Oxime and Hydrazone Bioconjugation Reactions

Introduction

Bioconjugation is the process of joining two molecules, at least one of which is a biomolecule (e.g., carbohydrate, cofactor, nucleic acid, peptide, or protein), through a covalent bond to form a complex, new molecule.^[1],[2],[3] The reactions that form oxime and hydrazone bonds are among the oldest, most intensively studied bioconjugation reactions. Bioconjugation reactions must be compatible with the biomolecules upon which they act. Hydrazone and oxime bond-forming reactions are biocompatible. They are also versatile, leading to the formation of widely diverse products, with usually rapid reaction rates. Indeed, these reactions have proven useful in bioconjugate chemistry, polymer chemistry, chemical biology, combinatorial chemistry, and organic chemistry. In this article, we will explore the reaction mechanisms that form oxime and hydrazone bonds and then discuss how discrete PEG products improve these reactions.

 

The General Reaction Scheme for Oximes and Hydrazones

Oximes and the reactions leading to their formation were investigated beginning in 1882. Hydrazones and their reactions were studied as early as 1888.^[4] Figure 1, below, shows the general reaction scheme for the formation of hydrazone and oxime bonds.

 

 

In oxime and hydrazone bond-forming reactions, an aldehyde or ketone reacts readily with a nucleophilic alkoxyamine (also known as aminooxy or oxyamine) (Figure 1.a) or hydrazine (Figure 1.b.), to yield an oxime (Figure 1.c.) or hydrazone (Figure 1.d.), respectively. These reactions are chemoselective. Water forms as a byproduct of the reaction. In general, aldehydes react more rapidly than ketones. In biomolecular reactions, aryl aldehydes may be preferred over aliphatic aldehydes because of their unique reactivity (discussed below).

 

Stability

Hydrazones and oximes are much more hydrolytically stable than imines (Figure 2), which also possess a carbon-nitrogen double bond. The hydrolytic stability of hydrazones and oximes is due partly to the α-effect of the heteroatom (N or O) adjacent to the sp² nitrogen and partly due to inductive effects that reduce the basicity of the sp² nitrogen.^[5],[6]

 

Catalysis

At pH < 7, these reactions proceed under general acid catalysis and the reaction rate increases as pH decreases. However, if pH < 3, the reaction rate slows down. Also, at pH ≥ 7, the reaction rate for hydrazone and oxime bond formation slows dramatically. A catalyst is needed to drive the reaction quickly to completion at neutral or higher pH. Furthermore, in biomolecular conjugations and biomolecular labeling, the concentrations and quantities of biomolecules are low enough that the biomolecules are limiting reagents in the reaction. This necessitates the use of an efficient catalyst for hydrazone and oxime bond formation in order to achieve high yields.^[2],[4] Catalysts will be discussed in detail below.

Hydrazone Bond Formation

The reaction of hydrazine- or hydrazide-containing compounds with an aldehyde or ketone forms hydrazone bonds. Such bonds are a type of Schiff base. They are less acid-labile than a standard Schiff base. However, they are more labile than oxime bonds (discussed below). Moreover, the degree of lability to acid depends on whether the hydrazone bond was formed from an aldehyde or a ketone.^[7] Furthermore, hydrazone bonds formed from aromatic aldehydes have been shown to be more stable than hydrazone bonds formed from aliphatic aldehydes.^[8] Figure 3 illustrates the formation of hydrazone bonds.

 

Applications of Hydrazones

Because they are stable under neutral to alkaline conditions (pH 7 – 9) and labile under slightly acidic conditions (pH 4 – 5), a hydrazone bond can be used to create a cleavable linkage. Kale and Torchilin proved the usefulness of cleavable hydrazone bonds in developing drug delivery agents.^[8] In addition, immobilized affinity ligands can be made cleavable by the use of hydrazone bonds.^[9],[10] Also, hydrazide-containing compounds react chemoselectively with aldehydes and ketones – though they are not perfectly bioorthogonal.^[11] Aldehydes and ketones can be introduced into biomolecules in several ways, and they appear naturally under specific conditions.^[4],[11]

Oxime Bond Formation

 

Aldehydes and ketones react with oxyamine groups (R-ONH₂) to form an oxime bond. Specifically, the type of oxime bond formed depends upon the type of carbonyl reactant used. If the oxyamine reacts with an aldehyde, the product is called an aldoxime. If the oxyamine reacts with a ketone, the product is called a ketoxime.^[1],[7] As with hydrazone bond formation, aldehydes react more rapidly than ketones.

 

Oxime bonds are more stable than hydrazone bonds.^[2],[4-8] As explained above, both α-effect and inductive effects contribute to the stability of the Schiff base that is formed in hydrazones and oximes. However, mechanistic studies by Kalia and Raines showed that the greater electronegativity of oxygen compared to nitrogen explains the greater stability of the sp² nitrogen in an oxime bond relative to the sp² nitrogen in a hydrazone bond.^[5] Indeed, the rate constant for oxime hydrolysis is almost 1,000-fold lower than the rate constant for hydrazone hydrolysis.² Consequently, the C=N bond in an oxime does not need to be reduced with sodium cyanoborohydride in order to maintain a stable conjugate. In contrast, it may be advantageous to reduce the C=N bond in a hydrazone so that the conjugate remains stable.^[7]

Applications of Oximes

Because they are more stable than hydrazone bonds, oxime bonds are preferred in conjugates where stability is required. Stability notwithstanding, oxime bonds can be reversed in vivo through the action of oxime hydrolase.

Several prodrugs incorporate oxime bonds in their structure. When oxime hydrolase reverses the bond, the prodrugs become active. Prodrugs that contain strongly basic amidoxime or guanidoxime groups have displayed enhanced membrane permeability and absorption.^[12] Oximes also are highly useful in creating biomolecular constructs for cell targeting, lectin targeting, imaging applications, and synthetic vaccines, and in ¹⁸F labeling of peptides and proteins for positron emission tomography (PET).^[13],[14]

Catalysis of Hydrazone and Oxime Bond Formation

Aniline has long been known to catalyze the formation of hydrazone and oxime bonds.^{[15],[16],[17]} Aniline's toxicity^[18],[19] makes it unsuitable for use with living cells. Moreover, at physiological pH, the amount of aniline needed to catalyze hydrazone and oxime bond formation ranges from 10 – 100 mM in reactions where the biomolecules being reacted may have a far lower concentration. Acetate also catalyzes hydrazone and oxime bond formation, and it is non-toxic. However, at physiological pH, acetate is inferior to aniline as a catalyst.^[20]

 

 

Numerous catalysts (see Figure 5) for the formation of hydrazone and oxime bonds have been tested over time. In 2013, Crisalli and Kool published two papers showing that anthranilic acid, certain anthranilic acid derivatives, and 3,5-diaminobenzoic acid, provided superior catalysis of hydrazone and oxime bond formation at physiological pH compared to aniline. The concentrations of these catalysts were remarkably lower than the concentration of aniline needed to affect catalysis.^[21],[22]

Finding Better Catalysts

Furthermore, in 2014, Michaela Wendeler and colleagues showed that p-substituted anilines with electron-donating ring substituents were superior catalysts to aniline at acidic and neutral pH values. One catalyst, p-phenylenediamine, proved superior to all other catalysts studied. Additionally, p-phenylenediamine effectively catalyzed hydrazone and oxime bond formation at lower concentrations of catalyst (2 mM) than aniline (10 mM).^[23] See also, A Better Catalyst for Oxime-Based Bioconjugations. Kölmel and Kool published a detailed review of the mechanism and catalysis in 2017.^[4]

Another paper from the Kool lab in 2018 showed that the use of commercially available amine buffers catalyzed the formation of hydrazone and oxime bonds at 50 mM concentration.^[24] The goal of the study was to find biocompatible (i.e., low toxicity) amine buffers that catalyze hydrazone and oxime bond formation effectively. This permits the use of hydrazine, hydrazide, and oxyamine reagents in living cells for crosslinking and labeling purposes. Of twenty buffers that were tested, four buffers were found to catalyze bond formation effectively; however, one of the four buffers (p-phenylenediamine) was found to be toxic to living cells. Figure 6 shows the three low toxicity amine buffers that best catalyzed the bond formation.

 

Forming Oxime and Hydrazone Bonds Requires Insertion of Aldehyde, Hydrazide, Hydrazine, or Oxyamine Functional Groups into Biomolecules

Aldehydes and ketones appear only infrequently in biomolecules. To create hydrazone and oxime bonds in biomolecules, the appropriate reaction partners must be introduced. Hermanson has published several protocols for introducing these groups into biomolecules.^[25] For example, aldehyde residues can be introduced into carbohydrates through periodate oxidation. Hydrazide residues can be introduced through derivatization.

Small molecular weight PEG linkers and spacers can enhance bioconjugation chemistry that forms oxime or hydrazone bonds. Quanta BioDesign offers several discrete PEG (dPEG^®) reagents for introducing aryl aldehyde and oxyamine groups. We also offer hydrazide and oxyamine products for labeling or for introducing additional functionality to a biomolecule. If you do not know what a dPEG^® product is, or if you have questions about the advantages and utility of dPEG® reagents, please visit the two pages that are linked immediately below.

What is dPEG^®?

Frequently Asked Questions (about dPEG^® products)

Aryl Aldehyde Products

 

 

For example, 4-formyl-benzamido-dPEG^®₂₄-TFP ester, product number 10082 (Figure 7), is a crosslinker that reacts with free amines through the tetrafluorophenyl ester, leaving an aryl aldehyde available to react with an oxyamine, hydrazine, or hydrazide moiety. The aryl aldehyde is less reactive than an aliphatic aldehyde. Consequently, the aryl aldehyde is less likely to react with free amines to form a Schiff base. Moreover, a Schiff base between an aryl aldehyde and a free amine is easily reversed. This means that the aldehyde is free to react with a hydrazide or oxyamine group.^[26]

 

Protected Oxyamine Products for Oxime Bond Formation

 

 

As another example, phthalimidooxy-dPEG^®₁₂-NHS ester, product number 11135 (Figure 8), contains an amine-reactive N-hydroxysuccinimidyl (NHS) ester and a protected oxyamine group separated by a 41-atom (52.2 – 53.2 Å) dPEG^® spacer. The phthaloyl moiety stably protects the oxyamine group. The NHS ester end of the molecule must be reacted first, because it will not survive the removal of the phthaloyl protecting group. Following amide bond formation, the phthalimidooxy group can be removed using 50% aqueous hydrazine^[27],[28] or 50% aqueous hydroxylamine^{[29],[30],[31]}, leaving the oxyamine group free to react.

A Caution Regarding Hydrazine

Please note that hydrazine is a toxic substance.^[32] Stringent precautions must be observed to avoid exposure via skin or the respiratory tract. Where possible, use aqueous hydroxylamine instead of hydrazine, as hydroxylamine is considerably less toxic than hydrazine.^[33] However, do not use hydroxylamine with proteins containing heme, as hydroxylamine will irreversibly coordinate with the central iron atom, deactivating the heme protein. Moreover, hydroxylamine should be completely removed from serine proteases, as it has been shown to accelerate enzymatic activity of these enzymes.^[34]

A list of the products offered by Quanta BioDesign for hydrazone and oxime bond formation is given below.

dPEG^® Products for Creating Oxime or Hydrazone Bonds

Aldehyde Partners

PN10075, 4-formyl-benzamido-dPEG^®₁₂-EDA-MAL

 

PN10081, 4-formyl-benzamido-dPEG^®₁₂-TFP ester

PN10082, 4-formyl-benzamido-dPEG^®₂₄-TFP ester

 

Biotin Labeling of Aldehydes

PN10219, Biotin-dPEG^®₄-hydrazide

 

PN11100, Biotin-dPEG^®₃-oxyamine•HCl

PN11102, Biotin-dPEG^®₁₁-oxyamine•HCl

For more information about biotinylation with dPEG^® products, please click here.

 

Products for Hydrazone Bond Formation

PN10210, MAL-dPEG^®₄-t-boc hydrazide

PN10961, MAL-dPEG^®₈-t-boc hydrazide

PN10962, MAL-dPEG^®₁₂-t-boc hydrazide

 

PN10041, Amino-dPEG^®₄-t-boc hydrazide

PN10957, Amino-dPEG^®₈-t-boc hydrazide

PN10958, Amino-dPEG^®₁₂-t-boc hydrazide

 

Products for Oxime Bond Formation

PN10011, Phthalimidooxy-dPEG^®₄-NHS ester

PN11135, Phthalimidooxy-dPEG^®₁₂-NHS ester

 

PN11112, Amino-dPEG^®₁₁-ONH-t-boc

REFERENCES

 

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[2] Kalia, J.; Raines, R. T. Advances in Bioconjugation. Curr. Org. Chem. 2010, 14(2), 138–147. http://www.eurekaselect.com/70683/article (accessed Apr 9, 2019).

[3] Hermanson, G. T. Chapter 1, Introduction to Bioconjugation. Bioconjugate Techniques, 3rd ed. New York: Academic Press, 2013, 1-126. The definition of "bioconjugation" is on page 1. Greg's book is considered the best reference on the subject of bioconjugation. Quanta BioDesign makes this book available to our customers, and we endorse it without reservation. Click here to learn more about this essential reference for bioconjugation and to purchase a copy.

[4] Kölmel, D. K.; Kool, E. T. Oximes and Hydrazones in Bioconjugation: Mechanism and Catalysis. Chem. Rev. 2017, 117(15), 10358–10376. https://doi.org/10.1021/acs.chemrev.7b00090.

[5] Kalia, J.; Raines, R. T. Hydrolytic Stability of Hydrazones and Oximes. Angewandte Chemie International Edition 2008, 47(39), 7523–7526. https://doi.org/10.1002/anie.200802651.

[6] Ulrich, S.; Boturyn, D.; Marra, A.; Renaudet, O.; Dumy, P. Oxime Ligation: A Chemoselective Click-Type Reaction for Accessing Multifunctional Biomolecular Constructs. Chemistry – A European Journal 2014, 20(1), 34–41. https://doi.org/10.1002/chem.201302426.

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[8] Kale, A. A.; Torchilin, V. P. Design, Synthesis, and Characterization of PH-Sensitive PEG−PE Conjugates for Stimuli-Sensitive Pharmaceutical Nanocarriers: The Effect of Substitutes at the Hydrazone Linkage on the PH Stability of PEG−PE Conjugates. Bioconjugate Chem. 2007, 18(2), 363–370. https://doi.org/10.1021/bc060228x.

[9] Hermanson, G. T. Chapter 14, Microparticles and Nanoparticles. Bioconjugate Chemistry, 3^rd ed. New York: Academic Press, 2013, 549-588.

[10] Hermanson, G. T. Chapter 15, Immobilization of Ligands on Chromatography Supports. Bioconjugate Chemistry, 3^rd ed. New York: Academic Press, 2013, 589-740.

[11] Hermanson, G. T. Chapter 17, Chemoselective Ligation Bioorthogonal Reagents. Bioconjugate Chemistry, 3^rd ed. New York: Academic Press, 2013, 757-786, particularly pages 760-766.

[12] a. Rautio, J.; Kumpulainen, H.; Heimbach, T.; Oliyai, R.; Oh, D.; Järvinen, T.; Savolainen, J. Prodrugs: Design and Clinical Applications. Nature Reviews Drug Discovery 2008, 7(3), 255–270. https://doi.org/10.1038/nrd2468. b. Barot, M.; Bagui, M.; R. Gokulgandhi, M.; K. Mitra, A. Prodrug Strategies in Ocular Drug Delivery. Medicinal Chemistry 2012, 8(4), 753-768. https://doi.org/info:doi/10.2174/157340612801216283. c. R. Kokil, G.; V. Rewatkar, P. Bioprecursor Prodrugs: Molecular Modification of the Active Principle. Mini Reviews in Medicinal Chemistry 2010, 10(14), 1316-1330. https://doi.org/info:doi/10.2174/138955710793564179.

[13] Ulrich, S.; Boturyn, D.; Marra, A.; Renaudet, O.; Dumy, P. Oxime Ligation: A Chemoselective Click-Type Reaction for Accessing Multifunctional Biomolecular Constructs. Chemistry – A European Journal 2014, 20(1), 34–41. https://doi.org/10.1002/chem.201302426.

[14] Li, X.-G.; Haaparanta, M.; Solin, O. Oxime Formation for Fluorine-18 Labeling of Peptides and Proteins for Positron Emission Tomography (PET) Imaging: A Review. Journal of Fluorine Chemistry 2012, 143, 49–56. https://doi.org/10.1016/j.jfluchem.2012.07.005.

[15] Dirksen, A.; Hackeng, T. M.; Dawson, P. E. Nucleophilic Catalysis of Oxime Ligation. Angewandte Chemie International Edition 2006, 45(45), 7581–7584. https://doi.org/10.1002/anie.200602877.

[16] Dirksen, A.; Dawson, P. E. Rapid Oxime and Hydrazone Ligations with Aromatic Aldehydes for Biomolecular Labeling. Bioconjugate Chem. 2008, 19(12), 2543–2548. https://doi.org/10.1021/bc800310p.

[17] Thygesen, M. B.; Munch, H.; Sauer, J.; Cló, E.; Jørgensen, M. R.; Hindsgaul, O.; Jensen, K. J. Nucleophilic Catalysis of Carbohydrate Oxime Formation by Anilines. J. Org. Chem. 2010, 75(5), 1752–1755. https://doi.org/10.1021/jo902425v.

[18] National Research Council (US) Subcommittee on Acute Exposure Guideline Levels. Aniline Acute Exposure Guideline Levels; National Academies Press (US), 2000. https://www.ncbi.nlm.nih.gov/books/NBK222410/ (accessed April 11, 2019).

[19] ATSDR - ToxFAQs^TM: Aniline https://www.atsdr.cdc.gov/toxfaqs/tf.asp?id=449&tid=79 (accessed Apr 11, 2019).

[20] Wang, S.; Gurav, D.; Oommen, O. P.; Varghese, O. P. Insights into the Mechanism and Catalysis of Oxime Coupling Chemistry at Physiological PH. Chemistry – A European Journal 2015, 21(15), 5980–5985. https://doi.org/10.1002/chem.201406458.

[21] Crisalli, P.; Kool, E. T. Water-Soluble Organocatalysts for Hydrazone and Oxime Formation. J. Org. Chem. 2013, 78(3), 1184–1189. https://doi.org/10.1021/jo302746p.

[22] Crisalli, P.; Kool, E. T. Importance of Ortho Proton Donors in Catalysis of Hydrazone Formation. Org. Lett. 2013, 15(7), 1646–1649. https://doi.org/10.1021/ol400427x.

[23] Wendeler, M.; Grinberg, L.; Wang, X.; Dawson, P. E.; Baca, M. Enhanced Catalysis of Oxime-Based Bioconjugations by Substituted Anilines. Bioconjugate Chem. 2014, 25(1), 93–101. https://doi.org/10.1021/bc400380f.

[24] Larsen, D.; Kietrys, A. M.; Clark, S. A.; Park, H. S.; Ekebergh, A.; Kool, E. T. Exceptionally Rapid Oxime and Hydrazone Formation Promoted by Catalytic Amine Buffers with Low Toxicity. Chem. Sci. 2018, 9(23), 5252–5259. https://doi.org/10.1039/C8SC01082J.

[25] Hermanson, G. T. Chapter 2, Functional Targets for Bioconjugation. Bioconjugate Techniques, 3rd ed. New York: Academic Press, 2013, 127-228. See pages 203-215 for the discussions and protocols specific to introducing aldehyde, hydrazine, hydrazide, and oxyamine functional groups into biomolecules.

[26] Hermanson, G. T. Chapter 18, PEGylation and Synthetic Polymer Modification. Bioconjugate Techniques, 3rd ed. New York: Academic Press, 2013, 787-838. See specifically pages 802-803.

[27] Maryanoff, B. E.; Greco, M. N.; Zhang, H.-C.; Andrade-Gordon, P.; Kauffman, J. A.; Nicolaou, K. C.; Liu, A.; Brungs, P. H. Macrocyclic Peptide Inhibitors of Serine Proteases. Convergent Total Synthesis of Cyclotheonamides A and B via a Late-Stage Primary Amine Intermediate. Study of Thrombin Inhibition under Diverse Conditions. J. Am. Chem. Soc. 1995, 117(4), 1225–1239. https://doi.org/10.1021/ja00109a006.

[28] Khan, M. N. Kinetic Evidence for the Occurrence of a Stepwise Mechanism in the Hydrazinolysis of Phthalimide. J. Org. Chem. 1995, 60(14), 4536–4541. https://doi.org/10.1021/jo00119a035.

[29] Mootoo, D. R.; Fraser-Reid, B. N-Pentenyl 2-Amino-2-Deoxy Glycosides Undergo Stereoselective Coupling under Mild, Chemospecific Conditions. Tetrahedron Letters 1989, 30(18), 2363–2366. https://doi.org/10.1016/S0040-4039(01)80399-3.

[30] Knight, D. W.; Leese, M. P. A Survey of Suitable Protecting Groups for the Synthesis of Hydroxylamines by Mitsunobu Reactions. Tetrahedron Letters 2001, 42(13), 2593–2595. https://doi.org/10.1016/S0040-4039(01)00234-9.

[31] Toyokuni, T.; Walsh, J. C.; Dominguez, A.; Phelps, M. E.; Barrio, J. R.; Gambhir, S. S.; Satyamurthy, N. Synthesis of a New Heterobifunctional Linker, N-[4-(Aminooxy)Butyl]Maleimide, for Facile Access to a Thiol-Reactive ¹⁸F-Labeling Agent. Bioconjugate Chem. 2003, 14(6), 1253–1259. https://doi.org/10.1021/bc034107y.

[32] a. ATSDR - Toxic Substances - Hydrazines https://www.atsdr.cdc.gov/substances/toxsubstance.asp?toxid=89 (accessed Apr 26, 2019). b. CDC - Hydrazine - NIOSH Workplace Safety and Health Topic https://www.cdc.gov/niosh/topics/hydrazine/default.html (accessed Apr 26, 2019). c. CDC - NIOSH 1988 OSHA PEL Project Documentation: List by Chemical Name: HYDRAZINE https://www.cdc.gov/niosh/pel88/302-01.html (accessed Apr. 26, 2019). d. Hydrazine. Wikipedia; 2019. (accessed Apr. 26, 2019). e. Hydrazine. EPA, document number 302-01-2. https://www.epa.gov/sites/production/files/2016-09/documents/hydrazine.pdf (accessed Apr. 26, 2019).

[33] This does not mean, however, that hydroxylamine is entirely benign. It irritates skin, mucous membranes, and eyes. It is toxic if swallowed. See, PubChem. Hydroxylamine https://pubchem.ncbi.nlm.nih.gov/compound/787 (accessed Apr 26, 2019); Hydroxylamine. Wikipedia; 2018 (accessed Apr 26, 2019); see also, Case Details > Explosion and fire of highly-concentrated hydroxylamine at a re-distillation unit http://www.shippai.org/fkd/en/cfen/CC1000050.html (accessed Apr 26, 2019).

[34] Hermanson, G. T. Chapter 2, Functional Targets for Bioconjugation. Op. cit., page 228, and citations therein.