Palladium-unleashed proteins: Gentle aldehyde decaging for site-selective protein modification

Article abstract of DOI:10.1039/c7cc07740h

Protein bioconjugation frequently makes use of aldehydes as reactive handles, with methods for their installation being highly valued. Here a new, powerful strategy to unmask a reactive protein aldehyde is presented. A genetically encoded caged glyoxyl aldehyde, situated in solvent-accessible locations, can be rapidly decaged through treatment with just one equivalent of allylpalladium(ii) chloride dimer at physiological pH. The protein aldehyde can undergo subsequent oxime ligation for site-selective protein modification. Quick yet mild conditions, orthogonality and powerful exposed reactivity make this strategy of great potential in protein modification.

Full text of DOI:10.1039/c7cc07740h

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Palladium-unleashed proteins: gentle aldehyde
decaging for site-selective protein modification†
Cite this:DOI: 10.1039/c7cc07740h
a
a
a
Robin L. Brabham,
Richard J. Spears,
Julia Walton,
*
Swati Tyagi,b
Edward A. Lemkeb and Martin A. Fascione
a
Received 6th October 2017,
Accepted 4th December 2017
DOI: 10.1039/c7cc07740h
rsc.li/chemcomm
Protein bioconjugation frequently makes use of aldehydes as reac-
tive handles, with methods for their installation being highly valued.
Here a new, powerful strategy to unmask a reactive protein aldehyde
is presented. A genetically encoded caged glyoxyl aldehyde, situated
in solvent-accessible locations, can be rapidly decaged through
treatment with just one equivalent of allylpalladium(II) chloride dimer
at physiological pH. The protein aldehyde can undergo subsequent
oxime ligation for site-selective protein modification. Quick yet mild
conditions, orthogonality and powerful exposed reactivity make this
strategy of great potential in protein modification.
Aldehydes are a powerful yet underutilised tool for bioorthogo-
nal chemistry, where the high electrophilicity coupled with
good stability and low abundance in nature make them an
attractive handle for protein modification.1 Bioorthogonal reactions
involving aldehydes have been developed to take advantage of the
unique reactivity of this functional group2–8 and an impressive array
of bioconjugates are synthetically accessible, including antibody–drug
conjugates,9 protein–protein conjugates10 and labelled live cells.11
Access to such aldehydes, however, can be an impediment to their
usage, with incorporation methods either requiring enzyme recogni-
tion sequences, location at a protein terminus, or both. Use of
Fig. 1 Selected methods for the installation of aldehydes in proteins.
formylglycine-generating enzyme (FGE), for example, will only form pyrrolysine (Pyl) tRNACUA/pyrrolysyl-tRNA synthetase (RS) pair
an aldehyde on the side chain of a cysteine in a CXPXR sequence from several species of archaeal methanogens for amber stop
(Fig. 1a).12 Some strategies are less flexible for aldehyde positioning: codon (TAG) suppression has allowed access to proteins con-
periodate-mediated oxidative cleavage of serine or threonine residues taining a wide range of non-canonical functionality, including
(Fig. 1b)13 or transamination of glycine residues14 occurs only at such alkenes,16–18 alkynes,19–21 azides,22 and aryl halides,23 with gener-
N-terminal residues, whilst tubulin tyrosine ligase (‘‘Tub’’) requires a ally excellent levels of site specificity; indeed, UAA mutagenesis has
Tub tag on a protein C-terminus to append tyrosine derivatives such become a widely utilised tool in chemical biology.24 Notably, the
as m-formyl-L-tyrosine 1 (Fig. S1, ESI†).15
aldehyde-containing UAA m-formyl-L-phenylalanine 2 (Fig. S1,
The technique of unnatural amino acid (UAA) mutagenesis ESI†) has been genetically encoded using an engineered pylRS
has become a standard tool in chemical biology. Use of the variant from Methanosarcina mazei.25 The wild-type Methanosarcina
barkeri pyrrolysine tRNA-RS pair genetically encodes 2-thiazolidine
derivative ThzK, with the methyl ester 3 used as a suitable
a Department of Chemistry, University of York, Heslington Road, YO31 5DD, York,
precursor for incorporation into proteins (Fig. 1c).26 The thiazo-
UK. E-mail: martin.fascione@york.ac.uk
lidine group has seen use in peptide synthesis as an aldehyde-
based protecting group for 1,2-aminothiols such as cysteine with
various deprotection strategies,27–29 and a 4-thiazolidine derivative
b EMBL, Meyerhofstrasse 1, 69117 Heidelberg, Germany
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c7cc07740h
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has been used as a caged pseudo-N-terminal cysteine for protein The new aldehydes are subsequently shown to be amenable to
CBT condensation ligations,30 showing the potential for a 2- site-selective modification.
thiazolidine to be used to smuggle an aldehyde group into a
Green fluorescent protein (GFP) and superfolder green
protein. Notably, a thiazolidine will decage to yield a glyoxyl fluorescent protein (sfGFP) are highly useful test systems for
group, a highly reactive aldehyde to the extent that more reliable, protein modification due to ease of visualisation and highly
reproducible and standardised modification methodologies have optimised expression systems for use with UAA mutagenesis.
been established for this protein aldehyde than any other.1 As Two GFP mutants containing amber stop codons at surface-exposed
reactive carbonyls have been documented as forming undesired sites were selected: sfGFP(N150TAG)37 and GFP(Y39TAG).38 An
adducts with 1,2-aminothiols in biological media,31,32 the use of a advantage of using GFP is the facile confirmation and assaying of
protection/deprotection strategy avoids such side reactions which successful stop codon suppression through green fluorescence
may suppress genetic incorporation or conjugation yields. Indeed, of harvested cell pellets.39 Genetic encoding of ThzK has
UAA mutagenesis has been shown to exhibit excellent synergy previously made use of the M. barkeri pyrrolysine tRNA-RS
with decaging strategies, where photodecaging33 and metal- pair,26 although the promiscuity of the corresponding M. mazei
mediated decaging34–36 reactions have expanded the paradigm pair is generally sufficiently similar to the extent that either pair
of functionality which can be genetically encoded. In this work a would be likely to genetically encode ThzK.30 Protected ThzK 3
rapid yet mild palladium glyoxyl-decaging strategy is presented was synthesised in four straightforward steps via 4–9 with a
which reveals protein aldehydes from surface-exposed ThzK cumulative yield of ca. 45% (Scheme S1, ESI†) following litera-
residues in a protein without the need for enzyme recognition ture precedent.26 Separate saponification is unnecessary as this
sequences, using just a single equivalent of palladium (Fig. 1c). amino acid can be delivered into protein expression systems in
Fig. 2 (a) Reagents screened to decage protein thiazolidine GFP(Y39ThzK) 10 and sfGFP(N150ThzK) 11 to afford protein aldehyde/hydrate 12/13
(calculated masses shown). Table shows decaging conditions and conversions for protein thiazolidine 10. (b) Complete decaging of 10 (upper) using 16
to form 12 (lower) as the aldehyde (experimental masses shown). (c) Complete decaging of 11 (upper) using 16 to form 13 (lower) as the hydrate
(experimental masses shown).
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a mildly basic stock solution, exposing the C-terminus as and rapid, requiring only limited exposure to a very low loading
needed for translation. Using an adapted general method for of palladium reagent.
amber stop codon suppression in GFP expression,38 both
As further confirmation of the exposed aldehyde reactivity,
mutants were individually expressed with the supplementation aniline-catalysed oxime ligation was performed upon protein
of 3 at 1.6 mM in growth media and the green fluorescence of glyoxyl species 12 and 13 using an aminooxy biotin probe 18 to
the harvested cell pellets confirmed the presence of full-length afford biotinylated proteins 19 and 20 respectively (Fig. 3a).
protein, demonstrating that ThzK can be encoded by the Pleasingly, complete ligation was observed with both proteins
M. mazei pyrrolysine tRNA-RS pair. Following nickel affinity within 24 h, with a Western blot confirming the incorporation
purification of the cell lysate, both GFP(Y39ThzK) 10 and of the biotin probe in 19 (Fig. 3b). Further oxime ligation was
sfGFP(N150ThzK) 11 could be isolated and their purity con- performed using the fluorescent aminooxy dansyl probe 21
firmed by SDS-PAGE and ESI-FTICR-MS (Fig. S2 and S3, ESI†). with 12 and 13 to afford dansylated proteins 22 and 23
Following successful ThzK incorporation, 10 and 11 were used respectively. Again full conversion was observed and through
as test beds for novel decaging strategies to yield GFP(Y39GlyoxylK) denatured protein in-gel fluorescence the presence of a dansyl
12 and sfGFP(N150GlyoxylK) 13 (Fig. 2a). Inspired by examples of group in 23 could be unequivocally visualised (Fig. 3c).
palladium-mediated deprotection in aqueous conditions,35,36 Irrespective of differences in protein and aldehyde/hydrate
including thiazolidine cleavage,40 we opted to screen commercially distribution, the aldehydes uncaged by the method reported
available palladium sources as potential biologically compatible here can be modified to completion following established
reagents for unmasking of glyoxyl aldehydes. Palladium complexes protocols.
14–17 were trialled at 37 1C, pH 7.4 in order to maintain biocom-
patibility over a range of concentrations and time intervals. These
complexes are a mixture of Pd(0) and Pd(II)41 and at 100 equiva-
lents have been used previously to decage thiazolidine peptides.40
Addition of 100 equiv. Pd(OAc)2 14 and allylpalladium(II) chloride
dimer 16 led to protein precipitation, although some decaging was
observed with 16. PdCl2(amphos)2 15 was found to be unreactive,
neither decaging nor inducing precipitation. Success was first
achieved using tris(dibenzylideneacetone)dipalladium(0) 17, with
complete decaging observed after 48 hours. Further optimisation
of conditions led to complete decaging within 24 hours. Curiously,
under these conditions decaging of ThzK-containing 10 largely
afforded glyoxyl-containing 12 as the aldehyde form, whilst glyoxyl
aldehydes in aqueous conditions generally exist in the hydrated
form. On closer inspection, the observation that allylpalladium(II)
chloride dimer 16 induced precipitation of GFP seemed unusual
given its successful use for other types of decaging with GFP.36
Subsequently, further screening using this palladium source
showed that at concentrations greater than 0.5 mM, 16 will cause
GFP to precipitate, but not at lower concentrations. Hence further
screening was carried out using fewer equivalents with an identical
concentration of GFP(Y39ThzK) 10 and pleasingly full decaging of
the ThzK group to a glyoxyl could be observed within one hour
using just one equivalent of allylpalladium(II)chloride dimer 16
(Fig. 2b) at room temperature. Notably, use of tris(dibenzylidene-
acetone)dipalladium(0) 17 under the same conditions afforded no
detectable decaging. One equivalent of 16 strikes the balance
between minimal protein precipitation and maximum extent of
decaging, with substoichiometric quantities of palladium resulting
in poorer conversion. These optimised conditions were then
applied to sfGFP(N150ThzK) 11 (Fig. 2c) and complete decaging
was also achieved with no protocol alterations necessary, despite
Fig. 3 (a) Oxime ligation of protein aldehydes 12/13 using biotin probe 18
or dansyl probe 21 (calculated masses shown). (b) Complete formation of
biotinylated GFP 19 and biotinylated sfGFP 20 confirmed by MS (experi-
mental masses shown) and accompanying Coomassie-stained SDS-PAGE
(upper) and Western blot (lower). (c) Complete formation of dansyl
proteins 22/23 confirmed by MS and accompanying Coomassie-stained
SDS-PAGE (upper) & denatured protein in-gel fluorescence (lower).
the altered position of the ThzK moiety within the protein scaffold.
In this example, the decaged glyoxyl exists almost exclusively as the
hydrate, indicating that differences in the microenvironment
surrounding the thiazolidine may affect the electrophilicity and
reactivity of the resulting glyoxyl species. The final optimised
procedure using allylpalladium(II) chloride dimer 16 is simple
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In summary, a new way to uncage a genetically encoded
glyoxyl aldehyde precursor at physiological pH has been
demonstrated using stoichiometric Pd(II), facilitating access
to internally-modified proteins through aldehyde ligations with-
out the need for an enzyme recognition sequence and hence
minimising structural perturbations. This method requires only
short reaction times under gentle conditions and the resulting
aldehyde can be modified in completion. It is hoped that this
latest addition to the chemical biologist’s toolbox will open up
opportunities for creating exciting new bioconjugates, achieving
a greater understanding of complex biological systems.
We thank Dr Ed Bergstrom and the CoEMS for support with
protein MS. This work was supported by the University of York
(R. L. B.) and an EPSRC DTG studentship (EP/M506680/1, R. J. S.).
Conflicts of interest
R. L. B., R. J. S., & M. A. F. are authors on PCT/GB/2017/052896
application which in one claim covers Pd-mediated decaging.
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