Amino-Acid-Catalyzed Direct Aldol Bioconjugation

Article abstract of DOI:10.1021/acs.orglett.8b02265

A site-specific bioconjugation was developed based on direct aldol coupling using amino-acid-derived organocatalysts. The functionalization exhibits fast kinetics and occurs under mild, biocompatible conditions (viz., aqueous media, moderate temperature, and neutral pH). The resulting bioconjugates were found to be stable toward abundant aldolase enzymes, as well as acidic and basic pH. The methodology was demonstrated through conjugation of a variety of small molecules, dyes, and peptides to proteins, including a single-domain antibody, which was then used for cellular imaging.

Full text of DOI:10.1021/acs.orglett.8b02265

Letter
pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
Amino-Acid-Catalyzed Direct Aldol Bioconjugation
Tiauna S. Howard,† Ryan D. Cohen,†,‡ Ogonna Nwajiobi,∥ Zilma P. Muneeswaran,† Yonnette E. Sim,†
Neelam N. Lahankar,† Johannes T.-H. Yeh,§ and Monika Raj*,∥
†Department of Chemistry and Biochemistry, Seton Hall University, South Orange, New Jersey 07079, United States
‡Department of Process Research and Development, Merck & Co., Inc., Rahway, New Jersey 07065, United States
§Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, United States
∥Department of Chemistry and Biochemistry, Auburn University, Auburn, Alabama 36849, United States
S
* Supporting Information
ABSTRACT: A site-specific bioconjugation was developed based
on direct aldol coupling using amino-acid-derived organocatalysts.
The functionalization exhibits fast kinetics and occurs under mild,
biocompatible conditions (viz., aqueous media, moderate temper-
ature, and neutral pH). The resulting bioconjugates were found to
be stable toward abundant aldolase enzymes, as well as acidic and
basic pH. The methodology was demonstrated through conjugation
of a variety of small molecules, dyes, and peptides to proteins,
including a single-domain antibody, which was then used for cellular
imaging.
ost-translational modification of proteins to install new
Pfunctionality is widely known in nature, yet similar
selectivity and efficiency is currently unattainable from the
available chemical methods.1,2 Single-site biomolecule func-
tionalization to generate homogeneous constructs in the
presence of amino acids with diverse reactivity in both a
chemo- and regioselective manner is an exceptional challenge,
which is further exacerbated by the requirement for non-
denaturing conditions.3 Nevertheless, bioorthogonal chemical
reactions have emerged for modification of proteins with both
natural and unnatural amino acids. For example, aldehydes and
ketones can be readily installed onto a protein’s N-terminus by
well-established methods.4 They then become key handles for
further transformation, such as the commonly employed
conjugations with hydrazines and hydroxylamines to generate
hydrazones and oximes, respectively.5,6 However, this
approach has limitations, such as slow kinetics, acidic (pH
5−6) conditions, high concentrations of harmful catalysts,
cross-reactivity with metabolites, and poor stability of the
resulting conjugates due to reversibility.6,7 This leads to the
development of more advanced aldehyde bioconjugations,
which exhibit improved stability of conjugates, such as Pictet−
Spengler,8 indium-mediated allylation,9 and Wittig reactions.10
We considered that amino acids and/or peptides could be
used for the conjugation of proteins by catalyzing the aldol
reaction, which would lead to carbon−carbon bond formation
at the site of conjugation (Figure 1).11,12 Existing bioconju-
gation methods utilizing the aldol reaction are limited by poor
substrate scope and slow kinetics.13 While the efficiency and
selectivity of the proposed amino-acid-catalyzed reaction may
be impeded by the presence of a variety of functional groups
Figure 1. (a) Proposed Aldol bioconjugation reaction using amino-
acid-derived catalysts. (b) Key reactive intermediates.
within complex biomolecules, we envisioned that careful use of
particular amino acids or peptides would serve as highly
reactive catalysts for the desired transformations under
physiologically relevant conditions. The interaction between
the amino group of the catalyst and the aldehyde/ketone
would guide its selectivity by formation of an iminium ion-
enamine complex. The ability to achieve high levels of chemo-
and regioselectivity and fast reaction kinetics with aldehydes
and ketones under aqueous conditions by using well-designed
amino-acid-derived organocatalysts suggests that they could be
very attractive for the development of a new site-selective
bioconjugation method leading to the modification at the N-
terminus of proteins. In addition, this method has the potential
to generate a chiral center at the site of bioconjugation with
high stereoselectivity through judicious organocatalyst selec-
Received: July 19, 2018
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.8b02265
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Letter
tion, which may be of therapeutic significance (see Supporting
Information).
Here we report an organocatalyzed aldol bioconjugation
reaction that is applicable to a wide range of peptides and
proteins. The organocatalyzed aldol bioconjugation reaction is
chemoselective, site-specific, and rapid and occurs under mild
reaction conditions. We illustrate the diversity of this reaction
for a series of expressed and commercially available proteins
and antibodies.
We started our investigation with proline as catalyst because
a multitude of reports have shown that proline can form an
iminium ion 1 with the ketone followed by rearrangement to
an enamine 2, as needed for carrying out nucleophilic attack on
the electrophilic aldehyde 3 (Figure 1).14 A simple amino acid
aldehyde, phenylalanine aldehyde 4, was used to optimize
reaction conditions and to explore substrate scope with
different ketones (5−7, Figure 2). First, we compared the
Figure 3. Chemoselective nature of aldol bioconjugation. (a)
Reaction conditions: peptide aldehydes 9−11 (7 mM), hydroxyace-
tone 6 (84 mM), and proline (100 mM) in 25 mM phosphate buffer
(pH 7.5) and up to 15% DMSO at 60 °C. (b) Peptide 11 containing
Lys can form macrocyclic imine 14a, but this did not impede the
formation of aldol bioconjugate 14. (c) Stability of 14 with acid, base,
or aldolase at 37 °C.
11, which were generated from N-terminal serine-containing
peptides by sodium periodate (NaIO4) oxidation,4a were
reacted with hydroxyacetone 6 resulting in high conversions to
the desired conjugated products 12−14 within 15 to 30 min.
No interference was observed from the presence of other
reactive amino acid side chains. In the case of lysine, self-
condensation of 11 resulted in the formation of a macrocyclic
imine 14a (Figure 3b). However, reaction of 14a with 6
ultimately leads to the desired aldol bioconjugate 14,
presumably due to the high rate of transamination reaction
of the imine 14a with proline catalyst.15
The stability of the aldol bioconjugation product was then
evaluated against acidic and basic conditions, as well as
aldolase enzymes (Figure 3c). As expected, incubation of 14
for 24 h at pH 4.3, 7.3, and 10.3 at both room temperature and
37 °C leads to no detectable degradation. Notably, 14 was also
found to be stable toward the retro-aldol reaction catalyzed by
aldolases, which are highly abundant in nature. The stability of
the aldol bioconjugate toward aldolases is presumably due to
the enzyme’s high substrate specificity.16
We next explored the aldol bioconjugation reaction with
horse heart myoglobin (Figure 4). The N-terminal glycine
residue was converted to an aldehyde using the well-known
pyridoxal 5′-phosphate (PLP) reagent.4b Hydroxyacetone 6
was then reacted with myoglobin aldehyde 15 at 37 °C in
buffer using proline resulting in bioconjugated myoglobin 16
with 80% conversion within 30 min as determined by LC-MS.
The analysis of the resulting myoglobin bioconjugate 16 using
CD and UV showed that the structure of modified protein 16
remained intact after the bioconjugation (see Supporting
Information). Two control reactions were then performed
using (1) unmodified myoglobin (i.e., no aldehyde handle)
and (2) no organocatalyst. In both cases, no bioconjugation
product was observed after 24 h.
Peptide ketones, GIRVF 17 and ACF 18, were generated
from reaction of PLP with peptides, AGIRVF and AACF,
respectively. These were then reacted with myoglobin
aldehyde 15 resulting in desired protein bioconjugation
products, 19 (66%) and 20 (83%), respectively, when using
O-tBu-Thr as organocatalyst in place of proline. Peptide
ketones gave high conversion to bioconjugated products with
Figure 2. Model aldol bioconjugation between (a) phenylalanine
aldehyde 4 and ketones (5−7) catalyzed by proline. (b) Comparison
of uncatalyzed and proline-catalyzed (100 mM) reaction rates in
DMSO at 37 °C between 4 (1 equiv., 7 mM) and hydroxyacetone 6
(12 equiv., 84 mM). (c) Comparison of reaction in 100% DMSO
versus 1:1 sodium phosphate buffer (pH 7.4):DMSO. (d) Proposed
role of water in increasing reaction kinetics.
rates of catalyzed versus uncatalyzed reactions between 4 and
hydroxyacetone 6 (Figure 2b). No product was detected in the
absence of an organocatalyst. While most aldehyde bio-
conjugations operate at slightly acidic pH (5.5 to 6.5),5,6,8 86%
conversion occurred for the pH 7.4 phosphate-buffered
reaction. Furthermore, the rate was considerably faster with
aqueous buffer than in neat DMSO (Figure 2c).
The increase in the rate of the organocatalyzed reaction in
buffer might be due to the dual role of water in activating the
aldehyde for nucleophilic attack through hydrogen bonding 8a
and by accelerating the hydrolysis of the iminium ion
intermediate 8b formed between the aldol bioconjugate and
the organocatalyst, resulting in the release of the desired
bioconjugate (Figure 2d).14 Different amino-acid-based
organocatalysts were then screened for the reaction of either
4 or peptide aldehyde, VF 9, with 6, and the highest
conversion was observed with proline (see Supporting
Information). After optimizing catalyst loading and temper-
ature, 96% product formed within 15 min using 100 mM
proline at 60 °C and pH 7.5. The optimized reaction
conditions (100 mM proline, 5% DMSO, 25 mM phosphate
buffer, pH 7.5 at 60 °C) were then used for evaluating
substrate scope (Figure 3). Unprotected peptide aldehydes 9−
B
DOI: 10.1021/acs.orglett.8b02265
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Figure 4. Organocatalyzed protein modification. (a) Ligation of N-
terminal myoglobin aldehyde 15 (0.15 mM) with different ketones (6,
17, and 18 (10 mM)) using either proline (b, c) or O-tBu-Thr (d, e)
(100 mM) as catalysts in 25 mM phosphate buffer (pH 7.5) and up to
15% DMSO at 37 °C. Deconvoluted ESI-MS spectra of (b)
unconjugated myoglobin aldehyde 15, (c) myoglobin hydroxyacetone
bioconjugate 16 generated by reaction with 6, and (d and e)
myoglobin−peptide bioconjugates 19 (60%) and 20 (40%) generated
by reaction with peptide ketones GIRVF 17 and ACF 18.
Figure 5. Nanobody−drug conjugates. (a) Synthesis of nanobody−
drug conjugates (NDCs) at 1:1 drug-to-nanobody ratio (DNR).
Reaction conditions: G110 21 (0.15 mM), ketones 17 or 18 (10
mM), O-tBu-Thr (100 mM) in 25 mM phosphate buffer, pH 7.5, and
up to 15% DMSO at 37 °C. Deconvoluted ESI-MS spectra of (b)
NDC 22 (66%), (c) NDC 24 (83%), and (d) dual labeled NDC 25
(99%). (e) Fluorescent imaging with 25 indicates that these
conjugates bind to cell surface receptors. (f) Control reaction: cells
treated with dye but no nanobody or nanobody lacking dye do not
fluoresce.
O-tBu-Thr as compared to proline (see Supporting Informa-
tion). Further, reaction of myoglobin aldehyde with drug,
FITC, and biotin ketones (see Supporting Information)
demonstrated the flexibility of this approach with various
ketones which is in contrast to known bioconjugation
techniques, where reaction works with only one type of
ketone.13
Antibody−drug conjugates (ADCs) are utilized for targeted
drug delivery, and the synthesis of homogeneous conjugates is
critical for both the safety and efficacy of this therapy.17 We
aimed to conjugate a peptide ketone, GIRVF 17, to a single
domain antibody G110. Single domain antibodies, also referred
to as nanobodies, have been shown to be equally specific as
normal antibodies and in some cases more bioactive.18,19 We
chose a nanobody for conjugation because of their small size,
robust stability, and simpler expression and purification in E.
coli cells. The bioconjugation of 17 to nanobody G110
containing an N-terminal aldehyde 21 was catalyzed by O-tBu-
Thr and produced homogeneous nanobody-drug conjugate
NDC 22 (1:1) with 66% conversion (Figure 5).
The consistent nanobody−peptide conjugate ratio is in
contrast to some other bioconjugation techniques generating
heterogeneous mixtures of ADCs with variable drug to
antibody ratio (DAR).20 The mild conditions required for
this bioconjugation method maintained the structurally critical
disulfide bond in G110, which was confirmed by reaction with
a maleimide dye 23 that resulted in no cysteine modification.
While traditional ADCs use a variety of linkers to attach drug
molecules to antibodies, our method has the capability of being
linker-free.
Modification of proteins in the presence of free cysteines is
particularly challenging due to the strong nucleophilicity of the
thiol group. To assess the compatibility of our approach with
free cysteines, we synthesized peptide ketone ACF 18 and
conjugated it to 21, which formed nanobody−peptide
conjugate 24 (83% conversion, Figure 5c). Cys remained
unreacted and allowed for further ligation with a maleimide
dye 23 generating multiply labeled G110 25 (Figure 5d).
Fluorescence microscopy showed that this labeled G110
nanobody 25 can bind to an EGF receptor-expressing breast
cancer cell line, MDA-MB468, which confirms the stability of
aldol bioconjugates in cellular assays (Figure 5e). Thus, this
method can be used when the chemistry for Cys modification
is not compatible or for applications that require specific
labeling at multiple locations through orthogonal methods.
In all the above experiments, a peptide or protein with an
electrophilic N-terminal aldehyde was conjugated to a small
molecule containing a nucleophilic ketone, also reported by
Spears and colleagues while our manuscript was under
review.21 We report that these roles can be reversed. A ketone
was generated on protein α-lactalbumin 26 containing
glutamic acid at the N-terminus, followed by reaction with 2-
pyridine carboxyaldehyde (2-PCA) 27 and peptide aldehyde
VF 9 using O-tBu-Thr as organocatalyst, yielding aldol
bioconjugates 28 and 29 (57%), respectively (Figure 6).
Notably, the resulting aldol bioconjugate 28 further underwent
dehydration to the more stable α,β-unsaturated enone 30
(50%) in 1 h. The condensation product was only observed
with 2-PCA 27 presumably due to stabilization of the resulting
double bond by conjugation with the aromatic ring of 2-PCA.
Further, reaction with other peptide ketones and aldehydes
(see Supporting Information) demonstrated the broad
applicability of this approach for modification of different
proteins under mild conditions.
We note that the ease of utilizing amino acids as catalysts
makes them particularly attractive for routine modification of
biomolecules for application in chemical biology, in medicine,
and for generating peptide−polymer conjugates in material
science. Here, we demonstrated an organocatalyzed bioconju-
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.8b02265.
■
S
Supporting figures, experimental procedures, and ana-
lytical data for new compounds (PDF)
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: mzr0068@auburn.edu.
ORCID
Monika Raj: 0000-0001-9636-2222
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was supported by start-up funds granted to M.R.
by Auburn University. T.S.H. was supported by the Gates
Millennium Scholars Program. J.Y. was supported by Cold
Spring Harbor Laboratory and Northwell Health Affiliation.
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DOI: 10.1021/acs.orglett.8b02265
Org. Lett. XXXX, XXX, XXX−XXX