Late-Stage Functionalization of Peptides and Cyclopeptides Using Organozinc Reagents

Article abstract of DOI:10.1002/anie.201902454

We report a new late-stage functionalization of small peptides and cyclopeptides relying on the Negishi cross-coupling of readily prepared iodotyrosine- or iodophenylalanine-containing peptides with aryl-, heteroaryl-, and alkylzinc pivalates or halides.

Full text of DOI:10.1002/anie.201902454

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Accepted Article
Title: Late-stage Functionalization of Peptides and Cyclopeptides using
Organozinc reagents
Authors: Marcel Leroux, Thomas Vorherr, Ian Lewis, Michael Schaefer,
Guido Koch, Konstantin Karaghiosoff, and Paul Knochel
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Angewandte Chemie International Edition
10.1002/anie.201902454
COMMUNICATION
Late-stage Functionalization of Peptides and Cyclopeptides using
Organozinc reagents
[
a]
[b]
[b]
[b]
[b]
Marcel Leroux , Thomas Vorherr* , Ian Lewis , Michael Schaefer , Guido Koch , Konstantin
[a]
[a]
Karaghiosoff , Paul Knochel*
Dedication ((optional))
Abstract: We have reported a new late-stage functionalization of
small peptides and cyclopeptides using readily prepared iodotyrosine
or iodophenylalanine containing peptides and performing Negishi
cross-couplings with aryl-, heteroaryl- and alkyl-zinc pivalates or
halides. In-silico and in-vitro determinations of membrane
permeability data of the modified cyclopeptides showed that in most
cases, the solubility was improved while the cell-membrane
permeability was maintained by the introduction of polar pyridyl units.
Scheme 1. Preparation of modified tyrosine-based peptides (4) starting from a
tyrosine containing peptides of type 2.
In preliminary experiments, we have determined the optimum
cross-coupling conditions, varying the temperature, concentration,
rate of addition of the organozinc reagent and Pd-catalyst system
on the protected iodotyrosine derivative 3a[15]. Both electron-rich
or electron-poor arylzinc pivalates (1a–b) as well as pyridylzinc
The late-stage functionalization of peptides is an important and
practical method for preparing modified peptide derivatives.[1] The
possibility of combinatorial modification of drug targets is
essential for an efficient search of optimal properties like
bioavailability, stability, membrane permeability and to achieve
improved structure-activity relationships.[2] A wide range of
transition-metal catalyzed C-H activations have been used for
late-stage activations.[3] Barluenga[4] and Davis[5] have
demonstrated the utility of Suzuki-Miyaura cross-couplings for the
modification of amino-acids and small peptides. As an alternative
to the Suzuki-Miyaura reaction, the Negishi cross-coupling
involving organozinc reagents often requires milder reaction
pivalates[16] (1c–e) gave satisfactory results using 4% Pd(OAc)
and 8% SPhos[17] leading to functionalized tyrosines (4a–e) in 50–
7% yield (Scheme 2).
2
8
[
6]
conditions and less sophisticated ligands . Therefore, the
Negishi-coupling should be well suited for the late-stage
functionalization of complex molecules. Furthermore, organozinc
halides are readily prepared and tolerate the presence of various
[
7]
functional groups.
Recently, we have shown that organozinc pivalates of type 1
[
8]
Scheme 2. Negishi cross-coupling of tyrosine derivative 3a with aryl- and
heteroarylzinc pivalates (1e–e) leading to the tyrosines 4a–e.
[9]
with the general formula RZnX⋅Mg(OPiv) ⋅LiCl (abbreviated
2
RZnOPiv) display enhanced moisture- and air-stability. They
have already found useful applications in high-throughput
screenings[ and various cobalt-catalyzed reactions.[11]
An attempt to extend this cross-coupling to alkylzinc pivalates was
difficult and a new optimization study was required. We have
found, that in this case, the best zinc organometallics were
alkylzinc halides prepared by the direct oxidative insertion of zinc
powder in the presence of LiCl.[18]
10]
Herein, we wish to report
a
quite general late-stage
functionalization of small peptides and cyclopeptides bearing an
ortho-iodotyrosine amino-acid or an iodophenylalanine using
Negishi cross-couplings with organozinc pivalates (1).
Thus, the iodination of tyrosine containing peptides[12] of type 2
[
13]
with NaI, chloramine-T in DMF at 0–25 °C (Method A) or with
2 4 2 2
IPy BF in CH Cl
at 25 °C (Method B)[ produces the mono-
14]
iodotyrosine derived peptides of type 3, which after Pd-catalyzed
cross-couplings with various organozinc pivalates (1) gave
modified peptides of type 4 (Scheme 1).
[
[
a]
b]
Marcel Leroux, Prof. Dr. K. Karaghiosoff, Prof. Dr. P. Knochel;
Department Chemie, Ludwig-Maximilians-Universität München
Butenandtstrasse 5–13, Haus F, 81377 München (Germany)
E-mail: paul.knochel@cup.uni-muenchen.de
Dr. Thomas Vorherr, Dr. Ian Lewis, Dr. Michael Schaefer, Dr. Guido
Koch; Novartis Pharma AG, CH-4002 Basel (Switzerland)
E-Mail: thomas.vorherr@novartis.com
Scheme 3. Cross-coupling reactions of tyrosine derivatives (3a–b) with
organozinc halides (5a–e) leading to alkylated tyrosine derivatives (6a–f).
As catalyst, the system developed by Fu[19] with 4% Pd(dba)
dba = dibenzylideneacetone) and 8% PtBu gave the best results.
Thus, various functionalized primary and secondary alkylzinc
2
Supporting information for this article is given via a link at the end of
the document.
(
3
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COMMUNICATION
halides (5a–e) bearing a chloro-, a carbethoxy- or a cyano
function led to the modified tyrosines (6a–e) including the
dipeptide 6f (Scheme 3). Remarkably, the used Fu-catalyst[19] led
to relatively fast cross-coupling reactions. Combined with the slow
Additionally, we have extended this late stage functionalization to
four iodocyclopeptides (one derived from tyrosine; see 3a and
three derived from phenylalanine 11–13).
Cyclopeptides are an important class of metabolically quite stable
addition[ of the organozinc reagent , an excellent compatibility
20]
[21]
22]
peptides with
a
broad range of medicinal applications.[
with the acidic amides and phenolic protons was achieved.
Especially proline containing cyclopeptides have been
[
23]
With these optimized conditions in hand, we have applied these
late-stage functionalizations to dipeptides (3b and 3c) and a
tetrapeptide (3d). As indicated in Scheme 4, a range of arylzinc
pivalates bearing both electron-donating or accepting groups as
well as heteroaryl-zinc pivalates were used leading after Negishi
cross-couplings to the expected products (7a–f, 8a–d and 9a–c)
in 38–92% yield.
increasingly investigated due to their high bioactivity . They
have found medical applications e.g. as immunosuppressant,
[
24]
anti-HIV, antibacterial or antiviral drugs
.
Thus, the
[
25]
iodocyclopeptide 3e prepared via method B was treated with 2-,
3- and 4-pyridylzinc pivalates derivatives. Hence, using standard
cross-coupling conditions, the expected cyclopeptide products
(10a–c) were obtained in 54–82% yield (Scheme 5).
Scheme 5. Modification of cyclic peptide 3e bearing an iodotyrosine via cross-
coupling reactions with heteroarylzinc pivalates (Pyr: various pyridyl scaffolds)
leading to functionalized peptides of type 10.
Similarly, we submitted iodophenylalanine cyclopeptides (11–13)
to a cross-coupling with the same pyridylzinc pivalates and have
obtained the desired coupling-products (14a–c, 15a–c and 16a–
c) in 38–84% yield (Table 1). We noticed that in the case of the
cross-coupling of ortho-phenylalanine cyclopeptide 11 with
2-pyridylzinc pivalate (1c) harsher reaction conditions were
required (60 °C, 24 h instead of 25 °C, 6 h).
A set of experiments was designed to gain more insights
regarding steric limitations of the reactions and the influence of
differentially substituted pyridyl cores. The latter modification has
been suggested to promote permeability, if the pyridyl nitrogen
can act as an intramolecular H-bond acceptor.[26] In general, this
kind of assessment is of importance when fine-tuning properties
and structure-activity relationships during the drug discovery
process. The membrane permeability- and solubility-parameters
of the modified cyclopeptides 14–16 were compared to the results
of in-silico calculations. Thus, this set of nine cross-coupling
products (14–16) had been submitted for MD (molecular
dynamics) simulations followed by calculation of the solvent-
accessible polar surface area (abbreviated SAPSA) to result in an
in-silico assessment regarding their permeability properties in the
first instance (Table 2) [27]
.
Scheme 4. Cross-coupling reactions of the iodotyrosine derived oligopeptides
(3b–3d) with organozinc pivalates (1) leading to the functionalized tyrosine
containing peptides (7–9).
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COMMUNICATION
Table 1. Modification of iodophenylalanine-cyclopeptides (11–13) via cross-
Table 2. In-silico and in-vitro profiling of the iodophenylalanine starting materials
(11–13) and the pyridyl cross-coupling products (14–16).
coupling reactions with 2-, 3- and 4-pyridylzinc pivalates (1c–e).
iodo-cyclopeptide
modified cyclopeptide
Entry
Peptide
SAPSA
Sol. pH=6.8
(µM)
PAMPA
(pH 6.8)
MDCK
SFC
(min)
2
-
(Å )
(10
6
cm/s)
62
74
75
76
84
83
74
101
94
84
< 4
3
-4.2
-4.3
-5.4
-4.7[b]
-4.3
-4.5
-4.5
-4.7
-4.8
-4.5
-4.6
-4.6
n.d. [a]
n.d. [a]
n.d. [a]
n.d. [a]
15.7
19.3
3.3
3.15
3.14
3.23
3.37
3.31
3.39
3.51
3.60
3.76
3.61
3.72
3.89
1
2
3
4
5
6
7
8
9
11
12
13
< 4
24
46
52
21
26
31
9
4a: R = 2-pyridyl 38%[
a]
b]
b]
14a
14b
14c
15a
15b
15c
16a
16b
16c
1
1
[
4b: R = 3-pyridyl 62%
14c: R = 4-pyridyl 67%[
1
1
3.2
13.6
1.4
10
5a: R = 2-pyridyl 72%[
b]
b]
b]
1
[
1
5b: R = 3-pyridyl 62%
11
99
< 4
22
2.1
12
15c: R = 4-pyridyl 77%[
12
106
2.9
[
[
a]
b]
not determined due to QC or recovery problems
at pH 8.0
In summary, this methodology including selective tyrosine
iodination and Negishi cross-coupling reaction tolerates peptides
based on several amino acids[28]. A wide range of organozinc
reagents bearing various functional groups as well as
heterocycles can be used. Furthermore, this new late-stage
1
6a: R = 2-pyridyl 84%[
b]
b]
b]
[
16b: R = 3-pyridyl 62%
16c: R = 4-pyridyl 80%[
functionalization of
readily available iodotyrosine
or
13
iodophenylalanine containing peptides offers opportunities for a
detailed site-specific exploration and probing of the property
space. In addition, for intracellular targets or when an oral route is
required, the ability to manipulate properties with the aim to obtain
permeable molecules retaining some polarity enables a drug
discovery process not compromised by poor solubility. Further
extensions of this approach are underway.
[
[
a]
b]
RZnOPiv (3.0 equiv), 8% Pd(OAc)
2
, 16% SPhos, THF, 60 °C, 24 h.
, 8% SPhos, THF, 25 °C, 6 h.
RZnOPiv (2.5 equiv), 4% Pd(OAc)
2
Interestingly, different SAPSA values were observed, and as
expected in the cases, in which the pyridyl moiety is more
exposed (meta and para substituted phenylalanine residues)
more polar surface area is accessible. Next, solubility and
permeability parameters were assessed. The low solubility
measured for the starting materials (11–13) was in most of the
cases improved by the pyridyl core (see Table 2, entries 4–9 and
Acknowledgements
1
2), and on the basis of the PAMPA (Parallel Artificial Membrane
Preliminary experiments of Dr. Andreas Benischke are kindly
acknowledged. We thank the SFB 749 for financial support. We
also thank BASF (Ludwigshafen) and Albemarle (Frankfurt a. M.)
for the generous gift of chemicals.
Permeability Assay) results, all cyclic peptides display high
permeability. However, in the cellular assessment (MDCK; Madin
Darby canine kidney cells assay), the position of the pyridyl
linkage seemed to be sensitive regarding transport across. As for
the in-silico analyses, the para- phenylalanine substituted
analogues showed reduced transport rates.
Keywords: peptides ⋅ cyclopeptides ⋅ organozinc ⋅ cross-
coupling ⋅ membrane permeability
However, this assay reflects the sum of active and passive
transport properties, whereas the SFC (supercritical fluid
chromatography) method rank-orders only for passive
permeability. Nevertheless, the cross-coupling products resulting
from ortho-phenylalanine substitutions clearly showed a higher
passive permeability. This finding was in line with the MD
simulations followed by SAPSA calculation of the preferred
conformational clusters.
[
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COMMUNICATION
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Entry for the Table of Contents (Please choose one layout)
Layout 2:
COMMUNICATION
Marcel Leroux, Thomas Vorherr, Ian
Lewis, Michael Schaefer, Guido Koch,
Konstantin Karaghiosoff and Paul
Knochel*
Page No. – Page No.
Late-stage Functionalization of
Peptides and Cyclopeptides using
Organozinc reagents
Late-stage peptide functionalization: We have reported a new late-stage functionalization of small peptides and cyclopeptides using
readily prepared iodotyrosine or iodophenylalanine containing peptides and performing Negishi cross-couplings with aryl-, heteroaryl-
and alkyl-zinc pivalates or halides. In-silico and in-vitro determinations of membrane permeability data of the modified cyclopeptides
showed that in most cases, the solubility was improved while the cell-membrane permeability was maintained by the introduction of
polar pyridyl units
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