ortho-Methoxyphenols as Convenient Oxidative Bioconjugation Reagents with Application to Site-Selective Heterobifunctional Cross-Linkers

Article abstract of DOI:10.1021/jacs.6b12966

The synthesis of complex protein-based bioconjugates has been facilitated greatly by recent developments in chemoselective methods for biomolecular modification. The oxidative coupling of o-aminophenols or catechols with aniline functional groups is chemoselective, mild, and rapid; however, the oxidatively sensitive nature of the electron-rich aromatics and the paucity of commercial sources pose some obstacles to the general use of these reactive strategies. Herein, we identify o-methoxyphenols as air-stable, commercially available derivatives that undergo efficient oxidative couplings with anilines in the presence of periodate as oxidant. Mechanistic considerations informed the development of a preoxidation protocol that can greatly reduce the amount of periodate necessary for effective coupling. The stability and versatility of these reagents was demonstrated through the synthesis of complex protein-protein bioconjugates using a site-selective heterobifunctional cross-linker comprising both o-methoxyphenol and 2-pyridinecarboxaldehyde moieties. This compound was used to link epidermal growth factor to genome-free MS2 viral capsids, affording nanoscale delivery vectors that can target a variety of cancer cell types.

Full text of DOI:10.1021/jacs.6b12966

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Article
ortho-Methoxyphenols as Convenient Oxidative Bioconjugation Reagents
with Application to Site-Selective Heterobifunctional Crosslinkers
Adel M. ElSohly, James Irving MacDonald, Nina B Hentzen, Ioana
Laura Aanei, Kareem M. El Muslemany, and Matthew B Francis
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.6b12966 • Publication Date (Web): 16 Feb 2017
Downloaded from http://pubs.acs.org on February 16, 2017
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ortho-Methoxyphenols as Convenient Oxidative Bioconjugation
Reagents with Application to Site-Selective Heterobifunctional
Crosslinkers
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Adel M. ElSohly, James I. MacDonald, Nina B. Hentzen, Ioana L. Aanei, Kareem M. El Muslemany, and
a,b,*
Matthew B. Francis
a
b
Department of Chemistry, University of California, Berkeley, California 94720ꢀ1460, Materials Sciences Division, Lawrence Berkeley
⊥
National Laboratories, Berkeley, California 94720ꢀ1460, These authors contributed equally to this work.
ABSTRACT: The synthesis of complex proteinꢀbased bioconjugates has been facilitated greatly by recent developments in
chemoselective methods for biomolecular modification. The oxidative coupling of oꢀaminophenols or catechols with aniline
functional groups is chemoselective, mild, and rapid; however, the oxidatively sensitive nature of the electronꢀrich aromatics
and the paucity of commercial sources pose some obstacles to the general use of these reactive strategies. Herein, we identify
oꢀmethoxyphenols as airꢀstable, commercially available derivatives that undergo efficient oxidative couplings with anilines in
the presence of periodate as oxidant. Mechanistic considerations informed the development of a preꢀoxidation protocol that
can greatly reduce the amount of periodate necessary for effective coupling. The stability and versatility of these reagents was
demonstrated through the synthesis of complex proteinꢀprotein bioconjugates using a siteꢀselective heterobifunctional
crosslinker comprising both oꢀmethoxyphenol and 2ꢀpyridinecarboxaldehyde moieties. This compound was used to link
epidermal growth factor to genomeꢀfree MS2 viral capsids, affording nanoscale delivery vectors that can target a variety of
cancer cell types.
INTRODUCTION
Siteꢀspecific and chemoselective bioconjugation remains a challenging task for many proteins. The reactions used for this
purpose must be compatible with nearꢀneutral aqueous solutions and be kinetically competent at low micromolar
concentrations. They also push the limits of functional group tolerance to proceed in the presence of amines, carboxylic acids,
thiols, and electron rich aromatic rings that are not intended for modification. Despite these challenges, an increasing number
of techniques have been developed to achieve desired high levels of selectivity through the modification of both native and
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artificiallyꢀintroduced amino acids or the use of tag strategies. In our own lab, these reactions have enabled the synthesis
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of a variety of complex proteinꢀbased materials, including drug delivery systems,
water remediation materials, and
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artificial light harvesting systems.
To build these materials, our group has devoted considerable attention to the advancement of reliable and efficient
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oxidative coupling reactions (Figure 1a).
These strategies rely on the addition of either anilines or protein Nꢀtermini to oꢀ
26
iminoquinones and oꢀquinones that are generated in situ using potassium ferricyanide or sodium periodate. These reactions
exhibit high chemoselectivities and can proceed in aqueous media at high dilution, but there are a few limitations of the oꢀ
aminophenol and catechol substrates that have potentially limited the widespread adoption of these methods. As both types of
substrates are oxidized in the presence of mild oxidants (such as ferricyanide), these electron rich aromatic rings show
considerable instability to storage under ambient conditions. This requires the preparation of the oꢀaminophenol through
chemoselective dithionite reduction of a precursor oꢀnitrophenol moiety immediately prior to use. As such, the requisite NHS
22
esters and/or amines are not commercially available and must be synthesized from the parent phenol through nitration.
Furthermore, the necessity to reduce the nitrophenol group can limit the types of molecules that can be used with this
conjugation procedure since some reductively sensitive moieties (e.g. azo dyes, alkoxyamines, and oximes) will be
incompatible with this protocol. Similar issues are associated with the catechol substrates.
Herein, we describe the use of oꢀmethoxyphenol substrates as airꢀstable, commercially available reagents that can be used
in oxidative coupling reactions with anilines in the presence of sodium periodate with short reaction times (c.a. 10 min) and
excellent chemoselectivity. We further demonstrate the versatility of this reaction by applying these reagents to the
development of a siteꢀselective heterobifunctional linker that also involves an Nꢀterminal selective condensation reaction of
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ꢀpyridinecarboxaldehydes (2PCA) recently reported by our laboratory. The synthetic tractability and storability of these oꢀ
methoxyphenol reagents addresses many of the challenges associated with previous oxidative coupling reactions. Thus, these
reagents substantially improve the practicality of this useful class of bioconjugation reactions.
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RESULTS AND DISCUSSION
One useful variant of these oxidative coupling strategies is the application of oꢀazidophenols as iminoquinone precursors
that can be activated through photolysis. As this reaction was being developed, an informative control experiment was
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performed wherein an oꢀazidophenol was treated in the absence of light with periodate and toluidine, and the reaction was
monitored by RPꢀHPLC. Surprisingly, ~40% conversion to the coupled product was observed, wherein the azido group in the
starting material had been converted to the carbonyl group (Figure 1b). We hypothesized that this occurred through the
nucleophilic addition of water to the periodateꢀactivated aromatic ring, leading to an azidohydrin intermediate that expels
hydrazoic acid to generate the presumed oꢀquinone for coupling. This experiment suggested that the placement of other
leaving groups ortho to the phenol could potentially allow for airꢀstable oxidative coupling partners that would be easier to
access. Crucial to the success of such an approach, however, would be the identification of a substituent that was both
electron rich enough to promote oxidation and also capable of serving as a good leaving group.
Previous studies from the Snyder and Quideau groups indicated that such oxidative dearomatizations of o-
methoxyphenols are efficient in the presence of hypervalent iodine species. To apply this in an oxidative coupling context, 2ꢀ
methoxyꢀpꢀcresol was treated with sodium periodate and pꢀtoluidine in 10 mM pH 7.0 phosphate buffer. After 30 min, HPLC
analysis indicated that the desired coupling had occurred, but the conversion was quite low (~25%, data not shown). Careful
examination of the reaction revealed that the oꢀmethoxyphenol was not fully soluble in the reaction conditions, suggesting
that the use of a lower ionic strength buffer, such as BisTris, would promote a more efficient coupling. This hypothesis was
validated, as the buffer switch resulted in a near quantitative coupling after 30 min at room temperature (Figure 1c). Other
orthoꢀleaving groups were examined, such as bromo, chloro, and fluoro; however, no reaction was observed in any of those
cases. Furthermore, since orthoꢀmethoxyphenols are ubiquitous, airꢀstable, commercially available, and inexpensive (see
sample substrates in Figure 1d), the optimization of this new version of the coupling reaction was pursued.
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Reaction Optimization in Macromolecular Couplings
While the above couplings on small molecule substrates were promising, the combination of poor solubility of the simple
cresol derivative in phosphate buffer and the ultimate application of the reaction to proteins led us to pursue optimization of
the reaction within the macromolecular context. For these studies, an oꢀmethoxyphenolꢀPEGꢀ5kꢀOMe (MPꢀPEG) was readily
synthesized (see Supporting Information) and used in gel shift assays for coupling to bacteriophage MS2 viral capsids
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bearing aniline moieties introduced using amber stop codon suppression techniques (Figure 2a). The 180 protein monomers
of each 27 nm capsid contained one instance of this residue on the exterior surface. Previous work in our group has shown
MS2 to be an attractive scaffold for creating nanoscale biomaterials for drug delivery and cellular imaging, due to its ability
to carry and protect cargo within its protein shell.
First, a time screen (Figure 2b) with 200 µM PEG, 20 µM protein, and 1 mM periodate revealed that the reaction reached
completion in 10 min, and thus 10 min coupling times were used in all subsequent screens and experiments unless noted
otherwise. The reaction was quite tolerant of a variety of buffers, Figure 2c. Tris buffer was a notable exception, likely
because its 1,2ꢀaminoalcohols participate in competing oxidative cleavage reactions with sodium periodate. Similarly, the
corresponding oꢀaminophenol oxidative coupling reaction also proceeded with reduced yield with periodate in this buffer
(
see Supplementary Figure S1e). As such, phosphate buffer was used in all remaining experiments. An equivalent screen
indicated that high levels of conversion could be reached with as little as 2.5 equiv of PEG reagent; however, a notable
increase in yield was observed when 5 equiv were used instead (Figure 2d). The use of 10 equiv provided little improvement
in yield. Similar to our previously reported oxidative couplings, the efficiency of this reaction was found to be pH
independent over the range from 6ꢀ8 (Figure 2e).
In terms of storage, the MPꢀPEG reagent was left under ambient conditions (i.e. room temperature with no precautions
taken to exclude oxygen) for over a year and showed identical reactivity over that time period, in sharp contrast to the
aminophenol reagents, which showed noticeable color changes in a matter of hours under identical conditions. This tradeoff
in air stability, however, is also reflected in the greater difficulty in oxidation: potassium ferricyanide is completely incapable
of oxidizing the oꢀmethoxyphenol congener even when present in great excess, while the oꢀaminophenols are oxidized
essentially instantaneously. Although many proteins are quite compatible with sodium periodate (especially over the short
reaction times and low equivalents generally used in these couplings), a screen was carried out to identify other oxidants
capable of initiating the transformation. Despite an extensive screen of oxidants (see Supporting Information), we were
unable to identify another waterꢀsoluble, commercially available oxidant to effect the desired oxidation controllably.
Methods to Limit Periodate Exposure
The large difference in reaction time required to reach completion for the oꢀaminophenol (<2 min) and oꢀmethoxyphenol
couplings (10 min) and the necessity for a strong oxidant suggested that oꢀquinone generation from the oꢀmethoxyphenol
couplings was rate determining. This mechanistic insight presented a method to limit protein exposure to periodate for cases
in which proteins of interest are highly susceptible to oxidation. In particular, if oꢀquinone generation were rate limiting, then
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use of a preꢀoxidation protocol to generate the oꢀquinone with subsequent introduction of the anilineꢀcontaining protein could
provide a considerable decrease in both the reaction time and the amount of periodate used.
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To probe this hypothesis, an oxidative coupling reaction was performed under a stricter set of conditions (20 µM paF MS2,
00 µM PEG, and 500 µM sodium periodate final concentrations, compared to the 1 mM periodate used previously). As
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shown in Figure 3b, preꢀoxidation for as little as 2 min prior to introduction of the MS2 allowed for a dramatic increase in
yield of the coupled products relative to a reaction that was set up under identical conditions but without preꢀoxidation. Thus,
use of a preꢀoxidation protocol enabled use of half the periodate (i.e. a total of 2.5 equiv relative to the oꢀmethoxyphenol
since the overall reaction involves two 2ꢀelectron oxidations) and reaction times could also be reduced by at least half.
Such an approach was also examined for oꢀaminophenolꢀbased couplings with limited success (see Supplementary Figure
S2b)— a fact that can be rationalized if oxidation is also rateꢀlimiting in those couplings. Slow oxidation would produce oꢀ
iminoquinones in the presence of remaining oꢀaminophenols that could serve as nucleophile in the coupling reaction.
Evidence for this pathway was found in small molecule couplings where masses corresponding to dimers and trimers were
observed in the absence of toluidine as a nucleophile.
The success in implementing a preꢀoxidation strategy indicated that a method to further reduce periodate exposure could
be possible. In particular, if a suitable reductant could be identified to quench excess periodate without reducing the
generated oꢀquinone, then a preꢀoxidation followed by mild reductive quench would dramatically lower the amount of
periodate present in solution. Our previous buffer screen indicated that Tris was incompatible with this oxidative coupling
reaction (vide supra) and suggested that vicinal diols could serve as an appropriate reductant. Indeed, mannose was found to
be effective at rapidly quenching periodate (Figure 3c) while also allowing for useful levels of conversion to be achieved in a
preꢀoxidation/mannose quenching protocol (Figure 3d). This procedure is likely to find utility with proteins that show
sensitivity to oxidative conditions.
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Application to Heterobifunctional Linkers
To exploit the latent reactivity of the oꢀmethoxyphenol, the functional group was incorporated into a heterobifunctional
crosslinker. This class of reagents is commonly used to join oligonucleotides, proteins, molecular complexes, and surfaces to
32ꢀ36
protein substrates using two differentially reactive ends (Figure 4a).
Furthermore, the ability to tune the length and
properties of the spacer arm and to target specific functional groups on a protein surface by altering the reactive ends allows
for these reagents to be used in a diverse array of applications.
Recently, our group has identified 2ꢀpyridinecarboxaldehyde (2PCA) reagents to be highly specific for the modification of
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protein Nꢀtermini under mild conditions. Since oꢀmethoxyphenol and 2PCA reactions demonstrate exceptional siteꢀ
selectivity and require different reaction conditions, incorporating these two functional groups into a single
heterobifunctional linker has the potential to be a powerful tool for making wellꢀdefined protein conjugates (Figure 4b). First,
the 2PCA group can be reacted with the Nꢀterminus of a protein of interest to install a single oxidative coupling partner. After
removal of the excess linker, the Nꢀterminally modified protein can be coupled to anilines on a second biomolecule. The oꢀ
aminophenol and oꢀcatechol versions of these reagents would be expected to condense with the free aldehyde groups, thus
highlighting the advantages of the methoxy derivatives.
Two heterobifunctional crosslinkers with hydrophilic (5) and hydrophobic (6) spacers were synthesized (Figure 4c). To
evaluate their reactivity, proofꢀofꢀconcept studies were first conducted with RNase A and paF MS2 viral capsids. RNase A
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has previously demonstrated high levels of Nꢀterminal modification by 2PCA reagents.
Reactions with 25 µM RNase A and 10 mM crosslinker in pH 7.5 phosphate buffer at 37 °C for 16 h showed good
conversions to oꢀmethoxyphenol linkerꢀprotein conjugates (MPꢀRNase A), as analyzed by ESIꢀTOF mass spectrometry
Figures 5aꢀc). After purification to remove excess crosslinker, SDSꢀPAGE analysis showed that the MPꢀRNase A conjugate
(
was successfully conjugated to paF MS2 by oxidative coupling in the presence of NaIO (Figure 5d). A screen of varying
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concentrations of NaIO for the oxidative coupling reaction showed a strong dependence on oxidant concentration with the
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highest conversion occurring at 2 mM. Most importantly, the selectivity of this strategy was confirmed by the lack of RNase
AꢀMS2 conjugates when reactions were conducted with no NaIO , with wildꢀtype MS2 (lacking paF residues), or with
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unmodified RNase A (see Supplementary Figure S3).
Strategy Validation: A Receptor-Targeted Imaging Agent
With the ability to attach protein substrates to the exterior of MS2 capsids using the heterobifunctional crosslinkers, this
strategy was applied to the construction of a targeted imaging agent by attaching the human epidermal growth factor (EGF).
EGF has become a popular targeting biomolecule for cancer therapy because it is known to bind to the epidermal growth
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factor receptor (EGFR), which is overexpressed in human carcinomas, at low nanomolar affinity.
Commercially
available EGF was readily modified with oꢀmethoxyphenol crosslinkers 5 or 6, as determined by ESIꢀTOF mass
spectrometry (Figures 6aꢀc). In addition to the anticipated modification, a second addition of the crosslinkers without the loss
of water was observed. This is most likely the result of a competing aldolꢀtype reaction, also at the Nꢀterminal position.
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Previous investigations into this type of product have been consistent with this outcome.
Surprisingly, the optimal oxidative coupling conditions (4:1 protein to MS2 monomer ratio, 2 mM NaIO , pH 6.5, 5 min)
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found for the conjugation of the linkerꢀRNAse A conjugates to paF MS2 afforded low yields when MPꢀEGF linker
conjugates were used. Optimization studies with the MPꢀlinker conjugates found that yields could be improved by increasing
the concentration of NaIO to 5 mM or 8 mM and extending the reaction time to 10 min (Supplementary Figure S6). Similar
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to studies with RNAse A, performing the reaction at pH 5.5–6.5 afforded the highest yields. Of note, the addition of 150 mM
NaCl increased the yield of the reaction. NaCl may serve to minimize any repulsive electrostatic interactions between EGF
and the MS2 viral capsid, as both have a negative surface charge. With these optimal conditions, only two equivalents of the
MPꢀEGF conjugate were required to achieve sufficient yields. Selectivity was confirmed by the lack of coupling in reactions
performed with no NaIO , with wildꢀtype MS2, or with unmodified EGF (Supplementary Figure S6).
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The levels of modification were quantified by optical densitometry based on fluorescence of an Oregon Green 488 dye
covalently attached on the interior surface (vide infra). Modification levels were found to range between 19% and 31% (55ꢀ
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6 copies per capsid), depending on the amount of the MPꢀEGF that was used (Figure 6d). Importantly, excess MPꢀEGF
conjugate could be readily removed by using 100 kDa MWCO ultracentrifugation spin concentrators, as demonstrated by the
lack of an EGF protein band in the Coomassieꢀstained gel.
Transmission electron microscopy of the EGFꢀMS2 conjugates confirmed that the capsids remained intact and were not
distorted by the reaction conditions (Supplementary Figure S8). Additionally, size exclusion chromatography (SEC) and
dynamic light scattering (DLS) studies indicated an increase in capsid diameter that was consistent with the addition of EGF
to the exterior surfaces (Supplementary Figure S8).
The stability of the EGFꢀMS2 conjugates was assessed over time (Supplementary Figure S9). Over 48 h, storage of these
conjugates at 4 °C resulted in minimal decomposition. Surprisingly, storage at 23 °C resulted in the loss of close to half of the
EGF attached to MS2 over the same time period. These results are in contrast to previous stability studies performed with the
product of 2PCA and RNase A, which was stable at 23 °C. The increased steric bulk or repulsive electrostatic interaction of
EGF with MS2 may account for the reduced stability of the 2PCAꢀEGF linkage.
Evaluation of EGF-MS2 Conjugates
The cellꢀtargeting ability of the EGFꢀMS2 conjugates was evaluated with flow cytometry studies. A fluorescently labeled
EGFꢀMS2 conjugate was synthesized by first alkylating the mutated cysteine residue inside of paF N87C MS2 capsids with
Oregon Green 488 maleimide dye (EGFꢀMS2ꢀOG). Then, EGF was attached to the exterior of the MS2 capsid via
heterobifunctional crosslinker 5. Cell binding studies were conducted with an EGFRꢀpositive human epidermoid carcinoma
cell line, A431, and an EGFRꢀnegative breast cancer cell line, MCF7. These cell lines were incubated with varying
concentrations of MS2ꢀOG conjugates for 1 h at 4 °C (Figures 7aꢀb). Doseꢀdependent binding of EGFꢀMS2ꢀOG to A431
cells was observed, while A431 cells incubated with MS2ꢀOG (lacking EGF) under the same conditions showed no binding.
The specificity of the EGFꢀMS2ꢀOG agent was also verified by lack of binding to EGFRꢀnegative MCF7 cells even at the
highest concentration of MS2 conjugates. Finally, incubation of the A431 cells with EGFꢀMS2ꢀOG and increasing
concentrations of free soluble EGF decreased the binding of EGFꢀMS2ꢀOG (see Supplementary Figure S10), confirming the
specific EGF/EGFR interaction.
Confocal microscopy images of live cells further verified that EGFꢀMS2ꢀOG conjugates bind A431 cells (Figure 7c).
Increased binding was observed over time at 37 °C. After 90 min of incubation, images showed accumulation of green
fluorescence inside the cells, suggesting that EGFꢀMS2ꢀOG may be internalized through receptorꢀmediated endocytosis.
Similar to the flow cytometry findings, MS2ꢀOG revealed no binding to the cells even after 2 h at 37 °C.
CONCLUSIONS
The oxidative coupling of oꢀmethoxyphenols and anilines detailed above provides a convenient platform for the effecient
preparation of complex bioconjugates. The excellent air stability of these derivatives and their convenient commercial
availability make them an attractive option for bioconjugation. The short reaction times (10 min) and high effciencies mirror
the previouslyꢀreported oxidative couplings of oꢀaminophenols while also enabling preꢀoxidation protocols that are
ineffective with the latter substrates. The improved storability and unique reactivity facilitated the development of a
heterobifunctional linker strategy that has the potential to enable the synthesis of complex bioconjugates. The ability to
construct wellꢀdefined proteinꢀprotein conjugates rapidly with predictable attachment sites is difficult to achieve and relies
heavily on protein engineering techniques. This crosslinker strategy lessens the need for protein engineering and offers the
ability to incorporate endogenous or commercially available proteins into these conjugates. Further optimization and
application of both reaction types continue in our laboratories.
AUTHOR INFORMATION
Corresponding Author
mbfrancis@berkeley.edu
Notes
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The authors declare no competing financial interest.
ACKNOWLEDGEMENTS
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The Nꢀterminal chemistry described in this work was supported by the NSF (CHEꢀ1413666). The studies of MS2ꢀEGF
conjugates were supported by the NIH (R21 EB018055). AME was supported by the DOD Breast Cancer Research Program
(
W81XWHꢀ14ꢀ0400). JIM and ILA were supported by the Berkeley Chemical Biology Graduate Program (Training Grant 1
T32 GMO66698). NBH was supported by a Fulbright Scholarship.
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0
SUPPORTING INFORMATION AVAILABLE
Supporting information, including full experimental details, characterization of small molecules, and supplementary figures,
is provided. This information is available free of charge via the Internet at http://pubs.acs.org.
REFERENCES
1.Stephanopoulos, N.; Francis, M. B. Nat. Chem. Biol. 2011, 7, 876−884.
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.Boutureira, O.; Bernardes, G. J. L. Chem. Rev. 2015, 115, 2174−2195.
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.Gautier, A.; Juillerat, A.; Heinis, C.; Correa Jr., I. R.; Kindermann, M.; Beaufils, F.; Johnsson, K. Chem. Biol. 2008,
5, 128–136.
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.Halo, T. L., Appelbaum, J.; Hobert, E. M.; Balkin, D. M.; Schepartz, A. J. Am. Chem. Soc. 2009, 131, 438–439.
.Zhang, C.; Spokoyny, A. M.; Zou, Y.; Simon, M. D.; Pentelute, B. L. Angew. Chem. Int. Ed. 2013, 52, 14001–14005.
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120–128.
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1. Kovacs, E. W.; Hooker, J. M.; Romanini, D. W.; Holder, P. G.; Berry, K. E.; Francis, M. B. Bioconjugate Chem.
007, 18, 1140ꢀ1147
2
1
1
2. Wu, W; Hsiao, S. C.; Carrico, Z. M.; Francis, M. B. Angew. Chem. Int. Ed. 2009, 48, 9493ꢀ9497.
3. Tong, G. J.; Hsiao, S. C.; Carrico, Z. M.; Francis, M. B. J. Am. Chem. Soc. 2009, 131, 11174ꢀ11178.
14. Stephanopoulos, N.; Tong, G. J.; Hsiao, S. C.; Francis, M. B.; ACS Nano. 2010, 4, 6014ꢀ6020.
1
5. Netirojjanakul, C.; Witus, L. S.; Behrens, C. R.; Weng, C. H.; Iavarone, A. T.; Francis, M. B. Chem. Sci. 2013, 4,
66ꢀ272.
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1
6. Finbloom, J. A.; Han, K.; Aanei, I. L.; Hartman, E. C.; Finley, D. T.; Dedeo, M. T.; Fishman, M.; Downing, K. H.;
and Francis, M. B. Bioconjugate Chem. 2016, 27, 2480ꢀ2485.
17. EsserꢀKahn, A. P.; Francis, M. B. Angew. Chem. Int. Ed. 2008, 47, 3751ꢀ3754.
18. Miller, R. A.; Presley, A. D.; Francis, M. B. J. Am. Chem. Soc. 2007, 129, 3104ꢀ3109.
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9. Ma, Y.; Miller, R. A.; Fleming, G. R.; Francis, M. B. J. Phys. Chem. B. 2008, 112, 6887ꢀ6892.
0. Stephanopoulos, N.; Carrico, Z. M.; Francis, M. B. Angew. Chem. Int. Ed. 2009, 48, 9498ꢀ9502.
1. Miller, R. A.; Stephanopoulos, N.; McFarland, J. M.; Rosko, A. S.; Geissler, P. L.; Francis, M. B. J. Am. Chem. Soc.
2
010, 132, 6068ꢀ6074.
22. Behrens, C. R.; Hooker J.M.; Obermeyer, A.C.; Romanini, D.W.; Katz, E. M.; Francis MB. J Am Chem Soc. 2011,
33, 16398−16401.
3. Obermeyer, A. C.; Jarman, J. B.; Netirojjanakul, C.; El Muslemany, K.; Francis, M. B. Angew. Chem. Int. Ed. 2014,
3, 1057−1061.
4. Obermeyer, A. C.; Jarman, J. B.; Francis, M. B. J. Am. Chem. Soc. 2014, 136, 9572−9579.
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25. ElSohly, A. M.; Francis, M. B. Acc. Chem. Res. 2015, 48, 1971ꢀ1978.
26. Liu, B.; Burdine, L.; Kodadek, T. J. Am. Chem. Soc. 2006, 128, 15228–15235.
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7. MacDonald, J. I.; Munch, H. K.; Moore, T.; and Francis, M. B. Nature Chem. Bio. 2015, 11, 326ꢀ331.
8. El Muslemany, K. M.; Twite, A. A.; ElSohly, A. M.; Obermeyer, A. C.; Mathies, R. A.; Francis, M. B. J. Am.
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Chem. Soc. 2014, 136, 12600−12606.
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30. Deffieux, D.; Gossart, P.; Quideau, S. Tetrahedron Lett. 2014, 55, 2455ꢀ2458.
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1. Carrico, Z. M.; Romanini, D. W.; Mehl, R. A.; Francis, M. B. Chem. Commun. 2008, 1205ꢀ1207.
2. Hermanson, G. T. Heterobifunctional Crosslinkers. Bioconjugate Techniques, 3rd Edition; Academic Press: San
Diego, CA, 2013, pp 299ꢀ339.
3
3. Friedhoff, P. Anal. Bioanal. Chem. 2004, 381, 78–80
0
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34. Rappsilber, J.; Siniossoglou, S.; Hurt, E. C.; Mann, M. Anal. Chem. 2000, 72, 267–275.
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5. ShriverꢀLake, L. C.; Donner, B.; Edelstein, R.; Breslin, K.; Bhatia, S. K.; Ligler, F. S. Biosens. Bioelectron. 1997,
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17.
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37. Normanno, N.; De Luca, A.; Bianco, C.; Strizzi, L.; Mancino, M.; Maiello, M. R.; Carotenuto, A.; De Feo, G.;
Caponigro, F.; Salomon, D. S. Gene 2006, 366, 2–16.
3
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Figure Captions:
Figure 1. Previously reported oxidative couplings and initial experiments with a methoxyphenol variant. (a) Oxidative
couplings of aminophenols in the presence of sodium periodate and potassium ferricyanide are summarized. (b) Control
experiments from a related lightꢀactivated azidophenol coupling exhibited notable conversion to coupled product in the
presence of a strong oxidant but in the absence of light, prompting consideration of other derivatives for oxidative couplings.
c) A 2ꢀmethoxyꢀpꢀcresol substrate undergoes highly efficient oxidative coupling with toluidine in the presence of sodium
(
periodate as the oxidant (see HPLC trace of the crude reaction mixture to the right). (d) Orthoꢀmethoxyphenols are
ubiquitious, airꢀstable, and commercially available, making them convenient reagents for oxidative coupling reactions.
Figure 2. Reaction optimization in the context of macromolecular couplings. All reactions were optimized for the conversion
of the reaction described in scheme (a) by gel shift assays and quantified by optical densitometry. (b) Time screens reveal
that the reaction reaches completion in 10 min. (c) Most buffers are compatible with this reaction, with Tris serving as a
notable exception (due to the presence of a vicinal aminoalcohol). (d) Five equiv of oꢀmethoxyphenol are sufficient to
achieve high levels of conversion. (e) The reaction is essentially pH independent over the range of 6–8.
Figure 3. Development of methods to reduce periodate equivalents/exposure to proteins. All reactions were optimized for the
conversion of the reaction described in scheme (a) by gel shift assays and quantified by optical densitometry. (b) Preꢀ
oxidation of the oꢀmethoxyphenol component for as little as 2 min prior to addition of the anilineꢀcontaining protein reduces
the concentration of periodate required for the reaction (500 µM vs 1 mM) while maintaining high reaction conversions. (c)
Orthoꢀaminophenol PEG (APꢀPEG) was used as a control to assess the effectiveness of mannose in quenching periodate.
Incubation of periodate with mannose prior to addition of the PEG reagent completely inhibits the reaction. (d) Combining a
preꢀoxidation and mannose quench provides useful levels of conversion after addition of paFꢀMS2.
Figure 4. Heterobifunctional crosslinkers. (a) Representative heterobifunctional crosslinkers that bear orthogonally reactive
groups at the end of a spacer region. Common reactive moieties employed in this class of reagents are depicted. (b) Selective
Nꢀterminal reactivity of 2ꢀpyridinecarboxaldehyde (2PCA) and design of a new siteꢀselective heterobifunctional crosslinker
comprised of 2PCA and oꢀmethoxyphenol moieties. (c) Synthesis of oꢀmethoxyphenol/2PCA heterobifunctional crosslinkers
5
and 6. Two different crosslinkers were synthesized with variable hydrophilic (5) or hydrophobic (6) spacers.
Figure 5. Synthesis of a proteinꢀprotein conjugate using oꢀmethoxyphenol/2PCA heterobifunctional crosslinkers. (a) RNase
A is modified to good conversions with crosslinker 5 or 6, as demonstrated by reconstructed ESIꢀTOF mass spectra (b) and
c), respectively. (d) Conjugation of RNase A to MS2 in the presence of NaIO is confirmed by SDSꢀPAGE analysis.
(
4
Figure 6. Synthesis of EGFꢀMS2 conjugates using oꢀmethoxyphenol/2PCA heterobifunctional crosslinkers. (a) EGF is
modified to good conversions with crosslinker 5 or 6, as demonstrated by reconstructed ESIꢀTOF mass spectra (b) and (c),
respectively. (d) Conjugation of MPꢀEGF with a PEG spacer to MS2 in the presence of NaIO is confirmed by SDSꢀPAGE
4
analysis after spin concentration to remove unbound EGF.
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Figure 7. Binding studies of EGFꢀMS2 conjugates. Flow cytometry data of EGFꢀMS2ꢀOregon Green 488 indicate that
conjugates bind to EGFRꢀpositive cells (a) and do not bind to EGFRꢀnegative cells (b). (c) Live cell confocal microscopy
indicates that these conjugates bind EGFRꢀpositive cells and are rapidly internalized. Cells treated with no agent or capsids
lacking EGF do not exhibit significant fluorescence signal in the green channel.
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4
4
4
4
4
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4
4
5
5
5
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5
5
5
5
6
a
O
R2
0
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6
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0
1
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9
0
NaIO4
O
CN
O
(
X = NH2 only)
N
NH2
R2
H
R1
OH
R2
K3Fe(CN)6
OH
R1
N
X
R1
OH
O
O
O
N
O
O
NH2
3
K Fe(CN)
6
X = NH2 or OH
R2
R2
R1
O
H
K3Fe(CN)6
O
N
N
R1
OH
R1
NH2
OH
b
N3
NaIO4
R2
O
+
no light
N
R2
R1
~
40% by HPLC
H
O
I(VII)
[
]
H
O
O
O
OH
–HN3
N3
O
N3
R1
R1
R1
c
d
OH
Me
NH2
OH
coupled
product
OMe
+
NaIO4
Me
O
N
pH 7
BisTris
Me
Me
5
10
15
20
~quant. by HPLC
time (min)
OH
OH
OH
OMe
OMe
OMe
–or–
–or–
H
O
NH2
HO
O
vanillin
3-O-methyldopamine
ferulic acid
[
convenient, air-stable sources]
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a
b
OH
OH
10
11
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13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
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37
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40
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44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
OMe
O
[
Conditions]
+
NH2
N
paF MS2
PEG5k
PEG5k
80
70
c
80
70
60
60
50
40
50
40
30
20
10
30
2
0
0
1
0
0
0
10
20
30
Time (min)
Buffer
Conditions: MP-PEG (200 µM),
Conditions: MP-PEG (200 µM),
MS2 (20 µM), NaIO (1 mM),
10 mM pH 7 buffer, 10 min
MS2 (20 µM), NaIO (1 mM),
4
4
1
0 mM pH 7 phos
d
80
e 80
70
60
50
40
30
20
10
0
7
6
5
4
0
0
0
0
30
20
10
0
2.5
5
10
6
6.5
7
pH
7.5
8
equivalents
Conditions: MP-PEG (20x µM),
MS2 (20 µM), NaIO (1 mM),
10 mM pH 7 phos, 10 min
Conditions: MP-PEG (100 µM),
MS2 (20 µM), NaIO (1 mM),
4
4
10 mM phos, 10 min
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2
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2
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3
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4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a
b
OH
OH
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
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8
9
0
1
2
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4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
OMe
O
[
Conditions]
+
NH2
N
paF MS2
PEG5k
PEG5k
9
0
0
0
0
0
8
7
6
5
40
3
2
10
0
0
0
0
2
4
6
8
10
pre-oxidation time (min)
Conditions: MP-PEG (100 µM),
NaIO (500 µM), 10 mM pH 7 phos,
4
then paF-MS2 (20 µM), 10 min
c
90
80
d 70
OH
60
7
6
0
0
NH2
50
40
30
50
4
3
0
0
PEG5k
(AP-PEG)
2
0
0
20
1
10
0
0
0
5
10
0
2
5
10
quench time (min)
quench time after pre-oxidation (min)
Conditions: NaIO (500 µM),
mannose (5 mM), then AP-PEG (100µM),
10 mM pH 7 phos,
Conditions: MP-PEG (100 µM),
4
NaIO (500 µM), 5 min then
4
mannose (5 mM) 10 mM pH 7 phos
then paF-MS2 (20 µM), 10 min
then paF-MS2 (20 µM), 10 min
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4
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4
4
4
4
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5
5
5
5
5
5
5
5
5
6
a
b
R
O
O
O
O
R
O
O
O
H
HN
N
O
N
O
O
N
H
O
N
N
+
2
H N
N
N
N
H
O
O
N
4
H
O
O
O
O
0
1
2
3
4
5
6
7
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9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
O
O
O
O
H
N
O
O
NH2
O
N3
N
N
N
N
Br
MeO
HO
spacer
O
H
N
N
O
O
H
O
N
O
O
O
N
H
aldehyde reactive
CuAAC reactive
thiol reactive
amine reactive
aniline selective
N-terminus selective
O
c
N
1
) NH -(PEG) -COOtBu
Et N, DMF, 23 °C, 16 h
OH
N
H
2
3
HN
OH
OMe
O
3
OMe
O
2
) TFA, CH Cl , 23 °C, 2 h
4
O
2
2
N
N
H
O
OH
N
H
HCTU, DIPEA
DMF, 23 °C, 16 h
O
N
3
N
H
OH
O
3
O
OMe
2
5
6
O
O
O
N
N
O
6-aminohexanoic acid
N
H
OH
OH
HN
O
Et N, DMF
3 °C, 16 h
1
3
OMe
O
OMe
O
O
2
4
N
N
H
OH
N
N
H
HCTU, DIPEA
DMF, 23 °C, 16 h
N
H
O
O
3
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
OH
25 µM
RNase A
Lys
a
OMe
O
5
or 6
HN
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
(
10 mM)
pH 7.5
N
N
N
37 °C
spacer
c
N
12-16 h
N
H
O
b
with compound 5:
14251 (P)
with compound 6:
14161 (P)
1
3682 (SM)
1
3682 (SM)
13.0
13.4 13.8 14.2 14.6
15.0 13.0
13.4 13.8 14.2 14.6
15.0
mass (kDa)
mass (kDa)
d
OH
Lys
O
O
HN
O
N
N
N
N
O
N
N
H
3
O
kDa
60
50
40
30
RNase A-MS2
MP-RNase A
2
0
5
1
MS2 monomer
NaIO4 (mM):
rel. mod.:
2
1
0.5 0.2 0.1
0
0
%
100 95 72 21 12
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a
b
OH
Asn
2
EGF
5 µM
OMe
O
5
or 6
HN
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
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45
46
47
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49
50
51
52
53
54
55
56
57
58
59
60
O
(
10 mM)
N
pH 7.5
3
N
N
7 °C
spacer
c
N
12-16 h
N
H
O
with compound 5: 6784 (P)
with compound 6:
6694 (P)
(
SM+2(5)-H O)
(SM+2(6)-H O)
6
215 (SM)
2
2
6
215 (SM)
7370
7191
5.0
6.0
7.0
8.0
5.0
6.0
7.0
mass (kDa)
8.0
mass (kDa)
OH
d
Asn
O
O
HN
O
N
N
N
N
spacer
N
N
H
O
Coomassie fluorescence
kDa
6
0
50
40
30
20
EGF-(MS2)2
EGF-MS2
15
MS2 monomer
10
EGF
3.5
1
2
equiv EGF/MS2
19 31 % EGF-MS2
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 14 of 15
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2
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a
A431 (EGFR+)
b
MCF7 (EGFR-)
1
00
100
80
80
60
40
20
0
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
4
2
0
0
0
0
0
1
2
3
4
0
1
2
3
4
1
0
10
10
10
10
10
10
Fluorescence
MS2-OG
10
10
10
Fluorescence
no agent
111 nM EGF-MS2-OG
2
2 nM EGF-MS2-OG
222 nM EGF-MS2-OG
c
2
0 µm
20 µm
20 µm
20 µm
20 µm
2
0 µm
ACS Paragon Plus Environment

Page 15 of 15
Journal of the American Chemical Society
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40
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50
51
52
53
54
55
56
57
58
59
60
OH
R1
NH2
OH
R1
OMe
NaIO4
R2
O
+
10 min
N
R2
air stable,
commercial
high yield,
single product
OH
OMe
O
O
aniline
selective
N
N-terminus
H
N
selective
spacer
N
N
H
O
site-selective heterobifunctional crosslinker
ACS Paragon Plus Environment