Microfluidic preparation of89 zr-radiolabelled proteins by flow photochemistry

Article abstract of DOI:10.3390/molecules26030764

⁸⁹ Zr-radiolabelled proteins functionalised with desferrioxamine B are a cornerstone of diagnostic positron emission tomography. In the clinical setting,⁸⁹ Zr-labelled proteins are produced manually. Here, we explore the potential of

Full text of DOI:10.3390/molecules26030764

molecules
Communication
89
Microfluidic Preparation of Zr-Radiolabelled Proteins by
Flow Photochemistry
Daniel F. Earley 1 , Amaury Guillou 1 , Dion van der Born , Alex J. Poot 3 and Jason P. Holland 1,*
2
1
Department of Chemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland;
daniel.earley@chem.uzh.ch (D.F.E.); amaury.guillou@chem.uzh.ch (A.G.)
FutureChemistry Agro Business Park 10, 6708 PW Wageningen, The Netherlands;
2
d.vanderborn@futurechemistry.com
Department of Radiology and Nuclear Medicine, UMC Utrecht, Heidelberglaan 100, 3584 CX Utrecht,
3
The Netherlands; a.j.poot@umcutrecht.nl
*
Correspondence: jason.holland@chem.uzh.ch
Abstract: 89Zr-radiolabelled proteins functionalised with desferrioxamine B are a cornerstone of
diagnostic positron emission tomography. In the clinical setting, 89Zr-labelled proteins are produced
manually. Here, we explore the potential of using a microfluidic photochemical flow reactor to
prepare 89Zr-radiolabelled proteins. The light-induced functionalisation and 89Zr-radiolabelling of
8
9
human serum albumin ([ Zr]ZrDFO-PEG -Et-azepin-HSA) was achieved by flow photochemistry
3
with a decay-corrected radiochemical yield (RCY) of 31.2
±
1.3% (n = 3) and radiochemical purity
>
90%. In comparison, a manual batch photoreactor synthesis produced the same radiotracer in a
decay-corrected RCY of 59.6
±
3.6% (n = 3) with an equivalent RCP > 90%. The results indicate that
photoradiolabelling in flow is a feasible platform for the automated production of protein-based 89Zr-
radiotracers, but further refinement of the apparatus and optimisation of the method are required
before the flow process is competitive with manual reactions.
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ꢁꢂꢃꢄꢅꢆ
Citation: Earley, D.F.; Guillou, A.;
van der Born, D.; Poot, A.J.; Holland,
J.P. Microfluidic Preparation of
Keywords: flow chemistry; photochemistry; radiochemistry; protein conjugation
89Zr-Radiolabelled Proteins by Flow
Photochemistry. Molecules 2021, 26,
7
64. https://doi.org/10.3390/
1. Introduction
molecules26030764
Due to their unique structures and functions, high affinity and target specificity,
protein-based drug-conjugates have fast become essential tools in medical imaging. Protein-
derived immunoglobulin fragments and monoclonal antibodies (mAbs) are frequently
Academic Editor: Luca D. D’Andrea
Received: 18 January 2021
utilised for the development of both therapeutic and diagnostic agents [
1]. For example,
Accepted: 29 January 2021
Published: 2 February 2021
in the clinic, mAbs functionalised with metal-binding chelates such as desferrioxamine B
89
(
DFO) and radiolabelled with zirconium-89 ( Zr-mAbs) provide sophisticated, rationally
designed, radiopharmaceuticals for use in positron emission tomography (PET) [2].
The preparation of radiolabelled protein-conjugates requires the formation of a new
covalent bond between the protein and the ligand, which must be achieved without dis-
rupting the structural and biological properties of the target protein. Standard conjugation
methods require the use of pre-installed reactive groups such as activated esters or benzyl-
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published maps and institutional affil-
iations.
isothiocyanates, prior chemical modification and/or pre-activation of the protein [
rely on thermochemically-driven reactions (at room temperature to 37 C) with amino-acid
3
], and
◦
side chains or glycans [4–7]. These processes often include time consuming multi-step
syntheses that are difficult to automate, and can be incompatible with protein formulation
buffers which mandate pre-purification of the protein vector.
Copyright: © 2021 by the authors.
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/
In the last decade, flow chemistry has been combined with radiochemistry to prepare
a variety of radiotracers with different radionuclides, including 1 C [
1
8,9
] and
18
F [10–13].
Small reaction volumes, efficient mixing and reproducible control over all essential reac-
tion parameters are some of the features that make microfluidic reactions attractive for
radiochemistry. In 2016, Wright et al. [14] demonstrated the microfluidic radiolabelling
4
.0/).
Molecules 2021, 26, 764. https://doi.org/10.3390/molecules26030764
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 764
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of 89Zr-mAbs. In 2019, Poot et al. [15] reported proof-of-concept studies demonstrating
89
that automated radiolabelling of Zr-mAbs in a batch reactor can also be combined with
automated purification. However, both approaches relied on the use of pre-functionalised
proteins bearing the DFO chelate. In contrast, we provide a proof-of-concept showing
8
9
that flow-based photochemistry can be combined with radiochemistry to produce Zr-
radiolabelled proteins direct from the unfunctionalised (native) protein source.
Light-induced functionalisation of proteins with metal-binding chelates bearing pho-
tochemically active groups presents an alternative to traditional protein conjugation
chemistries [
4,16]. The aryl azide (ArN ) group absorbs various wavelengths of light
3
(302–400 nm) to generate highly reactive nitrenes [17], which can be utilised to form new
covalent bonds to a target protein [16]. This light-induced process is compatible with bio-
logically relevant media, and occurs extremely rapidly (lifetimes of reactive intermediates
are in the nanosecond to microsecond range) compared with traditional bioconjugation
methods that react directly with native functional groups on the protein [18]. The proposed
reaction mechanism favours the formation of a seven-membered azepine ring species
kinetically which can then react rapidly with nucleophiles like primary amines to form a
new covalent bond [16].
We recently reported the photoradiosynthesis of several viable 68Ga3+ and 89Zr4+
protein-conjugate PET radiotracers, from photoactivatable metal-binding chelates function-
alised with an ArN group [19–22]. Importantly, this photochemical conjugation process
3
occurs at wavelengths that do not disrupt protein structure or function. Photoradiolabelling
to produce 89Zr-mAbs is compatible with several different antibody formulations (with
mixtures containing large quantities of histidine; ascorbic acid; sugars such as α,α-trehalose;
and surfactants such as polysorbate 20) which allows for direct protein-conjugation without
the need for pre-purification of the protein before performing the bioconjugation step.
To enhance the water-solubility of the photoactivatable DFO derivatives, we recently
introduced two new compounds (including DFO-PEG -Et-ArN
1; Scheme 1) that link
3
3
the metal binding chelate to the ArN group via a polar tris-polyethylene glycol (PEG )
3
3
linker [22]. Compound
diosynthesis of Zr-radiolabelled protein conjugates via photochemistry in flow (Figure 1).
1
is an excellent ligand for exploring the potential for automated ra-
89
Scheme 1. Synthesis of DFO-PEG -Et-ArN3 (1). Reagents and conditions: (a) imidazole-1-sulphonyl azide HCl,
3
CuSO4
·
5H O, K CO , MeOH, rt, 18 h; (b) N-Boc-4,7,10-trioxa-1,13-tridecanediamine (3), HATU, DIPEA, anhyd. DMF, rt,
2 2 3
2
4 h; (c) TFA, CH Cl , rt, 1 h; (d) succinic anhydride, anhyd. DMF, rt, 48 h; (e) DFO-mesylate, HATU, DIPEA, anhyd. DMF,
2
2
rt, 48 h.

Molecules 2021, 26, 764
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Figure 1. Illustration depicting the general concept of the light-activated preparation of 89Zr-radiolabelled protein conjugates
in a microfluidic photochemistry device.
Here, we present the synthesis of a radiolabelled protein conjugate prepared by light-
induced photoconjugation using a microfluidic photochemical reactor in continuous flow.
89
89
[
Zr]ZrDFO-PEG -Et-azepin-HSA was isolated after photoactivation of [ Zr]ZrDFO-
3
PEG -Et-ArN and conjugation to human serum albumin (HSA) in a custom microfluidic
3
3
photochemical apparatus. The results provide confidence that an automated procedure
can be developed for the radiosynthesis of 89Zr-labelled proteins for future applications in
PET starting from the native unfunctionalised protein.
2. Results and Discussion
2
.1. Synthesis of DFO-PEG -Et-ArN , (1)
3
3
The functionalised DFO-PEG -Et-ArN metal binding chelate (compound 1; Scheme 1)
3
3
was synthesised with an overall yield of 24% [22]. Briefly, treatment of 3-(4-aminophenyl)
propanoic acid with imidazole-1-sulphonyl azide HCl [23], potassium carbonate and a
catalytic amount of copper(II) sulphate pentahydrate furnished the corresponding para-
substituted aryl azide compound
2
with a 76% yield. In parallel, the preparation of
was achieved by treating 4,7,10-trioxa-1,13-
tridecanediamine with di-tert-butyl dicarbonate [24]. Then, the amide coupling of the car-
boxylic acid functionalised aryl azide derivative with the mono-protected amine was ac-
the mono-protected PEG linker derivative
3
3
2
3
complished by the treatment of O-(7-azabenzotriazol-1-yl)-N,N,N”,N”-tetramethyluronium
hexafluorophosphate (HATU) and N,N-diisopropylethylamine (DIPEA) in anhydrous
DMF. Purification by flash column chromatography afforded compound
Deprotection of compound with trifluoroacetic acid (TFA) and subsequent treatment
with succinic anhydride furnished compound (via compound ; 66% yield over two
steps). Finally, HATU-mediated coupling of compound with DFO mesylate afforded
)—58% yield. Full experimental details and
4 (82% yield).
4
6
5
6
the target compound DFO-PEG -Et-ArN (
characterisation data, including H and C{ H} NMR spectroscopy and high-resolution
1
3
3
1
13
1
electrospray ionisation mass spectrometry for compound
1 and associated intermediates,
are provided in the Supplementary Materials (Figures S1–S17).
2.2. Chip Design and Instrumentation
Flow photoradiochemistry was performed by using a FutureChemistry FlowStart
B-222 photochemistry module (Figure 2A) equipped with a twin light-emitting diode (LED)
light source (365 nM; LedEngin, Inc.) connected in series (Figure 2B). Light intensity was
set to 100% power and was controlled by using a prototype FutureChemistry B-271 photo-
chemistry module. The emission profile was measured experimentally with an emission
maximum observed at 366.5 nm (Figure 2C; full-width at half-maximum
≈
14 nm). The

Molecules 2021, 26, 764
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photochemical flow reaction was performed using a mounted FutureChemistry borosilicate
glass microfluidic chip with approximately 700 wide and 500 µm deep channels and a total
internal volume of 112 µL (Figure 2D).
Figure 2. (A) FutureChemistry FlowStart B-222 photochemistry module. (B) LedEngin, Inc twin LEDs connected in series.
(
C
) Emission spectra for the twin LED light source (combined plots). (
D
) Microfluidic chip (top), oriented in the direction of
1
flow (left to right); and microfluidic chip holder with standard inch-28 screw-connectors for input and output.
4
The chip is comprised of two segments including a shorter split and recombine mixing
section (which splits the flow and recombines it multiple times to ensure homogeneity of
solutions injected), and a longer linear reaction channel. Due to the small channels, the
surface area of the microfluidic chip is much larger compared to standard batch reactors,
which ensures efficient heat transfer and exposure of the reagents to light for sufficient
time to complete the photochemical activation of the ArN group [25,26].
3
2
.3. Flow Radiochemistry
89
The [ Zr]ZrDFO-PEG -Et-azepin-HSA protein conjugate was prepared by flow pho-
3
8
9
toradiochemistry on a microfluidic chip by reaction of a pre-labelled solution of [ Zr]ZrDFO-
PEG -Et-ArN ( Zr-
89
+
1 ; solution A) at a pH of 8.0 to 8.5, and a solution of native (unfunc-
3
3
–
1
tionalised) HSA in Chelex-treated water (solution B; 45 mg mL protein concentration;
Scheme 2). As an example, solution A was prepared by incubating ligand (20 L; 2 mM
L; 5.532 MBq) in
L; pre-treated with Chelex-100 resin) at room temperature and at a pH of 8.0 to
.5 (the optimal range for ArN photoconjugation) [19
20]. Quantitative 89Zr-radiolabelling
was characterised by radio-instant thin layer
1
µ
89
4–
stock solution <1% DMSO/H O) with aliquots of [ Zr][Zr(C O ) ] (40
µ
2
2
4 4
H O (40
8
µ
2
,
3
89
+
yields were obtained in <5 min and Zr-
1
chromatography (radio-iTLC; Figure 3A) and radio-HPLC methods (Figure 3B). The chemi-
cal identity and radiochemical purity (RCP) of 89Zr-
1
+
(Figure 3B; blue trace) was confirmed

Molecules 2021, 26, 764
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nat
by comparison of the elution profile of the corresponding [ Zr]ZrDFO-PEG -Et-ArN
3
3
nat
+
89
+
(
Zr-
1
) complex (Figure 3B; green trace). The Zr-
1
complex was then used in the
microfluidic photoconjugation reaction without further purification. Irradiation of a solu-
nat
+
tion of Zr-
1 with a powerful LED confirmed the complex was photochemically active
(
Figure 3B; green trace).
89
Scheme 2. Preparation of the [ Zr]ZrDFO-PEG -Et-azepin-HSA protein conjugate via sequential radiolabelling and flow
3
photoconjugation.
Then, by using a pair of syringe pumps operated by independent drive units, 100 µL
of solution A and 100
chip at a flow rate of 5
µ
L of solution B were injected simultaneously onto the microfluidic
L/min. The reaction vessel was then irradiated at a wavelength of
L total volume of chip). After this time, 150 L of H O (for each
µ
3
65 nm for 20 min (100
µ
µ
2
syringe) was injected on to the chip (to flush the system) with irradiation continuing for
a further 20 min at the same flow rate. The crude product was collected in an Eppendorf
tube, reaching a final volume of approximately 500 µL.

Molecules 2021, 26, 764
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8
9
Figure 3. Radioactive chromatography: (
A
) Radio-ITLC chromatograms of [ Zr]ZrDFO-PEG -Et-
3
8
9
−
ArN before (blue) and after (black) photolysis at 395 nm. The elution profile of [ Zr][Zr(DTPA)]
3
(
(
red) is shown as a control. (
B
) HPLC chromatograms show the elution profiles of compound
1
nat
black); the non-radioactive [ Zr]Zr-complexes before (green) and after (red) photolysis; and the
Zr]ZrDFO-PEG -Et-ArN complex (blue).
[89
3
3
89
Aliquots of the crude [ Zr]ZrDFO-PEG -Et-azepin-HSA protein conjugate mixtures
3
were retained for analysis and fractions purified by size-exclusion gel filtration (PD-10)
chromatography. Crude and purified samples were then characterised by radio-iTLC, ana-
lytical PD-10 size-exclusion chromatography (SEC), and automated radio-HPLC equipped
with a SEC gel-filtration column (Figure 4). After optimisation of the reaction conditions,
89
the decay-corrected radiochemical yield (RCY) for the isolated [ Zr]ZrDFO-PEG -Et-
3
azepin-HSA product was 31.2
concentration in the reaction mixture of 135
±
1.3% (n = 3 independent experiments; with final protein
M; errors reported as 1 standard deviation).
µ
For each reaction the radiochemical purity (RCP) of the isolated product was >90% (deter-
mined by HPLC). The fraction of protein aggregation (indicated by an asterisk in Figure 4C)
was <10%. Experimental data confirm that the 89Zr-radiolabelled proteins can be produced
by photochemical methods in an automated, microfluidic system.
8
9
Figure 4. Radioactive chromatography depicting: (
A
) radio-ITLC chromatograms of purified (black) [ Zr]ZrDFO-PEG -Et-
3
89
−
azepin-HSA (RCP > 95%) and control (blue) [ Zr][Zr(DTPA)] . (
B
) Analytical SEC (PD-10) elution profiles displaying
) Radioactive (black) and electronic
absorption (blue) SEC gel-filtration HPLC chromatograms of the purified [89Zr]ZrDFO-PEG -Et-azepin-HSA (RCP > 90%).
89
crude (blue) and purified (black) samples of [ Zr]ZrDFO-PEG -Et-azepin-HSA. (
C
3
3
Note that (*) designates aggregated protein.
For comparison, manual reactions were also performed by using direct, top-down
irradiation a stirred reaction mixture in a
previously reported by Guillou et al., [22] [ Zr]ZrDFO-PEG -Et-azepin-HSA was produced
≈
1 mL glass vial. In this manual approach
89
3
with a decay-corrected RCY of 59.6
of the microfluidic apparatus and optimisation of the chemical methods is required before
±
3.6% (n = 3) and with a RCP > 90%. Further refinement
the process is competitive with manual reactions.

Molecules 2021, 26, 764
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3
. Conclusions
89
The synthesis of [ Zr]ZrDFO-PEG -Et-azepin-HSA, was achieved by light-induced
3
flow photoconjugation by using commercially available photochemistry modules. Using a
microfluidic chip comprised of a split and recombine mixing section and a linear reaction
89
section, coupled to a twin LED light source, [ Zr]ZrDFO-PEG -Et-azepin-has was prepared
3
in a radiochemical yield of 31.2
results provide an encouraging proof-of-concept that continuous flow procedures can be
developed to produce protein-based radiotracers by automated instrumentation.
±
1.3% (n = 3) in high radiochemical purity (>90%). The
4. Experimental
4.1. General
All reagents and anhydrous solvents were purchased from commercial sources (Sigma-
Aldrich (St. Louis, MO, USA), Merck (Darmstadt, Germany), Tokyo Chemical Industry
Eschborn, Germany) or abcr (Karlsruhe, Germany)) and were used without any further
(
purification unless otherwise stated. All aqueous reactions were carried out using MilliQ
◦
H O (>18.2 M MΩ·cm at 25 C, Merck, Darmstadt, Germany). All anhydrous reactions
2
were carried out in oven-dried glassware under an inert atmosphere. Reactions (where
possible) were monitored using thin layer chromatography (TLC) analysis on aluminium
plates coated with Silica Gel 60 F254 (E. Merck), and were visualised by short-wave ultra-
violet irradiation (254 nm; where applicable), stained with ninhydrin in EtOH or propargyl
◦
alcohol and Cu(I)Br in EtOH, followed by charring at
≈
200 C. Purification was carried
out by flash chromatography on a column of silica gel 60 (0.040–0.063 mm) or by reversed-
phase C18 column chromatography using a Teledyne Isco CombiFlash Rf+ Lumen flash
chromatography system fitted with RediSep Rf Gold reversed-phase C18 columns (5 to
®
®
50 g), eluting in a gradient of 0 to 100% of solvent B (MeOH with 0.1% TFA added). Solvent
A: MilliQ H O with 0.1% TFA added. Evaporation of solvents was performed under
2
reduced pressure by using a rotary evaporator (Rotavapor R-300, Büchi Labortechnik AG,
Flawil, Switzerland).
1
13
1
H NMR and C{ H} NMR experiments were performed using deuterated solvents
1
13
(
Sigma-Aldrich, St. Louis, MO) on a Bruker AV-400 ( H: 400 MHz, C: 100.6 MHz) or a
1
13
1
Bruker AV-500 ( H: 500 MHz, C: 125.8 MHz) spectrometer. Chemical shifts (δ) for H
and 13C spectra are reported in parts per million (ppm) and are relative to the residual
solvent peak. Coupling constants (J) are reported in Hz. Peak multiplicities are abbreviated
as follows: s (singlet), d (doublet), dd (doublet of doublets), t (triplet), q (quartet), quint
1
1
(
quintet), m (multiplet), and br s (broaden singlet). Two-dimensional H- H correlation
spectroscopy (COSY) and 13C heteronuclear single quantum coherence (HSQC) NMR
1
13
experiments were also performed to aid in the assignment of the H and C spectra,
respectively. High-resolution electrospray ionisation mass spectrometry (HR-ESI-MS) was
performed in either positive or negative ionisation mode (as indicated) using a Bruker
MaXis QTOF-MS instrument (Bruker Daltronics GmbH, Bremen, Germany) and was
measured by the mass spectrometry service at the Department of Chemistry, University
of Zurich.
Analytical high-performance liquid chromatography (HPLC) experiments were per-
formed using a Hitachi Chromaster Ultra Rs system fitted with a reversed-phase VP 250/4
Nucleodur C18 HTec (4 mm ID
to a FlowStar LB 514 radioactivity detector (Berthold Technologies, Zug, Switzerland)
×
250 mm, 5 µm) column. This system was also fitted
2
equipped with a 20 µL PET cell (MX-20-6, Berthold Technologies) for analysing radio-
chemical reactions. Size-exclusion high-performance liquid chromatography (SEC-HPLC)
experiments (for protein samples) were performed using a Rigol HPLC system (Contrec
AG, Dietikon, Switzerland) equipped with an Enrich SEC 650 size-exclusion column (24 mL
volume, 10 mm ID
absorption was measured at 280 nm.
×
300 mm, Bio-Rad Laboratories, Basel, Switzerland). Electronic

Molecules 2021, 26, 764
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Purities of synthetic intermediates after chromatographic purification were judged to
1
13
be >90% by analysis of H and C NMR spectra. Purities of final compounds were
NMR or HPLC analysis), after reverse-phase C18 chromatography.
≥
95%
(
4
.2. Synthesis of DFO-PEG -Et-ArN (1)
3 3
Synthesis of compound 2. 3-(4-Aminophenyl)propanoic acid (1.00 g, 6.05 mmoL) was
taken up in MeOH (30 mL) to which imidazole-1-sulphonyl azide HCl (1.52 g, 7.26 mmol),
K CO (2.26 g, 16.3 mmoL) and CuSO 5H O were added and the reaction mixture was
·
2
3
4
2
stirred for 16 h at rt. The reaction was monitored by TLC and on complete conversion
of the starting materials, the mixture was concentrated under reduced pressure. The
resulting crude residue was dissolved in H O (60 mL), acidified with concentrated HCl
2
and extracted with EtOAc (3
×
50 mL). The organic layers were combined, dried over
NaSO and concentrated under reduced pressure. The residue was then co-evaporated
4
1
with cyclohexane to give compound
400 MHz, CDCl3):
H, CH ), 2.67 (t, J = 7.7 Hz, 2H, CH ). C{ H} NMR (101 MHz, CDCl3):
3
(875 mg, 76% yield) as a pale-yellow solid. H NMR
(
δ 7.23–7.17 (m, 2H, CH ), 7.00–6.92 (m, 2H, CH ), 2.94 (t, J = 7.6 Hz,
Ar Ar
13
1
2
δ
178.5 (C=O),
2
2
1
38.4 (Cqt), 137.0 (Cqt), 129.8 (CH ), 119.3 (CH ), 35.6 (CH ), 30.1 (CH ). HR-ESI-MS
Ar
Ar
2
2
−
(
negative mode): m/z calcd. for C H N O [M − H] 190.0622, found 190.0621.
9
9
3
2
Synthesis of compound 4. Compound
2 (300 mg, 1.57 mmol) and N-[(dimethylamino)-
1
H-1,2,3-triazolo-[4,5- ]pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophos-
β
phate N-oxide (HATU; 895 mg, 2.36 mmoL) were taken up in dry DMF (4 mL) and stirred
under N for 20 min at rt. To a solution of the N-Boc-4,7,10-trioxa-1,13-tridecanediamine,
2
compound
3 (755 mg, 2.36 mmoL) dissolved in dry DMF (1 mL) was then added and the
mixture stirred for a further 10 min. At this time, DIPEA (1.09 mL, 6.28 mmol) was added,
and the reaction mixture was stirred at rt for 16 h under N (g). The reaction was monitored
2
by TLC, and on completion, the mixture was concentrated under reduced pressure. The
crude residue was dissolved in EtOAc (25 mL) and washed with H O (2
aqueous layers were then back-extracted with EtOAc (50 mL); the organic layers were
×
50 mL). The
2
combined and washed with brine, dried over NaSO and concentrated under reduced
4
pressure. The crude residue was then purified by flash chromatography on a bed of silica
[
1/1 (v/v) EtOAc/hexane] to give compound 4 (638 mg, 82% yield) as a yellow to orange
1
oil. H NMR (400 MHz, CDCl3):
δ 7.21–7.15 (m, 2H, CH ), 6.97–6.89 (m, 2H, CH ),
Ar Ar
6
.25 (s, 1H, NH), 4.92 (s, 1H, NH), 3.64–3.45 (m, 12H, CH ), 3.33 (q, J = 6.0 Hz, 2H, CH ),
2
2
3
.19 (q, J = 6.3 Hz, 2H, CH ), 2.92 (dd, J = 8.5, 6.9 Hz, 2H, CH ), 2.46–2.37 (m, 2H, CH ),
2
2
2
13
1
1
.75–1.69 (m, 4H, CH ), 1.42 (s, 9H, C(CH ) ). C{ H} NMR (101 MHz, CDCl3):
δ
171.9
2
3 3
(C=O), 156.2 (C=O), 138.1 (Cqt), 138.0 (Cqt), 129.9 (CHAr), 119.1 (CHAr), 79.1 (
C(CH ) ), 70.6
3 3
(
CH ), 70.6 (CH ), 70.3 (CH ), 70.1 (CH ), 69.6 (CH ), 38.6 (CH ), 38.4 (CH ), 38.1 (CH ),
2
2
2
2
2
2
2
2
3
1.2 (CH ), 29.8 (CH ), 28.9 (CH ), 28.6 (C(CH ) ). HR-ESI-MS (positive mode): m/z calcd.
2 2 2 3 3
+
for C H N O [M + Na] 516.2796, found 516.2792.
24
39
5
6
Synthesis of compound 5. Compound
4 (638 mg, 1.29 mmoL) was dissolved in
◦
CH Cl (10 mL) and cooled to 0 C, and then TFA (2 mL) was added drop-wise. The
2
2
reaction mixture was then allowed to warm slowly to rt and then stirred for 1 h. At this
time, TLC (10% MeOH in EtOAc) showed complete consumption of the starting material.
The reaction mixture was then concentrated under reduced pressure and purified by flash
column chromatography (C18, H O to 100% MeOH) to give compound
5
as a yellow oil
2
(
387 mg, 76% yield). 1H NMR (400 MHz, CDCl3):
d, J = 8.0 Hz, 2H, CH ), 6.59 (m, J = 5.6 Hz, 1H, NH), 3.68–3.60 (m, 7H, CH ), 3.57–3.53
δ 7.18 (d, J = 8.0 Hz, 2H, CH ), 6.93
Ar
(
Ar
2
(
m, 2H, CH ), 3.46 (t, J = 5.7 Hz, 2H, CH ), 3.28 (q, J = 6.4 Hz, 2H, CH ), 3.02 (t, J = 5.9 Hz,
2 2 2
2
H, CH ), 2.96–2.87 (m, 3H, CH ), 2.45 (t, J = 7.7 Hz, 2H, CH ), 1.85 (quint, J = 5.9 Hz, 2H,
2 2 2
13
1
CH ), 1.70 (quint, J = 6.2 Hz, 2H, CH ), 1.36–1.20 (m, 2H, NH ). C{ H} NMR (101 MHz,
2
2
2
CDCl3):
δ 172.7 (C=O), 138.0 (Cqt), 138.0 (Cqt), 129.9 (CHAr), 119.2 (CHAr), 70.5 (CH ), 70.5
2
(
2
CH ), 70.0 (CH ), 69.9 (CH ), 69.2 (CH ), 40.3 (CH ), 38.2 (CH ), 37.2 (CH ), 31.2 (CH ),
9.2 (CH ), 29.2 (CH ). HR-ESI-MS (positive mode): m/z calcd. for C H N O [M + H]
2 2 19 31 5 4
2 2 2 2 2 2 2 2
+
394.2449, found 394.2453.

Molecules 2021, 26, 764
9 of 12
Synthesis of compound 6. Compound
5
(224 mg, 0.55 mmoL) was taken up in dry
DMF (5 mL). Succinic anhydride (110 mg, 1.10 mmoL) was added and the reaction mixture
was stirred for 16 h at rt. The reaction was monitored by TLC, and on completion, the
solvent was removed under reduced pressure and the crude mixture was purified by flash
column chromatography (C18, H O to 100% MeOH) to give compound
6 (235 mg, 87%
2
1
yield) as an orange to brown oil. H NMR (400 MHz, MeOD):
δ 7.24 (d, J = 8.0 Hz, 2H,
CHAr), 6.98 (d, J = 8.0 Hz, 2H, CHAr), 3.66–3.47 (m, 11H, CH ), 3.39 (t, J = 6.2 Hz, 2H, CH ),
2
2
3
2
.23 (dt, J = 19.5, 6.7 Hz, 4H, CH ), 2.90 (t, J = 7.5 Hz, 2H, CH ), 2.58 (t, J = 7.0 Hz, 2H, CH ),
.50–2.42 (m, 4H, CH ), 1.71 (dquint, J = 30.2, 6.4 Hz, 4H, CH ). C{ H} NMR (101 MHz,
2 2
2 2 2
13
1
MeOD):
δ 176.2 (C=O), 174.9 (C=O), 174.4 (C=O), 139.4 (CHqt), 139.2 (CHqt), 131.0 (CHAr),
1
20.0 (CH ), 71.5 (CH ), 71.2 (CH ), 69.8 (CH ), 69.7 (CH ), 38.8 (CH ), 37.8 (CH ), 37.7
Ar 2 2 2 2 2 2
(
CH ), 32.2 (CH ), 31.6 (CH ), 30.4 (CH ), 30.3 (CH ). HR-ESI-MS (positive mode): m/z
2 2 2 2 2
+
calcd. for C H N O [M+H] 494.2609, found 494.2611.
23
35
5
7
Synthesis of DFO-PEG -Et-ArN (1). Compound
6
(222 mg, 0.45 mmoL) and HATU
3
3
(232 mg, 0.61 mmoL) were dissolved in dry DMF (6 mL) and stirred for 20 min under
N (g). DFO mesylate (267 mg, 0.41 mmoL) was then dissolved in DMF (4 mL) and added
2
to the reaction mixture with stirring for a further 10 min. At this time, DIPEA (0.29 mL,
1
.63 mmoL) was added and the was reaction stirred under N (g) for 16 h at rt. The
2
reaction was monitored by TLC, and on completion the mixture was concentrated under
reduced pressure and the crude residue was purified by flash column chromatography (C18,
H O/MeOH 0% MeOH to 100%) followed by washing with ice-cold acetone (6 × 5 mL
;
2
separated by centrifugation between each wash) to give compound
1 (DFO-PEG -Et-ArN ;
3 3
2
44 mg, 58% yield) as an off-white solid. 1H NMR (500 MHz, DMSO-d6):
δ 9.62 (m, 2H,
OH), 7.77 (q, J = 5.0, 4.6 Hz, 4H, NH), 7.23 (d, J = 8.4 Hz, 2H, CHAr), 7.02 (d, J = 8.4 Hz, 2H,
CH ), 3.57–3.42 (m, 14H, CH ), 3.37 (t, J = 6.4 Hz, 2H, CH ), 3.32 (t, J = 6.4 Hz, 2H, CH ),
Ar
2
2
2
3
.10–2.94 (m, 10H, CH ), 2.79 (t, J = 7.6 Hz, 2H, CH ), 2.57 (t, J = 7.4 Hz, 4H, CH ), 2.33 (t,
2 2 2
J = 7.7 Hz, 3H, CH ), 2.29–2.23 (m, 7H, CH ), 1.96 (s, 3H, CH ), 1.58 (dt, J = 13.7, 6.8 Hz,
2
2
3
4
H, CH ), 1.49 (t, J = 7.2 Hz, 5H, CH ), 1.41–1.34 (m, 5H, CH ), 1.24–1.17 (m, 5H, CH ).
2 2 2 2
1
3
1
C{ H} NMR (126 MHz, DMSO-d6):
δ 172.0 (C=O), 171.3 (C=O), 171.2 (C=O), 171.1 (C=O),
171.0 (C=O), 170.1 (C=O), 162.3 (C=O), 158.6–157.7 (TFA), 138.4 (CHqt), 136.9 (CHqt), 129.8
(
CHAr), 118.9 (CHAr), 69.7 (CH ), 69.5 (CH ), 68.0 (CH ), 68.0 (CH ), 47.1 (CH ), 46.8 (CH ),
2 2 2 2 2 2
3
8.4 (CH ), 38.4 (CH ), 36.9 (CH ), 35.8 (CH ), 35.7 (CH ), 30.9 (CH ), 30.9 (CH ), 30.4
2 2 2 2 2 2 2
(
2
CH ), 29.9 (CH ), 29.4 (CH ), 29.3 (CH ), 28.8 (CH ), 27.6 (CH ), 26.0 (CH ), 23.5 (CH ),
2 2 2 2 2 2 2 2
2
+
0.3 (CH3). HR-ESI-MS (positive mode): m/z calcd. for C H N O [M + H] 518.8055,
48 81 11 14
found 518.8057.
4.3. Flow Photochemistry
Flow photoradiochemistry was performed using a FlowStart B-222 (Future Chemistry,
Nijmegen, The Netherlands) photochemistry module equipped with a twin light-emitting
diode (LED; LedEngin Inc., San Jose, CA, USA) light source (365 nM), connected in series.
Light intensity was set to 100% power and controlled using a prototype Future Chemistry
B-271 (Future Chemistry, Nijmegen, The Netherlands) photochemistry module. LED in-
tensity was measured by using a S470C Thermal Power Sensor Head Volume Absorber,
0
.25–10.6 µm, 0.1 mW–5 W, Ø15 mm. Light intensity for each LED was 366.5 nm (FWHM
of 10 nm). The photochemical flow reactions were performed using a mounted Micronit
microfluidics E3 custom borosilicate glass chip (Future Chemistry, Nijmegen, The Nether-
lands) with an internal diameter width of less than 700 m, depth of 500 m and a 112
≈
µ
µ
µL
total volume. The temperature of all photochemical conjugation reactions was typically
◦
2
3 ± 2 C (ambient conditions).
4.4. Radioactivity and Radioactive Measurements
All instruments for measuring radioactivity were calibrated and maintained in accor-
8
9
4−
dance with previously reported routine quality control procedures. [ Zr][Zr(C O ) ]
4 4
2
was obtained as a solution in
≈
1.0 M aq oxalic acid from PerkinElmer (Boston, MA, manu-

Molecules 2021, 26, 764
10 of 12
factured by the BV Cyclotron VU, Amsterdam, The Netherlands) and was used without
further purification. Radioactive reactions were monitored by using instant thin-layer chro-
matography (radio-iTLC). Glass-fibre iTLC plates impregnated with silica-gel (iTLC-SG,
Agilent Technologies) were developed in using aqueous mobile phases containing DTPA
(
50 mM, pH7.1) and were analysed on a radio-TLC detector (SCAN-RAM, LabLogic Sys-
tems Ltd, Sheffield, United Kingdom). Radiochemical conversion (RCC) was determined
by integrating the data obtained by the radio-TLC plate reader and determining both
8
9
the percentage of radiolabelled product (R = 0.0) and ‘free’ Zr (R = 1.0; present in the
f
f
89
−
analyses as [ Zr][Zr(DTPA)] ). Integration and data analysis were performed by using the
software Laura version 5.0.4.29 (LabLogic). Appropriate background and decay corrections
were applied as necessary. Radiochemical purities (RCPs) of labelled protein samples
were determined by size-exclusion chromatography (SEC) using two different columns
and techniques. The first technique used an automated size-exclusion column (Bio-Rad
Laboratories, ENrich SEC 70, 10
HPLC system (Contrec AG, Dietikon, Switzerland) equipped with a UV/visible detector
±
2
µ
m, 10 mm ID
×
300 mm) connected to a Rigol
2
(absorption measured at 220, 254 and 280 nm) and a radioactivity detector (FlowStar LB
514, Berthold Technologies, Zug, Switzerland). Isocratic elution with phosphate buffered
saline (PBS, pH7.4) was used. The second method used a manual procedure involving size-
exclusion column chromatography and a PD-10 desalting column (Sephadex G-25 resin,
8
5–260
µ
m, 14.5 mm ID
×
50 mm, >30 kDa, GE Healthcare). For analytical procedures,
200 L fractions were collected up to
PD-10 columns were eluted with PBS. A total of 40
×
µ
a final elution volume of 8 mL. Note that the loading/dead-volume of the PD-10 columns
was precisely 2.5 mL, which was discarded prior to aliquot collection. For quantification
of radioactivity, each fraction was measured on a gamma counter (HIDEX Automatic
Gamma Counter, Hidex AMG, Turku, Finland) using an energy window between 480 and
5
58 keV for 89Zr (511 keV emission) and a counting time of 30 s. Appropriate background
and decay corrections were applied throughout. PD-10 SEC columns were also used for
preparative purification and reformulation of radiolabelled products (in sterile PBS; pH 7.4)
by collecting a fraction of the eluate corresponding to the high molecular weight protein
(
>30 kDa fraction eluted in the range 0.0 to 1.6 mL as indicated for each experiment).
4
.5. 89Zr-Radioactive Stocks
89
4–
Stock solutions of [ Zr][Zr(C O ) ] were prepared on several occasions using the
2
4 4
89
4−
same procedure. As an example, a stock solution of [ Zr][Zr(C O ) ] was prepared
2
4 4
by adding 89Zr radioactivity from the source (89.11 MBq, 150
acid; PerkinElmer) to an Eppendorf tube. The solution was neutralized by the addition of
aliquots of 1.0 M aq Na CO (total volume of 180 L added, final pH 7.5–7.7, final volume
L, final activity = 82.98 MBq). Caution: Acid neutralization with Na CO releases
µL in
≈
1.0 M aq oxalic
µ
≈
2
3
≈
345
µ
2
3
CO (g) and care should be taken to ensure that no radioactivity escapes the microcentrifuge
2
tube. After CO evolution ceased, several different reactions were performed by using the
2
same stock solutions.
Supplementary Materials: The following are available online, Figure S1. 1H (400 MHz, CDCl3,
1
3
1
2
98 K) NMR spectrum of compound
of compound
CDCl , 298 K) NMR spectrum of compound
2
. Figure S2. C{ H} (101 MHz, CDCl , 298 K) NMR spectrum
3
1
2
. Figure S3. HRMS (negative mode) spectrum of compound
2
. Figure S4. H (400 MHz,
1
3
1
4
. Figure S5. C{ H} (101 MHz, CDCl , 298 K) NMR
. Figure S6. HRMS spectrum of compound
. Figure S8. C{ H} (101 MHz, CDCl , 298 K) NMR
. Figure S9. HRMS spectrum of compound
3
3
1
spectrum of compound
CDCl , 298 K) NMR spectrum of compound
spectrum of compound
MeOD, 298 K) NMR spectrum of compound
4
4
. Figure S7. H (400 MHz,
1
3
1
5
3
3
1
5
5
. Figure S10. H (400 MHz,
1
3
1
6
. Figure S11. C{ H} (101 MHz, MeOD, 298 K) NMR
. Figure S12. HRMS (negative mode) spectrum of compound . Figure
. Figure S14. C{ H} (125 MHz,
. Figure S15. 2D-COSY (DMSO-d ) NMR spectrum
spectrum of compound
6
6
1
13
1
S13. H (500 MHz, DMSO-d , 298 K) NMR spectrum of compound
1
6
DMSO-d , 298 K) NMR spectrum of compound
1
6
6
of compound 1. Figure S16. 2D-hetereonuclear HSQC (DMSO-d ) NMR spectrum of compound 1.
6

Molecules 2021, 26, 764
11 of 12
Figure S17. HRMS spectrum of compound
1. Figure S18. Characterisation data for the radiochemical
89
synthesis of [ Zr]ZrDFO-PEG -Et-ArN .
3
3
Author Contributions: D.F.E. synthesised and characterised the ligand DFO-PEG -EtArN and
3
3
intermediates. D.F.E. and A.G. performed the radiochemistry, characterisation, optimisation and
analysis of the 89Zr-radiolabelled DFO-PEG -Et-azepin-HSA protein conjugate. D.v.d.B. provided the
3
flow photochemistry modules. D.v.d.B., A.J.P. and J.P.H. conceived of the project. J.P.H. supervised
the project. D.F.E. and J.P.H. prepared the manuscript which was reviewed by all authors. All authors
have read and agreed to the published version of the manuscript.
Funding: This research was funded by Swiss National Science Foundation (SNSF Professorships
PP00P2_163683 and PP00P2_190093), the Swiss Cancer League (Krebsliga Schweiz; KLS-4257-08-
2
017) and the University of Zurich (UZH). This project also received funding from the European
Union’s Horizon 2020 research and innovation programme/from the European Research Council
grant Agreement numbers 676904, ERC-StG-2015, NanoSCAN; 101001734, ERC-CoG-2020, Pho-
(
toPHARMA).
Data Availability Statement: The data presented in this study are available on request from the
corresponding author. All relevant data are presented in the manuscript and supporting information.
Conflicts of Interest: D.v.d.B. is an employee of FutureChemistry. The other authors declare no
conflict of interest.
Sample Availability: Samples of the compound DFO-PEG -Et-ArN
1 are available from the corre-
3
3
sponding author.
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