Bioorthogonal Diversification of Peptides through Selective Ruthenium(II)-Catalyzed C–H Activation

Article abstract of DOI:10.1002/anie.201609631

Methods for the chemoselective modification of amino acids and peptides are powerful techniques in biomolecular chemistry. Among other applications, they enable the total synthesis of artificial peptides. In recent years, significant momentum has been gained by exploiting palladium-catalyzed cross-coupling for peptide modification. Despite major advances, the prefunctionalization elements on the coupling partners translate into undesired byproduct formation and lengthy synthetic operations. In sharp contrast, we herein illustrate the unprecedented use of versatile ruthenium(II)carboxylate catalysis for the step-economical late-stage diversification of α- and β-amino acids, as well as peptides, through chemo-selective C?H arylation under racemization-free reaction conditions. The ligand-accelerated C?H activation strategy proved water-tolerant and set the stage for direct fluorescence labelling as well as various modes of peptide ligation with excellent levels of positional selectivity in a bioorthogonal fashion. The synthetic utility of our approach is further demonstrated by twofold C?H arylations for the complexity-increasing assembly of artificial peptides within a multicatalytic C?H activation manifold.

Full text of DOI:10.1002/anie.201609631

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International Edition: DOI: 10.1002/anie.201609631
German Edition: DOI: 10.1002/ange.201609631
Peptide Chemistry
Hot Paper
Bioorthogonal Diversification of Peptides through Selective
Ruthenium(II)-Catalyzed C–H Activation
Alexandra Schischko, Hongjun Ren, Nikolaos Kaplaneris, and Lutz Ackermann*
À
Abstract: Methods for the chemoselective modification of
amino acids and peptides are powerful techniques in biomo-
lecular chemistry. Among other applications, they enable the
total synthesis of artificial peptides. In recent years, significant
momentum has been gained by exploiting palladium-catalyzed
cross-coupling for peptide modification. Despite major advan-
ces, the prefunctionalization elements on the coupling partners
translate into undesired byproduct formation and lengthy
synthetic operations. In sharp contrast, we herein illustrate the
unprecedented use of versatile ruthenium(II)carboxylate cat-
alysis for the step-economical late-stage diversification of a-
and b-amino acids, as well as peptides, through chemo-selective
platform.[4,5,9] Palladium(II)-catalyzed C H functionalization
in particular has been identified as an enabling tool in
molecular amino acid synthesis,[10] with key contributions by
the groups of Yu,[11] Lavilla/Albericio,[12] and Daugulis,[13]
among others.[14a–e,10] Despite these undisputable advances,
auxiliary-based strategies (Scheme 1a) frequently require
stoichiometric quantities of silver(I) salts, and are positionally
limited to C-terminal residues. While tryptophan is utilized
for protein quantification and to study protein dynamics,
folding, and binding, as well as protein–protein interac-
tions,[15] tryptophan arylations are restricted by the need for
super-stoichiometric amounts of highly reactive hypervalent
À
C H arylation under racemization-free reaction conditions.
À
The ligand-accelerated C H activation strategy proved water-
tolerant and set the stage for direct fluorescence labelling as
well as various modes of peptide ligation with excellent levels
of positional selectivity in a bioorthogonal fashion. The
synthetic utility of our approach is further demonstrated by
À
twofold C H arylations for the complexity-increasing assem-
À
bly of artificial peptides within a multicatalytic C H activation
manifold.
T
he chemoselective ligation of peptides is of prime impor-
tance in biomolecular chemistry for the chemical synthesis of
peptides and proteins.[1–6] Non-natural peptides are of key
relevance for numerous applied research areas, ranging from
proteomics and diagnosis to asymmetric synthesis and drug
delivery.[1b,6,7] For instance, peptides derived from non-
proteinogenic amino acids are central to pharmaceutical
and medicinal chemistry, since they are largely characterized
by improved bioactivity and pharmacokinetics compared to
their natural counterparts.[8] Palladium-catalyzed cross-cou-
pling chemistry has proven instrumental for the late-stage
diversification of synthetically engineered peptides.[2] How-
ever, the power of this cross-coupling approach continues to
be comprised by its restriction to the synthesis and use of
prefunctionalized structural scaffolds, which leads to unde-
sired byproduct formation and lengthy syntheses. To address
the limitations of conventional cross-couplings through the
À
interconversion of preexisting functional groups, C H acti-
vation chemistry has recently emerged as a transformative
[*] A. Schischko, Dr. H. Ren, N. Kaplaneris, Prof. Dr. L. Ackermann
Institut fꢀr Organische und Biomolekulare Chemie
Georg-August-Universitꢁt Gçttingen
Tammannstrasse 2, 37077 Gçttingen (Germany)
E-mail: Lutz.Ackermann@chemie.uni-goettingen.de
À
Scheme 1. Strategies for C H activation in peptide diversification.
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under http://dx.doi.org/10.
1002/anie.201609631.
a) Auxiliary assistance on C-terminal residues. b) Palladium(II/IV)
catalysis with hypervalent iodine(III) or silver(I) reagents. c) Mild
ruthenium(II) catalysis for peptide diversification (this work).
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iodine reagents, boronic acid derivatives, and/or costly silver-
(I) salts, which limits further practical applications (Scheme
1b).[10] In stark contrast, we report herein the first site- and
chemo-selective ruthenium(II)-catalyzed[3,16] C H function-
À
alization of peptides. Notable features of our strategy include
1) the development of a uniquely versatile ruthenium(II)
À
catalyst for the C H functionalization of tryptophan-based
peptides with easily accessible arylating agents, and 2) race-
À
mization-free C H arylations, which set the stage for efficient
À
fluorescence labelling, b-amino acid C H modifications, and
different modes of bioorthogonal peptide ligation (Scheme
1c).
À
Our studies on peptide C H functionalizations com-
menced with establishing reaction conditions for the pre-
À
requisite C H arylation of amino acid 1 with the readily
available aryl bromide 2a (Table S2 in the Supporting
Information). Detailed optimization studies revealed the
most efficient catalysis to be realized with user-friendly
[RuCl2(p-cymene)]2 as the catalyst, along with the mild base
K3PO4. In contrast to our previous methods for ruthenium-
[16b]
À
catalyzed C H arylations,
the best results were achieved
with a novel catalytic system derived from electron-deficient
F5C6CO2H, which guarantees efficient carboxylate-assisted
C H activation.[17] With the optimized reaction conditions for
À
À
the amino acid C H diversification in hand, we explored its
versatility with a representative set of challenging aryl
bromides (2; Scheme 2a). We were pleased to observe that
a variety of decorated substrates 2 possessing electrophilic
functional groups, such as ester, nitrile, enolizable ketone, and
pyridine substituents, were fully tolerated by the ruthenium-
À
(II)-catalyzed C H activation. Specifically, chemoselective
transformation of the NH2-free aniline bromide indicated the
unique synthetic potential of our approach for a bioorthogo-
À
nal peptide diversification. The robustness of the C H
activation approach is demonstrated by, among others, the
installation of the redox-active ferrocene motif (3l) and
a pyrene-based fluorescent label (3m). It is particularly
noteworthy that racemization of the amino acids 3 did not
occur under the rather mild[18] C H arylation conditions (see
À
À
the Supporting Information). The potential of the C H
activation strategy for fluorescent labelling is highlighted by
À
the considerably improved fluorescent properties of the C H
arylated products 3 compared to the parent tryptophan
1 (Scheme 2b). b-Amino acids are key structural motifs of
non-natural b-peptides and represent versatile intermediates
in organic syntheses.[19] Therefore, we were delighted to
À
observe that the versatile ruthenium(II)-catalyzed C H
À
activation approach also enabled the first C H functionaliza-
tion of b3-tryptophan (4) with broad functional-group toler-
ance in a positional-selective fashion (Scheme 2c).
Subsequently, we evaluated the ruthenium(II)-catalyzed
À
C H arylation strategy for the envisioned bioorthogonal
À
diversification of peptides 6 (Scheme 3). The C H function-
À
Scheme 2. Expedient ruthenium(II)-catalyzed amino acid C H aryla-
alization approach proved amenable to efficient syntheses of
peptides 7 involving both a- and b-amino acids. Notably, the
optimized catalyst allowed the introduction of fluorescence
labels (7a,b) as well as the chemoselective transformation and
the preparation of NH-free tryptophan-containing peptides
(7b–h). These findings highlight the outstanding chemo-
À
tion. a) Versatile C H arylation of a-amino acid 1. b) Fluorescent
À
properties of C H arylated products 3 versus substrate 1, and a 3D
excitation/emission graph for 3m. c) Unprecedented ruthenium(II)-
3
À
catalyzed C H activation of b -amino acid 4.
2
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À
Scheme 3. Ruthenium(II)-catalyzed C H activation for the fluorescence
labeling and late-stage modification of peptides 6.
selectivity of our ruthenium(II) catalysis, which is inherently
complementary to previously reported methods (see below).
À
The robustness of the ruthenium(II) C H activation
À
method enabled twofold C H arylations for the homo-
arylation/ligation of peptides 6 and delivered the di- and
tetra-peptides 9 in high yields of isolated product (Scheme
À
4a). Based on these findings, the ruthenium(II) C H
arylation catalysis approach set the stage for the chemo-
selective ligation of peptides 6 and peptides 10, which again
proceeded without any notable racemization of the stereo-
genic centers (Scheme 4b). The bioorthogonal ligation of two
distinct peptides was accomplished by means of a novel
À
À
multicatalytic C H activation sequence that exploits the
Scheme 4. Peptide ligation by means of ruthenium(II)-catalyzed C H
complementary nature of palladium(II)[14d] and the
À
activation. a) A twofold C H arylation/ligation strategy. b) Chemical
À
ligation of peptides 6 by C H arylation. c) Peptide ligation by using
a sequential palladium(II)- and ruthenium(II)-catalyzed C H function-
À
ruthenium(II) catalysis (Scheme 4c). This twofold C H
À
arylation approach enabled the direct preparation of struc-
turally complex peptides 14 with excellent levels of positional-
and chemoselectivity control.
alization strategy.
À
Finally, this ruthenium(II) catalysis approach for bioor-
thogonal peptide diversification was shown to be fully
tolerant of water[20] (Scheme 5a), which should prove invalu-
able for further applications. In addition, the outstanding
robustness of the ruthenium(II)-catalyzed C H arylation
À
method was reflected by a chemoselectivity screen for C H
activation in the presence of specifically added amino acids
(Scheme 5b).
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and industry, particularly since the ruthenium(II)-catalyzed
À
C H arylation was shown to be fully tolerant of water and
could thus be performed in aqueous media.
Acknowledgements
Generous support by the European Research Council under
the European Communityꢀs Seventh Framework Program
(FP7 2007–2013)/ERC Grant agreement no. 307535, and the
Onassis Foundation (fellowship to N.K.) is gratefully
acknowledged.
Conflict of interest
The authors declare no conflict of interest.
À
Keywords: C H activation · labeling · ligation · peptides ·
ruthenium
[1] a) P. E. Dawson, T. W. Muir, I. Clark-Lewis, S. B. H. Kent,
Science 1994, 266, 776 – 779; b) E. Saxon, C. R. Bertozzi, Science
2000, 287, 2007 – 2010.
[2] J. M. Chalker, G. J. L. Bernardes, B. G. Davis, Acc. Chem. Res.
2011, 44, 730 – 741.
[3] a) S. De Sarkar, W. Liu, S. I. Kozhushkov, L. Ackermann, Adv.
Synth. Catal. 2014, 356, 1461 – 1479; b) P. B. Arockiam, C.
Bruneau, P. H. Dixneuf, Chem. Rev. 2012, 112, 5879 – 5918;
c) L. Ackermann, R. Vicente, Top. Curr. Chem. 2009, 292, 211 –
229; d) L. Ackermann, R. Vicente, A. R. Kapdi, Angew. Chem.
Int. Ed. 2009, 48, 9792 – 9826; Angew. Chem. 2009, 121, 9976 –
10011.
[4] a) X.-C. Wang, W. Gong, L.-Z. Fang, R.-Y. Zhu, S. Li, K. M.
Engle, J.-Q. Yu, Nature 2015, 519, 334 – 338; b) A. J. Hickman,
M. S. Sanford, Nature 2012, 484, 177 – 185; c) R. G. Bergman,
Nature 2007, 446, 391 – 393; d) J. Wencel-Delord, F. Glorius, Nat.
Chem. 2013, 5, 369 – 375.
[5] D.-H. Wang, K. M. Engle, B.-F. Shi, J.-Q. Yu, Science 2010, 327,
315 – 319.
[6] E. V. Vinogradova, C. Zhang, A. M. Spokoyny, B. L. Pentelute,
S. L. Buchwald, Nature 2015, 526, 687 – 691.
Scheme 5. Chemoselectivity of the bioorthogonal ruthenium(II)-cata-
À
À
lyzed amino acid C H arylation. a) C H arylation in the presence of
H2O. b) Chemoselectivity screen with different amino acids.
[7] E. A. C. Davie, S. M. Mennen, Y. Xu, S. J. Miller, Chem. Rev.
2007, 107, 5759 – 5812.
[8] F. Albericio, H. G. Kruger, Future Med. Chem. 2012, 4, 1527 –
1531.
[9] X. Chen, K. M. Engle, D.-H. Wang, J.-Q. Yu, Angew. Chem. Int.
Ed. 2009, 48, 5094 – 5115; Angew. Chem. 2009, 121, 5196 – 5217.
[10] A. F. M. Noisier, M. A. Brimble, Chem. Rev. 2014, 114, 8775 –
8806.
[11] a) W. Gong, G. Zhang, T. Liu, R. Giri, J.-Q. Yu, J. Am. Chem.
Soc. 2014, 136, 16940 – 16946; b) J. He, S. Li, Y. Deng, H. Fu,
B. N. Laforteza, J. E. Spangler, A. Homs, J.-Q. Yu, Science 2014,
343, 1216 – 1220.
[12] a) L. Mendive-Tapia, S. Preciado, J. Garcꢁa, R. Ramꢂn, N.
Kielland, F. Albericio, R. Lavilla, Nat. Commun. 2015, 6, DOI:
10.1038/ncomms8160; b) J. Ruiz-Rodrꢁguez, F. Albericio, R.
Lavilla, Chem. Eur. J. 2010, 16, 1124 – 1127.
[13] L. D. Tran, O. Daugulis, Angew. Chem. Int. Ed. 2012, 51, 5188 –
5191; Angew. Chem. 2012, 124, 5278 – 5281.
In summary, we have developed an unprecedented
À
ruthenium(II)-catalyzed peptide C H functionalization strat-
egy for fluorescent labelling and bioorthogonal chemical
ligation.
Powerful
ligand-accelerated
ruthenium(II)-
3
À
carboxylate catalysis enabled C H arylations of a- and b -
amino acids with broad scope and high catalytic efficacy. The
unique positional and chemoselectivity set the stage for
À
bioorthogonal C H functionalizations for the synthesis of
diversely decorated peptides. In this way, novel modes of
À
peptide ligation were introduced by means of twofold C H
activation. Our findings illustrate the outstanding synthetic
À
utility of ruthenium(II)-catalyzed C H activation in complex-
ity-increasing peptide synthesis. These methods should prove
instrumental for future applications in fluorescence labelling,
late-stage diversification, and peptide ligation in academia
4
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Angewandte
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[14] For representative examples of peptide modification, see: a) Y.
[17] L. Ackermann, Chem. Rev. 2011, 111, 1315 – 1345.
Seki, T. Ishiyama, D. Sasaki, J. Abe, Y. Sohma, K. Oisaki, M.
Kanai, J. Am. Chem. Soc. 2016, 138, 10798 – 10801; b) G. He, B.
Wang, W. A. Nack, G. Chen, Acc. Chem. Res. 2016, 49, 635 – 645;
c) T. J. Osberger, D. C. Rogness, J. T. Kohrt, A. F. Stepan, M. C.
White, Nature 2016, 537, 214 – 219; d) Y. Zhu, M. Bauer, L.
Ackermann, Chem. Eur. J. 2015, 21, 9980 – 9983; e) T. J. Wil-
liams, A. J. Reay, A. C. Whitwood, I. J. S. Fairlamb, Chem.
Commun. 2014, 50, 3052 – 3054; f) Y. Zhu, M. Bauer, J. Ploog, L.
Ackermann, Chem. Eur. J. 2014, 20, 13099 – 13102; g) L. S.
Witus, M. B. Francis, Acc. Chem. Res. 2011, 44, 774 – 783; h) B. V.
Popp, Z. T. Ball, J. Am. Chem. Soc. 2010, 132, 6660 – 6662;
i) B. V. Subba Reddy, R. R. Leleti, E. J. Corey, Org. Lett. 2006, 8,
3391 – 3394.
[18] T. Gensch, M. N. Hopkinson, F. Glorius, J. Wencel-Delord,
Chem. Soc. Rev. 2016, 45, 2900 – 2936.
[19] a) D. Seebach, J. Gardiner, Acc. Chem. Res. 2008, 41, 1366 –
1375; b) R. P. Cheng, S. H. Gellman, W. F. DeGrado, Chem. Rev.
2001, 101, 3219 – 3232.
[20] a) L. Ackermann, J. Pospech, H. K. Potukuchi, Org. Lett. 2012,
14, 2146 – 2149; b) P. B. Arockiam, C. Fischmeister, C. Bruneau,
P. H. Dixneuf, Angew. Chem. Int. Ed. 2010, 49, 6629 – 6632;
Angew. Chem. 2010, 122, 6779 – 6782; c) L. Ackermann, Org.
Lett. 2005, 7, 3123 – 3125.
[15] R. W. Sinkeldam, N. J. Greco, Y. Tor, Chem. Rev. 2010, 110,
2579 – 2619.
[16] a) K. Devaraj, C. Sollert, C. Juds, P. J. Gates, L. T. Pilarski,
Chem. Commun. 2016, 52, 5868 – 5871; b) L. Ackermann, A. V.
Lygin, Org. Lett. 2011, 13, 3332 – 3335.
Manuscript received: October 1, 2016
Revised: November 27, 2016
Final Article published: && &&, &&&&
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Communications
Peptide Chemistry
A. Schischko, H. Ren, N. Kaplaneris,
L. Ackermann*
&&& — &&&
Bioorthogonal Diversification of Peptides
through Selective Ruthenium(II)-
Catalyzed C–H Activation
Making a difference: A versatile
under racemization-free reaction condi-
À
ruthenium(II)carboxylate catalysis strat-
egy was developed that enables the step-
economical late-stage diversification of a-
and b-amino acids, as well as peptides,
tions. This ligand-accelerated C H acti-
vation is water-tolerant and sets the stage
for direct fluorescence labelling as well as
various modes of peptide ligation.
À
through bioorthogonal C H arylation
6
www.angewandte.org
ꢀ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2017, 56, 1 – 6
These are not the final page numbers!