Tuning Tetrazole Photochemistry for Protein Ligation and Molecular Imaging

Article abstract of DOI:10.1002/chem.202100061

Photochemistry provides a wide range of alternative reagents that hold potential for use in bimolecular functionalisation of proteins. Here, we report the synthesis and characterisation of metal ion binding chelates derivatised with disubstituted tetrazoles for the photoradiochemical labelling of monoclonal antibodies (mAbs). The photophysical properties of tetrazoles featuring extended aromatic systems and auxochromic substituents to tune excitation toward longer wavelengths (365 and 395 nm) were studied. Two photoactivatable chelates based on desferrioxamine B (DFO) and the aza-macrocycle NODAGA were functionalised with a tetrazole and developed for protein labelling with ⁸⁹Zr, ⁶⁴Cu and ⁶⁸Ga radionuclides. DFO-tetrazole (1) was assessed by direct conjugation to formulated trastuzumab and subsequent radiolabelling with ⁸⁹Zr. Radiochemical studies and cellular-based binding assays demonstrated that the radiotracer remained stable in vitro retained high immunoreactivity. Positron emission tomography (PET) imaging and biodistribution studies were used to measure the tumour specific uptake and pharmacokinetic profile in mice bearing SK-OV-3 xenografts. Experiments demonstrate that tetrazole-based photochemistry is a viable approach for the light-induced synthesis of PET radiotracers.

Full text of DOI:10.1002/chem.202100061

Communication
doi.org/10.1002/chem.202100061
Chemistry—A European Journal
&
Photochemistry
Tuning Tetrazole Photochemistry for Protein Ligation and
Molecular Imaging
Rachael Fay and Jason P. Holland*[a]
genic, generating emissive pyrazoline products.[5] Subsequent
Abstract: Photochemistry provides a wide range of alter-
native reagents that hold potential for use in bimolecular
functionalisation of proteins. Here, we report the synthesis
and characterisation of metal ion binding chelates deriva-
tised with disubstituted tetrazoles for the photoradio-
chemical labelling of monoclonal antibodies (mAbs). The
photophysical properties of tetrazoles featuring extended
aromatic systems and auxochromic substituents to tune
excitation toward longer wavelengths (365 and 395 nm)
were studied. Two photoactivatable chelates based on
desferrioxamine B (DFO) and the aza-macrocycle NODAGA
were functionalised with a tetrazole and developed for
protein labelling with 89Zr, 64Cu and 68Ga radionuclides.
DFO-tetrazole (1) was assessed by direct conjugation to
formulated trastuzumab and subsequent radiolabelling
with 89Zr. Radiochemical studies and cellular-based bind-
ing assays demonstrated that the radiotracer remained
stable in vitro retained high immunoreactivity. Positron
emission tomography (PET) imaging and biodistribution
studies were used to measure the tumour specific uptake
and pharmacokinetic profile in mice bearing SK-OV-3 xen-
ografts. Experiments demonstrate that tetrazole-based
photochemistry is a viable approach for the light-induced
synthesis of PET radiotracers.
work used genetically encoded artificial amino acids bearing
alkenes[6] and cyclopropenes[7] for site-specific, photo-induced
labelling of pre-modified proteins in vitro and in vivo. One of
the attractive features of tetrazole-alkene photochemistry is
that reactivity can be modified to increase coupling rates via
structural and frontier orbital control on the tetrazole[8–11] or
the alkene partners.[12,13] However, a drawback of tetrazole
photochemistry is the need to use short wavelength light
(ꢀ302 nm) to form the nitrile imine which is potentially dam-
aging to proteins like mAbs.[14,15]
The photo-click reaction between tetrazoles and alkenes was
reported to be bioorthogonal.[5,16] However, Huisgen et al.
noted the reactivity of thermally induced nitrile imines with
thiophenol in 1961.[3] More recently, the biorthogonality of the
‘photo-click’ chemistry has been questioned. Detailed experi-
mental evidence confirmed that the electrophilic nitrile imine
produced via photochemical activation from tetrazoles reacts
with nucleophilic amino acid residues in proteins.[17,18] Li et al.
demonstrated the photochemical induced reactivity of tetra-
zoles toward a range of carboxylic acids including pent-4-enoic
acid, AcOH, free alanine and proteins labelled at Glu resi-
dues.[17] Whilst the authors also reported reactivity toward a
range of biologically relevant nucleophiles such as thiols,
amines and alcohols, they noted that at physiological pH, car-
boxylic acids reacted preferentially with the nitrile imine, even
outcompeting the cycloaddition process. Similarly, Zhao et al.
reported the photo-induced reactivity of tetrazoles with pro-
pionic acid and a protected Asp derivative.[18] Competition ex-
periments with amino acids containing phenol, thiol, and
amino functional groups, reduced the yield of the reaction be-
tween their model tetrazole and Boc-Asp-OtBu, indicating that
other nucleophiles can also react with the nitrile imine.[18] Fol-
lowing these studies, the photo-induced reactivity of tetrazoles
with thiols (mercaptoethanol) and amino acids (Trp, Pro, His,
Ser, Asn, Tyr, Asn) was reported.[19–21]
The photochemical reactivity of tetrazoles was first reported
by Huisgen et al. in 1967.[1] Light-induced activation of diaryl-
tetrazoles populates an electronic excited state that undergoes
rapid loss of N2(g), to form a highly reactive nitrile imine. This
nitrile imine intermediate can behave as a 1,3-dipole and par-
ticipate in cycloaddition reactions[2] or as an electrophile, react-
ing with native nucleophiles on protein.[3] In 2008, the group
of Qing Lin reported the use of tetrazoles for labelling of post-
translationally modified proteins via photoinduced [3+2] cyclo-
additions with alkene substituents.[4] This remarkable and fast
reaction was described as a ‘photo-click’process and is fluoro-
Until recently, the combination of radiochemistry and photo-
chemistry has received minimal attention. Early work estab-
lished that compounds radiolabelled with 99mTc, 18F, 188Re and
111In radionuclides could be functionalised with substituted
(perfluoro-, nitro-) and non-substituted aryl azide (ArN3) for
photochemically induced labelling of different bioactive mole-
cules.[22–31]Sun et al. used the photo-induced cycloaddition pro-
cess and labelled an alkene-functionalised, tumour-specific
peptide with a modified tetrazole which allowed 68Ga and 64Cu
radiolabelling for PET and optical imaging in a glioblastoma
model.[32] Our group has also explored the use of photoactivat-
[a] R. Fay, Prof. Dr. J. P. Holland
Department of Chemistry
University of Zurich
Winterthurerstrasse 190, 8057 Zurich (Switzerland)
E-mail: jason.holland@chem.uzh.ch
Homepage: www.hollandlab.org
Supporting information and the ORCID identification numbers for the
authors of this article can be found under:
https://doi.org/10.1002/chem.202100061.
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Chemistry—A European Journal
able chelates bearing an aryl azide to make viable 68Ga and
89Zr PET radiotracers via direct functionalisation of mAbs.[33–36]
Here, we demonstrate that the photophysical properties of
tetrazoles can be tuned through synthetic control over the ary-
lene substituents to facilitate the rapid and direct, bimolecular
protein labelling at wavelengths in the UVA region of the spec-
trum. Two-step photochemical conjugation and radiolabelling
produced 89Zr-labelled trastuzumab for PET imaging of human
epidermal growth-factor receptor 2 (HER2/neu) expression in
tumours.
With six tetrazole scaffolds in hand, we measured their pho-
tophysical and photochemical properties by using electronic
absorption spectroscopy (UV/vis) analytical high-performance
liquid chromatography (HPLC) to analyse the formation of pho-
tolysis products. (Figure 2, Figures S37–S38, and Table S1). Tet-
razoles 10 and 12 displayed no photoactivity when irradiated
at 365 nm. For compound 9, minor decomposition was ob-
served under 365 nm irradiation but this was attributed to ho-
molytic cleavage of the bromo-substituent.[38] Whilst the naph-
thalene derivative (12) was not photo-reactive at 365 nm,
naphthylene analogues functionalised with auxochromic
groups (OMe, SMe, OH) exhibited bathochromic shifts that al-
lowed photolysis of the tetrazole to occur at longer wave-
lengths. In general, changes in the absorption spectra for com-
pounds 7, 8 and 13 followed the Woodward–Fieser–Kuhn
rules where the methyl-mercaptan substituent (9) induced the
greatest shift in Amax to 332 nm (+42 nm with respect to the
unsubstituted naphthalene-derivative (12), Amax =290 nm; Fig-
ure 2A).[39,40] Kinetic studies on the photoactivation revealed
that the rates of photolysis increased with increasing molecular
absorption coefficient (Table S1). Irradiation of compound 7
Tetrazole compounds 7, 9, 10, and 12 (Figure 1) were syn-
thesised by reacting phenylsulfonylhydrazones with arylene di-
azonium salts (generated in situ) and isolated in moderate
yields (23- 42%).[37] Compound 8 was synthesised from com-
pound 9 by nucleophilic aromatic substitution of the bromo-
substituent by using NaSMe. Compound 13 was synthesised in
two-steps involving isolation and deprotection of the corre-
sponding OBn derivative (14). Efforts to synthesise the anthra-
cene analogue (11) were not successful. Full experimental de-
tails and characterisation data are given in the supporting in-
formation (Schemes S1–S8, Figures S1–S36).
Figure 1. General synthetic Scheme to yield long-wavelength tetrazole analogues.
Figure 2. Photophysical and photochemical data on tetrazoles 7, 9 and 13. A) Electronic absorption spectra recorded in DMSO. B) HPLC chromatograms
before and after photolysis of compound 7. C) Kinetic plots showing the relative change in the concentration of compound 7 versus time during extended
photolysis at 365 and 395 nm.
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(e365 =298mÀ1 cmÀ1) with light at 365 nm induced a rapid first-
order decomposition with an observed rate constant,
kobs(365 nm)=0.44Æ0.12 minÀ1. Although compound 8 dis-
played the largest red-shift and fastest rate of photolysis, thio-
ethers are susceptible to oxidation in vivo.[41] Therefore, com-
pound 7 was selected for further applications in protein liga-
tion.
Next, we performed the photochemical conjugation be-
tween compound 1 and trastuzumab (formulated as Hercep-
tinTM; 10-to-1 chelate-to-mAb ratio, pH7) by irradiating reac-
tions at either 365 or 395 nm for 10 min at room temperature
(Figure 4A). Aliquots of the crude mixtures were retained for
analysis and the DFO-mAb conjugates were isolated from
small-molecule impurities by preparative size-exclusion chro-
matography (SEC). Samples of the crude photoconjugation
mixtures were radiolabelled with 89Zr4+ giving decay-corrected
To apply compound 7 in the photoradiochemical synthesis
of 89Zr-labelled mAbs, we synthesised the desferrioxamine-tet-
razole derivative, compound 1 (Scheme S8 and Figures S31– radiochemical yields (RCYs) of 27.9Æ10.5%, (n=3) and 34.8Æ
S36). DFO is a hexadentate chelate which is used in clinical PET
with 89Zr-mAbs to coordinate the 89Zr4+ metal ion (half-life,
3.0% (n=2), at 365 and 395 nm, respectively (measured by
manual size-exclusion chromatography with PD-10 columns;
Figures S46). For a more accurate quantitative analysis we also
measured the radiolabelled mixtures by radio-SEC which
showed that 15% of activity was associated with trastuzumab
(Figure 4, panel B, blue trace). Combined with the initial 10-to-
1 stoichiometric ratio of reagents, the isolated DFO-mAb con-
jugate used in future studies had an average of 1.5 accessible
DFO ligands per mAb. For in vitro and in vivo experiments, the
purified DFO-Np(OMe)-Tz-trastuzumab conjugate was radiola-
belled with an excess of 89Zr at pH 8.5 and purified by SEC
to give [89Zr]ZrDFO-Np(OMe)-Tz-trastuzumab in sterile PBS
(pH 7.4) with a RCY of 39.8%, a radiochemical purity (RCP)
>95%, and a molar activity of Am =4.01 MBq nmolÀ1 of mAb
(Figure 4B).
The stability of [89Zr]ZrDFO-Np(OMe)-Tz-trastuzumab was as-
sessed in PBS and human serum (Figure S47–S48). Data indi-
cated that the radiotracer was stable for up to 24 h at 378C in
serum with the main peak in radio-SEC still associated with the
89Zr-mAb. In sterile PBS, the radiotracer was stable for 3 days
with no change in RCP. Cellular binding studies using SK-OV-3
cells were performed to assess the biochemical integrity of the
radiotracer after the photoconjugation and radiolabelling.
Binding curve analysis indicated that the [89Zr]ZrDFO-Np(OMe)-
Tz-trastuzumab sample had an immunoreactive fraction of
82Æ4%, and retained specificity toward HER2/neu (Fig-
ure 4C).[43]
t
1/2 =78.41 h).[42] In addition, to increase radiochemical scope,
we also synthesised a NODAGA-based tetrazole (4) for 68Ga
and 64Cu radiochemistry (Scheme S7 and Figures S29–S31). Full
details of synthesis and radiosynthesis of
nat/64Cu]Cu-4À and [nat/68Ga]Ga-1 are presented in the support-
[
nat/68Ga]Ga-4,
[
ing information (Figures S39-S41). Details on the photochemi-
cal conjugation of Tz-4 to human serum albumin (HSA) and di-
nutuximab (a chimeric mAb against the disialoganglioside GD2
in neuroblastoma) and radiolabelling with 68Ga and 64Cu, re-
spectively, are also shown (Figures S42–S45).
The DFO analogue (compound 1) was synthesised from (7)
in 4-steps with an overall yield of 31%. Briefly, compound 7
was saponified and a Boc protected PEG linker was coupled to
yield compound 3 (Scheme S8). Deprotection of the Boc group
revealed the free amine (2) which was then coupled with DFO-
succinate via amide bond formation to yield the desired com-
pound 1. Photophysical experiments with (1) indicated that
the compound retained the photoactivity seen for the tetra-
zole core (e365 =312mÀ1 cmÀ1, kobs(365 nm)=1.88Æ1.09 minÀ1
;
e395 =200mÀ1 cmÀ1,
kobs(395 nm)=0.19Æ0.09 minÀ1,
Fig-
ure S38). The calculated photochemical quantum yields (fp) for
(1), which is a measure of the efficiency of light-induced activa-
tion, were 2.2Æ0.4% and 0.23Æ0.04% at 365 and 395 nm, re-
spectively. Complexation of natZr4+ with (1) was achieved by
using standard methods and the natZr-1+ complex was charac-
terised by HRMS and HPLC. The radioactive complex, 89Zr-1+
was prepared at 238C in ꢁ10 minutes and was characterised
by using radio-iTLC, and by radio-HPLC which gave a single
peak in the chromatogram (Figure 3). Co-injection of 89Zr-1+
with the non-radioactive complex measured by electronic ab-
sorption showed coincident retention times in HPLC.
PET imaging and biodistribution studies were performed to
measure the pharmacokinetics, tumour specificity and stability
of [89Zr]ZrDFO-Np(OMe)-Tz-trastuzumab in athymic nude mice
bearing SK-OV-3 xenografts (Figure 5, Figures S50–S55,
Table S2–S3). All animals received the same amount of activity
(0.53Æ0.02 MBq) but three different mass doses of
89[Zr]ZrDFO-Np(OMe)-Tz-trastuzumab. Group 1 received the
highest molar activity dose (20 mg, Am =4.01 MBqnmolÀ1),
group 2 received a 13-fold increase in protein dose (Am =
0.30 MBqnmolÀ1) and group 3 received a full blocking dose
(72-fold excess, Am =0.06 MBqnmolÀ1). SK-OV-3 tumours dis-
play extremely high expression of the HER2/neu protein and as
consequence, no differences were observed in the PET images
and biodistribution data between groups 1 and 2. For exam-
ple, biodistribution data revealed that 72 h post-administration
the tumour associated activity was 45.8Æ14.0%ID gÀ1 for
group 1 and 47.0Æ7.4%ID gÀ1 for group 2. In contrast, the
tumour uptake in the blocking group was reduced by ꢀ67%
to 15.5Æ5.2%ID gÀ1, indicating specific tumour uptake. The
tumour-to-tissue contrast was high although activity in the
Figure 3. Chromatographic data on the complexation of nat/89Zr4+ by com-
pound 1. A) Radio-iTLC data 89Zr-1+ before (black) and after (blue) irradiation
and a control (red, [89Zr][Zr(DTPA)]À. B) Analytical HPLC chromatograms for 1
(blue), natZr-1+ (green) and 89Zr-1+ (black).
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Figure 4. Photochemical conjugation and 89Zr-radiolabelling of DFO-Np(OMe)-Tz-trastuzumab. A) Schematic showing the structure of compound 1 and the
photochemical conjugation reaction with trastuzumab. B) SEC chromatograms showing the crude (blue) and purified (black) samples of the 89Zr-mAb, the
electronic absorption (280 nm) profile of trastuzumab, and the elution of [89Zr][Zr(DTPA)]À as a control. C) Cellular binding assays showing the specific binding
of [89Zr]ZrDFO-Np(OMe)-Tz-trastuzumab to SK-OV-3 ovarian cancer cells (HER2/neu-positive). Control study: n.s.=non-specific binding (achieved by pre-block-
ing with a 1025-fold excess of non-labelled trastuzumab).
bone was observed in all three groups (11.8Æ4.0%ID gÀ1,
10.1Æ5.7%ID gÀ1, 9.1Æ3.9%ID gÀ1 for groups 1, 2, and 3, re-
spectively). Bone uptake was in line with previous on 89Zr-tras-
tuzumab.[44]
corbic acid, histidine, etc.). Some drawbacks of the existing tet-
razole compound 1 species include the low water solubility
and the lack of chemo- and regioselective functionalisation.
Further experiments are underway to address these limitations.
This work expands the scope of photoradiolabelling and pro-
vides an encouraging benchmark demonstrating that 89Zr-
mAbs can be produced using tetrazole photochemistry.
Whole-body excretion studies were also used to measure
the effective half-life of [89Zr]ZrDFO-Np(OMe)-Tz-trastuzumab in
each mouse (Figure S56). Measurements indicated that no sig-
nificant difference in pharmacokinetics was observed between
animals in three different dose groups with t1/2(eff) equal to
29.8Æ0.8 h, 35.5Æ0.6 h, and 35.2Æ7.4 h, respectively. Values
were also consistent with prior measurements using 89Zr-radio-
labelled trastuzumab prepared via simultaneous photoradio-
labelling with an aryl azide species.[45]
Experimental Section
Animal imaging was conducted under an approved license from
the Veterinary Department of the Canton of Zurich. Approved pro-
tocol number 32357. Full experimental details and characterisation
data are given in the Supporting Information.
In conclusion, the experiments provide irrefutable evidence
that a direct photochemical conjugation occurs between the
light-induced nitrile imine and the protein. The nature of the
conjugate bonds formed remain uncertain but others have re-
ported convincing evidence that carboxylate, amine and
sulfhydryl nucleophiles from amino acids including Glu, Asp,
Lys and Cys can attack the nitrile imine intermediate
(Scheme S9).[17–21] Tetrazole photochemistry can be adapted for
labelling mAbs in the presence of standard formulation buffers
(containing high concentrations of trehalose, polysorbate, as-
Acknowledgements
J.P.H. thanks the Swiss National Science Foundation (SNSF Pro-
fessorship PP00P2 163683 and PP00P2 190093), the European
Research Council (ERC-2015-StG, NanoSCAN-676904; ERC-2020-
CoG, PhotoPHARMA, 101001734), the Swiss Cancer League
(Krebsliga Schweiz; KLS-4257-08-2017), and the University of
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Keywords: antibodies · photochemistry · positron emission
tomography · radiochemistry · tetrazoles
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Manuscript received: January 7, 2021
Accepted manuscript online: January 11, 2021
Version of record online: February 11, 2021
Chem. Eur. J. 2021, 27, 4893 –4897
www.chemeurj.org
4897
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