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Final Report

Particular

I. Award No.: DE-FG02-93ER61571II. PI: Jon Zubieta111.Institution: Department of Chemistry, Syracuse UniversityIV. Title: The Coordination Chemistry of Technetium and Rhenium

to Nuclear Medicine.and Applications

The Role of Coordination Chemistry of Technetium in the Development ofRadiopharmaceuticals. Whale ~c-based radiopharmaceutical agents and their targetsdisplay a remarkable diversity, an understanding of the underlying coordination chemistry oftechnetium is pivotal to their future development. The structural characterization of the Tc-ligand complex is fundamental both to the understanding of the chemical and physicalproperties of the agent and to the design of novel ligands possessing the appropriate donoratoms, complex stability, and three-dimensional structure to allow specificity in targeting.Consequently, investigations on the macroscopic WC level of the structural chemistry ofsuch complexes is crucial in the assessment of those characteristics which influence theirrelevant chemical and physical properties. The proposed research will focus on thedevelopment of broadly interrelated aspects of the coordination chemistry of technetium ofrelevance to bifunctional linker design, and to the general development of ligand types forspecific binding. The analogous chemistry of rhenium will be developed with a view tounderstanding the structural correlation to technetium.

WORK IN PROGRESS.This work evolves from the demonstration that bifunctional hydrazines can be used for

the properties of-c labeled protein conjugates. Since the elaboration of the coordinationchemistry is driven by the studies on the radiolabeled peptide derivatives, a summary of the mostrecent results of our collaborators on these systems will be reviewed, with an emphasis on thoseobservations which require development of the ligand or core fictional group chemistry of Tcand Re.

C.I. 99mTc-Labeled Chemotactic Peptide Derivatives. The results of our collaborators’investigations cited in Section B.11.3 have addressed several significant areas in the elaboration ofthese reagents including: (a) synthesis and chemical characterization of chemotactic peptide

‘mTc labeling; (b) in vitro characterizationreceptor agonists and antagonists that are suitable forof the peptides with respect to bioactivity and receptor binding; (c) rdolabeling with -c; (d)

in vivo studies of the effects of chemotaetic peptides on leukocyte leveIs in monkeys; (e)biodistribution and gamma camera imaging studies of focal sites of E. coli tiection in rats, rabbitsand dogs; and (f) the mechanisms by which radioIabeled chemotactic peptide analogues localize at

DISCLAIMER

This report was prepared as an account of work sponsoredby an agency of the United States Government. Neitherthe United States Government nor any agency thereof, norany of their empioyees, make any warranty, express orimplied, or assumes any legal liability or responsibility forthe accuracy, completeness, or usefulness of anyinformation, apparatus, product, or process disclosed, orrepresents that its use would not infringe privately ownedrights. Reference herein to any specific commercialproduct, process, or service by trade name, trademark,manufacturer, or otherwise does not necessarily constituteor imply its endorsement, recommendation, or favoring bythe United States Government or any agency thereof. Theviews and opinions of authors expressed herein do notnecessarily state or reflect those of the United StatesGovernment or any agency thereof.

DISCLAIMER

Portions of this document may be illegiblein electronic image products. Images areproduced from the best available originaldocument.

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sites of infection. Several more recent observations which will serve to drive a significant fictionof the coordination chemistry are presented in more detail.

C.11. E#ect of Co-Ligand on Infection Imag”ngand Biodistribution in Rabbits. The ‘Tc-complex (co-ligand) used for radiolabeling can have profound effects on biodistribution. In thisstudy, the distribution of molecular species formed with a variety of “co-ligands” was correlatedwith infection localization and biodistribution in l?. coli infected rabbits.J4ethoak Five ~c-co-ligand complexes (mannitol, glucamine, glucarate, tricine andglucoheptonate) were used to label f-MLFK-HYNIC and radiolabeled species were characterizedby reverse phase HPLC. One mCi of each ‘Tc-co-ligand-peptide complex was injected into”rabbits 24h after infection and imaging was performed 3-4 and 16-17 hrs later. After acquiringthe final images, the animals were sacrificed and biodistribution was measured.Results: With all 5 coligands, ‘Tc labeled peptide was obtained in > 90’XOradiochemical purity.Multiple radiolabeled species were obtained with each reagent, however, mannitol yielded themost homogeneous product. Image analysis demonstrated that, ‘Tc-mannitol-f-MLFK-HYNICproduced the highest target to background ratios (T/B). At 16-17 hrs, the T/B’s were: 3.9 M0.99,6.15* 1.07,2.8 A1.07, 2.57* 1.07, 3.41A1.07, and 1.8*1.07 at 3-4 h and 11.9*0.99, 3.94+1.07,3.79*1 .07, 4.81*1 .17, 7.&H.3 1 and 5.32*1.07 at 16-17, for mannitol, tricine, glucamine,glucarate-~ glucarate-b, and glucoheptonate, respectively. At both imaging times, %c-mannitol-f-MLFK-HYNIC had the lowest relative levels of accumulation in bowel.Biodistribution measurements demonstrated that the mannitol preparation had the highest Ievelof accumulation (%ID/g) in tiected tissue; mannitol > glucoheptonate = glucarate-b > glucarate-a> glucarnine> t.ricine;p<O.O1, The infected to normal muscle ratio for *Tc-mannitol-f-MLFK-HYNIC was -50:1. Conclusions: These results support ourpreviousfindings in rats that co-ligands can markedly e#iect the biodistribution and infection localization of 9%Tclabeled H17VICchemotactic peptides. For Mection imaging, the mannitoI preparation had the most favorabIecombination of accumulation in infected tissue, T/B ratio and biodistribution in uninfected

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LFfgure 1: Rdb-t-ffzc Chfclrllatogrerllof~c-ghcarete-f-MLFK-HYNIC preparatim-m~=wfwtlw=~ ‘~c-mza “-f-MtJ=K-t+YNIC,bg%c-’glm~ti “-f-MLFK-HYNIC,

c +C-%fucarate, d 99%04- ,RADIOACTIWTY e unlebeledLMLFK-HYNIC.

J b Uv

TIME

,,

Figure 2 Representativeantarkxirnageaoftheinfecfimciteinrabbitaat 17 IIOuraaftarinjactirmof f-MLFK-HYNICradiofabeledwith%C-

rnannitd(A)and %c-bicine (6). The anirnaiewereinfecfed24 hoursbeforaradk@amacauticefedrniniatratiorLInter-sebowelaccmulatbn ianotedwith@5ne aawellaa a Ieeaintenaainfectimbcakatmcomparedwiththernannitdprep(A).=

Ffgure 3: Garnrnacamera irnageec4a rabbitat 5 (left)ad15 (right) hcura after injectionof 99MTC-HP2 mkkbakdusingmannitolaacdigand.

C.11. 1. Optimization of Sn(IJ.)-A4anito1Formulation to Control Radiolabeled Speciation.As discussed above, the nature of the starting Tc-species used to label Formyl-M-L-F-K-HYNICsignificantly influences the biological fate and infection localizing characteristics of the peptideconjugate. To date, Tc-mannitol appears to offer significant advantages over several other co-Iigands we have studied with regard to the number of radiolabeled species generate~ normaltissue distribution and infection localization. As seen with other polyhydric Iigands, the reactionof Tc-mannitol with formyI-ML-FK-HYNIC generates several radiolabeled peptide species. Thisis the likely result of formation of isomers between Tc and the polyhydric ligand. As ourobjective is to produce a clinically usefil tracer for infection imaging, we undertook a study tooptimize the Sn(II) MannitoI formulation so as to achieve rapid and quantitative peptide IabeIingwhile limiting the number of radiolabeled species generated.

Mannitol concentration, Sn(II) concentration and pH were studied as a fimction of time todetermine the effect on labeling yield and speciation, as determined by reverse phase HPLC(0.1% TFA in a MeCN varying concentrations of mannitol [80-0. 10 mghn.1]and a fixed amountof SnC12/H20 [100 ughnl] at pH 5.0-5.2. The effect of SnC12/H20concentration was studied at80 mg/ml and 10 mg/ml mannitol at pH 5.0-5.2. Kits contained 0.5 cc of the Sn-mannitol solution -and Tc-mannitol was prepared by addition of 2.0 ml NaTc04. Formation of Tc-mannitol was .monitored by radio-TLC using ITLC-sg eluted with acetone (IW= 0.0) and saline @f= 1.0).

The rapid formation of labeled peptide was favored at low concentrations of mannitol[S 2.5 rngkt] with 2 predominant Tc-peptide peaks. However, at mannitol concentrations <0.5mg/kit, greater than 80% of @e Tc-peptide was found in the more hydrophobic peak. Sn(II)concentrations did not affect labeling yields or speciation at mannitol concentrations of 0.5 mg/kitbut an inverse correlation was found between Sn(II) concentration and labeling yield at mannitol ~

. ...

concentrations of 40 mglkit. These results indicate the optimal formulation of Sn(II)-mannitol tobe 0.5 mgkit mannitol 50 ugkit SnC12/H20at pH 5.0-5.2.

flGURE 4 RP-HPLCshowingseverslradbkbelw.

i-9mn—wuw Tc+.

“nYJac@Hc4wTR.@-I.m=Filx=

I III I / ...”“i

ce~rm Muw-IN

/

cu+ct?4 KAlicw

““”””’““””i.LA-..

/-. ”,.,

iti

Il,lm. IYwIu t 1%1 *I-.-I.

FIGURE 5 RP-HPLC shoving eeverslrsdkdsbeledSw”es.

C.1 1.2. Pyridinethiols and Pyrirnidinethiols (Pi) as Co-Ligan& for Tc-99m LubeledHydrazinonicoti;mide (H17VIC)Conjugated Peptizes.’ As previ&s st&ies have demonstrate~Tc-99m-mannitol gives superior imaging results when used as the starting species (or coligand) tolabeI For-MLFK-HYNIC, as compared with other polyhydric ‘coligands’. However, radio-HPLC

‘Tc-peptide speeies. We are interested in producing ‘Tc-indicated the presence of severalHYNIC-peptides as homogeneousradiochemical species in an effort to simpli@ formulation andmechanistic studies. Previous work at the ‘carrier’level in our laboratory showed these ligandsform stable and reproducible complexes with Tc-hydrazinopyridine. To this end, the potentiaIof pyrimidinethiols and pyridinethiols as coligands was investigated.

The Tc-mannitol-HYNIC-peptide was converted to the corresponding pyrimidine-2-thiol, 4-methyl-pyrimidine-2-thiol(P2T), pyridine-2-thiol, and 3-trimethylsilyl-pyridine-2-thiolcontaining complexes. Forrnyl-MLFK-HYNIC was labeled by incubating equal volumes of‘Tc-mannitol with peptide [20 pghnl] in 50mM acetate btier pH 5.2 for 1 hr. Labeling wasdetermined by reverse phase-HPLC eluted with O.1% TFA and a water - acetonitrile gradient.Conversion to the PT containing complex was accomplished by incubation of equal volumes ofPT [1 mg/mI DMSO] and Tc-peptide at room temperature for 1 hr.

For all PTs, HPLC analysis showed the formation of unique hydrophobic species withretention times greater than the Tc-mannitol labeled peptide. In the case of P2T a primary

f“.

.

homogeneouspeak was obtained which when isolated and re-chromatographed eluted at the same~. Initial imaging experiments with this complex were petiormed in rabbits with E. coliifiections in the Iefi posterior thigh. 24 hrs after infection, rabbits (n= 3) were injected iv. with0.5 mCi of Tc-P2T-peptide and imaged at 4 and 18 h pi. The animals were sacrificed at 18 h andtissue activity determined. ROI analysis gave target/background of 4.26t0.97 and 9.83*1 .6, at 4and 18 h, respectively. Tissue counting (18 h) resulted in pus:normal muscle >150:1 and infectedmuscle: normal muscle >50:1. At 4 and 18h GI accumulation was evident. PTs offer excellentinfection localization, however further work is necessary to limit GI accumulation. In summary,the PT’s provide improved radiochemical formulations by limiting the radioactive species present,as compared to polyhydric ligantk

deartyk delineatedat 3 h andthe activityintheinfectedhigh iaintenseat 18h.

C.1 1.3. NJW3is(2-mercaptoethy~methylamine ~S2): A New Co-Ligandfor 99”TcLabeling of Hydrazino-Nicotinamide (HYNIC) Peptides. With the aim of limiting radiochemicalspeciation of *Tc-HYNIC-peptides radiochemical species with more predictable radiochemicaland biological properties we previously investigated bidentate aminothiol Iigands,pyrirnidinethiols and pyridinethiols, as coligands. These ligands resulted in an improvedradiochemical formulation by limiting the number of radiochemical species observed with reversephase HPLC. However, the use of these ligands resulted in undesirable significant GI tractaccumulation of rad.iolabel

Tridentate ligands, such as NS2, may constrain the possible coordination geometries andimprove overall stability. To investigate this, we synthesized NS2, converted the Tc-mannitol-For-MLFK-HYNXC to the corresponding NS2 containing complex (Tc-NS2-For-MLFK-HYNIC) and compared its tiection imaging and biodistribution properties with Tc-mannitol-For-MLFK-HYNIC. Formyl-MLFK-HYNIC was labeled by incubating equal volumes of Tc- .99m-mannitoI with peptide [20 pg/ml] in 50mM acetate buffer pH 5.2 for 1 hr. Conversion tothe NS2 containing complex was accomplished by addition of NS2 [1 m~ml DMSO] to the Tc- “mannitol-For-MLFK-HYNIC solution at room temperature. HPLC showed a single uniquehydrophobic species with retention time greater than the Tc-mannitol-For-MLFK-HYNIC.Imaging experiments with this complex were performed in rabbits with 1?.coli inkctions in theleft thigh. 24 hrs after infectio~ rabbits (n = 6) were injected iv. with 0.5 mCi of Tc-NS2-peptide or Tc-mannitol-peptide and imaged at 4 and 18 h pi. The animals were sacrificed at 18 hand tissue activity determined.

.

ROI analysis gave target/background ratios of 3.91N.32 and 11.89 f 2.21 for ‘“Tc-mannitol-For-MLFK(HYNIC) and 3.93 * 0.69 and 22.40 *5.52 for *Tc-NS2-For-MLFK(HYNIC) at 3 and 18 h. Tissue radioactivity measurements (18h) demonstrated thatcompared to Tc-mannitol-peptide, accumulation of Tc-NS2-peptide was lower in blood, heartand normal muscle and higher in spleen, infected muscle and pus (p <0.01). There was nosignificant difference in accumulation into the GI tract. These results indicate that the Tc-NS2-peptide complex is more chemically homogeneousthan the Tc-mannitol peptide and exhibitssubstantially improved infection localization and biodistribution properties.

C.12. HKWC-Maleimide, a Thiol-Spec@c Linker for Labeling Peptides with 99mTc:Conjugation, Labeling and In Vivo Evaluation. CurrentIy, the chemistry of attachment toproteins and peptides involves the acylation of primary amino groups via the N-hydroxysuccinimidyl ester of HYNIC (SHNH). Since many peptides of biological interestpossess more than one primary amino group, use of SHNH for conjugation can lead toheterogeneous products. In some cases, this lack of site-specificity may lead to derivatization ofresidues criticaI for biological activity. To overcome these limitations we have developed amethod which allows site directed conjugation of a stable ‘mTc chelating agent. The methodmakes use of a novel HYNIC-maleimide (HM) bifunctional linker and free thiols, of Cys residuesat non-critical sequence positions. The utility of HM was tested by conjugation to a chemotactic

*Tc-mannitol and comparison of its infection imagingpeptide analog, For-MLFC, labeling viaand biodistribution properties with For-MLFK-HYNIC, in a rabbit modeI of infection. Sites ofitiection were produced in New Zealand white rabbits (n = 6/peptide) by injection of asuspension of E. coli in a posterior thigh. 24 hrs after infection, the rabbits were injected iv.

‘mTc-peptide and imaged at 3 and 18 h pi. The animals werewith 0.5 mCi of HPLC purifiedsacrificed at 18h and tissue activity determined. At 3 and 18h pi., the organ distribution of bothpreparations was qualitatively similar, with high accumulation in liver, spleen and rnmrow andminimal bowel accumulation. ROI analysis gave target/background ratios of 3.9M0.32 and11.89fl.21 for For-MLFK(HYNIC) and 3.67k0.47 andl 1.18t0.89 for For-MLFC(HM), at 3and 18 h, respectively. Tissue radioactivity measurements ( 18h) demonstrated that compared toFor-MLFK(HYNIC), the accumulation of For-MLFC(HM) was greater in liver, spleen andmarrow and lower in he@ kidney, adrenal, stomach, normtd muscle and testes (p < 0.01).However, there was no significant difference in accumulation into GI trac~ idected muscle orpus. Reverse-phase HPLC indicated that HM increases peptide Iipophilicity as compared withSHNH, which may account for minor differences in biodistribution. These results indicate thatthe HYNIC-maleirnide bifimctional chelator can be successfully used to incorporate HYNIC intoa Cys containing peptide. This derivative can be readily labeled with Tc-99m and exhibits in vivocharacteristics similar to peptide conjugated with HYNIC.

C.L3. Summary and Signl@cance. The results to date demonstrate that structuralmodtications to the peptide and its radiolabeled analog can be made so that alterations inpharmacological characteristics (i.e. potential unwanted side effects) and localizing characteristics .can be improved over the initial peptides and their radioIabeled complexes. This is significantbecause it demonstrates the feasibility of developing a diagnostic peptide analog with lowpotential for side effects and improved localizing ability.

The structural modifications to the peptide MLF included both amino-terminal andcarboxy-terrninal modifications. Amino terminal modifications being critical for receptor bindingand activation characteristics and the carboxy terminus being important for chemical modificationto produce radiolabeled analogs. The peptide was able to tolerate these modifications and

maintain biological and receptor binding characteristics. This fmdlng is critical for developmentof a successfid diagnostic drug.

Studies of the influence of coligand on the biological fate of the radiolabeled peptidecomplex is also significant. While it is important to preserve receptor binding afilnity (asdescribed above) receptor affinity alone will not predict localizing properties nor will its assuregood localization. The demonstration that the coligand can markedly influence the distribution ofthe peptide complex in various tissues suggests that peptides which have good binding affinitiescan be prepared with suitable coligands to ‘tailor’their distribution, thereby enhancing signal tonoise of the tracer. This is a novel finding which we believe is a direct effect of utilizinghydrazinonicotinamide as the iadiometal chelating group.

C.II. Coordination Chemistry of Technetium and Rhenium. The work evolves fromour demonstration that bifimctional hydrazines can be used for the preparation of ‘mTc labeledprotein conjugates. However, despite the manifest potential of bifimctional organohydrazinoligands in protein radiolabeling for nuclear medicinal applications, the nature of the metalcoordination to the HYNIC linker has not been elucidated. In order to define some structuralpossibilities for the coordination chemistry relevant to these reagents, we investigated thechemistry of Tc and Re with hydrazinopyridine, which not only established the formation of therobust {M=NNR} core, but also implicated chelate formation through the pyridine nitrogen as asignificant structured deterrninant.58

During the course of these studies, we also noted that the identity of the coligandscoordinated to the Tc site may profoundly influence the structures of complexes with the Tc-hydrazido core, {TcNNR}, and the radiochemicai yield of the protein conjugate. Sulfur Iigandswere particulady effective in the stabilization of the Tc-hydrazido core as a consequence of thehigh affinity of Tc for sull%rdonors. These observations suggested a systematic study of Tc andRe coordination chemistry with hydrazine ligand types and with thiolate ligands both ascoligands in the preparation of hydrazido-linked protein conjugates and for the development ofbifunctional polythiolate ligands.

C.111. Technetium and Rhenium Coordination.C.11I. 1. Models for (Tc-(coligand)-HH?IC-peptide) Radiopharmaceuticals. In an eflort

to model the interaction of Tc and Re precursors with the hydrazinonicotinamide unit of themost effective linker (HYNIC), the reactions of perrhenate with hydrazinopyridine wereinvestigated. Significantly, hydrazinopyridine acts as both Iigand and reductant to give thecompound ~C13(HNNpy)(NNpyH)] (M= Tc (la), Re (lb)) in quantitative yield. While theparticipation of the pyridyl nitrogen in chelate formation is significant in complex stabilization, itis also noteworthy that the protonation pattern excludes the hydrazido(2-) unit as a contributorto the overall structure. Furthermore, the preferential protonation of the cwitrogen and the

pyridyl nitrogen establishes the relatively weak basicity of the ~-nitrogen site.

(A) neutml organodiazatido (B) hydrazido(2-) (C) neutral pyridiniumdiazcnido

These complexes are stablein organic solvents and inmixed organickpxnlsmedi~sg demonstrating therobust nature of the metal-organohydrazine corewhich extends to a varietyof substituents such as the

[Tc(?12-NHNCJ&N)(r@NCJ&N)

(~2-C~N-2-S)(Tll-C~N-2-S)] (4a)

.

,,,

hydrazinoimidazoles of which [ReC13(NNC31&N2H)(HNNHCJH4NZH)](2) is representative.However, detailed synthetic and structural investigations of this class of compounds revealed acomplex coordination chemistry, manifested in both monodentate terminal linkage and chelationby organohydrazine ligands and by several distinct patterns for the relative orientations of theligands within the {M(TI1-NNR)(q2-HNNR)}cores, which reflect the metal electronicrequirements and the degree of ligand protonation. This latter observation is illustrated by thestructure of ~Et3] [ReCIJ(NNCsHqN)(HNNCsHqN] (3). Thus, it is not surprising that thelabeled protein conjugates are mixtures of materials as determined by radio-HPLC. While thechemistry of the pyridylhydrazino Iigand demonstrates the facility of complex formation and therobust nature of such materials, it also suggests a variety of coordination modes for theradiopharmaceutical materkils.

Since formulation and mechanistic studies of ‘TC-HYNIC-peptide radiochemicalspecies require homogeneous materials with favorable pharmacokinetics, the synthesis of highpurity compositions required some attention. The addition of thiol Iigands such as pyrimidine -2-thiol resulted in complexes exhibiting a primary homogeneous peak, which when isolated andrechromatographed eluted at the same tR.

In order to provide a structural basis for such radiopharmaceutical preparations, the

chemistry of the {M(TI1-NNR)(q2-HNNR)} core with thiolate coligands was investigated. The

prototypical structure is represented by ~e(HNNpy)(NNpyH)( q2-SC5HdN)(q1-SCSH4N)](4).However, it was subsequently noted that while Tc-chemotactic peptide radlochemical speciesusing pyridinethiol and pyriddinethiol coligands exhibited excellent infection localization, the GIuptake would hinder the use of such a ligand system.

Further elaboration of the coligand theme demonstrated that tridentate dithiolamine ligandtypes not only provide more homogeneous radiopharrnaceutical preparations with improvedstability, but also exhibited rapid localization and retention at the site of infection, constant ordecreased concentrations in normal tissues, and no adverse biological effects.a Structural modelsfor the metal coordination geometry were developed and ~(NNpyH)(HNNpy){SCH2CH2N(Me)CH2CH2S}] (M= Tc (5a), Re (5b)) and ~e(NNpyH)(HNNpy){(SCH2)2C&13N}](6) provide representative examples.

C.II. 1.2. ‘Technetium Essential” Complexes. The neutral character, lipophilicity, andsmall molecular weight of the model complexes suggested an evaluation of their biodistributionswith a view toward brain-imaging agents. While Tc-hydrazido core complexes of the typesdiscussed above did not exhibit useful brain uptalceproperties, thiolate complexes of the Tc-oxoand Tc-imido cores, {Tc=O} and {Tc-NR}, respectively, traverse the blood-brain barrier andaccumulate in the brain. In designing these materials, we have exploited the”3 + 1” concept ofusing a tridentate dithiolate with a -2 charge in combination with a monothiol of -1 charge topreserve the oxo-metaI core and the forrrd +5 oxidation state of the metal.61SQ Representative -S&UCtUrM are provided by ~O(SCHzCHzOCHzCHzS)(SCHzCb~] (M= Tc (7a), Re (7b))and ~eO(SCI-12CH2SCH2CH2S){SCH2CH(OH)CH(OH)CH2SH}] (8). The pendant arm of such .structure types may, of course, be functionaliid to link metal centers into binuclear complexes,such as ~e202(SCH2CH2SCH2CH2S)3 ] (9).

Since the monothiolate ligand may be derivatized at will, the”3 + 1“ concept may also beexploited for the synthesis of cationic species, such as ~eO(SCH2CH2SCH2CH2S)(SCH2CH2NH3)]+C1-(10). This observation suggests a route to potential heart-imaging agents.These themes will be tier elaborated in the following sections.

,,,

lwT12-NHNc5H4N)(+NmH4NH){q3-(SCH2CHJ.JWH,)l (5b)

pte(T12-NHNc3H4N)(T@’Nc5H4NH)

{q3-(C5H3N-2,qCH,SMI (6)

~eO{q3-(SCH,CH,),S) {q’- ~eO{q3-(SCH,CH,~O) (C&OMe-4-CH,S)] (7b)SCH*CH(OH)CH(OH)CH,SH}](8)

pko{q3-(scH,cH2).2s)(scH,cH,NH3)~(lo) ~~02{p-(SCH2CH2~O]{q3-(SCH2CH&O}~ (9)

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C.Z12. Synthesis of Ligandr.C.112.1. B@mctional Poi’ythiolates. We have observed that I?unctionalizedthiols add

rapidly and in excellent yield under flee radical conditions to mono, tris and tetravinyl sikines.Encouraged by these results, we have successfidly prepared the polythiolateHOOCCH2Si(CH2CF12SH)3(11) by hydrosilation of a fictionalized alkene using trichlorosilane,replacement of the chlorines by vinyl groups, fkeeradical additions of protected thiol equivalentsand deprotection.

Si(CH=CH~ AcSHH&

Si(CH2CH2SAc)3He ~

.

f’ SH

H6;i <SH ~ H0~Si(CH2CH2SAch

(11)L SH

An alternative approach to potential linkers, is provided by the NS3 tripodal ligands.G3

Hcl-N(cH~cH2cl)3KSC(0)CH3 N(CH2CH2SC(0)CH3h

!M

RN(CH2CH&)31 (12)+ Q ~(~2~2sC(O~3)314

We have also developed a new reagent for peptide labeling with-c, 6-mereaptomethylpyridme-3-earboxylic acid (MEMNIC). As sho~ below (Schematic 3), the N-epsilonMEMNIC derivative of for-MLFK was prepared using the N-hydroxyniccinimidyl ester ofMEMNIC. Biodistribution and imaging studies of ““Tc labeled for-MLFK-MEMNIC exhibitlower aceurmdation in most organs and normal muscle compared to for-MLFK-HYNIC and good “tiection localization. These results indicate that MEMNIC and related thiolate-based linkers can .be effketive for -c labeling of peptides.

/.1.

#

HSTr,DBU,

--

“3’*;R :: “ m.++!!.‘“’c”“’r,+%

I

.,,+;..$ &CH2C12

for-MLFK

I

oDMF,DIPEA

‘+,., *for-MLFK-N

‘H

/

MeOH

KOH

0+,”for-MLFK-N

‘H (13)

Schematic 3. Preparation of the MEMMC-Chemotactic Peptide Derivative.