Palladium-Catalyzed Aminocarbonylation in Solid-Phase Peptide Synthesis: A Method for Capping, Cyclization, and Isotope Labeling

Article abstract of DOI:10.1021/acs.orglett.7b01068

A new synthetic approach for introducing N-capping groups onto peptides attached to a solid support, combining aminocarbonylation under mild conditions using a palladacycle precatalyst and solid-phase peptide synthesis, is reported. The use of a silacarboxylic acid as an in situ CO-releasing molecule allowed the reaction to be performed in a single vial. The method also enables versatile substitution of side chains, side-chain-to-side-chain cyclizations, and selective [¹³C] acyl labeling of modified peptides.

Full text of DOI:10.1021/acs.orglett.7b01068

Letter
pubs.acs.org/OrgLett
Palladium-Catalyzed Aminocarbonylation in Solid-Phase Peptide
Synthesis: A Method for Capping, Cyclization, and Isotope Labeling
Anna Skogh,† Stig D. Friis,‡ Troels Skrydstrup,‡ and Anja Sandstrom*,†
̈
†Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry, BMC, Uppsala University, Box 574, SE-751 23 Uppsala,
Sweden
‡Carbon Dioxide Activation Center (CADIAC), Interdisciplinary Nanoscience Center (iNANO) and Department of Chemistry,
Aarhus University, Gustav Wieds Vej 14, 8000 Aarhus C, Denmark
S
* Supporting Information
ABSTRACT: A new synthetic approach for introducing N-
capping groups onto peptides attached to a solid support,
combining aminocarbonylation under mild conditions using a
palladacycle precatalyst and solid-phase peptide synthesis, is
reported. The use of a silacarboxylic acid as an in situ CO-
releasing molecule allowed the reaction to be performed in a
single vial. The method also enables versatile substitution of side chains, side-chain-to-side-chain cyclizations, and selective [13C]
acyl labeling of modified peptides.
well-established, three-component, Pd-catalyzed coupling be-
eptides have been neglected in drug discovery for many
Pyears due to their generally poor oral bioavailability and tween (hetero)aryl halides, an amine, and carbon monoxide.15
The development of CO-releasing molecules (CORMs) for in
situ16 and ex situ17 generation of CO has improved handling and
safety aspects significantly in carbonylation chemistry. To our
knowledge, aminocarbonylation has so far never been applied in
SPPS.
We foresee several benefits to combining an amino-
carbonylation protocol with SPPS. First, it would facilitate the
possibility of using excess reagents to drive reactions to
completion due to easy workup using filtration. Second, the
use of aryl halides instead of reactive acyl-transfer reagents like
acid chlorides allows mild conditions but also new opportunities
for cyclization of peptides without the demand of orthogonally
protected carboxylic acids. Moreover, such methods would
enable 13C- and potentially 11C-isotope labeling of peptides,
which would be useful in the study of bioactive peptides in
modern drug discovery.18−21
limited stability. However, the situation is rapidly changing due
to the comparatively higher approval rate of peptide-based drugs
as compared to small-molecule drugs in recent years.1−4 Indeed,
peptides and peptidomimetics beyond the rule of five have been
suggested as potential future therapeutics for addressing difficult
targets and diseases with unmet medical needs.1,2 Several
chemical approaches have been used to improve bioavailability
and stability of peptides, for example, N-methylation of the
backbone,5 various cyclizations strategies,3,6 substitution by
constrained and unnatural amino acids,7 and modifications of the
C- and N-terminal ends.1 N-Terminal capping of peptides with
acyl, aroyl, or heteroaroyl groups can, besides masking the basic
amine, improve target interactions, selectivity, cell permeability,
and plasma stability.8−11 Aroyl N-capping groups are found in
several pharmaceutically relevant peptide-based compounds
(Figure 1) and have proven useful for 18F-labeling and as
photoactivatable probes in peptides.12,13
Herein, we demonstrate for the first time the facile conversion
of aryl and heteroaryl iodides into their corresponding amides
using solid-phase bound amino acid/peptide nucleophiles and in
situ generation of CO from a crystalline silacarboxylic acid18 at
room temperature.
N-Capping groups are usually incorporated through acylation
using carboxylic acid derivatives in solid-phase peptide synthesis
(SPPS).10,13,14 An alternative approach to amides is through the
In order to develop a safe, mild, and easy-to-execute
aminocarbonylation compatible with the polystyrene resin the
mode of mixing, choice of CORM, and reaction temperature
were investigated. First, mixing by agitation rather than magnetic
stirring was preferred in our SPPS protocol to reduce the risk of
resin fragmentation. Second, during preliminary studies,
silacarboxylic acid 3 proved to be a clean and efficient CO
precursor for aminocarbonylations upon fluoride treatment at
Figure 1. Examples of pharmaceuticals with N-capping groups marked
in bold style.
Received: April 13, 2017
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.7b01068
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
room temperature, even when used in situ. Third, a low reaction
temperature was desired to avoid racemization of the amino
acids. Previously, the employment of a XantPhos ligated
palladacycle precatalyst22 in aminocarbonylation reactions has
been demonstrated to convert (hetero)aryl bromides to their
corresponding amide in 14−16 h at only 45 °C due to the
enhanced reactivity of the active Pd-catalyst generated.23 We
therefore envisioned that using the more reactive (hetero)aryl
iodides could enable formation of the corresponding amides
through agitation already at room temperature using this
palladacycle precatalyst and silacarboxylic acid 3 even if the
nucleophile is attached to a solid phase. Our investigation
commenced with the reaction of 4-iodoanisole with preloaded
phenylalanine Wang polystyrene (PS) resin (1a) in dry DMF in
the presence of 2 mol % of XantPhos-ligated palladacycle
precatalyst 2 and 2 equiv of MePh2SiCOOH (3) and KF (entry
1, Table 1). The solid-phase-bound amine 1a was set as the
limiting reagent, and the yield was calculated from the loading of
the resin. Aminocarbonylation of 4-iodoanisole proceeded
efficiently and afforded 4a in 95% yield after 15 h with
subsequent cleavage from the resin using 95% TFA in the
presence of triethylsilane (TES) followed by precipitation in cold
diethyl ether. Furthermore, the method was successfully applied
to a range of other solid-phase-bound amino acid nucleophiles
(entries 2−8). Thus, the secondary amine of proline (1b) (entry
2) gave 4b in an isolated yield of 94%. Moreover, the β-branched
amino acids 1c and 1d (entries 3 and 4) were transformed into
products 4c and 4d in 90% and 95% yields, respectively.
To investigate whether racemization had occurred, the optical
rotation of selected products from Table 1 were compared with
those synthesized from the conventional coupling of p-anisic acid
using N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)uranium
hexafluorophosphate (HBTU) and N,N-diisopropylethylamine
(DIPEA). In all cases, equivalent values for optical rotation were
obtained, indicating the present method is performing equally
well in this aspect. The fact that no epimers where formed in the
synthesis of longer peptides (vide infra) further supports
negligible racemization (see the Supporting Information).
Next, an array of (hetero)aryl iodides were explored to
demonstrate the generality of this reaction (Scheme 1). The
electron-rich aryl iodides showed good isolated yields using a low
catalytic loading and only 2 equiv of 3 (5a,b, Scheme 1), while
aminocarbonylation of iodobenzene to yield product 5c required
a higher catalytic loading of 5 mol % to reach full conversion of 1a
according to 1H NMR analysis.
Table 1. Aminocarbonylation of 4-Iodoanisole with Solid-
Phase-Bound Amino Acid Nucleophiles
a
a
b
1a−h (0.3 mmol, 0.075 M). Isolated yield after precipitation (purity
Electron-poor aryl iodides showed diminished levels of
reactivity (5d−f, Scheme 1). Increasing the catalytic loading
(2) was beneficial, but full conversion of the amine was reached
only after the amount of 3 was increased to 3 equiv. This result is
in agreement with the observation that CO insertion into
electron-poor aryl palladium(II) intermediates is slower when
compared to more electron-rich substrates.24 Furthermore, the
aminocarbonylation of 1-bromo-3-iodobenzene exhibited good
chemoselectivity, which would allow further functionalization of
the aryl bromide product 5g.
In general, meta- and para-substituted aryl iodides were
successfully transformed into their corresponding products 5a−g
(82−94% yields, Scheme 1). However, applying the reaction
protocol to ortho-substituted substrates mainly resulted in
recovery of unreacted 1a. An increase in the reaction temperature
to 45 °C, under ultrasonication, however, resulted in 5h in 80%
yield. The same was observed for ortho-substituted products 5i−
k, which all worked well at this temperature.
c
ca. 87−100%). Optical rotation of products was compared with the
corresponding products synthesized from p-anisic acid, HBTU, and
d
DIPEA. 1.5 equiv (0.45 mmol) of MePh2SiCOOH did not give full
conversion of the amino acid nucleophile, according to 1H NMR
analysis.
Because of the importance of heteroaryl cores in pharmaceuti-
cally relevant compounds, the use of heteroaryl halides as
substrates was next examined. Both 5n and 5o were synthesized
from the corresponding 4- or 5-substituted 2-bromothiazole.
The aminocarbonylation of 2,4-dibromothiazole gave clean
conversion with complete selectivity for the bromide positioned
in the more electron poor 2-position, thus securing 5n in an
isolated yield of 85%. The ethyl ester containing product 5o was
successfully isolated in 86% yield and hence was unaffected by
the treatment with aqueous TFA and TES to cleave the product
from the solid-phase resin. Furthermore, due to the unfavorable
B
DOI: 10.1021/acs.orglett.7b01068
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
Scheme 1. Aminocarbonylation of a Variety of Aryl and
Heteroaryl Iodides with Solid-Phase-Bound Phenylalanine
Scheme 2. Application of the Optimized Combined
Aminocarbonylation and Fmoc-SPPS Method
a
a
a
b
See the Supporting Information for experimental details. Isolated
yields after preparative RP-HPLC. Isolated yields not compensated for
c
possible water content. MePh2Si13COOH ([13C]-3*) was used for
the isotopic labeling. 8 equiv of 1-bromo-4-iodobenzene and 4 equiv
d
of 3 and KF were used in the aminocarbonylation.
CH2Cl2, was chosen. In order to ensure complete conversion of
the amines to their corresponding amides, 8 equiv of 1-bromo-4-
iodobenzene were used together with an increased amount of the
CORM. Peptide 6b was isolated using preparative RP-HPLC in a
yield of 61%.28
a
1a (0.3 mmol, 0.075 M). Isolated yield after precipitation (purity ca.
b
c
92−100%). Five mol % of 2 was used. Three equiv (0.6 mmol) of 3
and KF was used. 45 °C, under ultrasonication. Synthesized from the
corresponding heteroaryl bromide. Synthesized from Trt-protected 4-
iodoimidazole. Synthesized from Boc protected 3-iodoindole.
d
e
f
g
4-Iodophenylalanine and lysine protected with the highly acid-
labile N-methyltrityl (Mtt) group were incorporated into the
precursor peptides to 6c and [13C]-6c* in order to achieve an
intramolecular aminocarbonylation after selective deprotection.
Indeed, the side-chain-to-side-chain cyclized products 6c and
[13C]-6c* were formed as major components (approximately
70% HPLC purity) and isolated in 11% and 6% yields,
respectively (Scheme 3). Elevated temperature was needed for
the cyclization to occur, in accordance to the synthesis of a cyclic
RGD peptide in solution using a Pd-catalyzed aminocarbonyla-
tion protocol.29 In agreement with the RGD case and with our
previously reported aminocarbonylations of peptide substrates
no epimerization was observed, even if heating and long reaction
times were required in these cases.21,29,30 However, N-terminal
Fmoc proved to be unstable during these conditions,
necessitating an exchange to Boc protection. Minor amount of
intermolecular cyclization was observed, possibly as a result of
the relatively high loading of the Rink amide resin (0.68 mmol/g)
(see the Supporting Information).31
coordination of the unprotected basic nitrogen atoms in the
palladium-catalyzed coupling reactions,23,25 the acid-labile trityl
(Trt)- and Boc-protecting groups, respectively, were used for the
imidazole and indole substrates (to be simultaneous cleaved
upon treatment with 95% TFA). This resulted in good isolated
yields of 5p and 5q (80% and 86%, respectively).
The applicability of the method was later demonstrated in the
preparation of biologically and pharmaceutically relevant
peptides (Scheme 2).10,26 The peptides were synthesized using
standard Fmoc-SPPS from Rink Amide PS resin on a 0.1 mmol
scale. The N-capping group in the bortezomib analogue 6a was
incorporated using the optimized aminocarbonylation protocol
as the last step before final cleavage from the resin and resulted in
the product as the major component in an isolated yield of 78%
after preparative RP-HPLC. To avoid C-terminal amide
hydrolysis it is important to not prolong the cleavage time in
TFA further.27
The method was further extended to selective [13C] acyl
labeling using silacarboxylic acid [13C]-3*, thus securing peptide
[13C]-6a* in an isolated yield of 73% (Scheme 2).
In order to functionalize the nucleophilic lysine in the
precursor peptide to 6b (peptide part of the anticancer agent
CR1166) the orthogonal protecting group Alloc, which is
selectively deprotected using Pd(PPh3)4 and PhSiH3 in dry
In conclusion, a mild and efficient protocol for the conversion
of aryl and heteroaryl iodides to their corresponding amides
through Pd-catalyzed aminocarbonylation using solid-phase-
bound amino acid/peptide nucleophiles has been developed.
The use of a palladacycle precatalyst enabled the reaction to be
performed at room temperature, while the use of a silacarboxylic
acid as an in situ CO source, compatible with the resin, enables
C
DOI: 10.1021/acs.orglett.7b01068
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
(4) Uhlig, T.; Kyprianou, T.; Martinelli, F. G.; Oppici, C. A.; Heiligers,
D.; Hills, D.; Calvo, X. R.; Verhaert, P. EuPa Open Proteomics 2014, 4, 58.
(5) Chatterjee, J.; Rechenmacher, F.; Kessler, H. Angew. Chem., Int. Ed.
2013, 52, 254.
Scheme 3. Intramolecular Cyclization Using the Optimized
Aminocarbonylation Method
a
(6) Hill, T. a; Shepherd, N. E.; Diness, F.; Fairlie, D. P. Angew. Chem.,
Int. Ed. 2014, 53, 13020.
(7) Hanessian, S.; McNaughton-Smith, G.; Lombart, H. G.; Lubell, W.
D. Tetrahedron 1997, 53, 12789.
(8) Lin, K. C.; Ateeq, H. S.; Hsiung, S. H.; Chong, L. T.; Zimmerman,
C. N.; Castro, A.; Lee, W. C.; Hammond, C. E.; Kalkunte, S.; Chen, L. L.;
Pepinsky, R. B.; Leone, D. R.; Sprague, A. G.; Abraham, W. M.; Gill, A.;
Lobb, R. R.; Adams, S. P. J. Med. Chem. 1999, 42, 920.
(9) Zhou, H. J.; Aujay, M. A.; Bennett, M. K.; Dajee, M.; Demo, S. D.;
Fang, Y.; Ho, M. N.; Jiang, J.; Kirk, C. J.; Laidig, G. J.; Lewis, E. R.; Lu, Y.;
Muchamuel, T.; Parlati, F.; Ring, E.; Shenk, K. D.; Shields, J.; Shwonek,
P. J.; Stanton, T.; Sun, C. M.; Sylvain, C.; Woo, T. M.; Yang, J. J. Med.
Chem. 2009, 52, 3028.
(10) Patra, C. R.; Rupasinghe, C. N.; Dutta, S. K.; Bhattacharya, S.;
Wang, E.; Spaller, M. R.; Mukhopadhyay, D. ACS Chem. Biol. 2012, 7,
770.
(11) Lim, H. A.; Ang, M. J. Y.; Joy, J.; Poulsen, A.; Wu, W.; Ching, S. C.;
Hill, J.; Chia, C. S. B. Eur. J. Med. Chem. 2013, 62, 199.
(12) Xu, T.; Khanna, H.; Coward, J. K. Bioorg. Med. Chem. 1998, 6,
1821.
a
See the Supporting Information for experimental details. Synthesized
b
from Rink Amide PS resin (0.68 mmol/g). 6c was run at 65 °C and
c
[13C]-6c* at 45 °C, under ultrasonication. Isolated yields after
d
preparative RP-HPLC. Isolated as cis/trans rotamers.
(13) Sutcliffe-Goulden, J. L.; O’Doherty, M. J.; Marsden, P. K.; Hart, I.
R.; Marshall, J. F.; Bansal, S. S. Eur. J. Nucl. Med. Mol. Imaging 2002, 29,
754.
the reaction to be agitated in a single vial. The presented method
is versatile, high yielding, has a broad scope, and comprises a new
solid-phase synthetic approach to N-modifications, isotopic
labeling, and cyclization of peptides. Altogether, because of its
easy-to-execute and efficient nature, we foresee that this strategy
will be useful in peptide-related drug discovery.
(14) Merrifield, R. B. J. Am. Chem. Soc. 1963, 85, 2149.
(15) Brennfuhrer, A.; Neumann, H.; Beller, M. Angew. Chem., Int. Ed.
̈
2009, 48, 4114.
(16) Wannberg, J.; Larhed, M. J. Org. Chem. 2003, 68, 5750.
(17) Friis, S. D.; Lindhardt, A. T.; Skrydstrup, T. Acc. Chem. Res. 2016,
49, 594.
(18) Friis, S. D.; Taaning, R. H.; Lindhardt, A. T.; Skrydstrup, T. J. Am.
Chem. Soc. 2011, 133, 18114.
(19) Cornilleau, T.; Audrain, H.; Guillemet, A.; Hermange, P.;
Fouquet, E. Org. Lett. 2015, 17, 354.
(20) Friis, S. D.; Andersen, T. L.; Skrydstrup, T. Org. Lett. 2013, 15,
1378.
(21) Andersen, T. L.; Nordeman, P.; Christoffersen, H. F.; Audrain, H.;
Antoni, G.; Skrydstrup, T. Angew. Chem., Int. Ed. 2017, 56, 4549.
(22) Bruno, N. C.; Niljianskul, N.; Buchwald, S. L. J. Org. Chem. 2014,
79, 4161.
(23) Friis, S. D.; Skrydstrup, T.; Buchwald, S. L. Org. Lett. 2014, 16,
4296.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.orglett.7b01068.
■
S
Experimental procedures, NMR spectra, and RP-HPLC
chromatograms (PDF)
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: anja.sandstrom@orgfarm.uu.se.
ORCID
(24) Wu, X. F.; Neumann, H.; Spannenberg, A.; Schulz, T.; Jiao, H.;
Beller, M. J. Am. Chem. Soc. 2010, 132, 14596.
(25) Dufert, M. A.; Billingsley, K. L.; Buchwald, S. L. J. Am. Chem. Soc.
2013, 135, 12877.
(26) Teicher, B. A.; Ara, G.; Herbst, R.; Palombella, V. J.; Adams, J.
Clin. Cancer Res. 1999, 5, 2638.
̈
Troels Skrydstrup: 0000-0001-8090-5050
Anja Sandstrom: 0000-0001-5720-2904
̈
Notes
(27) Samaritoni, J. G.; Copes, A. T.; Crews, D. K.; Glos, C.; Thompson,
A. L.; Wilson, C.; O’Donnell, M. J.; Scott, W. L. J. Org. Chem. 2014, 79,
3140.
(28) Isolated yield was affected by LC−MS analysis of resin aliquots to
follow each step of the synthesis and also due to poor solubility of the
crude peptide.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We gratefully acknowledge financial support from the Anna
Maria Lundin Travel Grant (Smalands Nation), the Danish
■
̊
National Research Foundation (Grant No. DNRF118), the
Villum Foundation, and the Danish Council for Independent
Research: Technology and Production Sciences. The authors
thank Alfred Svan for assistance with the HRMS analysis.
(29) Doi, T.; Kamioka, S.; Shimazu, S.; Takahashi, T. Org. Lett. 2008,
10, 817.
(30) Skogh, A.; Fransson, R.; Skold, C.; Larhed, M.; Sandstrom, A. J.
̈
̈
Org. Chem. 2013, 78, 12251.
(31) The isolated yields of 6c and [13C]-6c* were affected by LC−MS
analysis of resin aliquots to follow each step of the synthesis and by
successive HPLC runs in order to separate the desired product from the
coeluting side product, formed from intermolecular cyclization, as well
as minor loss during Mtt cleavage. See the Supporting Information.
REFERENCES
■
(1) Vlieghe, P.; Lisowski, V.; Martinez, J.; Khrestchatisky, M. Drug
Discovery Today 2010, 15, 40.
(2) Doak, B. C.; Over, B.; Giordanetto, F.; Kihlberg. Chem. Biol. 2014,
21, 1115.
(3) Craik, D. J.; Fairlie, D. P.; Liras, S.; Price, D. Chem. Biol. Drug Des.
2013, 81, 136.
D
DOI: 10.1021/acs.orglett.7b01068
Org. Lett. XXXX, XXX, XXX−XXX