Beyond Traditional SPAAC: Achieving Orthogonality and Rapid Kinetics with Fluoroalkyl Azides

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However, its relatively slow kinetics and limited selectivity hinder the simultaneous labelling of multiple targets. Here, we report on a systematic study of fluoroalkyl azides as novel SPAAC reagents that display enhanced reactivity with electron-rich cyclooctynes, while showing significantly reduced reactivity with electron-deficient dipolarophiles. Kinetic measurements revealed over 100-fold rate differences depending on the azide–alkyne pair, enabling orthogonal bioconjugation in both purified proteins and living cells. Experimental and computational data support the rate trends and mechanistic rationale based on electronic complementarity. Fluorescently labelled fluoroalkyl and alkyl azide probes demonstrate the selective labeling of modified antibodies and lectin protein in vitro and the selective labelling of organelles in living cells. This dual-selectivity strategy enables orthogonal SPAAC labeling. Physical sciences/Chemistry/Organic chemistry/Synthetic chemistry methodology Physical sciences/Chemistry/Biochemistry/Chemical modification azide fluorine click reaction bioconjugation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Bioorthogonal reactions are reactions that do not interfere with biological systems. 1 – 3 The reactants of such reactions must react under biocompatible conditions selectively and at high reaction rates. Moreover, both the starting materials and products must be inert towards functional groups commonly present in biomolecules and non-toxic to the organism. Despite these stringent requirements, however, bioorthogonal reactions have found broad applications, 4 ranging from bioconjugations 5 and bioimaging 6 to drug delivery. 7 One of the most popular bioorthogonal reactions is strain-promoted azide-alkyne cycloaddition (SPAAC). 8 In this variant of the Huisgen cycloaddition, 9 an azide reacts with a strained cycloalkyne to form a triazole (Scheme 1 ). The strain release during the cycloaddition allows the reaction to be performed at ambient temperature and, unlike copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), does not require a metal catalyst. The most broadly utilized cycloalkynes are the cyclooctynes DIBAC 10 and BCN, 11 whereas DIFO 12 is not easily available and BARAC 13 and cycloheptyne TMTH 14 suffer from limited stability. An additional complication arising from the use of asymmetric reagents (such as DIBAC) is the formation of isomers of products. As for azide reagents, structurally diverse aromatic and aliphatic azides have been evaluated and the reaction has found applications in chemical biology, bioconjugation, and material science. SPAAC reactions are relatively slow, with reaction rate constants typically in the range of 0.01–1 M – 1 ·s – 1 . 15 However, the reaction rate of the click reaction with benzyl azide is affected by the structure of cyclooctyne and it has been shown that a few-fold rate increase can be achieved when switching from DIFO or BCN to the more electron-deficient DIBAC (Table 1). 10 , 11 , 16 – 18 Comparison of the reactivity of various azides with BCN and DIBAC has revealed that a judicious choice of azide structure can speed up the SPAAC reaction with electron-rich BCN up to 10-fold when switching from the electron-rich benzyl azide to the electron-deficient 4-azidopyridine (a further 3-fold increase was achieved with 4-azido-1-methylpyridinium salt) (Table 2). 19 However, azidopyridines or their salts are not typically used as Table 1. Absolute rate constants of SPAAC reactions of benzyl azide with various cyclooctynes. Table 2. Absolute rate constants of SPAAC reactions of azides with cyclooctynes. labelling reagents. Nevertheless, electron-poor DIBAC reacted the fastest with benzyl azide and 2-azido-1,3-diisopropylbenzene, showcasing the importance of both elec- tronic and steric factors. These differences in reaction rate were then applied in the three-component labelling of a BCN-labelled protein with a diazide and a DIBAC-labelled fluorescent dye. 19 We have developed a new group of organic alkyl azides featuring a difluoro substitution in the α-carbon position (RCF 2 N 3 ). 20 , 21 These fluoroalkyl azides have been shown to undergo CuAAC efficiently 22 – 26 as well as nitrene formation for the synthesis of N -trifluoromethyl-aziridines (from CF 3 N 3 ). 27 An additional benefit of fluorinated azides compared to alkyl azides is their improved stability. For example, CF 3 N 3 is a highly thermally stable molecule, and even N 3 CF 2 N 3 is quite stable 28 compared to the explosive N 3 CH 2 N 3 . Whilst there is one example of the usage of a fluorinated azide derived from azidodifluoroacetic acid bound to a uridine phosphate amino sugar in SPAAC, 29 no systematic research of the reactivity of fluorinated azides and their applicability in bioorthogonal labelling has been conducted to date. Due to the strong electronegativity of fluorine atoms, fluorinated azides are expected to react preferentially with electron-rich cyclooctynes whereas alkyl azides should react faster with electron-deficient cyclooctynes. If the difference in reactivity is sufficiently high, selectivity can be achieved in bioconjugation of a mixture of reaction constituents. Thus, one kind of bioconjugation reaction (SPAAC) would be used in the bioorthogonal ligation of two appropriate reaction partners. To this end, however, knowledge of the reaction rates of the two azide families is essential for the development of selective SPAAC for application in bioconjugation and molecular biology (Fig. 1 ). Results and discussion At the outset of the study, we evaluated preparative SPAAC reactions of commercially available or synthesized eight- to ten-membered cycloalkynes 1 with fluoroalkyl azides 2 in an organic solvent (Scheme 2). Reactions with a BCN derivative were complete within 1 hour at ambient temperature, yielding products 3a–3d in quantitative amounts, while reactions with a DIBAC derivative required up to 4 hours under the same conditions and produced SPAAC products 3e–3h in nearly quantitative yields. These products formed as regioisomers at a ratio of approximately 2:1. The NMR characterization is further complicated by restricted rotation around the amide bonds, which meant that rotamers were also observed by 1 H NMR. Reaction with cyclooct-2-yn-1-ol was less efficient than with BCN or DIBAC derivatives. The nine-membered cyclic sulfur derivative afforded cycloaddition product 3j in high yield but required prolonged heating to 100°C. Finally, a ten-membered, but still a rather strained derivative synthesized from 4,4’-biphenol underwent the SPAAC reaction at 100°C to give 3k in a high yield. Next, the model fluoroalkyl azide PhOCF 2 CF 2 N 3 ( 2a ) and its non-fluorinated analogue PhOCH 2 CH 2 N 3 ( 4 ) were used together with the most reactive derivatives 1a (BCN, R = H) and 1b (DIBAC, R = CH 2 CH 2 NHAc) derivatives for the determination of absolute rate constants for the SPAAC reaction. Both azides are easy to handle liquids, unlike gaseous azidomethanes. For applications in bioorthogonal chemistry, it is essential that reactions proceed with a sufficiently high rate. Neither 1 H nor 19 F NMR spectroscopy could be used to obtain kinetic data, because a preliminary test reaction of BCN with 2a revealed that it was too fast for precise reaction monitoring. UV spectroscopy was also considered, but the absorption differences between the starting alkynes, azides and the triazole products were insufficient for the method to be utilized. By contrast, infrared spectroscopy allowed for the precise monitoring of azide concentration in time as azide stretching vibration (~ 2100 cm – 1 ) is spectral region silent for other functional groups (Fig. 2). The determined absolute rate constants of the second-order SPAAC reactions of azides 2a and 4 with cyclooctynes BCN and DIBAC (at room temperature and a solvent mixture of THF and water (9:1)) (Fig. 3) revealed that, indeed, fluorinated azide 2a reacted faster than non-fluorinated azide 4 with BCN. Specifically, a 16-fold difference was observed. Conversely, a 20-fold rate increase was observed for 4 compared to 2a in cycloaddition with the DIBAC derivative. When comparing BCN versus DIBAC for azide 2a a 126-fold rate increase was observed, while DIBAC over BCN was only 2.5-times more reactive for alkyl azide 4 . In terms of absolute values, 2a displayed reactivity that was almost identical to that of 4-azidopyridine. Note that the absolute rate constants of SPAAC reactions are sensitive to temperature and the solvent used. The reaction was also characterized with the help of ab initio calculations. The structures of azides ( 2a and 4 ) and cyclooctynes BCN and DIBAC, as well as all four transition states of the azide-alkyne cycloadditions, were optimized at the DFT/CAM-B3LYP/6-31g* level, and activation energies were calculated at the highly accurate DLPNO-CCSD(T)/cc-pVTZ level 30 , 31 with the extrapolation to a complete PNO (pair natural orbital) space 32 (Table 3 ). The results are qualitatively in line with kinetic experiments, where the reaction of BCN with 2a is faster than with 4 and the reaction of DIBAC with 4 is faster than with 2a (see the SI for further details). Previous studies gauged SPAAC reactivity by comparing the HOMO–LUMO energies of the azide and cycloalkyne partners, using these frontier-orbital gaps to assign the cycloaddition to either a normal- or inverse-electron-demand regime. 19 , 33 In the present system, however, such frontier-orbital descriptors do not reproduce the experimentally observed rate order. This shortcoming implies that a purely one-electron view of the transition state is too simplistic; when the activation barriers differ by only a few kilocalories per mole, additional factors – including Table 3 Activation energies calculated at the DLPNO-CCSD(T)/cc-pVTZ level with the extrapolation to a complete PNO space. Transition states and reactants were optimized at the CAM-B3LYP/6-31g* level. Entry Reactants E A (eV) 1 BCN + 4 0.43 2 BCN + 2a 0.38 3 DIBAC + 4 0.27 4 DIBAC + 2a 0.39 electronic polarization, entropic contributions, distortion/strain energy, and solvent effects – contribute comparably. A detailed discussion of these contributions is provided in the Supporting Information. Alkyl and aryl azides are widely used in SPAAC reactions in bioconjugations; however, fluoroalkyl azide 2a has not been evaluated in this setting. The compatibility of 2a with aqueous media and its possible reactivity with naturally occurring functional groups were evaluated in a series of control experiments. To this end, azide 2a was dissolved in a mixture of deuterated PBS buffer (pH 7.4) and MeCN. After 3 or 120 hours at room temperature, no decomposition was observed and azide 2a was fully recovered from the buffer solution by extraction (Scheme 3A). The SPAAC reaction was also tested in almost pure water (traces of THF were used to solubilize 2c ) in 3mM concentration of BCN and 2c . Extraction of the reaction mixture after 1 h of stirring afforded only product 3b , indicating that the click reaction is not affected by the presence of a large excess of water (Scheme 3B). Next, the compatibility of the click reaction with fluorinated azide in the presence of functional groups that are prevalent in biological systems was tested. Fluorinated azides are known to react with primary alkyl amines to form tetrazoles. 26 However, this reaction typically requires a non-aqueous solvent and an organic base (e.g. Et 3 N). To investigate whether this reaction would proceed in water, azide 2a was treated with L-lysine, and after 24 hours no reaction or decomposition was observed by 19 F NMR. Only in the presence of Et 3 N at 40°C for three days, a low conversion of azide to tetrazole 5 and amide 6 was observed by 19 F NMR (Scheme 3C). In the presence of excess lysine, a SPAAC reaction between 2a and BCN took place selectively and no tetrazole 5 or amide 6 were observed (Scheme 3D). The cysteine-containing tripeptide glutathione plays a vital role as an antioxidant in living organisms. The SPAAC reaction was repeated in the presence of a 10-fold excess of this strong sulfur-containing nucleophile, and again, only the triazole product 3c formed with no traces of fluorinated side-products detectable by 19 F NMR (Scheme 3D). To successfully apply the established differences in the reactivity of fluoroalkyl and alkyl azides in SPAAC bioconjugation and imaging followed by fluorescent spectroscopy or confocal fluorescent microscopy, a set of fluorescent fluorinated azide-containing probes was needed. Therefore, a new azidotetrafluoroethoxy-substituted benzoic acid 10 was synthetized in three steps from ethyl 4-hydroxybenzoate ( 7 ) by (i) the fluoroalkylation of the phenolate derived from 7 using 1,2-dibromotetrafluoroethane, (ii) magnesiation with Turbo-Grignard and azidation according to a literature precedent, 23 and (iii) alkaline hydrolysis. Azide 10 was then coupled to form a coumarin-based dye 11 or converted to an NHS ester 12 , to form a BODIPY probe 13 (Scheme 4 A). Commercial fluorescein- and rhodamine-tagged alkyl azides 14 and 15 , respectively, were used as fluorescent alkyl azide probes (Scheme 4 B). The reactivity of coumarin probe 11 with BCN and DIBAC was tested in separate experiments and in a mixture of both cyclooctynes. UPLC-MS analysis revealed that although probe 11 (Fig. 4 A) reacted efficiently with BCN (Fig. 4 B), it reacted less efficiently with DIBAC (Fig. 4 C). A competition experiment with both cyclooctynes revealed that 11 reacted predominantly with BCN over DIBAC (Fig. 4 D), suggesting that the selectivity predicted from the comparison of absolute rate constants was retained in the competition experiment and might be applicable in bioconjugation or bioimaging. To investigate the selectivity of the labelling reaction on model biomolecules, we prepared trastuzumab (TzMab), a monoclonal anti-HER2 antibody, 34 and concanavalin A (ConA), 35 a mannose-binding lectin, each modified with either BCN or DIBAC via active ester chemistry (for details, see the SI). This resulted in four conjugates: TzMab-BCN, TzMab-DIBAC, ConA-BCN, and ConA-DIBAC. The degree of labelling (DOL) of each protein was determined by intact mass spectrometry. These DOL measurements were then used to normalize the protein concentrations in subsequent experiments so that each sample contained an equivalent amount of strained dipolarophile. The two proteins were selected due to their difference in size, which allows their distinction and separation by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). To distinguish between the azide dyes, a green fluorescent BODIPY fluoroalkyl probe 13 was used in combination with the red alkylazide TAMRA dye 15 . Initial experiments (lanes 2–5 in experiment 1 and 10–13 in experiment 2), confirmed the presence of the modification on both proteins, which were successfully labelled with both azide probes (Fig. 5; controls and Figures SI11–SI13 in the SI). The fluorescently labelled TzMab runs at around 150 kDa, while ConA, which is a tetramer, appears on the non-reducing denaturating gel mainly in two bands corresponding to a monomer and a dimer (ca. 26 kDa and 50 kDa, respectively). In the first competition experiment, we combined the two azide probes ( 13 and 15 ) with individual proteins carrying either BCN or DIBAC. Because the two dyes have different fluorescence properties, the band intensities cannot be compared directly, but the outcome was clear: BCN-modified proteins (TzMab-BCN and ConA-BCN) gave mixtures of click products (labelled ‘m’ in Fig. 5, lanes 7 and 9), whereas DIBAC-modified proteins (TzMab-DIBAC and ConA-DIBAC) reacted selectively with the alkyl azide 15 (labelled ‘s’ in Fig. 5, lanes 6 and 8), showing virtually no reactivity with azide 13 . In the second competition experiment, we instead combined the two proteins, each bearing a different dipolarophile, with a single azide probe. Interestingly, we saw strong selectivity: ConA-BCN, in the presence of equimolar TzMab-DIBAC, reacted exclusively with fluoroalkyl azide 13 (Fig. 5, lane 16). However, when the pairing was reversed (ConA-DIBAC with TzMab-BCN), azide 13 produced a mixture of products. These results demonstrate that both the dipolarophile and the protein context influence the selectivity of the SPAAC reaction. We next carried out one-pot competition experiments with both proteins and both azides (Figure SI13). A representative outcome is shown in Fig. 5, lane 18: azide 13 was first added to a mixture of ConA-BCN and TzMab-DIBAC, followed by alkyl azide 15 . The results demonstrated clear selectivity: fluoroalkyl azide 13 reacted preferentially with ConA-BCN, whereas alkyl azide 15 reacted with TzMab-DIBAC. To verify that the two proteins remained active after modification, we used the mixture of the labelled proteins from the last competition experiment to label live SKBR3 cancer cells. This cell line expresses the HER2 antigen, to which TzMab binds. In addition, ConA can label parts of glycoconjugates containing mannose residues on cells of the same line. SKBR3 cells treated with the mixture of fluorescent proteins exhibited distinct fluorescent signal on the cell surface in both channels, as confirmed by fluorescence confocal microscopy and flow cytometry analysis (Figure SI16). Therefore, it can be concluded that, after modification, both proteins successfully recognize their targets. Next, we tested whether selective labelling of BCN and DIBAC with fluoroalkyl and alkyl azides could be achieved in live cells. For this purpose, we treated the osteosarcoma cancer cell line U2OS with a BCN–triphenylphosphonium (TPP) conjugate and DIBAC–sulfo-NHS ester. BCN–TPP is enriched in mitochondria due to the inherent ability of the TPP group to accumulate in this organelle. 36 The use of the cell-impermeable DIBAC–sulfo-NHS ester ensures that the DIBAC moiety modifies cell surface components. In this way, we achieved selective installation of BCN groups inside the cells, specifically in mitochondria, and DIBAC groups on the cell surface. Control experiments performed with cells treated with only one of the dipolarophile-containing reagents confirmed their specific localization inside the cells (BCN–TPP) and on the cell membrane (DIBAC–sulfo-NHS ester), respectively (Fig. 6C). In the competition experiment, U2OS cells treated with both dipolarophiles were labelled with fluoroalkyl azide 11 and, after washing, with TAMRA azide 15 . After an additional washing step, images of the cells were obtained under a confocal microscope (Fig. 6C). The reaction led to the efficient and specific labeling of the two dipolarophiles in the cells. Fluoroalkyl azide 11 reacted specifically with the intracellular BCN–TPP probe whereas TAMRA azide 15 reacted with the DIBAC moiety on the cell surface. Additional experiments with fluorescein azide 14 produced similar results (Figures SI19 and SI20). These experiments demonstrate that excellent selectivity in the SPAAC reaction can be achieved using fluoroalkyl and alkyl azides in combination with BCN and DIBAC dipolarophiles. The difference in reactivity of the probes enables preferential click labeling, providing practical strategies for the labelling and modification of two different targets via the SPAAC reaction, even in the context of living cells. Conclusions The results of our work demonstrate that fluoroalkyl azides (RCF 2 N 3 ) exhibit highly selective and accelerated reactivity in SPAAC with electron-rich cyclooctynes, such as BCN, while reacting significantly more slowly with electron-deficient counterparts, such as DIBAC. Kinetic studies revealed up to a 126-fold rate enhancement in the reaction of PhOCF 2 CF 2 N 3 with BCN over DIBAC and a 16-fold increase in rate compared to non-fluorinated alkyl azide analogue in reaction with BCN. Ab initio calculations confirmed the selectivity of alkynes with respect to the azides used. Dual labelling experiments using BCN- and DIBAC-modified proteins and live-cell imaging further validated the orthogonal reactivity of fluorophore-modified fluoroalkyl and alkyl azides in biological systems. Together, these findings significantly expand the capabilities of bioorthogonal chemistry, offering future potential applications in dual-target labelling, advanced imaging, and next-generation molecular tools for biomedical applications. Declarations DATA AVAILABILITY The data supporting this article are included as part of the Supplementary Information. The mass spectrometry proteomic data have been deposited to the ProteomeXchange Consortium via the PRIDE 37 partner depository with the dataset identifier PXD066546. AUTHOR INFORMATION Corresponding Author * [email protected] Author Contributions M.T. conceived the idea, performed experiments, and partially wrote the manuscript, V.Š. performed experiments and partially wrote the manuscript, M.V. partially wrote the manuscript, J.L. prepared compounds 3i–3k , J.F. and P.S. performed calculations and coined their interpretations, L.B. measured kinetics by IR, F.F. measured and calculated DOL of the proteins, and P.B. conceived the idea, acquired funding, administered and supervised the project and partially wrote the manuscript. All authors have given approval to the final version of the manuscript. † These authors contributed equally. ACKNOWLEDGMENT This research was financially supported by the Czech Academy of Science (Research Plan RVO: 61388963) and the Czech Science Foundation (Project 23-04659S). J.F. and P.S. were supported by project 24-11566S. References Bird, R. E.; Lemmel, S. A.; Yu, X.; Zhou, Q. A. 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Supplementary Files SI.pdf Supplementary material Tableofcontent.docx scheme1.jpg Scheme 1. The SPAAC reaction and typical cycloalkynes and azides. scheme2.jpg Scheme 2. Testing SPACC reactions of cycloalkynes 1 with fluorinated azides 2 on a preparative scale. *Regioisomeric mixture formed. scheme3.jpg Scheme 3. Control experiments. A: Stability of azide 2a in aqueous buffer, B: SPAAC reaction in water at low concentration of azide and cyclooctyne, C: Reactivity of azide 2a with L-lysine, D: SPAAC reaction with L-lysine or glutathione additives. scheme4.jpg Scheme 4. A: Synthesis of fluorescent fluorinated azide probes 11 and 13, B: Structures of fluorescent alkyl azide probes 14 and 15. 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16:39:52","extension":"png","order_by":37,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":953,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7630137/v1/e4263f68fae7b5c8a4ccdf64.png"},{"id":92735789,"identity":"60ef8a35-c0f9-47d6-9784-5d3485f48f18","added_by":"auto","created_at":"2025-10-03 16:31:52","extension":"png","order_by":38,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1028,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7630137/v1/7ea5eb2181661c9fb5fb12eb.png"},{"id":92735770,"identity":"7ee25c3d-8903-4eb7-9f6b-4da8d5c981f1","added_by":"auto","created_at":"2025-10-03 16:31:52","extension":"png","order_by":39,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1006,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7630137/v1/59ee986453f8c4d68013b432.png"},{"id":92737994,"identity":"8097031b-8bca-486d-98ad-a00ca59da503","added_by":"auto","created_at":"2025-10-03 16:47:52","extension":"png","order_by":40,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1059,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7630137/v1/26d66123f3d24f4f0f807764.png"},{"id":92737995,"identity":"150a9a79-13fc-455a-affd-4e28332752f3","added_by":"auto","created_at":"2025-10-03 16:47:52","extension":"png","order_by":41,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":903,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-7630137/v1/92fbd29b74afd72ce76cfb11.png"},{"id":92735780,"identity":"c3fb66a2-1b2a-4a0e-8a92-db4e89e3aff8","added_by":"auto","created_at":"2025-10-03 16:31:52","extension":"xml","order_by":42,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":115266,"visible":true,"origin":"","legend":"","description":"","filename":"COMMSCHEM2508760structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7630137/v1/c3763c976083f7b0fb621007.xml"},{"id":92735792,"identity":"6966a5a3-efce-47cb-bc40-f85d0cea8f40","added_by":"auto","created_at":"2025-10-03 16:31:53","extension":"html","order_by":43,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":127335,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7630137/v1/1e30cf91a5ef06560083c2fb.html"},{"id":92735735,"identity":"f8cd5d7c-88f5-460d-bf03-005e6d3f85fd","added_by":"auto","created_at":"2025-10-03 16:31:50","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":48477,"visible":true,"origin":"","legend":"\u003cp\u003eProposed selective SPAAC cycloaddition of fluorinated and nonfluorinated alkyl azides (EDG – electron-donating group, EWG – electron-withdrawing group).\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7630137/v1/7aeea5389294ca41df137c10.jpg"},{"id":92735739,"identity":"ae8e163b-838b-4565-ac50-cd1a020c1ac3","added_by":"auto","created_at":"2025-10-03 16:31:51","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":53915,"visible":true,"origin":"","legend":"\u003cp\u003eIR spectra (in chloroform) of the starting reaction constituents used in the kinetic study. Signals corresponding to azide stretching vibrations are highlighted in yellow.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7630137/v1/87f74cec930b778c566895b9.jpg"},{"id":92737989,"identity":"5a518e9d-9460-4181-b151-ec232a32f539","added_by":"auto","created_at":"2025-10-03 16:47:51","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":72773,"visible":true,"origin":"","legend":"\u003cp\u003eAbsolute rate constants of SPAAC reactions of azides 2a and 4 with cyclooctynes 1a (BCN) and 1b (DIBAC).\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7630137/v1/3c204cee0d959410e94fdb68.jpg"},{"id":92737037,"identity":"22679586-5ca5-4c76-b571-352975b56475","added_by":"auto","created_at":"2025-10-03 16:39:51","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":48242,"visible":true,"origin":"","legend":"\u003cp\u003eSPAAC reaction with fluorescent probe 11 analyzed by UPLC-MS (UV-vis traces at 360 nm). A: 11. B: 11 + BCN (2 equiv.), MeCN, rt, 30 min. C: 11 + DIBAC (2 equiv.), MeCN, rt, 30 min. D: 11 + BCN (2 equiv.) + DIBAC (2 equiv.), MeCN, rt, 1 h.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7630137/v1/ac67bec1fa43da32d0ab0cbd.jpg"},{"id":92735747,"identity":"04dcd3e2-3e5b-4875-930c-e3717e77dde3","added_by":"auto","created_at":"2025-10-03 16:31:51","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":84231,"visible":true,"origin":"","legend":"\u003cp\u003eControl and competition labelling experiments using azides 13 and 15, performed on the proteins TzMab and ConA labelled either with BCN or DIBAC.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7630137/v1/cfef656f553c65a25d93a322.jpg"},{"id":92735754,"identity":"7357f510-dfc2-4395-845a-da0841d8109c","added_by":"auto","created_at":"2025-10-03 16:31:51","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":97769,"visible":true,"origin":"","legend":"\u003cp\u003eCompetition labelling experiments using azides 11 and 15, performed on live U2OS cancer cells treated with the mitochondrial BCN-TPP probe and cell surface-targeted sulfo-DIBAC-NHS ester. A: General scheme of the experiment, B: Structures of the reagents, C: Images from a confocal microscope. The indicated combinations of reagents were used.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7630137/v1/42c67850a638d28c7e858fd3.jpg"},{"id":108391158,"identity":"0d8fc957-09ad-4d0a-8b8c-a0ea325b2fe2","added_by":"auto","created_at":"2026-05-04 07:05:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":755067,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7630137/v1/4602222d-22c2-42cc-8447-1db0d976455b.pdf"},{"id":92737990,"identity":"28b802f5-99c3-4f2b-9505-40396ff142d5","added_by":"auto","created_at":"2025-10-03 16:47:51","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":6505876,"visible":true,"origin":"","legend":"Supplementary material","description":"","filename":"SI.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7630137/v1/a7abd7528ba98e30e16841c4.pdf"},{"id":92735740,"identity":"ae2d3ee6-4af0-44fd-afc3-24fc7d2e218f","added_by":"auto","created_at":"2025-10-03 16:31:51","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":61965,"visible":true,"origin":"","legend":"","description":"","filename":"Tableofcontent.docx","url":"https://assets-eu.researchsquare.com/files/rs-7630137/v1/0f0259d97edc50942b4378b6.docx"},{"id":92735742,"identity":"468dc9f8-f0a0-402d-b5c9-396136d567aa","added_by":"auto","created_at":"2025-10-03 16:31:51","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":43830,"visible":true,"origin":"","legend":"\u003cp\u003eScheme 1. The SPAAC reaction and typical cycloalkynes and azides.\u003c/p\u003e","description":"","filename":"scheme1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7630137/v1/27f34e6c0f5b4ea2757c5f9f.jpg"},{"id":92735744,"identity":"399f135e-bcc0-400a-b258-e328f70e67e4","added_by":"auto","created_at":"2025-10-03 16:31:51","extension":"jpg","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":86533,"visible":true,"origin":"","legend":"\u003cp\u003eScheme 2. Testing SPACC reactions of cycloalkynes 1 with fluorinated azides 2 on a preparative scale. *Regioisomeric mixture formed.\u003c/p\u003e","description":"","filename":"scheme2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7630137/v1/833ea4f2fbd5b9da102b16fd.jpg"},{"id":92737038,"identity":"fbe17b58-3b8c-4c60-9873-6986c06525b6","added_by":"auto","created_at":"2025-10-03 16:39:51","extension":"jpg","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":93859,"visible":true,"origin":"","legend":"\u003cp\u003eScheme 3. Control experiments. A: Stability of azide 2a in aqueous buffer, B: SPAAC reaction in water at low concentration of azide and cyclooctyne, C: Reactivity of azide 2a with L-lysine, D: SPAAC reaction with L-lysine or glutathione additives.\u003c/p\u003e","description":"","filename":"scheme3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7630137/v1/af3e1a707b5b87484ad97e80.jpg"},{"id":92737039,"identity":"eac18d23-694e-499d-b9d2-8442042dc6ce","added_by":"auto","created_at":"2025-10-03 16:39:51","extension":"jpg","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":112389,"visible":true,"origin":"","legend":"\u003cp\u003eScheme 4. A: Synthesis of fluorescent fluorinated azide probes 11 and 13, B: Structures of fluorescent alkyl azide probes 14 and 15.\u003c/p\u003e","description":"","filename":"scheme4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7630137/v1/59a9b8041e36bacccc438dd9.jpg"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Beyond Traditional SPAAC: Achieving Orthogonality and Rapid Kinetics with Fluoroalkyl Azides","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBioorthogonal reactions are reactions that do not interfere with biological systems.\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e The reactants of such reactions must react under biocompatible conditions selectively and at high reaction rates. Moreover, both the starting materials and products must be inert towards functional groups commonly present in biomolecules and non-toxic to the organism. Despite these stringent requirements, however, bioorthogonal reactions have found broad applications,\u003csup\u003e4\u003c/sup\u003e ranging from bioconjugations\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e and bioimaging\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e to drug delivery.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eOne of the most popular bioorthogonal reactions is strain-promoted azide-alkyne cycloaddition (SPAAC).\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e In this variant of the Huisgen cycloaddition,\u003csup\u003e9\u003c/sup\u003e an azide reacts with a strained cycloalkyne to form a triazole (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The strain release during the cycloaddition allows the reaction to be performed at ambient temperature and, unlike copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), does not require a metal catalyst. The most broadly utilized cycloalkynes are the cyclooctynes DIBAC\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e and BCN,\u003csup\u003e11\u003c/sup\u003e whereas DIFO\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e is not easily available and BARAC\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e and cycloheptyne TMTH\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e suffer from limited stability. An additional complication arising from the use of asymmetric reagents (such as DIBAC) is the formation of isomers of products. As for azide reagents, structurally diverse aromatic and aliphatic azides have been evaluated and the reaction has found applications in chemical biology, bioconjugation, and material science.\u003c/p\u003e\u003cp\u003eSPAAC reactions are relatively slow, with reaction rate constants typically in the range of 0.01\u0026ndash;1 M\u003csup\u003e\u0026ndash;\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e\u0026middot;s\u003csup\u003e\u0026ndash;\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e.\u003csup\u003e15\u003c/sup\u003e However, the reaction rate of the click reaction with benzyl azide is affected by the structure of cyclooctyne and it has been shown that a few-fold rate increase can be achieved when switching from DIFO or BCN to the more electron-deficient DIBAC (Table\u0026nbsp;1).\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e Comparison of the reactivity of various azides with BCN and DIBAC has revealed that a judicious choice of azide structure can speed up the SPAAC reaction with electron-rich BCN up to 10-fold when switching from the electron-rich benzyl azide to the electron-deficient 4-azidopyridine (a further 3-fold increase was achieved with 4-azido-1-methylpyridinium salt) (Table\u0026nbsp;2).\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e However, azidopyridines or their salts are not typically used as\u003c/p\u003e\u003cp\u003eTable 1. Absolute rate constants of SPAAC reactions of benzyl azide with various cyclooctynes.\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\" style=\"width: 439px; height: 228.485px;\" width=\"439\" height=\"228.485\"\u003e\u003c/p\u003e\n\u003cp\u003eTable 2. Absolute rate constants of SPAAC reactions of azides with cyclooctynes.\u003c/p\u003e\n\u003cp\u003e\u003cimg 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LJWVlXjuuecmTUxSUlKwa9cuNDU1ITMzE52dnRgZGUFaWhra7Kw019TUNGGJ9uDgYLz77rtuuzGdXhMSBROTxYdJCREtGNeuXcPu3bvl8Iy88MILqK2tVaeLBwcHo7a2Ft/73vfkqqirq8P27duxZMkSfPjhh4iLi8PatWuxZMkS7N69G62trcD4YnuKt956C7W1tTbPMubFF1/Ea6+9Jodd7tixY4COExKFkpg8++yzTEwWASYlRKRLtt0zISEhmrKDBw9qypWWibq6OjzyyCNqvameQ/arX/0KX/nKVzSxzZs3271NQXNzs3qcBx98ENXV1erKxOfOnVPr2bb8nDhxYsLzA0BISIjdZMWVurq6UFFRofuERBEVFYVPP/0U//iP/ygX0QLDpISIdMm2e8ZsNmvK5O4bpXXj7NmzCLeZszzZc4yOjuLixYuTds1Mp7u7G0uXLgUAREdHIycnB+vWrcP27dsRExMjV5/SkiVL7CY+rrR27Vr87ne/k8O69rvf/Q7f+MY35DAtMExKiGhBaGpqQlJSkhy2Kzs7G5988gmamprUgaYBAQH4zW9+o6l37do1BAQEaGL2ZGRkQAiB5uZmDA0NycW65Ovr61H3JRoYGICvr68cpgWGSQkRLQj/8i//omklmcrWrVuRkZGBV199Vb179ZkzZxATE6N2BbW1tSEmJgbf/e53pb0Bf39/jIyMAONdIWvXrsXo6Ci6urpw9uxZzWyg6YyOjs4o8XE2T0pKent74efnJ4dpAWJSQkQLwrlz57Bt2zY5bFd0dDT6+/tx4MABHDlyBAAQHh6OxsZG7NixAwaDATt27MCFCxewc+dOeXds3boVb731FjDeFbJ7924sXboU69atQ21trboq8HRTkAHgvffem3WXjzN4UlJy8+ZNrF+/Xg7TAsTF04ho0enq6kJSUhLOnj0755spbt26FfX19Q7fGiE+Ph4lJSXzfnuDoqIifPzxxygsLJSLdCc9PR3Lli1TZwzRwsWkhIgWna1bt2L79u1YtmwZHnzwQc2dqWeqra0Nra2tOODAHQqd8Rxz1dvbi4iICHz00Udyke589atfRWNjI7twFgF23xDRopOYmIhly5bJ4VkJDg52OJlwxnPMlZ+fH7y9vWGxWOQiXWltbYWvry8TkkWCLSXkcsq3QcWyZcsQGxurafbu6urCO++8A4w3ZytN2cqS3bbfZOvq6vCVr3xFnQZqq6mpCSEhIXjvvffw6aefasq+8Y1vzLmp3t4xldi6detgNptnPMiSSC/0vqIrAKSmpmLVqlVISUmRi2gBYksJuVxraytOnTqlPm5qasKmTZvUx4cPH8a6detw9+5d3L17FytXrlSTkXPnzmlmRCgx2yRH0d/fj4sXL2LJkiU4d+4c3n33XbXs7t27WLdu3ZyX87Z3TCW2ZMkSXLx4cc7PTeQu+/fvx/nz53W7UurAwABqamqwf/9+uYgWKCYlNC82btyIAwcO4MCBA3jjjTfwq1/9Cl1dXWhra8PJkycxMjKCjIwMZGRkoLOzE2lpaeq+SUlJM/qW9Nprr8FkMqmPt23bph4zIyMDcXFx6j1SnM1kMrllqXByvtHRUTm0YPn6+mLfvn3Izs6Wi3QhPT0dWVlZvEvwIsKkhObF9evXUVJSgpKSEuzduxc5OTlYu3YtqqqqkJaWpunKWbt2Lbq6utTHQUFBCA0NnfLuqwBw8uRJu106U+nv71dfl+1mz7vvvqupc/36dbUsODgYJ0+e1NQnz3P48GGcP39eDi9oKSkpqKqq0t30YIvFAovFMud7GZFnYlJCblFfX4/R0VF0d3fPaABbVlYW0tLSJv0W29bWhri4OE3MNok4fPgwqqurZ7Wo1WzFxcXZvTss6Vd/fz/6+/vR1dWF/v5+tLa2ore3d9LzbCHy8vLC0aNHddVaYrVakZqaitLSUrmIFjgmJTQv5O4bAKipqUFISAh6e3vl6urNzRRLlizBd7/73Tl9i+3t7cXJkyfR2dnp0FoQtt1BBw4cwMaNG+Uq5GEuX76MAwcOICkpCdeuXUNLSwtqa2vR19cnV13QlDEbZWVlcpFbpKamIiEhAaGhoXIRLXBMSsitIiMjcfLkSU0C0tXVhXXr1mnqAcDOnTtx6tQpTbfJVJQk4sSJE8jJyZnxfVFocbl+/Tqam5sRHR2NuLg4HDp0aM6ztDxZYWEhamtr0dTUJBfNq6KiInh5eXFw6yLFpITmhe2YksOHD6OlpQXbtm1DcHAw0tLSsHTpUuTm5iI3N1ddqtveSpmVlZV276gaHByM6upqOazKyMjA/fffj9zcXE18xYoVmtYPZZuL6urqWY9pIfdji9cYLy8vVFZWIjU1FTdv3pSL50VVVRWuXLniEavMkmt84RU9T1CnBeELX/gCHnjgAfXxqlWrNMtqP/HEE/irv/orDA4OwsvLC2+88QZCQkIAAH/0R3+EP/uzP8PKlSsBACtXrsSaNWsQFBSkxhT/5//8H3zxi1+En5/fhP0A4NFHH8VHH32EP/uzP8MXv/hFzb7Tsfd8trGmpibcf//9eOKJJzT7kb5du3YNH330ERISEgAAly5dwurVq/Hwww/LVRcFLy8vREVF4bnnnsOXv/zleb3fTHp6Ot5//31UVlbiv/7X/yoX0yLBxdNowejv78fLL7+sjlmZT3v37sWrr77q0JgVmn8lJSV499131aniJSUlaG9vR1FRkd2WusVieHgYe/bswUMPPeTyhdWsVit27NiBv/iLv+C9bYjdN7RwrFixAjt37pz3mROjo6PYuXMnExIP9OCDD6qtchhf9ffOnTswm82aeouNt7e32h0aERExYeFAZ6mqqsKGDRuwa9cuJiQEsKWEiIim0tTUhPT0dPj6+qKwsHBGU/in09raitTUVPj5+aGwsBC+vr5yFVqkmJSQanR0FJ2dnZqY7cDN0dFR9PX1qYubjYyMqGUrV66c0FKgPN9kgz+VNT0mK1d0dXVh5cqVE16bvWPOhb3X0dXVhaVLl2qe315MpvzMS5cu1czgaGtrw7p16zRdAvZitmXyz6fUBzDh9zrd77qrq2tRzigh56mpqUF6ejrWr1+PmJgYxMbGwtvbW642qd7eXtTU1OD8+fPw8vJCYWEhp/zSBExKSNXW1oaQkBB1EbLf//73aGlpQWdnJ9auXYu2tjbk5OTg8uXLiI+Px/Xr19WZC9XV1bhw4QJ27typPt/Fixfx3HPPqfsr6urqEBMTg7CwMADAb3/7W/zkJz+xe9G8ePEi7rvvPnzlK1/RvDaMH7O4uHjOs2Wmeh3x8fHqlGKFvZit3NxcZGZmIi4uTv3dvPnmm1iyZAkMBgPMZrMmabAXsy0LCwtDc3OzJqZ0K4SEhMD2o6u8d5N9nA8fPoxvf/vbdn/HRDNltVrR0NCA2tpa1NTUIDAwEI899hgATLgh5fDwMCwWC/793/8dNTU1sFqtiI2Nxa5duxAYGKipS6QStCCMjIwIs9ks+vr61FhnZ6canwmz2SzkU+LChQtqzGw2i7i4OCGEEHFxcaK4uFit19jYOGHfsLAwUVxcLHJyctRYX1+fAKB5TbW1tSIgIEB9rBgZGRFhYWFCTPLa7B1zpqZ7HfLPN1lMoexr+/tPS0sTaWlpQoxlChPeB3sxBQARFhYmLly4oImZzWa7vwt7MVt9fX3qe0fkLI2NjSIrK0tkZWWJ8PBwzRYbGyuysrJEXl6euHHjhrwrkV1sKVkAlMXGwsLC1BUpo6OjNUuq207Bncxk37Ztv6HbtpT4+/sjMTERGB+wZjQakZGRAYy/pqSkJNTX12PTpk3qvWxyc3PR09MzYYaMve6Fixcv4u7duzhw4IDd12YvpsRlcjfJdK9D/vkwfo+QxMREuy0lW7duxb59+zQtRaOjoxgZGcGKFStgMBhw4cIFbNiwQS0PCQmZsqWksbERL7zwAn75y19qWlts91XcuHEDzz333ITfha34+HgcP358wu+ZiEgvOPtmASgoKEBaWhqam5thNpsRExOjlvn7++Py5cvTJiRTke8po6itrUVOTg5ycnJQW1urWYTqRz/6EQ4fPowlS5YgJiZGXSXSbDYjKCjI5lnG2LtQNjU1TehzbmtrU7fS0lK7r015TbabvGz4TF6H7c+Xk5ODlpYWTV1bLS0tmoQD40vj2/7ey8vLNc83naVLl+K73/0uXn/9dbkIkH7O8vJyuXiCbdu24Re/+IUcJiLSDSYlC8CdO3fw1FNPATaDNZXWAmeMlJ9spdRDhw7h8uXLuHz5Mn7yk58gJiYG/f39AIBz587hvvvuQ1tbG/z8/HDx4kVgPEm6e/eu9Ez23blzRw6pF2FlGuebb74pV0FmZuaETV5obSavw/bnu3z5siYBKikpQXx8vHrn4oCAAM3AX3uKioo0z6cYHR3F4cOHER8fr7k7MsaX1q+vr1d/r7Zsn6uoqEiNd3V1Ye/evdi7d++E6dHT/cxERO7EpGSB+PWvfw2MLyCG8ZkpzjDT51NaGPr6+tSE6PXXX0dOTg7effddnD17Fv39/di6dSvOnTun2Xd0dBQGg8HuhVemXITNZjPOnj07oQUEM2wpceR1dHV1ob29HZmZmThx4gT6+/sRExODt956S1Ovra3NbguQrKamBn5+fnjxxRft3p+nqKjIbpfRZAoKCrBz506Eh4fr6s6vRETTYVKyAGzbtg3f+9730NTUhNdeew1hYWEOddco3SNNTU345je/iZycHLvP19vbq9ZV7ikTHByMqqoqfPe739V8k09LS8O7776L6Oho/Omf/ini4+PR1NSEpqYmbN++3e4xQkJC8Jvf/EYTUwQHB+PChQtISkqa0Bpge1xlk5OD2bwO2cqVK/Hqq68iODgY999/PwDgpZdewsmTJ3H48GG0tbWhrq4OISEhOHPmjLz7BNu2bcOuXbs0i3jZCg4Oho+Pjxye1JEjRxAeHj6hO6m3txdf+9rXNDEiIj3hvW8WgIcffhj/5b/8F1RVVcHHxwc//OEP8cUvfhHd3d0ICAiwe8dde0ZHR/Gb3/wGzc3NaG5uxvvvv4/nnntO/ZY+OjqK4eFhbN26Fd3d3fjwww/Vun/0R3+EiooKLF++HP/wD/+Ab37zm1i+fLn63MuXL8e7776LJ554At/85jfxhz/8AVVVVXj//fdx4MAB7N271+aVjPnjP/5j/M//+T8RHR2tvjblHiUA8N//+3/HjRs38Nlnn834Z7Q11euw97tTYn/+538Og8GA5ORkJCQkICQkBEuXLsW+fftw9epV1NbW4pNPPsEPf/hDPPLIIwCAf/3Xf8VTTz2l+Z0oMT8/P/T29uLAgQP4u7/7OyxfvnxC/c2bN6OnpwdPPfUUvvSlL034Xdj+fpYvX46mpiacPHkSp06dUu/zc/DgQWRnZ8/6vj9ERPOFs29I1+Lj42c0c2g+jY6O4vnnn8eLL744YW2Guejq6sKxY8ecNjOmrq4OdXV1mvu31NXV4dNPP51VNxAR0Xxj9w3pmnLDND157733cP36dbz++ut2B6fO1g9/+EP8/ve/x7FjxzTTuOcqLS0Nd+7cwfPPP692q12/fp0JCRHpHltKiGapv79fM3BWXgNltuQl++2tWzIbtuu0yMvdExHpGZMSIiIi0gV23xAREZEuMCkhIiIiXXBK943BYJBDRERERKqZpBtOSUqIiIiIHMXuGyIiItIFJiVERESkC0xKiIiISBeYlBAREZEuMCkhIiIiXWBSQkRERLrApISIiIh0wW1JSWJiIgwGw5RbS0uLvBsREREtUG5LSqqqqhASEgKMr/Jmu5WVlcnViYiIaIFzW1ICAMuXL5dDAIB9+/apCQsREREtDm5NSqZy7do1hIWFyWEiIiJaoHSblNjavHkzDAYDfHx8cPv2bSQnJ8PHxwcGgwHJyclydSIiIvJAuktKEhMTUVBQoIldu3YNISEh8PHxwcsvv4y0tDTcuXMHJpMJpaWlKC8v19QnIiIiz6OLpMR2xs2lS5fkYmB8/El3dzfS0tKwevVqYHzsCQDcvXtXqk1ERESeRhdJie3Mm4SEBLlYQ0lIAOChhx4CALz33ntqrKCgYMLUYiIiItI/XSQltqqqqnDkyBE5TERERAuc7pISRx05cmTCuidERESkf7pNSqKiovD222/LYSJyg6qqKnXGmx4Hlre0tCAxMRE+Pj6IioqSixeklpYWGAwGbN68GQUFBbh9+7ZcxW30fr7MVk9PD5KTk7FmzZpFNSTAYDBgzZo1SE5ORk9Pj1zsEm5NSib7EFVVVeHKlSu477771JhS13Yf5Zc02fMQkXMkJibi2LFjAIDQ0FC52K3efvttbN26FT4+PvjVr36FhoYGAEB5eblmbNlkf1R7enrUOj4+Pujo6JCr2FVVVYU1a9bMy+0wlIuibcIVFhYGIQSys7Nx/PhxXSVjej5fZqunpwchISG4du0afvrTn6qt764+v1paWhAVFTVhNqor3L59GwUFBfDx8dHEhRD48Y9/rM6AnexndCa3JSWJiYkwm82ANPvGYDBgx44dk9b9i7/4CwBAR0eHuuqr2WxeEOuVTHY/IB8fHyQnJ8/4ZJZ973uAwTC22VuPLjHx8/LERLmUaExvby+MRqM6wFwvXnzxRURGRuL06dOaVaL37duHjIwM9fGPfvQj9f+2Tp48qf7/4sWLM/r5kpOTcd9996G7u1sucrq333570tcOAE8++SQuXrwIs9mMqqoqudht9Hq+zJZyfjQ0NGgmWrjy/CooKMCvf/3refnC3dHRgddeew3Lli3D0NCQXIyHHnoIDQ0NGBoa0vwsLiNIV0JCQoTt2zI4OCjKysoEAGE0GkV3d7em/ky98IIQwNiWnS2XCvG1r41tRJPx9/cXkZGRctitmpubBQBRWVkpFwkhhMjPzxeRkZHC399f+Pv7y8VicHBQGI1GERkZKQCI5uZmucqU5rLPXCk/y2SUn0Mv9Hi+zAUAYTKZ5LAQ83B+RUZGivz8fDnsEspnaTIJCQlTljuL21pKyD75fkDLly9XM/KhoaFJs/HpLF0KfO1rwH33AT/4AfDpp9pyb++xjcie27dvo7u7G48//rhc5Fb/8i//AgB44IEH5CKNF154Ad3d3RPGqf3oRz+Cv7+/7n6uuXj44Ydx5coVOewWej1fZkvpmvPz85OLNBbD+bVp0ybA5nfiKkxKPMSyZcvk0Kx5ewN/+7fAH/4AvPCCXEo0uffffx8A8Mgjj6gxZdCfwWBw+R+qySgLJ053n6xnn30WAPCTn/xEEz9x4gT+9m//VhNzBeX2GLa/s82bNzu1u0W58M1Hk/909Hq+zNYf/vAHQPo57HH3+YXxLh/lliyG8QGqmZmZcrU5U34Hyu/EVZiUeIje3l4AQGRkpFw0K9/9LvD1rwMNDcCPf/x5fJovmrTI/fznPwcAbNiwQY0pCx2WlZXh17/+9YSxUJs3b9aMg5LvW+Xj46NelJULlu3FShljNdWFu62tTQ7ZtXr1aiQkJKC0tFS9aL/99tu4c+cOEu0MpFJmtsjbXC+mL7/8MqqqqnDu3DkIIdDc3Izu7m588skngJ1xdQaDYc4DHG/cuCGH5t1szxdl3JxtQpWcnAzD+MUV4wOL5d+RfI7Z7mvvfcX4xds2QZrqwn39+nU5ZNdszy84+T2vqqrC0aNHsXfvXggh0N3dDaPRqH4+oqKiJhxrrgOjZ/o7mSsmJTqnjIouLS1FZWXljAZJTeeVV8b+TU39vBvHXlKSlgYsWzY2AHb79oldPrR4tLW1ISQkRO1erKqqwqVLl2CxWLBv3z4kJiYiMjIS+fn5EEJgcHAQ/v7+OHr0qPoc3/rWt3Dt2jX86le/ghACiYmJ6kX53Llz8Pf3x+7du9X6Bw8eRGRk5KR/1GcrNjYWsBmQ+Prrr6szRGTKzBZ5m65FBjYXT9vB9z09PfDx8VEv0mFhYTCZTGq5fBwhhEcvIjnb86WxsRHXrl1DVFSUelE/ffo0MH5uYDxJne4cU/T09ODSpUsTWo0KCgpw/Phx/PjHP4YQApWVlTNObKczm/MLDrznymweJVkDoH6OlJlOq1evRnZ2tlre0NAw4VjKLDW9YVKiU0o2++UvfxlnzpxBd3e30/44/+VfjnXffPopcOqUXDomLQ348EPg7l2grQ34l38BKirkWrRYXLlyBZs3b0ZPTw8SExNRU1OD999/f9Ikefny5WofNMZH+F+5cgWXLl1SL1SnT5/W/BF+YbxP0VXrWiQmJsJoNOLEiRPo6enBlStX1Gb3uZAveFN5/PHH0d3djb/4i79AeXk5enp6kJOTM6OLkMKT7vE12/NFmeFhNpvxzjvvyMV2yeeY4u2338bu3bsREhKidiMpjh8/jtOnT6uvIzEx0WkXZ2efX5jFOaZ0rURERCAzMxMdHR148sknZ/WzubpbZqaYlOiUks3m5+eju7vb7lQsuTlO3qZqCvzOd4AHHxwb9PpP/ySXAidPAvX1Y//ftGmsrs2yMbSIKF0WpaWl8Pf3x6VLlxAbGzthULatnp4e/OM//iNefPFFAEBrayv8/f01UyrtOXfuHNLT03H79m1N07+zmEwmdHd3IyEhASaTadrXM5nMzEx1eYLo6GhNU3hiYiKEEOo3fYyvNP3WW2/BaDRi//798Pf3n7LbwFZHRweioqKQm5urXuyn6tJyt7mcLxhPMiIjI9Vv/dORzzHFm2++iW984xv467/+a80Yj46ODgwNDeHhhx/W1HcmZ51fVVVV2Lx5M8xmM44ePYqoqCi1m2r16tUQQuDWrVtq/bCwMFgsFjzxxBPIzc1FYGCgptVpOlFRUdi5cycw3pU603PTFZiU6NyRI0cQGRmJ0tLSCSO75eY4eZvqW9iDD37ejaP8a88vfznWavL1rwMpKXIpLQYffvghAMBisUAIAX9/f3znO9+RqwEAjh49CoPBAH9/f2zevBlPPvkkMP4t37a5eTJhYWF44okn8PLLL097EZuLpKQkYHxtI+Uu43ORk5ODW7duQQiBO3fuzOgb6ZNPPolr167BYrEgISEBubm5U35xUCitCMrn+tq1a05rNXWF2ZwvczHZOQablgUlwfnpT3+qlo2MjADSTV2dzVnnV2JiIq5du6a+5w0NDZO2MikeeughVFVVobu7G/n5+bhy5Qq+9a1vydXsamhowJ07d9RkJycnR64yb5iUeADlAy1/I3DU3/zNWLLx858Dra1y6dhA2OBg4Ic/HJtSTItTY2MjYHNX7smmPwJQ+/u7u7vR09Oj+cY1029tubm5KC0ttTuA0VGrV69GZGQkIiMjp/0j70wGg0H9+ZWLB6Q7nC8Uszlf5mKqc+ydd97BpUuXYDAYEBgYiO7u7gnn0UzPw7lw1/lle1uW1atXq19m9TJFfDaYlHiAsLAwREZGoru72+nNtq+/PtYtMz5YXuOZZ8aWW/v7vwf+4R/GVoalxeenP/2pZtaX0k/++uuv29TSWr16NR5//HGUlpYCADZu3Aiz2TyjC8Lq1auRn59vdwCjLDg4WA5p3L17F7/4xS80x21oaJjQsqHMbnNlv/rLL7+svg5l3ExcXJxUy3Gu6PaajbmcLxhPFq5cuYKNGzfKRXbJ5xgA/OAHP8Dg4KDawpCQkKBemJXfizzOZCrTvRY9nV9ZWVnqMvAtLS34xS9+oc54cqbpfieOYlKiM8rJLd9jID8/Hxif6iaXzcTICPDBBxNn0GzaBHz729qY7JlngG9+E7BpCaVFoqenB0NDQ5qLvzL98cqVK1izZs2Eb6IY3++Xv/wlnnjiCWC868Lf31+9MN8enx5s231hO5AzKSkJv/jFL9THk1HW77E3Vbe8vBy5ubkYGhpSx3/Yk5ycrF7Ydu7caffncZTJZMJPf/pTfPnLX4bBYMCJEyeQn5/vUBO/TGl1cUW310zN9XxRxs1ERkZqumOmIp9jPT09WL58uebn37RpE/7xH/8RGP+9mEwmzcW7vLx8yqmxyv3XlEX6bOnp/FKSW39/fxgMBkRHRyMxMRElJSVy1TlTfge296RzCXmJV3IfZRlfZUl5i8WiKc/IyFDLJltW257s7M+XmH/wQbl0zIMPCvH1r3/++G//dmxpeiGEaGsbK7e3PD2REEJUVlaq566yJSQkaM5hi8WiLrcNQPj7+6vnsb+/vwCgqV9ZWTntMuXK0thlZWVy0aLjScu6y+eLv7+/yMjIEIODg2odk8mkltnbx/Ycs1gswmg0CtjccsBisajnlbJM/ODgoPq8GP9bmpGRoR7THuU4i53ye3M1gxj7pbuFwWBAfn7+lAMyyT3+6Z/GZuY0NIx17yQmAi6aqUnkkDVr1sBoNOLatWty0aLR0tKCrVu3orKyUteDYD2R0tIxODjo1lYod7p9+zYCAgKQmJiomVXmCm7rvrHX3Er68Zd/OTYlWIixtUqYkJBevf766+qdwufStenpWlpaEB0djZCQECYkLpCWlgaj0aiZlruY9PT0qF1caWlpcrHTuSUpyczMRHR0NGAzvcswvuxtVVWVugx1QUEBWlpa1PX8o6Ki1DKDtKKdsiSxwWDgB5NoEXnyySfR3NyMO3fuICQkZMoxAguJshR+amoqjh07NmFwJTnH6tWrYTabsXnzZjzzzDMwGAxylQXLYDDgiSeeUNdMceV0aoXbum+U5kZ73Tdvv/02nnrqKXUZ5qeffho7d+7Eww8/jNOnT8Pf3x8Y69zS7Ld582YAWNTNuERERJ7KLS0l01FG9965cwenT5/Gk08+qSYoq1evVv9v21R7+/ZtmM1mFBYWqjEiIiLyHLpMShS29zXIyclRmyf/x//4H4DNjY8A4OzZs4iMjJzRDbOIiIhIf3SdlEwmLCwM/v7+OHPmjBo7c+aMU5cyJiIiovnlkUkJAHz/+99Xly6uqqqC0WhkKwkREZEHc3tSMtfbcX/jG9+A0WjEm2++ie985zv427/9W7kKEREReRC3JSVhYWEwGo24dOkSbt++jY6ODvXGSsr9AaZKWJYvX47ExERcunQJGL+rIhEREXkutyUlAHD69GncuXMHX/7yl1FeXo6XXnoJHR0deOqpp4Dxu4Xa3gFSptw34vvf/75cRERERB7GbeuUOENLSwt2796NW7duyUVERETkYdzaUjIXtqvppaamspWEiIhogfC4lhKDwQCTyYQ7d+6gu7ubq7cSEREtEB7XUmI0GlFaWgoAvNcDERHRAuJxLSVERES0MHlcSwkREREtTExKiIiISBeYlBAREZEuMCkhIiIiXWBSQkRERLrApISIiIh0wSlTgm1XWSUiIiKSzSTdcEpSQkREROQodt8QERGRLjApISIiIl1gUkJERES6wKSEiIiIdIFJCREREekCkxIiIiLSBSYlREREpAtMSoiIiEgXmJQQERGRLjApISIiIl1gUkJERES6wKSEiIiIdIE35KNZ+9d//Vc8//zzMBqNctGc/du//RueeeYZHDlyRC4iIqJFgkkJzdqOHTsQEBCAxx9/XC6as9HRUbz00kv453/+Z/j6+srFRES0CDApoVlpbW1Famoqrl69Khc5rKysDB0dHSgtLZWLiIhoEWBSQrMSFBSEiooKBAYGykVO4ernJyIi/WJSQjNmryWjpqYGFotFU282vLy8kJKSAi8vL8DFLTFERKRvnH1DMzI8PIxTp04hKytLE/f29tY8ni0vLy81IQGA0NBQ+Pn5oaqqSlOPiIgWPraU0IykpqZi1apVSElJkYucbmBgAFu2bMGNGzc0CQsRES1sbCmhaVksFrS2tmL//v1ykUv4+vpi3759yM7OlouIiGgBY0sJTWv79u04dOgQoqKi5CKXsVqtCAoKQn19Pfz8/ORiIiJagNhSQlOqqamBl5fXvCYkGB9rkpeXh9TUVLmIiIgWKF0kJT09PUhOTp73Cx9NzWq1Ij09HYWFhXLRvIiNjYXVakVDQ4NcRG5QXl6ONWvWwGAwwMfHB5mZmbh9+7Zcza6Z7FtQUACDwTDlRp5jJu+5PS0tLUhOTlb3NRgMSExMREdHh1xV4/bt29i8eTPPE08n3Ki7u1uYTCYBQAAQkZGRchVyo7y8PHHs2DE5PK/a29tFaGiouHfvnlxE8yg/P18AEJWVlUIIIZqbm2f8mZ3pvkq9ybaEhARNfdKvmb7nsrKyMgFAmEwmMTg4qIkZjUY1Zk9GRoZ6rpDnctu7Nzg4KCIjI0Vzc7MYHByc0QlL86e/v1/4+fnpIhlISUkRhYWFcpjmyeDgoDAajcJkMmniyheK5uZmTdzWbPbNz8+fUE/YPMdUxyH9mM17LsvPzxf+/v4Tko/IyMgp921ubhZGo1GEhIQwKfFwbuu+Wb58ORoaGhAWFobly5fLxeRmqampyMvL08WU3KysLJSXl2NgYEAumrO2NuDiRTkKlJQA/f1ydHF75513MDQ0hEcffVQTf/rppwEA9fX1mrit2ex75MgRnD592qbWmLNnz+Lhhx9GWFiYXEQ6NJv3XHbkyBHcunVrVteE27dvIzo6GhcvXpzVfqRPbktKSL9aW1vR29uLxMREucgtvL29cejQIadOEW5tBZ57Dqir08YPHgT6+rSxxe6DDz4AADzwwAOa+H333QcAaGtr08RtObIvxi84x48fx4svvigXkU45+p7bc+vWLRiNRruJ6YEDB/DEE0/gySeflIvIAzEpoQlMJpPuboq3f/9+tLa2OrSkvT0xMcDoqBwlW5NdROxdIGSO7AsAr732Gnx8fHjB8SCOvuey8vJydHd3o07+BgHg7bffxk9/+lOUlJTIReShmJSQRllZGUJDQ3V5Q7zS0lKYTCY5PGdxcQJJSQKvvy6XkB709PQgNzcX3//+9+UiWiTefvtt7N+/H5WVlROSmp6eHuzcuRN1dXXstllAmJSQarL72+iFK+6L8+qrBmRmAl1dcgm528mTJ+Hv76+bbkSaXx0dHdi5cycqKyvtngPJyckwmUwTkhVFYmIiWlpa5DDpHJMSUn3729/G9u3b4evrq8ZeeeUV9Pb2aurNJ5PJBKvVqj4+fvw4du3aNWH9ipluGzZs0DzfihVAcTFw7JhQY4qSEiA+XqCpSS5ZXCa76eJ060bAgX17enpQWlqKF154QS4inZvre26ro6MDEREROH36tN2EpKWlBVeuXEFubq7m833lyhUAgMFgwKVLl+TdyAMwKSHVq6++isbGRs1FOzAw0G2rqtbU1GBgYEAzA6iiogLp6ekYn84+683eTf4OHACuX4cm+WhqAtrbBTIzDXjhBbGox51s2rQJAPDhhx9q4iMjIwCAxx9/XBO3Ndd9T548CaPRiKSkJLmIdG6u77mip6dnyoQE4+NT5M+2EAKRkZHA2JxgCCEmbUUh/WJSQqr169cjPDwcZWVlaiw2NhYYTxDmk9VqRWpqqmbA7cDAAM6fP49jx45p6jpDZeVY8qFYtw4oKjIgOBjYuBEY/3u6KCl/6BsbGzVxZWrns88+q4nbmsu+SivJsWPHOFbAA83lPbeldMvICUliYiIKCgo0MVqA5IVL3MFisQgAwt/fXy6ieXbv3j3h5+cn+vv71diNGzdEYGDgvC6klpWVJbKysjSx2NhYUV1drYnNVXGxEHFxn2licXGfCUAIs/nzWFjYZyIpSVtvMVIWviorKxPCZrGqjIwMTT17K2rOdF+FUr+7u1suIg8x0/dcPl8qKyvVmL0tPz9fs7/M399fAJiw+Bp5DrcmJRkZGeoKfMpmNBpFZGSkujwxzb+Kigqxe/duTWw+V1W1t5psY2OjCA8P19RzhNksRG2tNtbXN5as9PV9HjObhUhL+2xC3cUoPz9fGI1GgfEvEPYuEPJFRjGTfRUYX2acPNtM3nP5fFFWbp1sm2xFV2GTkCjHI89kEGMnBpFGUFAQKioq1KnBVqsVGzZswNWrVzUDYV0hLi4Ou3btUruOYOf1uFpdHXDffUB4+Nj4kg8/HBt7QkRErsMxJWRXRUUF9uzZoz728vJCVlYW0tPTNfWcrampCcPDw5qEpKioCOHh4fOWkADA5s1ARMTY7Juxf+UaRETkbGwpoUnt2bMHjz32GHbv3q3GXN1iIT//wMAAtm/fjvr6epe30MhGR4HOzrFBr0uWyKVERORsTEpoUgMDA9iyZYtmGq3FYsGePXvQ3t4uV3dYUVERPv74YxQWFqqx1NRUrFq1CikpKZq6RES08DApoSnZSxTstaA4yl6LiCsTICIi0h8mJTQlq9WKLVu2oLKyEuvXrwcmaUFxlL0WkYiICGRlZSE8PFxTl4iIFiYOdKUp2Rvg6uvri0OHDjlt0KvFYkFTU5MmIampqYG3tzcTEiKiRYRJCU3L3qqu+/fvR1NTE27evGlTc25SU1M13UP2VnMlIqKFj0kJzUheXh6ys7PV++LYa0GZC3stIspN9+Z7tg0REbkXkxKakcnui/PQQw9heHhYU3c2bt68OW/3tyEiIn3jQFeasflY1dXeaq5ERLQ4sKWEZsxZXTaTsbeaKxERLR5sKaFZk1dddRZXPS8REXkGtpTQrJ08eRKbN2+GwWBw6vbnf/7nTEiIiBYxtpQQERGRLrClhIiIiHSBSQkRERHpApMSIiIi0gUmJURERKQLTEqIiIhIF5iUEBERkS4wKSEiIiJdYFJCREREusCkhIiIiHSBSQkRERHpApMSIiIi0gUmJURERKQLTEqIiIhIF5iUEBERkS4wKSEiIiJdYFJCREREusCkhIiIiHSBSQkRERHpApMSIiIi0gUmJURERKQLTEqIiIhIF5iUEBERkS4wKSEiIiJdYFJCREREusCkhIiIiHSBSQkRERHpgkEIIeQgLT7Dw8PIyMjAn/zJn8hFczY8PIxnn30WYWFhchEREdEEbCkhAMDLL7+MTz/9VA47xGq14sUXX4TVapWLiIiIJmBLCaG3txfbt29He3s7vLy85GKHpKamYtWqVUhJSZGLiIiINJiUEOLi4rBr1y7ExsbKRQ4bGBjA9u3bUV9fD19fX7mYiIhIxaRkkWtqakJ2djYaGxvVWG9vL3p7ezX1ZsPLywuhoaHq46KiInz88ccoLCzU1CMiIrLFpGSRCwoKQkVFBQIDA9VYUVERamtrNfVmw9vbG5WVlWpXkNVqRUREBEpLSzXHISIissWkZBErKytDR0cHSktL5SKnq6mpwfnz51FdXS0XERERAUxKFi+r1YoNGzbg6tWr8zbWw5VjV4iIyPO5dUpwS0sLkpOTsWbNGhgMBhgMBiQmJqKjo0OuSk6Wnp6OQ4cOzVtCAgB5eXnIzs7mFGEPVV5ern5WfXx8kJmZidu3b8vVJpjt53yuxyF9ceR9nOm+yrnl4+Oj1k1OTkZPT49clTyFcJOysjIBQJhMJjE4OKiJGY1GNUbO99FHH4n169eLe/fuyUUul5KSIgoLC+Uw6Vx+fr4AICorK4UQQjQ3NwsAIjIyUq6qMdvP+VyPQ/riyPs4032nOrf8/f01dclzuC0pyc/PF/7+/hP+KEVGRgoAorm5WRMn54mNjRXV1dVyeF4MDQ2J9evXi/7+frmIdGpwcFAYjUZhMpk0cZPJNO1ndTafc0eOQ/rhyPs4m32Vc0tm79wiz+G27psjR47g1q1bWL58uVxELtTa2oqBgQG3jevw9vbGvn37kJ+fLxeRTr3zzjsYGhrCo48+qok//fTTAID6+npN3NZsPueOHIf0w5H3cTb7KufWZFauXCmHyAO4LSmZzK1bt2A0Gnm/FBcxmUzzMttmKikpKWhqaoLFYpGLSIc++OADAMADDzygid93330AgLa2Nk18Jux9zl1xHJp/jryPjuwLAB0dHbhy5QpMJhNWr14tF5MH0FVSUl5eju7ubtTV1clF5ATnzp1DYGCgLtYKycvLQ3p6uhwmHZrsQjDXLw6Tfc6dfRxyD0fex7nu29HRgYKCAjzzzDPIz8/H6dOn5SrkIXSTlLz99tvYv38/Kisrpz0BafasViuys7ORl5cnF7lFVFQUvLy8UFNTIxc5rK6uDnv37kV8fDxKSkowOjqqlsXHx6Orq2tC/dzcXE3MVl0dsHevgM3TAADi4wWkp6Jp8HNOzhYVFYXAwEAcPXpUjdmbqUOeQRdJSUdHB3bu3InKykokJibKxeQE2dnZ2Ldv37xOAZ6OK6YIHz58GCdOnEB0dDQyMzPR3t6O7du3q4lJdXU1RkZGNPt8+umnMJvNmpitTz8Fzp414Px5bby62gDpqWgK/JyTKzQ0NEAIge7ubrzwwgs4evQooqKi5GrkIdyelHR0dCAiIgKnT5/mHyoXGRgYQFVVle7u1Lt+/XqEh4ejrKxMLpqTtrY2nDx5EvX19YiOjkZwcDDeeOMN3H///VMmHTMRECBw8CDQ3y+XLHze3t5yCBj/7M7UTD7nzjgOuZ8j76Mj+65evRpHjhyByWSC2WxGVVWVXIU8gFuTkp6enmn/UJHjTCYTCgsL1XvR6EleXh5OnTqFgYEBuWjWrly5gqSkJCxZskQTv3z5MsLDwzWx2dq4ESguBg4cEHLRgrdp0yYAwIcffqiJKy1Ojz/+uCYum+nn3NHjkD448j46sq/Cz88PAPDJJ5/IReQB3JqUJCcnw2QyTfhDlZiYiIKCAk2M5uatt96aMAXYGQmAI2yP7+XlhaNHj+Lb3/42mpqa5rQpz2c2mxEUFGRzJPtSUlIQHx+vbqdOnZKr2LVrF3D9OtDUJJcsbJGRkQCguZM0bKZnPvvss5q4bKafc0ePQ/rgyPs4m30LCgqwefNmm1pj7t69CwB45JFH5CLyBPLCJfOlsrJSAJh0y8/Pl3ehOfibv/mbCQsR7d69W10tcb4pi6cNDQ2psfb2drFkyRIRHh4+p035WdLS0kRaWprN0SYCIC5cuCDMZrO6paWlibi4OCGEEJ2dnSIgIEAAEDk5OUIIIYqLhYiL+0wIIURjoxABAWP/B4Qwm8eeNydn7HFc3GdiZEQ52sKhLF5VVlYmxPgqm0ajUWRkZGjqKZ9fxWw/5zM9DunbTN9H+XwRs9hXWfk1IyNDXZyvsrJSGI3GCau/kudwW1KirLo32cbV+Jzj3r17IjAwUNy4cUON9ff3Cz8/P90sMx8eHi4aGxs1sbm4cOGCCAsLk8MiJydHXLhwQYjxP4JmJZMYV1xcrCYlcXFxorOzU4yMjIiAgADR19enSUrG6nwmios/T0r6+j4vz8kRorZWrbqg5OfnC6PRKDC+jLecUAg7F5m5fM5nchzSv5m8j/L5opjJvt3d3SIjI0OEhISozxMSEmK3LnmOiWcDLTjV1dUiNjZWE8vKyhJZWVmamKvduHFDBAYGapIhe69trkZGRgQAUWuTFXR2dgrYJCK2/1fYJiW2wsLC7CYlfX1jCYltS4kiLe2zCTEiIpoZt44pofkRGxuL4eFhNNkMhjh27BjOnz8/r+NL0tPTkZWVpQ64tVqtSE9PR2FhoVx1TpYsWYLOzk6kpaVh7dq1iI+Px7p161BbW4vg4GC5+pTq6uqwfft2rFixQi7CihVATo4cHVu3pLYWWLpULiEiopkwiLFvj7TAWSwW7NmzB+3t7WqsqqoKtbW1qKys1NR1hYaGBpw6dUpz74qioiJ8/PHHTktKbCkrQ65bt04zG6etrW1CrL+/HyMjI1i7di0AIDc3F0NDQzhx4sR4OTAyAowXAwBGR4HOTmDdOsB2sk9XF1BQIPDGG4bPg0RENCNMShYRk8mEhx56CPv371djQUFBqKiocPnS8/JxBgYGsGXLFty4cUNXU5VLSkpw9+5dZGRkyEWT6uoCjh0TePNNAzo7gdJSJiVERHPBpGQRsZcI2GtBcTZ7LSL2EiQ9MBi0yYTZbJ5R109uLpCZObbI2k9+YtC0qhAR0cwwKVlk7CUIcXFx2LVrl2YtE2exWq3YsGEDrl69qi5xPx+JEBEReR4mJYuM1WpFUFAQ6uvr1ZUP7bWgOEtqaipWrVqlWeI+IiICWVlZDq+ySkRECwtn3ywyXl5eyMvLQ2pqqhrz9fVFYmIiioqKNHUd1dvbi4aGBk0XTU1NDby9vZmQEBHRBGwpWaS2bNmCwsJChIaGApN0szhK7hay10pDRESkYEvJIlVaWgqTyaQ+9vLyQlZWFtLT0zX15qqpqQnDw8OacSplZWWIiopiQkJERHaxpWQR27NnDx577DHs3r1bjclTd+dKfh5XjlshIqKFgUnJImYvUWhtbUVqaiquXr0qV5+xsrIydHR0oLS0VI3pdQowERHpB7tvFjFfX1/s27cP2dnZaiw0NBQxMTGwWq2aurNhtVqRl5enPrZYLGhtbWVCQkREU2JLySLnigGuMk4BJiKimWBLySJnb4qwM3EKMBERzRRbSgiwMzDVGTgFmIiIZoMtJQQAOHnyJDZv3gyDweC07Y/+6I+wefNmJiRERDQjbCkhIiIiXWBLCREREekCkxIiIiLSBSYlREREpAtMSoiIiEgXmJQQERGRLjApISIiIl1gUkJERES6wKSEiIiIdIFJCREREekCkxIiIiLSBSYlREREpAtMSoiIiEgXmJQQERGRLjApISIiIl1gUkJERES6wKSEiIiIdIFJCREREekCkxIiIiLSBSYlREREpAtMSoiIiEgXmJQQERGRLhiEEEIOErlKS0sLfvSjH8Hb21sumrP/+I//wMqVK5GcnCwXERGRB2FLCc2r73znOxgcHJTDDsvPz0dvb68cJiIiD8KWEpo3NTU1OH/+PKqrq+Uih7nyuYmIaH6wpYTmhdVqRXp6OgoLC+Uip4iNjYXVakVDQ4NcREREHoItJTQvjh8/jrt37yIvL0+N3bx5EwMDA5p6sxUeHq7+32KxwGQyobGxEV5eXpp6RESkf0xKyOUGBgawZcsW3LhxQ5MspKeno7W1VVN3Nry8vFBRUQFfX181lpqailWrViElJUVTl4iI9I9JCbncjh07EBMTg8TERLnI6YaHh7FlyxY0NjZqkhUiItI/jikhl2ptbUVvb++8JCQA4O3tjUOHDiE7O1suIiIinWNLCblUUFAQKioqEBgYKBe5lLuOS0REc8eWEnKZsrIyhIaGuiUxKC0thclkksNERKRjbCkhl9DD2I75HMtCRESOc2tLSUtLC5KTk+Hj4wODwQAfHx8kJyejp6dHrkoeJjs7G/v27XNbQgIAhYWFSE9Ph9VqlYtoDsrLy7FmzRr1s5qZmYnbt2/L1SbV09OD5ORkREVFyUUqpY7yN8FgMCAxMREtLS1yVdI5R86Xuexrez3hFxEPJtykrKxMABAmk0kMDg5qYv7+/nJ18iDt7e0iNDRU3Lt3Ty6ad3l5eeLYsWNymGYpPz9fABCVlZVCCCGam5sFABEZGSlXnaC7u1uYTCYBYMp9LBaLMBqNIiQkRHR3dwsxvm9ISIgAIJqbm+VdSKccOV9mu+/g4KAwmUzCaDSKyspK9dwhz+S2pCQ/P99u8hEZGck/QB4uKipK1NfXy2G3uHfvnli/fr346KOP5CKaocHBQWE0GoXJZNLElURjqs/q4OCgiIyMFM3NzWJwcHDKi0tCQoIAMOGi0t3dLQCIhIQETZz0ydHzZbb7hoSEiJCQEPXLLXk2t3XfHDlyBLdu3ZLDqpUrV8qhRaerqwvx8fHIzc2Vi3D48GHEx8ejq6tLLrKrv78fubm5iI+Px969e1FXVydXcYqamhp4eXlN2UQ/n7y8vJCXl4fU1FS5yCG5uWObra4uID5eaIMLwDvvvIOhoSE8+uijmvjTTz8NAKivr9fEbS1fvhwNDQ0ICwvD8uXL5WKN4eFhAMDq1as1ceWxUk765sj5Mtt9CwoKYDab8cYbb0x7fpFncFtSYk9HRweuXLkCk8k04Q/TYjQyMoLq6mpkZmZidHRUjff396O2thbV1dUYGRnR7GNPV1cXVq5ciaGhIWRmZiI6OhppaWk4fPiwXNUhrr6/zVy54r44ZrNAZibQ1vZ5bGQEqK422FZbED744AMAwAMPPKCJ33fffQCANttfggO8vb2B8b8DtpSxBI8//rgmTvrkyPkym31v376N48ePw2Qy4aGHHrKpTZ5MF0lJR0cHCgoK8MwzzyA/Px+nT5+WqyxqaWlpeO+999TH7777Lnbv3q2pAwAlJSWIj4/H4cOHNUnMsWPHUFxcjBMnTiA4OBjR0dH42c9+hpMnT075B2K2ioqKEBsbCz8/P7nI7fLy8pCdne3UQa8BAQI7diy8lhHZZOdIWFiYHHJIeno6jEYj9u7dqyYmPT09+Na3voWQkBAkJSXJu5AOOXK+zGZf21aV5ORkdWDsmjVrUFBQIFcnD+H2pCQqKgqBgYE4evSoGptulPVi89RTT2m6W5qamvDII49o6uTm5qKqqgqZmZkAgO3btwPjrSrV1dWIj4/X1F+xYgXS0tJQVVWlic/VwMAAysvLkZWVJRfpQmBgIEJDQ1FWViYXzdmhQwZs3AiUlMglNBcPPfQQzGYzuru7ERgYCIPBAH9/f9y6dQuXLl1i8zxp/PM//zMA4Ac/+AGefvpp3Lp1C4ODg3jiiSdw9OhRp/1to/mlm3VKenp68KMf/QhHjx5FSEgIrl27JldZdNra2hASEoKRkREsXboUQgiMjo6q/zcYDDCbzQgODobBYEBjY6N611ylDABCQkJg720uKSnBu+++i8uXL8NqtaKoqGjOLQlvv/02XnrpJc1UvN7eXre2mihjEJRugeHhYQQGBiIuLg7Lli2Tas9MeHg4wsPDER8vsG2bAd/4BrBuHdDXN7aFhAB2ftUeLSoqCleuXEFzc/OEb6wGgwGRkZEz7hqbqn5HRweeeeYZhISEIDc3F6tXr0ZPTw8SEhLQ3d2NxsZGNtN7AEfOl9nsO1VdHx8fPPzww5Meh/TL7S0litWrV+PIkSMwmUwwm83Mcm0sWbIEcXFxaGpqwnvvvTdpM/bLL7+M+Ph4tVVkNnfgHRgYmHNCAgC3bt3C+vXr1cdWqxXbt29Hb2+vpt582rNnD5qamtTH3t7e+I//+A/87ne/09SbDfnnWbsWyMkBXntNm4mMjo4NejUYJg6I9TRKUieTx344Ki8vD93d3SgpKVHHlK1evRoNDQ0YGhrC3r175V1Ihxw5XxzZ19bDDz+MK1euyGHyBPJ0HHdT5qjn5+fLRYuO2WwWylt04cIFkZaWJpKSkkRtba0QY00fwmw2q/9vbGwUZrNZ3fr6+jRlsrCwMHHhwgU5PCft7e0iMDBQE6uurhaxsbGa2Hyxd+zCwkKRkpKiic1VXNxnorh47P8jI0IAQhQXj/0rhBC1tWOPhRAiLOwzMf5WeCTlM1lWVqaJK+tHzOazOtWUYIyvY2LPVGWkL46cL7PZd6ppwpGRkcJoNMph8gBuaykpKCjA5s2b5TDu3r0LABPGTCx227Ztw8mTJ3H27Fm7sxACAgLw61//GsHBwVi3bh1ycnLUmTnFxcV4+eWX0d/fr9a/ePEiWlpaEBsba/MscxcYGIjAwECcO3dOjbli1stMWK1WZGdnIy8vT40NDAzg/PnzmrFLzrJkCVBbCxw8+HksOho4cGCsxeT++4GlS2338CyRkZEAgMbGRk1cmZ757LPPauJzFRISAox35dpSxpgp5aRvjpwvs9lXmTb84YcfqjGMny9Xrlzhqq6eSs5S5ouSEWdkZKiL3lRWVgqj0TjpN6nFxralRAghAgICRFhYmPoYNi0lnZ2dAoCIi4sTAERaWppaTwghcnJy1PKAgAAREBAgOjs7NXUc1d/fL/z8/DQrubpjdVd7LSIpKSmisLBQE3OEbUuJIizsM7WlRAGM1R0Z0cY9jfKtVPkG29zcLIxGo8jIyNDUm6pFw2KxCEyxYvNbb70lAExY0VU59ltvvSXvQjrlyPky033F+MJpRqNRbS3p7u4WCQkJwmg0TliEjzyD/b8e86C7u1tkZGSoS0grf4ymatpbbEZGRtSkQ4wnHkqXjBhPWkZsrnZK/cmSjenKnWE+EoKpTJYYyV1LjursFBO6ZPr6hLB5u1QXLoxtni4/P18YjUY1sbD3WbV3kZE/5wDULx/KUuKK5uZmdWVXpV5CQoLdJnrSt7meL2KG+wppiXnlfDGZTExIPJhuZt/QwmC1WrFlyxZUVlaqA1/n847Be/bswWOPPaZZxyUiIgJZWVnqzKT5cPEi8MknQEbG2JThZcuAnTvlWkREZMttY0poYfLy8kJWVhbS09PVmLe3Nw4dOoTs7GxNXWezWCywWCyahKSmpgbe3t7zmpAAQGzs2KqvBgPw7rsCThq6Q0S0oLGlhFwiLi4Ou3bt0gykDQoKQkVFBQIDAzV1nUV+fqvVig0bNuDq1asub6EhIiLHsaWEXCIrKwv5+fmatU9KS0thMpk09ZylqqoK69ev1yQ8RUVFSExMZEJCROQh2FJCLpOamopVq1YhJSVFje3YsQMxMTFOna5nr0VkYGAAW7ZswY0bN+Dl5SXvQkREOsSkhFzG3gBXVyQL6enpWLZsGY4dO6bGXJH8EBGRazEpIZcqKytDR0cHSktL1djx48dx9+5dzeJmc9Xb24vt27ejvb1dTXJaW1uRmpqKq1evytWJiEjHmJSQy9kbgBoUFIT6+nqHb9jnjgG1RETkGhzoSi4nD3D18vJCXl4eUlNTNfVmq6GhAVarVZOQlJWVITQ0lAkJEZEHYlJCLhcaGgo/Pz/NnZ9jY2MRExOjqTdbXl5emm6h4eFhnDp1CllZWZp6RETkGdh9Q/PCFQNcZfZm+xARkedgSwnNC19fX+zatQvHjx+Xi5zi5s2baGpqwv79++UiIiLyEGwpoXmjrCfygx/8AD4+PnKxQ/Lz87Fv3z7N+BIiIvIsTEpoXr3xxhs4deoUvvzlL8tFc/Yf//Ef+NKXvoR/+qd/kouIiMiDMCkhIiIiXeCYEiIiItIFJiVERESkC0xKiIiISBeYlBAREZEuMCkhIiIiXWBSQkRERLrApISIiIh0gUkJERER6QKTEiIiItIFJiVERESkC0xKiIiISBeYlBAREZEuMCkhIiIiXWBSQkRERLrApISIiIh0gUkJERER6QKTEiIiItIFJiVERESkC0xKiIiISBeYlBAREZEuMCkhIiIiXWBSQkRERLpgEEIIOUjkSkVFRRgYGICXl5dcNGe9vb3Izs7GqlWr5CIiIvIQbCmheTUwMIBTp07JYYf9+7//Ow4fPiyHiYjIg7ClhOZVamoqVq1ahZSUFLnIYUFBQaioqEBgYKBcREREHoBJCc0bi8WCPXv2oL29XS5yClc/PxERuRaTEpo327dvx6FDhxAVFaXGmpqaNHVmy8/PD35+furjHTt2ICYmBomJiZp6RESkf0xKaF7U1NTg/PnzqK6uVmMDAwPYs2cPrFarpu5sxMTEaLqCBgYGsGXLFty4ccOpA2mJiMj1mJSQy1mtVgQFBaG+vl7TquEqr7zyiuZfIiLyDExKyOWKiorw8ccfo7CwUC5yCavVig0bNuDq1avw9fWVi4mISKeYlJBLuas7paqqCrW1taisrJSLiIhIp7hOCblUdnY2jh49Oq8JCQAkJibi5s2bsFgschEREekUW0rIZdw9RdfdxyciotlhSwm5jMlkQmlpqRyeN4GBgVi/fj2qqqrkItKBzZs3yyEiWuR0k5Tcvn0bmzdvhsFgkIvIA9XU1MDX1xehoaFy0bwqLCxEenq6Q9OOaUx5eTnWrFkDg8EAHx8fZGZm4vbt23K1CQwGg93NbDbLVdHS0oLk5GT4+Piox0lOTkZPT49clXRurucLZrGvcr4odQ0GAxITE9HR0SFXJU8hdCIjI0MAEDp6STRH9+7dE35+fqK/v18ucousrCyRlZUlh2kW8vPzBQBRWVkphBCiublZABCRkZFy1QmUz7W9zVZZWZkAIEwmkxgcHNTE/P39NXVJ3xw5X2a671Tni9FoVGPkWXSRATQ3Nwuj0ShCQkIm/KGiifr6+kROTo6Ii4sTSUlJora2Vq4yQW1trUhKShIjIyOaeFxcnOjs7NTEHKW3JMAVSVJOzthmq7NTiLi4z7TBBWBwcFAYjUZhMpk0cZPJJACI5uZmTVw2kzpi/GJkL/mIjIyc8XOQ+zlyvsxmX+V8kZMPni+eze3dN7dv30Z0dDQuXryI5cuXy8Uk6erqwsqVKzE0NITMzExER0cjLS1t2jvkfvrppzh79izOnz+viVdXV2NkZEQTc8TAwADOnz+PY8eOyUVu4+Xlhby8PKSmpspFc2Y2C2RmAm1tn8dGRoDq6oXX/fjOO+9gaGgIjz76qCb+9NNPAwDq6+s18bk6cuQIbt26JYdVK1eulEOkQ46cL7PZVzlfeN1YWNyelBw4cABPPPEEnnzySbmI7Dh27BiKi4tx4sQJBAcHIzo6Gj/72c9w8uRJtNleIe0ICAjAwYMH0d/fLxc5TWpqKvLy8uZ9CvB0XDFFOCBAYMcOIYcXnA8++AAA8MADD2ji9913HwBMe945oqOjA1euXIHJZMLq1avlYtIhR84XR/ZV3Lp1C0ajEWFhYXIReQC3JiVvv/02fvrTn6KkpEQuIjv6+/tRXV2N+Ph4TXzFihVIS0ubdpbJxo0bUVxcjAMHDshFTmGxWHDz5k3d3gyvoqICe/bskcNzduiQARs3Agv99J3sQjCbP/rFxcWagYvJycl2By4qOjo6UFBQgGeeeQb5+fk4ffq0XIV0ypHzxZF9MT5Atru7G3V1dXIReQi3JSU9PT3YuXMn6urq2Pw2Q319fcB4EiLz8/NDd3e3HJ5g165duH79ut278w4MDKCpqWnOW0JCAioqKiY8pzvZHl+ZIvzSSy9NeO0z3eSWluPHDTh4EHBh45PHS0hIwKZNm/D+++9DCIG8vDyUlpZq7hZtKyoqCoGBgTh69KgamyqBIcL4l9z9+/ejsrJyxkkM6ZA8yGS+REZGioyMjAkx5SUlJCRwoJLEbDZPOhC4uLhYxMXFCTE+qBXjsxuUQbC25Y2NjSIgIECI8UGIZrNZCCFEZWWlCA8Pn9P2yCOPCG9vb81g0o8++kgEBgaKe/fuqbH51NjYKKKiojSxI0eOiD/5kz+Z8Ppnuu3evVsIMTagtbh47DlzcoRIS/tMmM1CKG/PyMhYHWDigFhPM9XAQdiZFTETymw7ZYaFPd3d3epMjJCQELmYdMqR82Wu+1osFmE0Gqc8n8gz2L/CuZgyxWu6zd6JudgBEI2NjXJYhIWFiQsXLgghhAgICBAjIyOis7NTTT5skxIxPuumuLhYwCYpcVRhYaFISUnRxFJSUkRhYaEmNl8CAwNFe3u7+ri/v18EBgY6ZRaObVIyMjKWjBQXf56U1NaOPRZCiLCwz0Rf3+f7epqEhAS7n0eLxTLlhWIqb731lgAg8vPz5aIJlJkXvOB4BkfOl7nsy4RkYXFL901YWBjGEyLNFhkZCQDqYzbBTVRcXIyXX35ZM1j14sWLaGlpQWxsLDA+Q2fJkiVTzlYoKSnBwYMH5bBD9u/fj9bWVk0XR1ZWFsrLy+e9G6esrAyhoaEIDAxUY/n5+di1a5fT7xy8ZAlQWwvY/jqjo4EDB4DRUeD++4GlS2338CybNm0CAHz44YeauDJr6/HHH9fEnc3Pzw8A8Mknn8hFpEOOnC+z3benpwcRERE4ffq0bsey0ey4JSmhuTtw4AC2b9+OlStXIj4+HmvXrsX3vvc9dHZ2YsmSJZq62dnZOHPmjCamWLFiBXJycuSwQ7y8vJCVlYX09HQ15u3tjX379iE/P19T15WsVivy8/ORlZWlxiwWC1pbW7F//35NXWeJjgbCwoQc9uhkRKF8WWhsbNTElemZzz77rCZuq6CgAGvWrJHD+PnPfw6MD75WFBQU2F16/u7duwCARx55RC4iHXLkfJntvsnJyTCZTBMSksTERBQUFGhi5CHkphN38vf3FwAmLIZDE42MjAiz2Wx34bORkRERFxenWVStr69vQl3lOeQF1RwVGxsrqqurNTG5K8WV7HUZRUVFifr6ek3MEZ2dYkKXTF+fEPZ6wi5cGNs8mdKFUlZWJoTNgofyuDCl61WhjAmxXXWzsrLSblO8UjcjI0NT12g0TqhL+jbX80XMYl/lPJpsm0nXIOmPbpISJSEBl5R2WFxcnN1xJ/Plxo0bEwa4NjY2ivDwcE09V7B37OrqahEbG6up52oXLnw+wLW42POTEjGeNBiNRvUzau+PvnyRsVgswmQyaT7fISEhdvft7u4WGRkZ6srOU9Ul/ZvL+aKYyb7KoNjJNnlcCnkGgxg7MWiB6Orqwrp16zQxd7zFqampWLVqFVJSUtRYXFwcdu3apY59cQX5GFarFVu2bEFlZSXWr18vV3eZ0VHg+ecFqqsNiIsTePNNA6TeNSIikjApIZewWq3YsGEDrl69qg4s7e3txfbt29He3u6SFV8bGhpw6tQpzVLURUVF+Pjjj1FYWKipS0RE+sOkhFymrKwMHR0dKC0tVWPp6elYtmyZ0++NY7VaERERgdLSUnXGzcDAACIiInD16lV4e3vLuxARkc4wKSGXCgoKQkVFhZoo2GtBcQZ7LSL2upCIiEi/mJSQS7W2tiI1NRVXr15VY+fOncPPfvazCUvSz9XAwAC2b9+O+vp6NdGxWCzYs2cP2tvb5epERKRTXKeEXCo0NBS+vr6oqalRY7t374bFYplwH5m5srcoWmpqKseREBF5GCYl5HKlpaVITU2F1WrVxEwmk6beXNhbFK2mpgbe3t4IDw/X1CUiIn1jUkIu5+vri127duH48eNqLDQ0FH5+fqiqqtLUna309HRkZWWps3msVivS09PZSkJE5IGYlNC8OHbsGM6fP6+5B46jiYPVakVMTAyioqLUWFlZGaKiotT7pRARkefgQFeaN1VVVaitrUVlZaVc5BQDAwPYsmULbty44ZJ1UIiIyLXYUkLzJjExEb29vWhtbZWLnCI7OxtHjx5lQkJE5KHYUkLzymKx4Pnnn8frr78uFznkt7/9LY4fP84pwEREHoxJCc27v/zLv8S9e/fwxS9+US6as1//+tf4+7//e2zdulUuIiIiD8GkhIiIiHSBY0qIiIhIF5iUEBERkS4wKSEiIiJdYFJCREREusCkhIiIiHSBSQkRERHpApMSIiIi0gUmJURERKQLTEqIiIhIF5iUEBERkS4wKSEiIiJdYFJCREREusCkhIiIiHSBSQkRERHpApMSIiIi0gUmJURERKQLTEqIiIhIF5iUEBERkS4wKSEiIiJdYFJCHqekpARnzpyBwWDQbGvXrkVdXZ1cnYiIPIRBCCHkIJGedXV14be//S0effRR2J6+bW1tCAkJQV9fH1asWKHZh4iI9I8tJeRxjh07hj/+4z+WwwgODkZYWBj6+vrQ1tYGg8GAvXv3wmAwyFWJiEiHmJSQR2lra5NDqra2NrS0tGDdunVqLDw8XNOaQkRE+sWkhDzKjRs3EBISoj62HVOyY8cONDY2YsmSJWr5tm3b1P8TEZG+MSkhj9LR0YGNGzeqj4UQ6tbV1YXw8HBNfY4tISLyHExKyKO0trZi/fr1cpiIiBYAJiXkUVpaWrB27Vo5TERECwCTEvIYbW1tiIuLk8NERLRAcJ0S8hijo6Po6+tjSwkR0QLFlhLSvePHj8NqtWLJkiVMSIiIFjAmJaRrTU1NuHLlCry8vOQiIiJaYNh9Q7oWFBSEiooKBAYGykVERLTAsKWEdKusrAyhoaFMSIiIFgm2lJAuWa1WbNiwAVevXoWvr69cTERECxBbSkiX0tPTcejQISYkRESLCFtKSHd6e3uxfft2tLe3c4ArEdEiwpYS0p3U1FTk5eUxISEiWmSYlJCuNDU1YXh4GLGxsXIREREtcOy+IV3hFGAiosWLLSWkG5wCTES0uLGlhHSBU4CJiIhJCc3au+++iy984Qty2CHFxcUICwtDSkqKXERERIsEkxKalaamJuzcuRMBAQFykUMsFgsuXbqEJ554Qi4iIqJFgkkJzYqrBqJaLBbs2bMH7e3tchERES0SHOhKM+bKgaiBgYEIDQ1FWVmZXERERIsEW0poRoaHh7FlyxY0Nja6bCDqwMAAtmzZghs3bnDhNCKiRYgtJTQj2dnZ2LdvnyYhKSoqgsFgcGirqalRn8/X1xeHDh1Cenq6GiMiosWDLSU0rZs3b2LHjh24evWqy1swrFYrgoKCUF9fDz8/P7mYiIgWMLaU0LTS09ORlZXl8oQEALy8vJCXl4fU1FS5iIiIFjgmJTQlpXtlPu9FExsbi+HhYTQ1NclFRES0gOmy+2bz5s24du2aHKZ5ZrVaERERgdLSUpfMuJkKpwgTES0+bm0pkQc9KpvZbJarkhu4cgrwdDhFWH/Ky8uxZs0aGAwG+Pj4IDMzE7dv35arTdDS0oLk5GT4+Pio+yYnJ6Onp0euCjhwHNKXubyPBQUFE64H8jaXuuRBhBsBmHQj9+rv7xfr168XQ0NDctG80cNroDH5+fkCgKisrBRCCNHc3CwAiMjISLmqRllZmQAgTCaTGBwc1MT8/f3l6nM+DunLXN9HZb/JtoSEhDnVJc/h1qs/ANHc3CyHSQdSUlJEYWGhHJ53hYWFIiUlRQ7TPBocHBRGo1GYTCZN3GQyTfsZzs/Pt5t8REZGTtjXkeOQfjjyPubn50/YT9g8p+2+s6lLnsOt3TekTxaLBU1NTbq4Od7+/fvR1NSEmzdvykU0T9555x0MDQ3h0Ucf1cSffvppAEB9fb0mbuvIkSO4deuWHFatXLlS/b8jxyH9cOR9PHLkCE6fPi2HcfbsWTz88MMICwtTY7OpS56DSQlNkJqaisLCQjnsFl5eXsjKynL6gmp1dYA8uae/Hygp0cYI+OCDDwAADzzwgCZ+3333AQDa2to08el0dHTgypUrMJlMWL16tRp39nHIPZz9Pt6+fRvHjx/Hiy++KBdNMJu6pE9uT0qKi4s1g6GSk5OnHQxFrlNTUwNvb2+Eh4fLRW4TGxsLq9WKhoYGuWjOzp0TiIgYS0QUfX3AwYO2tQhTXERm+020o6MDBQUFeOaZZ5Cfnz/hW66zjkPu5ez38bXXXoOPjw+efPJJuWiC2dQlfXJrUpKQkIBNmzbh/fffhxACeXl5KC0tRVRUlFyV5oHVakV6erpuWkls5eXlOb21BAAOHBByiFwgKioKgYGBOHr0qBrjlw+aTk9PD3Jzc/H9739fLppgNnVJv9yalFRVVeHIkSNYvnw5AGDfvn3IyMiA2WxGVVWVXJ1crKioCLGxsbpc3j0wMBDh4eEoKiqSi+YsJwe4fn1iNw45X0NDA4QQ6O7uxgsvvICjR4/yywdN6+TJk/D390diYqJcNMFs6pJ+uTUpsefrX/86AOCTTz6Ri8iFBgYGUF5ejqysLLlIN44ePYry8nIMDw/LRXOybBlw5owBL7wgMDoql5LC29tbDgHj3TGztXr1ahw5cgQmk2nClw9nHofcx1nvY09PD0pLS/HCCy/IRRPMpi7pm+6SEnKPl156CX/1V3+lub/NK6+84tal3hsaGnD8+HH1sa+vL77yla/AaDROWCRppltERITmGOHhwMaNwPnzmjCAsUGv8fFi0bekbNq0CQDw4YcfauIjIyMAgMcff1wTnwmlNc72y4crjkPzz1nv48mTJ2E0GpGUlCQXTTCbuqRvbktKCgoKsGbNGjmMn//85wCAjRs3ykXkQq+99hr+9//+37BarWosNjbWbTfGs1qtSE1N1TTFDgwM4De/+Q2GhoYwvsbOrLfGxkbNcQCgpMSAgweB3/zm81hTE9DeLpCZyZaUyMhIAJjwu1Omdj777LOauK2CggJs3rxZDuPu3bsAgEceeUSNOXIc0g9nvI9Ky8exY8fU7v3JzKYueQB54ZL5oqzGZ7vSY2VlpcAMVv0j17C3UJm7FlFz9WuJi/tMFBd//ri4WIiAgM+E8ono6xNiZOTzun19n9ddjJSFr8rKyoQYX6HTaDSKjIwMTT1lNU2F8jnPyMjQfM6NRqPdz/lMj0P6NtP3UT5fFMr+3d3dctEEs6lL+jfxbJgnFotFmEwm4e/vr56YISEhIj8/X65K8+TevXti/fr14qOPPlJj7ljqvb+/X/j5+Yl79+6psfb2dhEYGKip5wg5KRkZEQIY22yFhX0mkpI+0wYXqfz8fGE0GgXGl4i391mVLzLd3d0iIyNDhISEzPhzPpPjkP7N5H2UzxfbuL3VWu2ZTV3SP13eJZjcp6amBufPn0d1dbUaKyoqwscffzxvU4VNJhMeeugh7N+/X41t374dhw4dctqMjbo64CtfAYKDP4+1tQGtrcCBA9pYVZXA1q0GREd/HiciIudz25gS0id7C5Xt378fra2tsFgsmrquYLFY0NraqklIampq4OXl5bSEBACio7UJCTD2WElIlBVfg4OBp54y4NNPtXWJiMj52FJCE1gsFphMJjQ2Nqqzcey1oLhCREQEsrKy1BVlrVYrgoKCUF9fP6/rp/T3AytXAnFxAtXVBvT1AStWyLWIiMiZ2FJCEwQGBiI0NBRlZWVqLDY2FhhPTlzF3hL3ZWVliIqKmteEBBhLQEZGgMxMA0ZGmJAQEc0HtpSQXQMDA9i+fTvq6+vh6+sLALh58yZ27NiBq1evatYzcQZ7LSIDAwPYsmULbty44fTjERGR/rClhOzy9fXFrl27kJ+fr8bWr1+P8PBwTQuKs9hb4j49PR1ZWVlMSIiIFgm2lNCUgoKCUFFRgcDAQADA8PAwtmzZgsbGRrUFxVH2WkQsFgv27NmD9vZ2uToRES1QbCmhKRUWFmpWdfX29sahQ4eQnZ2tqecIey0iJpMJpaWlmnpERLSwMSmhKYWHh8Pb21szwNWZU4QtFgssFgt2796txmpqauDr64vQ0FBNXSIiWtjYfUPT6u3txfbt29He3q62ZrS2tiI1NRVXr16Vq8/Kli1bUFhYqCYgVqsVGzZswNWrV53WPURERJ6BLSU0LT8/P8TGxqKoqEiNhYaGws/PDzdv3tTUnY3W1lZ1+rHi+PHj2LVrFxMSIqJFiC0lNCPz0YJhb8ArEREtHmwpoRnx8vJCXl6eZtCrs6WmpiIvL48JCRHRIsWkhGYsMTERN2/edMoAV5nFYsHNmzeRmJgoFxER0SLB7huaFYvFgm984xvYuHGjXOSQX/7yl3jnnXfw8MMPy0VERLRIMCmhWWtubsZ//ud/ymGH/K//9b+wcuVKvPLKK3IREREtEkxKSBfmYyAtERHpG8eUkC7Mx0BaIiLSNyYlpBuJiYno7e1Fa2urXERERIsAu29IV3gjPiKixYstJaQrgYGBCAwMxLlz5+QiIiJa4NhSQrrDlV2JiBYntpSQ7vj6+mLfvn3Izs6Wi4iIaAFjSwnpktVqRVBQEOrr6+Hn5ycXExHRAsSWEtIlThEmIlp8mJSQbsXGxmJ4eBhNTU1yERERLUDsviFd4xRhIqLFgy0lpGuBgYEIDw9HUVGRXERERAsMkxLSvaNHj6K8vFx9XFJSgjNnzsBgMGi2tWvXoq6uTrMvERF5DnbfkEcYHh6Gt7c3AKCrqwu//e1v8eijj8L29G1ra0NISAj6+vqwYsUKm72JiMgTsKWEPIKSkADAsWPH8Md//MeacgAIDg5GWFgY+vr60NbWBoPBgL1798JgMMhViYhIh5iUkEdpa2uTQ6q2tja0tLRg3bp1aiw8PFzTmkJERPrFpIQ8yo0bNxASEqI+th1TsmPHDjQ2NmLJkiVq+bZt29T/ExGRvjEpIY/S0dGBjRs3qo+FEOrW1dWF8PBwTX2OLSEi8hxMSsijtLa2Yv369XKYiIgWACYl5FFaWlqwdu1aOUxERAsAkxLyGG1tbYiLi5PDRES0QHCdEvIYo6Oj6OvrY0sJEdECxaSEiIiIdIHdN0RERKQLTEqIiIhIF5iUEBERkS4wKSEiIiJdYFJCREREusCkhIiIiHSBSQkRERHpApMSIiIi0gUmJURERKQLTEqIiIhIF5iUEBERkS4wKSEiIiJdYFJCREREusC7BJPDPv74Y2RlZcHPz08umrPh4WFs2bIFCQkJchERES1QbCkhhx0+fBj//u//LocdlpmZCavVKoeJiGiBYksJOcRisWDPnj1ob2+XixyWmpqKVatWISUlRS4iIqIFiEkJOSQoKAgVFRUIDAyUixxmtVqxYcMGXL16Fb6+vnIxEREtMExKaM6qqqpQW1uLyspKNXbz5k0MDAxo6s2Gt7e3JsEpKytDR0cHSktLNfWIiGjhYVJCczJZK0Z6ejpaW1s1dWfDz88PFRUVmpgrW2OIiEg/mJTQnLzyyiuaf12pqakJ2dnZaGxslIuIiGgBYVJCszYwMIAtW7bgxo0b8PLykotdIi4uDrt27UJsbKxcRERECwSTEpq1HTt2ICYmBomJiXKRy/T29mL79u1ob2+ft0SIiIjmF9cpoVmxWCy4efPmvCYkGB9rEhUVhbKyMrmIiIgWCLaU0Ky4c9DpZINriYhoYWBLCc1YVVUV1q9f75aEBAC8vLxw9OhRZGdny0VERLQAsKWEZkRPrRTubK0hIv3bvHkzrl27JofJA+impaSlpQXJycnw8fGZ9/EKND0vLy9UV1e7PSEBgMLCQqSmpsphcrHy8nKsWbMGBoMBPj4+yMzMxO3bt+VqU5ruc97T06OWGwwGGAwGJCYmoqWlRa5KOufI+TLTfZVzRN7MZrNclTyFcLPBwUFhMpmE0WgUlZWVoru7W65C86Szs1PExcWJzs5OTTwnJ0fU1tZqYu4WGxsrqqur5fCc1dYKkZT0mRgZ0cbj4j4T0q9jUcrPzxcARGVlpRBCiObmZgFAREZGylXtmsnn3GKxCKPRKEJCQtTy7u5uERISIgCI5uZmeRfSKUfOl9nsC2DSjTyT29+5kJAQERISIgYHB+Uimmdms1kAEHFxcZp4XFycKC4u1sTc7aOPPhLr168X9+7dk4vmpLhYCGDsX1uAEGazNrbYDA4OCqPRKEwmkyZuMplmnCzM5HOekJAgAExIWLq7uwUAkZCQoImTPjlyvsx2X3sx8mxu7b4pKCiA2WzGG2+8geXLl8vF5CbXr19HXV2dHNYVV0wRDggQOHgQ6O+XSxa3d955B0NDQ3j00Uc18aeffhoAUF9fr4nLZvo5Hx4eBgCsXr1aE1ceK+Wkb46cL47sSwuD25KS27dv4/jx4zCZTHjooYfkYnKjyspKxMTEYHR0VC7Slby8PJw6dcqhGwDa2rgRKC4GDhwQctGi9sEHHwAAHnjgAU38vvvuAwC0tbVp4rZm8zn39vYGAHR0dGjiyliCxx9/XBMnfXLkfHFkX1oY3JaU2GbEycnJ6qCmNWvWoKCgQK5O8yg4OBhJSUl4/fXX5SK3s01AlCnC3/72t9HU1DSnTb554K5dwPXrQFOTJryoTXYhCAsLk0MTzOZznp6eDqPRiL1796qJSU9PD771rW8hJCQESUlJmvqkT46cL3PZt7i4WDMoNjk52e6gWPIQcn/OfFH6CENCQsRbb70lhM1gONgMcqL5o4wpEUKIvr4+AUD09fXpZkxJf3+/CAwMFENDQ2qsvb1dLFmyRISHh89pi42NFffu3RPFxWODWoUQorFRiICAsf8rY0pGRsbKASFyctTDLwqRkZGT9t1jkgGIitl+zru7u4XRaBSwGbDo7+8/YZwJ6Zcj58ts901ISBD5+fnqWKWysjL1fCPP5LakZKqTz2g0Tjj5yPVskxIhhCguLhZxcXG6SUpSUlJEYWGhJhYeHi4aGxs1sbmwTUrE+KwbZfCr2Tw2O0f5FYSFfSb6+j7fd6Gb6rNq70Jha6p95c+5xWIR/v7+IiEhYcLsG6PRKCwWi83epFdTveeOnC/T7avIyMgQsJPwkmdwW/fNVB5++GFcuXJFDtM8O3DgAK5fv47q6mq5aN5ZLBa0trZi//79aqympgbe3t4IDw/X1HWGkhIDDh78/HF0NHDgADA6Ctx/P7B0qW3thU0Z6yGTx37Mlvw5z8vLQ3d3N0pKStTBratXr0ZDQwOGhoawd+9em71Jrxw5XxzZV/H1r38dAPDJJ5/IReQB3JaUyCPsZUajUQ6RG5w5c0YOuUV2djaOHj2q3iHYarUiPT0dhYWFclWnWLECyMmRo4srGVFs2rQJAPDhhx9q4iMjI8A0A1Bn8zm/dOkSAEyYoaM85oJYnsGR88WRfWlhcFtSokz5kk++27dv48qVK3ZXeyTXWrdu3YQ//OHh4TCbzYiPj9fEBwYGUFVVpYm5Sk1NDQAgNjZWjRUVFSE2NhZ+fn42NecuPh44ftygib34ImA2A+vWfR4TAoiPN2D8JS0KkZGRAIDGxkZNXJme+eyzz2ritmbzOQ8JCQHGB7faUgYtKuWkb46cL7PZt6CgAGvWrLGpNebnP/85AGDjxo1yEXkCuT9nPil9xUr/YXd3t0hISBBGo5ED23Tu3r17IjQ0VLS3t8tFTnXv3j0RGBgobty4ocb6+/uFn5+f0xZOm4kLFz4f4FpcPPZ4MVEGppaVlQkxvsqm0WgUGRkZmnqws5rmTD/nb731ljpI0XZMiXJsZaAs6Z8j58tM91VWfjWZTOpA18rKyhmPPSF9cmtSYrv0NAB1JT8mJPpUWVmpSQSqq6tFbGyspo6zFRYWipSUFE1s9+7doqKiQhNzNdvZN3FxE5ejXwzy8/PVz6q/v7/Iz8+Xq9i9yMzmc97c3Kyu7KrUTUhIsDvwkfRtrueLmOG+FotFmEwm4e/vrz5PSEiI3brkOXiXYJqx9PR0LFu2DMeOHVNjcXFx2LVrl6ZrxVkGBgYQERGBq1evqgPgLBYL9uzZg/b2drk6ERF5OCYlNGNWqxUbNmzA1atX1bsFWywWmEwmNDY2qoNQnUVZBdR2xs2WLVtQWFiI0NBQTV0iIvJ8bhvoSp7Hy8sLWVlZSE9PV2OBgYEIDQ116j1oMMUUYF9fXyYkREQLFFtKaNaCgoJQUVGBwMBAYPxGaVu2bEFjY6PaguKoiIgIZGVlqWuQ2GulISKihYUtJTRrpaWlMJlM6mNvb2/s27cP+fn5mnpzZW9RtOPHj2PXrl1MSIiIFjC2lNCc2BvgKregzIXVakVQUBDq6+vVNUgGBgawZcsW3Lhxw+njVoiISD+YlNCc2EsUGhoacOrUKXWho7k4fvw47t69i7y8PDW2Y8cOxMTEcEE9IqIFjt03NCe+vr5ITExEUVGRGouKikJMTAysVqum7mx4e3sjKytLfWyxWHDz5k0mJEREiwBbSmjO5mPwqTO6hIiIyDOwpYTmzN4UYWeqqqrC+vXrmZAQES0SbCkhhwUFBeGll17Cgw8+KBfN2f/9v/8X+/btc2krDBER6QuTEnLYm2++iby8PKcmD8PDw4iMjMTx48flIiIiWqCYlBAREZEucEwJERER6QKTEiIiItIFJiVERESkC0xKiIiISBeYlBAREZEuMCkhIiIiXWBSQkRERLrApISIiIh0gUkJERER6QKTEiIiItIFJiVERESkC0xKiIiISBeYlBAREZEuMCkhIiIiXWBSQkRERLrApISIiIh0gUkJERER6QKTEiIiItIFJiVERESkC0xKiIiISBeYlBAREZEuMCkhIiIiXWBSQkRERLrApISIiIh0gUkJERER6QKTEiIiItIFJiVERESkC0xKyOWCgoJgMBicsv3d3/2d/PRERLRAMCkhl2poaICvry+EEA5vQ0NDOH36NAYGBuTDEBHRAmAQQgg5SOQMVqsVERERKC0tRWBgoFw8J2VlZejo6EBpaalcREREHo5JCblMUVERPv74YxQWFgIAent7ce7cObnatLy8vJCSkgIvLy9gvDuooqLCaYkOERHpA7tvyCUGBgZw6tQp5OXlqbHh4WFNnZmyWq2wWq3q44qKCuzZs0dTh4iIPB9bSmjWbt68ifXr18thjT179uCxxx7D7t275SKncPXzExHR/GNSQrO2Z88eVFRUyGGVxWLBnj170N7eLhc5zcDAALZs2YIbN26o3TpEROTZvvDKK6+8IgeJ7GlqasK5c+fws5/9DAMDAxgeHrbbYrJ9+3ZUVFTA19dXLnKaJUuWAAB+/OMfIyoqSi4mIiIPxJYSmrWpWkqUpGWycmeyWq3YsmULKisr7SZHRLQ4bd68GdeuXZPD5AHcNtC1oKBgwsJY8kb6tG7dOjkEjCcJ2dnZmsGtruTl5YW8vDykpqbKReRkLS0tSE5Oxpo1a9TPZ2JiIjo6OuSqdpWXl6v7+vj4IDMzE7dv35ar0QLiyHs+033la4aymc1muSp5CuEm+fn5AsCkW0JCgrwL6VxeXp4oLS2Vwy4XGxsrqqur5TA5SVlZmQAgTCaTGBwc1MSMRqMam4zyWa+srBRCCNHc3CwAiMjISLkqLRCOvOez2Ve+bthu5Jnc9s7l5+cLk8kkh8Xg4KAwGo2iublZLiKy66OPPhLr168X9+7dk4vICfLz84W/v/+E5CMyMlIAmPKzqnye5c+6yWSadl/yTI6857Pd116MPJvbum+OHDmC06dPy2GcPXsWDz/8MMLCwuQimkZubi727t2riTU1NSE+Pl4Tc1Rubi7q6uo0MWcfYzb8/PwQGxuLoqIiuWhW6uqA3FxtLDcX6OrSxhabI0eO4NatW1i+fLlcNK133nkHQ0NDePTRRzXxp59+GgBQX1+viZPnc+Q9d2RfWhjclpTYc/v2bRw/fhwvvviiXEQzYDabcfbsWXTZXEUvXryI6upqTT1Hmc1mpKWlob+/X405+xizlZWVhfLycofui/Ppp0BmJtDU9HnMbBYYGbGtRYpbt27BaDRO+QXigw8+AAA88MADmvh9990HAGhra9PEyfM58p47si8tDLpKSl577TX4+PjgySeflItohpKSkvDOO+8AAEZHR+VitLW1Ye/evdi7d68meZmt7373uzhw4IAcdhtnDXrNyQFeflnAzq+ObJSXl6O7u3tCi5lssovIVIkMeTZH3vO57FtcXKwZFJucnGx3UCx5Bt1MCe7p6YG/vz8qKyuRmJgoF9MMxMfHY/fu3Thx4gSam5vVC0ZMTAyEEOjv78fKlSvR2NgIAHj99ddRUlKCFStWoKmpCU22TQSTSElJwd/8zd8gMzMTra2tWLZsGXbu3AmDwQB3nEpNTU0IDAyEt7c3AOBrX/saNmzYgI0bN8pVpxQeHo4PPwwHAISGAqWlAm+8YUB8vEBmpgHBwUB/P9DXBwQHy3svLm+//TaeeuqpGX1Wo6KicOXKFTQ3N0+4sBgMBkRGRqKhoUETJ8/myHs+230TExOxadMmJCUlYfny5SgvL8f+/fsREhLCKcEeSjctJSdPnoS/v/+0f+Roal/5ylcAAP39/Whubsbjjz+ulvX19SEgIACvv/46Xn/9dVy/fh19fX0AoF7Up+Ll5aVZPfXAgQO4fPmyphtnPg0MDMBkMmli/+///T+1qXc2bH/+4GBg9WqDphunvx84cEAgJ0dg69b5T770oqOjAzt37pxRQkLkalVVVThy5Ig63mnfvn3IyMiA2WxGVVWVXJ08gTzy1R26u7sFAJGfny8X0SzExcUJs9kscnJyxIULF0RaWpoQ4yPUhRDCbDaLpKQkYTab1a2vr096lukpxxFCiM7OThEXF+eWKXj79+/XTEEuLCwUKSkpmjqzUVw8tgkhxMiIEGFhn4mwsM/E+I8qhBDCbBYiKemzzwOLiMViEUajUZ2qORMJCQl2Z0hYLBaBSaZ5kmdz5D13ZF/FW2+9xeuJB9NFS8nJkydhNBqRlJQkF9EcPPvss3juuefw1FNPaeLBwcG4c+cOUlJSkJOTg9LSUixdulRTZ7bWrl2Lbdu2yWGXs1gsaG1txf79+4HxVpPz58/j6NGjctU5WbIEKCoyoKVFu4hfTs5YK8liG3PS09ODiIgInD59elYtJJs2bQIAfPjhh5r4yPjoYduWPFoYHHnPHdmXFgg5S5lvbCVxns7OTjEyMiLEeKuIwvb/Yrye2WxW686W7XEU8jGGhoZcuqBZaGiouHr1qvo4JSVFFBYWaurMVl/f2Gars3Os1WRk5POy4uKxFpPFJDIyUmRkZMhhkZCQMOVnV/mGKy+GmJGRIQCI7u5uTZw8nyPv+Wz2VdbPkSl133rrLbmIPIDbkxJlUZypTlTyPPfu3ROBgYHixo0bcpHDKisrRWJiovq4vb1dBAYGauo4W2fnWHeO2Tz2b2enXGPhqqysFLCzYqay2SYlSsyW8hkvKysTYnyFTqPRaDfJoYVhpu+5I+eLsvKr7UrDyrk6k24e0ie3JyXKSUWer729XVRUVKiPq6urRWxsrKaOo+7duyf8/PxEf3+/GgsPDxeNjY2aeq5QXCxEXNxnYh4OpSvKyq2Tbbb9//YuMmL8AmI0GgUA4e/vP2XrCi0MM3nPHTlfLBaLMJlMwt/fX32ekJAQu3XJc+hmSjB5PqvViqCgINTX18PPzw8AEBcXh127diE2NlauPiepqalYtWoVUlJSAAA1NTU4f/682xdvIyIixzEpIaeSk4SbN29ix44duHr1qmY68VzIz2W1WrFhwwZcvXoVvr6+cnUiIvIwuph9QwtHbGwsrFarusDR+vXrER4ejrKyMrnqrKWnpyMrK0tNbo4fP45du3YxISEiWiDYUkJTioiI0DyurKycNgmwWCwwmUxobGx0WouG3AIzMDCALVu24MaNGw63wBARkT4wKSHV6OgoRkZGsGLFCrlo1uSxH+fOncPPfvYzVFRUyFWnZbVasWXLFlRWVmL9+vWAC8aqEBGR+7H7hlSdnZ24fPmyHJ4T+a69u3fvhsVigcVikatOq6ioCFFRUWpC0tTUhOHhYSYkREQLDFtKSNXW1obW1lan3f23rKwMHR0dKC0tBca7XLy9vWfd3dLb26vO5rFarYiIiEBpaSkCAwPlqkRE5MHYUkIus3//frS2tqqtI76+vrNOSACoCQnGE53Q0FAmJERECxCTEnKp0tLSCXfynauBgQGUl5cjKytLLiIiogWASQm5VGhoKL70pS/BYDA4vK1YsQK7d++Gt7e3fBgiIloAmJSQyzU1NWH8lgYOb866CzAREekPkxJSLV26FA8++KAcJiIimhecfUNERES6wJYSIiIi0gUmJURERKQLTEqIiIhIF5iUEBERkS4wKSEiIiJdYFJCREREusCkhIiIiHSBSQkRERHpApMSIiIi0gUmJURERKQLTEqIiIhIF5iUEBERkS4wKSEiIiJdcMpdgg0GgxwiIiIiUs0k3XBKUkJERETkKHbfEBERkS4wKSEiIiJdYFJCREREusCkhIiIiHSBSQkRERHpApMSIiIi0gUmJURERKQLTEqIiIhIF5iUEBERkS4wKSEiIiJd+P8BVZxXC4HfqTgAAAAASUVORK5CYII=\" style=\"width: 475px; height: 545.082px;\" width=\"475\" height=\"545.082\"\u003e\u003c/p\u003e\u003cp\u003elabelling reagents. Nevertheless, electron-poor DIBAC reacted the fastest with benzyl azide and 2-azido-1,3-diisopropylbenzene, showcasing the importance of both elec- tronic and steric factors. These differences in reaction rate were then applied in the three-component labelling of a BCN-labelled protein with a diazide and a DIBAC-labelled fluorescent dye.\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eWe have developed a new group of organic alkyl azides featuring a difluoro substitution in the α-carbon position (RCF\u003csub\u003e2\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003e).\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e These fluoroalkyl azides have been shown to undergo CuAAC efficiently\u003csup\u003e\u003cspan additionalcitationids=\"CR23 CR24 CR25\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e as well as nitrene formation for the synthesis of \u003cem\u003eN\u003c/em\u003e-trifluoromethyl-aziridines (from CF\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003e).\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e An additional benefit of fluorinated azides compared to alkyl azides is their improved stability. For example, CF\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003e is a highly thermally stable molecule, and even N\u003csub\u003e3\u003c/sub\u003eCF\u003csub\u003e2\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003e is quite stable\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e compared to the explosive N\u003csub\u003e3\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003e. Whilst there is one example of the usage of a fluorinated azide derived from azidodifluoroacetic acid bound to a uridine phosphate amino sugar in SPAAC,\u003csup\u003e29\u003c/sup\u003e no systematic research of the reactivity of fluorinated azides and their applicability in bioorthogonal labelling has been conducted to date. Due to the strong electronegativity of fluorine atoms, fluorinated azides are expected to react preferentially with electron-rich cyclooctynes whereas alkyl azides should react faster with electron-deficient cyclooctynes. If the difference in reactivity is sufficiently high, selectivity can be achieved in bioconjugation of a mixture of reaction constituents. Thus, one kind of bioconjugation reaction (SPAAC) would be used in the bioorthogonal ligation of two appropriate reaction partners. To this end, however, knowledge of the reaction rates of the two azide families is essential for the development of selective SPAAC for application in bioconjugation and molecular biology (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cp\u003eAt the outset of the study, we evaluated preparative SPAAC reactions of commercially available or synthesized eight- to ten-membered cycloalkynes \u003cb\u003e1\u003c/b\u003e with fluoroalkyl azides \u003cb\u003e2\u003c/b\u003e in an organic solvent (Scheme 2). Reactions with a BCN derivative were complete within 1 hour at ambient temperature, yielding products \u003cb\u003e3a\u0026ndash;3d\u003c/b\u003e in quantitative amounts, while reactions with a DIBAC derivative required up to 4 hours under the same conditions and produced SPAAC products \u003cb\u003e3e\u0026ndash;3h\u003c/b\u003e in nearly quantitative yields. These products formed as regioisomers at a ratio of approximately 2:1. The NMR characterization is further complicated by restricted rotation around the amide bonds, which meant that rotamers were also observed by \u003csup\u003e1\u003c/sup\u003eH NMR. Reaction with cyclooct-2-yn-1-ol was less efficient than with BCN or DIBAC derivatives. The nine-membered cyclic sulfur derivative afforded cycloaddition product \u003cb\u003e3j\u003c/b\u003e in high yield but required prolonged heating to 100\u0026deg;C. Finally, a ten-membered, but still a rather strained derivative synthesized from 4,4\u0026rsquo;-biphenol underwent the SPAAC reaction at 100\u0026deg;C to give \u003cb\u003e3k\u003c/b\u003e in a high yield.\u003c/p\u003e\u003cp\u003eNext, the model fluoroalkyl azide PhOCF\u003csub\u003e2\u003c/sub\u003eCF\u003csub\u003e2\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003e (\u003cb\u003e2a\u003c/b\u003e) and its non-fluorinated analogue PhOCH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003e (\u003cb\u003e4\u003c/b\u003e) were used together with the most reactive derivatives \u003cb\u003e1a\u003c/b\u003e (BCN, R\u0026thinsp;=\u0026thinsp;H)\u003c/p\u003e\u003cp\u003eand \u003cb\u003e1b\u003c/b\u003e (DIBAC, R\u0026thinsp;=\u0026thinsp;CH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003eNHAc) derivatives for the determination of absolute rate constants for the SPAAC reaction. Both azides are easy to handle liquids, unlike gaseous azidomethanes. For applications in bioorthogonal chemistry, it is essential that reactions proceed with a sufficiently high rate.\u003c/p\u003e\u003cp\u003eNeither \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH nor \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003eF NMR spectroscopy could be used to obtain kinetic data, because a preliminary test reaction of BCN with \u003cb\u003e2a\u003c/b\u003e revealed that it was too fast for precise reaction monitoring. UV spectroscopy was also considered, but the absorption differences between the starting alkynes, azides and the triazole products were insufficient for the method to be utilized. By contrast, infrared spectroscopy allowed for the precise monitoring of azide concentration in time as azide stretching vibration (~\u0026thinsp;2100 cm\u003csup\u003e\u0026ndash;\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e) is spectral region silent for other functional groups (Fig.\u0026nbsp;2).\u003c/p\u003e\u003cp\u003eThe determined absolute rate constants of the second-order SPAAC reactions of azides \u003cb\u003e2a\u003c/b\u003e and \u003cb\u003e4\u003c/b\u003e with cyclooctynes BCN and DIBAC (at room temperature and a solvent mixture of THF and water (9:1)) (Fig.\u0026nbsp;3) revealed that, indeed, fluorinated azide \u003cb\u003e2a\u003c/b\u003e reacted faster than non-fluorinated azide 4 with BCN. Specifically, a 16-fold difference was observed. Conversely, a 20-fold rate increase was observed for \u003cb\u003e4\u003c/b\u003e compared to \u003cb\u003e2a\u003c/b\u003e in cycloaddition with the DIBAC derivative. When comparing BCN versus DIBAC for azide \u003cb\u003e2a\u003c/b\u003e a 126-fold rate increase was observed, while DIBAC over BCN was only 2.5-times more reactive for alkyl azide \u003cb\u003e4\u003c/b\u003e. In terms of absolute values, \u003cb\u003e2a\u003c/b\u003e displayed reactivity that was almost identical to that of 4-azidopyridine. Note that the absolute rate constants of SPAAC reactions are sensitive to temperature and the solvent used.\u003c/p\u003e\u003cp\u003eThe reaction was also characterized with the help of \u003cem\u003eab initio\u003c/em\u003e calculations. The structures of azides (\u003cb\u003e2a\u003c/b\u003e and \u003cb\u003e4\u003c/b\u003e) and cyclooctynes BCN and DIBAC, as well as all four transition\u003c/p\u003e\u003cp\u003estates of the azide-alkyne cycloadditions, were optimized at the DFT/CAM-B3LYP/6-31g* level, and activation energies were calculated at the highly accurate DLPNO-CCSD(T)/cc-pVTZ level\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e with the extrapolation to a complete PNO (pair natural orbital) space\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The results are qualitatively in line with kinetic experiments, where the reaction of BCN with \u003cb\u003e2a\u003c/b\u003e is faster than with \u003cb\u003e4\u003c/b\u003e and the reaction of DIBAC with \u003cb\u003e4\u003c/b\u003e is faster than with \u003cb\u003e2a\u003c/b\u003e (see the SI for further details). Previous studies gauged SPAAC reactivity by comparing the HOMO\u0026ndash;LUMO energies of the azide and cycloalkyne partners, using these frontier-orbital gaps to assign the cycloaddition to either a normal- or inverse-electron-demand regime.\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e In the present system, however, such frontier-orbital descriptors do not reproduce the experimentally observed rate order. This shortcoming implies that a purely one-electron view of the transition state is too simplistic; when the activation barriers differ by only a few kilocalories per mole, additional factors \u0026ndash; including\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eActivation energies calculated at the DLPNO-CCSD(T)/cc-pVTZ level with the extrapolation to a complete PNO space. Transition states and reactants were optimized at the CAM-B3LYP/6-31g* level.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEntry\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eReactants\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eE\u003c/em\u003e\u003csub\u003eA\u003c/sub\u003e (eV)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBCN\u0026thinsp;+\u0026thinsp;\u003cb\u003e4\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.43\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBCN\u0026thinsp;+\u0026thinsp;2a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.38\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDIBAC\u0026thinsp;+\u0026thinsp;\u003cb\u003e4\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.27\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDIBAC\u0026thinsp;+\u0026thinsp;\u003cb\u003e2a\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.39\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eelectronic polarization, entropic contributions, distortion/strain energy, and solvent effects \u0026ndash; contribute comparably. A detailed discussion of these contributions is provided in the Supporting Information.\u003c/p\u003e\u003cp\u003eAlkyl and aryl azides are widely used in SPAAC reactions in bioconjugations; however, fluoroalkyl azide \u003cb\u003e2a\u003c/b\u003e has not been evaluated in this setting. The compatibility of \u003cb\u003e2a\u003c/b\u003e with aqueous media and its possible reactivity with naturally occurring functional groups were evaluated in a series of control experiments. To this end, azide \u003cb\u003e2a\u003c/b\u003e was dissolved in a mixture of deuterated PBS buffer (pH 7.4) and MeCN. After 3 or 120 hours at room temperature, no decomposition was observed and azide \u003cb\u003e2a\u003c/b\u003e was fully recovered from the buffer solution by extraction (Scheme 3A). The SPAAC reaction was also tested in almost pure water (traces of THF were used to solubilize \u003cb\u003e2c\u003c/b\u003e) in 3mM concentration of BCN and \u003cb\u003e2c\u003c/b\u003e. Extraction of the reaction mixture after 1 h of stirring afforded only product \u003cb\u003e3b\u003c/b\u003e, indicating that the click reaction is not affected by the presence of a large excess of water (Scheme 3B). Next, the compatibility of the click reaction with fluorinated azide in the presence of functional groups that are prevalent in biological systems was tested. Fluorinated azides are known to react with primary alkyl amines to form tetrazoles.\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e However, this reaction typically requires a non-aqueous solvent and an organic base (e.g. Et\u003csub\u003e3\u003c/sub\u003eN). To investigate whether this reaction would proceed in water, azide \u003cb\u003e2a\u003c/b\u003e was treated with L-lysine, and after 24 hours no reaction or decomposition was observed by \u003csup\u003e19\u003c/sup\u003eF NMR. Only in the presence of Et\u003csub\u003e3\u003c/sub\u003eN at 40\u0026deg;C for three days, a low conversion of azide to tetrazole \u003cb\u003e5\u003c/b\u003e and amide \u003cb\u003e6\u003c/b\u003e was observed by \u003csup\u003e19\u003c/sup\u003eF NMR (Scheme 3C). In the presence of excess lysine, a SPAAC reaction between \u003cb\u003e2a\u003c/b\u003e and BCN took place selectively and no tetrazole \u003cb\u003e5\u003c/b\u003e or amide \u003cb\u003e6\u003c/b\u003e were observed (Scheme 3D). The cysteine-containing tripeptide glutathione plays a vital role as an antioxidant in living organisms. The SPAAC reaction was repeated in the presence of a 10-fold excess of this strong sulfur-containing nucleophile, and again, only the triazole product \u003cb\u003e3c\u003c/b\u003e formed with no traces of fluorinated side-products detectable by \u003csup\u003e19\u003c/sup\u003eF NMR (Scheme 3D).\u003c/p\u003e\u003cp\u003eTo successfully apply the established differences in the reactivity of fluoroalkyl and alkyl azides in SPAAC bioconjugation and imaging followed by fluorescent spectroscopy or confocal fluorescent microscopy, a set of fluorescent fluorinated azide-containing probes was needed. Therefore, a new azidotetrafluoroethoxy-substituted benzoic acid 10 was synthetized in three steps from ethyl 4-hydroxybenzoate (\u003cb\u003e7\u003c/b\u003e) by (i) the fluoroalkylation of the phenolate derived from \u003cb\u003e7\u003c/b\u003e using 1,2-dibromotetrafluoroethane, (ii) magnesiation with Turbo-Grignard and azidation according to a literature precedent,\u003csup\u003e23\u003c/sup\u003e and (iii) alkaline hydrolysis. Azide \u003cb\u003e10\u003c/b\u003e was then coupled to form a coumarin-based dye \u003cb\u003e11\u003c/b\u003e or converted to an NHS ester \u003cb\u003e12\u003c/b\u003e, to form a BODIPY probe \u003cb\u003e13\u003c/b\u003e (Scheme \u003cspan refid=\"Sch2\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Commercial fluorescein- and rhodamine-tagged alkyl azides \u003cb\u003e14\u003c/b\u003e and \u003cb\u003e15\u003c/b\u003e, respectively, were used as fluorescent alkyl azide probes (Scheme \u003cspan refid=\"Sch2\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eThe reactivity of coumarin probe \u003cb\u003e11\u003c/b\u003e with BCN and DIBAC was tested in separate experiments and in a mixture of both cyclooctynes. UPLC-MS analysis revealed that although probe \u003cb\u003e11\u003c/b\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) reacted efficiently with BCN (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), it reacted less efficiently with DIBAC (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). A competition experiment with both cyclooctynes revealed that \u003cb\u003e11\u003c/b\u003e reacted predominantly with BCN over DIBAC (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003eD), suggesting that the selectivity predicted from the comparison of absolute rate constants was retained in the competition experiment and might be applicable in bioconjugation or bioimaging.\u003c/p\u003e\u003cp\u003eTo investigate the selectivity of the labelling reaction on model biomolecules, we prepared trastuzumab (TzMab), a monoclonal anti-HER2 antibody,\u003csup\u003e34\u003c/sup\u003e and concanavalin A (ConA),\u003csup\u003e35\u003c/sup\u003e a mannose-binding lectin, each modified with either BCN or DIBAC via active ester chemistry (for details, see the SI). This resulted in four conjugates: TzMab-BCN, TzMab-DIBAC, ConA-BCN, and ConA-DIBAC. The degree of labelling (DOL) of each protein was determined by intact mass spectrometry. These DOL measurements were then used to normalize the protein concentrations in subsequent experiments so that each sample contained an equivalent amount of strained dipolarophile. The two proteins were selected due to their difference in size, which allows their distinction and separation by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).\u003c/p\u003e\u003cp\u003eTo distinguish between the azide dyes, a green fluorescent BODIPY fluoroalkyl probe \u003cb\u003e13\u003c/b\u003e was used in combination with the red alkylazide TAMRA dye \u003cb\u003e15\u003c/b\u003e. Initial experiments (lanes 2\u0026ndash;5 in experiment 1 and 10\u0026ndash;13 in experiment 2), confirmed the presence of the modification on both proteins, which were successfully labelled with both azide probes (Fig.\u0026nbsp;5; controls and Figures SI11\u0026ndash;SI13 in the SI). The fluorescently labelled TzMab runs at around 150 kDa, while ConA, which is a tetramer, appears on the non-reducing denaturating gel mainly in two bands corresponding to a monomer and a dimer (ca. 26 kDa and 50 kDa, respectively).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn the first competition experiment, we combined the two azide probes (\u003cb\u003e13\u003c/b\u003e and \u003cb\u003e15\u003c/b\u003e) with individual proteins carrying either BCN or DIBAC. Because the two dyes have different fluorescence properties, the band intensities cannot be compared directly, but the outcome was clear: BCN-modified proteins (TzMab-BCN and ConA-BCN) gave mixtures of click products (labelled \u0026lsquo;m\u0026rsquo; in Fig.\u0026nbsp;5, lanes 7 and 9), whereas DIBAC-modified proteins (TzMab-DIBAC and ConA-DIBAC) reacted selectively with the alkyl azide \u003cb\u003e15\u003c/b\u003e (labelled \u0026lsquo;s\u0026rsquo; in Fig.\u0026nbsp;5, lanes 6 and 8), showing virtually no reactivity with azide \u003cb\u003e13\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eIn the second competition experiment, we instead combined the two proteins, each bearing a different dipolarophile, with a single azide probe. Interestingly, we saw strong selectivity: ConA-BCN, in the presence of equimolar TzMab-DIBAC, reacted exclusively with fluoroalkyl azide \u003cb\u003e13\u003c/b\u003e (Fig.\u0026nbsp;5, lane 16). However, when the pairing was reversed (ConA-DIBAC with TzMab-BCN), azide \u003cb\u003e13\u003c/b\u003e produced a mixture of products. These results demonstrate that both the dipolarophile and the protein context influence the selectivity of the SPAAC reaction.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe next carried out one-pot competition experiments with both proteins and both azides (Figure SI13). A representative outcome is shown in Fig.\u0026nbsp;5, lane 18: azide \u003cb\u003e13\u003c/b\u003e was first added to a mixture of ConA-BCN and TzMab-DIBAC, followed by alkyl azide \u003cb\u003e15\u003c/b\u003e. The results demonstrated clear selectivity: fluoroalkyl azide \u003cb\u003e13\u003c/b\u003e reacted preferentially with ConA-BCN, whereas alkyl azide \u003cb\u003e15\u003c/b\u003e reacted with TzMab-DIBAC.\u003c/p\u003e\u003cp\u003eTo verify that the two proteins remained active after modification, we used the mixture of the labelled proteins from the last competition experiment to label live SKBR3 cancer cells. This cell line expresses the HER2 antigen, to which TzMab binds. In addition, ConA can label parts of glycoconjugates containing mannose residues on cells of the same line. SKBR3 cells treated with the mixture of fluorescent proteins exhibited distinct fluorescent signal on the cell surface in both channels, as confirmed by fluorescence confocal microscopy and flow cytometry analysis (Figure SI16). Therefore, it can be concluded that, after modification, both proteins successfully recognize their targets.\u003c/p\u003e\u003cp\u003eNext, we tested whether selective labelling of BCN and DIBAC with fluoroalkyl and alkyl azides could be achieved in live cells. For this purpose, we treated the osteosarcoma cancer cell line U2OS with a BCN\u0026ndash;triphenylphosphonium (TPP) conjugate and DIBAC\u0026ndash;sulfo-NHS ester. BCN\u0026ndash;TPP is enriched in mitochondria due to the inherent ability of the TPP group to accumulate in this organelle.\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e The use of the cell-impermeable DIBAC\u0026ndash;sulfo-NHS ester ensures that the DIBAC moiety modifies cell surface components. In this way, we achieved selective installation of BCN groups inside the cells, specifically in mitochondria, and DIBAC groups on the cell surface.\u003c/p\u003e\u003cp\u003eControl experiments performed with cells treated with only one of the dipolarophile-containing reagents confirmed their specific localization inside the cells (BCN\u0026ndash;TPP) and on the cell membrane (DIBAC\u0026ndash;sulfo-NHS ester), respectively (Fig.\u0026nbsp;6C). In the competition experiment, U2OS cells treated with both dipolarophiles were labelled with fluoroalkyl azide \u003cb\u003e11\u003c/b\u003e and, after washing, with TAMRA azide \u003cb\u003e15\u003c/b\u003e. After an additional washing step, images of the cells were obtained under a confocal microscope (Fig.\u0026nbsp;6C). The reaction led to the efficient and specific labeling of the two dipolarophiles in the cells. Fluoroalkyl azide \u003cb\u003e11\u003c/b\u003e reacted specifically with the intracellular BCN\u0026ndash;TPP probe whereas TAMRA azide \u003cb\u003e15\u003c/b\u003e reacted with the DIBAC moiety on the cell surface.\u003c/p\u003e\u003cp\u003eAdditional experiments with fluorescein azide \u003cb\u003e14\u003c/b\u003e produced similar results (Figures SI19 and SI20). These experiments demonstrate that excellent selectivity in the SPAAC reaction can be achieved using fluoroalkyl and alkyl azides in combination with BCN and DIBAC dipolarophiles. The difference in reactivity of the probes enables preferential click labeling, providing practical strategies for the labelling and modification of two different targets via the SPAAC reaction, even in the context of living cells.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe results of our work demonstrate that fluoroalkyl azides (RCF\u003csub\u003e2\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003e) exhibit highly selective and accelerated reactivity in SPAAC with electron-rich cyclooctynes, such as BCN, while reacting significantly more slowly with electron-deficient counterparts, such as DIBAC. Kinetic studies revealed up to a 126-fold rate enhancement in the reaction of PhOCF\u003csub\u003e2\u003c/sub\u003eCF\u003csub\u003e2\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003e with BCN over DIBAC and a 16-fold increase in rate compared to non-fluorinated alkyl azide analogue in reaction with BCN. \u003cem\u003eAb initio\u003c/em\u003e calculations confirmed the selectivity of alkynes with respect to the azides used. Dual labelling experiments using BCN- and DIBAC-modified proteins and live-cell imaging further validated the orthogonal reactivity of fluorophore-modified fluoroalkyl and alkyl azides in biological systems. Together, these findings significantly expand the capabilities of bioorthogonal chemistry, offering future potential applications in dual-target labelling, advanced imaging, and next-generation molecular tools for biomedical applications.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eDATA AVAILABILITY\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe data supporting this article are included as part of the Supplementary Information. The mass spectrometry proteomic data have been deposited to the ProteomeXchange Consortium via the PRIDE\u003csup\u003e37\u003c/sup\u003e partner depository with the dataset identifier PXD066546.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAUTHOR INFORMATION\u003c/p\u003e\n\u003cp\u003eCorresponding Author\u003c/p\u003e\n\u003cp\u003e* [email protected]\u003c/p\u003e\n\u003cp\u003eAuthor Contributions\u003c/p\u003e\n\u003cp\u003eM.T. conceived the idea, performed experiments, and partially wrote the manuscript, V.\u0026Scaron;. performed experiments and partially wrote the manuscript, M.V. partially wrote the manuscript, J.L. prepared compounds \u003cstrong\u003e3i\u0026ndash;3k\u003c/strong\u003e, J.F. and P.S. performed calculations and coined their interpretations, L.B. measured kinetics by IR, F.F. measured and calculated DOL of the proteins, and P.B. conceived the idea, acquired funding, administered and supervised the project and partially wrote the manuscript. All authors have given approval to the final version of the manuscript. \u0026dagger; These authors contributed equally.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eACKNOWLEDGMENT\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis research was financially supported by the Czech Academy of Science (Research Plan RVO: 61388963) and the Czech Science Foundation (Project 23-04659S). J.F. and P.S. were supported by project 24-11566S.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBird, R. E.; Lemmel, S. A.; Yu, X.; Zhou, Q. A. Bioorthogonal Chemistry and Its Applications. \u003cem\u003eBioconjug. Chem. \u003c/em\u003e\u003cstrong\u003e2021\u003c/strong\u003e, \u003cem\u003e32\u003c/em\u003e, 2457\u0026ndash;2479. DOI: 10.1021/acs.bioconjchem.1c00461.\u003c/li\u003e\n\u003cli\u003eScinto, S. L.; Bilodeau, D. A.; Hincapie, R.; Lee, W.; Nguyen, S. S.; Xu, M.; am Ende, C. W.; Finn, M. G.; Lang, K.; Lin, Q.; Pezacki, J. P.; Prescher, J. A.; Robillard, M. 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DOI: 10.1093/nar/gkae1011.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Schemes","content":"\u003cp\u003eSchemes 1 to 4 are available in the Supplementary Files section.\u003c/p\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":"azide, fluorine, click reaction, bioconjugation","lastPublishedDoi":"10.21203/rs.3.rs-7630137/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7630137/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eStrain-promoted azide\u0026ndash;alkyne cycloaddition (SPAAC) is a cornerstone of bioorthogonal chemistry, offering metal-free and biocompatible ligation for applications ranging from bioconjugation to live-cell imaging. However, its relatively slow kinetics and limited selectivity hinder the simultaneous labelling of multiple targets. Here, we report on a systematic study of fluoroalkyl azides as novel SPAAC reagents that display enhanced reactivity with electron-rich cyclooctynes, while showing significantly reduced reactivity with electron-deficient dipolarophiles. Kinetic measurements revealed over 100-fold rate differences depending on the azide\u0026ndash;alkyne pair, enabling orthogonal bioconjugation in both purified proteins and living cells. Experimental and computational data support the rate trends and mechanistic rationale based on electronic complementarity. Fluorescently labelled fluoroalkyl and alkyl azide probes demonstrate the selective labeling of modified antibodies and lectin protein in vitro and the selective labelling of organelles in living cells. This dual-selectivity strategy enables orthogonal SPAAC labeling.\u003c/p\u003e","manuscriptTitle":"Beyond Traditional SPAAC: Achieving Orthogonality and Rapid Kinetics with Fluoroalkyl Azides","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-03 16:31:46","doi":"10.21203/rs.3.rs-7630137/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":"4c08bb87-2794-41b2-a9a3-df09a4595746","owner":[],"postedDate":"October 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":55107579,"name":"Physical sciences/Chemistry/Organic chemistry/Synthetic chemistry methodology"},{"id":55107580,"name":"Physical sciences/Chemistry/Biochemistry/Chemical modification"}],"tags":[],"updatedAt":"2026-05-04T07:05:43+00:00","versionOfRecord":{"articleIdentity":"rs-7630137","link":"https://doi.org/10.1038/s42004-026-01927-6","journal":{"identity":"communications-chemistry","isVorOnly":false,"title":"Communications Chemistry"},"publishedOn":"2026-03-13 04:00:00","publishedOnDateReadable":"March 13th, 2026"},"versionCreatedAt":"2025-10-03 16:31:46","video":"","vorDoi":"10.1038/s42004-026-01927-6","vorDoiUrl":"https://doi.org/10.1038/s42004-026-01927-6","workflowStages":[]},"version":"v1","identity":"rs-7630137","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7630137","identity":"rs-7630137","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}