A universal and ready-to-use heterotrifunctional cross-linking reagent for facile synthetic access to sophisticated bioconjugates

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A universal and ready-to-use heterotrifunctional cross-linking reagent for

facile synthetic access to sophisticated bioconjugates† ‡

Guillaume Clave´,^a,bHerve´ Volland,^cMe´lanie Flaender,^eDidier Gasparutto,^eAnthony Romieu*^a,band

Pierre-Yves Renard*^a,b,d

Received 17th May 2010, Accepted 17th June 2010

DOI: 10.1039/c0ob00133c

We describe for the ﬁrst time, the synthesis and some bioconjugation applications of an original

heterotrifunctional cross-linking reagent (also named tripod) bearing three different bioorthogonal

functional groups which are fully compatible amongst themselves. Contrary to the ﬁrst generation

tripod recently reported by us (Org. Biomol. Chem., 2008, 6, 3065), the use of an azido group instead of

the nucleophile-sensitive active carbamate moiety enables us to reach the targeted chemical

orthogonality without the use of temporary aminooxy- and thiol protecting groups. Thus, the

preparation of sophisticated bioconjugates through the sequential derivatisation of the tripod by means

of copper-mediated 1,3-dipolar cycloaddition, oxime ligation and aqueous compatible mild

thiol-alkylation reactions, is signiﬁcantly simpler and more convenient. The chemoselective

bioconjugation protocols were optimised through the preparation of FRET cassettes based on cyanine

and/or xanthene ﬂuorescent dye pairs and subsequent anchoring to fragile biomolecules. The

applicability of this universal cross-linking reagent was also illustrated by the preparation of biochips

suitable for aﬂatoxin B1 detection through the SPIT-FRI method.

applications including diagnostic assays, multimodality optical

Introduction

imaging and drug delivery.⁴The most popular methods to

During the past decade, considerable efforts have been devoted

construct such bioconjugates encompass simple second-order

to the development of efﬁcient, cutting edge and highly practical

reactions that selectively target the functionalities present (or pre-

viously introduced) within the native biomolecule/biopolymer.⁵

Primary amines and sulfhydryls are the most commonly modiﬁed

functional groups. The use of a commercially available homo-

or heterobifunctional cross-linking reagent often enables to selec-

tively introduce and/or derivatise these bioconjugable groups.

In recent years, new elegant bioconjugation approaches based

on original reactions belonging to the “click” chemistry repertoire

have been developed.⁶Thus, reactions that meet the “click”

chemistry requirements are effective for selective labelling of

biomolecules as they are high-yielding under biological conditions

and are bioorthogonal since the involved precursors are stable

to water and do not react with common functional groups

present in biological molecules. These reactions include the

very popular azide-alkyne Huisgen cycloaddition, the Diels–

Alder-type cycloadditions, the Staudinger ligation of triarylphos-

phines and azides, and condensation of aldehyde or ketone with

a-nucelophiles such as aminooxy and hydrazide derivatives.

The availability of such efﬁcient bioorthogonal reactions and

their presumed compatibility with some classic bioconjugation

methods should enable us to consider the construction of so-

phisticated bioconjugates, especially those resulting from the

covalent association of three different (bio)molecular partners.

Indeed, there is a growing need for three-component biomolecular

systems which are useful 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,⁷the energy transfer terminators (i.e.,

dideoxynucleotides labelled with FRET cassettes) for DNA

sequencing,⁸and the biopolymer microarrays currently used for

bioconjugation methods for the chemical modiﬁcation of biologi-

cal molecules with various reporter groups (e.g., ﬂuorescent labels,

nanoparticles, nanomaterials or other biopolymers).^1,2

Indeed, there is a growing need of multicomponent biomolec-

ular systems with uniquely combined properties of the individual

components, especially in the following disciplines: biotechnol-

ogy, nano(bio)technology, bio-organic and medicinal chemistry.³

Among the myriad of chemically engineered biomolecular tools

that are currently designed, biosensors, molecular bio-probes and

drug vectors are of particular interest for various biomedical

^aEquipe de Chimie Bio-Organique, COBRA-CNRS UMR 6014 & FR 3038,

rue Lucien Tesnie`re, 76131, Mont-Saint-Aignan, France. E-mail: pierre-

yves.renard@univ-rouen.fr, anthony.romieu@univ-rouen.fr; Fax: +33 2-35-

52-29-71; Tel: +33 2-35-52-24-14 (or 24-15)

^bUniversite´ de Rouen, Place Emile Blondel, 76821, Mont-Saint-Aignan,

France

^ciBiTecS, Laboratoire d’Etudes et de Recherches en Immuno-analyse,

Commissariat a` l¢Energie Atomique, Gif-sur-Yvette, F-91191, France

^dInstitut Universitaire de France, 103 Boulevard Saint-Michel, 75005, Paris,

France

^eLaboratoire des Le´sions des Acides Nucle´iques, CEA, INAC, SCIB

(UMR_E 3 CEA-UJF & FRE 3200 CEA-CNRS), 17 Rue des Martyrs,

F-38054, Grenoble Cedex 9, France

† Such heterotrifunctional cross-linking reagents were covered by a patent

entitled “Preparation of trifunctional pseudopeptide reagents, especially

luminescent reagents, and their bioconjugates, and their use for the

functionalization of solid supports for the detection of biomolecules”,

G. Clave´, P.-Y. Renard, A. Romieu and H. Volland, PCT Int. Appl., 2009,

WO 2009043986.

‡ Electronic supplementary information (ESI) available: Detailed synthetic

procedures for compound 26. Characterisation data for compounds 3, 10

and 22–25. See DOI: 10.1039/c0ob00133c

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Fig. 1 Structures of heterotrifunctional cross-linkers 1 and 2 developed by us and Watzke et al. respectively, and the ready-to-use azido tripod 3 studied

in this work.

the rapid analysis of various biological events.⁹However, due to

the lack of universal heterotrifunctional cross-linking reagents

(i.e., cross-linkers equipped with a triply (bio)orthogonal set

of functional groups), synthetic access to these (bio)molecular

architectures is still tricky or impossible in most cases. To ﬁll

this gap, we have recently reported a heterotrifunctional linker

molecule 1 (also named tripod, see Fig. 1 for the corresponding

structure) based on a dipeptidyl scaffold (i.e., lysine-cysteine) that

contains an amine-reactive N-hydroxysuccinimidyl carbamate, an

aldehyde/ketone-reactive aminooxy group, a thiol group and a

water-solubilising pseudo-PEG spacer.¹⁰The potential utility of

this bioconjugation reagent was demonstrated by the preparation

of biochips suitable for substance P and microcystin-LR detection

through the SPIT-FRI (for Solid-Phase Immobilised Tripod for

Fluorescent Renewable Immunoassay) method.^10,11However, in

this ﬁrst generation tripod the chemical orthogonality between

the active carbamate and the nucleophilic aminooxy and thiol

functionalities, and so the chemical stability of the cross-linking

reagent was obtained only by using temporary protecting groups

for these two latter reactive groups (i.e., phthaloyl (Pht) for

aminooxy and SEt for thiol). Thus, bioconjugation schemes

involving the use of this reagent always require two additional

deprotection steps which must be performed immediately after the

acylation reaction of the amine-containing biomolecule with its N-

hydroxysuccinimidyl carbamate. Since these deprotections require

the use of hydrazine monohydrate in MeOH and dithiothreitol

(DTT) in aq. buffer, some side-reactions may occur with fragile

biomolecules and chemical reporter groups (e.g., ﬂuorophores)

exhibiting nucleophile- and/or reductant-sensitive moieties, caus-

ing signiﬁcant losses of bioconjugate material. Furthermore, the

multi-step bioconjugation procedures are also complicated by

tedious and time-consuming reversed-phase HPLC (RP-HPLC)

puriﬁcations. Thus, the overall yield of the bioconjugation strate-

gies based on the use and sequential derviatisation of 1 is often

poor to modest.

bioorthogonal functional groups which are expected to be fully

compatible amongst themselves without using temporary protect-

ing groups. The availability of such an unprecedented cross-linking

reagent should allow straightforward access to sophisticated and

fragile bioconjugates because the three bioorthogonal reactions

leading to the covalent association of (bio)molecular partners

will be performed directly without any previous deprotection

step. In this context, we have chosen to replace the nucleophile-

sensitive active carbamate moiety of 1 by an azido group aimed

at using Staudinger ligation or copper-catalysed azide-alkyne 1,3-

dipolar cycloaddition (CuAAC) as one of the three derivatisation

reactions. Surprisingly, despite numerous recent work about the

use and optimisation of “click” chemistry reactions in the context

of biomolecule labelling,⁶only one study has been published on

the synthesis and applications of heterotrifunctional cross-linking

reagents one of whose three free reactive moieties is an azido

group (or its complementary functional group: alkyne or tri-

arylphosphine). This work describes the design and synthesis of an

aromatic building block 2 for C- and N-terminal protein labelling

and protein immobilisation.¹²The availability of an azido group,

an S-protected cysteine residue and a carboxylic acid onto the

benzene ring, enables 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; no example of sequential triple derivatisation of

reagent 2 with a ﬂuorescent reporter group, a protein and ﬁnally

a phosphane-modiﬁed surface has been reported to date. In the

present paper, we want to describe the synthesis of the original

azido tripod 3 bearing three free reactive groups that can be

directly and sequentially derivatised. To illustrate the versatility

of this novel bioconjugation reagent, the construction of three

different FRET cassettes based on cyanine and/or xanthene dye

pairs and their subsequent grafting to fragile biomolecules (i.e.,

aﬂatoxin B2, AFB2) and biopolymers (i.e., DNA fragments) were

explored. Furthermore, azido tripod 3 was also used to improve

the implementation of the original immunoassay format called

To improve the universality of bioconjugation reagent 1, we have

explored the chemistry of a new tripod bearing three different

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SPIT-FRI (for Solid-Phase Immobilised Tripod for Fluorescent

Renewable Immunoassay),¹³especially in the context of aﬂatoxin

B1 (ABF1) detection.

three different (bio)molecular partners, through the use of only

three reactions belonging to the repertoire of “wet chemistry”.

Concerning the core structure of our second generation tripod

3, the previously designed PEG-peptide scaffold based on the

(lysine-cysteine) sequence was kept unchanged because it exhibits

a good hydrophilic-hydrophobic balance which enables us to get a

cross-linking reagent signiﬁcantly soluble in water and related aq.

buffers, a key feature for bioconjugation applications.

Results and discussion

General considerations for the design of the second generation

tripod suitable for sequential bioconjugation of three different

(bio)molecular partners

Synthesis of the building blocks A–C for the preparation of the

second generation tripod

In the context of peptide bioconjugation, we have recently

proved that the aminooxy and thiol groups of tripod 1, once

deprotected, were orthogonal to each other and could be used

for a sequential derivatisation of substance P and microcystin-LR

analogues. However, the exempliﬁcation of the published ﬁve-step

bioconjugation protocol to other biomolecules (or biopolymers)

more fragile than peptides is not trivial and in some cases led to the

desired ﬁnal bioconjugate with a very poor yield. For instance, our

attempts to synthesise biochips suitable for detection of aﬂatoxins,

by means of ﬂuorescent labelling and immobilisation of AFB2

through the sequential tripod derivatisation gave unsatisfactory

results. Indeed, we observed that the lactone moiety of this myco-

toxin is highly reactive toward hydrazine used for the removal of

the Pht aminooxy protecting group, causing signiﬁcant alteration

of the AFB2-tripod conjugate during this ﬁrst deprotection step

and so decreasing the desired product recovery to a ca. 40% yield.¹⁴

The second generation tripod 3 was synthesised from three dif-

ferent building blocks A–C through a highly convergent synthetic

strategy similar to this developed for the preparation of 1 (Fig. 2).¹⁰

However, some modiﬁcations have been brought to the synthesis

of PEG-based building block A to incorporate the azido moiety

onto the “N-terminal” side of this pseudo-dipeptide. Furthermore,

the temporary aminooxy protecting group was revisited because

our ﬁrst synthetic attempts toward the second generation tripod

3 using the ﬁrst generation building block B have shown the

non-compatibility of the hydrazine-mediated deprotection of

phthaloyl with the reductant-sensitive azido function. Indeed,

formation of a signiﬁcant amount of amino tripod was observed

and so we suspected once again that traces of diimine in the

hydrazine solution induced this azido reducing reaction. Finally,

the structure and so the synthesis of the ﬁrst generation cysteine-

based building block C were kept unchanged.

In addition to its a-nucleophile character, the contamination

of commercial hydrazine solutions with trace amounts of diimine

can also negatively affect the structural integrity (and so the cor-

responding spectral properties) of some ﬂuorescent organic dyes

currently used for bio-labelling. As an example, during our syn-

thesis of a cyanine-based amino acid, an unwanted side-reaction

of reduction of the polymethene chain of this ﬂuorophore took

place during the deprotection of the phthaloyl group according

to the hydrazine commercial source.¹⁵These selected experimental

observations have led us to think that the use of hydrazine was

not compatible with the development of versatile bioconjugation

schemes involving our tripod technology. Consequently, to avoid

this non-selective deprotection step, we decided to explore a second

generation of heterotrifunctional cross-linking reagents bearing a

free aminooxy functional group. Since this a-nucleophile is free

to attack the active carbamate found in the structure of the ﬁrst

generation tripods, this latter electrophilic moiety must be replaced

by a bioconjugable, bioorthogonal and non-nucleophile sensitive

functional group.

We have naturally chosen the azido group which is known

to be inert toward most N-, O- and S-nucleophiles under

physiological conditions and easily and selectively derivatisable

through Staudinger ligation or CuAAC reaction.¹⁶Indeed, the

expected lack of cross-reactivity between these three bioconjugable

handles (i.e., aminooxy, azido and thiol groups) will open up a

new avenue for designing an original, easy-to-use and universal

bioconjugation strategy leading to the covalent association of

Fig. 2 Building block approach to the solution-phase synthesis of

ready-to-use heterotrifunctional cross-linking reagent 3.

Synthesis of azido-PEG-acid spacer A. Hydrophilic linker

A was synthesised in 48% overall yield through a four-step

procedure depicted in Scheme 1. The main feature of this synthetic

route is its shortness made possible by the re-use of two key

intermediates involved in the preparation of the ﬁrst generation

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Scheme 1 Reagents and conditions: (a) Benzyl chloroformate, TEA, DMAP, CH₂Cl₂, overnight; (b) TFA, CH₂Cl₂, 4 ^◦C to rt, 53% (overall yield

for steps a–b); (c) BOP, DIEA, CH₃CN–DMF (1 : 1, v/v), overnight; (d) 1 M aq. LiOH, MeOH, rt, 91% (overall yield for steps c–d). BOP =

benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexaﬂuorophosphate.

of building block A (i.e., N-Boc amino- and azido acids 4 and

6), as starting materials. Firstly, the N-Boc amino acid 4 was

converted to the corresponding benzyl ester by using unusual

conditions reported by Kim et al. (i.e., treatment of carboxylic

acid with benzyl chloroformate in the presence of TEA and

DMAP, in dry CH₂Cl₂).¹⁷The benzyl moiety was preferred to

standard alkyl esters (i.e., ethyl or methyl ester) to get a more

hydrophobic compound which was easily puriﬁed by conventional

liquid–liquid extraction and column chromatography. Further

treatment of this resulting benzyl ester with a 20% solution of

TFA in CH₂Cl₂gave the amine 5 in 53% overall yield for the

two steps. Amidiﬁcation of 5 with azido acid 6 has been readily

achieved with BOP phosphonium salt in the presence of DIEA,

in CH₃CN–DMF (1 : 1).¹⁸Finally, the benzyl ester was removed

by short treatment of the protected pseudo-PEG linker 7 with

1 M LiOH in H₂O–MeOH to give the target building block A

in 49% overall yield from 4. All spectroscopic data, in particular

NMR and mass spectrometry, were in agreement with the structure

assigned.

Synthesis of aminooxy-containing lysine building block B. As

mentioned above, the choice of azido moiety as the third bioconju-

gable function involves the replacement of the phthaloyl aminooxy

functionality by another protecting group which enables: (1) the

Scheme 2 Reagents and conditions: (a) Benzyl bromide, Cs₂CO₃, DMF, rt;

prevention of the N-overacylation side-reaction frequently en-

(b) Boc₂O, DMAP, CH₃CN, rt; (c) 10% Pd/C, H₂, MeOH, rt, 74% (overall

countered during aminooxy peptide synthesis,¹⁹and (2) the release

of the aminooxy reactive group under conditions compatible with

CH₃CN, 40% (overall yield for steps d–e).

yield for steps a–c); (d) DCC, NHS, CH₃CN, rt, 1h; (e) Fmoc-Lys-OH,

the stability of alkyl azides. Since the azido moiety is stable toward

treatment with triﬂuoroacetic acid, the N,N-bis-Boc protection

of aminooxyacetic acid (Aoaa) was considered. The use of this

acid-labile bis-carbamate protecting group led us to modify the

original synthetic route leading to the building block B (Scheme 2).

(Boc)₂–Aoaa–OH 9 was readily prepared from commercial mono-

Boc building block 8 by using the three-step procedure reported

by Brask and Jensen and involving transient protection of the

carboxylic acid function of 8 as a benzyl ester.²⁰Thereafter, 9

was activated as the N-hydroxysuccinimidyl ester (NHS) and

subsequently coupled to the free e-amino group of lysine. This

pre-activation procedure avoided an additional protection step

of the lysine carboxylic acid function. (Boc)₂–Aoaa–OH was

converted into the corresponding NHS ester by treatment with

DCC-NHS in CH₃CN. This crude mixture of active ester was

directly reacted with Fmoc-Lys-OH to provide the corresponding

building block B in a moderate yield (40%). It is interesting to

note that during the course of our work, Foillard et al. reported

an alternative acide-labile aminooxy protecting group (i.e., 1-

ethoxyethylidene, Eei) removable under milder conditions than

the bis-Boc moiety (i.e., 1% or 5% TFA solution in CH₃CN-H₂O

(1:1)) and more adequate for stepwise SPPS of Aoaa-containing

peptides.²¹Thus, the synthesis and use of Fmoc–Lys(Eei-Aoaa)–

OH building block could be further considered especially to speed

up the production of the targeted heterotrifunctional peptide-

based cross-linking reagents through standard automated solid-

phase synthesis techniques.

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with building block A to obtain the fully protected trifunctional

pseudo-peptide 10, (2) subsequent treatment of 10 with a 4.5%

solution of TFA in CH₂Cl₂to remove the two Boc groups, to give

the free aminooxy pseudo-peptide, and ﬁnally (3) the deprotection

of the thiol group by treatment with a large excess of DTT in

0.1 M aq. NaHCO₃buffer. Puriﬁcation was achieved by RP-HPLC

to give the ready-to-use azido tripod 3 as a TFA salt and in a

moderate yet not optimised overall yield (5%) from BC. Indeed,

the use of RP-HPLC for puriﬁcations (followed by lyophilisation)

and the small-scale chosen for the two latter deprotection steps

(<5 mg) induced signiﬁcant losses of material whereas these

reactions were found to be almost complete. Analysis of 3 using

ESI mass spectrometry indicated the presence and integrity of the

free aminooxy, azido and sulfhydryl reactive groups within the

pseudo-peptide architecture (see ESI‡). Interestingly, this reagent

could be stored at -20 ^◦C under an argon atmosphere for several

months without detectable degradation. The inert atmosphere

was found to be essential for long-time storage, to avoid both

the oxidative dimerisation of its sulfhydryl moiety and aminooxy

carbonatation.

Synthesis of aminooxy-azido-sulfhydryl-heterotrifunctional

cross-linking reagent 3 using amino acid building blocks

Due to the availability of the three amino acid building blocks

A, B and C, the azido tripod 3 was synthesised using the same

highly convergent solution-phase synthesis that we used to prepare

the ﬁrst generation heterotrifuntional cross-linking reagent 1

(Scheme 3). 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 10%

diethylamine solution in CH₃CN to give building block BC in 59%

overall yield. This latter pseudo-dipeptide was then subjected to a

further three-step reaction sequence: (1) BOP-mediated coupling

Bioconjugation applications of azido tripod 3

To demonstrate the broad utility of azido tripod 3 in bioconjuga-

tion chemistry, it was essential to develop a reliable high-yielding

protocol enabling the sequential and chemoselective derivatisation

of the three reactive groups with three different (bio)molecular

partners. Since the full chemical orthogonality between free

aminooxy and thiol functions and the superior chemical inertia

of the azido moiety are well established, we planned to make the

three reactions, namely, thiol alkylation (through Michael addition

or S_N2 reaction), oxime ligation and CuAAC²²in this order.

Furthermore, the choice of a “wet chemistry” sequence involving

CuAAC as the ultimate bioconjugation reaction was supported

by the fact that: (1) free sulfhydryl reactive groups could be

otherwise oxidatively damaged by copper and sodium ascorbate,

which are currently used to generate “in situ” the catalytically

active copper(I) species required in the CuAAC reaction, especially

through the generation of reactive oxygen species,²³and (2) copper

salts are known to catalyse the cleavage of the N–O bond and so

could lead to the degradation of free aminooxy moieties.²⁴To

illustrate the efﬁcacy of this three-step bioconjugation procedure,

the preparation of FRET cassette bio-probes and immunosensors

was considered.

Preparation of FRET cassettes based on cyanine and/or xan-

thene dyes and their use in labelling of fragile biomolecules and

biopolymers. Fluorescent organic dyes are widely used as non-

radioactive labels in biological analysis and as a key component of

optical bio-probes designed for various bio-imaging applications.²⁵

However, the undesirable spectral properties of various ﬂuo-

rophores still constrain the full potential of their applications. For

instance, many bright organic dyes including cyanine and xanthene

derivatives have the serious disadvantage of very small Stokes

shifts (typically less than 30 nm), which can lead to signiﬁcant self-

quenching and ﬂuorescence detection errors because of excitation

backscattering effects. To overcome such a limitation, the single

ﬂuorophore can be replaced by a more sophisticated energy-

donor–acceptor architecture based on a ﬂuorescence resonance

energy transfer phenomenon.²⁶Such FRET dyads (also named

Scheme 3 Reagents and conditions: (a) BOP, DIEA, CH₃CN, rt, 2 h, 62%;

(b) Et₂NH, CH₃CN, 2 h, 96%.; (c) building block A, BOP, DIEA, DMF,

rt, 2 h; (d) TFA, CH₂Cl₂, rt; (e) DTT, 0.1 M aq. NaHCO₃(pH 8.5), rt, 5%

(overall yield for steps c-e after RP-HPLC puriﬁcation).

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FRET cassettes) result from the covalent association of two

complementary dyes through a non-conjugated spacer and exhibit

through-space energy transfer. Thus, the pseudo-Stokes shift

(i.e., the wavelength discrepancy between the donor absorption

and the acceptor emission in an energy transfer system with

almost 100% energy transfer efﬁciency) of FRET-based energy

cassettes are larger than the Stokes shifts of either the donor or

acceptor dyes. Interestingly, these energy transfer ﬂuorescent labels

have found numerous applications in the ﬁeld of DNA analysis,

including sequencing, fragment sizing, and short tandem repeat-

based cancer diagnosis.⁸However, the energy transfer ﬂuorescent

primers and terminators required for such applications are not

synthetically easily accessible. Indeed, their preparation often

relies on solution or solid-phase peptide (or oligonucleotide)

chemistry involving the use of ﬂuorescent building blocks and

harsh conditions (especially for the deprotection steps) not fully-

compatible with the moderate stability of some ﬂuorophores.²⁷

In this context, we wished to use tripod 3 to develop a general

and versatile method for FRET cassettes synthesis and their

subsequent grafting to biological material. Thus, we planned

the synthesis of two different energy transfer dyes through the

sequential derivatisation of the two reactive aminooxy and thiol

groups of 3; the azido moiety being free for further grafting

to a biomolecule/biopolymer by means of a CuAAC reaction.

Either sulfoindocyanine dye Cy 3.0²⁸or R6G-WS (a water-soluble

analogue of rhodamine 6G recently developed by us)²⁹were chosen

as the donor and sulfoindocyanine Cy 5.0²⁸as the acceptor.

The carboxylic acid moieties of these dyes was converted into

a function which is able to readily react with thiol or aminooxy

reactive groups. Thus, the iodoacetamide derivatives of Cy 3.0

and R6G-WS 11 and 12 were prepared by using the three-step

synthetic procedure reported by Bouteiller et al.³⁰

Scheme 4 Reagents and conditions: (a) ethylenediamine dihydrochloride,

DMF–H₂O (9 : 1, v/v), 4 ^◦C to rt; (b) NMP, 4 ^◦C to rt, 11% (overall yield

for steps a–b after RP-HPLC puriﬁcation).

synthesis and biomolecular labelling of FRET cassettes through

the three-step sequential derivatisation described in Schemes 5

and 7. First, cross-linking reagent 3 readily reacted through its

free thiol with a slight excess of iodoacetyl derivative 11 (or

12) in aqueous sodium bicarbonate buffer (pH 8.5) to obtain

mono-ﬂuorescently labelled tripod 16 (or 17). This S_N2 reaction

was found to be complete within 1 h and puriﬁcation by RP-

HPLC enabled us to recover 16 (or 17) in almost quantitative

yield. Thereafter, 16 (or 17) and ﬂuorescent aldehyde 15 were

linked together through chemoselective oxime ligation. Such a

reaction was performed in aqueous sodium acetate buffer (pH

4.2 in the range 4–5 which is known to be optimal for oxime

bond formation)³²and found to proceed essentially to completion

within 3 h to yield exclusively the corresponding FRET cassettes

18 and 19. These original energy transfer ﬂuorescent azido-

containing labels were puriﬁed by RP-HPLC and their structures

conﬁrmed by ESI mass spectrometry (see ESI‡). Finally, we exam-

ined their covalent attachment to biomolecules and biopolymers

(Scheme 7). As an example of a fragile biomolecule, we have

chosen to work with mycotoxin AFB2 which exhibits a highly

nucleophile-sensitive lactone moiety. Indeed, the preparation of

a ﬂuorescent probe of AFB2 is of particular interest especially to

demonstrate the efﬁcacy of our three-step bioconjugation protocol

and to get a useful tool for ﬂuorescence immunoassays aimed

at detecting aﬂatoxins. Since ABF2 possesses only a keto group

whose chemical modiﬁcation through oxime ligation does not alter

its biological properties, it was essential to introduce the alkyne

moiety onto this position. Thus, we have used a two-step synthetic

sequence involving AFB2 carboxylic acid derivative 20 as the key

intermediate (Scheme 6). This latter compound was obtained

by reaction of commercial AFB2 with aminooxyacetic acid in

The synthesis of a ﬂuorescent aldehyde 15 derived from Cy 5.0

and reactive toward the aminooxy moiety was also considered.

This original compound was readily synthesised by using a two-

step method (Scheme 4). Cy 5.0 NHS ester 13 generated by

reaction with TSTU uronium salt in the presence of DIEA, in

dry NMP, was reacted with ethylenediamine (dihydrochloride

salt) under the conditions reported by Gruber et al. to give Cy

5.0 amine 14.³¹Finally, acylation of the primary amino group

of 14 with the NHS ester of 4-formylbenzoic acid (generated by

treatment with TSTU-DIEA in dry NMP) provided after RP-

HPLC puriﬁcation, the aminooxy-reactive Cy 5.0 analogue 15

(overall yield for the three steps 11%). The easy availability of

these monofunctional reactive dyes has enabled us to explore the

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Scheme 5 Reagents and conditions: (a) Thiol-reactive ﬂuorophore 11 or 12, CH₃CN, 0.1 M aq. NaHCO₃(pH 8.5), rt; (b) Cy 5.0 aldehyde 15, aq. sodium

acetate buffer (pH 4.2), rt.

MeOH–H₂O–pyridine (4 : 1 : 1) under reﬂux, by using a published

procedure.³³Conversion into NHS ester by treatment with TSTU-

DIEA in dry NMP and subsequent acylation reaction with

propargylamine led to the targeted alkyne derivative of AFB2 21 in

a moderate 32% yield. CuAAC reaction between this latter alkyne

and azido FRET cassette 19 was performed under the standard

conditions using CuSO₄and sodium ascorbate as the catalytic

system. This bioconjugation reaction was found to work well

without altering the ABF2 lactone core and the reporter cyanine

and xanthene dyes. The corresponding tri-molecular conjugate 22

was isolated in a pure form by RP-HPLC and its structure was

conﬁrmed by ESI mass spectrometry (see ESI‡). Interestingly, the

success of the present CuAAC reaction enables us to expand the

list of clickable ﬂuorophores for biological labelling to cyanine and

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HPLC in almost quantitative yields and their structures conﬁrmed

by ESI mass spectrometry (see ESI‡). Furthermore, these two

modiﬁed oligonucleotides were studied by UV-vis absorption and

ﬂuorescence spectroscopy (see ESI‡). The distance between the

two cyanine dye molecules within ﬂuorescent conjugate 24 was

determined by applying a methodology previously described for

a FRET ﬂuorogenic caspase-3 probe³⁹and was found to be 44

˚

1 A (for an energy transfer efﬁciency E = 0.75). These successful

examples support the fact that the successive three bioconjugation

reactions involving tripod reagent 3 can be performed on each case

with high yields, through easily reproducible fool-proof protocols

(especially for non-chemists), and at a small scale compatible with

highly valuable bio-chemical samples.

Preparation of ﬂuorescent tripod-functionalised surfaces suit-

able for the detection of aﬂatoxin B1 through the SPIT-FRI

immunoassay. In addition to these bioconjugations performed in

homogeneous solution, we planned to use azido tripod 3 toprepare

biochips suitable for mycotoxin AFB1 detection through the SPIT-

FRI method. Since, the implementation of such immunoassay

requires the ﬂuorescent labelling of AFB2 (a validated analogue

of AFB1 which is well recognised by anti-AFB1 monoclonal

antibodies (mAbs)) and its subsequent immobilisation on a glass

surface, we have performed the following derivatisations of 3: (1)

S_N2 reaction of its thiol group with iodoacetyl derivative of R6G-

WS 12, (2) oxime ligation between its free aminooxy moiety and an

aldehydic glass surface, and ﬁnally (3) CuAAC reaction between

the grafted azido and the alkyne derivative of AFB2 21, directly on

solid surface and according to the mild and non-denaturing “click”

conditions.⁴⁰However, under these conditions, we have observed

the deterioration of the tripod-functionalised surface and severe

quenching of R6G-WS ﬂuorescence. Thus, we have modiﬁed the

chronological sequence of derivatisation events (Scheme 8). After

ﬂuorescent labelling, we performed the CuAAC reaction with the

tripod bearing the free aminooxy moiety to get 25. We were pleased

to observe the formation of the desired product but the yield was

found to be modest (ca. 20%) and explained by the formation

of side-products probably stemming from the copper-catalysed

degradation of the free aminooxy moiety. The resulting ﬂuorescent

tripod was efﬁciently immobilised on an aldehydic glass surface

through oxime ligation performed in aqueous sodium acetate

buffer (pH 4.2). Before starting SPIT-FRI experiments through

ﬂuorescence signal observations, a capping step carried out in the

presence of the in-house-developed aminooxy acid pseudo-PEG

linker 26 (for its synthesis, see ESI‡) has enabled us to mask the

remaining reactive aldehyde functions.

Scheme 6 Reagents and conditions: (a) Aoaa, MeOH–H₂O-pyridine,

reﬂux; (b) TSTU, DIEA, DMF, rt, 1 h then propargylamine, rt,

1 h, 32% (after RP-HPLC puriﬁcation). TSTU = O-(N-succinimidyl)-

1,1,3,3-tetramethyluronium tetraﬂuoroborate.

xanthene derivatives, recently reported.³⁴Furthermore, the mild

conditions used for the three derivatisation steps did not lead to

signiﬁcant degradation of intermediary conjugates contrary to the

bioconjugation scheme published for the ﬁrst generation tripod

1, by avoiding deleterious chemicals such as hydrazine. We then

extended the scope of this labelling approach with energy transfer

dyes to DNA fragments: a synthetic 16-mer oligonucleotide 5¢-

d(TGA ACT GCA GCT CCT U) bearing the alkyne group as

a nucleotide modiﬁcation at the 2¢-position of uridine (16-mer

ODN).³⁵Indeed, Berndl et al. have recently demonstrated that the

CuAAC reaction is a valuable tool for the post-synthetic mod-

iﬁcation of oligonucleotides with brightly emitting ﬂuorophores

that are not stable under the typically strong basic conditions

involved during DNA cleavage and deprotection.³⁶Fluorescent

labelling of various synthetic DNA fragments were successfully

performed with base-labile phenoxazinium and coumarin dyes

but not yet with FRET cassettes. However, such constructions

should enable to get more powerful and sophisticated ﬂuorescent

labels for nucleic acids in screening assays or cell biology. In

this context, we decided to explore the “click” modiﬁcation of

16-mer ODN with FRET cassettes 18 and 19.³⁷Thus, CuAAC

reactions between 50–100 micromolar concentrations of energy

transfer azido dye and alkyne were performed in H₂O–DMSO

(1 : 1) with the catalytic system (CuSO₄-sodium ascorbate) and

DIEA and tris-(benzyltriazolylmethyl)amine (TBTA), a well-

known stabilising ligand for copper(I) species.³⁸The corresponding

ﬂuorescent oligonucleotides 23 and 24 were isolated by RP-

Indeed, we have already encountered some issues when we per-

formed SPIT-FRI experiments after aldehyde-capping with BSA

protein, especially non-speciﬁc adsorption of mAbs. Therefore,

we decided to saturate the surface through the formation of stable

oxime covalent linkages. In this context, an aqueous solution of

hydroxylamine could be used as an efﬁcient capping agent, but a

PEG derivative such as 26 was preferred because some recent work

has clearly underlined the positive effect of such a hydrophilic

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Scheme 7 Reagents and conditions: (a) AFB2–alkyne 21, H₂O–tBuOH (9 : 1, v/v), 0.1 M aq. CuSO₄, 0.1 M aq. sodium ascorbate, rt, 6 h; (b) 16-mer

ODN, H₂O-DMSO (1 : 1, v/v), 10 mM aq. CuSO₄, 10 mM TBTA in DMSO, 50 mM aq. DIEA, 10 mM aq. sodium ascorbate, rt, overnight.

spacer in decreasing the non-speciﬁc adsorption of proteins on

solid supports.⁴¹After washing, an intense ﬂuorescence signal

was observed and proved the efﬁcacy of the oxime ligation to

covalently graft ABF1 related tripods to the solid surface (Fig. 3A–

B, left). In a second step, FP647-labelled anti-AFB1 mAbs, which

are able to recognise both AFB1 and its analogue AFB2, were

with the SPIT-FRI method. Thus, the tripod-functionalised

glass surface previously prepared was transposed to a portable

biosensor.⁴²At the beginning of the experiment the ﬂuorescence

signal was very strong and stable despite continuous washing with

buffer (Fig. 4), conﬁrming the stability of the oxime linkage on

the solid-phase. Then, anti-AFB1 mAbs were introduced and

we observed a signiﬁcant decrease of the ﬂuorescence intensity

(Fig. 4, activation step). The quenching level is stable during the

subsequent washings thanks to the strong afﬁnity of the mAb

for AFB2. Finally, a solution containing original AFB1 was used

to displace mAbs interacting with the immobilised tripod. The

increase of the ﬂuorescence signal is modest but signiﬁcant enough

to conclude the integrity of our biochip (Fig. 4, competition step).

Further experiments are in progress to improve this detection-

quantiﬁcation immunoassay method and to expand its scope to a

larger number of natural toxins such anatoxins, microcystins and

saxitoxins.

R

added. As Fluo Probe^ꢀ647 acts as a quencher of R6G-WS, a

decrease in the ﬂuorescence emission of R6G-WS via FRET was

observed (Fig. 3A–B, middle). Finally, competition experiments

using original AFB1 aimed at detecting and quantifying this

mycotoxin have been done. As expected, the competitive process

occurring between the solid-phase tripod and AFB1 in solution

for the binding to the quencher-labelled mAb, led to an increase

of the measured ﬂuorescence (Fig. 3A–B, right). We observed

partial recovery (around 25%) of the original ﬂuorescence level. In

addition to this preliminary experiment, we tried to quantify AFB1

samples in the context of a continuous-ﬂow sensor compatible

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Scheme 8 Reagents and conditions: (a) Thiol-reactive ﬂuorophore 12, CH₃CN, 0.1 M aq. NaHCO₃(pH 8.5), rt; (b) AFB2-alkyne 21, H₂O-DMSO (1 : 1,

v/v), 10 mM aq. CuSO₄, 10 mM TBTA in DMSO, 50 mM aq. DIEA, 10 mM aq. sodium ascorbate, rt, overnight; (c) aq. sodium acetate buffer (pH 4.2),

rt, 2 h.

one pot procedure and so avoiding any intermediate RP-HPLC

puriﬁcation. Interestingly, such a one pot synthesis has been

recently reported by Mhidia et al. for the preparation of peptide-

protein conjugates by means of combining two reactions namely

Conclusion

In this paper, we have described for the ﬁrst time the synthesis

and four applications of an heterotrifunctional cross-linking

reagent whose full-orthogonality between its three bioconjugable

functions was obtained without using temporary protecting

groups. Furthermore, we have demonstrated that the sequential

derivatisation of this tripod can be performed with good to

high yields under mild conditions fully-compatible with various

fragile (bio)molecules and biopolymers. The chosen derivatisation

reactions (i.e., S_N2 reaction, oxime ligation and CuAAC reaction)

performed in aqueous buffers, are easy to perform and have

enabled us to establish an efﬁcient, and reproducible protocol

which opens up a new avenue for the synthetic access to highly

sophisticated bioconjugates. However, to speed up the three-

step bioconjugation methods involving the use of azido tripod

3, further work is in progress in our lab to perform the three

derivatisation reactions (or at least two of them) through a

amine acylation with N-succinimidyl carbamate (NSC chemistry)

and a-oxo semicarbazone ligation.⁴³

Experimental section‡

General

Column chromatography puriﬁcations were performed on

R

Geduran^ꢀSi 60 silica gel (40–63 mm) from Merck. 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.,

l = 254 nm), (2) spray with a 0.2% (w/v) ninhydrin solution in

absolute ethanol, (3) spray with a 3.5% (w/v) phosphomolybdic

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Fig. 3 (A) Immobilisation of ﬂuorescent tripod 25 on the aldehyde-functionalised silica surface by oxime ligation as described in Scheme 8 (left); binding

of FP647-labelled anti-AFB1 monoclonal antibodies (middle) and competition with AFB1 (right). (B) Relative ﬂuorescence intensity of the spot for each

step.

Fig. 4 SPIT-FRI experiment in the context of a continuous-ﬂow sensor: step 1 = washing with EIA buffer (0.1 M phosphate buffer (pH 7.4) containing

0.15 M NaCl, 0.1% tween 20, 0.1% PVP and 0.1 M sodium azide); step 2 = activation with a 1.5 mM solution (in EIA buffer) of anti-AFB1 mAbs; step

3 = washing with EIA buffer: step 4 = competition with a 0.5 mM solution (in EIA buffer) of AFB1. Inset: zoom for the competition step.

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acid solution in absolute ethanol. All solvents were dried following

standard procedures (CH₃CN: distillation over CaH₂, CH₂Cl₂:

distillation over P₂O₅, DMF and NMP: distillation over BaO).

DIEA and TEA were distilled from CaH₂and stored over

BaO. N-Sulfosuccinimidyl-(4-iodoacetyl)-aminobenzoate (sulfo-

SIAB) and tris-(benzyltriazolylmethyl)amine (TBTA) were pur-

chased from Pierce and Sigma-Aldrich respectively. N^a-(9-

Fluorenylmethoxycarbonyl)-L-lysine (Fmoc–Lys–OH) was pre-

pared by TFA-mediated removal of the N^a-Boc-protecting group

of commercially available N^a-(9-ﬂuorenylmethoxycarbonyl)-

N^e-(tert-butyloxycarbonyl)-L-lysine (Fmoc–Lys(Boc)–OH, Iris

Biotech). (Boc)₂-Aoaa-OH, AFB2 carboxylic acid derivative 20,

and the sulfoindocyanine dyes Cy 3.0 and Cy 5.0 were synthesised

following the literature procedures.^20,28,33Cysteine building block

C, pseudo-PEG derivatives 4 and 6, the water-soluble analogue

of rhodamine 6G (R6G-WS) and its thiol-reactive derivative 12

were prepared according to protocols recently reported by us.^10,29

The HPLC-gradient grade acetonitrile (CH₃CN) and methanol

(CH₃OH) were obtained from Acros or Fisher Scientiﬁc. Buffers

and aq. mobile-phases for HPLC were prepared using water puri-

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. Dual UV

detection was achieved at 220 and 260 nm. System B: RP-HPLC

(Thermo Hypersil GOLD C₁₈column, 5 mm, 4.6 ¥ 150 mm) with

CH₃CN and 0.1% aq. triﬂuoroacetic acid (aq. TFA, 0.1%, v/v, pH

2.2) as eluents [0% CH₃CN (5 min), followed by linear gradient

from 0 to 60% (30 min) then from 60 to 90% (10 min) of CH₃CN]

at a ﬂow rate of 1.0 mL min^-1. Triple UV detection was achieved

at 210, 260 and 285 nm. System C: RP-HPLC (Waters XTerra MS

C₁₈column, 5 mm, 7.8 ¥ 100 mm) with CH₃CN and aq. TFA, 0.1%

as eluents [0% CH₃CN (5 min), followed by linear gradient from

0 to 60% (40 min) of CH₃CN] at a ﬂow rate of 2.5 mL min^-1. Dual

UV-visible detection was achieved at 230 and 550 nm. System

D: RP-HPLC (Thermo Hypersil GOLD C₁₈column, 5 mm, 10 ¥

250 mm) with CH₃CN and aq. TFA, 0.1% as eluents [0% CH₃CN

(5 min), followed by linear gradient from 0 to 20% (5 min) then

from 20 to 60% (40 min) of CH₃CN] at a ﬂow rate of 4 mL

min^-1. Visible detection was achieved at 675 nm. System E: system

D with visible detection at 550 nm. System F: system D with

dual visible detection at 550 and 675 nm. System G: RP-HPLC

(Thermo Hypersil GOLD C₁₈column, 5 mm, 10 ¥ 100 mm) with

CH₃CN and aq. triethylammonium acetate buffer (aq. TEAA,

25 mM, pH 7.0) as eluents [0% CH₃CN (5 min), followed by linear

gradient from 0 to 20% (5 min) then from 20 to 60% (40 min) of

CH₃CN] at a ﬂow rate of 3 mL min^-1. Triple UV-visible detection

was achieved at 220, 550 and 675 nm. System H: system B with

the following gradient [0% CH₃CN (5 min), followed by linear

gradient from 0 to 60% (40 min) of CH₃CN] at a ﬂow rate of 1 mL

min^-1. Visible detection was achieved at 550 nm. System I: system

C with the following gradient [0% CH₃CN (5 min), followed by

linear gradient from 0 to 30% (15 min) then from 30 to 80%

(100 min) of CH₃CN] at a ﬂow rate of 2.5 mL min^-1. UV detection

was achieved at 260 nm. System J: system I with the following

gradient [5% CH₃CN (5 min), followed by linear gradient from

5 to 45% (40 min) of CH₃CN]. System K: RP-HPLC (Thermo

Hypersil GOLD C₁₈column, 5 mm, 4.6 ¥ 100 mm) with CH₃CN

and aq. TEAA 25 mM as eluents [0% CH₃CN (5 min), followed

by linear gradient from 0 to 80% (40 min) of CH₃CN] at a ﬂow

rate of 1.0 mL min^-1. Triple UV-visible detection was achieved

at 260, 550 and 650 nm. System L: RP-HPLC (Thermo Hypersil

GOLD C₁₈column, 5 mm, 2.1 ¥ 50 mm) with CH₃CN and aq.

AcOH, 0.1% as eluents [0% CH₃CN (5 min), followed by linear

gradient from 0 to 80% (40 min) of CH₃CN] at a ﬂow rate of

0.2 mL min^-1. Triple UV-visible detection was achieved at 220, 550

and 650 nm. ESI-MS detection was achieved both in negative and

positive modes. System M: RP-HPLC (Thermo Hypersil GOLD

C₁₈column, 5 mm, 10 ¥ 100 mm) with CH₃CN and aq. TFA, 0.1%

as eluents [0% CH₃CN (5 min), followed by linear gradient from

0 to 20% (5 min) then from 20 to 60% of CH₃CN] at a ﬂow rate

of 2.5 mL min^-1. Triple UV-visible detection was achieved at 220,

550 and 650 nm. System N: system C with the following column

Thermo Hypersil GOLD C₁₈(5 mm, 10 ¥ 100 mm).

ﬁed with a Milli-Q system (puriﬁed to 18.2 MX.cm). ¹H and ¹³

C

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(d_H= 7.26, d_C= 77.36) or CD₃OD (d_H= 3.31, d_C= 49.00).⁴⁴

J values are in Hz. Infrared (IR) spectra were recorded as a thin-

ﬁlm on sodium chloride plates or KBr pellets using a Perkin Elmer

FT-IR Paragon 500 spectrometer with frequencies given in recip-

rocal centimetres (cm^-1). UV-visible spectra were obtained on a

Varian Cary 50 scan spectrophotometer. Fluorophore-containing

compounds were quantiﬁed by UV-visible spectroscopy at the l_max

using the corresponding tabulated molar extinction coefﬁcient

(i.e., 65 000 dm³mol^-1cm^-1for R6G-WS, 150 000 dm³mol^-1

cm^-1for Cy 3.0 and 250 000 dm³mol^-1cm^-1for Cy 5.0).

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-visible 2000 detector. Mass spectra were obtained with either

a Thermo Finnigan LCQ Advantage Max (ion-trap), or a LCQ

Duo (ion-trap), or a LTQ Orbitrap XL apparatus equipped

with an electrospray source. The iodoacetamide derivative of

sulfocyanine dye Cy 3.0 11 and the intermediates NHS ester

and Cy 3.0 amine involved in its synthesis were characterised by

MALDI-TOF mass spectrometry on a Voyager DE PRO in the

reﬂector mode with a-cyano-4-hydroxycinnamic acid (CHCA) as

amatrix. Modiﬁedoligonucleotide 16-mer ODN was characterised

by MALDI-TOF mass spectrometry measurements on a Brucker

Biﬂex spectrometer using 3-hydroxypicolinic acid as a matrix. The

monoclonal antibodies anti-AFB1 were obtained as previously

described and the corresponding cross-reactivity experiments were

performed by using standard conditions.⁴⁵

High-performance liquid chromatography separations

Oligonucleotide synthesis

Several chromatographic systems were used for the analytical

experiments and the puriﬁcation steps. System A: RP-HPLC

(Thermo Hypersil GOLD C₁₈column, 5 mm, 10 ¥ 250 mm) with

The synthesis of oligonucleotide 16-mer ODN was carried out

on an Applied Biosystems 392 DNA/RNA synthesizer using

the phosphoramidite chemistry on a scale of 1 mmol. The

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insertion of 2¢-O-propargyl-uridine residue was performed by

using the corresponding phosphoramidite unit prepared following

the previously described methodologies.⁴⁶Upon completion, the

alkyne function-containing oligonucleotide has been deprotected

in concentrated aqueous ammonia for 16 h at 55 ^◦C. After speed-

vac evaporation of ammonia, the crude 5¢-DMTr oligonucleotide

was detritylated and puriﬁed on-line by RP-HPLC using a poly-

meric support.⁴⁷After desalting by size exclusion chromatography,

the oligonucleotide was then quantiﬁed by UV measurements

at 260 nm. The purity and the integrity of the synthetic DNA

oligomer were checked by RP-HPLC analyses together with

MALDI-TOF mass measurements. Finally the alkyne-modiﬁed

DNA fragment was lyophilised and freezed at -20 ^◦C until use in

the “click” reaction.

mixture was evaporated to dryness, taken up with AcOEt (10 mL),

washed by aq. 10% citric acid (10 mL), aq. sat. NaHCO₃(10 mL)

and brine (10 mL). The organic layer was dried over Na₂SO₄,

ﬁltrated and evaporated to dryness. The resulting residue was

puriﬁed by chromatography on a silica gel column (10 g) with

a mixture of CH₂Cl₂–MeOH (98 : 2, v/v) as the mobile phase, to

give azido-PEG-benzyl ester 7 as a yellow oil (314 mg, 0.74 mmol,

yield 91%). R_f(CH₂Cl₂–MeOH, 9 : 1, v/v) 0.47; n_max(neat)/cm^-1

593, 700, 753, 846, 1121, 1201, 1284, 1346, 1456, 1542, 1664, 1751,

2109, 2874, 3411 (broad); d_H(300 MHz; CDCl₃) 3.38–3.76 (m,

16H, CH₂PEG), 4.00 (s, 2H, CH₂PEG), 4.19 (s, 2H, CH₂PEG),

5.19 (s, 2H, CH₂Bn), 7.36 (s, 5H, Bn); d_C(75.5 MHz; CDCl₃) 38.6,

50.6, 66.6, 68.7, 69.9, 71.2, 70.3, 70.7, 70.9(2C), 128.5, 128.6, 128.7,

135.4, 169.9, 170.3; MS (ESI+): m/z 425.13 [M + H]⁺, calcd for

C₁₉H₂₈N₄O₇: 424.19.

(2-(2-Aminoethoxy)ethoxy)acetic acid benzyl ester (5)

Azido-PEG-acid spacer (A). Azido-PEG-benzyl ester spacer 7

(75 mg, 0.18 mmol) was dissolved in MeOH (2 mL) and aq. 1.0 M

LiOH (720 mL, 0.72 mmol) was added dropwise. The resulting

reaction mixture was stirred at room temperature for 2 h. The

reaction was checked for completion by TLC (CH₂Cl₂–MeOH,

9 : 1, v/v). The solution was acidiﬁed to pH ~ 4 by adding aq. 1.0 M

KHSO₄. Thereafter, volatiles were removed by evaporation under

reduced pressure and the resulting aq. residue was lyophilised to

give a white powder. This amorphous powder was suspended in

(a) Esteriﬁcation. Boc-protected amino acid 4 (600 mg,

2.28 mmol) was dissolved in dry CH₂Cl₂(10 mL). The resulting

solution was cooled to 4 ^◦C, and DMAP (17 mg, 0.14 mmol),

TEA (216 mL, 1.54 mmol) and benzyl chloroformate (239 mL,

1.68 mmol) were sequentially added. The resulting reaction

mixture was stirred at room temperature overnight and checked

for completion by TLC (CH₂Cl₂–AcOEt, 9 : 1, v/v). Volatiles were

removed by evaporation under reduced pressure; the resulting

residue was taken up in AcOEt (30 mL) and washed by aq. 10%

citric acid (30 mL), aq. sat. NaHCO₃(30 mL) and brine (30 mL).

The organic layer was dried over Na₂SO₄, ﬁltrated and evaporated

to dryness. The resulting residue was puriﬁed by chromatography

on a silica gel column (10 g) with a mixture of CH₂Cl₂–AcOEt

(9 : 1, v/v) as the mobile phase, giving benzyl ester as colorless oil

(414 mg, 1.2 mmol, yield 53%). R_f(CH₂Cl₂–AcOEt, 9 : 1, v/v) 0.18;

R

CH₂Cl₂and ﬁltrated through a Celiteꢀ 545 pad. The solvent was

removed by rotatory evaporation to give the azido pseudo-PEG-

acid spacer A as colorless oil (58 mg, 0.17 mmol, quantitative

yield). This compound was used in the next coupling reaction step

without further puriﬁcation. R_f(CH₂Cl₂–MeOH, 9 : 1, v/v) 0.20.

d_H(300 MHz; CD₃OD) 3.25–3.27 (m, 2H, CH₂PEG), 3.35–3.42

(m, 4H, CH₂PEG), 3.53 (t, J = 5.5 Hz, 2H, CH₂PEG), 3.59–3.66

(m, 10H, CH₂PEG), 3.96 (s, 2H, CH₂PEG), 4.02 (s, 2H, CH₂

PEG); d_C(75.5 MHz; CDCl₃): d 39.6, 51.7, 69.4, 70.4, 71.1, 71.2,

71.3, 71.3, 71.6, 72.0, 172.8, 174.5; MS (ESI-): m/z 333.47 [M -

H]^-, 667.00 [2M - H]^-, calcd for C₁₂H₂₂N₄O₇: 334.14.

n

_max(neat)/cm^-1699, 755, 864, 1121, 1149, 1251, 1365, 1391, 1455,

1517, 1711, 1755, 2926, 2925, 3370 (br); d_H(300 MHz; CDCl₃)

1.41 (s, 9H, tBu), 3.26–3.30 (m, 2H, CH₂PEG), 3.0 (t, J = 5.1 Hz,

2H, CH₂PEG), 3.60–3.63 (m, 2H, CH₂PEG), 3.68–3.73 (m, 2H,

CH₂PEG), 4.16 (s, 2H, CH₂PEG), 5.04 (bs, 1H, NH), 5.16 (s,

2H, CH₂Bn), 7.32 (s, 5H, Bn); d_C(75.5 MHz; CD₃OD) 28.4, 40.3,

66.6, 68.6, 70.3 (2C), 70.9, 79.1, 128.5, 128.6, 135.4, 156.0, 170.3;

MS (ESI+): m/z 353.93 [M + H]⁺, 376.07 [M + Na]⁺, calcd for

C₁₈H₂₇NO₆: 353.18.

Fmoc-Lys((Boc)₂-Aoaa)-OH (B). (Boc)₂-Aoaa-OH 9 (300 mg,

1.03 mmol) was dissolved in dry CH₃CN (10 mL). N-

hydroxysuccinimide (130 mg, 1.13 mmol) and DCC (233 mg,

1.13 mmol) were sequentially added and the resulting reaction

mixture was stirred at room temperature for 1 h. Thereafter, a

solution of Fmoc-Lys–OH (417.4 mg, 1.13 mmol) in dry CH₃CN

was added and the resulting reaction mixture was stirred at room

temperature. The reaction was checked for completion by TLC

(CH₂Cl₂–MeOH, 96 : 4, v/v) and evaporated to dryness. The

resulting residue was taken up with AcOEt (15 mL), washed by

aq. 10% citric acid (15 mL) and brine (15 mL). The organic layer

was dried over Na₂SO₄, ﬁltrated and evaporated to dryness. The

resulting residue was puriﬁed by chromatography on a silica gel

column (40 g) with a step gradient of MeOH (0–8%) in CH₂Cl₂as

the mobile phase, giving the targeted building block B as a white

foam (266 mg, 0.42 mmol, yield 40%). R_f(CH₂Cl₂–MeOH, 96 : 4,

v/v) 0.19; n_max(KBr)/cm^-1641, 732, 846, 912, 1041, 1125, 1350,

1455, 1538, 1670, 2254, 2938, 2983, 3321; d_H(300 MHz; CDCl₃)

1.36–2.06 (m, 24H, CH₂b, d, g Lys, 2 x tBu), 3.29 (t, J = 6.2 Hz,

2H, CH₂e Lys), 4.19 (t, J = 6.8 Hz, 1H, CH a Lys), 4.36 (d, 2H,

J = 7.2 Hz, CH₂Fmoc), 4.46 (s, 2H, CH₂Aoaa), 5.74 (d, J =

7.9 Hz, 1H, CH Fmoc), 7.26–7.91 (m, 8H, Fmoc); d_C(75.5 MHz;

CDCl₃) 22.4, 28.0, 28.8, 31.7, 38.8, 47.1, 53.7, 67.1, 76.4, 85.4,

(b) Boc removal. Benzyl ester obtained previously (287 mg,

0.81 mmol) was dissolved in CH₂Cl₂(20 mL). The solution was

cooled to 4 ^◦C and TFA (4.8 mL, 65 mmol) was added dropwise.

The mixture was stirred at room temperature for 1 h and checked

for completion by TLC (CH₂Cl₂–MeOH, 9 : 1, v/v). The mixture

was evaporated to dryness; the resulting residue was taken up in

deionised water (20 mL) and lyophilised to give the compound

5 as a yellow oil (quantitative yield). This compound was used

in the next coupling reaction step without further puriﬁcation or

analysis. R_f(CH₂Cl₂–MeOH, 9 : 1, v/v) 0.41.

Azido-PEG-benzyl ester spacer (7). Benzyl ester 5 (297 mg,

0.81 mmol) and (2-(2-azidoethoxy)ethoxy)acetic acid 6 (153 mg,

0.81 mmol) were dissolved in a mixture of DMF–CH₃CN (1 : 1,

v/v, 8 mL). BOP reagent (430 mg, 0.97 mmol) and DIEA

(402 mL, 2.43 mmol) were sequentially added. The resulting

reaction mixture was stirred at room temperature for 12 h and

checked for completion by TLC (CH₂Cl₂–MeOH, 9 : 1, v/v). The

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120.0, 125.2, 127.1, 127.7, 141.3, 143.8, 144.0, 150.5, 156.3; MS

dissolved in a mixture of CH₃CN–H₂O (2 : 1, v/v, 3 mL) and

puriﬁed by semi-preparative RP-HPLC (system A, 2 injections).

The product-containing fractions were lyophilised to give fully-

protected tripod 10 as a white amorphous powder (22 mg, 24 mmol,

yield 20%). d_H(300 MHz; CDCl₃) 1.31 (t, J = 7.4 Hz, 3H, CH₃SEt),

1.48–2.01 (m, 24H, CH₂b, d, g Lys, 2 x tBu), 2.71 (q, J = 7.4 Hz,

2H, CH₂SEt), 3.03–3.20 (m, 2H, CH₂b Cys), 3.30 (q, J = 6.6 Hz,

2H, CH₂e Lys), 3.41–3.71 (m, 16H, CH₂PEG), 4.00 (s, 2H, CH₂

PEG), 4.04 (s, 2H, CH₂PEG), 4.35–4.42 (m, 3H, CH a Cys, CH₂

Aoaa), 4.68–4.75 (m, 1H, CH a Cys), 5.50 (bs, 1H, NH), 6.72 (bs,

1H, NH), 7.40 (d, J = 7.0 Hz, 1H, NH), 7.67–7.71 (m, 1H, NH);

d_C(75.5 MHz; CDCl₃) 14.5, 23.0, 28.2, 29.1, 31.2, 32.6, 38.6, 38.8,

39.3, 50.7, 52.5, 53.7, 70.2, 70.3, 70.5, 70.7, 70.9, 71.1, 85.4, 150.6,

167.9, 170.3, 171.1, 172.0, 172.4; MS (ESI+): m/z 898.33 [M +

H]⁺, 920.20 [M + Na]⁺, calcd for C₃₅H₆₃N₉O₁₄S₂: 897.39.

(ESI-): m/z 640.47 [M - H]^-, calcd for C₃₃H₄₃N₃O₁₀: 641.29.

Fmoc-Lys((Boc)₂-Aoaa)-Cys(SEt)-NH₂(BC). Lysine building

block B (247 mg, 0.41 mmol) and the TFA salt of H-Cys(SEt)–NH₂

C (134 mg, 0.46 mmol) were dissolved in dry CH₃CN (3 mL). BOP

reagent (224 mg, 0.51 mmol) and dry DIEA (159 mL, 0.92 mmol)

were sequentially added and the resulting reaction mixture was

stirred at room temperature. After 5 min, dry NMP was added

(300 mL) to dissolve a newly formed precipitate and the reaction

stirred for a further 2 h. The reaction was checked for completion

by TLC (CH₂Cl₂–MeOH, 9 : 1, v/v) and evaporated to dryness.

The resulting residue was taken up with AcOEt (10 mL), washed

by aq. 10% citric acid (10 mL), aq. sat. NaHCO₃(10 mL) and

brine (10 mL). The organic layer was dried over Na₂SO₄, ﬁltrated

and evaporated to dryness. The resulting residue was puriﬁed by

chromatography on a silica gel column (20 g) with a step gradient

of MeOH (0–8%) in CH₂Cl₂as the mobile phase, to give full-

protected dipeptide BC as a white foam (208 mg, 259 mmol, yield

62%). R_f(CH₂Cl₂–MeOH, 8 : 2, v/v) 0.48; n_max(KBr)/cm^-1647,

733, 847, 911, 1034, 1122, 1371, 1455, 1530, 1693, 1756, 1790,

2249, 2870, 1932, 2981, 3064, 3322 (br); d_H(300 MHz; CDCl₃)

1.24 (t, J = 7.2 Hz, 3H, CH₃SEt), 1.41–1.91 (m, 24H, CH₂b,

d, g Lys, 2 x tBu), 2.66 (q, J = 7.4 Hz, 2H, CH₂SEt), 3.00–3.35

(m, 4H, CH₂b Cys, CH₂e Lys), 4.17–4.53 (m, 4H, CH a Lys,

CH Fmoc, CH₂Fmoc), 4.74–4.80 (m, 1H, CH a Cys), 7.27–7.76

(m, 8H, Fmoc); d_C(75.5 MHz, CDCl₃) 14.3, 19.0, 22.4, 27.9, 29.0,

31.3, 32.3, 32.4, 38.2, 39.5, 47.0, 52.0, 52.5, 53.5, 55.3, 67.1, 71.1,

76.5, 77.6, 85.2, 85.4, 119.9, 125.1, 127.1, 127.7, 141.2, 143.68,

143.73, 150.4, 156.7; MS (ESI+): m/z 804.07 [M + H]⁺, calcd for

C₃₈H₅₃N₅O₁₀S₂: 803.32.

Azido-tripod (3)

(a) Boc removal. Fully-protected tripod 10 (5.5 mg, 6 mmol)

was dissolved in dry CH₂Cl₂(800 mL) and the solution was cooled

to 4 ^◦C. TFA (37 mL, 480 mmol) was added dropwise and the

resulting reaction mixture was stirred at room temperature for 2 h.

The reaction was checked for completion by RP-HPLC (system

B) and then evaporated to dryness. The resulting oily residue

was dissolved in deionised water and lyophilised to give the free

aminooxy tripod (4.9 mg, 6 mmol, quantitative yield) as a yellow

oil. This compound was used in the next deprotection step without

further puriﬁcation or analysis.

(b) Removal of the SEt group. Aminooxy pseudo-peptide

(4.9 mg, 6 mmol) was dissolved in aq. 0.1 M NaHCO₃buffer

(pH 8.5, 500 mL). DTT (18 mg, 120 mmol) 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 B). 0.1% aq. TFA (2 mL) was added and the resulting

aq. solution was puriﬁed by semi-preparative RP-HPLC (system

C, 2 injections). The product-containing fractions were lyophilised

to give the targeted azido tripod 3 (0.9 mg, 1.4 mmol, yield 24%).

MS (ESI+): m/z 638.33 [M + H]⁺, 1275.00 [2M + H]⁺, calcd for

C₂₃H₄₃N₉O₁₀S: 637.28.

Fully-protected heterotrifunctional cross-linker (10)

(a) Fmoc removal. Fully-protected dipeptide BC (104 mg,

129 mmol) was dissolved in dry CH₃CN (3 mL). Diethylamine

(300 mL, 2.9 mmol) was added dropwise and the resulting mixture

was stirred at room temperature for 2 h. The reaction was

checked for completion by TLC (CH₂Cl₂–MeOH, 9 : 1, v/v) and

evaporated to dryness. The resulting residue was puriﬁed by

chromatography on a silica gel column (10 g) with a step gradient

of MeOH (0–50%) in CH₂Cl₂as the mobile phase, giving amine

building block as a white foam (72 mg, 124 mmol, yield 96%). This

intermediate compound was immediately used without further

analyses. R_f(CH₂Cl₂–MeOH, 8 : 2, v/v) 0.43.

Preparation of thiol-reactive Cy 3.0 derivative (11)

(a) Preparation of Cy 3.0 carboxylic acid, succinimidyl ester.

Cy 3.0 carboxylic acid (5.4 mg, 8.5 mmol) was introduced into a

Reacti-Vial^TMand dissolved in 145 mL of dry NMP. TSTU reagent

(2.55 mg, 8.5 mmol) and DIEA (3 mL, 16.9 mmol) in solution in

75 mL of dry NMP were added and 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

H) and the resulting N-hydroxysuccinimidyl ester was used in the

(b) Peptide coupling. Free N-terminal dipeptide (72 mg,

124 mmol) and azido-PEG-acid spacer A (50 mg, 149 mmol) were

dissolved in dry DMF (2 mL). BOP reagent (66 mg, 149 mmol)

and dry DIEA (43 mL, 248 mmol) were sequentially added and

the resulting reaction mixture was stirred at room temperature for

2 h. The reaction was checked for completion by TLC (CH₂Cl₂–

MeOH, 9 : 1, v/v) and the mixture was evaporated close to dryness.

The resulting residue was taken up with AcOEt (10 mL), washed

by aq. 10% citric acid (10 mL), aq. sat. NaHCO₃(10 mL) and brine

(10 mL). The organic layer was dried over Na₂SO₄, ﬁltrated and

evaporated to dryness. The resulting residue was ﬁrstly puriﬁed by

chromatography on a silica gel column (10 g) with a step gradient

of MeOH (0–10%) in CH₂Cl₂as the mobile phase but the purity

of recovered product was not satisﬁed. Then, this product was

next step without further puriﬁcation. HPLC (system H): t_R

=

26.1 min.

(b) Synthesis of Cy 3.0 amine. Ethylenediamine dihydrochlo-

ride (131 mg, 0.98 mmol) was dissolved in a mixture of DMF–H₂O

(85 : 15, v/v, 10.2 mL). The crude reaction mixture containing the

NHS ester of Cy 3.0 and a solution of DIEA (82.4 mL, 0.47 mmol)

in DMF (0.8 mL) were sequentially added. The resulting reaction

mixture was protected from light and stirred at room temper-

ature for 30 min. The reaction was checked for completion by

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RP-HPLC (system H) and the mixture was evaporated to dryness.

The resulting residue was dissolved in 0.1% aq. TFA (3 mL)

and puriﬁed by RP-HPLC (system I, 2 injections). The product-

containing fractions were lyophilised to give Cy 3.0 amine as a

pink amorphous powder. HPLC (system H): t_R= 21.1 min, purity

> 95%; MS (MADI-TOF+): m/z 673.40 [M + H]⁺, calcd for

C₃₃H₄₄N₄O₇S₂: 672.26.

AFB2-alkyne (21). AFB2 carboxylic acid derivative 20 (12 mg,

31 mmol) was dissolved in dry DMF (800 mL). TSTU reagent

(18.6 mg, 62 mmol) and DIEA (11 mL, 62 mmol) were sequentially

added. The mixture was stirred at room temperature for 1 h.

Thereafter, propargylamine (4 mL, 62 mmol) was added and the

mixture was stirred at room temperature for a further 1 h. Aq.

AcOH 0.1% was added to quench the reaction (1.2 mL) and then

the mixture was puriﬁed by RP-HPLC (system A, 2 injections).

The product-containing fractions were lyophilised to give AFB2-

alkyne 21 as a white powder (5 mg, 12 mmol, yield 32%). MS

(ESI+): m/z 425.13 [M + H]⁺, 447.27 [M + Na]⁺, calcd for

C₂₂H₂₀N₂O₇: 424.12.

(c) Preparation of Cy 3.0 SIAB derivative. Cy 3.0 amine

(1.0 mg, 1.35 mmol) was dissolved in 0.1 M aq. borate buffer

(100 mL, 50 mM + 5 mM EDTA, pH 8.2). A solution of sulfo-

SIAB reagent (0.96 mg, 1.92 mmol) in 0.1 M aq. borate buffer

(50 mL) was added. The resulting reaction mixture was protected

from light and stirred at room temperature for 2 h. The reaction

was checked for completion by RP-HPLC (system H). A further

amount of sulfo-SIAB reagent (0.35 mg, 0.69 mmol) in 0.1 M

aq. borate buffer (30 mL) and borate buffer (100 mL) were added

and the mixture was stirred again for 1 h. Finally, the reaction

mixture was quenched by dilution with 0.1% aq. TFA (3 mL)

and puriﬁed by RP-HPLC (system J, 2 injections). The product-

containing fractions were lyophilised to give the thiol-reactive Cy

3.0 SIAB derivative 11 as a pink powder (0.5 mg, 0.52 mmol, overall

yield for the three steps 3.5%). HPLC (system H): t_R= 24.9 min,

purity > 95%; MS (MADI-TOF+): 960.23 [M + H]⁺, calcd for

C₄₂H₅₀IN₅O₉S₂: 959.20.

FRET cassette (R6G-Cy5.0) labelled AFB2 (22)

(a) S_N2 reaction with iodoacetyl derivative of R6G-WS. Azido

tripod 3 (1.5 mg, 2.3 mmol) was dissolved in a mixture of CH₃CN

and 0.1 M aq. NaHCO₃buffer (1 : 3, v/v, 800 mL). Thiol-reactive

R6G-WS derivative 12 (2.8 mg, 2.6 mmol) was added and the

reaction mixture was stirred at room temperature for 4 h. The

reaction was checked for completion by RP-HPLC (system B)

and then quenched by addition of aq. TFA 0.1% (2 mL). The

mixture was puriﬁed by RP-HPLC (system E, 2 injections).

The product-containing fractions were lyophilised to give the

ﬂuorescent aminooxy tripod 17 as a pink amorphous powder.

MS (ESI+): m/z 807.60 [M + 2H]²⁺, 1613.20 [M + H]⁺, calcd for

C₆₈H₉₂N₁₆O₂₄S₃: 1612.56.

Aminooxy-reactive Cy 5.0 derivative (15)

(b) Oxime ligation with Cy 5.0 aldehyde. Fluorescent tripod

17 (0.5 mg, 310 nmol) was dissolved in acetate buffer (pH 4.2,

350 mL). Cy 5.0 aldehyde (0.42 mg, 620 nmol) was added and

the resulting mixture was stirred at room temperature for 4 h.

The reaction was checked for completion by RP-HPLC (system

B) and then quenched by addition of aq. TFA 0.1% (2 mL). The

mixture was puriﬁed by RP-HPLC (system F, 2 injections). The

product-containing fractions were lyophilised to give the energy

transfer azido dye 19 as a purple amorphous powder. MS (ESI+):

m/z 1214.53 [M + 2H]²⁺, calcd for C₁₁₁H₁₄₀N₂₀O₃₂S₅: 2424.85.

(a) Synthesis of Cy 5.0 amine. Cy 5.0 carboxylic acid (7.5 mg,

11 mmol) was dissolved in dry NMP (400 mL). TSTU reagent

(7 mg, 22 mmol) and DIEA (8 mL, 44 mmol) were sequentially

added. The resulting reaction mixture was stirred at room

temperature for 1 h. Meanwhile, ethylenediamine dihydrochloride

(65 mg, 0.49 mmol) was dissolved in a mixture of DMF–H₂O

(9 : 1, v/v, 10 mL) and DIEA (174 mL, 1 mmol) was then added.

Thereafter the mixture was cooled to 4 ^◦C and NHS ester of Cy 5.0

13 was added dropwise. The reaction was checked for completion

by RP-HPLC (system B) and then quenched by addition of glacial

acetic acid to reach pH 3-4. The mixture was evaporated to dryness

and then dissolved in aq. TFA 0.1%. The resulting solution was

puriﬁed by RP-HPLC (system D, 2 injections). The product-

containing fractions were lyophilised to give Cy 5.0 amine 14 as a

blue amorphous powder. The compound was used in the next step

without further puriﬁcation or analysis.

(c) CuAAC reaction with AFB2-alkyne. FRET cassette 19 (22

mg, 9 nmol) and AFB2-alkyne (5 mg, 12 nmol) were dissolved in

H₂O–tBuOH (9 : 1, v/v, 100 mL). 10 mL of a 0.1 M aq. CuSO₄

solution and 10 mL of a 0.1 M aq. sodium ascorbate solution

were added. The reaction mixture was stirred at room temperature

for 6 h, with further additions of 10 mL of aq. sodium ascorbate

solution every 90 min. The reaction was checked for completion by

RP-HPLC (system B) and then quenched by addition of aq. TFA

0.1% (2 mL). The mixture was puriﬁed by RP-HPLC (system F, 2

injections). The product-containing fractions were lyophilised to

give the ﬂuorescent conjugate of AFB2 22 as a purple amorphous

powder (16.2 mg, 8.1 nmol). MS (ESI-): m/z 1424.73 [M - 2H]^2-,

calcd for C₁₃₃H₁₆₂N₂₂O₃₉S₅: 2850.99.

(b) Preparation of Cy 5.0 aldehyde. 4-Formylbenzoic acid

(3.7 mg, 25 mmol) was dissolved in dry NMP (300 mL). TSTU

reagent (7.5 mg, 25 mmol) and DIEA (17 mL, 100 mmol) were

sequentially added. The resulting reaction mixture was stirred at

room temperature for 1 h. Cy 5.0 amine was dissolved in dry

NMP (100 mL) and then cooled to 4 ^◦C. The mixture containing

the NHS ester of 4-formylbenzoic acid was added dropwise to the

Cy 5.0 amine solution. The reaction was checked for completion

by RP-HPLC (system B) and then quenched by addition of aq.

TFA 0.1% (2 mL). The mixture was puriﬁed by RP-HPLC (system

D, 2 injections). The product-containing fractions were lyophilised

to give Cy 5.0 aldehyde 15 as a blue amorphous powder (0.93 mg,

1.2 mmol, overall yield for the two steps 11%). HPLC (system K):

t_R= 22.8 min, purity > 95%; MS (ESI+): m/z 831.40 [M + H]⁺,

calcd for C₄₃H₅₀N₄O₉S₂: 830.30.

FRET cassette (R6G-WS-Cy5.0) labelled oligonucleotide (23)

CuAAC reaction with alkyne-oligonucleotide. FRET cassette

19 (11.1 mg, 5 nmol) and alkyne-oligonucleotide 16-mer ODN (39

mg, 8 nmol) were dissolved in H₂O-DMSO (1 : 1, v/v, 80 mL). 8 mL

of a 10 mM aq. CuSO₄solution, 8 mL of 10 mM TBTA solution

in DMSO, 4 mL of a 50 mM aq. DIEA solution, and 8 mL of

10 mM aq. sodium ascorbate solution were sequentially added.

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The resulting reaction mixture was stirred at room temperature

for 90 min. Thereafter, further amounts of CuSO₄(8 mL) and

sodium ascorbate (8 mL) were added. The reaction mixture was

stirred at room temperature overnight. Further amounts of CuSO₄

(8 mL) and sodium ascorbate (8 mL) were added again and the

reaction was stirred at room temperature for 90 min. The crude was

desalted by size exclusion chromatography on a NAP-5 column

with deionised water as eluent. The fractions containing coloured

product were dried in a speed-vac for 4 h. The resulting residue

was taken up in a mixture of CH₃CN and 0.1 M aq. TEAA buffer

pH ~ 7 (1 : 9, v/v, 1 mL) and puriﬁed by RP-HPLC (system G, 1

injection). The product-containing fractions were dried in a speed-

vac to give the FRET cassette labelled oligonucleotide as a purple

v/v, 20 mL). 10 mL of a 10 mM aq. CuSO₄solution, 10 mL

of a 10 mM TBTA solution in DMSO, 4 mL of a 50 mM aq.

DIEA solution, and 10 mL of 10 mM aq. sodium ascorbate

solution were sequentially added. The resulting reaction mixture

was stirred at room temperature for 90 min. Further amounts of

CuSO₄(10 mL) and sodium ascorbate (10 mL) were added and

the reaction was stirred at room temperature overnight. Further

amounts of CuSO₄(10 mL) and sodium ascorbate (10 mL) were

added again and the reaction was stirred at room temperature

for a further 90 min. 0.1% aq. TFA (1 mL) was added and the

crude product was puriﬁed by RP-HPLC (system M, 1 injection).

The product-containing fractions were lyophilised to give the

ﬂuorescent aminooxy derivative 25 as a pink amorphous powder.

HPLC (system N): t_R= 28.0 min, purity 93%; MS (ESI+): m/z

1039.64 [M + H + K]²⁺, calcd for C₉₀H₁₁₂N₁₈O₃₁S₃: 2036.69

amorphous powder (16.2 mg, 8.1 nmol). HPLC (system K): t_R

=

21.8 and 21.9 min (mixture of two racemic diastereomers), purity

> 95%; MS (ESI-): m/z 728.76 [M - 10H]^10-, 809.84 [M - 9H]^9-,

911.20 [M - 8H]^8-, calcd: 7298.97.

(c) Immobilisation of ﬂuorescent AFB2 conjugate. A 40 mM

solution of the aldehyde-reactive ﬂuorescent AFB2 derivative 25

was prepared in sodium acetate buffer and 1 mL of this solution

was put over the freshly prepared aldehydic surface. After 2 h

of incubation in a humid atmosphere at room temperature, the

reaction solution was carefully removed and the surface was

washed with deionised water, 0.2% aq. SDS, deionised water and

dried. Fluorescence scanning was performed with an Olympus

inverted microscope model IX71 (4X objective) equipped with a

camera PCO 1600, and the resulting images were analysed using

ImageJ software (http://rsbweb.nih.gov/ij/).

FRET cassette (Cy3.0-Cy5.0) labelled oligonucleotide (24)

(a) S_N2 reaction with iodoacetyl derivative of Cy 3.0. Azido

tripod 3 (0.38 mg, 0.6 mmol) was dissolved in a mixture of CH₃CN

and 0.1 M aq. NaHCO₃buffer (1 : 3, v/v, 500 mL). Thiol-reactive

Cy 3.0 derivative 11 (0.5 mg, 0.52 mmol) was added and the reaction

mixture was stirred at room temperature for 2 h. The reaction was

checked for completion by LC-MS (system L) and then quenched

by addition of aq. TFA 0.1% (2 mL). The mixture was puriﬁed

by RP-HPLC (system M, 2 injections). The product-containing

fractions were lyophilised to give the ﬂuorescent aminooxy tripod

16 as a pink amorphous powder. MS (ESI+): m/z 850.4 [M + 2H

+ 2TFA]²⁺, calcd for C₆₅H₉₂N₁₄O₁₉S₃: 1468.58.

SPIT-FRI experiment

The detection of AFB1 through the SPIT-FRI method was

performed by using the thick layer previously prepared (vide

supra) and a continuous-ﬂow device (ﬂow rate of 100 mL min^-1,

thermostated at 22 ^◦C) previously described.⁴²Once the layer ﬁxed

into the device, the EIA buffer (0.1 M phosphate buffer (pH 7.4)

containing 0.15 M NaCl, 0.1% BSA and 0.01% sodium azide)

was pumped through the microchannels for 30 min. Then, the

activation step was performed by passing the anti-AFB1 mAb

solution (1.5 mM in EIA buffer) for 40 min. A further washing

using EIA buffer was done for 40 min. Finally, the competition

step was performed by passing the AFB1 solution (0.5 mM in EIA

buffer) was performed for 50 min.

(b) Oxime ligation with Cy 5.0 aldehyde. Fluorescent tripod 16

was dissolved in acetate buffer (pH 4.2, 350 mL). Cy 5.0 aldehyde

15 (62.3 mg, 75 nmol) was added and the resulting mixture was

stirred at room temperature for 4 h. The reaction was checked for

completion by LC-MS (system L) and then quenched by addition

of aq. TFA 0.1% (2 mL). The mixture was puriﬁed by RP-HPLC

(system M, 2 injections). The product-containing fractions were

lyophilised to give the energy transfer azido dye 18 as a purple

amorphous powder (2.6 mg, 8.2 nmol). MS (ESI+): m/z 1141.61

[M + 2H]²⁺, calcd for C₁₀₈H₁₄₀N₁₈O₂₇S₅: 2280.87.

(c) CuAAC reaction with alkyne-oligonucleotide. The same

conditions as those described for the synthesis and puriﬁcation of

ﬂuorescent oligonucleotide conjugate 23 were used. HPLC (system

K): t_R= 19.5 min, purity > 95%; MS (ESI-): m/z 714.36 [M -

10H]^10-, 793.96 [M - 9H]^9-, 893.45 [M - 8H]^8-, 1021.09 [M - 7H]^7-,

calcd: 7154.92.

Acknowledgements

This work was supported by the Agence Nationale de la Recherche

(Programme Emergence et Maturation 2005, ANR-2005-EMPB-

027-04), Commissariat a` l’Energie Atomique, Institut Univer-

sitaire de France (IUF) and Ose´o (A0905002P, especially for

the postdoctoral fellowship of Dr Guillaume Clave´). We thank

the “Re´gion Haute-Normandie” for ﬁnancial support via the

CRUNCh program (CPER 2007-2013). We thank Elisabeth Roger

(INSA de Rouen) for IR measurements, Dr Franc¸ois Fenaille

(CEA Saclay) for MS measurements on LTQ Orbitrap XL

apparatus, Christine Saint-Pierre (CEA Grenoble) and Dr Je´roˆme

Leprince (U413 INSERM/IFRMP 23) for MALDI-TOF mass

measurements, and QUIDD company for the open access to their

HPLC instruments.

Fluorescent labelling and immobilisation of mycotoxin AFB2 on

silica surfaces

(a) Preparation of the aldehyde-functionalised silica surface.

This was achieved from a silicon substrate (square, 10 ¥ 10) doped

with a thick SiO₂layer (thickness 500 nm) by using experimental

conditions already reported by us.¹⁰

(b) CuAAC reaction with alkyne derivative of AFB2. R6G-

WS ﬂuorescent labelled tripod 17 (100 mg, 60 nmol) and AFB2-

alkyne 21 (34 mg, 80 nmol) were dissolved in H₂O-DMSO (1 : 1,

4344 | Org. Biomol. Chem., 2010, 8, 4329–4345

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A universal and ready-to-use heterotrifunctional cross-linking reagent for facile synthetic access to sophisticated bioconjugates

DOI: 10.1039/c0ob00133c

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