Cucurbit[6]uril-Promoted Click Chemistry for Protein Modification

Article abstract of DOI:10.1021/jacs.7b05164

Azide-alkyne cycloaddition is a powerful reaction for the formation of bioconjugates. When catalyzed by Cu(I) or strain promotion, this cycloaddition is considered to be a click reaction with many applications in chemical biology and materials science. We report a new type of azide-alkyne click chemistry for the synthesis of protein conjugates using cucurbit[6]uril (CB6) supramolecular chemistry. CB6-promoted azide-alkyne cycloaddition has been previously used for the synthesis of rotaxanes but has not been applied to the development of complex bioconjugates. By developing new substrates for CB6 click that do not contain any cross-reactive functional groups and by optimizing reaction conditions, we converted CB6 click chemistry from a rotaxane synthesis tool into a useful bioconjugation technique. Using these new parameters, we synthesized a series of protein conjugates including protein-peptide, protein-DNA, protein-polymer, and protein-drug conjugates. We further demonstrated that CB6 click can be used in conjunction with strain-promoted azide-alkyne cycloaddition to generate distinct bioconjugates in protein mixtures. CB6 click is a promising new reaction for the development of protein conjugates and can be applied toward the synthesis of complex biomaterials for a wide range of applications.

Full text of DOI:10.1021/jacs.7b05164

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Article
Cucurbit[6]uril-promoted click chemistry for protein modification
Joel A. Finbloom, Kenneth Han, Clancy C. Slack, Ariel L Furst, and Matthew B Francis
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b05164 • Publication Date (Web): 26 Jun 2017
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Journal of the American Chemical Society
Cucurbit[6]uril-promoted click chemistry for protein modification
Joel A. Finbloom,† Kenneth Han,† Clancy C. Slack,†, ‡ Ariel L. Furst,† and Matthew B. Francis.*,†, ‡
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†Department of Chemistry, University of California, Berkeley ‡Materials Sciences Division, Lawrence Berkeley National
Laboratories, Berkeley, California 94720.
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ABSTRACT: Azide-alkyne cycloaddition is a powerful reaction for the formation of bioconjugates. When catalyzed by Cu(I) or
strain promotion, this cycloaddition is considered to be a “click” reaction with many applications in chemical biology and materials
science. We report a new type of azide-alkyne click chemistry for the synthesis of protein conjugates using cucurbit[6]uril (CB6)
supramolecular chemistry. CB6-promoted azide-alkyne cycloaddition has been previously used for the synthesis of rotaxanes, but
has not been applied to the development of complex bioconjugates. By developing new substrates for CB6 click that do not contain
any cross-reactive functional groups and by optimizing reaction conditions, we converted CB6 click chemistry from a rotaxane
synthesis tool into a useful bioconjugation technique. Using these new parameters, we synthesized a series of protein conjugates
including protein-peptide, protein-DNA, protein-polymer, and protein-drug conjugates. We further demonstrated that CB6 click can
be used in conjunction with strain-promoted azide-alkyne cycloaddition to generate distinct bioconjugates in protein mixtures. CB6
click is a promising new reaction for the development of protein conjugates, and can be applied toward the synthesis of complex
biomaterials for a wide range of applications.
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Since Mock’s discovery and subsequent kinetic studies,
INTRODUCTION
CB6-AAC has seen use for the synthesis of pseudorotaxanes
and rotaxanes.13,14 In particular, Stoddart and coworkers re-
Protein modification with synthetic molecules allows for the
development of useful analytical platforms, therapeutic agents,
and imaging tools with functions that cannot be achieved by
the individual components alone.1–3 The highly valuable set of
chemical reactions used to functionalize biomolecules must
take place in aqueous systems at near-neutral pH and with low
concentrations of substrates.1–3 Of these strategies, the highly
exothermic Huisgen 1,3 dipolar azide-alkyne cycloaddition
(AAC) has emerged as a particularly powerful reaction to
couple two components together under the most demanding of
circumstances.4 When catalyzed by Cu(I), Cu-AAC is consid-
ered to be a “click” reaction,5 owing to its speed of reactivity,
high conversion rates, and virtually complete functional group
tolerance.5,6 Application of the copper catalyst also establishes
the 1,4-substitution pattern of the triazole product.
cently reported a cooperative capture method for the synthesis
of CB6 rotaxanes, in which a cyclodextrin or pillarene host
forms a hydrogen bonding network with the CB6.14 This tem-
plating of substrates increases CB6 solubility in pure water
and allows for quantitative rotaxane synthesis on the minutes
to hours timescale.14–16
Since its first report in 2002, Cu-AAC has been widely used
in the fields of materials chemistry and chemical biology.7
However, copper can perturb protein function, cause DNA
damage, and is toxic to cells, rendering it less desirable for
many biological applications.7,8 As an alternative, Bertozzi and
coworkers have developed a strain-promoted azide-alkyne
cycloaddition (SP-AAC) that has suitable kinetics and reac-
tivity for biological applications without the need for copper
as a catalyst.9 This reaction is typically performed between an
azide installed on the biomolecule and a cyclooctyne on the
synthetic substrate. This method overcomes many of the bio-
logical limitations of Cu-AAC, albeit through the use of sub-
strates that can be limited by instability, reduced water solubil-
ity, and synthetic accessibility.7,8
An unexplored bioconjugation alternative for SP-AAC is
the cucurbit[6]uril-promoted AAC (CB6-AAC), Figure 1. In
this reaction, CB6 forms a heteroternary complex with propar-
gylamine and azidoethylamine derivatives, whereby the proto-
nated amine moieties form a hydrogen bonding network with
the carbonyl portal of the CB6. This aligns the azide and al-
kyne components to facilitate triazole product formation. The
concept of CB6-AAC was first reported by Mock and cowork-
ers in 1983, and affords the 1,4-regioisomer of the triazole
product selectively.10–12
Figure 1. A protein bioconjugation strategy using CB6 click
chemistry. An azidoethylpiperidine can be installed via NHS ester
chemistry, providing a functional handle for subsequent modifica-
tion via the CB6 click reaction.
Many features of the CB6-AAC reaction suggest it will
have the high functional group tolerance and water compatibil-
ity that bioconjugation strategies require. However, to date it
has not been explored in the context of biomolecule modifica-
tion. Herein we report the use of CB6 click chemistry as an
efficient strategy for the synthesis of highly functionalized
protein conjugates. We first adapted the reactive substrates to
allow facile introduction on biomolecules, and optimized the
conditions to produce protein conjugates in hours using rela-
tively low equivalents of the synthetic coupling partners.
When this reaction is performed with bulky substrates, pro-
tein-rotaxane conjugates are synthesized due to the formation
of a mechanical bond14 between the CB6 host and the triazole
axle of the conjugate (Figure 1). Protein-rotaxane conjugates
are a relatively unexplored class of protein-based materials,
with our lab recently reporting on the first synthesis of such
conjugates using thiol-maleimide chemistry.17 Using this op-
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timized CB6 click reaction, we synthesized a series of protein product existed as a stable pseudorotaxane. In line with this
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conjugates, ranging in substrate scope from small molecule
and synthetic polymer coupling partners to biomolecules, such
as DNA and peptides.
interpretation, subsequent incubation with a known CB6 guest
(spermine) caused a mixture of TMV-CB6-triazole and TMV-
triazole peaks to be observed by LC/MS (Supporting Infor-
mation Figure S1). Although CB6 is retained on the protein
conjugate product, CB6 is not anticipated to interfere with any
downstream applications. Cucurbiturils have been used in a
wide range of drug delivery and other biological applications,
and no toxicity or unwanted downstream effects have been
observed.12 Additionally, we have previously demonstrated
that the CB6 moiety does not interfere with enzyme-substrate
binding, even when the substrate is located on the CB6 rotax-
ane axle.19 Importantly, no cyclodextrin or secondary supra-
molecular host other than CB6 was required in this reaction, in
contrast to the cooperative capture method previously report-
ed.14 This may be due to the substantially increased CB6 solu-
bility in buffer, relative to the pure water solvent that was used
previously for rotaxane synthesis.
RESULTS AND DISCUSSION
Development of a CB6 click bioconjugation strategy. In
order to apply CB6 click for bioconjugation, new substrates
had to be developed that did not contain cross-reactive amines.
While azidoethylpyridinium substrates were previously used
for CB6 click in the synthesis of rotaxanes,14 they do not offer
the synthetic modularity desired for bioconjugation. We there-
fore used previously unreported piperidine derivatives as the
substrates. The synthesis of azidoethylpiperidine NHS ester 1
provided an azide CB6 click handle that can be installed onto
proteins easily using NHS ester chemistry (Figure 2a). This
would not be possible with the reactive secondary amines of
azidoethylamine derivatives previously used in the synthesis
of CB6 rotaxanes.
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Figure 2. Development of CB6 click as a bioconjugation strategy.
(a) NHS ester 1 can be reacted with free amines on proteins to
install click handles. Following incubation with a propargylamine
derivative and CB6, the protein-CB6-triazole product is formed.
(b) RR-TMV-T104K double disks were used as a model protein
for reaction development studies. TMV assemblies were modified
to ~60% (20 additions per double disk) using 10 equiv of NHS
ester 1, as observed by LC/MS. (c) Virtually complete conversion
to the triazole-CB6 product was observed after 24 h of incubation
with 10 equiv of CB6 and propargylamine at 37 ºC.
Optimization studies were performed on a recombinantly
expressed, double arginine mutant of the tobacco mosaic virus
capsid protein (RR-TMV) that assembles from 34 monomers
into a stable disk-shaped nanocarrier for drug delivery applica-
tions.18 The specific protein used for these studies also had a
T104K mutation that, after double disk assembly, provided
modification sites on the protein surface. NHS ester 1 was
conjugated to the RR-TMV-T104K coat proteins with ~60%
conversion using 10 equiv of NHS ester (Figure 2b). We then
monitored via LC/MS the disappearance of the TMV-pipN3
peak and the emergence of a new TMV-CB6-triazole peak for
subsequent CB6 click optimization studies. Incubation of
TMV-pipN3 with 10 equiv of CB6 and propargylamine at 37
°C in 50 mM BisTRIS, pH 6, led to approximately 95% con-
version after 24 h, as measured by comparing the TMV-pipN3
peak to the newly formed TMV-CB6-triazole peak (Figure
2c). Interestingly, the observed mass included a CB6 molecule
that was still associated with the protein, suggesting that the
Figure 3. Optimization studies of CB6 click chemistry on TMV
coat protein disks modified with an azidoethylpiperidine handle.
(a) Buffer and pH conditions were screened for CB6 click reac-
tions on TMV. All reactions were performed with 5 equiv of CB6
and 10 equiv of propargylamine for 24 h at 37 °C. (b) The reagent
equivalents were screened for the CB6 click reaction on TMV. All
reactions were carried out for 6 h at 37 °C in 50 mM BisTRIS, pH
6. (c) A time course study was performed for the CB6 click reac-
tion on TMV. Both reactions were conducted with 5 equiv of CB6
and 10 equiv of alkyne in 50 mM BisTRIS, pH 6.
Studies of buffer conditions with CB6 click revealed Bis-
TRIS to be an ideally suited buffer for this reaction, leading to
near-quantitative conversion within 24 h when the components
were incubated at 37 °C (Figure 3a). While the reaction does
proceed to a degree in both sodium phosphate (NaPhos) and
potassium phosphate (KPhos) buffers, the presence of large
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concentrations of small ions impedes the speed of the CB6
click reaction. Both Na+ and K+ are known to interact with the
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carbonyl portal of cucurbiturils.20,21 Additionally, higher over-
all buffer strengths may lead to an inhibitory effect on the
speed of the reaction, again due to unwanted interactions be-
tween the buffer ions and the CB6 ring. The reaction proceeds
better at pH 6 vs 7, as lowering the pH increases the ratio of
protonated to non-protonated amines on the substrates. These
interactions are presumably essential for CB6 binding and
subsequent triazole formation. A study of substrate equivalents
revealed that between 5 and 20 equiv (50 – 200 µM) of pro-
pargylamine and CB6 were suitable to achieve good levels of
conversion after 6 h (Figure 3b). A time course study was run
at both 37 °C and ambient temperature. Near quantitative con-
version was achieved at 37 °C, with high degrees of conver-
sion (~85%) still being observed at ambient temperatures
(Figure 3c). In all conditions tested, the CB6 click substrates
displayed excellent solubility. This enhanced water solubility
over the cyclooctyne SP-AAC substrates could lend to in-
creased ease of protein modification.
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PEGylation of proteins via CB6 click chemistry. Poly-
mer-protein conjugates are a class of materials that have seen
increasing use in pharmaceuticals and drug delivery applica-
tions.22,23 Polyethylene glycol (PEG) is the most commonly
used polymer in the synthesis of these materials, and was
therefore chosen as a model polymer substrate for protein con-
jugation via CB6 click chemistry. To facilitate substrate de-
sign for this reaction, propargylpiperidine NHS ester 2 was
synthesized (Figure 4a). This substrate allows for the conver-
sion of any amine-bearing small molecule or polymer into a
reactive alkyne handle for CB6 click chemistry. Additionally,
this substrate can be attached to proteins to install a propargyl
CB6 click handle instead of the azide handle 1. Lysozyme was
reacted with NHS ester 1, leading to 2-5 modifications of ly-
sozyme with the azidoethylpiperidine handle (Figure 4c). Ly-
sozyme-pipN3 was then reacted with 10 equiv of CB6 and 10
equiv of PEG5k-propargylpiperidine 3a for 24 h at 37 °C. Ap-
proximately 34% PEGylation was observed via gel electro-
phoresis (Figure 4d). PEGylation was tested with other pro-
teins and similarly modest levels of conversion were observed.
Figure 4. PEGylation of lysozyme via CB6 click. (a) Methoxy-
terminated PEG5k-amine was reacted with NHS ester 2 to form
PEG5k-propargylpiperidine 3a, while modification with propar-
gylglycine led to PEG5k-propargylglycine 3b. (b) Incubation of
lysozyme-pipN3 with 3a or 3b in the presence of CB6 leads to
modification of the protein. (c) Lysozyme was treated with 10
equiv of NHS ester 1 to install an azide handle for subsequent
CB6 click chemistry. (d) Incubation of lysozyme-pipN3 with 10
equiv of CB6 and 10 equiv of 3a or 3b in 50 mM BisTRIS, pH 6
for 24 h at 37 °C led to ~34% PEGylation with 3a and ~98%
PEGylation with 3b, as observed by gel electrophoresis.
Protein-peptide and protein-DNA conjugation via CB6
click chemistry. Bioconjugates that combine different classes
of biomolecules are valuable in the fields of biomaterials and
chemical biology. As such, it was important to determine that
the CB6 click tolerates peptide and DNA substrates for protein
conjugation. To do this, a heptameric peptide 4 containing an
RGDS sequence was synthesized via solid phase peptide syn-
thesis (Figure 5a). RGD peptides bind to the integrin recep-
tors of cells, and are widely used for targeted delivery of
chemotherapeutics.25 N-propargylglycine was incorporated at
the N terminus of peptide 4 as a reactive handle for CB6 click
chemistry. We previously used N-propargylglycine to synthe-
size rotaxanes bearing a protease cut sequence.19 However,
those syntheses were enhanced through cyclodextrin-
templated CB6 click, and it remained to be determined if CB6
click could facilitate conjugation between peptide and protein
substrates. Treatment of TMV-pipN3 with 10 equiv of peptide
4 and 10 equiv of CB6 led to 70% conversion to the protein-
peptide conjugate after 24 h at 37 ºC (Figure 5b).
The limited modification may be due to the presence of two
piperidine substrates, rather than the azidoethylpiperidine and
the propargylamine substrates used in optimization experi-
ments. To test this hypothesis, another PEG substrate 3b was
synthesized using commercially available propargylglycine
(Figure 4a). Significantly enhanced modification of lysozyme
was observed after incubation with CB6 and PEG 3b, with
near quantitative conversion to multiple PEGylation products
closely resembling the mass spectrum ladder of the az-
idoethylpiperidine modification of lysozyme. These yield dif-
ferences reflect a known trend in binding efficiencies of pro-
pargylamine derivatives for CB6.14,24 When a single alkyl sub-
stitution occurs on the propargylamine derivative leading to a
secondary amine substrate, only minimal perturbation in CB6
binding affinities is observed. However, when two alkyl sub-
stitutions occur, the subsequent tertiary amine displays over
1000-fold weaker binding affinities for CB6.24 Therefore, CB6
click substrates containing the propargylglycine derivative,
rather than the propargylpiperidine derivative, lead to excel-
lent conversions to the protein-CB6-triazole products.
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Figure 6. Using CB6-click to access protein-DNA conjugates. (a)
A 22 nt 5'-aminoDNA strand was reacted with NHS ester 2 to
form DNA 5 for subsequent CB6 click chemistry onto proteins.
(b) Lysozyme-pipN3 was incubated with 1 equiv of 5 and CB6 for
24 h at 37 °C. Approximately 50% modification was observed by
gel electrophoresis.
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CB6 click for the development of protein-drug conju-
gates. Protein-drug conjugates are becoming increasingly im-
portant in the areas of medicine and chemical biology. For
example, antibody-drug conjugates are used to target chemo-
therapeutic drugs to sites of interest.28,29 Additionally, protein-
based nanomaterials, such as the recently reported RR-TMV
double disk, can be used as nanocarriers with potential appli-
cations in drug delivery.18,30 Using CB6 click chemistry, we
developed protein-drug conjugates using the RR-TMV double
disk and doxorubicin (DOX) as a model chemotherapeutic.
DOX-SS-propargylamine 7 was synthesized from DOX-SPDP
6 and N-propargylcysteamine to install a CB6 click handle
onto DOX via a redox-cleavable disulfide linker (Figure 7a).
Doxorubicin disulfide derivatives have been previously syn-
thesized through reaction at the glycosamine of the drug, and
the cleaved DOX-SH have been shown to retain activity
against cancer cells.31,32
Figure 5. Using CB6-click to prepare protein-peptide conjugates.
(a) An RGDS peptide 4 was synthesized, bearing a propargylgly-
cine amino acid for CB6 click chemistry onto proteins. (b) TMV-
pipN3 was incubated with 10 equiv of 4 and CB6 for 24 h at 37
°C. Approximately 70% modification was observed by LC/MS.
Protein-DNA conjugates are used for many applications,
including therapeutic targeted delivery with aptamers26 and the
immobilization of proteins through DNA complementation.27
We therefore sought to determine if CB6 click would be a
suitable reaction for protein-DNA conjugation. A 22-base
oligomer ssDNA-amine bearing a binding motif for the TATA
binding protein (TBP) transcription factor was reacted with
propargylpiperidine-NHS 2 to yield DNA 5 with a reactive
alkyne for CB6 click (Figure 6a). Lysozyme was modified
with 1 equiv of NHS ester 1 to yield lysozyme-pipN3 with
predominantly a single azidopiperidine modification (Support-
ing Information Figure S2). Lysoyzme-pipN3 was incubated
with 1 equiv of 5 and 1 equiv of CB6. After 24 h at 37 ºC,
approximately 50% conversion to singly and doubly modified
lysozyme-CB6-DNA was observed by gel electrophoresis
(Figure 6b). When TMV was modified with DNA 5, only
modest modification was observed (Supporting Information
Figure S3), likely due to the charge repulsion present between
the DNA strands on the tightly packed TMV double disks.
These results demonstrate that CB6 click chemistry can be
used with a variety of substrates, ranging from small mole-
cules and polymers, to complex biomolecules, such as pep-
tides and DNA.
DOX-SS-propargylamine 7 was reacted with TMV-pipN3
and CB6 to synthesize TMV-CB6-DOX conjugates (Figure
2e). Interestingly, the reaction proceeded to 99% conversion
after 1 h incubation at 37 °C while only using 2 equiv of CB6
and 7 (Figure 7b,c). Additionally, this reaction proceeded
quantitatively in both BisTRIS and phosphate buffers. To test
the modularity of this reaction, human serum albumin (HSA)
was also conjugated to doxorubicin via CB6 click chemistry.
HSA-doxorubicin conjugates are used clinically, as are other
HSA-drug conjugates,33,34 and therefore HSA was an im-
portant protein system to test with this chemistry. HSA-DOX
conjugation via CB6 click also demonstrated the enhanced
reactivity observed with the TMV-DOX conjugation (Support-
ing Information Figure S4). This enhancement in reactivity
may be due to a hydrogen bond network formed between the
DOX and the CB6, similar to the cooperative capture rotaxane
synthesis using cyclodextrins or pillarenes and CB6.14
To determine if hydrogen bonding from the anthracene
core of the DOX led to the rate enhancement, 3,4-
dihydroxybenzyl propargylamine was synthesized and reacted
with TMV-pipN3. Interestingly, a 2-fold enhancement in mod-
ification was observed for this substrate when compared to
propargylamine (Supporting Information Figure S5). It is
therefore likely that the phenol groups on the DOX align with
the CB6 carbonyl portal to form a hydrogen-bond network,
leading to the observed rate enhancement.
The TMV-CB6-DOX conjugates were treated with DTT to
cleave the disulfide linkage and release the DOX-SH payload.
The release of ~90% DOX-SH was achieved after 2 h at ambi-
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ent temperature, as monitored by LC/MS (Figure 7d). The pargylamine derivative of Alexa Fluor 488 was synthesized.
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CB6 click chemistry is therefore a particularly promising reac-
tion for the synthesis of protein-conjugates, as the presence of
the CB6 does not interfere with release of the chemotherapeu-
tic cargo.
N-propargylcysteamine was reacted with AF488-maleimide to
form the AF488-propargylamine product 8 (Figure 8a). The
propargylcysteamine derivative allows for facile installation of
CB6 click handles onto any maleimide molecule of interest.
The TMV disks were reacted with AF488-propargylamine 8
and CB6, followed by reaction with AF594-maleimide (Figure
8b). This modification strategy yielded TMV disks that were
orthogonally labeled with AF488 donors at the 104K position
and AF594 acceptors at the 123C position (Supporting Infor-
mation Figure S6). Fluorescence spectroscopy revealed a
strong FRET response (54% FRET efficiency as measured by
quenching of AF488 fluorescence) for the TMV disks upon
excitation at 450 nm (Figure 8b).
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Figure 8. Development of FRET system using CB6 click chemis-
try. (a) Alexa Fluor 488 C5 maleimide was reacted with propar-
gylcysteamine to form AF488-propargylamine 8. (b) RR-TMV
disks were modified with Alexa Fluor 488 (AF488) donors via
CB6 click chemistry, followed by modification with Alexa Fluor
594 maleimide acceptors. Excitation at 450 nm led to a significant
FRET response (54% efficiency) of the TMV-CB6-AF488-AF594
conjugates, as determined by comparing the emission spectra.
Figure 7. Synthesis of doxorubicin-protein conjugates via CB6
click chemistry. (a) DOX-SS-propargylamine 7 was synthesized
to contain a CB6 click handle and a cleavable disulfide linker for
drug release. (b) TMV double disks were modified with az-
idoethylpiperidine at the T104K sites, followed by subsequent
modification with CB6 and 7. (c) RR-TMV assembles into double
disks composed of 34 monomers. (d) Model of a TMV-CB6-
doxorubicin (DOX) monomer (based on PDB ID 1EI7). (e) Incu-
bation of TMV-pipN3 with 2 equiv of CB6 and 7 for 1 h at 37 °C
led to 99% conversion to the TMV-CB6-DOX product. (f) Cleav-
age and release of ~90% DOX-SH was achieved after 2 h of incu-
bation with 5 mM DTT.
We next sought to take advantage of the inherent substrate
specificity of the CB6 click reaction to demonstrate its com-
plementarity to the SP-AAC reaction. Since CB6 only binds to
propargylamine and azidoethylamine derivatives, it should
therefore be possible to conduct the CB6 click reaction in the
presence of an alkyl azide on the protein surface. To test this,
azidopentanoic acid NHS ester was synthesized37 and reacted
with TMV to install an alkyl azide handle (TMV-N3, Support-
ing Information Figure S7). TMV-N3 was then mixed 1:1 with
TMV-pipN3 and reacted with CB6 and 8 (Figure 9a). Com-
plete conversion to the TMV-CB6-AF488 product was ob-
served, while no side reactions between 8 and TMV-N3 were
observed (Figure 9b). Subsequent incubation with DBCO-Cy5
modified the TMV-N3, forming two separate protein conju-
gates in one pot (Figure 9c). Incubation of the starting TMV
mixture with DBCO-Cy5 alone led to modification of both
TMV-pipN3 and TMV-N3 (Supporting Information Figure S9),
as both of these substrates are reactive toward strained al-
kynes. Taken together, these results demonstrate that the CB6
click is a bioorthogonal reaction that offers complementarity
to the SP-AAC for the separate modification of protein mix-
tures in solution.
The orthogonality of CB6 click for protein modification.
It is often important in bioconjugate synthesis to have multiple
functional handles for two orthogonal reactions to occur. One
of the most common bioconjugation techniques is thiol-
maleimide coupling, and we therefore sought to confirm that
the CB6 click reaction is orthogonal to reactive thiols. To test
this, an RR-TMV mutant bearing a reactive cysteine at posi-
tion 123 (123C) and the reactive lysine at position 104 (104K)
was reacted with NHS ester 1 to install a pipN3 handle for CB6
click (Supporting Information Figure S6). We have previously
reported on the dual-modification of TMV disks with FRET
dyes for light harvesting applications.35,36 To make a FRET
pair using the CB6 click and maleimide-thiol reactions, a pro-
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Journal of the American Chemical Society
Page 6 of 17
tate site-selective protein modification, and expand CB6 click
chemistry for in vivo applications in drug delivery and disease
imaging.
1
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3
4
5
6
7
8
9
ASSOCIATED CONTENT
Supporting Information
Experimental details, synthetic procedures,38–40 characterization of
synthetic compounds, LC/MS of spermine competition, LC/MS of
lysozyme-pipN3, DNA modification of RR-TMV, LC/MS of
HSA-DOX conjugates, modification of TMV with 3,4 dihy-
droxybenzyl propargylamine, LC/MS of TMV FRET pair, LC/MS
of TMV-N3, LCMS of SP-AAC and CB6-AAC of TMV mixture.
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The Supporting Information is available free of charge on the
ACS Publications website.
AUTHOR INFORMATION
Corresponding Author
* mbfrancis@berkeley.edu
Author Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENT
M.B.F. acknowledges the U.S. Department of Energy, Office of
Science, Basic Energy Sciences, Materials Sciences and Engineer-
ing Division, under Contract No. DE-AC02-05CH11231 for reac-
tion development, as well as the Hana Jabsheh Initiative for the
doxorubicin studies. J.A.F. was supported under contract FA9550-
11-C-0028 and awarded by the Department of Defense, Air Force
Office of Scientific Research, National Defense Science and En-
gineering Graduate (NDSEG) Fellowship, 32 CFR 168a. A.L.F.
was supported by the Arnold O. Beckman Foundation.
Figure 9. SP-AAC and CB6-AAC complementarity for the modi-
fication of protein mixtures. (a) Two TMV populations bearing
pipN3 or alkyl-N3 handles were mixed together for modification
via CB6-AAC followed by SP-AAC. (b) LC/MS spectra revealed
specific modification of the azidopiperidine handles with CB6 and
propargylamine-AF488 8, while no TMV-N3 modification was
observed. (c) Subsequent reaction of the TMV mixture with
DBCO-Cy5 led to specific TMV-N3 labeling via SP-AAC. En-
larged versions of the LC/MS traces appear in Supporting Infor-
mation Figure S8.
ABBREVIATIONS
CB6, cucurbit[6]uril; AAC, azide-alkyne cycloaddition; TMV,
tobacco mosaic virus; DOX, doxorubicin; LC/MS, liquid chroma-
tography/mass spectrometry; AF, Alexa Fluor.
CONCLUSIONS
REFERENCES
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mine and azidoethylamine, CB6 click handles can be easily
installed onto virtually any substrate of interest. The reaction
itself is operationally simple and occurs under sufficiently
mild conditions to maintain biomolecular structures. We tested
this system with a variety of proteins and coupling partners,
and demonstrated its utility for the synthesis of complex pro-
tein bioconjugates. The reaction is particularly effective for
the installation of small molecules and peptides, and still pro-
vides good yields when large macromolecules are used. Im-
portantly, this strategy can achieve modification of specific
subsets of azides in the presence of others.
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Authors are required to submit a graphic entry for the Table of Contents (TOC) that, in conjunction with the manuscript title,
should give the reader a representative idea of one of the following: A key structure, reaction, equation, concept, or theorem,
etc., that is discussed in the manuscript. Consult the journal’s Instructions for Authors for TOC graphic specifications.
ACS Paragon Plus Environment

Journal of the American ChemicPalaSgeoc8ieotfy17
R
N
1
2
3
4
5
6
7
H2
O
O
O
O
O
O
N
N
N
N
N
N
O
O
N
N
H
N
H
N
N
N
N
N
N
N
N
R
N3
H
N
H
N
N
N
N
N
N
N
O
O
H2
O
N
N
N N
O
N
N
O
ACS Paragon Plus Environment
O
CB6
Cucurbit[6]uril (CB6)

O
a
PaJgoeu9rnoaf
lO1o7f the American Chemical Society
N
O
O
N3
NH2
O
NH2
N
O
1
N
H
N
H
N
N
N
H
NH
N
N
H
NH3
H
N3 CB6
N
O
RR-TMV-T104K
1
2
c
b
TMV-pipN3
(17755)
TMV-CB6-triazole
(18807)
3
4
5
6
7
TMV
(17575)
TMV
(17575)
TMV-pipN3
(17755)
817000
9
18000
mass (Da)
19000
17000
18000
mass (Da)
19000
10
11
12
13
14
15
16
17
ACS Paragon Plus Environment

a
Journal of the American ChemiPcalgSeo1c0ieotfy17
100
80
60
40
20
1
2
3
4
0
KPhos NaPhos KPhos KPhos BisTRIS BisTRIS BisTRIS
100 mM 100 mM 100 mM 10 mM 100 mM 50 mM 10 mM
pH 7
pH 6
pH 6
pH 6
pH 6
pH 6
pH 6
5
6
7
8
9
10
11
b
100
80
60
40
20
0
CB6: 1 equiv 5 equiv 5 equiv 10 equiv 20 equiv
Alkyne: 10 equiv 10 equiv 20 equiv 10 equiv 20 equiv
c
12
13
14
15
16
17
18
19
20
21
22
100
80
60
40
37 °C
25 °C
20
0
ACS Parago1n0 Plus Env2ir4onment
0
5
15
20
30
Time (h)

a
O
PaJgoeu1r1naolfo1f7the American Chemical Society
O
O
MeO
O
N
O
N
O
n
H
O
N
N
2
3a
MeO
O
O
NH2
n
--or--
O
H
mPEG5k-NH2
1
2
3
4
5
MeO
O
N
1. Boc-N-propargylglycine NHS
O
N
H
n
2. TFA
3b
b
O
N
O
O
OMe
N
O
H
N
N
N
NH
n
H
N
H
O
N
3a or 3b
6N
7H
N
--or--
N3
CB6
O
N
O
H
8
9
N
H2
N
NH
O
OMe
N
N
H
O
n
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
c
d
+4
(15026)
100 kDa
75
+3
(14846)
50
37
+5
(15206)
25
20
15
+2
(14665)
10
-
PEG:
3a
3b
14000
15000
mass (Da)
16000
% mod: 0% 34% 98%
ACS Paragon Plus Environment

a
OH
H
N
Journal of the American ChemiPcalgSeo1c2ieotfy17
O
O
O
O
H2
N
O
H
N
H
N
N
N
N
O
N
H
H
H
H
O
O
O
NH
O
N3
4
HN
O
1
H2N
NH2
CB6
2
3
4
5
6
7
O
OH
N
O
O
O
O
O
H
N
N
H2
N
H
N
H
H
NH
N
N
N
N
N
N
H
O
H
H
O
O
O
O
HN
H2
N
NH2
b
TMV-CB6-RGDS
(19394)
8
9
TMV
(17575)
10
11
12
13
14
15
16
17
18
TMV-pipN3
(17755)
ACS Paragon Plus Environment
17000
18000
mass (Da)
19000
20000

a
b
O
Lysozyme
O
PaJgoeu1r3naolfo1f7the NAmerican Chemical Society
37 kD
H
N
N
H
H
5
NH
N3
25
20
CB6
1
15
10
O
2
O
-
-
N
DNA:
CB6:
+
+
N
H
3
N
N
N
NH
H
N
H
4
5
6
7
8
9
% mod: 0% 50%
10
11
12
13
14
ACS Paragon Plus Environment

a
b
O
OH
O
O
OH
O
OH
OH
SH
N
Journal of the American ChemiPcalgSeo1c4ieotfy17
OH
OH
H
OMe O
OH
O
OMe O
OH
O
O
O
7
6
H
HO
N
HN
S
HN
S
HO
S
S
N
1
2
3
4
5
6
O
O
O
OH
O
O
HO
HO
O
OH
NH
OMe
O
N
N
O
H2
N
N
7, CB6
H
H
N
S
N
H
OH
N
S
N3
H
N
O
RR-TMV-pipN3
O
7
8
c
d
9
DOX
10
11
12
13
14
15
16
17
18
19
20
21
22
K104
CB6
RR-TMV-T104K
18 nm
e
f
TMV-CB6-DOX
(19497)
TMV-CB6-SH
(18868)
TMV
(17575)
TMV
(17575)
DTT
- DOX-SH
TMV-CB6-DOX
(19497)
17000
23
18000
mass (Da)
19000
20000
17000
18000
19000
20000
ACS Paragon Plus Environment
mass (Da)
24
25
26

a
SO3
SO3
SO3
SO3
NH2
O
H2
N
NH2
O
H
N
PaJgoeu1r5naolfo1f7the American Chemical Society
2
H
N
HS
CO2
CO2
O
O
N
H2
N
N
1
2
3
4
5
6
7
8
9
O
N
O
N
4
H
S
4
H
O
O
AF488-maleimide
AF488-propargylamine (8)
b
O
TMV-CB6-AF488
TMV-CB6-AF488-AF594
HN
N
N
H2
N
NH
AF488
N
S
O
S
AF594
FRET
N
O
RR-TMV
11040K-CB6-AF488
480
530 580
630
680 730
123C-AF594
11
wavelength (nm)
12
13
14
15
16
17
18
ACS Paragon Plus Environment

a
Journal of the American CThMeVmiPcalgSeo1c6ieotfy17
a
b
H
N3
N
H
H
N3
N
N
3
O
O
1
2
TMV-N3 (b)
(17700)
TMV-pipN (a)
(177553)
3
4
5
6
7
8
9
17500
18500
mass (Da)
19500
CB6-AAC (CB6 + 8)
b
S
H
N
N
N
TMV
AF488
H2
H
N
N N
b
O
H
N3
N
3
O
c
10
TMV-CB6-AF488 (c)
11(19565)
TMV-N3 (b)
12
13
14
17500
TMV
18500
mass (Da)
19500
SP-AAC (DBCO-Cy5)
c
15
16
17
S
H
N
N
N
AF488
H2
H
N N
N
O
TMV-CB6-AF488 (c)
18
19
20
21
22
23
24
25
26
27
28
d
c
N
N
H
N
H
N
N
N
Cy5
O
O
TMV-DBCO-Cy5 (d)
17500
18500
mass (Da)
19500
(18709)
ACS Paragon Plus Environment

O
OH
O
HO
HO
PaJgoeu1rn7aolfo1f7the American Chemical Society
O
O
O
OH
O
OMe
O
N
N
O
N
H
N
N
H
NH
H
N
N
H2
N
NH
N
N
S
OH
NH
H
N
S
O
CB6
CB6
O
N
H
1
2
3
4
5
6
7
DNA
Small molecules
Peptides
N
H
N3
CB6 Click
O
O
O
O
N
N
N
O
N
O
O
N
N
N
N
N
N
N
N
R
Polymers
N
N
N
N
N
N
N
N
O
O
O
N
H2
N
O
N
N
O
O
O
ACS Paragon Plus Environment
N
N
H
O
H
O
O
O
O
N
N
H2
N
N
N
H2
N
NH
H
H
N
NH
O
OMe
N
N
N
N
O
n
N
N
H
H
H
O
O
O
O
CB6
CB6
HN
H2N
NH2