used routinely for bioconjugation and tagging molecules, there remain several limitations with the activated acid strategy. First, the reagents are commercially expensive. Second, the activated acids are unstable in moisture or aqueous conditions and rapidly hydrolyze. Third, the reaction proceeds with 1 equiv of leaving group that requires purification. Fourth, the leaving group is a significant byproduct and may be toxic depending on the bioconjugation application. Fifth, there is very poor atom economy as the formation of the activated acid requires activating reagents (e.g., DCC and NHS) as well as the loss of the leaving group after reaction (e.g., NHS group). Herein, we develop and employ a new primary-amineconjugation strategy based on dialdehyde click chemistry. We show that the dialdehyde group is stable, does not need activation, and reacts rapidly with primary amines at mild conditions in organic solvents or aqueous solution to generate a dihydropyridine conjugate. Furthermore, no catalyst or activating group is required, and the only byproduct generated is water. The click reaction proceeds with high yield, no purification, and efficient atom economy. We characterize the dialdehyde click chemistry reaction with physical organic chemistry principles, including kinetics and NMR. To demonstrate its broad utility, we developed a straightforward synthetic route to generate a range of molecules containing the dialdehyde conjugate group. In one demonstration, we synthesized a dialdehyde lipid molecule that is used to generate dialdehydepresenting liposomes that are conjugated with a range of amineterminated ligands for delivery and installation onto cell surfaces via liposome fusion. In a second demonstration, we synthesized biotin and dinitro phenol (DNP) dialdehyde reagents that are conjugated to liposomes presenting primary amines. These liposomes presenting biotin and DNP ligands through the dialdehyde click-chemistry reaction were then successfully delivered and installed onto mammalian and bacteria cell surfaces via liposome fusion. As a third example, we generated a dialdehyde-presenting glass surface to immobilize primary-amine-containing ligands for material science applications. We report the broad utility of the dialdehyde click chemistry as a complementary and alternative method for amine conjugation.
Figure 1. Schematic of a general click-reaction strategy to ligate a primary amine group (2) with a dialdehyde group (1). At room temperature and aqueous conditions, a primary-amine-containing molecule reacts with a dialdehyde substrate to generate a dihydro pyridine conjugate (3). The dialdehyde contains a quaternary carbon consisting of several groups (R1 and R2). The dialdehyde group is stable in aqueous conditions and does not require an activating group or catalyst to react with a primary amine. The dialdehyde click reaction has efficient atom economy, requires no purification step, is fastreacting and stable, and proceeds with high yield, and water is the only byproduct.
To develop a new stable dialdehyde click-conjugation strategy for primary amines that was to have broad scope, react at mild conditions, have fast kinetics with high yield and high atom economy, and be synthetically flexible, we considered several key design criteria. First, it should modify the glutaraldehyde backbone to form a stable reagent to inhibit aldol-type self-reaction. Second, the two linked dialdehydes would be at the correct distance to intramolecularly trap the primary amine into a six-member ring without the need for activation or a catalyst. We envisioned a molecule that would initially react with an amine via a single aldehyde to form a classic imine but then have a rapid intramolecular reaction with the second nearby aldehyde to snare the amine into a stable ring. Third, the proposed dialdehyde reagents must have straightforward synthesis and be amenable to presenting a broad range of molecules. To limit or inhibit the ability for 1,5-dialdehydes to self-react via aldol-type chemistry, we reasoned that by generating a quaternary carbon at the 3 carbon position, self-reaction would be sterically unfavorable and the dialdehyde would be stable in aqueous media via an intramolecular hydrate (hemiacetal cyclic pyran). This steric bulk prevents aldol-type reactions at physiological conditions and also provides a linkage for tethering a range of molecules to the dialdehyde (see the Supporting Information). To demonstrate the broad utility of the dialdehyde group as a primary-amine-conjugation strategy, we synthesized a suite of dialdehyde containing molecules. Scheme 1 shows the general synthetic route to produce a lipid-like dialdehyde molecule (9) that has broad utility in biomembrane biophysical and cell biology studies. The synthetic design requires only five steps, and each proceeds with high yield. The route is straightforward and can be scaled to generate vast amounts of the dialdehyde (9). To understand the conjugation reaction, we studied the model reaction of a quaternary carbon−dialdehyde (9) with propyl amine in aqueous conditions at room temperature. Figure 2 shows a proton NMR of the starting dialdehyde (9) and the final conjugate product (10). The dialdehyde (9) is stable at physiological conditions and can be stored in solution or neat at room temperature or at −20 °C for months. NMR analysis shows that in aqueous conditions the dialdehyde (9) is in equilibrium and exists as a cyclic hydrate pyran diol (Figure S1). However, once the dialdehyde (9) is exposed to a primary-amine-containing molecule, the amine initially reacts with one aldehyde in a typical imine reaction, but because of
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RESULTS AND DISCUSSION Figure 1 shows the general schematic for the conjugation of a disubstituted 1,5-pentanedial (1) with a primary-amine-containing molecule (2) to form a disubstituted 1,4-dihydropyridine (3) conjugate. The inspiration for developing the dialdehyde conjugation reaction comes from the well-known ability of glutaraldehyde (a 1,5 pentanedial) to act as a cross-linker with biomolecules in biological systems.37−44 Unlike single ketones or aldehydes, which react with primary amines to form unstable imines in aqueous conditions, the glutaraldehyde is a dialdehyde and has unique cross-linking reactivity due to the interaction between the two aldehydes. In aqueous environments and at high concentrations, glutaraldehyde self-reacts via aldol type reactions to generate a variety of undefined complex polymeric species that contain sites for reaction with amineand thiol-containing molecules.42,44−47 B
DOI: 10.1021/acs.bioconjchem.7b00106 Bioconjugate Chem. XXXX, XXX, XXX−XXX
Article
Bioconjugate Chemistry Scheme 1. Synthetic Route to Generating a Lipid-like Dialdehyde Reagenta
(i) cis-1,4-Dichloro-2-butene (1.0 equiv), Na (2.2 equiv), anhydrous t-butanol, 12 h, 95%. (ii) LiAlH4 (3.0 equiv), −78 °C, dry THF, 2 h, CH3COOH/H2O 1 M, 3 h, 95%. (iii) NaH (excess), dry THF, Ar, 1-bromododecane (2.2 equiv) 24 h, 80%. (IV) OsO4, N-methylmorpholine N-oxide (excess), CH3CN/acetone/H2O, 12 h, 90%. (V) NaIO4, Acetone/H2O, 2 h, 75%. a
Figure 2. NMR spectra comparison of the dialdehyde (9) and conjugate (10). The dialdehyde reacts rapidly with propyl amine to generate the conjugate. The NMR spectra show the key protons found in the dialdehyde and conjugate, respectively, that are monitored to measure reaction kinetics. Key proton peaks (9.6 Ha aldehyde proton), 4.7−5.3 proton peaks for the dialdehyde cyclic pyran hydrate (Supporting Information), and proton peaks Hc at 6.0 and Hb at 4.3 vinylic protons and Hd α protons to nitrogen.
Figure 3. Rate of reaction of the dialdehyde (9) with a primary amine (propyl amine) to form a dihydro pyridine conjugate (10). The reaction is fast (25 °C, pH 7.0), with a t1/2 life of less than 5 s (kobs = 0.14 s−1 ± 0.02) followed by a slow-dehydration step (see Figure S2).
reagent (dialdehyde) and releases no byproduct other than water. This reaction is traceless, rapid, requires no purification, does not require a catalyst, and proceeds with high atom economy. To demonstrate the utility of the lipid−dialdehyde conjugation strategy, we used liposome nanoparticle technology and cell-surface engineering. Liposomes are ubiquitous in biological research, and the synthetic generation, modification, and delivery of these nanoparticles has been of intense interest in fundamental biophysical research as drug and probe delivery systems and as cell membrane model systems.50−54 We have used the liposome-fusion strategy in combination with bioorthogonal chemistry to deliver bio-orthogonal-like lipid molecules to cell surfaces for a range of studies and applications.53−59 We used this approach to engineer the cell surface to display molecules that gave the cell new capabilities including fluorescent, redox, and photoswitchable properties.57,58,60 We also used this system to deliver complementary bio-orthogonal groups to multiple different cell types to generate scaffold-free coculture spheroids and functional 3D liver and cardiac tissue.57,61,62 Figure 4 shows how the lipid dialdehyde (9) may be used to conjugate a range of ligands containing primary amines to cell surfaces. Because of the flexibility of the bioconjugation dialdehyde methodology two different pathways may be used to tailor cell surfaces with a variety of ligands. Pathway A shows the formation of a liposome containing background lipids and
the close proximity, it then reacts with the second nearby aldehyde to trap the amine and generate a six-member diol piperidine-type molecule that then dehydrates to form a 1,4-dihydropyridine conjugate (10) (Figure S1). This trapping is an intramolecular process and is fast and stable. The NMR spectrum shows the formation of the 1,4-dihydropyridine (10) over time by following the appearance of the vinylic protons and the alpha protons to the primary amine (Figures S2 and S3). Figure 3 shows that the conjugation reaction is fast with a t1/2 of less than 5 s (kobs = 0.14 s−1 ± 0.02) followed by a slow dehydration step to generate the 1,4-dihydropyridine product. Normally, without the second aldehyde group a standard single imine would form and then hydrolyze back to the starting molecules (amine and aldehyde) due to the equilibrium being far to the left and greatly favoring the starting reactants.48,49 The dihydropyridine conjugate (10) formed is stable in organic solvents and at physiological conditions. The conjugate (10) can be stored at physiological conditions or neat at −20 °C for months. However, the conjugate (10) is unstable at low pH (
