Key words IEDDA - bioorthogonal - 2-pyrone - fluorogenic probes - protein labeling
With its range of emerging reactions, bioorthogonal chemistry has revolutionized the
field of chemical biology in the last two decades.[1 ] Manipulation of biomolecules through these rapid, high yielding, biocompatible,
and highly selective reactions has greatly facilitated our ultimate goal of understanding
biological processes. Among the existing bioorthogonal reactions, the inverse-electron-demand
Diels–Alder (IEDDA) reactions of tetrazines and strained cyclooctenes or cyclooctynes
deserve special attention for its remarkable reaction kinetics and full biocompatibility.[2 ] Moreover, the tetrazine moiety is able to quench the fluorescence of suitable fluorescent
frames. This two-in-one feature of the tetrazine moiety was harnessed in the development
of bioorthogonally applicable fluorogenic probes.[3 ] While the field of bioorthogonal chemistry and imaging probes have benefited greatly
from tetrazines, the need for mutually orthogonal bioorthogonal reactions, for example
in multi-color labeling schemes, called for the development of alternative bioorthogonal
reactions.[4 ] This includes development of novel dienes for IEDDA schemes with substantially different
reactivities toward strained alkenes/alkynes.[5a ]
[b ] Such needs were addressed recently by the development of triazines that can also
react with strained dienophiles in IEDDA reactions.[5c,d ] Sydnones can also react with strained alkynes in thermal [3 + 2] cycloadditions
and, similarly, they can render fluorescent cores fluorogenic.[5e ] These are very important additions to the bioorthogonal toolbox, offering more options
to develop mutually orthogonal bioorthogonal chemistries.[5f ]
[g ] IEDDA reactions of 2-pyrones have been known for quite a while[6 ] and even used to access bioorthogonal functions, such as in the synthesis of a reactive
cyclooctyne;[7 ] yet, to our knowledge, the use of 2-pyrones as bioorthogonally applicable dienes
remained unexplored.
Our research group has been heavily involved[8 ] in the design and synthesis of bioorthogonally applicable fluorogenic probes for
protein labeling purposes and, in this context, the 2-pyrone moiety seems a particularly
useful platform. We reasoned that 2-pyrones form a benzene ring with suitable alkynes
via an IEDDA–retroDA reaction sequence (Scheme [1 ]), which, upon careful design, would directly result in scaffolds with extended conjugation,
giving rise to dramatic changes in spectroscopic properties. In line with the above
discussed need for surrogate bioorthogonal functions, and to establish the viability
of our hypothesis regarding the fluorogenic probe design potential of 2-pyrones, we
set forth a study aiming at exploring the applicability of the 2-pyrone scaffold from
these two aspects. Literature examples describe related reactions of 2-pyrones with
electron-deficient alkynes that required long reaction times at higher temperatures;[9 ] however, no studies under physiological conditions are reported. Therefore, we first
set up a pilot experiment using commercially available methyl coumalate (1 ) with strained cyclooctyne BCN (Scheme [1 ]) in aqueous solution at room temperature. We chose BCN as suitable dienophile because
it produces an aromatic system (i.e., a substituted benzene ring) upon IEDDA reaction.
Although more reactive trans -cyclooctenes could also be considered as complementary bioorthogonal platforms, their
reaction would yield a cyclohexadiene. Since our goal is to fabricate fluorogenic
systems taking advantage of the possible 2-in-1 feature of the pyrone scaffold, we
foresaw that generation of a fully aromatic system would profoundly affect the spectroscopic
characteristics of heterocycles.
Scheme 1 Reaction of methyl coumalate (1 ) with BCN
To our delight, the overnight reaction of 1 and BCN resulted in the desired 1-BCN product in 90% yield following chromatography. Inspired by this finding, we moved
onto our ultimate goal; namely, to explore the feasibility of using the pyrone moiety
in the fabrication of bioorthogonally applicable fluorogenic probes. To this end,
we designed and synthesized a set of probes 2 –6 furnished with a 2-pyrone moiety. The probes were designed in a way that in the product
of the reaction with BCN the benzene ring becomes directly attached to a conjugated
system, giving rise to π-extended structures (Scheme [2 ]).
Scheme 2 Target bioorthogonal probes 2 –6
As stated above, we anticipated that the electronic differences between the pyrone
and the benzene ring would dramatically change the photophysical properties of the
probes, resulting in highly fluorogenic probes. For synthetic considerations, 4-hydroxy-6-methyl-pyrone
(7 ) was chosen as a readily available starting material, which offers a conjugation
site via its hydroxyl group. We believed that cross-coupling reactions of pseudohalogenated
pyrones would allow a versatile approach for the synthesis of a wide variety of 2-pyrone
derivatized scaffolds.[10 ] Therefore, 7 was converted into its triflate derivative 8 ,[10a ] through treatment with PhNTf2 . After optimizing the reaction conditions, 8 could be prepared in good yields on the multi-gram scale and served as a common building
block for further probes. First, 8 was coupled to 4-vinylpyridine in a Heck reaction to access 9 , which was N-alkylated with methyl iodide to yield probe 2 (Scheme [3 ]).
Scheme 3 Synthesis of probe 2 . Reagents and conditions : (a) PhNTf2 , NEt3 , DCM, 40 °C, 1 h, 88%; (b) 4-vinylpyridine, Pd2 (dba)3 , QPhos, Cy2 NMe, DMF, 100 °C, N2 , 1 h, MW, 50%; (c) MeI, MeCN, 60 °C, 1 h, 20%.
Next, 4-methyl-7-hydroxycoumarin (10 ) was converted into its pinacol boronic ester counterpart 12
[11 ] in two steps. This was further reacted with 8 in a Suzuki cross-coupling reaction to result in target probe 3 (Scheme [4 ]).
Scheme 4 Synthesis of probe 3 . Reagents and conditions : (a) PhNTf2 , NEt3 , DCM, 40 °C, 1 h, 85%; (b) B2 pin2 , KOAc, Pd(dppf)Cl2 , dioxane, N2 , 100 °C, 2 h, 90%; (c) 8 , Pd(dppf)Cl2 , KOAc, 1,4-dioxane, N2 , 100 °C, 2 h, 81%.
To further extend the conjugated system, 7-diethylaminocoumarin (13 ) was prepared according to reported procedures.[12 ] Site-selective bromination was effected with N -bromosuccinimide (NBS) to yield 3-bromo derivative 14 , which was subjected to a Sonogashira cross-coupling with TMS-acetylene to deliver
15 . Removal of the TMS group gave 16 ,[13 ] which was immediately used in a second Sonogashira coupling reaction with pyrone
triflate 8 , to give acetylene-linked probe 4 in excellent yield (Scheme [5 ]).
Scheme 5 Synthesis of probe 4 . Reagents and conditions : (a) NBS, NH4 OAc, MeCN, 25 °C, 2 h, dark, 30%; (b) TMS-acetylene, Pd(PPh3 )2 Cl2 , CuI, EDIPA, DMF, N2 , 45 °C, 1 h, 91%; (c) 1 M TBAF in THF, 25 °C, 2 h, 26%; (d) 8 , Pd(PPh3 )2 Cl2 , CuI, EDIPA, MeCN, N2 , 45 °C, 1 h, quant.
To access vinylene extended probe 5 , intermediate 18 was synthesized through a Vilsmeier–Haack formylation,[12 ] Wittig reaction sequence, via
17 . Finally, the pyrone ring was installed in a Heck reaction to yield 5 (Scheme [6 ]).
Scheme 6 Synthesis of probe 5 . Reagents and conditions : (a) POCl3 , DMF, N2 , 0 °C/30 min, 60 °C/16 h, 54%; (b) i . Ph3 P+ Me Br– , n -BuLi/hexane, THF, N2 , 0 °C, 20 min; ii . 17 , THF, 25 °C, 18 h, 47%; (c) 8 , Pd2 (dba)3 , QPhos, Cy2 NMe, DMF, 100 °C, N2 , 1 h, MW, 32%.
To study the effects of different connectivity of the pyrone ring and to be able to
install an electron-withdrawing group, which facilitates IEDDA reaction, we also prepared
bromo-pyrone 19 .[14 ] Vinyl coumarin 18 was then allowed to react with 19 to yield probe 6 in good yield (Scheme [7 ]).
Scheme 7 Synthesis of probe 6 . Reagents and conditions : (a) PBPB, AcOH, 100 °C, 12 h, 41%; (b) 18 , Pd2 (dba)3 , QPhos, Cy2 NMe, DMF, 100 °C, N2 , 1 h, MW, 85%.
With the desired molecules in hand, we tested their reactions with BCN and monitored
the changes in their fluorescence spectra. Results showed that all compounds reacted
with BCN; however, the change in the fluorescence properties varied considerably (Table
[1 ] and see the Supporting Information for spectra). Compound 2 and its reaction product with BCN (2-BCN ) both showed large Stokes shifts; however, the intensity of the fluorescence increased
by only about threefold upon reaction. Probes 3 and 3-BCN showed similar absorption and emission properties, with somewhat larger (13-fold)
increase in fluorescence. These latter two probes, however, required UV excitation,
which is not ideal for biological applications. Probe 4 , on the other hand, showed significantly redshifted excitation and emission maxima,
both of which were slightly blueshifted upon reaction with BCN. Notably, however,
this reaction was accompanied by a huge increase in the intensity of fluorescence
(over 100-fold). Reaction of probe 5 with BCN resulted in decreased fluorescence intensity and the disappearance of the
originally large Stokes shift. Changing the substitution pattern of the 2-pyrone frame
and introducing an electron-withdrawing group onto it (6 ) resulted in a hypsochromic shift compared to 5 , and only a slight increase in fluorescence was observed upon reaction. These results
indicate that the position and the nature of the linkage between the 2-pyrone and
the fluorescent frame should be carefully considered during the design process.
Table 1 Excitation and Emission Maximaa of the New Fluorescent Dyes and the Change in Fluorescence Intensity upon Reaction
of BCN
Compound
λmax (ex) (nm)
λmax (em) (nm)
Change in fluorescence
2
360
491
slight increase (ca. 3.3×)
3
335
429
increase (ca. 12–13×)
4
465
536
huge increase (>100×)
5
469
593
decrease
6
407
490
slight increase (ca. 2.5×)
a Measurement conditions: 50% MeCN in H2 O, 25 °C.
Based on these findings, we chose 4 for further experiments. First, we quantified its reaction speed with BCN and found
the second-order rate constant to be k
2 = 0.095 M–1 s–1 , which is in the same order of magnitude as the well-established strain-promoted
cycloaddition reactions of azides and strained alkynes (e.g., DIFO and BCN have a
k
2 of 0.076 and 0.14, respectively).[15 ] We also measured the molar absorption coefficients, fluorescence quantum yields,
and the fluorescence enhancement (Table [2 ]).
Table 2 Spectral Propertiesa (ε and φ values) of 4 and 4-BCN and the Calculated Fluorescence Enhancement upon Reaction with BCN
4
4-BCN
ε
465nm (M–1 cm–1 )
φ
b
ε
426nm (M–1 cm–1 )
φ
b
52730
0.009
33150
1.0
Fluorescence enhancement (φ
4-BCN
/φ
4
):
111×
a Measurement conditions: 50% MeCN in H2 O, 25 °C.
b Using coumarin-153 in EtOH (φ = 0.544) as reference standard.[16 ]
To test whether the pyrone-derivatized bioorthogonally applicable fluorogenic probe
4 is suitable for protein labeling, we first functionalized a human serum protein,
Transferrin (TF, 76 kDa) with BCN using BCN-NHS. Following removal of unreacted reagents,
probe 4 was added to the samples at different concentrations (i.e., 125, 250 or 500 μM).
Following 24 hour incubation, the reaction mixtures were worked up, then the samples
were subjected to an SDS-polyacrylamide gel, and in-gel fluorescence detection (Figure
[1 ]). Fluorescent Transferrin bands occurred in the lanes only where Transferrin was
co-incubated both with NHS-BCN and 4 . As expected, fluorescence intensity was proportional to the concentration of the
probe added. These results indicated that the fluorogenic probe 4 indeed reacted specifically with transferrin-conjugated BCN, in a concentration-dependent
manner. As control experiments we mixed probe 4 with Transferrin that was not treated with BCN previously. Only a very low fluorescent
signal was observed, indicating that fluorogenic 4 in its quenched (i.e., 2-pyrone) form does not contribute to background fluorescence
even when it is adhered non-specifically to the protein by adsorption. We have also
studied the labeling scheme of further proteins including β-lactoglobulin B, α1-acid
glycoprotein, myoglobin and a trypsin inhibitor, each of which showed similar results
to transferrin labeling (see the Supporting Information). To explore the labeling
efficacy, we have also labeled a BCN-tagged 17-mer oligonucleotide and a BCN-tagged
cyclic peptide, phalloidin. The reactions were followed either by capillary electrophoresis
or by LC-MS. The labeling reactions were accomplished either in 50% DMSO-water (oligonucleotide)
or in methanol (phalloidin). Mass spectrometry proved the formation of the expected
products; however, the conversion rates were very different, i.e., 33% vs. 100% for
the aqueous solution and methanol, respectively, which is attributed to the limited
solubility of compound 4 (see the Supporting Information).
Figure 1 In-gel SDS PAGE representation of the ability of the fluorogenic coumarin-pyrone
derivative 4 to label the serum protein Transferrin (TF) modified with NHS-BCN. Top image is the
fluorescent channel detected using 460–490 nm excitation and emission detection with
a 532/28 nm band pass filter. Coomassie staining to indicate equal protein loading
is shown in the lower image. Molecular weights corresponding to the visible bands
of the marker are indicated.
The stability of compound 4 was assessed in the presence of excess amounts of GSH (1–10 mM). LC-MS analysis of
the samples indicated no substantial change of the pyrone in the presence of up to
5 mM GSH after 24 hours. Higher amounts of GSH (10 mM), however, led to ca. 20% decomposition
of 4 after 24 hours and MS indicated the presence of a 4 -GSH adduct. We also checked fluorescence spectra of these samples and, to our delight,
found no change in the emission spectrum, indicating that the product is not fluorescent.
We also explored the mutual orthogonality of the 2-pyrone moiety in the presence of
other bioorthogonal functions. We assumed that, similar to tetrazines, 2-pyrones are
also inert towards sterically demanding dibenzocyclooctynes, such as DBCO.[5f ] Preliminary studies confirmed that 4 indeed does not react with DBCO. We then combined excess amounts of DBCO and BCN
and added 2-pyrone, bearing probe 4 , to this solution. Next, all the remaining BCN was consumed by adding a tetrazine
bearing fluorogenic probe, PheCou,[17a ] in excess. Finally, we added an azide-bearing probe, CBRD.[17b ] The reaction mixture was then analyzed by LC-MS. To our delight, only the three
expected products could be detected; namely, 4 -BCN, PheCou-BCN, and CBRD-DBCO (see the Supporting Information).
In conclusion, we have explored the potential of the 2-pyrone moiety in bioorthogonal
reaction schemes in combination with strained alkyne, BCN. The inverse-electron-demand
Diels–Alder reaction follows a moderate kinetics with second-order rate constant around
0.1 M–1 s–1 , which is similar to, for example, SPAAC reactions of azides with BCN. We also prepared
several 2-pyrone appended probes, one of which showed remarkable fluorescence increase
upon IEDDA reaction with BCN. We also demonstrated the applicability of our new 2-pyrone
probe in protein labeling schemes in vitro with various BCN-modified proteins. Sequential addition of 4 , a tetrazine- and an azide-appending probe, to a mixture of BCN and DBCO revealed
that the SPAAC reaction of azides and the IEDDA of 2-pyrones can be conducted orthogonally
upon careful selection of reaction partners.
Since tetrazines are very hard to connect directly to fluorescent frames, as only
a few examples are reported,[3b ]
[18 ] we believe that the finding that the 2-pyrone moiety allows several cross-coupling
reactions (e.g., Heck, Suzuki, Sonogashira) giving rise to various 2-pyrone-appending
profluorescent frames, further highlights the significance of this study. The pyrone
moiety is stable, easy to handle, and, similar to the tetrazine function, can also
participate in IEDDA reactions, which upon careful design, can result in fluorogenic
probes. Upon reacting with alkynes, the formed aromatic moiety allows direct extension
of conjugated systems. Furthermore, by adding the pyrone moiety to the bioorthogonal
reaction tool-box, we envision that it can be applied to develop mutually orthogonal
bioorthogonal reactions taking advantage of its substantially different reaction kinetics.
All starting materials were obtained from commercial suppliers (Sigma–Aldrich, Fluka,
Merck, Alfa Aesar, Reanal, Molar Chemicals, Fluorochem) and used without further purification.
(1R ,8S ,9S )-Bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN) and the corresponding succynimidyl carbonate
(BCN-NHS) was obtained from Sigma. Analytical thin-layer chromatography (TLC) was
performed on silica gel 60 F254 precoated aluminum TLC plates from Merck. Flash column
chromatography was performed with a Teledyne Isco CombiFlash® Rf+ automated flash chromatographer with silica gel (25–40 μm) from Zeochem. Microwave
experiments were carried out with an AntonPaar (Graz, Austria) Monowave 300 microwave
reactor (maximum power 850 W). NMR spectra were recorded with a Varian Inova 500 MHz
spectrometer. Chemical shifts (δ) are given in parts per million (ppm) using solvent
signals or TMS as the reference. Coupling constants (J ) are reported in hertz (Hz). Analytical RP-HPLC-UV/Vis-MS measurements were conducted
with a Shimadzu LCMS-2020 instrument applying a Gemini C18 column (100 × 2.00 mm I.D.)
in which the stationary phase was 5 μm silica with a pore size of 110 Å. The chromatograms
were detected with UV/Vis diode array (190–800 nm) and ESI-MS detectors. Linear gradient
elution (0 min 0% B; 2.0 min 100% B; 3.5 min 100% B; 4.5 min 0% B; 5.0 min 0% B) with
eluents A (2% HCOOH, 5% MeCN, and 93% H2 O) and B (2% HCOOH, 80% MeCN, and 18% H2 O) was used at a flow rate of 1.0 mL min–1 at 30 °C. The samples were dissolved in MeCN–H2 O mixture. Spectroscopic measurements were performed with a Jasco FP 8300 spectrofluorometer.
Quartz cuvettes with path length of 1 cm were used. The exact masses were determined
with an Agilent 6230 time-of-flight mass spectrometer.
Methyl 1-(Hydroxymethyl)-1a,2,3,8,9,9a-hexahydro-1H -benzo[a ]-cyclopropa[e ][8]annulene-5-carboxylate (1-BCN)
Methyl 1-(Hydroxymethyl)-1a,2,3,8,9,9a-hexahydro-1H -benzo[a ]-cyclopropa[e ][8]annulene-5-carboxylate (1-BCN)
In a round-bottom flask, methyl coumalate (1 ; 5.0 mg, 0.033 mmol, 1 equiv) was dissolved in 50% MeCN–H2 O (1 mL) and BCN (15 mg, 0.10 mmol, 3 equiv) was added. The reaction mixture was stirred
at 25 °C for 18 hours, then concentrated in vacuo on a rotary evaporator. The crude product was purified by flash column chromatography
(0→60%, EtOAc–hexane) to give the desired product.
Yield: 7.6 mg (90%); white solid.
1 H NMR (CDCl3 , 500 MHz): δ = 7.80–7.75 (m, 2 H), 7.16 (d, J = 8.4 Hz, 1 H), 3.89 (s, 3 H), 3.70 (dd, J = 7.8, 1.3 Hz, 2 H), 3.06–2.95 (m, 2 H), 2.81 (m, 2 H), 2.31–2.20 (m, 2 H), 1.49
(m, 2 H), 1.13–1.02 (m, 1 H), 0.91 (m, 2 H).
13 C NMR (CDCl3 , 126 MHz): δ = 167.5, 147.7, 142.2, 131.3, 130.4, 128.0, 127.5, 59.9, 52.0, 33.6,
33.4, 24.54, 24.53, 22.1, 19.4.
LC-MS (ESI): m /z = 261 (C16 H21 O3 ) [M + H]+ .
6-Methyl-2-oxo-2H -pyran-4-yl Trifluoromethanesulfonate (8)[10a ]
6-Methyl-2-oxo-2H -pyran-4-yl Trifluoromethanesulfonate (8)[10a ]
4-Hydroxy-6-methyl-pyrone (7 ; 631 mg, 1.20 mmol, 1 equiv) and N -phenyl-bis(trifluoromethanesulfonimide) (1.97 g, 1.32 mmol, 1.1 equiv) were dissolved
in DCM (40 mL, stabilized with amylene), then triethylamine (1.00 mL, 1.80 mmol, 1.5
equiv) was added, and the reaction mixture was stirred at 40 °C for 1 h. After cooling
to r.t., it was diluted with EtOAc (200 mL), washed with sat. NaHCO3 (3 × 100 mL) and dried over MgSO4 . After filtration and evaporation, the crude product was purified by flash column
chromatography (hexane–EtOAc, 10:1 v/v) to give the desired product.
Yield: 1.14 g (88%); colorless oil.
Repeating the same procedure with 7 (3.00 g) under similar conditions gave 8 (5.29 g, 86%) as a pale-yellow oil, which solidified in the freezer.
It is important to note, that 8 slowly decomposed in the fridge, but was stable at –20 °C.
Rf
0.76 (hexane–EtOAc, 1:1 v/v).
IR (neat): 3104, 1743, 1646, 1575, 1429, 1318, 1206, 1134, 1109, 961, 803 cm–1 .
1 H NMR (CDCl3 , 500 MHz): δ = 6.10 (s, 1 H), 6.05 (s, 1 H), 2.31 (s, 3 H).
13 C NMR (CDCl3 , 126 MHz): δ = 165.7, 161.7, 161.1, 120.6, 102.5, 99.7, 20.4.
HRMS (ESI): m /z [M + H]+ calcd for C7 H6 F3 O5 S: 258.9888; found: 258.9893.
(E )-6-Methyl-4-(2-(pyridin-4-yl)vinyl)pyrone (9)
(E )-6-Methyl-4-(2-(pyridin-4-yl)vinyl)pyrone (9)
In a microwave pressure tube with a magnetic stir bar, 4-vinylpyridine (122 mg, 1.16
mmol, 3 equiv) and 8 (100 mg, 0.39 mmol, 1 equiv) were dissolved in abs. DMF (4 mL) under N2 . N ,N -Dicyclohexylmethylamine (410 μL, 1.94 mmol, 5 equiv), QPhos (14 mg, 0.02 mmol, 0.05
equiv) and Pd2 (dba)3 (17 mg, 0.02 mmol, 0.05 equiv) were added and the reaction mixture was heated in
a microwave reactor at 100 °C for 1 hour. The solvent was evaporated in vacuo and the crude product was purified by flash chromatography twice (first with DCM–MeOH,
20:1, then hexane–EtOAc, 1:1 v/v eluent) to give the product.
Yield: 42 mg (50%); sticky white solid; Rf
0.31 (DCM–MeOH, 20:1 v/v).
IR (neat): 2925, 2852, 2781, 1737, 1594, 1448, 1261, 1199, 1049, 890 cm–1 .
1 H NMR (CD3 CN, 500 MHz): δ = 8.59 (d, J = 6.1 Hz, 2 H), 7.50 (d, J = 6.1 Hz, 2 H), 7.31 (d, J = 16.4 Hz, 1 H), 7.17 (d, J = 16.4 Hz, 1 H), 6.47 (s, 1 H), 6.16 (s, 1 H), 2.25 (s, 3 H).
13 C NMR (CD3 CN, 126 MHz): δ = 163.13, 153.34, 152.25, 151.05, 144.28, 134.76, 130.08, 122.51,
111.42, 101.25, 20.12.
HRMS (ESI): m /z [M + H]+ calcd for C13 H12 NO2 : 214.0868; found: 214.0865.
(E )-1-Methyl-4-(2-(6-methyl-2-oxo-2H -pyran-4-yl)vinyl)pyridin-1-ium Iodide (2)
(E )-1-Methyl-4-(2-(6-methyl-2-oxo-2H -pyran-4-yl)vinyl)pyridin-1-ium Iodide (2)
Compound 9 (52 mg, 0.24 mmol, 1 equiv) was dissolved in anhydrous acetonitrile (4 mL) in a pressure
tube, then methyl iodide (150 μL, 2.40 mmol, 10 equiv) was added and the vial was
sealed. The reaction mixture was stirred at 60 °C for 1 h, then cooled to r.t. and
the solvent was evaporated. The crude product was suspended in EtOAc, filtered, and
washed with EtOAc to give the desired product, which required no further purification.
Yield: 18 mg (20%); yellow powder.
IR (neat): 3041, 3012, 1734, 1705, 1639, 1517, 1312, 1228, 984, 846 cm–1 .
1 H NMR (D2 O/CD3 CN, 500 MHz): δ = 9.05 (s, 2 H), 8.49 (s, 2 H), 7.88 (s, 2 H), 7.03 (s, 1 H), 6.77
(s, 1 H), 4.67 (s, 3 H), 2.68 (s, 3 H).
13 C NMR (D2 O/CD3 CN, 126 MHz): δ = 165.1, 162.6, 150.9, 150.6, 144.5, 134.6, 130.4, 124.6, 111.1, 100.9,
47.0, 18.5.
HRMS (ESI): m /z [M]+ calcd for C14 H14 NO2 : 228.1025; found: 228.1016.
4-Methyl-coumarin-7-yl Trifluoromethanesulfonate (11) and 4-Methyl-7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)coumarin
(12)
4-Methyl-coumarin-7-yl Trifluoromethanesulfonate (11) and 4-Methyl-7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)coumarin
(12)
Prepared according to reported procedures[11 ] with slight modifications.
7-Hydroxy-4-methyl-coumarin (10 ; 176 mg, 1.0 mmol, 1 equiv) and N -phenyl-bis(trifluoromethanesulfonimide) (393 mg, 1.1 mmol, 1.1 equiv) were dissolved
in DCM (20 mL, stabilized with amylene), then triethylamine (0.21 mL, 1.5 mmol, 1.5
equiv) was added and the reaction mixture was stirred at 40 °C for 1 h. After cooling
to r.t., it was diluted with EtOAc (200 mL), washed with sat. NaHCO3 (3 × 100 mL) and dried over MgSO4 . After filtration and evaporation of the solvent, the crude product was purified
by flash column chromatography (0→50%, EtOAc–hexane) to give 11 .
Yield: 262 mg (85%); white crystalline solid; Rf
0.63 (hexane–EtOAc, 1:1 v/v).
1 H NMR (CDCl3 , 500 MHz): δ = (d, J = 8.8 Hz, 1 H), 7.29 (d, J = 2.4 Hz, 1 H), 7.24 (dd, J = 8.8, 2.4 Hz, 1 H), 6.36 (d, J = 1.2 Hz, 1 H), 2.46 (d, J = 1.2 Hz, 3 H).
LC-MS (ESI): m /z = 309 (C11 H8 F3 O5 S) [M + H]+ .
Then compound 11 (202 mg, 0.66 mmol, 1 equiv), bis(pinacolato)diboron (200 mg, 0.79 mmol, 1.2 equiv)
and anhydrous KOAc (159 mg, 1.62 mmol, 3.6 equiv) were dissolved in abs. 1,4-dioxane
(6 mL) under N2 atmosphere. Pd(dppf)Cl2 (30 mg, 0.04 mmol, 0.06 equiv) was added and the reaction mixture was stirred at
100 °C for 2 hours. After cooling to r.t., water (100 mL) was added and the mixture
was extracted with EtOAc (3 × 75 mL). The combined organic phases was washed with
brine (100 mL) and dried over MgSO4 . The crude product was purified by flash column chromatography (0→5%, DCM–MeOH) to
give the product as a mixture of the pinacolatoboron and the free boronic acid derivatives,
which was used in the next step.
Yield: 133 mg (90%); off-white solid.
IR (neat): 2979, 1730, 1622, 1508, 1338, 1280, 1125, 850 cm–1 .
LC-MS (ESI): m /z = 287 (C16 H20 BO4 ) [M + H]+ for 12 and m /z = 203 (C10 H8 BO4 ) [M–H]– for the free boronic acid derivative.
4-Methyl-7-(6-methyl-2-oxo-2H -pyran-4-yl)coumarin (3)
4-Methyl-7-(6-methyl-2-oxo-2H -pyran-4-yl)coumarin (3)
Compound 12 (128 mg, 0.45 mmol, 1 equiv), 8 (115 mg, 0.45 mmol, 1 equiv), and anhydrous KOAc (232 mg, 2.36 mmol, 3.6 equiv) were
dissolved in abs. 1,4-dioxane (6 mL). Pd(dppf)Cl2 (19.8 mg, 0.027 mmol, 0.06 equiv) was added and the reaction mixture was stirred
at 100 °C for 2 hours. After cooling to r.t., water (100 mL) was added and the mixture
was extracted with EtOAc (3 × 75 mL). The combined organic phases was washed with
brine (100 mL) and dried over MgSO4 . The crude product was purified by flash column chromatography (0→5%, DCM–MeOH) to
give the desired product.
Yield: 98 mg (81%); pale-yellow solid; Rf
0.26 (hexane–EtOAc, 1:1 v/v).
IR (neat): 3051, 2921, 1707, 1634, 1323, 1026, 871 cm–1 .
1 H NMR (CDCl3 , 500 MHz): δ = 7.70 (d, J = 8.0 Hz, 1 H), 7.52–7.48 (m, 2 H), 6.40 (s, 1 H), 6.37 (d, J = 1.0 Hz, 1 H), 6.28 (s, 1 H), 2.48 (d, J = 1.1 Hz, 3 H), 2.35 (s, 3 H).
13 C NMR (CDCl3 , 126 MHz): δ = 163.7, 162.1, 153.6, 151.6, 150.6, 125.6, 122.5, 121.6, 116.5, 115.3,
110.7, 109.5, 103.0, 101.5, 20.4, 18.7.
HRMS (ESI): m /z [M + H]+ calcd for C16 H13 O4 : 269.0814; found: 269.0807.
3-Bromo-7-(diethylamino)-coumarin (14), 7-(Diethylamino)-3-((trimethylsilyl)ethynyl)-coumarin
(15) and 7-(Diethylamino)-3-ethynyl-coumarin (16)
3-Bromo-7-(diethylamino)-coumarin (14), 7-(Diethylamino)-3-((trimethylsilyl)ethynyl)-coumarin
(15) and 7-(Diethylamino)-3-ethynyl-coumarin (16)
Prepared according to reported procedures[13 ] with slight modifications.
In a round-bottom flask 7-(diethylamino)-coumarin (13 ; 2.60 g, 12.0 mmol, 1 equiv) and NH4 OAc (92 mg, 1.2 mmol, 0.1 equiv) were dissolved in acetonitrile (250 mL) and N -bromosuccinimide (2.60 g, 14.3 mmol, 1.2 equiv) was added while stirring. The reaction
mixture was stirred at r.t. for 2 hours in the dark (covered with aluminum foil),
then concentrated onto silica. The crude product was partially purified by column
chromatography (hexane–EtOAc, 4:1 v/v), then recrystallized from acetonitrile and
washed with cold Et2 O to give 14 .
Yield: 1.03 g (30%); light-brown solid; Rf
0.76 (hexane–EtOAc, 1:1 v/v); Rf
0.50 (hexane–EtOAc, 3:1 v/v).
LC-MS (ESI): m /z = 296 and 298 (C13 H15 BrNO2 ) [M + H]+ .
Compound 14 (500 mg, 1.7 mmol, 1 equiv), CuI (74 mg, 0.39 mmol, 0.2 equiv), and Pd(PPh3 )2 Cl2 (119 mg, 0.17 mmol, 0.1 equiv) was mixed in a round-bottom flask under N2 atmosphere. Then anhydrous DMF (15 mL), EDIPA (1.2 mL, 6.8 mmol, 4 equiv) and trimethylsilylacetylene
(0.70 mL, 5.1 mmol, 3 equiv) were added. The reaction mixture was stirred at 45 °C
for 1 h, then water (150 mL) was added and the mixture was extracted with DCM (3 ×
100 mL). The combined organic phases was washed with sat. EDTA (100 mL), brine (100
mL) and dried over MgSO4 . The crude product was purified by flash column chromatography (hexane–EtOAc, 4:1
v/v) to give 15 .
Yield: 482 mg (91%); brown solid; Rf
0.81 (hexane–EtOAc, 1:1 v/v); Rf
0.53 (hexane–EtOAc, 3:1 v/v).
LC-MS (ESI): m /z = 314 (C18 H24 NO2 Si) [M + H]+ .
To TMS-protected coumarin 15 (197 mg, 0.63 mmol, 1 equiv) was added TBAF (1 M in THF, 1.26 mL, 1.26 mmol, 2 equiv)
and the mixture was stirred at r.t. for 2 hours. The reaction mixture was concentrated
onto Celite and purified by flash column chromatography (0→2% MeOH–DCM) to give the
product 16 . It is important to note that 16 quickly decomposed and was used immediately in the next step.
Yield: 40 mg (26%); yellow-brownish oil; Rf
0.20 (DCM).
LC-MS (ESI): m /z = 242 (C15 H16 NO2 ) [M + H]+ .
7-(Diethylamino)-3-((6-methyl-2-oxo-2H -pyran-4-yl)ethynyl)coumarin (4)
7-(Diethylamino)-3-((6-methyl-2-oxo-2H -pyran-4-yl)ethynyl)coumarin (4)
In a round-bottom flask, 8 (36 mg, 0.138 mmol, 1 equiv), Pd(PPh3 )2 Cl2 (5.0 mg, 0.007 mmol, 0.05 equiv), and CuI (3.0 mg, 0.014 mmol, 0.1 equiv) were mixed
and flushed with N2 . Then coumarin 16 (40 mg, 0.16 mmol, 1.2 equiv) dissolved in anhydrous acetonitrile (5 mL) was added,
followed by EDIPA (96 μL, 0.552 mmol, 4 equiv). The reaction mixture was stirred at
45 °C for 1 h, then the solvent was evaporated and the crude product was purified
by flash column chromatography (0→3% DCM–MeOH) to give the desired product.
Yield: 48 mg (quant.); off-white solid; Rf
0.35 (hexane–EtOAc, 1:1 v/v).
IR (neat): 2969, 2931, 2192, 1708, 1578, 1513, 1414, 1280, 1134, 816 cm–1 .
1 H NMR (CDCl3 , 500 MHz): δ = 7.83 (s, 1 H), 7.27 (d, J = 8.9 Hz, 1 H), 6.62 (dd, J = 8.9, 2.5 Hz, 1 H), 6.49 (d, J = 2.5 Hz, 1 H), 6.27 (s, 1 H), 6.08 (t, J = 1.0 Hz, 1 H), 3.45 (q, J = 7.1 Hz, 4 H), 2.24 (d, J = 1.0 Hz, 3 H), 1.24 (t, J = 7.1 Hz, 6 H).
13 C NMR (CDCl3 , 126 MHz): δ = 162.4, 161.9, 157.1, 152.2, 147.9, 138.9, 129.8, 113.9, 109.8, 108.4,
105.5, 97.4, 94.8, 89.5, 45.2, 20.0, 12.6.
HRMS (ESI): m /z [M + H]+ calcd for C21 H20 NO4 : 350.1392; found: 350.1386.
7-(Diethylamino)coumarin-3-carbaldehyde (17)
7-(Diethylamino)coumarin-3-carbaldehyde (17)
Synthesized as described in the literature.[12 ]
POCl3 (4.7 mL, 50.4 mmol, 3.1 equiv) under N2 atmosphere in a round-bottom flask was cooled to 0 °C in an ice-water bath and anhydrous
DMF (4.8 mL, 61.8 mmol, 3.8 equiv) was added dropwise and the mixture was stirred
for 30 minutes. Then 7-(diethylamino)coumarin (13 ; 3.53 g, 16.2 mmol, 1 equiv) was dissolved in anhydrous DMF (20 mL) and slowly added.
The reaction mixture was stirred at 60 °C for 16 hours, then poured onto ice and the
pH was adjusted to 6 with 20% NaOH solution. The precipitate was filtered, washed
with cold EtOH, then recrystallized from abs. EtOH to give the product.
Yield: 2.16 g (54%); orange powder; Rf
0.52 (hexane–EtOAc, 1:1 v/v); Rf
0.18 (hexane–EtOAc, 3:1 v/v).
1 H NMR (CDCl3 , 500 MHz): δ = 10.14 (s, 1 H), 8.26 (s, 1 H), 7.41 (d, J = 9.0 Hz, 1 H), 6.64 (dd, J = 9.0, 2.5 Hz, 1 H), 6.49 (d, J = 2.5 Hz, 1 H), 3.48 (q, J = 7.2 Hz, 4 H), 1.26 (t, J = 7.2 Hz, 6 H).
LC-MS (ESI): m /z = 246 (C14 H16 NO3 ) [M + H]+ .
7-(Diethylamino)-3-vinyl-coumarin (18)
7-(Diethylamino)-3-vinyl-coumarin (18)
Methyltriphenylphosphonium bromide (464 mg, 1.30 mmol, 1.3 equiv) was dissolved in
anhydrous THF (4 mL) under N2 , cooled to 0 °C and n -BuLi (2.5 M in hexane, 0.65 mL, 1.30 mmol, 1.3 equiv) was added dropwise, then stirred
for 20 minutes. Compound 17 (245 mg, 1.00 mmol, 1 equiv) was dissolved in anhydrous THF (3 mL) and added dropwise
to the above reaction mixture at 0 °C, then stirred at 25 °C for 18 hours. The reaction
was quenched with sat. NH4 Cl (50 mL) and extracted with EtOAc (3 × 50 mL) and dried with brine and MgSO4 to give the product. It should be noted that 18 quickly decomposes on dry silica and in the fridge over time.
Yield: 115 mg (47%); off-white sticky solid; Rf
0.57 (hexane–EtOAc, 3:1 v/v).
1 H NMR (CDCl3 , 500 MHz): δ = 7.53 (s, 1 H), 7.22 (d, J = 8.8 Hz, 1 H), 6.64 (dd, J = 17.6, 11.3 Hz, 1 H), 6.54 (dd, J = 8.8, 2.5 Hz, 1 H), 6.44 (d, J = 2.5 Hz, 1 H), 5.99 (dd, J = 17.6, 1.3 Hz, 1 H), 5.26 (dd, J = 11.3, 1.3 Hz, 1 H), 3.37 (q, J = 7.1 Hz, 4 H), 1.17 (t, J = 7.1 Hz, 6 H).
13 C NMR (CDCl3 , 126 MHz): δ = 161.3, 155.8, 150.6, 138.6, 131.2, 128.9, 117.9, 115.7, 109.1, 108.8,
97.1, 44.8, 12.5.
HRMS (ESI): m /z [M + H]+ calcd for C15 H18 NO2 : 244.1338; found: 244.1336.
(E )-7-(Diethylamino)-3-(2-(6-methyl-2-oxo-2H -pyran-4-yl)vinyl)coumarin (5)
(E )-7-(Diethylamino)-3-(2-(6-methyl-2-oxo-2H -pyran-4-yl)vinyl)coumarin (5)
Compound 18 (75 mg, 0.30 mmol, 2 equiv) and 8 (39 mg, 0.15 mmol, 1 equiv) were dissolved in abs. DMF (2 mL) under N2 . N ,N -Dicyclohexylmethylamine (130 μL, 0.60 mmol, 4 equiv), QPhos (11 mg, 0.015 mmol, 0.1
equiv) and Pd2 (dba)3 (14 mg, 0.015 mmol, 0.1 equiv) were added and the reaction mixture was heated in
a microwave reactor at 100 °C for 1 hour. The solvent was evaporated in vacuo and the crude product was purified by flash chromatography (0→100%, EtOAc–hexane)
to give the desired product.
Yield: 17 mg (32%); orange solid; Rf
0.38 (hexane–EtOAc, 1:1 v/v).
IR (neat): 3114, 2923, 1705, 1651, 1626, 1528, 1444, 1363, 1250, 1141, 841 cm–1 .
1 H NMR (CDCl3 , 500 MHz): δ = 7.75 (s, 1 H), 7.31 (d, J = 8.9 Hz, 1 H), 7.17 (s, 2 H), 6.61 (dd, J = 8.9, 2.6 Hz, 1 H), 6.50 (d, J = 2.6 Hz, 1 H), 6.26 (s, 1 H), 6.08 (s, 1 H), 3.45 (q, J = 7.1 Hz, 4 H), 2.27 (s, 3 H), 1.24 (t, J = 7.1 Hz, 6 H).
13 C NMR (CDCl3 , 126 MHz): δ = 163.9, 161.3, 156.5, 152.3, 151.7, 142.8, 141.2, 136.4, 131.3, 129.8,
125.3, 115.7, 109.7, 109.1, 100.9, 97.3, 45.2, 20.2, 12.6.
HRMS (ESI): m /z [M + H]+ calcd for C21 H22 NO4 : 352.1549; found: 352.1549.
Methyl 3-Bromo-2-oxo-2H -pyran-5-carboxylate (19)
Methyl 3-Bromo-2-oxo-2H -pyran-5-carboxylate (19)
Pyridinium bromide perbromide (PBPB; 2.16 g, 6.76 mmol, 1.3 equiv) was dissolved in
glacial acetic acid (60 mL) and heated to 100 °C. Meanwhile methyl coumalate (800
mg, 5.20 mmol, 1 equiv) was dissolved in glacial acetic acid (40 mL) and added dropwise
to the hot solution. The reaction mixture was stirred at 100 °C for further 12 hours,
then cooled to r.t. and most of the acetic acid was removed on a rotary evaporator.
Water (200 mL) was added, the mixture was extracted with EtOAc (3 × 100 mL), and the
combined organic phases were washed with brine and dried over MgSO4 . The crude product was purified by flash chromatography (0→30%, EtOAc–hexane) to
give the product.
Yield: 484 mg (41%); off-white solid; Rf
0.68 (hexane–EtOAc, 1:1 v/v).
IR (neat): 3075, 2961, 1744, 1707, 1436, 1283, 1103, 955, 857 cm–1 .
1 H NMR (CDCl3 , 500 MHz): δ = 8.26 (d, J = 2.2 Hz, 1 H), 8.13 (d, J = 2.2 Hz, 1 H), 3.87 (s, 3 H).
13 C NMR (CDCl3 , 126 MHz): δ = 162.5, 156.8, 156.6, 142.7, 112.9, 111.7, 52.9.
LC-MS (ESI): m /z = 233 and 235 (C7 H6 BrO4 ) [M + H]+ .
HRMS (ESI): did not ionize.
Methyl (E )-3-(2-(7-(Diethylamino)-coumarin-3-yl)vinyl)-2-oxo-2H -pyran-5-carboxylate (6)
Methyl (E )-3-(2-(7-(Diethylamino)-coumarin-3-yl)vinyl)-2-oxo-2H -pyran-5-carboxylate (6)
Compound 18 (84 mg, 0.34 mmol, 2 equiv) and 19 (40 mg, 0.17 mmol, 1 equiv) were dissolved in abs. DMF (4 mL) under N2 . N ,N -Dicyclohexylmethylamine (146 μL, 0.68 mmol, 4 equiv), QPhos (12 mg, 0.017 mmol, 0.1
equiv) and Pd2 (dba)3 (16 mg, 0.017 mmol, 0.1 equiv) were added and the reaction mixture was heated in
a microwave reactor at 100 °C for 1 hour. The solvent was evaporated in vacuo and the crude product was purified by flash column chromatography (0→20% EtOAc–hexane)
to give the desired product.
Yield: 58 mg (85%); reddish brown solid; Rf
0.55 (hexane–EtOAc, 1:1 v/v).
IR (neat): 2975, 2931, 1714, 1603, 1589, 1509, 1412, 1134, 998, 733 cm–1 .
1 H NMR (CDCl3 , 500 MHz): δ = 8.20 (d, J = 2.2 Hz, 1 H), 7.87 (d, J = 2.2 Hz, 1 H), 7.71 (s, 1 H), 7.62 (d, J = 16.1 Hz, 1 H), 7.49 (d, J = 16.1 Hz, 1 H), 7.29 (d, J = 8.8 Hz, 1 H), 6.60 (dd, J = 8.8, 2.3 Hz, 1 H), 6.50 (d, J = 2.3 Hz, 1 H), 3.89 (s, 3 H), 3.43 (q, J = 7.1 Hz, 4 H), 1.22 (t, J = 7.1 Hz, 6 H).
13 C NMR (CDCl3 , 126 MHz): δ = 163.7, 161.1, 159.8, 156.0, 154.9, 151.1, 141.1, 135.7, 129.7, 129.4,
124.3, 123.3, 116.9, 113.0, 109.5, 97.2, 52.6, 45.1, 12.6.
HRMS (ESI): m /z [M + H]+ calcd for C22 H22 NO6 : 396.1447; found: 396.1441.
7-(Diethylamino)-3-((1-(hydroxymethyl)-7-methyl-1a,2,3,8,9,9a-hexahydro-1H -benzo[a ]cyclopropa[e ][8]annulen-5-yl)ethynyl)-coumarin (4-BCN)
7-(Diethylamino)-3-((1-(hydroxymethyl)-7-methyl-1a,2,3,8,9,9a-hexahydro-1H -benzo[a ]cyclopropa[e ][8]annulen-5-yl)ethynyl)-coumarin (4-BCN)
In a round-bottom flask 4 (20 mg, 0.057 mmol, 1 equiv) was dissolved in acetonitrile (5 mL) and BCN (17 mg,
0.114 mmol, 2 equiv) was added. The reaction mixture was stirred at 40 °C for 6 hours,
then 2.5 days at 25 °C. After concentration onto silica, the crude product was purified
by flash column chromatography (0→60%, EtOAc–hexane) to give the desired product.
Yield: 23 mg (88%); bright-yellow, sticky solid; Rf
0.21 (hexane–EtOAc, 1:1 v/v).
IR (neat): 3414, 2970, 2924, 1716, 1614, 1516, 1254, 1131, 1014, 727cm–1 .
1 H NMR (CDCl3 , 500 MHz): δ = 7.78 (s, 1 H), 7.29 (d, J = 8.9 Hz, 1 H), 7.26 (s, 1 H), 7.22 (s, 1 H), 6.61 (dd, J = 8.9, 2.1 Hz, 1 H), 6.53 (d, J = 2.1 Hz, 1 H), 3.76 (m, 2 H), 3.46 (q, J = 7.1 Hz, 4 H), 3.02 (m, 1 H), 2.96 (m, 1 H), 2.81 (m, 2 H), 2.34 (s, 3 H), 2.32–2.21
(m, 2 H), 1.71 (s, 1 H), 1.64 (m, 2 H), 1.60–1.44 (m, 2 H), 1.25 (t, J = 7.1 Hz, 6 H), 1.11 (m, 2 H).
13 C NMR (CDCl3 , 126 MHz): δ = 161.1, 156.3, 151.1, 145.0, 136.1, 131.7, 131.2, 128.9, 125.2, 119.9,
110.1, 109.3, 108.7, 105.4, 97.6, 94.1, 83.7, 60.0, 45.1, 34.0, 32.4, 26.6, 23.6,
20.0, 12.6.
HRMS (ESI): m /z [M + H]+ calcd for C30 H34 NO3 : 456.2539; found: 456.2531.
Activation and Conjugation of Transferrin for SDS-PAGE and In-Gel Fluorescence Study
Activation and Conjugation of Transferrin for SDS-PAGE and In-Gel Fluorescence Study
Human Transferrin (TF, 70 μM) and BCN-NHS (350 μM; from 20 mM stock solution in DMSO)
were mixed and incubated for 60 min at r.t. in 110 mM Na2 CO3 /NaHCO3 buffer (pH 9.0). The excess reagents were removed by using a SpinPrep column (Sigma,
St Louis, MO, USA) filled with Sephadex G-25 ‘Fine’ desalting gel (Pharmacia Fine
Chemicals, Sweden). This procedure resulted in a buffer exchange also to PBS (pH 7.4).
In the next step, 4 (500, 250 or 125 μM) was added and co-incubated for 24 hours. The excess reagents
were removed again by using a SpinPrep column filled with Sephadex G-25 ‘Fine’ desalting
gel. The labeled proteins were subjected to sodium dodecyl-sulfate polyacrylamide
gel electrophoresis (SDS-PAGE).
SDS Polyacrylamide Gel Electrophoresis and In-Gel Fluorescence Detection
SDS Polyacrylamide Gel Electrophoresis and In-Gel Fluorescence Detection
The samples were diluted with a sample buffer (250 mM Tris-HCl (pH 6.8), 10% SDS,
40% glycerol, 0.02% bromophenol blue, 400 mM DTT) in a 3:1 ratio. The size of the
polyacrylamide gels was 8.5 cm × 7.5 cm × 0.1 cm. They were the combination of 4%
concentration and 8% separation PAGE gels (acrylamide/bisacrylamide ratio was 29:1;
and concentration gel buffer was 125 mM Tris-HCl + 0.1% SDS (pH 6.8) and the separation
gel buffer was 375 mM Tris-HCl + 0.1% SDS (pH 8.8)). PageRuler Plus Prestained Protein
Ladder (Thermo Fisher Scientific) was applied as the molecular weight standard. Separations
were carried out in a Mini-Protean Tetra Cell (Bio-Rad, Hercules, CA, USA) using 25
mM Tris /192 mM glycine + 0.1% SDS (pH 8.3) as the running buffer and 150 V voltage
for 70 min at 25 °C.
The gels were documented by using a Biorad Bio-Rad ChemiDoc™ Imager. 4/4-BCN fluorescence was detected in the Pro-Q Emerald 488 channel using blue led epi illumination
(460–490 nm) and emission detection with a 532/28 nm band pass filter. Afterwards,
the gels were stained for proteins with Coomassie Brilliant-Blue and documented using
the colorimetric setting with white epi illumination.
