Journal of the American Chemical Society
Article
poly adenine adsorption to gold rather than utilizing gold−
(abTyr), features a binuclear copper active site and is
responsible for the formation of melanin. abTyr is isolated
from the common button mushroom,Agaricus bisporus, and is
commercially available in its active form. Past work has
demonstrated that abTyr can oxidize phenols on a variety of
substrates, including small molecules, peptides, and proteins.
Importantly, the enzymatic version of this reaction allows
thiols to be used as a nucleophile without competing side
2
1
thiol chemistry. Additionally, the enhanced stability of
polydentate ligands has led to the development of DNA with
22
bi- or even trithiol anchoring groups. However, these
techniques require that commercial DNA is modified prior
to conjugation to AuNPs. In addition, techniques used for
DNA conjugation may not be feasible for protein conjugation
due to the greater sensitivity of proteins to temperature and
pH. Another limitation of current DNA-AuNP conjugation
methods is that relatively dense layers of DNA are often
required to maintain colloidal stability, making low density
DNA-AuNP conjugates difficult to produce. Thus, while many
successful strategies exist for generating DNA-AuNP con-
jugates, there is a need for methods that are compatible with
both proteins and DNA and allow the production of low-
density DNA-AuNPs.
34
reaction pathways. Our lab has also demonstrated successful
oxidative couplings with a smaller tyrosinase (35.5 kDa versus
35
∼120 kDa for abTyr) fromBacillus megaterium.
There are a number of features of the oxidative coupling
strategy that are particularly attractive for AuNP modification.
Chief among them is the ability to use a nonreactive phenol
functional group as a surface coating that does not decompose
or otherwise react until the desired reaction takes place. In
addition, the oxidative coupling reaction can proceed at the
low concentrations that are frequently encountered in the
context of metal nanoparticle modification. To demonstrate
the utility of this approach, AuNPs are first functionalized with
a monolayer of phenols that, upon enzymatic activation with
abTyr, can be coupled to a variety of proteins bearing thiol,
proline, or aniline functional groups. This highlights how a
single type of functional AuNP is compatible with multiple
functional handles and can easily be applied for versatile
protein conjugation. We also demonstrate the effective
coupling of thiol-DNA by producing DNA-AuNP conjugates
with varying DNA densities. Finally, to highlight the utility of
protein-AuNP constructs, we use this enzymatic bioconjuga-
tion technique to conjugate dye-labeled and aniline-containing
circular permutant tobacco mosaic virus (cpTMV) protein
disks to AuNPs and demonstrate energy transfer between the
dyes and the AuNPs. The novelty of this approach is the direct
enzymatic activation of AuNP surfaces with a view toward
subsequent bioconjugation, which has not been reported to
date.
Enzymes could offer a unique addition to the AuNP
conjugation toolbox by allowing timely and efficient activation
of stable, prefunctionalized AuNPs. The use of enzymes in
some aspects of metal surface modification has been reported.
For example, aggregates of β-agarase were tethered to magnetic
particles via the initial oxidation of engineered tyrosine
23
residues on the surface of the protein aggregates. In another
instance, AuNPs of two different sizes were functionalized with
DNA, which was then activated with a restriction endonu-
clease. The activated AuNPs were subsequently ligated to form
24
nanoparticle-based structures. However, direct enzymatic
activation of AuNP surfaces for subsequent bioconjugation has
not been reported.
Over the years, our lab has developed a rapid and efficient
oxidative coupling approach for biomolecules through the
reactions of reactive ortho-quinoid intermediates with specific
25
RESULTS AND DISCUSSION
■
Generating Phenol Monolayers on 5 nm AuNPs.
Previous work in our lab has demonstrated oxidative coupling
on AuNPs by decorating the particles with anilines and
conjugating the functionalized AuNPs to aminophenol-
36
containing proteins and DNA. However, we were also
interested in immobilizing the phenolic coupling partner on
AuNPs to allow oxidative coupling with native nucleophiles on
proteins, such as cysteines and N-terminal prolines (Figure 1).
This would negate the need for prior functionalization with
aminophenols and would also allow the coupling of
commercially available thiol-DNA. In the previous iteration
Figure 1. Oxidative coupling reactions. A variety of oxidants can be
used to oxidize phenol based moieties to ortho-quinoid intermediates
of the oxidative coupling reaction (involving K Fe(CN) and
(green). These intermediates can undergo subsequent reactions with
3
6
catechols), it was necessary to immobilize the aniline partners
because catechols are not stable in solution due to oxidation by
air, leading to self-coupling and subsequent polymerization.
However, we reasoned that the ability to oxidize phenols
enzymatically would allow us to generate stable AuNPs that
could be activated only when desired.
both amine- and thiol-based nucleophiles.
be accessed by potassium ferricyanide (K Fe(CN) ) or sodium
periodate oxidation of ortho-phenolic compounds, such as
catechols, aminophenols, and methoxyphenols.
3
6
2
6−30
This
versatile chemistry has enabled the construction of various
bioconjugates that tether proteins, peptides, and nucleic acids
to small molecules, other biomolecules, and even surfaces, such
To this end, we designed a thiol-PEG5k-phenol ligand for
AuNP functionalization. The long PEG linker was chosen to
shield the AuNP surfaces and prevent aggregation, and the 5k
length aided in quick purification with 3k MWCO spin filters
during the synthetic manipulations. Synthesis of the phenol
ligand was based on an analogous aniline ligand previously
3
1
32
as glass and gold electrodes. More recently, we have
developed an enzymatic version of the oxidative coupling
reaction that allows ortho-quinones to be accessed from water-
33
36
soluble and highly stable phenols. The enzyme, tyrosinase
reported by our lab. Briefly, DCC/NHS was used to
7
343
J. Am. Chem. Soc. 2021, 143, 7342−7350