Site-specific incorporation of quadricyclane into a protein and photocleavage of the quadricyclane ligation adduct

Article abstract of DOI:10.1016/j.bmc.2018.04.009

The quadricyclane (QC) ligation is a bioorthogonal reaction between a quadricyclane moiety and a nickel bis(dithiolene) derivative. Here we show that a QC amino acid can be incorporated into a protein site-specifically using the pyrrolysine-based genetic

Full text of DOI:10.1016/j.bmc.2018.04.009

Accepted Manuscript
Site-Specific Incorporation of Quadricyclane into a Protein and Photocleavage
of the Quadricyclane Ligation Adduct
Frederick M. Tomlin, Chelsea G. Gordon, Yisu Han, Taia S. Wu, Ellen M.
Sletten, Carolyn R. Bertozzi
PII:
S0968-0896(17)32453-7
https://doi.org/10.1016/j.bmc.2018.04.009
BMC 14292
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
22 December 2017
30 March 2018
3 April 2018
Please cite this article as: Tomlin, F.M., Gordon, C.G., Han, Y., Wu, T.S., Sletten, E.M., Bertozzi, C.R., Site-Specific
Incorporation of Quadricyclane into a Protein and Photocleavage of the Quadricyclane Ligation Adduct, Bioorganic
& Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.04.009
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Site-Specific Incorporation of Quadricyclane into a Protein and Photocleavage of the
Quadricyclane Ligation Adduct
Frederick M. Tomlin,a,b Chelsea G. Gordon,b Yisu Han,b Taia S. Wu,a Ellen M. Sletten,b Carolyn
R. Bertozzi*a,b,c
a Department of Chemistry, Stanford University, Stanford, California 94305, United States
b Department of Chemistry, University of California, Berkeley, Berkeley, California 94720,
United States
c Howard Hughes Medical Institute, Stanford University, Stanford, California 94305, United
States
*Email: bertozzi@stanford.edu
Abstract
The quadricyclane (QC) ligation is a bioorthogonal reaction between a quadricyclane
moiety and a nickel bis(dithiolene) derivative. Here we show that a QC amino acid can be
incorporated into a protein site-specifically using the pyrrolysine-based genetic code expansion
platform, and subsequently used for ligation chemistry. Additionally, we exploited the
photolability of the QC ligation product to render the adduct cleavable with a handheld UV lamp.
We further developed a protein purification method that involves QC ligation of biotin to a
protein of interest, capture on streptavidin resin, and finally release using only UV light. The QC
ligation thus brings novel chemical manipulations to the realm of bioorthogonal chemistry.
Keywords: quadricyclane; bioorthogonal; pyrrolysine; unnatural amino acid; photocleavage;
photolysis; protein purification
1. Introduction
The current scope of the bioorthogonal toolbox is much smaller than the repertoire of
conventional synthetic chemical reactions. This is in part because the field is young, established
less than two decades ago, but also because the constraints on a bioorthogonal reaction are
formidable compared to those surrounding traditional organic transformations.1 The solvent is

limited to water at near physiological pH, temperature is restricted to 37 ºC or lower, and cross-
reactivity with biological functionalities and other bioorthogonal reagents is not tolerated. In
addition, the ligation has to be rapid enough to proceed on the timescale of the biological process
of interest and one of the reagents must be capable of incorporation into a target biomolecule via
the organism’s own biosynthetic machinery. Together, these requirements make the design of
new bioorthogonal reactions a challenging pursuit.
Several bioorthogonal reactions involving phosphines,2–4 azides and other 1,3-dipoles,
alkynes, alkenes and tetrazines are now in widespread use to generate covalent adducts under
physiological conditions.5–7 However, there is a strong need for new bioorthogonal chemistries
due to the demands of multiplexed imaging experiments and the lack of chemical manipulations
accessible via the current bioorthogonal repertoire.6,8 Multiplexed imaging requires mutual
orthogonality across bioorthogonal reactions, meaning the chemical functionalities unique to
each ligation do not react with each other. Often, this requirement is a challenge due to inherent
cross-reactivity between reagents.1,9–11
Lack of access to reactions other than ligations is the second driver of bioorthogonal
reaction development. The majority of bioorthogonal reactions in the current toolbox produce a
stable, covalent bond between two entities. Notable exceptions include uncaging reactions, bond
cleavage reactions, and a recent report from Addy et al. in which the adduct of the bioorthogonal
coupling of 5-hydroxytryptophan and an aromatic diazonium ion could be cleaved with the
addition of the small molecule dithionite.12–14 The conventional synthetic toolbox contains
additional diverse chemistries that are reversible, stereoselective, or triggered by environmental
cues (i.e. photochemistries, pH sensitive reactions, etc.). The field of bioorthogonal chemistry
has just begun to branch into these areas of new chemical functionality, and innovations in the

design of unique ligations will drive this growth.
Rising to meet these needs, we proposed a new bioorthogonal reaction — the
quadricyclane (QC) ligation.15 The reaction is unique because it exploits a chemical space
previously unexplored through the lens of bioorthogonality. It is a formal [2 + 2 + 2]
cycloaddition proceeding between the strained hydrocarbon quadricyclane (QC, 1) and nickel
bis(dithiolene) (2) to form adduct 3 (Scheme 1). We found that this reaction meets many of the
requirements of bioorthogonality including rapid rate, selectivity, and compatibility with
physiological conditions. Most importantly, the QC ligation was orthogonal to important protein
bioconjugation chemistries such as oxime formation with aldehydes and aminooxy groups and
copper-free click reaction.15
Scheme 1. The quadricyclane (QC) ligation. The QC ligation is a formal [2 + 2 + 2] reaction between QC (1)
and nickel bis(dithiolene) (2) to form adduct 3. This work describes the photocleavage of 3 with UV light to produce
norbornadiene (4) and the original nickel bis(dithiolene) 2 applied in a biological context.
Here, we show that QC can be site-specifically incorporated into a protein of interest
using the pyrrolysine-based genetic code expansion platform16,17 and employed for protein
chemical modification. We also demonstrate that protein-bound QC adducts can be cleaved by
mild photolysis to form norbornadiene 4 and the original nickel complex 2 (Scheme 1).
Together, the QC ligation and photocleavage chemistries enabled a new avenue for protein
purification.

2. Results
2.1 Site-specific incorporation of a QC amino acid into a protein
We first addressed the challenge of incorporating QC into a protein of interest, an
important capability for bioorthogonal applications, and for this we chose the pyrrolysine method
for unnatural amino acid incorporation. The PylRS/tRNACUA pair has proven a useful tool for
non-natural amino acid incorporation in E. coli, mammalian cells, and even model organisms.18–
24
We envisioned using PylRS/tRNACUA to site-specifically install a QC-containing amino acid
in response to an in-frame amber stop codon in a protein of interest. Accordingly, we designed
QC amino acid 6 (Figure 1A) based on its synthetic accessibility from known intermediate p-
nitrophenyl QC carbonate (5, Figure 1A)15 and its structural similarity to norbornene amino acids
that have been successfully incorporated into proteins using the PylRS/tRNACUA system.25–27
The previously reported N--Boc-L-lysine (7, Figure 1B), a known substrate of wildtype PylRS,
was used as a positive control in this study.25

Figure 1. Site-specific incorporation of QC amino acid into a protein. A) QC amino acid 6 was synthesized in a
2-step procedure from p-nitrophenyl carbonate 5: (1) N-Fmoc-L-lysine, DMF, rt, 16 h. (2) piperidine, DMF, rt, 2 h.
B) N--Boc-L-lysine (7), a known substrate of WT PylRS. C) Expression of full-length sfGFP-FLAG in the
presence of 6 or 7. E. coli cells were transformed with plasmids pBKPylRS and psfGFP150TAGFLAGPylT (+RS+T)
or with psfGFP150TAGFLAGPylT only (–RS+T) (see Supplementary data for plasmid maps and sequences) and
cultures were supplemented with 1 mM 6, Boc-lysine (7), or vehicle (–). Protein was expressed for 3.5 h, after
which lysates were analyzed by western blot probing with α-GFP and α-FLAG antibodies. D) High-resolution
electrospray ionization (ESI) mass spectrometry (MS) analysis of purified QC-sfGFP-FLAG. Red represents the
mass of the full-length protein and the green represents the mass of the full-length protein minus methionine. Black
numbers represent neutral loss of H2O, which may occur during fragmentation using ESI-MS.28 QC-sfGFP-FLAG
observed mass: 28147 Daltons, calculated mass: 28147.61 Daltons.
We next sought to determine if 6 is recognized by PylRS and incorporated into a
recombinant protein. DH10β E. coli were transformed with pBKPylRS which encodes the gene

for PylRS from Monosarchina barkeri, and psfGFP150TAGFLAGPylT which encodes tRNACUA
and a FLAG-tagged superfolder GFP (sfGFP) with an in-frame amber stop codon at position
150. This site is known to be amenable to unnatural amino acid incorporation.29 Protein
expression was induced in the presence of approximately 1 mM 6, 1 mM 7, or vehicle. For these
experiments, vehicle was 0.1 M NaOH in ddH2O, the solvent in which the non-natural amino
acid was dissolved for addition to E. coli culture. The solubility of 6 was not complete in 0.1 M
NaOH, however, after a large screen of solvents it was found to be optimal. Following
expression, cells were pelleted and subjected to sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE). Proteins were then transferred to nitrocellulose and analyzed via
western blot for the presence of full-length QC- or Boc-sfGFP-FLAG by probing with an -GFP
antibody.
We expected to see production of full-length sfGFP-FLAG only in samples treated with 6
or 7 and transformed with psfGFP150TAGFLAGPylT and pBKPylRS. For bacteria transformed
with both plasmids (+RS+T), a dark band was visible at ~28 kDa in the +6 and +7 lanes whereas
only a faint band was apparent without the amino acid supplements (–) lane (Figure 1C). This
band represents full-length sfGFP-FLAG, and its presence in the +6 and +7 lanes suggested
successful translational read-through at the amber site in both samples. These observations
suggest that 6 is accepted by wildtype PylRS and incorporated site-specifically into proteins via
the PylRS/tRNACUA system. The small amount of full-length sfGFP formed in the absence of
amino acid supplement may be caused by misacylation of tRNACUA with native amino acids by
PylRS. As expected, E. coli that were not transformed with the gene for PylRS (–RS+T) did not
generate full-length sfGFP-FLAG, showing that PylRS is necessary for read-through of the
amber stop codon. We further characterized full-length QC-sfGFP-FLAG by high-resolution

ESI-MS and found that it matched the predicted mass of 28147 Daltons (Figure 1D).
The lower band in the blot shown in Figure 1C at ~14 kDa, present in all samples,
represents the amber termination product, confirmed by mass spectrometry (Figure S1). This is a
common byproduct of the unnatural amino acid incorporation process as the amber codon still
acts as a stop codon and is recognized by release factors such as RF1 in E. coli.30
After confirming the presence of 6 in sfGFP-FLAG, we sought to label the QC handle
with nickel bis(dithiolene) biotin 8 (Figure 2A). To start, we subjected purified QC-sfGFP-
FLAG or Boc-sfGFP-FLAG to 200 M 6 in PBS supplemented with 1mM K3Fe(CN)6 or with
vehicle (1 mM K3Fe(CN)6 in PBS) for 1 h. The K3Fe(CN)6 is used as an additive to prevent
reduction of Ni bis(dithiolene) adducts.15 After 1 h, the reaction was quenched with 100 equiv. of
QC acetate (9, Figure 2B, hereafter referred to as QCOAc) relative to 8 followed by addition of
sodium diethyldithiocarbamate (Na-DEDTC), which prevents photocleavage of the adduct.15
Ligation product was analyzed by western blot with an -biotin-horseradish peroxidase (HRP)
antibody (Figure 2C). We observed robust labeling of QC-sfGFP-FLAG in the presence of 8
(lane 1) with minimal signal from the Boc-sfGFP-FLAG negative control (lane 3). The smear
observed in lane 1 may be due to the addition of a large PEG-containing group to QC-sfGFP,
rather than off-target reactivity since no biotin signal is observed when Boc-sfGFP was
incubated with 8, shown in lane 3. These data suggest selective reaction of 6 in sfGFP with 8 and
minimal side reactivity of 8 with other amino acids in sfGFP-FLAG. Additional blot exposure
times are shown in Figure S2.

Figure 2. Labeling QC-sfGFP-FLAG via the quadricyclane ligation. A) Schematic showing labeling QC-sfGFP-
FLAG with 8. B) Structure of QC acetate (QCOAc, 9). C) Purified QC-sfGFP-FLAG or purified Boc-sfGFP-FLAG
was incubated with 200 μM 8 in PBS with 1 mM K3Fe(CN)6 (+8) or with 1 mM K3Fe(CN)6 in PBS (–8) for 1 h at rt
in the dark. The reaction was quenched with QCOAc (100 equiv. relative to 8) followed by treatment with sodium
diethyldithiocarbamate (Na-DEDTC). Protein was analyzed by western blot with an α-biotin-HRP antibody. D)
Various quantities of purified QC-sfGFP-FLAG or Boc-sfGFP-FLAG (5.0, 2.5, 1.0, 0.5, or 0 μg) were added to 10.0
μg bacterial lysate. The mixtures were incubated with 8 (200 μM) in K3Fe(CN)6 (1 mM) in PBS for 1 h at rt,
quenched via addition of QCOAc (100 equiv. relative to 8), and treated with Na-DEDTC. Mixtures were analyzed
via western blot with an -biotin-HRP antibody.

We next sought to achieve selective labeling of QC-sfGFP-FLAG in the more complex
environment of bacterial lysate. For this experiment, we doped varying amounts of purified QC-
sfGFP-FLAG or Boc-sfGFP-FLAG into 10 g samples of bacterial lysate (Figure 2D). Each
sample was then incubated with Ni bis(dithiolene) biotin 8 (200 M) and K3Fe(CN)6 (1 mM) in
PBS for 1 h at 22 °C. The reaction was quenched with QCOAc (100 equiv. relative to 8), treated
with Na-DEDTC, and samples were analyzed by western blot and the presence of biotin was
probed with an -biotin-HRP antibody. Results are shown in Figure 2D, where QC-sfGFP-
FLAG can be seen to be selectively labeled by 8 in the presence of complex cell lysate at all QC-
sfGFP-FLAG:lysate ratios, with more robust labeling at higher ratios (5.0 or 2.0 g QC-sfGFP-
FLAG added to 10 g lysate). Samples with Boc-sfGFP-FLAG showed minor labeling at only
the highest concentration (5.0 g), suggesting occurrence of background labeling on sfGFP-
FLAG. Thus, while it is clear that we have achieved labeling of QC-sfGFP-FLAG in the
presence of bacterial lysate, background reactivity, both on Boc-GFP-FLAG as well as on lysate,
remains a liability of the QC ligation.
In summary, we achieved site-specific incorporation of a QC amino acid into a protein of
interest using wildtype PylRS and tRNACUA. Subsequently, this QC-labeled protein could be
selectively modified in the presence of bacterial cell lysate. These developments demonstrate the
potential applications of the QC ligation to native biological systems.
2.2 Photocleavage of the QC/nickel bis(dithiolene) adduct
Early reports of the photolability of the QC/metal bis(dithiolene) adduct piqued our
interest for expanding the types of reactions accessible via the QC ligation. In 1965, Schrauzer

and Mayweg were the first to describe the decomposition of adduct 10 (Figure 3A) when
exposed to daylight.31 Later studies by Wing and Sugimori describe the photolysis reaction to be
that shown in Figure 3A, where adduct 10 is cleaved to form norbornadiene 11, an isomer of QC,
and the starting nickel bis(dithiolene) 12.32–34 We envisioned taking advantage of the
photolability of adducts such as 10 to render the product of the QC ligation cleavable. While
there are numerous examples of cleavable bioconjugations that take advantage of native
functionalities such as primary amines and thiols,35–42 or slowly reversible reactions such as
aldehyde or ketone condensations,28,43–46 there is only one example of a fast and reversible
bioorthogonal reaction that utilizes only non-native reactive groups.14 The ligation adduct of the
chemoselective rapid azo-coupling reaction (CRACR) can be cleaved upon exposure to the small
molecule dithionite.14 This requirement inherently limits the spatial control of this reaction in
biological systems. A cleavable QC ligation reaction would enable selective tagging and
photochemically controlled untagging of target biomolecules in a system, presenting
opportunities for temporally and spatially controlled protein modification. Additionally, a
cleavable bioorthogonal adduct could have applications in protein enrichment and purification.
Thus, we sought to optimize the QC/nickel bis(dithiolene) photocleavage reaction for use on
proteins.

Figure 3. Photocleavage of the QC/nickel bis(dithiolene) adduct. A) Reaction scheme for the model
photocleavage. Adduct 10 was dissolved in CDCl3 to a concentration of (5 mM) and placed in a borosilicate glass
tube. The solution was placed in a foil-lined container and irradiated with 365 nm light from a handheld lamp for 0-
25 min. B) Representative 1H NMR spectra from Figure 3A at the indicated times. Diagnostic peaks are labeled with
colors corresponding to the stars in Figure 3A. C) The progress of photocleavage was determined by comparing the
integration of individual peaks of 10 to 11. Each measurement is the average of three separate experiments. Error
bars represent one standard deviation.
We began by analyzing the efficiency of the photocleavage reaction in a flask, aiming to
determine its half-life upon irradiation with a handheld UV lamp. Analyses were performed via
1H NMR, and we selected adduct 10 due to its relative stability to reduction in solution (the
anionic form of the nickel bis(dithiolene) adduct is paramagnetic and challenging to analyze by
NMR). Photocleavage was performed using a 365 nm light because of the high absorbance
coefficient associated with this wavelength.33,34 We observed clean conversion of 10 to 11 and

12 within minutes (Figure 3B). Product formation was quantified by comparing the integration of
1H NMR peaks of product and starting material, and the half-life of this photocleavage was
found to be ~8 min at 5 mM in CDCl3 (Figure 3C). A reaction occurring in minutes is useful on
biological time-frames, and 8 min compares favorably to the half-life of the well-established
bioorthogonal copper-free click reaction between the cyclooctyne BARAC and benzyl azide,
with a maximum half-life of ~3.5 min when both reactants are present at 5 mM in CD3CN at
room temperature.47 Given the rapid photolysis of adduct 10 at room temperature using a
handheld lamp, we decided to test this approach on proteins labeled via the QC ligation.
To this end, we non-specifically labeled bovine serum albumin (BSA) with p-nitrophenyl
quadricyclane carbonate (5, Figure 4A) as we previously reported15 to give QC-BSA, our model
protein for optimization of the photocleavage reaction. QC-BSA was then labeled via QC
ligation with nickel bis(dithiolene) biotin 8 to yield biotin-labeled BSA 13 (Figure 4A).15 As a
negative control, BSA that was not QC-modified was exposed to the same reaction conditions
with 8 (hereafter referred to as 8-treated BSA). We irradiated 13 and 8-treated BSA using a 365
nm handheld lamp and probed for release of 8 by western blot using an -biotin-HRP antibody
(Figure 4A and B). To obviate the possibility of the precedented reaction between the nickel
bis(dithiolene) 8 and norbornadiene 14 (Figure 4A) released by the photocleavage,33 our samples
were treated with scavenging QCOAc to sequester 8. When samples of 13 were irradiated with
UV light, the amount of biotin signal was significantly reduced with increasing exposure times
(Figure 4B). No biotin signal was observed with 8-treated BSA.

Figure 4. Photocleavage of the QC/nickel bis(dithiolene) adduct on a protein. A) Lysines on bovine serum
albumin (BSA) were capped via reaction with p-nitrophenyl quadricyclane carbonate 5. The resulting QC-BSA and
untreated BSA (negative control) were treated with nickel bis(dithiolene) biotin 8 (50 M in PBS) for 30 min to
form adduct 13 or 8-treated BSA. The reactions were quenched with QCOAc and protein was purified from excess
small molecule via centrifugal filtration with an Amicon Ultra 10 kDa molecular weight cutoff filter. 13 and 8-
treated BSA in PBS were irradiated with a 365 nm handheld UV lamp for 0, 10, 30, or 60 min to cleave the adduct,
providing 14 and 8. Protein products were separated from free 8 via centrifugal filtration as described above. C)
Protein from Figure 4A was analyzed via western blot with an -biotin-HRP antibody. Q = 13, B = 8-treated BSA.
With a readily cleavable biotin attached to a protein, we next sought to utilize the
photocleavage capability of the QC ligation as a novel protein purification method. A schematic
of purification using this photocleavage for QC-BSA or BSA is presented in Figure 5A. In the
presence of just PBS or cell lysate, QC-BSA or BSA were incubated with 8 for 14 h in the dark.
After removal of remaining small molecule by centrifugal filtration, the mixture was added to
streptavidin resin for 30 min in the dark, and the beads were treated with PBS until no more
protein washed from the resin (Washes). The beads were then irradiated with 365 nm light from
a handheld lamp in 5-minute increments for a total of 20 minutes with elutions of PBS after
every 5-minute mark (Elutions). Finally, the beads were washed with 8 M guanidine HCl (pH
1.5) to remove any remaining protein (not shown schematically). Following the collection of all

washes and elutions, the proteins were visualized by SDS-PAGE and Coomassie staining. When
either QC-BSA or BSA was used, protein was present in the initial washes (Figure 5B, Wash 1;
Wash 2,3), but not in the final washes (Wash 6,7). After UV irradiation, a very faint band was
observed when BSA was used (BSA, Elution 1 and 2). However, UV irradiation of the QC-BSA-
containing mixture provided a significant amount of protein in the elution lanes (QC-BSA,
Elution 1 and 2). This suggests that biotin attached to the protein specifically through the QC
ligation was removed upon UV irradiation, thus releasing the protein from the streptavidin resin.
Very little protein was left on the beads after UV irradiation (QC-BSA and BSA, Elution B).
Figure 5. QC photocleavage as a method for protein purification. A) Schematic showing general protein
purification procedure using QC-BSA and cell lysate. QC-BSA in a mixture with cell lysate was incubated with 8
for 14 h in the dark and subsequently quenched with QCOAc. The samples were transferred to Amicon Ultra
centrifugal filters and washed with PBS three times. The concentrated samples were incubated with Pierce™

Streptavidin Agarose for 30 min. The resin was washed with PBS by spin filtration seven times (Washes 1-7).
Following the washes, the resin was suspended in PBS and irradiated with 365 nm light by a handheld lamp for 5
min and the supernatant was eluted (Elution 1). The irradiation was repeated three more times with additional PBS
each time (Elutions 2-4). The beads were then stripped of all remaining protein with 8 M guanidine HCl (pH = 1.5, 4
washes) and this was concentrated on an Amicon Ultra centrifugal filter (Elution B). Washes 1; 2,3; 6,7 and Elutions
1; 2; B were separated by gel electrophoresis and visualized with Coomassie staining. B) The general procedure
from Figure 5A was followed with the following quantities of reagents: QC-BSA or BSA (100 µL in PBS, 1
mg/mL), 8 (100 µL, 50 µM in PBS), QCOAc (2.5 µL, 200 mM in DMSO), PBS rinse (200 µL, then twice with 350
µL), Pierce Streptavidin Agarose (100 µL), PBS washes (50 µL each, lanes Wash 1; 2,3; 6,7), elutions (50 µL PBS
each, lanes Elution 1 and 2), guanidine HCl (50 µL washes, Elution B). C) Protein from Wash 1 and Elution 1 of
BSA and QC-BSA samples from Figure 5B were subjected to ESI intact mass analysis. Unmodified BSA: BSA with
no QC modifications. The numbers represent the number of QC moieties attached to BSA. D) The general procedure
from Figure 5A and 5B was performed in the presence of bacterial cell lysate (10 µL, 4.94 g/mL) and K3Fe(CN)6
(10 µL, 22 mM). K3Fe(CN)6 and lysate were added sequentially to the solutions of QC-BSA or BSA before
addition of 8. After QC ligation with 8, the capture, wash, and elutions sequence depicted in Figure 5A was
performed. Lanes are as described in Figure 5B.
Curiously, when the QC-BSA mixture was incubated with the streptavidin beads, most of
the total protein was present in the initial wash, rather than eluting only after irradiation. We
hypothesized that since QC-BSA was non-specifically labeled with QC, the washes might
contain BSA that was not labeled with QC, and thus could not undergo reaction with 8. We
performed intact mass analysis of the protein present in Wash 1 and Elution 1 of both QC-BSA
and BSA samples from Figure 5B. When the mixture with BSA was incubated with the
streptavidin resin, only unmodified BSA was observed to wash off the resin (Figure 5C, BSA
Wash 1). A small amount of unmodified BSA was also observed eluting from the column in the
elution of BSA (Figure 5C, BSA Elution 1). Notably, the incubation of the QC-BSA mixture

with streptavidin resin showed a large amount of unmodified BSA in addition to some QC-BSA
in the initial wash (Figure 5C, QC-BSA Wash 1). This suggests that a significant portion of the
QC-BSA sample was BSA that was not labeled by 5, and thus would not react specifically with
8. After irradiation of QC-BSA-containing resin, however, the elution contained only QC-BSA
(Figure 5C, QC-BSA Elution 1). This suggests that BSA modified with QC could bind to the
streptavidin after treatment with 8 and be subsequently released upon exposure to UV light. No
protein was observed to be modified by 8 in the intact mass analysis. This could be due to
quantitative photocleavage, or perhaps the QC ligation adduct does not ionize well or is not
stable under the conditions present in ESI.
To further demonstrate the utility of this photocleavage, the isolation of a QC-modified
protein from a complex mixture of biological material was attempted. To a solution of QC-BSA
or BSA was added K3Fe(CN)6 and bacterial cell lysate. To this mixture, 8 was added and,
following an incubation in the dark, the solutions were quenched with QCOAc and washed with
PBS. The samples were then incubated on Pierce™ Streptavidin Agarose, followed by the
washes and elutions described above. When BSA was used, most protein was found to come off
the resin in the washes (BSA Washes, Figure 5D), while very little protein was observed in the
elutions (BSA, Elutions). In contrast, when QC-BSA was used the bacterial lysate was only
present in the washes (QC-BSA, Washes). Following irradiation, only QC-BSA was present in
the elutions (QC-BSA, Elutions). In the complex mixture of cell lysate, QC-modified protein
could be labeled and released selectively using the QC ligation and subsequent photocleavage.
This presents a promising new protein purification method that is based on a photocleavable
click chemistry adduct.

3. Conclusion
Over the past two decades, much attention has been given to bioorthogonal reactions that
form adducts between two molecules. Here we have described the development of the QC
ligation to be more biologically accessible and chemically useful beyond typical adduct-forming
bioorthogonal reactions. As the number of chemical transformations that are adapted for use in
biological systems increases, the impetus for discovery will shift to the creative application of
these reactions. Using these new reactions, the ability to augment the complexity of life, rather
than study it, will be a transformative force in the third decade of bioorthogonal chemistry.
4. Experimental
4.1 General Chemical Methods
Reagents and solvents were obtained commercially and used without further purification
with the exception of anhydrous solvents which were prepared by the method developed by
Pangborn et al.48 Anhydrous dimethylformamide was obtained from Sigma Aldrich in a
Sure/Seal™ container. Flash column chromatography was performed according to the method
described by Still et al. using Silicycle Silica Flash P60 silica (40-63 mm, 230-400 mesh).49 All
high pressure liquid chromatography purifications were performed on a Varian ProStar HPLC
equipped with UV/vis detector using a 100 Å C18 column (250 x 21.4 mm) at a flow rate of 20
mL/min.
Reaction progress was monitored by TLC with Silicycle glass backed extra hard layer
TLC plates (60 Å, 250 m) visualized with ultraviolet light or with potassium permanganate or
p-anisaldehyde staining followed by brief heating on a hot plate. Unless otherwise noted,
anhydrous magnesium sulfate was used to dry organic extracts and solutions were concentrated
using a Buchi Rotovapor-114 equipped with a Welch 2026 self-cleaning dry vacuum system.
Compounds were dried under reduced pressure on a Schlenk line equipped with an Edwards high
vacuum. Lyophilization was performed using a Labconco FreeZone Plus 4.5 Liter benchtop
lyophilizer equipped with a Labconco vacuum (Model 117).
1
All H and 13C spectra were obtained with a Bruker AV-600, AV-500, AVB-400, or
AVQ-400 spectrometer. Peaks are reported in ppm and referenced to the solvent. Data are
presented as follows: chemical shift in ppm, multiplicity (s = singlet, d = doublet, t = triplet, q =
quartet, quin = quintet, m =multiplet), coupling constant in Hz, and integration value. High-
resolution electrospray ionization (ESI) mass spectrometry data were obtained through the UC
Berkeley Mass Spectrometry Facility on a Linear trap quadrupole fourier transform ion cyclotron
resonance (LTQ-FT-ICR) mass spectrometer from Thermo Fisher. High-resolution electron

ionization (EI) mass spectrometry data were obtained through the UC Berkeley Mass
Spectrometry Facility on an AutoSpec mass spectrometer from Waters.
4.2 Synthesis of quadricyclane amino acid (6)
4.2.1 Fmoc-protected QC amino acid
To a flame dried round bottom flask was added 30 mL dry dimethylformamide followed
by N-Fmoc-L-lysine (455 mg, 1.23 mmol, 1.00 equiv.). p-nitrophenyl carbonate 5 (508 mg,
1.86 mmol, 1.50 equiv.), prepared via the route published by Sletten et al.15 was added, and the
reaction mixture immediately turned yellow. The solution was stirred at room temperature
overnight, and the following day was concentrated to a yellow oil. Crude product was purified by
flash column chromatography on silica gel that was pre-treated with 0.1% acetic acid in hexanes
followed by washing with ~4 column volumes of hexanes. Product was eluted with
hexanes:EtOAc:MeOH (10:1:0, 2:1:0, 0:1:0, 0:95:5, 0:9:1) to give the product in 85% yield (526
1
1
mg as a mixture with 140 mg DMF as determined by H NMR). Rf = 0.54 in 100% EtOAc; H
NMR (600 MHz, MeOD)  7.78 (d, J = 7.5 Hz, 2H), 7.67 (dd, J = 11.3, 7.5 Hz, 2H), 7.38 (t, J =
7.5 Hz, 2H), 7.30 (t, J = 7.5 Hz, 2H), 5.53–5.46 (m, 1H), 4.34 (qd, J = 10.6, 7.1 Hz, 2H), 4.21 (t,
J = 7.0 Hz, 1H), 4.14 (dd, J = 9.5, 4.6 Hz, 1H), 3.16–3.06 (m, 2H), 1.87–1.84 (m, 1H), 1.81–1.65
13
(m, 3H), 1.62–1.36 (m, 8H); C NMR (151 MHz, MeOD)  176.0, 159.6, 158.7, 145.3, 145.1,
142.6, 142.5, 128.8, 128.8, 128.2, 128.1, 126.3, 126.2, 120.9, 120.9, 83.7, 67.9, 55.3, 48.4, 41.4,
32.3, 30.5, 26.8, 26.8, 24.2, 16.8, 15.3; HRMS (ESI) calcd for C29H30O6N2Na [M+Na]+:
525.1996; found: 525.1993.
4.2.2 QC amino acid (6)
Fmoc-protected QC amino acid (598 mg, 1.19 mmol, 1.00 equiv.) was dissolved in
anhydrous dimethylformamide (10 mL) in a round bottom flask. Piperidine (400 L, 4.05 mmol,
3.40 equiv.) was added, causing an immediate color change to yellow followed by cloudiness in
the reaction mixture. The reaction mixture was stirred at room temperature for 2 hours at which
point it was concentrated to an off white solid. The solid was triturated with cold EtOAc and
filtered. The remaining white solid was washed with cold EtOAc to yield pure 6 (263 mg, 78%
1
yield). Rf = 0.44 in 3:1 MeOH:EtOAC; H NMR (600 MHz, D2O) 5.50 (s, 1H), 3.70 (s, 1H),
3.14 (t, J = 6.9 Hz, 2H), 1.85–1.80 (m, 4H), 1.63 (s, 2H), 1.57–1.46 (m, 4H), 1.42–1.32 (m, 2H);
13C NMR (151 MHz, DMSO)  169.5, 156.5, 81.0, 54.1, 40.1, 30.8, 29.2, 25.5, 22.5, 15.6, 13.9;
HRMS (ESI) calcd for C14H20O4N2Na [M+Na]+: 303.1315; found: 303.131
4.3 Synthesis of nickel bis(dithiolene) biotin (8)
4.3.1 Dithiocarbonate ligand (S2)
Dithiocarbonate S1 (Scheme S1) was synthesized according to the route developed by
Sletten et al.15 EZ-link amine-PEG3-biotin from Thermo Scientific (53 mg) was transferred to a
round bottom flask as a solution in water, concentrated to a solid on a rotary evaporator, and
azeotroped from toluene (2 x 2 mL). The amine-PEG3-biotin was then dissolved in 1 mL dry
dimethylformamide under an atmosphere of nitrogen. In a separate flask S1 (24.2 mg, 0.06
mmol, 1.00 equiv.) was dissolved in 1 mL dry dimethylformamide. 500 L of the amine-PEG3-
biotin solution (26.5 mg, 0.07 mmol, 1.17 equiv.) was added to the solution of S1 followed by
addition of EDCHCl (22.6 mg, 0.12 mmol, 2.00 equiv.), HOBt hydrate (13.0 mg) and

triethylamine (20 L, 0.14 mmol, 2.33 equiv.). The reaction mixture was stirred at room
temperature for 3 days, then concentrated to a yellow oil. Crude product was dissolved in 1:2
MeOH:H2O, filtered through a 0.2 micron filter, and purified by HPLC on a C18 prep column,
30 to 95% MeOH in H2O over 30 min, 20 mL/min flow rate. The product eluted as a broad peak
1
between 8 and 12 min, yielding S2 as a yellow oil (28 mg, 0.035 mmol, 58% yield). H NMR
(600 MHz, MeOD)  7.76 (t, J = 8.5 Hz, 4H), 7.34 (dd, J = 11.2, 8.1 Hz, 4H), 4.49–4.46 (m,
1H), 4.30–4.28 (m, 1H), 3.65–3.61 (m, 10H), 3.57–3.53 (m, 5H), 3.50–3.48(m, 2H), 3.21–3.17
(m, 2H), 3.15–3.11 (m, 1H), 2.92–2.87 (m, 2H), 2.70–2.68 (m, 1H), 2.49–2.48 (m, 1H), 2.20 (t, J
13
= 7.5 Hz, 2H), 1.75–1.69 (m, 1H), 1.66–1.54 (m, 3H), 1.44–1.39 (m, 2H); C NMR (151 MHz,
MeOD)  190.7, 176.1, 169.1, 166.0, 147.2, 136.2, 135.8, 134.4, 130.9, 130.7, 130.2, 129.8,
129.0, 127.7, 71.5, 71.5, 71.3, 71.2, 70.6, 70.5, 63.4, 61.6, 57.0, 49.9, 43.4, 41.1, 41.0, 40.4, 40.3,
40.3, 37.2, 36.7, 35.9, 32.3, 29.7, 29.5, 27.0, 26.8, 19.3, 15.7. HRMS (ESI) calcd for
C34H41O10N4S4 [M–H]–: 793.1711; found: 793.1698
4.3.2 Nickel bis(dithiolene) biotin (8)
Dithiocarbonate S2 (17.3 mg, 0.02 mmol, 1.00 equiv.) was dissolved in 400 L THF and
400 L H2O. Tetramethylammonium hydroxide pentahydrate (8.4 mg, 0.05 mmol, 2.5 equiv.)
was added to the reaction mixture as a solution in 60 L MeOH and the mixture quickly became
a dark yellow/orange color. After stirring the resulting mixture at room temperature for 30
minutes, nickel(II) chloride hexahydrate (2.8 mg, 0.01 mmol, 0.5 equiv.) was added. The
solution immediately became dark red, and was stirred at room temperature overnight. The
following morning, iodine (3.2 mg, 0.01 mmol, 0.5 equiv.) was added and the reaction mixture
immediately became dark green. After stirring at room temperature for 3 h, the reaction mixture
was concentrated to a green solid on a rotary evaporator. The crude product was purified by flash
column chromatography and eluted with acetonitrile:H2O (10:1, 5:1, 3:1, 2:1). Fractions
containing product were pooled, acetonitrile was evaporated on a rotary evaporator, and the
remaining aqueous solution was lyophilized overnight to yield 8 as a dark green, fluffy solid (7.2
mg, 0.005 mmol, 21% yield). Rf = 0.64 in 5:1 acetonitrile:H2O; UV/vis/NIR 225–900 nm
(water): 859 nm (1.3 au), 311 nm (3.9 au), 282 nm (4.1 au), 232 nm (44.3 au); HRMS (ESI)
calcd for C66H82O18N8NiS8 [M–2H]2–: 794.1439; found: 794.1417.
4.4 Non-natural amino acid incorporation and QC ligation labeling experiments
4.4.1 General methods
N-Boc-L-lysine (Boc-lysine) was purchased from Chem-Impex and used without further
purification. 6 was synthesized and purified as described above. Phosphate-buffered saline 1X, –
calcium –magnesium (PBS) was obtained from Corning Cellgro. All protein expression
experiments were performed using DH10 cells from Invitrogen (MAX efficiency, chemically
competent). A New Brunswick Scientific C25 incubator shaker was used to incubate E. coli
liquid cultures at 37 ºC with shaking at 225 rpm. A Lab-line Imperial II incubator was used to
incubate LB agar plates at 37 ºC. LB agar plates were prepared using Luria Broth Base (Miller’s
LB Broth Base, Invitrogen) and agar (granulated, Fisher Scientific). LB liquid media and LB
agar plates were supplemented with kanamycin to 50 g/mL and/or tetracycline to 25 g/mL
depending on the combination of plasmids used. Sample centrifugation was performed in a
Thermo Scientific Sorvall RC 6+ centrifuge, Sorvall RC 5C Plus centrifuge, a Sorvall Legent RT
centrifuge, or an Eppendorf centrifuge 5417C.

Gel electrophoresis was performed using BioRad Criterion gel electrophoresis chambers
equipped with a BioRad Power Pac 200. All gels (Biorad Criterion XT pre-cast) were run in
MES buffer. Western blot transfers were performed using BioRad Criterion blotter transfer
boxes equipped with a BioRad Power Pac 200. Gels were transferred to nitrocellulose (BioRad
0.45 m) in tris-glycine buffer (20% MeOH). See Blue Plus 2 pre-stained protein ladder from
Life Technologies was used for all gels and western blots. -GFP (4B10) Mouse mAb (#2955),
-Mouse IgG horseradish-peroxidase-linked (HRP) antibody (#7076), and -FLAG®-HRP
rabbit polyclonal antibody (DYKDDDDK Tag antibody HRP conjugate, #2044) were obtained
from Cell Signaling Technologies. Mouse -biotin-HRP was obtained from Southern Biotech.
Protein loading was assessed via staining with Ponceau (5% acetic acid, 0.1% (w/v) ponceau).
Blots were visualized on Premium X-ray film (blue) from Phenix Research Products.
Densitometry analyses were performed using ImageJ 1.48v (National Institutes of Health). Fast
protein liquid chromatography (FPLC) was performed on a Pharmacia Biotech Akta explorer 10.
High resolution mass spectrometry data were obtained on a Linear Trap Quadrupole LTQ-
Orbitrap-XL mass spectrometer (Thermo Scientific) equipped with an electrospray ionization
(ESI) source.
4.4.2 General Procedure for Transformation of E. coli with plasmids psfGFP150TAGFLAGPylT
and/or pBKPylRS
DH10 E. coli were transformed with psfGFP150TAGFLAGPylT and pBKPylRS
(+RS+T) or with psfGFP150TAGFLAGPylT and sterile ddH2O (–RS+T) by heat shock (30 sec,
42 ºC, 2 min, wet ice) and recovered in 250 L super optimal broth with 20 mM glucose (S.O.C.
medium) for 35 min at 37 ºC and 225 rpm. Cells were then plated on kanamycin/tetracycline
(+RS+T E. coli) or tetracycline (–RS+T E. coli) LB agar plates and incubated at 37 ºC overnight.
The following day, 5 mL liquid cultures of LB media supplemented with kanamycin/tetracycline
(+RS+T E. coli) or tetracycline (–RS+T E. coli) were inoculated with one colony from +RS+T
and one colony from –RS+T respectively. Cultures were incubated at 37 ºC and 225 rpm
overnight. The following morning, glycerol stocks were prepared (375 L culture and 125 L
sterile 80% glycerol in ddH2O) and frozen to –80 ºC for future use.
4.4.3 Expression of QC- and Boc-sfGFP-FLAG
DH10 cells were transformed with the relevant plasmids (+RS+T or –RS+T) as
described above and the resulting 5 mL overnight cultures were used immediately. Alternatively,
glycerol stabs of transformed DH10 cells stored at –80 ºC were used to streak fresh LB agar
plates (supplemented with the requisite antibiotics). The plates were grown overnight at 37 ºC,
and 5 mL liquid media cultures (supplemented with the requisite antibiotics) were inoculated
with single colonies from each plate. These 5 mL cultures were then incubated overnight at 37 ºC
and 225 rpm and used the next day to inoculate larger cultures for protein expression. All protein
expression protocols begin with a 5 mL overnight culture.
4.4.3.1 Expression of QC-sfGFP-FLAG and Boc-sfGFP-FLAG for use in the experiment shown
in Figure 1C
DH10 E. coli were transformed with pBKPylRS and/or psfGFP150TAGFLAGPylT and
5 mL overnight cultures were prepared as described in previous experiments. The following
morning, 2 mL from each culture was used to inoculate 50 mL LB media cultures (supplemented
with the requisite antibiotics). Each culture was incubated at 37 ºC and 225 rpm to OD600 ~0.4 at

which point the cultures were divided into three separate 15 mL polystyrene tubes (5 mL/tube)
and supplemented with 6 or BocLys (from 22 mM stock solution of amino acid in sterile 0.1 M
NaOH ddH2O, actual concentration may be lower due to limited solubility) to ~1 mM or
supplemented with the same volume of vehicle (0.1 M NaOH) and incubated at 37 ºC and 225
rpm for 30 min. Protein expression was induced via addition of 20% L-arabinose in sterile
ddH2O to 0.2% and expressed for 3.5 hours. Following expression, cultures were transferred to
15 mL falcon tubes and pelleted at 3500 rom and 4 ºC for 10 min. Pellets were washed 3x via
resuspension in LB media followed by pelleting at 9,700 rpm for 5 min and aspiration of the
supernatant. Washed pellets were stored at –80 ºC until further use.
Samples were thawed on ice, then warmed to rt. 1/6 of each pellet was isolated and
resuspended in 40 L 1X SDS loading buffer supplemented with 2.5% -mercaptoethanol. The
suspension was centrifuged at 14,000 rpm for 15 min, and samples were loaded onto two, 18-
well 4–12% bis-tris Criterion XT precast polyacrylamide gels (15 l sample/per well). Gels were
treated as described in the General Western Blotting Procedure section with -GFP Mouse mAb
and -Mouse-HRP or with -FLAG-HRP rabbit polyclonal antibody.
4.4.3.2 Expression of QC-sfGFP-FLAG and Boc-sfGFP-FLAG for use in the experiment shown
in Figures 1D and 2C-D.
+RS+T DH10 and –RS+T DH10 E. coli 5 mL overnight cultures were prepared as
described above using pBKPylRS and psfGFP15TLAGFLAGPylT plasmids. The following
morning, 2 mL from each culture was used to inoculate 100 mL LB media (supplemented with
the requisite antibiotics). Cultures were grown to OD600 = 0.3–0.5 and supplemented with Boc-
Lys or 6 (from 50 mM amino acid stock solution in 0.1 M NaOH ddH2O, actual concentration
may be lower due to limited solubility) to ~1 mM. Flasks were incubated for an additional 30
min at 37 ºC and 225 rpm after which protein expression was induced via addition of 20% L-
arabinose in sterile ddH2O to 0.2%. Protein was expressed for 3.5 hours and then each culture
was separated into two 50 mL falcon tubes and pelleted at 3,500 rpm for 10 min at 4 ºC. The
supernatant was aspirated and pellets from each culture were combined and washed 3x by
resuspension in 20 mL LB media, pelleting at 3,500 rpm for 10 min at 4 ºC, and aspiration of
supernatant. Pellets were stored at –20 ºC until lysis, at which point they were resuspended in 3
mL lysis buffer (tris-HCl (10 mM), NaCl (200 mM), imidazole (20 mM), pH = 8, Roche
protease inhibitor) and, lysed via sonication (Misonix Sonicator 3000, power level 2, 4 min on
total: 10 sec on, 10 sec off). The resulting lysate was clarified by centrifugation (20 min, 21,000
x g, 4 ºC).
FLAG-tagged protein was then purified via affinity column at room temperature using
ANTI-FLAG® M2 Affinity Gel from Sigma Aldrich. The gel was equilibrated with 5 column
volumes of TBS buffer (150 mM NaCl, 50 mM tris-HCl, pH = 7.4), lysate was loaded via three
passes through the column, and unbound protein was washed off the column with 20 column
volumes of TBS buffer. Bound protein was eluted with five column volumes 100 µg/mL
3XFLAG peptide (Sigma Aldrich) in TBS. Elutions were pooled and concentrated to ~500 L
using an Amicon Ultra 4 mL, 10 kDa molecular weight cutoff centrifugal filter. Protein was
further purified by fast protein liquid chromatography (FPLC) at 4 ºC on a Superdex 75 10/300
GL (GE Healthcare Life Science) column using FPLC buffer (tris-HCl (20 mM), NaCl (100
mM), pH = 7.4) at a flow rate of 0.5–0.6 mL/min Fractions (0.5 mL each) were analyzed by
polyacrylamide gel electrophoresis followed by protein visualization with Coomassie stain
(0.25% (w/v) Coomassie, 40% methanol, 10% acetic acid). Following FPLC purification, protein

was transferred to PBS via solvent exchange using Amicon Ultra 10 kDa molecular weight
cutoff filters.
4.4.4 General QC ligation procedure for sfGFP
Purified QC- or Boc-sfGFP (~0.2 mg/mL) in the presence or absence of E. coli lysate
(0.33 mg/mL) was combined with 6 (200 M) and K3Fe(CN)6 (1 mM) in PBS and incubated in
the dark at room temperature for 1-2 h. Reactions were quenched via addition of 100 equiv.
QCOAc (200 mM solution in DMSO) relative to sfGFP. Samples were then combined with Na-
DEDTC (100 mM solution in PBS).
4.4.5 General Western Blotting Procedure
All samples were combined with 4X sodium dodecyl sulfate (SDS) loading buffer
(supplemented with 10% -mercaptoethanol) to a final loading buffer concentration of 1X for
separation via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). All
SDS-PAGE was performed using 4–12% bis-tris Criterion XT precast polyacrylamide gels. Gels
were run at 175 V for 40 min and then were transferred to nitrocellulose at 75 V for 90 min. The
blots were treated with Ponceau stain to visualize proteins.
Blots to be analyzed with -biotin-HRP were blocked with 5% bovine serum albumin
(BSA) in PBST at 4 ºC in the dark overnight. All other blots were blocked with 5% milk in
PBST at 4 ºC in the dark overnight. After blocking, -biotin-HRP blots were incubated in a
1:200,000 dilution of -biotin-HRP in 5% BSA, PBST for 15 min at room temperature in the
dark and then washed 3 x 10 min with PBST. -FLAG blots were incubated in a 1:3,000 dilution
of -FLAG-HRP in 5% milk PBST followed by washing 3 x 10 min PBST. -GFP blots were
incubated in a 1:3,000 dilution of-GFP in 5% milk, PBST for 1 h at room temperature
followed by washing 3 x 10 min with PBST. After washing, blots were incubated in a 1:10,000
dilution of -mouse-HRP in 5% milk, PBST for 1 h at room temperature followed by washing 3
x 10 min with PBST.
For all blots, detection was performed by chemiluminescence using SuperSignal West
Pico Chemiluminescent Substrate (Thermo Scientific). Blots were developed on a Kodak X-
OMAT 500RA processor.
4.4.6 Analysis of Termination Product
We expressed His6-tagged QC-sfGFP and Boc-sfGFP according to the procedure
described in section 4.4.3.2 using plasmids psfGFP150TAGHis6PylT and pBKPylRS. Upon lysis,
His6-tagged proteins were purified on a Ni2+-NTA column. Qiagen Ni2+-NTA beads (500 l)
were washed 3x via suspension in binding buffer (imidazole (5 mM), tris-HCl (10 mM), NaCl
(200 mM), pH = 8) followed by centrifugation and aspiration of supernatant. Beads were then
added to clarified lysate as a suspension in 500 L binding buffer, and the mixture was incubated
with agitation for 1–1.25 h at 4 ºC, after which beads were transferred to a fritted column. At 4
ºC, the beads were washed 3 x 3 mL wash buffer (tris-HCl (10 mM), imidazole (20 mM), NaCl
(200 mM), pH = 8) followed by elution with 6 x 1 mL wash buffer supplemented with imidazole
to 200 mM. SDS-PAGE analysis of the fractions indicated co-elution of termination product
along with full length QC- and Boc-sfGFP-His6. Elutions were pooled and concentrated to ~500
L using an Amicon Ultra 4 mL, 10 kDa molecular weight cutoff centrifugal filter. Full length
QC- and Boc-sfGFP-His6 was separated from amber termination product via FPLC purification
according to the protocol described in section 4.4.3.2. Purified proteins were further

characterized by high resolution mass spectrometry (Figure S1).
4.5 Cloning to exchange His6 tag for FLAG tag
The sfGFP gene from psfGFP150TAGHis6PylT (sequence below, plasmid obtained from
Jason Chin Lab, Medical Research Council, Cambridge, UK) was amplified using forward
primer and reverse primers purchased from Elim Biopharma with PCR. Forward primer: 5'-
CAGGAGGAATTAACCATGGTTAGCAAAG-3',
reverse
primer:
5'-
ATCGTCGTCCTTGTAGTCGCTGCCTTTATACAGTTCATCCATACC-3'. The PCR reaction
was purified by agarose gel electrophoresis, and the band corresponding to the PCR product was
excised from the gel and purified using a Zymoclean Gel DNA Recovery Kit (Zymo Research)
to give gene sfGFP150TAGFLAG1. Gene sfGFP150TAGFLAG1 was further amplified with
forward and reverse primers to form full-length insert sfGFP150TAGFLAG2. Forward primer:
as
above,
reverse
primer:
5'-
TTCGCTCGAGCTTTACTTGTCATCGTCGTCCTTGTAGTCGCTG-3'. The insert was
isolated by agarose gel electrophoresis as described above, and the gene ends were digested for 1
h 45 min at 37 ºC with XhoI and NcoI-HF (both from New England Biolabs) and purified using
a Zymo DNA Clean & Concentrator Kit (Zymo Research). Vector psfGFP150TAGHis6PylT was
linearized with XhoI and NcoI-HF (both from New England Biolabs) at 37 ºC for 1 h 40 min,
and linearized vector was isolated by agarose gel electrophoresis and treated with Antarctic
phosphatase (New England Biolabs) at 37 ºC for 1 h followed by purification with a Zymo DNA
Clean & Concentrator Kit. The full-length sfGFP150TAGFLAG insert was ligated to linearized
vector with T4 DNA ligase (New England Biolabs) at rt for 50 min to form
psfGFP150TAGFLAGPylT.
One Shot, chemically competent DH5 E. coli (Life Technologies) were transformed
with 2 L ligation reaction containing the full plasmid psfGFP150TAGFLAGPylT. E. coli were
transformed by heat shock (30 sec, 42 ºC, 2 min, wet ice) and recovered in 250 L super optimal
broth with 20 mM glucose (S.O.C. medium) for 35 min at 37 ºC and 225 rpm. Cells were then
plated on tetracycline LB agar plates (~100–200 L S.O.C. culture per plate) and incubated at 37
ºC overnight. Single colonies were selected for inoculation of 5 mL liquid LB media cultures
(supplemented with tetracycline) and cultures were incubated overnight at 37 ºC and 225 rpm.
The resulting cultures were used to prepare psfGFP150TAGFLAGPylT plasmid DNA using a
Qiagen Miniprep kit. The sfGFP150TAGFLAG portion of the plasmid was sequenced using
forward
and
reverse
primers
by
Elim
Bioharma.
reverse
Forward
primer:
5'-
5'-
CAGGAGGAATTAACCATGGTTAGCAAAG-3',
primer:
TTCGCTCGAGCTTTACTTGTCATCGTCGTCCTTGTAGTCGCTG-3'.
4.6 1H NMR Studies of Photocleavage of 10
QC-Ni-bis(dithiolene) adduct 10 was prepared as reported by Sletten et al.15 A 5 mM
solution of 10 in CDCl3 was added to 5 NMR tubes (600 µL each) wrapped in foil. An NMR was
taken of one sample (0 min), and the others were placed in a dark container and irradiated with
365 nm light from a handheld UV lamp (Spectroline model ENF-260c, 254/365 nm). At 5, 10,
15, and 25 minutes a sample was removed from the box and a 1H NMR spectrum was taken. The
amount of product was determined by measuring the integration of a product peak and
comparing it to a starting material peak. This procedure was replicated 3 times, and the amount
of product formation at each time point was averaged.

4.7 Preparation of QC-BSA
BSA (10 mg) was dissolved in PBS (0.5 mL) and to this mixture was added p-nitrophenyl
carbonate 5 (1.4 mg, 0.056 mmol) dissolved in DMSO (83.3 µL) with a small amount of DMF
(16.7 µL). An addition 200 uL of DMSO was added and the resulting yellow mixture was
incubated at 22 °C for 3 h with gentle agitation. The protein was purified in illustra NAP-5
columns (GE Healthcare Life Sciences, Pittsburgh) as per manufacturer’s protocol, eluting in
PBS. Protein concentration was measured by Pierce BCA Protein Assay (Thermo Fisher
Scientific, Waltham). Resulting protein solutions were stored at 4 °C until use.
4.8 Photocleavage of the QC/nickel bis(dithiolene) adduct on a protein
A solution of QC-BSA or BSA (80 µL, 1 mg/mL in PBS) was combined with a solution
of nickel bis(dithiolene) biotin 8 (80 µL of 100 µM in PBS, 50 µM final concentration 8).
Reactions were incubated in the dark at room temperature for 30 min, then quenched via addition
of 4 µL of a QCOAc in DMSO (200 mM, 100 equiv QCOAc relative to 8). Each reaction was
diluted with 300 µl PBS, then transferred to an Amicon Ultra centrifugal filter (10k MW cutoff)
and centrifuged at 14,000 x g for 5 min. Concentrated reactions were diluted with 350 µl PBS
followed by centrifugation in a centrifugal filter at 14,000 x g for 5 min. This wash procedure
was repeated one additional time to give the resulting proteins as solutions in ~50 µl PBS.
In fresh 1.5 mL eppendorf tubes, 50 µL of each sample was diluted with 380 µL PBS.
The resulting solutions were aliquoted into borosilicate glass tubes (50 µL protein solution/tube).
The protein in each tube was then diluted with an additional 550 µl PBS and 15 µl 200 mM
QCOAc in DMSO to a final QCOAc concentration of 5 mM (solution is cloudy due to minimal
solubility of QCOAc in PBS at this concentration). Tubes were irradiated at 365 nm with a
handheld UV lamp or were irradiated with ambient light for 0, 10, 30, or 60 min (0 min samples
were kept in the dark for the duration of the experiment).
From each sample, 400 µL was transferred to a 0.5 ml Amicon Ultra centrifugal filter and
centrifuged at 14,000 x g for 5 min. The remaining sample (~200 µL) was then added to each
filter and centrifuged at 14,000 x g for 5 min. PBS (350 µL) was added to each sample and they
were centrifuged at 14,000 x g for 5 min. This process was repeated one additional time. The
samples were subjected to western blot using an α-biotin HRP antibody were performed
according to the general procedures.
4.9 Purification of biotin-labeled protein from streptavidin resin by photocleavage
QC-BSA or BSA (100 µL, 1 mg/mL in PBS) was mixed with a solution of 8 (100 µL, 50
µM in PBS) and gently agitated for 14 h in the dark. In purification experiments where cell
lysate was used, K3Fe(CN)6 (10 µL of 22 mM in ddH2O) and bacterial cell lysate (10 µL, 4.94
mg/mL) were added sequentially prior to the addition of 8. QCOAc (2.5 µL, 200 mM in DMSO)
was added to quench the reaction. The mixtures were transferred to Amicon Ultra centrifugal
filters, 10k MW cutoff, containing 200 µL PBS, then centrifuged at 14000 x g, 5 min. PBS (350
µL) was added and the samples were spun at 14000 x g, 5 min. This was repeated once more
before the samples were collected, spun 1000 x g, 2 min.
For each sample, Pierce™ Streptavidin Agarose (100 µL, ThermoFisher Scientific) was
added to a Micro Bio-Spin™ Chromatography Column (Bio-Rad, Hercules). The storage buffer
was removed via centrifugation 500 x g, 1 min. The gel was washed three times with PBS (100
µL), and centrifuged 500 x g, 1 min each wash. The samples were then loaded onto the beads
and allowed to incubate in the dark for 30 min. The supernatant was removed with

centrifugation, 500 x g, 1 min (Wash 1). The beads were washed 6 more times with PBS (50 µL)
and collected by centrifugation, 500 x g, 1 min, (Washes 2-7). Washes 2 and 3, 4 and 5, and 6
and 7 were combined as pairs. Each sample was resuspended in PBS (50 µL) and irradiated with
365 nm light from a handheld UV lamp (Spectroline model ENF-260c, 254/365 nm) in a foil-
lined container for 5 min. The supernatant was collected by centrifugation (500 x g, 1 min,
Elution 1). This irradiation procedure was repeated three more times (Elutions 2-4). Finally, the
beads were stripped of any remaining bound protein with the procedure above with five washes
of 8 M guanidine HCl (pH 1.5, 50 µL each). These were combined and transferred to an Amicon
Ultra spin concentrator (10k MW cutoff) and centrifuged at 14000 x g, 5 min. The concentrated
solution was washed twice with PBS (250 µL) before being collected (Beads, lane B). The
samples were then separated by gel electrophoresis and visualized with Coomassie staining.
Acknowledgments
We thank Jason Chin for providing plasmids encoding the pyrrolysine synthetase and
transferase. C.G.G was supported by a predoctoral fellowship from the National Science
Foundation. E.M.S. was supported by a predoctoral fellowship from the American Chemical
Society. This work was supported by a grant from the National Institutes of Health (GM058867)
to C.R.B.
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The Quadricyclane Ligation
Handhe
Ar
Ar’
Ar
S S
Ni
S S
O
Ar’
Covalent Adduct
Formation
protein
of interest