From DNA cross-linking to peptide labeling: On the versatility of the furan-oxidation-conjugation strategy

Article abstract of DOI:10.1039/b817447d

Based on the incorporation of commercially available N-Fmoc-furylalanine, a new method for peptide labeling is proposed, relying on the selective oxidative transformation of the furan moiety into a reactive aldehyde and subsequent reductive amination. The

Full text of DOI:10.1039/b817447d

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From DNA cross-linking to peptide labeling: on the versatility
of the furan-oxidation–conjugation strategyw
Annelies Deceuninck and Annemieke Madder*
Received (in Cambridge, UK) 6th October 2008, Accepted 11th November 2008
First published as an Advance Article on the web 1st December 2008
DOI: 10.1039/b817447d
Based on the incorporation of commercially available N-Fmoc-
furylalanine, a new method for peptide labeling is proposed,
relying on the selective oxidative transformation of the
furan moiety into a reactive aldehyde and subsequent reductive
amination.
labeling context, most existing site-selective techniques rely on
amine derivatization, where selectivity issues need to be solved
18
by either specifically modifying N-termini or using carefully
balanced orthogonal protection strategies.19–22
Recently, a few elegant methodologies have been developed
based on orthogonal chemistries such as enzyme-mediated
peptide modification23 or Pd-catalyzed coupling reactions.24
However, substrate selectivity counteracts generality in the
former method while the latter method is limited to arylations.
Site-selective labeling of peptides would therefore greatly
benefit from an easily applicable inverse chemistry, using
nucleophilic labels to react with a specifically located electro-
philic moiety in the peptide.
Modern chemical biology oriented research frequently relies
on the ability to site-selectively modify biomacromolecules
such as peptides, carbohydrates and oligonucleotides.
Although the so called ‘‘click’’ 1,3-dipolar cycloaddition bet-
ween alkynes and azides has revolutionized the field of
bioconjugation chemistry, it does require the introduction of
unnatural functionality in both partners.1–4 As for methods
relying on only one unnatural reaction partner, mostly one
makes use of the naturally available nucleophilic biomolecule
functionalities such as amines and alcohols. Also, maleimide
conjugation with thiols is frequently used,5,6 but has recently
been found to lead to heterogeneous conjugates due to
susceptibility of the imido group to spontaneous hydrolysis.7
Carbonyl condensation reactions represent an often used
alternative.8,9 However, site-selective introduction of selec-
tively modifiable carbonyl moieties into biomolecules is not
straightforward, in particular the introduction of aldehydes
still represents a major synthetic hurdle.10
Our proposed strategy for the generation of aldehyde
functionality (Scheme 1) involves incorporation of a furan
moiety as a masked reactive aldehyde. Oxidative ring opening
of the furan moiety generates an enal functionality with
various possibilities for conjugation, labeling and cross-
linking. The furan moiety can be introduced by capping
the sequence with 3-(2-furyl)propionic acid or by using
commercially available Fmoc-b-2-furylalanine, allowing
introduction at any desired position in the peptide.
First experiments were conducted on TentaGel resin using a
photocleavable linker for easy reaction monitoring at every
stage. A small test peptide 1 was synthesized and capped with
Recently random incorporation of aldehyde functionality into
proteins has been demonstrated.11 Existing methods for
site-selective introduction of aldehyde functionalities are centered
around oxidation of N-terminal serine residues.12 Internal or
C-terminal aldehyde functionality can be generated only through
the use of building blocks requiring lengthy syntheses and/or
making use of orthogonal protection schemes.13–16
We earlier developed
a methodology for site-selective
DNA-cross-linking based on the in situ oxidation of an
incorporated furan moiety, generating a reactive aldehyde in a
site-selective way.17 We here report on our results indicating
that a similar strategy can be applied in a totally different
chemical context, namely that of peptide synthesis. We describe
the application of this furan-oxidation principle for the site-
selective introduction of aldehyde functionality in peptides. As
a first proof of principle, we set out to explore the potential of
the proposed methodology via reductive amination based label-
ing of solid-phase bound peptide aldehydes. In a peptide
Laboratory for Organic and Biomimetic Chemistry, Department of
Organic Chemistry, Krijgslaan 281, S4; B-9000 Gent, Belgium.
E-mail: annemieke.madder@ugent.be; Fax: (+)32-9-264 49 98
w Electronic supplementary information (ESI) available: Experimental
procedures, RP-HPLC and MS data for all labelled peptides. NMR
data for labelled peptide 8. See DOI: 10.1039/b817447d
Scheme 1 General strategy for peptide labeling on solid phase
(SPPS = solid phase peptide synthesis).
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340 | Chem. Commun., 2009, 340–342

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Fig. 1 Labeling on Rink resin: HPLC analysis of crude cleaved
peptide. Initial conditions (top) and optimized conditions (bottom).
a MW corresponding to 2. Reductive amination was then
performed with 30 equiv. of 1-aminopyrene (AP) and 60 equiv.
of sodium cyanoborohydride in THF–water–15% aq. acetic
acid (3 : 1 : 1) for 20 h. Cleavage of the resulting product
and LC-MS analysis showed that a labeled product was
formed.
Under these initial conditions, however, this turned out to be
a product of over-reduction, where next to the imine func-
tionality in 3, 1,2- and 1,4-reduction had occurred yielding a
mixture of 4, 5 and 6 (see Scheme 2 and Table 1, entry 1). When
reaction times were reduced (entry 2) or the reaction was run in
the absence of acetic acid (entry 3), higher yields of amine 4 were
obtained, but next to products of over-reduction the unreduced
imine 3 was also still present. In all cases low recovery was a
serious problem and we reasoned this was caused by quenching
of the light by the excess of pyrene label used, thus interfering
with the photolytic cleavage from the resin.
Scheme 2 Labeling of TentaGel bound peptides and photolytic
cleavage.
3-(2-furyl)propionic acid (Scheme 2). In the earlier reported
DNA cross-linking strategy N-bromosuccinimide (NBS)
was used for the oxidative transformation of the furan
moiety.17
This reagent seems unsuitable in this peptide context as it
was described in the early 1960s for fragmentation of poly-
peptides particularly at the level of tryptophan and tyrosine
residues.25 It was however later shown that in fully protected
peptides bound to a hydrazide resin, selective oxidation of the
hydrazide linkage is possible under mild NBS conditions.26
The method was even shown to be compatible with oxidation-
sensitive residues, provided suitable protecting groups and
oxidative conditions are used.27
In a second series of experiments we therefore turned to the
use of the acid labile Rink amide resin. Initially, labeling was
performed using 30 equiv. of aminopyrene and 60 equiv. of
NaBH3CN. After cleavage a complex reaction mixture was
observed (entry 4 and Fig. 1, top). Since the reaction product
contained doubly labeled products as well as products of over-
reduction, reaction times and the number of equiv. amino-
pyrene and NaBH3CN added were gradually lowered.
Surprisingly, reductive amination with only 1 equiv. of amino-
pyrene and 1 equiv. of NaBH3CN in THF–water–15% aq.
acetic acid (3 : 1 : 1) for a period of 1 h led to a perfectly clean
labeled product as evidenced by the crude chromatogram in
Fig. 1 (bottom).
In test peptide 1, the furan moiety could be selectively
oxidized without side reactions using only 3 equiv. of NBS
in THF–acetone–water (5 : 4 : 2). ESI-MS analysis after
cleavage confirmed the presence of an oxidation product with
Table 1 Optimization of labeling conditions on TentaGel
Upon ESI-MS analysis (Fig. 2), the molecular weight of the
labeled peptide turned out to be 18 mass units lower than
Equiv.
Entry AP
Equiv. Reaction
NaBH3CN time
Solventa Productsb
1
2
30
30
60
60
20 h
4 h
A
A
4 (16%)
5 or 6 (84%)
3 (15%)
4 (60%)
5 or 6 (25%)
3 (22%)
4 (58%)
3
30
60
20 h
B
5 or 6 (20%)
Complex
8 (83%)
4c
5c
30
1
60
1
4 h
1 h
A
A
a
Solvent A = THF–H2O–15% aq. CH3COOH (3 : 1 : 1); solvent B =
b
THF–H2O (3 : 1). Percentages refer to relative amounts of labeled
c
peptides as determined via HPLC. Experiments on Rink amide AM
resin involving acidic release of peptides.
Fig. 2 ESI-MS analysis of crude labeled peptide 8.
Chem. Commun., 2009, 340–342 | 341
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importantly, incorporation of this residue can be easily achieved
at any position, including the C-terminus. Furthermore, the
current labeling technique is orthogonal to earlier developed
methods based on N-terminal labeling or incorporation of
protected lysine derivatives. For this and the previous reason,
the current method offers attractive opportunities for the
straightforward synthesis of doubly labeled peptides which
are often used in FRET based screening strategies.
We thank the FWO Vlaanderen (project 1.5.146.06) and
Ghent University (GOA 01G01507 and BOF 01D29405) for
financial support.
Notes and references
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Scheme 3 Labeling and pyrenylpyrrole formation on Rink amide
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Table 2 Labeling tests on functionalized resins
Entry
Peptide
Resina
Purityb
1
2
3
Ac–FurAla–Ala–Ala–Gly
Fur–Ala–Cys(StBu)–Gly
Fur–Ala–Met–Gly
R
R
R
R
R
R
R
W
W
W
W
80%
70%
85%
68%
80%
64%
78%
93%
79%
80%
71%
4
5
Fur–Ala–Arg(Pbf)–Gly
Fur–Ala–Ser(tBu)–Gly
6
7
Fur–Ala–Trp(Boc)–Gly
Fur–Ala–Tyr(tBu)–Gly
8
9
10
11
Fur–Phe–Gln(Trt)–Gly–Ile–Ile
Fur–Ile–Leu–Pro–Glu(tBu)–Ile
Fur–Ala–His(Trt)–Asn(Trt)–Leu–Ala
Ac–Leu–FurAla–Gly–Lys(Boc)–Val
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a
b
R: Rink amide AM resin; W: Wang resin. As determined from the
crude chromatogram.
expected. Mechanistically, attack of the amine on the enone
functionality is possible, yielding a cyclized product which can
aromatize upon loss of H2O (see Scheme 3). The structure of
1
the obtained material was confirmed by H-NMR analysis of
the cleaved and isolated peptide 8 (see ESIw). Upon NaBH4
treatment of the resin before cleavage, pyrenylpyrrole forma-
tion was suppressed and the labeled peptide can be cleaved as
the aminopyrene derivative.
To demonstrate the scope of this approach in a more
general context, a series of peptides was made, incorporating
different functionalized amino acids (see Table 2). Incorpora-
tion of an internal furan moiety (using N-Fmoc-furylalanine)
evenly provided labeled peptides in good yield. As for labeling
using amines other than aminopyrene, peptides labeled with
7-amino-4-methylcoumarine have been generated with similar
purity (see ESIw).
In summary we have developed a novel strategy for site-
selective fluorescent labeling on solid phase via transformation
of an incorporated furan moiety into an aldehyde functionality.
Selective oxidation of the furan moiety can be conducted using
3 equiv. of NBS and releases the caged electrophilic keto-enal at
any required position in very short time. The methodology
is very straightforward in terms of the building blocks
used (commercially available N-Fmoc-2-furylalanine). More
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342 | Chem. Commun., 2009, 340–342