5’-Maleimide Modifier Phosphoramidite
The 5’-Maleimide Modifier Phosphoramidite is a Diels-Alder adduct that enables the incorporation of a maleimide cycloadduct into oligonucleotides, which are stable to ammonium hydroxide at room temperature. This phosphoramidite enables incorporation into both DNA and RNA oligonucleotides during synthesis, with both phosphate and phosphorothioate linkages. A retro Diels-Alder reaction deprotects the maleimide immediately before a follow-up conjugation reaction, for example, to a thio-group-containing molecule such as a terminal cysteine in a peptide or oligonucleotide.
Additon of the maleimide modifier to an oligonucleotide using automated solid phase oligonucleotide synthesis
Unmasking the maleimide group via retro-Diels-Alder reaction
The oxanorbornene phosphoramidite can be added to both DNA and RNA with both phosphate and phosphorothioate linkages. A retro Diels-Alder reaction allows deprotection of the maleimide immediately before conjugation, as shown in the above reaction schemes.
Diels-Alder Reaction
The normal-electron Diels-Alder reaction is a pericyclic reaction that occurs via a single cyclic transition state and relies on orbital symmetry. The highest occupied molecular orbital (HOMO) of an electron-rich diene reacts with the lowest unoccupied molecular orbital (LUMO) of an electron-deficient dienophile.
The Diels-Alder reaction is a concerted [4+2] cycloaddition between a conjugated diene of 4 p-electrons and a substituted alkene or alkyne, the dienophile with 2 p-electrons, to form a six-membered cyclohexene ring as illustrated below.
The interaction between HOMO of the diene and the LUMO of the dienophile leads to the formation of the Diels-Alder adduct. The LUMO is a p*-antibonding orbital, typically p2* for simple alkenes, characterized by high energy and electron-accepting capability, often lowered in energy by electron-withdrawing substituents. This stereo-selective reaction forms two new carbon-carbon σ-bonds, and one p-bond.
thio-Diels–Alder reaction
The thio-Diels–Alder reaction is a [4+2] cycloaddition in which a sulfur-containing compound acts as either the diene or dienophile to allow the synthesis of sulfur-based heterocycles, typically dihydrothiopyrans, with high regio- and stereo-selectivity, allowing the creation of complex bicyclic structures.
Maleimide Diels-Alder Reaction
The maleimide Diels-Alder reaction is a [4+2] cycloaddition involving a concerted reaction between a conjugated diene, such as a furan or cyclopentadiene, and a maleimide acting as a strong, electron-deficient dienophile. This thermally allowed reaction forms a bicyclic cyclohexene derivative, often favoring the endo isomer at lower temperatures and the exo isomer at higher temperatures. The reaction occurs in a single, concerted step in which the p-electrons of the dienophile in the maleimide group attack the diene and the bonds shift simultaneously to form two new σ-bonds and one new p-bond.
Maleimides are exceptionally reactive in Diels-Alder reactions due to the strong electron-withdrawing nature of the two adjacent carbonyl groups, making them excellent dienophiles. The kinetically controlled reaction often favors the endo-adduct at lower temperatures because the reaction proceeds faster. However, the Exo-Adduct formed by a thermodynamic reaction is generally more stable and favored at higher temperatures. The reaction is often reversible, especially with furan as the diene, where higher temperatures around 60 to 100 ºC can trigger a reverse reaction known as the retro-Diels-Alder reaction. In the retro-Diels-Alder reaction, the adduct is cleaved into the maleimide and the diene.
retro-Diels-Alder
The retro-Diels-Alder (rDA) reaction is the reverse of a Diels-Alder [4+2] cycloaddition, as a concerted, thermally induced cycloreversion that breaks a cyclohexene ring into a diene and a dienophile. The result is the decomposition of dicyclopentadiene into two molecules of cyclopentadiene upon heating. rDA is highly favored by high temperatures and entropy, often used for deprotection or synthesis at high temperatures (>200 ºC) to produce stable products like aromatic rings or gases, for example, CO2 and N2. The reaction typically proceeds in a single step, with electron pairs shifting simultaneously. The two σ-bonds at C1-C6 and C3-C4 break, and the single p-bond migrates to form a diene and a dienophile.
Starting from the cyclohexene, the σ-bond between C1 and C6 breaks, pushing electrons to form a p-bond between C1 and C2. Simultaneously, the σ-bond between C3 and C4 breaks, forming a p-bond between C3 and C4. The remaining p-bond between C5 and C6 breaks, pushing electrons to form a p-bond between C5 and C6. High temperatures promote the rDA reaction, whereas lower temperatures favor the forward Diels-Alder product.
Thiol-maleimide reaction
The thiol-maleimide reaction allows the addition of chemical labels to biomolecules via thiol conjugation, such as fluorescent dyes, PEG, radiolabels, and small molecules. The advantages of this reaction are the rapid reaction between maleimides and thiols and the preference for reaction at neutral pH. However, various side reactions are possible during thiol-maleimide conjugation, including thiazine rearrangement. The instability of the maleimide-cysteine conjugate possibly explains these side reactions.
Fluorescein-5-maleimide allows the modification of sulfhydryl groups to form thioester bonds.
A general reaction scheme of maleimides with thiol group for labeling of biomolecules.
Diels-Alder Cycloaddtion
Marchan et al. in 2006 reported the synthesis of peptide–oligonucleotide conjugates with high purity and yield via the Diels-Alder reaction. The cycloaddition between a diene-modified oligonucleotide and a maleimide-derivatized peptide produced the desired peptide–oligonucleotide conjugate. This reaction occurs in mild conditions and in aqueous solution at 37°C and is complete in 8–10 h by reacting the diene-oligonucleotide with a small excess of maleimide-peptide.
Oligonucleotide-peptide conjugation through Diels-Alder reaction.
Because maleimide is unstable during ammonia deprotection of oligonucleotides, Paris et al. developed an alternative method for introducing the maleimide group into oligonucleotides, using 2,5-dimethylfuran as a protecting group for maleimide.
Diels-Alder protection of maleimide with a furan. This reaction is reversible when heated.
Sanchez et al. in 2011 showed that the reaction of maleimide-containing compounds with 2,5-dimethylfuran results in a mixture of exo and endo isomers from which the exo cycloadduct can be easily isolated, taking advantage of its stability in concentrated aqueous ammonia. Retro-Diels can remove the protecting group (Alder maleimide deprotection), affording the maleimido-oligonucleotide, now suitable for conjugation to various thiols.
Oxanorbornene derivatives allow the introduction of protected maleimides into oligonucleotides, peptides and proteins.
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| Modifier for oligonucleotide conjugation. | Modifier for peptide conjugation. |
Synthesis of cyclic oligonucleotides
In 2013, Sanchez et al. showed that the synthesis of cyclic oligonucleotides is possible with the help of the thiol-maleimide reaction. Subsequent removal of the thiol and maleimide protecting groups from 5′-maleimido-3′-thiol-derivatized linear precursors affords the cyclic molecules under Retro–Diels–Alder conditions in high yields.
Antibody drug conjugation
St Amant, in 2018, showed that the Diels-Alder reaction also enables the production of antibody conjugates and that oxanorbornene conjugates are more stable in serum than thiol-maleimide conjugates.
Linking biomolecules to each other
More recently, Agramunt et al. in 2020 incorporated 7-oxanorbornene as a dienophile to link biomolecules via tetrazines, using the inverse-electron-demand Diels-Alder cycloaddition reaction. This reaction represents a general method for linking peptides, oligonucleotides, biotin, GalNAc, and fluorophores, as well as other biomolecules of interest.
Inverse electron demand Diels-Alder (IEDDA) cycloaddition for multiple bioconjugations.
References
5-maleimide-modifier-phosphoramidite
Agramunt J., Ginesi R., Pedroso E., Grandas A. Inverse Electron-Demand Diels-Alder Bioconjugation Reactions Using 7-Oxanorbornenes as Dienophiles. J. Org. Chem. 2020;85:6593–6604. doi: 10.1021/acs.joc.0c00583. [PubMed]
Marchán V, Ortega S, Pulido D, Pedroso E, Grandas A. Diels-Alder cycloadditions in water for the straightforward preparation of peptide-oligonucleotide conjugates. Nucleic Acids Res. 2006 Feb 14;34(3):e24. [PMC]
Sánchez A, Pedroso E, Grandas A. Maleimide-dimethylfuran exo adducts: effective maleimide protection in the synthesis of oligonucleotide conjugates. Org Lett. 2011 Aug 19;13(16):4364-7. [PubMed]
Sánchez A, Pedroso E, Grandas A. Conjugation reactions involving maleimides and phosphorothioate oligonucleotides. Bioconjug Chem. 2012 Feb 15;23(2):300-7. [ACS]
Sánchez A, Pedroso E, Grandas A. Oligonucleotide cyclization: the thiol-maleimide reaction revisited. Chem Commun (Camb). 2013 Jan 11;49(3):309-11. [RSC]
St Amant AH, Lemen D, Florinas S, Mao S, Fazenbaker C, Zhong H, Wu H, Gao C, Christie RJ, Read de Alaniz J. Tuning the Diels-Alder Reaction for Bioconjugation to Maleimide Drug-Linkers. Bioconjug Chem. 2018 Jul 18;29(7):2406-2414. [escholarship]
The Diels-Alder Reaction in March. Advanced organic chemistry. 6th Editon. Pp. 1194-1215. WILEY 2007.
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