The first ready-to-use benzene-based heterotrifunctional cross-linker for multiple bioconjugation

Article abstract of DOI:10.1039/c3ob40086g

The synthesis and applications of the first water-soluble benzene derivative bearing a set of three different and orthogonal bioconjugatable groups (aminooxy, azido and thiol) are described. The combined use of a 5-amino isophthalic acid scaffold and unusual acid-labile protecting groups for temporarily masking aminooxy and thiol moieties has enabled the development of a highly convergent approach towards the synthesis of such a trivalent bioconjugation platform in good yields. The potential utility of this ready-to-use cross-linking reagent for creating complex and fragile tri-component (bio)molecular systems was illustrated through (1) the rapid preparation of a three-colour FRET cascade with valuable spectral properties and (2) the luminescent/fluorescent labelling of peptides and peptide- oligonucleotide conjugates. Thus, such (bio)molecular assemblies were readily obtained via a three-step process or in a one-pot manner, both involving oxime ligation, thiol-alkylation (S_N2 or Michael addition) and copper-catalysed azide-alkyne 1,3-dipolar cycloaddition (CuAAC) reactions.

Full text of DOI:10.1039/c3ob40086g

Organic &
Biomolecular Chemistry
View Article Online
View Journal | View Issue
PAPER
The first “ready-to-use” benzene-based
heterotrifunctional cross-linker for multiple
Cite this: Org. Biomol. Chem., 2013, 11,
2693
bioconjugation†
Guillaume Viault,a Sébastien Dautrey,a Nicolas Maindron,a Julie Hardouin,b
Pierre-Yves Renard*a and Anthony Romieu*a
The synthesis and applications of the first water-soluble benzene derivative bearing a set of three
different and orthogonal bioconjugatable groups (aminooxy, azido and thiol) are described. The com-
bined use of a 5-amino isophthalic acid scaffold and unusual acid-labile protecting groups for tempor-
arily masking aminooxy and thiol moieties has enabled the development of a highly convergent
approach towards the synthesis of such a trivalent bioconjugation platform in good yields. The potential
utility of this “ready-to-use” cross-linking reagent for creating complex and fragile tri-component (bio)
molecular systems was illustrated through (1) the rapid preparation of a three-colour FRET cascade with
valuable spectral properties and (2) the luminescent/fluorescent labelling of peptides and peptide–oligo-
nucleotide conjugates. Thus, such (bio)molecular assemblies were readily obtained via a three-step
process or in a “one-pot” manner, both involving oxime ligation, thiol-alkylation (SN2 or Michael
addition) and copper-catalysed azide-alkyne 1,3-dipolar cycloaddition (CuAAC) reactions.
Received 22nd October 2012,
Accepted 12th February 2013
DOI: 10.1039/c3ob40086g
www.rsc.org/obc
different functional groups allowing sequential bioconjuga-
tions through biocompatible and/or bioorthogonal “click”
Introduction
Heteromultifunctional molecular scaffolds equipped with reactions.3,6,7
three (or more) (bio)orthogonal reactive handles are now
It is also worth mentioning the recent work of the Santoyo-
regarded as versatile and unique tools to make easier access to Gonzalez group related to the facile synthesis of alkyne-vinyl
a myriad of chemically engineered bioconjugates,1 through sulfone derivatised tags (AVST) from a PEGylated bis-vinyl
synthetic methods involving a sequential combination of chemo- sulfone.8 Indeed, these non-peptidyl trivalent cross-linkers
selective ligations.2,3 Indeed, there is a growing need for have enabled the convenient and efficient dual labelling of
such multicomponent biomolecular systems with uniquely proteins (e.g., glycoprotein horseradish peroxidase (HRP)) with
combined properties of the individual components, especially a biotin and a fluorescent dye, through effective reactions
for the development of innovative and valuable diagnostic belonging to the “click chemistry” repertoire (i.e., CuAAC and
and/or therapeutic tools (e.g., multimodality molecular Michael-type addition using a vinyl sulfone as acceptor).
imaging probes, “smart” drug delivery systems, etc.). The vast
The preparation of such multifunctional architectures relies
majority of heterotrifunctional cross-linking reagents (also almost exclusively on classical stepwise peptide synthesis in
named by us “tripods”) commercially available and/or reported solution, and so, it is not always easy to rapidly expand their
in the literature are based on amino acid or peptidyl scaffolds structural diversity (especially for fine-tuning of both physio-
that contain either a set of two different functional groups and chemical properties and bioconjugation ability) as well as to
a biotin affinity tag (e.g., Sulfo-SBED, TRICEPS),4,5 or three greatly simplify their synthetic access. Thus, there is an urgent
need for “tripods” derived from structurally simpler molecular
scaffolds, and easily accessible using flexible and highly con-
aNormandie Univ, COBRA, UMR 6014 & FR 3038; UNIV Rouen; INSA Rouen; CNRS,
1 Rue Tesnières, 76821 Mont St Aignan Cedex, France.
vergent synthetic routes. In that context, our recent research
efforts have been focused on design and evaluation of the bio-
conjugation ability of a non-peptidic cross-linker based on a
benzenic core decorated with three different conjugation
handles: an aminooxy functional group for oxime ligation, a
thiol for reactions with maleimide or α-haloacetyl derivatives,
and an azido for copper-mediated azide-alkyne 1,3-dipolar
E-mail: pierre-yves.renard@univ-rouen.fr, anthony.romieu@univ-rouen.fr;
http://ircof.crihan.fr; Fax: +33 (0)2 35 52 29 71; Tel: +33 (0)2 35 52 24 76 (or 24 27)
bLaboratory PBS UMR 6270, Bât. Chimie, 76821 Mont St Aignan Cedex, France
†Electronic supplementary information (ESI) available: Detailed synthetic pro-
cedures for compounds 10–12, 16, 18 and 22 and characterisation/spectral data
for all compounds and (bio)conjugates. See DOI: 10.1039/c3ob40086g
This journal is © The Royal Society of Chemistry 2013
Org. Biomol. Chem., 2013, 11, 2693–2705 | 2693

View Article Online
Paper
Organic & Biomolecular Chemistry
Fig. 1 Examples of trivalent benzene derivatives bearing two or three identical (photo)reactive groups, published in 2012.
aminooxy and thiol moieties, both labile under the same
acidic conditions were used, and (2) a cheap benzenic building
block 2 whose two functional groups (i.e., amino and car-
boxylic acid) can be easily unveiled through standard and
easily implemented reactions (i.e., hydrogenation and saponifi-
cation) was chosen as starting material. Thus, a short six-step
synthetic route was designed as depicted in Scheme 1. First,
the nitro group of 2 was converted into primary amine by Pd/C
cat. hydrogenation, and the resulting aniline was used as an
anchoring point to graft the aminooxy-containing building
block. Aminooxyacetic acid (Aoaa) N-protected with the acid-
labile 1-ethoxyethylidene group (Eei) was preferred to the stan-
dard di-Boc-derivative because it has been shown recently that
this oxime is more adequate for stepwise SPPS of Aoaa-con-
taining peptides, especially in preventing the N-overacylation
side-reactions frequently encountered during such syntheses.11
Thus, acylation of the aniline derivative with the known NHS
active ester 3,11 provided oxime derivative 4 in 55% overall
yield for the two steps. Amidification of its carboxylic acid
moiety with azido-PEG-amine spacer 5 (for its synthesis, see
ESI†) has been readily achieved with BOP/DIEA, in dry CH3CN
to provide 6 in 94% yield. Thereafter, methyl ester 6 was sapo-
nificated by treatment with 1.0 M aq. LiOH in MeOH to give
the targeted benzoic acid 7. This acid was coupled to S-pro-
tected mercaptoamine linker 8 (for its synthesis, see ESI†)
under the same conditions used for 5, to afford fully protected
heterotrifunctional cross-linker 9 in 91% yield. Finally, a single
treatment of 9 with a mixture of TFA/TES/H2O has led to het-
erotrifunctional benzenic derivative 1. The best way to purify
this polar compound is to make semi-preparative RP-HPLC
which enables to recover 1 as a TFA salt and in a moderate not
yet optimised yield (23%). Indeed, the use of RP-HPLC for
purifications (followed by lyophilisation) and the small-scale
Fig. 2 Structure of heterotrifunctional benzene-based cross-linker 1.
cycloaddition (CuAAC) or Staudinger ligation, and pseudo-PEG
moieties as neutral water-solubilising moieties.9 To the best of
our knowledge, only trivalent benzene derivatives bearing two
or three identical (photo)reactive groups have already been
reported in the literature, especially for applications in
peptide/protein bioconjugation (see Fig.
examples).10
1 for selected
In this article, we report the rapid and straightforward syn-
thesis of the first benzene-based “tripod” 1 (Fig. 2) and its
application for the preparation of sophisticated three-com-
ponent conjugates including a three-colour FRET cascade
based on cyanine dyes and luminescent peptide–oligonucleo-
tide conjugates (POCs), through sequential mild and biocom-
patible reactions performed under mild aq. conditions fully
compatible with the moderate stability of selected fluorescent
labels and biopolymers. Dual fluorescent labelling of peptides
through a sequential one-pot derivatisation of 1 that avoids
intermediate chromatographic purifications is also presented.
Results and discussion
Synthesis of heterotrifunctional benzene-based cross-linker 1
Compared to the convergent synthetic strategies previously chosen for the deprotection step induced significant loss of
developed to prepare heterotrifunctional peptide-based tem- material whereas this reaction was found to be almost com-
plates,3,6,7 our basic idea is to dramatically simplify the ortho- plete. But compared to the previously reported peptidyl ana-
gonal protection/deprotection strategy required to ensure the logue isolated in
a
very low yield (<5%),6 the single
correct assembly of functionalised building blocks. To this deprotection step and the presence of a UV chromophore have
end, (1) unusual protecting groups suitable for masking made easier the chromatographic purification and helped to
2694 | Org. Biomol. Chem., 2013, 11, 2693–2705
This journal is © The Royal Society of Chemistry 2013

View Article Online
Organic & Biomolecular Chemistry
Paper
Scheme 1 Synthesis of benzene-based heterotrifunctional bioconjugate linker 1. Reagents and conditions: (a) H2(g), Pd/C, EtOH, rt, 3 h; (b) 3, DIEA, CH3CN, rt,
12 h, 55% for 2 steps; (c) 5, BOP, DIEA, CH3CN, rt, 12 h, 94%; (d) 1.0 M aq. LiOH, MeOH, rt, 5 h, 93%; (e) 8, DIEA, CH3CN, rt, 3 h, 91%; (f) TFA–TES–H2O
(95 : 2.5 : 2.5, v/v/v), rt, 3 h, 23% after RP-HPLC purification.
dramatically increase the isolated yield. The structure of 1 was first triple-colour labelling strategy for proteins, using a combi-
unambiguously confirmed by detailed measurements, includ- nation of site-specific labelling via an unnatural amino acid
ing ESI-MS and NMR analyses (see ESI†). We have observed (para-acetylphenylalanine for oxime ligation) and statistical
that this free aminooxy derivative readily reacts with acetone labelling of two cysteine residues (by Michael addition), has
commonly found in chemistry labs’ atmosphere, leading to been reported,14 but this approach is not entirely satisfactory
blocking of its third bioconjugatable group as an unreactive due to the lack of three independent chemoselective fluo-
oxime. Consequently, this reagent was stored under an argon rescent tagging reactions easily implemented to a wide range
atmosphere, and at −20 °C to limit also both partial reduction of bioconjugatable fluorophores. Consequently, most three-
of its azido group through a thiol-mediated redox process,12 colour single-molecule FRET studies were performed on fluo-
and oxidative dimerisation to disulfide. Indeed, under these rescently-labelled nucleic acids (i.e., DNA duplexes or DNA
storage conditions, such self-reactivity was highlighted by origami).15 In this context, the sequential derivatisation of 1
periodical HPLC analyses of 1 and about 30% degradation with three different complementary fluorophores appears to
occurred within two months (see ESI†). In this context, it is be a very promising way and still an unexplored approach to
generally better to perform deprotection of 9 only a few days achieve this ambitious goal. Sulfoindocyanine dyes Cy 3.0,
prior to the use of 1 in bioconjugation.
Cy 5.0 and Cy 7.0 from GE Healthcare (CyDye™) were chosen for
the practical implementation of this synthetic strategy.16 The
carboxylic acid function of these dyes was used to introduce
the required conjugatable handle α-iodoacetyl (IAc), aldehyde
Synthesis of fluorescent/luminescent (bio)molecular
assemblies by sequential triple derivatisation of 1
To demonstrate the facile applicability of the novel bioconjuga- and terminal alkyne respectively (for their synthesis, see ESI†).
tion reagent 1 toward the construction of three-component Despite the drawbacks related to the limited fluorescence
(bio)molecular systems under mild conditions, the preparation quantum yield in aqueous solution and poor (photo)chemical
of a three-colour or triple FRET cascade was first considered stability of cyanine dye Cy 7.0,17 we have chosen to use this
(Scheme 2). Such constructs, in which an intermediate energy fragile fluorescent label rather than more stable NIR fluoro-
acceptor relays the energy gained from the donor to a lower- phores, especially to demonstrate that the use of cross-linker 1
energy acceptor, offer the advantage that they yield infor- enables the design of effective bioconjugation schemes under
mation about the position of the three chromophores relative very mild aqueous conditions that maintain the structural
to each other, they extend the working range compared to con- integrity of each molecular partner involved in the final tar-
ventional FRET systems, and the spectral shift between the geted assembly. Furthermore, the symmetrical dicarboxylic
excitation of the donor and the emission of the lower-energy acid cyanine dye Cy 7.0 was used because the unsymmetrical
acceptor is much more pronounced.13 In addition, fewer analogue (bearing N-ethyl and N-carboxypentyl substituents) is
labelled sample molecules are required to measure relative dis- prone to aggregation in aqueous environments.18 As depicted
tances. Despite these obvious advantages, no general synthetic in Scheme 2, sequential derivatisation of 1 was readily and
method for their preparation is now available. Recently, the easily achieved under mild aqueous conditions and in the
This journal is © The Royal Society of Chemistry 2013
Org. Biomol. Chem., 2013, 11, 2693–2705 | 2695

View Article Online
Paper
Organic & Biomolecular Chemistry
Scheme 2 Bioconjugation schemes involving 1. Reagents and conditions: (a) 0.1 M aq. NaOAc buffer pH 4.2, Cy 5.0–aldehyde 10, or peptide–aldehyde 16, rt, 4 h,
32% and 45%; (b) CH3CN and 0.1 M aq. NaHCO3 buffer pH 8.5 (1 : 3, v/v), Cy 3.0–IAB 11 or Eu(III) chelate–IAB 18, rt, 3 h, 36% and 30%; (c) H2O, 10 mM aq. CuSO4,
10 mM aq. sodium ascorbate, Cy 7.0–alkyne 12, rt, 2 h, 47%; (d) H2O, DMSO 10 mM aq. CuSO4, 10 mM aq. sodium ascorbate, 10 mM TBTA, 50 mM aq. DIEA, ODN–
alkyne 17, rt, 24 h, 66%.
following order: (1) oxime ligation with Cy 5.0–aldehyde 10, (2) exposure to “atmospheric” acetone (vide supra), it makes sense
SN2 thio-alkylation with the α-IAc derivative of Cy 3.0 11 and to perform oxime ligation prior to the other two reactions. Fur-
(3) CuAAC reaction with Cy 7.0–alkyne 12. To avoid the pre- thermore, as demonstrated by Galibert et al., it is essential to
mature “capping” of an aminooxy moiety of 1 by prolonged achieve CuAAC after thiol alkylation to avoid copper-mediated
2696 | Org. Biomol. Chem., 2013, 11, 2693–2705
This journal is © The Royal Society of Chemistry 2013

View Article Online
Organic & Biomolecular Chemistry
Paper
Fig. 3 (a) UV-vis absorption spectrum of 15 in PBS (concentration: 3.5 μM) at 25 °C; (b) normalised fluorescence emission spectra of 15 in PBS at 25 °C upon exci-
tation at 500 nm ( ), 600 nm ( ) and 730 nm ( ); (c) picture of PBS solutions (concentration: 5.0 μM) of Cy 3.0 dye (left) and FRET cascade 15 (right).
oxidation of thiopeptide scaffolds.3 In addition to the final for the determination of the corresponding quantum yield
FRET cascade 15, we have chosen to isolate the fluorescent (ΦF) values of Cy 3.0). Thus, a large pseudo-Stokes’ shift (up to
derivatives 13 and 14 formed from their ligation, in a pure 229 nm) was observed, which is, to the best of our knowledge,
form to determine their spectral features under physiological one of the few examples of FRET-based systems emitting in
conditions. Thus, each fluorescent “tripod” (i.e., mono- the NIR region upon green excitation, and particularly valuable
labelled, FRET pair and FRET cascade) was isolated by semi- for various applications in the field of DNA analysis and mole-
preparative RP-HPLC and their structures confirmed by ESI-MS cular imaging.19,20 Interestingly, the second carboxylic acid
(see ESI†). In order to confirm the presence and integrity of function of final acceptor Cy 7.0 remains free for further conju-
the three cyanine units grafted to the same benzene template, gation of this cassette to a biomolecule or a biological target.
the photophysical properties of 15 were determined in phos-
To provide additional examples of sequential triple derivati-
phate buffered saline (PBS, pH 7.5). As illustrated in Fig. 3a, 15 sation of cross-linker 1, we have decided to apply this biocon-
exhibits three characteristic absorption maxima at 550, 648 jugate assembly strategy to the preparation of luminescent
and 761 nm assigned to the three different cyanine labels. peptide–oligonucleotide conjugates (POCs) (Scheme 2).
Thus, the resulting solution colour of 15 is dramatically Indeed, it is now well established that linking peptides to
different than that of the donor Cy 3.0 unit (Fig. 3c). Further- ODNs improves some of the desired properties of these DNA
more, the absorption ratios are in good agreement with the fragments to be used as therapeutic agents (cellular delivery,
expected dye : tripod molar ratios equal to 1, and are further stability to exonucleases, improved binding to complementary
evidence for the high purity of this fluorescent three-com- sequences and greater rate of hybridization).21 To gain relevant
ponent conjugate. Upon excitation at 500 nm (Cy 3.0 core), 15 in cellulo or in vivo biodistribution data of such potential (anti-
exhibits strong red and NIR emission bands centered at 668 sense) drugs, one of the most practical solutions relies on the
and 779 nm respectively, and a weak green emission band at use of bis-conjugates of ODNs which combine a luminescent/
561 nm, indicating efficient energy transfer between Cy 3.0 fluorescent moiety with the grafted peptide sequences, the
and Cy 5.0 (Fig. 3b), this latter cyanine unit serving as an fluorophore serving as a probe to detect the transport of the
energy relay toward the final acceptor Cy 7.0. The energy trans- POC.22 The vast majority of that type of optical bioprobes are
fer efficiency (E.T.E) was calculated based on the equation: E.T.E readily obtained by sequential derivatisation of a 3′,5′-bifunc-
= {100 × [1 − (ΦF (donor Cy 3.0 in the FRET cascade))/(ΦF tionalised oligonucleotide (produced by solid-phase synthesis)
(free donor Cy 3.0))]%} and found to be equal to 95% (see ESI† with a peptide and a fluorescent reporter group, for instance
This journal is © The Royal Society of Chemistry 2013
Org. Biomol. Chem., 2013, 11, 2693–2705 | 2697

View Article Online
Paper
Organic & Biomolecular Chemistry
Fig. 4 ESI mass spectrum of luminescent peptide–ODN conjugate 21 recorded in the negative mode (calcd mass 7918.47, found: 7919.20). Under our ionisation
conditions, only the −4 charge state was observed (inset: zoom).
through the sequential formation of two oxime bonds.23 between the luminescent peptide conjugate and ODN–alkyne
However, the success of this approach has required the use of 17 was performed in H2O–DMSO (1 : 1) with the catalytic
an acid-labile protecting group to temporarily mask the system (CuSO4–sodium ascorbate) and DIEA and tris-(benzyl-
second reactive functionality of ODN (trityl for the aminooxy triazolylmethyl)amine (TBTA), a well known stabilising ligand
moiety or benzaldehyde acetal for the diol used as an alde- for copper(I) species.26 The corresponding luminescent POC 21
hyde-precursor). In order to shorten the synthetic routes of was isolated by RP-HPLC under non-acidic conditions (to
these useful bioconjugates, “tripod” 1 appears to be a success- avoid premature decomplexation of lanthanide cations) in a
ful solution to “cross-link” rapidly and effectively the three good yield (66%) and its structure was unambiguously con-
substrates involved. The dodecapeptide Ac-Lys-Gly-Arg-Ala-Asn- firmed by ESI-MS (Fig. 4). The presence and integrity of a
Leu-Arg-Ile-Leu-Ala-Arg-Tyr-OH whose ε-amino group was deri- grafted Eu(III) chelate were also confirmed by detailed photo-
vatised with the NHS ester of para-formylbenzoic acid aldehyde physical measurements (see ESI†). Interestingly, a relative
(peptide–aldehyde 16) and a synthetic 16-mer oligonucleotide quantum yield of 9.5% was found in aqueous buffer. This
(ODN) 5′-d(TGA ACT GCA GCT CCT U) bearing the alkyne good value is similar to those previously reported for protein
group as a nucleotide modification at the 2′-position of conjugates labeling with the same Eu(III) chelate.24 This indi-
uridine (ODN–alkyne 17) were chosen as representative bio- cates that the benzene core of 1 does not negatively affect the
polymers (for their synthesis, see ESI†). As a luminescent tag, an luminescence properties of such grafted labels.
original thiol-reactive Eu(III) bispyridinylpyrazine-based chelate
Synthesis of fluorescent bioconjugates by sequential one-pot
derivatisation of 1
18 was selected, especially for further time-resolved imaging of
such POCs within living cells. This bioconjugatable lanthanide
(III) complex was easily synthesised by acylation of the parent The great utility of the trifunctional scaffold 1 as a “ready-to-
amino compound recently reported by us,24 with the commer- use” and user-friendly bioconjugation reagent was ultimately
cially available heterobifunctional cross-linker SulfoSuccinimi- demonstrated through the one-pot sequential chemoselective
dyl(4-Iodoacetyl)AminoBenzoate (i.e., Sulfo-SIAB) in aqueous ligations of peptide 16 and cyanine dyes Cy 7.0–alkyne 12 and
NaHCO3 buffer (pH 8.5), and subsequently purified by semi- Cy 5.0–maleimide 22 (Scheme 3). For this latter fluorescent
preparative RP-HPLC (see ESI†). Since of the three (bio)molecu- marker, maleimide was preferred to α-iodoacetyl as the thiol-
lar partners involved in the targeted POC, 16 exhibits the reactive moiety because the Michael addition (leading to its
highest chemical stability, oxime ligation was performed conjugation to the peptidyl architecture) proceeds rapidly and
first.25 Thereafter, SN2 thio-alkylation was readily achieved in good yields in neutral aqueous solutions whereas this is not
with luminescent IAc derivative 18. Finally, CuAAC reaction the case for the SN2 thio-alkylation involving the
2698 | Org. Biomol. Chem., 2013, 11, 2693–2705
This journal is © The Royal Society of Chemistry 2013

View Article Online
Organic & Biomolecular Chemistry
Paper
Scheme 3 One-pot bioconjugation scheme involving 1. Reagents and conditions: (a) 0.1 M aq. NaOAc buffer pH 4.2, peptide–aldehyde 16, rt, 2 h, then Cy 5.0–
maleimide 22, H2O, rt, 2 h and finally Cy 7.0–alkyne 12, Cu(0) microsized powder, tBuOH, rt, 12 h, 28% overall yield.
corresponding α-IAc fluorescent derivative, which effectively heterotrifunctional aromatic scaffold suitable for various chal-
works only in the pH range 8.0–8.5.27,28 Indeed, for this one- lenging bioconjugation applications. We have demonstrated
pot approach, we have chosen to conduct the two last derivati- that the selected set of three reactive groups (aminooxy, azido
sations (Michael addition and CuAAC) at neutral pH because it and thiol) is particularly well-suited for rapid derivatisation/
was not easy to accurately adjust the pH mixture as required by functionalisation of fragile and high-value added biopolymers,
the triptych “oxime ligation (performed at pH 4.2), SN2 thio- through biocompatible reactions easily performed on small
alkylation (performed at pH 8.5) and CuAAC (performed at scales, either in three steps or in a one-pot sequential manner.
neutral pH)”, especially when the reactions were conducted in We think that this novel cross-linking reagent represents: (1) a
small volumes and with coloured reagents such as cyanine significant innovation in the field of chemical biology
dyes. Chemoselective ligations were carefully monitored by especially to get highly sophisticated and fragile (bio)conju-
RP-HPLC analysis (Fig. 5). Oxime ligation between 1 and gates which are not accessible by derivatisation of commer-
peptide–aldehyde 16 was found to be complete within 2 h cially available bioconjugation reagents (mainly hetero- or
(Fig. 5a). Then, Cy 5.0–maleimide 22 (for its synthesis see homobifunctional cross-linkers), and (2) a valuable tool to
ESI†) was added to the reaction mixture. Thiol-mediated make easier the preparation of biological composite nanoma-
Michael addition was observed at rt providing within 2 h the terials through addressing challenges related to the controlled
Cy 5.0–labelled peptide (Fig. 5b). For this latter reaction, we display of biomolecules on nanoparticles.29 Thus, our current
have noticed the formation of side-products that result from efforts are devoted to expanding the functional diversity of
hydrolysis of maleimide derivatives, which may make the use heterotrifunctional benzenic scaffolds for the implementation
of this “tripod” system in some practical one-pot biomolecule of new and promising “click” reactions such as tetrazine
labelling and cross-linking applications difficult. The ligation30 and related cycloadditions.31
implementation of an alternative bioconjugation reaction not
involving thiols is probably the best way to solve this problem
(vide infra, Conclusions section). Finally, CuAAC with Cy 7.0–
alkyne 12 was carried out by adding a Cu(0) microsized
powder and at rt overnight (Fig. 5c). The expected FRET cas-
Experimental section†
General
sette (Cy 5.0–Cy 7.0) labelled peptide 23 was isolated by Column chromatography purifications were performed on
RP-HPLC in a satisfying 28% overall yield. The presence and Geduran® Si 60 silica gel (40–63 μm) from Merck. TLC was
integrity of cyanine labels grafted to the peptide were con- carried out on Merck DC Kieselgel 60 F-254 aluminium sheets.
firmed by ESI mass spectrometry (Fig. 5e), UV-vis absorption The spots were visualised by illumination with a UV lamp (λ =
and fluorescence analyses (see ESI†). In the present case, the 254 nm or 365 nm), by immersion in ninhydrin solution or
isolated yield of the three-component bioconjugate is signifi- potassium permanganate solution. Small scale syntheses (i.e.,
cantly higher than those of compounds 15 and 21 synthesised bioconjugation experiments) were performed in standard
according to a three-step bioconjugation protocol involving single-use microtubes (0.5 or 1.7 mL). All solvents were dried
chromatographic isolation of each intermediate (overall yield following standard procedures (CH3CN: distillation over CaH2,
5.5% and 9.0% respectively).
CH2Cl2: distillation over P2O5, THF: distillation over Na°/benzo-
phenone). Anhydrous DMF was obtained from Carlo Erba-
SdS or Fisher Scientific. TEA was distilled from CaH2 and
stored over BaO. The HPLC-gradient grade acetonitrile
(CH3CN) was obtained from VWR. Buffers and aq. mobile-
Conclusions and future work
In summary, we have described the easy and high-yielding phases for HPLC were prepared using water purified with a
functionalisation of benzene core to get the first Milli-Q system (purified to 18.2 MΩ cm). Triethylammonium
a
This journal is © The Royal Society of Chemistry 2013
Org. Biomol. Chem., 2013, 11, 2693–2705 | 2699

View Article Online
Paper
Organic & Biomolecular Chemistry
Fig. 5 (a–c) RP-HPLC elution profiles (system K) for the preparation of FRET cassette (Cy 5.0–Cy 7.0) labelled peptide 23 through a sequential one-pot approach; (d)
RP-HPLC elution profile (system K) of purified 23; (e) ESI mass spectrum of purified 23 recorded in the negative mode (calcd mass 3845.73). Under ionisation con-
ditions, only the −2 and −3 charge states were observed.
2700 | Org. Biomol. Chem., 2013, 11, 2693–2705
This journal is © The Royal Society of Chemistry 2013

View Article Online
Organic & Biomolecular Chemistry
Paper
acetate (TEAA, 2.0 M) and triethylammonium bicarbonate chromatogram at absorbance maximum for each compound)
(TEAB, 1.0 M) buffers were prepared from distilled triethyl- mode (220–750 nm). System B: semi-preparative RP-HPLC
amine and glacial acetic acid or CO2 gas.
(Thermo Hypersil GOLD C18 column, 5 μm, 21.2 × 250 mm)
with CH3CN and aq. TFA (0.1%, v/v, pH 2.2) as eluents [100%
TFA (5 min), followed by linear gradient from 0 to 20%
(12.5 min) and 20 to 60% (80 min) of CH3CN] at a flow rate of
15 mL min−1. Dual UV detection was achieved at 240 and
297 nm. System C: semi-preparative RP-HPLC (Thermo Hyper-
sil GOLD C18 column, 5 μm, 10 × 100 mm) with CH3CN and
aq. TFA (0.1%, v/v, pH 2.2) as eluents [100% TFA (5 min), fol-
lowed by linear gradient from 0 to 25% (17.5 min) and 25 to
75% (100 min) of CH3CN] at a flow rate of 4 mL min−1. Dual
UV detection was achieved at 240 and 330 nm. System D:
system A with CH3CN and aq. triethylammonium acetate
buffer (TEAA, 25 mM, pH 7.0) as eluents. Visible detection at
268, 343 or 646 nm. System E: semi-preparative RP-HPLC
(Thermo Hypersil GOLD C18 column, 5 μm, 10 × 100 mm) with
CH3CN and aq. triethylammonium bicarbonate buffer (TEAB,
50 mM, pH 7.5) as eluents [100% TEAB (5 min), followed by
linear gradient from 0 to 20% (10 min) and 20 to 80%
(120 min) of CH3CN] at a flow rate of 4 mL min−1. Dual visible
detection was achieved at 520 and 610 nm. System F: system E
with the following gradient [100% TEAB (5 min), followed by
linear gradient from 0 to 20% (10 min) and 20 to 40% (80 min)
of CH3CN]. Dual visible detection was achieved at 647 and
700 nm. System G: system C with the following gradient [100%
TFA (5 min), followed by linear gradient from 0 to 20%
(12.5 min) and 20 to 40% (60 min) of CH3CN]. Dual UV detec-
tion was achieved at 225 and 280 nm. System H: system E with
the following gradient [100% TEAB (5 min) followed by linear
gradient from 0 to 25% (10 min) and 25 to 70% (100 min) of
CH3CN]. Dual UV detection was achieved at 230 and 340 nm.
System I: RP-HPLC (Thermo Hypersil GOLD C18 column, 5 μm,
4.6 × 100 mm) with CH3CN and TEAA (100 mM, pH 7.0) as
eluents [100% TEAA (10 min), followed by linear gradient from
Instruments and methods
The synthesis of dodecapeptide Ac-Lys-Gly-Arg-Ala-Asn-Leu-
Arg-Ile-Leu-Ala-Arg-Tyr-OH was carried out on an Applied Bio-
systems 433A synthesizer using the standard Fmoc/tBu chem-
istry32 as previously described by us.33 The synthesis of ODN–
alkyne 17 was carried out on an Applied Biosystems 392 DNA/
RNA synthesizer as previously described by us.6 1H and 13C
NMR spectra were recorded on a Bruker DPX 300 spectrometer
(Bruker, Wissembourg, France). Chemical shifts are expressed
in parts per million (ppm) and relative to tetramethylsilane
from CDCl3 (δH = 7.26, δC = 77.16) or D2O (δH = 4.79).34 Infra-
red (IR) spectra were recorded with a universal ATR sampling
accessory on a Perkin Elmer FT-IR Spectrum 100 spectrometer.
Analytical HPLC was performed on a Thermo Scientific Sur-
veyor Plus instrument equipped with a PDA detector. Semi-pre-
parative HPLC was performed on
a Thermo Scientific
SPECTRASYSTEM liquid chromatography system (P4000)
equipped with a UV-visible 2000 detector. Low-resolution mass
spectra (LRMS) were obtained with a Finnigan LCQ Advantage
MAX (ion trap) apparatus equipped with an electrospray
source. High-resolution mass spectra (HRMS) were recorded
on an LTQ Orbitrap Elite (Thermo Scientific). UV-visible
spectra were obtained on a Varian Cary 50 scan spectropho-
tometer by using a rectangular quartz microcell (Hellma,
108.002-QS, light path: 10 mm, 500 μL). Fluorescence spectro-
scopic studies (emission/excitation spectra) were performed
with a Varian Cary Eclipse spectrophotometer with a fluor-
escence quartz ultra-microcell (Hellma, 105.251-QS, light path:
3 × 3 mm, 45 μL). Emission spectra were recorded under the
same conditions after excitation at the corresponding wave-
length (see Table S1,† excitation and emission filters: auto,
excitation and emission slit = 5 nm) in PBS. Relative quantum
yields were measured in PBS at 25 °C by a relative method
using a suitable standard (see Table S1†). The following
equation was used to determine the relative fluorescence
quantum yield:
0 to 100% (25 min) of CH3CN] at a flow rate of 1.0 mL min−1
.
UV detection was achieved at 260 nm. System J: LC-MS under
the following conditions: RP-HPLC (Thermo Hypersil GOLD
C18 column, 5 μm, 2.1 × 150 mm) with CH3CN and TEAB
(25 mM, pH 7.5) as eluents [100% TEAB (5 min) followed by
linear gradient from 0 to 80% (40 min) of CH3CN] at a flow
rate of 0.25 mL min−1. Dual UV detection was achieved at 260
and 345 nm. ESI-MS detection in the negative mode (full scan,
150–1500 a.m.u., data type: centroid, sheat gas flow rate: 60
arb unit, aux/sweep gas flow rate: 20, spray voltage: 4.5 kV,
capillary temp: 270 °C, capillary voltage: −10 V, tube lens
offset: −50 V). System K: system A with the following gradient
[100% TFA (5 min), followed by linear gradient from 0 to 100%
(35 min) of CH3CN] at a flow rate of 0.25 mL min−1. UV-vis
2
ΦFðxÞ ¼ ðAS=AXÞðFX=FSÞðnX=nSÞ ΦFðsÞ
where A is the absorbance (in the range 0.01–0.1 A.U.), F is the
area under the emission curve, n is the refractive index of the
solvents (at 25 °C) used in measurements, and the subscripts s
and x represent standard and unknown, respectively.
High-performance liquid chromatography separations
Several chromatographic systems were used for the analytical detection with the “Max Plot” (i.e., chromatogram at absor-
experiments and the purification steps: System A: RP-HPLC bance maximum for each compound) mode (220–750 nm).
(Thermo Hypersil GOLD C18 column, 5 μm, 2.1 × 100 mm) System L: RP-HPLC (Thermo Hypersil GOLD C18 column, 5 μm,
with CH3CN and 0.1% aq. trifluoroacetic acid (aq. TFA, 0.1%, 4.6 × 100 mm) with CH3CN and aq. TFA (0.1%, v/v, pH 2.2) as
v/v, pH 2.2) as eluents [100% TFA (5 min), followed by linear eluents [100 TFA (5 min), followed by a linear gradient from 0
gradient from 0 to 80% (40 min) of CH3CN] at a flow rate of to 20% (10 min) and 20 to 60% (80 min) of CH3CN]. Visible
0.25 mL min−1. UV-vis detection with the “Max Plot” (i.e., detection was achieved at 630 and 700 nm.
This journal is © The Royal Society of Chemistry 2013
Org. Biomol. Chem., 2013, 11, 2693–2705 | 2701

View Article Online
Paper
Organic & Biomolecular Chemistry
Synthesised compounds
The layers were separated and the aqueous layer was extracted
with CH2Cl2 (4 × 10 mL). The combined organic layers were
3-Amino-5-(methoxycarbonyl)benzoic acid was synthesised dried (over anhydrous Na2SO4) and concentrated in vacuo to
from nitrobenzene derivative 2 by using a recent literature pro- afford benzoic acid 7 as a colourless oil (100 mg, yield 93%)
cedure.35 An N-protected derivative of aminooxyacetic acid directly used without purification. νmax(neat)/cm−1 3330, 2936,
(Eei-Aoaa-OH) and its NHS active ester were synthesised 2108, 1644, 1546, 1451, 1308, 1098; δH(300 MHz; CDCl3) 9.85
according to published procedures.11 See ESI† for the synthesis (bs, 1 H), 8.56 (s, 1 H), 8.42 (s, 1 H), 8.27 (s, 1 H), 8.18 (s, 1 H),
of bioconjugatable cyanine dyes 10–12 and 22, peptide–alde- 7.72 (s, 1 H), 4.42 (s, 2 H), 3.91 (q, J 6.9, 2 H), 3.70–3.55 (m, 10
hyde 16 and thiol-reactive Eu(III) bispyridinylpyrazine-based H), 3.30–3.25 (m, 2 H), 1.97 (s, 3 H), 1.18 (t, J 6.9, 3 H);
chelate 18.
δC(75 MHz; CDCl3) 169.4, 168.2, 166.5, 165.0, 137.9, 135.3,
3-(2-(1-Ethoxyethylideneaminooxy)acetamido)-5-(methoxy- 131.1, 124.1, 124.0, 123.7, 72.9, 70.2, 69.9, 62.8, 50.5, 40.0,
carbonyl)benzoic acid (4). To a stirred solution of 3-amino-5- 14.2, 14.0; HRMS (ESI+): m/z 481.2045 [M + H]+, calcd for
(methoxycarbonyl)benzoic acid (1.23 g, 6.31 mmol) in dry C20H29N6O8+ 481.2047.
CH3CN (50 mL) were added NHS ester of Eei-Aoaa-OH 3 (2.0 g,
Full-protected tripod (9). To a stirred solution of benzoic
7.75 mmol) and DIEA (3.3 mL, 18.9 mmol). The resulting reac- acid 7 (280 mg, 0.58 mmol) in dry CH3CN (10 mL) were added
tion mixture was stirred at rt for 12 h before it was concen- S-trityl amino-PEG linker 8 (240 mg, 0.58 mmol), BOP reagent
trated in vacuo and diluted with EtOAc (50 mL). The organic (260 mg, 0.59 mmol) and DIEA (0.3 mL, 1.7 mmol). The result-
layers were washed with 10% aq. citric acid (3 × 20 mL), dried ing reaction mixture was stirred at rt for 3 h before it was con-
(over anhydrous Na2SO4) and concentrated in vacuo. Flash- centrated in vacuo and diluted with EtOAc (20 mL). The
column chromatography (silica gel, CH2Cl2–MeOH, 9 : 1, v/v) organic layers were washed with 10% aq. citric acid (3 ×
afforded benzoic acid 4 as a colourless solid (1.2 g, yield 56%). 10 mL), sat. aq. NaHCO3 (3 × 10 mL), brine (20 mL), dried
Rf 0.30 (CH2Cl2–MeOH, 9 : 1, v/v); νmax(neat)/cm−1 3248, 2928, (over anhydrous Na2SO4) and concentrated in vacuo. Flash-
1736, 1695, 1679, 1639, 1546, 1416, 1309, 1211, 1105, 885, 747, column chromatography (silica gel, CH2Cl2–MeOH, 98 : 2, v/v)
702; δH(300 MHz; CDCl3) 10.48 (bs, 1 H), 8.50–8.45 (m, 3 H), afforded the full-protected tripod 9 as a colourless oil (460 mg,
4.51 (s, 2 H), 4.00 (q, J 7.1, 2 H), 3.94 (s, 3 H), 2.06 (s, 3 H), 1.27 yield 91%). Rf 0.15 (CH2Cl2–MeOH, 98 : 2, v/v); νmax(neat)/cm−1
(t, J 7.1, 3 H); δC(75 MHz; CDCl3) 170.0, 169.4, 165.8, 165.3, 3306, 2867, 2099, 1645, 1596, 1540, 1444, 1306, 1098, 744, 700;
137.9, 131.6, 130.8, 127.0, 125.6, 125.4, 73.0, 63.0, 52.6, 14.2, δH(300 MHz; CDCl3) 8.55 (s, 1 H), 8.22–8.18 (m, 2 H), 7.97 (s,
14.1; HRMS (ESI+): m/z 339.1190 [M
+
H]+, calcd for 1 H), 7.45–7.40 (m, 6 H), 7.30–7.20 (m, 11 H), 4.51 (s, 2 H),
4.03 (q, J 7.0, 2 H), 3.70–3.60 (m, 16 H), 3.50–3.45 (m, 2 H),
C15H19N2O7+ 339.1192.
Azido derivative (6). To a stirred solution of benzoic acid 4 3.40–3.30 (m, 4 H), 2.43 (t, J 6.4, 2 H), 2.09 (s, 3 H), 1.30 (t,
(1.1 g, 3.2 mmol) in dry CH3CN (30 mL) were added azido J 6.9, 3 H); δC(75 MHz; CDCl3) 169.1, 166.3, 164.9, 144.7, 138.0,
amino-PEG linker 5 (0.57 g, 3.2 mmol), BOP reagent (1.44 g, 135.6, 129.5, 127.8, 126.6, 121.1, 73.0, 70.5, 70.3, 70.2, 70.1,
3.2 mmol) and DIEA (1.7 mL, 9.7 mmol). The resulting reac- 70.0, 69.7, 69.6, 66.6, 62.9, 50.6, 40.0, 31.6, 14.3, 14.1; HRMS
tion mixture was stirred at rt for 3 h before it was concentrated (ESI+): m/z 870.3885 [M
in vacuo and diluted with EtOAc (50 mL). The organic layers 870.3860.
+
H]+, calcd for C45H56N7O9S+
were washed with 10% aq. citric acid (3 × 20 mL), sat. aq.
NaHCO3 (3 × 20 mL), brine (20 mL), dried (over anhydrous 0.15 mmol) was dissolved in
Benzenic “tripod” (1). The full-protected tripod 9 (130 mg,
mL of TFA–TES–H2O
5
Na2SO4) and concentrated in vacuo. Flash-column chromato- (95 : 2.5 : 2.5, v/v/v) and the resulting reaction mixture was
graphy (silica gel, CH2Cl2–MeOH, 99 : 1, v/v) afforded azido stirred at rt for 3 h. Thereafter, the product was isolated after
derivative 6 as a colourless oil (1.37 g, yield 84%). Rf 0.1 removal of volatiles under reduced pressure, precipitation
(CH2Cl2–MeOH, 99 : 1, v/v); νmax(neat)/cm−1 3315, 2864, 2098, from Et2O and semi-preparative RP-HPLC purification (system
1723, 1642, 1538, 1450, 1306, 1259, 1116, 1092, 1018, 968, 883, B) to yield the TFA salt of tripod 1 as a white amorphous
825; δH(300 MHz; CDCl3) 8.44 (s, 1 H), 8.31 (s, 1 H), 8.11 (s, powder (30 mg, yield 23%). IR (neat) νmax(neat powder)/cm−1
1 H), 8.05 (s, 1 H), 7.07 (bs, 1H), 4.38 (s, 2H), 3.90 (q, J 7.1, 2H), 3316, 2872, 2109, 1645, 1596, 1547, 1447, 1344, 1295, 1201,
3.81 (s, 3H), 3.60–3.55 (m, 10 H), 3.26 (t, J 5.1, 2 H), 1.95 (s, 1132, 896, 836, 799, 721; δH(300 MHz; D2O) 7.95 (s, 2 H), 7.86
3 H), 1.17 (t, J 7.1, 3 H); δC (75 MHz; CDCl3) 169.1, 166.2, 165.9, (s, 1 H), 4.74 (s, 2 H), 3.67–3.50 (m, 20 H), 3.33 (t, J 5.1, 2 H),
164.9, 138.0, 135.7, 131.1, 123.4, 123.3, 122.9, 72.9, 70.4, 70.2, 2.55 (t, J 6.2, 2 H); δC(75 MHz; CDCl3) 168.8, 167.7, 162.8 (q,
1
69.9, 69.6, 62.8, 52.3, 50.5, 39.9, 14.2, 13.9; HRMS (ESI+) 2JCF 35.5, TFA), 137.1, 135.0, 134.9, 122.7, 122.4, 116.3 (q, JCF
495.2205 [M + H]+, calcd for C21H31N6O8+ 495.2203.
291.8, TFA), 72.1, 72.0, 69.5, 69.4, 69.2, 69.1, 68.7, 68.6, 50.0,
Benzoic acid derivative (7). To a stirred solution of azido 39.6, 23.0; HPLC (system A): tR = 23.8 min (purity 91%); LRMS
derivative 6 (110 mg, 0.22 mmol) in MeOH (5 mL) was added (ESI+): m/z 558.20 [M + H]+, 1115.07 [2M + H]+; HRMS (ESI+):
1.0 M aq. LiOH (1 mL). The resulting reaction mixture was m/z 558.2350, calcd for C22H36N7O8S+ 558.2346.
stirred at rt for 5 h before it was concentrated in vacuo and
Preparation of three-colour FRET cascade (15)
diluted with CH2Cl2 (10 mL) and 1.0 M aq. NaOH (10 mL). The
aqueous layer was washed with CH2Cl2 (2 × 10 mL), acidified (a) Oxime ligation with Cy 5.0 aldehyde: Benzenic “tripod” 1
with 1.0 M aq. HCl (15 mL) and diluted with CH2Cl2 (10 mL). (2.5 mg, 4.48 μmol) was dissolved in 0.1 M aq. NaOAc buffer
2702 | Org. Biomol. Chem., 2013, 11, 2693–2705
This journal is © The Royal Society of Chemistry 2013

View Article Online
Organic & Biomolecular Chemistry
Paper
(pH 4.2, 1.0 mL). Cy 5.0 aldehyde 10 (2.8 mg, 3.38 μmol) was reaction was checked for completion by RP-HPLC (system A)
added and the resulting mixture was stirred at rt for 4 h. The and then quenched by addition of aq. TFA 0.1% (2 mL). The
reaction was checked for completion by RP-HPLC (system A) mixture was purified by semi-preparative RP-HPLC (system G,
and then quenched by addition of aq. TFA 0.1% (2 mL). Then, 2 injections). The product-containing fractions were lyophi-
the mixture was purified by semi-preparative RP-HPLC (system lised to give the peptide–tripod conjugate 19 as a white amor-
C, 2 injections). The product-containing fractions were lyophi- phous powder (1.7 mg, yield 45%). HPLC (system D): tR
=
lised to give the red-fluorescent tripod 13 as a blue amorphous 27.2 min (purity > 95%); LRMS (ESI+): m/z 715.53 [M + 3H]3+,
powder (1.5 mg, yield 32%). HPLC (system D): tR = 28.2 min 1072.60 [M + 2H]2+, calcd for C95H150N30O25S 2143.11.
(purity > 95%); LRMS (ESI−): m/z 1368.40 [M − H]−, calcd for
(b) SN2 reaction with an iodoacetyl derivative of Eu(III)
C65H83N11O16S3 1369.52; λmax (PBS) nm 658 (ε/dm3 mol−1 chelate: Peptide–tripod conjugate 19 (4.0 mg, 1.87 μmol) was
cm−1 250 000); λmax em (PBS) nm 668 (ΦF 0.40 in PBS + 5% dissolved in a mixture of CH3CN and 0.1 M aq. NaHCO3 buffer
BSA).
(1 : 3, v/v, 1.0 mL, pH 8.5). Thiol-reactive Eu(III) chelate 18 was
(b) SN2 reaction with an iodoacetyl derivative of Cy 3.0: Cy added and the reaction mixture was stirred at rt for 3 h. The
5.0–labelled tripod 13 (1.5 mg, 1.1 μmol) was dissolved in a reaction was checked for completion by RP-HPLC (system D)
mixture of CH3CN and 0.1 M aq. NaHCO3 buffer (1 : 3, v/v, and the mixture was dissolved in 50 mM aq. TEAB (2 mL) and
1.0 mL, pH 8.5). Thiol-reactive Cy 3.0 derivative 11 (2.1 mg, purified by semi-preparative RP-HPLC (system H). The product
2.2 μmol) was added and the resulting reaction mixture was containing fractions were lyophilised to give the luminescent
stirred at rt for 3 h. The reaction was checked for completion peptide–tripod conjugate 20 as a white amorphous powder
by RP-HPLC (system D) and the mixture was dissolved in aq. (0.23 mg, yield 30%). HPLC (system D): tR = 26.6 min (purity >
TEAB (5 mL, 50 mM, pH 7.5) and purified by semi-preparative 95%); MS (ESI−): m/z 1522.48 [M
RP-HPLC (system E). The product-containing fractions were 132H1842EuN38O35S 3046.27; λmax (H2O) nm 262 and 347; λmax
− , calcd for
2H]2−
C
lyophilised to give the doubly fluorescently-labelled azido em (H2O) nm 615 (only the most intense emission band corre-
tripod 14 as a dark blue amorphous powder (0.86 mg, yield sponding to the 5D0→7F2 transition is reported, ΦF 0.080 in
36%). HPLC (system D): tR = 27.3 min (purity >95%); LRMS H2O). Please note: semi-preparative purification was per-
(ESI−): m/z 1099.93 [M − 2H]2−, calcd for C107H132N16O25S5 formed under non-acidic conditions (i.e., TEAB) to avoid pre-
2200.81; λmax (PBS) nm 549 (ε/dm3 mol−1 cm−1 150 000) and mature decomplexation of lanthanide cations.
653 (ε/dm3 mol−1 cm−1 250 000), λmax em (PBS) nm 565 and
(c) CuAAC reaction with ODN–alkyne: Luminescent
670. Please note: semi-preparative purification was performed peptide–tripod conjugate 20 (30 μg, 10 nmol) and ODN–alkyne
under non-acidic conditions (i.e., TEAB) to avoid severe degra- 17 (82 μg, 17 nmol) were dissolved in H2O–DMSO (1 : 1, v/v,
dation of the Cy 3.0 unit.
160 μL). 16 μL of a 10 mM aq. CuSO4 solution, 16 μL of a
(c) CuAAC reaction with Cy 7.0–alkyne. Doubly fluores- 10 mM TBTA solution in DMSO, 8 μL of a 50 mM aq. DIEA solu-
cently-labelled azido tripod 14 (0.86 mg, 0.39 μmol) and Cy tion, and 16 μL of 10 mM aq. sodium ascorbate solution were
7.0–alkyne 12 (0.26 mg, 0.33 μmol) were dissolved in ultrapure sequentially added. The resulting reaction mixture was stirred
water (0.4 mL). 10 μL of a 10 mM aq. CuSO4 solution and at rt for 5 h. The reaction was checked for completion by
10 μL of a 10 mM aq. sodium ascorbate solution were added. RP-HPLC (system I). Thereafter, further amounts of ODN–
The resulting reaction mixture was stirred at room temperature alkyne 17 (82 μg, 17 nmol), CuSO4 solution (16 μL) and
for 2 h. The reaction was checked for completion by RP-HPLC sodium ascorbate solution (16 μL) were added. The reaction
(system D) and then quenched by addition of aq. TEAB (2 mL, mixture was stirred at rt overnight. Further amounts of ODN–
50 mM, pH 7.5). The mixture was purified by semi-preparative alkyne 17 (82 μg, 17 nmol), CuSO4 solution (16 μL) and
RP-HPLC (system F, 2 injections). The product-containing frac- sodium ascorbate solution (16 μL) were added again and the
tions were lyophilised to give the three-colour FRET cascade 15 reaction mixture was stirred at rt for a further 2 h. The result-
as a dark blue amorphous powder (0.45 mg, yield 47%). HPLC ing residue was taken up in 0.1 M aq. TEAA buffer pH ∼ 7
(system D): tR = 26.1 min (purity > 95%); LRMS (ESI−): m/z (3 mL) and purified by RP-HPLC (system I, 1 injection). The
1001.73 [M
C
− , 1502.80 [M − , calcd for product-containing fractions were dried using a concentrator
3H]3− 2H]2−
149H183N19O34S7 3006.12; λmax (PBS) nm 550 (ε/dm3 mol−1 (GeneVac miVac) to give luminescent peptide–ODN conjugate
cm−1 150 000), 650 (ε/dm3 mol−1 cm−1 250 000) and 761 21 as a white amorphous powder (52 μg, yield 66%). LC-MS
(ε/dm3 mol−1 cm−1 200 000); λmax em (PBS) nm 560, 670 and 776. (system J): tR = 22.7 min; MS (ESI−): m/z 1978.03, 1978.80 and
See Table S1† for ΦF and E.T.E. Please note: semi-preparative 1979.70 [M − 4H]4−, found 7919.20, calcd 7918.47; λmax (TEAA)
purification was performed under non-acidic conditions (i.e., nm 262 and 343; λmax em (TEAA) nm 615 (only the most
TEAB) to avoid severe degradation of the Cy 7.0 unit.
intense emission band corresponding to the 5D0→7F2 tran-
sition is reported, ΦF 0.095 in TEAA). Please note: semi-pre-
parative purification was performed under non-acidic
Preparation of luminescent POC (21)
(a) Oxime ligation with peptide–aldehyde: Benzenic “tripod” 1 conditions (i.e., TEAB) to avoid premature decomplexation of
(2.5 mg, 4.48 μmol) was dissolved in 0.1 M aq. NaOAc buffer lanthanide cations.
(pH 4.2, 1.0 mL). Peptide–aldehyde 16 (2.8 mg, 1.74 μmol) was
Sequential one-pot approach – Preparation of FRET cassette
added and the resulting mixture was stirred at rt for 4 h. The (Cy 5.0–Cy 7.0) labelled peptide (23). Oxime ligation with
This journal is © The Royal Society of Chemistry 2013
Org. Biomol. Chem., 2013, 11, 2693–2705 | 2703

View Article Online
Paper
Organic & Biomolecular Chemistry
peptide aldehyde: Benzenic “tripod” 1 (0.7 mg, 1.2 μmol) was
dissolved in 0.1 M aq. NaOAc buffer (pH 4.2, 0.4 mL). Peptide
aldehyde 16 (2.0 mg, 1.2 μmol) was added and the resulting
reaction mixture was stirred at rt for 2 h. The reaction was
checked for completion by RP-HPLC (system K). Then, Michael
addition with Cy 5.0–maleimide: To the previous mixture, a
solution of Cy 5.0–maleimide 22 (1.2 mg, 1.3 μmol) in ultra-
pure water (0.3 mL) was added and the resulting reaction
mixture was stirred at rt for 2 h. The reaction was checked for
completion by RP-HPLC (system K) and ESI-MS; LRMS (ESI+):
m/z 1014.84 [M + 3H]3+, 1521.28 [M + 2H]2+, calcd for
5 A. P. Frei, O.-Y. Jeon, S. Kilcher, H. Moest, L. M. Henning,
C. Jost, A. Pluckthun, J. Mercer, R. Aebersold,
E. M. Carreira and B. Wollscheid, Nat. Biotechnol., 2012, 30,
997.
6 G. Clavé, H. Volland, M. Flaender, D. Gasparutto,
A. Romieu and P.-Y. Renard, Org. Biomol. Chem., 2010, 8,
4329.
7 D. M. Beal, V. E. Albrow, G. Burslem, L. Hitchen,
C. Fernandes, C. Lapthorn, L. R. Roberts, M. D. Selby and
L. H. Jones, Org. Biomol. Chem., 2012, 10, 548.
8 J. Morales-Sanfrutos, F. J. Lopez-Jaramillo, F. Hernandez-
Mateo and F. Santoyo-Gonzalez, J. Org. Chem., 2010, 75,
4039.
9 For comprehensive reviews about bioorthogonal chemistry,
see: (a) M. D. Best, Biochemistry, 2009, 48, 6571;
(b) E. M. Sletten and C. R. Bertozzi, Angew. Chem., Int. Ed.,
2009, 48, 6974; (c) J. C. Jewett and C. R. Bertozzi, Chem. Soc.
Rev., 2010, 39, 1272; (d) R. K. V. Lim and Q. Lin, Chem.
Commun., 2010, 46, 1589.
C141H201N35O35S3 3040.42. Finally, CuAAC reaction with Cy
7.0–alkyne. To 100 μL of the previous mixture, a solution of Cy
7.0–alkyne 12 (0.5 mg, 0.6 μmol) in ultrapure water (40 μL),
tBuOH (100 μL) and Cu(0) microsized powder (5 equiv.) were
added. The resulting reaction mixture was stirred at rt for 12 h.
The reaction was checked for completion by RP-HPLC (system
K) and then quenched by addition of aq. TFA 0.1% (2 mL). The
mixture was purified by RP-HPLC (system L, 2 injections). The
product-containing fractions were lyophilised to give the FRET 10 (a) A. Angelini, P. Diderich, J. Morales-Sanfrutos,
cascade (Cy 5.0–Cy 7.0) labelled peptide 23 as a dark green
amorphous powder (0.19 mg, yield 28%). HPLC (system K): tR
= 24.0 min (purity >95%); LRMS (ESI−): m/z 1281.24 [M − 3H]3
−, 1921.25 [M − 2H]2−, calcd for C183H252N38O44S5: 3845.73;
λmax (H2O) nm 649 (ε/dm3 mol−1 cm−1 250 000) and 758 (ε/
dm3 mol−1 cm−1 200 000); λmax em (PBS) nm 669 and 773.
S. Thurnheer, D. Hacker, L. Menin and C. Heinis, Bioconju-
gate Chem., 2012, 23, 1856; (b) M. B. Baker, I. Ghiviriga and
R. K. Castellano, Chem. Sci., 2012, 3, 1095;
(c) L. E. J. Smeenk, N. Dailly, H. Hiemstra, J. H. van Maar-
seveen and P. Timmerman, Org. Lett., 2012, 14, 1194;
(d) A. Welle, F. Billard and J. Marchand-Brynaert, Synthesis,
2012, 44, 2249.
11 S. Foillard, M. Ohsten Rasmussen, J. Razkin, D. Boturyn
and P. Dumy, J. Org. Chem., 2008, 73, 983.
12 A. L. Handlon and N. J. Oppenheimer, Pharm. Res., 1988, 5,
297.
Acknowledgements
Financial support from FEDER (TRIPODE, no 33883) for a
postdoctoral fellowship to G.V., the CRUNCh program (CPER 13 R. Roy, S. Hohng and T. Ha, Nat. Methods, 2008, 5, 507.
2007–2013), l’Agence Nationale de la Recherche (Programme 14 S. Milles, C. Koehler, Y. Gambin, A. A. Deniz and
PIRIBio 2009, ANR “CLICKMASSLINK”) for a postdoctoral fel-
E. A. Lemke, Mol. BioSyst., 2012, 8, 2531.
lowship to S.D. and CNRS for the Ph.D. grant of N.M. are 15 For recent examples, see: (a) I. H. Stein, C. Steinhauer and
greatly acknowledged. We thank Dr Didier Gasparutto (LAN/
SCIB/INAC, CEA-Grenoble) for the gift of ODN–alkyne 17 and
Dr Guillaume Clavé for helpful discussions about bioconjuga-
tion reactions in the context of “tripods”.
P. Tinnefeld, J. Am. Chem. Soc., 2011, 133, 4193;
(b) A. Altevogt, R. Flehr, S. Gehne, M. U. Kumke and
W. Bannwarth, Helv. Chim. Acta, 2012, 95, 543.
16 R. B. Mujumdar, L. A. Ernst, S. R. Mujumdar, C. J. Lewis
and A. S. Waggoner, Bioconjugate Chem., 1993, 4, 105.
17 (a) C. M. S. Yau, S. I. Pascu, S. A. Odom, J. E. Warren,
E. J. F. Klotz, M. J. Frampton, C. C. Williams,
V. Coropceanu, M. K. Kuimova, D. Phillips, S. Barlow,
J.-L. Bredas, S. R. Marder, V. Millar and H. L. Anderson,
Chem. Commun., 2008, 2897; (b) N. Karton-Lifshin,
L. Albertazzi, M. Bendikov, P. S. Baran and D. Shabat,
J. Am. Chem. Soc., 2012, 134, 20412.
Notes and references
1 D. M. Beal and L. H. Jones, Angew. Chem., Int. Ed., 2012, 51,
6320.
2 For the most relevant examples, see: (a) M. Galibert,
P. Dumy and D. Boturyn, Angew. Chem., Int. Ed., 2009, 48,
2576; (b) P. Kele, G. Mezo, D. Achatz and O. S. Wolfbeis, 18 The aggregation propensity of non-symmetrical Cy 7.0 in
Angew. Chem., Int. Ed., 2009, 48, 344; (c) R. Mhidia, water was highlighted by us but never published.
A. Vallin, N. Ollivier, A. Blanpain, G. Shi, R. Christiano, 19 S. Kumar, C.-Y. Chen and C. W. Fuller, Synthesis and spec-
L. Johannes and O. Melnyk, Bioconjugate Chem., 2010, 21,
219.
3 M. Galibert, O. Renaudet, P. Dumy and D. Boturyn, Angew.
Chem., Int. Ed., 2011, 123, 1941.
4 G. T. Hermanson, in Bioconjugate Techniques, Academic
Press, London, Editon edn., 2008, p. 337.
troscopic analysis of energy transfer dye-labeled nucleotides
and oligonucleotides for multiplex applications, S.G. Panda-
lai, 2004.
20 (a) E. A. Jares-Erijman and T. M. Jovin, Curr. Opin. Chem.
Biol., 2006, 10, 409; (b) T. Ueno and T. Nagano, Nat.
Methods, 2011, 8, 642.
2704 | Org. Biomol. Chem., 2013, 11, 2693–2705
This journal is © The Royal Society of Chemistry 2013

View Article Online
Organic & Biomolecular Chemistry
Paper
21 For recent reviews, see: (a) K. Lu, Q.-P. Duan, L. Ma and
D.-X. Zhao, Bioconjugate Chem., 2010, 21, 187;
(b) A. D. Baldwin and K. L. Kiick, Bioconjugate Chem., 2011,
22, 1946.
(b) R. L. Juliano, X. Ming and O. Nakagawa, Acc. Chem. Res., 29 W. R. Algar, D. E. Prasuhn, M. H. Stewart, T. L. Jennings,
2012, 45, 1067.
J. B. Blanco-Canosa, P. E. Dawson and I. L. Medintz,
22 P. Järver, T. Coursindel, S. EL Andaloussi, C. Godfrey,
Bioconjugate Chem., 2011, 22, 825.
M. J. A. Wood and M. J. Gait, Mol. Ther. Nucleic Acids, 2012, 30 (a) M. L. Blackman, M. Royzen and J. M. Fox, J. Am. Chem.
1, e27.
Soc., 2008, 130, 13518; (b) N. K. Devaraj and R. Weissleder,
Acc. Chem. Res., 2011, 44, 816; (c) J. D. Thomas, H. Cui,
P. J. North, T. Hofer, C. Rader and T. R. Burke, Bioconjugate
Chem., 2012, 23, 2007.
23 O. P. Edupuganti, Y. Singh, E. Defrancq and P. Dumy,
Chem.–Eur. J., 2004, 10, 5988.
24 N. Maindron, S. Poupart, M. Hamon, J.-B. Langlois, N. Plé,
L. Jean, A. Romieu and P.-Y. Renard, Org. Biomol. Chem., 31 (a) H. Stockmann, A. A. Neves, S. Stairs, K. M. Brindle and
2011, 9, 2357.
F. J. Leeper, Org. Biomol. Chem., 2011, 9, 7303; (b) C. Sabot,
E. Oueis, X. Brune and P.-Y. Renard, Chem. Commun., 2012,
48, 768.
25 For a comprehensive review about the synthesis of POCs
through site-specific bioconjugation, see: Y. Singh,
P. Murat and E. Defrancq, Chem. Soc. Rev., 2010, 39, 2054.
26 T. R. Chan, R. Hilgraf, K. B. Sharpless and V. V. Fokin,
Org. Lett., 2004, 6, 2853.
27 For a recent minireview about bioconjugation using thiols,
see: M. H. Stenzel, ACS Macro Lett., 2013, 2, 14.
28 The main drawbacks associated with the use of malemide-
thiol adducts in bioconjugation are poor hydrolytic stability
32 G. B. Fields and R. L. Noble, Int. J. Pept. Protein Res., 1990,
35, 161.
33 T. Priem, C. Bouteiller, D. Camporese, A. Romieu and
P.-Y. Renard, Org. Biomol. Chem., 2012, 10, 1068.
34 G. R. Fulmer, A. J. M. Miller, N. H. Sherden, H. E. Gottlieb,
A. Nudelman, B. M. Stoltz, J. E. Bercaw and K. I. Goldberg,
Organometallics, 2010, 29, 2176.
and propensity for retro and exchange reactions in redu- 35 N. Fujimoto, M. Matsumura, I. Azumaya, S. Nishiyama,
cing biological environments, see (a) J. Kalia and
R. T. Raines, Bioorg. Med. Chem. Lett., 2007, 17, 6286;
H. Masu, H. Kagechika and A. Tanatani, Chem. Commun.,
2012, 48, 4809.
This journal is © The Royal Society of Chemistry 2013
Org. Biomol. Chem., 2013, 11, 2693–2705 | 2705