Application of Methyl Bisphosphine-Ligated Palladium Complexes for Low Pressure N-11C-Acetylation of Peptides

Article abstract of DOI:10.1002/anie.201700446

A mild and effective method is described for ¹¹C-labeling of peptides selectively at the N-terminal nitrogen or at internal lysine positions. The presented method relies on the use of specific biphosphine palladium–methyl complexes and their high reactivity towards amino-carbonylation of amine groups in the presence [¹¹C]carbon monoxide. The protocol facilitates the production of native N-¹¹C-acetylated peptides, without any structural modifications and has been applied to a selection of bioactive peptides.

Full text of DOI:10.1002/anie.201700446

Angewandte
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International Edition: DOI: 10.1002/anie.201700446
German Edition: DOI: 10.1002/ange.201700446
11C-Labeling
Application of Methyl Bisphosphine-Ligated Palladium Complexes for
Low Pressure N-11C-Acetylation of Peptides
Thomas L. Andersen+, Patrik Nordeman+, Heidi F. Christoffersen, Hꢀlꢁne Audrain,
Gunnar Antoni,* and Troels Skrydstrup*
Abstract: A mild and effective method is described for 11C-
labeling of peptides selectively at the N-terminal nitrogen or at
internal lysine positions. The presented method relies on the
use of specific biphosphine palladium–methyl complexes and
their high reactivity towards amino-carbonylation of amine
groups in the presence [11C]carbon monoxide. The protocol
facilitates the production of native N-11C-acetylated peptides,
without any structural modifications and has been applied to
a selection of bioactive peptides.
peptides or proteins (11C, t1/2 = 20.4 min) could provide
a valuable tool for their PET studies. Previous efforts into
N-11C-acetylation have been focused on small molecule
labeling. For example, Rahman and Lꢀngstrçm reported on
the use of reactive lithium amide species and their addition to
in-situ-prepared PdMeI-complexes in the presence of pres-
surized 11CO gas.[9–12] Although expedient in delivering
several simple 11C-labeled acetamides, these strong basic
conditions are incompatible with the peptide core structure.
In this communication, we demonstrate the feasibility of
N-terminal 11C-acetylation, and lysine N-11C-acetylation in
peptides through a Pd-mediated carbonylation reaction
employing [11C]CO. Importantly, this radiolabeling method-
ology allows access to the native N-acetylated peptide without
structural modification.
Neutral methyl palladium(II) complexes possessing the
structure LnPdMeX (X = halide, L = P- or N-based ligands)
have previously been employed as palladium hydride pre-
cursors in olefin isomerization processes.[13] However, PdX-
(COMe)(tmeda) complexes (X = I, Br, or Cl) were reported
to form rapidly from the Pd–Me complexes in the presence of
CO gas.[14] As such, we envisioned that this reactivity could be
exploited for the rapid and chemoselective 11C-acetylation of
amines through an aminocarbonylation (Scheme 1).[15] For
this transformation to be successful, the protocol would have
to be streamlined for the short half-life of 11C and the pico- to
nanomole quantities of 11CO available in a labeling setup.
Furthermore, mild conditions are necessary to avoid epime-
rization of the peptide of interest.
T
he use of radiolabeled peptides and proteins as imaging
tools for drug development programs and as clinical diag-
nostics and therapeutics has recently attracted considerable
attention.[1–6] Specific radiolabeling of such biotherapeutics
with short-lived radioisotopes for positron emission tomog-
raphy (PET) studies would provide valuable information on
their pharmacokinetic and targeting properties. Different
techniques have been applied for the radioisotope-labeling of
peptides and/or proteins, including 18F-labeling of amino acid
side-chains, introduction of 18F-containing prosthetic groups
or chelating groups for metals 68Ga or 64Cu, and 11C-
methylation of homocysteine residues.[7] However, with the
exception of the latter, these techniques result in structural
modifications of the native peptide/protein structure, which
can lead to altered pharmacokinetic properties.
N-Acetylation is a post-translational covalent modifica-
tion of proteins either at the N-terminal position or on lysine
residues.[8] Terminal N-acetylation is found in many antimi-
crobial peptides, and is a common functionalization for
peptides representing an internal portion of a protein
sequence. Hence, methods for the rapid N-11C-acetylation of
[*] T. L. Andersen,[+] H. F. Christoffersen, Prof. Dr. T. Skrydstrup
Carbon Dioxide Activation Center (CADIAC), Department of
Chemistry and the Interdisciplinary Nanoscience Center (iNANO),
Aarhus University
Gustav Wieds Vej 14, 8000 Aarhus C (Denmark)
E-mail: ts@chem.au.dk
Dr. P. Nordeman,[+] Prof. Dr. G. Antoni
Department of Medicinal Chemistry, Uppsala University
75123 Uppsala (Sweden)
E-mail: gunnar.antoni@akademiska.se
Dr. H. Audrain
Department of Nuclear Medicine and PET Center
Aarhus University Hospital
8000 Aarhus (Denmark)
[+] These authors contributed equally to this work.
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.201700446.
Scheme 1. General strategy for the N-11C-acetylation of terminal
amines and lysine residues.
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To assess the validity of this labeling strategy, an N-
acetylation protocol was first developed using CO and
peptide 1 as the nucleophilic coupling partner (Table 1).
This peptide serves as a pentameric model structure for the
was not obtained in the absence of a supporting phosphine
ligand (entry 10). To evaluate whether the bis-alkene ligand
of Pd-1 or the rate of ligand substitution had any influence on
the reaction outcome, the phosphine ligated complexes Pd-2
and Pd-3 were prepared by ligand exchange as described by
Mazet et al.[13a] Employing these complexes led to a small
yield increase (entries 11 and 12). Gratifyingly, the yield
increased to 57% when substoichiometric CO was employed
(CO/Pd = 1:2, entry 13).
Table 1: Screening of conditions for the N-acetylation of H2N-LULUPhol
(1) with Pd-complexes Pd-1, Pd-2, and Pd-3.[a]
With these results, we turned to a 11C-labeling setting.
Generation and delivery of 11CO to the reaction vessel was
conducted in a previously described low-pressure setup.[23]
When adjusting the reaction timescale to a realistic labeling
scenario (8 min) at 608C under otherwise unaltered condi-
tions, the 11C-acetylated peptide [11C]2 could be formed with
13% radiochemical conversion from 11CO as determined by
analytical radio-HPLC (Table 2, entry 1). Once again, acety-
lation of the C-terminal OH-group was not observed. Under
these conditions XantPhos was superior to dppf as the
supporting ligand, both as an additive with Pd-1 and when
used directly as in the case of Pd-2 and Pd-3 (entries 2–4). A
minor improvement was obtained by increasing the amount
of peptide and Pd-source, whereas the addition of amine
bases did not prove beneficial (entries 5–7).
Entry Pd-complex
Ligand
HPLC-yield of 2 (isolated yield)[b]
[c]
1
2
3
4
5
6
7
8
9
10
11
12
13
Pd-1
Pd-1
Pd-1
Pd-1
Pd-1
Pd-1
Pd-1
Pd-1
Pd-1
Pd-1
Pd-2
Pd-3
Pd-1
HBF4P(tBu)3
2%
5%
2%
<1%
1%
13%
5%
35%
39%
CataCXium A[c]
[c]
Cy2tBuPHBF4
tBu2CyPHBF4
[c]
dppbz
dppp
dppb
dppf
XantPhos
none
none
none
XantPhos
In contrast, a profound effect was observed when a stock
solution of peptide 1 in DMF was dried extensively by storage
over molecular sieves prior to the 11C-acetylation step. This is
consistent with the finding that [11C]AcOH was commonly
observed as a side product in these reactions, suggesting that
even minute traces of water may compromise the 11C-
<2%
45% (44%)
48%
57%[d]
[a] All reactions were run in a two-chamber system. See Supporting
Information for details on the reaction setup. [b] Yields determined by
UV-HPLC calibrated against an internal standard. [c] 2.2 equiv of ligand
used. [d] Yield based on 0.5 equiv of COgen, reaction conducted at 808C.
Table 2: Adaption of N-acetylation conditions to a PET-setting with
11CO.[a]
Entry
Complex
(mmol)
Additive
(mmol)
TE
RCP
RCC
peptaibol family;[16] a class of naturally occurring peptides
possessing antimicrobial properties.[17] The inherent ability of
such peptides to accumulate at infection sites has generated
an interest in their potential use as infection-selective PET-
tracers.[18]
For the acetylation of 1, commercially available Pd-
1 (Table 1) was employed as the methyl donor. To mimic the
low partial pressure and trace amounts of 11CO available in
a 11C-labeling setting, test reactions were performed with only
a slight excess of CO gas (22 mmol, 1.1 equiv) generated
ex situ in a separate reaction chamber.[19] Of the several
phosphine ligands commonly employed for carbonylation,
most only afforded the desired N-acetylated peptide 2 in trace
amounts (entries 1–7).[20] Better results were observed with
either dppf or XantPhos, leading to 2 in 35% and 39%,
respectively (entries 8 and 9).[21] Acetylation of the C-
terminal OH group was not observed, as determined by
comparison with an O-acetylated reference.[22] N-Acetylation
[%][b]
[%][c]
[%][d]
1
2
3
4
5
Pd-1 (10)
Pd-1 (10)
Pd-3 (10)
Pd-2 (10)
Pd-2 (20)
Pd-2 (20)
Pd-2 (20)
Pd-1 (20)
Pd-2 (20)
Pd-1 (20)
XantPhos (10)
dppf (10)
88
11
18
90
92
90
99
15
–
5
18
20
10
3
70Æ6
74
44
13
–
1
16
18
–
–
–
6
7
Et3N (180)
DBU (170)
9
3
8[e]
9[e]
10[e,g]
XantPhos (20) 88Æ3
62Æ1[f]
–
94
99
69
XantPhos (20)
43
[a] Reactions performed in Aarhus, Denmark. See Supporting Informa-
tion for details on the generation and delivery of 11CO. [b] TE=Trapping
efficiency; fraction of radioactivity remaining in the vial after purging.
[c] RCP=Radiochemical purity; determined by radio-HPLC of the crude
reaction. [d] RCC=Radiochemical conversion from 11CO
(RCC=TE·RCP). [e] Stock solution of 1 in DMF dried on molecular
sieves. [f] Average of 3 reactions. [g] Reaction performed in Uppsala,
Sweden.
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acetylation yield owing to a competing hydroxycarbonylation
Because the choice of amino acid at the e-position of
pathway. Interestingly, a similar trend was not observed when
stoichiometric amounts of CO were employed (Table 1). With
this precaution, [11C]2 was obtained with a 62 Æ 1% radio-
chemical conversion from 11CO in a reproducible manner
(Table 2, entry 8).[24] A slight increase in the yield was
obtained by employing Pd-2 directly (entry 9).
To establish the reproducibility of the described protocols,
the 11C-labeling of 1 was conducted at a separate PET-center
in Uppsala, Sweden. Under conditions similar to those
reported in Table 2, entry 8, this led to the formation of
[11C]2 in a radiochemical conversion of 43% as determined by
analytical radio-HPLC (entry 10). Preparative HPLC-purifi-
cation afforded 1.09 GBq (27.2 mCi) of [11C]2 as a pure
sample (RCP = 99%), in a decay-corrected radiochemical
yield of 33% (total synthesis time = 30 min). The molar
activity was determined to be 281 GBqmmolÀ1 indicating that
isotopic dilution is minimal. This finding is in line with
previous results.[23b]
pentameric cyclo-RGD peptides does not have a significant
effect on receptor interaction, a cyclo-RGDfK version has
been developed in which covalent modifications can be
carried out at the lysine residue for various purposes including
radiolabeling and medical imaging.[25] In the present case, N-
11C-acetylation of the cyclo-RGDfK lysine residue was
possible under unaltered conditions from Pd-1 to produce
[11C]acetyl cRGDfK ([11C]3) with a radiochemical conversion
from 11CO of 63 Æ 11% (n = 3). Virtually complete trapping
of the starting activity was observed. Again, the general
reproducibility was assessed by repeating the labeling at the
Uppsala PET-center. Starting from Pd-1 under non-modified
labeling conditions, 1.48 GBq (40 mCi) of highly pure (RCP =
99%) [11C]3 could be isolated, resulting in an isolated
radiochemical yield of 37%.
In a similar fashion, the labeling of an amino-acid-derived
pharmaceutical, lacosamide, was evaluated. Starting from the
corresponding amine, [11C]lacosamide ([11C]4) could be
reproducibly generated with a radiochemical conversion
from 11CO in 63 Æ 5% (n = 3) by performing the reaction at
808C (Scheme 2). Preparative HPLC purification afforded
2.45 GBq (66.2 mCi) of [11C]4 in a decay-corrected radio-
chemical yield of 46%, (RCP = 98%). The isotopic dilution
of the radioactive isotope was again found to be minimal as
reflected by the molar activity (404 GBqmmolÀ1).
We sought to extend the method to the labeling of further
motifs with peptide core-structures. To this end, we consid-
ered first the synthesis of [11C]3 (Scheme 2). Cyclic pentame-
ric peptides displaying an arginine–glycine–aspartic acid
tripeptide sequence (RGD) have been developed as ligands
for the aVb3 integrin receptor, which is involved in tumor-
induced angiogenesis and metastasis in human cancers.
As a final evaluation, we examined the labeling of a larger
peptide. Such motifs must be expected to react slower in the
acetylation step as a consequence of its folding properties. In
the present scenario, we focused our examination on the 14-
meric antimicrobial peptide, SPF-5506-A4 (6) as depicted in
Scheme 3. This peptide belongs to the peptaibol A4-subgroup
Scheme 2. N-11C-acetylation of peptide and amino acid motifs. [a]
Radiochemical conversion based on analytical radio-HPLC, decay
corrected. [b] Conditions: Pd-1 (10 mmol), XantPhos (10 mmol), non-
acetylated peptide (10 mmol) in DMF (0.35 mL) at 808C for 8 min. [c]
Conditions: Pd-1 (20 mmol), XantPhos (20 mmol), non-acetylated pep-
tide (20 mmol) in DMF (0.70 mL) at 608C for 8 min. [d] Decay-
corrected isolated yield based on radioactivity delivered to the reaction
vial at t=0 min.
Scheme 3. Carbonylative acetylation of 5 with CO and 11CO. [a]
reaction setup similar to Table 1; yield of 6 determined by UV-HPLC
calibrated against an internal standard. [b] Radiochemical conversion
based on analytical radio-HPLC, decay corrected.
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and has also been reported to inhibit the formation of amyloid
b-peptide (Ab) in the cerebral cortex of primary guinea
pigs.[26]
Keywords: 11C-labeling · carbonylation · palladium · peptides
The precursor 5 for SPF-5506-A4 (Scheme 3) was pre-
pared through solid-phase peptide synthesis using a previously
described method.[16a] N-Acetylation studies were conducted
in a two-chamber system, employing 1.1 equivalents of ex situ
generated CO. At 408C with the XantPhos ligated Pd-2, N-
acetylation occurred in 32% (isolated yield 25%) as deter-
mined by HPLC analysis (Scheme 3). Acetylation of the C-
terminal alcohol or the internal serine residue was not
observed, as confirmed by comparing with genuine O-
acetylated samples by HPLC. Increasing the temperature to
608C proved beneficial, affording the N-acetylated peptide 6
in a 62% yield. The N-selectivity was not compromised by
this increase in temperature.
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We proceeded to prepare [11C]6 under the optimized
labeling conditions as described in Table 2, entry 9. Prelimi-
nary attempts led to the formation of [11C]6 with a radio-
chemical conversion from 11CO of 7% (not shown). Although
the main fraction of the initial radioactivity was trapped by
the reaction components, incorporation of 11CO into the
product had occurred only to a small extent, whereas several
radiolabeled side-products were observed. A considerable
improvement was obtained by increasing the labeling temper-
ature to 808C.[27] Once again, it was found that careful
exclusion of water from the reaction medium was beneficial.
With this fine tuning, formation of [11C]6 occurred with a 15%
radiochemical conversion when employing Pd-1 and with
a 21% radiochemical conversion when employing Pd-2.[28] As
before, acetylation of the C-terminal alcohol or the internal
serine residue was not observed.
[10] A low yielding 11C-labeling of N-phenyl piperazine has been
reported by photoinitiated carbonylation with [11C]CO and MeI.
Although interesting, this strategy has not been extended to any
other substrates: O. Itsenko, T. Kihlberg, B. Lꢀngstrçm, J. Org.
Chem. 2004, 69, 4356 – 4360.
[11] The 11C-acetylation of aryl motifes is more developed and can be
achieved with either [11C]MeI: a) K. Dahl, M. Schou, C. Halldin,
Eur. J. Org. Chem. 2016, 2775 – 2777; with 11CO: b) O. Rahman,
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4791; e) T. Arai, M. R. Zhang, M. Ogawa, T. Fukumura, K. Kato,
K. Suzuki, Appl. Radiat. Isot. 2009, 67, 296 – 300.
In conclusion, this method provides access to 11C-labeled
peptides through N-terminal or internal lysine residue N-11C-
acetylation. The protocol is characterized by a mild reaction
profile, which facilitates the labeling of peptides, as well as
small molecules displaying an amine. The method provides
the labeled molecules in useful radiochemical yields and with
high specific activity suitable for in vivo studies. These results
highlight the characteristics of performing optimization
reactions with minute amounts of CO (mmol range) as
a model system for 11C-labeling. Such an approach led to
transferrable results in a time and cost-saving manner while
minimizing the often restricted cyclotron time.
[12] For reviews applying 11CO in PET-labeling see: B. H. Rotstein,
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[15] For an example on the Pd-catalyzed aminocarbonylation of
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Acknowledgements
We appreciate generous financial support from the Danish
National Research Foundation (grant no. DNRF118), and
from Aarhus University.
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Conflict of interest
Troels Skrydstrup is co-owner of SyTracks a/s, which com-
mercializes the two-chamber technology and COgen.
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[27] Increasing the temperature or modifiying the loading or the ratio
of the reactants did not improve the radiochemical yield.
[28] The isolation of [11C]6 was complicated owing to difficulties in
handling the peptide, and thus the isolated radiochemical yield
was lowered in comparison to Scheme 3. In a single run, we
managed to isolate 3.46 mCi of [11C]6 (RCY= 2.1%, RCP =
98%). See Supporting Information.
[21] Epimerization of the peptide does not occur under the applied
conditions (see Supporting Information).
[22] The acetylation proved to be N-terminal selective in the
presence of tryptophan and histidine but not in the presence of
cysteine, in which case S-acetylation is a competing reaction. See
Supporting Information.
[23] a) K. Dahl, M. Schou, N. Amini, C. Halldin, Eur. J. Org. Chem.
2013, 1228 – 1231; b) T. L. Andersen, S. D. Friis, H. Audrain, P.
Manuscript received: January 14, 2017
Revised: February 12, 2017
Final Article published: && &&, &&&&
Angew. Chem. Int. Ed. 2017, 56, 1 – 6
ꢀ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!

Angewandte
Communications
Chemie
Communications
11C-Labeling
T. L. Andersen, P. Nordeman,
H. F. Christoffersen, H. Audrain,
G. Antoni,*
T. Skrydstrup*
&&&& — &&&&
Application of Methyl Bisphosphine-
Ligated Palladium Complexes for Low
Pressure N-11C-Acetylation of Peptides
PET tracers from 11CO: 11C-Labeling of
peptides by selective N-acetylation at the
terminal nitrogen or at internal lysine
positions is described applying biphos-
phine palladium–methyl complexes in the
presence [11C]carbon monoxide.
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!