Gold-catalyzed direct alkynylation of tryptophan in peptides using TIPS-EBX

Article abstract of DOI:10.3762/bjoc.12.74

The selective functionalization of peptides containing only natural amino acids is important for the modification of biomolecules. In particular, the installation of an alkyne as a useful handle for bioconjugation is highly attractive, but the use of a carbon linker is usually required. Herein, we report the gold-catalyzed direct alkynylation of tryptophan in peptides using the hypervalent iodine reagent TIPS-EBX (1-[(triisopropylsilyl)ethynyl]-1,2-benziodoxol-3(1H)-one). The reaction proceeded in 50-78% yield under mild conditions and could be applied to peptides containing other nucleophilic and aromatic amino acids, such as serine, phenylalanine or tyrosine.

Full text of DOI:10.3762/bjoc.12.74

Gold-catalyzed direct alkynylation of tryptophan in peptides
using TIPS-EBX
Gergely L. Tolnai1,2, Jonathan P. Brand1,3 and Jerome Waser*1
Letter
Open Access
Address:
Beilstein J. Org. Chem. 2016, 12, 745–749.
1Laboratory of Catalysis and Organic Synthesis, Ecole Polytechnique
Fédérale de Lausanne, EPFL SB ISIC LCSO, BCH 4306, 1015
Lausanne, Switzerland, 2Department of Organic Chemistry, Arrhenius
Laboratory, Stockholm University, SE-106 91 Stockholm, Sweden and
3Givaudan, Chemin de la parfumerie 5, 1214 Vernier, Switzerland
doi:10.3762/bjoc.12.74
Received: 07 February 2016
Accepted: 06 April 2016
Published: 19 April 2016
Email:
This article is part of the Thematic Series "C–H
Functionalization/activation in organic synthesis".
Jerome Waser* - jerome.waser@epfl.ch
* Corresponding author
Guest Editor: R. Sarpong
Keywords:
© 2016 Tolnai et al; licensee Beilstein-Institut.
License and terms: see end of document.
alkynes; C–H functionalization; gold catalysis; hypervalent iodine;
peptides
Abstract
The selective functionalization of peptides containing only natural amino acids is important for the modification of biomolecules. In
particular, the installation of an alkyne as a useful handle for bioconjugation is highly attractive, but the use of a carbon linker is
usually required. Herein, we report the gold-catalyzed direct alkynylation of tryptophan in peptides using the hypervalent iodine
reagent TIPS-EBX (1-[(triisopropylsilyl)ethynyl]-1,2-benziodoxol-3(1H)-one). The reaction proceeded in 50–78% yield under mild
conditions and could be applied to peptides containing other nucleophilic and aromatic amino acids, such as serine, phenylalanine
or tyrosine.
Introduction
Alkynes have always been important building blocks in synthe- desired molecules. In this context, the modification of natural
tic organic chemistry. Recently, they have attracted also strong peptides and proteins is highly attractive, and it has been the
interest for applications in materials science and chemical target of intensive research in the last decades (Figure 1) [6-11].
biology [1]. One of the most important transformations of The functionalization of highly reactive cysteine, lysine and the
alkynes is the copper-catalyzed [3 + 2] cycloaddition with N-terminus has been particularly successful [12-17], whereas
azides, which can be performed under mild conditions in the the more challenging modification of the electron-rich aromatic
presence of multiple functional groups, and has therefore found residues of tyrosine [18-20] and tryptophan [21-31] has been
broad applications for the modification of biomolecules and the focus of recent interest. As tryptophan is a rare amino acid,
polymers [2-5]. But before the unique reactivity of the triple its functionalization is especially interesting. It has been
bond can be unravelled, it is necessary to introduce it onto the achieved in the past for example by Francis and co-workers and
745

Beilstein J. Org. Chem. 2016, 12, 745–749.
Ball and co-workers using rhodium-catalyzed carbene-insertion
reactions [21-23] or via direct C–H arylation [24-29]. If the
installation of alkynes on peptides or proteins is desired, an
indirect method using a linker is used, for example an alkyl-
ation reaction of cysteine. The direct introduction of an alkyne
onto the biomolecule would be interesting to profit from modi-
fied electronic and spectroscopic properties. However, the
direct alkynylation of peptides or proteins is usually based on
the use of the Sonogashira reaction, which requires modified
non-natural amino acids [32,33].
Figure 1: Functionalization of natural peptides and proteins: state of
the art.
In 2013, our group reported the alkynylation of thiols using the
hypervalent iodine reagent TIPS-EBX (1a, 1-[(triisopropyl-
silyl)ethynyl]-1,2-benziodoxol-3(1H)-one) (Scheme 1A) [34].
The reaction was almost instantaneous. It was highly chemose-
lective for thiols in the presence of other nucleophilic func-
Scheme 1: Alkynylation with EBX reagents.
tional groups. The alkynylation could be therefore applied to ed to tryptophan-containing peptides. Even if the reaction gave
cysteine-containing peptides. The scope of the reaction could be C3-alkynylation for C3-unsubstituted indoles, we demonstrated
later extended to a broad range of aliphatic and aromatic that C2-alkynylation could be achieved on skatole (2a,
alkynes [35]. In 2015, the efficiency of the reaction for the Scheme 1B) [37]. Very recently, Hansen et al. indeed reported a
functionalization of proteins both in cell lysates and in the modified protocol using a gold catalyst and TIPS-EBX (1a) for
living cell was finally demonstrated [36].
the alkynylation of tryptophan-containing peptides and even
proteins (Scheme 1C) [39]. This recent disclosure motivated us
Even if the alkynylation of cysteines is an important method, to report our own work on this transformation, resulting in an
thiols are often part of disulfide bonds in folded proteins, and efficient direct alkynylation of tryptophan-containing peptides.
therefore difficult to access. Reduction and unfolding, or pro-
tein engineering to install more accessible cysteines, are usually Results and Discussion
required. For these reasons, it is important to develop selective We started our investigation by attempting the alkynylation of
alkynylation methods in order to functionalize other amino valine-tryptophan dipeptide 4a as model substrate (Table 1). An
acids. The direct C–H functionalization of aromatic compounds often used carboxybenzyl (Cbz, Z) protecting group was
is an attractive method for the modification of biomolecules chosen. Examining this substrate will tell if C2-alkynylation is
without the need for non-natural amino acids. However, the possible in the presence of an ester, a carbamate and an amide
multiple functional groups present in biomolecules make such a protecting group. A promising result was obtained with
process highly challenging. Based on our previous work on the 5 mol % gold chloride as catalyst at room temperature in aceto-
alkynylation of indoles using TIPS-EBX (1a) and a gold cataly- nitrile (Table 1, entry 1). Although the reaction did not go to
sis [37,38], we wondered if this transformation could be extend- completion even after two days, the desired C2 alkynylation
746

Beilstein J. Org. Chem. 2016, 12, 745–749.
product 5a was obtained in 44% yield. The yield could be in- obtained in 64% and 53% yield, respectively. The reaction was
creased to 72% when the reaction was performed at 40 °C (Ta- therefore general for dipeptides bearing tryptophan at the
ble 1, entry 2). No further improvement was observed at higher C-terminus. On the other hand, only traces of alkynylated
temperature (Table 1, entry 3). The product 5a could also be ob- dipeptide 5g with a tryptophan at the N-terminus could be ob-
tained in a broad range of other solvents, as long as the solu- tained under these reaction conditions. A first example of
bility of the substrate 4a and TIPS-EBX (1a) was sufficient (Ta- valine–tryptophan–valine tripeptide was also examined, and
ble 1, entries 4–8). The best yield was obtained in acetonitrile product 5h was isolated in 50% yield, demonstrating that
(Table 1, entry 2). Although the presence of water slowed down alkynylation of tryptophan inside a peptide chain was possible.
the reaction, the desired product could still be obtained in 41% Unfortunately, only limited conversion was observed with N- or
yield (Table 1, entry 9). Monitoring the reaction over time C-terminus unprotected peptides. Nevertheless, Hansen and
showed that 34% of product 5a was already formed after 20 co-workers recently demonstrated that N- and C-termini unpro-
min (Table 1, entry 10), but the reaction then slowed down sig- tected peptides, as well as more complex peptides and even pro-
nificantly, with 67% yield after 10 h and 78% after 24 h teins, could be alkynylated using modified reaction conditions
(Table 1, entries 11 and 12). At this point, a conversion higher (10 mol % AuCl(SMe2), three equivalents TIPS-EBX (1a) and
than 90% was achieved, with no significant improvement after a 2 mol % trifluoroacetic acid as co-catalyst) [39]. They also
longer reaction time.
demonstrated that the obtained silylalkyne products can be
easily deprotected with fluoride sources to allow bioconjuga-
tion via cycloaddition with azides.
Table 1: Optimization of the alkynylation of dipeptide 4a.
Conclusion
In conclusion, our work combined with the results of Hansen
and co-workers has demonstrated that the gold-catalyzed
alkynylation of indoles could be extended to tryptophan in
peptides. When considering the scarcity of methods allowing
the modification of tryptophan under mild conditions without
requiring the installation of non-natural amino acids, the trans-
formation will be highly useful for bioconjugation. A current
limitation of the developed alkynylation reaction is the require-
ment for organic solvents. Investigations are currently ongoing
in our laboratory for the development of water-compatible
reagents and catalysts.
entry
solvent
time (h)
T (°C)
yielda
1
2
3
4
5
6
7
8
9
CH3CN
CH3CN
CH3CN
iPrOH
48
48
48
48
48
48
48
48
48
23
40
60
40
40
40
40
40
40
44%
72%
67%
60%
50%
39%
63%
38%
41%
MeOH
Experimental
General procedure for the gold-catalyzed
alkynylation
acetone
CH2Cl2
DMSO
CH3CN
The starting peptide 4 (0.20 mmol, 1 equiv) and TIPS-EBX (1a,
0.240 mmol, 103 mg, 1.2 equiv) were added into a 5 mL test
tube equipped with a stirring bar. Acetonitrile (2 mL) was
added, then the reaction mixture was stirred at 40 °C for 2 min.
Gold(I) chloride (2.3 mg, 10 µmol, 0.05 equiv) was added in
one portion. The reaction tube was sealed and stirring was
continued for 24 h at 40 °C. Afterwards, the mixture was
diluted with EtOAc (50 mL), and the organic layer was washed
with a mixture of water (2.5 mL) and conc. NaHCO3 solution
(2.5 mL), and then with brine (20 mL), and dried over MgSO4.
The solvent was evaporated under reduced pressure and the re-
sulting yellow oil was purified by column chromatography
(SiO2, hexane/EtOAc 3:1 to 2:3). The product was dried under
reduced pressure, and washed into a vial with Et2O. The sol-
vent was evaporated under vacuum and dried under high
vacuum (ca. 10−2 mbar) for several hours.
5% H2O
10
11
12
CH3CN
CH3CN
CH3CN
0.3
10
24
40
40
40
34%
67%
78%
aReaction conditions: 0.20 mmol 4a, 0.24 mmol TIPS-EBX (1a),
0.010 mmol AuCl in 2 mL solvent were stirred at the indicated temper-
ature and time. Isolated yields after column chromatography are given.
With the optimized conditions in hand, we investigated the
scope of the reaction with different amino acids in the dipep-
tide (Scheme 2). With glycine as second amino acid, the desired
product 5b could be obtained in 66% yield. The reaction was
selective for tryptophan in the presence of other aromatic amino
acids, such as phenylalanine or tyrosine (products 5c and 5d).
Serine and proline containing dipeptides 5e and 5f could also be
747

Beilstein J. Org. Chem. 2016, 12, 745–749.
Scheme 2: Alkynylation of tryptophan-containing peptides.
References
1. Diederich, F.; Stang, P. J.; Tykwinski, R. R., Eds. Acetylene Chemistry:
Chemistry, Biology and Material Science; Wiley-VCH: Weinheim,
Germany, 2005.
Supporting Information
Supporting Information File 1
Experimental procedure and characterization data for all
compounds. NMR spectra of new compounds.
[http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-12-74-S1.pdf]
2. Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B.
Angew. Chem., Int. Ed. 2002, 41, 2596.
doi:10.1002/1521-3773(20020715)41:14<2596::AID-ANIE2596>3.0.CO
;2-4
3. Lutz, J.-F. Angew. Chem., Int. Ed. 2007, 46, 1018.
doi:10.1002/anie.200604050
4. Meldal, M.; Tornøe, C. W. Chem. Rev. 2008, 108, 2952.
doi:10.1021/cr0783479
Acknowledgements
We thank the EPFL for funding and F. Hoffmann-La Roche
Ltd. for an unrestricted research grant. The work of G.L.T. was
supported by a Sciex-NMSch fellowship of the Swiss confedera-
tion.
5. McKay, C. S.; Finn, M. G. Chem. Biol. 2014, 21, 1075.
doi:10.1016/j.chembiol.2014.09.002
6. Stephanopoulos, N.; Francis, M. B. Nat. Chem. Biol. 2011, 7, 876.
doi:10.1038/nchembio.720
748

Beilstein J. Org. Chem. 2016, 12, 745–749.
7. Takaoka, Y.; Ojida, A.; Hamachi, I. Angew. Chem., Int. Ed. 2013, 52,
4088. doi:10.1002/anie.201207089
34.Frei, R.; Waser, J. J. Am. Chem. Soc. 2013, 135, 9620.
doi:10.1021/ja4044196
8. Patterson, D. M.; Nazarova, L. A.; Prescher, J. A. ACS Chem. Biol.
2014, 9, 592. doi:10.1021/cb400828a
35.Frei, R.; Wodrich, M. D.; Hari, D. P.; Borin, P.-A.; Chauvier, C.;
Waser, J. J. Am. Chem. Soc. 2014, 136, 16563.
doi:10.1021/ja5083014
9. Yang, M.; Li, J.; Chen, P. R. Chem. Soc. Rev. 2014, 43, 6511.
doi:10.1039/C4CS00117F
36.Abegg, D.; Frei, R.; Cerato, L.; Prasad Hari, D.; Wang, C.; Waser, J.;
Adibekian, A. Angew. Chem., Int. Ed. 2015, 54, 10852.
doi:10.1002/anie.201505641
10.Boutureira, O.; Bernardes, G. J. L. Chem. Rev. 2015, 115, 2174.
doi:10.1021/cr500399p
11.Koniev, O.; Wagner, A. Chem. Soc. Rev. 2015, 44, 5495.
doi:10.1039/C5CS00048C
37.Brand, J. P.; Charpentier, J.; Waser, J. Angew. Chem., Int. Ed. 2009,
48, 9346. doi:10.1002/anie.200905419
12.Chalker, J. M.; Bernardes, G. J. L.; Davis, B. G. Acc. Chem. Res. 2011,
44, 730. doi:10.1021/ar200056q
38.Tolnai, G. L.; Ganss, S.; Brand, J. P.; Waser, J. Org. Lett. 2013, 15,
112. doi:10.1021/ol3031389
13.Kundu, R.; Ball, Z. T. Chem. Commun. 2013, 49, 4166.
doi:10.1039/C2CC37323H
39.Hansen, M. B.; Hubálek, F.; Skrydstrup, T.; Hoeg-Jensen, T.
Chem. – Eur. J. 2016, 22, 1572. doi:10.1002/chem.201504462
14.Toda, N.; Asano, S.; Barbas, C. F., III. Angew. Chem., Int. Ed. 2013,
52, 12592. doi:10.1002/anie.201306241
15.Abbas, A.; Xing, B.; Loh, T.-P. Angew. Chem., Int. Ed. 2014, 53, 7491.
doi:10.1002/anie.201403121
License and Terms
16.Obermeyer, A. C.; Jarman, J. B.; Francis, M. B. J. Am. Chem. Soc.
2014, 136, 9572. doi:10.1021/ja500728c
This is an Open Access article under the terms of the
Creative Commons Attribution License
17.Vinogradova, E. V.; Zhang, C.; Spokoyny, A. M.; Pentelute, B. L.;
Buchwald, S. L. Nature 2015, 526, 687. doi:10.1038/nature15739
18.Joshi, N. S.; Whitaker, L. R.; Francis, M. B. J. Am. Chem. Soc. 2004,
126, 15942. doi:10.1021/ja0439017
(http://creativecommons.org/licenses/by/2.0), which
permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
19.Tilley, S. D.; Francis, M. B. J. Am. Chem. Soc. 2006, 128, 1080.
doi:10.1021/ja057106k
The license is subject to the Beilstein Journal of Organic
Chemistry terms and conditions:
20.Ban, H.; Gavrilyuk, J.; Barbas, C. F., III. J. Am. Chem. Soc. 2010, 132,
1523. doi:10.1021/ja909062q
(http://www.beilstein-journals.org/bjoc)
21.Antos, J. M.; Francis, M. B. J. Am. Chem. Soc. 2004, 126, 10256.
doi:10.1021/ja047272c
22.Antos, J. M.; McFarland, J. M.; Iavarone, A. T.; Francis, M. B.
J. Am. Chem. Soc. 2009, 131, 6301. doi:10.1021/ja900094h
23.Popp, B. V.; Ball, Z. T. J. Am. Chem. Soc. 2010, 132, 6660.
doi:10.1021/ja101456c
The definitive version of this article is the electronic one
which can be found at:
doi:10.3762/bjoc.12.74
24.Ruiz-Rodríguez, J.; Albericio, F.; Lavilla, R. Chem. – Eur. J. 2010, 16,
1124. doi:10.1002/chem.200902676
25.Preciado, S.; Mendive-Tapia, L.; Albericio, F.; Lavilla, R. J. Org. Chem.
2013, 78, 8129. doi:10.1021/jo400961x
26.Mendive-Tapia, L.; Preciado, S.; Garcia, J.; Ramon, R.; Kielland, N.;
Albericio, F.; Lavilla, R. Nat. Commun. 2015, 6, No. 7160.
doi:10.1038/ncomms8160
27.Williams, T. J.; Reay, A. J.; Whitwood, A. C.; Fairlamb, I. J. S.
Chem. Commun. 2014, 50, 3052. doi:10.1039/c3cc48481e
28.Reay, A. J.; Williams, T. J.; Fairlamb, I. J. S. Org. Biomol. Chem. 2015,
13, 8298. doi:10.1039/C5OB01174D
29.Zhu, Y.; Bauer, M.; Ackermann, L. Chem. – Eur. J. 2015, 21, 9980.
doi:10.1002/chem.201501831
30.Perekalin, D. S.; Novikov, V. V.; Pavlov, A. A.; Ivanov, I. A.;
Anisimova, N. Yu.; Kopylov, A. N.; Volkov, D. S.; Seregina, I. F.;
Bolshov, M. A.; Kudinov, A. R. Chem. – Eur. J. 2015, 21, 4923.
doi:10.1002/chem.201406510
31.Siti, W.; Khan, A. K.; de Hoog, H.-P. M.; Liedberg, B.; Nallani, M.
Org. Biomol. Chem. 2015, 13, 3202. doi:10.1039/C4OB02025A
32.Kodama, K.; Fukuzawa, S.; Nakayama, H.; Sakamoto, K.; Kigawa, T.;
Yabuki, T.; Matsuda, N.; Shirouzu, M.; Takio, K.; Yokoyama, S.;
Tachibana, K. ChemBioChem 2007, 8, 232.
doi:10.1002/cbic.200600432
33.Li, N.; Lim, R. K. V.; Edwardraja, S.; Lin, Q. J. Am. Chem. Soc. 2011,
133, 15316. doi:10.1021/ja2066913
749