Organic &
Biomolecular Chemistry
PAPER
Highly specific, multi-branched fluorescent reporters
for analysis of human neutrophil elastase†
Cite this: Org. Biomol. Chem., 2013, 11,
4414
Nicolaos Avlonitis,‡a,b Manuelle Debunne,‡b Tashfeen Aslam,a Neil McDonald,b
Chris Haslett,b Kevin Dhaliwalb and Mark Bradley*a
Received 30th January 2013,
Accepted 29th April 2013
Human neutrophil elastase (HNE) is a serine protease implicated in the pathogenesis of acute and
chronic inflammatory disease. Here a series of, internally quenched, single fluorophore fluorescent repor-
ters were synthesised that allowed the rapid, highly specific and sensitive analysis of HNE activity over
closely related proteases.
DOI: 10.1039/c3ob40212f
on two dyes as required in conventional FRET systems. Here
this approach was applied to the synthesis of substrates for
Introduction
Human neutrophil elastase (HNE) is a proteolytic enzyme purified HNE, as a fore-runner for the analysis of HNE activity
stored within the primary azurophilic granules of polymorpho- in or on cells. The multi-valent fluorescent peptides were
nuclear cells (PMN).1 It is a protease whose major function is readily synthesised by solid-phase techniques,11,12 allowing the
the proteolytic destruction of bacteria, but when uncontrolled generation of mono-disperse products,13 in addition, they
is involved in the pathogenesis of acute and chronic inflamma- demonstrated high biocompatibilities, low toxicities and an
tory diseases. Excessive HNE degrades matrix, cleaves cellular ability to access intracellular compartments.14,15
receptors, activates profibrogenic mediators and contributes to
epithelial and endothelial damage,2–5 and thus tight control
and monitoring of HNE activity is important therapeutically.
Assessment of HNE activity requires probes with subtle
molecular specificity that permit specific detection of HNE
Results and discussion
over other closely related serine proteases such as proteinase 3
and cathepsin G.6,7 Gauthier et al.8 has previously described
FRET (Förster resonance energy transfer) labelled peptides
such as Abz-APEEIMRRQ-EDDnp (Abz: ortho-aminobenzoyl,
EDDnp: N-(2,4-dinitrophenyl)ethylenediamine) as substrates
for the analysis of neutrophil serine proteases on the surfaces
of neutrophils, but these have limitations, notably because of
their short emission wavelengths.
Previously we reported an assay for proteases based on
multi-branched fluorescently-labelled scaffolds which display
the phenomenon of internal or self-quenching.9,10 This strategy
offers a single fluorophore-based method for the analysis of
proteolytic activity, where amplification of signal upon sub-
strate cleavage is possible, without the need of having to rely
The three multi-branched peptides S1, S2 and S3 shown in
Fig. 1, were based on the peptide sequences optimised for
HNE cleavage by Gauthier and co-workers6 (APEEIMDRQ
and APEEIMRRQ) but utilised a single fluorophore fluorescein
(λex = 494 nm and λem = 518 nm), with S1 and S2 based on a
trivalent scaffold, whilst S3 was hexa-valent.
Synthesis required the preparation of the monomer (6)
which was synthesised in six steps11 as shown in Scheme 1
and monomer (12) which was synthesised in five steps as
shown in Scheme 2. Monomer (6) was prepared by the 1,4
addition of the hydroxy groups of 1,1,1-tris(hydroxymethyl)-
amino-methane onto acrylonitrile, followed by amino protec-
tion (Boc). Reduction of the nitrile groups with borane–THF
complex gave (3) which was treated with Dde-OH to give the
tris-Dde (2-acetyl-dimedone) protected amine (4). Following
removal of the Boc protecting group, the isocyanate (6) was
prepared following the procedure of Knölker.16 Monomer
(12)17 was synthesised from α-resorcylic acid by esterification
followed by alkylation with 2-(Boc-amino)ethyl bromide in the
presence of potassium carbonate in DMF to yield (9). Saponifi-
cation (NaOH in MeOH/dioxane), followed by removal of the
Boc protecting groups and reprotection with Fmoc-N-hydroxy-
succinimide gave the branched monomer (12).
aEaStChem, School of Chemistry, University of Edinburgh, West Mains Road, EH9 3JJ
Edinburgh, UK. E-mail: mark.bradley@ed.ac.uk
bMRC/Centre of Inflammation Research, Queen’s Medical Research Institute,
University of Edinburgh, 47 Little France Crescent, EH16 4TJ Edinburgh, UK
†Electronic supplementary information (ESI) available: The experimental details
of the synthesis of monomers (6), (12) and probes S1–S3 are described in details.
Biological experimental also described. See DOI: 10.1039/c3ob40212f
‡The authors NA and MD contributed equally to this work.
4414 | Org. Biomol. Chem., 2013, 11, 4414–4418
This journal is © The Royal Society of Chemistry 2013