A novel heterotrifunctional peptide-based cross-linking reagent for facile access to bioconjugates. Applications to peptide fluorescent labelling and immobilisation

Article abstract of DOI:10.1039/b807263a

A convenient, versatile and straightforward synthesis of a novel heterotrifunctional peptide-based linker molecule is described. This generic bio-labelling reagent contains an amine-reactive N-hydroxysuccinimidyl carbamate moiety, an aldehyde/ketone-reactive aminooxy group and a thiol group with a propensity to form urea, oxime and thioether linkages respectively. The full chemical orthogonality between the free aminooxy and thiol functionalities was demonstrated through the preparation of a fluorescent reagent suitable for the selective staining of a carboxaldehyde-modified surface by means of oxime ligation. The absence of reactivity of these two functions toward the nucleophile-sensitive active carbamate was obtained by using temporary aminooxy- and thiol-protecting groups removable under mild conditions. The utility of the linker molecule to cross-link three different molecular partners has been illustrated by the preparation of fluorescent tripod-functionalised surfaces which may be useful in developing new peptide microarrays and related immunosensors.

Full text of DOI:10.1039/b807263a

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
A novel heterotrifunctional peptide-based cross-linking reagent for facile
access to bioconjugates. Applications to peptide fluorescent labelling and
immobilisation†‡
Guillaume Clave´,a,b Herve´ Boutal,c Antoine Hoang,d Franc¸ois Perraut,e Herve´ Volland,c
Pierre-Yves Renard*a,b, f and Anthony Romieu*a,b
Received 29th April 2008, Accepted 2nd June 2008
First published as an Advance Article on the web 30th June 2008
DOI: 10.1039/b807263a
A convenient, versatile and straightforward synthesis of a novel heterotrifunctional peptide-based
linker molecule is described. This generic bio-labelling reagent contains an amine-reactive
N-hydroxysuccinimidyl carbamate moiety, an aldehyde/ketone-reactive aminooxy group and a thiol
group with a propensity to form urea, oxime and thioether linkages respectively. The full chemical
orthogonality between the free aminooxy and thiol functionalities was demonstrated through the
preparation of a fluorescent reagent suitable for the selective staining of a carboxaldehyde-modified
surface by means of oxime ligation. The absence of reactivity of these two functions toward the
nucleophile-sensitive active carbamate was obtained by using temporary aminooxy- and
thiol-protecting groups removable under mild conditions. The utility of the linker molecule to cross-link
three different molecular partners has been illustrated by the preparation of fluorescent
tripod-functionalised surfaces which may be useful in developing new peptide microarrays and related
immunosensors.
The conjugation of two or more (bio)molecules is most fre-
Introduction
quently achieved through two sequential steps: (1) the incorpo-
Recent advances in the field of bioconjugate chemistry have
ration of mutually reactive groups into the individual molecular
spurred the construction of novel engineered molecules based
components, and (2) their bio-orthogonal coupling in solution
on chemoselective coupling reactions either between two (or
leading to the formation of a stable chemical linkage such as
more) different biopolymers or between a biopolymer and various
chemical reporter groups. As the resulting bioconjugates possess
carboxamide, disulfide, hydrazone, oxime, thioether, carbo- or
heterocycle (i.e., Diels–Alder cycloadducts, thiazolidine or triazole
the combined and unaltered properties of their individual compo-
moieties). The use of a heterobifunctional cross-linking reagent
nents, bioconjugation appears to be the best approach to introduce
is often required for the implementation of such biocompatible
a high level of chemical diversity within complex biopolymers and
synthetic strategies. Indeed, numerous commercially available
provide sophisticated chemically engineered biomolecular tools
heterobifunctional coupling agents enable the conversion of free
essential for various applications in diagnostics, therapeutics and
related biomedical fields.1
amino (or sulfhydryl) groups of a biopolymer into carboxamides
(or thioethers) bearing various terminal reactive moieties, includ-
ing activated mixed disulfide, azido, carboxaldehyde, iodoacetyl,
maleimide and succinimidyl esters. Thereafter, the resulting adduct
aEquipe de Chimie Bio-Organique, COBRA – CNRS UMR 6014, rue
is conjugated to other (bio)molecular components through a
Lucien Tesnie`re, 76131, Mont-Saint-Aignan, France. E-mail: pierre-
second chemoselective reaction to give the targeted bioconjugate.
However, such a strategy is not suitable for the preparation of
bioconjugates resulting from the covalent association of three
different molecular partners. In this case, the use of a cross-
linking reagent bearing three different orthogonal reactive groups
is thus required. These trifunctional building blocks are key
components in the preparation of molecular tools for applications
in proteomics and genomics. As illustrative examples, one can
mention: the activity-based probes for the identification of enzyme
activities from complex proteomes;2 the energy transfer termina-
tors (i.e., dideoxynucleotides labelled with FRET cassettes) for
DNA sequencing;3 and the biopolymer microarrays currently used
for the rapid analysis of various biological events.4 However,
only few cross-linkers equipped with a triply orthogonal set
of functional groups have been reported to date. Some het-
erotrifunctional reagents suitable for bio-labelling applications
yves.renard@univ-rouen.fr, anthony.romieu@univ-rouen.fr; Fax: +33 2-35-
52–29-59
bUniversite´ de Rouen, Place Emile Blondel, 76821, Mont-Saint-Aignan,
France
cCEA, iBiTecS, Laboratoire d’Etudes et de Recherches en Immuno-analyse,
Gif sur Yvette, F-91191, France
dLaboratoire de Fonctionnalisation Chimique de Microcomposants, Com-
missariat a` l’Energie Atomique, 17 rue des Martyrs, F-38054, Grenoble,
France
eLaboratoire d’Imagerie et des Syste`mes d’Acquisition, Commissariat a`
l’Energie Atomique, 17 rue des Martyrs, F-38054, Grenoble, France
f
Institut Universitaire de France
† Such heterotrifunctional cross-linking reagents were covered by a patent
entitled “Re´actif pseudo peptidique trifonctionnel, ses utilisations et
applications” filed on 11th July 2007 at the European Patent Office:
G. Clave´, P.-Y. Renard, A. Romieu and H. Volland, no. 07/0518.
‡ Electronic supplementary information (ESI) available: Detailed synthetic
procedures for compounds 4, 7–13 and A; characterisation data for
compounds A, B, C, 5, 19 and 23. See DOI: 10.1039/b807263a
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are commercially available from Pierce (e.g., Sulfo-SBED biotin
label reagent 1, see Fig. 1 for the corresponding structure)
but one of the three available functionalities found in these
trifunctional reagents is always the biotin moiety, which is used
as an affinity probe (through its strong interaction with avidin,
neutravidin or streptavidin protein) and not as a reactive group
for subsequent reactions with a biopolymer or a reporter group.5
Recently, Watzke et al. reported the design and synthesis of an
aromatic building block 2 for C- and N-terminal protein labelling
and protein immobilisation.6 The presence of an azido group,
an S-protected cysteine residue and a carboxylic acid onto the
benzene ring, enable both the fluorescent labelling of proteins and
their subsequent immobilisation on a phosphane-functionalised
surface by means of the Staudinger ligation. However, the full
orthogonality between the three functional groups was not clearly
demonstrated, and no example of sequential triple derivatisa-
tion of reagent 2 with a fluorescent reporter group, a protein
and finally a phosphane-modified surface has been reported to
date.
architecture (i.e., lysine–cysteine) that contains an amine-reactive
N-hydroxysuccinimidyl carbamate, an aldehyde/ketone-reactive
aminooxy group,7 a thiol group and an hydrophilic pseudo-PEG
spacer (Fig. 1). In this paper, we describe the general synthesis of
a representative heterotrifunctional cross-linking reagent 3, used
in a protected form 5, and to illustrate its efficacy in coupling a
representative biomolecule (i.e., a neuropeptide) to two different
molecular partners (namely, a chemical reporter group and a
solid phase). The first targeted application is thus the detection of
neuropetide substance P (SP) through the original immunoassay
SPIT-FRI (for Solid-Phase Immobilised Tripod for Fluorescent
Renewable Immunoassay) recently developed by us.8
Results and discussion
Considerations for the design and synthesis of heterotrifunctional
cross-linking reagents suitable for bio-labelling applications
The two obvious synthetic strategies to obtain heterotrifuntional
linkers consist in derivatising either a triply substituted ben-
zene (the strategy used by Watzke et al.) or a functionalized
amino acid (e.g., aspartic acid, lysine),9 readily available from
commercial sources. As we suspect that an aromatic core could
With the goal in mind to develop a universal, versatile
and ready-to-use bio-labelling reagent, compatible with fragile
biopolymers, we have explored the chemistry of a new family
of heterotrifunctional linker molecules based on a dipeptidyl
Fig. 1 Structures of Sulfo-SBED biotin label reagent 1, heterotrifunctional cross-linker 2 developed by Watzke et al.6 and peptide-based cross-linking
reagents 3–5 studied in this work.
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impede some of the physicochemical properties of the resulting
bioconjugates (e.g., fluorescence properties), we decided to use
a pseudo-peptidyl architecture as the scaffold of our tripodal
systems. The choice of its constituting amino acids was guided by
the following requirements: (1) their reactive side-chains should
be easily and selectively converted into bioconjugatable groups,
preferably through standard peptide coupling reactions, (2) the
corresponding heterotrifunctional reagents must display good
water solubility properties achieved by the presence of hydrophilic
moieties within their core structures and/or side-chains, and (3)
the synthetic accessibility of the suitably protected building blocks
must be as easy as possible, especially for the non-natural amino
acids. Thus, we have selected L-cysteine, L-lysine and an original
amino-PEG-acid spacer. A key parameter in the design of our
heterotrifunctional linker lies in its shape and length versatility:
the chosen hydrophilic pseudo-PEG spacer can be introduced
between any of the functional groups (and two spacers can be
introduced at different positions), and its length can be easily
adapted to any targeted use for the linker. Moreover, in order to
have a flexible and easy synthetic access to the heterotrifunctional
linker, we chose a highly convergent and versatile synthetic strategy
(Fig. 2), based on fully protected building blocks and well-
established solution-phase peptide coupling reactions. Concerning
the choice of the three bioconjugatable groups, we have focused
on the well-known bioconjugate chemistry of the aminooxy and
thiol groups.10 The aminooxy group was chosen since we have
already experimented in its use in biomolecule immobilisation on
the solid phase,11,12 and since we suspected (and proved in this
study, vide infra) that its particular “super-nucleophile” character
would allow an orthogonal reactivity compared with the sulfhydryl
group. We also took advantage of the original and unprecedented
“wet chemistry” of the N-hydroxysuccinimidyl carbamate moiety,
whose potential in the construction of biconjugates (through the
formation of urea linkages) has never been reported,13 although
the activated carbamates are more stable than their parent
activated esters. The sequential derivatisation of the tri-orthogonal
set of functional groups of on reagent 3 should enable the covalent
association of three different (bio and/or macro)molecules in a
highly efficient manner respectively through chemoselective N-
acylation reaction (under Schotten–Bauman conditions), Michael
addition (or SN2 reaction) and oxime ligation. Despite the full
orthogonality between the aminooxy and thiol groups (vide infra),
these bioconjugatable moieties were kept protected subsequent
to their introduction onto the peptide scaffold, to avoid self-
degradation of the heterotrifunctional reagent through side re-
actions with the reactive electrophilic carbon center of the third
functional group. It thus appeared essential to use aminooxy-
and thiol-protecting groups removable under mild conditions
(aqueous buffers, pH 6–9 and room temperature are preferred)
prior to their derivatization in order to keep the integrity of
the (bio)molecules previously anchored onto the trifunctional
peptide-based linker. The aminooxy phthalimide protection and
the sulfhydryl protection as mixed ethyl disulfide (SEt)14 were used
for the temporary masking of these nucleophilic moieties, and so
Fig. 2 Building block approach to the solution-phase synthesis of heterotrifunctional cross-linking reagent 5.
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the synthesis of fully protected derivative 5 was achieved§. As
illustrated in Fig. 2, the use of three functionalised amino acid
building blocks A–C enabled us to develop a highly convergent
synthetic strategy based on solution-phase peptide couplings,
suitable for the generation of a large set of heterotrifunctional
peptide based-linkers derived from 5. Indeed, the re-use of some
building blocks avoided having to start the synthesis from the
beginning with the commercially available lysine and cysteine
derivatives, and additional joining of a pseudo-PEG linker can
be achieved on any of the building blocks.
Synthesis of building blocks A–C used for the preparation of
heterotrifunctional cross-linking reagents
Synthesis of Boc-protected amino-PEG-acid spacer A. The
PEG-based building block was introduced within the peptide scaf-
fold both as a flexible spacer between the anchored biomolecule
and the two other molecular components of the resulting biocon-
jugate, and in order to increase reagent solubility in water and
related aqueous buffers. Linker A was synthesised in 29% overall
yield through a 7-step synthetic procedure depicted in Scheme 1.
Commercially available 2-(2-(2-chloroethoxy)ethoxy)ethanol 6
was allowed to react with sodium azide in the presence of sodium
iodide as a catalyst, under refluxing ethanol, producing azido
compound 7 in quantitative yield. This alcohol was subjected
to Jones’ oxidation to produce carboxylic acid 8. Conversion
of the azido group into primary amine was achieved by Pd/C
catalytic hydrogenation. Two units of the resulting amino acid
9 were assembled by using standard peptide chemistry. The Boc
urethane and the methyl ester were chosen respectively as N- and
C-terminal protecting functions, and BOP phosphonium salt in
the presence of DIEA was used as coupling reagent.15 As the final
step, the methyl ester was removed by short treatment of the fully
protected pseudo-PEG linker with 1 M LiOH in H2O–MeOH, to
give the target building block A. All spectroscopic data (see ESI‡),
in particular NMR and mass spectrometry, were in agreement
with the structure assigned.
Scheme 1 Reagents and conditions: a) NaN3 (1.2 equiv), NaI (0.1 equiv),
EtOH, reflux, 5 days, quant. yield; b) Jones’ reagent (3 equiv), ace-
tone, 4 ◦C to rt, 1 h, 91%; c) 10% Pd/C, H2, EtOH, 12 h, quant.
yield; d) Boc2O (1.1 equiv), 2 M aq. NaOH, THF, 4 ◦C to rt, 63%.
e) 2,2-dimethoxypropane, 37% HCl, 1 h, 79%. f) BOP (1 equiv), DIEA
(3 equiv), CH3CN, overnight; g) 1 M aq. LiOH, MeOH, rt, 51% (overall
yield for steps f–g). DIEA = N,N-diisopropylethylamine, Boc2O =
tert-butyl dicarbonate, BOP = ((benzotriazole-1-yl)oxy)tris(dimethyl-
amino)phosphonium hexafluorophosphate.
derivatisation of reagent 5. In this context, we first explored the
use of carbamate-type protecting groups removable under mild
reducing conditions (i.e., azidomethyloxycarbonyl (Azoc),18 2-
pyridyldithioethyloxycarbonyl (Pydec)19), but the corresponding
N-protected Aoaa derivatives were found to be too unstable to
be easily handled. Thus, we turned our attention to the use of
the hydrazine-labile phthalimide protecting group (Pht), which
represents a good compromise between stability and lability. The
phthaloyl protection of this a-nucleophilic moiety has already been
successfully used in the field of bioconjugate chemistry especially
for the solid-supported synthesis of oligomeric bioconjugates and
for the surface immobilisation of biopolymers through oxime
ligation.20 Furthermore, in contrast to the single carbamate-based
protecting groups (i.e., Aloc or Boc), the phthalimide moiety
ensures complete protection of the nitrogen of aminooxyacetic
acid, therefore preventing the N-overacylation side-reaction fre-
quently encountered during aminooxy peptide synthesis.21,22 To
incorporate the Aoaa moiety within the dipeptidyl architecture
of our heterotrifunctional reagent, we designed building block B,
which corresponds to Na-Boc-L-lysine acylated by Pht-Aoaa-OH
12 (Scheme 2). This N-phthaloyl building block was activated as
the hydroxybenzotriazole (OBt) ester and subsequently coupled
to the free e-amino group of lysine. This pre-activation procedure
avoided an additionnal protection step of the lysine carboxylic acid
function. Pht-Aoaa-OH was converted into the corresponding
OBt esters by treatment with DCC–HOBt in CH3CN–DMF
Synthesis of aminooxy-containing lysine building block B. The
introduction of the super-nucleophile aminooxy moiety within
synthetic biopolymers (especially synthetic peptides) is often
achieved through coupling reaction between a free amino group
on the target (bio)macromolecule and a N-protected derivative
of aminooxy acetic acid (Aoaa).16 Carbamate protecting groups
such as Aloc or Boc are often used for amine protection of
Aoaa.17 However, their removal conditions, under Pd(0) catalysis
and harsh acidic conditions respectively, are not compatible with
the stability of the bioconjugates targeted through the sequential
§ Synthesis of N-Fmoc aminooxy heterotrifunctional cross-linking reagent
4 was also achieved (see ESI‡). Preliminary bio-derivatisation attempts
were performed with this Fmoc derivative, especially through acylation
reactions of its active carbamate moiety with various amine-containing
peptides, but the resulting conjugates were found to be highly hydrophobic.
Thus, the purification and handling of such mono derivatised reagents,
especially in aqueous buffers, are not trivial tasks. Furthermore, the
premature cleavage of the Fmoc group of reagent 4 was observed
during some bioconjugation reactions performed in alkaline buffers.
Consequently, in the present article, we have focused on the derivatisation
of the phthalimide derivative 5, but bioconjugate applications of 4 could be
envisaged, especially those involving more hydrophilic biopolymers such
as nucleic acids.
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Scheme 2 Reagents and conditions: a) DCC (1.2 equiv), HOBt·H2O
(1.2 equiv), CH3CN–DMF (1 : 1, v/v), rt, 2 h; b) Boc-Lys-OH (1 equiv),
DMF, rt, 2 h, quant. yield. DCC = N,Nꢀ-dicyclohexylcarbodiimide,
HOBt·H2O = hydroxybenzotriazole monohydrate.
Scheme 3 Reagents and conditions: a) isobutyl chloroformate (1 equiv),
NMM (1 equiv), ethyl acetate, −15 ◦C, 10 min then 20% aq. NH3 (3 equiv),
4 ◦C, 30 min, quant. yield; b) TFA–H2O (95 : 5, v/v), 4 ◦C to rt, 1 h, quant.
yield. NMM = N-methylmorpholine, TFA = trifluoroacetic acid.
(1 : 1).23 This crude mixture of active esters was directly reacted
with Boc-Lys-OH to provide the corresponding building blockB in
quantitative yield. It is important to note that during the course of
our work, Dumy et al. reported the use of 1-ethoxyethylidene (Eei)-
protected Aoaa for the synthesis of aminooxy peptides suitable
for oxime ligation.22,24 The Eei protecting group prevents N-
overacylation and can be removed under mild acidic conditions
(i.e., aqueous solution of 1% or 5% TFA in CH3CN). Thus, the use
of this Aoaa building block in the synthesis of heterotrifunctional
reagent such as 5 could also have been considered, but would have
required the choice of an alternative protection scheme for the
three building blocks involved in the synthesis of 5 (e.g., Fmoc
strategy).
Synthesis of N-phthaloyl aminooxy heterotrifunctional
cross-linking reagent 5 using amino acid building blocks
Heterotrifunctional reagent 5 was synthesised as shown in
Scheme 4. Due to the availability of the three amino acid building
blocks A, B and C, a highly convergent solution-phase synthesis
from S-protected amino acid H-Cys(SEt)-NH2 was developed.
Firstly, the lysine building block B was coupled to S-ethylthio
cysteine carboxamide C in the presence of BOP–DIEA, and the
resulting fully protected dipeptide was treated with a 12% TFA
solution in CH2Cl2 to give building block D in quantitative yield.
This latter pseudo-dipeptide was then subjected to the same two-
step reaction sequence: BOP-mediated coupling with building
block A to obtain the fully protected trifunctional pseudo-peptide
15, and subsequent Boc removal to give the free N-terminal PEG-
peptide 16 in 35% overall yield for the two steps. Conversion of
the primary amino group into N-hydroxysuccinimidyl carbamate
was performed with N,Nꢀ-disuccinimidyl carbonate (DSC) and
TEA in dry DMF.27 Purification was achieved by reverse-phase
HPLC to give the fully protected heterotrifunctional reagent 5
in quantitative yield. For this chromatographic purification, the
use of an acidic mobile phase (i.e., aq. TFA, 0.1%) was found
to be essential to prevent premature hydrolysis of the active
carbamate moiety. All spectroscopic data, especially NMR and
mass spectrometry, were in agreement with the structure assigned
Synthesis of cysteine building block C. In our first attempts
to prepare trifunctional-bioconjugable cross-linking reagents, we
investigated the synthesis of a pseudo-dipeptide (i.e., lysine–
lysine) containing the hydrophilic pseudo-PEG spacer derived
from building block A, the amine-reactive N-hydroxysuccinimidyl
carbamate, the Aoaa moiety and a thiol-reactive maleimide group.
The corresponding heterotrifunctional reagent 13 was obtained
but the yield was impaired by the poor stability exhibited by the
maleimide group during the chemical assembly process of building
blocks. Indeed, the propensity of the imido group of maleimides
to undergo spontaneous hydrolysis or related nucleophilic ring-
opening reactions has already been reported in the literature25 and
was observed by us during the coupling reaction between Boc-
protected amino-PEG-acid spacer A and the two lysine building
blocks.
In this context, we decided to use a thiol group as the third
bioconjugatable reactive group of our reagents. The commercial
availability of N,S-diprotected L-cysteine derivatives has enabled
the obtaining of the required building block C in only two synthetic
steps. The mixed ethyl disulfide (SEt) was used as thiol protection
moiety because of its high stability under acidic and basic
conditions and its lability under mild reducing conditions fully
compatible with sophisticated bioconjugation schemes involving
fragile biopolymers. Furthermore, in contrast to the correspond-
ing aryl derivatives (e.g., 3-nitro-2-pyridylthio, Npys), this mixed
disulfide is not prone to hydrolytic cleavage under the alkaline
conditions (i.e., pH 8–9) currently used in some bioconjugation
reactions. As depicted in Scheme 3, the synthesis of building block
C started with the conversion of the free carboxylic acid function
of Boc-Cys(SEt)-OH into the non-reactive carboxamide moiety
by treatment of the pre-formed isobutyloxy mixed anhydride
with aqueous ammonia.26 Finally, the Boc group on carboxamide
14 was removed by treatment with trifluoroacetic acid to yield
building block C as a TFA salt in quantitative yield.
◦
(see ESI‡). Interestingly, this reagent could be stored at −20 C
for several months without detectable degradation.
Bioconjugation applications of fully protected heterotrifuntional
cross-linking reagent 5
To demonstrate the potential utility of reagent 3 in the field of
bioconjugate chemistry, the development of a reliable protocol
enabling the sequential and chemoselective derivatization of the
three reactive groups with three different (bio)molecular partners
was achieved.
Firstly, we have studied both the chemical compatibility between
the free aminooxy and thiol functions and the orthogonality
of their respective protecting groups. As mentioned in the
introduction, a possible application of our heterotrifunctional
reagent concerns biochips and biosensors and thus immobilisation
and visualisation of biopolymers on solid surfaces.28 In order to
validate the immobilisation step, we planned the synthesis of the
fluorescent aminooxy reagent 19 bearing a rhodamine 6G label
and suitable for immobilisation on a aldehyde-functionalised sur-
face by means of oxime ligation (Fig. 3A).29 To avoid interferences
with the active carbamate, N-Boc-protected derivative 15 was
employed in the corresponding derivatisation reactions. As the
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Scheme 4 Reagents and conditions: a) BOP (1.1 equiv), DIEA (3.1 equiv), CH3CN–DMF (1 : 1, v/v), rt, 3 h, quant. yield; b) 12% TFA, CH2Cl2, 4 ◦C to
rt, 3 h, quant. yield; c) building block A (1 equiv), BOP (1.1 equiv), DIEA (3 equiv), CH3CN–DMF (1 : 1, v/v), rt, 3 h, 45% after RP-HPLC purification;
d) 7.5% TFA, CH2Cl2, 4 ◦C to rt, 1 h, 78%; e) DSC (2.5 equiv), TEA (2.5 equiv), DMF, 2 h, quant. yield after RP-HPLC purification.
R
iodoacetyl derivatives are known to exhibit excellent selective reac-
tivity toward sulfhydryl groups especially when the corresponding
SN2 alkylation reaction is performed in aqueous media at slightly
alkaline pH,30 we have decided to perform a fluorescent labelling
reaction between a iodoacetamide-based fluorescent reagent and
the pseudo-peptide 17 bearing free aminooxy and thiol groups
(Scheme 5).
532 and Fluo Probeꢀ 532 labels. The thiol-reactive derivative
18 was prepared by using the three-step synthetic procedure
reported by Bouteiller et al. for sulfocyanine dyes.32 The HPLC
elution profile of the reaction mixture has clearly shown the
absence of the doubly labelled pseudo-peptide resulting from
a competitive non-selective anchoring of rhodamine dye onto
the aminooxy and thiol functions.33 The moderate 28% isolated
yield is explained by significant losses of fluorescent conjugate
during the chromatographic purification. For the immobilisation
of the fluorescent reactive label 19 by means of oxime ligation,
a silica surface had to be decorated with an appropriately
functionalised carboxaldehyde. The introduction of these required
aldehyde functions onto solid supports was achieved by using
original surface chemistry developed by us.11,34 For the spotting
experiments, the aminooxy reagent 19 (positive control) as well
as the non-reactive oxime derivative 20 (obtained by mixing 19
with acetone) as a negative control were dissolved in deionised
water at 50 lM. Subsequent to the manual spotting process, the
slides were incubated in a humid atmosphere for 15 min. After
removal of excess reagent and some washings, the fluorescent
Firstly, removal of the phthaloyl and SEt protecting groups
were achieved successively under standard conditions: treatment
with 1 equiv of hydrazine monohydrate in MeOH and treatment
with an excess of dithiothreitol (DTT) in a mixture of NMP
and aqueous sodium bicarbonate buffer (pH 8.5) respectively.
The resulting N-Boc-protected pseudo-peptide 17 was isolated
by reverse-phase HPLC in a satisfactory yield (overall yield for
the two steps: 45%). The iodoacetamide derivative of rhodamine
dye R6G-WS 18 was attached to the free cysteine of the pseudo-
peptide 17 through a SN2 reaction performed in aqueous sodium
bicarbonate buffer (pH 8.5). R6G-WS is a water-soluble analogue
of rhodamine 6G recently developed by us,31 whose spectral
R
properties are similar to commercially available Alexa Fluorꢀ
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Fig. 3 Silica surface staining with the fluorescently labelled aminooxy reagent 19. A) Principle of immobilisation by oxime ligation. B) Results.
Scheme 5 Reagents and conditions: a) Hydrazine monohydrate (1 equiv), MeOH, rt, 1 h, 63% after RP-HPLC purification; b) DTT (30 equiv), 0.1 M aq.
NaHCO3 buffer (pH 8.5), NMP, rt, 3 h, 71% after RP-HPLC purification; c) thiol-reactive fluorophore 18 (1 equiv), 0.1 M aq. NaHCO3 buffer (pH 8.5),
rt, 1 h, 28%.
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signals were recorded and quantified. The obtained results are
shown in Fig. 3B. The immobilisation of 19 gave clear fluorescent
signals, whereas poor signals were obtained for the negative
control. These results demonstrate that the immobilisation of the
aminooxy pseudo-peptide 19 onto the aldehyde-modified silica
surface was successful.
Furthermore, the full chemical orthogonality between free
aminooxy and thiol functions within the same molecule was
demonstrated for the first time, and this should be a valuable
tool for the design of new bioconjugation strategies.
ronments has already been validated by several methods including
immunoassays.36 SPIT-FRI detection of this neuropeptide has
already been achieved using a peptide construction derived from
SP, bearing a fluorescent label and immobilised on solid surface
through the neutravidin–biotin interaction.8 Such a modified
peptide was readily prepared by solid-phase synthesis but its non-
general structure restricted its use for the preparation of biochips
for detection of SP only. To demonstrate the general applicability
of our heterotrifunctional reagent, we wished to prepare a similar
tripod bearing the SP peptidyl sequence, a fluorescent label and
the aminooxy function for subsequent surface immobilisation by
means of oxime ligation, through a simple and sequential solution-
phase derivatisation approach. As described for the previous SPIT-
FRI experiments on SP, we worked with a validated analogue of
this neuropeptide in which all reactive amino acid residues were re-
placed by non-reactive amino acids and the C-terminal carboxylic
acid converted into carboxamide: H-Arg-Pro-Ala-Pro-Gln-Gln-
Phe-Phe-Gly-Ala-Met-NH2. This latter peptide was reacted with a
slight excess of reagent 5 in NMP in the presence of DIEA to obtain
linker-peptide 21. As expected, the acylation reaction was found
to be fast and efficient. Purification was achieved by reverse-phase
HPLC to give 21 in almost quantitative yield. It is also possible to
perform this N-terminalmodification reaction ina slightly alkaline
buffer (pH 7–8) compatible with the stability of most proteins, but
in the present case the resulting urea derivative 21 was obtained
in a lower yield. Thereafter, the Pht and SEt protecting groups
Preparation of fluorescent tripod-functionalized surfaces suitable
for the detection of neuropeptide substance P through the SPIT-FRI
immunoassay. In addition to these preliminary experiments and
to probe the ability of heterotrifunctional cross-linking reagents
such as 3 to conjugate three different molecular partners, we have
prepared fluorescent tripod functionalised surfaces suitable for
the detection of neuropeptide SP through the original SPIT-FRI
immunoassay.8 SP is a neuropeptide composed of eleven amino
acid residues with the following sequence: H-Arg-Pro-Lys-Pro-
Glu-Glu-Phe-Phe-Gly-Leu-Met-OH determined by Chang and
Leeman.35 This short peptide is both a neurotransmitter and
a neuromodulator. In the central nervous system, SP has been
associated with the regulation of mood disorders, anxiety, stress,
reinforcement, neurogenesis, respiratory rhythm, neurotoxicity,
nausea, emesis and pain. Its detection in complex biological envi-
Scheme 6 Reagents and conditions: a) H-Arg-Pro-Ala-Pro-Gln-Gln-Phe-Phe-Gly-Ala-Met-NH2 (1 equiv), DIEA (6 equiv), NMP, rt, 6 h then acetic acid
(4 equiv), 92% after RP-HPLC purification; b) hydrazine monohydrate (1 equiv), MeOH, rt, 1 h, RP-HPLC purification; c) DTT (20 equiv), 0.1 M borate
buffer (pH 8.2), NMP, rt, 2 h then acetic acid (2 equiv), RP-HPLC purification; d) FluoProbeꢀR 532A maleimide (1 equiv), DIEA (3 equiv), NMP, rt, 2 h,
RP-HPLC purification, 15% (overall yield for b + c + d). NMP = N-methylpyrrolidone.
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were cleanly removed by using the two-step synthetic procedure
previously used for the preparation of fluorescent aminooxy
reagent 19 (see Scheme 6). Finally, the site-selective fluorescent
labelling of peptide 22 was achieved by Michael addition of
the thiol group to the maleimide of a water-soluble analogue of
efficient methodology for the solution-phase synthesis of this new
class of pseudo-peptides relies on the use of three pre-synthesised
building blocks. The linker reported herein could be of general
utility to cross-couple three different molecular partners and to
obtain sophisticated (bio)conjugates that are not accessible by
derivatisation of commercially available cross-linking reagents.
Thus, it provides a valuable tool for the introduction of different
functional groups of interest into biopolymers. This has been
demonstrated in the present work by utilising the linker molecule
5 for the covalent attachment of an aminooxy function and a
fluorescence marker to the N-terminus of neuropeptide substance
P, which could be further immobilised on aldehyde-modified glass
slides via highly chemoselective oxime ligation. This new strategy
for the site-specific immobilisation and visualisation of peptides
(and proteins) onto a silica surface is a promising method that
may prove useful in the next future for the ready preparation of
new generations of peptide (or protein) microarrays suitable for
immunoassay applications.37
R
rhodamine 6G (Fluo Probeꢀ 532, FP532). The rhodamine dye–
peptide conjugate 23 was isolated in a pure form by reverse-phase
HPLC (overall yield for the three steps: 15%). Its structure was
confirmed by ESI mass spectrometry (see ESI‡). Interestingly, as
already observed for the iodoacetamide derivative 18, no further
reaction between the thiol-reactive maleimide moiety and the
aminooxy group was observed. To obtain biochips suitable for SP
detection through SPIT-FRI method, solid-phase immobilisation
of fluorescent tripod 23 was performed in the same manner as
described for the fluorescent aminooxy reagent 19. After washings,
an intense fluorescence signal was observed and proved the efficacy
of the oxime ligation to covalently graft SP related tripods to
a solid surface (Fig. 4). In a second step, FP647-labelled anti-
SP monoclonal antibodies (mAb) which are able to recognise
R
both SP and its analogue, were added. As Fluo Probeꢀ 647 acts
Experimental‡
as a quencher of FP532, a decrease of fluorescence emission of
FP532 via FRET was observed. Competition experiments using
original SP aimed at detecting and quantifying this neuropetide
are in progress, and the corresponding results will be reported in
due course. Indeed, as previously reported with the biotinylated
tripod,8 the added analyte (i.e., SP) will compete with the solid-
phase tripod for the binding to the specific antibody, further
leading to the displacement of the tripod–antibody complex and
thus to an increase of the tripod-related fluorescence proportional
to the amount of the analyte.
General
Column chromatography purifications were performed on silica
gel (40–63 lm) from SdS. TLC were carried out on Merck DC
Kieselgel 60 F-254 aluminium sheets. Compounds were visualised
by one or more of the following methods: (1) illumination with
a short wavelength UV lamp (i.e., k = 254 nm), (2) spray with
a 0.2% (w/v) ninhydrin solution in absolute ethanol, (3) spray
with a 3.5% (w/v) phosphomolybdic acid solution in absolute
ethanol. All solvents were dried following standard procedures
(CH3CN: distillation over CaH2, CH2Cl2: distillation over P2O5,
DMF: distillation over BaO). Anhydrous ethanol was obtained
by storing absolute ethanol (EtOH, VWR PROLABO, AnalaR
NORMAPUR) over anhydrous Na2SO4. Ethyl acetate (AcOEt,
˚
Riedel de Hae¨n, extra pure) was dried by storage over 4 A
molecular sieves. DIEA and TEA were distilled from CaH2 and
stored over BaO. The water-soluble analogue of rhodamine 6G
(R6G-WS) was prepared by using literature procedures.31 N-
Sulfosuccinimidyl[4-iodoacetyl]aminobenzoate (Sulfo-SIAB) and
R
FluoProbesꢀ 532A maleimide were purchased from Pierce
and Interchim respectively. Na-(tert-Butyloxycarbonyl)-L-lysine
(Boc-Lys-OH) was prepared by hydrogenolysis of commercially
available Na-(tert-butyloxycarbonyl)-Ne-(benzyloxycarbonyl)-L-
lysine (Boc-Lys(Z)-OH, Bachem). N-(tert-Butyloxycarbonyl)-S-
(ethylthio)-L-cysteine dicyclohexylammonium salt (Boc-Cys(SEt)-
OH). DCHA was purchased from Bachem. Synthetic analogue
of substance P was synthesised in the “Laboratoire d’Etudes et
de Recherche en Immunoanalyse” using a ABI 433A apparatus
(Applied Biosystems) and standard Fmoc chemistry. The HPLC-
gradient grade solvents (acetone, MeOH and CH3CN) were
obtained from Acros or Fisher Scientific. Buffers and aqueous
mobile-phases for HPLC were prepared using water purified with
Fig. 4 A) Immobilisation of fluorescent tripod 23 on the aldehyde-func-
tionalised silica surface by oxime ligation as described in Fig. 3A. B)
Binding of FP647-labelled anti-SP monoclonal antibodies.
These preliminary results clearly show that fluorescent tripods
such as 23, readily obtained through step-by-step bioconjugation
protocols, are promising tools for both immobilisation and
visualisation of biopolymers.
1
a Milli-Q system (purified to 18.2 MX cm). H and 13C NMR
spectra were recorded on a Bruker DPX 300 spectrometer (Bruker,
Wissembourg, France). Chemical shifts are expressed in parts per
million (ppm) relative to the residual solvent signal: CD3CN (dH =
1.96, dC = 1.79 (CH3), 118.26 (CN)), CDCl3 (dH = 7.26, dC = 77.36),
Conclusion
A new heterotrifunctional cross-linking reagent based on a
hydrophilic peptide scaffold and bearing an original set of three
orthogonal reactive groups has been developed. The flexible and
CD3OD (dH = 3.31, dC = 49.00 (CH3), or D2O (dH = 4.79).38
J
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values are in Hz. Infrared (IR) spectra were recorded as thin-film
on sodium chloride plates or KBr pellets using a Perkin Elmer FT-
IR Paragon 500 spectrometer with frequencies given in reciprocal
centimetres (cm−1). UV-visible spectra were obtained on a Varian
Cary 50 scan spectrophotometer. Chromophore-containing com-
pounds were quantified by UV-visible spectroscopy at the kmax
using the corresponding tabulated molar extinction coefficient.
Fluorescence spectroscopic studies were performed with a Varian
Cary Eclipse spectrophotometer. Analytical HPLC was performed
on a Thermo Electron Surveyor instrument equipped with a PDA
detector. Semi-preparative HPLC was performed on a Finnigan
SpectraSYSTEM liquid chromatography system equipped with
UV-vis 2000 detector. Mass spectra were obtained with a Thermo
Finnigan LCQ Advantage Max (ion-trap) apparatus equipped
with an electrospray source or a Voyager DE PRO MALDI-
TOF mass spectrometer (reflector mode, by using a-cyano-4-
hydroxycinnamic acid (CHCA) as matrix).
Hydroxybenzotriazole monohydrate (162 mg, 1.2 mmol) and
DCC (250 mg, 1.1 mmol) were sequentially added and the
resulting reaction mixture was stirred at room temperature for
1 h under an argon atmosphere. Thereafter, a solution of Na-Boc-
L-lysine (317 mg, 1.2 mmol) in dry DMF (1 mL) was added and
the resulting reaction mixture was stirred at room temperature.
The reaction was checked for completion by RP-HPLC (system
A) and TLC (CH2Cl2–MeOH, 8 : 2, v/v). The mixture was
evaporated to dryness. The resulting residue was taken up with
ethyl acetate, washed by 10% aq. citric acid and brine. The organic
layer was dried over Na2SO4, filtrated and evaporated to dryness.
The resulting residue was purified by chromatography on a silica
gel column with a step gradient of MeOH (0–5%) in CH2Cl2 as
the mobile phase, to give the lysine building block B as a white
foam (512 mg, 1.1 mmol, quantitative yield). Rf (CH2Cl2–MeOH,
8 : 2, v/v) 0.68; IR (KBr): mmax 518, 588, 703, 784, 878, 912, 964,
1030 (broad), 1081, 1164 (broad), 1249, 1368, 1469, 1520, 1732,
1
1792, 2936, 3357 (broad) cm−1; H NMR (300 MHz, CD3OD):
High-performance liquid chromatography separations
d 1.59–1.87 (m, 15H, CH2 b, d, c Lys, tBu), 3.31–3.37 (q, J =
6.3 Hz, 2H, CH2 e Lys), 4.25–4.29 (t, J = 5.5 Hz, 1H, CH a
Lys), 4.72 (s, 2H, CH2 phthalimidooxy-acetyl), 7.74–7.87 (m,
4H, phthalimide), 10.74 (s, 1H, CO2H); 13C NMR (75.5 MHz,
CD3OD): d 22.5, 28.3, 28.7, 32.0, 39.1, 53.3, 53.5, 80.0, 124.1,
128.5, 135.2, 163.2, 167.8, 175.6; MS (ESI+): m/z 449.33 [M +
H]+, 472.13 [M + Na]+, calcd for C21H27N3O8 449.46.
Several chromatographic systems were used for the analytical
experiments and the purification steps. System A: RP-HPLC
(Thermo Hypersil GOLD C18 column, 5 lm, 4.6 × 150 mm)
with CH3CN and 0.1% aqueous trifluoroacetic acid (aq. TFA,
0.1%, v/v, pH 2.0) as the eluents [0% CH3CN (5 min), followed
by linear gradient from 0 to 60% (30 min) of CH3CN] at a flow
rate of 1 mL min−1. Triple UV detection was achieved at 210,
260 and 285 nm. System B: System A with the following gradient
[20% CH3CN (5 min), followed by linear gradient from 20 to
90% (35 min) of CH3CN]. UV detection was achieved at 254 nm.
System C: System A with visible detection at 530 nm. System D:
RP-HPLC (Varian Kromasil C18 column, 10 lm, 21.2 × 250 mm)
with CH3CN and deionised water as eluents [10% CH3CN (5 min),
followed by linear gradient from 10 to 40% (15 min) and 40 to
70% (40 min) of CH3CN] at a flow rate of 20 mL min−1. Dual
UV detection was achieved at 254 and 305 nm. System E: RP-
HPLC (Thermo Hypersil GOLD C18 column, 5 lm, 10 × 250 mm)
with CH3CN and 0.1% aq. TFA as eluents [10% CH3CN (5 min),
followed by linear gradient from 10 to 40% (15 min) and 40 to
70% (40 min) of CH3CN] at a flow rate of 5 mL min−1. Dual UV
detection was achieved at 254 and 305 nm. System F: System E
with the following gradient [0% CH3CN (5 min), followed by linear
gradient from 0 to 30% (30 min), then from 30 to 60% (15 min)
of CH3CN] at a flow rate of 4 mL min−1. Visible detection was
achieved at 550 nm. System G: HPLC (Thermo Hypersil GOLD
C18 column, 5 lm, 10 × 250 mm) with CH3CN and 0.1% aq. acetic
acid (aq. AcOH, 0.1%, v/v, pH 3.3) as eluents [0% CH3CN (5 min),
followed by linear gradient from 0 to 30% (30 min) of CH3CN]
at a flow rate of 4 mL min−1. UV detection was achieved at 220
and 260 nm. System H: RP-HPLC (MS C18, Waters XTerra, 5 lm,
7.8 × 100 mm) with CH3CN and aq. TFA as eluents [0% CH3CN
(5 min), followed by linear gradient from 0 to 50% (50 min) of
CH3CN] at a flow rate of 2 mL min−1. UV detection was achieved
at 305 nm. System I: System H with the following gradient [0%
CH3CN (5 min), followed by linear gradient from 0 to 60% (40 min)
of CH3CN]. Visible detection was achieved at 550 nm.
N-(tert-Butyloxycarbonyl)-S-(ethylthio)-L-cysteine carboxam-
ide (14). Boc-Cys(SEt)-OH. DCHA (0.5 g, 1.08 mmol) was dis-
solved in dry ethyl acetate (15 mL) and the resulting solution was
cooled to −15◦C (bath of ethylene glycol–dry ice). NMM (119 lL,
1.08 mmol) and isobutyl chloroformate (140 lL, 1.08 mmol) were
sequentially added and the resulting mixture was stirred at −15 ◦C
for 10 min under an argon atmosphere. To this mixed anhydride
solution was added 20% (v/v) aq. ammonia solution (0.38 mL,
3.24 mmol) and the mixture was stirred at 4 ◦C for 30 min.
Thereafter, the reaction mixture was evaporated to dryness; the
resulting residue was taken up in ethyl acetate and washed with
deionised water. The organic layer was dried over Na2SO4, filtrated
and evaporated to dryness. 302 mg (1.08 mmol) of Boc-Cys(SEt)-
NH2 14 was obtained as a white solid (quantitative yield). Rf
(CH2Cl2–AcOEt, 1 : 1, v/v) 0.47; IR (KBr): mmax 623, 653, 714,
759, 778, 822, 860, 947, 969, 1021, 1046, 1115, 1169, 1255, 1270,
1320, 1370, 1393, 1429, 1517, 1662, 1686, 2781, 2871, 2930, 2975,
3186, 3342, 3389 cm−1; 1H NMR (300 MHz, CDCl3): d 1.24–1.29
(t, J = 6.8 Hz, 3H, CH3(SEt)), 1.39 (s, 9H, tBu), 2.63–2.70 (q, J =
7.1 Hz, 2H, CH2(SEt)), 2.99–3.01 (d, J = 6.0 Hz, 2H, CH2 b),
4.36–4.39 (m, 1H, CH a), 5.25–5.28 (d, J = 9.0 Hz, 1H, NH), 5.63
(bs, 1H, NH), 6.31 (bs, 1H, NH); 13C NMR (75.5 MHz, CDCl3): d
13.8, 27.8, 32.0, 39.9, 53.0, 80.1, 155.1, 172.6; MS (MALDI-TOF,
positive mode): m/z 303.13 [M + Na]+, calcd for C10H20N2O3S2
280.41.
S-(Ethylthio)-L-cysteine carboxamide (C). Boc-Cys(SEt)-NH2
14 (336 mg, 1.2 mmol) was slowly dissolved in a stirred TFA–H2O
mixture (95 : 5, v/v, 14 mL) while being warmed from 4 ◦C to
rt over 1 h. The reaction was checked for completion by TLC
(CH2Cl2–MeOH, 9 : 1, v/v) and the mixture was evaporated
almost to dryness. Deionised water was added and the resulting
solution was lyophilised to give the cysteine building block C
as a white powder (353 mg, 1.2 mmol, quantitative yield). Rf
Na-(tert-Butyloxycarbonyl)-Ne-(phthalimidooxyacetyl)-L-lysine
(B). Phthalimidooxyacetic acid 12 (259 mg, 1.1 mmol) was
dissolved in a mixture of dry CH3CN–DMF (1 : 1, v/v, 10 mL).
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(CH2Cl2–MeOH, 9 : 1, v/v) 0.09; IR(KBr): mmax 582, 676, 725, 801,
and TLC (CH2Cl2–MeOH, 9 : 1, v/v). Thereafter, the reaction
mixture was evaporated to dryness. The resulting residue was
taken up with ethyl acetate, washed by 10% aq. citric acid, sat.
aq. NaHCO3 and brine. The organic layer was dried over Na2SO4,
filtrated and evaporated to dryness. The resulting residue was
dissolved in a mixture of CH3CN–H2O (2: 1, v/v, 4.5 mL) and
purified by RP-HPLC (system D, 3 injections, tR = 31.3–32.6 min).
The product-containing fractions were lyophilised to give the
coupling product 15 as a white amorphous powder (133 mg,
0.15 mmol, yield 45%). Rf (CH2Cl2–MeOH, 9 : 1, v/v) 0.50; IR
(KBr): mmax 703, 755, 878, 1127 (broad), 1253, 1367, 1456, 1339,
844, 869, 955, 1136, 1185, 128, 1302, 1346, 1441, 1531, 1574, 1615,
1
1682, 2361, 2979, 3188, 3314, 3391 cm−1; H NMR (300 MHz,
D2O): d 1.26–1.31 (t, J = 7.1 Hz, 3H, CH3(SEt)), 1.39 (s, 9H),
2.72–2.80 (q, J = 7.1 Hz, 2H, CH2(SEt)), 3.25–3.26 (d, J = 6.0 Hz,
2H, CH2 b), 4.30–4.35 (m, 1H, CH a); 13C NMR (75.5 MHz, D2O):
d 13.8, 30.1, 38.3, 52.2, 170.8; MS (MALDI-TOF, positive mode):
m/z 181.00 [M + H]+, calcd for C5H12N2OS2 180.29.
Na-(Phthalimidooxyacetyl)-L-lysinyl-S-(ethylthio)-L-cysteine car-
boxamide (D). (a) Coupling reaction: Lysine building block B
(215 mg, 0.48 mmol) and TFA salt of H-Cys(SEt)-OH C (141 mg,
0.48 mmol) were dissolved in a mixture of dry CH3CN–DMF
(1 : 1, v/v, 5 mL). BOP reagent (233 mg, 0.53 mmol) and freshly
distilled DIEA (275 lL, 1.5 mmol) were sequentially added and
the resulting reaction mixture was stirred at room temperature for
3 h under an argon atmosphere. The reaction was checked for
completion by RP-HPLC (system A) and TLC (CH2Cl2–MeOH,
9 : 1, v/v). Thereafter, the mixture was evaporated to dryness.
The resulting residue was taken up with ethyl acetate, washed
by 10% aq. citric acid, aq. sat. NaHCO3 and brine. The organic
layer was dried over Na2SO4, filtrated and evaporated to dryness.
The resulting residue was purified by chromatography on a silica
gel column with a step gradient of MeOH (0–5%) in CH2Cl2 as
the mobile phase, to give the coupling product as a white foam
(288 mg, 0.47 mmol, quantitative yield). Rf (CH2Cl2–MeOH, 9 :
1, v/v) 0.58; IR (KBr): mmax 518, 588, 702, 786.2, 846, 878, 912,
1030 (broad), 1082, 1132, 1167 (broad), 1251, 1288 (broad), 1367,
1467, 1524 (broad), 1667 (broad), 1737, 1792, 2868, 1929, 3321
(broad) cm−1; 1H NMR (300 MHz, CDCl3): d 1.27–1.32 (t, 3H, J =
7.3 Hz, CH3(SEt) Cys), 1.32–2.03 (m, 15H, CH2 b, d, c Lys, tBu),
2.66–2.74 (q, 2H, J = 7.3 Hz, CH2(SEt) Cys), 3.05–3.19 (m, 2H,
CH2 bCys), 3.29–3.45 (m, 2H, CH2 e Lys), 3.99–4.05 (m, 1H, CH a
Lys), 4.70 (s, 2H, CH2 phthalimidooxy-acetyl), 4.70–4.77 (m, 1H,
CH a Cys), 5.52–5.54 (d, J = 5.6 Hz, 1H, NH), 5.75 (bs, 1H, NH),
6.95 (bs, 1H, NH), 7.79–7.83 (m, 4H, phthalimide); 13C NMR
(75.5 MHz, CDCl3): d 14.4, 22.7, 28.4, 28.9, 29.8, 32.5, 38.4, 39.5,
52.4, 53.6, 124.3, 128.5, 135.3, 163.9, 167.4, 172.6; MS (ESI+):
1
1662.3 (broad), 1733, 1791, 2926, 3344 (broad) cm−1; H NMR
(300 MHz, CDCl3): d 1.27–1.32 (t, 3H, J = 7.3 Hz, CH3(SEt)
Cys), 1.43–1.95 (m, 15H, CH2 b, d, c Lys, tBu), 2.66–2.73 (q, 2H,
J = 7.3 Hz, CH2(SEt) Cys), 3.04–3.15 (m, 2H, CH2 b Cys), 3.39–
4.02 (m, 18H, CH2 e Lys + 8 × CH2 linker), 4.03 (s, 4H, 2 ×
CH2 linker), 4.39–4.45 (m, 1H, CH a Lys), 4.68–4.72 (m, 3H, CH2
phthalimidooxy-acetyl + CH a Cys), 5.23 (bs, 1H, NH), 6.00 (bs,
2H, NH2), 6.16 (bs, 1H, NH), 6.97 (bs, 1H, NH), 7.46 (bs, 2H,
2 x NH), 7.79–7.83 (m, 4H, phthalimide); 13C NMR (75.5 MHz,
CDCl3): d 14.4, 22.8, 28.5, 28.8, 31.4, 32.5, 38.8, 38.9, 39.2, 40.5,
52.6, 53.5, 70.1, 70.3, 70.4, 71.1, 71.2, 124.3, 128.6, 135.3, 163.9,
167.5, 171.0, 171.2, 172.0, 175.7; HPLC (system A): tR = 26.9 min;
•
MS (ESI+): m/z 902.00 [M + H]+, 919.00 [M + H2O]+ , 924.33
[M + Na]+, calcd for C38H59N7O14S2 902.06. (b) Removal of the
Boc group: Fully protected pseudo-peptide 15 (25 mg, 28 lmol)
was dissolved in dry CH2Cl2 (2 mL). The solution was cooled
◦
to 4 C and TFA (164 lL, 2.2 mmol) was added dropwise. The
resulting reaction mixture was stirred at room temperature for 1 h.
The reaction was checked for completion by RP-HPLC (system
A) and the mixture was evaporated to dryness. A minimum of
deionised water was added (ca. 3 mL) and the resulting aq. solution
was purified by RP-HPLC (system D, 2 injections, tR = 31.4–
35.0 min). The product-containing fractions were lyophilised to
give the PEG-peptide 16 as a white amorphous powder (20 mg,
22 lmol, yield 78%). HPLC (system A): tR = 22.7 min, purity 95%;
MS (ESI+): m/z 802.60 [M + H]+, calcd for C33H51N7O12S2 801.94.
(c) Conversion into N-hydroxysuccinimidyl carbamate: TFA salt
of PEG-peptide 16 (20 mg, 22 lmol) was dissolved in dry DMF
(500 lL). TEA (3.7 lL, 26.4 lmol) and 50 lL of a solution of
DSC reagent in dry DMF (14 mg, 55 lmol) were sequentially
added and the resulting reaction mixture was stirred at room
temperature for 2 h. The reaction was checked for completion by
RP-HPLC (system A). Finally, the reaction mixture was quenched
by dilution with aq. TFA 0.1% (pH 2, ca. 2 mL) and purified
by RP-HPLC (system E, 2 injections, tR = 25.9–27.8 min). The
product-containing fractions were lyophilised to give 5 as a white
amorphous powder (20 mg, 21 lmol, quantitative yield). 1H NMR
(300 MHz, CDCl3): d 1.23–1.27 (t, 3H, J = 7.2 Hz, CH3(SEt) Cys),
1.44–1.93 (m, 6H, CH2 b, d, c Lys), 2.65–2.73 (q, 2H, J = 7.2 Hz,
CH2(SEt) Cys), 2.82 (s, 4H, 2 × CH2 succinimide), 2.98–3.18 (m,
2H, CH2 b Cys), 3.33–3.77 (m, 18H, CH2 e Lys + 8 × CH2 linker),
4.03 (s, 4H, 2 × CH2 linker), 4.43–4.45 (m, 1H, CH a Lys), 4.68–
4.71 (m, 3H, CH2 phthalimidooxy-acetyl + CH a Cys), 6.06 (bs,
1H, NH), 6.90 (bs, 1H, NH), 7.00–7.04 (m, 3H, NH + NH2),
7.78–7.83 (m, 4H, phthalimide); 13C NMR (75.5 MHz, CDCl3): d
14.4, 22.8, 25.6, 28.8, 31.5, 32.5, 38.9, 39.0, 39.2, 41.9, 52.6, 53.3,
69.5, 70.1, 70.2, 70.3, 71.0, 71.1, 77.4, 124.2, 128.6, 135.3, 152.0,
163.9, 167.7, 170.4, 171.3, 171.3, 172.1, 173.5; HPLC (system A):
•
m/z 611.67 [M + H]+, 629.00 [M + H2O]+ , calcd for C26H37N5O8S2
611.74. (b) Removal of the Boc group: Fully protected dipeptide
(223 mg, 0.36 mmol) was dissolved in dry CH2Cl2 (12 mL) and the
solution was cooled to 4 ◦C. TFA (1.6 mL, 21.9 mmol) was added
dropwise and the resulting reaction mixture was stirred at room
temperature for 3 h. The reaction was checked for completion by
TLC (CH2Cl2–MeOH, 9 : 1, v/v) and the mixture was evaporated
to dryness. The resulting oily residue was dissolved in deionised
water and the resulting aqueous solution was lyophilised to give
the dipeptide building block D as a white amorphous powder
(221 mg, 0.35 mmol, quantitative yield). This compound was used
in the next coupling reaction step without further purification.
Fully protected heterotrifunctional cross-linker (5). (a) Cou-
pling reaction: The TFA salt of dipeptide D (269 mg, 0.43 mmol)
and Boc-protected amino-PEG-acid spacer A (175 mg, 0.43 mmol)
were dissolved in dry CH3CN–DMF (1 : 1, v/v, 5 mL). BOP
reagent (209 mg, 0.47 mmol) and dry DIEA (222 lL, 1.29 mmol)
were sequentially added and the resulting reaction mixture was
stirred at room temperature for 3 h under an argon atmosphere.
The reaction was checked for completion by RP-HPLC (system A)
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tR = 25.2 min, purity 92%; MS (ESI+): m/z 943.27 [M + H]+,
temperature for 2 h. The reaction was checked for completion by
RP-HPLC (system C) and the mixture was evaporated to dryness.
The resulting residue was dissolved in 0.1% aq. TFA (pH 2, ca.
965.20 [M + Na]+, calcd for C38H54N8O16S2 943.03.
Sulfhydryl aminooxy pseudo-peptide (17). (a) Removal of the
phthaloyl group: Fully protected pseudo-peptide 15 (11.0 mg,
12.2 lmol) was dissolved in MeOH (1.5 mL) and a solution of
hydrazine monohydrate (12.2 lmol) in MeOH (250 lL) was added.
The resulting reaction mixture was stirred at room temperature
for 1 h. The deprotection reaction was checked for completion
by RP-HPLC (system A). Thereafter, volatiles were removed
under reduced pressure without warming. 0.1% aq. TFA (pH 2,
1 mL) and CH3CN (1 mL) were added and the resulting solution
was purified by RP-HPLC (system G, 2 injections, tR = 32.8–
39.2 min). The product-containing fractions were lyophilised to
give aminooxy pseudo-peptide as a white amorphous powder
(5.9 mg, 7.6 lmol, yield 63%). 1H NMR (300 MHz, D2O): d 1.23–
1.28 (t, 3H, J = 7.3 Hz, CH3(SEt) Cys), 1.40–1.86 (m, 15H, CH2
b, d, c Lys, tBu), 2.67–2.74 (q, 2H, J = 7.3 Hz, CH2(SEt) Cys),
3.19–3.27 (m, 4H, CH2 b Cys, CH2 e Lys), 3.44–3.48 (t, J = 5.5 Hz,
2H, CH2 linker), 3.56–3.60 (t, J = 5.3 Hz, 2H, CH2 linker), 3.64–
3.74 (m, 8H, 4 × CH2 linker), 4.00 (s, 4H, 2 × CH2 linker), 4.06
(s, 2H, CH2 linker), 4.15 (s, 2H, CH2 linker) 4.10 (s, 2H, CH2
linker), 4.35–4.37 (t, J = 2.0 Hz, 1H, CH a Lys), 4.40–4.63 (t, J =
4.6 Hz, 1H, CH a Cys); HPLC (system A): tR = 23.7 min. MS
(ESI+): m/z 772.00 [M + H]+, calcd for C30H57N7O12S2 771.96.
(b) Removal of the SEt group: Aminooxy pseudo-peptide (5.9 mg,
7.6 lmol) was dissolved in a mixture of 0.1 M aq. NaHCO3 buffer
(pH 8.5, 500 lL) and NMP (400 lL). DTT (20.0 mg, 130 lmol)
was added and the resulting reaction mixture was stirred at room
temperature for 1 h. The reaction was checked for completion
by RP-HPLC (system A). Further amount of DTT (15.0 mg,
97 lmol) was added and the mixture was stirred for 2 h. Finally,
the reaction mixture was quenched by dilution with 0.1% aq.
AcOH (pH 3.3, 10 mL) and the solution was lyophilised. The
resulting residue was dissolved in a mixture of CH3CN–0.1% aq.
AcOH (1 : 2, v/v, 3 mL) and purified by RP-HPLC (system G, 2
injections, tR = 31.5–34.7 min). The product-containing fractions
were lyophilised to give the sulfhydryl aminooxy pseudo-peptide
17 as a white powder (3.0 mg, 4.2 lmol, yield 71%). MS (ESI+):
m/z 719.33 [M + Li]+, calcd for C28H53N7O12S 711.84.
3 mL) and purified by RP-HPLC (system F, 2 injections, tR
=
25.7–29.2 min). The product-containing fractions were lyophilised
to give R6G-WS amine as a pink powder. HPLC (system C): tR =
25.7 min, purity > 95%; MS (ESI+): m/z 817.27 [M + H]+, calcd
for C36H43N6O12S2 815.24.
(c) R6G-WS SIAB derivative. R6G-WS amine (3.4 lmol) was
dissolved in 0.1 M aq. borate buffer (500 lL). A solution of
Sulfo-SIAB reagent (4.2 mg, 8.4 lmol) in 0.1 M aq.borate buffer
(50 lL, 50 mM + 5 mM EDTA, pH 8.2) was added. The resulting
reaction mixture was protected from light and stirred at room
temperature for 1 h. The reaction was checked for completion
by RP-HPLC (system C). Further amount of Sulfo-SIAB reagent
(4.2 mg, 8.4 lmol) was added and the mixture was stirred for
one more hour. Finally, the reaction mixture was quenched by
dilution with 0.1% aq. TFA (pH 2, ca. 3 mL) and purified by RP-
HPLC (system F, 2 injections, tR = 22.8–26.4 min). The product-
containing fractions were lyophilised to give the thiol-reactive
R6G-WS derivative 18 as a pink powder (2.5 mg, 2.2 lmol,
over yield for the three steps 27%). HPLC (system C): tR
=
27.7 min, purity 93%; MS (ESI+): m/z 1104.07 [M + H]+, calcd
for C45H50IN7O14S2: 1103.97.
R6G-WS labelled aminooxy reagent (19). Sulfhydryl amino-
oxy pseudo-peptide 17 (3.0 mg, 4.7 lmol) was dissolved in 0.1 M
aq. NaHCO3 buffer (400 lL) and a solution of thiol-reactive R6G-
WS derivative 18 (5.3 mg, 4.7 lmol) in 0.1 M aq. NaHCO3 buffer
(pH 8.5, 400 lL) was added. The resulting reaction mixture was
protected from light and stirred at room temperature for 1 h.
Finally, the reaction mixture was quenched by dilution with 0.1%
aq. AcOH (pH 3.3, 2 mL) and purified by RP-HPLC (system
F, 2 injections, tR = 50.1–55.6 min). The product-containing
fractions were lyophilised to give the fluorescent aminooxy reagent
19 as a pink powder. Quantification was achieved by UV-vis
measurements at kmax = 531 nm of R6G-WS by using the e value
65000 L mol−1 cm−1 (yield estimated after RP-HPLC purification
28%). HPLC (system B): tR = 27.6 min, purity 97%; MS (ESI−):
m/z 1686.47 [M − H]−, 1670.73 [M − NH2]−, 834.87 [M − NH2 −
H]2−, calcd for C73H102N14O26S3 1687.90.
Preparation of thiol-reactive R6G-WS derivative (18).
(a) N-Hydroxysuccinimidyl ester of R6G-WS. The sulfonated
analogue of rhodamine 6G (5.3 mg, 8.7 lmol) was dissolved in dry
NMP (200 lL). A solution of TSTU reagent (3.0 mg, 10 lmol)
in dry NMP (38 lL) and a solution of DIEA (2.7 lL) in dry
NMP (17 lL) were sequentially added. The resulting reaction
mixture was protected from light and stirred at room temperature
for 75 min. The reaction was checked for completion by RP-HPLC
(system C). The resulting N-hydroxysuccinimidyl ester was used
in the next step without further purification. HPLC (system C):
tR = 26.9 min (compared to tR = 25.9 min for R6G-WS carboxylic
acid); MS (ESI−): m/z 870.40 [M - H]−, calcd for C38H42N5O15S2
870.8.
(b) R6G-WS amine. Ethylenediamine dihydrochloride
(96.7 mg, 738 lmol) was dissolved in a mixture of DMF-H2O
(9 : 1, v/v, 10 mL). The crude reaction mixture containing the
N-hydroxysuccinimidyl ester and a solution of DIEA (64 lL)
in DMF (150 lL) were sequentially added and the resulting
reaction mixture was protected from light and stirred at room
Substance P-tripod conjugate (21). N-Hydroxysuccinimidyl
carbamate cross-linking reagent
5
(5.0 mg, 5.4 lmol)
and undecapeptide H-Arg-Pro-Ala-Pro-Gln-Gln-Phe-Phe-Gly-
Ala-Met-NH2 (8 mg, 5.4 lmol) were dissolved in dry NMP
(400 lL). DIEA (3.8 lL, 21.6 lmol) was added and the resulting
reaction mixture was stirred at room temperature for 4 h. Further
DIEA (1.9 lL, 10.8 lmol) was added and the mixture was stirred
for 2 h. The reaction was checked for completion by RP-HPLC
(system A). Acetic acid (1.2 lL, 21.6 lmol) and 0.1% aq. TFA
(pH 2, 1 mL) were sequentially added and the aqueous mixture
was purified by RP-HPLC (system H, 2 injections, tR = 32.5–
33.2 min). The product-containing fractions were lyophilised to
give peptide conjugate 21 as a white powder (10.3 mg, 5.0 lmol,
yield 92%). HPLC (system B): tR = 26.2 min; MS (ESI+): m/z
1056.4 [M + 2H2O + 2H]2+, calcd for C91H136N24O26S3 2078.44.
R
FluoProbesꢀ 532A labelled substance P-tripod conjugate (23).
(a) Removal of the phthaloyl group: Substance P-tripod conjugate
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21 (10.3 mg, 5 lmol) was dissolved in MeOH (500 lL) and a
solution of hydrazine monohydrate (5 lmol) in MeOH (100 lL)
was added. The resulting reaction mixture was stirred at room
temperature for 1 h. The deprotection reaction was checked
for completion by RP-HPLC (system A). Thereafter, 0.1% aq.
TFA (pH 2, 2 mL) was added and the resulting solution was
purified by RP-HPLC (system H, 2 injections). The product-
containing fractions were lyophilised to give the aminooxy tripod
as a white powder. (b) Removal of the SEt group: Aminooxy-
peptide (3.8 mg, 1.8 lmol) was dissolved in dry NMP (100 lL).
A solution of DTT (5.6 mg, 36 lmol) in 0.1 M aq. borate
buffer (pH 8.2, 300 lL) was added and the resulting reaction
mixture was stirred at room temperature for 2 h. The reaction
was checked for completion by RP-HPLC (system A). Acetic
acid (0.2 lL, 3.6 lmol) and 0.1% aq. TFA (pH 2, 500 lL)
were sequentially added and the aqueous mixture was purified
by RP-HPLC (system I, 2 injections). The product-containing
fractions were lyophilised to give the sulfhydryl tripod 22 as a white
powder. (c) Fluorescent labelling: Sulfhydryl tripod 22 (1.5 mg,
0.7 lmol) was dissolved in dry NMP (300 lL). A solution of
pH 7.4) and 1 lL of the resulting solution was put over the freshly
prepared aldehydic surface. After 4 h of incubation in the dark
and in a humid atmosphere at room temperature, the reaction
solution was carefully removed and the surface was washed for
10 min with phosphate buffer (10 mM + 0.05% Tween, pH 7.4).
Finally, slide was stored with EIA buffer (0.1 M phosphate buffer
(pH 7.4) containing 0.15 M NaCl, 0.1% BSA and 0.01% sodium
azide) overnight before observation. Fluorescence scanning was
performed with an Olympus inverted microscope model IX71 (4X
objective) equipped with a camera PCO 1600. Thereafter, FP647-
labelled anti-SP mAb (1.5 nmol mL−1) was added and after 30 min
of incubation, fluorescence scanning of plots was again performed.
Acknowledgements
This work was supported by the Agence Nationale de la Recherche
(Programme Emergence et Maturation 2005), Commissariat a`
l’Energie Atomique and Institut Universitaire de France (IUF).
We thank Elisabeth Roger (INSA de Rouen) for IR mea-
surements, Dr Je´roˆme Leprince (U 413 INSERM-IFRMP 23)
for MALDI-TOF mass spectrometry measurements, Dr Herve´
Bernard (INRA) for peptide synthesis, Dr Christophe Portal
(QUIDD) for English corrections of the manuscript and QUIDD
company for the open access to their HPLC instruments.
R
FluoProbesꢀ 532A maleimide (0.5 mg, 0.7 lmol) in dry NMP
(200 lL) and a solution of DIEA (0.7 lmol) in dry NMP (50 lL)
were sequentially added and the resulting reaction mixture was
protected from light and stirred at room temperature for 1 h.
The labelling reaction was checked for completion by RP-HPLC
(system C). After 2 h, further DIEA (1.4 lmol) in dry NMP (41 lL)
was added. Finally, 0.1% aq. TFA (pH 2, ca. 2 mL) was added
and the aqueous mixture was purified by RP-HPLC (system I, 2
injections, tR = 26.5–27.3 min). The product-containing fractions
were lyophilised to give fluorescent tripod 23 as a pink amorphous
powder. Quantification was achieved by UV-vis measurements at
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