Synthesis and incorporation of unnatural amino acids to probe and optimize protein bioconjugations

Article abstract of DOI:10.1021/acs.bioconjchem.5b00424

The utilization of unnatural amino acids (UAAs) in bioconjugations is ideal due to their ability to confer a degree of bioorthogonality and specificity. In order to elucidate optimal conditions for the preparation of bioconjugates with UAAs, we synthesized 9 UAAs with variable methylene tethers (2-4) and either an azide, alkyne, or halide functional group. All 9 UAAs were then incorporated into green fluorescent protein (GFP) using a promiscuous aminoacyl-tRNA synthetase. The different bioconjugations were then analyzed for optimal tether length via reaction with either a fluorophore or a derivatized resin. Interestingly, the optimal tether length was found to be dependent on the type of reaction. Overall, these findings provide a better understanding of various parameters that can be optimized for the efficient preparation of bioconjugates.

Full text of DOI:10.1021/acs.bioconjchem.5b00424

Communication
pubs.acs.org/bc
Synthesis and Incorporation of Unnatural Amino Acids To Probe and
Optimize Protein Bioconjugations
Johnathan C. Maza, Jaclyn R. McKenna, Benjamin K. Raliski, Matthew T. Freedman,
and Douglas D. Young*
Department of Chemistry, College of William & Mary P.O. Box 8795, Williamsburg, Virginia 23187, United States
S
* Supporting Information
ABSTRACT: The utilization of unnatural amino acids (UAAs) in bioconjugations is ideal due to their ability to confer a degree
of bioorthogonality and specificity. In order to elucidate optimal conditions for the preparation of bioconjugates with UAAs, we
synthesized 9 UAAs with variable methylene tethers (2−4) and either an azide, alkyne, or halide functional group. All 9 UAAs
were then incorporated into green fluorescent protein (GFP) using a promiscuous aminoacyl-tRNA synthetase. The different
bioconjugations were then analyzed for optimal tether length via reaction with either a fluorophore or a derivatized resin.
Interestingly, the optimal tether length was found to be dependent on the type of reaction. Overall, these findings provide a
better understanding of various parameters that can be optimized for the efficient preparation of bioconjugates.
growing area of interest in chemical biology is the ability
a reactive handle to facilitate the generation of a stable
Ato perform chemistry on biological systems, particularly bioconjugate in a well-defined fashion.
Multiple techniques exist for the introduction of UAAs that
conjugation reactions between bioactive molecules and either
molecular probes or surfaces. These bioconjugates can be
employed as therapeutic agents or diagnostic tools, and toward
fundamental research.1−6 One of the most useful strategies for
bioconjugation involves the formation of strong covalent
linkages between the bioactive molecule and its partner (as
opposed to processes such as adsorption or encapsulation).7,8
Due to the breadth of their applicability, there is a constant
impetus for the optimization and development of methods for
their preparation.
In order for such chemistry to be feasible, certain conditions
must be met by the chemical reaction employed. Most
importantly, the chemistry must be compatible with a biological
setting. That is, the reaction must proceed at relatively rapid
rates under physiological conditions (typically at 37 °C and pH
∼7) as well as being inert to the myriad of chemical
functionalities encountered in biological systems to provide a
degree of chemoselectivity.9 Should the conditions above be
met, the chemistry is deemed bioorthogonal. While a range of
bioorthogonal reactions have been elucidated, several issues
preclude their widespread use.10 These bioorthogonal reactions
include 1,3-dipolar cycloadditions, Sonogashira and Suzuki
couplings, oxime ligations, photo-cross-linking reactions, and,
most recently, Glaser-Hay couplings.11−18 One primary
obstacle is the introduction of novel functionality into the
biomolecule/protein; however, this can rapidly be accom-
plished via the site-specific incorporation of unnatural amino
acids (UAAs).19 This novel chemical functionality can serve as
range from completely synthetic to exploitation of endogenous
translational machinery.20−22 The evolution of orthogonal
aminoacyl-tRNA synthetase/tRNA pairs has found a wide
degree of success in bioconjugations and various other
applications.22 Specifically, several examples of the use of
unnatural amino acids to generate well-defined bioconjugates
include the reaction of an azide containing calmodulin onto
carbon nanotubes and nanoparticles via the Staudinger-Bertozzi
ligation,23,24 the immobilization of other proteins to M-20
Dynabeads in a cell-free system with a click reaction,25,26 and
the labeling of proteins with an agarose resin and azido UAA
for secretome MS analysis (however, in this case the UAA was
not site-specifically incorporated into the pool of proteins).27
More recently, we demonstrated the optimization of protein
immobilization utilizing an incorporated azido UAA, conferring
increased stability to organic solvents.28 The combination of
these studies demonstrates the utility of site-specific bio-
conjugations, and the stabilizing benefits of the solid support.
Other studies have used similar techniques to label proteins
with molecular probes or generate novel therapeutic bio-
conjugates.4,29 Given the power of this technology, we aimed to
expand the utility of UAA bioconjugations through the
alteration of the reactive UAAs to optimize reaction parameters.
Received: July 28, 2015
Revised: August 18, 2015
© XXXX American Chemical Society
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Initial investigations attempted to further utilize UAAs to
optimize bioconjugations through several different bioorthog-
onal reactions. Our previous studies immobilizing GFP
demonstrated that the UAA residue location on the protein
dictated its immobilization efficiency.28 Thus, the spatial
configuration of the UAA handle is vital for its reactivity with
its conjugation partner. While it is possible to alter where the
UAA can be incorporated, this requires extensive genetic
manipulation. An alternative approach is to simply vary the
distance of the UAA reactive group from the protein’s surface.
By extending the distance from the protein, the handle is less
hindered by the protein’s steric bulk, which may increase its
propensity to react with its orthogonal partner. Moreover,
removing the reactive partner from conjugation with the
aromatic ring may alter its chemical reactivity. Thus, it becomes
important to ascertain the ideal distance of the functional
handle from the protein for efficient reactivity. In order to
probe novel bioorthogonal reactions utilizing UAA technology,
a variety of UAAs with new functional groups were synthesized
and designed with variable tether lengths to separate the
reactive species from the bulk of the protein. These groups
included an azide, an alkyne, and a bromide, respectively, all of
which can be derived from a modified tyrosine (Scheme 1).
These functionalities afford access to the Cu(I)-catalyzed 1,3-
dipolar cycloaddition, the Glaser-Hay coupling, and a
nucleophilic substitution, respectively.
Typically with the new synthesis of UAAs a unique aaRS
must be evolved to recognize and incorporate the UAA;
however, based on the previous demonstration of the
promiscuity of the pCNF-aaRS in E. coli,30,31 it was our hope
that the structural similarity of the 9 prepared UAAs could
facilitate incorporation without the need of a double sieve aaRS
selection. With respect to the bromo-derivatives, a previous
report used a similar approach to their incorporation in
mammalian cells.32,33 Based on our previous studies we
employed GFP as a model protein for incorporation due to
its well-documented fluorescent properties facilitating rapid
visualization and quantitation. Our previous findings indicated
that residue 151 conferred the best conjugation efficiencies, and
was thus selected for this study.28 BL21(DE3) E. coli were
cotransformed with pEVOL-pCNF-aaRS and pET-GFP-151-
TAG plasmids to introduce both the GFP protein and the
translational machinery necessary for TAG suppression with
UAAs. Following protein expression (30 °C, 16 h) in the
presence or absence of the UAA, cells were lysed and GFP was
purified via Ni-NTA chromatography. Gratifyingly, the
application of the polyspecific aaRS was successful, as the 9
UAAs were able to be incorporated into GFP, albeit with
differential expression levels (Table 1). The incorporation of
Table 1. Protein Expression Yields for the Tethered UAAs
Scheme 1. Synthesis of Tethered UAAs
To optimize the immobilization process, UAAs were
synthesized with various numbers of methylene units extending
from a tyrosine precursor. Derivatives containing 2, 3, and 4
methylenes of the azide, alkyne, and bromide functionalities
previously mentioned are readily accessible via a SN2 reaction
(Scheme 1).
the UAA was visualized both by fluorescence and SDS-PAGE
analysis (see Supporting Information). As the pCNF synthetase
was evolved to recognize pCNF, it was hypothesized that the
p2yneY-, p3yneY-, and p4yneY-UAA series would be most
easily incorporated due to the structural similarity between the
cyano group and alkyne group in size and polarizability.
This result was observed, with the p3yneY (n = 2) producing
a larger quantity of mutant-GFP, and the p4yneY producing the
lowest quantity of mutant-GFP within the alkyne series. These
qualitative observations make sense based on the evolution of
the synthetase. We hypothesize that the increased tether
distance may have more difficulty fitting into the binding
pocket of the tRNA synthetase relative to smaller derivatives,
preventing the UAA from being incorporated into the
polypeptide chain. Thus, a trade-off may exist between the
advantages of distancing the functional group from the core of
the protein and the reduced protein yields of these same
compounds. This trend was observed for all of the UAAs: as the
tether length increased, the expression yield decreased.
Synthesis of the nine UAAs began from a common N-Boc-
OMe protected tyrosine precursor. The goal was to synthesize a
bromotyrosine derivative via a substitution reaction with either
dibromoethane, dibromopropane, or dibromobutane. These
bromo-tyrosines (1−3) would then have alkyl chains of varying
lengths, and a good leaving group in future reactions. Then, a
secondary SN2 reaction with either an azide (4−6) or alkynyl
nucleophile would install the desired novel reactivity to the
tyrosine derivatives. However, synthesis of the alkynyl-tyrosine
series (7−9) proved more difficult than initially anticipated, and
the series was prepared directly via SN2 reactions with
commercially available bromo-alkynes (see Supporting In-
formation). Deprotection with LiOH followed by 2% TFA,
afforded the 9 UAAs in moderate overall yields (see Supporting
Information).
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However, all 9 UAAs were capable of being incorporated using
a single aaRS, not only obviating the necessity of a tedious
selection, but also increasing the versatility of this approach.
Given the ability to incorporate three different functionalities
with variable tether lengths into GFP, we next sought to
employ these new UAAs in bioorthogonal conjugation
reactions. Studies were initiated with the azido-GFP series, as
the cycloaddition reaction is the most common and well-
understood bioorthogonal reaction. Moreover, the various
tethers could be directly compared to the commonly utilized p-
azidophenylalanine (pAzF) that has no methylene tether and
contains the azido group in direct conjugation with the
aromatic ring. We hypothesized that in addition to providing
distance between the protein and the conjugation partner, the
removal of the azido conjugation may also alter its reactivity.
The various azido-containing GFP mutants were conjugated
with a fluorescent Alexafluor 488 alkyne dye using a CuSO4/
TBTA/TCEP catalyst system based on previously reported
conditions. Gratifyingly, all tethered derivatives could be
conjugated to the dye, as determined by both fluorimetry and
SDS-PAGE analysis (Figure 1). Control reactions were also
functionality that could be employed as a reaction partner with
the azido-GFP series.
It was expected that by extending the tethering distance of
the functional handle of the UAA, and thus the site of
bioorothogonal reactivity, the steric hindrance due to the
protein’s bulk could be decreased and immobilization efficiency
could be improved. As such, the p2AzY, p3AzY-, and p4AzY-
mutated GFPs were exposed to copper click conditions (50
mM CuSO4, 50 mM TCEP, and 5 mM TBTA) at fixed
concentration of GFP (roughly 0.250 mg/mL). As a positive
control, the pAzF GFP, which has no tether between the
aromatic ring and the azide functional group, was also subjected
to the same copper click conditions. We have previously
immobilized the p-azidophenylalanine mutated GFP on the
alkyne-functionalized sepharose resin using similar condi-
tions.28 As a negative control, wild-type GFP, in which there
is no azido-functional group present, was also subjected to the
same copper click conditions. This control provided a means to
rule out nonspecific interactions as the reason for immobiliza-
tion rather than the selective copper click reaction. Results
indicated that that copper click-based immobilization strategy
was effective for all of the mutants in the series (Figure 2).
Figure 1. Expression and conjugation of GFP-151 mutants with an
AlexaFluor488 dye. The Coomassie stained gel (top) indicates the
successful incorporation of all three azides in the series as an
appropriate band is observed for GFP at ∼26 kDa. The fluorescence of
the same gels (bottom) indicates that each azido-GFP mutant also was
able to undergo a successful bioconjugation with an AlexaFluor-488
alkyne. No coupling was observed in the absence of copper catalyst
despite the inclusion of the fluorophore alkyne, as no fluorescence is
detected in the absence of catalyst.
Figure 2. Click immobilization of GFP-151 mutants on a sepahrose
resin with either an azide tethered tyrosine UAAs or a p-
azidophenylalanine. (A) Immobilization of GFP at a fixed concen-
tration of 0.246 mg/mL of each mutant protein. Fluorscence signal at
528 nm on a plate reader indicates that the n = 2 GFP-151 azide
tethered variant performed best for immobilization (immobilizations
conducted in triplicate, average standard deviation 296). (B) Image
of the immobilized resins as irradiated at 365 nm on a transilluminator.
Overall, the tethered variants appeared to have a greater immobiliza-
tion efficiency than the traditional pAzF.
performed in the absence of catalyst system to ensure that
protein labeling was not due to nonspecific reactions or
noncovalent association by the dye. These reactions yielded no
fluorescent product following protein denaturation, and thus
indicated that any fluorescence is indeed a result of the
coupling to form the triazole ring. Interestingly, the p3AzY
handle appears to have the best conjugation efficiency,
compared to the more sterically hindered p2AzY and the
more flexible p4AzY handles.
With successful bioorthogonal reaction conditions in hand,
we next sought to apply the chemistry for immobilization
purposes. As mentioned previously, the most robust and ideal
mechanisms for protein immobilization involve covalent
linkage. However, this technique is often confounded by the
potential for multiple reaction sites on a given protein, which
could render proteins that are oriented incorrectly for their
function. UAA mutagenesis allows for the development of a
bioorthogonal handle in a predetermined site.34 Based on
previous UAA immobilization results, an epoxy-activated
sepharose resin was derivatized with propargyl alcohol in the
presence of trimethylamine (TEA) by stirring overnight at
room temperature.28 This afforded a resin with an alkyne
Interestingly, the tether distance did factor into the efficiency of
the immobilization reaction but not as expected, as increased
distance from the protein did not necessarily lead to improved
immobilization as measured by relative fluorescence intensity
on a microplate reader (Figure 2). However, confirming the
results observed when coupling to the Alexafluor488 dye, the
p3AzY (n = 2) derivative again had the highest coupling
efficiency, indicating that the interplay of structural rigidity and
distance is important when optimizing the bioconjugation
reaction. This 3-carbon tether seems to exploit the best
properties of each factor, conferring a high coupling efficiency.
Based on these results, we next investigated the role of the
methylene tether on alternate reactions. Recently, we reported
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the first Glaser-Hay bioconjugation, linking two terminal alkyne
functionalities to yield a conjugated diyne.18 Consequently, this
reaction was selected to explore the reactivity of the alkyne-
GFP series. The alkyne-containing GFPs were first conjugated
to the same Alexafluor 488 alkyne dye previously employed in
the click reactions. All mutant proteins were subjected to
conjugation conditions in the presence of CuI (50 mM) and
TMEDA (50 mM) and a control in the presence of the alkyne
fluorophore but in the absence of catalyst system. After running
for 4 h at 4 °C, the proteins were denatured at 98 °C for 15
min and then analyzed via SDS-PAGE (Figure 3). These
the resin after reaction with CuI/TMEDA (Figure 3B).
However, it appears that there is no relative benefit to
increasing the tether length, in contrast to the trends observed
with the azide tethers. Interestingly, the p-propargylphenylala-
nine, which has an alkynyl moiety closest to the phenyl ring of
phenylalanine, performed the best. Based on the proposed
mechanism for the Glaser-Hay reaction, we hypothesize that
the enhanced rigidity of the pPrF helps properly orient the
protein−resin interactons as the copper binds in a η1 fashion.
Moreover, the enhanced flexibility afforded by the novel
alkynyl-UAAs may diminish their propensity to react, as the
steric bulk of the protein may be more predisposed to interact
with the resin. An odd/even effect is observed as the p3yneY-
containing GFP performed the best among the UAA-mutated
GFPs in the immobilization experiment (Figure 3C). This
suggests that there may be some benefit, although slight, to
increasing the distance of the protein from the site of reactivity.
This increase drops once the alkyl chain gets too flexible, as
evidenced by the drop in the p4ynY-containing GFP.
Interestingly, within the series, the conjugation partner also
plays a role in efficiency as the Glaser-Hay reaction with the
small fluorophore was most efficient with the longer tether
(Figure 3A), whereas reaction with the bulky resin required
decreased flexibility.
Finally, the bromo-GFP series was investigated utilizing a
SN2 reaction. The incorporation of this series with a different
synthetase, and their reaction with thiols, has previously been
reported in a mammalian system;32,33 however, we were
interested to see if these synthetic intermediates could also be
incorporated with the pCNF aaRS. Due to the presence of
many nucleophilic functional groups within biological settings,
this reaction is not truly bioorthogonal, as cross-reactivity is
likely. Nevertheless, we reasoned that successful nucleophilic
substitution with an amine resin, thereby immobilizing the GFP
onto the resin, was an ideal means to demonstrate the
successful incorporation of the bromo-UAAs and the
generation of the p2BrY-, p3BrY-, and p4BrY-GFPs. An
amino-functionalized sepharose resin was generated using
etheylenediamine. This resin was then subjected to the
bromo-GFP series in the presence of a carbonate buffer (pH
= 9.5). As a control, wild-type GFP was subjected to the same
conditions. Gratifyingly, all of the bromo-containing GFPs were
successfully immobilized, with p4BrY performing the best
(Figure 4). Compared to the other functionalities and
reactions, in which the three carbon alkyl chain variant of the
Figure 3. Glaser-Hay reactions of alkyne-containing GFP mutants. (A)
Bioconjugation of mutant GFPs with AlexaFluor488 under Glaser-Hay
conditions. The Coomassie stained gel (top) indicates the
incorporation of all tethered mutants into GFP-151. Moreover, the
successful Glaser-Hay coupling with the fluorophore was confirmed via
fluorescent imaging of the same gel (bottom), as AlexaFluor
fluorescence is only observed in reactions containing the fluorophore
and the CuI/TMEDA system (immobilizations conducted in triplicate,
average standard deviation 774). (B) Immobilization of alkyne-GFP
mutants on a sepharose resin under Glaser-Hay conditions. Measuring
resin fluorescence on a plate-reader (Ex 395/Em 512), all GFPs were
able to be immobilized. (C) Immobilization was also confirmed by
GFP fluorescent imaging on a transilluminator.
couplings all were successful, as demonstrated by a fluorescent
band at ∼26 kDa; however, the different tethered derivatives
reacted with different efficiencies. Furthermore, no coupling
was observed in the absence of the Glaser-Hay catalyst system,
even in the presence of the fluorophore, indicating that
bioorthogonal Glaser-Hay chemistry is responsible for the
conjugation between the fluorophore and the dye.
The alkyne series was also reacted with the alkyne-derivatized
sepharose resin to assess coupling efficiencies. All of the
alkynyl-UAA containing GFP mutants were able to be
immobilized, as indicated by the presence of fluorescence on
Figure 4. Immobilization of bromide-containing UAAs on a
derivatized sepharose resin. Fluoresence was determined on a plate
reader (Ex 395/Em 512), with the increased tether increasing
immobilization efficiency (immobilizations conducted in triplicate,
average standard deviation 1401).
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propensity to immobilize. This is to be expected, as this bromo-
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AUTHOR INFORMATION
Corresponding Author
*E-mail: dyoung01@wm.edu.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors would like to thank the Jeffress Memorial Trust
and the College of William & Mary for financial support. Also,
we thank Dr. Peter G. Schultz for providing plasmids necessary
for incorporation of unnatural amino acids, and Dr. JC Poutsma
and Kathy Huynh for assistance with mass spectrometry.
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DOI: 10.1021/acs.bioconjchem.5b00424
Bioconjugate Chem. XXXX, XXX, XXX−XXX