Direct Dual-Functionalisation of Amine-Oligonucleotides for Conjugation Purposes

Direct Dual-Functionalisation of Amine-Oligonucleotides for Conjugation Purposes | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Direct Dual-Functionalisation of Amine-Oligonucleotides for Conjugation Purposes Annemieke Madder, Jan Meffert, Monica Lopes, Enrico Cadoni, Martin Bollmark, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7265337/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 10 Jan, 2026 Read the published version in Communications Chemistry → Version 1 posted You are reading this latest preprint version Abstract The optimisation and further expansion of methods for the synthesis of oligonucleotide conjugates is receiving increased attention due to their importance for further advancement of therapeutic and diagnostic nucleic acid-based applications. Current methodologies, particularly those relying on maleimide-type linkers, are often hampered by multi-step synthetic procedures and linker instability. Herein, we present a versatile method for the direct functionalisation of readily available amino-modified oligonucleotides (AONs), where a 5-hydroxy-1,5-dihydro-2H-pyrrol-2-ones (5HP2O) Michael acceptor is directly formed in a rapid and efficient manner on a free primary amine. The methodology demonstrates broad applicability, tolerating various amino-modifiers and their positions within different oligonucleotide types, including DNA, LNA, PNA, and phosphorothioate-modified oligonucleotide strands. Most importantly, the possibility to introduce an additional second orthogonal reactive handle uniquely enables a direct dual-functionalisation of AONs for the assembly of complex constructs, as exemplified by the synthesis of a fluorescent peptide-oligonucleotide construct. Physical sciences/Chemistry/Chemical biology/Chemical modification Physical sciences/Chemistry/Organic chemistry/Synthetic chemistry methodology bioconjugation oligonucleotide conjugates oligonucleotide functionalisation dual-functionalisation 5HP2O maleimide alternative Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Oligonucleotide (ON) conjugates, mainly to proteins 1,2 or peptides 3 , are indispensable tools for a range of therapeutic 4–6 and diagnostic applications 7 . Conjugation to other modalities allows overcoming several hurdles known in the field of naked oligonucleotide therapeutics (e.g. antisense oligonucleotides or RNA interference), such as lack of cell specificity, or ineffective and insufficient cellular uptake 8 . In a diagnostic context, the combination of different modalities in one construct allows for imaging applications such as DNA-PAINT 2,9 or for biomarker detection such as immuno-PCR 10 . The synthesis of such hybrid molecules typically requires the combination of two orthogonal chemical strategies (peptide/protein versus oligonucleotide chemistry) through one bifunctional linker molecule 11 . A prevalent site-selective approach, as cysteines are of low abundance, involves the conjugation of a thiol-containing protein or peptide to a maleimide-functionalised oligonucleotide. The required thiol functionality on the biomolecule can be readily accessed by reducing native disulfide bonds (e.g. via Tris-(2-carboxyethyl)-phosphine, TCEP) 12,13 , through site-specific genetic incorporation of a cysteine residue 14 or by lysine modification to introduce thiols (e.g. via N-succinimidyl acetylthioacetate, SATA) 15,16 . 7 However, the preparation of the complementary functionalised oligonucleotide partner is typically less straightforward. Due to its reactivity towards ammonia and inherent instability, a maleimide moiety cannot be kept intact upon resin cleavage after solid-phase synthesis. While protected phosphoramidites containing a maleimide precursor are commercially available, they are prone to hydrolysis (as any phosphoramidite), require the use of ultra-mild oligonucleotide synthesis and an additional deprotection step in toluene at high temperature (see Fig. 1a, 1) 17,18 . Consequently, the maleimide functionality is mainly introduced via post-synthetic amide-couplings in solution-phase, where amino-modified oligonucleotide (AON) reacts with activated esters (see Fig. 1b, 3-5). AONs are readily available through solid-phase oligonucleotide synthesis and therefore represent an ideal starting point for most oligonucleotide conjugations. Primary amines can be introduced at multiple positions within the ON strand, at the 3’ or 5’ terminus and nucleobases, leading to a great flexibility for subsequent modification 19 . Nevertheless, this strategy does not allow circumventing the intrinsic instability of the maleimide moiety, which is prone to hydrolysis during its introduction via amide-coupling, during purification and bioconjugation, limiting the overall functionalisation yield 20,21 . Moreover, maleimide-based conjugates suffer from thiol-exchange and retro-Michael instabilities. 20,22 Surprisingly, only a few oligonucleotide conjugates based on alternative Michael acceptors have been described. Vinylpyrimidine moieties [4] , introduced via phosphoramidite chemistry (see Fig. 1a, 2) or benzoylacryl [5] moieties, introduced through active ester-mediated amide coupling (see Fig. 1b, 4), have been reported to overcome known issues of maleimide. However, their introduction still relies on conventional linker-based ON modification – requiring synthesis and purification of the linker molecules before reaction with the oligonucleotide, which then introduces additional linker length and hydrophobicity and possible immunogenicity. Beyond the synthetic considerations of oligonucleotide modification, critical linker properties such as length 23 , attachment site 24–26 , hydrophobicity 23 , chemical stability 27,28 and immunogenicity 29,30 heavily impact the performance of the final conjugate. Therefore, the development of novel and versatile methodologies for modifying AONs with precise control over these critical features remains a significant endeavour. 24 Gothelf and coworkers recently described the direct formation of a maleimide, respectively bromomaleimide moiety, on AONs and their subsequent use for bioconjugation to proteins via a reaction with N-methoxycarbonylmaleimide, respectively N-methoxycarbonylbromomaleimide (see Fig. 1c, 6) 21 . This method to selectively introduce and form a maleimide directly on the AON allows for a shorter linker length, greater control over the position of modification and avoids pre-synthesis of heterobifunctional linkers. High conversions (>80%) were achieved under optimized alkaline conditions, but the process remains sensitive to hydrolysis of both the reagent and the product, demanding fine-tuned procedural control. To obtain stable conjugates bromomaleimides were used in combination with an additional post-conjugation hydrolysis step. 21 Previously, we described a series of bioconjugation reagents based on the 5-hydroxy-1,5-dihydro-2H-pyrrol-2-ones (5HP2O) scaffold (see Fig. 1b, 5), which can be synthesised starting from modified furans in a one-pot synthesis 31,32 . Compared to maleimides, these reagents exhibit an enhanced stability towards hydrolysis and the resulting conjugates proved to be more resistant towards retro-Michael and thiol-exchange reactions 31 . Herein, we leverage the synthetic flexibility of the 5HP2O methodology to create a more robust and versatile method for direct oligonucleotide functionalisation. Inspired by Gothelf’s work (vide supra ), we investigated the use of the AON itself as an amine component in the preparative one-pot synthesis of the 5HP2O moiety. This approach provides a direct dual-functionalisation procedure converting AONs into stable multi-reactive ONs ready for further orthogonal downstream modification while avoiding known issues of maleimide and complex linker synthesis. Results and Discussion Condition screening for the direct formation of 5HP2Os on amine-oligonucleotides In the reported 5HP2O synthetic process, a substituted furan is photo-oxidised in the presence of singlet oxygen and methylene blue (MB) in methanol, followed by a reduction with dimethylsulfide (DMS). Lastly, the 5HP2O product gets formed upon addition of an amine catalysed by methylene blue as a redox catalyst (see Fig. 2a). 31 To evaluate the feasibility of direct modification of an AON with activated furan – towards a 5HP2O bearing ON – based on the modular three-step one-pot synthetic procedure, we decided to perform the first two steps as previously described (1 mmol substituted furan, 83 mM, see Supplementary Scheme 1) and then dilute the reaction mixture to a concentration which is relevant for AON modification (40 nmol AON, 100-200 µM). This allows, on the one hand, to use the activated furan (keto-enal) solution as a stock solution and, on the other hand, to screen reaction conditions solely for the relevant third reaction step (see Fig. 2a). It is to note here, that the keto-enal stock solution contains dimethylsulfide (reducing agent) and methylene blue (photosensitizer and redox catalyst). Both reagents, arising from the first two steps, are again required to form the final 5HP2O-modified ON in the third step (see precise mechanism in Supplementary Scheme 1). The aim here was to stick as close as possible to the known conditions, ultimately screening the equivalents of base (triethylamine, Et 3 N), keto-enal stock solution and determining an adequate solvent system compatible with oligonucleotides. Water is well tolerated in the reaction so that a mixture of MeOH:water (9:1 v\v) was used as the starting point in combination with an overnight reaction. An oligonucleotide of 21 nucleobases bearing the terminal 5’-amino modifier D1 (see Fig. 3) was chosen as model compound given the similar size of many therapeutic oligonucleotides. HPLC analysis showed that increasing keto-enal and triethylamine amounts lead to a consumption of starting material AON and to the formation of two new peaks in a concentration-dependent manner. The full consumption of the starting material was observed above 800 equiv. of keto-enal and triethylamine, which however, led to the formation of two major products (see Fig. 2b, chrom. i to vi). The first peak could be identified as the desired directly modified 5HP2O-ON (see Fig. 2e+f and Supplementary Fig. 4). The side product could not be identified given the vast plethora of theoretically possible side products (see Supplementary Scheme 2 and Supplementary Fig. 6). Finally, by reducing the amount of keto-enal 16-fold to 50 equiv., the amount of side product could be reduced to nearly zero and mainly conversion to the desired product peak was observed (see Fig. 2b, chrom. vii). The large amount of required base, likely reflects the need of a deprotonated amine and it is furthermore required for the reaction mechanism. Further experiments showed that the amount of keto-enal stock solution (also including MB, DMS) could not be further reduced, as only incomplete AON consumption was observed (see Fig. 2c, chrom. i-ii). Increasing the amount of base even further had no impact on the reaction profile (see Fig. 2c, chrom. iii-iv). Finally, increasing the AON concentration from 100 µM to 200 µM was identified to be very beneficial and 86% yield of the desired product was observed (see Fig. 2c, chrom. vi). In summary, straightforward and efficient direct modification of the AON was achieved with 50 equiv. of keto-enal stock solution, 800 equiv. of triethyvlamine at a AON concentration of 200 µM at room temperature for 30 min in Water:MeOH (1:9 v/v ). Isolated yield of this reaction was 32% on a 40 nmol scale and is on par with isolated yields of NHS-mediated functionalisation using 5HP2O-C5-NHS (see Supplementary Fig. 8). Notably, traditional NHS-mediated maleimide functionalisation leads to a maximum in reaction yield of 70% due to constant maleimide hydrolysis (see Supplementary Fig. 8) and therewith is below the herein observed yield. Furthermore, the keto-enal stock solution is stable for months when stored in the freezer, allowing for plug-and-play modifications of AON (see Supplementary Fig. 5). Negative controls show no undesired modification Aside from the condition screening, emphasis was set on negative controls to evaluate the possible degradation of the AON under the reaction conditions. Upon incubating the AON in solutions without keto-enal but containing MB, Et 3 N and DMS, degradation was evaluated through HPLC analysis. An onset of degradation is only noticeable when increasing the reagent concentration two-fold and does not occur under the identified reaction conditions (see Fig. 2d, chrom. 1-3). This leads to a window, in which the desired 5HP2O-functionalisation can be performed in the absence of any degradation pathway driven through DMS or MB. Furthermore, reducing the amount of methanol in the solvent system significantly slows down the formation of 5HP2O on the AON, as shown by HPLC experiments. Evaporation of methanol and DMS followed by redissolving in pure water stops the reaction and therefore proves a successful way to quench the reaction (see Supplementary Fig. 7). Our group previously reported on the modification of exocyclic amines of nucleobases with activated furan (keto-enal) 33,34 . To exclude any undesired modification of the AON, we performed control experiments with an oligonucleotide, lacking the 5’-aminohexyl modification. As hoped, we did not observe any peak shift (see Fig. 2d, chrom. 5-6) nor mass change (see Supplementary Fig. 27+28) of the control reaction. Moreover, a potential backbone modification with 5HP2O would interfere with or at least influence base pairing to the complementary strand. Therefore, melting assays were performed upon hybridizing the functionalised AON with its complement (see Fig. 2g and Supplementary Fig. 29). No difference in melting temperature between the non-functionalised and directly functionalised oligonucleotide was observed. Contrastingly, functionalisation of the oligonucleotide via a classical NHS-mediated amide coupling (see Supplementary Fig. 8) led to a significant increase in melting temperature. This could be caused by the increased linker length (see Fig. 1), as five extra carbons are introduced. Although we cannot fully explain this phenomenon, it nonetheless demonstrates that linker length and or introduction methodology indeed play a role in ON properties. In summary, tweaking the reaction conditions to find a window in which the AON can be modified but not degraded was crucial to allow for this AON functionalisation protocol. A variety of amine modifiers and oligonucleotide backbones are accepted With optimised conditions in hand, we set out to evaluate the substrate scope of the methodology regarding other amino-modified oligomers. Next to the initial model substrate D1 bearing a 5’-aminohexyl moiety, three phosphorothioate oligodeoxynucleotides (including multiple LNA modifications) bearing different 5’-modifications were investigated. Both, 4-aminomethylbenzoic acid-containing (PAMBA) sequences (D2+3) and an anomeric aminoribose-containing sequence (D4) were well tolerated and full conversion was observed via LCMS (see Supplementary Figs. 11-16). Conveniently, aminoribose D4 in combination with the herein discussed methodology, enables the formation of a Michael acceptor closely located to the natural backbone of an oligonucleotide without introduction of additional carbons. This could be of special interest in applications like DNA-PAINT, reducing a potential linkage error. Internal amino-modification of oligodeoxynucleotides bearing either one (Int1) or two amines (Int2) proceeded equally smoothly, yielding the products with one and two 5HP2Os, respectively (see Supplementary Figs. 17-20). Lastly, we applied the methodology to peptide nucleic acids (PNAs). PNAs feature a neutral peptidic backbone and therefore solubility is a key issue when utilizing or conjugating these oligomers. To circumvent solubility problems, especially when additionally modifying the PNA with hydrophobic linkers (e.g. SMCC, see Fig. 1b), charged amino acids or ethylene glycol spacers are usually introduced into the sequence. We decided to synthesise and evaluate four different PNAs (see Supplementary Figs. 21-24), bearing different modifications next to the free N-terminus. Direct 5HP2O functionalisation on N-terminally introduced charged amino acids (Glu or Arg in P1, P3) was not possible. This could be due to a change in local pH microenvironment leading to a non-reactive protonated amine-PNA or due to steric hindrance. However, formation of 5HP2O on P2 and P4, containing an ethylene glycol spacer after the charged amino acids, was confirmed by a peak shift in HPLC, and the correct product mass could be observed in MALDI-TOF-MS (see Supplementary Figs. 25+26). Starting with modified furan leads to direct dual-functionalisation of oligonucleotides Showing the broad applicability of this methodology by functionalising eight different primary amine-bearing oligomers (phosphodiester-, phosphorothioate- or peptidic backbone, LNA modifications, internal amines and different terminal amines), we next evaluated the dual-functionalisation of AONs – a unique approach allowed through the 5HP2O synthetic methodology. This required the synthesis of substituted furan building blocks, bearing a modified side chain rather than a methyl group. Several clickable handles like a (protected) ketone (MF5 respectively MF6), azide (MF4) and alkyne (MF2) were chosen for incorporation in order to allow for post-labelling by biorthogonal CuAAC reactions or oxime ligations. Moreover, we aimed for commercially available furan propionic acid (MF1) as a precursor for a straightforward synthetic approach to these modified keto-enals. Starting from MF1, all other modified furans were obtained via short synthetic routes (see Supplementary Section 7 and 11), and standard amide coupling showed to be the most straightforward approach to these building blocks (as exemplified by MF2 and MF3). As initially discussed for methylfuran, the synthesised modified analogues were converted to the modified keto-enal by means of a photooxidation followed by reduction in methanol. Applying the previously described reaction conditions to an azide-bearing keto-enal (generated from MF4) and AON D1 shows unexpected degradation of the AON. Detailed investigation of different reaction conditions (see Supplementary Fig. 30+31) highlighted the influence of the reaction time and especially of the solvent system. Dual functionalisation with the azide-bearing keto-enal required a longer reaction time (overnight rather than 30 min) and a reduced amount of methanol (50% vs. 90%). Interestingly, no conversion was observed in 20% methanol, showcasing the relevance of the solvent in this functionalisation reaction. This influence of the solvent system is in accordance with the aforementioned results for unmodified keto-enal (see Supplementary Fig. 7). Overnight incubation of AON D1 with the azide-containing keto-enal (see Supplementary Fig. 30) leads to the desired dual-functionalised product in a 60% yield. Employing the alternative alkyne-containing keto-enal generated from MF2 (see Fig. 4c-e and Supplementary Fig. 35) led to a dual-modification yield of 71%. We continued to use this keto-enal as model compound for further bioconjugation and CuAAC labelling. Other dual-functionalisation reactions were analysed by LCMS, obtaining the desired constructs in medium to good yield depending on the building block (see Fig. 4 b and Supplementary Fig. 32-34). We suspect that the differences in 5HP2O functionalisation yields observed for the different building blocks (especially when compared to methylfuran), arise through a difference in photooxidation efficiency of the modified furans in the first step. These conditions were kept unchanged and are based on the earlier reactions with 2-methylfuran. However, other 2-modified furans might require an increased amount of methylene blue or an increased irradiation time to drive full photooxidation. An extreme case was the ketone-bearing MF5 which led to no conversion, whereas the acetal-protected version thereof (MF6) led to 56% yield. Consequently, we did not investigate subsequent oxime ligations, which would require additional deprotection steps but rather focused on CuAAC labelling. Lastly, it can be stated that the discussed dual-functionalisation yields are around the same level as an NHS-mediated maleimide functionalisation (see Supplementary Fig. 8). Follow up bioconjugation and multi-functionalisation After direct 5HP2O functionalisation of AON D1, the conjugation to thiol-bearing model compounds was evaluated. Therefore, purified mono-functionalised 5HP2O-D1-ON was incubated overnight with glutathione (GSH) as a model peptide and with an alphabody ADDIN CitaviPlaceholder{{"$id":"1","$type":"SwissAcademic.Citavi.Citations.WordPlaceholder, SwissAcademic.Citavi","Entries":[{"$id":"2","$type":"SwissAcademic.Citavi.Citations.WordPlaceholderEntry, SwissAcademic.Citavi","Id":"da740330-52a9-4373-a0c0-28b61c14c0e8","RangeLength":2,"ReferenceId":"9debe18e-e2be-469f-8309-7737dea93ee3","IsNonrecurringReferenceInMultipleCitation":true,"PageRange":{"$id":"3","$type":"SwissAcademic.PageRange, SwissAcademic","EndPage":{"$id":"4","$type":"SwissAcademic.PageNumber, SwissAcademic","IsFullyNumeric":false,"NumberingType":0,"NumeralSystem":0},"NumberingType":0,"NumeralSystem":0,"StartPage":{"$id":"5","$type":"SwissAcademic.PageNumber, SwissAcademic","IsFullyNumeric":false,"NumberingType":0,"NumeralSystem":0}},"Reference":{"$id":"6","$type":"SwissAcademic.Citavi.Reference, 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The employed alphabody is a triple-helical protein of small size (around 11.5 kDa) targeting Interleukin-23 35 . Reduction and charachterisation was performed as previously described 31 . Both conjugates were obtained in high yields and could be characterised by mass spectrometry (see Fig. 5a-c and Supplementary Fig. 37). It has to be noted here, that the longer reaction time (vs. traditional maleimide chemistry) is not an issue in view of the non-hydrolysable 5HP2O scaffold 31 . Finally, to obtain an efficient platform for AON conjugation, we aimed to minimise purification after direct functionalisation. Filtration through a size exclusion column before the addition of cysteine-containing molecules to the functionalised AON proved to be sufficient, yielding the desired conjugate. In combination with an additional alkyne tag, leveraging the functionalised side chain of the 5HP2Os, we were able to obtain a fluorescently labelled glutathione oligonucleotide conjugate in three steps from the readily available AON (see Fig. 5e-h). In another batch, the intermediate GSH-5HP2O(Alkyne)-D1-ON got isolated and also characterised (see Supplementary Fig. 39). Conclusion We herein present a methodology for the direct modification of amino-modified oligonucleotides with 5HP2Os, which are stable Michael acceptors ready to be employed for bioconjugation purposes. This procedure is based on methylene blue, first acting as a photosensitizer on preparative scale (1 mmol) in the initial singlet oxygen-mediated oxidation of 2-substituted furans and subsequently - upon dilution - acting as a redox catalyst during the formation of 5HP2Os on the AON (40 nmol scale). The here described methodology extends the currently available methods for cumbersome preparation of “bioconjugation-ready” oligonucleotides, which mainly relies on synthesis of heterobifunctional linkers followed by NHS- or phosphoramidite-mediated ON modification. Several common amine modifiers (aminohexyl, PAMBA) and backbones (PS, PO and PNA) are readily accepted at different positions within the oligonucleotide, and full conversion with nearly no degradation is seen in short reaction times (30 min). Synthesis of modified furan building blocks followed by their photooxidation allows for the introduction of an extra reactive handle (e.g. azide and alkyne) into the AON aside the Michael acceptor. Both the theoretical yields and the isolated yields are comparable to standard linker-based conjugations. Ready-to-use stock solutions of activated furan derivatives were shown to be stable, allowing for a plug-and-play modification of AON depending on the desired application. Finally, the possibility of follow-up bioconjugation in combination with additional follow-up click chemistry greatly simplifies the generation of more complex peptide/protein oligonucleotide conjugates, which are quickly becoming important constructs in advanced therapeutic or diagnostic applications such as targeted oligonucleotide delivery or DNA-PAINT. Methods Information on the synthesis and characterisation of small molecules and oligonucleotides can be found in the Supporting Information of this article. Activation of 2-substituted furan towards substituted keto-enal stock solution In a test tube with 10 mL methanol was added 2-substituted furan (1 mmol) and catalytic amounts of methylene blue (0.2 mol%, 0.64 mg, 0.002 mmol). Pressured air was bubbled through the solution while it got irradiated for 10 minutes at room temperature using the indicated lamp (see Supporting Information Section 1.1). The reaction mixture was then transferred into a round-bottomed flask, DMS (4.0 equiv, 300 µL) and Et 3 N (4.2 µL, 0.03 mmol) were added and the flask was sealed for further 40 minutes at room temperature. Subsequently, additional methylene blue (2.0 mol%, 6.4 mg, 0.02 mmol) was added and the keto-enal stock solution was stored in the freezer after concentration adjustment to 50 mM. Direct functionalisation of AON via activated 2-methylfuran Direct oligonucleotide functionalisation was performed at 200 µM AON concentration in water:methanol (1:9 v/v, 100 µL) for 30 minutes at room temperature. Therefore, to an amber screw-cap HPLC vial was added in the following order: methanol, triethylamine (800 equiv., c fin = 80 mM), keto-enal stock solution (50 equiv., c fin = 5 mM) and amine-oligonucleotide (1 equiv., c fin = 200 µM). The reaction was incubated for 30 min at room temperature before concentration under reduced pressure and redissolving in water. Reactions were either purified by RP-HPLC, or by SEC-spin columns (MicroBio Spin 6 kDa cutoff). Direct Dual-functionalisation of AON via activated 2-substituted furan Direct dual oligonucleotide functionalisation was performed at 200 µM AON concentration in water:methanol (1:1 v/v, 100 µL). Therefore, to an Eppendorf vial was added in the following order: methanol, water, triethylamine (800 equiv., c fin = 80 mM), keto-enal stock solution (50 equiv., c fin = 5 mM) and amine-oligonucleotide (1 equiv., c fin = 100 µM). The reaction was shaken overnight at 37 °C before concentration under reduced pressure and redissolving in water. Reactions were either purified by RP-HPLC, or by SEC-spin columns (MicroBio Spin 6 kDa cutoff). Follow-Up Bioconjugation of 5HP2O-functionalised oligonucleotides Bioconjugation of functionalised oligonucleotide was usually performed at 40 µM 5HP2O-ON concentration in buffer (Carmody buffer pH 9 or 10, 100 µL), overnight at 37 °C. Therefore, to an Eppendorf vial was added in the following order: buffer, 5HP2O-ON and cysteine-containing peptide or protein. Glutathione (40 equiv., c fin = 1.6 mM) was added without prior reduction from a freshly prepared stock solution. In contrast, alphabody protein (10 equiv., c fin = 0.4 mM) was pre-treated with TCEP (5 equiv. in regard to the protein) at 400µM for 30 min at room temperature. Subsequently, the solution was buffer exchanged (Carmody buffer pH 10) and the excess TCEP was removed using a MicroBio Spin (6 kDa cutoff) before its addition to the 5HP2O-ON. Follow-Up CUAAC Labelling To the crude bioconjugation reaction mixture was added in the following order: NBD-azide (40 equiv, c fin = 1.6 mM), THTPA:CuSO 4 (25 equiv., pre-incubation for 5 min in a ratio 1:2 CuSO 4 :THTPA at 50 mM CuSO 4 , c fin = 1.6 mM), sodium ascorbate (40 equiv., c fin = 1.6 mM). The reaction was incubated for 30 minutes at room temperature before purification by RP-HPLC. Declarations Acknowledgments This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie Grant Agreement No. 956070 (OLIGOMED) (J.M., M.L., A.M.). E.C. acknowledges a FWO fellowship (12B1923N). We thank Dr. Chloe Howells and Prof. Eugen Stulz for LCMS measurement of crude functionalisation reactions. We thank Pieter Surmont and Prof. Frederic Lynen for HRMS Orbitrap measurement of purified oligonucleotide constructs. We thank Jan Goeman for LCMS measurements and Stijn Tanghe for general lab support. We thank the NMR expertise centre (Ghent University) for providing support and access to its NMR infrastructure. The 400 MHz NMR used in this work has been funded by a grant/project of the Research Foundation Flanders (FWO I006920N) and the ‘Bijzonder Onderzoeksfonds’ (BOF.BAS.2022.0023.01). Author contributions Jan H. Meffert was responsible for the overall conceptualization of the study, designed and conducted all experiments including condition screening, oligonucleotide mono- and dual-functionalisation, product analysis and small molecule synthesis, and wrote the manuscript, with support from the other authors. Enrico Cadoni provided PNA constructs (P1 to P4). Mónica Lopes, Martin Bollmark and UIf Tedebark provided phosphorothioated amine oligonucleotides (D2-4). Annemieke Madder was responsible for the funding and overall guidance, contributed to the conceptualisation and assisted in manuscript writing, proofreading and correction. Competing interests Annemieke Madder has a pending patent application relating to 5HP2Os application for bioconjugation (Bioconjugation reagent and methods, WO2020174086A2, Annemieke MADDER, Ewout DE GEYTER, Eirini ANTONATOU, Sabina SMOLEN, Dimitris Kalaitzakis, Georgios VASSILIKOGIANNAKIS, Europe&US). All other authors declare no competing interest. 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Development of Phosphoramidite Reagents for the Synthesis of Base-Labile Oligonucleotides Modified with a Linear Aminoalkyl and Amino-PEG Linker at the 3′-End. Molecules 27, 8501 (2022). Ravasco, J. M. J. M., Faustino, H., Trindade, A. & Gois, P. M. P. Bioconjugation with Maleimides: A Useful Tool for Chemical Biology. Chemistry 25, 43–59 (2019). Kjærsgaard, N. L., Hansen, R. A. & Gothelf, K. V. Preparation of Maleimide-Modified Oligonucleotides from the Corresponding Amines Using N-Methoxycarbonylmaleimide. Bioconjug Chem (2022). Fontaine, S. D., Reid, R., Robinson, L., Ashley, G. W. & Santi, D. V. Long-term stabilization of maleimide-thiol conjugates. Bioconjug Chem 26, 145–152 (2015). Ou, L. et al. Assessment of Crosslinkers between Peptide Antigen and Carrier Protein for Fusion Peptide-Directed Vaccines against HIV-1. Vaccines (Basel) 10 (2022). Rocha Tapia, A. et al. 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Comparison of four bifunctional reagents for coupling peptides to proteins and the effect of the three moieties on the immunogenicity of the conjugates. J Immunol Methods 120, 133–143 (1989). Kafi, K. et al. Maleimide conjugation markedly enhances the immunogenicity of both human and murine idiotype-KLH vaccines. Mol Immunol 46, 448–456 (2009). Geyter, E. de et al. 5-Hydroxy-pyrrolone based building blocks as maleimide alternatives for protein bioconjugation and single-site multi-functionalization. Chem Sci 12, 5246–5252 (2021). Kalaitzakis, D., Kouridaki, A., Noutsias, D., Montagnon, T. & Vassilikogiannakis, G. Methylene Blue as a Photosensitizer and Redox Agent: Synthesis of 5-Hydroxy-1H-pyrrol-2(5H)-ones from Furans. Angew Chem Int Ed Engl 54, 6283–6287 (2015). Halila, S., Velasco, T., Clercq, P. de & Madder, A. Fine-tuning furan toxicity: fast and quantitative DNA interchain cross-link formation upon selective oxidation of a furan containing oligonucleotide. Chem Commun (Camb) , 936–938 (2005). Beeck, M. op de & Madder, A. Sequence specific DNA cross-linking triggered by visible light. J Am Chem Soc 134, 10737–10740 (2012). Desmet, J. et al. Structural basis of IL-23 antagonism by an Alphabody protein scaffold. Nat Commun 5, 5237 (2014). Additional Declarations There is NO Competing Interest. Supplementary Files MeffertMadderCommChem2025Supporting.docx Supporting information Onlinefloatimage1.png Cite Share Download PDF Status: Published Journal Publication published 10 Jan, 2026 Read the published version in Communications Chemistry → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7265337","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":497214196,"identity":"40b08cc2-2169-4993-961f-f0bd8b666f6c","order_by":0,"name":"Annemieke Madder","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0003-0179-7608","institution":"Ghent University","correspondingAuthor":true,"prefix":"","firstName":"Annemieke","middleName":"","lastName":"Madder","suffix":""},{"id":497214197,"identity":"c612fee7-2a11-4552-aac5-6cd2877f9f90","order_by":1,"name":"Jan Meffert","email":"","orcid":"https://orcid.org/0000-0003-4342-5618","institution":"Ghent University","correspondingAuthor":false,"prefix":"","firstName":"Jan","middleName":"","lastName":"Meffert","suffix":""},{"id":497214198,"identity":"17a42492-5c3f-4e83-b6cc-1120dce22a2e","order_by":2,"name":"Monica Lopes","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Monica","middleName":"","lastName":"Lopes","suffix":""},{"id":497214199,"identity":"ec7cc25f-849d-4f49-a1fa-f9a6c5636749","order_by":3,"name":"Enrico Cadoni","email":"","orcid":"https://orcid.org/0000-0001-5585-7579","institution":"Ghent University","correspondingAuthor":false,"prefix":"","firstName":"Enrico","middleName":"","lastName":"Cadoni","suffix":""},{"id":497214200,"identity":"20b4dc2b-d55e-4e09-a7a7-55cd5cfdb5b6","order_by":4,"name":"Martin Bollmark","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Martin","middleName":"","lastName":"Bollmark","suffix":""},{"id":497214201,"identity":"cad90744-19c2-4462-a29f-aec931410b0e","order_by":5,"name":"Ulf Tedebark","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Ulf","middleName":"","lastName":"Tedebark","suffix":""}],"badges":[],"createdAt":"2025-07-31 19:55:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7265337/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7265337/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s42004-025-01882-8","type":"published","date":"2026-01-10T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":88997294,"identity":"be5d68e4-f2f8-4402-bca6-881c45af8f65","added_by":"auto","created_at":"2025-08-13 14:45:09","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":49227,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverview of existing approaches for oligonucleotide functionalisation for the purpose of bioconjugation to cysteine-containing proteins or peptides.\u003c/strong\u003e (\u003cstrong\u003ea\u003c/strong\u003e) Phosphoramidites generally suffer from poor stability combined with a complex synthesis. The commercially available maleimide phosphoramidite requires a deprotection step to liberate reactive maleimide. (\u003cstrong\u003eb\u003c/strong\u003e) Heterobifunctional linkers allow for the introduction of thiol-reactive handles (like maleimide, benzoacryl or 5HP2O) into AON. (\u003cstrong\u003ec\u003c/strong\u003e) Direct Functionalisation allows for the introduction of the reactive handle without additional linker. The thiol-reactive handle is directly formed on the primary amine of the oligonucleotide.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7265337/v1/ffbc2b0b5b5c30784afe4e16.png"},{"id":88996344,"identity":"a5eb6b7d-d157-4ca2-8550-fa5fd169aa40","added_by":"auto","created_at":"2025-08-13 14:37:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":94606,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDirect 5HP2O-functionalisation of amino-modified oligonucleotides.\u003c/strong\u003e (\u003cstrong\u003ea\u003c/strong\u003e) General reaction scheme. The first two steps are performed on 1 mmol scale (83 mM) yielding the keto-enal stock solution. The third step (actual AON functionalisation) is then performed at reduced concentration (see Supplementary Scheme 1 for mechanism). (\u003cstrong\u003eb-d\u003c/strong\u003e) Chromatograms (chrom.) in each panel are numbered from top to bottom. Full chromatograms can be found in Supplementary Fig. 1-3 (\u003cstrong\u003eb+c\u003c/strong\u003e) Crude HPLC chromatograms of condition-screening reactions of AON modification. Reactions were performed overnight at 100 µM (besides C, chrom. 5+6) at room temperature. Product yield (as stated in %) was calculated by integrating product signal against other relevant signals. \u0026nbsp;(\u003cstrong\u003ed\u003c/strong\u003e) HPLC chromatograms of negative control reactions without keto-enal (chrom. 1-4) or without free primary amine in the ON (chrom. 5-6). Non-keto-enal driven consumption of AON (=degradation) is only onsetting when doubling the amount of reagent, proving that the AON is stable at reaction conditions against DMS, MB and ET\u003csub\u003e3\u003c/sub\u003eN. Under the identified final reaction conditions, the corresponding non-amine bearing ON is not reactive. (\u003cstrong\u003ee\u003c/strong\u003e) ORBITRAP ESI- spectrum of purified product peak. See Supplementary Fig. 4 for corresponding chromatogram. (\u003cstrong\u003ef\u003c/strong\u003e) Deconvoluted mass corresponds to the desired calculated mass. (\u003cstrong\u003eg\u003c/strong\u003e) Comparison of melting temperatures (find underlying melting curves in Supplementary Fig. 29) between non-modified AON D1, directly-modified AON D1 and conventionally NHS-modified AON D1 with NC1 (find sequences in Supplementary Table 2 and data on NHS-mediated functionalisation in Supplementary Fig. 8) to evaluate potential nucleobase modification. No difference in melting temperature (AON and 5HP2O-D1-ON) could be observed, showcasing selectivity of the keto-enal for the primary amine.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7265337/v1/11707304767e668572217066.png"},{"id":88996349,"identity":"62e8244f-5d1d-4378-ad19-a9e5cd25216c","added_by":"auto","created_at":"2025-08-13 14:37:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":41364,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSubstrate scope in terms of amino-position, -linker and oligonucleotide backbone. \u003c/strong\u003eNext to aminohexyl-containing AON D1, also para-aminomethylphenyl-containing sequences D2 and D3, and the anomeric aminomethylribose AON D4 can be functionalised in high yields. The described methodology can be applied on classical phosphodiester AONs (D1) and on phosphorothioate bearing AONs (D2-4). Internal amines (Int1) are tolerated and multiple 5HP2Os can be introduced on one strand (Int2). See list of sequences in Supplementary Table 2. When switching to a peptidic backbone (list of sequences in Supplementary Table 1) and analysing several N-terminal amino PNAs, 5HP2O formation could not be achieved on the amine of an amino acid (P1 or P3) but an additional hydrophilic amino ethylene glycol spacer allowed formation of 5HP2O on that amine (P2 or P4). All indicated yields are calculated based on integration of product signal against other signal arising from oligomers. All products were successfully characterised by LCMS (see Supplementary Sections 4 and 5).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7265337/v1/b2e1394a9722c66dc23425fa.png"},{"id":88996347,"identity":"27f5d465-1399-4072-8c77-6b72a5e0aba0","added_by":"auto","created_at":"2025-08-13 14:37:09","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":83151,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDirect dual-modification of amino-modified oligonucleotides via modified furans (MF).\u003c/strong\u003e (\u003cstrong\u003ea\u003c/strong\u003e) Schematic representation of synthetic workflow. Synthesis of several 2-modified furans (see Supplementary Section 7) followed by their photoactivation yielding modified keto-enals which are employed for the modification of AON D1. (\u003cstrong\u003eb\u003c/strong\u003e) Overview of obtained dual-modified constructs stating modification yield and mass as observed by LCMS (Molecular Weight) or Orbitrap (Exact Mass as highlighted with asterisk). See Supplementary Section 8 for product synthesis and Supplementary Section 11 for product characterisation. (\u003cstrong\u003ec\u003c/strong\u003e) HPLC Chromatograms of of alkyne-keto-enal mediated dual modification of AON D1. (\u003cstrong\u003ed\u003c/strong\u003e) ORBITRAP ESI- spectrum of purified product peak. (\u003cstrong\u003ee\u003c/strong\u003e) Deconvoluted mass corresponds to the desired calculated mass.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7265337/v1/db8d666b890c0971aa8dfb3a.png"},{"id":88997297,"identity":"7f5899d7-9747-4b9a-89c3-3ffc45b31854","added_by":"auto","created_at":"2025-08-13 14:45:10","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":127257,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFollow-up bioconjugation and labelling of 5HP2O functionalised oligonucleotides.\u003c/strong\u003e (\u003cstrong\u003ea\u003c/strong\u003e) Reaction scheme of bioconjugation of mono-functionalised 5HP2O-D1-ON with glutathione (GSH) and alphabody protein. (\u003cstrong\u003eb\u003c/strong\u003e) HPLC chromatograms of crude reactions of purified 5HP2O-D1-ON with GSH and alphabody protein. (\u003cstrong\u003ec\u003c/strong\u003e) ORBITRAP ESI- spectrum of isolated alphabody conjugate (Ab-5HP2O-D1-ON). (\u003cstrong\u003ed\u003c/strong\u003e) Deconvoluted mass corresponds to the desired calculated mass of Ab-5HP2O-D1-ON. (\u003cstrong\u003ee\u003c/strong\u003e) Direct dual functionalisation of D1-AON with alkyne-bearing keto-enal followed by purification via spin column, conjugation and CUAAC labelling allows for the assembly of a fluorescent conjugate in three steps (\u003cstrong\u003ef\u003c/strong\u003e) HPLC chromatograms of crude reactions of 5HP2O-D1-ON with GSH followed by CUAAC labelling with NBD fluorophore. Find full chromatograms in Supplementary Fig. 38. In another batch, the intermediate (GSH-5HP2O(Alkyne)-D1-ON) got isolated and charachterised (see Supplementary Fig. 39). (\u003cstrong\u003eg\u003c/strong\u003e) ORBITRAP ESI- spectrum of isolated fluorescent conjugate (GSH-5HP2O(Alkyne-NBD)-D1-ON). (\u003cstrong\u003eh\u003c/strong\u003e) Deconvoluted mass corresponds to the desired calculated mass of GSH-5HP2O(Alkyne-NBD)-D1-AON.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7265337/v1/b459ec02ee17845493069302.png"},{"id":102488423,"identity":"daf56daa-26b0-46ac-a719-b18c0744163a","added_by":"auto","created_at":"2026-02-12 08:12:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1361231,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7265337/v1/6b383800-b651-4ef0-a3f0-9b01d21eaa89.pdf"},{"id":88996368,"identity":"b76dd381-63c2-4ea6-8b70-f519c8298ba6","added_by":"auto","created_at":"2025-08-13 14:37:10","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":23457060,"visible":true,"origin":"","legend":"Supporting information","description":"","filename":"MeffertMadderCommChem2025Supporting.docx","url":"https://assets-eu.researchsquare.com/files/rs-7265337/v1/c64f891610962af314026f37.docx"},{"id":88996348,"identity":"2f849bd4-c4cb-40f1-a9b3-540d00c0afe9","added_by":"auto","created_at":"2025-08-13 14:37:09","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":17542,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7265337/v1/3dd7add7803ac862fee3188d.png"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Direct Dual-Functionalisation of Amine-Oligonucleotides for Conjugation Purposes","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOligonucleotide (ON) conjugates, mainly to proteins\u003csup\u003e1,2\u003c/sup\u003e or peptides\u003csup\u003e3\u003c/sup\u003e, are indispensable tools for a range of therapeutic\u003csup\u003e4\u0026ndash;6\u003c/sup\u003e and diagnostic applications\u003csup\u003e7\u003c/sup\u003e. Conjugation to other modalities allows overcoming several hurdles known in the field of naked oligonucleotide therapeutics (e.g. antisense oligonucleotides or RNA interference), such as lack of cell specificity, or ineffective and insufficient cellular uptake\u003csup\u003e8\u003c/sup\u003e. In a diagnostic context, the combination of different modalities in one construct allows for imaging applications such as DNA-PAINT\u003csup\u003e2,9\u003c/sup\u003e or for biomarker detection such as immuno-PCR\u003csup\u003e10\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe synthesis of such hybrid molecules typically requires the combination of two orthogonal chemical strategies (peptide/protein versus oligonucleotide chemistry) through one bifunctional linker molecule\u003csup\u003e11\u003c/sup\u003e. A prevalent site-selective approach, as cysteines are of low abundance, involves the conjugation of a thiol-containing protein or peptide to a maleimide-functionalised oligonucleotide. The required thiol functionality on the biomolecule can be readily accessed by reducing native disulfide bonds (e.g. via Tris-(2-carboxyethyl)-phosphine, TCEP)\u003csup\u003e12,13\u003c/sup\u003e, through site-specific genetic incorporation of a cysteine residue\u003csup\u003e14\u003c/sup\u003e or by lysine modification to introduce thiols (e.g. via N-succinimidyl acetylthioacetate, SATA)\u003csup\u003e15,16\u003c/sup\u003e.\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eHowever, the preparation of the complementary functionalised oligonucleotide partner is typically less straightforward. Due to its reactivity towards ammonia and inherent instability, a maleimide moiety cannot be kept intact upon resin cleavage after solid-phase synthesis. While protected phosphoramidites containing a maleimide precursor are commercially available, they are prone to hydrolysis (as any phosphoramidite), require the use of ultra-mild oligonucleotide synthesis and an additional deprotection step in toluene at high temperature (see Fig. 1a, 1)\u003csup\u003e17,18\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConsequently, the maleimide functionality is mainly introduced via post-synthetic amide-couplings in solution-phase, where amino-modified oligonucleotide (AON) reacts with activated esters (see Fig. 1b, 3-5). AONs are readily available through solid-phase oligonucleotide synthesis and therefore represent an ideal starting point for most oligonucleotide conjugations. Primary amines can be introduced at multiple positions within the ON strand, at the 3\u0026rsquo; or 5\u0026rsquo; terminus and nucleobases, leading to a great flexibility for subsequent modification\u003csup\u003e19\u003c/sup\u003e. Nevertheless, this strategy does not allow circumventing the intrinsic instability of the maleimide moiety, which is prone to hydrolysis during its introduction via amide-coupling, during purification and bioconjugation, limiting the overall functionalisation yield\u003csup\u003e20,21\u003c/sup\u003e. Moreover, maleimide-based conjugates suffer from thiol-exchange and retro-Michael instabilities.\u003csup\u003e20,22\u003c/sup\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSurprisingly, only a few oligonucleotide conjugates based on alternative Michael acceptors have been described. Vinylpyrimidine moieties\u003csup\u003e[4]\u003c/sup\u003e, introduced via phosphoramidite chemistry (see Fig. 1a, 2) or benzoylacryl\u003csup\u003e[5]\u003c/sup\u003e moieties, introduced through active ester-mediated amide coupling (see Fig. 1b, 4), have been reported to overcome known issues of maleimide. However, their introduction still relies on conventional linker-based ON modification \u0026ndash; requiring synthesis and purification of the linker molecules before reaction with the oligonucleotide, which then introduces additional linker length and hydrophobicity and possible immunogenicity.\u003c/p\u003e\n\u003cp\u003eBeyond the synthetic considerations of oligonucleotide modification, critical linker properties such as length\u003csup\u003e23\u003c/sup\u003e, attachment site\u003csup\u003e24\u0026ndash;26\u003c/sup\u003e, hydrophobicity\u003csup\u003e23\u003c/sup\u003e, chemical stability\u003csup\u003e27,28\u003c/sup\u003e and immunogenicity\u003csup\u003e29,30\u003c/sup\u003e heavily impact the performance of the final conjugate. Therefore, the development of novel and versatile methodologies for modifying AONs with precise control over these critical features remains a significant endeavour.\u003csup\u003e24\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eGothelf and coworkers recently described the direct formation of a maleimide, respectively bromomaleimide moiety, on AONs and their subsequent use for bioconjugation to proteins \u003cem\u003evia\u003c/em\u003e a reaction with N-methoxycarbonylmaleimide, respectively N-methoxycarbonylbromomaleimide (see Fig. 1c, 6)\u003csup\u003e21\u003c/sup\u003e. This method to selectively introduce and form a maleimide directly on the AON allows for a shorter linker length, greater control over the position of modification and avoids pre-synthesis of heterobifunctional linkers. High conversions (\u0026gt;80%) were achieved under optimized alkaline conditions, but the process remains sensitive to hydrolysis of both the reagent and the product, demanding fine-tuned procedural control. To obtain stable conjugates bromomaleimides were used in combination with an additional post-conjugation hydrolysis step.\u003csup\u003e21\u003c/sup\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePreviously, we described a series of bioconjugation reagents based on the 5-hydroxy-1,5-dihydro-2H-pyrrol-2-ones (5HP2O) scaffold (see Fig. 1b, 5), which can be synthesised starting from modified furans in a one-pot synthesis\u003csup\u003e31,32\u003c/sup\u003e. Compared to maleimides, these reagents exhibit an enhanced stability towards hydrolysis and the resulting conjugates proved to be more resistant towards retro-Michael and thiol-exchange reactions\u003csup\u003e31\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eHerein, we leverage the synthetic flexibility of the 5HP2O methodology to create a more robust and versatile method for direct oligonucleotide functionalisation. Inspired by Gothelf\u0026rsquo;s work \u003cem\u003e(vide supra\u003c/em\u003e), we investigated the use of the AON itself as an amine component in the preparative one-pot synthesis of the 5HP2O moiety. This approach provides a direct dual-functionalisation procedure converting AONs into stable multi-reactive ONs ready for further orthogonal downstream modification while avoiding known issues of maleimide and complex linker synthesis.\u0026nbsp;\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003e\u003cstrong\u003eCondition screening for the direct formation of 5HP2Os on amine-oligonucleotides\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the reported 5HP2O synthetic process, a substituted furan is photo-oxidised in the presence of singlet oxygen and methylene blue (MB) in methanol, followed by a reduction with dimethylsulfide (DMS). Lastly, the 5HP2O product gets formed upon addition of an amine catalysed by methylene blue as a redox catalyst (see Fig. 2a).\u003csup\u003e31\u003c/sup\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo evaluate the feasibility of direct modification of an AON with activated furan \u0026ndash; towards a 5HP2O bearing ON \u0026ndash; based on the modular three-step one-pot synthetic procedure, we decided to perform the first two steps as previously described (1 mmol substituted furan, 83 mM, see Supplementary Scheme 1) and then dilute the reaction mixture to a concentration which is relevant for AON modification (40 nmol AON, 100-200 \u0026micro;M). This allows, on the one hand, to use the activated furan (keto-enal) solution as a stock solution and, on the other hand, to screen reaction conditions solely for the relevant third reaction step (see Fig. 2a). It is to note here, that the keto-enal stock solution contains dimethylsulfide (reducing agent) and methylene blue (photosensitizer and redox catalyst). Both reagents, arising from the first two steps, are again required to form the final 5HP2O-modified ON in the third step (see precise mechanism in Supplementary Scheme 1). \u0026nbsp;The aim here was to stick as close as possible to the known conditions, ultimately screening the equivalents of base (triethylamine, Et\u003csub\u003e3\u003c/sub\u003eN), keto-enal stock solution and determining an adequate solvent system compatible with oligonucleotides. Water is well tolerated in the reaction so that a mixture of MeOH:water (9:1 v\\v) was used as the starting point in combination with an overnight reaction.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAn oligonucleotide of 21 nucleobases bearing the terminal 5\u0026rsquo;-amino modifier D1 (see Fig. 3) was chosen as model compound given the similar size of many therapeutic oligonucleotides. HPLC analysis showed that increasing keto-enal and triethylamine amounts lead to a consumption of starting material AON and to the formation of two new peaks in a concentration-dependent manner. The full consumption of the starting material was observed above 800 equiv. of keto-enal and triethylamine, which however, led to the formation of two major products (see Fig. 2b, chrom. i to vi). The first peak could be identified as the desired directly modified 5HP2O-ON (see Fig. 2e+f and Supplementary Fig. 4). The side product could not be identified given the vast plethora of theoretically possible side products (see Supplementary Scheme 2 and Supplementary Fig. 6). \u0026nbsp;Finally, by reducing the amount of keto-enal 16-fold to 50 equiv., the amount of side product could be reduced to nearly zero and mainly conversion to the desired product peak was observed (see Fig. 2b, chrom. vii). The large amount of required base, likely reflects the need of a deprotonated amine and it is furthermore required for the reaction mechanism. Further experiments showed that the amount of keto-enal stock solution (also including MB, DMS) could not be further reduced, as only incomplete AON consumption was observed (see Fig. 2c, chrom. i-ii). Increasing the amount of base even further had no impact on the reaction profile (see Fig. 2c, chrom. iii-iv). Finally, increasing the AON concentration from 100 \u0026micro;M to 200 \u0026micro;M was identified to be very beneficial and 86% yield of the desired product was observed (see Fig. 2c, chrom. vi). In summary, straightforward and efficient direct modification of the AON was achieved with 50 equiv. of keto-enal stock solution, 800 equiv. of triethyvlamine at a AON concentration of 200 \u0026micro;M at room temperature for 30 min in Water:MeOH (1:9 \u003cem\u003ev/v\u003c/em\u003e). Isolated yield of this reaction was 32% on a 40 nmol scale and is on par with isolated yields of NHS-mediated functionalisation using 5HP2O-C5-NHS (see Supplementary Fig. 8). Notably, traditional NHS-mediated maleimide functionalisation leads to a maximum in reaction yield of 70% due to constant maleimide hydrolysis (see Supplementary Fig. 8) and therewith is below the herein observed yield. Furthermore, the keto-enal stock solution is stable for months when stored in the freezer, allowing for plug-and-play modifications of AON (see Supplementary Fig. 5).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNegative controls show no undesired modification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAside from the condition screening, emphasis was set on negative controls to evaluate the possible degradation of the AON under the reaction conditions. Upon incubating the AON in solutions without keto-enal but containing MB, Et\u003csub\u003e3\u003c/sub\u003eN and DMS, degradation was evaluated through HPLC analysis. An onset of degradation is only noticeable when increasing the reagent concentration two-fold and does not occur under the identified reaction conditions (see Fig. 2d, chrom. 1-3). This leads to a window, in which the desired 5HP2O-functionalisation can be performed in the absence of any degradation pathway driven through DMS or MB.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFurthermore, reducing the amount of methanol in the solvent system significantly slows down the formation of 5HP2O on the AON, as shown by HPLC experiments. Evaporation of methanol and DMS followed by redissolving in pure water stops the reaction and therefore proves a successful way to quench the reaction (see Supplementary Fig. 7).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOur group previously reported on the modification of exocyclic amines of nucleobases with activated furan (keto-enal)\u003csup\u003e33,34\u003c/sup\u003e. To exclude any undesired modification of the AON, we performed control experiments with an oligonucleotide, lacking the 5\u0026rsquo;-aminohexyl modification. As hoped, we did not observe any peak shift (see Fig. 2d, chrom. 5-6) nor mass change (see Supplementary Fig. 27+28) of the control reaction.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMoreover, a potential backbone modification with 5HP2O would interfere with or at least influence base pairing to the complementary strand. Therefore, melting assays were performed upon hybridizing the functionalised AON with its complement (see Fig. 2g and Supplementary Fig. 29). No difference in melting temperature between the non-functionalised and directly functionalised oligonucleotide was observed. Contrastingly, functionalisation of the oligonucleotide via a classical NHS-mediated amide coupling (see Supplementary Fig. 8) led to a significant increase in melting temperature. This could be caused by the increased linker length (see Fig. 1), as five extra carbons are introduced. Although we cannot fully explain this phenomenon, it nonetheless demonstrates that linker length and or introduction methodology indeed play a role in ON properties.\u003c/p\u003e\n\u003cp\u003eIn summary, tweaking the reaction conditions to find a window in which the AON can be modified but not degraded was crucial to allow for this AON functionalisation protocol.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA variety of amine modifiers and oligonucleotide backbones are accepted\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWith optimised conditions in hand, we set out to evaluate the substrate scope of the methodology regarding other amino-modified oligomers. Next to the initial model substrate D1 bearing a 5\u0026rsquo;-aminohexyl moiety, three phosphorothioate oligodeoxynucleotides (including multiple LNA modifications) bearing different 5\u0026rsquo;-modifications were investigated. Both, 4-aminomethylbenzoic acid-containing (PAMBA) sequences (D2+3) and an anomeric aminoribose-containing sequence (D4) were well tolerated and full conversion was observed via LCMS (see Supplementary Figs. 11-16). Conveniently, aminoribose D4 in combination with the herein discussed methodology, enables the formation of a Michael acceptor closely located to the natural backbone of an oligonucleotide without introduction of additional carbons. This could be of special interest in applications like DNA-PAINT, reducing a potential linkage error.\u003c/p\u003e\n\u003cp\u003eInternal amino-modification of oligodeoxynucleotides bearing either one (Int1) or two amines (Int2) proceeded equally smoothly, yielding the products with one and two 5HP2Os, respectively (see Supplementary Figs. 17-20).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLastly, we applied the methodology to peptide nucleic acids (PNAs). PNAs feature a neutral peptidic backbone and therefore solubility is a key issue when utilizing or conjugating these oligomers. To circumvent solubility problems, especially when additionally modifying the PNA with hydrophobic linkers (e.g. SMCC, see Fig. 1b), charged amino acids or ethylene glycol spacers are usually introduced into the sequence. We decided to synthesise and evaluate four different PNAs (see Supplementary Figs. 21-24), bearing different modifications next to the free N-terminus. Direct 5HP2O functionalisation on N-terminally introduced charged amino acids (Glu or Arg in P1, P3) was not possible. This could be due to a change in local pH microenvironment leading to a non-reactive protonated amine-PNA or due to steric hindrance. \u0026nbsp;However, formation of 5HP2O on P2 and P4, containing an ethylene glycol spacer after the charged amino acids, was confirmed by a peak shift in HPLC, and the correct product mass could be observed in MALDI-TOF-MS (see Supplementary Figs. 25+26).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStarting with modified furan leads to direct dual-functionalisation of oligonucleotides\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eShowing the broad applicability of this methodology by functionalising eight different primary amine-bearing oligomers (phosphodiester-, phosphorothioate- or peptidic backbone, LNA modifications, internal amines and different terminal amines), we next evaluated the dual-functionalisation of AONs \u0026ndash; a unique approach allowed through the 5HP2O synthetic methodology.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis required the synthesis of substituted furan building blocks, bearing a modified side chain rather than a methyl group. Several clickable handles like a (protected) ketone (MF5 respectively MF6), azide (MF4) and alkyne (MF2) were chosen for incorporation in order to allow for post-labelling by biorthogonal CuAAC reactions or oxime ligations. Moreover, we aimed for commercially available furan propionic acid (MF1) as a precursor for a straightforward synthetic approach to these modified keto-enals. Starting from MF1, all other modified furans were obtained via short synthetic routes (see Supplementary Section 7 and 11), and standard amide coupling showed to be the most straightforward approach to these building blocks (as exemplified by MF2 and MF3). As initially discussed for methylfuran, the synthesised modified analogues were converted to the modified keto-enal by means of a photooxidation followed by reduction in methanol.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eApplying the previously described reaction conditions to an azide-bearing keto-enal (generated from MF4) and AON D1 shows unexpected degradation of the AON. Detailed investigation of different reaction conditions (see Supplementary Fig. 30+31) highlighted the influence of the reaction time and especially of the solvent system. Dual functionalisation with the azide-bearing keto-enal required a longer reaction time (overnight rather than 30 min) and a reduced amount of methanol (50% vs. 90%). Interestingly, no conversion was observed in 20% methanol, showcasing the relevance of the solvent in this functionalisation reaction. This influence of the solvent system is in accordance with the aforementioned results for unmodified keto-enal (see Supplementary Fig. 7). Overnight incubation of AON D1 with the azide-containing keto-enal (see Supplementary Fig. 30) leads to the desired dual-functionalised product in a 60% yield. Employing the alternative alkyne-containing keto-enal generated from MF2 (see Fig. 4c-e and Supplementary Fig. 35) led to a dual-modification yield of 71%. We continued to use this keto-enal as model compound for further bioconjugation and CuAAC labelling. Other dual-functionalisation reactions were analysed by LCMS, obtaining the desired constructs in medium to good yield depending on the building block (see Fig. 4 b and Supplementary Fig. 32-34).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe suspect that the differences in 5HP2O functionalisation yields observed for the different building blocks (especially when compared to methylfuran), arise through a difference in photooxidation efficiency of the modified furans in the first step. These conditions were kept unchanged and are based on the earlier reactions with 2-methylfuran. However, other 2-modified furans might require an increased amount of methylene blue or an increased irradiation time to drive full photooxidation. An extreme case was the ketone-bearing MF5 which led to no conversion, whereas the acetal-protected version thereof (MF6) led to 56% yield. \u0026nbsp;Consequently, we did not investigate subsequent oxime ligations, which would require additional deprotection steps but rather focused on CuAAC labelling. Lastly, it can be stated that the discussed dual-functionalisation yields are around the same level as an NHS-mediated maleimide functionalisation (see Supplementary Fig. 8).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFollow up bioconjugation and multi-functionalisation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter direct 5HP2O\u0026nbsp;functionalisation of AON D1, the conjugation to thiol-bearing model compounds was evaluated. Therefore, purified mono-functionalised 5HP2O-D1-ON was incubated overnight with glutathione (GSH) as a model peptide and with an alphabody\u003c!--[if supportFields]\u003e\u003cspan style='mso-element:field-begin'\u003e\u003c/span\u003e\u003cspan class=msoIns\u003e\u003cins cite=\"mailto:Jan%20Heinrich%20Meffert\" datetime=\"2025-07-30T19:49\"\u003eADDIN CitaviPlaceholder{{"$id":"1","$type":"SwissAcademic.Citavi.Citations.WordPlaceholder, SwissAcademic.Citavi","Entries":[{"$id":"2","$type":"SwissAcademic.Citavi.Citations.WordPlaceholderEntry, SwissAcademic.Citavi","Id":"da740330-52a9-4373-a0c0-28b61c14c0e8","RangeLength":2,"ReferenceId":"9debe18e-e2be-469f-8309-7737dea93ee3","IsNonrecurringReferenceInMultipleCitation":true,"PageRange":{"$id":"3","$type":"SwissAcademic.PageRange, SwissAcademic","EndPage":{"$id":"4","$type":"SwissAcademic.PageNumber, 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Meffert","CreatedOn":"2021-10-07T08:56:26","ModifiedBy":"_Jabme","Id":"9debe18e-e2be-469f-8309-7737dea93ee3","ModifiedOn":"2025-07-23T20:15:14","Project":{"$ref":"8"}},"UseNumberingTypeOfParentDocument":false}],"FormattedText":{"$id":"34","Count":1,"TextUnits":[{"$id":"35","FontStyle":{"$id":"36","Superscript":true},"ReadingOrder":1,"Text":"35"}]},"Tag":"CitaviPlaceholder#11b0cedc-d664-4854-bfa3-98043ba8d65a","Text":"35","WAIVersion":"6.19.2.1"}}\u003c/del\u003e\u003c/span\u003e\u003cspan style='mso-element:field-separator'\u003e\u003c/span\u003e\u003c![endif]--\u003e\u003csup\u003e35\u003c/sup\u003e\u003c!--[if supportFields]\u003e\u003cspan style='mso-element:field-end'\u003e\u003c/span\u003e\u003c![endif]--\u003e protein (see Fig. 5a). The employed alphabody is a triple-helical protein of small size (around 11.5 kDa) targeting Interleukin-23\u003csup\u003e35\u003c/sup\u003e. Reduction and charachterisation was performed as previously described\u003csup\u003e31\u003c/sup\u003e. Both conjugates were obtained in high yields and could be characterised by mass spectrometry (see Fig. 5a-c and Supplementary Fig. 37). It has to be noted here, that the longer reaction time (vs. traditional maleimide chemistry) is not an issue in view of the non-hydrolysable 5HP2O scaffold\u003csup\u003e31\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFinally, to obtain an efficient platform for AON conjugation, we aimed to minimise purification after direct functionalisation. Filtration through a size exclusion column before the addition of cysteine-containing molecules to the functionalised AON proved to be sufficient, yielding the desired conjugate. In combination with an additional alkyne tag, leveraging the functionalised side chain of the 5HP2Os, we were able to obtain a fluorescently labelled glutathione oligonucleotide conjugate in three steps from the readily available AON (see Fig. 5e-h). In another batch, the intermediate GSH-5HP2O(Alkyne)-D1-ON got isolated and also characterised (see Supplementary Fig. 39).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eWe herein present a methodology for the direct modification of amino-modified oligonucleotides with 5HP2Os, which are stable Michael acceptors ready to be employed for bioconjugation purposes. This procedure is based on methylene blue, first acting as a photosensitizer on preparative scale (1 mmol) in the initial singlet oxygen-mediated oxidation of 2-substituted furans and subsequently - upon dilution - acting as a redox catalyst during the formation of 5HP2Os on the AON (40 nmol scale). The here described methodology extends the currently available methods for cumbersome preparation of “bioconjugation-ready” oligonucleotides, which mainly relies on synthesis of heterobifunctional linkers followed by NHS- or phosphoramidite-mediated ON modification. Several common amine modifiers (aminohexyl, PAMBA) and backbones (PS, PO and PNA) are readily accepted at different positions within the oligonucleotide, and full conversion with nearly no degradation is seen in short reaction times (30 min). Synthesis of modified furan building blocks followed by their photooxidation allows for the introduction of an extra reactive handle (e.g. azide and alkyne) into the AON aside the Michael acceptor. Both the theoretical yields and the isolated yields are comparable to standard linker-based conjugations. Ready-to-use stock solutions of activated furan derivatives were shown to be stable, allowing for a plug-and-play modification of AON depending on the desired application. Finally, the possibility of follow-up bioconjugation in combination with additional follow-up click chemistry greatly simplifies the generation of more complex peptide/protein oligonucleotide conjugates, which are quickly becoming important constructs in advanced therapeutic or diagnostic applications such as targeted oligonucleotide delivery or DNA-PAINT.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eInformation on the synthesis and characterisation of small molecules and oligonucleotides can be found in the Supporting Information of this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eActivation of 2-substituted furan towards substituted keto-enal stock solution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn a test tube with 10 mL methanol was added 2-substituted furan (1 mmol) and catalytic amounts of methylene blue (0.2 mol%, 0.64 mg, 0.002 mmol). Pressured air was bubbled through the solution while it got irradiated for 10 minutes at room temperature using the indicated lamp (see Supporting Information Section 1.1). The reaction mixture was then transferred into a round-bottomed flask, DMS (4.0 equiv, 300 µL) and Et\u003csub\u003e3\u003c/sub\u003eN (4.2 µL, 0.03 mmol) were added and the flask was sealed for further 40 minutes at room temperature. Subsequently, additional methylene blue (2.0 mol%, 6.4 mg, 0.02 mmol) was added and the keto-enal stock solution was stored in the freezer after concentration adjustment to 50 mM.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDirect functionalisation of AON via activated 2-methylfuran\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDirect oligonucleotide functionalisation was performed at 200 µM AON concentration in water:methanol (1:9 v/v, 100 µL) for 30 minutes at room temperature. Therefore, to an amber screw-cap HPLC vial was added in the following order: methanol, triethylamine (800 equiv., c\u003csub\u003efin\u003c/sub\u003e = 80 mM), keto-enal stock solution (50 equiv., c\u003csub\u003efin\u003c/sub\u003e = 5 mM) and amine-oligonucleotide (1 equiv., c\u003csub\u003efin\u003c/sub\u003e = 200 µM). The reaction was incubated for 30 min at room temperature before concentration under reduced pressure and redissolving in water. Reactions were either purified by RP-HPLC, or by SEC-spin columns (MicroBio Spin 6 kDa cutoff).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDirect Dual-functionalisation of AON via activated 2-substituted furan\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDirect dual oligonucleotide functionalisation was performed at 200 µM AON concentration in water:methanol (1:1 v/v, 100 µL). Therefore, to an Eppendorf vial was added in the following order: methanol, water, triethylamine (800 equiv., c\u003csub\u003efin\u003c/sub\u003e = 80 mM), keto-enal stock solution (50 equiv., c\u003csub\u003efin\u003c/sub\u003e = 5 mM) and amine-oligonucleotide (1 equiv., c\u003csub\u003efin\u003c/sub\u003e = 100 µM). The reaction was shaken overnight at 37 °C before concentration under reduced pressure and redissolving in water. Reactions were either purified by RP-HPLC, or by SEC-spin columns (MicroBio Spin 6 kDa cutoff).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFollow-Up Bioconjugation of 5HP2O-functionalised oligonucleotides\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBioconjugation of functionalised oligonucleotide was usually performed at 40 µM 5HP2O-ON concentration in buffer (Carmody buffer pH 9 or 10, 100 µL), overnight at 37 °C. Therefore, to an Eppendorf vial was added in the following order: buffer, 5HP2O-ON and cysteine-containing peptide or protein. Glutathione (40 equiv., c\u003csub\u003efin\u003c/sub\u003e = 1.6 mM) was added without prior reduction from a freshly prepared stock solution. In contrast, alphabody protein (10 equiv., c\u003csub\u003efin\u003c/sub\u003e = 0.4 mM) was pre-treated with TCEP (5 equiv. in regard to the protein) at 400µM for 30 min at room temperature. Subsequently, the solution was buffer exchanged (Carmody buffer pH 10) and the excess TCEP was removed using a MicroBio Spin (6 kDa cutoff) before its addition to the 5HP2O-ON.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFollow-Up CUAAC Labelling\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo the crude bioconjugation reaction mixture was added in the following order: NBD-azide (40 equiv, c\u003csub\u003efin\u003c/sub\u003e = 1.6 mM), THTPA:CuSO\u003csub\u003e4\u003c/sub\u003e (25 equiv., pre-incubation for 5 min in a ratio 1:2 CuSO\u003csub\u003e4\u003c/sub\u003e:THTPA at 50 mM CuSO\u003csub\u003e4\u003c/sub\u003e, c\u003csub\u003efin\u003c/sub\u003e = 1.6 mM), sodium ascorbate (40 equiv., c\u003csub\u003efin\u003c/sub\u003e = 1.6 mM). The reaction was incubated for 30 minutes at room temperature before purification by RP-HPLC.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis project has received funding from the European Union\u0026rsquo;s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie Grant Agreement No. 956070 (OLIGOMED) (J.M., M.L., A.M.). E.C. acknowledges a FWO fellowship (12B1923N). We thank Dr. Chloe Howells and Prof. Eugen Stulz for LCMS measurement of crude functionalisation reactions. We thank Pieter Surmont and Prof. Frederic Lynen for HRMS Orbitrap measurement of\u0026nbsp;purified oligonucleotide constructs. We thank Jan Goeman for LCMS measurements and Stijn Tanghe for general lab support. We thank the NMR expertise centre (Ghent University) for providing support and access to its NMR infrastructure. The 400 MHz NMR used in this work has been funded by a grant/project of the Research Foundation Flanders (FWO I006920N) and the \u0026lsquo;Bijzonder Onderzoeksfonds\u0026rsquo; (BOF.BAS.2022.0023.01).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJan H. Meffert was responsible for the overall conceptualization of the study, designed and conducted all experiments including condition screening, oligonucleotide mono- and dual-functionalisation, product analysis and small molecule synthesis, and wrote the manuscript, with support from the other authors. Enrico Cadoni provided PNA constructs (P1 to P4). M\u0026oacute;nica Lopes, Martin Bollmark and UIf Tedebark provided phosphorothioated amine oligonucleotides (D2-4). Annemieke Madder was responsible for the funding and overall guidance, contributed to the conceptualisation and assisted in manuscript writing, proofreading and correction.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnnemieke Madder has a pending patent application relating to 5HP2Os application for bioconjugation (Bioconjugation reagent and methods, WO2020174086A2, Annemieke MADDER, Ewout DE GEYTER, Eirini ANTONATOU, Sabina SMOLEN, Dimitris Kalaitzakis, Georgios VASSILIKOGIANNAKIS, Europe\u0026amp;US). All other authors declare no competing interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eJiao, J. \u003cem\u003eet al.\u003c/em\u003e Overcoming Limitations and Advancing the Therapeutic Potential of Antibody-Oligonucleotide Conjugates (AOCs): Current Status and Future Perspectives. \u003cem\u003ePharmacol Res\u003c/em\u003e, 107469 (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChung, K. K. 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Sequence specific DNA cross-linking triggered by visible light. \u003cem\u003eJ Am Chem Soc\u003c/em\u003e 134, 10737\u0026ndash;10740 (2012).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDesmet, J. \u003cem\u003eet al.\u003c/em\u003e Structural basis of IL-23 antagonism by an Alphabody protein scaffold. \u003cem\u003eNat Commun\u003c/em\u003e 5, 5237 (2014).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"bioconjugation, oligonucleotide conjugates, oligonucleotide functionalisation, dual-functionalisation, 5HP2O, maleimide alternative ","lastPublishedDoi":"10.21203/rs.3.rs-7265337/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7265337/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe optimisation and further expansion of methods for the synthesis of oligonucleotide conjugates is receiving increased attention due to their importance for further advancement of therapeutic and diagnostic nucleic acid-based applications. Current methodologies, particularly those relying on maleimide-type linkers, are often hampered by multi-step synthetic procedures and linker instability. Herein, we present a versatile method for the direct functionalisation of readily available amino-modified oligonucleotides (AONs), where a 5-hydroxy-1,5-dihydro-2H-pyrrol-2-ones (5HP2O) Michael acceptor is directly formed in a rapid and efficient manner on a free primary amine. The methodology demonstrates broad applicability, tolerating various amino-modifiers and their positions within different oligonucleotide types, including DNA, LNA, PNA, and phosphorothioate-modified oligonucleotide strands. Most importantly, the possibility to introduce an additional second orthogonal reactive handle uniquely enables a direct dual-functionalisation of AONs for the assembly of complex constructs, as exemplified by the synthesis of a fluorescent peptide-oligonucleotide construct.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e","manuscriptTitle":"Direct Dual-Functionalisation of Amine-Oligonucleotides for Conjugation Purposes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-13 14:37:05","doi":"10.21203/rs.3.rs-7265337/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-chemistry","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commschem","sideBox":"Learn more about [Communications Chemistry](http://www.nature.com/commschem/)","snPcode":"","submissionUrl":"","title":"Communications Chemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a6141662-5afd-4901-a0e1-858bf6d8aefa","owner":[],"postedDate":"August 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":52819036,"name":"Physical sciences/Chemistry/Chemical biology/Chemical modification"},{"id":52819037,"name":"Physical sciences/Chemistry/Organic chemistry/Synthetic chemistry methodology"}],"tags":[],"updatedAt":"2026-02-12T08:11:59+00:00","versionOfRecord":{"articleIdentity":"rs-7265337","link":"https://doi.org/10.1038/s42004-025-01882-8","journal":{"identity":"communications-chemistry","isVorOnly":false,"title":"Communications Chemistry"},"publishedOn":"2026-01-10 05:00:00","publishedOnDateReadable":"January 10th, 2026"},"versionCreatedAt":"2025-08-13 14:37:05","video":"","vorDoi":"10.1038/s42004-025-01882-8","vorDoiUrl":"https://doi.org/10.1038/s42004-025-01882-8","workflowStages":[]},"version":"v1","identity":"rs-7265337","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7265337","identity":"rs-7265337","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}