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A Flow Platform for Degradation-Free Cuaac Bioconjugation

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A Flow Platform for Degradation-Free Cuaac Bioconjugation ARTICLE DOI: 10.1038/s41467-018-06551-0 OPEN A flow platform for degradation-free CuAAC bioconjugation Marine Z.C. Hatit1, Linus F. Reichenbach1, John M. Tobin2, Filipe Vilela 2, Glenn A. Burley 1 & Allan J.B. Watson 3 The Cu-catalyzed azide-alkyne cycloaddition (CuAAC) reaction is a cornerstone method for the ligation of biomolecules. However, undesired Cu-mediated oxidation and Cu- 1234567890():,; contamination in bioconjugates limits biomedical utility. Here, we report a generic CuAAC flow platform for the rapid, robust, and broad-spectrum formation of discrete triazole bio- conjugates. This process leverages an engineering problem to chemical advantage: solvent- mediated Cu pipe erosion generates ppm levels of Cu in situ under laminar flow conditions. This is sufficient to catalyze the CuAAC reaction of small molecule alkynes and azides, fluorophores, marketed drug molecules, peptides, DNA, and therapeutic oligonucleotides. This flow approach, not replicated in batch, operates at ambient temperature and pressure, requires short residence times, avoids oxidation of sensitive functional groups, and produces products with very low ppm Cu contamination. 1 Department of Pure and Applied Chemistry, University of Strathclyde, 295 Cathedral Street, Glasgow G1 1XL, UK. 2 Chemical Sciences, Heriot-Watt University, Edinburgh EH14 4AS, UK. 3 School of Chemistry, University of St Andrews, North Haugh, St Andrews KY16 9ST, UK. Correspondence and requests for materials should be addressed to G.A.B. (email: [email protected]) or to A.J.B.W. (email: [email protected]) NATURE COMMUNICATIONS | (2018) 9:4021 | DOI: 10.1038/s41467-018-06551-0 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-06551-0 he Cu-catalyzed azide-alkyne cycloaddition (CuAAC) of a copper tube with the formation of a highly active CuAAC reaction (Scheme 1a) is a method of widespread utility catalyst under laminar flow conditions. This enables the facile T – throughout medicinal chemistry, chemical biology, and the formation (tR ca. 1 10 min) of discrete ligation products and material sciences1–6. The pervasiveness of this methodology can bioconjugates not possible using conventional batch conditions. be attributed to the rapid, chemo- and regiospecific generation of Significantly, the level of Cu present in products is well below the 1,2,3-triazole products and bioconjugates. reported mammalian cellular toxicity thresholds (e.g., <20 μM for A significant limitation of the CuAAC reaction conducted DNA)5,11,24 with no associated oxidative damage observed on a under batch conditions is the need for a Cu catalyst; this can be series of representative labile biomolecules, including peptides problematic in a number of applications5,6. Cu-mediated oxida- and DNA strands. tive damage of sensitive functional groups can result in product mixtures, which may complicate purification or lead to issues with bioassays due to the need for deconvolution of data or Results unknown pharmacology (Fig. 1b). In biomolecule tagging Reaction design. Flow-based technologies offer distinct advan- CuAAC modification of azide/alkyne biomolecules requires tages over batch, such as enhanced mass transfer, which is par- (super)stoichiometric loadings of Cu catalyst due to the presence ticularly advantageous for large molecular weight biomolecules of a number of Cu-chelating sites (e.g., N/S sites of peptides7,N7 where accessibility of functional groups is significantly compro- of purines in nucleic acids8), which can result in catalyst inhibi- mised in the batch regime25–29. Despite these advantages, appli- tion and the need for higher concentrations of Cu in the reaction cation of flow-based CuAAC bioconjugation has not been (Fig. 1b)6. In addition, oxidative damage of biomolecules is a reported due to the need for (i) excess Cu catalyst, which pro- significant issue associated with current CuAAC-based bio- – motes biomolecule degradation, (ii) ionic scavengers, which can conjugation strategies, severely limiting development9 11. These result in residual Cu trapped in bioconjugates, (iii) elevated issues have inspired the development of a series of alternative Cu- temperatures, which promotes biomolecule degradation, and (iv) free click approaches such as strain-promoted azide-alkyne organic solvents, which typically limits biocompatibility. This has cycloadditions (SPAAC)12 and inverse electron demand Diels- limited flow CuAAC applications to small molecules and pre- Alder (IEDDA) approaches using tetrazines13. Despite their vented the widespread development of flow-assisted synthesis of moderate to fast kinetics14, these processes have their own issues; discrete bioconjugates30–41. for example, lacking the chemo- and regiospecificity afforded by Whilst elemental Cu is an effective catalyst for flow CuAAC, the CuAAC reaction due to the reactive (electrophilic) nature of elevated temperature and pressures are required, likely in order to 15–21 the requisite cyclic alkynes/alkenes , which are susceptible to solubilize some Cu in the eluent. However, H2O/organic mixtures side reactions with nucleophilic residues (e.g., thiol residues in are extremely effective and biocompatible solvent mixtures for glutathione). Furthermore, the installation of these large lipo- CuAAC-mediated bioconjugation. In addition, the surface of Cu philic groups has a significant impact on the overall physico- pipes is typically covered in a protective oxide layer, which are chemical properties of the bioconjugate (Fig. 1c)22. generally poorly soluble in organic solvents but more soluble in Whilst efforts have been made to overcome the oxidation and H2O. Indeed, erosion of Cu tubes with H2O is a well-studied Cu contamination issues of the CuAAC reaction by the develop- engineering phenomenon, with Cu leaching a known problem in ment of bespoke ligands, conducting these reactions under anae- flow chemistry31. robic conditions, the addition of oxidation inhibitors, and Cu Based on this, we hypothesized that an aqueous/organic scavengers, these issues extend from the requirement for high [Cu] mixture (for example, H2O/MeCN) would offer a blend of to overcome slow catalytic turnover as a result of the numerous sufficient solubility of the (bio)organic components while Lewis-basic groups typically found in proteins and nucleic acids23. promoting controlled erosion of surface Cu salts under laminar Here we describe the development of a rapid flow-assisted flow conditions, with in situ Cu(I)/Cu(II) disproportionation CuAAC reaction that overcomes these problems (Fig. 1d). Our providing the mechanistically essential Cu(I) required for the operationally simple strategy couples solvent-induced erosion CuAAC reaction. Whilst the level of solubilized Cu was likely to a The CuAAC reaction b Cu-mediated oxidative damage and substrate ligation Oxidation N Cu N Cu Cu(I) O N N N Cu 3 NH Oxidation N NH O H Alkyne Azide Triazole N N RO O N NH N 2 H Chemoselective transformation using non-biological functional groups - bioorthogonal O O HS Regiospecific formation of 1,4-disubstituted 1,2,3-triazoles Cu Broad application in chemical, biological, and material sciences RO OR Cu Cu Cu Cu Oxidation cdAlternative azide ‘click’ processes: e.g., SPAAC Flow CuAAC via solvent-mediated Cu-erosion N N N N Nuc N 3 N3 N N Nuc R R e.g., thiol R Solvent-induced Cu erosion Enhanced CuAAC kinetics vs. batch Undesired Strained alkyne No substrate oxidation, low ppm Cu in products by-products Regioisomers Ambient temperature and pressure, short residency times Fig. 1 Azide-alkyne cycloaddition strategies. a The archetypical CuAAC reaction; b Examples of oxidatively labile and ligating functional groups found in biomolecules; c The SPAAC reaction and and the formation of regioisomeric triazole products; d The flow-assisted CuAAC reaction (this work). CuAAC Cu-catalyzed azide-alkyne cycloaddition, Nuc nucleophile, SPAAC strain-promoted azide-alkyne cycloaddition 2 NATURE COMMUNICATIONS | (2018) 9:4021 | DOI: 10.1038/s41467-018-06551-0 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-06551-0 ARTICLE a 22,000.0 110 Effect of added H2O on flow CuAAC reaction efficiency and Cu leaching Cu, RT, 1 atm 20,000.0 100 N 18,000.0 90 N Bn N 16,000.0 80 3 N Bn 14,000.0 70 12,000.0 60 1a-c2 MeCN/H2O 3a-c 1 atm, 1 mL/min 10,000.0 50 8000.0 40 Cu] (ppb or ug/L) 63 [ 6000.0 30 Me N 4000.0 20 O product to CuAAC Conversion 2000.0 10 Me N 0.0 0 Me 100/0 99/1 98/1 95/1 90/1 80/1 60/1 40/1 5/1 1/1 1/5 1/40 0/100 1a 1b 1c MeCN/H2O ratio Average copper leaching Conversion 1a to 3a Conversion 1b to 3b Conversion 1c to 3c bc Cu erosion as a function of H2O: reactor vs. control Batch vs. flow comparison of CuAAC reaction efficiency using 14 ppm Cu N 20,000 Me N N N N 18,000 N [Cu] Flow: 94% (tR = 10 min) Bn N Bn 16,000 Me N 3 Flask: 53% (72 h) 14,000 1a 2 Me 3a 12,000 Me 10,000 8000 O [Cu] O N Flow: 90% (tR = 10 min) Cu] (ppb or ug/L) Bn N N 63 6000 3 [ Flask: 0% (72 h) N 4000 1b 2 3b Bn 2000 N 0 [Cu] N Flow: 88% (t = 10 min) 100/0 99/1 98/1 95/1 90/1 80/1 60/1 40/1 5/1 1/1 1/5 1/40 0/100 Bn N Me R 3 N Flask: 0% (72 h) MeCN/H2O ratio Bn Used reactor Control reactor Me 1c 2 3c Fig. 2 Development of a flow CuAAC process based on H2O Cu erosion. a The effect of H2O on the CuAAC reaction efficiency using three representative alkynes; b Correlation of [Cu] vs. solvent composition ((H2O]); c Demonstration of the flow effect—14 ppm Cu CuAAC reactions in flow and in flask be very low; the increased circulation established under the flow Scope of the flow platform.
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