Palladium(II)-Catalyzed Site-Selective C(sp3)?H Alkynylation of Oligopeptides: A Linchpin Approach for Oligopeptide–Drug Conjugation

Article abstract of DOI:10.1002/anie.201706367

The palladium(II)-catalyzed C(sp³)?H alkynylation of oligopeptides was developed with tetrabutylammonium acetate as a key additive. Through molecular design, the acetylene motif served as a linchpin to introduce a broad range of carbonyl-containing pharmacophores onto oligopeptides, thus providing a chemical tool for the synthesis and modification of novel oligopeptide–pharmacophore conjugates by C?H functionalization. Dipeptide conjugates with coprostanol and estradiol were synthesized by this method for potential application in targeted drug delivery to tumor cells with overexpressed nuclear hormone receptors.

Full text of DOI:10.1002/anie.201706367

A Journal of the Gesellschaft Deutscher Chemiker
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Accepted Article
Title: Pd(II)-Catalyzed Site-Selective C(sp3)-H Alkynylation of
Oligopeptides: A Linchpin Approach for Oligopeptide-Drug
Conjugation
Authors: Tao Liu, Jennifer X Qiao, Michael A Poss, and Jin-Quan Yu
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201706367
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10.1002/anie.201706367
Angewandte Chemie International Edition
DOI: 10.1002/anie.201((will be filled in by the editorial staff))
Peptide C–H Functionaliation
Pd(II)-Catalyzed Site-Selective C(sp3)–H Alkynylation of Oligopeptides:
A Linchpin Approach for Oligopeptide-Drug Conjugation
Tao Liu,[a] Jennifer X. Qiao,[b] Michael A. Poss,[b] and Jin-Quan Yu*[a]
Abstract: The palladium(II)-catalyzed C(sp3)–H alkynylation of
oligopeptides was developed with tetrabutylammonium acetate as a
key additive. Through molecular design, the acetylene motif serves as
a linchpin to introduce a broad range of carbonyl-containing
pharmacophores onto oligopeptides, thus providing a chemical tool
for the synthesis and modification of novel oligopeptide-
pharmacophore conjugates via C–H functionalization. Dipeptide
Figure 1. Linchpin approach for oligopeptide-drug conjugation.
conjugates with coprostanol and estradiol has been synthesized using
Herein, we report palladium(II)-catalyzed selective C(sp3)–H
alkynylation of unactivated side chains of oligopeptides with alkyne
coupling partners. Through this coupling reaction, a wide range of
pharmaceutically relevant ketones including biologically active
coprostanol, estradiol and fatty acid are covalently attached to
peptides. Pharmacologically important amidyl N-methylated
oligopeptides are also alkynylated using this protocol.
this method for potential application in targeted drug delivery to
tumor cells with overexpressed nuclear hormone receptors.
Substantial progress has been made in the field of selective post
synthetic modification of oligopeptides and proteins through
bioorthogonal modification of prefunctionalized amino acid residues
and reactive side chains.[1] Combining both chemical and genomics
tools,[1b] methods ranging from ones focusing on cysteine or lysine[1c-
d] to others utilizing metal-catalyzed C–C and C–N bond formation[1e,
Table 1. Optimization of reaction conditions.[a,b]
1g]
have been developed, and successively utilized in medicinal
chemistry in the past decade.
However, due to the inevitable restriction of 20 proteinogenic
amino acids, and limited prefunctionalized amino acids and
oligopeptides in the toolbox of peptide synthesis, new methods to
functionalize unactivated side chains of oligopeptides are still in
urgent need. Transition-metal-catalyzed C(sp2)–H functionalization
on phenylalanine and tryptophan residues has been elegantly
demonstrated on bioactive oligopeptides.[2] C(sp3)–H arylation of
amino acid residues at the N-terminus of oligopeptides was also
developed recently.[3] In the context of developing technologies for
oligopeptide-drug conjugation, we became interested in developing
C(sp3)–H alkynylation of side chains as alkynes can serve as a
versatile linchpin for conjugation with various ketone molecules.
These peptide-ketone conjugates offer a number of potential
Entry Coupling Partner
Additive
K3PO4
K3PO4
NMR Yield
79%
1
2a
2b
2b
2b
2b
2b
2b
2b
2b
2b
2b
2b
2b
2b
2
3
4
5
6
7
8
9
23%
8%
61%
39%
34%
27%
35%
21%
19%
-
NBu4OAc
NBu4I
NBu4Br
NBu4Cl
NBu4BF4
NBu4PF6
HOAc
LiOAc
NaOAc
KOAc
10
11
12
13
14[c]
<5%
24%
41%
pharmaceutical applications (Figure 1). Despite a few recent reports
[4]
on alkynylation of unactivated C(sp3)–H bonds,
C(sp3)–H
84%
NBu4OAc
alkynylation of peptides has not been developed to date. Moreover,
only silyl acetylene derivatives have proven to be suitable reactive
coupling partners in C(sp3)–H alkynylation in general.
[a] Conditions: 1a (0.1 mmol), 2a or 2b (1.5 equiv), Pd(OAc)2 (10 mol%), AgOAc
(1.1 equiv), additive (1.0 equiv), t-butyl alcohol (1.0 mL), 100 °C in air, 14 h. [b]
NMR yields were determined with CH2Br2 as internal standard. [c] 2b (3.0 equiv).
We initiated our investigation of Pd(II)-catalyzed C(sp3)–H
alkynylation of oligopeptides with the commonly used coupling
partner triisopropylsilane acetylene bromide 2a, and found that
dipeptide 1a could be alkynylated in 79% NMR yield (entry 1, Table
1). In order to establish a broadly useful linchpin strategy for peptide-
drug conjugation through this C(sp3)–H alkynylation, we proposed to
covalently link the acetylene to ketones via a simple nucleophilic
addition reaction. Hence, the conjugation of carbonyl-containing
pharmacophores with oligopeptides can be accomplished via the C–
H alkynylation. To test this hypothesis, we prepared propargyl silyl
ether 2b (Table 1, entry 2). Under the same optimized conditions as
that for 2a (Table 1, entry 1), alkynylation of dipeptide 1a with the
[a]
T. Liu and Prof. Dr. J.-Q. Yu
Department of Chemistry, The Scripps Research Institute
(TSRI)
10550 North Torrey Pines Road, La Jolla, CA 92037 (USA)
E-mail: yu200@scripps.edu
[b]
Dr. J. X. Qiao and Dr. M. A. Poss
Department of Discovery Chemistry, Bristol-Myers Squibb, P.O.
Box 5400, Princeton, NJ 08543 (USA)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201xxxxxx.
1
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10.1002/anie.201706367
Angewandte Chemie International Edition
new coupling partner 2b afforded the desired product 3b only in 23%
NMR yield (entry 2).
during the synthesis. The dipeptide conjugates 3m-3o can then be
elongated to form linear or cyclic peptides of interest.
Table 2. Alkynylation with different propargyl silyl ether coupling partners.[a,b]
Subsequent investigation of additives led to a surprising finding.
In the presence of tetrabutylammonium acetate (NBu4OAc), the yield
was significantly increased from 23% to 61% (Table 1, entry 4).
Other quaternary ammonium salts generally showed lower reactivity
(entries 59, 21%39%), as did acetic acid or common acetate salts
provided comparable low yields (entries 1013, <5%41%). We
propose that the acetate anion in NBu4OAc facilitates C–H activation,
and the quaternary ammonium cation enhances the stability of the
palladium species during C–H activation.[5] The combined effects
granted NBu4OAc the highest reactivity in the C(sp3)–H alkynylation
of oligopeptides using propargyl silyl ether 2b as the coupling
partner.
After further optimization, we finalized the conditions for the
alkynylation of dipeptide 1a with the propargyl silyl ether coupling
partner 2b: 10 mol% of Pd(OAc)2, 1.1 equivalent of AgOAc, 1.0
equivalent of NBu4OAc, and t-butyl alcohol as the solvent (Table 1,
entry 14).
With optimized conditions in hand, we investigated the synthesis
of dipeptide conjugates from dipeptide 1a and a wide range of
coupling partners derived from ketones (Table 2). In the presence of
NBu4OAc, the isolated yield of the C(sp3)–H alkynylation of
dipeptide 1a with 2a was increased to 84% (compared with 79%
NMR yield without NBu4OAc, Table 1, entry 1). Propargyl silyl
ethers 2b2l were prepared from representative ketones of
pharmaceutical interest. The oxane-containing coupling partner 2c
and fluoro-containing coupling partner 2d showed good reactivity,
offering the desired product in good isolated yield. Admantane (2e),
monoterpenoid camphor (2f), and menthone (2g) are privileged
structures in medicinal chemistry research. The corresponding
propargyl silyl ethers and dipeptide conjugates were obtained in high
yields (2e2g, 7482%). Cycloheptane 2h and cyclooctane 2i are
widely found in bio-imaging agents, and alkynylation with each gave
high yields of 84% and 87%, respectively. Alkynylation with diethyl
substituted propargyl silyl ether 2j afforded the desired product in
63% yield, while coupling with the highly sterically hindered
diisobutyl propargyl silyl ether 2k gave an excellent yield of 90%.
We selected the benzophenone derivative 2l as a model example of
propargyl silyl ethers containing aromatic rings. To our delight,
alkynylation of 2l afforded the desired product in 79% yield.
Overexpressed nuclear hormone receptors have proven to be ideal
targets for targeted drug delivery. For instance, coprostanol (4m,
[a] Conditions: 1a (0.1 mmol), 2 (3.0 equiv), Pd(OAc)2 (10 mol%), AgOAc (1.1
equiv), NBu4OAc (1.0 equiv), t-butyl alcohol (1.0 mL), 100 ºC in air, 14 h. [b]
Isolated yield.
To extend the above protocol to other peptide substrates,
dipeptides containing various amino acids at the C-terminus were
prepared from the corresponding natural or unnatural amino acids
(Table 3). Selective alkynylation proceeded smoothly on dipeptides
with glycine at the C-terminus (3p, 67% yield). Dipeptides with alkyl
side chains reacted with 2b to give the desired products in high yields
(3q3t, 74%78%). Desired product 3u was obtained in 84% yield
from the dipeptide containing threonine at the C-terminus.
Furthermore, phenylalanine (3v), para-carboxy phenylalanine (3w)
and tyrosine (3x) containing dipeptides showed good reactivity.
Desired product 3y was obtained in 67% yield from the dipeptide
containing tryptophan at the C-terminus. Alkynylation of the
dipeptide derived from 2-aminoisobutyric acid also proceeded in high
yield (3z, 84%) using only 5 mol% of Pd(OAc)2.
Amidyl N-methylation has been used as a powerful tool to modify
the pharmacological properties of oligopeptides.[8] To test whether
C(sp3)–H alkynylation method is capable of selectively modifying N-
methylated alanine residues, we prepared the N-methylated dipeptide
substrates in Table 3. N-Acyl was used to mimic N-terminus upstream
amino acid residues in these peptides. To our delight, Pd-catalyzed
alkynylation under slightly modified conditions gave the desired
products selectively (3aa3ae). It is worth noting that the extra
coordinating group on the side chain of aspartic acid (3ab) did not
impair proper coordination of the Pd-catalyst and subsequent C(sp3)–
H alkynylation of the dipeptide. N-Methylated dipeptides derived
from para-carboxy phenylalanine (3ac, 3ad), as well as tyrosine
(3ae), were alkynylated in reasonable yields.
Figure 2) selectively binds overexpressed androgen receptor (AR) on
[6a]
prostate tumor cells,
while estradiol (4n) selectively binds
overexpressed estrogen receptor (ER) on breast tumor cells. [6b] We
demonstrated application of the C(sp3)–H alkynylation method to
synthesize the dipeptide-based conjugates 3m and 3n in good yields
regardless of the steric hindrance introduced by the steroids. A
cytotoxic payload can then be attached to either the N-terminus or C-
terminus of the dipeptide in 3m and 3n. It is also known that long
chain fatty acids are effective as pharmacokinetic enhancers.[7]
Oligopeptide-fatty acid conjugate 3o was obtained in 68% yield while
the free carboxylic acid group on oxostearic acid 4o was tolerated
2
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10.1002/anie.201706367
Angewandte Chemie International Edition
Figure 2. Synthesis of oligopeptide-pharmacophore conjugates. AR: Androgen receptor. ER: Estrogen receptor.
Table 3. Alkynylation of various dipeptides 1 with alkynyl bromide 2b.[a, b]
[a] Conditions: 1 (0.1 mmol), 2b (3.0 equiv), Pd(OAc)2 (10 mol%), AgOAc (1.1 equiv), NBu4OAc (1.0 equiv), t-butyl alcohol (1.0 mL), 100 ºC in air, 14 h. [b] Isolated
yield. [c] 5 mol% of Pd(OAc)2 was used. [d] Conditions: 120 ºC, 4 h. NMR yields in round brackets. [e] Using 2a (3.0 equiv) instead of 2b as the coupling partner.
Amino acid residues embedded in peptide backbones can
potentially offer multiple amidyl coordinating sites to bind to the
palladium catalyst in a N,N-biscoordinating mode. Tripeptide 5a
(Figure 3) containing three alanine residues was prepared to
investigate the possibility of selective labeling of just one alanine. To
our delight, the alanine residues at the C-terminus facilitated the
selective activation of C(sp3)–H on the alanine at the N-terminus.
Alkynylated tripeptide 6a was obtained in 47% isolated yield.
In conclusion, we have developed a Pd(II)-catalyzed C(sp3)–H
alkynylation of oligopeptides. Through molecular design, the
acetylene motif serves as a linchpin to introduce a broad range of
privileged carbonyl-containing pharmacophores onto oligopeptides.
This straightforward C(sp3)–H functionalization offers rapid access
to novel oligopeptide-pharmacophore conjugates, and has the
potential to accelerate the development of peptide-conjugate based
therapeutics. Further investigation is ongoing in our laboratory to
modify native oligopeptides and cyclic peptides through direct C–H
functionalizations.
Received: ((will be filled in by the editorial staff))
Published online on ((will be filled in by the editorial staff))
Figure 3. Pd-Catalyzed alkynylation on tripeptide by N,N-biscoordination.
3
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10.1002/anie.201706367
Angewandte Chemie International Edition
Acknowledgement
[3] a) W. Gong, G. Zhang, T. Liu, R. Giri, J.-Q. Yu, J. Am. Chem. Soc.
2014, 36, 16940; b) A. F. M. Noisier, J. García, I. A. Ionut, F. Albericio,
Angew. Chem. Int. Ed. 2017, 56, 314; Angew. Chem. 2017, 129, 320.
[4] For examples of Pd-catalyzed C(sp3)−H alkynylation with silyl
acetylenes, see: a) Y. Ano, M. Tobisu, N. Chatani, J. Am. Chem. Soc.
2011, 133, 12984; b) J. He, M. Wasa, K. S. L. Chan, J.-Q. Yu, J. Am.
Chem. Soc. 2013, 135, 3387; c) B. Wang, C. Lu, S.-Y. Zhang, G. He,
W. A. Nack, G. Chen, Org. Lett. 2014, 16, 6260; d) B. Wang, G. He, G.
Chen, Sci. China Chem. 2015, 58, 1345; e) X. Ye, C. Xu, L. Wojtas, N.
G. Akhmedov, H. Chen, X. Shi, Org. Lett. 2016, 18, 2970; f) H. Fu, P.-
X. Shen, J. He, F. Zhang, S. Li, P. Wang, T. Liu, J.-Q. Yu, Angew. Chem.
Int. Ed. 2017, 56, 1873; Angew. Chem. 2017, 129, 1899.
We gratefully acknowledge The Scripps Research Institute, the NIH
(NIGMS, 2R01GM084019) and Bristol-Myers Squibb for their financial
support.
Keywords: palladium··C–H activation··alkynylation··pharmacophore
··peptide
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10.1002/anie.201706367
Angewandte Chemie International Edition
Peptide C–H Functionaliation
T. Liu, J. X. Qiao, M. A. Poss, J.-Q.
Yu* __________ Page – Page
Pd(II)-Catalyzed Site-Selective
C(sp3)–H Alkynylation of
Oligopeptides: A Linchpin Approach
for Oligopeptide-Drug Conjugation
The palladium(II)-catalyzed C(sp3)–H alkynylation of oligopeptides was developed with
tetrabutylammonium acetate as a key additive. Through molecular design, the acetylene motif
serves as a linchpin to introduce a broad range of carbonyl-containing pharmacophores onto
oligopeptides, thus providing a chemical tool for the synthesis and modification of novel
oligopeptide-pharmacophore conjugates via C–H functionalization. Dipeptide conjugates with
coprostanol and estradiol have been synthesized using this method for potential application in
targeted drug delivery to tumor cells having overexpressed nuclear hormone receptors.
5
This article is protected by copyright. All rights reserved.