Phosphorescent proteins for bio-imaging and site selective bio-conjugation of peptides and proteins with luminescent cyclometalated iridium(iii) complexes

Article abstract of DOI:10.1039/c3cc48376b

A new bio-conjugation reaction for site selective modification of proteins and peptides with phosphorescent iridium(iii) complexes has been developed; the Ir(iii)-modified proteins and peptides display long emission lifetimes and large Stoke shifts that can be used for bio-imaging studies.

Full text of DOI:10.1039/c3cc48376b

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Phosphorescent proteins for bio-imaging and site
selective bio-conjugation of peptides and proteins
with luminescent cyclometalated iridium(III)
complexes†
Cite this: Chem. Commun., 2014,
0, 4375
5
Received 1st November 2013,
Accepted 7th March 2014
ab
c
c
ab
Hoi-Yan Shiu, Hiu-Chi Chong, Yun-Chung Leung, Taotao Zou and
DOI: 10.1039/c3cc48376b
ab
Chi-Ming Che*
www.rsc.org/chemcomm
A new bio-conjugation reaction for site selective modification of
proteins and peptides with phosphorescent iridium(III) complexes
has been developed; the Ir(III)-modified proteins and peptides dis-
play long emission lifetimes and large Stoke shifts that can be used
for bio-imaging studies.
Phosphorescent metal complexes are increasingly used in the
1
–5
design of biological probes and labels
owing to their long
Scheme 1 Chemo-selective cysteine modification by electron-deficient
alkynes.
emission lifetimes (100 ns to ms), high photostability, and large
6,7
Stokes shift between absorption and emission (hundreds of nm).
In this area, luminescent cyclometalated iridium(III) complexes
8
11
have been extensively studied, and we have previously reported as a bioprobe for bioimaging. Our interest in this area is to
+
the luminophore [Ir(ppy)
2
(phen-NHCO)] to develop FRET-based develop site selective bioconjugation of cellular proteins with
9
probes for the detection of Cys and Hcy.
luminescent metal complexes to give phosphorescent reporter
Among the biological probes, green fluorescent proteins (GFP), proteins. Previously, we reported a selective chemical modification
isolated from the bioluminescent jellyfish Aequorea victoria, are the method, involving conjugate addition of the thiol on the residue of
10
most widely used reporter probes in bio-imaging studies. Green cysteine of biomolecules to an electron-deficient alkynoic amide
12
fluorescent proteins are based on fluorescent organic lumino- (Scheme 1). This chemo-selective cysteine modification has been
phores with emission lifetimes in the nanosecond time regime. used to modify unprotected peptides and proteins with organic com-
One way to expand the applications of luminescent reporter pounds in aqueous media, giving a vinyl sulfide linkage attached to
12
proteins is to develop artificial phosphorescent proteins by site the exposed cysteine(s) of the peptides/proteins. In this work, the
+
2
selective bio-conjugation of natural proteins with luminescent phosphorescent complex [Ir(ppy) (phen-propiolamide)] (1) contain-
transition metal complexes. Such artificial phosphorescent pro- ing a substituted 1,10-phenanthroline ligand incorporated with an
teins have emission lifetimes significantly longer than that of the electron-deficient alkynoic amide motif was prepared (Fig. 1).
natural green fluorescent proteins by orders of magnitude. In the
The alkynoic amide group of 1 is conceived to react with the
literature, there have been examples on the modification of bio- cysteine residue on proteins to generate phosphorescent pro-
molecules with phosphorescent metal complexes though most of teins via the formation of a vinyl sulfide linkage (Scheme 1).
the methods used for bioconjugation are usually site non-specific. The alkynoic amide group was chosen because in the reported
Ma and coworkers recently reported an artificial luminescent peptide modification by the alkynoic amide, complete conver-
protein, apoferritin-encapsulated luminescent europium complex, sion could be achieved when the alkynoic amide was used in
excess and the modification was exclusively on the cysteine
1
2
a
residue. Besides, the amide bond of an alkynoic amide was
State Key Laboratory of Synthetic Chemistry, Institute of Molecular Functional
Materials and Department of Chemistry, The University of Hong Kong,
Pokfulam Road, Hong Kong, China. E-mail: cmche@hku.hk; Fax: +852 2857-1586
HKU Shenzhen Institute of Research and Innovation, Shenzhen 518053, China
Department of Applied Biology and Chemical Technology, The Hong Kong
Polytechnic University, Hung Hom, Kowloon, Hong Kong, China
1
2
reported to remain intact in the cellular environment.
The coupling of an unprotected peptide STSSSCNLSK (2)
with 1 (1 equiv.) in 100 mL of pH 8.0 phosphate buffered saline
b
c
PBS/CH
3
CN = 9/1 at room temperature for 24 h was examined
(Scheme 2). Selective attachment of 1 to the cysteine sulfhydryl
†
Electronic supplementary information (ESI) available: Syntheses and characteri-
zation of 1 and the conjugates. See DOI: 10.1039/c3cc48376b
group of 2 was confirmed by LC-MS/MS. Complete conversion
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2
Fig. 1 Structure of complex 1 [Ir(ppy) (phen-propiolamide)] .
Scheme 3 Structures of 1-modified CAYEMWFHQK, CALNN and
SSCSSCPLSK.
Scheme 2 Structure of 1-modified STSSSCNLSK.
of 1 to 1-modified STSSSCNLSK was observed and neither the
N-terminal a-amino group nor the side-chains of serine and
lysine were modified (ESI†).
To examine the compatibility of the electron-deficient alkynoic
amide with other nucleophilic amino acids, we conducted a
coupling reaction of the unprotected peptide AYEMWCFHQK
(3) and 1 (88% conversion). The nucleophilic side-chains of
histidine, methionine, lysine and tyrosine on 1-modified 3 were
observed to be intact (Fig. 2).
Upon using 1 and in pH 8.0 PBS/CH CN, selective cysteine
3
modification of other unprotected peptides, including a peptide
with N-terminal cysteine CAYEMWFHQK (4) (complete conversion),
a short peptide with N-terminal cysteine CALNN (5) (complete
conversion) and a peptide with two cysteine units SSCSSCPLSK
(6) (95% conversion), was observed, generating phosphorescent
iridium(III)-modified peptides (Scheme 3).
Fig. 3 The QTOF MS/MS spectrum of 1-modified GK-21 at cysteine after
tryptic digestion of 1-modified BSA.
The site selective modification of cysteine-containing peptides
was extended to the preparation of phosphorescent proteins.
Selective conjugation of the single, surface exposed cysteine
residue of bovine serum albumin (BSA) to 1 (10 equiv.) in pH 8.0
PBS/CH CN = 9/1 was observed with 84% conversion for a reaction
3
time of 24 h at room temperature. As depicted in Fig. 3, selective
LC-MS/MS analysis of the tryptic digest of the reaction mixture of
1
and BSA. Similarly, the surface exposed cysteine residue of
human serum albumin (HSA) was selectively modified by 1 with
1
3
8
7% conversion (ESI†). Scheme 4 depicts the structure of
attachment of 1 to the cysteine residue of BSA was confirmed by
1-modified BSA and 1-modified HSA, and the corresponding
peptide sequences, GK-21 and AK-21, obtained after tryptic
digestion of 1-modifed BSA and 1-modified HSA respectively.
To exemplify the application of this modification method, a
therapeutically important protein which displays anti-cancer
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activity, BCArg, was allowed to react with 1 (10 equiv.) in pH 8.0
PBS/CH CN = 9/1 at 37 1C for 24 h. BCArg is a recombinant protein
3
expressed in E. coli, where the gene is from Bacillus caldovelox, an
organism found in the hot spring. BCArg contains the nucleophilic
amino acid residues including 14 lysine, 11 histidine, 12 methionine
and 8 tyrosine. A single cysteine was incorporated to BCArg through
site-mutagensis. A subsequent site selective bioconjugation
reaction of 1 led to a complete conversion of BCArg to 1-modified
BCArg. Tryptic digestion of 1-modifed BCArg revealed that the
Fig. 2 The QTOF MS/MS spectrum of 1-modified 3.
4376 | Chem. Commun., 2014, 50, 4375--4378
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quantum yield of 0.12 and a lifetime of 0.61 ms in degassed
CH CN upon excitation at 380 nm (ESI†). Upon the bio-
3
conjugation reactions of 1 with proteins (BSA or BCArg), any
excess of 1 present in the reaction mixture was removed by
micro-spin column separation (MicroSpin G-50 Columns, GE
Healthcare). The emission spectra of 1, 1-modified BSA and
1-modified BCArg are similar with their emission peak maxima
at 590 nm, 588 nm, and 570 nm, respectively (Fig. S15, ESI†).
Comparing the photophysical data of 1 with that of conven-
tional fluorescent probes (emission lifetimes 0.1–20 ns; Stokes
Scheme 4 Structure of 1-modified BSA and 1-modified HSA (above);
structure of peptide sequences: GK-21 and AK-21 (below).
1
7,18
shifts tens of nm),
1 displays a long emission lifetime
(
(
in microsecond range) and a comparatively large Stokes shift
210 nm), both of which could allow effective elimination of
auto-fluorescence and scattering light from the samples and
nearby optics that are fast decaying and have small Stokes
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shifts. Complex 1 could also serve as a cell imaging agent
according to fluorescence microscopic analysis and it mainly
localized in the mitochondria (Fig. S23, ESI†).
In summary, a phosphorescent iridium(III) complex containing
a polyvinyl ligand appended with an electron-deficient alkynoic
amide was synthesized. This complex could be selectively intro-
Fig. 4 SDS-PAGE of native BCArg and 1-modified BCArg.
cysteine-containing peptide, named LK-48: LGVIWYDAHGDVN duced into peptides and proteins with high conversion yields by
TAETSPSGNIHGMPLAASLGFGHPALTQIGGYCPK was modified its reaction with the free cysteine residue on these biomolecules
À
by one molecule of 1 (ESI†) while the other typsin-digested providing a stable vinyl sulfide linkage.
peptide fragments remained intact. Upon modification of BCArg
This work was supported by the University Grants Committee
with 1 (Fig. 4), the intrinsic bioactivity of BCArg to breakdown (Area of Excellence Scheme AoE/P-03/08) of the Hong Kong Special
À1
arginine was preserved with the enzymatic activity (U mg ) of Administrative Region of China, the National Key Basic Research
BCArg and 1-modifed BCArg being 252.2 and 224.6, respectively. Program of China (no. 2013CB834802) and The University of
Since cysteine was engineered to the surface of BCArg, conjugation Hong Kong (University Development Fund).
of a small molecule like 1 would minimize the steric hindrance
and thus preserving the enzymatic activity after conjugation. This
finding suggested that 1 could act as an imaging tool which is able
to retain the protein functionality and enzyme activity.
Notes and references
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