Conjugation of the closo-Borane Mercaptoundecahydrododecaborate (BSH) to aTumour Selective Peptide

Walter Miera,*, Detlef Gabelb, Uwe Haberkorna, and Michael Eisenhutc

a Heidelberg, Universitätsklinikum Heidelberg, Abteilung für Nuklearmedizin,b Bremen, Universität Bremen, Fachbereich Chemiec Heidelberg, Deutsches Krebsforschungszentrum, Abteilung Radiopharmazeutische Chemie

Received February 12th, 2004.

Professor Reinhard Schmutzler zum 70. Geburtstag gewidmet

Abstract. The non-toxic disodium mercaptoundecahydrododeca-borate (BSH) is one of the most promising agents known forboron-neutron-capture therapy (BNCT). In order to enhance theintratumoral concentration of the boron cluster by receptormediated endocytosis, a synthetic method for the conjugation ofborane clusters to peptides was developed. A Michael addition wasperformed to link BSH to the thiol reactive maleimido modifiedTyr3-octreotate. Tyr3-octreotate, synthesized by Fmoc-solid phasepeptide synthesis, is a selective ligand for somatostatin receptors.

Konjugation des closo-Boran-Mercaptoundecahydrododecaborat (BSH) an eintumorselektives Peptid

Inhaltsübersicht. Das untoxische Mercaptoundecahydrododecabo-rat (BSH) ist einer der vielversprechendsten Wirkstoffe für die Bor-Neutroneneinfangtherapie (BNCT). Um die intratumorale Kon-zentration dieser Substanz durch Rezeptor-vermittelte Endocytosezu erhöhen, wurde eine synthetische Methode zur Konjugation vonBorclustern mit Peptiden entwickelt. Eine Michael-Addition er-folgte, um BSH mit dem Thiol-reaktiven Maleimido-modifiziertenTyr3-Octreotat umzusetzen. Tyr3-Octreotat, ein selektiver Ligand

Introduction

For more than 50 years, chemotherapy is considered to beone of the most favourable methods for the treatment ofcancer. Unfortunately the systemic toxicity of antineoplas-tic drugs restricts the application of chemotherapy. Thechemotherapeutic agents used in systemic cancer therapyexert their cytotoxic effect mainly on proliferating cells sothat not only tumours but also normal proliferating tissue,such as bone marrow, intestinal or dermal epithelia is affec-ted. Consequently, the systemic side effects of chemothera-

* PD Dr. Walter MierRadiopharmazeutisches LaborAbteilung für NuklearmedizinUniversitätsklinikum HeidelbergIm Neuenheimer Feld 40069120 HeidelbergTel.: 0049-6221-567720E-mail: walter�mier@med.uni-heidelberg.de

1258 2004 WILEY-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim DOI: 10.1002/zaac.200400064 Z. Anorg. Allg. Chem. 2004, 630, 1258�1262

The modified boron cluster was obtained in 68 % yield after purifi-cation. Characterisation of the BSH conjugate was performed withHPLC, MALDI-TOF and electrospray mass spectrometry. TheBSH-Tyr3-octreotate conjugate is considered to target receptor-positive tumours which might further improve the potential ofBNCT.

Keywords: Closo-borane; Boron neutron capture therapy; Tar-geting; Somatostatin; Mercaptoundecahydrododecaborate

für Somatostatin-Rezeptoren wurde mittels Fmoc-Festphasensyn-these hergestellt. Nach Aufreinigung wurde der modifizierte Bor-cluster in 68 %-Ausbeute erhalten. Die Charakterisierung des BSH-Konjugats erfolgte mittels HPLC, MALDI-TOF und Electrospray-Massenspektrometrie. Das BSH-Tyr3-Octreotat-Konjugat ist geeig-net, Rezeptor-positive Tumoren zu adressieren, um das Potentialder BNCT zu erweitern.

peutic agents in healthy tissues represent one of the fore-most problems in cancer chemotherapy.

Neutron capture therapy represents an alternative tochemotherapy [1]. For neutron capture therapy isotopeswhich have a high capacity for absorbing thermic neutrons(neutrons in the 20 keV energy range which are able toreach deep-seated tumours) are incorporated into cancercells. Examples are 157Gd [2] or the readily available stableand non-toxic boron isotope 10B (used for boron-neutron-capture therapy, BNCT). Upon irradiation with neutrons,10B undergoes the following nuclear reaction:

105B � 1

0n � 115B* � 7

3Li � 42He

Thus, the boron atom decays to yield energetic short-rangealpha particles and lithium ions which deposit most of theirenergy within the cell containing the boron. The almost ex-clusive deposition of energy in the nearest proximity of thecaptured neutron allows the selective destruction of the tar-get cell.

Conjugation of the closo-Borane Mercaptoundeca-hydrododecaborate (BSH) to a Tumour Selective Peptide

Estimates for the concentration of 10B required for a suc-cessful therapy of tumours are about 20 µg/gram tissue [3].However, it was impossible to attain a sufficient target tonontarget ratios of 10B at this concentration in clinicalBNCT applications. Carrier molecules which would help toovercome these problems are currently under scrutiny fortargeted drug delivery. Peptides suitable as carriers are som-atostatin (SST), gastrin releasing peptide, epidermal growthfactor (EGF), neurotensin, substance P and insulin-likegrowth factor (IGF). Conjugates of EGF with boronatedstarburst dendrimers have been successfully used for thetargeting of EGF receptor expressing tumours [4, 5]. In or-der to extend the possibilities for peptide receptor targetingwith drugs for BNCT, SST receptor-selective peptides wereinvestigated. These peptides have found widespread appli-cation in nuclear medicine. For example DOTA--Phe1-Tyr3-octreotide (DOTATOC) complexed with the beta par-ticle-emitting radionuclide 90Y3� [6] was successfully usedin clinical trials for the therapy of SST receptor-expressingtumours [7].

We herein present a method for the conjugation of bor-ane clusters to peptides. A Michael addition was performedto link the non-toxic [8] mercaptoundecahydrododecaborate(BSH) to maleimido modified Tyr3-octreotate. The inor-ganic BSH is one of the most promising compounds knownfor BNCT [9]. The targeting approach through receptor me-diated endocytosis might further improve the potential ofthis drug.

Results and Discussion

Synthesis

Scheme 1 shows the synthesis pathway for the synthesis ofthe peptide moiety of the conjugate. The peptide moietychosen was Tyr3-octreotate. Thus, H--Phe-Cys(Acm)-Tyr(tBu)--Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Thr(tBu)-OH(1) was assembled using SPPS on a Wang resin. The pep-tide was cyclized using Tl(TFA)3 to obtain H--Phe-cyclo-[Cys-Tyr(tBu)--Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys]-Thr(tBu)-Wang-resin (2). The resin bound peptide was then coupledwith maleimido-caproic acid. After drying the resin invacuo, the protecting-group cleavage was performed withTFA containing the scavengers triisopropylsilane and water.Under these conditions, the protecting groups of the aminoacids were cleaved simultaneously releasing peptide 4 fromthe resin.

The chemical synthesis of the BSH-octreotate conjugatewas performed in solution according to the reaction se-quence outlined in Scheme 2.

Preliminary attempts for the conjugation of BSH toFmoc-Asp(OAll)-OH using ammonium peroxodisulfate(NH4)2[S2O8] as a catalyst indicated that the conjugation ofBSH to non-activated alkenes is not efficient for the syn-thesis of defined products. Therefore, the conjugation ofBSH to a maleimido modified peptide was chosen.

The thiol modified boron cluster was added to a solutionof compound 4 in aqueous buffer containing acetonitrile at

Z. Anorg. Allg. Chem. 2004, 630, 1258�1262 zaac.wiley-vch.de 2004 WILEY-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim 1259

Scheme 1 Synthesis of the thiol reactive Tyr3-octreotate. (a) Step-wise elongation using 4 equivalents of the Fmoc protected aminoacids activated with HBTU and DIPEA as the base, (b) cyclizationusing Tl(TFA)3, (c) conjugation of Mal-(CH2)5COOH activated byHBTU / DIPEA, (d) cleavage with TFA containing 2.5 % H2O,2.5 % phenol and 2.5 % triisopropylsilane.

Scheme 2 Conjugation reaction of BSH with the maleimido pep-tide 4 resulting in the boron-cluster-peptide conjugate 5.

pH 7.0 under an argon atmosphere. The reaction of BSHproceeded slowly as compared to aliphatic thiols. However,the conjugation had reached completeness after 24 h as de-termined by HPLC.

The chromatogram of the crude product mixture showedtwo major peaks. These peaks correspond to the excess ofBSH (I) and the conjugate (III). The conjugation of thecharged BSH moiety led to a decreased retention time ofthe BSH-octreotate conjugate, indicating that the boroncluster might improve the solubility in aqueous solution.Due to the increased polarity, the conjugate could be easilyseparated by HPLC from the side products all having longerretention times as shown in Figure 1.

Performing the reaction without air protection, the for-mation of the BSH dimer, BSSB, was observed. As shownin Figure 2 this compound eluted prior to BSH (Fig. 2).The oxidation rate of thiols is pH dependent and dimeris-ation had, therefore, to be expected under these conditions.In general, the reduction of the BSSB disulphides can beachieved by addition of dithiothreitol (DTT) or other re-

W. Mier, D. Gabel, U. Haberkorn, M. Eisenhut

Figure 1 Reverse phase HPLC chromatogram: Michael additionof the thiol-containing BSH to the maleimido-modified Tyr3-oc-treotate 4. (A): Reaction mixture immediately after addition of themaleimide and adjustment of the pH, I: BSH, II: maleimido-pepti-de(B): I: excess BSH and III: conjugate.

2004 WILEY-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim zaac.wiley-vch.de Z. Anorg. Allg. Chem. 2004, 630, 1258�12621260

ducing agents such as tris-(2-carboxyethyl)phosphine(TCEP). In this case, however, addition of a reducing agentis not allowed because of the disulphide bond of the cyclicpeptide.

Figure 2 Reverse phase HPLC chromatogram of a reaction mix-ture similar to that shown in Fig. 1B, except that the reaction wasnot refrained from air.

The product was thoroughly studied using mass spectro-metric methods. MALDI-TOF analysis was used to identifythe mass composition of compound 5. This study wasfurther confirmed by electrospray mass spectrometry on ahigher resolution level. The single positive charged boronion cluster at 1418.8 m/z (highest abundant mass), shownin Figure 3, corresponds to the protonated product, witha calculated mass of m/z 1419.68. The comparison of theobserved ESI spectrum, with a computer generated isotopepattern validates the assumption of an ion with the compo-sition [C59H87B12N11O15S3

2�� 3 H�]�. Further singly posi-tive charged boron cluster ions at 1440.8 and 1456.8 corre-spond with the sodium and the potassium containing clus-ters [M2� � 2 H� � Na�]� (C59H89B12N11NaO15S3: calcu-lated m/z � 1441.68) and [M2� � 2 H� � K�]�

(C59H89B12KN11O15S3: calculated m/z � 1457.68), respec-tively. The negative ion data showed masses of m/z � 708.0(highest abundant peak), corresponding to [M2� � H�]2�

and m/z � 1416.7, corresponding to [M2� � 2 H�]� (datanot shown).

Conjugation of the closo-Borane Mercaptoundeca-hydrododecaborate (BSH) to a Tumour Selective Peptide

Figure 3 Electrospray mass spectrometry of the boron cluster peptide conjugate 5 in the positive ion mode. Three groups of clusters (A �

protonated clusters, B � sodium containing and C � potassium containing clusters) could be resolved.

Compounds bearing a smaller number of boron atomsper molecule, such as carboxyphenylboronic acid are not aswell suited for this kind of targeting approach. Thus mostof the attempts made today focus on deltahedral boranes.In order to obtain defined compounds the postsyntheticconjugation of only one moiety was chosen in contrast tothe EGF-conjugates described in the literature [4, 5]. Inthese conjugates activated dendrimers were conjugated toan undefined number of boron clusters. There have beenseveral syntheses using carboranes as a boron moiety withinconjugates designed for BNCT [10�13]. Derivatives of car-boranes are uncharged and therefore lipophilic. Hence thein vivo characteristics of carboranes is challenging due tonon-specific binding and solubility problems. Deltahedralcloso boranes which do not include substituted skeletal het-eroatoms are doubly negative charged providing sufficientpolarity to be soluble in water. The borane cluster selectedhere was the closo-borane BSH ([B12H11SH]2�) which hasbeen thoroughly studied with respect to the pharmacokinet-ics and toxicity and in addition providing a thiol group,useful for conjugation reactions [14].

Experimental

General

All synthesis reagents and solvents were purchased from Merck(Darmstadt, Germany). The chemicals for peptide synthesis wereobtained from Novabiochem (Läufelfingen, Switzerland). Thal-lium(III) trifluoroacetate and triisopropylsilane (TIS) were ob-tained from Fluka (Buchs, Switzerland). A sample of high quality

Z. Anorg. Allg. Chem. 2004, 630, 1258�1262 zaac.wiley-vch.de 2004 WILEY-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim 1261

of BSH [15], was obtained from Boron Biologicals Inc., Raleigh,USA. A corresponding 10B enriched compound has been describedin the literature [16].Analysis and separation by liquid chromatography (HPLC) wasperformed on a Agilent 1100 system (Agilent, Waldbronn,Germany). The columns used were LiChrosorb RP-select B 5 µm,250 � 4 mm and 10 µm, 250 � 10 mm (Merck, Darmstadt,Germany). The peptides were synthesised manually with an in-house manufactured SPPS reactor. Lyophilisation was performedon a Christ (Osterode, Germany) α1-2 lyophilizator. MALDI-TOFmass spectrometry analysis was performed on a matrix assistedlaser desorption ionisation time-of-flight mass spectrometer(MALDI-3, Kratos Instruments, England). ESI-MS analyses wereperformed using a triple-quadrupole instrument (TSQ 7000 fromFinnigan, San Jose, USA) equipped with a nanoelectrospray source(EMBL, Heidelberg).

Synthesis of the Maleimido-Peptide 4

The peptide was assembled by Fmoc chemistry on 1 g of Fmoc-Thr(tBu)-Wang resin (0.61 mmol/g). Nα-Fmoc amino acids withthe following side chain protecting groups were employed: Cys-(Acm), Lys(Boc), Thr(tBu), -Trp(Boc) and Tyr(tBu). All couplingswere performed in DMF. The peptide chain was constructed manu-ally according to a modified in situ neutralisation cycle [17]. Briefly,this cycle consisted of a twofold decoupling (1 min and 5 min) with50 % piperidine in DMF and 10 min coupling with 4 eq of theFmoc-amino acid (0.4 M in DMF, incubated for 5 min with 3.9 eqof HBTU and 6 eq DIPEA). After completion, the resin (1.75 gdry weight) was treated with piperidine in DMF to deprotect theterminal α-aminogroup of the peptide. An aliquot was cleaved andanalysed by HPLC indicating formation of 1 with a yield > 90 %.200 mg of the resin-bound peptide 1 was cyclized at room tempera-ture with a 2-fold molar excess of thallium(III)trifluoroacetate in

W. Mier, D. Gabel, U. Haberkorn, M. Eisenhut

DMF. As determined by analysis of a small aliquot, formation of2 was essentially complete within 1 h. After thorough washing,N-maleimido-6-caproic acid was coupled as described above, theresin was washed and dried under vacuum overnight. Cleavage wasperformed with 5 mL of 37:1:1:1 TFA/H2O/phenol/TIS for 2 h atroom temperature. The resin was filtered and washed. The peptidewas precipitated by the gradual addition of tert-butyl-methyletherat 4 °C. Purification was accomplished by reversed-phase HPLCon the RP-selectB column using a gradient of 20 % B � 50 % Bin 7.5 min and 50 � 100 % B in 5 min (A � H2O and B � aceto-nitrile, both containing 0.1 % TFA), flow rate � 4 mL/min. Underthese conditions the peptide was eluted at 10.2 min. After lyophilis-ation, 44 mg of 4 (43.6 % overall yield) were obtained as a fluffypowder. The purified peptide was characterised by MALDI-TOFmass spectrometry. Calcd. for C59H75N11O15S2 [M � H]� m/z1243.4; found: 1244.2.13C NMR (CD3OD) δ � 20.02 (q), 20.58 (q), 22.89 (t), 26.28 (t), 27.14 (t),27.70 (2 C) (t), 29.24 (t), 31.42 (t), 36.75 (t), 38.40 (t), 39.34 (t), 40.19 (t),40.65 (t), 46.45 (t), 46.52 (t), 53.86 (d), 54.16 (d), 54.43 (d), 55.18 (d), 56.42(d), 57.68 (d), 59.68 (d), 60.55 (d), 68.55 (d), 69.09 (d), 110.41 (s), 112.33 (d),116.23 (2 C) (d), 119.53 (d), 120.05 (d), 122.57 (d), 124.83 (d), 127.74 (d),128.66 (s), 128.91 (s), 129.34 (2 C) (d), 130.66 (2 C) (d), 131.54 (2 C) (d),135.32 (2 C) (d), 137.99 (s), 138.56 (s), 157.35 (s), 171.26 (s), 172.11 (s),172.60 (2 C) (s), 172.74 (s), 173.26 (s), 173.57 (s), 174.20 (s), 174.35 (s),175.00 (s), 175.36 (s).

Synthesis of the Tyr3-octreotate-oligodeoxynucleotide conjugatedBSH (5)

A sample of 18.2 mg (80 µmol) of disodium mercaptoundecahydro-dodecaborate (BSH) was dissolved in double distilled water con-taining 50 % acetonitrile and added to 10 mg (8 µmol) of thelyophilised maleimido-peptide 5 under argon. The pH of this mix-ture was shifted stepwise within 30 min from ca. 4.0 to 7.0 by ad-ding 0.5 M phosphate buffer. The mixture was incubated at roomtemperature for 24 h, after which analytical HPLC indicated com-pletion of conjugation. Buffers A: 0.1 % TFA in H2O and B: 0.1 %TFA in acetonitrile were employed for purification of the conjugateby RP-HPLC. A linear gradient of 0 % � 50 % B (4 mL/min) in20 min was used. The conjugate eluting at 16.7 min was collectedand lyophilised. 7.85 mg of a white fluffy powder (68 % based onthe amount of the starting maleimido-peptide) was obtained.The conjugate was characterised by MALDI-TOF-analysis using2-(4-hydroxyphenylazo)benzoic acid as matrix in the positive ionmode 5: m/z � 1417.5 [M2� � 3 H�]� (C59H90B12N11O15S3:calculated 1419.68 g/mol); 1441.7 [M2� � 2 H� � Na�]�

(C59H89B12N11NaO15S3: calculated 1441.68 g/mol); 1456.9 [M2� �

2 H� � K�]� (C59H89B12KN11O15S3: calculated 1457.68 g/mol).More accurate characterisation was achieved with ESI MS in bothpositive m/z � 1418.8 [M2� � 3 H�]� (C59H90B12N11O15S3: calcu-lated 1419.68 g/mol); 1440.8 [M2� � 2 H� � Na�]�

(C59H89B12N11NaO15S3: calculated 1441.68 g/mol); 1456.8 [M2� �

2004 WILEY-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim zaac.wiley-vch.de Z. Anorg. Allg. Chem. 2004, 630, 1258�12621262

2 H� � K�]� (C59H89B12KN11O15S3: calculated 1457.68 g/mol)and negative ion mode: 1416.7 [M2� � H�]� (C59H88B12N11O15S3:calculated 1417.68 g/mol); 1438.6 [M2� � Na�]�

(C59H87B12N11NaO15S3: calculated 1439.68 g/mol); 1455.5 [M2� �

K�]� (C59H87B12KN11O15S3: calculated 1455.68 g/mol).

Acknowledgement. The authors are grateful to W.D. Lehmann andG. Erben (DKFZ Heidelberg) for the recording of ESI-mass spec-tra.

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