Applied Radiation and Isotopes 69 (2011) 410–414
`
`Contents lists available at ScienceDirect
`
`Applied Radiation and Isotopes
`
`journal homepage: www.elsevier.com/locate/apradiso
`
`Automated radiochemical synthesis of [18F]FBEM: A thiol reactive synthon
`for radiofluorination of peptides and proteins
`
`Dale O. Kiesewetter n, Orit Jacobson, Lixin Lang, Xiaoyuan Chen
`
`Laboratory of Molecular Imaging and Nanomedicine (LOMIN), National Institute of Biomedical Imaging and Bioengineering (NIBIB), National Institutes of Health (NIH), Building 10,
`Room 1C401, MSC 1180, Bethesda, MD 20892, USA
`
`a r t i c l e i n f o
`
`a b s t r a c t
`
`Article history:
`Received 14 June 2010
`Received in revised form
`30 August 2010
`Accepted 30 September 2010
`
`Keywords:
`PET
`Fluorine-18
`Thiol reactive synthon
`[18F]FBEM
`
`1.
`
`Introduction
`
`The automated radiochemical synthesis of N-[2-(4-[18F]fluorobenzamido)ethyl]maleimide ([18F]FBEM,
`IUPAC name: N-maleoylethyl-4-[18F]fluorobenzamide), a prosthetic group for radiolabeling the free
`sulfhydryl groups of peptides and proteins, is herein described. 4-[18F]fluorobenzoic acid was first
`prepared by nucleophilic displacement of a trimethylammonium moiety on a pentamethylbenzyl
`benzoate ester with [18F]fluoride. In the second step the ester was cleaved under acidic conditions.
`Finally, 4-[18F]fluorobenzoic acid was coupled to N-(2-aminoethyl)maleimide using diethylcyanopho-
`sphate and diisopropylethyl amine. Following high-performance liquid chromatography (HPLC)
`purification, [18F]FBEM was obtained in 17.3 7 7.1% yield (not decay corrected) in approximately
`95 min. Isolation from the HPLC eluate and preparation for subsequent use, which was conducted
`manually, required an additional 10–15 min. The measured specific activity for three batches was
`181.3, 251.6, and 351.5 GBq/ mmol at the end of bombardment (EOB).
`
`Published by Elsevier Ltd.
`
`Radiolabeled peptides and proteins are increasingly being studied
`as imaging agents for a wide variety of cellular targets in cancer
`research and other biological processes. The radionuclides employed
`for application to positron emission tomography (PET) imaging
`include fluorine-18, bromine-76,
`iodine-124, as well as several
`metallic radionuclides. Radiolabeling with fluorine-18 has been one
`of our interests because of the favorable nuclear decay properties of
`fluorine-18 (b + 0.635 MeV, 97% abundance, t½ 109.8 min).
`Peptides have been radiolabeled by electrophilic radiofluor-
`ination (Ogawa et al., 2003) and by nucleophilic substitution on
`aromatic rings highly activated toward nucleophilic aromatic
`substitution (Becaud et al., 2009). While there are a few examples
`of direct fluorination of appropriately functionalized peptides
`with [18F]fluorine sources, most peptides and proteins are not
`expected to be compatible with the conditions required. The use
`of small prosthetic groups, labeled with fluorine-18, that contain a
`functional group reactive toward either the e-amine of lysine
`residues or the sulfhydryl of cysteine residues has dominated the
`field of peptide and protein labeling (Wester and Schottelius,
`2007). N-hydroxysuccinimidyl esters of fluorinated carboxylic
`acids have been employed to form peptide and protein amides at
`terminal amines of lysine residues and the terminal alpha amines
`(Vaidyanathan and Zalutsky, 2006).
`
`n Corresponding author. Tel.: + 1 301 451 3531; fax: + 1 301 402 3521.
`E-mail address: dk7k@nih.gov (D.O. Kiesewetter).
`
`0969-8043/$ - see front matter Published by Elsevier Ltd.
`doi:10.1016/j.apradiso.2010.09.023
`
`Likewise, fluorinated maleimide prosthetic groups have been
`used to functionalize free sulfhydryls of cysteine residues. A number
`of
`these prosthetic groups have been reported. Shiue et al.
`(1988) reported two 18F-labeled maleimides, 1(4-[18F]fluorophenyl)
`pyrrole-2,5-dione ([18F]FPPD) and N-[3-(2,5-dioxo-2,5-dihydro-
`pyrrol-1-yl)phenyl]-4-[18F]fluorobenzamide ([18F]DDPFB) in abstract
`form, but no applications have been published. de Bruin et al. (2005)
`reported the preparation of the heteroaromatic [18F]maleimide,
`1-[3-(2-[18F]fluoropyridine-3-yloxy)propyl]pyrrole-2,5-dione
`([18F]FPyME). [18F]FPyME was prepared in three steps; the first step
`was nucleophilic heteroaromatic substitution by [18F]fluoride on a
`nitro or trimethylammonium substituted pyridine,
`followed by
`deprotection of a primary amine, and then formation of the
`maleimide. Optimized conditions provided 17–20% (uncorrected)
`radiochemical yield of [18F]FPyME from [18F]fluoride in 110 min,
`including high-performance liquid chromatography (HPLC) purifica-
`tion. The procedure has no mention of using an automated device.
`N-[4-[(4-[18F]fluorobenzylidene)aminooxy]butyl]-maleimide has
`also been prepared as an alternative for 18F-labeling of sulfhydryls
`(Toyokuni et al., 2003). This compound was prepared in two synthetic
`steps, the first was the synthesis of 4-[18F]fluorobenzaldehyde and
`subsequently coupling to N-[4-(aminooxy)butyl]maleimide. The
`synthesis, with no indication of automation, provided an overall
`radiochemical yield after the two synthetic steps and HPLC
`purification of  35% (decay corrected) in approximately 60 min.
`N-[6-(Aminooxy)hexyl]maleimide has been employed to make
`two other fluorinated maleimide prosthetic groups. One resulted
`from coupling to [18F]fluorobenzaldehyde (Berndt et al., 2007).
`The radiosynthesis, conducted in a Nuclear Interface fluorination
`
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`
`411
`
`module, provided the product following a two-step sequence in a
`single reaction vessel. The overall yield was  29% (decay
`corrected) in a synthesis time of 69 min. The second tracer
`resulted from coupling with glucose (Wuest et al., 2008). The
`reported radiochemical yield was 42% (decay corrected) from
`[18F]FDG in 45 min. The coupling with [18F]FDG may result in
`several isomeric products.
`There are two published syntheses of N-[2-(4-[18F]fluorobenza-
`mido)ethyl]maleimide ([18F]FBEM, IUPAC name: N-maleoylethyl-4-
`[18F]fluorobenzamide). One of the syntheses, published by Cai et al.
`(2006), first prepared N-hydroxysuccinimidyl [18F]fluorobenzoate
`([18F]SFB), which is a three-step reaction, and reacted that product
`with N-(2-aminoethyl)maleimide (upper scheme in Fig. 1). The
`reported radiochemical yield based on [18F]fluoride was 572% in a
`synthesis time of  150 min. We have developed an automated
`method to conduct our synthesis of [18F]FBEM based on our
`previously published manual procedure (lower scheme in Fig. 1)
`(Kiesewetter et al., 2008) and that of the previously published
`radiochemical synthesis of [18F]fluoropaclitaxel (Kiesewetter et al.,
`2003; Kalen et al., 2007). The radiochemical synthesis of [18F]FBEM
`was accomplished in three radiochemical steps using a two-pot
`[18F]Fluoride
`synthetic
`sequence (lower
`scheme of Fig. 1).
`displacement of the trimethylammonium moiety of the substrate
`was conducted in the first reaction vessel; acidolysis of
`the
`pentamethylbenzyl protecting group of
`the benzoic acid and
`the resulting [18F]fluorobenzoic acid with N-(2-
`coupling of
`aminoethyl)maleimide using diethylcyanophosphonate
`as
`the
`coupling reagent were conducted in the second reaction vessel. The
`resulting product was purified by HPLC, isolated from the HPLC
`eluate, and utilized for coupling with free sulfhydryl peptides.
`This procedure, described in detail below, clearly represented
`an improvement compared to the earlier procedure of Cai et al.
`(2006) for the preparation of the same maleimide prosthetic
`group. This procedure required fewer chemical synthesis steps,
`provided higher yield (1776% uncorrected), and required less
`time ( 115 min from [18F]fluoride availability) to have product
`ready for the peptide coupling reaction.
`
`2. Materials and methods
`
`2.1. Hardware and software
`
`We procured a multi-module configuration from Eckert & Ziegler
`Eurotope GMBH (Berlin) and the corresponding Modular Laboratory
`Software controller. Initial programming was suggested by Eckert &
`Ziegler personnel but was modified by us to result in the successful
`synthesis of FBEM. The modules acquired included two Peltier
`Reaction Modules (PRM) with pneumatic lifts, internal radioactivity
`detectors, and stirrer; one solenoid valve module (SVM, 2-way, 5
`valves per module); three SVMs (3-way, 5 valves per module); two
`single stopcock modules (SSM) with Teflon valves (3-way, 3 valves
`per module); two vial holder modules (VHM) with extra vial holder
`plates and connector for HPLC module; one KNF vacuum pump; one
`HPLC module (including injector, pump control, and radioactivity
`detector), one Knauer Model K120 isocratic HPLC pump (Knauer
`GmbH, Berlin, Germany); and one Knauer Model 200 UV detector
`(Knauer GmbH, Berlin, Germany).
`The Peltier Reactor Modules could be used with standard
`v-vials from Wheaton or Alltech of 2–5 mL sizes or with an 11 mL
`Sigradur reactor vial. We used the Sigradur reaction vial for the
`fluoride displacement reaction. The Peltier Reactor Modules can heat
`and cool rapidly; we employed it to heat as high as 120 1C and to cool
`to 0 1C. Evaporations were conducted with vacuum, argon flow, or a
`combination of the two methods. Two manifolds for argon, one at
`1.2 bar and the second at 0.6 bar, were set up. The higher pressure
`was used for evaporations and liquid transfers and the lower pressure
`was used for reagent addition. V-vials (1 mL) were used to contain
`reagents and solvents that were to be added according to the
`programmed method. Vials were named 1-x, for 1 mL vials and 2-x
`for larger volume vials by the software.
`
`2.2. Reagents and supplies
`
`Kryptofix 2.2.2 and K2CO3 were purchased from EM Sciences.
`Kryptofix 2.2.2 solutions could be prepared in bulk at 4.5 mg/
`
`O
`
`N
`
`O
`
`HN
`
`O
`
`NO O
`
`H2N
`
`18F
`
`18F
`
`O
`
`O
`
`N
`
`O
`
`O
`
`NO O
`
`H2N
`
`N
`
`iPr2NEt
`
`PO
`
`O
`
`O
`
`TSTU
`
`OH
`
`O
`
`N
`
`TfO-
`
`O
`
`O
`
`18F-/K222/K2CO3
`
`18F
`
`O
`
`O
`
`1) 0.1M NaOH
`2) 0.2M HCl
`
`18F
`
`TFA
`
`Cai et al.
`
`This publication
`
`N
`
`TfO-
`
`O
`
`O
`
`18F-/K222/K2CO3
`
`18F
`
`O
`
`O
`
`Fig. 1. Scheme of the chemical transformations during the radiochemical synthesis. The steps above the dashed line indicate the procedure of Cai et al. (2006), while those
`below the line are the steps of the procedure described in this manuscript.
`
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`412
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`
`0.1 mL in acetonitrile and stored in a freezer for up to several
`months. Aqueous K2CO3 stock solutions at 0.69 mg/80 mL were
`stable at room temperature for several months. All other reagent
`solutions were freshly prepared for each synthesis. Pentamethyl-
`benzyl 4-(N,N,N-trimethylammonium)benzoate trifluoromethane-
`sulfonate was synthesized as previously described (Lang et al.,
`1999). Diethylcyanophosphonate, diisopropylethyl amine, N-(2-
`aminoethyl)maleimide trifluoroacetate salt, and trifluoroacetic acid
`were obtained from Sigma-Aldrich. Ammonium acetate, petroleum
`ether, and ethyl ether were obtained from Mallinckrodt-Baker.
`Dichloromethane and acetonitrile were obtained from Fisher
`Scientific. We utilized Sep Pak Light Silica from Waters and Bond
`Elut cartridges from Varian Instruments.
`
`2.3. HPLC
`
`For semi-preparative HPLC, the software program of the Eckert
`& Ziegler module initialized the UV detector and the isocratic HPLC
`pump. The column was equilibrated with 20% CH3CN: 80% water
`mixture at 2 mL/min from the beginning of the automated process
`until the start time of the coupling reaction. At that time the flow
`was increased to 6 mL/min. After the peak was collected, the eluant
`was changed to 75% CH3CN: 25% water in order to elute more non-
`polar components of the reaction mixture from the column. The
`column was washed for 20 min with this stronger eluant.
`
`2.4. Automated radiochemical synthesis of FBEM
`
`Module cleaning: the module was plumbed and programmed to
`conduct the radiochemical synthesis. Prior to conducting synthesis,
`all vials were replaced with clean vials and the system was cleaned
`by running water through the vials and tubing that were exposed to
`[18F]fluoride solution and subsequently by passing
`the initial
`acetonitrile through all the vials and tubing on the system. Finally
`all the tubing and vials were dried with a stream of argon for 10 min.
`An automated method was written to conduct this cleaning routine.
`In order to extend the lifetime of the solenoid valves, at the end of
`every synthesis, acetonitrile was passed through the solenoid valves
`that carried [18F]fluoride, carbonate/Kryptofix, fluoride-displace-
`ment substrate, and diethyl ether solutions. To minimize radiation
`
`exposure, this valve flushing was set up to allow remote addition of
`acetonitrile to the necessary vials. The valves were actuated using
`the manual mode of the software.
`
`2.5. Setup of module
`
`A clean Sigradur reaction vial was installed in PRM1 (Fig. 2) for
`every 5–10 syntheses. A clean 5 mL v-vial was placed in PRM2 for
`each reaction. A Waters Sep Pak Light Silica SPE cartridge was
`installed in fittings between PRM1 and PRM2. The module was
`tested for vacuum and pressure leaks. Vial 1-2 was loaded with
`500 mL CH3CN, 4.5 mg Kryptofix 2.2.2, 80 mL water containing
`0.69 mg K2CO3. Vial 1-3 was loaded with 500 mL CH3CN. Vial 1-4
`was loaded with 400 mL CH3CN containing 5 mg pentamethylbenzyl
`(4-trimethylammonium)benzoate
`trifluoromethanesulfonic
`acid
`salt. Vials 1-5 and 1-6 were each loaded with 800 mL ethyl ether.
`Vial 1-7 was loaded with 150 mL trifluoroacetic acid. Vial
`1-8 was loaded with two solutions one of N-(2-aminoethyl)malei-
`mide trifluoroacetate salt (2.3 mg in 300 mL CH3CN) and a second of
`diethylcyanophosphonate (5.8 mg in 300 mL CH3CN). Due to some
`concern with the stability of this solution, it was prepared and
`placed into the vial during the evaporation of the trifluoroacetic acid
`during the processing sequence. We entered into the radiation field
`to place this solution into the module at the appropriate time. Vial 1-
`9 was loaded with 800 mL of 5% CH3CN in 50 mM NH4OAc. Vial 1-10
`was loaded with a solution of 20 mL diisopropylethyl amine and
`200 mL CH3CN. Finally, vial 2-1 was loaded with an aqueous solution
`of [18F]fluoride in a volume not greater than 0.5 mL.
`
`2.6. Processing steps
`
`The [18F]fluoride was transferred into PRM1 followed by
`addition of the K222/K2CO3 solution. PRM1 was heated to
`120 1C and argon flow (1.3 bar) with vacuum applied for 5 min.
`The system pressure was measured to be  0.5 bar. After two
`1 min cycles of full vacuum (0.99 bar) and then argon flow plus
`vacuum were applied, a portion of 0.5 mL of CH3CN was added to
`PRM1 and the drying cycle was repeated. The complete drying
`cycle requires about 20 min.
`
`Fig. 2. Schematic of the Eckert & Ziegler module setup that identifies all valves and vials.
`
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`
`413
`
`2.7. Fluoride displacement reaction
`
`measurement, the HPLC UV response at 230 nm was calibrated
`with authentic FBEM.
`
`PRM1 was cooled to 40 1C. The substrate for fluoride displace-
`ment was added and PRM1 heated to 105 1C for 10 min. PRM1
`was set for a temperature of 10 1C and allowed to cool toward that
`temperature for  2 min; diethyl ether was added to PRM1 and
`the lift lowered to allow the transfer of the ethereal solution using
`argon pressure through the Waters Sep Pak Light Silica and into
`PRM2. A second portion of ether was added and was transferred
`through the cartridge. PRM2 was heated to 35 1C and argon flow
`used to evaporate the ethereal solution. This evaporation
`proceeded for 4.5 min.
`
`2.8. Cleavage of protecting group
`
`PRM2 was cooled to 20 1C followed by the addition of 150 mL
`trifluoroacetic acid. After standing for 2 min, PRM2 was cooled to
`0 1C. TFA was allowed to evaporate under an argon stream for a
`total of 6.5 min. At various intervals during this time period the
`argon flow was stopped for a few seconds and then resumed to
`effect complete removal of TFA from the reactor.
`
`2.9. Coupling of 4-[18F]fluorobenzoic acid to N-(2-aminoethyl)maleimide
`
`A solution of N-(2-aminoethyl)maleimide (2.3 mg in 300 mL
`CH3CN) and diethylcyanophosphonate (5.8 mg in 300 mL CH3CN)
`was added to PRM2 followed by a solution of diisopropylethyl
`amine (20 mL in 200 mL CH3CN). PRM2 was heated to 75 1C for
`7 min and then cooled to 35 1C. The reaction solution was
`evaporated under a stream of argon for 3 min and then diluted
`with 800 mL of 5% CH3CN in 50 mM NH4OAc.
`
`2.10. HPLC purification
`
`The contents of PRM2 were loaded onto the HPLC system
`(20% CH3CN in water, 6 mL/min) employing a LUNA C-18(2)
`9.4 mm  250 mm column. The radioactive peak eluting at
`20 min was collected as [18F]FBEM. After peak collection, the
`HPLC eluant was changed to 75% CH3CN, 25% water at 6 mL/min
`for an additional 15 min to clean up the column for the next use.
`
`2.11. Product isolation
`
`The product was isolated from the HPLC eluant by diluting the
`fraction to 20 mL with water and passing through an activated
`(2 mL ethanol followed by 2 mL water) Varian Bond Elut C-18
`(500 mg) cartridge. The cartridge was washed with 1.5 mL
`petroleum ether and then the trapped [18F]FBEM was eluted
`with 1.5 mL CH2Cl2. The CH2Cl2 was evaporated under a stream of
`argon. The residue, which contained a small amount of water, was
`treated with 10 mL of ethanol and then utilized for further protein
`labeling.
`
`2.12. Determination of radiochemical purity and specific activity
`
`Because [18F]FBEM was used for subsequent coupling, the
`radiochemical purity and specific activity were not routinely
`measured. Analytical HPLC employed a Zorbax SB300 C-18
`column (4.6 mm  250 mm, 5 mm), a gradient eluant of 20% A,
`80% B at time¼0 to 50% A, 50% B at time¼20. Solvent A was 0.1%
`TFA in acetonitrile; solvent B was 0.1% TFA in water. The flow rate
`was 1 mL/min. [18F]FBEM was eluted at  7.9 min. 4-[18F]Fluor-
`obenzoic acid was eluted at 9.2 min. For specific activity
`
`3. Results
`
`[18F]FBEM was prepared using the sequence shown in Fig. 1 by
`the modular system shown in Fig. 2. Uncorrected radiochemical
`yield of [18F]FBEM was 17.377.1% (n¼21) from [18F]fluoride. The
`UV and radiochromatograms (Fig. 3) revealed baseline separation
`between a large UV impurity and the radioactive product. The
`procedure, from placing [18F]fluoride into position on the module
`until collection of the HPLC product peak, required 98 74 min
`(range 83–110). To date, the highest radioactivity level synthesis
`[18F]fluoride and provided
`employed 8.2 GBq (222 mCi) of
`1.87 GBq (50.6 mCi) [18F]FBEM in the HPLC fraction 96 min later
`(22.8% uncorrected; 41.7% corrected for decay). There were three
`total failures not included in these averages. One failure resulted
`from a plugged line that prevented loading of the HPLC loop; the
`other two failures were attributed to instability of the solution of
`N-(2-aminoethyl)maleimide trifluoroacetate salt and diethylcya-
`nophosphonate. The time range was due to continued monitoring
`and adjustment of the time allowed for complete TFA evaporation
`and to the small variability in the manual activation of HPLC
`injection and peak collection. Additional 10–15 min was required
`to manually prepare the compound for subsequent use in
`radioconjugation of proteins or peptides. Because the product
`was used immediately for protein radiolabeling reactions, the
`specific radioactivity was not routinely measured. However, the
`specific activity measured for three batches was 181.3, 251.6, and
`351.5 GBq/ mmol
`(4.9, 6.8, and 9.5 Ci/ mmol) at
`the end of
`bombardment (EOB).
`
`Fig. 3. Example HPLC chromatogram of [18F]FBEM purification; panel A: radio-
`chemical trace and panel B: UV trace at 254 nm.
`
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`
`4. Discussion
`
`The preparation of radiolabeled peptides is a common procedure
`in PET radiochemistry laboratories. Succinimidyl 4-[18F]fluoro-
`benzoate (SFB) is utilized to radiolabel many peptides and proteins
`by the formation of amide bonds with e-amine of lysine residues.
`We have been preparing [18F]FBEM as a prosthetic group for specific
`radiolabeling of cysteine containing proteins (Kiesewetter et al.,
`2008). The radiochemical synthesis route that we developed for
`[18F]FBEM was similar to that of [18F]fluoropaclitaxel (Kalen et al.,
`2007) We acquired a modular radiochemical synthesis system with
`appropriate complexity to conduct this multistep radiosynthesis
`from Eckert & Ziegler GmbH. Our initial difficulties with leaking
`solenoid valves have been ameliorated by prompt flushing of these
`valves following the conclusion of the synthesis.
`In the initial development of the automated synthesis, we
`evaluated the radiochemical yields of the various steps. The
`fluoride displacement reaction, which required anhydrous condi-
`tions, provided consistent yields that were somewhat lower than
`that obtained in our manual synthesis (Kiesewetter et al., 2008).
`The lower yield may be due to the larger reaction vial surface and
`a more dilute concentration of substrate. We saw this lower yield
`as an acceptable trade for lower radiation exposure. The removal
`of the pentamethylbenzyl protecting group with TFA proceeded
`quickly as expected, but the complete evaporation of this reagent
`through the large reaction vial and the long length of tubing
`proved more challenging. We eventually derived an evaporation
`sequence that removed the vapors sufficiently to allow the second
`reaction to proceed reliably. The time required was longer than
`that achieved manually.
`The coupling reaction between N-(2-aminoethyl)maleimide
`and 4-[18F]fluorobenzoic acid also required use of larger volumes.
`In the end the reagent amounts were increased by almost a factor
`of 2 over those of the manual procedure. Unfortunately, we could
`not combine diisopropylethyl amine and N-(2-aminoethyl)malei-
`mide in the same reagent vial as the maleimide decomposed. We
`observed that a premixing of the maleimide and diethylcyano-
`phosphonate was possible, but the length of time the solution
`stood was important. The best and most reliable yields were
`obtained if the solution was prepared as close as possible to the
`time the module would add the solution. We accomplished this
`by going into the cell to change the vial. Placing the vial into the
`module required less than 30 s when properly executed, but did
`require entry into the radiation area. Several options were
`considered; however we believe the best option would be to
`acquire a system with one more valve to allow separate addition
`of this reagent.
`The HPLC injection was accomplished using the HPLC module
`of the Eckert & Ziegler GmbH system. The system employs a
`bubble detector to signal the beginning and completion of the
`liquid delivery from PRM2. We programmed the system to wait
`for our manual signal to load the sample onto the injection loop.
`The operator waits for the end of the liquid flow and can
`simultaneously observe radioactivity increase on the loop, due to
`its proximity to the HPLC radioactivity detector, and radioactivity
`decrease in PRM2. When the liquid passed the bubble meter, the
`operator pressed a button on the interactive screen to inject the
`sample onto the column. Operator selected buttons were also
`programmed to allow selection for peak collection.
`This radiochemical synthesis, which involves a three-step
`sequence, is rather complicated for automation. However, the
`time required and the radiochemical yields obtained were
`appropriate for radiolabeling proteins for a large number of small
`
`animal imaging studies. The addition of one valve module will
`allow for the solid phase extraction of [18F]FBEM to be conducted
`as part of the automated procedure.
`
`5. Conclusion
`
`three-
`We have developed an automated radiochemical
`step procedure for the preparation of [18F]FBEM. The reproduci-
`bility of the automated method depends on the efficient evapora-
`tion of trifluoroacetic acid following the deprotection step and
`the stability of
`the mixture of diethylcyanophosphate and
`N-(2-aminoethyl)maleimide. The method provides [18F]FBEM
`with an uncorrected radiochemical yield of 17.377.1%, high
`radiochemical purity (4 99%), and with a synthesis time of
`approximately 95 min. The measured specific activity for three
`batches was 181.3, 251.6, and 351.5 GBq/ mmol at the end of
`bombardment (EOB).
`
`Acknowledgements
`
`This research was supported by the Intramural Research
`Program of the National Institute of Biomedical Imaging and
`Bioengineering, National Institutes of Health. Technical assistance
`was provided by Eckert & Ziegler Eurotope GmbH for program-
`ming suggestions.
`
`References
`
`Becaud, J., et al., 2009. Direct one-step[18]F-labeling of peptides via nucleophilic
`aromatic substitution. Bioconjug. Chem. 20, 2254–2261.
`Berndt, M., Pietzsch, J., Wuest, F., 2007. Labeling of low-density lipoproteins using
`the F-18-labeled thiol-reactive N-[6-(4-[F-18]fluorobenzylidene)aminooxy-
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`Petitioner GE Healthcare – Ex. 1042, p. 414
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