Mechanistic investigation and further optimization of the aqueous Glaser-Hay bioconjugation

Article abstract of DOI:10.1039/c9ob00327d

The Glaser-Hay bioconjugation has recently emerged as an efficient and attractive method to generate stable, useful bioconjugates with numerous applications, specifically in the field of therapeutics. Herein, we investigate the mechanism of the aqueous Glaser-Hay coupling to better understand optimization strategies. In doing so, it was identified that catalase is able to minimize protein oxidation and improve coupling efficiency, suggesting that hydrogen peroxide is produced during the aqueous Glaser-Hay bioconjugation. Further, several new ligands were investigated to minimize protein oxidation and maximize coupling efficiency. Finally, two novel strategies to streamline the Glaser-Hay bioconjugation and eliminate the need for secondary purification have been developed.

Full text of DOI:10.1039/c9ob00327d

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Biomolecular Chemistry
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Mechanistic investigation and further optimization
of the aqueous Glaser−Hay bioconjugation†
Cite this: DOI: 10.1039/c9ob00327d
Christopher R. Travis, Lauren E. Mazur, Emily M. Peairs, Gillian H. Gaunt and
Douglas D. Young
*
The Glaser−Hay bioconjugation has recently emerged as an efficient and attractive method to generate
stable, useful bioconjugates with numerous applications, specifically in the field of therapeutics. Herein,
we investigate the mechanism of the aqueous Glaser−Hay coupling to better understand optimization
strategies. In doing so, it was identified that catalase is able to minimize protein oxidation and improve
coupling efficiency, suggesting that hydrogen peroxide is produced during the aqueous Glaser−Hay bio-
conjugation. Further, several new ligands were investigated to minimize protein oxidation and maximize
coupling efficiency. Finally, two novel strategies to streamline the Glaser−Hay bioconjugation and elimin-
ate the need for secondary purification have been developed.
Received 9th February 2019,
Accepted 6th March 2019
DOI: 10.1039/c9ob00327d
rsc.li/obc
While the biological Glaser−Hay coupling is effective,
protein oxidation has been observed after around 6 hours of
Introduction
Protein bioconjugates, in which a protein is conjugated to reaction time.13 Our previous work reported two optimized
another molecule, represent a critical area of research with conditions for the Glaser−Hay bioconjugation: the use of the
widespread applications, most notably in the development of traditional TMEDA ligand in a pH 6.0 reaction to afford faster
strategies for site-specific drug delivery and improved cellular coupling, and the use of a carboxylated biphenyl ligand in a
imaging.1–5 Unnatural amino acid (UAA) technology introduces pH 8.0 reaction to minimize protein degradation.15 This bipyri-
novel chemical moieties into proteins and allows for the prepa- dyl ligand approach was also elucidated by others as a superior
ration of well-defined, homogenous protein bioconjugates, ligand system.21 Despite the utility of the Glaser−Hay biocon-
which display therapeutic advantages over the heterogenous jugation and this previous work, we hypothesized there was a
bioconjugate mixtures afforded through the reactivity of native potential for further optimization, which could be facilitated
amino acid residues.6–11
via an enhanced understanding of the reaction mechanism in
The Glaser−Hay coupling represents an attractive reaction aqueous solution.
to develop as a bioconjugation, given its many chemical advan-
tages, among them the formation of a highly stable carbon–
carbon bond in the form of a well-defined linear 1,3-diyne.
Results and discussion
Investigating the aqueous mechanism of the Glaser−Hay
Moreover, the reaction has been demonstrated to be tolerant
of a wide range of functional groups.12 Our previous work
reported the first successful biological Glaser−Hay coupling in
coupling
The mechanism of the Glaser−Hay coupling in organic sol-
vents has been studied in detail. In 1964, Bohlmann et al.
reported the formation of a dicopper(II)-diacetylide complex as
the rate-limiting step in the observed second-order kinetics.22
a
full-length protein under mild, aqueous reaction
conditions.13–15 The Glaser−Hay bioconjugation relies on the
incorporation of an UAA containing a terminal alkyne: p-pro-
pargyloxyphenylalanine (pPrF, 1) (Fig. 1). The diyne product of
the Glaser−Hay reaction has many useful downstream appli-
cations, including its potential to obtain bioconjugates with
diverse biological, photochemical, and optoelectronic
properties.16–20
Department of Chemistry, College of William & Mary, PO Box 8795, Williamsburg,
VA, USA 23185. E-mail: dyoung01@wm.edu
†Electronic supplementary information (ESI) available: Sample 1H NMR spectra,
kinetic data, UV-Vis and SDS-PAGE data. See DOI: 10.1039/c9ob00327d
Fig. 1 (A) Structure of alkynyl UAA, pPrF, which is incorporated into
protein for utilization in Glaser−Hay bioconjugations. (B) Generalized
Glaser−Hay bioconjugation between a protein and a soluble alkyne. The
blue star can represent a fluorophore, protein, surface, or resin.
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baseline to reflect the low concentrations of catalyst and
ligand when the reaction is conducted on a biological system.
Given these starting conditions, our results are translatable to
the previously developed aqueous Glaser−Hay bioconjugation
despite the higher concentrations of alkyne and lack of protein
partner.
In order to probe the reaction kinetics of the aqueous
Glaser−Hay coupling, the concentrations of reaction com-
ponents were systematically adjusted. Each trial was then com-
pared as a function of time for the rate of formation of a
methylene product peak observed at 50 ppm in the 13C NMR
relative to the corresponding starting material peak at approxi-
mately 49.3 ppm for each set of reaction conditions (Fig. 2A).
The 50 ppm peak corresponds to the methylene peak in the
diyne-containing product. The peak seen at approximately
49.3 ppm corresponds to the methylene peak in the alkyne
starting material.
Scheme 1 Potential Glaser−Hay mechanisms. (A) Previously proposed
Glaser−Hay mechanisms in organic solution.23,24 (B) Potential
Glaser−Hay mechanism in aqueous solution utilizing a single Cu atom.
Later work suggested that the mechanism progresses through
a dicopper(III) intermediate (Scheme 1A; bottom pathway).23
Under all reaction conditions tested, we observed the
instantaneous disappearance of the terminal alkyne peak in the
Most recently, Vilhelmsen et al. reported the Glaser−Hay 1H NMR (see ESI†), suggesting the rapid conversion to a copper
reaction as involving a Cu2+ intermediate, leading to the gene- acetylide intermediate during the aqueous Glaser−Hay coupl-
ration of hydroxyl radicals (Scheme 1A; top pathway).24 This ing. This was corroborated by the presence of a triplet in the
study utilized UV/Vis spectroscopy as well as 13C NMR to study 13C spectra due to the coupling of the carbon to the copper.
reaction progression under various conditions. They reported
that increased copper(I) and/or increased TMEDA resulted in
an increased reaction rate. However, they also notably reported
that the reaction rate slowed after some time, due to the build-
up of water in the reaction through its absorption from the air.
Given the detrimental effects of water on the rate of the
Glaser−Hay coupling in an organic solvent, we sought to inves-
tigate the kinetics of the aqueous Glaser−Hay coupling, as the
reaction pathway in water may differ from the pathways pre-
viously observed for the reaction in organic solvents.
For this study, we employed UV/Vis spectroscopy as well as
1
both 13C and H NMR to monitor the Glaser−Hay coupling of
the dimerization of propargyl alcohol in an aqueous solution.
For NMR experiments, deuterated water (D2O) was employed
as the solvent to allow for an NMR lock and for clarity of NMR
spectra. Notably, reaction progress was tracked via relative inte-
gration of 13C product and reactant peaks. Although such a
method for quantitative kinetics studies is abnormal, it has
been proven effective in quantitative analyses, specifically in
kinetic studies of in vivo and in vitro processes.25–30 Further,
Vilhelmsen et al. previously utilized integration of 13C NMR to
successfully track the kinetics Glaser−Hay coupling in organic
solvent.24 While poor signal to noise ratio and the effects of
the nuclear Overhauser effect are cited as pitfalls of integrating
13C NMR spectra, the strength of the 400 MHz NMR instru-
ment coupled with the deuterated solvent used appears to be
sufficient to overcome these limitations.24
The Glaser−Hay dimerization of propargyl alcohol was pre-
pared and air was bubbled through the solution for
10 minutes (see ESI†). The vial was then sealed and allowed to
stir at 80 °C. At specific timepoints, the reaction was moni-
tored by NMR. The relatively low equivalencies of copper(I)
Fig. 2 Effect of varying copper(I) iodide on the rate of the aqueous
Glaser−Hay reaction. (A) 13C NMR timecourse studies monitoring the
methylene carbon conversion between starting material and product.
(B) Normalized 13C integration data with increasing copper concen-
trations (1.2, 2.4, 4.8, and 24 mol% respectively) demonstrating decreas-
iodide (2.4 mol%) and TMEDA (4.0 mol%) were chosen as the ing rates of reaction.
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Varying the amount of copper(I) iodide in the reaction previously reported that the addition of the radical scavengers
yielded surprising results. We observed that increases in the ascorbic acid, cysteine, and oleic acid did not afford better
level of copper(I) in the reaction resulted in decreased reaction coupling additions and that ascorbic acid actually increased
rates and less conversion of starting material to product, even protein oxidation and inhibited coupling.15
at ten times (24 mol%) the original copper(I) iodide amount
Taking these results along with aforementioned obser-
(Fig. 2B). This finding is significant, as it suggests that the vations of the instantaneous formation of the copper acetylide
aqueous Glaser−Hay coupling may not progress through a and the potential lack of formation of a dicopper intermediate,
dicopper acetylide intermediate, as had been reported as the we hypothesized that the oxidative damage to protein in the
mechanism of the reaction in organic solution. This is logical aqueous Glaser−Hay bioconjugation is more likely due to
given the high coordination of water as the solvent; its ability damage from hydrogen peroxide (H2O2) generation rather than
to act as a good ligand to bind copper may make the dicopper free radicals.
complex less likely to form. Instead, these results suggest that
This hypothesis is also supported by our previous report
copper coordinates to just a single alkyne at a time to generate that ascorbic acid results in increased protein oxidation and
a copper acetylide, which has been reported to be fairly inhibits coupling. Previous studies have reported that ascorbic
stable.31 At this stage, the copper acetylide could coordinate a acid can be cytotoxic due to its production of the reactive
free alkyne or a second copper acetylide; however, water sol- oxygen species (ROS) hydrogen peroxide, which damages pro-
vation of the complex may inhibit diacetylide formation teins and other cellular components.33,34 However, it has also
(Scheme 1B). Conversely, if a mono-acetylide complex reacting been demonstrated that the presence of the enzyme catalase in
with a free alkyne is employed, increased copper in the cells can mitigate the cytotoxicity of ascorbic acid through the
aqueous reaction could result in most of the alkyne being con- breakdown of hydrogen peroxide. Given these results, we
verted to its copper acetylide form, leaving little free terminal sought to investigate whether catalase could decrease protein
alkyne starting material to react to form the dimer product. oxidation and improve coupling efficiency in the Glaser−Hay
Further, the addition of more copper may lead to the for- bioconjugation.
mation of larger copper acetylide clusters, possibly limiting
the reactivity of the catalyst in an aqueous solution.32
In order to probe the effects of catalase, we performed a
250 mL expression of GFP containing pPrF at residue 151.
Varying the amount of TMEDA in the reaction demon- Following purification, the protein was buffer exchanged into
strated kinetics similar to those found in organic solution. PBS and concentrated to a standard concentration of 1.0
Increases in the amount of TMEDA led to increased reaction mg mL−1 to remove variability in initial protein concentration.
rates, suggesting increases in nitrogenous ligand concen- With the GFP-151-pPrF in hand, a series of reactions to test the
tration may help facilitate quicker reaction and more efficient effectiveness of catalase in a coupling reaction between the
coupling (see ESI†). This could be due to the fact that with mutant protein and a Fluor-488 alkyne dye were prepared.
more TMEDA in the system, more of the copper is coordinated
The addition of catalase to the Glaser−Hay bioconjugation
to TMEDA and forming a reactive copper complex, rather than resulted in significantly less protein degradation. Better coup-
being solvated by just water.
ling efficiency was also observed when catalase was added to
UV/Vis spectroscopy indicated
a
shift in absorbance the reaction (Fig. 3). These results suggest that the previously
immediately after the addition of propargyl alcohol to the cata- observed protein oxidation in the Glaser−Hay bioconjugation
lyst mixture in aqueous solution. When only copper(I) iodide is likely due to the production of hydrogen peroxide, which
and TMEDA were present in an aqueous solution, the solution can be broken down by catalase.
was blue and had a maximal absorbance at a wavelength just
above 600 nm (see ESI†). When the propargyl alcohol was
added to the reaction, the color immediately converted to a
green-yellow, as the absorbance shifted to a wavelength of just
above 400 nm. This shift confirms the rapid formation of a
copper acetylide complex, which have been reported to be
yellow in color.31 The shift in color also indicates the changing
oxidation state of copper in the reaction, as the blue color of
the copper complex is lost upon coordination to propargyl
alcohol. Taking specific timepoints after the addition of pro-
pargyl alcohol showed that the maximum wavelength of
absorption did not change over the course of the reaction.
Optimizing the biological Glaser−Hay coupling
Fig. 3 Standard Glaser−Hay bioconjugation of GFP-151-pPrF with
Fluor-488 alkyne using the CuI/TMEDA system. The reaction was con-
ducted in the presence (Lane 1) and absence (Lane 2) of catalase. Both
reactions afforded fluorophore coupling to the protein (top gel);
however, less protein degradation was observed when catalase is
With our newly improved understanding of the potential
mechanism of the aqueous Glaser−Hay coupling, we sought to
further optimize the aqueous Glaser−Hay bioconjugation to
reduce protein oxidation and improve coupling efficiency. We employed (Coomassie stained gel, bottom).
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It has also been reported that hydroxyl radicals are gener-
ated through the interaction of Cu2+ ions and hydrogen per-
oxide in phosphate-buffered solution.32 Additionally, it has
been suggested that the level of radical formation is at least
partially dependent on the ligand chelating the Cu2+ ion in
solution. Given this as well as our previous report of improved
coupling with a carboxylated biphenyl ligand, we sought to
investigate whether other ligands could be employed to mini-
mize protein degradation in the Glaser−Hay bioconjugation.15
For the ligand, the traditionally used 2 as well as the
recently reported 3 were investigated along with biquinoline
(4), a dimethylated bipyridyl (5) and terpyridine (6) to examine
the electronic contributions of the ligand to the progress of
the reaction (Fig. 4). We hypothesized that altering the elec-
tronics of the coordinating ligand could impact the ability of
copper to facilitate the aqueous Glaser−Hay bioconjugation.
The electron donating/withdrawing capability of each ligand
may alter the ability of copper to coordinate alkyne and partici-
pate in the oxidative addition. Further, we hypothesized the
various substituents on these bipyridines could afford extra
rigidity, aiding in chelation and activation of the copper center
to improve the reaction. Finally, the commercial availability
and affordable cost of these ligands proffer practicality, improv-
ing the potential utilization of these ligands that do not require
additional synthetic steps to access. Due to varied solubilities, a
stock solution of ligand 2 was prepared as a 500 mM solution in
H2O. For ligand 3, a 500 mM solution was prepared in 1 M
NaOH. For ligands 4, 5, and 6, 500 mM stock solutions were pre-
pared in DMSO. To test these conditions, the reactions were again
conducted between the same mutant protein and alkyne-contain-
ing dye. When conducting Glaser−Hay reactions with 2, GFP-151-
pPrF at pH = 6.0 was employed for 4 hours, based on previously
reported optimized conditions.15 Similarly, when conducting reac-
tions with 3, the protein at pH = 8.0 was employed for 8 hours, in
accordance with previously optimized conditions. For reactions
with 4, 5, and 6, the same reaction conditions as 3 were employed
due to similarities in ligand structure.
Fig. 5 Investigation of the effects of catalase and various nitrogenous
ligands on the biological Glaser−Hay reaction. Protein degradation was
assessed in the presence (blue) and absence (red) of catalase via
SDS-PAGE normalization of protein concentrations for each reaction.
Moreover, coupling efficiency (grey) of each reaction was determined by
ratios of fluorescence resulting from conjugation of the fluorescent dye
to the total amount of protein in the lane. These ratios were normalized
to the previously reported conditions (ligand 2).
Gratifyingly, the addition of catalase improved coupling and
reduced protein oxidation for each of the ligands examined
(Fig. 5). However, differences in protein oxidation were
observed between the different ligands. Reactions with ligands
2, 3, and 4 demonstrated little protein oxidation in general,
especially in the presence of catalase. However, the reaction
with ligand 5 demonstrated significantly more protein oxi-
dation, especially in the absence of catalase. These results
indicate that ligand selection has a significant impact on
protein degradation during the aqueous Glaser−Hay bioconju-
gation. Further, we identified 2,2′-biquinoline (4) as a promis-
ing ligand choice.
Overall, we demonstrate that the addition of catalase as well
as appropriate ligand selection can greatly reduce protein
degradation and improve coupling in the Glaser−Hay
bioconjugation.
A total of ten reactions were prepared to test the effective-
ness of each ligand with and without the addition of catalase.
Streamlining the biological Glaser−Hay coupling
Bioconjugation reactions such as the Glaser−Hay on proteins
containing UAAs can be time-consuming and involve multiple
purification steps. Therefore, we sought to streamline the
process by attempting the Glaser−Hay bioconjugation reaction
directly on the lysate from purification as well as during the
protein purification process (Fig. 6). Each of these reaction
timepoints was analyzed with ligands 2, 3, and
4 in
Glaser−Hay bioconjugations with a Fluor-488 alkyne dye.
Preparation of ligand solutions and reaction duration
remained the same as the aforementioned conditions.
To examine the Glaser−Hay bioconjugation during the
protein purification process, protein purification was per-
formed using the terminal 6XHis tag and a Ni-NTA resin.
Purification was performed using a Qiagen Ni-NTA Quik Spin
Kit according to manufacturer’s protocol up until the elution
step. At this point, the GFP-151-pPrF remained bound to the
Fig. 4 Nitrogenous ligands employed in the Glaser−Hay optimization.
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Fig. 7 SDS-PAGE results from investigations of the Glaser−Hay biocon-
jugation. The best coupling was observed from conducting the reaction
on the completely purified protein (Lane 1). Coupling was observed with
both the Glaser−Hay reaction being performed on the Ni-NTA resin
(Lane 2), and directly on the lysate (Lane 3). No coupling is observed in
the absence of the CuI/TMEDA system as observed by the presence of
protein but lack of fluorescence in Lane 4.
Quik Spin Kit. pEvol plasmids were obtained from the labora-
tory of Dr Peter Schultz.
Dimerization of propargyl alcohol. The following was used
as a standard to monitor the aqueous Glaser−Hay coupling.
Amounts of Cu(I), TMEDA, and propargyl alcohol were varied
to analyze the effect of each on the reaction kinetics. To a
flame-dried vial, 8 mg of copper(I) iodide (2.4 mol%) was
added to 2 mL D2O, along with 10 µL of TMEDA (4.0 mol%).
Next, 100 µL of propargyl alcohol (1.7 mmol, 1 eq.) was added,
and air was bubbled through the reaction for 10 minutes. The
reaction vial was sealed and stirred at 80 °C for 12 hours.
Timepoints were taken at 1 hour, 3 hours, 6 hours, 8 hours,
10 hours, and 12 hours. At each timepoint, a sample was
removed from the vial and directly added to an NMR tube for
1H and 13C NMR analysis. Following data acquisition, these
samples were returned to their respective reactions.
Synthesis of p-dipropargylaminophenylalanine (pPrF). Boc-
Tyrosine-OMe (114 mg, 2 eq., 0.385 mmol) was added to a
flame-dried vial. Cesium carbonate (254 mg, 3 eq., 0.58 mmol)
was then added, followed by dry DMF (3 mL). This mixture was
stirred at 100 °C for 20 min. 5-Bromo-1-pentyne (20 µL, 1 eq.,
0.19 mmol) was then added to the mixture, as well as a cata-
lytic potassium iodide. The reaction was stirred overnight at
100 °C, then cooled to room temperature and washed with
brine (10 mL) and diethyl ether (10 mL). The ether layer was
then washed with brine (3 × 10 mL). The brine layer was then
back-extracted with ether (10 mL). The organic layers were
combined, dried with magnesium sulfate, filtered, and excess
solvent was removed in vacuo. Column chromatography (silica
gel, 5 : 1 hexanes/ethyl acetate) was performed to yield the pro-
tected product. The product was then dissolved in 1,4-dioxane
(2 mL). Then, 1 M lithium hydroxide (2 mL) was added and
the reaction was stirred at room temperature for 2 hours. 1,4-
Dioxane was then removed in vacuo and the resulting water
Fig. 6 Streamlining of the Glaser−Hay bioconjugation. Following
protein expression with the pPrF UAA, the Glaser−Hay reaction can be
conducted at various stages of the purification process. This reaction
can occur directly from the lysate (first column), from the Ni-NTA
immobilized protein (second column), or following elution from the Ni-
NTA resin (third column).
Ni-NTA resin, and Glaser−Hay reactants were added in the
hope of performing the Glaser−Hay bioconjugation with the
protein bound to the purification matrix. To test the
Glaser−Hay bioconjugation on cell lysate, protein expressions
were pelleted, resuspended and lysed using commercially avail-
able BugBuster for 20 minutes. After centrifugation, the lysate
was used directly in the Glaser−Hay bioconjugation.
Both reaction preparation strategies were successful, as
indicated by the presence of fluorescence bands on SDS-PAGE
(Fig. 7). Binding the mutant protein to the nickel purification
resin prior to conducting the Glaser−Hay bioconjugation
afforded better coupling than conducting the reaction on the
lysate. Nevertheless, each of these approaches allows for
increased applicability of the Glaser−Hay bioconjugation by
streamlining the reaction process and eliminating additional
purification steps.
Experimental
Materials and methods
General. Reactions were conducted under ambient atmo- solution was acidified through the dropwise addition of 6 M
sphere with non-distilled solvents. NMR data was acquired on HCl. The reaction was then extracted into ethyl acetate and the
a Varian Gemini 400 MHz. All GFP proteins were purified organic layer dried with magnesium sulfate and filtered.
according to manufacturer’s protocols using a Qiagen Ni-NTA Excess solvent was removed in vacuo to yield a colorless oil.
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This oil was dissolved in dichloromethane (DCM, 1.5 mL). stained for 3 hours using Coomassie Brilliant Blue, then
Trifluoroacetic acid (TFA, 0.5 mL) was added and the reaction destained overnight using a methanol solution (60% deionized
was stirred at room temperature for 1 hour. Excess solvent was H2O, 30% MeOH, 10% acetic acid). The gel was then analyzed
removed in vacuo to yield pPrF as a white solid (22 mg, again on the gel imager to indicate protein presence and rela-
1
0.061 mmol, 31.6% yield). H NMR (400 MHz, CDCl3): δ 7.02 tive degradation.
(d, J = 12 Hz, 2 H), 6.82 (d, J = 12 Hz, 2 H), 4.95 (d, J = 8 Hz,
Protocol for Glaser−Hay bioconjugation during protein puri-
1 H), 4.53 (d, J = 8 Hz, 1 H), 4.03 (t, J = 4 Hz, 2 H), 3.71 (s, 3 H), fication. A 250 mL expression of GFP/pPrF was spun down and
3.02 (m, J = 8 Hz, 1 H), 2.39 (t, J = 4 Hz, 2 H), 1.97 (m, J = 8 Hz, cells were lysed using commercially available BugBuster.
2 H), 1.55 (s, 1 H), 1.41 (s, 9 H). 13C NMR (400 MHz, CDCl3): 250 µL of lysate was added to 100 µL of Ni-NTA resin and
δ 172.4, 157.9, 130.3, 127.9, 114.5, 83.5, 79.9, 68.8, 66.0, 54.5, allowed to bind before being washed with an imidazole-con-
52.2, 37.4, 28.3, 28.2, 21.1, 15.1.
taining wash buffer according to manufacturer’s protocol.
Expression of pPrF-containing GFP-151. Escherichia coli Next, the resin was washed with PBS (5 × 200 µL) and then
BL21(DE3) cells were co-transformed with a pET-GFP-TAG-151 dried. 75 µL of PBS was then added to wet the resin. Next,
plasmid (2.0 µL) and a pEvol-pCNF plasmid (2.0 µL) using an 10 µL of a premixed 1 : 1 solution of CuI (500 mM in H2O) and
Eppendorf electroporator. Cells were then plated on LB-agar nitrogenous ligand (500 mM) was added. Finally, 40 µL of
plates supplemented with ampicillin (50 mg mL−1) and chlor- Fluor-488 Alkyne (1 mM in DMSO) was added. The reaction
amphenicol (34 mg mL−1) and grown at 37 °C. After 16 hours, was incubated at room temperature (22 °C) for the appropriate
a single colony was used to inoculate LB media (10 mL) sup- reaction duration. The nickel resin was then washed with PBS
plemented with ampicillin and chloramphenicol. The culture (8 × 200 µL) and imidazole-containing wash buffer (3 ×
was grown to confluence at 37 °C over 16 hours. This culture 200 µL); protein was eluted following manufacturer’s protocol.
was then used to begin an expression culture in LB media The reaction was analyzed by SDS-PAGE and imaged immedi-
(250 mL) at OD600 = 0.1, then incubated at 37 °C until it ately to analyze fluorescence. Fluorescence intensity indicated
reached an OD600 of between 0.7 and 0.8. At this point, mutant the effective coupling reaction, as previously explained. The gel
protein expression was induced through the addition of 1 M was then stained for 3 hours using Coomassie Brilliant Blue,
ITPG (250 µL) and 20% arabinose (250 µL), as well as 100 mM then destained overnight using a methanol solution (60% de-
pPrF (2.5 mL). Induced cells were grown for an additional ionized H2O, 30% MeOH, 10% acetic acid). The gel was then
16 hours at 30 °C, then harvested via centrifugation (10 min, analyzed again on the gel imager to indicate protein presence
5000 rpm). The media was decanted, and the cell pellet was and relative degradation.
stored in a −80 °C freezer for 20 minutes. Mutant GFP was
Protocol for Glaser−Hay bioconjugation on cell lysate.
then purified using commercially available Ni-NTA spin Expression of GFP/pPrF was spun down and cells were lysed
columns according to the manufacturer’s protocol. Protein using commercially available BugBuster. The lysed cells were
yield and purity was assessed via SDS-PAGE and spectrophoto- centrifuged again and the lysate was decanted. 20 µL of a pre-
metrically via a Nanodrop spectrophotometer. Protein was mixed 1 : 1 solution of CuI (500 mM in H2O) and nitrogenous
then transferred into phosphate buffered saline solution (PBS) ligand (500 mM) was added to 250 µL of lysate. Next, 40 µL of
using 10k MWCO spin columns prior to use in bioconjugation Fluor-488 Alkyne (1 mM in DMSO) was added. The reaction
reactions.
was incubated at room temperature (22 °C) for the appropriate
Protocol for Glaser−Hay bioconjugation. To a sterile 1.5 mL reaction duration. The lysate was then bound to Ni-NTA resin
Eppendorf tube, the following were added: 5 µL of a vigorously and protein was purified according to manufacturer’s protocol.
shaken solution of CuI (500 mM in H2O) and 5 µL of nitrogen- The reaction was analyzed by SDS-PAGE and imaged immedi-
ous ligand (500 mM). The two solutions were thoroughly ately to analyze fluorescence. Fluorescence intensity indicated
mixed by pipetting and allowed to incubate for ten minutes. the effective coupling reaction, as previously explained. The gel
30 µL of GFP containing a terminal alkyne UAA (GFP/pPrF; was stained for 3 hours using Coomassie Brilliant Blue, then
pH = 8.0, 1.04 0.03 mg mL−1) and 20 µL of Fluor-488 Alkyne destained overnight using a methanol solution (60% deionized
(1 mM in DMSO) were then added to the tube. Finally, 5 µL of H2O, 30% MeOH, 10% acetic acid). The gel was then analyzed
catalase (10 mg mL−1 in H2O) was added to the tube. For again on the gel imager to indicate protein presence and rela-
control reactions, 5 µL of PBS at the appropriate pH was added tive degradation.
in place of catalase. The reaction was incubated at room temp-
erature (22 °C) for the appropriate reaction duration. Excess
reactants were then removed by buffer exchange using 10k
MWCO concentrator columns. The reaction was washed with
Conclusions
PBS (8 × 200 µL) to a final volume of 50 µL. The reaction was Overall, a better understanding of the aqueous Glaser−Hay
analyzed by SDS-PAGE and imaged immediately to analyze reaction has been obtained, which facilitates the further
fluorescence. Fluorescence intensity indicated the effective optimization of the reaction to broaden its utility. Our results
coupling reaction, as the GFP had been denatured and thus suggest that the aqueous Glaser−Hay reaction may not pro-
was no longer fluorescent, while the coupling to the fluoro- gress through a dicopper intermediate, which would make it
phore re-establishes a fluorescent signal. The gel was then mechanistically different from the organic solvent based
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Glaser−Hay reaction. These mechanistic insights also led to a
better understanding of hydrogen peroxide as the source of
9 L. Wang and P. G. Schultz, Angew. Chem., Int. Ed., 2004, 44,
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Conflicts of interest
There are no conflicts to declare.
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Arnold and Mabel Beckman Foundation through the Beckman
Scholars Program. DDY would like to acknowledge funding
from the National Institute of General Medical Sciences of the
NIH (R15GM113203) and the Camille and Henry Dreyfus
Foundation (TH-17-020). We would also like to thank Prof.
Peter Schultz for providing the pEvol plasmids employed in
this study. We would also like to thank Prof. Robert Hinkle
and Prof. William McNamara for assistance with kinetic
experiments.
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