Exploiting furan's versatile reactivity in reversible and irreversible orthogonal peptide labeling

Article abstract of DOI:10.1039/c3cc40588e

A general method for the facile and versatile decoration of peptides is proposed exploiting furan based cycloaddition and electrophilic aromatic substitution reactions. Given the commercial availability of furylalanine derivatives for peptide synthesis, the current work significantly enlarges the toolbox of available methodologies for site specific labeling and conjugation of peptide probes. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c3cc40588e

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ARTICLE TYPE
Exploiting Furan’s Versatile Reactivity in Reversible and Irreversible
Orthogonal Peptide Labeling
Kurt Hoogewijs,a Dieter Buyst,a,b Johan M. Winne,a José C. Martinsb and Annemieke Madder*ab
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
5
DOI: 10.1039/b000000x
A general method for the facile and versatile decoration of
peptides is proposed exploiting furan based cycloaddition and
electrophilic aromatic substitution reactions. Given the
commercial availability of furylalanine derivatives for peptide
10 synthesis, the current work significantly enlarges the toolbox
of available methodologies for site specific labeling and
conjugation of peptide probes.
Site-specific and selective biomacromolecule modification has
become an area of research with ever-growing importance in
15 biological, biochemical, diagnostic and medicinal developments.1
Since peptides, proteins and nucleic acids feature a large variety
of functional groups with often overlapping reactivity profiles,
site-selectivity is a major concern. It drives the quest for the
development of compatible and orthogonal chemistries for
20 postsynthetic decoration with fluorescent labels, affinity tags,
spin probes and other types of biomolecules (e.g. peptide-
oligonucleotide, protein-carbohydrate conjugates).2
In this respect, chemoselective derivatisation via the Diels-Alder
cycloaddition reaction has naturally attracted attention as a highly
25 promising method. Recent reviews describe the versatility of
Diels-Alder ligation for the decoration of peptides3, proteins4 and
nucleic acids.5 Frequently employed Diels-Alder partners for
peptide and protein modification are the 2,4-hexadienyl or
cyclopentadienyl/maleimide3,4 and the tetrazine/norbornene or
30 cyclooctene couples.6 Besides these classical dienyl systems,7 a
furan moiety has also been used to partner up with the maleimide
dienophile in the case of covalent conjugation of nucleic acids.8,9
However, to the best of our knowledge, furan-based Diels-Alder
conjugations have not been explored yet for the decoration of
35 peptides and proteins, in spite of the fact that β-(2-furyl)-alanine
is a commercially available building block.
An explanation for this apparent oversight might be found in the
fact that, until recently, incorporation of furan moieties into
peptides was thought to present severe hurdles linked to furan
40 instability during resin cleavage and side chain deprotection, as
reported by Schulz and coworkers in 2004.10 In a more recent
contribution dealing with the site specific incorporation of furan
at various positions in a peptide, we showed that these hurdles
can be overcome by slightly adapting the reaction conditions.11
45 Additionally, the recently reported procedure of Davis and
coworkers on the Suzuki-Miyaura based introduction of furan
onto genetically encoded aryl halides12 opens the way to site-
specific furan incorporation into proteins. These recent synthetic
50 Fig.1 Relevant peptide structures and corresponding HPLC traces of
reaction mixtures after cleavage of: a) peptide 1; b) peptide 1 after
treatment with 20 equiv. N-phenylmaleimide in toluene, 48h at RT; c)
peptide 1 after treatment with 20 equiv. N-phenylmaleimide in toluene,
24h at 70°C; or d) retro-Diels-Alder after reaction c by heating the solid
55 supported products mixture in toluene for 24h at 70°C. Each reaction was
followed by cleavage and deprotection with TFA/TIS/H2O (95:2,5:2,5),
2h at RT for HPLC analysis.
advances prompted us to explore furan’s unique reactivity for
biomolecular labeling and conjugation strategies involving
60 peptides. In order to test the feasibility and usefulness of a furan-
based Diels-Alder peptide conjugation strategy, model peptide 1
featuring a furylalanine was synthesized on solid support (Figure
1). Inspired by the earlier successes of Graham and coworkers8 in
oligonucleotide labeling, we chose to evaluate maleimide as
65 potential dienophile partner. This choice is even more relevant as
numerous maleimide-derivatised fluorophores and other
frequently used labels have been developed and commercialized.
The solid supported and fully protected peptide 1 was thus
incubated with an excess of N-phenylmaleimide in a minimal
70 amount of toluene at room temperature for 48 hours. The
resulting product was analysed after cleavage from the acid labile
Rink amide resin and complete deprotection using TFA. As
expected, several products were formed, corresponding to the
various diastereoisomers formed during the cycloaddition
75 reaction (see Figure 1, HPLC trace b). As can be seen from the
HPLC traces in Figure 1, complete conversion to the desired
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Fig. 2 Reaction conditions: (i) PTAD in DCM, 15 min. at RT; (ii)
TFA/TIS/H2O (95:2,5:2,5), 2h at RT. HPLC traces of reaction mixtures
for: a) Peptide 1 after cleavage and deprotection with TFA/TIS/H2O
5 (95:2,5:2,5); b) Crude HPLC revealed complete conversion; c) Labeled
peptide 4 after HPLC purification.
50
Fig.3 a) Proposed structures for the furylalanine-PTAD adduct; b) 2
doublets between 6,2 and 6,6 ppm; c) broadened signal at 11,5 ppm
assigned to exchangeable proton 18 in 5; d) measurements at -50°C
sharpen and move the signal downfield by slowing down exchange.
adduct was not achieved. In an effort to drive the reaction to
completion, a fresh reaction mixture was heated to 70°C for 24
hours. Again, similar yields of conversion were observed, albeit
10 with higher selectivity (see Figure 1, HPLC trace c). The obtained
conversion of ~85% is consistent with earlier reported
experiments.13 The apparent reluctance of the reaction to achieve
completion could be explained by its reversible nature. The retro-
Diels-Alder reaction has been illustrated in various cases and
15 even quantitatively exploited for deprotection or cleavage
purposes.14 Indeed, we found that heating of the resin-bound
labeled peptide 3 to 70°C for 24 h in toluene, resulted in a
complete retro-Diels-Alder conversion. As can be derived from
Figure 1 (HPLC trace d) the retro reaction regenerates the starting
20 material with quasi-unaltered purity.
As such, this furan-maleimide Diels-Alder based methodology
allows for peptide labeling and conjugation purposes in a way
that is completely orthogonal to conventional peptide synthesis
strategies. Given the range of commercially available maleimide
25 derivatives, this Diels-Alder strategy thus significantly enlarges
the application potential of the maleimide-derivatives based
toolbox. The reversibility of this furan-maleimide Diels-Alder
reaction has recently been exploited, both in polymer synthesis
for the generation of thermoreversible materials15 as well as in the
30 construction of controlled-release systems for drug delivery16 or
triggered removal of fluorescent labels.17 This reversible peptide
conjugation methodology thus offers interesting perspectives in
the area of controlled- or slow-release peptide drugs.
The furan-maleimide reaction, while offering certain advantages
35 in terms of reversibility, still suffers from a non optimal
conversion (vide supra, ~85%). Therefore, our attention next
turned towards cycloadditions using the more reactive 1,2,4-
triazole-3,5-diones (TADs) from which the synthesis of
fluorescent derivatives was recently described.18 TADs have
40 previously shown their use in Diels-Alder type reactions with a
range of dienes.19 As for the reactivity with respect to the furan
diene, though Diels-Alder reactions have been described with
dialkyl-azo-dicarboxylates and the resulting Diels-Alder adducts
unambiguously characterized,20 to the best of our knowledge only
45 one example of a TAD-furan combination has been reported.21
Considering the application of TAD derivatives for selective
reaction with furylalanine within a peptide containing otherwise
natural amino acids, concerns may be raised regarding potential
side-reactions with other aromatic side chain functionalities.
55 Indeed, Barbas and coworkers, in a sole example of the use of 4-
phenyl-1,2,4-triazole-3,5-dione (PTAD) in a peptide context,18
recently showed PTAD to chemoselectively react with
unprotected tyrosine giving rise to a formal ortho substituted
electrophilic aromatic substitution product. We thus set out to
60 explore the potential usefulness of a furan-based Diels-Alder
labeling of furylalanine containing peptides with PTAD. Use of
peptide 1, containing a tyrosine residue, allows direct verification
of possible selectivity problems versus natural aromatic side
chain residues.
65 Using only 3 equivalents of 4-phenyl-1,2,4-triazole-3,5-dione
(PTAD) in dichloromethane, complete reaction was observed
with solid supported peptide 1 in only 15 minutes at room
temperature. Judging from HPLC analysis (Figure 2), only a
single product is formed. Considering the potential tyrosine
70 versus β-(2-furyl)-alanine selectivity issue the resulting peptide
was studied by detailed NMR analysis at 700 MHz. The main
concerns are possible side-reactions at the tyrosine residue and
the actual structure of the cycloaddition product with furan.
Although the tyrosine side chain is protected during the labeling
75 reaction on support, PTAD substitution on that residue cannot be
excluded and should thus be verified. Indeed, all aromatic protons
of the tyrosine residue are observed via their characteristic
doublets, thus eliminating the possibility of reaction at the
phenolic side chain (Figure S55). The inability to locate the
80 resonance for the bridgehead proton of the expected oxabicyclic
system casts considerable doubts on the supposed Diels-Alder
type structure (Figure 2). Therefore, to afford unambiguous
elucidation of the structure of the formed product, the spectral
complexity was reduced by repeating the reaction on a single,
85 solid phase supported β-2-furyl-alanine residue. By treating solid-
supported N-acetyl-β-2-furylalanine with 3 equivalents of PTAD
for 15 minutes in DCM, modified amino acid 5/6 (Figure 3a) was
obtained and further analyzed by NMR spectroscopy. Protons at
C-7 and C-8 are observed as mutually coupled doublets,
90 indicating there are no other neighbouring protons (Figure 3b).
Moreover, a signal broadened through exchange with residual
water in DMF-d7, can be seen at 11,61 ppm (Figure 3c), favoring
structure 5 over the Diels-Alder adduct 6, since only the former
features an exchangeable hydrogen in the side chain. This
95 hypothesis was confirmed by measurements at -50°C, where the
exchange broadening is sufficiently reduced (Figure 3d) to allow

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45 Notes and references
a Organic and Biomimetic Chemistry Research Group and b NMR and
Structure Analysis, Department of Organic Chemistry; Krijgslaan 281,
S4; B-9000 Gent, Belgium; Fax: (+) 32-9-264 49 98; E-mail:
annemieke.madder@ugent.be
50 † Electronic Supplementary Information (ESI) available: Experimental
procedures, HPLC and NMR data. See DOI: 10.1039/b000000x/
‡ We thank Prof. Dr. Alain Krief for valuable suggestions and BOF-
UGent (01J06111) for financial support. The 700 MHz equipment is part
of the Interuniversitary NMR Facility funded in part by the FFEU-ZWAP
55 initiative of the Flemish Government.
1
(a) C. Chamberlain, K.M. Hahn, Traffic 2000, 1, 755.; (b) M.
Sameiro, T. Goncalves, Chem. Rev. 2009, 109, 190; (c) H. Sahoo,
RSC Adv.2012, 18, 7017.
2
(a) E. M. Sletten and C. R. Bertozzi, Acc. Chem. Res., 2011, 44, 666-
676; (b) J. M. Chalker, G. J. L. Bernardes and B. G. Davis, Acc.
Chem. Res., 2011, 44, 730-741; (c) S. Ng, M. R. Jafari and R. Derda,
ACS Chem. Biol., 2012, 7, 123-138.
A. D. de Araujo, J. M. Palomo, J. Cramer, O. Seitz, K. Alexandrov
and H. Waldmann, Chem.--Eur. J., 2006, 12, 6095-6109.
Fig. 4 FRET-probe 7 with alpha-chymotrypsin (solid) and control without
enzyme (dashed) for 1h at 37°C in 0,02 M NH4HCO3 buffer.
60
the surrounding structure to be mapped through both nOe and
3
n
5
heteronuclear JCH correlation techniques (cfr. Electronic
65 4 J. M. Palomo, Eur. J. Org. Chem., 2010, 33, 6303-6314.
Supplementary Information). Having established the furan-PTAD
aromatic substitution as a useful tool for quantitative site-
selective and irreversible peptide labeling, we further explored
the application potential of the newly developed methodology in
5
6
S. H. Weisbrod and A. Marx, Chem. Commun., 2008, 44, 5675-5685.
(a) N. K. Devaraj, R. Weissleder and S. A. Hilderbrand, Bioconjugate
Chem., 2008, 19, 2297-2299; (b) M. L. Blackman, M. Royzen and J.
M. Fox, J. Am. Chem. Soc., 2008, 130, 13518
70 7 V. Marchan, S. Ortega, D. Pulido, E. Pedroso and A. Grandas,
10 dual orthogonal peptide labeling. Indeed, given the recent
development of fluorescent TAD derivatives,18 current furan-
PTAD conjugation allows for convenient construction of FRET-
probes. A simple FRET-probe 7 was synthesized using the
commercially available TAD based compound DMEQ-TAD (4-
15 [2-(3,4-Dihydro-6,7-dimethoxy-4-methyl-3-oxo-2-quinoxalinyl)-
ethyl]-3H-1,2,4-triazole-3,5(4H)-dione). Following completion of
the peptide synthesis and labeling through reaction between
DMEQ-TAD and the β-2-furylalanine containing peptide,
subsequent Fmoc deprotection and labeling with the DABSYL
20 quencher was carried out. The resulting peptide 7 was cleaved
from the resin and purified by HPLC (Figure 4). Enzymatic
digestion of the peptide induced by alfa-chymotrypsin was
monitored by fluorescence spectrometry at 440 nm during 1
hour.22 In the short dead time between enzyme addition and
25 recording of the first measurements, a significant increase in
fluorescence was already observed compared to the control
sample (without enzyme treatment). This increase in fluorescence
continued for 30 minutes during which the control sample
exhibited by comparison only negligible amounts of fluorescence
30 (Figure 4). An additional advantage of this type of DMEQ-TAD
conjugation, apart from ease of synthesis, is the fluorogenic
nature of the labeling procedure. The fluorescence arises only
after conjugation, thus avoiding any problems associated with
background fluorescence caused by remaining excess reagent.
Nucleic Acids Res., 2006, 34, e24.
8
9
D. Graham, A. Grondin, C. McHugh, L. Fruk and W. E. Smith,
Tetrahedron Lett., 2002, 43, 4785-4788.
A. H. El-Sagheer, V. V. Cheong and T. Brown, Org. Biomol. Chem.,
2011, 9, 232-235.
75
10 A. Schulz, A. Busmann, E. Kluver, M. Schnebel and K. Adermann,
Protein Pept. Lett., 2004, 11, 601-606.
11 K. Hoogewijs, A. Deceuninck and A. Madder, Org. Biomol. Chem.,
2012, 10, 3999-4002.
80 12 C. D. Spicer and B. G. Davis, Chem. Commun., 2011, 47, 1698-1700.
13 (a) A maximum yield of 86% was obtained in the reaction of 4,4-
diethoxybut-2-ynal with solid phase bound furan performed for the
development of a new Diels-Alder based linker strategy; (b) S.
Albert, L. Blanco and S. Deloisy, Arkivoc, 2009, 78-96.
85 14 A. Sanchez, E. Pedroso, A. Grandas, Org. Lett., 2011, 13, 4364-4367.
15 A.
Gandini,
Prog.
Polym.
Sci.,
2012.,
doi:
10.1016/j.progpolymsci.2012.04.002
16 S. Yamashita, H. Fukushima, Y. Niidome, T. Mori, Y. Katayama and
T. Niidome, Langmuir, 2011, 27, 14621-14626.
90 17 A. B. S. Bakhtiari, D. Hsiao, G. X. Jin, B. D. Gates and N. R. Branda,
Angew. Chem., Int. Ed., 2009, 48, 4166-4169.
18 H. Ban, J. Gavrilyuk and C. F. Barbas, J. Am. Chem. Soc., 2010, 132,
1523
19 (a) F. Fabris, L. Leoni and O. De Lucchi, Tetrahedron Lett., 1999,
95
40, 1223-1226.; (b) L. A. Paquette, P. Webber and I. Simpson, Org.
Lett., 2003, 5, 177-180.; (c) G. A. Abernethy, Anal. Bioanal. Chem.,
2012, 403, 1433-1440.
20 M. E. Gonzalez-Rosende, J. Sepulveda-Arques, E. Zaballos-Garcia,
L. R. Domingo, R. J. Zaragoza, W. B. Jennings, S. E. Lawrence and
D. O'Leary, J. Chem. Soc., Perkin Trans. 2, 1999, 1, 73-79.
21 (a) Only a suggestion for the actual structure of the adduct is
available, based upon X-Ray photoelectron spectroscopic analysis, as
proposed by Chen et al. (b) G. H. Chen, M. Gupta, K. Chan and K.
K. Gleason, Macromol. Rapid Commun., 2007, 28, 2205-2209.
100
35 Conclusions
In conclusion, as the incorporation of furan moieties in peptides
and proteins has recently been firmly established in various
contexts (vide supra),11,12 the current methodology ideally
complements the toolbox of bio-orthogonal labeling reactions. In
40 conjunction with our previously developed furan-oxidation based
peptide labeling23 and nucleic acid crosslinking methodologies24
the current work again testifies of the usefulness and versatile
application of a simple and small aromatic furan moiety for the
decoration and conjugation of different biomacromolecules.
105 22 A. Chersi, S. Ferracuti, G. Falasca, R. H. Butler and D. Fruci, Anal.
Biochem., 2006, 357, 194-199.
23 A. Deceuninck and A. Madder, Chem. Commun., 2009, 3, 340-342.
24 (a) K. Stevens and A. Madder, Nucleic Acids Res., 2009, 37, 1555-
1565. (b) M. Op de Beeck and A. Madder, J. Am. Chem. Soc., 2011,
110
133, 796-807. (c) A. M. Jawalekar, M. O. de Beeck, F. L. van Delft
and A. Madder, Chem. Commun., 2011, 47, 2796-2798.; (d) M. Op
de Beeck and A. Madder, J. Am. Chem. Soc., 2012, 134, 10737-
10740.