Self-cleavable chemiluminescent probes suitable for protease sensing

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Self-cleavable chemiluminescent probes suitable for protease sensing†

Jean-Alexandre Richard,^a,dLudovic Jean,^a,b,dCaroline Schenkels,^a,dMarc Massonneau,^dAnthony Romieu*^a,b

and Pierre-Yves Renard*^a,b,c

Received 23rd March 2009, Accepted 22nd April 2009

First published as an Advance Article on the web 1st June 2009

DOI: 10.1039/b905725k

A new generation of dioxetane-based chemiluminescent substrates suitable for detecting protease

activities is described. Our strategy involves the use of a self-cleavable spacer as the key molecular

component of these protease-sensitive chemiluminescent probes. Among the assayed strategies, the

PABA (para-aminobenzylic alcohol) linker associated with an ether linkage enables the release of the

light-emitting phenolic 1,2-dioxetane moiety through an enzyme-initiated domino reaction. To validate

this strategy, two proteolytic enzymes were chosen: penicillin amidase and caspase-3, and the

corresponding self-cleavable chemiluminescent substrates were synthesised. Their evaluation using an

in vitro assay has enabled us to prove the decomposition of the linker under physiological conditions

and the selectivity for the targeted enzyme.

triggered by a biochemical reaction within a living organism.⁵

Introduction

Despite many problems associated with the cell penetration of

From the human genome data, it is now known that more than 500

the probes, bioluminescence is now widely used as a biochemical

genes encode proteases.¹Proteases mediate non speciﬁc protein

hydrolysis, but also have a vital role in post-translational mod-

and diagnostic tool, especially in the context of emerging and

challenging biomedical applications such as in vitro and in vivo

imaging.⁶

iﬁcations of proteins, performing selective and efﬁcient cleavage

of speciﬁc substrates.²In addition, the expression and activity

of such proteolytic enzymes are signiﬁcantly up-regulated in

several pathologies, including cancer, arthritis and atherosclerosis.

They can hence be considered as key biological markers for

these pathologies. Synthetic probes for the detection of protease

activity are therefore essential, and most probes use ﬂuorescence

detection.³Fluorescence can provide very sensitive monitoring,

but this technique suffers from several drawbacks associated with

the use of photonic excitation to promote ﬂuorescent moieties

to their emitting excited state: susceptibility to photobleaching

of commonly used organic ﬂuorophores, background interfer-

ences due to Raman and Rayleigh scatterings and high levels

of background ﬂuorescence when analysing cellular extracts or

imaging enzyme activities in vivo. Thus, luminescence technologies

which require no light excitation source are becoming extremely

attractive for reaching ultra-low detection limits for the targeted

analytes, and result from signiﬁcantly improved signal-to-noise

ratios thanks to the reduction or suppression of autoﬂuorescence

issues.⁴For these reasons, during the past half-century, consider-

able research effort has been devoted to the understanding and

applications of bioluminescence, a luminescence phenomenon

The implementation of this molecular detection technique

requires the use of exogenous enzymes (e.g., aequorin or the

different classes of luciferase)^7,8which are produced in various

organisms through genetic engineering. However, the need to

produce, handle and target luminescent proteins, and to co-localise

the corresponding modiﬁed luminescent substrate (generally a

modiﬁed luciferin), complicates the design of simple and reli-

able bioluminescent enzyme assays. To circumvent this problem,

chemiluminescence appears to be a good alternative since the same

advantages as bioluminescence could be obtained without the need

for genetically encoded enzymes. Among the chemiluminescent

species already reported, the most famous are luminol and oxalate

esters because of their usual applications in forensic science (for

the detection of blood) and in “light stick devices” respectively

(Fig. 1A).⁹However, these chemiluminescent species require ROS

(reactive oxygen species) such as hydrogen peroxide for the

triggering of light emission, which is a severe limitation for their

wider use in biological systems. Thus, the use of other high-

energy molecules such as 1,2-dioxetanes 1, whose luminescence

can be simply triggered by the cleavage of a chemical bond under

mild conditions, appears to be an excellent alternative strategy

to consider for biological applications.^7,10Indeed, the chemical

or enzymatic cleavage of 1 results in the release of an aromatic

anion 2 which undergoes an electron transfer according to a

CIEEL (Chemically Initiated Electron Exchange Luminescence)

mechanism.¹¹This process results in the formation of the ester

3 in the excited state which is responsible for the light emission

when returning back to the ground state (Fig. 1B).¹²Such

reporter probes using 1,2-dioxetane moieties have already been

used for the detection of biologically relevant enzymes such as

alkaline phosphatase,¹³neuramidinase,¹⁴acetylcholine esterase¹⁵

^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-

52-29-59

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

France

^cInstitut Universitaire de France, 103, bd Saint-Michel, 75005 Paris, France

^dQUIDD, Technopoˆle du Madrillet, 50, rue Ettore Bugatti, 76800 Saint-

Etienne du Rouvray, France

† Electronic supplementary information (ESI) available: Table 1 and Fig. 6.

See DOI: 10.1039/b905725k

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bond releasing an amine and a carboxylic acid), to the release

of a phenol, we choose to use a self-cleavable linker (also named

a self-immolative linker or reactive chemical adaptor). This self-

cleavable linker will be bound to the peptidyl substrate at one

end and to the dioxetane at the other. The amide bond cleavage

should thus lead to a spontaneous decomposition of the linker

yielding the free phenolate (Fig. 3B).^18,19Three years ago, we

patented our ﬁrst results concerning the development of self-

cleavable chemiluminescent substrates for protease assays,²⁰and

such chemical adaptor strategy has since been applied by other

groups to the bioluminescent substrate luciferin for designing

alkaline phosphatase-sensitive bioluminogenic substrates²¹and

for the construction of releasable luciferin-transporter conjugates

used as tools for the real-time analysis of cellular uptake in the

context of drug delivery.²²However, those assays all require the

ancillary addition of luciferase enzyme. We recently reported

our preliminary results on chemiluminescent species based on

phenolic 1,2-dioxetanes;²³reported here is the full account of

this study, focused on the choice of the self-cleavable linker and

implications of the chosen spacer-technology in the design of a

viable and straightforward synthetic route leading to protease-

sensitive chemiluminescent molecular systems.

Fig. 1 Chemical structure of the main compounds currently used as

chemiluminescent labels. A) Compounds chemically activated by basic

hydrogen peroxide; B) CIEEL-type 1,2-dioxetanes.

Results and discussion

Choice of the targeted enzymes

In order to design a new general method to detect proteases

through chemiluminescence, two amide bond cleaving enzymes

were targeted. Firstly, penicillin amidase (also called penicillin G

acylase, PGA) was chosen because this enzyme is able to recognise

and cleave the simple phenylacetyl moiety and thus constitutes

an interesting amine releasing enzyme for the choice of the self-

cleavable linker. Furthermore, this enzyme is inexpensive and

readily available in an immobilised form which tolerates the use of

an organic co-solvent such as acetone or methanol. Therefore, the

ﬁrst model chemiluminescent probes with unoptimised properties,

such as water solubility, could be rapidly evaluated. Afterwards,

extension to caspase-3 was envisaged in order to apply the strategy

to a representative and biologically signiﬁcant protease, and open

new perspectives for biological applications. Indeed, caspases

are Cysteine-Aspartic-acid-ProteASEs that play a critical role

in the apoptotic cascade (programmed cell death). Caspase-3

has been speciﬁcally identiﬁed as being a key mediator of

apoptosis in mammalian cells:²⁴activation of caspase-3 indicates

that the apoptotic pathway has progressed to an irreversible stage.

Disorders in the apoptotic process are involved in many diseases or

disorders such as ischemia, neurodegenerative diseases or cancers.

This protease is thus an attractive target for diagnostic and

therapeutic applications through its detection in biological media

using smart optical bioprobes. The main strategy for both in vitro

and in cellulo sensing of caspase-3 relies on the use of ﬂuorogenic

probes based either on the Fluorescence Resonance Energy

Transfer (FRET) or pro-ﬂuorescence processes.^19,25All these

probes are based on the fact that upon binding with caspase-3,

protease-mediated cleavage of their peptide substrate occurs after

the aspartic acid residue at the C-terminal side. The preferred

sequence for caspase-3 cleavage being Asp-Glu-Val-Asp.

Fig. 2 Representative 1,2-dioxetane-based chemiluminescent substrates

used for detecting enzyme activities.

or catalytic antibodies¹⁶(Fig. 2). In order to widen the scope

of such chemiluminescent probes, our interest was focused on

the development of chemiluminescent assays for new classes of

enzymes, and we chose to focus our ﬁrst efforts on proteases.

Thus, we investigated the use of such 1,2-dioxetanes as

luminescent markers of peptide bond cleavage events. CIEEL-

active dioxetanes bearing an aryl moiety meta-substituted with

an amide have been described only in a patent application for

the detection of peptidases. However, as the resulting aniline

released is not negatively charged under physiological conditions,

the electron transfer process is poorly efﬁcient and the light

emitted is particularly low (Fig. 3A).¹⁷Thanks to their lower

pKa, phenolic or thiophenolic 1,2-dioxetanes are the most suit-

able energy sources for such enzyme-sensitive chemiluminescent

bioprobes. Therefore, we decided to use phenolic 1,2-dioxetanes

to develop a general method for the detection of proteases by

chemiluminescence. In order to transfer the information between

the enzymatic reaction itself (namely, the cleavage of an amide

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Fig. 3 General mechanisms of the activation of chemiluminescent substrates by proteolytic enzymes. A) Release of aniline-based 1,2-dioxetanes having

poor light-emitting efﬁciency; B) release of light-emitting phenolate-based 1,2-dioxetanes through a self-immolative strategy using either Waldmann

traceless linker or PABA linker.

Synthesis of the amide and peptide substrates

multi-step synthetic scheme enabling the versatile introduction

of self-cleavable linkers and 1,2-dioxetane units. A crucial aspect

for the synthesis of this tetrapeptide was the choice of a suitable

side-chain protecting group for the aspartic and glutamic acids.

It must be stable to the acidic and basic conditions encountered

during the synthetic process of peptide chain elongation and also

removable under mild conditions (especially as far as the pH range

is concerned), since it needs to be removed at the very end of the

synthesis without destruction of the self-cleavable linker. It should

also be compatible with the 1,2-dioxetane ring formation, usually

undertaken through [2 + 2] cycloaddition. Indeed, it is well known

that 1,2-dioxetanes are sensitive to acidic media and we suspected

that the linkage connecting the phenol moiety to the self-cleavable

linker within the targeted chemiluminescent probes would be

not fully stable under basic conditions. Thus, we chose to use

2-(trimethylsilyl)ethyl esters (TMSE),²⁸since they are described

to be stable enough to undergo the multi-step peptide synthesis

The synthesis of the PGA substrate was straightforward since

commercially available phenylacetyl chloride could directly react

with the amino group of the chosen self-cleavable linker (vide infra)

to afford a carboxamide derivative suitable for the subsequent in-

troduction of the 1,2-dioxetane moiety. More challenging was the

synthesis of the caspase-3 peptidyl substrate, since an orthogonal

protection of the carboxylic acid side-chains towards both the

backbone amine and carboxylic acid functions is required. A solid-

phase peptide synthesis using a Fmoc/tBu strategy²⁶could be en-

visioned but was hampered by the problematic Asp-Glu sequence

which is prone to form undesired aspartimide derivatives especially

during the Fmoc removal step.²⁷A solution peptide synthesis using

the Boc/benzyl strategy was thus preferred, both to avoid this

tricky issue and to get the side-chain protected tetrapeptide in

a multigram scale which could be further derivatised through a

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and could be removed by treatment with a ﬂuoride ion source

under mild conditions,²⁹which have already been used with 1,2-

dioxetanes.

phosphonium salt in the presence of DIEA,³¹to yield the dipeptide

10 in quantitative yield. At this stage, the Boc protecting group

was removed and the N-terminal amino group of the dipeptide

was converted into acetamide to protect the resulting caspase-3

substrate against endogenous exopeptidases. It is noteworthy that

it was essential to isolate 11 as an ammonium salt to avoid an

undesired cyclisation side-reaction leading to the formation of

diketopiperazine. Finally, the deprotection of the benzyl ester

allowed the obtention of the ﬁrst dipeptide Ac-Asp(TMSE)-

Glu(TMSE)-OH 13 in 51% overall yield for the 8 steps. The

second dipeptide was obtained from 8 through a shorter synthetic

route of sequential Boc removal, BOP-mediated peptide coupling

with commercially available Boc-Val-OH 15 and Boc removal to

afford ammonium 17 in 77% overall yield. The two dipeptides

Thus, a highly convergent solution peptide synthesis was

performed starting from commercially available Boc-Glu-OBn

4, Boc-Asp-OBn 7 and Boc-Val-OH 15 (Schemes 1–3). The

carboxylic acid functionalities of the side-chains were protected

as TMSE esters by treatment with 2-trimethylsilylethanol and

DCC, in the presence of pyridine, to give 5 and 8 in quan-

titative yield.³⁰Removal of the Boc group of 5 by treatment

with TFA and the benzyl group of 8 under standard Pd/C

hydrogenolysis conditions afforded H-Glu(TMSE)-OBn 6 and

Boc-Asp(TMSE)-OH 9 respectively in good yields. These two

amino acid building blocks were coupled together using BOP

Scheme 1 Preparation of dipeptide Ac-Asp(TMSE)-Glu(TMSE)-OH 13.

Scheme 2 Preparation of dipeptide H-Val-Asp(TMSE)-OH 17.

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Scheme 3 Preparation of side-chain protected tetrapeptide Ac-Asp(TMSE)-Glu(TMSE)-Val-Asp(TMSE)-OH 19.

Ac-Asp(TMSE)-Glu(TMSE)-OH 13 and H-Val-Asp(TMSE)-

OBn 17 (TFA salt) were then assembled by using BOP/DIEA,

affording the expected tetrapeptide Ac-Asp(TMSE)-Glu(TMSE)-

Val-Asp(TMSE)-OH 19 after ﬁnal Pd/C hydrogenolysis to remove

the benzyl ester. The side-chain protected tetrapetide Asp-Glu-

Val-Asp substrate of caspase-3 was thus obtained through an

efﬁcient and convergent 13-step route with a 93% average yield

for each step.

to investigate the ingenious traceless linker recently developed

by Waldmann and Grether for solid-phase organic synthesis

applications (Fig. 3B).³³Indeed, this enzyme-labile safety catch

linker allows the release of alcohols or amines through an original

enzyme-initiated lactam-cyclisation. First, the conjugation of the

phenylacetyl moiety to a phenolic 1,2-dioxetane moiety by means

of this reactive chemical adaptor was explored, with the aim

of synthesising a chemiluminescent probe for the detection of

PGA (Scheme 4). Phenylacetamide 20, readily prepared in 4

steps from homovanillic acid, was coupled to phenolic enol ether

21, a precursor of the targeted 1,2-dioxetane, in the presence of

BOP/TEA³⁴to give the aryl ester 22 in 53% yield. Thereafter, the

key step of the synthesis, the [2 + 2] cycloaddition of singlet oxygen

with the enol ether moiety of 22, was performed. ¹O₂was generated

Choice of the self-cleavable linker and its introduction onto the

peptide scaffold. Synthesis of the chemiluminescent probes

Among the numerous self-cleavable linkers already described

especially in the context of pro-drug design,³²we ﬁrst chose

Scheme 4 Preparation of PGA chemiluminescent probe 23.

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Fig. 4 Synthetic strategy used for the preparation of caspase-3 chemiluminescent probe based on the Waldmann enzyme-labile safety catch linker.

under mild conditions through a method involving reduction of

ozone with triphenylphosphite.³⁵Such unusual conditions were

preferred to standard type II photosensitisation (i.e., oxygen,

visible light and a photosensitiser such as methylene blue, rose

bengal or TPP)³⁶as the amount of ¹O₂generated could be checked

more carefully, thus avoiding side reactions. The resulting 1,2-

dioxetane 23 was found to be stable on silica gel but puriﬁcation by

semi-preparative RP-HPLC was preferred to resolve the complex

reaction mixture containing 23, triphenylphosphate and some

minor side-products resulting from premature degradation of

the dioxetane. The structure of 23 was conﬁrmed by ESI mass

spectrometry.

tripartate pro-drugs which are readily activated under physio-

logical conditions by enzymatic cleavage through an efﬁcient

azaquinone-methide 1,6-elimination process.³⁹The potential use

of PABA as a suitable reactive chemical adaptor for self-cleavable

chemiluminescent probes was ﬁrst evaluated through the synthesis

of a PGA-sensitive chemiluminescent substrate (Scheme 5). PABA

reacted with phenylacetyl chloride and the resulting benzylic

alcohol was activated as the mesylate ester 27 as previously

described.¹⁹Then, etheriﬁcation with sodium salt of phenol 21

was performed and enol ether 28 could be obtained in 54%

yield. Finally, the [2 + 2] cycloaddition reaction was achieved

under the same conditions as described for 22. The resulting 1,2-

dioxetane 29 was found to be stable enough to be puriﬁed by

silica gel column chromatography in a good yield. Comparatively,

this synthetic scheme and the yields obtained proved that PABA is

more practical than the Waldmann linker for the design of a simple

and versatile synthetic route leading to the targeted protease-

sensitive chemiluminescent probes, since this heterobifunctional

linker is commercially available and so 1,2-dioxetane 29 was

obtained in only 4 steps and 31% overall yield.

This interesting ﬁnding led us to envisage a further extension

of this synthetic strategy to the more functionalised peptidyl

substrate of caspase-3. For this purpose, PABA was introduced

onto the C-terminal side of tetrapeptide 19 through a peptide

coupling reaction, but this synthetic process was accompanied

by epimerisation at the asymmetric centre of the C-terminal

aspartic acid residue (Scheme 6). Indeed, the weak nucleophilicity

of the amino group of PABA was responsible for a competitive

cyclisation reaction leading to the formation of stereochemically

labile 5(4H)-oxazolone, which easily racemised under the coupling

reaction conditions giving rise to a mixture of diastereomers

30a and 30b.⁴⁰It is interesting to note that a recent publication

described similar problems for the coupling of a 7-aminocoumarin

ﬂuorescent label on a Asp-Glu-Val-Asp peptide sequence.⁴¹The

authors found that the use of HATU as a coupling reagent and

TBP as a base decreased the epimerisation ratio from 75% to 3%.

In our case, we found that the use of such a base had a deleterious

effect on the epimerisation level (see ESI,† Table 1, entries 1–3).

Surprisingly the addition of a stoichiometric amount of HOBt,

an additive well-known to reduce racemisation,⁴²did not improve

the diastereomeric ratio in favour of 30a (Table 1,† entries 3–4).

By using EEDQ,⁴³a coupling reagent which works under neutral

conditions, a better diastereoisomeric ratio (80:20) was obtained

(Table 1,† entry 5) but ﬁnally the best coupling conditions were

found as follows: the use of EDC and a slow warming of the

This ﬁrst success in the synthesis of a self-cleavable chemilu-

minescent probe led us to envisage the extension of the chosen

synthetic strategy to the more functionalised peptidyl substrate of

caspase-3 (Fig. 4). Thus, starting from 3,4-dihydroxyphenylacetic

acid 24, amino acid 25 was efﬁciently synthesised in three steps and

introduced onto the C-terminal carboxylic acid of tetrapeptide 19.

Further derivatisation of the resulting peptide–linker conjugate

with the phenolic enol ether 21, precursor of the targeted 1,2-

dioxetane, through the BOP-mediated esteriﬁcation reaction led to

compound 26. At this stage, we tested the deprotection conditions

for the TMSE esters in order to check the selectivity of their

removal towards the undesired hydrolysis of the aryl ester bond

between the linker and the enol ether moiety. Unfortunately, in

our hands all our efforts to remove the TMSE esters ended

up with a simultaneous release of enol ether 21. In addition,

acidic ﬂuoride ion sources such as TEA·3HF, HF·pyridine or

TBAF buffered with PTSA³⁷were not effective at inducing the

deprotection of TMSE esters. Basic ﬂuoride ion sources such as

TBAF (1.0 M in THF) or TAS-F³⁸were effective at obtaining an

efﬁcient removal of the TMSE esters but were accompanied by a

quantitative release of enol ether 21 in the reaction mixture. The

use of the Waldmann safety catch linker and of other associated

reactive linkers implying amine cyclisation onto an ester linkage

thus appeared incompatible with the use of TMSE protected

polypeptides. Other orthogonal protecting groups could have been

1

used, such as allyl esters, but they are sensitive to O₂[2 + 2]

cycloaddition conditions.

Thus, also taking into account the potential fragility of ester

linkages in the context of in cellulo and in vivo applications,

we have chosen to replace this “cyclisation” based self-cleavable

linker by an “elimination” based self-cleavable linker such as

para-aminobenzylic alcohol (PABA), a linker widely used in pro-

drug strategies.³²In addition, this spacer enables the design of

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Scheme 5 Preparation of PGA chemiluminescent probe 29.

Scheme 6 Preparation of peptide–PABA conjugate 30 by EDC-mediated peptide coupling reaction.

reaction mixture from 0 ^◦C to 20 ^◦C (Table 1,† entry 6). In

these conditions, only 10% of the undesired diastereoisomer was

detected by RP-HPLC. The synthesis was taken further using

the diastereoisomeric mixture and the small amount of undesired

epimer 30b was removed in the next steps (vide infra).

linkage cleaved. On the other hand, it showed that the stability

of the carbonate linkage was not compatible with the reaction

conditions required for the removal of the TMSE groups, and

could lead to a high non speciﬁc light emission.

A more stable linkage had thus to be investigated to connect the

1,2-dioxetane to the self-cleavable linker. Therefore, by analogy

with the preparation of the self-cleavable PGA chemiluminescent

probe 29 (vide supra) we decided to use again the ether function

even if the synthetic yield was expected to be modest (Scheme 8).

Conversion of the benzylic alcohol into the mesylate derivative

34 and its subsequent nucleophilic substitution with 21 afforded

enol ether 35 in a moderate 44% overall yield for the two steps.

As observed for carbonate derivative 32, the conversion rate

of the [2 + 2] cycloaddition was found to be close to 50%

and 1,2-dioxetane 36 was obtained with an unoptimised 25%

yield (moreover, unreacted enol ether 35 could be recovered and

recycled). Finally, the complete removal of the TMSE side-chain

protecting groups was successfully performed by treatment with

TBAF (1.0 M in THF), highlighting the higher stability of the

ether moiety compared to the previously used ester and carbonate

linkages. Puriﬁcation was achieved by semi-preparative RP-HPLC

to give the caspase-sensitive chemiluminescent substrate 37 in

quantitative yield. Importantly, for this chromatographic puriﬁ-

cation, the use of a slightly alkaline mobile phase (i.e., aq. TEAB

50 mM, pH 7.5) was found to be essential to prevent premature

cleavage of the 1,2-dioxetane moiety, observed in particular with

After the introduction of the PABA linker, several strategies

were investigated to connect it to the phenolic enol ether 21.⁴⁴

Knowing that the etheriﬁcation reactions of peptide–PABA

conjugate 27a with phenolic compounds were often tricky and

not always reproducible,¹⁹the ﬁrst synthetic route explored was

the activation of this benzylic alcohol 30a as a para-nitrophenyl

carbonate derivative to form a carbonate linkage with 21.⁴⁵Thus,

enol ether 21 was reacted with activated carbonate 31 to afford

1

32 in 58% yield. Then, the [2 + 2] cycloaddition of O₂to enol

ether 32 was performed. Contrary to the reaction of the enol

ether 28 derived from phenylacetic acid, only 50% conversion was

obtained but 1,2-dioxetane 33 was isolated in a pure form by semi-

preparative RP-HPLC. The removal of the undesired diastereoiso-

mer was achieved during this chromatographic puriﬁcation step.

Finally, the deprotection of the TMSE esters was carried out.

Unfortunately, in the presence of TBAF or TAS-F, the unstable

phenolate 1,2-dioxetane 2 was released which decomposed rapidly

to excited benzoate 3, emitting a persistent blue ﬂuorescent light

which was observed in the reaction ﬂask (Scheme 7). On the one

hand, this observation was encouraging, since it conﬁrmed that

such 1,2-dioxetanes were efﬁcient light emitters once the PABA

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Scheme 7 Attempted preparation of the caspase-3 chemiluminescent probe bearing a carbonate linkage between PABA and the light-emitting

1,2-dioxetane moiety.

deionised water. The structure of 37 was conﬁrmed by ESI mass

spectrometry.

to reach a maximum (after 90 s in the reaction conditions of

Fig. 5A) and thereafter slowly decreased. It is noteworthy that even

after 7.5 min, light emission still occurred. Finally, an increase in

the amount of light emitted with length of enzymatic reaction time

was observed, highlighting the efﬁciency of the enzymatic cleavage

and the subsequent decomposition of the self-cleavable spacer

(Fig. 5B). The same peptide cleavage reaction was performed with

a decreased amount of caspase-3 introduced into the reaction

buffer, and allowed us to ﬁnd a detection limit of around 1.0 pmol

of enzyme (Fig. 5C). Furthermore, the high degree of speciﬁcity of

the cleavage was demonstrated by two control reactions in which

the chemiluminescent peptide 37 was incubated with PGA and

caspase-9 respectively. Indeed, in both cases, no light emission

was detected (data not shown).

Chemiluminescent phenylacetamide derivatives 23 and 29 were

assayed against PGA and the same experimental conditions

required for unveiling the chemiluminescence of the released

phenolate 1,2-dioxetane 2 in aq. buffers (vide supra) were used.

Light emission and time-dependant changes in luminescence

intensity centred at 530 nm similar to those displayed in Fig. 5A,

were observed with 29, conﬁrming the efﬁciency of our strategy

based on a PABA self-cleavable linker and ether linkage to

detect different types of protease or related proteolytic enzymes.

Furthermore, we have shown that the light emitted was different

from the one generated through ﬁreﬂy bioluminescence. Indeed,

this latter emission is known to proceed via multiple short-lived

Characterization of chemiluminescent probes through in vitro

protease assay

With the three self-cleavable chemiluminescent probes 23, 29 and

37 in hand, their selective cleavage by the targeted protease (i.e.,

PGA for 23 and 29, and caspase-3 for 37) was investigated. As

already reported in the literature by several of us,¹⁵a ﬁnal time

detection was preferred to observe the chemiluminescence follow-

ing the peptide bond cleavage event. The addition of enhancers

proved critical for the observation of a signiﬁcant light signal in

aqueous media and so restricted the use of such chemiluminescent

probes to in vitro assays. Thus, some additives were introduced

in the enzyme buffer: a surfactant (i.e., cetyltrimethylammonium

bromide, CTAB) was used in order to create a hydrophobic

environment (through the formation of a micelle) around the

light-emitting 1,2-dioxetane moiety and a ﬂuorophore (i.e., 5-

(stearoylamino)ﬂuorescein, l_em= 530 nm) to perform an energy

transfer between the enzymatically released 1,2-dioxetane and the

xanthene-based ﬂuorophore.⁴⁶Thus, after incubation of 37 with

recombinant human capase-3, the light emitted was recorded and

a spontaneous emission was detected around 530 nm, highlighting

a complete energy transfer between 2 (normal emission at l_em

460 nm) and the ﬂuorophore. The luminescence rapidly increased

=

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Scheme 8 Preparation of caspase-3 chemiluminescent probe 37.

ﬂashes of light.⁷On the contrary, the light emission observed in our

case was a “glow” type chemiluminescence which is characteristic

of phenolate 1,2-dioxetane 2 (see ESI,† Fig. 6). This feature is

of great importance for subsequent applications since it would

allow the observation of a prolonged light signal (i.e., over

more than 10 min) in biological media. On the other hand, we

observed an anomalous behaviour of the PGA probe 23, whose

chemiluminescence was unveiled directly during incubation only

with the buffer containing enhancers, and without the enzyme.

This non-enzymatic activation of 23 was attributed to the release

of phenolate 1,2-dioxetane 2 through the hydrolytic cleavage of its

aryl ester linkage, conﬁrming that the Waldmann linker and related

self-cleavable spacers using a carboxyl functional group to graft the

phenol-based chemiluminescent species to the peptidyl substrate,

are not valuable candidates for our latent chemiluminophore

strategy.

dioxetane moiety. Our investigations have led us to identify the

self-cleavable linker PABA and its connection to the 1,2-dioxetane

by an ether linkage as the optimal combination for designing a

reliable and robust synthetic route for these smart optical bio-

probes. This ﬂexible strategy constitutes a general method for the

detection of proteolytic enzymes because a large set of ﬂuorogenic,

bioluminogenic or chemoluminogenic substrates can be produced

by changing only the enzyme recognition unit or the phenol-based

optical marker. It could also be easily adapted to other amine

releasing enzymes. Further work is under investigation to improve

the properties of chemiluminescent caspase-3 substrate 37 aimed

at using it in biological media such as cellular extracts. This work

is currently on-going in our laboratory and will be reported in due

course.

Experimental

Conclusion

General

We have described the synthesis of the ﬁrst self-cleavable chemilu-

minescent probes suitable for the detection of protease (or related

proteolytic enzymes) activities. An efﬁcient strategy was devised

thanks to the use of a reactive chemical adaptor which allowed us

to reconcile the detection of a protease involving a peptide bond

cleavage event and the release of a light-emitting phenolic 1,2-

Column chromatographies were performed on silica gel (40–

63 mm) from SdS. TLC were carried out on Merck DC Kieselgel

60 F-254 aluminium sheets. Compounds were visualised by one

or both of the following methods: 1) staining with a 0.2% (w/v)

ninhydrin solution in absolute ethanol, 2) staining with a 3.5%

(w/v) phosphomolybdic acid solution in absolute ethanol. All

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expressed in parts per million (ppm) from CDCl₃(d_H= 7.26,

d_C= 77.36) or CD₃OD (d_H= 3.34, d_C= 49.86).⁴⁷J values are

expressed in Hz. Optical rotations were measured with a Perkin

Elmer 341 polarimeter. Infrared (IR) spectra were recorded as

thin-ﬁlm on sodium chloride plates or KBr pellets using a Perkin

Elmer FT-IR Paragon 500 spectrometer. UV–visible spectra

were obtained on a Varian Cary 50 scan spectrophotometer.

Chemiluminescence 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 either with a Finnigan LCQ Advantage MAX (ion trap)

apparatus equipped with an electrospray source or by MALDI-

TOF mass spectrometry on a Voyager DE PRO in the reﬂector

mode with a-CHCA as a matrix.

High-performance liquid chromatography separations

Several chromatographic systems were used for the analytical

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

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

CH₃CN and triethylammonium acetate (TEAA, 100 mM, pH 7.0)

as the eluents [75% TEAA (2 min), then linear gradient from 25

to 100% (30 min) of CH₃CN] at a ﬂow rate of 1.0 mL min^-1. Dual

UV–visible detection was achieved at 254 and 285 nm. System B:

RP-HPLC (Macherey-Nagel Nucleosil C₁₈column, 5 mm, 10 ¥

250 mm) with CH₃CN and deionised water as the eluents [75%

H₂O (5 min), then linear gradient from 25 to 55% (15 min) and 55

to 90% (70 min) of CH₃CN] at a ﬂow rate of 5.0 mL min^-1. UV

detection was achieved at 260 nm. System C: RP-HPLC (Thermo

Hypersil GOLD C₁₈column, 5 mm, 4.6 ¥ 150 mm) with CH₃CN

and deionised water as the eluents [100% H₂O (5 min), then linear

gradient from 0 to 100% (40 min) of CH₃CN] at a ﬂow rate of

1.0 mL min^-1. Dual UV–visible detection was achieved at 210

and 254 nm. System D: RP-HPLC (Thermo Hypersil GOLD C₁₈

column, 5 mm, 10 ¥ 250 mm) with CH₃CN and TFA 0.1% as the

eluents [80% TFA (5 min), then linear gradient from 20 to 68%

(20min), 68to80%(10min)and80to100%(5min)ofCH₃CN]ata

ﬂow rate of 5.0 mL min^-1. Dual UV–visible detection was achieved

at 210 and 250 nm. System E: system C with the following gradient

[75% H₂O (2 min), then linear gradient from 25 to 100% (30 min)

of CH₃CN]. System F: RP-HPLC (Thermo Hypersil GOLD C₁₈

column, 5 mm, 10 ¥ 250 mm) with CH₃CN and deionised water

as the eluents [75% H₂O (3 min), then linear gradient from 25 to

65% (15 min) and 65 to 100% (60 min) of CH₃CN] at a ﬂow rate

of 5.0 mL min^-1. Dual UV–visible detection was achieved at 210

and 250 nm. System G: RP-HPLC (Thermo Hypersil GOLD C₁₈

column, 5 mm, 10 ¥ 250 mm) with CH₃CN and triethylammonium

bicarbonate (TEAB, 50 mM, pH 7.5) as the eluents [100% TEAB

(5 min), then linear gradient from 0 to 100% (65 min) of CH₃CN]

at a ﬂow rate of 4.0 mL min^-1. Dual UV–visible detection was

achieved at 210 and 250 nm.

Fig. 5 In vitro cleavage of self-cleavable chemiluminescent probe 37 by

recombinant human caspase-3. A) Chemiluminescence spectra of probe

37 (concentration: 30 mM) in buffer containing enhancers (CTAB, 2 mM,

ﬂuorescein 0.18 mM) recorded after incubation with recombinant human

caspase-3 (1.6 10^-3U, incubation time: 105 min); B) maximum light

emission of probe 37 as a function of the incubation time with recombinant

human caspase-3; C) determination of the detection limit of caspase-3:

light emission could be observed until a caspase-3 amount of 1.31 pmol.

solvents were dried following standard procedures (acetonitrile:

distillation over CaH₂, dichloromethane: distillation over P₂O₅,

DMF: distillation over BaO under reduced pressure). DIEA,

pyridine and triethylamine were distilled from CaH₂and stored

over BaO. Phenolic enol ether 21 derived from adamantanone

was synthesised using literature procedures⁴⁴and preparation of

phenylacetamide 20 and mesylate esters 27 and 34 was recently

described by us.^19,20Recombinant human caspase-3 and 9 enzymes

(respectively 5.52 U/mg and 7139.39 U/mg) were purchased

from Sigma. Recombinant A. faecalis PGA (0.63 U/mg) was

gratefully furnished by Pr. L. Fischer, Universita¨t Hohenheim

(Institut fu¨r Lebensmitteltechnologie, Fachgebiet Biotechnologie,

Stuttgart). The HPLC-grade acetone and acetonitrile (CH₃CN)

were obtained from Acros and Panreac respectively. Buffers

(caspase-3, caspase-9, alkaline and phosphate buffers) and aq.

mobile phases for HPLC were prepared using deionised water

puriﬁed with a Milli-Q system (puriﬁed to 18.2 MX.cm). Tri-

ethylammonium acetate (TEAA, 1.0 M) and triethylammonium

bicarbonate (TEAB, 1.0 M) buffers were prepared from distilled

triethylamine and glacial acetic acid and CO₂gas respectively.

¹H and ¹³C NMR spectra were recorded on a Bruker DPX 300

spectrometer (Bruker, Wissembourg, France). Chemical shifts are

General procedure for the side chain protection of glutamic and

aspartic acids Boc-Glu(TMSE)-OBn (5) and Boc-Asp(TMSE)-

OBn (8). Boc-Glu-OBn or Boc-Asp-OBn was dissolved in dry

CH₃CN (0.8 M) and dry DMF was added until complete

solubilisation. To this solution was added dry pyridine (2 equiv)

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and 2-(trimethylsilyl)ethanol (1.2 equiv). The mixture was cooled

to 0 ^◦C, then DCC (1.1 equiv) was added and the resulting reaction

mixture was stirred for 16 h at room temperature. Oxalic acid

(5.0 M in DMF) was added and the mixture was stirred for 20 min.

The DCU precipitate was ﬁltered, washed with AcOEt and the

combined ﬁltrates were evaporated to dryness. The resulting oily

residue was dissolved in AcOEt and washed successively with a

sat. solution of NaHCO₃, deionised water, aq. citric acid (10%)

and again with deionised water. The organic layer was dried

over Na₂SO₄, ﬁltered and concentrated under reduced pressure.

The crude product was puriﬁed by chromatography on silica gel

(CH₂Cl₂–AcOEt, 95 : 5, v/v) for 5 and pure CH₂Cl₂for 8, yielding

the corresponding TMSE ester.

CH), 4.15 (t, J = 8.0 Hz, 2H, OCH₂), 5.15 (s, 2H, ArCH₂O),

7.35 (s, 5H, 5ArH); ¹³C NMR (75.4 MHz, CDCl₃): d -1.2 (3C),

17.6, 30.0, 31.1, 54.2, 63.0, 67.1, 128.6–128.9 (3 peaks, 5C), 135.9,

173.6, 175.8; IR (neat): n_max3323, 3019, 2957, 2898, 1732, 1694,

1520, 1455, 1417, 1392, 1326, 1251, 1217, 1042; MS (MALDI-

TOF, positive mode, a-CHCA matrix): m/z 338.47 [M + H]⁺,

calcd for [C₁₇H₂₈NO₄Si]⁺338.18; elemental analysis: calcd (%) for

C₁₇H₂₇NO₄Si: C, 60.50; H, 8.06; N, 4.15; found: C, 60.34; H, 7.89;

N, 4.03.

H-Asp(TMSE)-Glu(TMSE)-OBn 11 (TFA salt, yellow oil,

82%): R_f(CH₂Cl₂–AcOEt, 1 : 1, v/v) 0.5; [a]²¹+5.1^◦(c 1.01

365

in CHCl₃); ¹H NMR (300 MHz, CDCl₃): d 0.02 (s, 9H, Si(CH₃)₃),

0.03 (s, 9H, Si(CH₃)₃), 0.91–1.02 (m, 4H, 2CH₂Si), 1.97–2.23 (m,

2H, CH_2Glu), 2.33 (t, J = 7.0 Hz, 2H, CH_2GluCO), 2.93 (d, J =

7.0 Hz, 2H, CH_2Asp), 4.10–4.24 (m, 4H, 2OCH₂), 4.49 (t, J =

7.0 Hz, 1H, C*H_Asp), 4.52–4.60 (m, 1H, C*H_Glu), 5.10 (Abd, J =

12 Hz, 1H, 0.5ArCH₂O), 5.18 (Abd, J = 12 Hz, 1H, 0.5ArCH₂O),

7.33 (s, 5H, 5ArH), 7.95 (d, J = 8.0 Hz, 1H, NH_Glu); ¹³C NMR

(75.4 MHz, CDCl₃): d -1.4 (3C), -1.3 (3C), 17.4 (2C), 26.8, 30.5,

35.2, 50.1, 52.6, 63.8, 65.1, 67.9, 128.6–129.0 (3 peaks, 5C), 135.2,

168.6, 171.0, 171.4, 174.1; IR (neat): n_max3361, 2954, 2886, 2360,

1737, 1682, 1506, 1455, 1386, 1250, 1172, 1063; MS (MALDI-

TOF, positive mode, a-CHCA matrix): m/z 575.46 [M + Na]⁺,

calcd for [C₂₆H₄₄N₂O₇Si₂Na]⁺575.26.

Boc-Glu(TMSE)-OBn 5 (colorless oil, quantitative yield): R_f

(CH₂Cl₂–AcOEt, 95 : 5, v/v) 0.5; [a]²¹+4.6^◦(c 1.12 in CHCl₃);

365

¹H NMR (300 MHz, CDCl₃): d 0.03 (s, 9H, Si(CH₃)₃), 0.95 (t,

J = 7.0 Hz, 2H, CH₂Si), 1.43 (s, 9H, C(CH₃)₃), 1.91–1.96 (m, 1H,

0.5CH₂), 2.15–2.19 (m, 1H, 0.5CH₂), 2.27–2.42 (m, 2H, CH₂CO),

4.14 (t, J = 7.0 Hz, 2H, OCH₂), 4.35–4.41 (m, 1H, CH), 5.10 (d,

J = 9.0 Hz, 1H, NH), 5.17 (s, 2H, ArCH₂O), 7.35 (s, 5H, 5ArH);

¹³C NMR (75.4 MHz, CDCl₃): d -1.3 (3C), 17.5, 27.9, 28.5 (3C),

30.7, 53.3, 63.1, 67.4, 80.2, 128.5–128.9 (3 peaks, 5C), 135.5, 155.6,

172.4, 173.0; IR (neat): n_max3371, 2955, 2899, 1732, 1500, 1455,

1391, 1367, 1251, 1169, 1049; MS (ESI+): m/z 460.27 [M + Na]⁺,

calcd for [C₂₂H₃₅NO₆SiNa]⁺460.21; elemental analysis: calcd (%)

for C₂₂H₃₅NO₆Si: C, 60.38; H, 8.06; N, 3.20; found: C, 60.36; H,

8.02; N, 3.44.

H-Asp(TMSE)-OBn 14 (yellow oil, 85%): R_f(CH₂Cl₂–AcOEt,

1 : 1, v/v) 0.5; [a]²¹₃₆₅-84.0^◦(c 1.01 in CHCl₃); ¹H NMR (300 MHz,

CDCl₃): d 0.02 (s, 9H, Si(CH₃)₃), 0.95 (t, J = 7 Hz, 2H, CH₂Si),

1.79 (s, 2H, NH₂), 2.70 (dd, J = 7.0 and 17.0 Hz, 1H, 0.5CH₂), 2.79

(dd, J = 5.0 and 17.0 Hz, 1H, 0.5CH₂), 3.82–3.86 (m, 1H, CH),

4.14 (t, J = 7.0 Hz, 2H, OCH₂), 5.16 (s, 2H, ArCH₂O), 7.35 (s,

5H, 5ArH); ¹³C NMR (75.4 MHz, CDCl₃): d -1.2 (3C), 17.6, 39.3,

51.6, 63.4, 67.3, 128.6–128.9 (2 peaks, 5C), 135.8, 171.6, 174.5; IR

(neat): n_max3196, 3065, 2954, 2897, 1735, 1681, 1498, 1455, 1250,

1172, 1042; MS (MALDI-TOF, positive mode, a-CHCA matrix):

m/z 324.48 [M + H]⁺, calcd for [C₁₆H₂₆NO₄Si]⁺324.16; elemental

analysis: calcd (%) for C₁₆H₂₅NO₄Si: C, 59.41; H, 7.79; N, 4.33;

found: C, 59.34; H, 7.39; N, 4.23.

Boc-Asp(TMSE)-OBn 8 (colorless oil, quantitative yield): R_f

(CH₂Cl₂) 0.5; [a]²¹

+12.7^◦(c 0.98 in CHCl₃); ¹H NMR

365

(300 MHz, CDCl₃): d 0.02 (s, 9H, Si(CH₃)₃), 0.93 (t, J = 9.0 Hz,

2H, CH₂Si), 1.43 (s, 9H, C(CH₃)₃), 2.79 (dd, J = 4.5 and 17.0 Hz,

1H, 0.5CH₂), 3.00 (dd, J = 4.5 and 17.0 Hz, 1H, 0.5CH₂), 4.11

(t, J = 9.0 Hz, 2H, OCH₂), 4.57–4.63 (m, 1H, CH), 5.14 (Abd,

J = 12 Hz, 1H, 0.5ArCH₂O), 5.22 (Abd, J = 12.0 Hz, 1H,

0.5ArCH₂O), 5.52 (d, J = 9.0 Hz, 1H, NH), 7.34 (s, 5H, 5ArH);

¹³C NMR (75.4 MHz, CDCl₃): d -1.2 (3C), 17.5, 28.6 (3C), 37.2,

50.4, 63.7, 67.7, 80.4, 128.5–128.8 (3 peaks, 5C), 135.6, 155.7,

171.3 (2C); IR (neat): n_max3376, 2955, 2899, 1724, 1499, 1456,

1412, 1391, 1367, 1342, 1251, 1169, 1046; MS (ESI+): m/z 446.33

[M + Na]⁺, calcd for [C₂₁H₃₃NO₆SiNa]⁺446.20; elemental analysis:

calcd (%) for C₂₁H₃₃NO₆Si: C, 59.55; H, 7.85; N, 3.31; found: C,

59.41; H, 7.76; N, 3.33.

H-Val-Asp(TMSE)-OBn 17 (TFA salt, white solid, 92%): R_f

(CH₂Cl₂–AcOEt, 1 : 1, v/v) 0.5; [a]²¹+38.7^◦(c 0.97 in CHCl₃);

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¹H NMR (300 MHz, CDCl₃): d 0.01 (s, 9H, Si(CH₃)₃), 0.88–0.99

(m, 8H, CH₂Si + 2CH_3Val), 2.13–2.19 (m, 1H, CHCH₃), 2.76 (dd,

J = 5.0 and 17.0 Hz, 1H, 0.5CH₂), 2.98 (dd, J = 5.0 and 17.0 Hz,

1H, 0.5CH₂), 3.85 (d, J = 5.0 Hz, 1H, C*H_Val), 4.05–4.13 (m,

2H, OCH₂), 4.91–4.94 (m, 1H, C*H_Asp), 5.09 (bd, J = 12 Hz, 1H,

0.5ArCH₂O), 5.17 (Abd, J = 12 Hz, 1H, 0.5ArCH₂O), 7.27–7.31

(m, 5H, 5ArH); 7.55 (d, J = 8.0 Hz, 1H, NH_Asp), 8.10 (s_br, 3H,

NH₃⁺); ¹³C NMR (75.4 MHz, CDCl₃): d -1.3 (3C), 17.4, 17.8,

18.0, 30.6, 35.9, 49.1, 59.0, 64.0, 68.1, 128.8–128.9 (2 peaks, 5C),

135.3, 168.3, 170.3, 171.7; IR (KBr): n_max3366, 2947, 1671, 1542,

1429, 1385, 1353, 1288, 1180, 1138, 1055; MS (ESI+): m/z 423.13

[M + H]⁺, calcd for [C₂₁H₃₅N₂O₅Si]⁺423.23; elemental analysis:

calcd (%) for C₂₃H₃₅F₃N₂O₇Si: C, 51.48; H, 6.57; N, 5.22; found:

C, 51.51; H, 6.43; N, 5.17.

General procedure for the removal of Boc protecting groups.

Amino acid 5 (or 8) or dipeptide 10 (or 16) building block was

dissolved in dry CH₂Cl₂(0.1 M). The solution was cooled to 0 ^◦C

and TFA (20 equiv) was added. The reaction mixture was stirred at

room temperature for 2 h. Then, the reaction mixture was cooled to

0 ^◦C and a solution of sat. NaHCO₃was added. The aqueous layer

was extracted twice with CH₂Cl₂and the combined organic layers

were washed with deionised water, dried over Na₂SO₄, ﬁltered and

concentrated under reduced pressure to afford the corresponding

primary amine (for the dipeptides 11 and 17, 1.2 equiv of TFA was

added to form the corresponding ammonium salt).

H-Glu(TMSE)-OBn 6 (yellow oil, 84%): R_f(CH₂Cl₂–AcOEt, 1 :

General procedure for the removal of Bn protecting groups.

Amino acid 8 or peptide 12 (or 18) building block _◦was dissolved

in AcOEt (0.06 M). The solution was cooled to 0 C and Pd/C

catalyst (10%, 40 mg for each mmol of amino acid or peptide) was

added. Then, the reaction mixture was stirred under a hydrogen

1, v/v) 0.5; [a]²¹-2.4^◦(c 1.12 in CHCl₃); ¹H NMR (300 MHz,

365

CDCl₃): d 0.03 (s, 9H, Si(CH₃)₃), 0.97 (t, J = 8.0 Hz, 2H, CH₂Si),

1.51 (s, 2H, NH₂), 1.81–1.91 (m, 1H, 0.5CH₂), 2.04–2.14 (m, 1H,

0.5CH₂), 2.43 (t, J = 7.5 Hz, 2H, CH₂CO), 3.49–3.54 (m, 1H,

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atmosphere at room temperature for 16 h. Thereafter, Pd/C

the mixture was stirred for 20 min in order to convert the excess of

acetic anhydride to AcOEt. The reaction mixture was concentrated

to dryness and the resulting oily residue was dissolved in CH₂Cl₂

(50 mL). The organic phase was successively washed with aq.

citric acid (10%), deionised water, sat. NaHCO₃and deionised

water (20 mL each). The combined organic layers were dried over

Na₂SO₄, ﬁltered and concentrated under reduced pressure. The

crude product was puriﬁed by chromatography on a silica gel

column using CH₂Cl₂–AcOEt (7 : 3, v/v) as the mobile phase,

affording Ac-Asp(TMSE)-Glu(TMSE)-OBn 12 as a colorless oil

which quickly crystallized (617 mg, 77%). R_f(CH₂Cl₂–AcOEt, 7 :

R

catalyst was removed by ﬁltration through a Celite^ꢀ545 pad,

and the ﬁltrate was concentrated under vacuum to yield the

corresponding carboxylic acid.

Boc-Asp(TMSE)-OH 9 (colorless crystals, quantitative yield):

R_f(CH₂Cl₂–CH₃OH, 95 : 5, v/v) 0.4; [a]²¹+106.3^◦(c 0.98 in

365

1

CHCl₃); H NMR (300 MHz, CDCl₃): d 0.03 (s, 9H, Si(CH₃)₃),

0.98 (t, J = 7.0 Hz, 2H, CH₂Si), 1.43 (s, 9H, C(CH₃)₃), 2.80 (dd,

J = 5.0 and 17.0 Hz, 1H, 0.5CH₂), 3.00 (dd, J = 4.0 and 17.0 Hz,

1H, CH₂), 4.18 (t, J = 7.0 Hz, 2H, OCH₂), 4.58–4.61 (m, 1H, CH),

5.56 (d, J = 9.0 Hz, 1H, NH), 11.05 (brs, 1H, COOH); ¹³C NMR

(75.4 MHz, CDCl₃): d 1.2 (3C), 17.6, 28.6 (3C), 37.0, 50.1, 63.9,

80.7, 155.9, 171.7, 176.5; IR (KBr): n_max3445, 2956, 1736, 1658,

1519, 1456, 1412, 1393, 1371, 1355, 1335, 1289, 1252, 1219, 1196,

1175, 1066, 1048; MS (MALDI-TOF, positive mode, a-CHCA

matrix): m/z 372.42 [M + K]⁺, calcd for [C₁₄H₂₆NO₆SiK]⁺372.12;

elemental analysis: calcd (%) for C₁₄H₂₆NO₆Si: C, 50.43; H, 8.16;

N, 4.20; found: C, 50.35; H, 8.13; N, 4.21.

3, v/v) 0.5; [a]²¹+5.5^◦(c 0.95 in CHCl₃); ¹H NMR (300 MHz,

436

CDCl₃): d 0.03 (s, 18H, 2Si(CH₃)₃), 0.93–1.02 (m, 4H, 2CH₂Si),

1.90–2.04 (m, 4H, CH₃+ CH_2Glu), 2.1–2.4 (m, 4H, CH_2GluCO +

CH_2Glu), 2.57 (dd, J = 6.0 and 17.0 Hz, 1H, 0.5CH_2Asp), 2.98 (dd,

J = 4.0 and 17 Hz, 1H, 0.5CH_2Asp), 4.11–4.21 (m, 4H, 2OCH₂),

4.52–4.62 (m, 1H, C*H_Glu), 4.77–4.87 (m, 1H, C*H_Asp), 5.15 (s,

2H, ArCH₂O), 6.78 (d, J = 8 Hz, 1H, NH_Asp), 7.34 (s, 5H, 5ArH);

¹³C NMR (75.4 MHz, CDCl₃): d -1.2 (6C), 17.5, 17.6, 23.6, 27.2,

30.6, 36.2, 49.4, 52.4, 63.4, 63.9, 67.6, 128.6–129.0 (3 peaks, 5C),

135.5, 170.4, 170.9, 171.4, 172.8, 173.3; IR (KBr): n_max3290, 3092,

2955, 2899, 1733, 1643, 1557, 1456, 1425, 1385, 1352, 1304, 1250,

1179, 1164, 1060; MS (MALDI-TOF, positive mode, a-CHCA

matrix): m/z 617.63 [M + Na]⁺, calcd for [C₂₈H₄₆N₂O₈Si₂Na]⁺

617.27; elemental analysis: calcd (%) for C₂₈H₄₆N₂O₈Si₂: C, 56.54;

H, 7.79; N, 4.71; found: C, 56.63; H, 7.65; N, 4.52.

Ac-Asp(TMSE)-Glu(TMSE)-OH 13 (colorless oil, quantitative

yield): R_f(100% AcOEt) 0.2; [a]²¹+32.1^◦(c 0.39 in CHCl₃);

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¹H NMR (300 MHz, CDCl₃): d 0.03 (s, 9H, Si(CH₃)₃), 0.06 (s,

9H, Si(CH₃)₃), 0.96–1.02 (m, 4H, 2CH₂Si), 1.96–2.10 (m, 4H,

CH₃+ 0.5CH_2Glu), 2.15–2.30 (m, 1H, 0.5CH_2Glu), 2.32–2.51 (m,

2H, CH_2GluCO), 2.63 (dd, J = 6.0 and 17.0 Hz, 1H, 0.5CH_2Asp),

2.98 (dd, J = 4.0 and 17.0 Hz, 1H, 0.5CH_2Asp), 4.09–4.22 (m, 4H,

2OCH₂), 4.46–4.53 (m, 1H, C*H_Glu), 4.83–4.89 (m, 1H, C*H_Asp),

7.03 (d, J = 8.0 Hz, 1H, NH_Asp), 7.56 (d, J = 8.0 Hz, 1H, NH_Glu);

¹³C NMR (75.4 MHz, CDCl₃): d -1.17 (6C), 17.5, 17.6, 23.5, 26.8,

30.9, 36.1, 49.6, 52.6, 63.5, 64.0, 171.3, 171.4, 172.7, 173.9, 174.0;

IR (neat): n_max3308, 2954, 1733, 1656, 1536, 1389, 1250, 1173,

1063; MS (MALDI-TOF, positive mode, a-CHCA matrix): m/z

527.58 [M + Na]⁺, calcd for [C₂₁H₄₀N₂O₈Si₂Na]⁺527.22; elemental

analysis: calcd (%) for C₂₁H₄₀N₂O₈Si₂, 0.14CH₂Cl₂: C, 49.13; H,

7.86; N, 5.42; found: C, 48.87; H, 7.84; N, 5.50

General procedure for the peptide coupling. The amine (1

equiv), the carboxylic acid (1 equiv) and BOP phosphonium salt

(1 equiv) were dissolved in dry CH₃CN (0.1 M). Then, DIEA (3

equiv) was added and the resulting reaction mixture was stirred

at room temperature for 45 min. The solvent was removed under

vacuum, the resulting oil was dissolved in CH₂Cl₂and washed

with a sat. solution of NaHCO₃. The aqueous layer was extracted

with CH₂Cl₂and the combined organic layers were washed with

deionised water, dried over Na₂SO₄, ﬁltered and concentrated

under reduced pressure. The crude product was puriﬁed over silica

gel with CH₂Cl₂–AcOEt as eluents.

Ac-Asp(TMSE)-Glu(TMSE)-Val-Asp(TMSE)-OH 19 (gray

solid, quantitative yield): R_f(CH₂Cl₂–MeOH, 95 : 5, v/v) 0.5;

[a]²¹₃₆₅+17.5^◦(c 0.94 in CHCl₃); ¹H NMR (300 MHz, CD₃OD): d

0.08 (s, 27H, 3Si(CH₃)₃), 0.96–1.08 (m, 12H, 3CH₂Si + 2CH_3Val),

1.91–2.05 (m, 4H, 0.5CH_2Glu+ CH₃), 2.12–2.24 (m, 2H, CHCH₃+

0.5CH_2Glu), 2.38–2.50 (m, 2H, CH_2GluCO), 2.65–2.93 (m, 4H,

2CH_2Asp), 4.16–4.28 (m, 7H, C*H_Val+ 3OCH₂), 4.40–4.45 (m, 1H,

C*H_Glu), 4.70–4.80 (m, 2H, 2C*H_Asp), 7.97 (d, J = 8.0 Hz, 1H,

NH_Val), 8.22 (d, J = 8.0 Hz, 1H, NH_Asp), 8.32 (d, J = 8.0 Hz, 1H,

NH_Asp), 8.41 (d, J = 8.0 Hz, 1H NH_Glu); ¹³C NMR (75.4 MHz,

CD₃OD): d -0.6 (9C), 19.0 (3C), 19.6, 20.6, 23.4, 28.8, 32.3, 32.6,

37.8, 38.1, 51.2, 52.2, 55.0, 61.0, 64.7, 65.0, 65.2, 173.1, 173.3,

173.8, 173.9, 174.0, 174.2(0), 174.2(4), 175.7; IR (KBr): n_max3926,

2958, 1732, 1694, 1668, 1645, 1564, 1538, 1393, 1360, 1251, 1177,

1064; MS (MALDI-TOF, positive mode, a-CHCA matrix): m/z

841.57 [M + Na]⁺, calcd for [C₃₅H₆₆N₄O₁₂Si₃Na]⁺841.39; elemental

analysis: calcd (%) for C₃₅H₆₆N₄O₁₂Si₃,1.2CH₂Cl₂: C, 47.20; H,

7.48; N, 6.08; found: C, 47.14; H, 7.22; N, 6.21.

Boc-Asp(TMSE)-Glu(TMSE)-OBn 10 (yellow oil, 98%): R_f

(CH₂Cl₂–AcOEt, 7 : 3, v/v) 0.5; [a]²¹+44.7^◦(c 1.06 in CHCl₃);

365

¹H NMR (300 MHz, CDCl₃): d 0.03 (s, 18H, 2Si(CH₃)₃), 0.93–

1.01 (m, 4H, 2CH₂Si), 1.45 (s, 9H, C(CH₃)₃), 1.97–2.04 (m, 1H,

0.5CH_2Glu), 2.19–2.37 (m, 3H, CH_2GluCO + 0.5CH_2Glu), 2.62 (dd,

J = 6.0 and 17.0 Hz, 1H, 0.5CH_2Asp), 2.99 (dd, J = 5.0 and 17 Hz,

1H, 0.5CH_2Asp), 4.11–4.20 (m, 4H, 2OCH₂), 4.46–4.54 (m, 1H,

C*H_Asp), 4.59–4.66 (m, 1H, C*H_Glu), 5.16 (s, 2H, ArCH₂O), 5.68

(d, J = 8.0 Hz, 1H, NH_Asp), 7.18 (d, J = 8.0 Hz, 1H, NH_Glu), 7.34 (s,

5H, 5ArH); ¹³C NMR (75.4 MHz, CDCl₃): d -1.2 (6C), 17.6 (2C),

27.7, 28.6 (3C), 30.4, 36.5, 50.8, 52.1, 63.2, 63.7, 67.6, 80.8, 128.5–

128.9 (3 peaks, 5C), 135.5, 155.7, 171.1, 171.6, 172.5, 173.1; IR

(neat): n_max3350, 2955, 2899, 1732, 1682, 1520, 1455, 1391, 1367,

1250, 1170, 1062; MS (MALDI-TOF, positive mode, a-CHCA

matrix): m/z 675.62 [M + Na]⁺, calcd for [C₃₁H₅₂N₂O₉Si₂Na]⁺

675.31; elemental analysis: calcd (%) for C₃₁H₅₂N₂O₉Si₂: C, 57.03;

H, 8.03; N, 4.29; found: C, 57.13; H, 7.71; N, 4.26.

Synthesis of Ac-Asp(TMSE)-Glu(TMSE)-OBn (12). TFA salt

of H-Asp(TMSE)-Glu(TMSE)-OBn 11 (892 mg, 1.34 mmol) was

dissolved in dry CH₃CN (7 mL). Then, acetic anhydride (505 mL,

5.35 mmol, 4 equiv) and dry pyridine (650 mL, 8.04 mmol, 6 equiv)

were added and the resulting reaction mixture was stirred at room

temperature for 1 h. Thereafter, EtOH (500 mL) was added and

Boc-Val-Asp(TMSE)-OBn 16 (colorless oil, quantitative yield):

R_f(CH₂Cl₂–AcOEt, 96 : 4, v/v) 0.5; [a]²¹+27.1^◦(c 0.99 in

365

1

CHCl₃); H NMR (300 MHz, CDCl₃): d 0.02 (s, 9H, Si(CH₃)₃),

0.83–0.93 (m, 8H, CH₂Si + 2CH_3Val), 1.43 (s, 9H, C(CH₃)₃),

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2.03–2.10 (m, 1H, CHCH₃), 2.78 (dd, J = 4.5 and 17.0 Hz, 1H,

0.5CH₂), 3.05 (dd, J = 4.5 and 17.0 Hz, 1H, 0.5CH₂), 3.94–3.98

(m, 1H, C*H_Val), 4.11 (t, J = 9.0 Hz, 2H, OCH₂), 4.86–4.90 (m,

1H, C*H_Asp), 5.09–5.21 (m, 3H, ArCH₂O + NH_Val), 6.82 (d, J =

8.0 Hz, 1H, NH_Asp), 7.29 (s, 5H, 5ArH); ¹³C NMR (75.4 MHz,

CDCl₃): d -1.2 (3C), 17.6, 17.8, 19.4, 28.6 (3C), 31.7, 36.6, 48.7,

59.9, 63.9, 67.9, 80.1, 128.7–128.9 (2 peaks, 5C), 135.4, 156.0,

170.7, 171.5, 171.6; IR (neat): n_max3324, 2960, 2900, 1732, 1661,

1505, 1456, 1391, 1366, 1289, 1250, 1173, 1046; MS (MALDI-

TOF, positive mode, a-CHCA matrix): m/z 545.63 [M + Na]⁺,

calcd for [C₂₆H₄₂N₂O₇SiNa]⁺545.27; elemental analysis: calcd (%)

for C₂₆H₄₂N₂O₇Si: C, 59.74; H, 8.10; N, 5.36; found: C, 59.24; H,

8.07; N, 5.31.

2.7 equiv) was introduced into a 2-neck round bottom ﬂask (10

mL) containing 2.5 mL of dry CH₂Cl₂. The solution was cooled

to -78 ^◦C and ozone (O₃) was bubbled until a blue color (excess of

O₃) appeared. Then, the reaction medium was purged with argon

gas until the solution became colorless (45 min). Enol ether 22

(10 mg, 16.8 mmol) in solution in dry CH₂Cl₂(0.5 mL) was added

and the resulting reaction mixture was stirred at -78 ^◦C for 30 min,

◦

warmed to -30 C and then stirred again for 1 h. The oxidation

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

the mixture was evaporated to dryness. The resulting yellow oily

residue was puriﬁed by semi-preparative RP-HPLC (system B).

The product-containing fractions were lyophilised to give the 1,2-

dioxetane 23 as a white powder (4.1 mg, 39%). A badly resolved ¹H

NMR spectrum was obtained. MS (ESI+): m/z 650.07 [M + H]⁺,

calcd for [C₃₇H₄₁NO₈]⁺650.27; HPLC (system A): t_R= 22.2 min,

purity 95%.

Ac-Asp(TMSE)-Glu(TMSE)-Val-Asp(TMSE)-OBn 18 (beige

solid, 96%): R_f0.5 (CH₂Cl₂–AcOEt, 1 : 1, v/v); [a]²¹-36.4^◦

365

1

(c 0.95 in CHCl₃); H NMR (300 MHz, CDCl₃): d 0.01 (s, 9H,

Si(CH₃)₃), 0.02 (s, 9H, Si(CH₃)₃), 0.03 (s, 9H, Si(CH₃)₃), 0.82–

1.01 (m, 12H, 3CH₂Si + 2CH_3Val), 2.05–2.22 (m, 6H, CHCH₃+

CH_2Glu+ CH₃), 2.42–2.49 (m, 2H, CH_2GluCO), 2.70–3.15 (m, 4H,

Synthesis of PGA chemiluminescent probe (29). (a) Coupling

of enol ether: The sodium salt of phenolic enol ether 21-Na

(0.15 mmol) was suspended in dry CH₃CN (1 mL). Crown ether

15-C-5 (5 mL, 0.025 mmol, 0.17 equiv) was added and the resulting

solution was stirred for 5 min. Then, a solution of mesylate ester

27 (0.2 mmol) in a mixture of dry CH₃CN (1 mL) and dry

DMF (500 mL) was added and the resulting reaction mixture was

stirred at room temperature for 6 h. Thereafter, the mixture was

concentrated under vacuum, AcOEt (15 mL) was added and the

solution was washed with aq. Na₂CO₃(10%, 5 mL) and deionised

water (5 mL). The organic phase was dried over Na₂SO₄, ﬁltered

and concentrated. The crude product was puriﬁed over a silica

gel pad using a mixture of cyclohexane–AcOEt (85 : 15, v/v),

affording 28 as a beige solid (40 mg, 54%). R_f(cyclohexane–

2CH_2Asp), 4.02–4.19 (m, 6H, 3OCH₂), 4.27–4.38 (m, 2H, C*H_Val

+

C*H_Glu), 4.72–4.76 (m, 1H, C*H_Asp), 4.87–4.95 (m, 1H, C*H_Asp),

5.11 (Abd, J = 12 Hz, 1H, 0.5ArCH₂O), 5.19 (Abd, J = 12 Hz, 1H,

0.5ArCH₂O), 6.91 (d, J = 8.0 Hz, 1H, NH_Asp), 7.07 (d, J = 8.0 Hz,

1H, NH_Asp), 7.12 (d, J = 8.0 Hz, 1H, NH_Val), 7.32 (s, 5H, 5ArH),

7.79 (d, J = 8.0 Hz, 1H, NH_Glu); ¹³C NMR (75.4 MHz, CDCl₃): d

-1.2 (9C), 17.5–17.6 (2 peaks, 3C), 18.0, 19.5, 23.4, 26.6, 30.7, 31.1,

36.2, 36.6, 49.0, 49.9, 54.5, 58.9, 63.7, 63.9, 64.0, 67.9, 128.7–128.9

(3 peaks, 5C), 135.5, 170.9, 171.0, 171.1, 171.3, 171.4, 171.7, 172.5,

175.0; IR (KBr): n_max3276, 2955, 1737, 1634, 1544, 1454, 1385,

1250, 1167; MS (MALDI-TOF, positive mode, a-CHCA matrix):

m/z 931.57 [M + Na]⁺, calcd for [C₄₂H₇₂N₄O₁₂Si₃Na]⁺931.44;

elemental analysis: calcd (%) for C₄₂H₇₂N₄O₁₂Si₃, 0.5CH₂Cl₂: C,

53.63; H, 7.73; N, 5.89; found: C, 53.36; H, 7.40; N, 5.89.

1

AcOEt, 75 : 25, v/v) 0.5; H NMR (300 MHz, CDCl₃): d 1.73–

1.98 (m, 12H, 2CH_Adam.+ 5CH_2Adam.), 2.61 (m, 1H, CH_Adam.), 3.23

(s, 1H, CH_Adam.), 3.27 (s, 3H, OCH₃), 3.75 (s, 2H, ArCH₂CO), 5.00

(s, 2H, ArCH₂O), 6.82–6.91 (m, 3H, 3ArH), 7.04 (s, 1H, ArH),

7.32-7-47 (m, 9H, 5ArH + 4ArH_PABA); ¹³C NMR (75.4 MHz,

CDCl₃): d 28.6 (2C), 30.5, 32.5, 37.5, 39.3 (2C), 39.5 (2C), 45.0,

58.0, 69.8, 114.3, 115.9, 120.2 (2C), 122.5, 128.0, 128.6 (2C),

129.2, 129.5 (2C), 129.8 (2C), 132.0, 133.3, 134.7, 137.2, 137.7,

143.6, 158.7, 169.5; IR (KBr): n_max3296, 2909, 2848, 2359, 1666,

1607, 1538, 1412, 1281, 1238, 1200, 1080, 1026; MS (ESI+): m/z

494.13 [M + H]⁺, calcd for [C₃₃H₃₆NO₃]⁺494.27. (b) Oxidation:

P(OC₆H₅)₃(56 mL, 0.21 mmol, 3.0 equiv) was introduced into

a 2-neck round bottom ﬂask (25 mL) containing 1.5 mL of dry

Synthesis of PGA chemiluminescent probe (23). (a) Coupling

of enol ether: Phenylacetamide 20 (656 mg, 1.91 mmol) was

dissolved in dry DMF (10 mL). Phenolic enol ether 21 (520 mg,

1.92 mmol), TEA (800 mL, 5.76 mmol) and BOP phosphonium

salt (950 mg, 2.15 mmol) were sequentially added. The resulting

reaction mixture was stirred at room temperature for 6 h. The

solvent was removed under vacuum, the resulting oily residue

was dissolved in AcOEt and washed with deionised water. The

organic phase was dried over Na₂SO₄, ﬁltered and concentrated.

The crude product was puriﬁed by chromatography on silica gel

using cyclohexane–AcOEt (4 : 6, v/v) as the mobile phase, yielding

the corresponding aryl ester 22 as a white solid (606 mg, 53%).

◦

CH₂Cl₂. The solution was cooled to -78 C and ozone (O₃) was

bubbled until a blue color (excess of O₃) appeared. Then, the

reaction medium was purged with argon gas until the solution

became colorless. Enol ether 28 (35 mg, 0.070 mmol) in solution

in dry CH₂Cl₂(2.5 mL) was added and the resulting reaction

mixture was allowed to warm at room temperature. After 2 h, the

formation of the 1,2-dioxetane was checked by TLC and showed

the complete conversion of the starting material. The reaction

mixture was concentrated under reduced pressure and the resulting

solid was puriﬁed by chromatography on a silica gel column using

a mixture of cyclohexane–AcOEt (7 : 3, v/v) as the mobile phase,

affording 29 as a white solid (27 mg, 73%). R_f(cyclohexane–

AcOEt, 7 : 3, v/v) 0.5; ¹H NMR (300 MHz, CDCl₃): d 1.52–1.95

(m, 12H, 2CH_Adam.+ 5CH_2Adam.), 2.11 (m, 1H, CH_Adam.), 3.01 (s,

1H, CH_Adam.), 3.20 (s, 3H, OCH₃), 3.75 (s, 2H, ArCH₂CO), 5.05

1

R_f(cyclohexane-AcOEt, 4 : 6, v/v) 0.45; H NMR (300 MHz,

CDCl₃): d 1.55–1.90 (m, 12H), 2.66 (br s, 1H), 3.25 (br s, 1H),

3.30 (s, 3H), 3.53 (s, 2H), 3.81 (s, 3H), 3.85 (s, 2H), 3.88 (s, 3H),

4.44 (d, J = 5.3 Hz, 2H), 6.77 (s, 1H), 6.80 (s, 1H), 6.84 (ddd,

J = 7.9 Hz, 2.3 Hz and 0.8 Hz, 1H), 6.91 (t, J = 2.3 Hz, 1H),

7.00-7.15 (m, 6H), 7.21 (t, J = 7.9 Hz, 1H); ¹³C NMR (75.4 MHz,

CDCl₃): d 28.3, 30.3, 32.2, 37.2, 38.3, 39.1, 39.3, 41.4, 43.9, 56.0,

56.1, 58.0, 112.9, 113.5, 120.4, 122.1, 124.1, 127.1, 127.3, 128.9,

129.1, 129.4, 132.9, 134.9, 137.3, 142.6, 148.5, 148.6, 150.6; MS

(MALDI-TOF, positive mode, a-CHCA matrix): m/z 618.30 [M +

Na]⁺, calcd for [C₃₇H₄₁NO₆Na]⁺618.28; elemental analysis: calcd

(%) for C₃₇H₄₁NO₆: C, 74.60; H, 6.94; N, 2.35; found: C, 74.25;

H, 7.09; N, 2.41. (b) Oxidation: P(OC₆H₅)₃(12.5 mL, 47.5 mmol,

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(s, 2H, ArCH₂O), 6.95–7.08 (m, 2H, 2ArH), 7.22-7-46 (m, 11H,

7ArH + 4ArH_PABA); ¹³C NMR (75.4 MHz, CDCl₃): d 26.2, 26.3,

31.8, 31.9, 32.6, 33.2, 33.4, 35.0, 36.7, 45.0, 50.2, 69.9, 95.8, 112.3,

116.5, 120.1 (2C), 120.4, 126.0, 128.0, 128.5 (2C), 129.5 (2C), 129.8

(2C), 130.2, 133.0, 134.7, 136.4, 137.8, 158.5, 169.4; MS (ESI+):

m/z 543.07 [M + H]⁺, calcd for [C₃₃H₃₇NO₆]⁺543.26.

NH_Val), 7.21 (d, J = 6.8 Hz, 1H, NH_Asp), 7.32–7.40 (m, 4H, 4ArH),

7.59 (d, J = 8.7 Hz, 1H, NH_Asp), 7.79 (d, J = 8.3 Hz, 2H, 2ArH),

8.27 (d, J = 8.3 Hz, 2H, 2ArH), 8.50 (d, J = 7.5 Hz, 1H, NH_Glu),

8.71 (s, 1H, ArNHCO); ¹³C NMR (75.4 MHz, CDCl₃): d -1.23

(9C), 17.6 (3C), 19.5 (2C), 23.3, 25.4, 29.4, 30.0, 31.5, 36.0, 36.8,

50.2, 51.0, 56.8, 60.6, 63.6, 64.0, 64.3, 71.1, 120.5, 122.2, 125.6,

129.9, 139.3, 145.6, 152.7, 155.9, 169.1, 171.3, 171.4, 171.8, 172.0,

172.3, 172.7, 172.9, 175.9; HPLC (system C) : t_R= 34.5 min, purity

>90%.

Ac-Asp(TMSE)-Glu(TMSE)-Val-Asp(TMSE)-PABA-OH (30a).

Side-chain protected tetrapeptide 19 (598 mg, 0.73 mmol) was

dissolved in dry DMF (7 mL) and the resulting solution was cooled

to 0 ^◦C. Then, PABA (90 mg, 0.73 mmol, 1 equiv) and the HCl salt

of EDC, (154 mg, 0.80 mmol, 1.1 equiv) were sequentially added

and the resulting reaction mixture was stirred at room temperature

for 1 h. The solvent was removed under vacuum, the resulting

solid was dissolved in CH₂Cl₂(40 mL), washed with aq. citric acid

(10%, 15 mL) and deionised water (20 mL). The organic phase

was dried over Na₂SO₄, ﬁltered and concentrated under reduced

pressure. The crude product was puriﬁed by chromatography on

a silica gel column using a mixture of CH₂Cl₂–AcOEt (3 : 7, v/v)

as the mobile phase, yielding the peptide–PABA conjugate as a

9:1 mixture of the two diastereoisomers 30a and 30b which was

resolved in the next step (yellow solid, 580 mg, 86%). For analytical

purpose, the mixture was puriﬁed by semi-preparative RP-HPLC

(system D) to get 30a in a pure form. R_f(CH₂Cl₂–AcOEt, 3 : 7,

Enol ether (32). Phenolic enol ether 21 (19 mg, 0.070 mmol,

1.5 equiv) and anhydrous K₂CO₃(13 mg, 0.094 mmol, 2 equiv)

were mixed in dry CH₃CN (400 mL) and the reaction mixture

was stirred at room temperature for 20 min. Then, activated

carbonate 28 (51 mg, 0.047 mmol) was added and the reaction

mixture was stirred at room temperature for 16 h. The solution was

concentrated and CH₂Cl₂(10 mL) was added. The organic phase

was washed with deionised water (5 mL), dried over Na₂SO₄,

ﬁltered and concentrated under reduced pressure. The resulting

solid was puriﬁed by chromatography on a silica gel column using

a mixture of CH₂Cl₂–AcOEt (1 : 1, v/v) as the mobile phase,

affording 32 as a white solid (33 mg, 58%). R_f(CH₂Cl₂–AcOEt,

1

1 : 1, v/v) 0.5; H NMR (300 MHz, CDCl₃): d 0.02 (s, 27H,

3Si(CH₃)₃), 0.90–1.05 (m, 12H, 3CH₂Si + 2CH_3Val), 1.55–2.00 (m,

17H, 5CH_2Adam.+ 2CH_Adam.+ CH₃+ CH_2Glu), 2.20–2.45 (m, 1H,

CHCH₃), 2.45–2.70 (m, 3H, CH_Adam.+ CH_2GluCO), 2.80–3.02 (m,

4H, 2CH_2Asp), 3.22 (s, 1H, CH_Adam.), 3.28 (s, 3H, OCH₃), 4.05–4.25

(m, 8H, C*H_Val+ C*H_Glu+ 3OCH₂), 4.68–4.82 (m, 1H, C*H_Asp),

4.92–5.05 (m, 1H, C*H_Asp), 5.20 (s, 2H, ArCH₂O), 7.05–7.38 (m,

6H, 4ArH + 2ArH_PABA), 7.70 (d, J = 8.7 Hz, 2H, 2ArH_PABA), 8.65

(s, 1H, ArNHCO); HPLC (system E) : t_R= 27.8 min, purity >90%.

◦

1

v/v) 0.5; [a]²¹-125.5 (c 1.1 in CHCl₃); H NMR (300 MHz,

365

CD₃OD): d 0.07 (s, 27H, 3Si(CH₃)₃), 0.99–1.04 (m, 12H, 3CH₂Si +

2CH_3Val), 1.95–2.10 (m, 4H, 0.5CH_2Glu+ CH₃), 2.10–2.18 (m, 2H,

CHCH₃+ 0.5CH_2Glu), 2.41–2.48 (m, 2H, CH_2GluCO), 2.7–3.1 (m,

4H, 2CH_2Asp), 4.05–4.25 (m, 7H, C*H_Val+ 3OCH₂), 4.36–4.42 (m,

1H, C*H_Glu), 4.58 (s, 2H, ArCH₂O), 4.70–4.76 (m, 1H, C*H_Asp),

4.80–4.95 (m, 1H, C*H_Asp), 7.33 (d, J = 8.0 Hz, 2H, 2ArH), 7.63

(d, J = 8.0 Hz, 2H, 2ArH), 7.94 (d, J = 7.0 Hz, 1H, NH_Val), 8.31

(d, J = 8.0 Hz, 1H, NH_Asp), 8.42–8.47 (m, 2H, NH_Glu+ NH_Asp),

9.48 (s, 1H, OH); ¹³C NMR (75.4 MHz, CD₃OD): d -0.6 (9C),

19.1 (3C), 20.0, 20.5, 23.4, 28.4, 32.1, 32.3, 37.7, 37.9, 52.3, 53.1,

55.7, 62.2, 64.8, 65.1, 65.3, 65.7, 122.2 (2C), 129.4 (2C), 139.3,

139.7, 171.4, 173.0, 173.4, 174.2(6), 174.3(4), 174.4, 175.2, 175.7;

IR (KBr): n_max3286, 2957, 1738, 1634, 1539, 1251, 1172; MS

(ESI+): m/z 946.40 [M + Na]⁺, calcd for [C₄₂H₇₃N₅O₁₂Si₃Na]⁺

946.45.

1,2-Dioxetane (33). P(OC₆H₅)₃(20 mL, 0.076 mmol, 2.7 equiv)

was introduced into a 2-neck round bottom ﬂask (25 mL)

containing 2 mL of dry CH₂Cl₂. The solution was cooled to -78 ^◦C

and O₃was bubbled until a blue color (excess of O₃) appeared.

Then, the reaction mixture was purged with argon gas until the

solution became colorless. Enol ether 32 (34 mg, 0.028 mmol)

in solution in dry CH₂Cl₂(2 mL) was added and the resulting

reaction mixture was allowed to warm at room temperature. After

1 h, the formation of the desired 1,2-dioxetane was checked by

RP-HPLC (system E): the RP-HPLC elution proﬁle showed ca.

50% conversion of the starting enol ether. The reaction mixture

was concentrated under reduced pressure and the resulting solid

was puriﬁed by semi-preparative RP-HPLC (system F) to give

after lyophilisation the corresponding 1,2-dioxetane 33 as a white

solid (7.0 mg, 20%). MS (ESI+): m/z 1156.27 [M - TMSEOH +

Na]⁺, calcd for [C₅₆H₇₉N₅NaO₁₆Si₂]⁺1156.50, under ionisation

conditions, the release of one unit of TMSE with subsequent

formation of an aspartimide was observed. HPLC (system E) :

t_R= 26.3 min, purity >90%.

4-Nitrophenyl carbonate derivative (31). para-Nitrophenyl

chloroformate (87 mg, 0.44 mmol, 4 equiv) and dry pyridine

(44 mL, 0.54 mmol, 5 equiv) were mixed in dry CH₂Cl₂and a

white precipitate was formed. Alcohol 30a-b (100 mg, 0.11 mmol)

was added and the reaction mixture turned slowly to yellow and

became homogenous. After 16 h of stirring at room temperature,

CH₂Cl₂was added and the solution was washed with aq. citric

acid (10%) and deionised water. The organic phase was dried

over Na₂SO₄, ﬁltered and concentrated under reduced pressure.

The resulting solid was puriﬁed by chromatography on a silica gel

column using a mixture of CH₂Cl₂–AcOEt (6 : 4, v/v) as the mobile

phase, affording activated carbonate 31 as a yellow solid (86 mg,

Synthesis of caspase-3 chemiluminescent probe (37). (a) Coup-

ling of enol ether: Phenolic enol ether 21 (46 mg, 0.17 mmol,

1 equiv) was dissolved in dry HPLC-grade acetone (1 mL).

Anhydrous K₂CO₃(47 mg, 0.34 mmol, 2 equiv) was added and the

resulting mixture was stirred for 20 min. Then, mesylate ester 34 in

solution in dry HPLC-grade acetone (1.5 mL) was added and the

mixture was stirred at room temperature for 16 h. The reaction

was checked to completion by TLC and the reaction mixture

1

72%). R_f(CH₂Cl₂–AcOEt, 6 : 4, v/v) 0.5; H NMR (300 MHz,

CDCl₃): d 0.02 (s, 9H, Si(CH₃)₃), 0.03 (s, 9H, Si(CH₃)₃), 0.05 (s,

9H, Si(CH₃)₃), 0.80–1.05 (m, 12H, 3CH₂Si + 2CH_3Val), 2.02–2.20

(m, 5H, CH_2Glu+ CH₃), 2.28–2.42 (m, 1H, CHCH₃), 2.45–2.70

(m, 2H, CH_2GluCO), 2.82–3.14 (m, 4H, 2CH_2Asp), 4.02–4.28 (m, 8H,

C*H_Val+ C*H_Glu+ 3OCH₂), 4.66–4.74 (m, 1H, C*H_Asp), 4.92–5.08

(m, 1H, C*H_Asp), 5.23 (s, 2H, ArCH₂O), 7.09 (d, J = 7.2 Hz, 1H,

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was diluted with CH₂Cl₂(15 mL) and washed with deionised

water (7 mL). The organic phase was dried over Na₂SO₄, ﬁltered

and evaporated under reduced pressure. The crude product was

puriﬁed by chromatography on a silica gel column using a mixture

of CH₂Cl₂–AcOEt (1 : 1, v/v) as the mobile phase, affording

35 as a pale yellow oil (88 mg, 44% over 2 steps from 27). R_f

(system G). The product-containing fractions were lyophilised

to give the target caspase-3 chemimuminescent probe 37 as a

colorless oil. A stock solution of this chemiluminescent probe was

prepared in DMSO and quantiﬁcation was achieved by UV–visible

absorption measurements in deionised water at l_max= 250 nm

(quantitative yield after RP-HPLC puriﬁcation). HPLC (system

C): t_R= 27.1 min, purity >90%; UV–vis (deionised water, 25 ^◦C):

l_max(e) = 250 nm (19 000 L mol^-1cm^-1); MS (ESI-): m/z 906.33

[M - H]^-, calcd for [C₄₅H₅₆N₅O₁₅]^-906.38.

(CH₂Cl₂–AcOEt, 1 : 1, v/v) 0.5; [a]²¹-23.7^◦(c 0.95 in CHCl₃);

365

¹H NMR (300 MHz, CDCl₃): d 0.02 (s, 9H, Si(CH₃)₃), 0.04

(s, 9H, Si(CH₃)₃), 0.06 (s, 9H, Si(CH₃)₃), 0.92–0.98 (m, 12H,

3CH₂Si + 2CH_3Val), 1.77–1.94 (m, 15H, 2CH_Adam.+ 5CH_2Adam.

+

CH₃), 2.31–2.42 (m, 3H, CHCH₃+ CH_2Glu), 2.50–2.63 (m, 3H,

CH_Adam.+ CH_2GluCO), 2.83–2.95 (m, 4H, 2CH_2Asp), 3.22 (s, 1H,

CH_Adam.), 3.27 (s, 3H, OCH₃), 3.84–3.89 (m, 1H, C*H_Asp), 4.05–

4.20 (m, 7H, C*H_Val+ 3OCH₂), 4.69–4.75 (m, 1H, C*H_Glu), 4.98

(s_br, 3H, ArCH₂O + C*H_Asp), 6.82–6.92 (m, 3H, 3ArH), 7.14 (d,

J = 8.0 Hz, 1H, NH_Glu), 7.19–7.25 (m, 2H, NH_Asp+ ArH), 7.37

(d, J = 9.0 Hz, 4H, 2ArH_PABA+ NH_Asp+ NH_Val), 7.68 (s, 2H,

2ArH_PABA), 8.62 (s, 1H, ArNH); ¹³C NMR (75.4 MHz, CDCl₃):

d -1.2 (9C), 17.6 (3C), 18.3, 19.5, 23.3, 26.7, 28.6 (2C), 29.8,

30.0, 30.5, 31.4, 32.5, 35.6, 36.0, 37.5, 39.4 (2C), 39.5 (2C), 49.8,

50.5, 54.1, 58.2, 63.4 (2C), 63.8, 70.0, 114.4, 115.8, 120.3 (2C),

122.5, 128.7 (2C), 129.2, 132.2, 133.1, 137.2, 138.1, 138.2, 143.6,

168.8, 170.2, 171.3, 171.7, 172.0, 173.3, 174.5, 175.8; IR (KBr):

n_max3322, 2925, 1720, 1655, 1536; MS (ESI+): m/z 1198.53 [M +

Na]⁺, calcd for [C₆₀H₉₃N₅O₁₃Si₃Na]⁺1198.60; HPLC (system C) :

t_R= 39.6 min, purity >98%. (b) Oxidation: P(OC₆H₅)₃(22 mL,

0.085 mmol, 2.7 equiv) was introduced into a 2-neck round bottom

ﬂask (25 mL) containing 2_◦mL of dry CH₂Cl₂. The resulting

solution was cooled to -78 C and O₃was bubbled until a blue

color (excess of O₃) appeared. Then, the reaction mixture was

purged with argon gas until the solution became colorless. Enol

ether 35 (37 mg, 0.031 mmol) in solution in dry CH₂Cl₂(2 mL) was

added and the resulting reaction mixture was allowed to warm at

room temperature. After 1 h, the formation of the 1,2-dioxetane

was checked by RP-HPLC (system E): the RP-HPLC elution

proﬁle showed ca. 50% conversion of the starting material. The

reaction mixture was concentrated under reduced pressure and

the resulting solid was puriﬁed by semi-preparative RP-HPLC

(system F), to give after lyophilisation the targeted 1,2-dioxetane

General procedure for the in vitro cleavage of chemiluminescent

probes (23), (29) and (37) by recombinant proteases

Enzymatic assay with recombinant human caspase-3: 1,2-

Dioxetane 37 was dissolved in caspase-3 buffer (200 mL, 10% (w/v)

sucrose, 50 mM PIPES, 100 mM NaCl, 10 mM DTT, 1 mM EDTA,

0.1% (w/v) CHAPS, pH 7.4, ﬁnal concentration 75 mM). 1 mL of

recombinant human caspase-3 (1.6 10^-3U) was added and the

enzymatic reaction mixture was incubated at 37 ^◦C for an overall

time of 120 min. Every 15 min, an aliquot (20 mL) was sampled

and added to an alkaline caspase-3 buffer (30 mL, same caspase-

3 buffer at pH 12.3, ﬁnal concentration: 30 mM) containing the

enhancers (CTAB, 2 mM, 5-(stearoylamino)ﬂuorescein 0.18 mM).

Luminescence spectra were recorded (into an ultra-micro ﬂuores-

R

cence cell Hellma^ꢀ, 105.51-QS, 10 ¥ 3 mm, 45 mL) for each aliquot

between 490 and 570 nm for 36 min under cycle scan mode (240

scans, scan time duration = 9 s) with the following parameters:

bio/chemiluminescence mode with gate time = 100 ms, emission

ﬁlter: open and PMT detector voltage level: high.

Caspase-3 detection limit: To determine the detection limit of the

self-cleavable chemiluminescent probe 37, a decreasing amount of

caspase-3 enzyme was incubated during 60 min with a solution of

37 (30 mM). The total light emission was recorded between 490 and

570 nm during 10 min and reported as a function of the enzyme

concentration. A detection limit of around 1.0 pmol was observed.

Representative control experiments with other proteases: To

demonstrate the speciﬁcity of the cleavage by caspase-3 enzyme,

the self-cleavable chemiluminescent probe 37 was incubated with

human recombinant caspase-9 (1 mL, 2.38 U in a caspase-9 buffer:

0.1 M MES, 10% PEG, 0.1% CHAPS, 10 mM DTT, pH 6.5) and

A. faecalis recombinant PGA (1.2 mg, 1.9 U in 50 mM phosphate

buffer, pH 7.5) for 60 min. Then, the enzymatic reaction mixture

was added onto the alkaline caspase-3 buffer (pH 12.3, ﬁnal

concentration of 34: 30 mM) containing the enhancers (CTAB,

2 mM, 5-(stearoylamino)ﬂuorescein 0.18 mM) and light emission

was recorded under the same conditions than described above. No

light could be detected for both enzymes.

1

36 as a white amorphous powder (10 mg, 25%, 50% bsrm). H

NMR (300 MHz, CDCl₃): d 0.03 (s, 27H, 3Si(CH₃)₃), 0.94–1.02

(m, 12H, 3CH₂Si + 2CH_3Val), 1.43–1.78 (m, 12H, 2CH_Adam.

+

5CH_2Adam.), 1.98 (s, 3H, CH₃), 2.22–2.52 (m, 6H, CHCH₃

+

CH_Adam.+ CH₂CO_Glu+ CH_2Glu), 2.78–3.02 (m, 4H, 2CH_2Asp), 3.20

(s, 1H, CH_Adam.), 3.21 (s, 3H, OCH₃), 3.80–3.92 (m, 1H, C*H_Asp),

4.08–4.22 (m, 7H, C*H_Val+ 3OCH₂), 4.75–4.82 (m, 1H, C*H_Glu),

5.05 (s_br, 3H, ArCH₂O + C*H_Asp), 6.36–6.40 (m, 1H, ArH), 6.99

(d, J = 6.0 Hz, 1H, ArH), 7.30–7.38 (m, 4H, 2ArH_PABA+ 2ArH),

7.71 (d, J = 8.0 Hz, 2H, 2ArH_PABA), 8.54–8.60 (m, 1H, NHAc);

IR (KBr): n_max3310, 2917, 1719, 1655, 1542, 1389, 1250, 1165;

MS (ESI+): m/z 1309.53 [M + H]⁺, calcd for [C₆₆H₁₀₉N₆O₁₅Si₃]⁺

1309.73; HPLC (system C) : t_R= 37.8 min, purity >98%. (c)

Removal of TMSE protecting groups: The latter fully-protected

1,2-dioxetane 36 (2.0 mg, 1.53 mmol) was dissolved in dry DMSO

(200 mL). TBAF (1.0 M in THF, 31 mL, 31 mmol, 20 equiv) was

added to the solution, allowing the quantitative removal of the

TMSE groups (followed by RP-HPLC analysis after 5 min of

reaction, system C). The mixture was diluted with TEAB buffer

(50 mM, pH 7.5) and puriﬁed by semi-preparative RP-HPLC

Enzymatic assay with PGA: 1,2-Dioxetane 23 or 29 was

dissolved in phosphate buffer (50 mM, pH 7.5, ﬁnal concentration:

75 mM). 10 mL of a solution of recombinant PGA in PBS (5.0 mg

in 3.0 mL of phosphate buffer, 1.05 10^-2U) were added and the

enzymatic reaction mixture was incubated at 37 ^◦C for an overall

time of 120 min. Every 5 min, an aliquot (20 mL) was sampled

and added to the same alkaline buffer (30 mL, ﬁnal concentration:

30 mM) used for 37. Luminescence spectra were recorded (into

R

an ultra-micro ﬂuorescence cell Hellma^ꢀ, 105.51-QS, 10 ¥ 3 mm,

45 mL) for each aliquot under the same conditions as described

for caspase-3 chemiluminescent probe.

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Abbreviations

spectrometry measurements. Recombinant A. faecalis PGA was

gratefully furnished by Pr. L. Fischer, Universita¨t Hohenheim

(Institut fu¨r Lebensmitteltechnologie, Fachgebiet Biotechnologie,

Stuttgart).

AcOEt

Asp

Boc

ethyl acetate

aspartic acid

tert-butyloxycarbonyl

Bn

benzyl

BOP

benzotriazol-1-yloxytris(dimethylamino)-

phosphonium hexaﬂuorophosphate

3-[(3-cholamidopropyl)dimethylammonio]-1-

propanesulfonate

a-cyano-4-hydroxycinnamic acid

chemically initiated electron exchange lumines-

cence

cetyltrimethylammonium bromide

N,N¢-dicyclohexylcarbodiimide

N,N¢-dicyclohexylurea

N,N-diisopropylethylamine

N,N-dimethylformamide

dimethylsulfoxide

dithiothreitol

N-(3-dimethylaminopropyl)-N¢-

ethylcarbodiimide

ethylenediaminetetraacetic acid

2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline

ﬂuorescence resonance energy transfer

glutamic acid

O-(7-azabenzotriazol-1-yl)-N,N,N¢,N¢-

tetramethyluronium hexaﬂuorophosphate

1-hydroxybenzotriazole

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Acknowledgements

This work was supported by La Re´gion Haute-Normandie via

the CRUNCh program (CPER 2007-2013), QUIDD and Institut

Universitaire de France (IUF). We thank Elisabeth Roger (INSA

de Rouen) for IR measurements, Annick Leboisselier (INSA de

Rouen) for the determination of elemental analyses, Dr Je´roˆme

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

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Self-cleavable chemiluminescent probes suitable for protease sensing

DOI: 10.1039/b905725k

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