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Applied Radiation and Isotopes 64 (2006) 645–650

www.elsevier.com/locate/apradiso

t-Butyloxycarbonyl: An ordinary but promising group for protectingpeptides from deiodination

Xin Sun, Taiwei Chu�, Xinqi Liu, Xiangyun Wang

Department of Applied Chemistry, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China

Received 9 September 2005; received in revised form 31 December 2005; accepted 31 December 2005

Abstract

In order to protect directly radioiodinated peptides from in vivo deiodination, a novel procedure was explored. Two peptides, Try-Gly-

Gly-Gly-Gly-Gly-Cys-Asn-Gly-Arg-Cys (YG5) and t-Boc-Try-Gly-Gly-Gly-Gly-Gly-Cys-Asn-Gly-Arg-Cys (t-BOC-YG5) were synthe-

sized and radiolabeled. A paired-label biodistribution study using [131I]t-BOC-YG5 and [125I]YG5 was undertaken in normal mice.

Compared to [125I]YG5, [131I]t-BOC-YG5 was quite resistant to in vivo deiodination, resulting in rapid reduction of the radioactive

background and negligible radioactivity accumulation in both thyroid and stomach. [131I]t-BOC-YG5 was also stable in human serum

even after 24 h. In conclusion, the t-BOC group has the potential to protect peptide from deiodination.

r 2006 Elsevier Ltd. All rights reserved.

Keywords: t-BOC; Radioiodination; Biodistribution; Deiodination; CNGRC

1. Introduction

During recent decades, considerable interest has beenfocused on the use of radiolabeled peptides as imagingagents or in vivo chemical probes. Iodine radionuclides, areespecially attractive for various applications because thetype of radiation is wide-ranging and well-suited for singlephoton emission tomography (123I, 124I), b-particle therapy(131I), and positron emission tomography (124I) (Larsonand Carrasquillo, 1988).

The direct radioiodine labeling of peptides or proteinsis, in fact, the radioiodination of tyrosine residues viaelectrophilic substitution. Unfortunately, such proceduresoften yield a product, which is susceptible to deiodinationin vivo (Bakker et al., 1990, 1991). Internalization ofpeptides, such as anti-EGFRvIII antibody (Wikstrand etal., 1995), anti-m mAb DA4-4 (Geissler et al., 1992), andcyclic RGD (Hart et al., 1994), after binding to the receptorexpressed on the cell membrane is an important phenom-enon. The internalization results in deiodination of directlyradioiodinated peptides. The low molecular weight radi-

e front matter r 2006 Elsevier Ltd. All rights reserved.

radiso.2005.12.019

ing author. Tel./fax: +86 10 62755409.

ess: twchu@pku.edu.cn (T. Chu).

olabeled catabolites formed, mainly monoiodotyrosine, arequickly removed from tumors, and significant uptake ofradioiodide in thyroid gland results (Geissler et al., 1992;Wikstrand et al., 1995).Alternative approaches for the prevention of in vivo

deiodination of radioiodinated peptides have been evalu-ated. The use of an acylation agent derived from theprototypical N-succinimidyl 3-iodobenzoate (SIB) forlabeling proteins decreases deiodination in mice comparedto proteins labeled by direct radioiodination methods(Pradeep et al., 1996; Reist et al., 1996; Shankar et al.,2004). However, the synthesis of such a prosthetic groupand its subsequent binding to a peptide could be labor-intensive and time-consuming. In addition, the acylationagents that have to be conjugated to the e-amino group of alysine moiety are not applicable when lysine is thefunctional group or when no lysine moiety exists in apeptide. Modification of tyrosine has also been reported toincrease the in vivo stability of a radioiodinated product(Krummeich et al., 1994). However, O-methylation oftyrosine reduces the reactivity of the aromatic nucleus andrequires a severe labeling reaction, which may alter thepeptide structure. Another approach to tyrosine modifica-tion, the methylation of tyrosine’s a-position, has not yet

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NH2-Gly-Gly-Gly-Gly-Gly-Cys-Asn-Gly-Arg-Cys-OH

NH2-Tyr-Gly-Gly-Gly-Gly-Gly-Cys-Asn-Gly-Arg-Cys-OH

O NH-Tyr-Gly-Gly-Gly-Gly-Gly-Cys-Asn-Gly-Arg-Cys-OH

O

G5:

t-BOC-YG5:

YG5:

Fig. 1. The amino acid sequences for G5, t-BOC-YG5, and YG5.

X. Sun et al. / Applied Radiation and Isotopes 64 (2006) 645–650646

been applied to protect peptide from deiodination for somereason.

The t-butyloxycarbonyl (t-BOC) group is a widely useda-amino-protecting group. The synthesis of t-BOC-pro-tected peptide has been thoroughly studied and now is aroutine technique. In addition, because the t-BOC group isstable over the pH range of 4–12, t-BOC-protected peptidescan survive at the low lysosomal pH of 5.

The NGR sequence motif (Asn-Gly-Arg) is an amino-peptidase N (CD13) ligand that targets activated bloodvessels in tumors (Pasqualini et al., 2000). In the presentstudy, two NGR—containing cyclic peptide analogues,Try-Gly-Gly-Gly-Gly-Gly-Cys-Asn-Gly-Arg-Cys (YG5)and t-Boc-Try-Gly-Gly-Gly-Gly-Gly-Cys-Asn-Gly-Arg-Cys(t-BOC-YG5) (Fig. 1), were synthesized and labeled with125I or 131I, respectively. A paired-label biodistribution studyof [131I]t-BOC-YG5 and [125I]YG5 was undertaken innormal mice in order to determine whether t-BOC couldprotect the peptide from in vivo deiodination.

2. Materials and methods

The cyclic peptide G5 (Fig. 1) was purchased from GLBiochem Ltd. (Shanghai, China). All other reagents wereof A. R. grade and used as supplied without further puri-fication. A Symmetry C18 column (5 mm, 3.9� 150mm,Waters, Massachusetts, USA) was used for sample analysisand separation. All reverse-phase high-performance liquidchromatography (RP-HPLC) analyses were performedwith a Waters 600E multisolvent delivery system (Massa-chusetts, USA). The elution was also monitored with aPackard 500 TR flow scintillation radioactivity detector(Connecticut, USA) in addition to the UV detector. Theradiochemical recovery was obtained from the added andrecovered radioactivity measured with a Capintec CRC-15R Dose Calibrator (New Jersey, USA). A PackardCobra II Series Counting System (Connecticut, USA) wasused to count the radioactivity of tissue samples. A WatersC18 Sep-Pak mini cartridge (Massachusetts, USA) wasemployed for desalination. Na131I and Na125I werepurchased from the China Institute of Atomic Energyand Beijing Atom Hightech Co. Ltd., respectively. The

matrix-assisted laser desorption ionization-time of flight(MALDI-TOF) mass spectra were provided by the BeijingMass Spectrometry Center, Chinese Academy of Sciences(CAS). Kunming mice (weighing 20–25 g) were purchasedfrom Institute of Zoology, CAS.

2.1. Synthesis of t-BOC-YG5

t-BOC-tyrosine-NHS was synthesized and purified byour laboratory according to the method of Iuchi et al.(1988). The cyclic peptide G5 (1 mmol in 16 mL DMF) wasadded to 400 mL DMF. N,N-diisopropylethylamine (2 mmolin 10 mL DMF) and t-BOC-tyrosine-NHS (5 mmol in100 mL DMF) were added to that solution. The mixturewas shaken at room temperature for 1 h. Hydrochloric acid(4 mmol in 40 mL DMF) was added to stop the reaction.The mixture was dried under vacuum and washed withacetonitrile (3� 1mL). Water (500 mL) was added todissolve the sample and ethyl acetate (5� 2mL) wasadded to remove the t-BOC-tyrosine-NHS completely.The MALDI-TOF-MS m/z calculated for t-BOC-YG5([M+H]+) was 1097.4, found: 1098.3, Fig. 2.

2.2. Synthesis of YG5

The YG5 was synthesized by the method of Huang andChen (1985). Briefly, the t-BOC-YG5 synthesized above(25 mL, 2� 10�3M) was dried under vacuum and then100 mL of formic acid (490%) was added to remove the t-BOC group at room temperature. After standing for 2 h,the formic acid was removed under vacuum. MALDI-TOF-MS m/z calculated for YG5 ([M+H]+) was 998.4,found: 998.3, depicted in Fig. 2.

2.3. Radioiodine labeling

Peptides were labeled with 125I or 131I by using theiodogen method (Wang et al., 1985). A solution of t-BOC-YG5 (25 mg, 50 mL in PBS, pH 7.4) was added to 300 mCi ofNa131I in a plastic vessel coated with 100 mg of iodogen.After incubation at room temperature for 10min, thereaction was stopped by removing the solution from the

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800 1000 1200 1400

YG5

t-BOC-YG5

Fig. 2. MALDI-TOF MS spectra of t-BOC-YG5 (up) and YG5 (down).

X. Sun et al. / Applied Radiation and Isotopes 64 (2006) 645–650 647

vessel. The solution of radiolabeled peptide was loaded onthe C18 Sep-Pak mini cartridge preconditioned with 5mLof methanol and the cartridge was washed with 5mL of1mM HCl to elute free 131I�. The radiolabeled peptide waseluted with 5mL of acetonitrile–water mixture (3:7, V/V).Radiochemical purity was determined by RP-HPLC on aC18 column. The gradient was run at a flow rate of 1mL/min using the following conditions: 0.1% trifluoroaceticacid (TFA)/water (solvent A) and 0.1% TFA/acetonitrile(solvent B). The gradient started with 90% solvent A for5min, changed to 40% solvent A over 15min. Since theradiochemical purity was usually higher than 95%, nofurther purification was needed.

The YG5 was radiolabeled, purified and determined withthe aforementioned method. Because the radiochemicalpurity was only 76%75%, another purification procedurewas required. The crude product without desalination wasinjected into RP-HPLC, eluted in as described above. Theeffluent was collected in fractions of 10 drops per tube,and detected with the dose-calibrator. The three tubesof effluent with the highest dose were mixed, and theradiochemical purity was determined again with RP-HPLC.

The acetonitrile in both purified solutions was removedunder vacuum, and the residue was dissolved in PBS(pH 7.4).

2.4. n-Octanol/PBS partition coefficient

Into a centrifugal tube were added 50 mL (1.5 mCi) of[131I]t-BOC-YG5 or [125I]YG5 and 350 mL of 0.1M PBS,and then mixed with 400 mL of n-octanol. The two-phasemixture was vortexed at room temperature for 3min andcentrifuged at 6000 rpm for 6min to separate the twophases. Aliquots of 100 mL n-Octanol and PBS werepipetted into separate tubes, and counted in an automatedg counter. The partition coefficient was calculated fromthree parallel runs.

2.5. Serum stability

To 400 mL of freshly prepared human serum was added100 mL (30 mCi) of the labeled peptide solution and the

mixture was shaken and incubated at 37 1C. At 1, 4, and24 h, a 100 mL specimen was taken out and passed througha 0.22 mm filter. The filtrate was determined by RP-HPLCwith the aforementioned method. PBS (5� 200 mL, 0.1M)was passed through the filter to wash out all the residualradiolabeled species not bound to serum proteins and thewashing fluid was collected. The radioactivity of thespecimen, the filtrate, and the washing fluid was measuredin the dose-calibrator, respectively.

2.6. Biodistribution

A paired-label experiment was performed after injectionof 0.1mL [131I]t-BOC-YG5 (1.5 mCi, 25 nmol/kg) and[125I]YG5 (1.5 mCi, 25 nmol/kg) into each normal maleKunming mouse via the tail vein. Groups of five animalswere sacrificed by cervical dislocation at 5min, 0.5, 1, 2,and 4 h post-injection. The organs or tissues of interestwere removed, washed with saline, and weighed prior toradioactivity counting in an automated g counter. Thewashing fluid from the stomach of the 1 h group wascollected and measured. A correction was applied for thecontribution of the 131I in the 125I counting window. Theresults were calculated and expressed as the mean7SD ofpercentage of the injected dose per gram of tissue (%ID/g)or percentage of the injected dose (%ID). All experimentswere carried out following the principles of laboratoryanimal care and the Chinese law on the protection ofanimals.

2.7. Thyroid gland blocking

A group of 5 normal mice were studied after blockingtheir thyroid glands by injection of 100 mL of 500 mg/mLNaI solution via the tail vein. After 6 h, each mouse wasinjected with 100 mL (1.5 mCi) of radioiodinated YG5 andsacrificed by cervical dislocation at 90min. As a compar-ison, another group of 5 normal mice were administered inthe same way without thyroid blocking. Thyroid glandswere removed and counted in an automated g counter.

3. Results and discussion

3.1. Radiolabeling and lipophilicity

The RP-HPLC analysis results showed that theradiochemical purity of [131I]t-BOC-YG5 and [125I]YG5was higher than 95%, and no radioiodide was found.The retention time of [131I]t-BOC-YG5 was 13.30min,[125I]YG5 9.60min, as depicted in Fig. 3, while theretention times of 131I� (125I�) and radioiodinated t-BOC-tyrosine were 1.90 and 17.20min, respectively. Then-octanol/PBS partition coefficients of [131I]t-BOC-YG5and [125I]YG5 were 0.03 and 0.01, respectively, indicatingthat the former was a little more lipophilic. The resultsagreed with their HPLC behavior: the less lipophilic

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adio

activ

ity

0 5 10 15 20 0 5 10 15 20Time (min)

[131I]t-BOC-YG5 [125I]YG5

Fig. 3. RP-HPLC of [131I]t-BOC-YG5 (left) and [125I]YG5 (right).

Rad

iact

ivity

0 5 10 15 20 25 0 5 10 15 20 25Retention time (min)

24 h

4 h

1 h

[131I]t-BOC-YG5 [125I]YG5

Fig. 4. RP-HPLC of [131I]t-BOC-YG5 and [125I]YG5 after 1, 4, and 24 h

incubation in human serum at 37 1C.

X. Sun et al. / Applied Radiation and Isotopes 64 (2006) 645–650648

[125I]YG5 having a shorter retention time than the morelipophilic [131I]t-BOC-YG5.

3.2. Serum stability

The total radioactivity of filtrate and washing fluid wasfound to be nearly equal to that of specimen at each timepoint, indicating that almost 100% of radiolabeled speciescould pass through the 0.22 mm filter and no serum peptidebinding occurred. Furthermore, the radioactivity recoveredin the filtrate was close to 100% for each specimen.Therefore, RP-HPLC results could represent the labeledspecies of each specimen. The [131I]t-BOC-YG5 and[125I]YG5 were quite stable in human serum as depictedby Fig. 4. In the case of [131I]t-BOC-YG5, only a smallportion was degraded into [131I]t-BOC-tyrosine and radio-iodide after 24 h incubation at 37 1C. The radioiodidemight be produced directly from deiodination of [131I]t-BOC-YG5 and indirectly by deiodination of [131I]t-BOC-tyrosine as well. As for [125I]YG5, only traces of radio-iodide were found after 24 h incubation at 37 1C.

3.3. Biodistribution in normal mice

The biodistribution results of [131I]t-BOC-YG5 and[125I]YG5 are shown in Fig. 5. Generally, [131I]t-BOC-YG5 had a significantly faster clearance rate than[125I]YG5 from the examined tissues. [131I]t-BOC-YG5was less avidly retained by blood, kidney, and liver.Compared to [125I]YG5, [131I]t-BOC-YG5 provided arapidly reduced radioactive background level.

The most striking difference in biodistribution between[131I]t-BOC-YG5 and [125I]YG5 was in the thyroid uptakeof radioactivity, Fig. 6. In the former case the radioactivityin thyroid remained a quite low level (o0.3%ID). Incontrast, it increased dramatically in the latter case,indicating that radioiodine was lost markedly from[125I]YG5. The differences were all significant or very

significant (po0:05 for 5min, po0:01 for the other timepoint). Since [131I]t-BOC-YG5 and [125I]YG5 were simul-taneously injected into the same mouse, the differencecould not be explained by individual difference among thetested animals.

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16

12

8

4

0

Liver32

24

16

8

0

Kidneys16

12

8

4

0

Blood

%ID

/g

4

3

2

1

05min 0.5h 1h 2h 4h

20

15

10

5

05min 0.5h 1h 2h 4h

16

12

8

4

05min 0.5h 1h 2h 4h

Muscle Stomach Sm. Intenstine

Fig. 5. Tissue distribution of [125I]YG5 (blank bar) and [131I]t-BOC-YG5 (black bar) in normal mice, expressed as %ID/g.

4

3

2

1

05min 0.5h 1h 2h 4h

Time

%ID

Fig. 6. Thyroid uptake of [125I]YG5 (blank bar) and [131]t-BOC-YG5

(black bar) in normal mice, expressed as %ID per organ.

4

2

0

no tyroid block thyroid block

%ID

Fig. 7. Left column: thyroid uptake of radioiodinated YG5 without

thyroid block. Right column: thyroid uptake of radioiodinated YG5 with

thyroid block.

X. Sun et al. / Applied Radiation and Isotopes 64 (2006) 645–650 649

In the thyroid blocking experiment, we measured thethyroid uptake of radioiodinated YG5 at 90min both withand without thyroid blocking, as depicted in Fig. 7. Theuptake in the former case was 0.5270.27%ID, while thatin the latter case arrived at 3.0170.78%ID. Therefore, theresults indicated that the t-BOC group protected theradioiodinated CNGRC from in vivo deiodination.

Further evidence came from the difference in gastro-intestinal retention between [131I]t-BOC-YG5 and[125I]YG5. The former gave rather low retention instomach (o1.70%ID/g after 30min post-injection), whilethe latter gave much higher retention: from 15.7%ID/g at30min to 4.13%ID/g at 4 h. Also the radioactivity ratio ofstomach-contents-to-stomach at 1 h post-injection was3.2370.92 for [125I]YG5 and 2.9270.72 for [131I]t-BOC-YG5. The corresponding ratio after i.v. administration ofNa125I was 1.9–3.2 due to direct secretion of iodide into thestomach by the Na+/I� symporter (NIS) (Josefsson et al.,

2002). Such a similarity suggested that the radioiodinefound in the stomach after i.v. administration of radio-iodinated peptides was also in the form of iodide. However,5–10% of administered dose was found in stomachcontents for [125I]YG5, but only 0.3–0.4% for [131I]t-BOC-YG5. The corresponding proportion for Na125I was9–16% (Josefsson et al., 2002). Therefore, it can beconcluded that a large amount of radioiodine was lostfrom the administered [125I]YG5, whereas only a trace of[131I]t-BOC-YG5 was deiodinated.However, perhaps due to different metabolic and

excretion mode from its unmodified counterpart, theradiotracer of [131I]t-BOC-YG5 remained largely in smallintestine, followed by temporary suspension in largeintestine. If radioiodide was the main chemical form inintestine, the NIS system could reabsorb it back intocirculatory system, resulting in low radioactivity inintestine (Josefsson et al., 2002). Therefore, in the case of

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[131I]t-BOC-YG5, a large part of the radioactivity inintestine could not be radioiodide.

It can be safely concluded that [125I]YG5 was definitelyunstable in vivo and dissociated to a large extent, leadingto significant uptake of radioiodide in thyroid andstomach. It reflected catabolism of label from radio-iodinated peptide and presented a metabolism patterndissimilar to [131I]t-BOC-YG5 (Zalutsky and Narula,1988). Structural similarity between the iodotyrosinecreated in conventional peptide iodinations and thyroidhormones can result in recognition of the labeled iodotyr-osyl group on [125I]YG5 by deiodinases (50DI, 50DII and5D) known to be involved in the metabolism of thyroidhormones (Kohrle, 1999). After the N-terminal of YG5was protected by t-BOC, in vivo deiodination was scarcelyfound even after 4 h. Other research has also shown thatconformal change of tyrosine can make the tyrosine orpeptide unrecognized by endogenous deiodinases (Foulonet al., 2000; Krummeich et al., 1994). Our results revealedthat modification of N-terminal tyrosine with an ordinaryprotection group, t-BOC, is a promising procedure toincrease peptide’s stability against in vivo deiodination.

4. Conclusions

Deiodination in vivo of the directly radioiodinatedpeptide is an important phenomenon, generating labeledcatabolites that can not be excreted rapidly from normaltissues. A simple procedure to prevent in vivo deiodinationof the labeled peptide is very attractive. Our study showedthat after N-termination with t-BOC-tyrosine, CNGRCbecame quite resistant to in vivo deiodination, resulting innegligible radioactivity accumulation in both thyroid andstomach. Moreover, such a t-BOC protected peptide wasstable in human serum even after 24 h. In short, t-BOCgroup has the potency to protect peptides from deiodina-tion and provide a reduced radioactive background fortumor imaging with radioiodinated peptide.

Acknowledgment

We thank the National Natural Science Foundation ofChina (no. 20301001) for financial support.

References

Bakker, W.H., Krenning, E.P., Breeman, W.A., Kooij, P.P.M., Reubi,

J.C., Koper, J.W., de Jong, M., Lameris, J.S., Visser, T.J., Lamberts,

S.W., 1991. In vivo use of a radioiodinated somatostatin analogue:

dynamics, metabolism, and binding to somatostatin receptor-positive

tumors in man. J. Nucl. Med. 32, 1184–1189.

Bakker, W.H., Krenning, E.P., Breeman, W.A., Koper, J.W., Kooij, P.P.,

Reubi, J.C., Klijn, J.G., Visser, T.J., Docter, R., Lamberts, S.W., 1990.

Receptor scintigraphy with a radioiodinated somatostatin analogue:

radiolabeling, purification, biologic activity, and in vivo application in

animals. J. Nucl. Med. 31, 1501–1509.

Foulon, C.F., Reist, C.J., Bigner, D.D., Zalutsky, M.R., 2000. Radio-

iodination via D-amino acid peptide enhances cellular retention and

tumor xenograft targeting of an internalizing anti-epidermal growth

factor receptor variant III monoclonal antibody. Cancer Res. 60,

4453–4460.

Geissler, F., Anderson, S.K., Venkatesan, P., Press, O., 1992. Intracellular

catabolism of radiolabeled anti-mu antibodies by malignant B-cells.

Cancer Res. 52, 2907–2915.

Hart, S.L., Knight, A.M., Harbottle, R.P., Mistry, A., Hunger, H.D.,

Cutler, D.F., Williamson, R., Coutelle, C., 1994. Cell binding and

internalization by filamentous phage displaying a cyclic Arg-Gly-Asp-

containing peptide. J. Biol. Chem. 269, 12468–12474.

Huang, W., Chen, C., 1985. The protection of amino group. In: Wu, T.

(Ed.), Peptide Synthesis. Science Press, Beijing, pp. 13–44.

Iuchi, K., Nitta, M., Ito, K., Molimoto, Y., Tsukamoto, G., 1988.

Synthesis and analgesic activity of cholecystokinin-heptapeptide

analogs with N-terminal substitution. Chem. Pharm. Bull. 36, 959–965.

Josefsson, M., Grunditz, T., Ohlsson, T., Ekblad, A., 2002. Sodium/

iodide-symporter: distribution in different mammals and role in

entero-thyroid circulation of iodide. Acta. Physiol. Scand. 175,

129–137.

Kohrle, J., 1999. Local activation and inactivation of thyroid hormones:

the deiodinase family. Mol. Cell Endocrinol. 151, 103–119.

Krummeich, C., Holschbach, M., Stocklin, G., 1994. Direct n.c.a.

electrophilic radioiodination of tyrosine analogues; their in vivo

stability and brain-uptake in mice. Appl. Radiat. Isot. 45, 929–935.

Larson, S.M., Carrasquillo, J.A., 1988. Advantages of radioiodine over

radioindium labeled monoclonal antibodies for imaging solid tumors.

Nucl. Med. Biol. 5, 231–234.

Pasqualini, R., Koivunen, E., Kain, R., Lahdenranta, J., Sakamoto, M.,

Stryhn, A., Ashmun, R.A., Shapiro, L.H., Arap, W., Ruoslahti, E.,

2000. Aminopeptidase N is a receptor for tumor-homing peptides and

a target for inhibiting angiogenesis. Cancer Res. 60, 722–727.

Pradeep, K.G., Kevin, L.A., Philip, C.W., Michael, R.Z., 1996. Enhanced

binding and inertness to dehalogenation of a-melanotropic peptides

labeled using N-succinimidyl 3-iodobenzoate. Bioconjugate Chem. 7,

233–239.

Reist, C.J., Garg, P.K., Alston, K.L., Bigner, D.D., Zalutsky, M.R., 1996.

Radioiodination of internalizing monoclonal antibodies using

N-succinimidyl 5-iodo-3-pyridinecarboxylate. Cancer Res. 56,

4970–4977.

Shankar, S., Vaidyanathan, G., Affleck, D.J., Peixoto, K., Bigner, D.D.,

Zalutsky, M.R., 2004. Evaluation of an internalizing monoclonal

antibody labeled using N-succinimidyl 3-[131I]iodo-4-phosphono-

methylbenzoate ([131I]SIPMB), a negatively charged substituent

bearing acylation agent. Nucl. Med. Biol. 31, 909–919.

Wang, S., Lin, H., Zhou, Q., 1985. The synthesis of labeled organic

compound. In: Wang, A., Ma, S. (Eds.), Nuclear Medicine and

Nuclear Biology: Fundamental and Application. Science Press,

Beijing, pp. 319–362.

Wikstrand, C.J., Hale, L.P., Batra, S.K., Hill, M.L., Humphrey, P.A.,

Kurpad, S.N., McLendon, R.E., Moscatello, D., Pegram, C.N., Reist,

C.J., Traweek, S.T., Wong, A.J., Zalutsky, M.R., Bigner, D.D., 1995.

Monoclonal antibodies against EGFRvIII are tumor specific and react

with breast and lungcarcinomas and malignant gliomas. Cancer Res.

55, 140–3148.

Zalutsky, M.R., Narula, A.S., 1988. Radiohalogenation of a monoclonal

antibody using an N-succinimidyl 3-(tri-n-butylstannyl) benzoate

intermediate. Cancer Res. 48, 1446–1450.