A Pictet-Spengler ligation for protein chemical modification

Article abstract of DOI:10.1073/pnas.1213186110

Aldehyde- and ketone-functionalized proteins are appealing substrates for the development of chemically modified biotherapeutics and protein-based materials. Their reactive carbonyl groups are typically conjugated with α-effect nucleophiles, such as substituted hydrazines and alkoxyamines, to generate hydrazones and oximes, respectively. However, the resulting C=N linkages are susceptible to hydrolysis under physiologically relevant conditions, which limits the utility of such conjugates in biological systems. Here we introduce a Pictet-Spengler ligation that is based on the classic Pictet- Spengler reaction of aldehydes and tryptamine nucleophiles. The ligation exploits the bioorthogonal reaction of aldehydes and alkoxyamines to form an intermediate oxyiminium ion; this intermediate undergoes intramolecular C-C bond formation with an indole nucleophile to form an oxacarboline product that is hydrolytically stable. We used the reaction for site-specific chemical modification of glyoxyl- and formylglycine-functionalized proteins, including an aldehyde-tagged variant of the therapeutic monoclonal antibody Herceptin. In conjunction with techniques for site-specific introduction of aldehydes into proteins, the Pictet-Spengler ligation offers a means to generate stable bioconjugates for medical and materials applications.

Full text of DOI:10.1073/pnas.1213186110

A Pictet-Spengler ligation for protein
chemical modification
Paresh Agarwala, Joep van der Weijdena, Ellen M. Slettena, David Rabukab, and Carolyn R. Bertozzia,c,d,1
Departments of aChemistry and cMolecular and Cell Biology, and dHoward Hughes Medical Institute, University of California, Berkeley, CA 94720;
and bRedwood Bioscience, Emeryville, CA 94608
Edited by David A. Tirrell, California Institute of Technology, Pasadena, CA, and approved November 7, 2012 (received for review July 31, 2012)
Aldehyde- and ketone-functionalized proteins are appealing sub-
strates for the development of chemically modified biotherapeutics
and protein-based materials. Their reactive carbonyl groups are
typically conjugated with α-effect nucleophiles, such as substituted
hydrazines and alkoxyamines, to generate hydrazones and oximes,
respectively. However, the resulting C=N linkages are susceptible to
hydrolysis under physiologically relevant conditions, which limits
the utility of such conjugates in biological systems. Here we intro-
duce a Pictet-Spengler ligation that is based on the classic Pictet-
Spengler reaction of aldehydes and tryptamine nucleophiles. The
ligation exploits the bioorthogonal reaction of aldehydes and
alkoxyamines to form an intermediate oxyiminium ion; this inter-
mediate undergoes intramolecular C–C bond formation with an
indole nucleophile to form an oxacarboline product that is hydro-
lytically stable. We used the reaction for site-specific chemical mod-
ification of glyoxyl- and formylglycine-functionalized proteins,
including an aldehyde-tagged variant of the therapeutic monoclo-
nal antibody Herceptin. In conjunction with techniques for site-spe-
cific introduction of aldehydes into proteins, the Pictet-Spengler
ligation offers a means to generate stable bioconjugates for med-
ical and materials applications.
modification by the formylglycine-generating enzyme (FGE), the
“aldehyde tag” method developed in our laboratory (21–25).
The diversity of methods for introducing reactive carbonyl
groups into proteins stands in contrast to the limited number of
reactions that have been adopted for their chemical modification.
Reductive amination has found some use, mainly with glycopro-
tein substrates in which aldehydes were introduced by glycan
oxidation (26). But the vast majority of examples use the hydra-
zone and oxime-forming reactions mentioned previously because
of their bioorthogonality, operational simplicity (i.e., no auxiliary
reagents are required), and good yields under mild aqueous
conditions. However, the resulting C=N bonds are susceptible to
hydrolysis (27), undermining the use of such conjugates in sit-
uations in which long-term stability is required. The oxime has
been identified as the most hydrolytically stable C=N linkage, but
it is still thermodynamically unstable to hydrolysis under dilute
conditions, decomposing via an acid-catalyzed process (28). Many
researchers have found that oxime conjugates that are kept under
ideal storage conditions—low temperature, high concentration,
and neutral or high pH—are kinetically stable and are therefore
suitable for short-term laboratory studies (23, 25, 29). However,
biological applications requiring extended persistence of the
conjugate at physiological temperatures and low concentrations
necessitate a significantly more stable covalent linkage than the
oxime provides.
The ideal bioconjugation reaction would form a stable C–C
bond with protein aldehydes and ketones. A few such reactions
have been reported, but they are limited by slow reaction kinetics
(30) or the need for organic cosolvents (31, 32). A C–C bond-
forming transformation possessing the kind of generality and
operational simplicity that led to the widespread adoption of
oxime bioconjugation has not yet been reported. Here we de-
scribe the development of the Pictet-Spengler ligation, a C–C
bond-forming reaction that capitalizes on the bioorthogonality of
oxime formation in an intermediate step. We used this reaction to
prepare hydrolytically stable conjugates with glyoxyl- and for-
mylglycine-modified proteins, including a monoclonal antibody.
bioorthogonal chemistry protein conjugation reaction methodology
|
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eaction methodology for protein modification has been an
Ractive area of research for decades. Early strategies focused
on global modification of native amino acids, providing access to
heterogeneously modified products (1). However, a variety of
applications necessitate site-specific modification of proteins:
biophysical studies requiring knowledge of the site of attachment
of a reporter molecule (2), preparation of protein microarrays
and functional materials requiring immobilization in a specific
orientation (3), and conjugation of protein drugs with poly(eth-
ylene glycol) or cytotoxic molecules, where the site of chemical
modification affects the pharmacokinetic and therapeutic prop-
erties of the resulting biologic (4, 5). Therefore, in recent years,
the field has refocused on methods to achieve site-specific pro-
tein modification, typically by introduction of a nonnative func-
tional group exhibiting bioorthogonal reactivity (6, 7).
Aldehydes and ketones are popular choices as chemical handles
for site-specific protein modification. Their unique reactivity as mild
electrophiles enables selective conjugation with α-effect nucleo-
philes such as substituted hydrazines and alkoxyamines, which
generate hydrazone and oxime-ligated products, respectively (8).
Several chemical, enzymatic, and genetic methods have been de-
veloped to introduce aldehydes and ketones into proteins site spe-
cifically. These include periodate oxidation of N-terminal serine
or threonine residues (9), pyridoxal phosphate-mediated N-terminal
transamination to yield an α-ketoamide or glyoxamide (10–13), ad-
dition of ketone-containing small molecules to protein C-terminal
thioesters generated by expressed protein ligation (14), genetically
encoded incorporation of unnatural amino acids containing ketones
via amber stop codon suppression (15–18), genetic encoding of
peptide tags that direct enzymatic ligation of aldehyde- or ketone-
bearing small molecules (19, 20), and genetic encoding of a site for
Results and Discussion
Design and Synthesis of Pictet-Spengler Ligation Reagents. For the
past century, the Pictet-Spengler reaction has played an im-
portant role in the synthesis of indole alkaloid natural products
(33). We hypothesized that the transformation (Fig. 1A), which
forms a C–C bond between tryptamine and an aldehyde or a
ketone, could be adapted for the purpose of irreversible bio-
conjugation. The canonical Pictet-Spengler reaction has previously
Author contributions: P.A., E.M.S., and C.R.B. designed research; P.A. and J.v.d.W. per-
formed research; D.R. contributed new reagents/analytic tools; P.A., J.v.d.W., and C.R.B.
analyzed data; and P.A., J.v.d.W., E.M.S., and C.R.B. wrote the paper.
Conflict of interest statement: C.R.B. is a cofounder and member of the Scientific Advisory
Board of Redwood Bioscience.
This article is a PNAS Direct Submission.
1To whom correspondence should be addressed. E-mail: crb@berkeley.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1213186110/-/DCSupplemental.
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cost, off-target reactivity, purification of the resulting conjugate,
and toxicity if applied to protein labeling on live cells.
In our design of the Pictet-Spengler ligation (Fig. 1B), we were
guided by a thorough kinetic study of the Pictet-Spengler reaction
indicating that formation of an iminium ion intermediate is par-
tially rate-limiting (34). Taking note of the rapid rates at which
α-effect amines condense with aldehydes and ketones (35), we
hypothesized that the rate of iminium ion formation could be
enhanced by replacing the aliphatic amine of tryptamine with an
aminooxy moiety. To further increase the rate of the reaction, we
moved the aminooxy substituent to the 2-position of the indole,
allowing the more nucleophilic 3-position to engage in electro-
philic substitution. Indoles that are substituted with aliphatic
amines at the 2-position are known to engage in “iso-Pictet-
Spengler” reactions in organic solvents (36, 37). Finally, we
methylated the aminooxy functionality to provide a reactive oxy-
iminium ion intermediate that would facilitate rapid C–C bond
formation via intramolecular electrophilic substitution. Pictet-
Spengler reactions of N-alkoxytryptamines to afford products with
exocyclic aminooxy functionality are known (38–40), but to the
best of our knowledge neither their kinetics nor their behavior in
aqueous media has been studied. With these precedents, we
expected aminooxy-functionalized indoles 1 to engage in a fast
Pictet-Spengler type reaction (Fig. 1B).
We prepared model indole 1a in a short, high-yielding syn-
thesis (Fig. 1C). First, an ester was installed as a masked func-
tionalization handle by protection of indole-2-methanol with
TBSCl followed by a 1,8-diazabicyclo[5.4.0]undec-7-ene–cata-
lyzed aza-Michael addition to methyl acrylate (41). Following
deprotection of the hydroxyl group to yield indole 2, the ami-
nooxy moiety was installed by reaction with 2-(trimethylsilyl)
ethoxycarbonyl (Teoc)-protected N-methylhydroxylamine under
modified Mitsunobu conditions (42, 43). Saponification of the
resulting product yielded compound 3, which was cleanly
deprotected with CsF to afford indole 1a in 66% yield over
six steps.
Reactivity of Model Indole 1a and Hydrolytic Stability of Products. To
validate the Pictet-Spengler ligation, we treated indole 1a with
either isobutyraldehyde or acetone in methanolic ammonium
acetate solutions at pH 4.5. Both reactions proceeded very
cleanly in less than 1 h to afford the desired dihydro-β-oxa-
γ-carboline (hereafter referred to as oxacarboline) products (SI
Appendix, Fig. S1). Analysis of the rate of the reaction of 1a with
isobutyraldehyde in D2O by 1H NMR spectroscopy revealed
a rate law that is first-order in the concentrations of 1a and
isobutyraldehyde at pD 7.0 (pD = pH meter reading + 0.41; SI
Appendix, Fig. S2 and Table S1), and a pD-rate constant profile
characteristic of aminooxy compounds under acidic conditions
(Fig. 1D) (44). These results show that our rate-enhancement
strategies were successful, as the Pictet-Spengler ligation is 4–5
orders of magnitude faster than the canonical Pictet-Spengler
reaction in aqueous media.*
Next, we compared the hydrolytic stability of the oxacarbo-
line generated by the Pictet-Spengler ligation with that of
a model oxime. We treated an aldehyde-derivatized fluorescein
4 (Fig. 2A) with benzylalkoxyamine or 1a to generate con-
jugates 5a and 5b, respectively. Buffered solutions at pH 4.5 or
5.0 containing 1 μM 5a or 5b were incubated at room temper-
ature and analyzed by liquid chromatography. Over the course
of 2 d, the majority of oxime 5a hydrolyzed, whereas more than
Fig. 1. Design and evaluation of the Pictet-Spengler ligation. (A) The Pictet-
Spengler reaction. (B) The Pictet-Spengler ligation. (C) Synthesis of aldehyde-
and ketone-reactive indoles used in this study. (D) Second-order rate
constants for the reaction of 1a with isobutyraldehyde in D2O solutions
containing 100 mM deuterated acetate (pD ≤ 5.5) or phosphate (pD ≥ 6.0)
buffers. Error bars represent SD of at least three replicate experiments.
ADDP, 1,1′-(azodicarbonyl)dipiperidine; DBU, 1,8-diazabicyclo[5.4.0]undec-
7-ene; DIPEA, diisopropylethylamine; PFP, pentafluorophenyl; TBAF, tetra-
butylammonium fluoride; TBS, tert-butyldimethylsilyl; Teoc, 2-(trimethylsilyl)
ethoxycarbonyl.
been used in this context (30); however, the reaction is slow
under protein-compatible conditions, proceeding with a second-
order rate constant of ∼10−4 M−1·s−1 at pH 4–5 (34). These slow
reaction kinetics necessitate high concentrations (e.g., 50 mM)
of the derivatizing reagent to achieve good yields of modified
protein, which can be problematic from the standpoints of reagent
*In an attempt to further increase the rate of the reaction, we also synthesized a variant
of 1a with a methoxy group at the 5-position of the indole (SI Appendix, Fig. S3A).
Kinetic experiments at pD 4.5 and 6.0 showed that the methoxylated indole reacts with
isobutyraldehyde about 30% faster than 1a, depending on the acidity of the solution (SI
Appendix, Table S2). Given the modest increase in reaction rate, we pursued further
studies with unsubstituted indole 1a.
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90% of oxacarboline 5b remained intact (Fig. 2B); no products
other than those arising from hydrolysis were detected (SI
Appendix, Fig. S4). Previous work has shown that oxime hy-
drolysis occurs on a similar timescale in the presence of excess
formaldehyde used as a trap to drive the reaction toward hy-
drolysis of the conjugate (28). Notably, our results indicate that
oxime hydrolysis can occur to an appreciable extent in aqueous
solution, even in the absence of a trap, underscoring the need
for irreversible bioconjugation reactions. These model experi-
ments establish that the Pictet-Spengler ligation proceeds
rapidly under acidic conditions to yield a hydrolytically stable
product.
Scope of the Reaction on Model Proteins. We next evaluated the
Pictet-Spengler ligation as a means to label aldehyde-func-
tionalized proteins. To facilitate the detection and manipula-
tion of labeled proteins, we prepared biotinylated indole 1b by
coupling indole 3 with amino-poly(ethylene glycol)-functional-
ized biotin, followed by deprotection of the Teoc group with
CsF. As a protein substrate for reaction with 1b, we generated
horse heart myoglobin with an N-terminal glyoxyl moiety
(glyoxyl-Mb) by pyridoxal phosphate-mediated transamination
(Fig. 3A) (10). In glyoxyl-Mb labeling experiments, conjugated
product was detected by SDS/PAGE and Western blotting with
a FITC-conjugated α-biotin antibody after quenching excess
labeling reagent with benzaldehyde. First, we established that
labeling occurs in a concentration- and time-dependent manner
(Fig. 3 B and C, respectively). Importantly, control samples of
Mb that were not aldehyde-functionalized showed negligible
labeling. We next studied the pH-dependence of the reaction,
observing a greater extent of biotinylation at more acidic pH
(Fig. 3D), as was also observed in kinetic studies of indole 1a.
Finally, we found that cotreatment with benzylalkoxyamine as
an aldehyde scavenger resulted in diminished biotinylation
Fig. 3. Optimization of the Pictet-Spengler ligation on glyoxyl-Mb. (A)
General scheme for biotinylation of glyoxyl-Mb. Indole 1b exhibits (B) con-
centration-dependent, (C) time-dependent, and (D) pH-dependent labeling
of glyoxyl-Mb. Additionally, (E) biotinylation can be diminished by cotreat-
ment with BnONH2. Mb (– aldehyde) or glyoxyl-Mb (+ aldehyde) was treated
with (B) 0–200 μM 1b for 3 h at pH 4.0, (C) 250 μM 1b for 0–2 h at pH 4.0, (D)
250 μM 1b for 3 h at pH 4.0–7.5, or (E) 100 μM 1b for 3 h at pH 4.5 in the
presence of 0–800 μM BnONH2. All reactions were run at 37 °C and quenched
with 10 mM benzaldehyde before resolution by SDS/PAGE. Biotinylation was
assessed with an FITC-conjugated α-biotin antibody and total protein load-
ing with Ponceau S.
(Fig. 3E). A similar series of experiments using commercial
aminooxy-biotin as a labeling reagent showed the same quali-
tative trends in labeling (SI Appendix, Fig. S5). Collectively,
these results establish that indole 1b specifically labels the al-
dehyde functionality in transaminated myoglobin, and, more
generally, behaves like a typical aminooxy reagent.†
We next studied the reaction of 1a with formylglycine-func-
tionalized maltose-binding protein (FGly-MBP), prepared using
the genetically encoded aldehyde tag method (Fig. 4A) (24).
Briefly, the 6-residue peptide sequence LCTPSR was engineered
at the C terminus of MBP, constituting residues 389–394 in the
recombinant protein. We also included a thrombin cleavage site
N-terminal to the aldehyde tag sequence. Coexpression of the
protein alongside the Mycobacterium tuberculosis FGE in Escher-
ichia coli resulted in oxidation of Cys390 to FGly (21). As a control,
we also expressed the C390A mutant, which is not a substrate for
FGE and lacks the FGly aldehyde. Incubation of FGly-MBP with
1 mM indole 1a at 37 °C for 12 h resulted in quantitative
conversion to the desired singly modified adduct, as judged by
†We also explored whether the Pictet-Spengler ligation could be accelerated by aniline
catalysis (45). Aniline did not increase the rate of the reaction of 1b with glyoxyl-Mb at
pH 4.5, and at higher pH (5.5 or 6.5) aniline was found to inhibit the reaction (SI Ap-
pendix, Fig. S5E), which was consistent with previous observations (23, 24).
Fig. 2. Hydrolytic stability of a model oxime and oxacarboline. (A) Scheme
showing hydrolysis of indoles 5a and 5b. (B) Liquid chromatography data
showing hydrolysis of 1 μM 5a and 5b at room temperature over 2 d.
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Fig. 4. Modification of FGly-MBP by the Pictet-Spengler ligation. (A) Scheme depicting Pictet-Spengler ligation with FGly-MBP followed by thrombin-cat-
alyzed cleavage of a C-terminal 8-mer peptide containing the oxacarboline. (B) Deconvoluted mass spectra of Pictet-Spengler ligations. FGly-MBP and MBP
C390A were incubated with 1 mM 1a at pH 5.0 for 12 h at 37 °C before analysis by ESI-MS. Expected masses (Da): FGly-MBP, 43256, and 43238 (M – H2O); FGly-
MBP + 1a, 43486; MBP C390A, 43242. (C) Thrombin-catalyzed cleavage of FGly-MBP conjugates. (D) Fluorescence polarization analysis of AF488-MBP con-
jugate hydrolysis; (Inset) polarization of solutions immediately following thrombin addition. Solutions containing 100 nM AF488 conjugate were incubated in
PBS (pH 7.2) at 37 °C for 1 wk before thrombin addition.
electrospray ionization mass spectrometry (ESI-MS), whereas the
C390A mutant showed no reaction (Fig. 4B). Additionally, when
an FGly-MBP conjugate of 1a was digested with trypsin, we were
able to identify the C-terminal 8-residue tryptic peptide contain-
ing the desired adduct by high-resolution ESI-MS (SI Appendix,
Fig. S6A). MS/MS fragmentation of the tryptic peptide by elec-
tron-transfer dissociation provided direct evidence for modifica-
tion of the FGly residue (SI Appendix, Fig. S6B). Reaction of FGly-
MBP with tryptophan methyl ester under identical conditions
resulted in only minimal (<3%) conversion to the modified
product (SI Appendix, Fig. S7), confirming that the Pictet-Spengler
ligation proceeds much more quickly than the canonical Pictet-
Spengler reaction on proteins.
To confirm that labeling occurred only at the FGly residue, we
exploited the thrombin cleavage site engineered directly upstream
of the aldehyde tag sequence. First, we prepared indole 1c by
coupling 3 with Alexa Fluor 488 (AF488) cadaverine followed
by deprotection with CsF. Next, we prepared oxacarboline- or
oxime-linked AF488 conjugates of FGly-MBP by treatment with
either 1c or AF488 hydroxylamine, incubated the conjugates with
various amounts of thrombin for 1 h, and then analyzed the
products by SDS/PAGE. The intensity of in-gel fluorescence from
the FGly-MBP band decreased at higher thrombin concen-
trations, consistent with labeling exclusively within the cleaved
C-terminal 8-residue peptide (Fig. 4C). Notably, the oxime- and
oxacarboline-linked AF488-MBP conjugates displayed qualita-
tively similar behavior, indicating that, relative to the oxime, the
larger oxacarboline moiety did not inhibit the protein’s ability to
serve as a substrate for thrombin. These experiments establish
that the Pictet-Spengler ligation exclusively labels the FGly resi-
due on the aldehyde-tagged protein.
Hydrolytic Stability of the Oxacarboline Linkage on a Protein. Next,
we assayed the hydrolytic stability of the oxacarboline linkage on
FGly-MBP. Fluorescence polarization is a technique that yields
information about the tumbling rate of a fluorophore in solution:
macromolecule-conjugated fluorophores tumble slowly and exhibit
high polarization values, whereas small-molecule fluorophores ex-
hibit low polarization values. Thus, fluorescence polarization is
ideally suited to monitor cleavage of protein-fluorophore con-
jugates (46). A solution of FGly-MBP was treated with 1c, AF488
hydroxylamine, or a lysine-reactive AF488-sulfodichlorophenol
ester to make oxacarboline-, oxime-, or amide-linked AF488-MBP,
respectively (SI Appendix, Fig. S8). The samples were then diluted
to 100 nM in AF488 conjugate and incubated at 37 °C. The fluo-
rescence polarization was monitored for 1 wk (Fig. 4D). The oxime
conjugate exhibited a steady drop in polarization, indicating nearly
complete hydrolysis of the conjugate over the course of 1 wk. In
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Fig. 5. Characterization of FGly-α-HER2 modified by the Pictet-Spengler ligation. (A) Reducing and nonreducing SDS/PAGE analysis of FGly-α-HER2 and
AF488-α-HER2. (B) Median fluorescence intensity of SKOV3 and Jurkat cell populations treated with human antibodies. Cells were treated with AF488-α-HER2,
FGly-α-HER2, or human isotype control and then fluorescently labeled with α-hIgG and α-AF488 antibodies. Error bars represent SD of three replicate
experiments. βME, beta-mercaptoethanol.
contrast, the oxacarboline and amide conjugates showed only a SKOV3 cells; furthermore, the AF488-α-HER2 conjugate had no
minimal change in polarization. To confirm that the oxacarboline- affinity for Jurkat T cells, which do not express HER2. Overall,
linked AF488 conjugate was still intact after 1 wk, we added
thrombin to the samples, which resulted in an immediate decrease
in polarization as the C-terminal peptide containing the fluo-
rophore was cleaved from the rest of the protein. The polari-
zation of the resulting solutions containing mixtures of free
and peptide-linked AF488 matched the polarization of indepen-
dently prepared solutions of the free fluorophores. The signal
from the amide-linked AF488 conjugate remained stable (no ly-
sine residues are present downstream of the thrombin cleavage
site), indicating that the decrease in polarization was not an
artifact of thrombin addition.
these experiments show that the Pictet-Spengler ligation can be
used to prepare a site-specifically labeled monoclonal antibody
without compromising binding activity.
Conclusion
The Pictet-Spengler ligation possesses the selectivity, kinetics,
and operational simplicity that originally popularized traditional
oxime and hydrazone protein conjugation reactions. However, its
oxacarboline product enables the persistence of bioconjugates
in hydrolytically demanding environments where C=N linkages
currently fail. We demonstrated the generality of the method
using a variety of aldehyde-functionalized proteins, including a
therapeutically relevant human IgG. Model reactions suggest
that ketones are potential substrates as well and suggest a future
direction to explore with respect to bioconjugation. We focused
here on the use of the Pictet-Spengler ligation for modification
of purified proteins, but applications extend to other biomol-
ecules that are amenable to functionalization with reactive car-
bonyl groups. Methods for metabolic (49–51), enzymatic (52),
and chemical (53, 54) functionalization of glycans with ketone
and aldehyde groups are well-established and are finding use in
proteomic analyses of glycosylated proteins. The Pictet-Spengler
ligation may enhance the performance of these methods as well
as others that seek to detect or manipulate carbonyl groups using
bioorthogonal chemistry (55, 56).
Application of the Pictet-Spengler Ligation to Site-Specific Modification
of a Monoclonal Antibody. To showcase the utility of the Pictet-
Spengler ligation in preparation of antibody conjugates, we used
an α-HER2 human IgG modified with an aldehyde tag sequence
at the C terminus of each of its two heavy chains (abbreviated
FGly-α-HER2). The parent antibody is a variant of the clinically
approved drug Herceptin (47) and of T-DM1, an antibody-drug
conjugate based on Herceptin that is presently in late-stage clinical
evaluation (48). FGly-α-HER2 was prepared as previously de-
scribed (24) and then labeled with indole 1c at pH 4.5 for 12 h; the
resulting conjugate (AF488-α-HER2) was cleanly modified on the
heavy chain (Fig. 5A) with an average of 1.0 0.13 fluorophores
per hIgG (SI Appendix, Fig. S9). We next assessed binding of
this antibody conjugate to the ovarian adenocarcinoma cell line
SKOV3, which overexpresses HER2, by flow cytometry. SKOV3
cells were treated with AF488-α-HER2 or FGly-α-HER2, fol-
lowed by a DyLight 649-conjugated α-hIgG secondary antibody to
measure total hIgG binding. We found no difference in binding
between AF488-α-HER2 and FGly-α-HER2 (Fig. 5B), suggesting
that neither the Pictet-Spengler ligation reaction conditions nor
the presence of the oxacarboline moiety negatively impacts the
antibody’s affinity for HER2. Incubation of the labeled cells with
a rabbit α-AF488 secondary antibody followed by a FITC-conju-
gated α-rabbit tertiary antibody resulted in increased fluorescence
on cells treated with AF488-α-HER2 but not with FGly-α-HER2
(Fig. 5B). This result confirms that the AF488 cargo was suc-
cessfully delivered to the cell surface by AF488-α-HER2. As
expected, an isotype control hIgG showed no significant binding to
Materials and Methods
Details concerning the synthesis and characterization of all new compounds
can be found in SI Appendix, SI Materials and Methods. Also included are details
of small molecule kinetics and hydrolysis experiments. Protocols for protein
conjugations and characterization of FGly-MBP and FGly-α-HER2 conjugates
are also included in SI Appendix, SI Materials and Methods.
ACKNOWLEDGMENTS. We thank Jason Hudak for preparing FGly-MBP, Tony
Lavarone and David King for technical assistance, Krishnan Palaniappan and
Leah Witus for helpful discussions, Benjamin Swarts for a critical reading of the
manuscript, and the laboratory of Prof. James Berger (University of California
at Berkeley) for use of instrumentation. P.A., J.v.d.W., and E.M.S. were sup-
ported by a National Science Foundation Graduate Research Fellowship, a Nora
Baart Foundation fellowship, and an American Chemical Society Organic Di-
vision graduate fellowship, respectively. This work was supported by National
Institutes of Health Grant GM59907 (to C.R.B.).
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