Fully deprotected alkynyl-functionalised mono- and oligosaccharides undergo CuAAC-based conjugation with water-soluble porphyrin azides in aqueous environments. The mild reaction conditions are fully compatible with the presence of labile glycosidic bonds. This approach provides an ideal strategy to conjugate tetrapyrroles to complex carbohydrates

Boyle, Ross W.

Bryden, Francesca

Daly, Robin

Giuntini, Francesca

Scanlan, Eoin M.

Organic & Biomolecular Chemistry

English

Repository@Hull - Worktribe

Organic &Biomolecular ChemistryCOMMUNICATIONCite this: Org. Biomol. Chem., 2014,12, 1203Received 21st November 2013,Accepted 23rd December 2013DOI: 10.1039/c3ob42306awww.rsc.org/obcHuisgen-based conjugation of water-solubleporphyrins to deprotected sugars: towards mildstrategies for the labelling of glycans†Francesca Giuntini,‡a Francesca Bryden,a Robin Daly,b Eoin M. Scanlanb andRoss W. Boyle*aFully deprotected alkynyl-functionalised mono- and oligosacchar-ides undergo CuAAC-based conjugation with water-soluble por-phyrin azides in aqueous environments. The mild reactionconditions are fully compatible with the presence of labile glycosi-dic bonds. This approach provides an ideal strategy to conjugatetetrapyrroles to complex carbohydrates.IntroductionAdvances in the field of functional glycomics shed light on thestructural and functional diversity of carbohydrates exposedon the cell membrane, and their fundamental role in a varietyof biological processes, including protein folding, self-recog-nition, cell migration, modulation of signalling pathways, andtrafficking.1,2 The weak but highly specific binding of surface-exposed glycans with carbohydrate-binding proteins (e.g.lectins) underpins diverse phenomena such as immunesystem modulation, pathogen invasion, cell migration and pro-liferation. Variations in the structure, expression, or bindingaffinity of glycans and glycoproteins have been associated withthe state and invasiveness of neoplastic lesions3,4 and non-malignant diseases,5 and with bacterial virulence and invasionof host tissues,6,7 as a consequence, the biochemistry ofglycan–lectin interactions has been object of investigation as apotential route for the identification of new therapeutictargets, and the development of expeditious diagnosticmethods.8–11Recently, the possibility of exploiting glycan–lectin inter-actions for analytical and sensing purposes has also received agreat deal of attention, with a consistent body of datademonstrating how viruses, bacterial strains, and bacterialspores can be detected by exploiting the specificity of glycan–lectin interactions. Many high-throughput strategies to probeglycan–lectin interactions have been devised, the majority ofwhich are based on fluorescence-assisted arrays, renewing theinterest in the synthesis of fluorescently labelled glycans andlectins.12–17 While labelling of proteins is readily achievable,the derivatisation of glycans remains less explored. Thecomplex structure of glycans and the recurring presence oflabile moieties (e.g. sialic acid)18 pose a challenge to theirchemical modification, and although various labelling strat-egies have been reported, a versatile and reliable approachremains to be devised.13,19,20Following our current interest in mild bio-orthogonalapproaches for the conjugation of tetrapyrrolic photosensiti-sers to macromolecules,21–24 we undertook a study aimed atidentifying a suitable method for the conjugation of porphyr-ins to glycans. A straightforward ligation strategy to labelglycans with porphyrins would not merely afford new lumines-cent lectin markers for in vitro assays, but could lead to photo-therapeutic agents targeted to cells over-expressing lectins, toviruses, and to bacteria organised in biofilms. Mannose con-taining glycopeptides labelled with fluorescein have previouslybeen used in cellular immunology screens for the developmentof carbohydrate vaccines.25 Endowed with high absorptioncoefficients and high luminescence and singlet oxygenquantum yields, porphyrins have found a variety of appli-cations in the biomedical field.26 Their versatility towardschemical modification, which allows the tuning of the mole-cule’s physico-chemical properties, contributed to make thesespecies excellent candidates as photodynamic therapy drugs,reporters for fluorescent diagnosis, and investigative tools.The association of porphyrins with carbohydrates has beenwidely explored with the purpose of obtaining water-soluble ortargeted species.27 The synthesis of porphyrin–carbohydrateconjugates is however hampered by their conflicting solubility,which often imposes the use of solvent mixtures, high temp-eratures, or alternatively requires the use of protectedcarbohydrates.28–34†Electronic supplementary information (ESI) available. See DOI:10.1039/c3ob42306a‡Present address: School of Pharmacy and Biomolecular Sciences, LiverpoolJohn Moores University, Liverpool L3 3AF, UK.aDepartment of Chemistry, University of Hull, Kingston-upon-Hull, East YorkshireHU6 7RX, UK. E-mail: R.W.Boyle@hull.ac.ukbSchool of Chemistry, Trinity Biomedical Sciences Institute, Trinity College, 152-160Pearse Street, Dublin 2, IrelandThis journal is © The Royal Society of Chemistry 2014 Org. Biomol. Chem., 2014, 12, 1203–1206 | 1203Open Access Article. Published on 02 January 2014. Downloaded on 27/08/2015 12:21:33.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article OnlineView Journal  | View IssueWhile these approaches prove efficient for the synthesis ofporphyrin–monosaccharides conjugates, mild methods thatpreserve the integrity of labile polysaccharides remain substan-tially unavailable.In a previous study23 we showed that water-soluble por-phyrin-azides can be conjugated to polyacrylamide nanoparti-cles in aqueous environment and in mild conditions, bycopper-catalysed azide–alkyne cycloaddition (CuAAC).35,36 Wereasoned that, in a similar way, CuAAC could provide theoptimal ligation chemistry for conjugation of porphyrins toglycans. CuAAC has proved to be an efficient tool for the conju-gation of porphyrins to peptides,37 polymers, and carbo-hydrates;30,32,34,38 furthermore, as a result of intenseinvestigations recently carried out in the field, the feasibility ofthis approach is facilitated by the improved synthetic accessi-bility of azide-containing porphyrins22,23,39 and alkynyl-carbohydrates.40We first assessed the efficiency of the cycloaddition usingwater-soluble porphyrin 1 and a simple deprotected monosac-charide bearing a terminal alkyne. Porphyrin 1 was syn-thesised from 5-(4-aminophenyl)-10,15,20-triphenylporphyrin,which was converted into the corresponding azide by treat-ment with sodium nitrite and sodium azide in water, accord-ing to a modified literature procedure.39 The resultingporphyrin-azide was treated with iodomethane, to afford thewater-soluble tricationic species. In order to avoid sequestra-tion of copper ions by the tetrapyrrole during the cyclo-addition, the porphyrin free base were converted into thecorresponding zinc complex.23Treatment of an aqueous solution of porphyrin 1 with1-α-propargyloxy mannose 439 in the presence of CuSO4·5H2Oand sodium ascorbate at room temperature, led to the com-plete conversion of 1 into cycloadduct 11 within 15 minutes.(Scheme 1, Table 1).Isolation of the desired product from the reaction mixturewas achieved by treating the reaction mixture with NH4PF6,which allowed the recovery of the water-insoluble hexafluoro-phosphate salt of the porphyrin by filtration, and subsequentconversion of the hexachlorophosphate salt into the corres-ponding trichloride by treatment of with tetra-n-butyl-ammonium chloride in acetone.23Encouraged by this positive result, we investigated the appli-cability of the conjugation to different deprotected carbohydrateand porphyrin substrates (Fig. 1). Gratifyingly, 1-β-propargyloxy-monosaccharides 5 and 6 and 1-β-propargyloxy-N-acetyl-lactosamine 7 underwent CuAAC with 1 to afford, respectively,adducts 10, 12 and 17 in excellent yields, and negativelycharged porphyrin 4 reacted with 1-α-propargyloxy mannose 4under the conditions described above, to afford the desiredconjugates in equally good yield (Table 1). Similarly, the pres-ence of a PEG chain as a spacer between the porphyrin andthe azide group did not affect the conjugation efficiency, asshown by the formation of cycloadducts 14–16 in high yieldsfrom the reaction of porphyrin 3 with 1-α-propargyloxy-hexoses4–6.39 The excellent yields indicate that our approach has thepotential of becoming a reliable bioconjugation tool of generalapplicability.We next proceeded to verify the compatibility of the ligationconditions with the presence of labile glycosidic bonds: wechose propargyloxy-bearing α-Fuc-(1–6)-GlcNAc 8 and Lewisxtrisaccharide 941 as models for a glycan substrate, as the linkα-(1–6) is known to be unstable to acidic and basic conditions,and high temperatures. When porphyrin 1 was treated with 8or 9 under the conditions reported above, the desired cycload-ducts were isolated (Table 2), and the spectroscopic dataScheme 1 CuAAC conjugation of porphyrin-azide 1 to 1-α-propargyl-mannose. Reaction conditions: 1 (1 equiv.), 4 (1.5 equiv.), CuSO4·5H2O(0.2 equiv.), Na ascorbate (0.5 equiv.), room temperature, 15 min.Table 1 Synthetic scope of the CuAAC-based cycloaddition: reactionof porphyrin-azides with 1-propargyloxy-monosaccharidesEntry R Ar Yield (%)10 9111 9312 9113 6314 9615 9316 97Communication Organic & Biomolecular Chemistry1204 | Org. Biomol. Chem., 2014, 12, 1203–1206 This journal is © The Royal Society of Chemistry 2014Open Access Article. Published on 02 January 2014. Downloaded on 27/08/2015 12:21:33.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article Onlineconfirmed that the oligosaccharide moieties were intact inboth 18 and 19. To the best of our knowledge, this is the firstreport of the conjugation of a porphyrin to a totally depro-tected α-Fuc-(1–6)-GlcNAc and Lewisx trisaccharide; the integ-rity of the labile link α-(1–6) in the conjugates further confirmsthe suitability this of ligation method for glycan labelling.ConclusionsIn conclusions, we devised a CuAAC-based method for the lig-ation of water-soluble azide-bearing porphyrins to alkyne-bearing fully deprotected carbohydrates. The reaction takesplace in aqueous solution and affords the desired conjugatesin good-to-excellent yields, and involves minimal manipulationfor the isolation of the cycloaddition product. Variations of thestructure of the carbohydrate and of the porphyrin are welltolerated and do not impair the efficiency of the reaction. Lig-ation of porphyrins to totally deprotected carbohydrates con-taining the labile α-(1–6) glycosidic bond, a recurrent featurein the structure of glycans, are achievable with this method. Itis envisaged that this approach will complement the recentchemo-enzymatic strategies for glycan modification.42Although it did not represent an issue in the present work, thepresence of the copper ion is certainly a caveat for any CuAAC-based labelling strategy to be carried out in biological environ-ment: we anticipate that the new porphyrins reported here willbe equally reactive under copper-free conditions,43 which hasalready been successfully applied to cyclooctyne-functionalisedcarbohydrate substrates.44 The application of this approach tomore complex carbohydrates is the object of current studies inour laboratories.AcknowledgementsThe authors thank EPSRC for funding the project (EP/H000151/1). Mass spectrometry data was acquired at theEPSRC UK National Mass Spectrometry Facility at SwanseaUniversity.Notes and references1 J. Li and J. Richards, in Functional Glycomics, ed. J. Li,Humana Press, 2010, vol. 600, pp. 1–8.2 G. W. Hart and R. J. Copeland, Cell, 2010, 143, 672–676.3 C. 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Downloaded on 27/08/2015 12:21:33.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article Online

Huisgen-based conjugation of water-soluble porphyrins to deprotected sugars: Towards mild strategies for the labelling of glycans

Fully deprotected alkynyl-functionalised mono- and oligosaccharides undergo CuAAC-based conjugation with water-soluble porphyrin azides in aqueous environments.</p

Francesca Giuntini

Francesca Bryden

Robin Daly

Eoin M. Scanlan

Ross W. Boyle

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Huisgen-based conjugation of water-soluble porphyrins to deprotected sugars: towards mild strategies for the labelling of glycans

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Huisgen-based conjugation of water-soluble porphyrins to deprotected sugars: Towards mild strategies for the labelling of glycans

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