PROTOCOL
`
`Conjugation of DOTA-like chelating agents to peptides
`and radiolabeling with trivalent metallic isotopes
`
`Jane K Sosabowski & Stephen J Mather
`
`Nuclear Medicine Research Laboratory, St. Bartholomew’s Hospital, London, EC1A 7BE, UK. Correspondence should be addressed to J.K.S.
`(jane.sosabowski@cancer.org.uk) or S.J.M. (stephen.mather@cancer.org.uk).
`
`Published online 3 August 2006; doi:10.1038/nprot.2006.175
`
`Peptides can be labeled with various trivalent radiometals for imaging or targeted radionuclide-therapy applications. The peptide
`is first conjugated to a chelating agent that is able to form stable complexes with the radionuclide of interest. This conjugation step
`can be carried out as part of the solid-phase peptide synthesis, or it can be undertaken in the solution phase after synthesis and
`purification of the peptide. The latter route, described here, involves reacting a molar excess of the activated tri-tert-butyl ester-
`derivatized chelator with a designated free amino group of a peptide analog, in which all other reactive amines are protected, in
`the presence of a coupling agent. The conjugate molecule is then purified prior to deprotection and further purification by HPLC.
`The product can be radiolabeled by addition of a suitable metal salt, followed, if necessary, by removal of the unchelated metal.
`The entire process of conjugation, purification and radiolabeling should take approximately 12.5 h.
`
`INTRODUCTION
`A number of trivalent metal radionuclides have physical properties
`suitable for radioisotope imaging (indium-111 (111In), gallium-
`67/68 (67/68Ga) and yttrium-86 (86Y)) or for targeted radionuclide
`therapy (90Y and lutetium-177 (177Lu)). These metal radionuclides
`can be combined with a targeting biomolecule (such as a peptide or
`antibody) in order to diagnose, monitor or treat disease. To obtain
`a radiolabeled biomolecule with the required stability, the peptide
`or protein must first be conjugated to a suitable chelator in order
`to complex the metal. The requirements of chelators for trivalent
`metals (such as In, Y, Ga and Lu) for labeling peptides are generally
`the same as those for labeling proteins1. The complexes should
`be stable in biological systems and their chelating ability should
`not be impaired by reaction with the peptide. Most often,
`diethylenetriaminepentaacetic acid (DTPA) and 1,4,7,10-tetra-
`azacyclododecanetetraacetic acid (DOTA) are used. Of the metals
`mentioned, the DOTA complexes are more thermodynamically and
`kinetically stable than the DTPA complexes. The drawback of using
`DOTA complexes is that they require a degree of heating to aid their
`formation, whereas the less-stable DTPA complexes can form at
`room temperature (19–25 1C). However, peptides (unlike proteins)
`are generally stable to heating; therefore, for peptide-labeling
`applications, DOTA is preferred. As DOTA is a tetra acid, it has
`four potential sites for conjugation, which can lead to a mixture
`of cross-linked conjugates. Fortunately, active-ester tri-t-butyl-
`protected DOTA compounds are commercially available along
`with other useful bifunctional chelators. For this protocol, we
`have described the use of 1,4,7,10-tetraazacyclododecane-1,4,7-tris
`(t-butyl acetate)-10-acetic acid (DOTA-(tBu)3; Fig. 1) a compound
`that requires activation of its free carboxyl group for the conjuga-
`tion to occur. The low aqueous solubility of this compound and the
`need for deprotection after conjugation make it unsuitable for
`chelation with large water-soluble antibodies or proteins, but it is
`ideal for reacting peptides in non-aqueous solvents.
`It is necessary to ensure that the conjugation of a chelator to the
`peptide does not substantially lower the peptide’s receptor-binding
`affinity. It is advantageous to use peptides in which the active
`sequence that confers binding affinity is known, so that the
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`conjugation can be directed away from this receptor binding site.
`If necessary, spacers can be used to increase the distance between
`the conjugation site and the receptor binding site. Usually, the
`peptide is synthesized in such a way that the receptor binding site is
`at the carboxy terminus and the chelator is conjugated to the
`amino-terminal group. This can be done either during fluorenyl-
`methoxycarbonyl (Fmoc) solid-phase peptide synthesis2–5 or in the
`solution phase after synthesis and purification of the peptide6,7.
`In the latter scenario, all free amino groups (such as those on
`lysine residues) other than the desired conjugation site should be
`tert-butyloxycarbonyl (BOC) protected, in order to prevent any
`undesirable side reactions. Chelators can also be conjugated to the
`free amino group of a lysine residue within the peptide sequence. As
`Fmoc solid-phase peptide synthesis is amply covered elsewhere3, this
`
`a
`
`O
`
`O
`
`O
`
`O
`
`b
`
`COOH
`
`COOH
`
`N
`
`N
`
`N
`
`N
`
`N
`
`N
`
`N
`
`N
`
`O
`
`O
`
`O
`
`OH
`
`COOH
`
`O
`
`Peptide
`
`Figure 1 | Chemical structures. (a) DOTA-tri-t-butyl ester. (b) The conjugated
`DOTA-peptide.
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`temperature (37–100 1C) and reaction time (10–60 min). The
`reaction kinetics vary depending on the metal8,9, and the peptide
`residues adjacent to the chelator can also have an effect. Therefore,
`the reaction conditions should be optimized for each peptide/
`chelator/metal combination.
`The amounts of the radioactive metal added will depend on the
`required specific activity (MBq nmol–1) of the product. For
`receptor imaging and therapy studies, it is usually the case that a
`high specific activity is required to prevent both saturation of the
`receptor and possible pharmacological side effects. Usually the
`peptide conjugate is in excess in comparison with the radionuclide,
`and the labeling will be highly efficient with almost 100% of the
`metal being complexed. If higher specific activity is required, then
`rigorous elimination of metal impurities (e.g., by performing the
`radiolabeling reaction in polypropylene instead of glass, using
`polypropylene weighing spatulas, and treating vials, pipette tips
`and so on with acid10) might become necessary, as well as some
`adjustments to the reaction conditions. As far as the reagents are
`concerned, it is important to start with high-grade materials with
`the lowest levels of metal impurities (for this reason, ammonium
`acetate buffer is used as opposed to sodium acetate). In addition,
`the buffers and solutions can be pretreated with Chelex chelating
`ion-exchange resin11 to remove trace multivalent metals. However,
`care should be taken with this approach if high radiolabeling
`efficiencies are required. It has been reported that treatment of
`radiolabeling reaction solutions with Chelex resin, while potentially
`increasing the specific activity of the labeled product, can also
`reduce the labeling efficiency. This is probably due to the presence
`of chelating material residue in the solvents, which has leached
`from the resin. To obtain radiolabeled products with the highest
`possible specific activities, the labeling efficiency might therefore
`have to be compromised and a post-labeling purification step (such
`as a Sep-Pak purification) might need to be introduced.
`This protocol describes the method required to conjugate a
`peptide to the DOTA chelator, and to label the conjugate with a
`radionuclide (such as 111In or 90Y) for use in radionuclide imaging
`or targeted therapy. In total, the entire process should take
`about 12.5 h.
`
`protocol provides a method for small-scale solution-phase conjuga-
`tion of the DOTA-tri-t-butyl ester to a peptide amino group.
`The conjugation method for peptides is quicker than that for
`proteins1, as the solubility of peptides in non-aqueous solvents
`(such as N,N-dimethylformamide (DMF) or N-methyl-2-pyrroli-
`dinone (NMP)) allows them to be reacted and purified more
`rapidly. In this protocol, a DOTA-tri-t-butyl ester is pre-incubated
`in NMP with equimolar amounts of
`the coupling agents,
`O-(7-azabenzotriazole-1-yl)-1,1,3,3,-tetramethyluronium hexa-
`fluorophosphate (HATU) and diisopropylethylamine (DIPEA),
`the last of which is an organic base. The peptide is added to this
`mixture in a peptide:chelator ratio of 1:3 to 1:4, which is suitable for
`small-scale reactions (using 1–5 mg peptide), such as those carried
`out early in the radiopharmaceutical development process. For
`reactions using a large amount of peptide, it is possible to use ratios
`close to 1:1 (ref. 7). After stirring for 4 h at room temperature, the
`product is purified by solid-phase extraction (or extracted into
`ethylacetate (EtOAc)) and the solvent is evaporated off under
`vacuum. The crude product is dissolved in a 94:4:2 (vol/vol)
`mixture of trifluoroacetic acid (TFA):thioanisole:water to remove
`the BOC-protecting groups. After 4 h, the residue is washed with
`ether, redissoved in acetonitrile (ACN)/water and purified by
`reversed phase (RP)-HPLC. The HPLC fractions are evaporated
`to dryness (overall yield, 60%). The final peptide product can then
`be dissolved in water or aqueous buffer (depending on the peptide)
`for radiolabeling.
`In the subsequent radiolabeling step, the metal chloride salt
`(dissolved in dilute HCl) is added to a reaction vial, followed by
`acetate buffer at pH 4–6. This pH range is chosen to ensure that the
`metal remains in solution during the subsequent labeling proce-
`dure. At pH 46, many metals form insoluble hydroxide complexes
`with water and become unavailable for binding to the chelator. The
`presence of weakly chelating ions in the buffer (such as acetate or
`citrate) will also reduce the likelihood of hydrolysis. Some anti-
`oxidants (such as ascorbic or gentisic acid) might also be added to
`prevent radiolysis of the peptide. The peptide is added to this weak
`metal acetate chelate solution and the vial is heated (usually in
`a heating block). The general reaction variables are pH (4–6),
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`MATERIALS
`REAGENTS
`. Peptide (typically 1–5 mg depending on scale; e.g., piCHEM, Bachem,
`BioSynthema)
`. DOTA-(tBu)3 (Macrocyclics, cat. no. B260)
`. NMP (Sigma-Aldrich)
`. HATU (Sigma-Aldrich)
`. DIPEA (Sigma-Aldrich)
`. Metal-free water (typical resistance, 18 mO cm–1; from an ELGA water-
`purification system)
`. TFA (Spectrophotometric grade; Sigma-Aldrich)
`. Thioanisole (Sigma-Aldrich)
`. ACN (HPLC grade; Sigma-Aldrich)
`. EDTA (Sigma-Aldrich)
`. Ammonium acetate (choose the grade with the lowest possible metal
`impurities; Sigma-Aldrich)
`. Glacial acetic acid (Sigma-Aldrich) ! CAUTION Highly corrosive.
`. Ammonia solution (35%; Sigma-Aldrich) ! CAUTION Vapors are irritating to
`respiratory system; corrosive.
`. Methanol (HPLC grade; Sigma-Aldrich)
`. Chelex-100 analytical grade resin, 100–200 mesh (Bio-Rad Laboratories)
`. Radionuclide as chloride salt in dilute hydrochloric acid.
`
`. 111In chloride (e.g., GE Healthcare, Tyco Healthcare, MDS Nordion)
`. 90Y chloride (e.g., MDS Nordion, Perkin Elmer)
`. 177Lu chloride (e.g., Perkin Elmer, IDB) ! CAUTION Radioactive; use
`appropriate radiation safety measures as laid out in local rules; standard
`shielding and radionuclide handling procedures should be employed; direct
`exposure to the radioactive dose should be kept to a minimum; individuals
`working with the material should monitor their radiation exposure with
`appropriate devices.
`. HPLC Solvent A: 0.1% TFA (vol/vol) in water (Sigma-Aldrich)
`. HPLC Solvent B: 0.1% TFA (vol/vol) in ACN (Sigma-Aldrich)
`EQUIPMENT
`. Calibrated pH meter
`. 1 ml Reacti-Vial with Reacti-Vial magnetic stirrer (Pierce and Warriner,
`cat. nos. 13221 and 16010)
`. Magnetic stirrer plate
`. C-18-Sep-Pak-Classic cartridge (Waters)
`. Metal-free round-bottomed polypropylene tubes, 2 ml (e.g., Nunc
`Cryo-tubes, Corning Inc.)
`. Centrifugal evaporator
`. RP-HPLC gradient system with UV and radiochemical detectors
`(Raytest GmbH)
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`. Jupiter C18 HPLC column, 5 mm, 300 A˚ , 250  4.6 mm internal diameter
`(i.d.) or similar (Phenomenex, cat. no. 00G-4053-E0)
`. Chromatographic support material, silica-gel-impregnated glass-fiber
`(ITLC-SG, Pall Corp.) cut into strips approximately 2  10 cm in size with
`a faint pencil mark 1.5 cm from one end
`. Gamma counter (e.g., Perkin Elmer)
`REAGENT SETUP
`0.1 M ammonium acetate buffer (pH 6) Dissolve 7.708 g ammonium acetate in
`1 l water, add 300 ml acetic acid. Mix well and check that the pH is 5.5–6.
`! CAUTION Acetic acid is highly corrosive.
`0.1 M ammonium acetate containing 50 mM EDTA Dissolve 185 mg EDTA in
`10 ml of 0.1 M ammonium acetate buffer (pH 6).
`1 M ammonium acetate buffer (pH 5.5–6) Prepare a 1-M solution of
`ammonium acetate by dissolving 3.854 g in 50 ml water. Prepare a 1-M solution of
`acetic acid by making 3 ml acetic acid up to 50 ml. Add one part 1 M acetic acid
`to eight parts 1 M ammonium acetate. Mix well and check that the pH is 5.5–6.
`If an extremely high specific activity radiolabeled product is required, this buffer can
`be treated with Chelex-100 resin prior to use (see INTRODUCTION for a
`discussion of the merits of this approach). ! CAUTION Acetic acid is highly corrosive.
`1:1 mixture of 3.5% (vol/vol) ammonia solution and methanol To prepare
`100 ml, using an appropriate measuring cylinder, measure 45 ml water and
`
`make up to 50 ml using 35% (vol/vol) ammonia solution. Add this to 50 ml
`methanol and mix. m CRITICAL This solution must be prepared fresh on
`the day of analysis to avoid evaporation of the solvents.
`EQUIPMENT SETUP
`Preparation of metal-free vials and disposables For preparation of an
`extremely high specific activity radiolabeled peptide, place vials, tubes, pipette
`tips and so on in a 500-ml bottle containing 0.1 M HCl (prepared with
`metal-free water), and place on a mixer overnight. Pour off the HCl and rinse
`thoroughly with metal-free water. Refill the bottle with metal-free water and
`place on a mixer for a further 2 h. Remove the vials and disposables from the
`water, and dry in an oven.
`HPLC method for peptide purification Set up a general HPLC method
`for peptide separation on a C18 column, as in the following example:
`0–20 min, 5–60% Solvent B; 20–25 min, hold at 60% Solvent B; 25–30 min,
`60–90% Solvent B; 30–35 min, 90–5% Solvent B. It might be necessary to
`optimize the separation of the conjugated peptide from any unconjugated
`peptide or chelator. Inject the starting materials to determine their elution
`times. Inject small amounts of the crude product and decrease the
`solvent gradient if necessary to improve the peak resolution. Try not to
`decrease the gradient to o1% ACN per min, as this might cause peak
`broadening.
`
`PROCEDURE
`Conjugation reaction
`1|
`Calculate the amount of DOTA-(tBu)3 to give a threefold to fourfold molar excess over the amount of peptide to be used
`(1–5 mg).
`
`2| Add equimolar amounts of HATU and DIPEA along with NMP (approximately 30 ml NMP per mg peptide to be used) to the
`DOTA-(tBu)3 chelator, and incubate at room temperature for 20 min.
`
`3|
`
`Transfer the mixture to a 1-ml Reacti-Vial.
`
`4| Dissolve the peptide in NMP (30 ml mg–1 peptide) and add the same amount of DIPEA as was added to the chelator.
`
`5|
`
`Transfer this solution to the 1-ml Reacti-Vial, add a Reacti-Vial magnetic stirrer and stir for 4 h at room temperature.
`
`Sep-Pak purification
`6| Activate a C-18-Sep-Pak cartridge with methanol and then pre-equilibrate the column with water.
`
`7|
`
`8|
`
`Load the reaction mixture onto the column and wash with 5 ml water.
`
`Elute the peptide conjugate into a 1.5-ml microcentrifuge tube with 1 ml of 0.1% TFA (vol/vol) in ACN.
`
`9|
`Evaporate the solvent using a centrifugal evaporator (this should take approximately 15 min at room temperature).
`’ PAUSE POINT The protected peptide-conjugate crude material can be stored in a refrigerator overnight at 4 1C.
`
`Deprotection
`10| Dissolve the crude product in 282 ml TFA, 12 ml thioanisole and 6 ml water, and react for 4 h.
`
`11| Evaporate off the deprotection mixture using a centrifugal evaporator (this should take approximately 15–20 min at room
`temperature).
`12| Wash the residue with ice-cold ether (3  1 ml) and re-dissolve in 20% ACN (vol/vol) in water.
`
`HPLC purification
`13| Purify the peptide conjugate by RP-HPLC using a Jupiter C18 column (or similar) equilibrated on 5% Solvent B.
`m CRITICAL STEP The first time this step is carried out for any peptide, it might be necessary to optimize the separation method,
`before injection of the entire crude mixture. Refer to the advice given in the EQUIPMENT SETUP. Be aware that any residual
`unconjugated DOTA-(tBu)3 might have also come through the SPE column, so there might be some deprotected DOTA in this HPLC
`purification mixture. If there is more than one unprotected primary amine group on the peptide, this will lead to a mixture of
`products, which will again complicate the purification procedure. In this case, the products should be separated by HPLC, collected
`and structural analysis carried out in order to identify the required product.
`
`14| Inject the crude product onto the HPLC and run the separation method.
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`15| Collect the HPLC eluate containing the peptide conjugate and evaporate to dryness in a weighed microcentrifuge tube
`using a centrifugal evaporator. Re-weigh the microcentrifuge tube to determine the yield of conjugate.
`
`16| Redissolve the product in water (with added base or acid to achieve solubility if necessary) to a concentration of
`1 mg ml–1, divide into suitable sized aliquots (10–50 mg depending on application) in metal-free tubes and store at below
`–20 1C until required.
`’ PAUSE POINT The peptide conjugate can be stored for months or years under these conditions.
`
`Radiolabeling
`! CAUTION Use appropriate radiation safety measures at this stage.
`17| Thaw out an aliquot of the peptide conjugate.
`
`18| Pipette the required volume of radionuclide solution into a metal-free polypropylene screw-top tube (such as a 2-ml
`Corning cryotube). The quantity will depend on the amount of radioactivity needed for the desired application.
`
`19| Add a volume of 1 M ammonium acetate buffer (pH 5.5–6) equal to 10% of the volume of the radioactive isotope.
`
`20| Add a volume of peptide conjugate corresponding to the required specific activity (MBq nmol–1) of the radiolabeled
`product. Check the pH of the labeling reaction using pH indicator strips (it should be in the range of 4 to 6).
`m CRITICAL STEP If the pH is o4, it is probable that 1 M ammonium acetate buffer has been omitted from the reaction mixture or
`insufficient buffer has been added. This will reduce the rate of metal complexation and more buffer should be added to compensate
`for this. If the pH is 46, the pH of the ammonium acetate buffer is probably incorrect. This could lead to the formation of insoluble
`metal hydroxides.
`
`21| Mix well and heat the labeling reaction in a dry-block heater for up to 30 min at up to 98 1C. The exact conditions depend
`on the radioisotope (e.g., 111In requires more heating than 90Y) and the peptide (the temperature or duration of heating might
`have to be minimized if there are any thermolabile residues, e.g., if it is necessary to prevent the oxidation of methionine).
`
`22| Remove the vial from the heating block and allow to cool for a few minutes. Quench the reaction by adding 10% of the
`total reaction volume of 0.1 M ammonium acetate/50 mM EDTA solution.
`
`Measurement of radiolabeling efficiency by thin-layer chromatography (TLC)
`23| Pour 0.1 M ammonium acetate containing 50 mM EDTA solution into a glass beaker (10–15 cm tall) or similar container
`until it is 0.5 cm deep. Cover the beaker with a Petri dish lid, aluminum foil or similar.
`
`24| Repeat this procedure using a 1:1 ratio of 3.5% (vol/vol) ammonia/methanol solution in a separate container.
`
`25| Place a 1-ml spot of the radiolabeled peptide sample to be analyzed onto the centre of the pencil mark on each of two
`chromatographic strips and allow the spots to dry.
`
`26| Using forceps, gently place the strips upright in each beaker with the pencil mark at the lower end just above the solvent
`level. Cover the beaker and allow the solvent to run up the support material.
`
`27| When the solvent is about 5 mm from the top of the strip, remove it from the beaker using forceps and lay it on a clean
`piece of tissue to dry.
`
`28| Cut the strips in two equal parts across the short axis. Place the upper and lower halves into separate gamma counter tubes
`and measure the radioactive counts from each tube in a gamma counter.
`
`29| In the case of the ammonium acetate/EDTA strip, unbound radionuclide migrates with a retardation factor (Rf) of 1, while
`labeled peptide remains at the origin. Calculate the proportion of unbound radionuclide as follows:
`100%
`% unbound radionuclideðAÞ ¼
`
`Counts on upper half
`Counts on lower plus upper half
`
`30| In the case of the 1:1 ratio of 3.5% (vol/vol) ammonia/methanol strip, calculate the amount of insoluble metal hydroxides
`(colloidal material) present in the radiolabeling mixture as follows:
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`% insoluble hydroxidesðBÞ ¼
`
`Counts on lower half
`Counts on lower plus upper half
`
`100%
`
`The peptide labeling efficiency is therefore 100 – (A + B)%.
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`Measurement of radiolabeling efficiency by HPLC
`31| Dilute radiolabeling reaction with water or saline. Inject on RP-HPLC using the same column and conditions
`as those used for the purification of the peptide conjugate.
`
` TIMING
`
`Steps 1–5: peptide conjugation, 5 h.
`
`Steps 9–15: deprotection and purification, 6.5 h.
`
`Steps 17–31: Radiolabeling and analysis, 1 h.
`
`? TROUBLESHOOTING
`Troubleshooting advice can be found in Table 1.
`
`TABLE 1 | Troubleshooting table.
`
`PROBLEM
`Poor peptide
`conjugate yields.
`
`POSSIBLE CAUSES
`If an EtOAc extraction step is used, the product can be lost. Use NMP as a reaction solvent instead of DMF, and use
`SPE purification instead of EtOAc extraction.
`
`Loss of peptide during HPLC purification. This can be due to peptide binding to free silanols on the C18 column.
`Use a column that tolerates low pH, such as Phenomenex Jupiter C18.
`
`Low labeling
`efficiency.
`
`Reaction mixture too dilute (i.e., peptide concentration too low).
`
`Reaction conditions not optimal for particular peptide-chelator-metal combination. Vary reaction time, temperature,
`volume and pH (do not exceed 6.5 due to formation of insoluble metal hydroxides).
`
`Contamination with metal ions. Make sure that all reagents and disposables are metal free. Use a polypropylene
`reaction vial for the radiolabeling reaction.
`
`Contamination of buffers with chelating material, such as Chelex. If high specific activities cannot be achieved
`without the use of Chelex, introduce a post-labeling Sep-Pak purification to remove the radiometal–Chelex complexes.
`
`High specific activity
`not achievable.
`
`Contamination with metal ions. Make sure that all reagents and disposables are metal free. Use a polypropylene
`reaction vial for the radiolabeling reaction.
`
`ANTICIPATED RESULTS
`Yields of conjugated peptide after deprotection and purification should be in the region of 60%. Radiolabeling efficiencies
`495% should be routinely achieved.
`
`COMPETING INTERESTS STATEMENT The authors declare that they have no
`competing financial interests.
`
`Published online at http://www.natureprotocols.com
`Rights and permissions information is available online at http://npg.nature.com/
`reprintsandpermissions
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` puorG gnihsilbuP erutaN 6002 ©
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`976 | VOL.1 NO.2 | 2006 | NATURE PROTOCOLS
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`Evergeen Ex. 1026
`5 of 5
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