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IntroDuctIonPET, a noninvasive imaging modality that has been used in clini-cal diagnostics for several decades, is able to visualize a variety of human afflictions such as cancer and cardiac and neurological dis-eases at a functional cell level and thus has increasing relevance in medical imaging. One of the most important radionuclides used in PET imaging is 18F, which possesses very favorable physical and chemical characteristics. However, the introduction of 18F into larger bioactive molecules such as peptides and proteins is gener-ally complex when using standard carbon-18F chemistry.

An alternative is the SiFA methodology, which is characterized by the application of building blocks that comprise a central silicon (Si) atom surrounded by two tertiary butyl (tBu) groups, a phenyl (Ph) moiety and a nonradioactive fluorine (F) atom1,2. The Ph moiety is amenable toward chemical modifications for further con-jugation of the SiFA moiety to biomolecules3. The two necessary tBu groups impose kinetic stability on the Si-F bond—the centerpiece of the SiFA group. Shielded against hydrolysis under physiological conditions resulting in physiologically stable products1,4,5, the Si-F bond easily exchanges its 19F atom with a radioactive 18F isotope. Notably, the isotopic exchange (IE) reaction proceeds at very low substrate concentrations6. An experimentally determined low acti-vation energy and a large pre-exponential factor for the IE7 confirm the results from density functional theory calculations in gas phase and solution8. This special feature of SiFA radiochemistry produces labeled compounds of high specific activities (SAs) and allows for a simple purification independent of HPLC. As the reaction condi-tions for the IE are very mild (room temperature and lower), no side reactions usually take place. Therefore, only unreacted 18F − has to be separated from the final product. The SiFA protocols are perfectly suited to label peptides4 in a single step (this protocol) and proteins with two different SiFA-labeling synthons3,9 while

requiring minimal technical skills. This was shown for several relevant peptides used in PET imaging of malignant diseases4,8.

Technical developments of the SiFA method for peptide labelingNormally, IE reactions do not deliver labeled compounds of high SA. However, fluorosilanes irrespective of their chemical deriva-tization rapidly undergo IE with 18F − in dipolar aprotic solvents such as acetonitrile, N,N-dimethyl formamide (DMF) and DMSO, although best results can normally be achieved using acetonitrile5. To impose kinetic stability to the SiFA unit under physiological conditions, two tBu groups have to be attached to the Si atom1,10–13. This structural prerequisite for hydrolytic stability increases the overall lipophilicity of the SiFA-conjugated peptides, leading to an unfavorable in vivo distribution of high liver uptake and reduced bioavailability4,12. To cope with high lipophilicity, sugar moieties and a PEG spacer are introduced, which alleviate this negative char-acteristic and improve the radioactivity accumulation in the target tissue (e.g., tumor). The most recent advancement in SiFA peptide labeling includes the additional introduction of hydrophilic aspar-tic acids counteracting the lipophilicity of a SiFA-derivatized pep-tide by introducing charged functional groups into the molecule. By using these three lipophilicity-reducing auxiliaries in variation or combination, the logD value of the peptide can be custom tai-lored. Lipophilicity is significantly reduced when aspartic acid is coupled to the peptide by standard solid-phase peptide synthesis before attaching the SiFA group. The labeling efficiency of these peptides with nucleophilic 18F − remains unchanged.

The general coupling of SiFA to peptides is achieved using a Ph group that is derivatized in accordance with the kind of click chemistry intended for bioconjugation. The most recent SiFA-labeling protocols for peptide labeling are easy to handle, even for

One-step 18F-labeling of peptides for positron emission tomography imaging using the SiFA methodologyCarmen Wängler1,2, Sabrina Niedermoser2,3, Joshua Chin4, Katy Orchovski4, Esther Schirrmacher4, Klaus Jurkschat5, Liuba Iovkova-Berends5, Alexey P Kostikov4, Ralf Schirrmacher4 & Björn Wängler3

1Biomedical Chemistry, Medical Research Center Mannheim, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany. 2Department of Nuclear Medicine, Hospital of the Ludwig-Maximilians-University, Munich, Germany. 3Molecular Imaging and Radiochemistry, Department of Clinical Radiology and Nuclear Medicine, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany. 4McConnell Brain Imaging Centre, Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada. 5Lehrstuhl für Anorganische Chemie II, TU University of Dortmund, Dortmund, Germany. Correspondence should be addressed to C.W. (carmen.waengler@medma.uni-heidelberg.de) or B.W. (bjoern.waengler@medma.uni-heidelberg.de).

Published online 4 October 2012; doi:10.1038/nprot.2012.109

Here we present a procedure to label peptides with the positron-emitting radioisotope fluorine-18 (18F) using the silicon-fluoride acceptor (siFa) labeling methodology. positron emission tomography (pet) has gained high importance in noninvasive imaging of various diseases over the past decades, and thus new specific imaging probes for pet imaging, especially those labeled with 18F, because of the advantageous properties of this nuclide, are highly sought after. N-terminally siFa–modified peptides can be labeled with 18F − in one step at room temperature (20–25 °c) or below without forming side products, thereby producing satisfactory radiochemical yields of 46 ± 1.5% (n = 10). the degree of chemoselectivity of the 18F-introduction, which is based on simple isotopic exchange, allows for a facile cartridge-based purification fully devoid of Hplc implementation, thereby yielding peptides with specific activities between 44.4 and 62.9 GBq mmol − 1 (1,200–1,700 ci mmol − 1) within 25 min.

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non-experts in the field of radiochemistry, especially when combin-ing SiFA labeling with the most recent advancements in 18F-drying chemistry. The solid-phase preparation of a no-carrier-added, nucleophilic and nonaqueous 18F − labeling cocktail14 required for the IE of the SiFA reaction allows for the whole labeling procedure to be completed independently of technical equipment.

SiFA labeling versus other 18F-labeling methodologies for peptidesThe most common way to introduce 18F into biomolecules entails forming a carbon-18F bond. The predominant domain of C-18F bond formation is associated with the radiosyntheses of small, low-molecular-weight compounds. Larger molecules such as peptides pose a challenge for radiochemists because of their structural com-plexity and need for either advanced protecting group strategies or complicated preceding prosthetic group syntheses. Advancements in C-18F bond formation that rely on efficient click chemistry have been reported, thereby providing radiochemists with powerful tools for peptide labeling15–21. As direct 18F-labeling approaches for com-plex biomolecules are rare22–24, prosthetic labeling groups based on active esters or various kinds of click chemistry have gained impor-tance25–36. Another convenient way to introduce 18F into various peptides is by on-resin solid-phase peptide synthesis37–39.

The recent advent of alternative 18F-labeling methods based on silicon-, boron- and aluminium-18F bond formations have intro-duced a new generation of PET imaging agents (for an overview, see refs. 40–42). These methodologies present not only new radiochem-istry but also include the strength of modern labeling concepts based on click chemistry43–45, which has proven to be extremely valuable in 18F-labeling of imaging probes. Besides the SiFA approach, another highly efficient Si-18F approach for peptide labeling10–12 was devel-oped over the past few years. This labeling concept is different, using leaving groups such as H, OH and alkoxyl to introduce 18F, but prin-cipally arrives at the same building block structure adopted by SiFA chemistry. This labeling strategy leads to compounds of high SA but still requires HPLC purification of the products because of the

contamination of the 18F-labeled product with the corresponding labeling precursor. Recently, others have used similar strategies to label peptides with silicon-based building blocks but still could not circumvent the need for HPLC purification13. Boron has also been under scrutiny as an alternative way to introduce 18F into biomolecules46,47, and it has been used for peptide labeling48. The formation of the B-18F bond has the intrinsic advantage of being able to work under aqueous labeling conditions. Another elaborate new strategy is the [18F]AlF method to label peptides49–52. The ease of labeling and the possibility to do aqueous 18F-labeling make this method one of the most efficient options. Similarly to the SiFA method, the [18F]AlF methodology is a true kit-labeling procedure devoid of HPLC purification, using solid-phase purification for the final product only.

Protocol outlineThis protocol describes the synthesis of [18F]SiFA peptides. In two related protocols, [18F]SiFA-labeling of unmodified proteins9 and [18F]SiFA-labeling of sulfo-SMCC-modified proteins3 are described. See also Supplementary Videos 1 and 2.

The stepwise synthesis of a SiFA-conjugated peptide (5) by com-mon Fmoc solid-phase peptide synthesis, the solution-phase con-jugation of SiFA-A (4) by oxime formation to the peptide and the one-step 18F-labeling yielding [18F]5 by IE is outlined in Figure 1. The final labeling precursor (5) bearing the SiFA group is obtained by assembling Fmoc-protected amino acids on a solid support, followed by introducing one or more auxiliaries to tailor the over-all lipophilicity of the final SiFA-conjugated peptide (5). The last component in the sequence synthesized on resin is bisBoc-pro-tected aminooxy acetic acid (Boc

2-Aoa), yielding the fully protected

peptide (2) on resin. The peptide is deprotected and at the same time cleaved from the solid support and subsequently purified by reversed-phase HPLC. The purified peptide 3 is conjugated to 4 via chemoselective oxime formation at pH 4 and subjected to a second HPLC purification32, which finally provides the labeling precursor 5

Asn(AcNH-β-Glc)

Aux

HN

O

OBoc2NPeptideAuxPeptideH2N PeptideAux

OH

SiF

SiFA-AHN

O

OH2N PeptideAux

pH 4HPLC

HN

O

ON PeptideAuxSiF

- Cleavage from resin- Deprotection- HPLC purification

+ Aux O

OBoc2N COOH

1

3

4

5

+ [18F]F–

– F–

[18F]5

Oxalic acid

=

Purified peptide

NH

OO

On

n = 1,2,3,5 and 27

PEG

,

O

O

HO

HN ,Asp

HN

O

O

HN OOH

OHOH

HN

OPeptide link

bisBoc aminooxy acetic acid (Boc2Aoa)

Aoa

Fmoc-SPPS 2

Synthesis of SiFA-peptides

Figure 1 | 18F-Labeling of peptides using SiFA. Synthesis of [18F]SiFA-labeled peptides ([18F]5) by IE. The SiFA lipophilicity can be counteracted by introducing one or more lipophilicity-reducing auxiliaries (Aux). The last synthon, SiFA-A (4), is conjugated in solution to the aminooxy-derivatized peptide 3, yielding the labeling precursor 5.

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for IE labeling with 18F. The precursor 5 can be aliquotted and stored before 18F-labeling. The 18F-labeling proceeds by oxalic acid (H

2C

2O

4)-supported reaction of 5 with azeotropically dried or

solid-phase–dried 18F − (Steps 26A and 26B). The purification of [18F]5 is achieved by simple solid-phase extraction (SPE).

A crucial step in 18F-labeling is the preparation of anhydrous and highly nucleophilic ‘naked’ 18F − in dipolar aprotic solvents such as DMSO and acetonitrile (Steps 26A and 26B). The classic method relies on the azeotropic drying of aqueous 18F − in the presence of the cryptand Kryptofix2.2.2 (ref. 53) and a base, usually K

2CO

3 or

K2C

2O

4. A serious drawback of this procedure is that not all of the

dried 18F − is available for the radiochemical reaction because of 18F surface adsorption in the drying vessel. By using a full produc-tion batch of 18F to synthesize high radioactivity amounts of SiFA-labeled peptides, an exact amount of oxalic acid must be added to partly neutralize the base in the labeling cocktail for SiFA-peptide labeling. It is difficult and unreliable to use an 18F-labeling solution

prepared by azeotropic drying; the exact amount of required oxalic acid cannot be determined because of 18F − and base adsorption on the drying vessel’s surface. The radiochemical yields of the SiFA reaction become far more reliable when the strong anion exchange (SAX) cartridge (Sep-Pak Light (46 mg)) SPE–based Munich method (Step 26A) is used for 18F drying. In this method, the pre-cise amount of base is known and no adsorption effects influence the exact addition of oxalic acid. Furthermore, no equipment in addition to the SAX cartridge is required, thereby simplifying the entire drying procedure enormously and reducing the drying time by more than 15 min. It is noteworthy that all SiFA reactions work well with azeotropically dried 18F − (Step 26B), although the yields are more variable and the amount of oxalic acid needed for base neutralization is more difficult to adjust. The highest radio-chemical yields (RCYs) for the SiFA IE are achieved using the Munich method and oxalic acid in combination, making this the preferable method.

MaterIalsREAGENTSSynthesis of SiFA-Aoa-AUX-peptides

DMF (≥ 99.8%, for peptide synthesis; Roth, cat. no. A529.1)Dichloromethane (ACS grade, Ph Eur.; Merck Chemicals, cat. no. 106050)Trifluoroacetic acid (TFA, for spectroscopy; Merck Chemicals, cat. no. 108262)Triisopropylsilane (99%; Aldrich, cat. no. 233781)N,N-Diisopropylethylamine (≥ 98.0%; Fluka, cat. no. 03440)Piperidine (≥ 99%, for peptide synthesis; Roth, cat. no. A122.1)Diethyl ether (Ph. Eur.; Merck Chemicals, cat. no. 100926)Water including 0.1% (vol/vol) TFA (LC-MS grade; Roth, cat. no. CP05.2)Water including 0.1% (vol/vol) formic acid (LC-MS grade, Roth, cat. no. CP03.2)Acetonitrile including 0.1% (vol/vol) TFA (LC-MS grade, Roth, cat. no. CP02.2)Acetonitrile including 0.1% (vol/vol) formic acid (LC-MS grade; Roth, cat. no. CP00.2)O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophos-phate (HBTU, ≥98.0%, Sigma-Aldrich, cat. no. 12804)Sodium methylate solution (0.5 M in methanol, ACS reagent; Fluka, cat. no. 71751)Fmoc-Thr(tBu)-Wang resin (100–200 mesh; Novabiochem, cat. no. 856017)Fmoc-Cys(Acm)-OH (Novabiochem, cat. no. 852006)Fmoc-Thr(tBu)-OH (Novabiochem, cat. no. 852000)Fmoc-Lys(Boc)-OH (Novabiochem, cat. no. 852012)Fmoc-D-Trp(Boc)-OH (Novabiochem, cat. no. 852164)Fmoc-Tyr(tBu)-OH (Novabiochem, cat. no. 852020)Fmoc-D-Phe-OH (Novabiochem, cat. no. 852148)Fmoc-Asp(OtBu)-OH (Novabiochem, cat. no. 852005)Fmoc-NH-(PEG)-COOH (9 atoms; Novabiochem, cat. no. 851037)Fmoc-Asn(Ac

3AcNH-β-Glc)-OH (Novabiochem, cat. no. 852135)

bisBoc-amino-oxyacetic acid (Novabiochem, cat. no. 851028)Thallium(III) trifluoroacetate (technical grade; Sigma-Aldrich, cat. no. 150533)Disodium hydrogen phosphate dodecahydrate (for analysis, EMSURE ISO, Reag. Ph. Eur., Merck, cat. no. 1065791000)n-Hexane (for liquid chromatography, Merck, cat. no. 1043911000)Hydrochloric acid (for analysis, 37%, Merck, cat. no. 1003172500)p-Toluenesulfonic acid (Sigma-Aldrich, cat. no. 402885-100G)Tetrabutylammonium difluorotriphenylstannate (97%, Sigma-Aldrich, cat. no. 418625-1G)phosphate buffer (0.5 M, pH = 4.0) (see Reagent Setup)

Preparation of [18F]fluorideKryptofix 2.2.2 (Aldrich, cat. no. 291110 or Merck Chemicals, cat. no. 814925)18F − from 18O(p,n)18F nuclear reaction using a cyclotron ! cautIon Prop-erly shield yourself against radiation by performing all reactions involving radioactivity in a lead-shielded fume hood.

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Tetrabutylammonium bicarbonate ([TBA]HCO3, 0.075 M aqueous

solution; ABX, cat. no. 808)Potassium hydroxide pellets (KOH, p.a.; Merck Chemicals, cat. no. 105033 or Aldrich, cat. no. 221473)Acetonitrile, anhydrous (Aldrich, cat. no. 271004 or puriss, absolute, over molecular sieve; Sigma-Aldrich, cat. no. 00709)Deionized water (Baxter, cat. no. JF7623)DMSO (extra dry; Acros Organics, cat. no. 348441000)

Peptide labelingAnhydrous [K + (2.2.2)]18F − in acetonitrile crItIcal After dissolving the [K + 2.2.2]OH− complex in acetonitrile, the preparation of anhydrous [K + (2.2.2)]18F − in acetonitrile should take place within 15 min.Ethanol (96% Ph. Eur.; Merck Chemicals, cat. no. 100971)Tracepur-H

2O (Merck Chemicals, cat. no. 100473)

Acetonitrile (puriss, absolute, over molecular sieve; Sigma-Aldrich, cat. no. 00709)DMSO (extra dry; Acros Organics, cat. no. 348441000)oxalic acid (≥ 99%; Sigma-Aldrich, cat. no. 241172)NaCl solution (0.9% (wt/vol); Braun, cat. no. 3820076)HEPES (GERBU Biotechnik, cat. no. 1009)SiFA-Aoa-AUX-peptide 5HEPES buffer (0.1 M), pH 2 and pH 4 (see Reagent Setup)

EQUIPMENTSynthesis of SiFA-Aoa-AUX-peptides

Waters SymmetryPrep C18 HPLC column (300 mm × 19 mm, 7 µm; Waters, cat. no. WAT066245)Semipreparative HPLC system A for peptide purification (see Equipment Setup)

Preparation of [18F]fluorideChromafix PS-HCO

3 cartridge (45 mg; GE Healthcare, cat. no. P5360LC;

see Equipment Setup)Sep-Pak Accell Plus QMA carbonate light (46 mg; Waters, cat. no. 186004540) (see Equipment Setup)Alpha 1-2LD plus lyophilization unit (Christ, cat. no. 101521)

Peptide labelingSep-Pak C18 light cartridge (Waters, cat. no. WAT022301; see Equipment Setup)Reaction vial: mini-vial 5.0 ml, 20/400 (Grace, cat. no. 95050)Septa, 20 mm, polytetrafluoroethylene(PTFE)/Sil (Wheaton, cat. no. W240586A)Analytical HPLC System A (Agilent, 1,200 series) equipped with a Gabi Star radioactivity detector (Raytest; see Equipment Setup)Chromolith performance RP18e (100 × 4.6 mm; Merck Chemicals, cat. no. 102129)Analytical HPLC system A for quality control (see Equipment Setup)

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REAGENT SETUPPhosphate buffer (0.5M, pH 4.0) Dissolve Na

2HPO

4 (179.1 g) in water

(1 liter). Adjust the pH to 4.0 using 37% (wt/vol) HCl.[K + 2.2.2]OH− elution cocktail (Step 26A) Dissolve 41 mg of Kryptofix2.2.2 (110 µmol) in 1 M KOH (100 µl), dilute with water to 1 ml and lyophilize to dryness overnight. Dissolve the dried cocktail in 0.5 ml of anhydrous acetonitrile for radiolabeling immediately before use. crItIcal The [K + 2.2.2]OH− complex can be stored in a solid lyophilized state in the freezer (less than − 20 °C) for up to 1 year. However, once it is dissolved in acetonitrile, it should be used within 5–10 min, as the solution quickly turns yellow and loses its elution activity.[TBA]HCO

3 elution cocktail (Step 26B) If azeotropic drying of 18F − is used,

mix 800 µl of acetonitrile and 300 µl of 0.075 M aqueous [TBA]HCO3 for

18F − elution. crItIcal Aqueous 0.075 M [TBA]HCO3 can be stored in

the refrigerator for up to 1 year. The prepared mixture of acetonitrile and [TBA]HCO

3 should be used within 15 min after preparation.

HEPES buffer (0.1 M), pH 2 and 4 Dissolve HEPES (2.38 g, 0.01 mol) in 100 ml of water. Adjust the pH to 2 and 4 with 37% (wt/vol) HCl

aq (1.2 ml

for pH 2 or 80 µl for pH 4). crItIcal HEPES buffers can be stored in the refrigerator for up to 6 months.

EQUIPMENT SETUPChromafix PS-HCO

3 cartridge preconditioning Precondition with 1 ml

of ethanol followed by 1 ml of water. crItIcal Do not dry the matrix with air at this point; instead, allow it to swell for 5 min and dry the matrix with 1 ml of air.Sep-Pak Light Accell Plus QMA cartridge preconditioning Precondition with 10 ml of water. crItIcal Do not dry the matrix with air.Sep-Pak C18 light cartridge preconditioning Precondition with 10 ml of ethanol followed by 10 ml of water. crItIcal Do not dry the matrix with air.HPLC system A Agilent 1200 HPLC series equipped with a Raytest Gabi Star radioactivity detector.Analytical HPLC system A for quality control Use a Chromolith perform-ance RP18e (100 × 4.6 mm) column with a gradient of 100% water including 0.1% (vol/vol) TFA to 100% acetonitrile including 0.1% (vol/vol) TFA over 5 min at a flow rate of 4 ml min − 1.Semipreparative HPLC system A for peptide purification Use a Waters SymmetryPrep C18 HPLC column (300 × 19 mm, 7 µm) with a gradient of 0–60% water including 0.1% (vol/vol) TFA to acetonitrile including 0.1% (vol/vol) TFA over 25 min at a flow rate of 8 ml min − 1.

proceDurepreparation of p-(di-tert-butylfluorosilyl)benzaldehyde (siFa-a, 4) ● tIMInG ~1 week1| Di-tert-butyldifluorosilane. Add di-tert-butyldichlorosilane (11.50 g, 53.92 mmol) to a suspension of tetrabutyl-ammonium difluorotriphenylstannate (68.00 g, 107.85 mmol) in dichloromethane (40 ml). The reaction is exothermic and the tetrabutylammonium difluorotriphenylstannate dissolves.

2| Continue stirring for 24 h and then remove the solvent by distillation.

3| Extract the residue three times with n-hexane (200 ml each) and evaporate the n-hexane of the combined extracts in vacuo.

4| Subject the oily residue to distillation. The product di-tert-butyldifluorosilane is obtained at a boiling point (b.p.) of 130–132 °C and in the form of a colorless liquid in 32% yields (3.13 g). (Another 1.42 g (15% yield) of the product can be obtained by continuous extraction of the residue from the reaction by using a Soxhlet apparatus, followed by distillation.) pause poInt Di-tert-butyldifluorosilane has a shelf life of up to 1 year at room temperature.

5| 2-[p-(Di-tert-butylfluorosilyl)phenyl]-1,3-dioxolane. At –78 °C and under magnetic stirring, add a solution of n-butyllithium in n-hexane dropwise (c = 1.6 mol per liter, 10.1 ml, 16.1 mmol) to a solution of 2-(p-bromphenyl)-1,3- dioxolane (3.69 g, 16.1 mmol) in THF (30 ml).

6| After the reaction mixture has been stirred for 2 h at –78 °C, add the resulting suspension dropwise over a period of 20 min to a cooled solution (–70 °C) of di-tert-butyldifluorosilane (3.05 g, 16.9 mmol) in THF (30 ml).

7| Stir the reaction mixture for 1 h at –70 °C and then allow it to warm to room temperature. Continue stirring for a further 3 d.

8| Distill off the solvent and add diethyl ether (150 ml) to the residue.

9| Hydrolyze the resulting mixture with water (150 ml) under ice cooling. ! cautIon This is an exothermic reaction. Add water dropwise.

10| Separate the organic layer and extract the aqueous layer four times with diethyl ether (50 ml each).

11| Dry the combined organic phases over magnesium sulfate, filter and remove the solvent in vacuo.

12| Purify the residue by Kugelrohr distillation. According to its 1H NMR spectrum, the major fraction (2.62 g) with a b.p. of 115–130 °C (5 × 10 − 4 mbar) contains 70% 2-[p-(di-tertbutylfluorosilyl)phenyl]-1,3-dioxolane (1.8 g, 52% yield) and 30% 2-(p-butylphenyl)-1,3-dioxolane. This mixture was used without further purification for the synthesis of p-(di-tertbutylfluoro-silyl) benzaldehyde. The compound should be stored in the freezer until further use.

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13| p-(Di-tert-butylfluorosilyl)benzaldehyde (SiFA-A, 4). Heat a solution containing 2.41 g of a mixture consisting of 2-[p-(di-tert-butylfluorosilyl)phenyl]-1,3-dioxolane (70%) and 2-(p-butylphenyl)-1,3-dioxolane in 50 ml of acetone and 15 mg of p-toluene sulfonic acid to reflux for 3 h.

14| Remove the solvent in vacuo and dissolve the residue in 50 ml of acetone.

15| Add another 15 mg of p-toluene sulfonic acid and heat the reaction mixture to reflux for a further 3 h, followed by solvent removal in vacuo.

16| Dissolve the residue in a mixture of n-hexane/acetic acid ethyl ester (8:2, 10 ml) and filter it through a column (height = 11 cm, diameter = 2.5 cm) filled with silica gel 60 (Geduran Si 60).

17| Remove the solvent of the filtrate in vacuo and purify the residue by Kugelrohr distillation to obtain 0.75 g (52% yield) of p-(di-tert-butylfluorosilyl)benzaldehyde 4 as a yellow oil, with a b.p. of 95–100 °C (5 × 10 − 4 mbar), which solidifies (m.p. 40–41 °C). pause poInt The substance has to be stored in the freezer at –20 °C and should be used within 1 year.

synthesis of siFa-aoa-auX-peptides 5 ● tIMInG ~3–4 d18| Synthesize the peptide on a solid support via the solid-phase peptide synthesis standard Fmoc strategy54. The reac-tion steps can be monitored by the Kaiser test (protocol provided in http://www.nature.com/nprot/journal/v5/n11/box/nprot.2010.146_BX1.html)55.

19| Remove the terminal Fmoc-protecting group using 50% (vol/vol) piperidine in DMF.

20| Auxiliary coupling. Couple Fmoc-NH-PEGX-COOH, Fmoc-Asp(OtBu)-OH, Fmoc-Asn(Ac3AcNH-β-Glc)-OH and finally bisBoc-Aoa-OH using a fourfold excess of the aforementioned reactants, HBTU (3.9 eq.) and N,N-diisopropylethylamine (4 eq.) in DMF at room temperature. crItIcal step Vary the combination of auxiliaries according to targeted overall lipophilicity of the peptide. Synthons, such as Fmoc-NH-PEG-COOH, Fmoc-Asp(OtBu)-OH and Fmoc-Asn(Ac3AcNH-β-Glc)-OH, lead to a significant increase of hy-drophilicity of the product peptide. crItIcal step If PEG auxiliaries with very long chain lengths are used, coupling efficiencies can decrease. Longer reac-tion times may be required for such long PEG linkers.? trouBlesHootInG

21| Cleave the peptide from the solid-phase support by incubating the resin with a mixture of neat TFA/triisopropylsilane/H2O = 95:2.5:2.5 for 1 h at room temperature, precipitate the product in diethyl ether and dry the crude intermediate.

22| Purify the peptide by semipreparative HPLC (semipreparative HPLC system A, using a Waters SymmetryPrep C18 HPLC column (300 × 19 mm, 7 µm)) with a gradient of 0–60% (vol/vol) water including 0.1% (vol/vol) TFA to acetonitrile including 0.1% (vol/vol) TFA over 25 min at a flow rate of 8 ml min − 1. Lyophilize the isolated product.

23| If the auxiliary Fmoc-Asn(Ac3AcNH-β-Glc)-OH is used, deprotect the carbohydrate moiety by dissolving the peptide in sodium methanolate solution in methanol (0.5 M) at pH 12–13 and at room temperature for 30–60 min. Stop the reaction by adding neat TFA and remove the volatile components by evaporation.

24| Dissolve the peptide in phosphate buffer (0.5 M, pH = 4.0) and add an excess of SiFA-A (4, 5 eq., 22 mg, 82.5 µmol) dissolved in acetonitrile to the peptide. Leave the reaction to stand at room temperature for 15 min.

25| Purify the product 5 by semipreparative HPLC, using the same conditions as mentioned above, and remove the solvent by lyophilization. pause poInt The SiFA-modified peptide can be stored in the freezer for up to 1 year.

preparation of [18F]fluoride ! cautIon Properly shield yourself against radiation by performing all reactions involving radioactivity in a lead-shielded fume hood.

26| [18F]Fluoride can be dried using either the Munich method (option A) or by azeotropic drying (option B))(a) Munich method ● tIMInG 3–5 min (i) Pass aqueous 18F − (purchased from a supplier or generated in on-site cyclotron by bombardment of 18O-enriched

water ([18O]H2O) with protons) through a SAX cartridge (Sep-Pak Accell Plus QMA Carbonate light (46 mg)), followed by 20 ml of air.

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crItIcal step This protocol requires QMA cartridges of 46-mg capacity. Higher-capacity cartridges require a higher volume of elution cocktail to release the 18F − . ? trouBlesHootInG

(ii) Rinse the cartridge with 5 ml of anhydrous acetonitrile and dry with 20 ml of air. crItIcal step Rinse the cartridge at a rate no faster than 1 ml per 10 s; water residues will lead to a low 18F-fluorination yield.

(iii) Elute with 0.5 ml of [K + 2.2.2]OH− (100 µmol) in acetonitrile (see Reagent Setup). The obtained solution can be used for radiolabeling. crItIcal step Elute the 18F − very slowly to achieve efficient ( > 95%) release of the 18F − from the cartridge. ? trouBlesHootInG

(B) azeotropic drying of 18F − using [tBa]Hco3 ● tIMInG 20–30 min (i) Load aqueous [18F]fluoride onto a Chromafix PS-HCO3 cartridge. (ii) Elute the [18F]fluoride from the cartridge with a mixture of 800 µl of acetonitrile and 300 µl of aqueous [TBA]HCO3

solution (see Reagent Setup). (iii) Dry this mixture azeotropically using a stream of nitrogen and reduced pressure of 600 mbar at 80 °C for 4 min. Add

800 µl of anhydrous acetonitrile and dry it again. Repeat this step once more, and then dry it at 20 mbar for 4 min (without stream of nitrogen) and dissolve the dried [TBA]18F complex in anhydrous acetonitrile (100 µl) for radiolabeling.

radiolabeling of siFa-aoa-auX-peptides 5 ● tIMInG 15–25 min27| 18F-Fluorination. Add 25 µl of oxalic acid in anhydrous acetonitrile (1 M, 25 µmol) to the cartridge eluate containing [K + (2.2.2)]18F − (obtained by Step 26A(iii) or 26B(iii)). Use the resulting solution as a whole, or aliquot it as needed for 18F-fluorination. crItIcal step Lower or higher amounts of oxalic acid addition could cause too high or too low a proton content in the labeling mixture and therefore result in lower RCY. If only a fraction of the prepared 18F-solution (obtained by Step 26A(iii) or 26B(iii)) is used, add the same fraction of oxalic acid solution in Step 27 (optimal ratio for peptide labeling is 4:1, [K + 2.2.2]OH−:H2C2O4; Fig. 2).? trouBlesHootInG

28| Add 10–25 µl of a 1 mmol per liter stock solution of SiFA-Aoa-AUX-peptide 5 (e.g., the somatostatin analog SiFA-Aoa-Asn(AcNH-β-Glc)-(Asp)2-PEG-Tyr3-octreotate prepared as described in Steps 18–25) in anhydrous DMSO to 450–500 µl of the [K + 2.2.2]18F − /H2C2O4 solution (obtained in Step 27).

29| Let the reaction stand for 5 min at room temperature without stirring, and then analyze the crude product by using the analytical HPLC system A.? trouBlesHootInG

30| Purification of peptide [18F]5. Condition a Sep-Pak C18 light cartridge (see Equipment Setup).

31| Add 9 ml of 0.1 M HEPES buffer, pH 2 (when the Munich method is used, Step 26A) or pH 4 (when the azeotropic drying method is used, Step 26B), to the reaction mixture containing [18F]5, and then trap the [18F]5 on the cartridge. crItIcal step The use of the Munich method (Step 26A) for peptide fluorination raises the pH value of the quenching solution because a stronger base (OH − ) is used. If the pH of the reaction mixture strongly differs from the isoelectric point of the peptide, the radiolabeled peptide may not be trapped on the C18 cartridge.? trouBlesHootInG

32| Remove solvent residues by washing the cartridge with water for injection (5–8 ml).

33| Elute the purified [18F]5 with ethanol (200–500 µl), dilute it with isotonic saline solution (final concentration of ethanol: maximal 10%) and, if necessary, filter it for sterility. crItIcal step Do not elute too fast. Adjust the amount of ethanol if necessary. ? trouBlesHootInG

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0 µmol oxalic acid25 µmol oxalic acid37.5 µmol oxalic acid50 µmol oxalic acid62.5 µmol oxalic acid75 µmol oxalic acid100 µmol oxalic acid

Figure 2 | Influence of the amount of oxalic acid on the radiochemical yield of peptide labeling with 18F − (labeled peptide = SiFA-Aoa-Tyr3-TATE). c.p.s., counts per second; Rel., relative.

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34| Carry out analytical HPLC for quality control to reveal the product’s radiochemical and chemical purity ( > 95%), and measure the recovered activity.

35| Determine the SA of the 18F-labeled peptide by HPLC system A or estimate it by dividing the measured radioactivity in the product by the amount of precursor used (equates to lower limit of SA)15.

? trouBlesHootInGTroubleshooting advice can be found in table 1.

● tIMInGSteps 1–17, synthesis of SiFA-A (4): within 1 weekSteps 18–25, synthesis of a peptide-like compound 5 including preparative HPLC purification: ~3–4 dStep 26A, drying of aqueous 18F − (in [18O]water from the cyclotron bombardment) using the Munich method: 3–5 min (oxalic acid can be added in exact quantities to ensure maximum efficiency of the IE.)Step 26B, conventional azeotropic drying: 20–30 min, depending on the individual setup in the laboratory.Steps 27–35, reaction of the SiFA-derivatized peptides 5 with 18F − (Steps 28 and 29): 5–10 min with 18F incorporation yields of 80–90% at room temperature (purification by SPE (Steps 30–33) can be completed within 10–15 min)

antIcIpateD resultsThe 18F-labeling of 5 to [18F]5 with addition of oxalic acid yields the labeled peptide in 46% overall RCY (not decay corrected) after SPE purification. The 18F incorporation of 18F − is > 98% as proven by radio-HPLC (Fig. 3). The radiochemical purity of the SPE-purified peptide [18F]5 is > 98% (Fig. 4). After SPE purification, the final product is devoid of any radioactive contami-nants, and SAs of 44.4–62.9 GBq µmol − 1 (1,200–1,700 Ci mmol − 1) can be achieved using 18F − starting activities of 3.3–6.7

taBle 1 | Troubleshooting table.

step problem possible reason solution

20 Inhomogeneous product Low coupling efficiency for very long PEG linkers

Extend coupling time of the PEG linker to 1–4 h

26A(i) 18F-fluoride is retained on QMA cartridge

High-capacity cartridge was used

Use a smaller-capacity cartridge (46 mg) or use more elution cocktail

26A(iii) 18F-fluoride is retained on QMA cartridge

Rate of elution is too fast Elute 18F-fluoride from the cartridge slower

27 Low yield of 18F-fluorination

Excess or dearth of H2C2O4 added

Use less/more oxalic acid to neutralize the base in elution cocktail. The optimal molar ratio of [K + 2.2.2]OH− to H2C2O4 is 4:1

29 Low yield of 18F-fluorination

Oxalic acid and the precursor peptide were added in the wrong order

Add oxalic acid and mix the solution before adding the precursor peptide

31 C18 cartridge does not retain radioactivity when diluted reaction mixture is passed through

pH rises due to the usage of OH − in the reaction mixture up to pH 6.5; pH of the reac-tion mixture strongly differs from the isoelectric point of the peptide

Decrease the pH value of the solution to pH 2–4 and trap the product on a new precondi-tioned C18 cartridge or adjust the pH value of the solution to a pH similar to the isoelec-tric point of the peptide

33 C18 cartridge retains radioactivity when eluted with ethanol

Not enough ethanol was used or solubility of the peptide in ethanol is not sufficient

Use more ethanol to elute [18F]-SiFA-peptide from the cartridge and adjust the dilution with isotonic saline solution to a total ethanol volume of 10% or use a mixture of water/ethanol 1:1 to elute [18F]-SiFA-peptide from the cartridge

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(90–180 mCi). If the amount of oxalic acid varies from the optimal ratio of [K + 2.2.2]OH−/H2C2O4 = 4:1, a decrease in peptide labeling yield can be observed, and the formation of a radioactive side product is clearly observed via HPLC (Fig. 2).

analytical dataDi-tert-butyldifluorosilane.

1H NMR (300.13 MHz, CDCl3): d 1.07 (t, 4J(1H–19F) = 1.1 Hz, 18H, CH3). 13C(1H) NMR (75.48 MHz, CDCl3): d 26.0 (s, CH3),

19.1 (t, 2J(13C–19F) = 13 Hz, CCH3). 19F NMR (282.38 MHz, CDCl3): d –160.1 (s, 1J(19F–29Si) = 326 Hz). 29Si(1H) NMR

(59.63 MHz, CDCl3): d –7.6 (t, 1J(29Si–19F) = 326 Hz).2-[p-(Di-tert-butylfluorosilyl)phenyl]-1,3-dioxolane.

1H NMR (300.13 MHz, CDCl3): d 7.59. (m, 4H, Harom), 5.83 (s, 1H, CH), 4.09 (m, 4H, CH2), 1.08 (d, 4J(1H–19F) = 1.1 Hz, 18H, CH3).

13C(1H) NMR (75.48 MHz, CDCl3): d 138.9 (s, Ci), 134.8 (d, 2J(13C–19F) = 14 Hz, Cp), 134.0 (d, 3J(13C–19F) = 4 Hz, Cm), 125.6 (s, Co), 103.6 (s, CH), 65.3 (s, CH2), 27.2 (s, CH3), 20.2 (d, 2J(13C–19F) = 12 Hz, CCH3).

19F NMR (282.38 MHz, CDCl3): d –191.5 (s, 1J(19F–29Si) = 298 Hz). 29Si(1H) NMR (59.63 MHz, CDCl3): d 14.1 (d, 1J(29Si–19F) = 298 Hz).p-(Di-tert-butylfluorosilyl)benzaldehyde (siFa-a, 4).

1H NMR (300.13 MHz, CDCl3): d 10.05. (s, 1H, CHO), 7.84 (m, 4H, Haromat), 1.07 (d, 4J(1H–19F) = 1.3 Hz, 18H, CH3). 13C(1H)

NMR (75.48 MHz, CDCl3): d 192.5 (s, CHO), 142.2 (d, 2J(13C–19F) = 13 Hz, Cp), 136.9 (s, Ci), 134.5 (d, 3J(13C–19F) = 4 Hz, Cm), 128.4 (s, Co), 27.2(s, CH3), 20.2 (d, 2J(13C–19F) = 12 Hz, CCH3).

19F NMR (282.38 MHz, CDCl3): d –191.1 (s,1J(19F–29Si) = 300 Hz). 29Si(1H) NMR (59.63 MHz, CDCl3): d 13.8 (d, 1J(29Si–19F) = 300 Hz). IR (KBr): n~(C-O) 1,705 cm − 1. Elemental analysis: calcu-lated (%) for C15H23FOSi (266.43 g mol − 1): C, 67.6; H, 8.7; found: C 67.8, H 8.7.

analytical data for peptides 5SiFA-Tyr3-octreotate: high-resolution electrospray ionization–mass spectrometry (HR-ESI-MS) (m/z): 1,370.58. [M + H] + ;

calculated for C66H88FN11O14S2Si: 1,369.57.SiFA-Aoa-Asn(AcNH-β-Glc)-Tyr3-octreotate: HR-ESI-MS (m/z): 1,686.70 [M + H] + ; calculated for C78H107FN14O21S2Si: 1,686.69.SiFA-Aoa-Asn(AcNH-β-Glc)-PEG-Tyr3-octreotate: HR-ESI-MS (m/z): 1,832.02 [M + H] + ; calculated for C83H116FN15O24S2Si: 1,832.77.SiFA-Aoa-Asn(AcNH-β-Glc)-(Asp)2-PEG-Tyr3-octreotate: matrix-assisted laser desorption/ionization–time of flight

(MALDI-TOF) (m/z): 2,064.97 [M + H] + ; calculated for C92H128FN17O30S2Si: 2,063.31.

0 1 2 3 4 5

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1,500

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Time (min)

0 1 2 3 4 5200

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Figure 3 | 18F-Labeling of SiFA-Aoa-Asn(AcNH-β-Glc)-(Asp)2-PEG-Tyr3-octreotate. The crude reaction mixture shows only minor radioactive side-product formation (black line, Rt = 1–2 min) and an almost quantitative formation of the labeled peptide (black line, Rt = 2.8 min). c.p.s., counts per second; Rel., relative.

0 1 2 3 4 5

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Figure 4 | HPLC chromatogram of SPE-purified [18F]SiFA-Aoa-Asn(AcNH-β-Glc)- (Asp)2-PEG-Tyr3-octreotate ([18F]5). The radiochemical purity is > 98%. c.p.s., counts per second.

Note: Supplementary information is available in the online version of the paper.

acknowleDGMents We thank the cyclotron crew of the McConnell Brain Imaging Centre for providing fluorine-18. The financial support of the following grant agencies is gratefully acknowledged: Canada Foundation for Innovation (CFI) project no. 203639 to R.S., Natural Sciences and Engineering Research Council of Canada (NSERC) Strategic Project Grant Program (STPGP) no. 412893 to R.S., Bayern-Quebec-Allianz to S.N., Deutsche Forschungsgemeinschaft (DFG) grant no. WA 2132/3-1 to B.W., and Fonds der Chemischen Industrie to C.W.

autHor contrIButIons A.P.K., S.N., J.C., K.O., E.S., L.I.-B., K.J. and C.W. performed the experimental work. C.W., B.W. and R.S. wrote the manuscript.

coMpetInG FInancIal Interests The authors declare no competing financial interests.

Published online at http://www.nature.com/doifinder/10.1038/nprot.2012.109. Reprints and permissions information is available online at http://www.nature.com/reprints/index.html.

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