Aldehyde-mediated bioconjugation: Via in situ generated ylides

Article abstract of DOI:10.1039/c9cc07443k

A technically simple approach for rapid, high-yielding and site-selective bioconjugation has been developed for both in vitro and cellular applications. This method involves the generation of maleimido-phosphonium ylides via 4-nitrophenol catalysis under physiological conditions followed by their Wittig reactions with aldehyde-appended biomolecules.

Full text of DOI:10.1039/c9cc07443k

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Aldehyde-mediated bioconjugation via in situ
generated ylides†
Cite this: Chem. Commun., 2019,
55, 14926
Sangeeta Parmar,‡ Sharad P. Pawar,‡ Ramkumar Iyer and Dimpy Kalia
*
Received 23rd September 2019,
Accepted 19th November 2019
DOI: 10.1039/c9cc07443k
rsc.li/chemcomm
A technically simple approach for rapid, high-yielding and site- limitations in aldehyde ligation chemistry. For example, the extensively
selective bioconjugation has been developed for both in vitro and used hydrazone/oxime chemistry proceeds slowly under physiological
cellular applications. This method involves the generation of conditions31 and forms conjugates that lack stability.36 Aniline
maleimido-phosphonium ylides via 4-nitrophenol catalysis under derivatives have been reported to speed up hydrazone/oxime bio-
physiological conditions followed by their Wittig reactions with conjugation but these catalysts are required in large amounts
aldehyde-appended biomolecules.
(as much as hundreds of millimolar concentrations or more in
some cases) for achieving acceptable reaction kinetics.15,31 Although
Bioconjugation reactions enable the site-specific attachment of alternate aldehyde ligation approaches32–41 have been reported, they
fluorophores, affinity tags and drugs onto biomolecules for diverse often proceed in suboptimal yields,33,35 form unstable linkages,37,41
applications in both basic and applied biological sciences.1,2
Over the past few years, several creative new approaches for site-
and require non-physiological acidic conditions.32,34
We sought to explore the potential of the facile reaction of
specific protein bioconjugation have been reported.3–20 A majority of aldehydes with maleimides in the presence of PPh3 and the
these approaches, however, entail the use of a large excess of 4-nitrophenol (4-NP) catalyst to form exocyclic maleimides42 for
bioconjugation reagents as compared to the target biomolecule to aldehyde-mediated bioconjugation (Fig. 1). Our experiments
achieve acceptable conversion rates. The use of excessive amounts focused on glyoxaldehydes as they can be easily installed into
of these organic compounds/metal complexes (amounting to tens or the N-termini of proteins via simple chemical treatments.27,28
even hundreds of millimolar concentrations) renders bioconjugate Incubating 1.5 mM glyoxaldehyde 1 with equimolar amounts of
purification challenging and is also not ideal for applications that N-ethylmaleimide (NEM) and PPh3 in the presence of 0.2 eq. of
entail bioconjugation within cells as it can cause cytotoxicity.21–23 4-NP at pH 7 at room temperature yielded the desired exocyclic
Moreover, subjecting cells to large amounts of fluorescent dyes can olefinic maleimide 4 in 95% conversion within 3 min (Fig. S1
lead to non-specific labelling.24,25 Another challenge is the prepara- and Table S1, ESI†). Remarkably, using 2.0 eq. of both NEM and
tion of bioconjugation reagents that typically entails executing PPh3 in the presence of 0.4 eq. 4-NP resulted in an even faster
tedious multi-step synthetic schemes.14,16–18,26 Thus, developing reaction, giving 4 in a quantitative yield within 1 min (Fig. 2,
technically straightforward bioconjugation approaches that pro- left panel). Similar results were obtained with glyoxaldehyde 2
ceed rapidly and in high yields with stoichiometric amounts/a to give 5 (Fig. 2, right panel). Notably, this chemistry proceeded
slight excess of commercially available/easily synthesizable quantitatively within 10 min with micromolar concentrations
reagents remains highly desirable.
of the aldehyde (50 mM), NEM (300 mM), PPh3 (300 mM) and
The availability of several facile methods for the site-specific 4-NP (60 mM) under neutral buffer conditions (Fig. S2, ESI†),
installation of aldehyde groups into proteins27–29 renders aldehyde
ligation11,17,27,28,30–35 extremely promising for protein bioconjugation.
The widespread use of this approach, however, is hampered by
Department of Chemistry, Indian Institute of Science Education and Research
(IISER) Bhopal, Bhauri, Bhopal Bypass Road, Bhopal 462066, Madhya Pradesh,
India. E-mail: dimpy@iiserb.ac.in
† Electronic supplementary information (ESI) available: Detailed experimental
Fig. 1 Proposed work plan for bioconjugation via aldehyde ligation involving
the in situ formation of maleimido-phosphonium ylides under aqueous
conditions.
methods, synthetic procedures, compound characterization, and MALDI mass
characterization of HPLC peaks. See DOI: 10.1039/c9cc07443k
‡ Equally contributed.
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Fig. 3 Aldehyde ligation on the TRAP6 peptide.
Fig. 2 HPLC analysis of our aldehyde ligation chemistry performed on
glyoxaldehydes 1 and 2. The control reaction in each case contained only
the glyoxaldehyde and 4-NP. Na-PB = sodium phosphate buffer.
reaction mixture demonstrated the presence of solely the expected
N-ethyl exocyclic maleimide–Mb conjugate 6 (Fig. 4b). Quantitative
protein labelling was also observed with the fluorophore
underlining its suitability for bioconjugation applications. N-dansylmaleimide (NDM) to form conjugate 7 (MALDI: Fig. 4b
Moreover, this chemistry is compatible with disulfide-rich and SDS-PAGE: lane 5, Fig. 4c). No fluorescence was observed in a
environments and proceeded efficiently in the presence of control experiment with native Mb not containing the aldehyde
5-fold excess oxidized glutathione (Fig. S3, ESI†).
functional group (lane 3, Fig. 4c left panel and MALDI: Fig. S6a,
We next tested the stability of conjugate 4 obtained as a result of ESI†), demonstrating the absence of the non-specificity of labelling.
our chemistry in the pH range of 5–8 by performing HPLC analysis. Aldehyde-specific labelling was further confirmed by trypsinization
These studies revealed that the conjugate was extremely stable and of conjugate 7 followed by the MS/MS of the labelled fragment
did not demonstrate any observable decomposition even after 2 (Fig. S7, ESI†). We were also successful in installing the alkyne
d-long incubations (Fig. S4, ESI†). These results are in stark contrast handle useful for azide–alkyne click reaction into Mb by treating
to a previously reported Wittig reaction-based bioconjugation strat- MbCHO with N-butynylmaleimide (NBM). This reaction also
egy that resulted in the formation of unstable olefinic esters that resulted in the exclusive formation of the expected exocyclic
hydrolyze over time, leading to the cleavage of the label from maleimide–Mb product, conjugate 8 (Fig. 4b). Upon screening
the bioconjugate.37 Moreover, our chemistry results exclusively bioconjugation conditions using lower equivalents of reagents
in the formation of the E isomer42,43 of the product as compared (10–25-fold excess), we found that labelling proceeded effectively
to existing approaches that form mixtures of E and Z isomers.35,37 even with a 10-fold excess of the reagents (Fig. S6, ESI†). Moreover,
In addition to glyoxaldehydes, this chemistry proceeds in the structural integrity of myoglobin was retained after protein
490% conversion rates on aromatic, aliphatic and conjugated bioconjugation as demonstrated by the circular dichroism (CD)
aldehydes with a merely 2-fold excess of NEM and PPh3 under spectra of myoglobin before and after modification (Fig. S8, ESI†).
physiological conditions (Fig. S5, ESI†). This diverse substrate
Another catalytic approach for aldehyde ligation that has been
scope renders our method amenable to labelling proteins recently developed utilizes aniline derivatives as catalysts to form
appended with not only glyoxaldehydes but also other alde- hydrazones and oximes.4,31 To compare our chemistry with this
hydes installed into them enzymatically.29,44
approach, we incubated glyoxaldehyde 1 with 2-fold excess benzyl
Next, we sought to utilize our chemistry for labelling a model alkoxyamine in the presence of a 5-fold excess of one of the most
peptide, the Thrombin Receptor Activator Peptide 6 (TRAP6) (Fig. 3). potent aniline-based catalysts (1,4-diaminobenzene)4,45 at neutral
Treatment of TRAP6 with NaIO4 resulted in the oxidation of its pH. HPLC analysis of this reaction demonstrated that this chemistry
N-terminal serine to a glyoxaldehyde to form TRAP6-CHO yields the desired oxime product (S31) in 55% conversion over 7.5 h
(Fig. 3).27 Incubation of TRAP6-CHO (500 mM, 1.0 eq.) with (Fig. S9, ESI†) in comparison to our method that yields the desired
NEM (2.0 eq.), PPh3 (2.0 eq.) and 4-NP (0.4 eq.) at pH 7 resulted conjugate quantitatively within just 1 min by employing a 2-fold
in its quantitative conversion to the desired exocyclic olefinic excess of NEM and PPh3 each and merely 0.4 equivalent of the 4-NP
maleimide product, TRAP6-EXO (Fig. 3) within 2 min (see Table S1 catalyst (Fig. 2). Moreover, our attempts at performing oxime-
for mass characterization, ESI†).
mediated aldehyde ligation on proteins by incubating MbCHO
To evaluate the scope of our chemistry for protein bioconjuga- (30 mM) with 25 fold-excess dansyl alkoxyamine (DanONH2) over a
tion, we generated N-terminal glyoxaldehyde-labelled myoglobin 2 h time period in the presence of either a 5-fold or 25-fold excess of
(MbCHO) by employing PLP chemistry28 (Fig. 4a). MbCHO the 1,4-diaminobenzene catalyst under neutral aqueous conditions
(30 mM) was incubated with NEM (750 mM), PPh3 (750 mM) and did not yield the desired fluorescent bioconjugate (Fig. S10, ESI†).
4-NP (150 mM) for 2 h at room temperature in 50 mM phosphate This observation justifies the requirements of extended reaction
buffer at pH 7. MALDI mass spectrometry analysis of the desalted times and a much larger molar excess of these aniline-based
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490 mM dansyl thiol (DanSH) in phosphate buffer at neutral pH
resulted in the exclusive formation of a dually-labelled conjugate, 9
(Fig. 4b). Similarly, conjugates 7 and 8 upon treatment with DanSH
were efficiently converted into dually-labelled conjugates 10 and 11
respectively (Fig. 4b). This result is consistent with SDS-PAGE
analysis that showed fluorescent bands for conjugates 11, 7 and
10 (lanes 4–6, Fig. 4c, right panel). We also employed this chemistry
to generate a protein conjugate appended with both a fluorescent
tag and the biotin affinity tag by incubating 7 with biotin thiol
(BioSH) under the conditions employed for dual labelling as
described above. MALDI analysis showed that this reaction resulted
in the formation of the desired dually-labelled conjugate 12 (Fig. 4b).
Thio-Michael adducts of bioconjugates are known to be prone to
thiol exchange-mediated breakdown.48,49 To test for the tendency of
our dually-labelled conjugates to undergo such breakdown, we
generated a dually-labelled GSH adduct of our TRAP6-EXO peptide,
conjugate S32 (Fig. S11, left panel, ESI†) and subjected it to a 20-fold
molar excess of N-acetyl cysteine (NAC). Even after 2 d of incubation,
no measurable thiol exchanged/ring hydrolyzed adducts were
observed (Fig. S11, right panel, ESI†).
The rapid rates of aldehyde ligation at neutral pH observed
without the use of a large excess of labelling reagents and high
conjugate stability render our method ideal for cellular applications.
To explore this aspect of our technology, we sought to fluorescently
label bacterial cell walls considering its applications in studying
bacterial cell wall remodeling50,51 and pathogenic bacterial
detection.40 Although the peptidoglycan layers of bacterial cell walls
have been demonstrated to undergo metabolic labelling with non-
natural D-amino acids,40,50 labelling with a glyoxaldehyde-tagged
D-amino acid has not been previously reported. To test this idea, we
synthesized D-Lys glyoxaldehyde (D-Lys-CHO, Fig. 5a) and adminis-
tered it to both Gram-positive (S. aureus) and Gram-negative (E. coli)
bacterial cells in culture (Fig. 5b). After 6 h, the excess amino acid
was washed off and aldehyde ligation was performed by incubating
cells with NDM (50 mM), PPh3 (50 mM) and 4-NP (10 mM) for 30 min.
Subsequent confocal microscopy imaging demonstrated robust
fluorescent labelling of both S. aureus and E. coli cell walls
(Fig. 5c). Control experiments with L-Lys-CHO yielded negligible
fluorescence (Fig. 5c). To more rigorously evaluate whether the
Fig. 4 Mono- and dual labelling of proteins: (a) general scheme; (b) MALDI
mass spectra and (c) SDS-PAGE analysis of myoglobin conjugates.
catalysts for hydrazone/oxime formation.15,31 In contrast to these modification was aldehyde-specific, cells metabolically labelled with
results, performing protein labelling by employing our approach D-Lys-CHO were first treated with a non-fluorescent aldehyde-
and subjecting MbCHO (30 mM) to exactly the same molar ratios of reactive compound (hydroxylamine hydrochloride) prior to treat-
bioconjugation reagents (5-fold excess of the 4-NP catalyst and ment with our fluorescent reagents. This experiment resulted in
25-fold excess of NEM and PPh3 each) for the same reaction time significantly reduced fluorescent labelling of cells (Fig. S13, ESI†),
resulted in the quantitative formation of the desired fluorescent establishing that our bioconjugation chemistry is aldehyde-specific
conjugate 7 (Fig. 4b, c and Fig. S10, ESI†).
not merely in in vitro settings but also in the cellular context.
A useful feature of our chemistry is that it results in the formation
In conclusion, we have developed a new method of bioconjuga-
of exocyclic olefinic maleimide moieties that can undergo facile tion that can be used to rapidly generate a wide range of both mono-
Michael addition with thiols,42 enabling the installation of two and dually labelled bioconjugates. We demonstrated the utility of
different labels at the same site (Fig. 4a). The generation of such our technology for cellular applications by employing it for fluores-
dually-labelled biomolecules is desirable as they can be employed for cently labelling bacterial cell walls. A major practical advantage of
fabricating antibody–drug conjugates with enhanced drug–antibody our technology is that it entails the use of readily commercially
ratios,46 and also for generating bifunctional protein conjugates such available reagents (maleimides, PPh3 and 4-NP), thereby obviating
as those labelled with affinity tags as well as with fluorophores.47
the requirement of their chemical synthesis. Taken together, we
To perform dual labelling of myoglobin, we treated our Mb believe that the technical simplicity, rapid kinetics, high product
conjugates 6–8 with thiol reagents. The treatment of 6 (20 mM) with yields, and amenability for facile dual functionalization render our
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applications.
This work was supported by funds to D. K. from IISER Bhopal,
DST-INSPIRE Faculty Award and SERB-ECRA. The imaging data
shown was obtained from the imaging facility at IISER Bhopal
supported by DST-FIST. We thank the Chemistry Department,
Savitribai Phule Pune University for hosting D. K. as a DST-
INSPIRE Faculty.
Conflicts of interest
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