N-Succinimidyl 3-((4-(4-[18F]fluorobutyl)-1H-1,2,3-triazol-1-yl)methyl)-5-(guanidinomethyl)benzoate ([18F]SFBTMGMB): A residualizing label for 18F-labeling of internalizing biomolecules

Article abstract of DOI:10.1039/c5ob02258d

Residualizing labeling methods for internalizing peptides and proteins are designed to trap the radionuclide inside the cell after intracellular degradation of the biomolecule. The goal of this work was to develop a residualizing label for the ¹⁸F-labeling of internalizing biomolecules based on a template used successfully for radioiodination. N-Succinimidyl 3-((4-(4-[¹⁸F]fluorobutyl)-1H-1,2,3-triazol-1-yl)methyl)-5-(bis-Boc-guanidinomethyl)benzoate ([¹⁸F]SFBTMGMB-Boc₂) was synthesized by a click reaction of an azide precursor and [¹⁸F]fluorohexyne in 8.5 ± 2.8% average decay-corrected radiochemical yield (n = 15). An anti-HER2 nanobody 5F7 was labeled with ¹⁸F using [¹⁸F]SFBTMGMB ([¹⁸F]RL-I), obtained by the deprotection of [¹⁸F]SFBTMGMB-Boc₂, in 31.2 ± 6.7% (n = 5) conjugation efficiency. The labeled nanobody had a radiochemical purity of >95%, bound to HER2-expressing BT474M1 breast cancer cells with an affinity of 4.7 ± 0.9 nM, and had an immunoreactive fraction of 62-80%. In summary, a novel residualizing prosthetic agent for labeling biomolecules with ¹⁸F has been developed. An anti-HER2 nanobody was labeled using this prosthetic group with retention of affinity and immunoreactivity to HER2.

Full text of DOI:10.1039/c5ob02258d

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N-Succinimidyl 3-((4-(4-[18F]fluorobutyl)-1H-1,2,3-triazol-1-
yl)methyl)-5-(guanidinomethyl)benzoate ([18F]SFBTMGMB):
A Residualizing Label for 18F-labeling of internalizing
biomolecules
Ganesan Vaidyanathan,a* Darryl McDougald,a Jaeyeon Choi,a Marek Pruszynski,a,b Eftychia
Koumarianou,a Zhengyuan Zhou,a and Michael R. Zalutsky.a
aDepartment of Radiology and Duke University Medical Center, Durham, North Carolina, USA;
bPresent address: Institute of Nuclear Chemistry and Technology, Warsaw, Poland.
For correspondence or reprints contact Ganesan Vaidyanathan, Box 3808, Department of
Radiology, Duke University Medical Center, Durham, North Carolina 27710, USA. Telephone:
(919) 684-7811; Fax (919) 684-7122. E-mail: ganesan.v@duke.edu.
1

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Abstract
Residualizing labeling methods for internalizing peptides and proteins are designed to
trap the radionuclide inside the cell after intracelular degradation of the biomolecule. The goal
of this work was to develop a residualizing label for the 18F-labeling of internalizing biomolecules
based on a template used successfully for radioiodination.
N-succinimidyl 3-((4-(4-
[18F]fluorobutyl)-1H-1,2,3-triazol-1-yl)methyl)-5-(bis-Boc-guanidinomethyl)benzoate
(Boc2-
[18F]SFBTMGMB) was synthesized by click reaction of an azide precursor and [18F]fluorohexyne
in 8.5 ± 2.8% average decay-corrected radiochemical yield (n =15). An anti-HER2 nanobody
5F7 was labeled with 18F using [18F]SFBTMGMB ([18F]RL-I), obtained by the deprotection of
Boc2-[18F]SFBTMGMB, in 31.2 ± 6.7% (n =5) conjugation efficiency. Thus labeled nanobody
had a radiochemical purity of >95%, bound to the HER2-expressing BT474M1 breast cancer
cells with an affinity of 4.7 ± 0.9 nM, and had an immunoreactive fraction of 62-80%. In
summary, a novel residualizing prosthetic agent for labeling biomolecules with 18F has been
developed. An anti-HER2 nanobody was labeled using this prosthetic group with retention of
affinity and immunoreactivity to HER2.
2

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Introduction
Radionuclide imaging using peptides and monoclonal antibodies (mAbs) is widely used
for a number of applications. These include diagnosing cancers, quantifying expression of a
particular target of interest, determination of radiation dosimetry prior to administering the same
vector labeled with a therapeutic radionuclide, and to determine the pharmacokinetics of the
labeled protein or peptide. The radiolabeling method has a significant influence on the
biodistribution of the tracer.1 For molecules that undergo internalization after binding to
receptors on the tumor cell, which subsequently undergo proteolysis within the lysosomal
compartment, residualizing labels (RL) are preferred. Residualizing labels can potentially
enable each of the radionuclide imaging applications noted above if the targeting vector
undergoes extensive internalization after receptor binding. If higher tumor uptake and hence
higher tumor-to-tissue ratios can be achieved using RLs compared with conventional prosthetic
agents, then that will certainly be an advantage. While radiometal labeling is generally
considered residualizing, radiohalogenation by the direct electrophilic approach is non-
residualizing. This reflects the fact that monoiodotyrosine and free iodide, the primary
radiolabeled catabolites from molecules radioiodinated by the direct method, wash out of the
cell rather quickly resulting in lower cumulated radioactivity in tumor.
To overcome this problem, we and others have developed residualizing labels for the
radiohalogenation of internalizing biomolecules.2-6 One of the agents we developed, N-
succinimidyl 4-guanidinomethyl-3-iodobenzoate (SGMIB), contains a guanidine function that
remains predominantly positively charged at lysosomal pH, and was designed on the basis that
positively charged radiolabeled catabolites will remain trapped within the cells. Excellent results
with respect to augmenting tumor retention of radioactivity from internalizing molecules
radioiohalogenated using this template have been obtained.7, 8
3

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Nanobodies (Nbs), a.k.a. VHH molecules and single domain antibody fragments
(SdAbs), are the antigen-binding fragments of heavy-chain-only antibodies from Camelidae.9-11
Their molecular weight (12–15 kDa) is an order of magnitude less than that of intact mAbs, and
considerably less than that of Fab (~50 kDa) or scFv (~25 kDa) fragments. SdAbs exhibit
nanomolar affinities, high thermal and chemical stability, are more water-soluble compared with
intact mAbs and their fragments, and have a lower tendency for aggregation. SdAbs specific to
a number of molecular targets such as the growth factor receptor tyrosine kinase HER2 as well
as epidermal growth factor receptor (EGFR) have been generated. There is an emerging
interest in exploiting the excellent properties of SdAbs as a platform for the development of
imaging agents for the quantification of HER2 and EGFR status by positron emission
tomography (PET) in cancer patients. Based on the results we have obtained, maximum tumor
uptake and contrast can be obtained within 2-4 hours of administration of labeled SdAbs,7, 12
making widely available 18F perhaps the ideal positron-emitter for labeling SdAbs.
18
Although a number of prosthetic agents have been reported for F-labeling of peptides
and proteins,13-15 N-succinimidyl 4-[18F]fluorobenzoate ([18F]SFB) — an agent originally
developed by us16 — remains the most widely used agent for this purpose. There have been a
few reports of [18F]SFB being utilized to label biomolecules reactive with internalizing molecular
targets, and in these cases 18F activity in tumor in vivo reduced with time.17, 18 It seems likely
that this behavior could in part reflect the lack of residualizing moieties in the structure of SFB,
making this reagent less than ideal for labeling internalizing biomolecules such as anti-HER2
SdAbs. To investigate the potential advantages of a residualizing label for 18F-labeling, we have
designed a new prosthetic agent combining the structural features of SGMIB and SFB.
Herein, we describe a method for the synthesis of N-succinimidyl 3-((4-(4-
[18F]fluorobutyl)-1H-1,2,3-triazol-1-yl)methyl)-5-(guanidinomethyl)benzoate ([18F]SFBTMGMB),
heretofore referred to as [18F]RL-I. An anti-HER2 SdAb 5F7 was labeled using [18F]RL-I and
4

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the integrity of the labeled SdAb as well as its affinity and immunoreactivity to HER2 were
retained.
Results and Discussion
The goal of this work was to develop a residualizing agent for labeling internalizing
18
biomolecules such as anti-HER2 nanobodies with F. Although we have developed a number
of residualizing labels for radioiodination, unlike the case with intact mAbs, the best tumor
targeting with SdAbs was seen when radioiodination was performed using the guanidine-
containing prosthetic agent SGMIB.7 Radiosynthesis of an analogous agent — N-succinimidyl
3-[18F]fluoro-4-guanidinomethylbenzoate ([18F]SFGMB) — wherein the radioiodine in SGMIB is
18
replaced with F, would be difficult. Introduction of 18F onto an aromatic ring by classical SNAr
substitution is facilitated by the presence of electron-withdrawing groups at the ortho- and para-
positions of the nucleofuge. In the SGMIB molecule, the iodine is present at the meta position
of the activating ester group. Furthermore, because the active ester is susceptible under 18F-
labeling conditions due to nucleophilic attack by fluoride or the constituent base,19
radiosynthesis of [18F]SFGMB by using SNAr substitution for 18F introduction will involve multiple
steps. While higher radiochemical yields theoretically can be obtained for an isomeric
compound wherein 18F is placed at the para-position of the ester group, its radiosynthesis will
still involve multiple steps due to the lability of the active ester to 18F-labeling conditions. For
these reasons, we resorted to a molecule containing a fluoroalkyl side chain because it is easier
to introduce 18F on an sp3 carbon by SN2 substitution. The logical approach for this would be to
have a precursor with an alkyl chain bearing a sulfonate leaving group, the displacement of
which with nucleophilic [18F]fluoride would deliver the required reagent in a single step.
18
However, because of the instability of the N-succinimidyl ester under F-labeling conditions as
noted above, an alternative approach involving a click reaction was utilized (Scheme 1). An
added advantage of this strategy is the presence of the polar triazole20 ring in its structure,
5

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which may contribute to its residualizing ability. Commercially available 3,5-dimethylbenzoic
acid was converted to its TMSE ester 2 in 79% yield. Benzylic bromination and subsequent
debromination of polybrominated side products with diethyl phosphite21, 22 rendered the bis-
bromomethyl derivative 3 in 43% yield. Monoguanidinylation of 3 adapting a reported
procedure23 delivered 4 in 33% yield. This guanidine derivative was treated with sodium azide
to obtain 5 in almost quantitative yields. Compound 5 was converted to 7, the precursor for 18F-
labeling, by fluoride-mediated deprotection of the TMSE ester, and in situ reesterification with N-
hydroxysuccinimide in 35% yield for the two steps. Two approaches were taken to synthesize
the protected standard 8. In the first approach, 5 was converted to 6 in 54% by its click reaction
with 6-fluorohex-1-yne, which was synthesized by the deoxofluorination24 of hex-5-yn-1-ol or
from the tosylate precursor as reported.25, 26 Removal of the TMSE group from 6 and in situ
reesterification with N-hydroxysuccinimide resulted in 8 in 40% yield for the two steps. In the
alternative approach, 7 was subjected to click reaction to obtain 8 in 44% yield. The final
deprotected compound SFBTMGMB (9) was synthesized by TFA-mediated deprotection of 8 in
96% yield.
The approach taken for the synthesis of [18F]SFBTMGMB-Boc2 is shown in Scheme 2.
Various conditions were tried to optimize radiochemical yields for the two steps. In the first step,
converting the tosylate precursor to 6-[18F]fluorohex-1-yne,26 initially, a large excess — up to 50
mg — of the tosylate precursor was used to offset its loss to side reactions like hydrolysis and β-
elimination.
Click reactions were initially performed without a ligand.
Considerable
improvement in radiochemical yields were obtained by including the water-soluble ligand
bathophenanthroline disulphonate (BPDS).27 TLC (1:4 ethyl acetate:hexanes) of the HPLC
injectate indicated [18F]SFBTMGMB-Boc2 (Rf = 0.5 – 0.6) was the predominant radioactive
species (50-60%) with about 25% of radioactivity remaining at Rf = 0; however, up to 50% of the
18F activity was retained in the normal phase HPLC column. The same phenomenon was
observed when HPLC-purified [18F]SFBTMGMB-Boc2 was reinjected onto the HPLC column.
6

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While the reason for this is not apparent, we think it might be due to the hydrolysis of the
compound in the column. It is also likely that the active ester could be reacting with the amino
groups on the stationary phase of the HPLC column, forming a covalent linkage. In future
studies, further refinements in methods will be introduced to avoid HPLC purification without
affecting the purity of the final product.
Under optimized conditions (vide infra),
[18F]SFBTMGMB-Boc2 was synthesized from aqueous fluoride in an overall decay-corrected
radiochemical yield of 8.5 ± 2.8% (n =15); 207.2 ± 66.6 MBq (5.6 ± 1.8 mCi) could be obtained
starting with 3.7 GBq (100 mCi) of aqueous [18F]fluoride in about 100 min, which includes HPLC
purification. HPLC-purified [18F]SFBTMGMB-Boc2 was more than 95% radiochemically pure
and generally no detectable UV peaks were seen in the quality control HPLC runs (Fig. 1). The
specific activity of purified [18F]SFBTMGMB-Boc2 was greater than 9.3 TBq (250 Ci)/mmol.
[18F]SFBTMGMB-Boc2 was deprotected by treatment with TFA to generate [18F]RL-I ([18F]9) and
used as such for coupling with SdAb.
Yields for coupling of [18F]SFBTMGMB to SdAb was erratic in early trials. It was thought
that this might be due to the consumption of the SdAb by either the co-eluting unalabeled carrier
potentially present in the mixture, or the azide precursor that could have bled into the
radioactive peak, although, there was considerable difference in the retention time of 5 and 8
(~10 min and 20 min, respectively). In initial experiments, up to 7 mg of the azide precursor
was used to facilitate the click reaction; subsequently, it was found that 3 mg was sufficient to
get similar click reaction yields. This amount itself was probably too large a quantity to avoid
HPLC co-elution. Attempts were made to scavenge the unreacted azide precursor by click
reaction with a polymer-bound alkyne that we synthesized by coupling propiolic acid to 4-
(bromomethyl)phenoxymethyl polystyrene following a reported procedure28. Parenthetically,
while this work was in progress, a similar strategy for scavenging excess alkyne-modified
peptide with a immobilized azide was reported.29 Scavenging unreacted azide precursor neither
7

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eliminated the presence of an unlabeled compound that closely eluted with [18F]SFBTMGMB-
Boc2 in quality control HPLC nor gave the labeled SdAb in reasonable yields consistently.
It was reasoned that the closely-eluting peak was not the azide precursor as suspected
but might be the product of click reaction between excess azide and the di-hexynyl ether (12;
Scheme 3). The occurrence of side reactions — β-elimination and hydrolysis — during
fluorination via SN2 reaction of aliphatic substrates, which result in the production of the
corresponding alkene and alcohol, respectively, is often reported in the literature; however,
although logical, formation of dialkyl ether has rarely been mentioned (Scheme 4). It can be
generated by the reaction of alcohol/alkoxide, formed by the hydrolysis of substrates such as
tosylates, with excess of the substrate. A careful search of the literature did lead to a report30
wherein such ether formation has been mentioned. During the synthesis of unlabeled 6-
fluorohex-1-yne by the reaction of tosylate precursor 10 with TBAF, di-hexynyl ether 12 was
isolated (see Electronic Supplementary Information) but no formation of the corresponding
alkene, hex-1-en-5-yne was seen. Formation of the alkene resulting from β-elimination has
been reported from 4-tosyloxy-1-butyne but not from 5-tosyloxy-1-pentyne upon treatment with
potassium [18F]fluoride,31 suggesting that it is even less likely that the 6-fluorohex-1-yne will be
formed from 6-tosyloxy-1-hexyne (hex-5-yn-1-yl 4-methylbenzenesulfonate).
To explore
whether 12 was formed during the radiochemical synthesis of 6-[18F]fluorohex-1-yne and
whether it underwent click reaction with 7 during the synthesis of [18F]SFBTMGMB-Boc2,
compound 13 was synthesized by the click reaction of azide 7 and 12, which was obtained by
the reaction of the potassium salt of hex-5-yn-1-ol with the tosylate precursor 10. Indeed, the
retention time of 13 on HPLC was the same as that of the byproduct that eluted very close to
[18F]SFBTMGMB-Boc2. Although circumstantial, this suggests that the byproduct may very well
be 13. Having an NHS moiety present in its structure, the deprotected derivative of 13 can
compete with [18F]RL-I for reaction with the nanobody, which might explain the less than
expected yields for the coupling of [18F]RL-I to SdAb. While there is a possibility of formation of
8

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the bis-adduct by the click reaction of 7 on both ends of 12, it was assumed that such a product
would be considerably more polar than [18F]SFBTMGMB-Boc2. In considering the options to
eliminate the nuisance byproduct, we first thought of clicking it with 4-azidomethyl- benzoic acid
or benzene sulfonic acid to increase its polarity. Similar approaches have been reported
recently for a peptide radiopharmaceutical32 and amino acids.33 We opted to evaluate a better
scavenging agent, 4-(azidomethyl)-N,N,N-trimethylbenzenaminium triflate (14) assuming that
the clicked product 15 can be easily washed away with water from the ethereal extract of
reaction mixture. Compound 14 was synthesized by quaternization of the known compound 4-
dimethylamino benzyl azide,34 which was synthesized starting from 4-dimethylamino
benzaldehdye as detailed in the experimental section. An additional in situ click reaction using
14 as the azide partner, after the click reaction involving the 6-[18F]flurohex-1-yne and the azide
precursor 7 considerably reduced the formation of the closely eluting byproduct in the synthesis
of Boc2-[18F]SFBTMGMB, and improved the efficiency of coupling of [18F]RL-I to SdAb. This
clearly indicates that the larger amounts of compound 13, which has an active ester in its
structure, was consuming most of the SdAb, making it less available for conjugation with
[18F]RL-I.
Even with the use of 14 for scavenging the putative 13, there were occasional failures in
the coupling reaction. As noted before, like others,26 we used a fairly large amount of the
tosylate precursor, which presumably led to the formation of 12 and in turn, 13. It was possible
to reduce the amounts of precursor 10 considerably without concomitant loss of radiochemical
yields for the initial labeling step. When only about 9 mg versus 50 mg of the tosylate precursor
was used, radiochemical yields for the first step were not affected considerably. Although
reduction in the amount of 10 itself may reduce the formation 13, we continued to do the second
click reaction with 14 to ensure the absence of 13 during the coupling reaction with the SdAb.
An anti-HER2 SdAb (5F7) having no C-terminal cysteine and that exists exclusively in
monomeric form was labeled with [18F]RL-I (Scheme 2). The efficiency for conjugation of 5F7
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with [18F]RL-I synthesized using the optimized method (including the use of reduced amount of
tosylate precursor) was 31.2 ± 6.7% based on the initial activity of [18F]RL-I (n = 5). The specific
activity of the labeled protein was 74-444 MBq (2-12 mCi)/mg. TCA precipitation, SDS-
PAGE/phosphor imaging (Fig. 2), and ITLC indicated that more than 95% of radioactivity was
associated with the SdAb. The 5F7 SdAb labeled using [18F]RL-I ([18F]RL-I-5F7) bound
specifically to the HER2-positive magnetic beads with an immunoreactive fraction (IRF) in the
range of 62 - 84%. The [18F]RL-I-5F7 SdAb conjugate bound to BT474M1 cells with a Kd value
of 4.7 ± 0.9 nM (Fig. 3). Similar low nanomolar affinity values have been obtained for another
anti-HER2 SdAb radioiodinated by various methods.7 A Kd value of 0.29 nM, determined by
surface plasmon resonance assay, has been reported for the unlabeled 5F7 SdAb.35 Our
results indicate that SdAb 5F7 can be labeled using [18F]RL-I in reasonable yields, high specific
activity and radiochemical purity, and with the preservation of affinity and immunoreactivity.
For comparative evaluation, SdAb 5F7 was also conjugated with [18F]SFB ([18F]SFB-
5F7) and [125I]SGMIB ([125I]SGMIB-5F7). SdAb 5F7 was conjugated with [18F]SFB in an average
conjugation efficiency of 37.8 ± 11.5% (n = 2) with a specific activity of 370-740 MBq (10-20
mCi)/mg. TCA precipitation (96.8 ± 1.5%), SDS-PAGE/phosphor imaging (100%; Fig. 2), and
ITLC (99.0 ± 0.5%) indicated that more than 95% of radioactivity was associated with the SdAb
and it gave an IRF of 94.3%. Affinity determination by saturation binding assay gave a Kd value
of 4.07 ± 0.55 nM (Fig. 3), not statistically different to that determined for [18F]RL-I-5F7. The
prosthetic agent [125I]SGMIB was conjugated to 5F7 in 35.0 ± 11.2% (n = 4) yields with specific
activity of 148 – 185 MBq (4-5 mCi)/mg. ITLC (97.6 ± 2.8%; n = 3) and TCA precipitation (96.7
± 2.6%; n = 3) of [125I]SGMIB-5F7 indicated that more than 95% of radioactivity was associated
with intact SdAb and its IRF was 79.3 ± 16.5% (n =2). Saturation binding assay of [125I]SGMIB-
5F7 gave a Kd value of 5.02 ± 1.32 nM (Fig. S1).
10

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Conclusion
A novel residualizing label for 18F-labeling was synthesized using a click reaction
between [18F]fluorohexyne and an azide precursor containing an active ester and a guanidine
moiety. An anti-HER2 SdAb was labeled with 18F using this prosthetic agent in reasonable
yields with the preservation of affinity and immunoreactivity for HER2. The overall decay-
corrected radiochemical yield of the final labeled nanobody from aqueous fluoride is only about
2-3%. A considerable improvement in radiochemical yield will be required before use of this
agent on a routine basis can be considered. As an alternative, it may be possible to modify the
protein with the deprotected derivative of azide precursor 7 first, and then perform the click
reaction on the derivatized protein using the [18F]fluorohexyne. The copper-free click reaction
using 18F-labeled cyclooctyne-containing derivatives that are reported in the literature36, 37 may
fare better with proteins and we are currently pursuing such this strategy. As will be published
elsewhere, higher retention of 18F activity in HER2 expressing tumor cells was observed both in
cell culture and in a murine model for [18F]RL-I-5F7 compared with [18F]SFB-5F7.
Experimental
Chemistry
General. Chemicals and reagents were purchased from Sigma Aldrich (St. Louis, MO) unless
noted otherwise. Sodium [125I]iodide in 0.1 N NaOH with specific activities of 81.4 TBq (2200
Ci)/mmol was purchased from Perkin-Elmer Life and Analytical Sciences (Boston, MA). The
Boc-protected tin precursor of SGMIB and radioiodinated SGMIB were synthesized as reported
before.8, 38 N-succinimidyl 4-[18F]fluorobenzoate ([18F]SFB)16, 39 was synthesized following a
modified one-pot procedure.40 Aluminum-backed sheets (Silica gel 60 F254) used for analytical
TLC and silica gel 60 for normal-phase column chromatography were obtained from EM
Science (Gibbstown, NJ). In some cases, chromatography was also performed with the Biotage
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Isolera chromatography system (Charlotte, NC) using their pre-packed columns. Preparative
thick layer chromatography was used for small-scale purification with plates obtained from
Whatman (Clifton, NJ) or EM Science. High pressure liquid chromatography (HPLC) was
performed using the following two systems: 1) for radiolabeled compounds: a Beckman Gold
HPLC system equipped with a Model 126 programmable solvent module, a Model 166 NM
variable wavelength detector, a Model 170 radioisotope detector and a Beckman System Gold
remote interface module SS420X; data were acquired using the 32 Karat® software (Beckman
Coulter, Inc., Brea, CA). Recently, the gamma detector in this system was replaced with a
ScanRam RadioTLC scanner/HPLC detector combination (LabLogic; Brandon, Fl) and later
radio HPLC analyses were performed with that detector. 2) for analytical and semi-preparative
HPLC of unlabeled compounds, a Waters Model Delta 600 semi-preparative system with a
Model 600 controller and a Model 2487 dual wavelength absorbance detector; data were
acquired using Millenium software.
Normal phase HPLC, used for purification of
[18F]SFBTMGMB-Boc2, was performed using a 9.4 × 250 mm 5 µm Zorbax NH2 semi-
preparative column obtained from Agilent Technologies (Santa Clara, CA). Reversed-phase
HPLC was performed using a Waters 4.6 × 250-mm XTerra RP18 (5 µm) column and a 19 ×
150-mm XTerra RP18 (7 µm) column for analytical and semi-preparative runs, respectively.
PD-10 desalting columns for gel filtration were purchased from GE Healthcare (Piscataway, NJ).
Instant thin layer chromatography (ITLC) was performed using silica gel impregnated glass fiber
sheets (Pall Corporation, East Hills, NY) eluted with PBS, pH 7.4. The intact SdAb stays at the
origin under these conditions. Developed sheets were analyzed for radioactivity either using the
TLC scanner described above, or cutting the sheet into small strips and counting them in an
automated gamma counter (LKB 1282, Wallac, Finland or Perkin Elmer Wizard II, Shelton, CT).
Proton NMR spectra were obtained on a Varian 400 and 13C spectra on a Varian 500 (125.8
MHz) NMR spectrometer (Palo Alto, CA); chemical shifts are reported in δ units using the
residual solvent peak as reference. Mass spectra were recorded using an Agilent LCMS-TOF
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with DART, a high resolution mass spectrometer used for ESI, DART and LC-MS. Elemental
analyses were performed by Atlantic Microlabs, Inc. (Norcross, GA).
SdAb, cells, and culture conditions. Anti-HER2 SdAb 5F7, was a gift from Hilde Revets of
Ablynx (Ghent, Belgium). All reagents used for cell studies were obtained from Invitrogen
(Grand Island, NY). HER2-expressing BT474M1 human breast carcinoma cells41 were cultured
in DMEM/F12 medium containing 10% fetal calf serum (FCS), streptomycin (100 µg/mL), and
penicillin (100 IU/mL) (Sigma Aldrich, MO). Cells were cultured at 37°C in a humidified
incubator under 5% CO2 with media changed every two days. When about 80% confluent, cells
were sub-cultured by trypsinization (0.05 % Trypsin- EDTA).
2-(Trimethylsilyl)ethyl 3,5-dimethylbenzoate (2). A mixture of 3,5-dimethylbenzoic acid (5.0
g, 33.3 mmol), 2-(trimethylsilyl)ethanol (3.9 g, 33.3 mmol), DMAP (1.5 mg, 0.01 mmol), and
o
EDC (7.7 g, 40.0 mmol) in ethyl acetate (75 mL) was stirred at 20 C overnight. The reaction
mixture was washed with water and then with saturated NaHCO3. The ethyl acetate solution
was separated, dried over MgSO4, and ethyl acetate removed by rotary evaporation. The
residue was purified by chromatography using 10:1 hexanes:ethyl acetate to obtain 2 (6.6 g,
26.4 mmol, 79 % yield) as a an oil: 1H-NMR (CDCl3) δ 0.00 (s, 9H), 1.03 - 1.07 (t, 2H), 2.27 (s,
6H), 4.30 - 4.33 (t, 2H), 7.08 (s, 1H), 7.57 (s, 2H). 13C-NMR (CDCl3) δ 0.00, 19.50, 22.33,
64.10, 128.85, 132.05, 136.10, 139.48, 168.20. No tangible mass spectral data could be
obtained for this compound. Anal. Calcd for C14H22O2Si: C, 67.15;. H, 8.86. Found: C, 67.28; H,
8.77.
2-(Trimethylsilyl)ethyl 3,5-bis(bromomethyl)benzoate (3). A mixture of 2 (4.0 g, 16.0 mmol),
NBS (11.4 g, 63.9 mmol), and benzoyl peroxide (0.4 g, 1.6 mmol) in dichloroethane (75 mL)
was heated at reflux for 6 h. The reaction mixture was concentrated to dryness and the residue
triturated with warm hexanes. The resultant free-flowing solid was filtered, and the filtrate was
concentrated to dryness to give a semi-solid. It was dissolved in THF (100 mL) and the solution
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cooled to 0-5 °C. Diethyl phosphite (11.1 g, 80 mmol) and N,N-diisopropylethylamine (10.4 g,
80 mmol) were added to the above, and the reaction mixture stirred at 20 oC for 72 h. THF was
evaporated, and the residue partitioned between water and ethyl acetate. The organic layer
was separated, dried over MgSO4 and concentrated to dryness. The residue was purified by
chromatography using 10:1 hexanes:ethyl acetate to afford 3 (2.8 g, 6.9 mmol, 42.9 % yield) as
an oil: 1H-NMR (CDCl3) δ 0.07 (s, 9H), 1.11 - 1.23 (t, 2H), 4.38 - 4.43 (t, 2H), 4.48 (s, 4H), 7.58
(s, 1H), 7.96 (s, 2H). 13C-NMR (CDCl3) δ 0.00, 18.92, 33.37, 65.19, 131.40, 133.38, 135.12,
140.31, 167.02. No tangible mass spectral data could be obtained for this compound. Anal.
Calcd for C14H20Br2O2Si: C, 41.19;. H, 4.94. Found: C, 41.25; H, 4.76.
2-(Trimethylsilyl)ethyl
3-((1,2-bis(tert-butoxycarbonyl)guanidino)methyl)-5-
(bromomethyl)benzoate (4). A solution of potassium tert-butoxide in THF (1 M; 11 mL, 11
mmol) was added to a solution of 1,3-bis(tert-butoxycarbonyl)guanidine (1.4 g, 5.5 mmol) in
DMF (25 mL), and the mixture stirred at 20 oC for 30 min. The above was added slowly over a
period of 30 min to a stirred solution of 3 (4.5 g, 11.0 mmol) in DMF (25 mL), and the mixture
stirred at 20 oC for 17 h. The mixture was partitioned between water and ethyl acetate, and the
organics were dried over sodium sulfate, and concentrated. The residue was purified by
chromatography using 5:1 hexanes:ethyl acetate to yield 4 (2.1 g, 3.6 mmol, 32.5 % yield) as
solid: 1H-NMR (CDCl3) δ 0.08 (s, 9H), 1.11 - 1.15 (t, 2H), 1.37 (s, 9H), 1.49 (s, 9H), 4.38 - 4.46
(t, 2H), 4.41 (s, 2H), 5.22 (s, 2H), 7.51 (s, 1H), 7.88 (s, 1H), 7.95 (s, 1H), 9.25 (bs, 1H), 9.42 (bs,
1H). 13C-NMR (CDCl3) δ 0.01, 18.91, 29.34, 29.76, 33.97, 48.51, 64.96, 80.44, 86.05, 129.97,
130.18, 132.63, 133.98, 139.58, 141.41, 155.14, 162.02, 166.06, 167.53. LRMS (DART) m/z:
588.2 and 586.2 (M+H)+, 532.1 and 530.1 (M-tBu); HRMS (DART) calcd for C25H4179BrN3O6Si
(M+H)+ 586.11948, found 586.1942 ± 0.0011 (n = 4).
2-(Trimethylsilyl)ethyl
3-(azidomethyl)-5-((1,2-bis(tert-
butoxycarbonyl)guanidino)methyl)benzoate (5). A mixture of 4 (1.2 g, 2.1 mmol) and
o
sodium azide (0.13 g, 2.1 mmol) in DMF (25 mL) was stirred at 80 C for 1.5 h. The reaction
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mixture was cooled to 20 oC, and partitioned between ethyl acetate and water. The pooled ethyl
acetate solution was dried with MgSO4, and concentrated to dryness to afford 5 (1.1 g, 2.1
mmol, 98 % yield) as a white foamy solid. 1H-NMR (CDCl3) δ 0.08 (s, 9H), 1.11-1.15 (t, 2H),
1.37 (s, 9H), 1.49 (s, 9H), 4.37-4.45 (t, 2H), 4.27 (s, 2H), 5.20 (s, 2H), 7.50 (s, 1H), 7.87 (s, 1H),
7.93 (s, 1H), 9.25 (bs, 1H), 9.42 (bs, 1H). 13C NMR (CDCl3) δ 0.00, 18.92, 29.32, 29.76, 48.51,
55.80, 64.97, 80.43, 86.00, 129.35, 130.00, 132.61, 133.11, 137.29, 141.45, 156.16, 162.06,
165.08, 167.65. LRMS (DART) m/z: 549.3 (M+H)+, 493.2 (M-tBu)+; HRMS (DART) calcd for
C25H41N6O6Si (M+H)+ 549.2857, found 549.2844 ± 0.0005 (n = 4).
2-(Trimethylsilyl)ethyl
3-((1,2-bis(tert-butoxycarbonyl)guanidino)methyl)-5-((4-(4-
fluorobutyl)-1H-1,2,3-triazol-1-yl)methyl)benzoate (6). A solution of 5 (50 mg; 0.09 mmol) in
DMF (3 mL) was mixed with a solution of 6-fluorohex-1-yne26 (excess) in dichloromethane. The
click reaction reagent was prepared as follows: A solution of CuSO4·5H2O (10 mg, 0.04 mmol)
in water (100 μL) was mixed with an aqueous solution of sodium ascorbate (30.0 mg, 0.15
mmol) (100μl). When the color of the mixture changed from black to yellow, a solution of the
disodium salt of bathophenanthrolinedisulfonic acid (hydrate) (12.0 mg, 20.0 μmol) in 1:4 (v/v)
DMF/H2O (200 μL) was added. A 100 μL portion of the click reaction reagent was added to the
substrate solution, and the final mixture was heated at 80 °C for 30 min in a sealed reaction
vessel. The reaction mixture was diluted with ethyl acetate and the solution washed with brine.
The pooled ethyl acetate solution was dried using anhydrous MgSO4, ethyl acetate evaporated
from the filtrate and the residue subjected to preparative TLC using 1:1 ethyl acetate:hexanes to
1
obtain 32 mg (0.03 mmol, 54.1 % yield) of 6 as a solid: H-NMR (CDCl3) δ 0.06 (s, 9H), 1.08 -
1.16 (t, 2H), 1.32 (s, 9H), 1.43 (s, 9H), 1.56 - 1.93 (m, 2H), 1.97 - 2.15 (m, 2H), 2.97 - 3.05 (t,
2H), 4.38 - 4.60 (m, 6H), 5.72 (s, 2H), 7.62 (s, 1H), 7.98 (s, 1H), 8.05 (s, 1H), 8.13 (s, 1H), 9.18
- 9.48 (bs, 2H). 13C NMR (CDCl3) δ 0.08, 18.79, 26.72, 29.44, 29.71, 49.52, 49.67, 55.16,
65.15, 80.55, 84.16, 86.13, 121.72, 122.34, 129.33, 130.44, 133.04, 136.70, 141.91, 149.89,
156.10, 162.03, 165.06, 167.48. LRMS (DART) m/z: 649.35 (M+H)+, 549.30 (M-Boc)+, 449.25
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(M-2Boc)+. HRMS (DART) calcd for C31H50FN6O6Si (M+H)+ 649.3545, found: 649.3537 ±
0.0002.
N-Succinimidyl
3-(azidomethyl)-5-((1,2-bis(tert-
butoxycarbonyl)guanidino)methyl)benzoate (7). A solution of tetrabutyl ammonium fluoride
in THF (1 M; 2.2 mL, 2.2 mmol) was added to a solution of 5 (1.0 g, 1.8 mmol) in THF (25 mL)
and the homogeneous mixture stirred at 20 oC for 16 h. THF was evaporated, and the residue
was partitioned between water and ethyl acetate. The pooled ethyl acetate solution was dried
over MgSO4, and the filtrate concentrated to dryness. The crude intermediate was taken in
ethyl acetate (25 mL), and N-hydroxysuccinimide (0.2 g, 1.8 mmol), DMAP (0.02 g, 0.2 mmol),
o
and EDC (0.35 g, 1.8 mmol) were added. The reaction mixture was stirred at 20 C for 16 h.
Water was added to the reaction mixture and layers were separated. The ethyl acetate layer
was dried with Na2SO4 and the filtrate concentrated using a rotary evaporator. The crude
product was purified by silica gel chromatography using 2:1 hexanes:ethyl acetate to obtain 7
1
(337 mg, 0.62 mmol, 33.9 % yield) as a white solid: H-NMR (CDCl3) δ 1.38 (s, 9H), 1.45 (s,
9H), 2.89 (s, 4H), 4.40 (s, 2H), 5.20 (s, 2H), 7.66 (s, 1H), 7.95 (s, 1H), 8.02 (s, 1H), 9.22 (bs,
1H), 9.42 (bs, 1H). 13C NMR (CDCl3) δ 25.69, 27.93, 28.28, 46.92, 54.04, 79.06, 84.93, 125.59,
128.70, 129.72, 133.97, 136.73, 140.63, 154.57, 160.40, 161.45, 163.52, 169.09. LRMS
(LCMS-ESI) m/z: 584.2 (M+K)+, 568.2 (M+Na)+, 546.2 (M+H)+, 490.2 (M-Bu)+, 434.1, 390.2.
HRMS (DART) calcd for C24H32N7O8 (M+H)+ 546.2312, found 546.2302 ± 0.0009 (n = 4).
N-succinimidyl 3-((1,2-bis(tert-butoxycarbonyl)guanidino)methyl)-5-((4-(4-fluorobutyl)-1H-
1,2,3-triazol-1-yl)methyl)benzoate (8). Method A. Tetrabutyl ammonium fluoride (1M in THF;
0.05 mL, 0.05 mmol) was added to a solution of 6 (30.0 mg, 0.05 mmol) in dry THF (25 mL) that
was cooled to 0-5°C and the resultant solution was stirred under argon overnight. THF was
evaporated and the residual material was partitioned between EtOAc and water. Pooled ethyl
acetate fractions were dried over MgSO4, and the filtrate was concentrated to dryness. DMAP
(0.85 mg, 6.94 µmol), EDC (13.30 mg, 0.07 mmol) and N-hydroxysuccinimide (7.98 mg, 0.07
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mmol) were added a solution of the crude intermediate product dissolved in 25 mL of
dichloromethane. The solution was stirred at 20oC for 4 h, washed sequentially with water and
saturated NaHCO3. Dichloromethane solution was dried over MgSO4, filtered and the filtrate
concentrated. The crude product was purified by preparative TLC using 1:1 hexanes:ethyl
acetate as the mobile phase to yield 12 mg (0.02 mmol, 40.2% yield) of 8 as a foamy solid.
Method B. A dichloromethane solution of 6-fluorohex-1-yne (excess) was added to a solution of
7 (35.0 mg, 0.06 mmol) in 3 mL of DMF. The click reagent (100 μL) prepared as described in a
previous section was added to the above mixture and the sealed vial was heated at 80 °C for 30
min. The reaction mixture was partitioned between a large volume of ethyl acetate and water.
The ethyl acetate solution was dried over MgSO4, filtered and the filtrate concentrated to
dryness. The crude product was purified by preparative TLC using 2:1 hexanes:ethyl acetate to
1
obtain 18 mg (0.03 mmol, 44 % yield) of 8. H-NMR (CDCl3) δ 1.36 (s, 9H), 1.46 (s, 9H), 1.57 -
1.62 (m, 2H), 1.68 - 1.76 (m, 2H), 2.70 - 2.73 (t, 2H), 2.88 (s, 4H), 3.60 - 3.64 (t, 2H) 5.16 (s,
2H), 5.51 (s, 2H), 7.22 (s, 1H), 7.63 (s, 1H), 7.86 (s, 1H), 8.05 (s, 1H), 9.30-9.45 (bs, 2H). 13C
NMR (CDCl3) δ 24.00, 24.16, 24.63, 26.86, 27.24, 28.90, 45.80, 52.29, 78.06, 82.09, 83.04,
83.95, 119.59, 125.92, 127.55, 129.14, 133.01, 134.95, 140.01, 153.40, 159.25, 160.15, 162.39,
167.99. LRMS (LCMS-ESI) m/z: 645.29 (M+). HRMS (DART) calcd for C30H41FN7O8 (M+H)+
646.3001, found 646.2990 ± 0.0012 (n = 4).
N-Succinimidyl
3-((4-(4-fluorobutyl)-1H-1,2,3-triazol-1-yl)methyl)-5-
(guanidinomethyl)benzoate (9). TFA (1 mL) was added to 50 mg (0.08 mmol) of 8 and the
resultant clear solution was left at 20oC. The reaction was followed by reversed-phase HPLC.
For this an analytical column was eluted with a gradient of mobile phase consisting of 0.1% TFA
in water (solvent A) and 0.1% TFA in acetonitrile (solvent B) at a flow rate of 1 mL/min; the
proportion of B was linearly increased from 5% to 100% over a period of 30 min. Under these
conditions, the retention times of 8 and 9 were 25.0 min and 22.2 min, respectively. HPLC of an
aliquot of reaction mixture at 30 min indicated a single peak (>98%) corresponding to the
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product. TFA was evaporated from the reaction mixture. To insure complete removal of TFA,
three 100 µL aliquots of dichloromethane were added, evaporating each time. The sample was
taken in acetonitrile and subjected to semi-preparative HPLC using the same gradient but at a
flow rate of 7 mL/min. HPLC fractions containing the product (tR = 14.3 min) were pooled, and
solvents evaporated to obtain 41 mg (95%) of a glassy solid of 9: Analytical HPLC of this
indicated a purity of 95.5%; a minor peak of lower tR, presumably the acid resulting from
hydrolysis of the product during HPLC purification or concentration was seen. 1H-NMR
(CD3CN) δ 1.72 (br m, 4 H), 2.72 (m, 2H), 2.86 (s, 4H), 4.35 – 4.45 (m, 4H), 5.64 (s, 2H), 6.60
(br s, 4H), 7.45-8.03 (m, 4H), 13.5 (br s, 1H). 13C NMR (CDCl3) δ 24.13, 24.98, 25.66, 29.5,
44.08, 53.08, 83.39, 84.80, 125.96, 128.88, 129.18, 133.31, 137.69, 138.82, 157.63, 161.74,
170.29. LRMS (LCMS-ESI) m/z: 446.3 (M+H)+. HRMS (DART) calcd for C20H25FN7O4 (M+H)+
446.1952, found 446.1948 ± 0.0003 (n = 4).
6-(Hex-5-yn-1-yloxy)hex-1-yne (12). The title compound42 was synthesized as follows:
Potassium tert-butoxide (1M in THF; 41.6 mL, 41.6 mmol) was added to a solution of hex-5-yn-
1-ol (3.27 g, 33.3 mmol) in 50 mL of DMF and the mixture stirred at 20oC for 30 min. Hex-5-yn-
1-yl 4-methylbenzenesulfonate (7.00 g, 27.7 mmol), prepared by adapting literature protocols,26,
43 was added to the above, and the mixture stirred at 20oC overnight. The reaction mixture was
partitioned between ether and water, the pooled ethereal layers were dried over Na2SO4 and the
filtrate concentrated to dryness. The residue was purified by silica gel chromatography using
5:1 (v/v) hexanes:ethyl acetate as the mobile phase to afford 12 (3.3 g, 18.51 mmol, 66.7 %
yield) as a clear oil. 1H-NMR (CDCl3) δ 1.53 - 1.69 (m, 8H), 1.91 (t, 2H), 2.17 - 2.21 (m, 4H),
3.39 (s, 4H).
N-Succinimidyl
3-((1,3-bis(tert-butoxycarbonyl)guanidino)methyl)-5-((4-(4-(hex-5-yn-1-
yloxy)butyl)-1H-1,2,3-triazol-1-yl)methyl)benzoate (13). Compounds 7 (50.0 mg, 0.09 mmol)
and 12 (49.0 mg, 0.28 mmol) were dissolved in 3.0 mL of DMF. A 100 μL aliquot of click
reaction reagent (vide infra) was added and the sealed vial was heated at 80 °C for 30 min. The
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reaction mixture was partitioned between ethyl acetate and water. The ethyl acetate layer was
dried with MgSO4, filtered, and the filtrate was concentrated to dryness. The crude product was
purified by preparative TLC using using 2:1 hexanes:ethyl acetate as the mobile phase to yield
21 mg (0.03 mmol, 31.7%) of a solid: 1H-NMR (CDCl3) δ 1.38 (s, 9H), 1.48 (s, 9H), 1.53 - 1.74
(m, 10H), 1.94 (t, 1H), 2.20 (m, 2H), 2.91 (s, 4H), 3.39 - 3.42 (tt, 4H) 5.18 (s, 2H), 5.62 (s, 2H),
7.22 (s, 1H), 7.65 (s, 1H), 7.93 (s, 1H), 8.09 (s, 1H), 9.22 (bs, 1H), 9.43 (bs, 1H). 13C NMR
(CDCl3) δ 18.24, 25.24, 25.49, 25.68, 27.81, 28.00, 28.19, 28.38, 28.77, 53.31, 70.24, 70.48,
79.09, 84.98, 120.23, 120.88, 125.94, 128.71, 130.08, 130.24, 133.97, 136.06, 141.05, 148.99,
154.46, 160.31, 161.22, 163.44, 169.04. LRMS (DART) m/z: 746.3 (M+Na)+, 724.4 (M+H)+,
624.3 (M-Boc)+. HRMS (DART) calcd for C36H50N7O9 (M+H)+ 724.3670, found 724.3655 ±
0.0012 (n = 4).
4-(azidomethyl)-N,N,N-trimethylbenzenaminium trifluoromethane sulfonate (14).
The
known compound 4-dimethylamino benzyl alcohol44, 45 was synthesized by sodium borohydride
reduction of the corresponding aldehyde. Sodium borohydride (1.18 g, 31.2 mmol) was added
to a cooled (0 - 5oC) solution of 4-(dimethylamino)benzaldehyde (3.10 g, 20.78 mmol) in THF
(55 mL) containing 100 µL MeOH. The mixture was stirred at 20oC overnight, partitioned
between ether and water and the ethereal layer was separated and dried over MgSO4. The
drying agent was filtered, and the solvents from the filtrate were evaporated to afford 3.0 g
1
(19.84 mmol, 95 % yield) of the alcohol as a pure solid: H-NMR (CDCl3) δ 2.2 (s, 6H), 3.81 (s,
2H), 6.61 (d, 2H), 7.05 (d, 2H). DBU (2.72 ml, 18.05 mmol) and 2-azido-1,3-dimethyl-4,5-
dihydro-1H-imidazol-3-ium hexafluorophosphate(V) (4.75 g, 16.67 mmol) were added to a
solution of the above alcohol (2.10 g, 13.89 mmol) in 65 mL THF, and the reaction mixture was
stirred at 20oC for 10 min. The reaction was quenched with the addition of saturated aqueous
NH4Cl, and organic materials were extracted from it with dichloromethane. The combined
extracts were washed with brine and then dried over anhydrous Na2SO4. The solvent from the
filtrate was removed in vacuo to afford the crude compound, which was purified by silica gel
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chromatography using 5:1 (v/v) hexanes/ethyl acetate to give 4-(azidomethyl)-N,N-
1
dimethylaniline34, 46 (2.1 g, 11.92 mmol, 86% yield) as a white solid: H-NMR (CDCl3) δ 2.87 (s,
6H), 3.78 (s, 2H), 6.66 (d, 2H), 7.25 (d, 2H). Methyl trifluoromethanesulfonate (1.02 g, 6.24
mmol) was added to a rapidly stirred solution of 4-(azidomethyl)-N,N-dimethylaniline (1.0 g, 5.67
mmol) in 60 mL of anhydrous ether. There was an immediate formation of an off white
precipitate. The heterogeneous mixture was stirred for 15 min and the precipitate was filtered,
washed with a large volume of ether, and dried under high vacuum to obtain 1.50 g (4.41 mmol,
78 % yield) of 14 as a tan solid: 1H-NMR (CD3OD) δ 3.65 (s, 9H), 4.49 (s, 2H), 7.62 (d, 2H),
7.92 (d, 2H). 13C-NMR (CD3CN) δ 52.98, 57.10, 120.66, 130.04, 138.98. LRMS (LCMS-ESI)
m/z: 191.1 (M+), 173.1, 163.1 (M-N2)+, 148.1. HRMS (DART) calcd for C10H15N4 (M+) 191.1291;
found 191.1295 ± 0.0001 (n = 4).
N-Succinimidyl
3-((4-(4-[18F]fluorobutyl)-1H-1,2,3-triazol-1-yl)methyl)-5-
(guanidinomethyl)benzoate ([18F]9)-Optimized procedure. A) Synthesis of 6-[18F]fluorohex-
1-yne. Fluorine-18 was obtained either by in house cyclotron irradiation of [18O]H2O as
described before47 or from PET-NET solutions (Durham, NC). For labeling reactions, 18F activity
trapped in a QMA cartridge was eluted with a mixture of Kryptofix (28 mg) and potassium
carbonate (2.4 mg) in 0.75 mL of 95% acetonitrile, and dried by azeotroping with acetonitrile
three times. A solution of hex-5-yn-1-yl 4-methylbenzenesulfonate (9 mg, 35 µmol) was added
to the reaction vial containing dried 18F activity (1.85 – 7.4 GBq; 50 – 200 mCi), and the reaction
vial heated at 110ºC for 20 min. The reaction vial was constantly purged with a gentle stream of
argon to hasten the distillation of the product. Collection of 6-[18F]fluorohex-1-yne was
facilitated using a 19-guage teflon tubing inserted into the headspace of the reaction vial
through a septum. The other end of the tubing was attached to the first of two 3 mL Wheaton
vials, each containing 200 µL of dimethylformamide. The two vials were connected with another
piece of the tubing. In each case, the tubing was immersed into the dimethylformamide so as to
trap 6-[18F]fluorohex-1-yne. After 20 min, the first Wheaton vial containing most of the
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radioactivity was used for the next step.
B) Click reaction. Compound 7 (3 mg, 11.00 µmol) was added to the above DMF solution of 6-
[18F]fluorohex-1-yne. The click reaction reagent prepared as described above (25 μL) was
added, and the mixture heated at 50°C for 15 min. A 7 mg aliquot of 4-(azidomethyl)-N,N,N-
trimethylbenzenaminium trifluoromethane sulfonate (14) and 100 μL of click reaction reagent
were added and the mixture heated for an additional 15 min. The reaction mixture was diluted
with ether, and the ethereal layer washed with brine and dried by passage over an anhydrous
sodium sulfate cartridge (Agilent Technologies, Santa Clara, CA; part no. 12132044). Ether
was evaporated to less than 500 µL using a stream of argon and the concentrate was purified
using normal phase HPLC. For this, the normal phase semi-preparative column was eluted at a
flow rate of 3 mL/min with a gradient consisting of 0.2% (v/v) acetic acid in both hexane
(solvent A) and ethyl acetate (solvent B); the composition of solvent B was linearly increased
from 30% to 100% over 30 min. A couple of experiments was performed to investigate whether
unreacted 7 could be scavenged with polymer-bound alkyne.28 For this, after the initial click
reaction with 7 and 6-[18F]fluoro-hex-1-yne, 10 mg (>1 equiv.) of the polymer and 100 μL of the
click reaction reagent were added and the mixture heated at 50oC for 15 min. The mixture was
processed as above but using ethyl acetate instead of ether for extraction. Ethyl acetate extract
was filtered to remove any residual polymer before concentration and injection onto HPLC.
Radiolabeling SdAb 5F7. SdAb 5F7 was radioiodinated using [125I]SGMIB as reported before7
and the SdAb was labeled with 18F using [18F]SFB adapting procedures used for conjugation
labeling of proteins with N-succinimidyl ester-containing prosthetic groups.48, 49 In these two
cases, 50 µL of 5F7 (2 mg/mL) solution in 0.1M borate buffer, pH 8.5 was used. For labeling
5F7 using [18F]RL-I, the HPLC fractions containing Boc2-[18F]SFBTMGMB were evaporated to
dryness using a stream of argon and the residual radioactivity was treated with 100 µL of TFA.
After 10 min at 20oC, TFA was evaporated from the mixture, and to insure complete removal of
TFA, 50 µL of ethyl acetate was added to the reaction mixture and evaporated three times.
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SdAb 5F7 in borate as above (100 µL; 200 µg) was added to [18F]RL-I as obtained above, and
the mixture incubated at 20oC for 20 min. The entire mixture was loaded onto a gel filtration
column (PD10; GE Healthcare, Piscataway, NJ), and the column eluted with PBS, pH 7.4,
collecting 250 µL fractions. The protein typically eluted in fractions 5-10.
Determination of radiochemical purity. Three tests were performed to determine protein-
associated radioactivity in a single, or when appropriate, in a paired-label format. TCA
precipitability was determined by incubating about 5 ng of each labeled SdAb with 800 μL of 2%
bovine serum albumin (BSA) and 100 μL of 20% TCA at 20 °C for 15 min. The mixtures were
pelletted, and both pellets and supernatants counted for radioactivity. Protein-associated
radioactivity was the percentage of total radioactivity (pellet plus supernatants) that was present
in the pellet. ITLC was performed by eluting silica gel-impregnated glass fiber sheets (Pall
Corporation, East Hills, NY) with PBS, pH 7.4. Under these conditions, the labeled protein
remained at the origin and small molecular weight components eluted with an Rf of 0.7 – 0.8.
The sheets were cut into small strips and counted for radioactivity. The integrity of labeled
proteins was further assessed by SDS-PAGE under nonreducing conditions and subsequent
phosphor imaging using a Storage Phosphor System Cyclone Plus phosphor imager (Perkin-
Elmer Life and Analytical Sciences, Downers Grove, IL, USA) as previously described.12
Immunoreactivity of 5F7 after labeling. The immunoreactivity of the labeled proteins was
determined by the Lindmo assay using magnetic beads coated with the extracellular domain of
HER2, or as control for nonspecific binding, with BSA.12, 48, 50
Determination of affinity (KD). The binding affinity of [18F]RL-I-5F7 and [18F]SFB-5F7 to HER2
on BT747M1 cells was determined using a saturation binding assay. BT474M1 cells were
plated in 24-well plates at a density of 8 × 104 cells/well and incubated at 37oC for 24 h. The
plates were kept at 4°C for 30 min, supernatants were removed, and the cells were washed
twice with 1 mL cold PBS. Cells were then incubated in triplicate with increasing concentrations
(0.05 – 100 nM; final volume 600 μL) of [18F]RL-I-5F7 or [18F]SFB-5F7 for 1 h at 4°C. A parallel
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assay was performed in which cells were co-incubated with 10 µM of HER2-specific
trastuzumab to determine nonspecific binding at each concentration. The medium containing
unbound radioactivity was removed, and the cells were washed twice with cold PBS. Finally,
the cells were solubilized by treatment with 1N NaOH (0.5 mL) at 37°C for 10 min. Cell-
associated 18F activity was determined using the automated gamma counter. The data were
fitted using GraphPad Prism software to determine KD values.
Acknowledgements
This work was supported in part by National Institutes of Health grants CA188177 and
CA42324. The authors want to thank Hilde Revets of Ablynx (Brussels, Belgium) for providing
5F7 SdAb and Marc Hens, who helped synthesizing some intermediates.
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Figure Captions
Figure 1. Quality Control HPLC of Boc2-[18F]SFBTMGMB.
Figure 2. SDS-PAGE (non-reducing)/Phophor image profile of [18F]RL-I-5F7 (lane 1)
and of [18F]SFB-5F7 (lane 2).
Figure 3. Saturation binding curves for [18F]SFB-5F7 and [18F]RL-I-5F7 obtained using
BT474M1 cells.
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O
O
O
O
SiMe3
COOH
a
1
2
b
O
O
SiMe3
SiMe3
c
Br
Br
Br
BocN
NH2
NBoc
4
3
d
O
O
O
O
SiMe3
e
SiMe3
NH2
N3
BocN
NH2
N
BocN
N
NBoc
NBoc
N
6
5
4
F
f
f
O
O
O
O N
O
O N
e
O
O
RN
NH2
NR
N3
BocN
NH2
NBoc
N
N
N
4
8
(R = Boc)
7
F
g
9
R = H
a) TMS-ethanol, EDC, DMAP b) i.NBS,
benzoyl peroxide ii. (EtO)2HPO, DIEA c)
Bis-Boc-guanidine, Pot. t-butoxide d) NaN3
e) 6-fluoro-hex-1-yne, CuSO4, sodium
ascorbate, BPDS f) i. TBAF ii. NHS, EDC,
DMAP g) TFA
Scheme 1. Synthesis of SFBTMGMB
and its azide precursor
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