Design, synthesis, in vitro and in vivo evaluation of heterobivalent sifalin-modified peptidic radioligands targeting both integrin αv β3 and the mc1 receptor—suitable for the specific visualization of melanomas?

Article abstract of DOI:10.3390/ph14060547

Combining two peptides addressing two different receptors to a heterobivalent peptidic ligand (HBPL) is thought to enable an improved tumor-targeting sensitivity and thus tumor visu-alization, compared to monovalent peptide ligands. In the case of melanoma, the Melanocortin-1 receptor (MC1R), which is stably overexpressed in the majority of primary malignant melanomas, and integrin α_v β₃, which is involved in lymph node metastasis and therefore has an important role in the transition from local to metastatic disease, are important target receptors. Thus, if a radiolabeled HBPL could be developed that was able to bind to both receptor types, the early diagnosis and correct staging of the disease would be significantly increased. Here, we report on the design, synthesis, radiolabeling and in vitro and in vivo testing of different SiFAlin-modified HBPLs (SiFA = silicon fluoride acceptor), consisting of an MC1R-targeting (GG-Nle-c(DHfRWK)) and an integrin α_v β₃-affine peptide (c(RGDfK)), being connected by a symmetrically branching framework including linkers of differing length and composition. Kit-like¹⁸ F-radiolabeling of the HBPLs 1–6 provided the labeled products [¹⁸ F]1–[¹⁸ F]6 in radiochemical yields of 27–50%, radiochemical purities of ≥95% and non-optimized molar activities of 17–51 GBq/μmol within short preparation times of 25 min. Besides the evaluation of radiotracers regarding log_D(7.4) and stability in human serum, the receptor affinities of the HBPLs were investigated in vitro on cell lines overexpressing integrin α_v β₃ (U87MG cells) or the MC1R (B16F10). Based on these re-sults, the most promising compounds [¹⁸ F]2, showing the highest affinity to both target receptors (IC_{50 (B16F10)} = 0.99 ± 0.11 nM, IC_{50 (U87MG)} = 1300 ± 288 nM), and [¹⁸ F]4, exhibiting the highest hy-drophilicity (log_D(7.4) = ?1.39 ± 0.03), were further investigated in vivo and ex vivo in a xenograft mouse model bearing both tumors. For both HBPLs, clear visualization of B16F10, as well as U87MG tumors, was feasible. Blocking studies using the respective monospecific peptides demon-strated both peptide binders of the HBPLs contributing to tumor uptake. Despite the somewhat lower target receptor affinities (IC_{50 (B16F10)} = 6.00 ± 0.47 nM and IC_{50 (U87MG)} = 2034 ± 323 nM) of [¹⁸ F]4, the tracer showed higher absolute tumor uptakes ([¹⁸ F]4: 2.58 ± 0.86% ID/g in B16F10 tumors and 3.92 ± 1.31% ID/g in U87MG tumors; [¹⁸ F]2: 2.32 ± 0.49% ID/g in B16F10 tumors and 2.33 ± 0.46% ID/g in U87MG tumors) as well as higher tumor-to-background ratios than [¹⁸ F]2. Thus, [¹⁸ F]4 demonstrates to be a highly potent radiotracer for the sensitive and bispecific imaging of malignant melanoma by PET/CT imaging and impressively illustrates the suitability of the un-derlying concept to develop heterobivalent integrin α_v β₃-and MC1R-bispecific radioligands for the sensitive and specific imaging of malignant melanoma by PET/CT.

Full text of DOI:10.3390/ph14060547

pharmaceuticals
Article
Design, Synthesis, In Vitro and In Vivo Evaluation of
Heterobivalent SiFAlin-Modified Peptidic Radioligands
Targeting Both Integrin α β and the MC1 Receptor—Suitable
for the Specific Visualizatvion3 of Melanomas?
Xia Cheng 1, Ralph Hübner 2 , Valeska von Kiedrowski 1, Gert Fricker 3, Ralf Schirrmacher 4
,
Carmen Wängler 2,
*
and Björn Wängler 1,
*
1
Molecular Imaging and Radiochemistry, Department of Clinical Radiology and Nuclear Medicine,
Medical Faculty Mannheim of Heidelberg University, Theodor-Kutzer-Ufer 1–3, 68167 Mannheim, Germany;
Xia.Cheng@medma.uni-heidelberg.de (X.C.); valeska.vonkiedrowski@medma.uni-heidelberg.de (V.v.K.)
Biomedical Chemistry, Department of Clinical Radiology and Nuclear Medicine, Medical Faculty Mannheim
of Heidelberg University, Theodor-Kutzer-Ufer 1–3, 68167 Mannheim, Germany;
Ralph.Huebner@medma.uni-heidelberg.de
Institute of Pharmacy and Molecular Biotechnology, University of Heidelberg, Im Neuenheimer Feld 329,
69120 Heidelberg, Germany; gert.fricker@uni-hd.de
Department of Oncology, Division of Oncological Imaging, University of Alberta, 11560 University Avenue,
Edmonton, AB T6G 1Z2, Canada; schirrma@ualberta.ca
2
3
4
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Cheng, X.; Hübner, R.; von
Kiedrowski, V.; Fricker, G.;
*
Correspondence: Carmen.Waengler@medma.uni-heidelberg.de (C.W.);
Bjoern.Waengler@medma.uni-heidelberg.de (B.W.)
Schirrmacher, R.; Wängler, C.;
Wängler, B. Design, Synthesis, In
Vitro and In Vivo Evaluation of
Heterobivalent SiFAlin-Modified
Peptidic Radioligands Targeting Both
Integrin αvβ3 and the MC1
Receptor—Suitable for the
Abstract: Combining two peptides addressing two different receptors to a heterobivalent peptidic
ligand (HBPL) is thought to enable an improved tumor-targeting sensitivity and thus tumor visu-
alization, compared to monovalent peptide ligands. In the case of melanoma, the Melanocortin-1
receptor (MC1R), which is stably overexpressed in the majority of primary malignant melanomas,
and integrin α β3, which is involved in lymph node metastasis and therefore has an important
v
role in the transition from local to metastatic disease, are important target receptors. Thus, if a
radiolabeled HBPL could be developed that was able to bind to both receptor types, the early di-
agnosis and correct staging of the disease would be significantly increased. Here, we report on
the design, synthesis, radiolabeling and in vitro and in vivo testing of different SiFAlin-modified
HBPLs (SiFA = silicon fluoride acceptor), consisting of an MC1R-targeting (GG-Nle-c(DHfRWK))
Specific Visualization of
Melanomas? Pharmaceuticals 2021, 14,
547. https://doi.org/10.3390/
ph14060547
Academic Editor: Gerald Reischl
and an integrin α β3-affine peptide (c(RGDfK)), being connected by a symmetrically branching
v
framework including linkers of differing length and composition. Kit-like 18F-radiolabeling of
Received: 6 May 2021
Accepted: 3 June 2021
Published: 7 June 2021
the HBPLs 1–6 provided the labeled products [18F]
radiochemical purities of
short preparation times of 25 min. Besides the evaluation of radiotracers regarding logD
1
–[18F]
6
in radiochemical yields of 27–50%,
95% and non-optimized molar activities of 17–51 GBq/ mol within
and
≥
µ
(7.4)
stability in human serum, the receptor affinities of the HBPLs were investigated in vitro on cell
lines overexpressing integrin α β (U87MG cells) or the MC1R (B16F10). Based on these re-
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
v
3
sults, the most promising compounds [18F]
2, showing the highest affinity to both target receptors
(IC50 (B16F10) = 0.99 ± 0.11 nM, IC50 (U87MG) = 1300 ± 288 nM), and [18F]
4, exhibiting the highest hy-
= −1.39 ± 0.03), were further investigated in vivo and ex vivo in a xenograft
drophilicity (logD
(7.4)
mouse model bearing both tumors. For both HBPLs, clear visualization of B16F10, as well as
U87MG tumors, was feasible. Blocking studies using the respective monospecific peptides demon-
strated both peptide binders of the HBPLs contributing to tumor uptake. Despite the somewhat
Copyright:
© 2021 by the authors.
lower target receptor affinities (IC50 (B16F10) = 6.00
±
0.47 nM and IC50 (U87MG) = 2034
: 2.58 0.86% ID/g in B16F10
0.49% ID/g in B16F10 tumors and
±
323 nM)
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
of [18F]
4
, the tracer showed higher absolute tumor uptakes ([18F]
4
±
tumors and 3.92 ± 1.31% ID/g in U87MG tumors; [18F]
2: 2.32
±
2.33 ± 0.46% ID/g in U87MG tumors) as well as higher tumor-to-background ratios than [18F]
2.
Thus, [18F]
4 demonstrates to be a highly potent radiotracer for the sensitive and bispecific imaging
of malignant melanoma by PET/CT imaging and impressively illustrates the suitability of the un-
Pharmaceuticals 2021, 14, 547. https://doi.org/10.3390/ph14060547
https://www.mdpi.com/journal/pharmaceuticals

Pharmaceuticals 2021, 14, 547
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derlying concept to develop heterobivalent integrin α β3- and MC1R-bispecific radioligands for the
v
sensitive and specific imaging of malignant melanoma by PET/CT.
Keywords: malignant melanoma; 18F; SiFAlin; MC1R; α β3; heterobivalent peptidic ligands;
v
PET/CT imaging
1. Introduction
With a global incidence increasing over the last decades and being among the tumor
types with the most increasing prevalence in Europe, malignant melanoma (MM) is the
most aggressive type of skin cancer. The probability of developing the disease is increasing
for people with a large number of melanocytic nevi, a fair skin type and genetic predisposi-
tion [1–
3]. Repeated exposure to strong UV (ultraviolet) radiation through recurrent intense
sun exposure is the most important environmental risk factor [
4
]. In most cases, an early
diagnosis enables a complete surgical removal and thus the patient to be cured. However,
early detection is often not possible since the disease has no particular symptoms, and the
tumors can rapidly progress from the fully encapsulated stage to infiltrative growth. In the
case of basal membrane penetration, the tumor has access to the blood and lymph vessels,
and metastases can be formed in organs or lymph nodes [5]. Since a cure is rarely possible
when metastasis has already occurred, an early, very sensitive and specific diagnosis of the
disease is of the highest importance. Moreover, the correct staging of the disease is critical,
as only, in this case, can an appropriate therapy, having the potential to cure the patient,
be chosen.
However, primary diagnosis using positron emission tomography (PET), which has
the highest sensitivity compared to other whole-body imaging techniques such as com-
puted tomography (CT) or magnetic resonance imaging (MRI), is often not suitable to
correctly identify MM lesions. One drawback of the commonly used radiotracer [18F]FDG
(2-[18F]fluoro-2-deoxyglucose) is its accumulation in inflamed tissues, giving false-positive
results. Furthermore, the detection of slowly growing lesions is often difficult as well,
resulting in possible false-negative imaging results [6]. Since the tumor visualization
sensitivity and specificity using [18F]FDG can be low, an early and correct diagnosis and
staging are often not possible. An alternative to unspecific, metabolically driven imaging is
addressing the tumor by a tumor-specific radiotracer. For this purpose, receptors that are
overexpressed in the tumor cell surface are especially useful. In the case of MM, the MC1R
is best suited, as this receptor type is overexpressed in about 80% of MM primaries [7,8]
and thus is a highly important target structure for MM-specific imaging. However, not
all lesions express the MC1R, resulting in an incomplete visualization of the tumor load
and thus false staging of the disease. In order to improve the diagnostic imaging of MM
and enable an adequate, early and sensitive diagnosis and correct staging, a reliable and
sensitive imaging method for MM is needed. Therefore, the development of target-specific
accumulating agents that are able to address more than just the MC1R is mandatory.
Such agents should be based on radiolabeled peptides being able to bind with high
affinity and specificity to surface receptors overexpressed by malignant cells and thus,
enable the distinction between benign and malignant tissue. Ideally, radiolabeled peptides
exhibit favorable tumor-to-background ratios, due to their tumor-specific accumulation,
and thus produce images of high quality. Furthermore, peptides exhibit low toxicity and
immunogenicity, are easily synthetically accessible and can be chemically modified at
defined sites. Their pharmacokinetics prove to be very advantageous due to rapid tissue
penetration, target accumulation and elimination from non-target tissues [9,10]. Therefore,
numerous radiolabeled peptides have been developed for both the diagnosis and therapy
of malignancies over the last decades [11,12].
Heterobivalent peptidic ligands (HBPLs), consisting of a radionuclide and two dif-
ferent peptides, each addressing its respective target receptor, have the advantage of a

Pharmaceuticals 2021, 14, 547
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higher target avidity compared to monovalent peptide ligands by being able to bind simul-
taneously or independently to different target receptors on the tumor surface, resulting
in stronger binding to the target cell [13]. Furthermore, HBPLs usually exhibit higher
metabolic stability than their respective monomers against peptidases, due to their higher
molecular weight and introduction of artificial structural elements [14]. The prerequisite for
an HBPL with high tumor visualization potential is at least a moderate binding affinity of
each of the included peptides to their target receptors. Ideally, both receptor types should
be present in high density to achieve a concomitant binding of both peptide binders of
the HBPL; however, the presence of only one target receptor is sufficient to achieve a high
tumor uptake [9,15–17], resulting in an overall improved imaging sensitivity.
In contrast to HBPLs, monovalent peptides, being able to address only one receptor
type and thus only visualize tumors that overexpress this particular receptor, can result in
limited tumor visualization sensitivity, as tumor cells can overexpress different receptor
types. In such cases of inhomogeneous receptor expression, which can further be caused
by tumor dedifferentiation, metastasis or triggered by therapy, the target receptor for the
monospecific binder can be absent or present in insufficient density [18–20]. This results
in an insufficient sensitivity of the peptides’ tumor delineation (Figure 1A). In contrast,
HBPLs have the advantage of binding to more than one receptor type and thus exhibit a
high tumor visualization efficiency (Figure 1B) [9,20].
Figure 1. Schematic depiction of the concept of HBPL application exemplified by a comparison of a radiolabeled monospe-
cific ( ) or heterobivalent peptidic ligand ( ) binding to tumor entities overexpressing different receptor types. In the case
of ( ), no binding is possible since the respective target receptor is only expressed to a low extent. In the case of ( ), the
HBPL can bind since at least one of the target receptors is expressed on the tumor surface.
A
B
A
B
For the development of HBPLs, some requirements have to be fulfilled. The peptides
have to be modified as little as possible in their chemical structure to preserve their binding
affinities to their corresponding receptors. In particular, the pharmacophoric site has
to remain unchanged. Furthermore, it is important to determine which receptor types
are overexpressed in a tumor entity and thus can be addressed by the radioligand to be
developed [9,20]. For this purpose, many studies have been performed within recent years
regarding the available receptor types on different human malignancies [21]. The results
obtained serve as a guideline for the choice of peptidic receptor ligands, yielding potent
tumor-targeting HBPLs with highly sensitive visualization properties.

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For MM, the MC1R represents one especially useful target structure for the specific
imaging of the disease (vide supra). Another receptor type that is of high potential for
MM imaging is integrin α β3, as it was shown that this receptor is overexpressed in the
v
blood vessels of many human tumors [22
–24]. Further studies revealed the involvement of
integrin α β in the progression of the disease and in the change of tumor growth from
v
3
radial to vertical (thus infiltrative) growth [25–30]. Therefore, integrin α β3, although
v
overexpressed in all neo-angiogenetic processes, is also an important marker protein for
MM targeting.
Thus, HBPLs based on MC1R- and integrin α β3-affine peptides would be most
v
promising for visualizing MM during all stages of the disease, enabling a highly sensitive
and especially correct assessment of the extent of the disease. This is of crucial importance
for choosing the optimal therapy approach, adapted to the extent of the disease: an
encapsulated tumor can be treated differently than an infiltratively growing or already
metastatic tumor. A high sensitivity to tumor imaging, surely identifying all tumor mass,
is thus the prerequisite for the choice of the best-suited therapy option.
So far, the concept to develop an HBPL based on an MC1R-specific peptide ([Cys3,4,10
DPhe7, Arg11]
MSH3-13) and an integrin α β3-affine peptide (c(RGDyD) (cyclic Arg-Gly-Asp-
,
α
v
DTyr-Asp)) has only been described once for the radiotracer 99mTc-RGD-Lys-(Arg11)CCMSH
intended for tumor therapy driven by caspase-3-induced apoptosis induction [31]. The
evaluation of this compound was performed in vitro on MC1R-exhibiting B16F1 cells and
in vivo in B16F1 melanoma-bearing mice. High binding affinity and tumor uptake, but also
a high renal uptake, were detected for this tracer. Therefore, structural modifications are
mandatory to obtain an HBPL with more favorable in vivo pharmacokinetics.
In the present study, we developed different radiolabeled MC1R- and α β3-bispecific
v
HBPLs. These were based on the α β3-affine peptide c(RGDfK) (cyclic Arg-Gly-Asp-DPhe-
v
Lys), showing high stability and integrin target affinity [32], and the macrocyclic lactam
GG-Nle-c(DHfRWK) (Gly-Gly-Nle-cyclic Asp-His-DPhe-Arg-Trp-Lys), giving excellent
results in terms of MC1R target affinity and stability against proteolytic degradation as
well [33,34].
As no HBPLs based on these peptidic ligands have been described so far, we intended
to assess the general feasibility of this concept and to develop different HBPLs, consisting
of the mentioned peptidic binders, a SiFAlin-moiety (for efficient radiolabeling of the HBPL
with the positron-emitting nuclide 18F) and a varying molecular design. The molecular
scaffold for the HBPLs was based on a symmetrical branching unit exhibiting linkers of
different lengths and compositions so as to be able to systematically determine the influence
of the used linker type and length on the biological parameters of the resulting HBPLs. The
developed agents were labeled with 18F and evaluated in vitro regarding their lipophilicity,
stability in human serum and especially their binding affinity to the respective target
receptors. Finally, the most promising 18F-labeled derivatives were evaluated in vivo, in
terms of their tumor visualization potential, in an appropriate preclinical tumor model
using PET/CT imaging and ex vivo biodistribution experiments.
2. Results and Discussion
2.1. General Considerations for the Design of the Heterobivalent SiFAlin-Modified Peptidic Ligands
The molecular design of the target compounds (Figure 2) included two different pep-
tides, each addressing specifically one of the two target receptors—c(RGDfK) for integrin
α β3 and GG-Nle-c(DHfRWK) for MC1R binding—and was based on the following con-
v
ditions: (i) The HBPLs should contain a SiFAlin-moiety exhibiting a permanent positive
charge. With this SiFAlin building block, the radionuclide 18F can be efficiently introduced
in one step [35]; (ii) the required molecular building blocks should be connected by a small
symmetrically branched framework resulting in homogeneous compounds [9,36]; (iii) a ly-
sine spacer should be introduced between the SiFAlin-moiety and the branched framework
to achieve a spatial distance between the SiFAlin and the peptides, preventing interference
with the peptide–receptor interaction, and to obtain the products in higher radiochemical

Pharmaceuticals 2021, 14, 547
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yields [
9,
20
,37
,
38]; (iv) as much as possible, the syntheses should be carried out on a solid
support to facilitate the assembly of the rather complex target molecules; (v) different linker
structures should be introduced between the peptides and the branching unit to systemati-
cally determine the optimal distance between both peptides. An optimal distance between
the peptide binders enables the binding of each peptide to the respective receptor while
remaining not interfered with by the second peptide and, at the same time, does not result
in a high entropy, limiting the benefits of peptide heterodimerization [15,39–43]. Since
the synthetic effort for the SiFAlin-linked framework is higher than that of the peptides,
the linkers should be introduced as bis-NHS (N-hydroxysuccinimide) active esters on the
peptidic side by derivatizing the Nε-amine of lysine of c(RGDfK) and the Nα-amine of
glycine of GG-Nle-c(DHfRWK), or the Nα-amine of glutamic acid for the EGEGE peptides.
Regarding the order of peptide-to-framework conjugations, the smaller c(RGDfK)-based
peptides should be reacted first, followed by the bulkier GG-Nle-c(DHfRWK) peptides, to
achieve higher product yields.
Figure 2. Depiction of the structures of the target HBPLs
black); the symmetrically branching framework (pink); linkers
PEG5, PEG8, DIG, Ox-EGEGE; PEG = polyethylene glycol; DIG = diglycolic acid; Ox = oxalic acid); the MC1R- and integrin
α β -affine peptides GG-Nle-c(DHfRWK) (green) and c(RGDfK) (orange).
1–
6
consisting of: a SiFAlin-moiety (blue); a short lysine linker
(
Y
of different lengths and compositions (PEG1, PEG3,
v
3
2.2. Synthesis of the Heterobivalent SiFAlin-Modified Peptidic Ligands
For the assembly of the SiFAlin-modified HBPLs , the monomeric peptides were
1–6
synthesized according to standard Fmoc-based solid-phase peptide synthesis (SPPS) proto-
cols. The c(RGDfK)-peptide was synthesized according to a known procedure [44] and was
obtained in an overall yield of 83%. For the synthesis of the peptide GG-Nle-c(DHfRWK)
(7) (Scheme 1A), all amino acids were coupled on a rink amide resin. After deprotection of
the acid-labile protecting groups (PG)–Mtt and O-2-PhiPr–under mildly acidic conditions,
the cyclization, deprotection and cleavage from the resin were performed. By optimizing
the reaction conditions, peptide 7 was isolated in an overall yield of 42%.

Pharmaceuticals 2021, 14, 547
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Scheme 1.
peptide ( ); isolated yield: 41%. (
ditions: ( ) deprotection of Fmoc-PG: piperidine/DMF (1/1, v/v), 2 + 5 min; (
DIPEA, 3.9 equiv. HBTU in DMF, 2 min, coupling, 60 min; (
) deprotection of Mtt- and O-2-PhiPr-PG: TFA/CH2Cl2
(1/99, v/v), 90 min; ( ) cyclization: 4.0 equiv. DIPEA, 3.9 equiv. PyBOP in DMF, 20 h; ( ) deprotection of Fmoc-PG:
piperidine/DMF (1/1, v/v), 2 10 min; ( ) cleavage from resin and deprotection: TFA/TIS/H2O (95/2.5/2.5, v/v/v),
3 h; ( ) conjugation: 4.0 equiv. DIPEA in CH2Cl2, 4 h; ( ) deprotection of All-PG: 24.0 equiv. PhSiH3, 0.25 equiv.
Pd(PPh3)4 in CH2Cl2, 3 30 min; ( ) cyclization: 4.0 equiv. DIPEA, 3.9 equiv. PyBOP in DMF, 12 h; ( ) deprotec-
tion of ivDde-PG: hydrazine/DMF (2/98, v/v), 2 10 min. DIPEA = N,N-diisopropylethylamine, DMF = dimethyl-
(A
) Synthesis of GG-Nle-c(DHfRWK)-peptide (
) Synthesis of EGEGE-GG-Nle-c(DHfRWK)-peptide (
) activation of amino acids: 4.0 equiv.
7
); isolated yield: 42%. (
B
) Synthesis of c(RGDfK)-EGEGE-
8
a
C
9); isolated yield: 43%. Con-
b
c
d
e
×
f
g
h
×
i
j
×
formamide, HBTU = 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, PyBOP = benzotriazol-1-
yloxytripyrrolidino-phosphonium hexafluorophosphate, TFA = trifluoroacetic acid, TIS = triisopropylsilane.
For the synthesis of the charged HBPL
6, further amino acids had to be added to
the monovalent peptides. For c(RGDfK)-EGEGE (
8
) (Scheme 1B), c(RGDfK) was first
synthesized according to a known procedure [45] and was then modified with the glutamic
acids and glycines at the Nε-amine of lysine (still on a solid support) before the deprotection
and cleavage from the resin were carried out. Peptide
8 was obtained in an overall yield of
41%. EGEGE-GG-Nle-c(DHfRWK) ( ) (Scheme 1C) was prepared in a different way than
9
GG-Nle-c(DHfRWK)-peptide. First, only the first six amino acids were conjugated to the
resin. Afterward, the cyclization was performed, and only then the following conjugation
of the remaining amino acids followed by the cleavage from the resin was performed.
Peptide
9 was obtained in an overall yield of 43% following this route (for analytical data,
see Supplementary Materials Figures S1–S3).
After successfully establishing the synthesis of the monovalent peptides c(RGDfK)
and 7–9, modification of the peptides with the different linkers (PEG1, PEG3, PEG5, PEG8
and DIG) was performed as follows.

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For the evaluation of the optimal conditions to obtain the peptide–linker conjugates
10–
21 (Scheme 2), which can be used for heterodimer synthesis, different solvent/base sys-
tems were tested during the reactions of the peptide c(RGDfK) and
7–
9
with the respective
bis-NHS esters of the linkers introduced to obtain the conjugates 10
–21. The best results in
terms of isolated yields were found for DIPEA in DMF. Besides the target NHS-PEGn pep-
tides 10 13 and 16 19, small amounts of hydrolyzed compound and homodimer were also
isolated. However, in the case of the DIG- and Ox-linker, only the hydrolyzed carboxylic
acids 14 15 20 and 21 could be isolated, although the reason for this is not obvious. For
–
–
,
,
further reactions of these agents with the framework structure, in order to obtain the target
HBPLs, these free carboxylic acid-comprising peptides had to be pre-activated with a suit-
able activation reagent (for analytical data, see Supplementary Materials Figures S4–S15).
Scheme 2. Depiction of the synthesis strategy to obtain the c(RGDfK)–linker conjugates 10
GG-Nle-c(DHfRWK)–linker conjugates 16 21. Conditions: ( ) c(RGDfK), DIPEA, DMF, yields: 41%
for 10, 29% for 11, 35% for 12, 32% for 13, 37% for 14; ( , DIPEA, DMF, 40% yield for 15; (c) 7,
–15 and
–
a
b)
8
DIPEA, DMF, yields: 43% for 16, 49% for 17, 42% for 18, 42% for 19, 61% for 20; (
d) 9, DIPEA, DMF,
44% yield for 21.
To obtain the SiFAlin building block 28 (Scheme 3), the acetal 26 was synthesized
following a published procedure [46 48] with some modifications. First, the hydroxyl func-
–
tion of the 4-bromobenzyl alcohol was protected with TBDMS-Cl (tert-butyldimethylsilyl
chloride) to produce 22, then the SiFA unit was introduced by an in-situ-preceding halogen–
metal exchange with subsequent transmetalation to produce 23. After the acidic depro-
tection of the TBDMS-PG, the resulting alcohol 24 was transferred to the bromide 25 by
an Appel reaction. Amination of 25 with 4,4-diethoxy-N,N-dimethylbutan-1-amine led to
the desired acetal 26. After the acidic deprotection of 26, the resulting aldehyde 27 was
oxidized using KMnO4 (potassium permanganate) to obtain the desired SiFAlin building
block 28.

Pharmaceuticals 2021, 14, 547
8 of 30
Scheme 3. Schematic depiction of the synthesis of SiFAlin acid 28. Conditions: (
a
) TBDMS-Cl,
t
imidazole, DMF, 98% yield; (
b
) SiF2(tBu)2, BuLi, Et2O, 92% yield; (
c) HCl, MeOH, 99% yield;
(
d
) CBr4, PPh3, CH2Cl2, 95% yield; (
e
) 4,4-diethoxy-N,N-dimethylbutan-1-amine, CH2Cl2, 93% yield;
(f) 95% TFA, 91% yield; (g) KMnO4, NaH2PO4, tBuOH/CH2Cl2, 69% yield.
Next, the SiFAlin-comprising symmetrically branching building block 29, being the
basis for the following peptide conjugation and thus peptide heterodimer synthesis,
was prepared. The branching unit was synthesized on a solid support using the same
standard protocols followed for peptide synthesis (Scheme 4). For this purpose, a rink
amide resin MBHA LL was first reacted with Fmoc-Lys(Mtt)-OH and N,N-bis[3[3 -(Fmoc-
amino)propyl]glycine. After the cleavage of the Mtt-PG under mildly acidic conditions,
28 was conjugated to the framework. After the deprotection and cleavage from the resin,
29 was isolated in overall yields of 43% (for analytical data, see Supplementary Materials
Figures S16–S46).
Scheme 4. Schematic depiction of the synthesis pathway towards the SiFAlin-modified branching
framework 29. Conditions: (
a
) deprotection of Fmoc-PG: piperidine/DMF (1/1, v/v), 2 + 5 min;
) activation of amino acids: 4.0 equiv. DIPEA, 3.9 equiv. HBTU in DMF, 2 min, coupling, 60 min;
) deprotection of Mtt-PG: TFA/CH2Cl2 (1/99, v/v), 90 min; ( ) deprotection of Fmoc-PGs: piperi-
) cleavage from the resin: TFA/TIS/H2O (95/2.5/2.5, v/v/v),
(b
(c
d
dine/DMF (1/1, v/v), 2
2 h; isolated yield: 43%.
×
10 min; (e
Finally, the synthesized building blocks 10
erobivalent target agents . For this purpose, 29 was first reacted with the c(RGDfK)
derivatives 10 15, which produced the monovalent intermediates 30 35. These were fur-
ther reacted with the GG-Nle-(DHfRWK) derivatives 16 21 into the final products
(Scheme 5), as this order gave better results (in terms of achievable isolated yields) than did
–21 and 29 were assembled into the het-
1–6
–
–
–
1–6
first conjugating the structurally more demanding peptides 16
ones (10–15).
–21 followed by the smaller

Pharmaceuticals 2021, 14, 547
9 of 30
Scheme 5. Schematic depiction of the synthesis of the MC1R- and integrin α β3-affine HBPLs
1–
6.
v
Conditions: (
DIPEA, DMF, 41% yield for 32; (
46% yield for 34; ( 15, DIPEA, PyBOP, DMF, 58% yield for 35; (
17, DIPEA, DMF, 66% yield for ; ( 18, DIPEA, DMF, 49% yield for
yield for ; ( 20, DIPEA, PyBOP, DMF, 24% yield for ; ( 21, DIPEA, PyBOP, DMF, 21% yield for
a
)
10, DIPEA, DMF, 60% yield for 30; (
13, DIPEA, DMF, 49% yield for 33; (
16, DIPEA, DMF, 77% yield for
; ( 19, DIPEA, DMF, 58%
b) 11, DIPEA, DMF, 67% yield for 31; (c) 12
,
d)
e) 14, DIPEA, PyBOP, DMF,
f
)
g)
1;
(h
)
2
i
)
3 j)
4
k
)
5
l
)
6.
The conjugation of the peptide–linker conjugates 10
analogously to the synthesis of the peptide–linker conjugates by directly reacting the
starting materials in DMF using DIPEA as a base. For the conjugation of 14 15 20 and 21,
–13 and 16–19 was conducted
,
,
which were obtained as free acids instead of the respective NHS esters, the linker-modified
peptides had to be activated before conjugation using PyBOP as the coupling agent.
The intermediates 30
Supplementary Materials Figures S47–S52). The isolated yields of the final products
varied depending on the reaction pathway. Whereas during the conjugation reactions
of the NHS-modified peptides 16 19 to 30 33 relatively high yields of 49–77% could be
–35 were obtained in yields of 41–67% (for analytical data, see
1–6
–
–
achieved, the yields during the reactions of 20 and 21 to 34 and 35 were considerably lower
at 24% and 21%, respectively. This might be attributable to the additional activation step
being required for the free acids 20 and 21 or the shorter linker structure, resulting in a
steric hindrance of the conjugation reaction.
For the HBPLs 1–6,
19F NMR spectra were recorded (for analytical data, see Supple-
mentary Materials Figures S53–S64) along with standard HR mass spectrometry to verify
that all agents contained the required fluorine atom in the SiFAlin building block, instead
of having formed the hydrolyzed hydroxy-comprising species. All spectra showed a signal
with a chemical shift between
intact SiFAlin-moiety [49,50].
δ
=
−
175– 177 ppm, which indicates the presence of an
−
2.3. 18F-Radiolabeling of
Human Serum
1–
6
and Determination of Lipophilicity and Stability of [18F]
1 6 in
–[18F]
In the following procedures, the HBPLs 1–6
were radiolabeled with [18F]fluoride as
instructed by previously published protocols on other SiFAlin-modified peptides [35,51].
Briefly, [18F]fluoride was dried using the “Munich method” [52] over a QMA carbonate
Sep-Pak SPE light cartridge, instead of applying an azeotropic drying, and the activity

Pharmaceuticals 2021, 14, 547
10 of 30
was eluted from the cartridge using a freshly prepared solution of K222 (Kryptofix222)
and KOH (potassium hydroxide) in acetonitrile (MeCN). After optimizing the reaction
and elution conditions, the pH of the obtained solution was adapted with oxalic acid,
preventing potential basic hydrolysis of the SiFAlin-moiety. To this mixture, small amounts
of the respective precursor molecules 1–6 at 25 nmol were added and incubated at ambient
temperature for 10 min. Afterward, the radiolabeled products [18F]1–[18F]6 were purified
using a C18 Sep-Pak SPE light cartridge and eluted with EtOH/H2O (ethanol/water, 9/1,
v/v). The 18F-labeled agents [18F]
1
–[18F]
6
were obtained in radiochemical yields (RCY) of
95% and non-optimized molar activities (Am) of
mol, starting from 0.8–2.2 GBq of [18F]fluoride (Table 1) within 25 min overall
preparation time.
27–50%, radiochemical purities (RCP) of
17–51 GBq/
≥
µ
Table 1. Summary of the results from 18F-radiolabeling and in vitro logD
Values are given as mean ± SD (standard deviation).
and stability evaluations. A0: starting activity.
(7.4)
Am
[GBq/µmol]
HBPL
Linker
RCP [%]
RCY [%]
A0 [GBq]
logD
Stability * [%]
(7.4)
18F]1
18F]2
18F]3
18F]4
18F]5
18F]6
PEG1
PEG3
PEG5
PEG8
DIG
≥98
≥98
≥97
≥97
≥95
≥97
27.2 ± 1.4
40.3 ± 2.6
50.4 ± 3.1
38.4 ± 0.6
27.3 ± 9.9
43.2 ± 1.6
17.5–28.6
20.0–31.2
25.1–49.8
25.7–42.7
17.0–47.6
39.3–51.4
1.0–1.6
0.8–1.2
0.9–2.0
1.1–1.9
1.8–2.2
1.2–1.9
−1.08 ± 0.07
−1.19 ± 0.05
−1.15 ± 0.01
−1.39 ± 0.03
−1.21 ± 0.01
−1.52 ± 0.01
87.0 ± 2.3
85.4 ± 2.8
81.4 ± 2.9
83.6 ± 0.8
83.2 ± 2.4
82.7 ± 1.8
[
[
[
[
[
[
Ox-EGEGE
* Intact radiotracer after 120 min of incubation in human serum.
Since high lipophilicity of peptidic radiotracers can lead to a high plasma protein
binding, resulting in unspecific organ and high liver uptakes, thus negatively impact tumor
visualization [46
imate estimation of the in vivo biodistribution behavior of the radioligands. Therefore,
the logD were determined via their
values of the SiFAlin-modified HBPLs [18F] –[18F]
,53,54], the lipophilicity of the HBPLs was determined to get an approx-
1
6
(7.4)
distribution coefficient between n-octanol and phosphate buffer at pH 7.4. The results
are also summarized in Table 1. In these experiments, [18F]4 (logD(7.4) = −1.39 ± 0.03) ex-
hibited the highest hydrophilicity of the PEGn-linker based HBPLs. Furthermore, it is
clear that the introduction of negative charges led to the expected substantially increased
hydrophilicity of [18F]
strated hydrophilicity suitable for further in vivo application.
6
(logD
=
−
1.52
±
0.01). Overall, all 18F-labeled HBPLs demon-
(7.4)
In addition to the investigation of the radiotracers’ lipophilicity, their stability in
human serum was evaluated in order to determine possible stability issues of the newly
developed agents. For this purpose, the respective 18F-labeled HBPLs were incubated in
human serum at 37 ◦C for 120 min. The results of these experiments are summarized in
Table 1, and the corresponding radio-HPLC chromatograms for [18F]
2 as representative ex-
amples for all compounds studied are depicted in Figure 3A (see Supplementary Materials,
Figure S91 for the results obtained for the other radioligands). In Figure 3B, the portions of
intact radiotracer over the course of the stability experiments are depicted.

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Figure 3. Depiction of the results of the in vitro serum stability evaluation experiments performed in
human serum. (
intact radiotracer over time. Values are depicted as mean (n = 3); error bars represent SD.
A 2 at certain time points and (B) portions of
) Radio-HPLC chromatograms for [18F]
As the results indicated the radiolabeled HBPLs [18F] –[18F]
6 to be sufficiently stable
1
in vitro (81–87% intact radioligand after 120 min), all radiotracers were found to be suitable
for in vivo imaging via PET/CT.
2.4. In Vitro Evaluation of 1–6 Regarding Their Binding Affinities to the Respective
Target Receptors
As in vitro receptor affinities represent an important parameter for the in vivo tumor
uptake of radiotracers, the binding affinities of
1–6 were determined to both target receptors—
integrin α β3 and the MC1R—in competitive displacement assays. During these evaluations
v
on MC1R-positive B16F10 cells [55] and integrin α β3-positive U87MG cells [56], α-MSH
v
(36), NDP (37), c(RGDfC) (38) and c(RGDyK) (39) (Figure 4) served as reference compounds,
and [125I]I-echistatin and [125I]I-NDP were used as integrin α β3-affine and MC1R-affine
v
competitors, respectively. Peptides 36–39 were synthesized using the same Fmoc-based SPPS
protocols used for the preparation of the other peptidic agents before.
Figure 4. Structures of
α
-MSH (36), NDP (37), c(RGDfC) (38) and c(RGDyK) (39), which were used as monospecific reference
peptides during the competitive displacement studies.
The resulting binding curves and determined IC50 values are depicted in Figure 5 and
summarized in Table 2.

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Figure 5. Depiction of the determined binding curves of the HBPLs
MC1R-positive B16F10 cells (
represent SD.
1
–
6
, obtained by competitive displacement assays on
A) and integrin α β3-positive U87MG cells (B). Values are depicted as mean (n = 9); error bars
v
Table 2. Summary of the IC50 values determined for the HBPLs 1–6 by competitive displacement
studies on MC1R-positive B16F10 cells and integrin α β3-positive U87MG cells. Values are given as
v
mean ± SD.
Compound
IC50 (B16F10) [nM]
Compound
IC50 (U87MG) [nM]
1
2
3
4
5
1.74 ± 0.25
0.99 ± 0.11
3.44 ± 0.09
6.00 ± 0.47
2.05 ± 0.35
4.18 ± 0.32
3.75 ± 0.61
0.17 ± 0.04
1
2
3
4
5
6
2881 ± 757
1300 ± 288
1911 ± 70
2034 ± 323
5895 ± 722
>100,000
6
α-MSH
NDP
c(RGDfC)
c(RGDfK)
1493 ± 210
427 ± 37
To ensure that the binding of each peptide binder to its target receptor was unaffected
by the other respective peptide of the HBPL, the monomeric peptides c(RGDfK) and GG-
Nle-c(DHfRWK), which cannot bind to the receptors MC1R and integrin α β3, respectively,
v
were also examined under the same conditions, showing—as expected—no receptor-
specific binding (see Supplementary Materials Figure S92 for details).
Considering the receptor affinity data with respect to the MC1R, none of the developed
agents was as potent as NDP (IC50 of 0.17
NDP is a superpotent synthetic analog of the endogenous ligand
a high potency. However, (IC50 of 3.44 0.09 nM), (IC50 of 2.05
of 1.74 0.25 nM) and (IC50 of 0.99 0.11 nM) showed considerably higher affinities
than the physiological reference -MSH (IC50 of 3.75 0.61 nM). In comparison, (IC50 of
, comprising the charged linker (IC50 of
0.32 nM), exhibited a fourfold higher IC50 value compared to its uncharged counter-
(same distance between both peptide binders but differing linker composition), and
±
0.04 nM), which is however not surprising as
-MSH, thus exhibiting
0.35 nM), (IC50
α
3
±
5
±
1
±
2
±
α
±
4
6.00 ± 0.47 nM) showed a decreased affinity and
6
4.18
part,
±
2
thus considerably decreased affinity.
The corresponding experiments on the integrin α β3-positive U87MG cells revealed
v
that neither
nor (IC50 of 1911
(IC50 of 427 37 nM; in accordance with former values obtained on these cells [57]),
whereas at least compound , showing an IC50 value of 1300 288 nM, demonstrated a
higher integrin affinity than the other reference c(RGDfC) (IC50 of 1493 210 nM; also in
accordance with literature data [44]). For HBPL , an IC50 value towards α β3 could not
be determined in the same concentration range of the other agents studied but showed
a substantially reduced affinity to the target receptor, compared to . This observed
5
(IC50 of 5895
±
722 nM), 1 (IC50 of 2881 of 757 nM), 4 (IC50 of 2034 of 323 nM)
3
±
70 nM) were as potent as the highly affine reference peptide c(RGDyK)
±
2
±
±
6
v
1–5
negative influence of anionic charges on the resulting receptor affinities was also described
in other studies [58] and could be confirmed here.
From the obtained results, it can be concluded that the introduction of a negatively
charged linker impairs binding to the MC1R, as well as to integrin α β3, and thus limits
v

Pharmaceuticals 2021, 14, 547
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the usefulness of the approach. Within the line of the other linkers used, a similar trend
can be observed on both cell lines with regard to the linker length used. In both cell lines
and thus for both receptor types, the affinities increased with increasing linker length up
to the PEG3-unit but then decreased with further increasing linker length, thus giving the
best results for the PEG3-modified analog 2 on both receptor types.
2.5. Evaluation of the In Vivo Pharmacokinetics and Ex Vivo Biodistribution of [18F]
For the evaluation of the in vivo pharmacokinetics, the two most promising HBPLs—
with the highest affinity to both target receptors and [18F]
with the highest hy-
2 4
and [18F]
[18F]
2
4
drophilicity and still reasonable binding affinities—were selected. For PET/CT imaging,
6-week-old male nude mice (Balb/cAnNRj-Foxn1nu/nu) were subcutaneously injected with
5
×
105 B16F10 cells into the right flank and 2.0–2.5
×
106 U87MG cells into the left flank to
generate the respective receptor-positive tumors. When the tumors reached a sufficient size
for imaging, each mouse was administered 4.15 or 3.95 2.06 MBq
2.28 MBq of [18F]
of [18F]
via the lateral tail vein under isoflurane anesthesia. To determine the receptor
±
2
±
4
specificity of both peptide parts of the labeled HBPLs and their relative contribution to
overall tumor uptake, blocking experiments were also performed. For these, the respective
radiotracer was coinjected with the corresponding blocking substance—20 µg NDP, 200 µg
c(RGDyK) or both for double blocking—via the lateral tail vein. After i.v. injection of the
tracers, a dynamic PET scan, followed by a CT scan, was performed. The resulting PET/CT
images and time–activity curves (TACs) are depicted in Figures 6–8. After completion of
the diagnostic scans, the mice were sacrificed, their organs (blood, spleen, liver, kidney,
pancreas, lung, heart, brain, bone, muscle, tail, tumors, stomach, colon and small intestine)
were collected and measured in a
γ-counter for ex vivo biodistribution (see Supplementary
Materials Table S1 for detailed results).
Figure 6. Depiction of the PET/CT images given as maximum intensity projections (MIPs) applying [18F]
(n = 3 for each group). From left to right: [18F]
without blocking; NDP blocking; c(RGDfK) blocking; blocking using both
2 as the radioligand
2
agents. Shown are MIPs obtained for 50–90 min PI (post-injection). Upper row: coronal slices; bottom row: transaxial slices
at the tumor level. Circled in orange: U87MG tumors; circled in green: B16F10 tumors.

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Figure 7. Depiction of the PET/CT images given as MIPs applying [18F]
4 as the radioligand (n = 3 for each group). From left
to right: [18F]
4 without blocking; NDP blocking; c(RGDfK) blocking; blocking using both agents. Shown are MIPs obtained
for 50–90 min PI (post-injection). Upper row: coronal slices; bottom row: transaxial slices at tumor level. Circled in orange:
U87MG tumors; circled in green: B16F10 tumors. In the transaxial sections, it was not always possible to depict both tumors
as some were out of plane.
Figure 8. Depiction of the TACs for B16F10 and U87MG tumors, kidneys, liver and heart over 90 min PI for [18F]
18F]4 (B) (n = 3). Values are depicted as mean; error bars represent SD.
2 (A) and
[
From the PET/CT scans (Figures 6 and 7) it was apparent that [18F]
2
and [18F]
accumulated
0.49% ID/g) as in the U87MG tumor
showed a higher accumulation in the U87MG
(difference not significant (ns), p = 0.17), which is at
to both receptor
4 both
could clearly visualize the B16F10 as well as the U87MG tumors. [18F]
2
in the B16F10 tumor to a similar extent (2.32
±
(2.33 ± 0.46% ID/g). In contrast, [18F]
4
tumor (3.92
±
1.31% ID/g) than [18F]
2
first glance astonishing, as lower receptor affinities were found for [18F]
4
types. Tumor uptakes of [18F]
4 in the B16F10 tumors were, however, comparable to those
of [18F]2 (2.58 ± 0.86% ID/g for [18F]4 and 2.32 ± 0.49% ID/g for [18F]2).
From the PET/CT data depicted in Figure 7, the visual impression obtained is that
[18F]
4
accumulates only to a low extent in B16F10 tumors. However, ex vivo biodistribution
data confirm the data of the TACs and the uptake to be comparatively high as in the case

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of [18F]
2
. Moreover, during the blocking experiments with c(RGDyK), not affecting the
uptake of [18F]
4
in B16F10 tumors, the tumor is clearly visible. Thus, the visually lower
in the B16F10 tumor, depicted in Figure 7, should be due to the
tumor uptake of [18F]
4
fact that the large tumor already had partially necrotic areas, showing no tracer uptake
anymore. This assumption is supported by the literature [59,60].
In the blocking experiments, the receptor specificity of the tracers could be demon-
strated, as blocking with NDP and c(RGDfK) resulted in a considerable decrease of the
respective tumor uptakes of both tracers in B16F10 and U87MG tumors. Coinjection with
NDP substantially reduced the accumulation in the MC1R-positive B16F10 tumors ([18F]
2:
reduction from 2.32
tion from 2.58 0.86% to 1.33
were also found for c(RGDyK) blocking, where U87MG tumor uptakes were reduced
from 2.33 0.46% to 1.48 (change significant, p = 0.04) and from
0.12% ID/g for [18F]
3.92 1.31% to 2.67 (change ns, p = 0.21). Despite these mostly
0.66% ID/g for [18F]
±
0.49% to 1.83
±
0.24% ID/g (change ns, p = 0.19) and [18F]
4: reduc-
±
±
0.27% ID/g (change ns, p = 0.07)). Corresponding results
±
±
2
±
±
4
insignificant changes observed for tumor uptakes of both tracers by blocking, the trends
are nonetheless clearly visible, confirming that both radiolabeled HBPLs bind specifically
to both target receptors, and each peptide part equally contributed to tumor visualization.
The TACs of [18F] and [18F]
2 4 show a comparable uptake pattern of both tracers over
time. The uptakes in kidneys and liver reached their maximum after 5–10 min, whereas
the curves of both tumors approached a plateau only after about 70 min. In the direct
comparison of both tracers, [18F]
4
showed a delayed accumulation in the tumors compared
to [18F]2, for which the reason is not obvious. [18F]
furthermore showed a considerably
lower uptake into kidneys and liver, resulting in higher tumor-to-organ ratios for [18F]
Additionally, for the blood and muscle, a lower unspecific uptake of [18F]
was found
4
4.
4
compared to [18F]
2, thus resulting in overall much more favorable and mostly significantly
higher tumor-to-organ ratios of [18F]4 (see Supplementary Materials Table S2 for details).
In summary, both radiotracers developed were able to clearly visualize both integrin
α β3-positive and MC1R-positive tumors, and both parts of the heterobivalent agents
v
contributed to receptor-specific tumor uptakes (see Supplementary Materials Figure S93).
However, [18F]
[18F]
in vitro receptor binding affinities to both target receptor types.
Therefore, [18F]
proved to be the more promising radiotracer for the bispecific imag-
4
demonstrated lower non-target organ uptakes and faster clearance than
2, thus resulting in considerably higher tumor-to-background ratios, despite its lower
4
ing of malignant melanoma by PET/CT, having a high potential for clinical translation.
3. Materials and Methods
3.1. General
3.1.1. Chemistry
All reagents and solvents for synthesis were at least of analytical grade and were
used without further purification. Dried solvents were obtained from Sigma-Aldrich
(Taufkirchen, Germany) and were stored under inert gas. Fmoc-protected amino acids and
resins were purchased from Novabiochem (Darmstadt, Germany). Water in HPLC grade
and spectroscopic trifluoroacetic acid (TFA) were obtained from Carl Roth (Karlsruhe, Ger-
many), and acetonitrile (MeCN) in HPLC grade from Häberle LABORTECHNIK (Lonsee-
Ettlenschieß, Germany). Moreover, 2-(bis(3-(((9H-fluoren-9-yl)methoxy)carbonylamino)-
propyl)amino)-acetic acid potassium hemisulfate ((Fmoc-NH-propyl)2Gly-OHxKHSO4)
and the bis-N-hydroxy-succinimide (NHS)-esters NHS-PEG1-NHS, NHS-PEG3-NHS and
NHS-PEG8-NHS were purchased from Iris Biotech (Marktredwitz, Germany) and NHS-
PEG5-NHS from BroadPharm (San Diego, CA, USA). Other chemicals and solvents were
obtained from commercial suppliers (Sigma-Aldrich (Taufkirchen, Germany), Merck (Darm-
stadt, Germany), TCI (Eschborn, Germany), abcr (Karlsruhe, Germany), Carl Roth (Karl-
sruhe, Germany) and Alfa Aesar (Schwerte, Germany)). The synthesis of c(RGDfK) was
carried out according to published procedures [44]. Thin-layer chromatography (TLC)
was performed on silica gel 60 F254 plates (MACHEREY-NAGEL; Düren, Germany) and

Pharmaceuticals 2021, 14, 547
16 of 30
visualized by exposure to ultraviolet (UV) light at 254 nm. Column chromatography was
performed on silica gel 60 (0.04–0.063 mm) (Carl Roth; Karlsruhe, Germany). NMR (nu-
clear magnetic resonance) spectra were recorded on a Varian NMR System Spectrometer
(500 MHz for 1H and 126 MHz for 13C) and a Varian Mercury Plus Spectrometer (282 MHz
for 19F) at room temperature. The chemical shifts ( ) of 1H- and 13C-spectra were inter-
δ
nally referenced to residual solvent signals and are expressed in parts per million (ppm).
For 19F-spectra, trifluoroacetic acid was used as external reference (
δ
=
−
76.55 ppm). All
coupling constants (J) are reported in Hertz (Hz), and the following notations indicate the
multiplicity of the signals: s (singlet), d (doublet), t (triplet) and m (multiplet). MS (mass
spectrometry) and HR (high-resolution) MS measurements were performed on a Bruker
Daltonics Microflex MALDI-TOF (MALDI: matrix-assisted laser desorption/ionization),
Jeol AccuTOF GCx FI/FD (FI: field ionization; FD: field desorption), Bruker ApexQe
DART/ESI Instrument (DART: direct analysis in real-time; ESI: electrospray ionization) and
Finnigan MAT95Q HR-ESI spectrometers. For HPLC (high-performance liquid chromatog-
raphy), a Dionex UltiMate 3000 system from Thermo Fisher Scientific was used together
with Chromeleon software (v7.11). For analytical chromatography and semipreparative
analyses, Chromolith Performance (RP-18e, 100–4.6 mm, Merck; Darmstadt, Germany)
and Chromolith (RP-18e, 100–10 mm, Merck; Darmstadt, Germany) columns were used,
respectively. All operations were performed with a flow rate of 4 mL/min using H2O +
0.1% TFA and MeCN + 0.1% TFA as solvents.
3.1.2. Radiolabeling
Tracepur H2O and Kryptofix222 (K222) were purchased from Merck. Anhydrous ace-
tonitrile (MeCN), dimethyl sulfoxide (DMSO), oxalic acid and n-octanol were obtained from
Sigma-Aldrich; ethanol (EtOH), sodium dihydrogen phosphate (NaH2PO4) and disodium
hydrogen phosphate (Na2HPO4) were obtained from Carl Roth; 0.9% sodium chloride
(NaCl)-solution was obtained from VWR (Bruchsal, Germany). Aqueous [18F]fluoride
solution was purchased from EuroPET in Freiburg or the University Hospital Tübingen.
The Sep-Pak Accell Plus QMA Carbonate (46 mg) and Sep-Pak C18 Plus SPE Light car-
tridges (130 mg) were obtained from Waters (Eschborn, Germany). For radioanalytical use,
a Dionex UltiMate 3000 system equipped with a Raytest Gabi Star radioactivity detector
was used together with a Chromolith Performance (RP-18e, 100–4.6 mm, Merck, Germany)
column. All operations were performed with a flow rate of 4 mL/min using H2O + 0.1%
TFA and MeCN + 0.1% TFA as solvents. Radioactivity was measured by an ISOMED
2010 activimeter.
3.1.3. Competitive Binding Studies
Murine melanoma cells (B16F10) and human glioblastoma cells (U87MG) as well as
Dulbecco’s Modified Eagle’s Medium (DMEM) and Eagle’s Minimum Essential Medium
(EMEM) were purchased from ATCC (Wesel, Germany). Fetal calf serum (FCS) was obtained
from Bio&SELL (Feucht, Germany); phosphate-buffered saline (PBS), 1,10-phenanthroline,
tris(hydroxymethyl)aminomethane hydrochloride (Tris HCl) and manganese chloride (MnCl2)
·
were obtained from Sigma-Aldrich (Taufkirchen, Germany); penicillin/streptomycin
(pen/strep) and 0.25% Trypsin with 0.02% EDTA-solution in PBS were obtained from Gibco
(Schwerte, Germany); 2-(4-(2-hydroxyethyl)-1-piperazinyl)-ethanesulfonic acid (HEPES)
was obtained from Gerbu (Heidelberg, Germany); bovine serum albumin (BSA), sodium
chloride (NaCl), calcium chloride (CaCl2) and magnesium chloride (MgCl2) were ob-
tained from Carl Roth (Karlsruhe, Germany). [125I]I-NDP (NEX352, 81.4 GBq/
[
Germany).
µ
mol) and
mol) were purchased from PerkinElmer (Rodgau,
γ
-counting was performed using a 2480 Wizard2 gamma counter system
125I]I-echistatin (NEX083, 81.4 GBq/
µ
from PerkinElmer.

Pharmaceuticals 2021, 14, 547
17 of 30
3.1.4. In Vivo PET Imaging
Briefly, 4–5-week-old male nude mice (Balb/cAnNRj-Foxn1nu/nu) were obtained from
Janvier. A dynamic PET scan over 90 min and a subsequent CT image over 20 min were
acquired using a triple-modality Bruker Albira II small-animal PET/CT/SPECT scanner.
Three animals were studied per group.
3.1.5. Statistical Analyses
For statistical analyses, unpaired, parametric two-tailed t-tests were performed. Statis-
tical significance is indicated as * (p < 0.05), ** (p < 0.01) or *** (p < 0.001).
3.2. Chemical Syntheses
3.2.1. Synthesis of Peptides 7–9
General procedure for peptide synthesis (GP1): Peptides were synthesized on a
solid support according to standard Fmoc-based solid-phase peptide synthesis (SPPS)
protocols. The resin was first swollen in CH2Cl2 for 60 min and then rinsed with DMF.
After deprotection of the Fmoc-protecting group (PG) with piperidine/DMF (1/1, v/v) for
2 + 5 min, the respective amino acid (4.0 equiv.) was pre-activated with DIPEA (4.0 equiv.)
and HBTU (3.9 equiv.) in DMF for 2 min and then coupled for 60 min. These steps were
repeated until the respective peptide sequence was complete.
GG-Nle-c(DHfRWK) (
was synthesized on rink amide resin AM LL (73.5 mg, 25
and then the Mtt- and O-2-PhiPr-PG were removed with TFA/CH2Cl2 (1/99, v/v) for
90 min. Subsequently, the cyclization was conducted using DIPEA (17 L, 100 mol,
4.0 equiv.) and PyBOP (50.7 mg, 97.5 mol, 3.9 equiv.) in DMF for 20 h. After Fmoc
deprotection, the cyclized peptide was cleaved from the resin and deprotected with
TFA/TIS/H2O (95/2.5/2.5, v/v/v) for 3 h. Briefly, was purified by HPLC (semiprepar-
ative, 0–30% MeCN + 0.1% TFA in 6 min, tR = 5.71 min) and isolated as a colorless
solid (11.5 mg, 10.5 mol, yield: 42%, purity: >99%). MS (MALDI) m/z calculated
for C52H74N17O10 [M + H]+: 1096.58, found: 1096.01; HR-ESI-MS m/z calculated for
C52H75N17O10 [M + 2H]2+
548.7936, found: 548.7928; HR-ESI-MS m/z calculated for
C52H74N17O10 [M + H]+: 1096.5799, found: 1096.5779.
c(RGDfK)-EGEGE ( ): According to GP1, the peptide sequence DGRKf was synthe-
sized on 2-Chlorotrityl chloride resin (164 mg, 200 mol, 1.0 equiv., 1.22 mmol/g), then the
Alloc-PG was removed with PhSiH3 (590 L, 519 mg, 4.8 mmol, 24.0 equiv.) and Pd(PPh3)4
(28.9 mg, 25 mol, 0.25 equiv.) in CH2Cl2 for 3 30 min. After deprotection of the Fmoc-PG
with piperidine/DMF (1/1, v/v) for 2 10 min, the peptide was cyclized using DIPEA
(136 L, 800 mol, 4.0 equiv.) and PyBOP (406 mg, 780 mol, 3.9 equiv.) in DMF for
12 h. Subsequently, the ivDde-PG of lysine was deprotected using hydrazine/DMF (2/98,
v/v) for 2 10 min and the remaining amino acids of the sequence EGEGE sequence
were coupled. The cyclized peptide was cleaved and deprotected with TFA/TIS/H2O
(95/2.5/2.5, v/v/v) for 3 h. Peptide was precipitated in cold Et2O, purified by HPLC
7): According to GP1, the peptide sequence GG-Nle-DHfRWK
µmol, 1.0 equiv., 0.34 mmol/g)
µ
µ
µ
7
µ
:
8
µ
µ
µ
×
×
µ
µ
µ
×
8
(semipreparative, 0–40% MeCN + 0.1% TFA in 8 min, tR = 3.88 min) and isolated as a
colorless solid (90.4 mg, 82 mmol, yield: 41%, purity: >99%). MS (MALDI) m/z calcu-
lated for C46H69N14O18 [M + H]+: 1105.49, found 1105.39; HR-ESI-MS m/z calculated
for C46H70N14O18 [M + 2H]2+
:
553.2491, found: 553.2486; HR-ESI-MS m/z calculated for
C46H69N14O18 [M + H]+: 1105.4909, found: 1105.4905.
EGEGE-GG-Nle-c(DHfRWK) ( ): According to GP1, the peptide sequence KWRfHD
was synthesized on rink amide resin AM LL (347 mg, 125 mol, 1.0 equiv., 0.36 mmol/g),
then the Mtt- and O-2-PhiPr-PG were removed with TFA/CH2Cl2 (1/99, v/v) for 90 min.
Subsequently, the cyclization was conducted using DIPEA (85 L, 500 mol, 4.0 equiv.)
and PyBOP (254 mg, 488 mol, 3.9 equiv.) in DMF for 15 h. After Fmoc-deprotection
9
µ
µ
µ
µ
for 2 × 10 min, the remaining amino acids of the EGEGE–GG–Nle sequence were cou-
pled. The cyclized peptide was cleaved and deprotected with TFA/TIS/H2O (95/2.5/2.5,
v/v/v) for 3 h. Peptide
9 was precipitated in cold Et2O, purified by HPLC (semiprepar-

Pharmaceuticals 2021, 14, 547
18 of 30
ative, 0–30% MeCN + 0.1% TFA in 8 min, tR = 7.18 min) and isolated as a colorless
solid (85.2 mg, 53.3
µmol, yield: 43%, purity: >88%). MS (MALDI) m/z calculated
for C71H101N22O21 [M + H]+: 1597.75, found: 1597.30; HR-ESI-MS m/z calculated for
C71H102N22O21 [M + 2H]2+
: 799.3789, found: 799.3790; HR-ESI-MS m/z calculated for
C71H102N22O21 [M + 2H]+: 1598.7590, found: 1598.7538.
3.2.2. Modification of the Peptides with Linker Structures to Obtain 10–21
General procedure for the synthesis of NHS-PEGn peptides (GP2): All steps were
carried out under an N2 atmosphere. A total of 1.0 equiv. of the respective peptide was
added to a solution of 1.0 equiv. bis-NHS-ester and 0.5–1.0 equiv. DIPEA in dry DMF.
Subsequently, the reaction mixture was stirred for 5–40 min at room temperature, while
reaction control was performed by HPLC (analytical, 0–50% MeCN + 0.1% TFA in 5 min).
After the removal of the solvent under reduced pressure, the corresponding NHS-PEGn-
peptide was obtained after purification via semipreparative HPLC. In addition to the
respective target product, small amounts of the hydrolyzed compound HO-PEGn-peptide
and the dimer peptide-PEGn-peptide were isolated (for analytical data, see Supplementary
Materials Figures S65–S80).
NHS-PEG1-c(RGDfK) (10): According to GP2, NHS-PEG1-NHS (3.3 mg, 8.3
1.0 equiv.), DIPEA (1.4 L, 1.1 mg, 8.3 mol, 1.0 equiv.) and c(RGDfK) (5.0 mg, 8.3
µ
mol,
mol,
µ
µ
µ
1.0 equiv.) were reacted in 5 mL dry DMF for 20 min. After purification by HPLC
(semipreparative, 0–30% MeCN + 0.1% TFA in 10 min, tR = 7.01 min), 10 was isolated as a
colorless solid (3.0 mg, 3.4 µmol, yield: 41%, purity: >93%). MS (MALDI) m/z calculated for
C39H56N10O14 [M]+: 888.40, found: 888.26; HR-ESI-MS m/z calculated for C39H57N10O14
[M + H]+: 889.4050, found: 889.4038.
NHS-PEG3-c(RGDfK) (11): According to GP2, NHS-PEG3-NHS (12.1 mg, 24.8
µmol,
1.0 equiv.), DIPEA (4.2 L, 3.2 mg, 24.8 mol, 1.0 equiv.) and c(RGDfK) (15.0 mg, 24.8 µ
µ
µ
mol,
1.0 equiv.) were reacted in 8 mL dry DMF for 15 min. After purification by HPLC
(semipreparative, 0–30% MeCN + 0.1% TFA in 10 min, tR = 4.84 min), 11 was isolated as a
colorless solid (7.0 mg, 7.2 µmol, yield: 29%, purity: >89%). MS (MALDI) m/z calculated for
C43H64N10O16 [M]+: 976.45, found: 976.68; HR-ESI-MS m/z calculated for C43H65N10O16
[M + H]+: 977.4575, found: 977.4567.
NHS-PEG5-c(RGDfK) (12): According to GP2, NHS-PEG5-NHS (9.6 mg, 16.6
1.0 equiv.), DIPEA (2.8 L, 2.2 mg, 16.6 mol, 1.0 equiv.) and c(RGDfK) (10.0 mg,
16.6 mol, 1.0 equiv.) were reacted in 6 mL dry DMF for 30 min. After purification
by HPLC (semipreparative, 15–30% MeCN + 0.1% TFA in 10 min, tR = 4.44 min), 12 was
µmol,
µ
µ
µ
isolated as a colorless solid (6.1 mg, 5.7 µmol, yield: 35%, purity: >91%). MS (MALDI) m/z
calculated for C47H73N10O18 [M + H]+: 1065.51, found: 1065.22; HR-ESI-MS m/z calculated
for C47H73N10O8 [M + H]+: 1065.5099, found: 1065.5099.
NHS-PEG8-c(RGDfK) (13): According to GP2, NHS-PEG8-NHS (13.5 mg, 19.1
µmol,
1.0 equiv.), DIPEA (3.3 L, 2.5 mg, 19.1 mol, 1.0 equiv.) and c(RGDfK) (11.5 mg, 19.1 µ
µ
µ
mol,
1.0 equiv.) were reacted in 6 mL dry DMF for 40 min. After purification by HPLC
(semipreparative, 0–30% MeCN + 0.1% TFA in 10 min, tR = 7.01 min), 13 was isolated as
a colorless solid (7.3 mg, 6.1 µmol, yield: 32%, purity: >82%). MS (MALDI) m/z calcu-
lated for C53H85N10O21 [M + H]+: 1197.59, found: 1197.34; HR-ESI-MS m/z calculated for
C53H84N10O21 [M + H]+: 1197.5885, found: 1197.5892.
NHS-PEG1-GG-Nle-c(DHfRWK) (16): According to GP2, NHS-PEG1-NHS (5.4 mg,
6.1 µmol, 1.0 equiv.), DIPEA (0.5 µL, 0.4 mg, 3.1 µmol, 0.5 equiv.) and 7 (6.7 mg, 6.1 µmol,
1.0 equiv.) were reacted in 5 mL dry DMF for 5 min. After purification by HPLC
(semipreparative, 15–50% MeCN + 0.1% TFA in 10 min, tR = 4.80 min), 16 was isolated
as a colorless solid (3.6 mg, 2.6 µmol, yield: 43%, purity: >93%). MS (MALDI) m/z calcu-
lated for C64H89N18O17 [M + H]+: 1381.66, found: 1381.34; HR-ESI-MS m/z calculated for
C64H89N18O17 [M + H]+: 1381.6648, found: 1381.6643.
NHS-PEG3-GG-Nle-c(DHfRWK) (17): According to GP2, NHS-PEG3-NHS (4.7 mg,
9.6 µmol, 1.0 equiv.), DIPEA (1.6 µL, 1.2 mg, 9.6 µmol, 1.0 equiv.) and 7 (10.5 mg, 9.6 µmol,

Pharmaceuticals 2021, 14, 547
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1.0 equiv.) were reacted in 6 mL dry DMF for 40 min. After purification by HPLC
(semipreparative, 10–30% MeCN + 0.1% TFA in 10 min, tR = 8.51 min), 17 was isolated
as a colorless solid (6.9 mg, 4.7 µmol, yield: 49%, purity: >91%). MS (MALDI) m/z cal-
culated for C68H97N18O19 [M + H]+: 1469.72, found: 1469.47; HR-ESI-MS m/z calculated
for C68H98N18O19 [M + 2H]2+: 735.3622, found: 735.3619; HR-ESI-MS m/z calculated for
C68H97N18O19 [M + H]+: 1469.7172, found: 1469.7154.
NHS-PEG5-GG-Nle-c(DHfRWK) (18): According to GP2, NHS-PEG5-NHS (5.3 mg,
9.1 µmol, 1.0 equiv.), DIPEA (1.6 µL, 1.2 mg, 9.1 µmol, 1.0 equiv.) and 7 (10.0 mg, 9.1 µmol,
1.0 equiv.) were reacted in 5 mL dry DMF for 30 min. After purification by HPLC
(semipreparative, 10–50% MeCN + 0.1% TFA in 8 min, tR = 5.86 min), 18 was isolated as
a colorless solid (5.9 mg, 3.8 µmol, yield: 42%, purity: >90%). MS (MALDI) m/z calcu-
lated for C72H104N18O21 [M]+: 1556.73, found: 1556.24; HR-ESI-MS m/z calculated for
C72H106N18O21 [M + 2H]2+: 779.3884, found: 779.3884.
NHS-PEG8-GG-Nle-c(DHfRWK) (19): According to GP2, NHS-PEG8-NHS (3.3 mg,
4.7 µmol, 1.0 equiv.), DIPEA (0.8 µL, 0.6 mg, 4.7 µmol, 1.0 equiv.) and 7 (5.1 mg, 4.7 µmol,
1.0 equiv.) were reacted in 3 mL dry DMF for 40 min. After purification by HPLC
(semipreparative, 0–50% MeCN + 0.1% TFA in 8 min, tR = 6.09 min), 19 was isolated
as a colorless solid (3.3 mg, 2.0 µmol, yield: 42%, purity: >90%). MS (MALDI) m/z calcu-
lated for C78H117N18O24 [M + H]+: 1689.85, found: 1689.09; HR-ESI-MS m/z calculated for
C78H118N18O24 [M + 2H]2+: 845.4278, found: 845.4275.
General procedure for the synthesis of HO-DIG- and HO-Ox-EGEGE peptides (GP3):
All steps were carried out under an N2 atmosphere. In total, 1.0–10.0 equiv. of the respective
peptide was added to a solution of 1.0–10.0 equiv. bis-NHS-ester and 1.0 equiv. DIPEA in
dry DMF. Subsequently, the reaction mixture was stirred for 5–50 min at room temperature,
while the reaction control was performed by HPLC (analytical, 0–50% MeCN + 0.1% TFA
in 5 min). After the removal of the solvent under reduced pressure, the corresponding HO-
DIG- or HO-Ox-EGEGE peptides were obtained after semipreparative HPLC purification.
In addition to the target products, small amounts of the dimers peptide-DIG/Ox-EGEGE-
peptide were isolated (for analytical data, see Supplementary Materials Figures S81–S84).
HO-DIG-c(RGDfK) (14): According to GP3, NHS-DIG-NHS (8.2 mg, 24.9
µmol,
1.5 equiv.), c(RGDfK) (10.0 mg, 16.6 mol, 1.0 equiv.) and DIPEA (2.8 L, 2.1 mg, 16.6 µ
µ
µ
mol,
1.0 equiv.) were reacted in 11 mL dry DMF for 50 min. After purification by HPLC
(semipreparative, 5–40% MeCN + 0.1% TFA in 10 min, tR = 3.80 min), 14 was isolated as a
colorless solid (4.4 mg, 6.1 µmol, yield: 37%, purity: >99%). MS (MALDI) m/z calculated
for C31H45N9O11 [M]+: 719.32, found: 719.59; HR-ESI-MS m/z calculated for C31H46N9O11
[M + H]+: 720.3311, found: 720.3309.
HO-Ox-EGEGE-c(RGDfK) (15): According to GP3, NHS-Ox-NHS (17.9 mg, 63
µmol,
5.0 equiv.), (13.9 mg, 12.6 mol, 1.0 equiv.) and DIPEA (10.7 L, 8.1 mg, 63 mol, 5.0 equiv.)
8
µ
µ
µ
were reacted in 8 mL dry DMF for 5 min. After purification by HPLC (semipreparative,
5–20% MeCN + 0.1% TFA in 8 min, tR = 4.29 min), 15 was isolated as a colorless solid (5.9 mg,
5.0 µmol, yield: 40%, purity: >99%). MS (MALDI) m/z calculated for C48H69N14O21
[M + H]+: 1177.48, found: 1177.02; HR-ESI-MS m/z calculated for C48H68N14O21 [M]+:
1176.4683, found: 1176.4913.
HO-DIG-GG-Nle-c(DHfRWK) (20): According to GP3, NHS-DIG-NHS (1.5 mg,
3.6 µmol, 1.0 equiv.), 7 (5.0 mg, 3.6 µmol, 1.0 equiv.) and DIPEA (0.6 µL, 0.5 mg, 3.6 µmol,
1.0 equiv.) were reacted in 2 mL dry DMF for 25 min. After purification by HPLC
(semipreparative, 10–40% MeCN + 0.1% TFA in 10 min, tR = 5.36 min), 20 was isolated
as a colorless solid (2.7 mg, 2.2 µmol, yield: 61%, purity: >99%). MS (MALDI) m/z calcu-
lated for C56H78N17O14 [M + H]+: 1212.59, found: 1212.16; HR-ESI-MS m/z calculated for
C56H79N17O14 [M + 2H]2+: 606.7991, found: 606.7991.
HO-Ox-EGEGE-GG-Nle-c(DHfRWK) (21): According to GP3, NHS-Ox-NHS (17.8 mg,
62.6
62.6
µ
mol, 10.0 equiv.),
9 (10.0 mg, 6.3 µmol, 1.0 equiv.) and DIPEA (10.7 µL, 8.1 mg,
µ
mol, 10.0 equiv.) were reacted in 5 mL dry DMF for 10 min. After purification
by HPLC (semipreparative, 0–40% MeCN + 0.1% TFA in 8 min, tR = 6.41 min), 21 was

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isolated as a colorless solid (4.5 mg, 2.7
µ
mol, yield: 44%, purity: >99%). MS (MALDI) m/z
calculated for C73H100N22O24 [M]+: 1668.73, found: 1668.90; HR-ESI-MS m/z calculated
for C73H102N22O24 [M + 2H]2+: 835.3713, found: 835.3716.
3.2.3. Synthesis of SiFAlin-Carboxylic Acid 28 and SiFAlin-Modified Symmetrically
Branching Framework 29
((4-Bromobenzyl)oxy)(tert-butyl)dimethylsilane (22): All steps were carried out under
an N2 atmosphere. TBDMS-Cl (5.81 g, 38.6 mmol, 1.2 equiv.) was added under ice-cooling
to a solution of 4-bromobenzyl alcohol (6.01 g, 32.1 mmol, 1.0 equiv.) and imidazole (5.47 g,
80.3 mmol, 2.5 equiv.) in 36 mL dry DMF. After stirring for 20 h at room temperature, the
reaction mixture was extracted with Et2O. The combined organic layers were washed with
H2O, dried over Na2SO4, concentrated under reduced pressure and the crude product was
purified by column chromatography (n-hexane/EtOAc 50/1
31.4 mmol, 98%) as a colorless liquid. H NMR (500 MHz, CDCl3)
→
10/1) to give 22 (9.47 g,
0.10 (s, 6H, SiCH3),
1
δ
0.94 (s, 9H, CH3), 4.68 (s, 2H, H-5), 7.19 (s, J = 8.2 Hz, 2H, H-3), 7.45 (d, J = 8.2 Hz, 2H, H-2)
ppm; 13C NMR (126 MHz, CDCl3) δ −5.12 (s, 2C, SiCH3), 18.54 (s, 1C, CqCH3), 26.07 (s, 3C,
CH3), 64.46 (s, 1C, C-5), 120.71 (s, 1H, C-1), 127.85 (s, 2C, C-3), 131.41 (s, 2C, C-2), 140.60 (s,
1C, C-4) ppm; MS (FI) m/z calculated for C13H22BrOSi [M + H]+: 301.1, found: 301.9.
Di-tert-butyl(4-(((tert-butyldimethylsilyl)oxy)methyl)phenyl)fluorosilane (23): The
t
reaction was carried out in heat-dried glassware under an N2 atmosphere. BuLi in pentane
◦
(1.6 M, 2.2 mL, 3.49 mmol, 2.1 equiv.) was added over a period of 15 min to a
−
78 C cooled
solution of 22 (500 mg, 1.66 mmol, 1.0 equiv.) in 2 mL dry Et2O. After stirring for 15 min at
78 ◦C, a solution of di-tert-butyldifluorosilane (385 mg, 2.14 mmol, 1.3 equiv.) in 1 mL
dry Et2O was added over a period of 15 min at
78 ◦C. The reaction mixture was stirred
for 2 d at room temperature, quenched with 10 mL saturated aqueous NaCl solution and
extracted with 3 15 mL Et2O. The combined organic layers were dried over Na2SO4 and
−
−
×
concentrated under reduced pressure. Purification by column chromatography (n-hexane)
gave compound 23 (582 mg, 1.52 mmol, 92%) as a colorless liquid. H NMR (500 MHz,
1
CDCl3)
δ 0.11 (s, 6H, SiCH3), 0.96 (s, 9H, CH3), 1.06 (s, 18H, H-1), 4.77 (s, 2H, H-7), 7.34
(d, J = 7.8 Hz, 2H, H-5), 7.57 (d, J = 7.8 Hz, 2H, H-4) ppm; 13C NMR (126 MHz, CDCl3)
δ −5.11 (s, 2C, SiCH3), 18.60 (s, 1H, CqCH3), 20.41 (d, J = 12.4 Hz, 2C, C-2), 26.13 (s, 3C,
CH3), 27.50 (d, J = 0.9 Hz, 6C, C-1), 65.00 (s, 1C, C-7), 125.33 (d, J = 0.9 Hz, 2C, C-5), 131.98
(d, J = 13.6 Hz, 1C, C-3), 134.07 (d, J = 4.2 Hz, 2C, C-4), 142.97 (s, 1C, C-6) ppm; 19F NMR
(282 MHz, CDCl3) δ −188.99 (s, 1F, SiF) ppm; MS (FD) m/z calculated for C21H39FOSi2
[M]+: 382.2, found: 382.2; MS (DART) m/z calculated for C21H38FOSi2 [M
found: 381.2440.
−
H]+: 381.2445,
(4-(Di-tert-butylfluorosilyl)phenyl)methanol (24): 6
to a colorless solution of 23 (72.7 mg, 190 mol, 1.0 equiv.) in 600
for 2 h at room temperature, the solvent was removed under reduced pressure. The crude
µL (1 vol.-%) conc. HCl was added
µ
µL MeOH. After stirring
product was purified by column chromatography (n-hexane/EtOAc 9/1
→
1/1) to give
1
compound 24 (50.8 mg, 189
µmol, 99%) as a colorless solid. H NMR (500 MHz, CDCl3)
δ
1.06 (s, 18H, H-1), 4.72 (s, 2H, H-7), 7.38 (d, J = 7.8 Hz, 2H, H-5), 7.61 (d, J = 7.8 Hz, 2H, H-4)
ppm; 13C NMR (126 MHz, CDCl3)
δ
20.40 (d, J = 12.5 Hz, 2C, C-2), 27.47 (d, J = 1.0 Hz, 6C,
C-1), 65.42 (s, 1C, C-7), 126.26 (d, J = 1.0 Hz, 2C, C-5), 133.10 (d, J = 13.7 Hz, 1C, C-3), 134.39
(d, J = 4.2 Hz, 2C, C-4), 142.26 (s, 1C, C-6) ppm; 19F NMR (282 MHz, CDCl3) δ −188.90
(s, 1F, SiF) ppm; MS (FD) m/z calculated for C15H25FOSi [M]+: 268.1, found: 268.1; MS
(DART) m/z calculated for C15H29FNOSi [M + NH4]+: 286.1997, found: 286.1996.
(4-(Bromomethyl)phenyl)di-tert-butylfluorosilane (25): All steps were carried out
under an N2 atmosphere. PPh3 (1.25 g, 4.70 mmol, 1.1 equiv.) was added in portions over a
period of 15 min to a solution of 24 (1.15 g, 4.27 mmol, 1.0 equiv.) and tetra-bromomethane
(1.57 g, 4.70 mmol, 1.1 equiv.) in 20 mL dry CH2Cl2. After stirring for 12 h at room
temperature, the solvent was removed under reduced pressure, and the crude product was
purified by column filtration (n-hexane) to give compound 25 (1.34 g, 4.04 mmol, 95%) as
1
a colorless solid. H NMR (500 MHz, CDCl3)
δ 1.05 (s, 18H, H-1), 4.50 (s, 2H, H-7), 7.40
(d, J = 7.9 Hz, 2H, H-5), 7.58 (d, J = 7.9 Hz, 2H, H-4) ppm; 13C NMR (126 MHz, CDCl3)
δ

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20.41 (d, J = 12.4 Hz, 2C, C-2), 27.45 (d, J = 0.9 Hz, 6C, C-1), 33.48 (s, 1C, C-7), 128.29 (d,
J = 1.0 Hz, 2C, C-5), 134.31 (d, J = 13.7 Hz, 1C, C-3), 134.54 (d, J = 4.3 Hz, 2C, C-4), 139.05
(s, 1C, C-6) ppm; 19F NMR (282 MHz, CDCl3) δ −188.83 (s, 1F, SiF) ppm; MS (FD) m/z
calculated for C15H24BrFSi [M]+: 330.1, found: 330.1.
N-(4-(Di-tert-butylfluorosilyl)benzyl)-4,4-diethoxy-N,N-dimethylbutan-1-aminium
bromide (26): All steps were carried out under an N2 atmosphere. Moreover, 4,4-diethoxy-
N,N-dimethylbutan-1-amine (0.76 g, 0.9 mL, 4.04 mmol, 1.0 equiv.) was added to a solution
of 25 (1.34 g, 4.04 mmol, 1.0 equiv.) in 20 mL dry CH2Cl2. After stirring for 12 h at room
temperature, the solvent was removed under reduced pressure and the crude product was
purified by column chromatography (CH2Cl2/MeOH, 20/1
(1.96 g, 3.77 mmol, 93%) as a colorless foam. H NMR (500 MHz, CDCl3)
→
5/1) to give compound 26
1.02 (s, 18H,
1
δ
H-1), 1.16 (t, J = 7.0 Hz, 6H, CH3), 1.66–1.71 (m, 2H, H-9), 1.90–1.97 (m, 2H, H-10), 3.25 (s,
6H, NCH3), 3.43–3.51 (m, 4H, OCH2), 3.59–3.66 (m, 2H, H-8), 4.50 (t, J = 4.6 Hz, 1H, H-11),
4.84 (s, 2H, H-7), 7.60 (d, J = 7.9 Hz, 2H, H-5), 7.67 (d, J = 7.9 Hz, 2H, H-4) ppm; 13C NMR
(126 MHz, CDCl3) δ 15.44 (s, 2C, CH3), 17.58 (s, 1C, C-10), 20.30 (d, J = 12.2 Hz, 2C, C-2),
27.34 (s, 6C, C-1), 30.40 (s, 1C, C-9), 50.00 (s, 2C, NCH3), 62.62 (s, 2C, OCH2), 63.51 (s, 1C,
C-8), 67.65 (s, 1C, C-7), 102.03 (s, 1C, C-11), 128.53 (s, 1C, C-6), 132.24 (s, 2C, C-5), 134.84
(d, J = 4.3 Hz, 2C, C-4), 137.53 (d, J = 13.9 Hz, 2C, C-3) ppm; 19F NMR (282 MHz, CDCl3)
δ −188.58 (s, 1F, SiF) ppm; MS (FD) m/z calculated for C25H47FNO2Si [M]+: 440.3, found:
440.1; HR-ESI-MS m/z calculated for C25H47FNO2Si [M]+: 440.3355, found: 440.3356.
N-(4-(Di-tert-butylfluorosilyl)benzyl)-N,N-dimethyl-4-oxobutan-1-aminium bromide
(
27): 1 mL of a TFA/H2O (v/v, 95/5) solution was added to 26 (48.4 mg, 93.0 mmol,
1.0 equiv.). After stirring for 30 min at room temperature, saturated aqueous NaCl solu-
tion was added, and the reaction mixture was acidified with neat HCl. The solution was
extracted with EtOAc, the combined organic layers were dried over Na2SO4 and concen-
trated under reduced pressure. Purification by column chromatography (CH2Cl2/MeOH
1
100/1
(500 MHz, CD3OD)
3.05 (s, 6H, NCH3), 3.34–3.38 (m, 2H, H-8), 4.54 (s, 2H, H-7), 7.61 (d, J = 8.0 Hz, 2H, H-5),
7.80 (d, J = 8.0 Hz, 2H, H-4) ppm; 13C NMR (126 MHz, CD3OD)
18.95 (s, 1C, C-9), 21.02
→
5/1) gave compound 27 (37.7 mg, 84.4 mmol, 91%) as a colorless oil. H NMR
δ
1.08 (s, 18H, H-1), 1.63–1.69 (m, 2H, H-10), 1.96–2.04 (m, 2H, H-9),
δ
(d, J = 12.1 Hz, 2C, C-2), 27.68 (s, 6C, C-1), 34.23 (s, 1C, C-10), 50.51 (s, 2C, NCH3), 65.54
(s, 1C, C-8), 68.61 (s, 1C, C-7), 130.30 (s, 1C, C-6), 133.25 (d, J = 3.4 Hz, 2C, C-5), 135.93 (d,
J = 4.2 Hz, 2C, C-4), 138.06 (s, 1C, C-3) ppm; 19F NMR (282 MHz, CD3OD) δ −189.05 (s, 1F,
SiF) ppm; MS (FD) m/z calculated for C21H37FNOSi [M]+: 366.2, found: 366.1; HR-ESI-MS
m/z calculated for C21H37FNOSi [M]+: 366.2623, found: 366.2624.
3-Carboxy-N-(4-(di-tert-butylfluorosilyl)benzyl)-N,N-dimethylpropan-1-aminium bro-
mide (28): 1 M KMnO4 (503
µ
L, 503
mol, 5.0 equiv.) were added to a solution of 27 (37.3 mg, 83.5
L CH2Cl2. After the violet reaction mixture was stirred for
µ
mol, 6.0 equiv.) and 1.25 M NaH2PO4 solution
(335
µ
L, 419
µ
µmol, 1.0 equiv.)
t
in 1.2 mL BuOH and 55
µ
100 min at room temperature, it was diluted with H2O and quenched with saturated aque-
ous Na2SO3 solution. The precipitated MnO2 in clear solution was dissolved by adding
12 M HCl. The solution was extracted with EtOAc, and the combined organic layers were
dried over Na2SO4 and concentrated under reduced pressure. After purification by HPLC
(semipreparative, 20–60% MeCN + 0.1% TFA in 8 min, tR = 4.54 min), compound 28 was
isolated as a colorless solid (26.6 mg, 57.5
(500 MHz, CD3OD) 1.08 (s, 18H, H-1), 2.10–2.23 (m, 2H, H-9), 2.47 (t, J = 6.8 Hz, 2H, H-10),
3.07 (s, 6H, NCH3), 3.35–3.47 (m, 2H, H-8), 4.56 (s, 2H, H-7), 7.63 (d, J = 7.9 Hz, 2H, H-5),
7.80 (d, J = 7.9 Hz, 2H, H-4) ppm; 13C NMR (126 MHz, CD3OD)
19.19 (s, 1C, C-9), 21.02
µ
mol, yield: 69%, purity: >99%). 1H NMR
δ
δ
(d, J = 12.3 Hz, 2C, C-2), 27.50 (s, 6C, C-1), 30.87 (s, 1C, C-10), 50.51 (s, 2C, NCH3), 65.00 (s,
1C, C-8), 68.84 (s, 1C, C-7), 130.21 (s, 1C, C-6), 133.31 (s, 2C, C-5), 135.94 (d, J = 4.4 Hz, 2C,
C-4), 138.50 (d, J = 13.9 Hz, 1C, C-3), 175.32 (s, 1C, C-11) ppm; 19F NMR (282 MHz, CD3OD)
δ −189.00 (s, 1F, SiF) ppm; MS (FD) m/z calculated for C21H35FNO2Si [M
−
2H]+ 380.2,
found 380.2; MS (MALDI) m/z calculated for C21H37FNO2Si [M]+: 382.26, found: 382.35;
HR-ESI-MS m/z calculated for C21H37FNO2Si [M]+: 382.2572, found: 382.2575.

Pharmaceuticals 2021, 14, 547
22 of 30
(S)-4-((6-Amino-5-(2-(bis(3-aminopropyl)amino)acetamido)-6-oxohexyl)amino)-N-(4-
(di-tert-butylfluoro-silyl)benzyl)-N,N-dimethyl-4-oxobutan-1-aminium bromide (SiFAlin-
APG) (29): According to GP1, Fmoc-Lys(Mtt)-OH (87.5 mg, 140
((Fmoc-NH-propyl)2Gly-OHxKHSO4 (108 mg, 140 mol, 4.0 equiv.) were coupled one
after the other using DIPEA (23.8 L, 140 mol, 4.0 equiv.) and PyBOP (71.4 mg, 140 mol,
3.9 equiv.) in DMF for 60 min on a rink amide resin MBHA LL (106 mg, 35 mol, 1.0 equiv.,
µmol, 4.0 equiv.) and
µ
µ
µ
µ
µ
0.33 mmol/g). After deprotection of the Mtt-PG with TFA/CH2Cl2 (1/99, v/v) for 90 min,
28 was coupled with DIPEA and PyBOP on the resin. After Fmoc-deprotection with piperi-
dine/DMF (1/1, v/v) for 2x10 min, the product was cleaved from resin with TFA/TIS/H2O
(95/2.5/2.5, v/v/v) for 2 h. Peptide 29 was purified by HPLC (semipreparative, 0–60%
MeCN + 0.1% TFA in 8 min, tR = 4.18 min) and isolated as a colorless solid (11.5 mg,
15.1 µ δ 1.07 (s, 18H, H-1),
mol, yield: 43%, purity: >99%). 1H NMR (500 MHz, D2O)
1.36–1.42 (m, 2H, H-14), 1.49–1.56 (m, 2H, H-13), 1.72–1.85 (m, 2H, H-15), 2.10–2.20 (m,
6H, H-9, H-20), 2.34 (t, J = 7.0 Hz, 2H, H-10), 3.08 (s, 6H, NCH3), 3.10 (d, J = 7.7 Hz, 4H,
H-21), 3.16 (t, J = 7.2 Hz, 2H, H-12), 3.22–3.29 (m, 2H, H-8), 3.33–3.40 (m, 4H, H-19), 4.16 (d,
J = 5.1 Hz, 2H, H-18), 4.26–4.30 (t, J = 7.3 Hz, 1H, H-16), 4.54 (s, 2H, H-7), 7.58 (d, J = 7.9 Hz
,
2H, H-5), 7.83 (d, J = 7.9 Hz, 2H, H-4) ppm; 13C NMR (126 MHz, D2O) δ 18.48 (s, 1C, C-9),
19.21 (d, J = 12.2 Hz, 2C, C-2), 21.88 (s, 2C, C-20), 22.36 (s, 1C, C-14), 26.37 (s, 6C, C-1), 27.91
(s, 1C, C-13), 30.50 (s, 1C, C-15), 31.75 (s, 1C, C-10), 36.35 (s, 2C, C-21), 39.04 (s, 1C, C-12),
49.82 (s, 2C, NCH3), 52.28 (s, 2C, C-19), 53.99 (s, 1C, C-16), 54.26 (s, 1C, C-18), 62.58 (s, 1C,
C-8), 67.82 (s, 1C, C-7), 128.30 (s, 1C, C-6), 131.82 (s, 2C, C-5), 134.64 (d, J = 4.2 Hz, 2C, C-4),
136.81 (d, J = 14.2 Hz, 2, C-3), 165.43 (s, 1C, C-17), 173.84 (s, 1C, C-11), 176.11 (s, 1C, CO)
ppm; 19F NMR (282 MHz, D2O) δ −188.04 (s, 1F, SiF) ppm; MS (MALDI) m/z calculated for
C35H67FN7O3Si [M]+:680.51, found: 680.35; HR-ESI-MS m/z calculated for C35H68FN7O3Si
[M + H]2+:340.7563, found: 340.7558; m/z calculated for C35H67FN7O3Si [M]+:680.5053,
found: 680.5042.
3.2.4. Conjugation of 10–21 to the SiFAlin-Modified Framework 29 to Obtain the Target
HBPLs 1–6 via the Intermediates 30–35
General procedure for the synthesis of SiFAlin-APG-PEGn peptides (GP4): All steps
were carried out under an N2 atmosphere. A total of 0.5–1.0 equiv. of the respective NHS-
PEGn-peptide and 0.5–2.0 equiv. DIPEA were added to a solution of 1.0 equiv. 29 in dry
DMF. Subsequently, the reaction mixture was stirred for 15–150 min at room temperature,
while a reaction control was performed by HPLC (analytical, 0–50% MeCN + 0.1% TFA in
5 min). After the removal of the solvent under reduced pressure, the respective SiFAlin-
APG-PEGn-peptide was obtained after semipreparative HPLC purification. In addition
to the target products, small amounts of the dimer SiFAlin-APG-[PEGn-peptide]2 were
isolated (for analytical data, see Supplementary Materials Figures S85–S88).
SiFAlin-APG-PEG1-c(RGDfK) (30): According to GP4, 29 (9.3 mg, 12.1
µmol, 1.0 equiv.),
10 (5.4 mg, 6.1 mol, 0.5 equiv.) and DIPEA (1.0 L, 0.8 mg, 6.1 mol, 0.5 equiv.) were
µ
µ
µ
reacted in 16 mL dry DMF for 80 min. After purification by HPLC (semipreparative, 15–50%
MeCN + 0.1% TFA in 10 min, tR = 5.64 min), 30 was isolated as a colorless solid (11.2 mg,
7.3 µmol, yield: 60%, purity: >96%). MS (MALDI) m/z calculated for C70H118N16O14Si
[M]+: 1453.88, found: 1453.58; HR-ESI-MS m/z calculated for C70H120FN16O14Si [M + 2H]3+
:
485.2969, found: 485.2967; HR-ESI-MS m/z calculated for C70H119FN16O14Si [M + 2H]2+:
727.4417, found: 727.4410.
SiFAlin-APG-PEG3-c(RGDfK) (31): According to GP4, 29 (5.0 mg, 6.5 mol, 1.0 equiv.),
µ
11 (6.1 mg, 6.5 mol, 1.0 equiv.) and DIPEA (2.2 L, 1.7 mg, 13.0 µmol, 2.0 equiv.)
µ
µ
were reacted in 7 mL dry DMF for 45 min. After purification by HPLC (semiprepar-
ative, 15–50% MeCN + 0.1% TFA in 10 min, tR = 5.95 min), 31 was isolated as a col-
orless solid (7.1 mg, 4.4
µmol, yield: 67%, purity: >96%). MS (MALDI) m/z calcu-
lated for C74H126FN16O16Si [M]+ 1541.93, found: 1541.82; HR-ESI-MS m/z calculated
for C74H128FN16O16Si [M + 2H]3+: 514.6477, found: 514.6474; HR-ESI-MS m/z calculated
for C74H127FN16O16Si [M + 2H]2+: 771.4679, found: 771.4676; HR-ESI-MS m/z calculated
for C74H126FN16O16Si [M]+: 1541.9286, found: 1541.9278.

Pharmaceuticals 2021, 14, 547
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SiFAlin-APG-PEG5-c(RGDfK) (32): According to GP4, 29 (12.0 mg, 15.8
µmol, 1.0 equiv.),
12 (8.7 mg, 8.2 mol, 0.5 equiv.) and DIPEA (2.7 L, 2.0 mg, 15.8 µmol, 1.0 equiv.) were
µ
µ
reacted in 14 mL dry DMF for 150 min. After purification by HPLC (semipreparative, 0–50%
MeCN + 0.1% TFA in 8 min, tR = 7.10 min), 32 was isolated as a colorless solid (10.6 mg,
6.5 µmol, yield: 41%, purity: >99%). MS (MALDI) m/z calculated for C78H134FN16O18Si
[M]+: 1629.98, found: 1629.16; HR-ESI-MS m/z for C78H137FN16O18Si [M + 3H]4+: 408.2507,
found: 408.2506; HR-ESI-MS m/z calculated for C78H136FN16O18Si [M + 2H]3+: 543.9985,
found: 543.9983; HR-ESI-MS m/z calculated for C78H135FN16O18Si [M + H]2+: 815.4941,
found: 815.4933.
SiFAlin-APG-PEG8-c(RGDfK) (33): According to GP4, 29 (7.3 mg, 9.6
µmol, 1.0 equiv.),
13 (5.9 mg, 4.9 mol, 0.5 equiv.) and DIPEA (1.6 L, 1.2 mg, 9.6 mol, 1.0 equiv.) were
µ
µ
µ
reacted in 10 mL dry DMF for 130 min. After purification by HPLC (semipreparative, 0–50%
MeCN + 0.1% TFA in 8 min, tR = 7.27 min), 33 was isolated as a colorless solid (8.7 mg,
4.7 µmol, yield: 49%, purity: >99%). MS (MALDI) m/z calculated for C84H146FN16O21Si
[M]+: 1762.06, found: 1762.21; HR-ESI-MS m/z calculated for C84H148FN16O21Si [M + 2H]3+
:
588.3592, found: 588.3585; HR-ESI-MS m/z calculated for C84H147FN16O21Si [M + H]2+:
881.5335, found: 881.5559.
General procedure for the synthesis of SiFAlin-APG-DIG- and SiFAlin-APG-Ox-EGEGE
peptides (GP5): All steps were carried out under an N2 atmosphere. Then, 0.5 equiv. PyBOP
and 0.5–2.0 equiv. DIPEA was added to a solution of 0.5 equiv. HO-DIG/Ox-EGEGE-
peptide in dry DMF and stirred for 15 min. Subsequently, 1.0 equiv. 29 was added and the
reaction mixture was stirred for 1–4 h at room temperature, while the reaction control was
performed by HPLC (analytical, 0–50% MeCN + 0.1% TFA in 5 min). After the removal of
the solvent under reduced pressure, the respective SiFAlin-APG-DIG/Ox-EGEGE-peptide
was obtained after semipreparative HPLC purification. In addition to the target product,
small amounts of the dimer SiFAlin-APG-[DIG/Ox-EGEGE-peptide]2 were isolated (for
analytical data, see Supplementary Materials Figures S89–S90).
SiFAlin-APG-DIG-c(RGDfK) (34): According to GP5, 29 (3.9 mg, 5.1
14 (1.9 mg, 2.6 mol, 0.5 equiv.), PyBOP (1.4 mg, 2.6 mol, 0.5 equiv.) and DIPEA (0.4
0.3 mg, 2.6 mol, 0.5 equiv.) were reacted in 2.5 mL dry DMF for 1 h. After purification
by HPLC (semipreparative, 5–40% MeCN + 0.1% TFA in 10 min, tR = 3.09 min), 34 was
µmol, 1.0 equiv.),
µ
µ
µL,
µ
isolated as a colorless solid (3.4 mg, 2.3 µmol, yield: 46%, purity: >97%). MS (MALDI) m/z
calculated for C66H110N16O13Si [M]+: 1381.82, found: 1381.45; HR-ESI-MS m/z calculated
for C66H112FN16O13Si [M + 2H]3+: 461.2777, found: 461.2772; HR-ESI-MS m/z calculated
for C66H111FN16O13Si [M + H]2+: 691.4129, found: 691.4125.
SiFAlin-APG-Ox-EGEGE-c(RGDfK) (35): According to GP5, 29 (1.3 mg, 1.7
1.0 equiv.), 15 (1.0 mg, 0.9 mol, 0.5 equiv.), PyBOP (0.4 mg, 0.9 mol, 0.5 equiv.) and
DIPEA (0.5 L, 0.4 mg, 3.4 mol, 2.0 equiv.) were reacted in 1.5 mL dry DMF for 4 h. After
purification by HPLC (semipreparative, 0–50% MeCN + 0.1% TFA in 8 min, tR = 6.56 min),
µmol,
µ
µ
µ
µ
35 was isolated as a colorless solid (1.9 mg, 1.0 µmol, yield: 58%, purity: >98%). MS
(MALDI) m/z calculated for C83H134FN21O23Si [M+H]+: 1839.97, found: 1839.06; HR-ESI-
MS m/z calculated for C37H69FN8O5Si [M-C46H64N13O18]+: 752.5139, found: 752.5257;
HR-ESI-MS m/z calculated for C44H81FN10O9Si [M-C44H52N11O14]2+: 940.5936, found:
940.4168.
General procedure for the synthesis of heterobivalent SiFAlin-modified HBPLs
1–
4
(GP6): All steps were carried out under an N2 atmosphere. 3.0–6.0 equiv. DIPEA
was added within a period of 0.5–6 h to a solution of 1.0 equiv. SiFAlin-APG-PEGn-
c(RGDfK) and 1.0–2.0 equiv. NHS-PEGn-GG-Nle-c(DHfRWK) in dry DMF. Subsequently,
the reaction mixture was stirred for 60 min at room temperature, while the reaction control
was performed by HPLC (analytical, 0–50% MeCN + 0.1% TFA in 5 min). After the removal
of the solvent under reduced pressure, the respective SiFAlin-APG-PEGn-c(RGDfK)/GG-
Nle-c(DHfRWK)-heterodimer was obtained after semipreparative HPLC purification.
SiFAlin-APG-PEG1-c(RGDfK)/GG-Nle-c(DHfRWK) (
1): According to GP6, 30 (5.1 mg,
3.3
µ
mol, 1.0 equiv.), 16 (6.1 mg, 4.4 mol, 1.3 equiv.) and DIPEA (6
µ
×
0.6 L, 2.6 mg,
µ

Pharmaceuticals 2021, 14, 547
24 of 30
19.8
µmol, 6.0 equiv.) were reacted in 9 mL dry DMF for 7 h. After purification by HPLC
(semipreparative, 15–50% MeCN + 0.1% TFA in 10 min, tR = 6.91 min), HBPL 1 was isolated
as a colorless solid (7.0 mg, 2.5 µ
mol, yield: 77%, purity: >99%). 19F NMR (282 MHz, D2O)
δ −176.21 (s, 1F, SiF) ppm; MS (MALDI) m/z calculated for C130H202FN33O28Si [M + H]+:
2720.51, found: 2720.11; HR-ESI-MS m/z calculated for C130H205FN33O28Si [M + 4H]5+:
544.9078, found: 544.9072; HR-ESI-MS m/z calculated for C130H204FN33O28Si [M + 3H]4+
:
680.8830, found: 680.8824.
SiFAlin-APG-PEG3-c(RGDfK)/GG-Nle-c(DHfRWK) (
mol, 1.0 equiv.), 17 (6.9 mg, 4.7 mol, 1.8 equiv.) and DIPEA (5x0.5
mol, 5.0 equiv.) were reacted in 11 mL dry DMF for 7 h. After purification by HPLC
(semipreparative, 15–50% MeCN + 0.1% TFA in 10 min, tR = 7.10 min), HBPL was isolated
2): According to GP6, 31 (4.3 mg,
2.7
µ
µ
µL, 1.9 mg,
14.7
µ
2
as a colorless solid (5.3 mg, 1.8 µ
mol, yield: 66%, purity: >99%). 19F NMR (282 MHz, D2O)
δ −176.21 (s, 1F, SiF) ppm; MS (MALDI) m/z calculated for C138H218FN33O32Si [M + H]+:
2896.62, found: 2896.11; HR-ESI-MS m/z calculated for C138H220FN33O32Si [M + 3H]4+:
724.9092, found: 724.9101; HR-ESI-MS m/z calculated for C138H219FN33O32Si [M + 2H]3+
:
966.2098, found: 966.2106; HR-ESI-MS m/z calculated for C138H218FN33O32Si [M + H]2+:
1448.8111, found: 1448.8124.
SiFAlin-APG-PEG5-c(RGDfK)/GG-Nle-c(DHfRWK) (
mol, 1.0 equiv.), 18 (4.6 mg, 3.0 mol, 1.2 equiv.) and DIPEA (2
mol, 4.0 equiv.) were reacted in 5 mL dry DMF for 3 h. After purification by HPLC
(semipreparative, 10–50% MeCN + 0.1% TFA in 8 min, tR = 7.26 min), HBPL was isolated
3): According to GP6, 32 (4.2 mg,
2.5
19.5
µ
µ
×
0.8 L, 3.3 mg,
µ
µ
3
as a colorless solid (3.8 mg, 1.2 µ
mol, yield: 49%, purity: >99%). 19F NMR (282 MHz,
D2O) δ −176.21 (s, 1F, SiF) ppm; MS (MALDI) m/z calculated for C146H233FN33O36Si [M]+:
3071.72, found: 3071.49; HR-ESI-MS m/z calculated for C146H237FN33O36Si [M + 4H]5+:
615.3498, found: 615.3494; HR-ESI-MS m/z calculated for C146H236FN33O36Si [M + 3H]4+
:
768.9354, found: 768.9353.
SiFAlin-APG-PEG8-c(RGDfK)/GG-Nle-c(DHfRWK) (
mol, 1.0 equiv.), 19 (11.2 mg, 6.6 mol, 1.9 equiv.) and DIPEA (6
mol, 6.0 equiv.) were reacted in 6 mL dry DMF for 4 h. After purification by HPLC
(semipreparative, 10–50% MeCN + 0.1% TFA in 8 min, tR = 7.55 min), HBPL was isolated
4): According to GP6, 33 (6.4 mg,
3.5
20.9
µ
µ
×
0.6 L, 2.7 mg,
µ
µ
4
as a colorless solid (3.0 mg, 2.0 µ
mol, yield: 58%, purity: >99%). 19F NMR (282 MHz,
D2O) δ −176.20 (s, 1F, SiF) ppm; MS (MALDI) m/z calculated for C158H258FN33O42Si [M]+
3336.88, found: 3336.07; HR-ESI-MS m/z calculated for C158H261FN33O42Si [M + 4H]5+:
668.1812, found: 668.1820; HR-ESI-MS m/z calculated for C158H260FN33O42Si [M + 3H]4+
:
835.2256, found: 835.2269.
General procedure for the synthesis of heterobivalent SiFAlin-modified HBPLs
5 and
6
(GP7): All steps were carried out under an N2 atmosphere. 1.3–3.0 equiv. PyBOP and
1.3–12.0 equiv. DIPEA were added to a solution of 1.3–3.0 equiv. HO-DIG-GG-Nle-
c(DHfRWK) or Ox-EGEGE-GG-Nle-c(DHfRWK) in dry DMF and stirred for 60 min. Subse-
quently, 1.0 equiv. SiFAlin-APG-DIG-c(RGDfK) (34) or SiFAlin-APG-Ox-EGEGE-c(RGDfK)
(35) were added, and the reaction mixture was reacted for 2–5 h at room temperature, while
the reaction control was performed by HPLC (analytical, 0–50% MeCN + 0.1% TFA in
5 min). After the removal of the solvent under reduced pressure, the SiFAlin-modified
HBPLs 5 and 6 were obtained after semipreparative HPLC purification.
SiFAlin-APG-DIG-c(RGDfK)/GG-Nle-c(DHfRWK) (
mol, 1.0 equiv.), 20 (2.0 mg, 1.6 mol, 1.3 equiv.), PyBOP (0.8 mg, 1.6
and DIPEA (0.3 L, 0.2 mg, 1.6 mol, 1.3 equiv.) were reacted in 2 mL dry DMF for
5 h. After purification by HPLC (semipreparative, 10–40% MeCN + 0.1% TFA in 11 min,
5): According to GP7, 34 (1.8 mg,
1.2
µ
µ
µmol, 1.3 equiv.)
µ
µ
tR = 10.18 min), HBPL
5 was isolated as a colorless solid (0.7 mg, 0.3 µmol, yield: 24%,
purity: >99%). 19F NMR (282 MHz, D2O) δ −176.21 (s, 1F, SiF) ppm; MS (MALDI) m/z
calculated for C122H185FN33O26Si [M]+: 2575.39, found: 2575.82; HR-ESI-MS m/z calculated
for C122H189FN33O26Si [M + 4H]5+: 516.0848, found: 516.0843; HR-ESI-MS m/z calculated
for C122H186FN33O26Si [M + 3H]4+: 644.8542, found: 644.8542.

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SiFAlin-APG-Ox-EGEGE-c(RGDfK)/GG-Nle-c(DHfRWK) (
(1.3 mg, 0.7 mol, 1.0 equiv.), 21 (3.4 mg, 2.0
3.0 equiv.) and DIPEA (4 0.3 L, 1.1 mg, 8.4
2.5 mL dry DMF for 3.5 h. After purification by HPLC (semipreparative, 0–50% MeCN +
0.1% TFA in 10 min, tR = 8.54 min; 20–40% MeCN + 0.1% TFA in 10 min, tR = 7.24 min),
6
): According to GP7, 35
µ
µ
mol, 3.0 equiv.), PyBOP (1.1 mg, 2.0
µmol,
×
µ
µ
mol, 12.0 equiv.) were reacted in
HBPL
6 was isolated as a colorless solid (0.5 mg, 0.15 µmol, yield: 21%, purity: >99%).
19F NMR (282 MHz, D2O) δ −176.19 (s, 1F, SiF) ppm; MS (MALDI) m/z calculated
for C156H231FN43O46Si [M]+: 3489.68, found: 3489.25; HR-ESI-MS m/z calculated for
C129H190FN35O39Si [M-C27H41N8O7]5+: 580.2744, found: 580.1284; HR-ESI-MS m/z calcu-
lated for C129H189FN35O39Si [M-C27H42N8O7]4+: 724.8403, found: 724.9088.
3.3. 18F-Radiolabeling, Evaluation of logD(7.4), Stability and Binding Affinities for HBPLs 1–6
3.3.1. 18F-Radiolabeling of the HBPLs 1–6
Aqueous [18F]fluoride solution (0.3–2.0 mL, 0.8–2.2 GBq) was flushed through an
anion exchange resin (Sep-Pak Accell Plus QMA Carbonate Plus SPE Light cartridge,
46 mg, Waters; Eschborn, Germany) which was preconditioned with 10 mL Tracepur
H2O. After drying with 20 mL air, removal of the remaining water with 5 mL dry MeCN
and repeated drying with 20 mL air, the radioactivity was eluted from the cartridge with
KOH (100
obtained dry [18F]KF-K222-hydroxide complex, a solution of oxalic acid in dry MeCN (25
25 mol, 1 M) was added first and then a solution of the respective HBPL precursor
in dry DMSO (25 L, 25 nmol, 1 mM) was added afterward. After reaction for 10 min at
µmol) and Kryptofix222 (K222, 41.4 mg, 110
µmol) in 500
µL dry MeCN. To the
µL,
µ
1–6
µ
room temperature, the mixture was analyzed by analytical radio-HPLC (0–100% MeCN +
0.1% TFA in 5 min). For purification, the reaction mixture was diluted with 9 mL 0.1 M
HEPES solution (pH = 2) and passed through a C18 cartridge (Sep-Pak C18 Plus SPE
Light cartridge, 130 mg, Waters; Eschborn, Germany), which was preconditioned with
10 mL EtOH and 10 mL Tracepur H2O. The cartridge was washed with 10 mL 0.05 M
phosphate buffer (pH = 7.4) and dried with 10 mL air. Finally, the radiotracer was eluted
with 500
µL EtOH/Tracepur H2O (9/1, v/v), and the RCPs of the radiolabeled products
were determined by analytical radio-HPLC (0–100% MeCN + 0.1% TFA in 5 min). The
radiotracers were obtained in RCYs of 27–50%, RCPs of 95–98% and Am of 17–51 GBq/
µmol
after an overall synthesis time of 25 min.
3.3.2. LogD(7.4) Determination of [18F]1–[18F]6
The radiotracer solutions were first diluted with 0.9% NaCl solution to give a final
EtOH concentration of <10%. A total of 5 MBq of the respective radiotracer in solution was
added to 1.6 mL of a mixture of n-octanol and 0.05 M phosphate buffer (pH = 7.4) (v/v, 1/1),
and the solution was vigorously shaken for 5 min. Subsequently, the phases were separated
by centrifugation at 13.4 rpm for 2 min, and 100 µL of organic and aqueous phases were col-
lected. The activity of each sample was measured using a gamma counter. The lipophilicity
of each compound was determined in triplicate in three independent experiments.
3.3.3. Determination of the Stability of [18F]1–[18F]6 in Human Serum
The radiotracer solutions were first diluted with 0.9% NaCl solution to give a final
EtOH concentration of <10%. Then, 6
human serum and incubated at 37◦C for 120 min. At defined time points (5, 15, 30, 60,
90 and 120 min), 125 L EtOH was added to one of the mixtures and the precipitation of
×
25
µL radiotracer solution was added to 6 × 100 µL
µ
serum proteins was supported by ice-cooling for 2 min. After centrifugation at 13.4 rpm
for 2 min, the supernatant was analyzed by analytical radio-HPLC (0–100% MeCN +
0.1% TFA in 5 min). The experiment was performed for each compound trice by three
independent experiments.

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3.3.4. Cell Culture
All cell lines were cultivated at 37 ◦C in a humidified incubator at 5% CO2. B16F10
cells were cultured in DMEM and the U87MG cells in EMEM, each medium supplemented
with 10% fetal bovine serum and 1% penicillin/streptomycin. The medium was exchanged
every 2–3 days and cells were split at 70–90% confluence using 0.25/0.02% Trypsin/EDTA
(w/v) in PBS. A medium change was performed 24 h before an experiment. For in vivo
experiments, the cell resuspension after centrifugation was performed in PBS. The cells
were homogenized in PBS to give a concentration of 5
U87MG cells/mL and were aliquoted and stored on ice upon use.
×
106 B16F10 cells/mL and 25
×
106
3.3.5. Competitive Displacement Studies on B16F10 and U87MG Cells
To determine the binding affinity to the respective receptor, competitive displacement
studies were performed on MC1R-expressing B16F10 and on integrin α β3-expressing
v
U87MG cells. Each compound was evaluated at least three times, each experiment being
performed in triplicate. As radioligands, [125I]I-NDP (81.4 GBq/ mol) and [125I]I-echistatin
µ
(81.4 GBq/
plate was incubated with 200
1 h. After preparing the binding buffers (DMEM with 25 mM HEPES, 0.3 mM 1,10-
phenanthroline and 0.2% BSA; EMEM with 20 mM Tris HCl, 150 mM NaCl, 2 mM CaCl2,
1 mM MgCl2, 1 mM MnCl2 and 0.1% BSA), the dilution series of the HBPLs 0.04–4 µM
and 2–400 M) and the reference agents 36 39 (0.01–1 M and 0.04–400 M) were prepared
µmol) were used as competitors. First, the Millipore MultiScreen 96-well filter
◦
L/well of a BSA/PBS (1/99, w/v) solution at 25 C for
µ
·
1–6 (
µ
–
µ
µ
in the respective binding buffer. The solution of the respective radioligand was prepared
by adding 55–75 kBq of the respective 125I-labeled competitor to 3.5 mL of binding buffer.
The respective cells were harvested and re-suspended in the binding buffer to give a cell
concentration of 2
Multiscreen vacuum manifold, 50
in each well. Subsequently, 25
L of the 125I-labeled competitor solution (0.018 kBq/
and 25 L of the respective compound to be tested were added. The compound to be tested
×
106/mL. After the BSA solution was filtered using the Millipore
µ
L of a cell suspension containing 105 cells were seeded
L)
µ
µ
µ
was added in eleven increasing concentrations, whereas the 12th well contained no test
compound to ensure the 100% binding of the 125I-labeled competitor. After incubation of the
plate for another hour at 25◦C, the solution was filtrated using the Millipore Multiscreen
vacuum manifold, and the cells were washed three times with cold PBS (1
2 × 100 µL). Using a Millipore MultiScreen disposable punch and a Millipore MultiScreen
punch kit, the filters of the well plate were collected in -counter tubes separately and
measured by -counting. The determination of the half-maximal inhibitory concentration
×
200 µL,
γ
γ
(IC50) values was performed by fitting the obtained data via nonlinear regression using
GraphPad Prism (v6.05).
3.4. In Vivo PET/CT Imaging and Ex Vivo Biodistribution of [18F]2 and [18F]4
Each male nude mouse (six weeks old) was injected subcutaneously with 5
×
105
B16F10 cells into the right flank and 2.0–2.5
×
106 U87MG cells into the left flank. The health
status and tumor growth of the mice were monitored regularly until the animals could be
examined after 15–21 days, depending on the tumor size. For the in vivo experiments, the
18F-radiolabeled compound was diluted in 0.9% saline to give a final EtOH concentration of
<10%. Each mouse was injected with 4.15
via the lateral tail vein under isoflurane anesthesia. For the blocking studies, the respective
±
2.28 MBq of [18F]
2
or 3.95
±
2.06 MBq of [18F]
4
radiotracer was coinjected with 20 µg NDP, 200 µg c(RGDyK) or both substances (double
blocking). Each 18F-labeled compound was studied with or without blocking in at least
three mice. Mice were measured under isoflurane anesthesia in a small PET/SPECT/CT
animal imaging system. First, a dynamic PET scan was performed over 90 min and the
scan was framed in 29 timeframes (10
×
1 min, 10
×
2 min, 6
×
5 min, 3
×
10 min). Images
were reconstructed using 12 iterations, a maximum likelihood expectation maximization
(MLEM) algorithm including corrections for scattered radiation and decay and a voxel
size of 0.5 mm. All PET scans were immediately followed by CT acquisition at a voltage

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of 45 kV and a current of 400
µA. The images were reconstructed using the filtered back
projection (FBP) algorithm with a voxel size of 250 µm. The analysis of the data, including
the generation of TACs of the kidneys, liver and tumors and the MIPs, was performed
via PMOD (v3.8). For ex vivo biodistribution, the mice were sacrificed directly after the
PET/CT scan. Organs (blood, spleen, liver, kidneys, pancreas, lungs, heart, brain, bone,
muscle, tail, tumors, stomach, colon and small intestine) were collected, weighed and their
radioactivity measured in a γ-counter. The percentage injected dose per gram (% ID/g) of
each tissue was calculated from the determined values, organ weights, reference values
and injected activity.
4. Conclusions
In the present study, different heterobivalent bispecific 18F-labeled agents for the sensi-
tive and receptor-specific imaging of malignant melanoma using PET/CT were developed.
After establishing the chemical and radio synthesis of the agents, the obtained tracers were
studied systematically in vitro, regarding their hydrophilicity, stability in human serum
and receptor-binding potential of both target receptor types, integrin α β and MC1R.
v
3
It was shown that the distance between the peptide binders strongly influences receptor
affinities and that the introduction of negatively charged linkers negatively affects the
receptor-binding potential of both receptor types. In vivo, the most potent tracers were
studied in direct comparison to PET/CT imaging and ex vivo biodistribution studies. These
experiments showed higher absolute tumor uptakes and tumor-to-background ratios and
thus more favorable in vivo pharmacokinetics for the agent, demonstrating slightly lower
affinities but comprising longer PEG linkers, though not for that agent exhibiting the high-
est receptor affinities. Heterodimer [18F]
tumor visualization ability and impressively illustrated the suitability of the underlying
4 thus demonstrated an excellent receptor-specific
concept to develop heterobivalent integrin α β3- and MC1R-bispecific radioligands for the
v
sensitive and specific imaging of malignant melanoma by PET/CT.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10
.3390/ph14060547/s1; analytical data, binding curves of c(RGDfK) and GG-Nle-c(DHfRWK) and ex
vivo biodistribution data of [18F]2 and [18F]4.
Author Contributions: Conceptualization: C.W. and B.W.; investigation: X.C., R.H. and V.v.K.;
writing—original draft preparation: X.C., C.W. and B.W.; writing—review and editing: R.S. and G.F.;
funding acquisition: C.W. and B.W. All authors have read and agreed to the published version of
the manuscript.
Funding: This research was funded by Wilhelm Sander-Stiftung under the funding code 2017.052.1.
Further, this research project is part of the Forschungscampus M2OLIE and was funded by the German
Federal Ministry of Education and Research (BMBF) within the framework “Research Campus —
public-private partnership for Innovation” under the funding code 13GW0388A. We acknowledge
financial support by the Open Access Publishing Fund of Ruprecht-Karls-Universität Heidelberg.
Institutional Review Board Statement: All animal experiments were performed in compliance with
the German animal protection laws and protocols of the local committee (approval number of the
animal testing application: 35-9185.81/G-271/19), approved on 18 November 2019.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available on request from the
corresponding author.
Acknowledgments: We thank T. Timmermann (RKU, Heidelberg) for performing NMR experiments
and W. Spahl (LMU, München) and J. Gross (RKU, Heidelberg) for performing the mass analyses.
Conflicts of Interest: The authors declare no conflict of interest.

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