Chapter 4 Synthesis, Characterization, and Evaluation of Radiometal-Containing Peptide Nucleic Acids Holger Stephan, Christian Foerster, and Gilles Gasser Abstract Peptide nucleic acids (PNAs) have very attractive properties for applications in nuclear medicine. Because PNAs have high selectivity for DNA/RNA recognition, resistance to nuclease/protease degradation, and high thermal and radiolytic stabilities, PNA bioconjugates could transform the areas of diagnostic and therapeutic nuclear medicine. In this book chapter, we report on the current developments towards the preparation of radiometal-containing PNA constructs and summarize the protocols for labeling these probes with 99mTc, 111In, 64Cu, 90Y, and 177Lu. Key words Peptide nucleic acid (PNA), Radiometal complexes, Bifunctional chelating agents, Molecular imaging, Endoradionuclide therapy
Abbreviations A Ac-GDAGG BFCA Bhoc Bipy Boc C Cbz DIPEA DMF DOTA Dpa-N3 DPA DPAm DTPA EDTA Fmoc G
Adenine TetrapeptideN-acetyl-glycine-D-alanine-glycine-glycine Bifunctional chelating agent Benzhydryloxycarbonyl 2,2′-bipyridine Di-tert-butyl dicarbonate Cytosine Benzyloxycarbonyl Diisopropylethylamine Dimethylformamide 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid 2-azido-N,N-bis((pyridin-2-yl)methyl)ethanamine N,N-bis(2-picolyl)amine N,N-bis(2-picolyl)amide Diethylenetriaminepentaacetic acid Ethylenediaminetetraacetic acid 9-fluorenylmethyloxycarbonyl Guanine
Peter E. Nielsen and Daniel H. Appella (eds.), Peptide Nucleic Acids: Methods and Protocols, Methods in Molecular Biology, vol. 1050, DOI 10.1007/978-1-62703-553-8_4, © Springer Science+Business Media New York 2014
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GDAGG HATU HCl HEPES HPLC ITLC MAG3 MAS3 PET PBS PNA PzDA SBTG2DAP SEC SPECT T TETA TFA TIS
1
Tetrapeptide glycine-D-alanine-glycine-glycine 2-(1H-7-azabenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate Hydrochloric acid 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid High-performance liquid chromatography Instant thin-layer chromatography S-acetylmercaptoacetyltriglycine S-acetylmercaptoacetyltriserine Positron emission tomography Phosphate-buffered saline Peptide nucleic acid N-(1-pyrazolyl)ethyl-ethane-1,2-diamine N,N′-bis(S-benzoyl-thioglycoloyl)diaminopropanoate Size-exclusion chromatography Single-photon emission computed tomography Thymine 1,4,8,11-tetraazacyclotetradecane1,4,8,11-tetraacetic acid Trifluoroacetic acid Triisopropylsilane
Introduction Radiolabeled molecules can be used in nuclear medicine for diagnostic imaging as well as for the delivery of targeted therapies to treat specific diseases [1–3]. In this area, there has been a steadily growing interest in applying radiolabeled peptide nucleic acids (PNAs) for monitoring gene expression [4, 5]. In addition to nucleic acid targets, radiolabeled PNA can be used to specifically direct the delivery of a radioisotope for therapeutic applications. For instance, Hnatowich et al. have proposed a “pretargeting using PNA” strategy to transport radionuclides to preceding targeted tissue [6–8]. In this approach, a single-stranded PNA (ssPNA) is attached to a protein/antibody that binds specifically to receptors that are overexpressed in cancer cells. After the initial administration of a PNA–antibody conjugate, sufficient time is allowed for any unbound PNA–antibody conjugates to be cleared from circulation, and then the complementary, radiolabeled PNA is administered. This technique allows radionuclides to be delivered to a tumor site with very high selectivity. Several recent reviews discuss this and other developments of radioactive PNA probes as well as potential applications in order to image gene expression in vivo [9–16]. In theory, radiometal-containing PNAs could be applied to a wide range of medical interventions such as early detection of diseases, monitoring of disease stages, patient selection for personalized medicine, and realtime assessment of therapeutic and surgical efficiency [1]. A radiometal-containing PNA consists of three units: a receptorspecific entity (necessary for cell binding and subsequent cellular
Synthesis, Characterization, and Evaluation of Radiometal-Containing Peptide Nucleic…
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uptake); a PNA oligomer (which binds to specific mRNA, DNA, or PNA sequences); and a radiometal complex which is stable in vivo (used for imaging (β+ and γ emitters) or therapy (β− emitters)). These three units are separated by spacers, and numerous studies have shown that the introduction of radiolabeled chelating agents does not significantly alter the hybridization properties of the parent PNA. Among the radiometal ions available for radiolabeling PNAs, the gammaemitters 99mTc and 111In have been the most commonly used isotopes [6–8, 17–32]. Both of these radionuclides have favorable decay characteristics, which allow prolonged in vivo evaluations and can be applied for single-photon emission computed tomography (SPECT). Radionuclides emitting positrons especially 64Cu were utilized for positron emission tomography to obtain biodistribution data of for quantification. Table 1 displays an overview of radiolabeled PNA sequences. Based on the results obtained with these PNAs, general guidelines have emerged for the proper design of PNA for use in vivo. For instance, by increasing the length of PNA sequence a significant higher uptake and retention in the kidneys based on rodent experiments was measured. Similar results have been observed for morpholino analogues, a different type of synthetic, uncharged oligonucleotide mimic [33, 34]. Also, the attachment of peptides necessary for targeting cell surfaces and promoting subsequent internalization into the cytosol may increase accumulation of PNA in the kidneys and the liver [35–37]. While radiolabeled PNAs typically show rapid blood clearance, they fortunately exhibit high metabolic and radiolytic stabilities. In general, PNA sequences of 18 bases seem to have an ideal length. Those sequences are synthetically feasible, possess high duplex stability, and exhibit good pharmacokinetics. To attach a radiometal to a PNA, a bifunctional chelating agent (BFCA) must first be coupled to the PNA so that formation of a kinetically and thermodynamically stable radiometal complex can occur. Many BFCAs have been specifically developed for 99mTc, which is the “workhorse” in nuclear medicine due to its daily availability based on a simple generator system and broad application in cost-efficient SPECT imaging. The first-generation chelators, MAG3, MAS3, GDAGG, and Ac-GDAGG (Fig. 1), were used to complex 99mTc as the [99mTcVO] oxo core [6–8, 17–23]. In addition to these standard BFCAs, SBTG2DAP has also been reported as a chelator for technetium [18, 19, 32], both in the thioester and the thiol forms [39]. Second-generation chelators, such as pyrazolyldiamine PzDA [25] and dipicolylamine DPA [26, 27] (Fig. 2), have also been developed. These two chelators rely on using the commercially available “Carbonyl Labeling Agent” pre-reduction Isolink® kit to generate the [99mTc(H2O)3(CO)3]+ precursor [24]. BFCAs for the radionuclide 111In rely on DTPA [28–30] and DOTA [31, 32], and when bound to PNA, both chelators yield complexes with kinetic and thermodynamic stability in vivo (Fig. 2). DOTA has also been used as the BFCA for 64Cu-, 90Y-, and 177Lu-radiolabeling (Fig. 2) [40–44]. When using either DOTA or SBTG2DAP [19, 32]
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Holger Stephan et al.
Table 1 Overview of radiolabeled PNA sequences that have been studied
PNA sequences
Radiometal nuclides (BFCAs)
Applications
References
TGT-ACG-TCA-CAA-CTA
99m
Evaluation of “Pretargeting”
[6–8]
TGT-ACG-TCA-CAA-CTA
99m
Biodistribution studies
[22]
CTG-GTG-TTC-CAT
99m
GCA-TCG-TCG-CGG
99m
GCA-TCG-TCG-CGG
99m
A-GAT-CAT-GCC-CGGCAT
99m
Serum stability studies Cellular internalization in human neuroblastoma cells (SH-SY5Y)
[23]
TGC-ATG-CAT-GCA
99m
Biodistribution studies
[26]
GCC-GCT-GTG-CGGTGC-GG
99m
Tc (DPAM)
Evaluation of a novel ligand for 99Tc labeling
[27]
TCT-CCC-AGC-GTGCGC-CAT TGT-GTT-GCG-ACCCTC-TTG
111
In (DOTA)
Plasma stability studies
[31]
CCA-GCG-TGC-GCC-AT
111
Biodistribution studies Small lymphotic lymphoma imaging
[32]
GTC-TCC-GCT-CCATCT-TGC GGA-GTC-TAC-GTATTT-ACC TAG-TTA-TCT-CTA-TCT
111
Biodistribution studies Brain cancer imaging
[28, 29]
DNA cleavage studies
[30]
TCT-CCC-AGC-GTGCGC-CAT
111
In, Y (DOTA)
In vitro binding to B-cell [43] lymphoma/leukemia-2 (bcl-2) cells
GCC-AAC-AGC-TCC
64
Pancreatic cancer imaging
[12, 19]
GCC-ATC-AGC-TCC
64
Biodistribution studies Pancreatic cancer imaging
[42]
TGG-TGT-GCT-TTGTGG-ATG CAT-CCA-CAA-AGCACA-CCA
64
Biodistribution studies
[40]
CTG-GTG-TTC-CAT
64
Biodistribution studies Breast cancer imaging
[41]
CCA-GCG-TGC-GCC-AT
177
In vitro evaluation of radiotherapy
[43]
Tc (MAG3) Tc (MAS3)
Tc (Ac-GDAGG) Biodistribution studies Breast cancer imaging Tc (GDAGG)
Biodistribution studies Breast cancer imaging
Tc (Ac-GDAGG) Biodistribution studies Breast cancer imaging Tc (PzDA)
Tc (DPA)
In (DOTA) In (DTPA)
111
In (DTPA) 90
Cu (SBTG2DAP) Cu (DOTA) Cu (DOTA)
Cu (DOTA) Lu (DOTA)
[18, 19] [17, 20, 22] [21]
41
Synthesis, Characterization, and Evaluation of Radiometal-Containing Peptide Nucleic…
resulting
chelating unit
99mTc-complex
O
O
O
HN
NH
N
N 99m
HN
NH
-
O
N
O H N
O
Tc N
O
O H N
O O
O
Ac-GDAGG O
O
O
HN
NH
N
N 99m
HN
SH
O H N
S
Tc N
O
O
MAG3 HO
-
O
O H N O
O
HO
-
O
OH O
OH O
HN
NH
N
N 99m
HN
SH
O H N
OH
Tc N
O
S
O
O H N O
OH
MAS3 O
O
O
O
HN
NH
N
N 99m
Tc
HN
NH2
N
N O H2
O H N O
O H N O
GDAGG + O S
O O
O
HN
HN
O
N
S O
99m
O HN
S
O
S
N H
Tc N
O
O
O
SBTG2DAP
+ N
OC
N
N N
H2N
N
99m
N
Tc
CO NH2
OC
PzDA + N N
DPA
N N
N
99m
OC
CO
Tc
N
CO
Fig. 1 Structures of BFCAs attached to PNA oligomers (left) for 99mTc-radiolabeling and their subsequent 99mTc-complexes (right); use Ac-GDAGG in this Figure
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Holger Stephan et al.
chelating unit
proposed complex structure under physiological conditions -
O
O
O N O
N 64
O N
N
N
N 90
O N
Y
O
N N H
O N
N
O
Cu
N
O
HO
O
O
O
N N H
O
O
O
HO O N
O
O
O
O N
OH
O
O
O
O
N
N O
111
In
DOTA
N
O
N
177
O N
Lu
N N H
O
O N
O
N H
O -
O OH OH O
N
N
H N
O
O
HO
O
N
O 111 In O N O
N O
HO
O
N
O
O
O
N H
O
DTPA 2+ O S
O O
O
HN
S
HN
O S
HN
O
O
O O
NH 64
S
Cu
N H
NH O
SBTG2DAP Fig. 2 Structures of BFCAs attached to PNA oligomers (left) for 64Cu/90Y/111In/177Lu-radiolabeling and of the subsequent radiometal complexes (right) (the structures of 64Cu-DOTA, 90Y-DOTA, 111In-DOTA, 177Lu-DOTA, and 111 In-DTPA complexes are based on crystal structure data) [64–67]
for 64Cu-radiolabeling, the in vivo stability of the complexes should be determined in particular cases as contrary opinions on complex stability have been reported based on transchelation phenomena predominantly occurring in liver tissue involving superoxide dismutase [45, 46]. There are four main approaches to attach a BFCA to a PNA oligomer. Since PNA possesses high chemical and thermal stabilities, harsh conditions can be applied during the labeling procedures without degradation of the PNA. The preparation of radiometal-containing PNAs consists of two mains parts: (1) preparation of the
Synthesis, Characterization, and Evaluation of Radiometal-Containing Peptide Nucleic…
43
BFCA-containing PNA oligomer and (2) radiolabeling. The methods described below may also be used to complex nonradioactive metals (e.g. Cu2+, Zn2+, Ni2+, Co2+, Zr4+) [47–57]. 1.1
Approach 1
The most common and straightforward method to couple a BFCA to a PNA oligomer was developed by Lewis et al. [43]. In this approach, a carboxylic acid on the BFCA is activated for amide coupling to the terminal amino group of the PNA oligomer on the solid support [4, 5, 17–45, 55]. This approach is illustrated in Scheme 1 (Subheading 1.1) exemplified by a Boc-protected derivative of the BFCA PzDA [25]. The resulting PzDA-PNA conjugate was purified by RP-HPLC and subsequently radiolabeled [60–62]. For specific applications peptides and/or spacer are required which can easily be introduced at the C-terminus (mainly for peptide grafting) or in between the chelating unit and the PNA sequence [4, 17, 19, 21, 23, 28, 32].
1.2
Approach 2
A second approach, which has been used by Hnatowic et al., involves coupling of BFCA to the PNA in solution (Scheme 1, Subheading 1.2) [6–8]. For example, the N-hydroxysuccinimide derivative of MAG3 (S-acetyl-NHS-MAG3) was coupled to a PNA using a large excess of the BCFA (20:1 molar ratio BCFA:PNA) [63]. After purification, the radiolabeling step is performed. This approach is rarely used because a large excess of the BCFA is always necessary for the reaction to proceed, two purifications are required (one after the synthesis of the PNA oligomer and another after the coupling of the BFCA), and the PNAs cannot have any additional unprotected amino groups as they would also react with the activated ester.
1.3
Approach 3
The third approach, which was developed by Gasser, Stephan, Metzler-Nolte et al., is in principle based on approach 1 Subheading 1.1 [26, 27] but relies on the preparation of an alkynecontaining PNA oligomer on the solid phase which is subsequently reacted with an azido-containing ligand using the Cu(I)-catalyzed [2 + 3] azide/alkyne cycloaddition reaction (i.e., Click reaction). As presented in Scheme 2, 4-pentynoic acid was coupled to the N-terminus of a PNA oligomer on the solid support. Then, a Click reaction with 2-azido-N,N-bis((pyridin-2-yl)methyl)ethanamine (Dpa-N3) afforded the expected Dpa-containing PNA bioconjugate (Dpa-PNA, Scheme 2). After cleavage from the resin, Dpa-PNA was purified by RP-HPLC [60–62]. For specific applications peptides and/or spacer are required which can easily be introduced at the C-terminus (mainly for peptide grafting) or in between the chelating unit and the PNA sequence. [4, 17, 19, 21, 23, 28, 32].
1.4
Approach 4
The fourth approach involves the insertion of a synthon that contains the BFCA within the PNA sequence during solid-phase synthesis. Lewis et al. have derivatized a lysine amino acid with a DOTA derivative to give N-α-(9-fluorenylmethoxycarbonyl)-N-ε-[tris(tert-butyl) DOTA]-L-lysine (FKD, Fig. 3) [31], which is compatible with the protocols of the Fmoc/Bhoc-protecting group strategy and can
NHFmoc
I.
NH2
Spacer
Spacer
O
O NH
Fmoc
NH
PNABhoc
Fmoc-SPPS O
NH
PNABhoc (a)
O
NH
NH
(b) N N (c) N NH O
O
O
NH Spacer
NH2
O
Spacer
NH O
Bhoc
PNA
II.
NH
O
O
PNA
NH
O
O
NH
OH NH2
O N
(c)
(d) N N
O
N N
N H
S N
O NH2
H N
O
O
PzDA-Boc
O
HN
O O
O
NH HN
NH NH Spacer
Spacer O
O PNA O NH2
Approach 1
HN
O O
O NH
NH
S
PNA
O N
O O S-Acetyl-NHS-MAG3
O NH2
Approach 2
Scheme 1 I. Schematic representation of the preparation on the solid phase (Approach 1) and in solution (Approach 2) of a PNA oligomer with a BFCA at the N-terminus. II. Structures of PzDA-Boc and S-acetyl-NHSMAG3. (a) (1) Piperidine in DMF (20 %); (2) washing with DMF, CH2Cl2, and DMF; (b) (1) PzDA-Boc, HATU, DIPEA, 2,6-lutidine in DMF; (2) washing with DMF, CH2Cl2, and DMF; (c) TFA:TIS:H2O 95:2.5:2.5 (v/v/v); (d) S-acetylNHS-MAG3 (in DMF): PNA (0.36 M sodium bicarbonate, 1.4 M sodium chloride, 1.4 mM DTPA, pH 9.3) 20:1 (molar ratio), 1 h, r.t.
45
Synthesis, Characterization, and Evaluation of Radiometal-Containing Peptide Nucleic…
N N
N
N
N
N
N
N
NH
Fmoc-SPPS
Boc
N
O
NHFmoc Fmoc
N
N
N
Spacer
Lys
(a)
(b)
Spacer
O
(c)
O
NH
Bhoc
NH
PNA
Boc
Lys
Spacer
Bhoc
Spacer
PNA
Bhoc
PNA
Boc
Lys
PNA
Boc
Lys
Lys O
NH2
Scheme 2 Synthesis of Dpa-PNA. (a) (1) Piperidine 20 % in DMF; (2) washing with DMF, CH2Cl2, and DMF; (3) 4-pentynoic acid, HATU, DIPEA, 2,6-lutidine, DMF; (4) washing with DMF, CH2Cl2, and DMF; (b) (1) CuI, Dpa-N3, DIPEA, DMF; (2) washing with DMF, CH3CN, EDTA 0.1 M, MeOH, and CH2Cl2; (c) TFA:H2O:TIS 95:2.5:2.5 v/v/v. Note that a Lys amino acid has been added to the C-terminus to increase water solubility O FmocHN
OH N N NH
N
O
N
N
O O
O BocHN
N
O
NH
O
N O
N
N
O
O OH
FmocHN
N
O OH
O O FKD
Bipy-PNA Monomer
Neocuprine-PNA Monomer
Fig. 3 Structures of N-α-(9-fluorenylmethoxycarbonyl)-N-ε-[tris(tert-butyl) DOTA]-L-lysine (FDK) [31], BipyPNA monomer [47], and Neocuproine-PNA monomer [48]
therefore be inserted anywhere within a PNA sequence. Alternative synthons have also been developed. For example: Balasubramanian, Achim et al. have prepared a series of BFCAs which are linked to a PNA backbone (see Bipy-PNA monomer [47] and NeocuproinePNA monomer [48] in Fig. 3) [47–51]. Of note, Spiccia et al. have recently reported the preparation of other BFCAs attached to a PNA backbone [58, 59]. The subsequent conjugates can then be prepared following protocols similar to standard solid-phase synthesis of PNA.
2
Materials All starting materials and reagents should be purchased at the highest commercially available purity, especially the inorganic salts. Use freshly prepared buffer solutions for each synthesis.
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Holger Stephan et al.
To handle the very low quantities of chelator-PNA oligomers, it is recommended to prepare stock solutions (0.1–2.0 mM) of the chelator-PNA derivatives in sterile and deionized water. Diligently follow all material safety data sheets, and follow proper chemical and waste disposal regulations. 2.1 99mTcRadiolabeling of PNA
1.
2.1.1 For MAG3-PNA [6–8, 22] and MAS3-PNA [22]
1. 250 mM ammonium acetate buffer (pH 5.2).
99m
Tc-pertechnetate eluate Mo/99mTc generator.
saline)
of
a
2. 50 mg/mL disodium tartrate in 500 mM sodium bicarbonate buffer (pH 9.3). 3. 1 mg/mL stannous 10 mM HCl.
2.1.2 For Ac-GDAGGPNA [18, 19, 32], GDAGG-PNA [17, 20], SBTG2DAP-PNA [18, 19, 32]
(physiological
99
chloride
dihydrate
solution
in
1. Buffer A: 50 mM phosphate buffer containing 0.1 % Tween-80. 2. Buffer B: 50 mM phosphate buffer (pH 4.5). 3. For Ac-GDAGG-PNA [18, 19, 32]: 1.3 mg/mL stannous chloride dihydrate solution in 50 mM HCl. 4. For GDAGG-PNA [17, 20]: 5 mg/mL stannous chloride dihydrate solution in 50 mM HCl. 5. For SBTG2DAP-PNA [18, 19, 32]: 1 mg/mL stannous chloride dihydrate solution in 50 mM HCl.
2.1.3 For PzDA-PNA [23] and DPA-PNA [26, 27]
1. Isolink® kit “Carbonyl Labeling Agent” (Mallinckrodt-Tyco, Inc.; the kit consists of 17 mg sodium tartrate, 3.2 mg sodium carbonate, and 8.1 mg potassium boranocarbonate in a nitrogen-purged and sealed 10 mL glass vial). 2. 200 mM phosphate buffer (pH 7.0).
2.2 111InRadiolabeling of DOTA-PNA and DTPA-PNA [20, 21, 32, 43]
1. [111In]InCl3 (typically dissolved in 10–100 mM HCl).
2.3 64CuRadiolabeling of DOTA-PNA [41, 42] and SBTG2DAP-PNA [19, 32]
1. [64Cu]CuCl2 (typically dissolved in 100 mM HCl).
2.4 90Y-Radiolabeling of DOTA-PNA [43]
2. 200 mM ammonium acetate buffer (pH 5.0) containing 1 mg/mL gentisic acid and 0.1 % Tween-80. 3. 10 mM phosphate buffer (pH 7.4) containing 150 mM sodium chloride and 0.05 % Tween-20 (see Note 1).
2. 100 mM ammonium acetate buffer (pH 5.5).
1. [90Y]YCl3 (typically dissolved in 10–100 mM HCl). 2. 200 mM ammonium acetate buffer (pH 5.0) containing 1 mg/mL gentisic acid.
Synthesis, Characterization, and Evaluation of Radiometal-Containing Peptide Nucleic…
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2.5 177LuRadiolabeling of DOTA-PNA [43]
1. [177Lu]LuCl3 (typically dissolved in 10–100 mM HCl).
2.6 Materials and Instrumentation for Purification and Analysis
1. UV and radionuclides detector.
2.6.1 HPLC Purification
1. Reverse-phase HPLC instrument (RP-HPLC), running at an isocratic flow rate of 0.5 or 1.0 mL/min. A linear gradient of 0–100 % solvent B over 30 min is recommended as appropriate starting conditions.
2. 200 mM ammonium acetate buffer (pH 5.0) containing 1 mg/mL gentisic acid and 0.1 % Tween-80.
2. C18 column, such as Jupiter 300 C18 (Phenomenex) or Eurosphere C18 Knauer. 3. Solvent A, aqueous 0.1 % v/v TFA. 4. Solvent B, 0.1 % v/v TFA in acetonitrile (HPLC grade). 2.6.2 Size-Exclusion Chromatography (SEC)
1. BioSep-SEC-S column (Phenomenex, 7.8 × 300 mm; 290 Å; 5 μm particles). 2. Mobile phase: 100 mM NaH2PO4/0.05 % NaN3 (pH 6.8). 3. Superdex 75 HR 10/30 column (GE Healthcare Life Sciences). 4. Mobile phase: 20 mM HEPES and 150 mM NaCl buffer (pH 7.3).
3
Methods After purification and isolation of the radiolabeled PNA derivative by HPLC, the solvent has to be removed by standard techniques (e.g. evaporation under reduced pressure or with the help by a gentle flow of gas stream). The resulting residue has to be re-dissolved into a biocompatible and sterile buffer (e.g. PBS). For in vivo applications, radiolabeled PNA must fulfill: (1) radiochemical purity of >98 % and (2) must be dissolved in non-toxic, biological compatible solvents/buffers. If no purification is required (radiochemical yields >98 %), the labeling has to be performed in biocompatible, sterile buffers.
3.1 99mTcRadiolabeling of MAG3-PNA [6–8, 22] and MAS3-PNA [22] Derivatives
1. Add 50 μL of the disodium tartrate solution to a solution of 30 nmol of MAG3-PNA in 100 μL of ammonium acetate buffer. 2. Add 200–350 MBq of 99mTc-pertechnetate solution (