Site-Specific Fluorogenic Protein Labelling Agent for Bioconjugation

Article Site-Speciﬁc Fluorogenic Protein Labelling Agent for Bioconjugation

Kelvin K. Tsao, Ann C. Lee, Karl É. Racine and Jeﬀrey W. Keillor * Department of Chemistry and Biomolecular Sciences, University of Ottawa, Ottawa, ON K1N 6N5, Canada; [email protected] (K.K.T.); [email protected] (A.C.L.); [email protected] (K.É.R.) * Correspondence: [email protected]; Tel.: +1-613-562-5800 (ext. 6314)

Received: 28 January 2020; Accepted: 25 February 2020; Published: 28 February 2020

Abstract: Many clinically relevant therapeutic agents are formed from the conjugation of small molecules to biomolecules through conjugating linkers. In this study, two novel conjugating linkers were prepared, comprising a central coumarin core, functionalized with a dimaleimide moiety at one end and a terminal alkyne at the other. In our first design, we developed a protein labelling method that site-specifically introduces an alkyne functional group to a dicysteine target peptide tag that was genetically fused to a protein of interest. This method allows for the subsequent attachment of azide-functionalized cargo in the facile synthesis of novel protein-cargo conjugates. However, the fluorogenic aspect of the reaction between the linker and the target peptide was less than we desired. To address this shortcoming, a second linker reagent was prepared. This new design also allowed for the site-specific introduction of an alkyne functional group onto the target peptide, but in a highly fluorogenic and rapid manner. The site-specific addition of an alkyne group to a protein of interest was thus monitored in situ by fluorescence increase, prior to the attachment of azide-functionalized cargo. Finally, we also demonstrated that the cargo can also be attached first, in an azide/alkyne cycloaddition reaction, prior to fluorogenic conjugation with the target peptide-fused protein.

Keywords: protein labelling; heterobifunctional; ﬂuorogenic; CuAAC

1. Introduction A number of clinically relevant therapeutic agents have been formed from the covalent attachment of synthetic polymers, drugs, and imaging probes to proteins or antibodies [1,2]. These protein-polymer or antibody-drug conjugates are formed through a number of linking strategies that connect the biomolecule to cargo. Appending cargo to these biomolecules relies on highly efficient and bioorthogonal “click” type reactions such as Michael addition, copper-catalyzed azide-alkyne cycloaddition (CuAAC), strained-promoted azide-cycloalkyne cycloaddition (SPAAC) and the Staudinger ligation [3–5]. The link between the protein and the cargo can be non-site specific, as in the case of modifying natural amino acids, or highly site specific, as is the case with labelling genetically fused tags or introducing unnatural amino acids [6,7]. Both the reactions used and the degree of site-specificity of the conjugation must be considered and optimized in order to effectively build new protein conjugates. Bioconjugates are most commonly synthesized by appending cargo to a protein or antibody through a linker that provides a site of attachment for the intended cargo. Examples of relevant cargo include polyethylene glycol [8–11], to improve pharmacokinetic properties, or a small molecule drug [12,13], for targeted delivery. Traditionally, the conjugation is monitored discontinuously (or ex situ), using methods such as sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE), high throughput liquid chromatography (HPLC), or mass spectrometry. More recently developed linkers allow for the continuous (or in situ) monitoring of the bioconjugation event by fluorescence,

Biomolecules 2020, 10, 369; doi:10.3390/biom10030369 www.mdpi.com/journal/biomolecules Biomolecules 2020, 10, 369 2 of 16 due to their fluorogenic nature. Many of these linkers have been reviewed by Liu and coworkers, but three significant methods stand out [14]. Most recently, Liu reported a fluorogenic linker that allowed the attachment of cargo through CuAAC on one end of the linker and reaction with a target protein’s native Cys thiol group through a Michael addition on the other [15]. O’Reilly et al. presented the fluorogenic conjugation of PEG onto salmon calcitonin (sCT) through an N-propargyldibromomaleimide linker [16]. Lastly, Cornelissen and coworkers developed a technique that conjugated PEG cargo to azide functionalized bovine serum albumin (BSA) through a fluorogenic 3-azidocoumarin linker [17]. However, drawbacks of the above linkers include dependence on the reactivity of an intrinsic Cys residue, reversible conjugation or the requirement of specific techniques to introduce unnatural amino acids into the protein. Thus, there remains a need for complementary methods that will expand the scope of biomolecules that can be easily and efficiently conjugated. The modular design of novel protein-cargo conjugates requires a linker that can be easily integrated into any protein. Furthermore, the conjugation method should also be site specific, so as not to disrupt the native function of the protein itself. Lastly, it would be advantageous for the linker to be fluorogenic such that the conjugation can be monitored in situ. Thus, we propose a fluorogenic linker that reacts site-specifically with a small peptide tag genetically fused to either terminus of a target protein. This approach eliminates the necessity of incorporating unnatural amino acid residues into a protein of interest, while also providing a specific site of modification that can be easily appended onto any protein with minimal disruption of that protein’s function. In previous work, we have developed a fluorogenic specific protein labelling method based on the conjugation between a pro-fluorescent probe and a small α-helical peptide genetically fused to the protein of interest [18,19]. The fluorogenic probes (referred to as FlARe probes) consist of a fluorophore covalently attached to two maleimide groups that quench the fluorophore’s emission through a photoinduced electron transfer (PeT) mechanism [20,21]. These bismaleimide probes have also been shown to react highly efficiently with the two activated cysteine thiols, located 10 Å apart, on a rationally designed α-helical target peptide (dC10*) in a Fluorogenic Addition Reaction (FlARe) [22,23]. When both maleimides react with the chemically tuned thiols, the PeT quenching is abolished, and the intrinsic fluorescence of the FlARe probe is restored. Herein, we report a method for the site-specific and fluorogenic introduction of an alkyne functional group onto a specific protein. This method uses a heterobifunctional pro-fluorescent linker that takes advantage of the FlARe protein labelling technology (Scheme1). The proposed linker consists of a coumarin fluorophore core, functionalized on one end with a dimaleimide moiety. These maleimide functional groups quench the coumarin’s fluorescence and allow the linker to react with the dC10* tag that is genetically fused to a protein of interest. This coumarin FlARe probe is additionally functionalized with a terminal alkyne on the other end, such that it modifies a specific protein bearing the dC10* tag by site-specifically introducing an alkyne group onto the exposed terminal target peptide sequence. Since the reaction between the linker and the dC10* tag is fluorogenic, the initial conjugation can be monitored in situ by fluorescence increase. After the initial labelling reaction is complete, the modified protein bearing the alkyne functional group can then be conjugated to a broad array of azide-functionalized cargo through CuAAC. Overall, this novel linker allows for a facile and effective method for the in vitro synthesis of fluorescent protein-conjugates that is easily amenable to engineered proteins of therapeutic significance. Biomolecules 2020, 10, 369 3 of 16

Biomolecules 2020, 10, 369 3 of 16

SchemeScheme 1. A 1 protein-cargo. A protein-cargo conjugate conjugate is is formed formed byby firstfirst labelling labelling a aprotein protein with with a heterobifunctional a heterobifunctional fluorogenicfluorogenic coumarin coumarin linker linker (A). Azide-functionalized(A). Azide-functionalized cargo cargo (B) ( isB) then is then attached attached to the to proteinthe protein through the alkynethrough moiety the alkyne on the moiety fluorogenic on the probe. fluorogenic Thus, probe. fluorogenic Thus, andfluorogenic site-specific and site-specific bioconjugation bioconjugation is achieved. is achieved.

2. Materials2. Materials and and Methods Methods

2.1. Expression2.1. Expression and and Purification Purification of of Test Test Protein Protein The plasmid coding for the tagged test protein maltose binding protein (MBP-dC10*) was The plasmid coding for the tagged test protein maltose binding protein (MBP-dC10*) was designed designed and created in a previously published study in which the reactivity of the original dC10 tag and created in a previously published study in which the reactivity of the original dC10 tag was was improved through the introduction of positively charged residues around the cysteines [24]. The improvedexpression through and purification the introduction of MBP-dC10* of positively followed charged the protocol residues from aroundthe same thestudy cysteines and consisted [22]. The expressionof transforming and purification the expression of MBP-dC10* plasmid into followed the BL21-Gold the protocol (DE3) fromstrain theof E. same coli cells study [24]. and Expression consisted of transformingof MBP-dC10* the expression was induced plasmid with IPTG into (0.3 the mM) BL21-Gold and purification (DE3) strain was performed of E. coli cells an amylose [22]. Expression resin of MBP-dC10*column and was elution induced with 10 with mM IPTG maltose. (0.3 According mM) and to purification the Bradford wasAssay, performed protein yields an amylose generally resin columnranged and around elution 12 with mg from 10 mM 500 maltose.mL of expression According culture. to the Bradford Assay, protein yields generally ranged around 12 mg from 500 mL of expression culture. 2.2. Determination of Fluorescence Enhancement Ratios 2.2. DeterminationEmission ofspectra Fluorescence and fluorescence Enhancement intensity Ratios measurements were recorded at 25 °C with a Synergy H4 Hybrid Multi-Mode Microplate Reader with excitation and emission monochromators Emission spectra and fluorescence intensity measurements were recorded at 25 ◦C with a Synergy set at 9-nm bandpass. The initial excitation and emission spectra were recorded for a solution of 10 H4 Hybrid Multi-Mode Microplate Reader with excitation and emission monochromators set at 9-nm µM labelling reagent (compounds 1, 6, 10, or 15) in 50 mM HEPES (I = 100 mM with NaCl, pH 7.4) µ bandpass.with 10% The DMSO initial and excitation 50 µM TCEP. and emission A solution spectra of 10 µM were labelling recorded reagent for ain solution 50 mM HEPES of 10 (IM = labelling100 reagentmM (compounds with NaCl, pH1, 6 7.4), 10 with, or 15 10%) in DMSO 50 mM and HEPES 50 µM (I TCEP= 100 with mM the with addition NaCl, of pH 10 7.4) µM with MBP-dC10* 10% DMSO and 50wasµ Mallowed TCEP. to A react solution for 4 h of (in 10 theµ Mcase labelling of 1 or 15 reagent) or 24 h in (in 50 the mM case HEPES of 6 or 10 (I).= After100 mMthe reaction with NaCl, pH 7.4)was with complete, 10% DMSO the final and excitation 50 µM TCEP and emission with the additionspectra were of 10 recorded.µM MBP-dC10* The ratio was of fluorescence allowed to react for 4 hintensity (in the caseat the of maximum1 or 15) or emission 24 h (in between the case ofthe6 initialor 10 ).and After final the spectra reaction gave was the complete, fluorescence the final excitationenhancement and emission (FE). spectra were recorded. The ratio of fluorescence intensity at the maximum emission between the initial and final spectra gave the fluorescence enhancement (FE). 2.3. Kinetic Studies 2.3. KineticKinetic Studies experiments were carried out at 25 °C using a Synergy H4 Hybrid Multi-Mode Microplate Reader with excitation and emission monochromators set at a 20-nm bandpass. Solutions Kinetic experiments were carried out at 25 C using a Synergy H4 Hybrid Multi-Mode Microplate containing 10 µM MBP-dC10* in 50 mM HEPES◦ buffer (I = 100 mM, pH 7.4) with 10% DMSO and 50 ReaderµM with TCEP excitation were prepared and emission in a 96-well monochromators plate. Labelling set atreagent a 20-nm was bandpass. added immediately Solutions containingbefore 10 µMrecording MBP-dC10* to a final in 50 concentration mM HEPES ofbu 10ff µM.er (I Samples= 100 mM, were pHexcited 7.4) at with 435 nm 10% for DMSO 10 and and 15 or 50 415µM nm TCEP werefor prepared 1 and fluorescence in a 96-well intensity plate. Labelling was followed reagent at 485 was nm addedfor 10 and immediately 15 or 460 nm before for 1 recordingas a function to of a final concentrationtime. All time-dependent of 10 µM. Samples fluorescence were excited curves at were 435 nm fitted for to10 a andsecond15 ororder 415 rate nm equation for 1 and to fluorescence obtain intensitythe second was followed order rate at constant 485 nm for(k2)10 forand the 15reactionor 460 between nm for the1 as label a function and MBP-dC10*. of time. All time-dependent fluorescence curves were fitted to a second order rate equation to obtain the second order rate constant 2.4. Bioconjugation of Rhodamine B Azide to MBP-dC10*-15 or 6 Adduct (k2) for the reaction between the label and MBP-dC10*. The protocol for performing the CuAAC between rhodamine B azide (RhB-N3) and MBP-dC10* 2.4. Bioconjugationlabelled with 6 of or Rhodamine 15 was adapted B Azide from to MBP-dC10*- procedures developed15 or 6 Adduct by Nasheri, et al. and Yang, et al. [25,26]. An aliquot of 5 µM 15 was incubated with 2.5 µM MBP-dC10* and 50 µM TCEP in HEPES The protocol for performing the CuAAC between rhodamine B azide (RhB-N3) and MBP-dC10* labelled with 6 or 15 was adapted from procedures developed by Nasheri, et al. and Yang, et al. [24,25]. An aliquot of 5 µM 15 was incubated with 2.5 µM MBP-dC10* and 50 µM TCEP in HEPES buffer Biomolecules 2020, 10, 369 4 of 16

(50 mM HEPES (I = 100 mM NaCl), pH 7.4) at 25 ◦C and a total volume of 200 µL for 1 h. The reaction was monitored using a Synergy H4 Hybrid Multi-Mode Microplate Reader with excitation (435 nm) and emission (485 nm) monochromators set at a 20-nm bandpass. After complete reaction, the mixture was transferred to a 500-µL 10-kDa MWCO Amicon filter and buffer exchanged into fresh HEPES buffer three times. After the third time, the solution was adjusted to 100 µL. A 500-µL click mixture (1.0 mM CuSO4, 100 µM TBTA, 1.0 mM TCEP-HCl, 50 µM RhB-N3, PBS pH 7.4, 0.25% DMSO) was prepared and 100 µL of this click mixture was added to the buffer exchanged solution containing the MBP-dC10*-15 adduct. The click reaction was allowed to progress for 2 h in the dark with gentle shaking at 25 C. After this, 500 µL of cold acetone (chilled to 80 C) was added and the protein was ◦ − ◦ precipitated overnight at 80 C. The protein was pelleted by spinning at 14,000 rpm for 15 min at 4 C − ◦ ◦ before removing the supernatant and drying off any excess acetone under a gentle stream of air. After, the pellet was washed with 100 µL MeOH chilled to 80 C before pelleting again at 14,000 rpm for − ◦ 15 min at 4 ◦C. The precipitate was then taken up in 35 µL SDS PAGE loading buffer (50 mM TCEP) and shaken for 15 min at room temperature. The samples were spun at 14,000 rpm for 5 min and then boiled at 90 ◦C for 5 min before 10 µL was loaded onto a 10%-acrylamide SDS-PAGE gel and run for 1.5 h at 120 V. In the case of 6, the reaction between MBP-dC10* and 6 was allowed to react for 24 h and the reaction was not monitored.

2.5. Bioconjugation of Rh-B-N3-15 Adduct to MBP-dC10*

The protocol for performing the CuAAC between Rhodamine B azide (RhB-N3) and 15 was adapted from procedures developed by Nasheri, et al. and Yang, et al. [24,25]. A 500-µL click mixture (1.0 mM CuSO4, 100 µM TBTA, 1.0 mM TCEP-HCl, 50 µM RhB-N3, PBS pH 7.4, 0.25% DMSO) was first prepared and 90 µL of this click mixture was incubated with 10 µL of a 200 µM 15 stock (final concentration 20 µM). The click reaction was allowed to progress in the dark at r.t. for 2 h with gentle shaking. After, 100 µL of a 10-µM MBP-dC10* solution was added to the mixture and the entire solution was allowed to react for 1 h at r.t. Next, 500 µL of cold acetone (chilled to 80 C) was added and the − ◦ protein was precipitated overnight at 80 C. The protein was pelleted by spinning at 14,000 rpm − ◦ for 15 min at 4 ◦C before removing the supernatant and drying off any excess acetone under a gentle stream of air. The pellet was washed with 100 µL MeOH chilled to 80 C before pelleting again at − ◦ 14,000 rpm for 15 min at 4 ◦C. The precipitate was taken up in 35 µL SDS PAGE loading buffer (50 mM TCEP) and sonicated for 3 minutes prior to being shaken for 15 min at room temperature. The samples were spun at 14,000 rpm for 5 min and then boiled at 90 ◦C for 5 min before 5 µL was loaded onto an 10% acrylamide SDS-PAGE gel and run for 1.5 h at 120 V.

2.6. Synthesis All reagents and solvents for reactions were used as received unless otherwise stated. Dichloromethane, dimethylformamide, methanol and acetonitrile were dried using a solvent puriﬁcation system from LC Technology Solutions Inc (Salisbury, USA). Reactions were monitored by thin layer chromatography (TLC) using E. Merck silica gel 60F254 precoated aluminum plates. Components were visualized by illumination with a short-wavelength ultraviolet light or long-wavelength visible light. Flash column chromatography was performed on ZEOCHEM® silica gel 60 (ECO 40–63 µm) using ethyl acetate/n-hexane or methanol/dichloromethane as eluting solvents. Nuclear magnetic resonance (NMR) spectra were recorded in deuterated DMSO or deuterated chloroform at ambient temperature unless otherwise stated. The experiments were performed either on a Bruker Avance 400 Fourier Transform Spectrometer operating at 400 MHz for 1H and at 100 MHz for 13C or on a Bruker Avance 600 Fourier Transform Spectrometer operating at 600 MHz for 1H and at 150 MHz for 13C. EI-MS spectra were recorded on a Kratos Concept mass spectrometer for both low resolution and high-resolution mass spectra. ESI-MS spectra were recorded on a Waters Micromass Biomolecules 2020, 10, 369 5 of 16

Q-TOF mass spectrometer. Melting points were measured on an EZ-Melt automated melting point apparatus and are uncorrected. Ultraviolet absorbance spectra and fluorescence spectroscopic studies were performed on a Synergy H4 Hybrid Multi-Mode Microplate Reader. Compound 5: Compound 5 was synthesized according to the procedures laid out by Chen and coworkers [19]. Compounds 2 and 3 were synthesized according to the procedures laid out by Abdizadeh and coworkers [26]. Compound 2: To a solution of 4-hydroxysalicylic acid (1.38 g, 10.0 mmol) and diethylmalonate (1.0 eq, 1.53 mL, 10.0 mmol) in EtOH (5 mL) was added piperidine (0.02 eq, 19.8 µL, 0.2 mmol) and acetic acid (0.02 eq, 11.4 µL, 0.2 mmol). The solution was stirred at room temperature for 20 min and then heated to 78 ◦C and stirred for 3 h. The reaction was cooled to room temperature and the product was crystallized as an orange solid in 81% yield. 1H NMR (400 MHz, DMSO) δ 11.04 (s, 1H), 8.73–8.53 (m, 1H), 7.71 (d, J = 8.6 Hz, 1H), 6.80 (dd, J = 8.6, 2.3 Hz, 1H), 6.75–6.47 (m, 1H), 4.22 (q, J = 7.1 Hz, 2H), 1.25 (t, J = 7.1 Hz, 3H). 13C NMR (100 MHz, DMSO) δ 164.5, 163.4, 157.6, 156.9, 149.9, 132.6, 114.5, 112.6, + 110.9, 102.3, 61.3, 14.6. HRMS (ESI): calcd for C12H10O5Na ([MNa] ): 257.0426, found: 257.0414. Compound 3: Compound 2 (50 mg, 0.159 mmol) was dissolved in acetonitrile (3.0 mL), to which a solution of NaOH (10.0 eq, 63.6 mg, 1.59 mmol) dissolved in water (2.0 mL) was added dropwise. The solution was stirred at room temperature for 3.5 h after which it was diluted with 10 mL water and then acidified with 10 mL 1 M HCl. The aqueous solution was extracted with EtOAc (3 30 mL) × and the combined organic layers were dried with MgSO4. After removing the solvent on the rotary evaporator, compound 3 was obtained as a yellow solid in quantitative yield. 1H NMR (400 MHz, DMSO) δ 12.00 (bs, 1H), 8.64 (s, 1H), 7.73 (d, J = 8.4 Hz, 1H), 6.83 (d, J = 8.4 Hz, 1H), 6.72 (s, 1H). 13C NMR (100 MHz, DMSO) δ 164.32 (d, J = 14.1 Hz), 157.75, 156.99, 148.96, 131.91, 114.17, 112.69, 110.53, 101.83. 13C NMR (100 MHz, DMSO) δ 164.3, 157.8, 157.0, 149.0, 131.9, 114.2, 112.7, 110.5, 101.8. HRMS + (ESI): calcd for C10H6O5Na ([MNa] ): 229.0113, found: 229.0128. Compound 1: To a solution of compound 3 (26.0 mg, 0.130 mmol) in anhydrous DMF (2.5 mL) was added HBTU (1.1 eq, 52.0 mg, 0.138 mmol). The mixture was stirred at room temperature for 10 min before 5 (1.5 eq, 59.0 mg, 0.188 mmol) was added. The mixture was stirred for an additional 20 min before trimethylamine (3.0 eq, 50 µL, 0.380 mmol) was added. It was then stirred overnight followed by diluting the mixture in EtOAc (100 mL) and washing with 1 M HCl (2 100 mL). The × organic layer was dried with MgSO4 before the solvent was removed using a rotary evaporator. The crude was purified by flash chromatography using a gradient elution system of 0%–5% MeOH in DCM to obtain compound 1 as a pale-yellow solid in 7% yield. 1H NMR (600 MHz, DMSO) δ 11.18 (bs, 1H), 10.86 (s, 1H), 8.83 (s, 1H), 7.86 (d, J = 8.7 Hz, 1H), 7.78 (d, J = 1.7 Hz, 2H), 7.12 (t, J = 1.8 Hz, 1H), 6.92 (dd, J = 8.6, 2.2 Hz, 1H), 6.85 (d, J = 2.0 Hz, 1H), 6.83 (q, J = 1.8 Hz, 2H), 2.09 (d, J = 1.7 Hz, 6H). 13C NMR (150 MHz, DMSO) δ 170.3, 169.4, 164.1, 161.2, 160.7, 156.5, 149.6, 148.4, 145.9, 138.7, 132.6, 132.3, 127.6, 120.0, 116.8, 114.6, 114.1, 111.2, 102.0, 10.9. HRMS (ESI): calcd for C26H17N3O8Na + ([MNa] ):522.0913, found: 522.0929. mp: decomp. at 267.7–271.1 ◦C. Compound 7: The synthesis of compound 7 was done according to Jiang, et al. [27]. A Finkelstein reaction was performed to synthesize 7 in which 6-chloro-1-hexyne (8.30 mmol, 1.00 mL) was added to dry acetone (100 mL) along with NaI (58.1 mmol, 8.71 g, 7 eq). The solution was brought to reflux and stirred overnight after which the acetone was removed using a rotary evaporator. The residue was then partitioned between hexanes (100 mL) and water (100 mL) and the organic layer was washed with water (100 mL) and brine (100 mL). The organic layer was then dried with MgSO4 and the solvent was removed using a rotary evaporator to obtain compound 7 as a transparent liquid in 94% yield 1 (7.78 mmol, 1.62 g). H NMR (400 MHz, CDCl3) δ ppm 3.20 (t, J = 6.91 Hz, 2 H), 2.22 (td, J = 6.98, 2.60 Hz, 3 H), 1.95 (m, 3 H), 1.63 (tt, J = 7.30 Hz, 3 H). Compound 8: Compound 8 (1.00 mmol, 234 mg) was dissolved in acetonitrile after which compound 7 was added. K2CO3 (1.2 mmol, 165 mg, 1.2 eq) and a catalytic amount of 18-crown-6 (0.1 mmol, 26.4 mg) was then added to the reaction mixture, which was brought to reflux and stirred Biomolecules 2020, 10, 369 6 of 16 overnight. The acetonitrile was removed using a rotary evaporator and the residue was partitioned between EtOAc (20 mL) and water (20 mL). The aqueous layer was extracted with EtOAc (3 30 mL) × and the combined organic layers were dried with MgSO4 and the solvent was removed. The residue was purified by flash chromatography using a gradient elution system of 10%–30% EtOAc in hexanes. Compound 8 was obtained as a white solid in 44% yield (0.437 mmol, 138 mg). 1H NMR (400 MHz, CDCl3) δ ppm 8.50 (s, 1 H), 7.49 (d, J = 8.72 Hz, 1 H), 6.88 (dd, J = 8.67, 2.20 Hz, 1 H), 6.80 (d, J = 2.06 Hz, 1 H), 4.40 (q, J = 7.15 Hz, 2 H), 4.08 (t, J = 6.22 Hz, 2 H), 2.30 (td, J = 6.91, 2.55 Hz, 2 H), 1.97 (m, 3 H), 13 1.74 (tt, J = 7.30 Hz, 2 H), 1.40 (t, J = 7.10 Hz, 3 H). C NMR (100 MHz, CDCl3) δ ppm 164.6, 163.5, 157.6, 157.2, 149.0, 130.7, 114.0, 114.0, 111.6, 100.8, 83.7, 68.9, 68.3, 61.7, 27.9, 24.9, 18.1, 14.3. HRMS + (ESI): calcd for C18H18O5Na ([MNa] ): 337.1052, found: 337.1074. mp: 92.6–93.6 ◦C. Compound 9: To a solution of 8 (0.437 mmol, 138 mg) dissolved in acetonitrile, NaOH (4.93 mmol, 197 mg, 10 eq) was added as a 2 M solution in water (2.5 mL). The reaction was stirred for 3.5 h after which it was partitioned between water (10 mL), 1 M HCl (10 mL) and EtOAc (30 mL). The aqueous phase was extracted with EtOAc (3 30 mL) and the combined organic phases were dried with MgSO . × 4 The remaining EtOAc was removed using a rotary evaporator to obtain compound 9 as a white solid in 97% yield (0.425 mmol, 122 mg). 1H NMR (400 MHz, DMSO) δ ppm 12.97 (br. s., 1 H), 8.71 (s, 1 H), 7.82 (d, J = 8.72 Hz, 1 H), 7.01 (m, 2 H), 4.14 (t, J = 6.22 Hz, 2 H), 2.79 (t, J = 2.55 Hz, 1 H), 2.24 (td, J = 6.69, 2.20 Hz, 2 H), 1.83 (tt, J = 8.50, 7.70 Hz, 2 H), 1.61 (tt, J = 7.30 Hz, 2 H). 13C NMR (100 MHz, DMSO) δ ppm 164.1, 163.9, 157.2, 156.8, 148.9, 131.5, 113.7, 113.5, 111.5, 100.6, 84.1, 71.4, 68.0, 27.4, 24.4, + 17.3. HRMS (ESI): calcd for C16H14O5Na ([MNa] ): 309.0739, found: 309.0727. mp: 170.0–170.9 ◦C. Compound 6: Compound 9 (0.349 mmol, 100 mg) was dissolved in SOCl2 (6 mL). The solution was brought to reflux and stirred for 6 h before removing excess SOCl2 using the rotary evaporator. The residue was dissolved in pyridine (5 mL) to which compound 5 (0.349 mmol, 149 mg, 1.0 eq) dissolved in pyridine (5 mL) was added and the mixture was left to stir overnight. The solution was then taken up in EtOAc (20 mL) and then washed with 1 M HCl (3 20 mL), 5% CuSO (20 mL) and × 4 brine (20 mL). The organic layer was dried with MgSO4 and the solvent was removed on a rotary evaporator to obtain the crude residue, which was purified by flash chromatography using a 5:4:1 hexanes/DCM/EtOAc mixture. Compound 6 was obtained as a pale-yellow solid in 12% yield (0.0431 1 mmol, 25 mg). H NMR (400 MHz, CDCl3) δ ppm 10.96 (s, 1 H), 8.92 (s, 1 H), 7.85 (d, J = 1.86 Hz, 2 H), 7.62 (d, J = 8.72 Hz, 1 H), 7.28 (t, J = 1.86 Hz, 1 H), 6.96 (dd, J = 8.72, 2.35 Hz, 1 H), 6.89 (d, J = 2.25 Hz, 1 H), 6.50 (q, J = 1.67 Hz, 2 H), 4.11 (t, J = 6.27 Hz, 2 H), 2.31 (td, J = 6.96, 2.65 Hz, 2 H), 2.18 (d, J = 1.86 13 Hz, 6 H), 1.99 (m, 3 H), 1.75 (m, 2 H). C NMR (100 MHz, CDCl3) δ ppm 170.1, 168.9, 164.7, 162.1, 160.1, 156.9, 149.3, 145.9, 138.9, 132.7, 131.2, 127.6, 118.4, 116.4, 114.7, 114.3, 112.4, 100.8, 83.7, 69.0, 68.4, + 27.9, 24.8, 18.1, 11.2. HRMS (ESI): calcd for C32H25N3O8Na ([MNa] ): 602.1539, found: 602.1550. mp: decomp. at 192.9–194.2 ◦C. Compound 12: To a solution of 3-aminophenol (1 g, 9.2 mmol, 2.0 eq) in THF (20 mL) was added 4-ethynylbenzaldehyde. The solution was stirred for 1.5 h at r.t. prior to adding STAB (1.36 g, 6.44 mmol, 1.4 eq). After stirring for 3 h at r.t., the reaction was quenched with sat. NaHCO3 (60 mL) and extracted with ether (3 20 mL). Compound 12 was purified using flash chromatography with a 2:8 × EtOAc/hexanes eluent and obtained as a transparent viscous oil in 75% yield. 1H NMR (400 MHz, CDCl3) δ ppm 7.51–7.39 (m, 2H), 7.31 (d, J = 8.4 Hz, 2H), 7.01 (t, J = 8.0 Hz, 1H), 6.20 (dddd, J = 10.4, 8.0, 13 2.3, 0.8 Hz, 2H), 6.08 (t, J = 2.3 Hz, 1H), 4.32 (s, 2H), 3.06 (s, 1H). C NMR (100 MHz, CDCl3) δ 156.8, 149.6, 140.4, 132.6, 130.4, 127.4, 121.1, 106.2, 104.9, 99.8, 83.6, 48.1. HRMS (EI): calcd for C15H13NO: 223.09971, found: 223.09831. Compound 13: Compound 12 (460 mg, 2.06 mmol) was dissolved in MeOH and cooled to 0 ◦C. Once cooled, acetaldehyde (4.0 eq, 0.461 mL, 8.24 mmol) was added and the solution was stirred for 2 h. After, the solution was removed from ice and then NaBH4 (6.0 eq., 468 mg, 12.4 mmol) was added and the mixture stirred at room temperature overnight. Purification by flash chromatography by gradient elution (0%–30% EtOAc in hexane) yielded compound 13 in 50% yield as a transparent viscous oil. 1H NMR (400 MHz, CDCl3) δ ppm 7.43 (d, J = 8.23 Hz, 2 H), 7.18 (d, J = 7.84 Hz, 2 H), 7.02 (t, J = 8.28 Hz, Biomolecules 2020, 10, 369 7 of 16

1 H), 6.25 (m, 1 H), 6.15 (m, 2 H), 4.62 (s, 1 H), 4.48 (s, 2 H), 3.43 (q, J = 7.09 Hz, 2 H), 3.04 (s, 1 H), 1.19 (t, 13 J = 7.05 Hz, 3 H). C NMR (100 MHz, CDCl3) δppm 156.7, 149.9, 140.1, 132.4, 130.2, 126.5, 120.5, 105.1, + 103.3, 99.2, 83.6, 53.8, 45.4, 12.2. HRMS (ESI): calcd for C17H18NO ([MH] ): 252.1388, found: 252.1362. Compound 11: The synthesis of compound 4 was adapted from a procedure developed by Takakura, et al. [28]. POCl3 (0.464 mL, 4.77 mmol, 4.0 eq) was added dropwise to DMF (1.06 mL, 13.7 mmol, 1.5 eq.) over ice and stirred for 5 min. Compound 13 was then added and the solution was stirred at r.t. for 1 h. Afterwards, the solution was heated to 70 ◦C and stirred for 1 h before cooling the solution to r.t. Hydrolysis was performed by adding H2O (10 mL) to the reaction and stirring for 30 min. The solution was then neutralized with NaOH and extracted with EtOAc (3 50 mL). × Compound 11 was purified by flash chromatography using a 2:8 EtOAc/hexanes elution system and 1 was obtained as a white solid in 44% yield. H NMR (400 MHz, CDCl3) δ ppm 11.57 (s, 1H), 9.57–9.18 (m, 1H), 7.45 (d, J = 8.3 Hz, 2H), 7.26 (t, J = 4.4 Hz, 2H), 7.13 (d, J = 8.5 Hz, 2H), 6.26 (dd, J = 8.9, 2.4 Hz, 1H), 6.12 (d, J = 2.4 Hz, 1H), 4.59 (s, 2H), 3.52 (q, J = 7.1 Hz, 2H), 3.06 (s, 1H), 1.26 (t, J = 7.1 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 192.4, 164.3, 154.9, 138.0, 135.5, 132.6, 126.2, 121.2, 112.1, 104.8, 97.5, 83.3, 53.4, 45.8, 12.2. HRMS (EI): calcd for C18H17NO2: 279.12593, found: 279.12498. mp: 97.1–97.8 ◦C. Compound 14: Compound 11 (184 mg, 0.658 mmol) and Meldrum’s acid (1.0 eq, 95 mg, 0.658 mmol) was dissolved in 99% EtOH (5 mL) to which piperidine (0.02 eq, 1.3 µL 0.0130 mmol) and AcOH (0.02 eq, 0.75 µL, 0.0130 mmol) was added. The solution was stirred at r.t. for 20 min before it was heated to 78 ◦C and stirred for 3 h. After, the mixture was cooled to r.t. and then to 0 ◦C and allowed to crystallize overnight. The product was filtered and collected as a yellow-green solid in 48% 1 yield. H NMR (400 MHz, CDCl3) δ ppm 12.30 (s, 1 H), 8.67 (s, 1 H), 7.46 (t, J = 8.52 Hz, 3 H), 7.13 (d, J = 8.33 Hz, 2 H), 6.72 (dd, J = 9.01, 2.45 Hz, 1 H), 6.57 (d, J = 2.25 Hz, 1 H), 4.67 (s, 2 H), 3.62 (q, 13 J = 7.15 Hz, 2 H), 3.08 (s, 1 H), 1.32 (t, J = 7.10 Hz, 3 H). C NMR (100 MHz, CDCl3) δ ppm 165.3, 164.1, 157.8, 154.3, 150.4, 136.8, 132.8, 132.0, 126.1, 121.7, 111.3, 109.2, 106.8, 97.7, 83.0, 53.9, 46.4, 12.1. HRMS + (ESI): calcd for C21H17NO4Na ([MNa] ): 370.1055, found: 370.1065. mp: 207.3–208.8 ◦C. Compound 10 and 15: POCl3 (3.0 eq, 40.4 µL, 432 µmol) was added to a solution of compound 5 (50 mg, 144 µmol) dissolved in DCE (1.4 mL) after which the solution was heated to 84 ◦C and stirred for 4 h to form the acyl chloride. The solvent was removed by rotary evaporation and the residue was dissolved in acetonitrile (1.4 mL). A separate solution containing 5 or 4 (to make 10 or 15 respectively) (1.2 eq., 49 mg, 173 µmol) and DIPEA (2.0 eq, 50 µL, 288 µmol) in acetonitrile (1.4 mL) was then added to the solution of the acyl chloride and the combined reaction mixture was stirred at room temperature overnight. The solvent was removed by rotary evaporation and the crude was purified by flash chromatography using a gradient elution system of 0%–50% EtOAc in hexanes elution system to afford the product. 1 Compound 10: yellow solid in 28% yield. H NMR (600 MHz, CDCl3) δ ppm 11.00 (s, 1H), 8.77 (s, 1H), 7.83 (d, J = 1.9 Hz, 2H), 7.51–7.41 (m, 3H), 7.23 (t, J = 1.8 Hz, 1H), 7.14 (d, J = 8.3 Hz, 2H), 6.67 (dd, J = 8.9, 2.5 Hz, 1H), 6.56 (d, J = 2.3 Hz, 1H), 6.48 (q, J = 1.7 Hz, 2H), 4.64 (s, 2H), 3.59 (q, J = 7.1 Hz, 13 2H), 3.07 (s, 1H), 2.17 (d, J = 1.8 Hz, 6H), 1.30 (t, J = 7.1 Hz, 3H). C NMR (150 MHz, CDCl3) δ 170.2, 169.1, 162.9, 161.2, 157.7, 153.6, 149.0, 146.0, 139.4, 137.5, 132.9, 132.7, 131.6, 127.7, 126.3, 121.6, 118.3, 116.5, 110.8, 109.4, 97.7, 83.3, 77.7, 54.0, 46.4, 12.3, 11.3. HRMS (ESI): C37H28N4O7 calcd for: 663.1856, + found: 663.1855 ([MNa] ). mp: decomp. at 330.0–331.2 ◦C. Compound 15: yellow solid in 37% yield. 1H NMR (600 MHz, DMSO) δ ppm 10.93 (s, 1H), 8.74 (s, 1H), 7.79 (d, J = 1.8 Hz, 2H), 7.74 (d, J = 9.1 Hz, 1H), 7.46 (d, J = 8.3 Hz, 2H), 7.23 (d, J = 8.4 Hz, 2H), 7.21 (s, 4H), 7.11 (t, J = 1.8 Hz, 1H), 6.86 (d, J = 11.2 Hz, 1H), 6.70 (s, 1H), 4.78 (s, 2H), 4.16 (s, 1H), 3.64 (q, J = 7.0 Hz, 2H), 1.19 (t, J = 7.1 Hz, 3H). 13C NMR (150 MHz, DMSO) δ 169.7, 162.0, 161.0, 157.1, 153.4, 148.3, 138.8, 134.8, 132.4, 132.0, 131.9, 127.7, 126.8, 126.0, 120.4, 120.2, 117.1, 110.9, 109.8, 108.4, + 96.8, 83.3, 80.8, 52.8, 45.7, 29.8, 12.2. HRMS (ESI): calcd for C35H24N4O7Na ([MNa] ): 635.1543, found: 635.1537. mp: decomp. at 184.8–184.9 ◦C. Biomolecules 2020, 10, 369 8 of 16

3. Results and Discussion

3.1. Initial Design

We initially envisioned attaching a dimaleimide quencher moiety at the C3 position of a coumarin fluorophore through an amide bond, while appending a hexyne tail at the C7 position through an Biomoleculesether linkage. 2020, 10 Thus,, 369 we first synthesized 1 (Scheme2) to confirm that the coumarin FlARe probe8 of 16 without the alkyne tail could be quenched by the dimaleimide quencher. Fluorogen 1 also allowed us probe without the alkyne tail could be quenched by the dimaleimide quencher. Fluorogen 1 also to subsequently determine the photophysical effects of adding the terminal alkyne tail. allowed us to subsequently determine the photophysical effects of adding the terminal alkyne tail.

Scheme 2. SyntheticSynthetic route route for for the the preparation preparation of fluorogen ﬂuorogen 1.

First the coumarin coumarin core core was was formed formed via via a a Knoeve Knoevenagelnagel condensation condensation between between 4-formylresorcinol 4-formylresorcinol and diethylmalonate, providing ester 2 in good yield (Scheme2 2).). CarboxylicCarboxylic acidacid 3 waswas then then obtained obtained in excellentexcellent yieldyield after after hydrolysis hydrolysis of 2.ofWe 2. originallyWe originally attempted attempted to couple to couple acid 3 withacid aniline3 with4 ;aniline however, 4; however,the instability the ofinstability the unsubstituted of the unsubstituted maleimide groups maleimide of aniline groups4 made of aniline this synthetic 4 made strategy this synthetic difficult strategyand impractical. difficult Therefore,and impractical. we switched Therefore, to aniline we 5switched, whose methyl-substitutedto aniline 5, whose maleimide methyl-substituted groups are maleimidemore stable. groups Aniline are5 morewas coupled stable. Aniline to carboxylic 5 was acidcoupled3 using to carboxylic HBTU toobtain acid 3 theusing FlARe HBTU probe to obtain1 [29]. theThe FlARe low yield probe of the1 [30]. final The coupling low yield reaction of the is probably final coupling due to thereaction low nucleophilicity is probably due of substitutedto the low nucleophilicityaniline 5. of substituted aniline 5. Probe 11was was then then reacted reacted with with an equimolaran equimolar concentration concentration of arepresentative of a representative target protein–namely,target protein– namely,maltose bindingmaltose proteinbinding genetically protein genetically fused to the fused dC10* to the target dC10* peptide target (MBP-dC10*), peptide (MBP-dC10*), in order to in validate order tothe validate activation the of activation the probe of by the FlARe probe (Figure by FlARe1)[22 (].Figure The reaction 1) [22]. ofThe1 withreaction MBP-dC10* of 1 with was MBP-dC10* observed wasto be observed fluorogenic to be with fluorogenic a fluorescence with a fluorescence enhancement enhancement of 12.7. This of is12.7. considerably This is considerably lower than lower the thanFE manifested the FE manifested by a previous by a previous FlARe probe FlARe comprised probe comprised of a 7-(diethyl)aminocoumarin of a 7-(diethyl)aminocoumarin instead instead of the of7-hydroxylcoumarin the 7-hydroxylcoumarin [19]. [19]. Notably, Notably, the the overall overall fluorescence fluorescence intensities intensities of of both both the the activatedactivated and quenched form of 1 are over 10-fold higher than those of the previously reported YC5 [19].[19]. Regarding Regarding reaction kinetics, the the majority majority of of 10 10 µMµM 1 reacts with 10 µMµM dC10* tag within 40 minutes, while the reaction was essentially complete after 2 hours (Figur (Figuree 11B).B). FromFrom thethe timetime coursecourse ofof thisthis reaction,reaction, thethe 1 1 second order raterate constantconstant waswas calculatedcalculated asask k2= = 245245 ± 88 MM−1s−1. .This This rate rate constant constant is is similar similar to to that that 2 ± − − measured for our previous 7-(diethyl)aminocoumarin7-(diethyl)aminocoumarin FlARe probeprobe,[19] [19], suggesting suggesting that that the change of substituent at the C7 position on the coumarin did not infl influenceuence reactivity. Encouraged by these results, we proceeded to append the terminal hexyne tail through an ether linkage at the C7 hydroxylhydroxyl group, in the synthesis of compound 6 (Scheme3 3).). Biomolecules 2020, 10, 369 9 of 16 BiomoleculesBiomolecules 20202020,, 1010,, 369369 99 ofof 1616

FigureFigure 1.1. (((AAA))) ExcitationExcitation Excitation (dotted(dotted (dotted line)line) line) andand and emissionemission emission (solid(solid (solid line)line) line) spectraspectra spectra ofof of 11 beforebefore (grey(grey line)line) andand afterafter reactingreacting withwith with MBP-dC10*MBP-dC10* MBP-dC10* (black(black (black line).line). line). ((BB ()B) KineticKinetic) Kinetic timetime time coursecourse course forfor for thethe the fluorogenicfluorogenic ﬂuorogenic reactionreaction reaction betweenbetween between 1010 µ µ µM10µM ofMof 1 of1 withwith1 with 1010 10µMµM Mofof ofMBP-dC10*MBP-dC10* MBP-dC10* (black),(black), (black), relativerelative relative toto to thethe the stabilitystability stability ofof of 11 1 ininin thethe the absenceabsence absence ofof of MBP-dC10*MBP-dC10* MBP-dC10* −1 1−1 1 (grey). Rate constant k2 == 245245 ± 8 8M M−1−s−1s.− . (grey). Rate constant k2 = 245 ±± 8 M s .

6, Et N, O 6, Et3N, O o 3o NaI, 56 o C,o.n. 100 o C, o.n. Cl NaI, 56 C,o.n. I 100 C, o.n. O Cl I O Acetone DMF Acetone DMF O O O 94% 44% O O O 94% 44%

78 78

O o O O O 1) SOCl2, 75 o C, 6 h O N O MeCN 1) SOCl2, 75 C, 6 h N MeCN 5 97% OH 2) 5, Pyridine, r.t., o.n. 97% OH 2) , Pyridine, r.t., o.n. O O O O 2 M NaOH, r.t., O O O 12% 2 M NaOH, r.t., O O O 12% N N 3.5 h NH N 3.5 h H O O O O O O O O 9 6 9 6 SchemeScheme 3.3. SyntheticSyntheticSynthetic routeroute route forfor thethe preparationpreparation ofof alkynealkyne 66.. In this approach, we attached the hexyne group before attaching the dimaleimide quenching InIn thisthis approach,approach, wewe attachedattached thethe hexynehexyne grougroupp beforebefore attachingattaching thethe dimaleimidedimaleimide quenchingquenching intermediate 5 (Scheme3). Thus, 7 was first synthesized from 6-chlorohexyne before using 2 to displace intermediateintermediate 55 (Scheme(Scheme 3).3). Thus,Thus, 77 waswas firstfirst synthesizedsynthesized fromfrom 6-chlorohexyne6-chlorohexyne beforebefore usingusing 22 toto the iodide in 7, obtaining 8 in decent yield. As performed in the synthesis of 1, ester 8 was then displacedisplace thethe iodideiodide inin 77,, obtainingobtaining 88 inin decentdecent yield.yield. AsAs performedperformed inin thethe synthesissynthesis ofof 11,, esterester 88 waswas hydrolyzed to provide 9 in excellent yield, which was then converted to the acid chloride using SOCl . thenthen hydrolyzedhydrolyzed toto provideprovide 99 inin excellentexcellent yield,yield, whichwhich waswas thenthen convertedconverted toto thethe acidacid chloridechloride usingusing2 TheSOCl acid2. The chloride acid chloride was then was reacted then with reacted aniline with5 in aniline pyridine 5 in to pyridine attach the to quenching attach the dimaleimide quenching SOCl2. The acid chloride was then reacted with aniline 5 in pyridine to attach the quenching moiety and form 6. This coupling of aniline 5 also gave a low yield, although slightly better than the dimaleimidedimaleimide moietymoiety andand formform 66.. ThisThis couplingcoupling ofof anilineaniline 55 alsoalso gavegave aa lowlow yield,yield, althoughalthough slightlyslightly HBTU-mediated reaction (Scheme2). betterbetter thanthan thethe HBTU-mediaHBTU-mediatedted reactionreaction (Scheme(Scheme 2).2). As before, this novel FlARe linker was incubated with an equimolar concentration of MBP-dC10* AsAs before,before, thisthis novelnovel FlAReFlARe linkerlinker waswas incubatedincubated withwith anan equimolarequimolar concentrationconcentration ofof MBP-dC10*MBP-dC10* (10 µM). However, no fluorescence increase was observed, even after 24 h of incubation (data not (10(10 µM).µM). However,However, nono fluorescencefluorescence increaseincrease waswas obobserved,served, eveneven afterafter 2424 hh ofof incubationincubation (data(data notnot shown). Since 6 only differs from 1 by the presence of the terminal hexyne tail, this functional group shown).shown). SinceSince 66 onlyonly differsdiffers fromfrom 11 byby thethe presencepresence ofof thethe terminalterminal hexynehexyne tail,tail, thisthis functionalfunctional groupgroup was suspected of disrupting the coumarin’s intrinsic fluorescence. Density Functional Theory (DFT) waswas suspectedsuspected ofof disruptingdisrupting thethe coumarin’scoumarin’s intrinintrinsicsic fluorescence.fluorescence. DensityDensity FunctionalFunctional TheoryTheory (DFT)(DFT) calculations of 6 showed that neither the LUMO nor the HOMO of the alkyne group were of appropriate calculationscalculations ofof 66 showedshowed thatthat neitherneither thethe LUMOLUMO nornor thethe HOMOHOMO ofof thethe alkynealkyne groupgroup werewere ofof energy to quench coumarin fluorescence through PeT (Figure S1). Thus, it is unlikely that the coumarin appropriateappropriate energyenergy toto quenchquench coumarincoumarin fluorescencefluorescence throughthrough PeTPeT (Figure(Figure S1).S1). Thus,Thus, itit isis unlikelyunlikely fluorescence of 6 was being quenched by this specific mechanism, as opposed to what has been thatthat thethe coumarincoumarin fluorescencefluorescence ofof 66 waswas beingbeing quenchedquenched byby thisthis specificspecific mechanism,mechanism, asas opposedopposed toto proposed to be the case for other alkyne-functionalized coumarins [30]. whatwhat hashas beenbeen proposedproposed toto bebe thethe casecase forfor otherother alkyne-functionalizedalkyne-functionalized coumarinscoumarins [31].[31]. Rather,Rather, differentdifferent studiesstudies havehave shownshown thatthat thethe coumcoumarinarin fluorophore’sfluorophore’s emissionemission isis veryvery sensitivesensitive to the functional group’s rigidity at the C7 position [32]. Indeed, this sensitivity is exploited in enzyme to the functional group’s rigidity at the C7 position [32]. Indeed, this sensitivity is exploited in enzyme Biomolecules 2020, 10, 369 10 of 16

Biomolecules 2020, 10, 369 10 of 16 Rather, different studies have shown that the coumarin fluorophore’s emission is very sensitive to activitythe functional probes group’sin which rigidity a carbohydra at the Cte7 linkedposition to [umbelliferone31]. Indeed, this at sensitivitythe C7 position is exploited is enzymatically in enzyme cleavedactivity to probes release in a whichhighly afluorescent carbohydrate 7-hydroxylcoum linked to umbelliferonearin fluorophore at the [33,34]. C7 position Therefore, is enzymatically in the case ofcleaved 6, the toterminal release hexyne a highly tail fluorescent at the C7 7-hydroxylcoumarin position offers a means fluorophore of rotational [32,33 ].relaxation Therefore, that in the could case severelyof 6, the diminish terminal hexynethe probe’s tail at fluorescence. the C7 position It is o ffstersriking a means to note of rotationalthat the 7-propargyl relaxation that coumarin could severely probe developeddiminish theby probe’sLiu, bearing fluorescence. a slightly It isshorter striking alky tol note chain that is theindeed 7-propargyl fluorescent coumarin [15]. Thus, probe extending developed theby alkyl Liu, bearing chain beyond a slightly three shorter carbons alkyl on chain 7-alkoxyco is indeedumarins fluorescent apparently [15]. Thus, has extendinga detrimental the alkyleffect chain on fluorescence.beyond three carbons on 7-alkoxycoumarins apparently has a detrimental effect on fluorescence. DespiteDespite the the lack lack of of fluorogenic fluorogenic acti activationvation by by FlARe, FlARe, we we tested tested whether whether 66 couldcould successfully successfully introduceintroduce an an alkyne alkyne group group site-specifically site-specifically to to a atarget target protein, protein, albeit albeit in in a anon-fluorogenic non-fluorogenic manner. manner. Specifically,Specifically, we we tested tested the the utility utility of of 66 toto couple couple RhB-N RhB-N3 3toto MBP-dC10* MBP-dC10* through through a aCuAAC CuAAC conjugating conjugating reaction.reaction. We We first first reacted reacted MBP-dC10* MBP-dC10* with with 66 forfor 24 24 h h to to ensure ensure complete complete labelling. labelling. Afterwards, Afterwards, we we performedperformed the the CuAAC CuAAC with with RhB-N RhB-N3 3andand evaluated evaluated the the bioconjugation bioconjugation by by fluorescent fluorescent SDS-PAGE SDS-PAGE (Figure(Figure 2).2). On On the the gel, gel, we we observed observed a a protein protein band band that that is is red red fluorescent fluorescent but but not not blue blue fluorescent fluorescent (Figure(Figure 2B,2B, lane 3). This This was was indicative of formation of the MBP-dC10*-RhB-NMBP-dC10*-RhB-N3 3conjugateconjugate using using compoundcompound 6 6asas the the linker. linker. The The lack lack of of any any blue blue fluoresc fluorescenceence is is consistent consistent with with the the non-fluorogenic non-fluorogenic naturenature of of 66 thatthat was was previously previously observed observed (Figure (Figure 2C).2C). Th Thee protein protein bands bands in in lane lane 5 5 of of Figure Figure 2 did not fluorescefluoresce at at all, all, confirming confirming that withoutwithout firstfirstintroducing introducing the the alkyne alkyne handle handle using using6, RhB-N6, RhB-N3 could3 could not notbe conjugatedbe conjugated to MBP-dC10*. to MBP-dC10*. In conclusion, In conclusion, although although compound compound6 was not 6 observedwas not observed to be fluorogenic to be fluorogenicas intended, as itintended, still introduced it still introduced an alkyne an group alkyne into group MBP-dC10*, into MBP-dC10*, to which to RhB-N which3 RhB-Ncould3 then could be thenconjugated be conjugated through through cycloaddition. cycloaddition.

FigureFigure 2. 2. SDSSDS PAGE PAGE gel gel of of protein protein conjugates conjugates obtained obtained after after first first reacting reacting 6 6withwith MBP-dC10* MBP-dC10* by by the the FlARe reaction followed by the conjugation of rhodamine B azide (RhB-N3) to MBP-dC10* through 6 FlARe reaction followed by the conjugation of rhodamine B azide (RhB-N3) to MBP-dC10* through using6 using CuAAC. CuAAC. A) Coomassie (A) Coomassie stained stained gel showing gel showing MBP-dC10* MBP-dC10* bands. bands. B) Unstained (B) Unstained gel excited gel excited with Greenwith GreenEpi Illumination Epi Illumination and imaged and imaged using using a 605/50 a 605 /filter.50 filter. C) (UnstainedC) Unstained gel gel excited excited with with Blue Blue Epi Epi IlluminationIllumination and and imaged imaged with aa 530530/28/28 filter.filter. Lanes: Lanes: (1) 1) Pre-stainedPre-stained protein protein standards standards (2) 2) MBP-dC10* MBP-dC10* (3) 3 3)MBP-dC10* MBP-dC10* reacted reacted with with6 and 6 and then then conjugated conjugated to RhB-N to RhB-N3 (4) MBP-dC10* 4) MBP-dC10* reacted reacted with with6 (5) MBP-dC10*6 5) MBP- 3 dC10*incubated incubated with RhB-Nwith RhB-N3 under under CuAAC CuAAC conditions. conditions.

3.2.3.2. Improved Improved Design Design AsAs previously previously discussed, discussed, the the non-fluorogenic non-fluorogenic nature nature of of 6 6mademade itit difficult difficult to to ascertain ascertain how how rapidlyrapidly and and efficiently efficiently the the alkyne alkyne group group was was introduc introduced.ed. To To address address this this issue, issue, we we designed designed a asecond second fluorogenicfluorogenic bioconjugating bioconjugating linker linker where where the the alkyne alkyne group group was was attached attached to the to coumarin the coumarin core coreat the at Cthe7 position C7 position as a as4-ethynylbenzylamine a 4-ethynylbenzylamine substituent. substituent. WeWe hypothesized hypothesized that that this this functional functional group group would present less rotational flexibility and maintain the coumarin’s intrinsic fluorescence. These modifications are comprised in the design of the fluorogenic linker 10 shown in Scheme 4. Biomolecules 2020, 10, 369 11 of 16 would present less rotational flexibility and maintain the coumarin’s intrinsic fluorescence. These modifications are comprised in the design of the fluorogenic linker 10 shown in Scheme4. Biomolecules 2020, 10, 369 11 of 16

SchemeScheme 4.4. Synthetic route for the preparationpreparation ofof ﬂuorogenicfluorogenic alkynesalkynes 1010 andand 1515..

PriorPrior toto thethe formationformation ofof 1010,, thethe salicylaldehydesalicylaldehyde 1111 waswas preparedprepared (Scheme(Scheme4 4).). First, First, the the terminal terminal alkynealkyne waswas attached attached by reactingby reacting 3-aminophenol 3-aminophenol with p -ethynylbenzaldehydewith p-ethynylbenzaldehyde in a reductive in a aminationreductive usingamination sodium using triacetoxyborohydride sodium triacetoxyborohydride (STAB). This (STAB). afforded This the afforded secondary the aminesecondary12 in amine good yield,12 in whichgood yield, was then which capped was then with capped acetaldehyde with acetaldehyde through a second through reductive a second amination reductive using amination STAB to using yield theSTAB tertiary to yield amine the tertiary13 in moderate amine 13 yield. in moderate A Vilsmeier–Haack yield. A Vilsmeier–Haack reaction was performedreaction was to performed obtain the functionalizedto obtain the functionalized salicylaldehyde salicylaldehyde11 in moderate 11 yield, in moderate which wasyield, then which followed was then by a followed Knoevenagel by a condensationKnoevenagel withcondensation Meldrum’s acidwith toMeldrum’s obtain coumarin acid 14to. Finally, obtain the coumarin di(methylmaleimide) 14. Finally, aniline the intermediatedi(methylmaleimide)5 was attached aniline to intermediate14 by first converting 5 was attached the latter to 14 to by its first acid converting chloride with the POCllatter3 toand its thenacid formingchloride thewith amide POCl bond3 and tothen aff ordforming10 in the good amide yield. bond to afford 10 in good yield. ReactingReacting equimolarequimolar amounts amounts of of10 10with with MBP-dC10* MBP-dC10* at at 10 10µ MµM resulted resulted in in a 37-folda 37-fold enhancement enhancement in fluorescencein fluorescence after after complete complete reaction reaction with with thetag the (Figure tag (Figures3A and 3A Figure and S2A). Not only diddid thisthis indicateindicate successfulsuccessful fluorescencefluorescence activationactivation byby FlARe,FlARe, itit isis alsoalso a significantsignificant improvementimprovement fromfrom thethe FEFE observed withwith probeprobe 1.1. However, nearlynearly 1010 hh werewere requiredrequired forfor thethe completecomplete reactionreaction ofof 1010 withwith MBP-dC10*MBP-dC10* atat 1 1 1010 µµM,M, providingproviding a a rate rate constant constant of ofk 2k2= =8.4 8.4 ± 0.70.7 MM−−1s−1, ,which which is is over over an an order order of magnitudemagnitude lowerlower ± thanthan thethe raterate constant constant measured measured for for1 (Figure1 (Figure S2B). S2B). This This suggests suggests that that the modifiedthe modified alkyne alkyne tail of tail6 may of 6 hindermay hinder the labelling the labelling reaction. reaction. On a practical On a level,practical this meanslevel, thatthis makingmeans micromolarthat making concentrations micromolar concentrations of a protein conjugate would require at least two days to complete, calling for a quicker alternative. As mentioned before, unsubstituted maleimides react more quickly than methylmaleimides [30]. Thus, the di(methylmaleimide) aniline moiety was replaced with dimaleimide aniline in the design of probe 15 (Scheme 4). Biomolecules 2020, 10, 369 12 of 16 of a protein conjugate would require at least two days to complete, calling for a quicker alternative. As mentioned before, unsubstituted maleimides react more quickly than methylmaleimides [29]. Thus, the di(methylmaleimide) aniline moiety was replaced with dimaleimide aniline in the design of probe 15 (Scheme4). Biomolecules 2020, 10, 369 12 of 16

FigureFigure 3. ((AA)) Excitation Excitation (dotted (dotted line) line) and and emissi emissionon (solid (solid line) line) spectra spectra of of 10 10 µMµM 1010 (grey(grey lines) lines) and and 1515 (blue(blue lines) lines) before before (light) (light) and and after after (d (dark)ark) reacting with 10 µMµM MBP-dC10*. ( (BB)) Kinetic Kinetic time time course course for for thethe fluorogenicfluorogenic reactionreaction between between 10 10µ MµM of of10 10(grey (grey lines) lines) and and15 (blue15 (blue lines) lines) with with 10 µ M10 ofµM MBP-dC10* of MBP- dC10*(dark lines)(dark (black),lines) (black), relative relative to the stability to the stability of 10 and of15 10in and the 15 absence in the of absence MBP-dC10* of MBP-dC10* (light lines). (light lines). Using the same synthetic strategy outlined in Scheme4, aniline 4 was coupled to 14 through the acylUsing chloride the same aff syntheticorded by strategy POCl3 to outlined obtain in15 Schemein decent 4, aniline yield. 4 Upon was coupled reacting to with 14 through equimolar the amounts of MBP-dC10* at 10 µM, the fluorescence intensity of 15 was found to increase 10-fold. The acyl chloride afforded by POCl3 to obtain 15 in decent yield. Upon reacting with equimolar amounts offinal MBP-dC10* MBP-dC10*- at 1015 µM,adduct the fluorescence displayed a intensity quantum of yield 15 was of Φfound= 0.093, to increase which 10-fold. is 57 times The higher final MBP- than dC10*-the quantum15 adduct yield displayed of the unreacted a quantum form yield of of15 Φ(Figure = 0.093, S3). which This is FE 57 wastimes three-fold higher than lower the compared quantum yieldto 10 ofbut the still unreacted comparable form to of the 15 FE(Figure observed S3). This with FE1 (Figurewas three-fold3A). The lower rate constantcompared of to the 10 reaction but still between the target peptide and 15 was calculated to be k = 180 10 M 1s 1, with completion at the comparable to the FE observed with 1 (Figure 3A). The rate2 constant± of the− reaction− between the target 10-µM concentrations achieved after only 30 minutes (Figure3B). Thus, switching to the unsubstituted peptide and 15 was calculated to be k2 = 180 ± 10 M−1s−1, with completion at the 10-µM concentrations achievedmaleimide after accelerated only 30 the minutes labelling (Figure reaction 3B). by overThus, an switching order of magnitude, to the unsubstituted making it comparable maleimide to acceleratedthe reaction the between labelling1 and reaction MBP-dC10*. by over an order of magnitude, making it comparable to the reaction betweenFurthermore, 1 and MBP-dC10*. the total time required to prepare a protein conjugate was reduced to a one-day process.Furthermore, Encouraged the bytotal both time the required fluorogenic to prepare nature ofa protein15 and itsconjugate rapid labelling was reduced reaction, to a the one-day utility process.of 15 for Encouraged bioconjugation by both was the assessed fluorogenic in a manner nature of similar 15 and to its6. rapid Thus, labelling 10 µM of reaction,15 was incubatedthe utility ofwith 15 for 10 µbioconjugationM MBP-dC10* was for 1asse h, afterssed whichin a manner RhB-N similar3 was thento 6. Thus, conjugated 10 µM to of MBP-dC10* 15 was incubated by CuAAC with through the alkyne handle introduced by 15. SDS-PAGE analysis showed a protein band that was 10 µM MBP-dC10* for 1 h, after which RhB-N3 was then conjugated to MBP-dC10* by CuAAC throughboth red theand alkyneblue fluorescent handle introduced (Figure4 B,C,by 15 lane. SDS-PAGE 4), indicating analysis that showed MBP-dC10* a protein was band conjugated that was to boththe red red fluorescent and blue fluorescent rhodamine (Figure B through 4B and the 4C, blue lane fluorescent 4), indicating15 coumarin that MBP-dC10* linker. Interestingly, was conjugated the toblue the fluorescence red fluorescent of this rhodamine protein bandB through is considerably the blue fluorescent dimmer than 15 coumarin MBP-dC10* linker. labelled Interestingly, only with the15 blue(Figure fluorescence4C, lane 2). of We this suspect protein this band is because is considerably of a FRET dimmer interaction than between MBP-dC10* the coumarin labelled only fluorophore with 15 (Figureof 15 and 4C, the lane rhodamine 2). We Bsuspect group ofthis the is modelbecaus ‘cargo’.e of a ThisFRET FRET interaction interaction between has beenthe identifiedcoumarin fluorophorepreviously in of the 15 literature,and the rhodamine and a comparison B group ofof the the coumarin model ‘cargo’. emission This spectrum FRET interaction of the adduct has been of 15 identifiedand the excitation previously spectrum in the literature, of rhodamine and a B comparison (see Figure S4)of the reveals coumarin the significant emission spectralspectrum overlap of the adductrequired of for 15 FRET and the [20 ,34excitation,35]. Furthermore, spectrum molecularof rhodamin modellinge B (see ofFigure the triazole S4) reveals adduct the formed significant after spectralCuAAC couplingoverlap required suggests thefor fluorophoresFRET [20,35,36]. would Furthermore, be separated bymolecular less than modelling 30 Å, allowing of the for etriazolefficient adductresonance formed energy after transfer. CuAAC The coupling protein bandsuggests in lane the 5 fl isuorophores neither red norwould blue be fluorescent, separated confirmingby less than that 30 Å,the allowing linker 15 foris requiredefficient toresonance successfully energy conjugate transfer. RhB-N The 3proteinto MBP-dC10* band in lane (Figure 5 is4 B,neither lane 5). red Therefore, nor blue fluorescent, confirming that the linker 15 is required to successfully conjugate RhB-N3 to MBP-dC10* (Figure 4B, lane 5). Therefore, 15 successfully introduced an exposed alkyne group in a site-specific and fluorogenic manner, which allowed for the attachment of the azide functionalized cargo, RhB- N3. Biomolecules 2020, 10, 369 13 of 16

15 successfully introduced an exposed alkyne group in a site-speciﬁc and ﬂuorogenic manner, which Biomoleculesallowed for2020 the, 10,attachment 369 of the azide functionalized cargo, RhB-N3. 13 of 16

FigureFigure 4. 4. SDSSDS PAGE PAGE gel gel of protein of protein conjugates conjugates obtained obtained after afterfirst reacting first reacting 10 µM 10 15µ withM 15 10with µM 10MBP-µM dC10*,MBP-dC10*, followed followed by the byconjugat the conjugationion of rhodamine of rhodamine B azide B (RhB-N azide (RhB-N3) to MBP-dC10*3) to MBP-dC10* through through 15 using15 CuAAC.using CuAAC. A) Coomassie (A) Coomassie stained stained gel showing gel showing MBP-dC10* MBP-dC10* bands. bands. B) Unstained (B) Unstained gel imaged gel imaged using using a 605/50a 605/ 50filter. filter. C) (UnstainedC) Unstained gel gelexcited excited with with Blue Blue Ep Epii Illumination Illumination and and imaged imaged with with a a530/28 530/28 filter. filter. Lanes:Lanes: 1) (1) MBP-dC10* MBP-dC10* 2) (2) MBP-dC10* MBP-dC10* reacted reacted with with 1515 3)(3) pre-stained pre-stained protein protein molecular molecular weight weight markers markers 4)(4) MBP-dC10* MBP-dC10* reacted reacted with with 1515 andand then then conjugated conjugated to to RhB-N RhB-N33 5)(5) MBP-dC10* MBP-dC10* incubated incubated with with RhB-N RhB-N33 underunder CuAAC CuAAC conditions. conditions.

InIn addition addition to to its rapid kinetics,kinetics, 1515was was also also noted noted to to be be resistant resistant to to hydrolysis hydrolysis over over the the course course of 3 ofh 3 under h under the the assay assay conditions. conditions. This This prompted prompted us to us perform to perform the CuAACthe CuAAC between between15 and 15 and RhB-N RhB-N3 prior3 priorto conjugation to conjugation of the of triazole the triazole product product to MBP-dC10*, to MBP-dC10*, as we as hypothesized we hypothesized that the that maleimides the maleimides would wouldremain remain intact afterintact the after CuAAC. the CuAAC. Thus, after Thus, performing after performing the CuAAC the betweenCuAAC 15betweenand RhB-N 15 and3, weRhB-N added3, weMBP-dC10* added MBP-dC10* in equimolar in equimolar concentrations concentrations to 15 and allowed to 15 and the allowed15-RhB triazolethe 15-RhB product triazole to react product with theto reactdC10* with tag the for 1dC10* h. As tag before, for 1 the h. bioconjugationAs before, the bioconjugation was analysed by was SDS-PAGE analysed(Figure by SDS-PAGE5). The presence (Figure 5).of The a red presence and blue of fluorescenta red and blue protein fluorescent band (Figure protein5B,C, band lane (Figure 2) indicates 5B and the C, successfullane 2) indicates formation the successfulof the MBP-dC10*-rhodamine formation of the MBP-dC10*-rhodamine conjugate. Like the previous conjugate. experiment, Like the previous in the lane experiment, representing in the the laneconjugation representing of the the RhB-N conjugation3 cargo to of MBP-dC10* the RhB-Nafter3 cargolabelling to MBP-dC10* the protein after with labelling15, the the blue protein fluorescence with 15of, the this blue band fluorescence is dimmer thanof this that band of MBP-dC10*is dimmer than labelled that of only MBP-dC10* with 15, labelled and no redonly fluorescence with 15, and is noobserved red fluorescence from the bandis observed in lane from 4. This the reflectsband in both lane the 4. FRETThis reflects interaction both occurringthe FRET betweeninteraction the occurringfluorophores between and thethe importancefluorophores of and15 for the successful importance bioconjugation. of 15 for successful bioconjugation. However,However, it it is is also also noteworthy noteworthy that that the the intensity intensity of of the the fluorescence fluorescence of of allall thethe protein protein bands bands shownshown in in Figure Figure 55 appearsappears to to be be lower lower than than those those from from the the previous previous experiment experiment (shown (shown in in Figure Figure 4).4). TheThe similarity similarity of of the the density density of of the the Coomassie Coomassie stained stained protein protein bands bands between between the the gels gels (Figure (Figure 4A4A vs. vs. FigureFigure 5A)5A) suggests this this is is not due to a difference di fference in in gel gel loading. loading. We We hypothesize hypothesize that that the the labelling labelling reactionreaction of of the the 15-RhB triazole adductadduct withwith MBP-dC10*MBP-dC10* (shown (shown in in Figure Figure5) 5) was was less less e ffi efficientcient than than the thereaction reaction of of15 15with with MBP-dC10* MBP-dC10* (shown (shown in in Figure Figure4 ),4), leading leading to to a a lower lower proportionproportion ofof protein being labelledlabelled after after 1 1 hour hour of of incubation, incubation, and and a a less less intense intense signal signal being being observed. observed. It It is is conceivable conceivable that that the the stericsteric bulk bulk of of the the adduct adduct pre-formed pre-formed between rhodaminerhodamine BB andand fluorogenfluorogen15 15decreases decreases the the e ffiefficiencyciency of ofthe the subsequent subsequent reaction reaction with with MBP-dC10*, MBP-dC10*, slowing slowin theg the labelling labelling reaction reaction in a in manner a manner similar similar to what to whatwas observedwas observed on comparing on comparing1 and 101 .and Therefore, 10. Therefore, although although we have we demonstrated have demonstrated that it is possible that it is to possibleperform to the perform FlARe the protein-labelling FlARe protein-labelling reaction either reaction before either or after before the or CuAAC after the reaction, CuAAC it reaction, appears it to appearsbe more to effi becient more to efficient label the to protein label the first protein and then first attach and then the cargoattach to the the cargo labelled to the protein labelled through protein the throughalkyne group.the alkyne group. Biomolecules 2020, 10, 369 14 of 16 Biomolecules 2020, 10, 369 14 of 16

Figure 5. SDS PAGE analysis of protein conjugates obtained after first reacting 15 with rhodamine B Figure 5. SDS PAGE analysis of protein conjugates obtained after first reacting 15 with rhodamine B azide (RhB-N3) in the CuAAC reaction, followed by conjugation to MBP-dC10* by the FlARe reaction. (azideA) Coomassie (RhB-N3) stainedin the CuAAC gel showing reaction, MBP-dC10* followed by bands. conjug (Bation) Unstained to MBP-dC10* gel excited by the with FlARe Green reaction. Epi IlluminationA) Coomassie and stained imaged gel using showing a 605 /MBP-dC10*50 filter. (C) bands. Unstained B) Unstained gel excited gel with excited Blue Epi with Illumination Green Epi andIllumination imaged with and aimaged 530/28 filter.using Lanes:a 605/50 (1) filter. Pre-stained C) Unstained protein standardsgel excited (2) with MBP-dC10* Blue Epi reactedIllumination with and imaged with a 530/28 filter. Lanes: 1) Pre-stained protein standards 2) MBP-dC10* reacted with the 15-RhB-N3 product of the CuAAC. (3) MBP-dC10* reacted with 15 subjected to a mock CuAAC the 15-RhB-N3 product of the CuAAC. 3) MBP-dC10* reacted with 15 subjected to a mock CuAAC without RhB-N3. (4) MBP-dC10* incubated with RhB-N3 under CuAAC conditions. (5) MBP-dC10*. without RhB-N3. 4) MBP-dC10* incubated with RhB-N3 under CuAAC conditions. 5) MBP-dC10*. 4. Conclusions 4. ConclusionsIn summary, we have developed a new bioconjugation method that first introduces an alkyne groupIn onto summary, a target we protein have developed in a site-specific, a new bioconjuga rapid andtion fluorogenic method that manner. first introduces This allows an foralkyne the subsequentgroup onto site-specifica target protein attachment in a site-specific, of a broad rapid array and of azide-functionalized fluorogenic manner. cargo. This allows Our initially for the designedsubsequent bifunctionalized site-specific attachment conjugating of agenta broad (6) provedarray of to azide-functionalized be non-fluorescent, presumablycargo. Our initially due to itsdesigned long, flexible bifunctionalized hexyne tail. conjugating Despite being agent non-fluorogenic,(6) proved to be non-fluorescent,6 was still capable presumably of introducing due to anits alkynelong, flexible group hexyne to the testtail. proteinDespite MBP-dC10*,being non-fluorogenic, onto which 6 was the modelstill capable cargo of RhB-N introducing3 could an then alkyne be attached.group to the In ourtest secondprotein design, MBP-dC10*, the long onto alkyne which tail the of model6 was cargo replaced RhB-N with3 could a shorter then and be attached. more rigid In alkynylour second amine design, substituent, the long whilealkyne the tail di(methylmaleimide) of 6 was replaced with aniline a shorter was and replaced more rigid with alkynyl dimaleimide amine aniline,substituent, resulting while in the the di(methylmaleimide) heterobifunctional fluorogenic aniline was linkerreplaced15. with These dimaleimide modifications aniline, resulted resulting in a linkerin the whose heterobifunctional fluorescence increasedfluorogenic 10-fold linker upon 15. reactingThese modifications with the dC10* resulted target peptide in a linker (Φ = 0.093),whose allowingfluorescence the introduction increased 10-fold of the upon exposed reac alkyneting with group the to dC10* be monitored target peptide in situ ( byΦ = fluorescence 0.093), allowing increase. the Subsequently,introduction of model the cargoexposed RhB-N alkyne3 could group be attachedto be monitored to MBP-dC10* in situ through by fluorescence the site-specifically increase. installedSubsequently, alkyne model handle cargo presented RhB-N by3 could15. Furthermore, be attached weto MBP-dC10* demonstrated through that azide-functionalized the site-specifically cargoinstalled can alkyne also be handle incorporated presented into by15 15by. CuAACFurthermore,prior towe reacting demonstrated with the that target azide-functionalized protein. Overall, ourcargo method can also allows be incorporated the site-specific into introduction 15 by CuAAC of an prior exposed to reacting alkyne with group the into target a protein protein. of Overall, interest geneticallyour method fused allows to the a dC10* site-specific tag, representing introduction an of approach an exposed that alkyne is easily group amenable into a protein for the of synthesis interest ofgenetically new, therapeutically fused to a dC10* important tag, representing protein-cargo an conjugates. approach that Additionally, is easily amenable the fluorogenic for the naturesynthesis of theof new, bioconjugating therapeutically linker important allows the protein-cargo initial protein co modificationnjugates. Additionally, step to be easily the fluorogenic monitored innature situ by of fluorescence.the bioconjugating Therefore, linker our allows linker the provides initial protein a facile, modification modular and step adaptable to be easily method monitored for the inin situ vitro by synthesisfluorescence. of new Therefore, protein-cargo our linker conjugates provides in thea facile, future. modular and adaptable method for the in vitro synthesis of new protein-cargo conjugates in the future. Supplementary Materials: The following are available online at http://www.mdpi.com/2218-273X/10/3/369/s1, Figure S1, Figure S2, Figure S3, Figure S4, Figure S5, synthetic protocols for RhB-N3, compound 4 and its Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1, Figure S1, Figure S2, intermediates, 1H-NMR and 13C-NMR spectra. Figure S3, Figure S4, Figure S5, synthetic protocols for RhB-N3, compound 4 and its intermediates, 1H-NMR and Author13C-NMR Contributions: spectra. Conceptualization, K.K.T. and J.W.K.; methodology, K.K.T., A.C.L. and K.É.R.; validation, K.K.T., and J.W.K.; formal analysis, K.K.T.; investigation, K.K.T, A.C.L. and K.É.R.; resources, J.W.K.; data curation, K.K.T.;Author writing—original Contributions: draftConceptualization, preparation, K.K.T.;K.K.T. writing—review and J.W.K.; me andthodology, editing, J.W.K.;K.K.T., visualization,A.C.L. and J.W.K.;K.É.R.; validation, K.K.T., and J.W.K.; formal analysis, K.K.T.; investigation, K.K.T, A.C.L. and K.É.R.; resources, J.W.K.; data curation, K.K.T.; writing—original draft preparation, K.K.T.; writing—review and editing, J.W.K.; Biomolecules 2020, 10, 369 15 of 16 supervision, K.K.T. and J.W.K.; project administration, K.K.T. and J.W.K.; funding acquisition, J.W.K. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Natural Sciences and Engineering Research Council of Canada, Discovery Grant number 05893. The APC was funded by the same grant. Acknowledgments: The authors would like to thank our departmental colleague John Pezacki and his research group for their assistance with the CuAAC bioconjugation. Conflicts of Interest: The authors declare no conflict of interest. Furthermore, the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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