Synthetic peptides caged on histidine residues with a bisbipyridyl ruthenium(ii) complex that can be photolyzed by visible light

Article abstract of DOI:10.1039/c4cc08049a

We report a light-sensitive histidine building block for Fmoc/tBu solid-phase peptide synthesis in which the imidazole side chain is coordinated to a ruthenium complex. We have applied this building block for the synthesis of caged-histidine peptides that can be readily deprotected by irradiation with visible light, and demonstrated the application of this approach for the photocontrol of the activity of Ni(ii)-dependent peptide nucleases. This journal is

Full text of DOI:10.1039/c4cc08049a

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Synthetic peptides caged on histidine residues
with a bisbipyridyl ruthenium(II) complex that can
be photolyzed by visible light†
Cite this:DOI: 10.1039/c4cc08049a
Received 11th October 2014,
Accepted 8th January 2015
Jesus Mosquera,‡ Mateo I. Sanchez,‡ Jose L. Mascarenas* and
´
´
´
˜
M. Eugenio Vazquez*
´
DOI: 10.1039/c4cc08049a
www.rsc.org/chemcomm
We report a light-sensitive histidine building block for Fmoc/tBu However, despite their wide range of applications, these caging
solid-phase peptide synthesis in which the imidazole side chain is groups are not particularly suited for biological studies, because
coordinated to a ruthenium complex. We have applied this building they require irradiation with harmful short-wavelength UV light
1
0
block for the synthesis of caged-histidine peptides that can be for photolysis (about 365 nm). Therefore, there has been a
readily deprotected by irradiation with visible light, and demon- great interest in the development of substitute long-wavelength
1
1,12
strated the application of this approach for the photocontrol of the sensitive caging groups.
In this context, photolabile bis-
activity of Ni(II)-dependent peptide nucleases.
bipyridyl ruthenium(II) complexes have been explored as alter-
native caging groups with promising spectroscopic properties
Caged peptides are bioactive species that include a photo- (i.e. a long photolysis wavelength and high uncaging quantum
1
3,14
cleavable protecting group masking a key functionality required yields).
With these premises, we decided to explore the
for their action. Photolysis of the caging group releases the application of ruthenium(II) bipyridyl complexes as photolabile
1
effector peptide, thus providing researchers with spatial and protecting groups for caging histidine residues, anticipating
2
,3
temporal control over biological processes. Peptides can be that the coordination of the imidazole side chain with these
4
caged by modifications in their backbone, or by introduction complexes should effectively impair any peptide requiring the
of photolabile groups in specific amino acid side chains, free imidazole for its activity. In addition to the spectroscopic
including amines and carboxylates in Lys or Asp/Glu residues, advantages afforded by the use of Ru(II) complexes as caging
5
e
thiols in cysteines, or hydroxyl groups in Ser, Thr and Tyr.
groups, relying on the coordination of the pros nitrogen (N ,
Oddly enough, the photocontrol of biological processes with Scheme 1) of the imidazole would also avoid potential synthetic
6
d
a
caged histidine peptides has not yet been described. This problems related with the known tendency of N to N acyl
1
5
constitutes a significant gap in caging technology because transfer during peptide elongation, as well as effectively block
histidine, although relatively uncommon in protein sequences the metal-coordinating nitrogen in the imidazole side-chain.
(
o2.5%), is a highly versatile amino acid that plays key roles in
The caged histidine building block, Fmoc–His(Ru)–OH (2),
the activity of many peptides and proteins, acting as an was efficiently synthesized by the two-step process outlined in
aromatic residue, a hydrogen bond donor or acceptor, or as a Scheme 1. In short, the commercially available cis-bis(2,2 -
coordinating ligand, and can even suffer posttranslational bipyridine)dichloro ruthenium(II) complex (Ru(bpy) Cl ) was
modifications. Therefore, given the functional plasticity and treated with triphenylphosphine and then with Boc–His–OH
biochemical relevance of this amino acid it would be highly
0
7
2
2
8
relevant to develop a practical method for the synthesis of
caged histidine peptide derivatives.
Most peptide caging approaches developed so far rely on the
1
,9
use of o-nitrobenzyl groups as photosensitive cleavable units.
Centro Singular de Investigaci o´ n en Qu ´ı mica Biol o´ xica e Materiais Moleculares
(
CIQUS) and Departamento de Qu ´ı mica Org a´ nica, Universidade de Santiago de
Compostela, 15782 Santiago de Compostela, Spain.
E-mail: eugenio.vazquez@usc.es
†
Electronic supplementary information (ESI) available. See DOI: 10.1039/
c4cc08049a
These authors contributed equally.
‡
Scheme 1 Synthesis of Fmoc–His(Ru)–OH.
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in a one-pot reaction to yield the Boc-protected intermediate 1.
Removal of the Boc protecting group with trifluoroacetic acid
followed by installation of the Fmoc group with 9-fluorenyl-
methyl N-succinimidyl carbonate (Fmoc–OSu), afforded the
desired Fmoc–His(Ru)–OH building block. The synthesis of the
trimethylphosphine analog of 2 was also attempted following
1
6
the same set of transformations, but deprotection of the Boc
intermediate with TFA resulted in partial decomplexation of the
histidine building block, which led us to focus our studies on the
more stable triphenylphosphine derivative.
Before its incorporation into peptides, we determined the
uncaging quantum yield of the Boc–His(Ru)–OH building block,
Fig. 1 Left: normalized HPLC traces at 220 nm of crude mixtures resulting
from the automated synthesis of test peptides. Purity (in brackets) was
estimated from the area of the HPLC peaks. (a) HAKAEAEAKAK (86%);
(
b) WLAHKYLQGGC (92%); (c) LFQFLGKIIHHVGNFVHGFSHVF (46%). Right:
which was obtained by comparing its photolysis rate with that of representative peptide uncaging; bottom trace: crude peptide YEGKH-
+
[
Ru(bpy)
source (see the ESI†).
yield (Func E 0.06) is comparable to the photolysis efficiency
2
PPh
3
–GABA] upon irradiation using a 455 nm LED SAEWG upper trace: HPLC after irradiation of the purified peptide,
showing the uncaged peptide (d) and the ruthenium photolysis byproducts
*). H represents the caged histidine.
1
3c,17
The resulting uncaging quantum
(
1
3c,18
reported for other Ru(II)-photolabile compounds,
and of
1
d
most organic cages. In addition to the expected uncaging attention on the Arg–Gly–His tripeptide (RGH), which has been
of the histidine side chain and release of the side chain- described as an efficient metal-chelating sequence with DNA
deprotected Boc–His–OH, the HPLC analysis also showed the binding and endonuclease properties in the presence of Ni(II)
2
1
competitive cleavage of the PPh ligand as a minor side reac- ions and oxidizing agents. Considering that the imidazole
3
tion (E5%), as well as peaks indicating the formation of group in the histidine side chain is required for chelation of the
+
2
+
[
Ru(bpy)
possibly resulting from the reaction of the [Ru(bpy)
photolysis byproduct with the HPLC solvent system (see ESI†).
2 3 2 3
PPh (MeCN)] and [Ru(bpy) PPh
(TFA)] complexes, Ni(II) ion, we reasoned that a caged histidine analog (rRGH)
+2
2
PPh
3
(H
2
O)]
should be unable to coordinate the metal ion and form the
19
catalytic metallopeptide. Furthermore, its nuclease activity
The stability of the ruthenium cage in the presence of various should be recovered upon irradiation and uncaging of the
potentially reactive species under physiological conditions (e.g. histidine residue (Fig. 2).
H O , histidine, glutathione), or competitive ions, such as nickel(II),
Once we synthesized the rRGH peptide following the
2
2
was confirmed by HPLC after 24 h incubation (see the ESI†).
procedures described before, we studied its uncaging: irradia-
With the desired building block in hand and having tion of a 10 mM solution of rRGH in Na-cacodylate buffer at pH
successfully demonstrated its photolabile properties, we tested 7.5 for 1 min using a 455 nm LED source results in quantitative
its integration in standard solid phase peptide synthesis (SPPS) photolysis of the caged rRGH peptide as shown by HPLC
protocols by synthesizing a series of test peptides. Coupling of (Fig. 3 left, top trace); in addition to the peak corresponding to
the caged histidine building block was conducted in all cases the uncaged peptide (RGH) that is eluted with the injection
using 5 equivalents of the Fmoc–His(Ru)–OH building block, peak, we also observed the ruthenium complexes arising from
+
2
2 3 2
and a mixture of HATU/HOAt (5 eq.) and DIEA (6 eq.) as base in the reaction of the photolyzed [Ru(bpy) PPh (H O)] with the
DMF. The stereochemical integrity of the caged residue is HPLC solvent system (Fig. 3 left, top trace, peaks labeled with
maintained under these conditions, optimized to avoid epimer- an asterisk). We next examined whether the uncaging event
ization of the Ca stereocenter (see ESI†). Cleavage of the
resulting peptides with a standard acidic TFA cocktail (TFA :
2
0
2 2 2
CH Cl : triisopropylsilane : H O: 90 : 5 : 2.5 : 2.5), afforded in
all cases the expected caged His peptides as major components
in the crude samples (Fig. 1, left); only the synthesis of the
longer peptide was problematic after the 15th coupling, resulting
in the appearance of significant secondary products, although
the desired peptide was obtained as the major product of the
synthesis (Fig. 1, left, trace c).
Irradiation of the purified peptides with visible light
resulted in all cases in complete uncaging and liberation of
the unprotected parent peptides, as well as formation of the
ruthenium photobyproducts (Fig. 1 right, and ESI†). Furthermore,
in contrast with the preliminary studies with the Boc–His(Ru)–OH
building block, no photodissociation of the PPh3 group was
observed in the uncaging of the peptides.
Fig. 2 Uncaging of the rRGH peptide yields the metal-chelating RGH
As a simple model system in which the newly developed
tripeptide, which displays nuclease activity in the form of a Ni(II) complex,
photolabile histidine building block is applied, we focused our RGH(Ni).
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DNA. We are also grateful to Prof. Wajih Al-Soufi, from the
Department of Physical Chemistry at the Universidad de Santiago
de Compostela, for his help in the construction of the LED
photolysis apparatus.
Notes and references
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Fig. 3 Left: uncaging of rRGH monitored by HPLC. Bottom trace: caged
peptide before photolysis; top trace: the photolyzed mixture showing the
complete disappearance of the caged peptide, and the formation of the
ruthenium byproducts (*); the uncaged peptide is eluted with the injection
peak (not shown). Right: nuclease activity of rRGH is monitored by 1%
agarose gel electrophoresis (see main text for reaction conditions). Lanes
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(a) M. I. S ´a nchez, J. Martinez-Costas, F. Gonzalez, M. A. Bermudez,
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–7: 27.6 mg mL of pcDNA 3.1 Neo plasmid; lane 2: 10 mM of RGH and
Ni(ClO ; lanes 3 and 4: 7.5 and 10 mM of rRGH and Ni(ClO ; lanes 5
and 6: 7.5 and 10 mM of rRGH and Ni(ClO after photolysis; lane 7:
Fmoc–His(Ru)–OH (10 mM) and Ni(ClO after photolysis. Photolysis was
by irradiation at 455 nm for 1 min in
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(
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Ni(ClO ) and 100 mM KHSO in Na-cacodylate buffer at pH 7.5
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to the supercoiled DNA (Fig. 3, lane 1) is completely converted
to a slower-migrating band, consistent with the formation of
the nicked-circular form of the DNA (Fig. 3, lane 2). In contrast,
the caged version of the peptide (rRGH) does not display
nuclease activity under the same conditions (Fig. 3, lanes 3 and 4).
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2
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6
1
(
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and 6). No degradation of the DNA band is observed in the
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8
control experiment in which the amino acid Fmoc–His(Ru)–OH
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In summary, we describe the first effective caged histidine
building block and its incorporation into peptides using standard
Fmoc/tBu SPPS protocols. In contrast with common UV-sensitive
o-nitrobenzyl groups, the photolabile Ru(II) bisbipyridyl complex
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9
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1
We are thankful for the support given by the Spanish grants
SAF2013-41943-R and CTQ2012-31341, the Xunta de Galicia
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