Bioconjugate Chem. 1997, 8, 572−584
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Biotin Reagents for Antibody Pretargeting. Synthesis, Radioiodination, and in Vitro Evaluation of Water Soluble, Biotinidase Resistant Biotin Derivatives D. Scott Wilbur,* Donald K. Hamlin, Pradip M. Pathare, and S. Ananda Weerawarna Department of Radiation Oncology, University of Washington, Seattle, Washington 98195. Received January 21, 1997X
As part of our development of antibody pretargeting for cancer therapy, an investigation has been conducted to examine the stability of water solubilized, radioiodinated biotin derivatives toward biotinidase degradation in mouse and human serum. Eight new biotin derivatives were synthesized to conduct the study. The biotin derivatives synthesized contained (1) the biotin moiety, (2) a water solubilizing linker moiety, (3) p-iodobenzoate or p-tri-n-butylstannylbenzoate moieties, and (4) in some of the compounds, N-methyl or R-methyl containing moieties were added to block biotinidase activity. The linker moiety, 4,7,10-trioxa-1,13-tridecanediamine, 5, was included in the biotin derivatives to improve their water solubility, and it also functioned as a 17 Å spacer between the biotin and the benzoyl moieties. Four of the new biotin derivatives (12, 14, 22, and 23) contained a p-tri-nbutylstannylbenzoyl moiety as precursors which could be radioiodinated in the last synthetic step. The other four biotin derivatives (13, 15, 24, and 25) contained p-iodobenzoyl moieties and were used as HPLC reference standards. Initial studies involved radioiodination of 12 to yield [125I]13. Radioiodinated 13, which did not contain a moiety for blocking biotinidase activity, was found to be rapidly degraded in both mouse and human serum at 37 °C. Derivatives which were designed to be stable to biotinidase incorporated N-methyl and R-methyl moieties adjacent to the biotin carboxylate group. In one set of biotin derivatives (14 and 15), the N-methyl moiety was obtained by incorporating N,N-dimethyl-4,7,10-trioxa-1,13-tridecanediamine, 9, as a linker in the place of 5. In the second set of biotin derivatives (22 and 24), the N-methyl moiety was introduced by incorporating a sarcosine (N-methylglycine) moiety between biotin and 5. The radioiodinated N-methyl containing biotin derivatives [125I]15 and [125I]24 were found to be very stable to biotinidase degradation. An R-methyl group was obtained in a pair of biotin derivatives (23 and 25) by incorporating a 3-aminobutyric acid moiety between biotin and 5. The radioiodinated R-methyl containing derivative, [125I]25, was found to have an intermediate stability with regards to biotinidase degradation.
INTRODUCTION
The very strong binding of biotin (vitamin H; coenzyme R) with the proteins avidin (1) and streptavidin (2) have made compounds containing biotin attractive for in vitro bioassays (3, 4) and in vivo medical applications. One important application under investigation by a number of research groups is the use of “pretargeted” monoclonal antibody conjugates with the biotin/(strept)avidin technology for imaging and therapy of cancer (5-7). A focus of our studies is the investigation of antibody conjugates with recombinant streptavidin (r-streptavidin)1 and radiolabeled biotin derivatives in tumor pretargeting protocols as a method of amplifying the amount of radioactivity delivered to cancer cells (8). We have hypothesized that the amount of radioactivity attached to a cancer cell which has been pretargeted with a biotinylated antibody might be increased by repeated administration of radio* Address correspondence to Department of Radiation Oncology, University of Washington, 2121 N. 35th Street, Seattle, WA 98103-9103. Phone: 206-685-3085. Fax: 206-685-9630. E-mail:
[email protected]. X Abstract published in Advance ACS Abstracts, July 1, 1997. 1 Abbreviations: ChT, chloramine-T; cpm, counts per minute; DIP, direct insertion probe, EDC, 1-(3-dimethylaminopropyl)3-ethylcarbodimide hydrochloride; EI, electron impact, 2-HEDS, 2-hydroxyethyl sulfide; HOHgBz, hydroxymercuribenzoic acid; nca, no-carrier-added; NCS, N-chlorosuccinimide; r-streptavidin, recombinant streptavidin; rt, room temperature; TFP, tetrafluorophenyl; TFP-OH, tetrafluorophenol; TFP-OTFA, tetrafluorophenyl trifluoroacetate.
S1043-1802(97)00085-2 CCC: $14.00
labeled biotin dimers or trimers that are capable of crosslinking streptavidin. To investigate this hypothesis, new biotin derivatives had to be prepared. Quite early in our studies in the preparation of new biotin derivatives, it became apparent that there were two problems, serum stability and aqueous solubility of biotin derivatives, that had to be surmounted before they could be applied to tumor pretargeting. In our initial design of biotin derivatives for pretargeting and in derivatizing other molecules (9), alkyl diamine linking moieties were used to conjugate biotin to the molecules of interest. However, low water solubility of biotin derivatives caused problems in their evaluation. For example, in one (unreported) study, a biotin dimer designed for use in cross-linking streptavidin on cancer cells was synthesized by conjugating two biotin moieties with p-iodobenzoyl-5-aminoisophthalate ditetrafluorophenyl ester (10) using two diaminopropane linkers. The biotin dimer obtained had very low water solubility (e.g., 98% pure stannylbenzoylbiotin derivatives. Interestingly, the radioiodination reactions of 12, 14, and 22 resulted in radioiodinated compounds that had retention times which were close to the iodinated standards, but did not coelute with them. Further, the initial radioactive peaks seen on HPLC analysis were transformed to more hydrophilic species within 30 min of isolation (as observed when reinjected onto the HPLC).3 We felt that there were a number of possible explanations for the apparent instability of radioiodinated material, but it seemed most likely that a radioiodinated impurity had been isolated rather than the desired product(s). The radioiodination of 22 gave a product which coeluted with the iodinated standard, but the yield was uncharacteristically low. Subsequently, 1 mg quantities of stannylbenzoate derivatives were highly purified (>99.5%) by multiple injections/isolations from an analytical HPLC column and were used in the radioiodination reactions. Use of the highly purified stannylbenzoylbiotin derivatives resulted in obtaining reasonable yields (24-76%) of nca radioiodinated products which coeluted with the iodobenzoylbiotin standards. The radioiodinated compounds obtained from the highly purified stannylbenzoylbiotin derivatives were stored at 4 °C for several weeks. In general, the compounds were found to be stable during storage. However, in one preparation of [125I]24, another compound slowly grew in with time. The new radioiodinated species formed had a HPLC retention time that indicated it might be the biotin sulfoxide. Streptavidin Binding. An evaluation of the binding of two radioiodinated biotin derivatives, 13 and 24, with streptavidin was conducted to demonstrate that 4 equiv bound, and to determine if nonspecific binding would be a problem in the biotinidase assay. Only biotin derivatives 13 and 24 were investigated as it was thought that the binding of the R-methyl biotinamide 25 should be similar to 13, and the N-methyl biotinamide 15 should be similar to 24. In the experiment, solutions of the nonradioactive biotin derivative (13 or 24) were mixed with tracer quantities of the same radioiodinated biotin derivative. From these stock solutions, varying quantities of the biotin derivative were added to three tubes containing a set amount of streptavidin. Quantities of the biotin derivative that were less than required to saturate the biotin binding sites on streptavidin were placed in two of the three tubes, and an excess of the biotin derivative was added to the third tube. The tubes were incubated for 1 h at room temperature, then the contents of each tube was transferred to a centrifuge filtration tube. After concentration and three washes with phosphate buffer, the top and bottom of the cen3 The radioactivity peak on the HPLC had a shorter retention time (tR) than expected in each radioiodination reaction. For example, in the radioiodination of 12 the iodo standard, 13, had a tR ) 7.0 min by UV detection, whereas the major radioactive peak had a tR ) 7.1 min. Due to the distance between the UV detector and the radioactivity detector on the HPLC used, the tR should be 0.3 min longer for the radioactive peak than the UV peak (previously determined). Reinjection of the isolated radioactivity (from peak at 7.1 min) after 30 min showed that the radioactive material had changed to one that eluted at tR ) 2.0 min. This change in tR most likely indicated that the radioiodine was not stably attached to the compound isolated.
Wilbur et al.
trifugation filter were counted separately in a well counter. The results, expressed as percent activity filtered (i.e., activity in filtrate plus three washes) are shown in Table 1. Since less than 4 equiv of each biotin derivative were added to some of the tubes containing r-streptavidin, it was expected in those examples that very little activity would pass through the centrifugation membrane. Indeed, in each tube containing less than the quantity of biotin derivative necessary for saturation of streptavidin (i.e., 8 nmol), only 3% of the activity passed through the membrane filter. Importantly, in those examples where an excess of biotin derivative was added, the amount in excess of 4 equiv filtered readily. The calculated excess percentages (33% for 13 and 21% for 24) were very close to the amounts filtered, indicating that there was very little if any nonspecific binding with streptavidin or the filtration membrane. The lack of nonspecific binding to the centrifugation filtration membrane and plastic parts was dramatically shown in that less than 0.5% of 13 and 24 were retained when centrifuged without streptavidin. Biotinidase Assay. Biotinidase activity results in cleavage of the amide bond linking biotin with the portion of the molecule containing the radiolabel. Therefore, biotinidase activity can be measured by evaluation of the amount of radioactivity that binds with an excess quantity of r-streptavidin after incubation in serum. In the initial experiment examining biotinidase activity, the biotin derivative 13, which did not contain a biotinidase blocking group, was incubated in dilute (10%) mouse serum at 37 °C for 2 h. Aliquots were taken at various time points and biotinidase activity was stopped with the addition of hydroxymercuribenzoic acid (HOHgBz), a chemical which is known to deactivate biotinidase (32). As a control, an equivalent sample of 13 was incubated in mouse serum that had been pretreated with HOHgBz. The results of a triplicate analyses for those studies are shown graphically in Figure 1. The results indicated that the biotinidase activity in mouse serum increased almost linearly over the 2 h period. Following the initial study with 13, evaluations of the stability of the water solubilized, N-methyl and R-methyl containing biotin derivatives 15, 24, and 25 toward biotinidase degradation were conducted. Since the most important issue to be determined was whether complete blocking of biotinidase activity could be obtained in the derivatives, it was only necessary to evaluate the biotinidase cleavage reaction (e.g., amount of radioactivity that did not bind with r-streptavidin) at a single time point. We chose to incubate for 2 h since over 50% specific cleavage4 had been seen at 2 h when 13 was incubated in dilute serum. In the analysis of each biotin derivative (i.e., 15, 24, and 25), equivalent samples of nonstabilized biotin derivative 13 and the biotin derivative being tested in serum pretreated with HOHgBz were also examined. All of the biotin derivatives were evaluated in human serum as well as mouse serum. We felt that it was important to evaluate the stability in mouse serum as our preclinical studies involving radiolabeled biotin derivatives in tumor pretargeting use athymic mice containing human tumor xenografts. It seemed equally important to evaluate the derivatives in human serum as the biotin derivatives are ultimately being developed for use in applications of human cancer therapy. The results of triplicate bioassays involving biotin derivatives 4 Specific cleavage is defined as the amount of radioactivity not bound with r-streptavidin after incubation for 2 h minus the amount of radioactivity not bound with r-streptavidin when HOHgBz is added prior to incubation.
Biotinidase Stabilized Biotin Derivatives
13, 15, 24, and 25 are provided in Table 2. The nonspecific binding of radioiodinated biotin derivatives in serum was assessed by the measuring the quantity of radioiodinated derivative retained when no r-streptavidin is present. That value is obtained by subtracting the amount filtered (from Table 2) from 100%. DISCUSSION
The primary purpose of this study was to gain information that would aid our development of radioiodinated biotin derivatives for application to radiotherapy of cancer using an antibody tumor “pretargeting” approach (8). It was apparent from initial studies that the biotin derivatives being developed needed to have improved water solubility. It also became apparent that the biotin derivatives synthesized needed to be designed in a manner that blocked the serum enzyme biotinidase (18, 22). Thus, the design of new biotin derivatives to be used in our studies had to incorporate functional groups which (1) permitted radioiodination, (2) improved water solubility, and (3) blocked biotinidase activity. An important consideration in the design of the new biotin derivatives was the functionality to be used to introduce radioiodine. As with all radioiodination reactions, it was desired that the radioiodine be introduced into the biotin derivatives in the last synthetic step, which meant that a iodine reactive intermediate had to be prepared. Some investigators have used biotin derivatives which contained phenolic moieties, such as tyrosine or tyramine, to allow incorporation of radioiodine (12). However, concern that mixtures of mono- and diiodo biotin derivatives would be obtained and concern that in vivo deiodination of phenolic biotin compounds would occur led us to choose an alternate labeling moiety. Our, and other investigators’, studies have shown that radioiodinated compounds containing iodophenyl derivatives are resistant to in vivo deiodination (31-33), so iodoarylbiotin derivatives were chosen. Introduction of high specific activity radioiodine into a biotin derivative through the use of a triazenyl intermediate had been reported (15), but the radiochemical yields were low. An alternative, more facile and higher yielding reactive intermediate was desired. Stannylbenzoyl intermediates were chosen for radioiodination of biotin because these intermediates had been used previously in many other applications and had proven to be facile and high yielding in radioiodination reactions. In the initial experiments with the stannylbenzoate derivatized biotin derivatives, radioiodinated products were obtained which appeared (by HPLC retention times) to be unstable. The identity of the unstable compounds were not determined because purification of the stannylbenzoate precursors resulted in obtaining the desired radioiodinated biotin derivatives. We were concerned about the potential for oxidation of the biotin thioether (to sulfoxide or sulfone) in the iodination reactions, but this had not been reported as a problem in a previous investigation using tri-n-butylstannyl-anilide as the moiety for radioiodination (17). To assure ourselves that the sulfoxide derivatives were not formed under the conditions of radioiodination, oxidation of 13 was conducted in HOAc with H2O2 to provide a biotin sulfoxide standard for HPLC analysis.5 Indeed, the HPLC retention time for the biotin sulfoxide derivative clearly ruled out formation of the sulfoxide in the iodination reaction. After purification of the stannylbenzoate intermediates, radioiodinated biotin derivatives were obtained which coeluted with the iodinated standards. Although the radiochemical yields were lower than anticipated, radioiodination of tri-n-butylstannylbenzoate derivatives, 12, 14, 22, and 23, resulted in
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acceptable yields (24-76%) of isolated biotin derivatives ([125I]13, [125I]15, [125I]24, and [125I]25). Although water solubilization of biotin derivatives had been largely ignored in previously reported radiohalogenated biotin derivatives, we felt that this was a very important aspect of the design of new derivatives. Many different water solubilizing linkers can be envisioned, however, we addressed the issue of increasing the water solubility by incorporating a neutral, nonionic, ether containing linker between the biotin moiety and the aryl moiety in the biotin derivatives of interest. In addition to providing water solubilizing properties, the linker (4,7, 10-trioxa-1,13-tridecanediamine) provided a 17 Å spacer6 between the biotin carboxylate and the radioiodination moiety. There have been many examples demonstrating that the binding avidity of biotin derivatives with avidin and streptavidin remains high when a linking molecule is used between the biotin carboxylate and the coupled compound (40). The most important aspect of the design of new biotin derivatives is modification such that serum biotinidase does not cleave biotin from the rest of the molecule. The new biotin derivatives could not be used for in vivo applications without being stable in the presence of biotinidase. Literature reports provided insight into the types of modifications that might be used to make water solubilized, radioiodinated biotin derivatives stable to biotinidase activity. It is important to note that biotinidase is not confined to a particular tissue, rather it is found in many tissues, with its activity being high in liver, kidney, adrenal glands, and serum (18, 22). Biotinidase appears to be involved in the transport and cellular uptake of biotin, and may function as a biotinyl transferase to biotinylate specific proteins or small molecules (24), but the function that is important in developing new biotin derivatives is its role in conserving biotin in the body by cleaving biotin from its lysine adduct, biocytin. Biocytin is produced upon catabolism of the four biotin-dependent enzymatic peptide carboxylases which utilize it in their catalytic active site (41). It has been reported that human serum biotinidase is specific for cleavage of the lysine portion of biocytin, and the fact that other biotin derivatives such as biotinylphenylalanine, biotinyl--aminocaproic acid, and biotinyl-γamino-n-butyric acid were not cleaved (22) tends to support this concept. However, the amide cleaving action of biotinidase cannot be truly specific to lysine since cleavage is observed with conjugated p-aminobenzoic acid and iodotyramine (42), as well as with L-lysine. Thus, it seems unlikely that simply changing the lysine to another amine containing molecule would block the action of biotinidase. Importantly, other investigators have re5 Formation of the biotin sulfoxide of 13 was accomplished using the reaction conditions reported by Melville (39). The H2O2 oxidation reaction with 13 resulted in formation of a single product; tR ) 11.0 min using Gradient I (tR ) 11.9 min for 13). The retention difference of 13 and the oxidized product (i.e., biotin sulfoxide) using the HPLC conditions for radioiodinated biotin derivatives was much more pronounced: 13, tR ) 10.7 min and oxidized product, tR ) 6.3 min. Investigation of the oxidation of 13 with NCS was also conducted. After 24 h with 1 equiv of 13 and NCS in MeOH/HOAc, there was about 22% of the same oxidized product (by coinjection on HPLC) as formed with H2O2. In the time period similar to the radioiodination reaction (i.e., 10 min), none of the oxidized product was seen. 6 This value is the distance between the two nitrogen atoms in the fully linearized 4,7,10-trioxa-1,13-tridecanediamine. The distance was obtained from the computer program ChemDraw 3D (CambridgeSoft Corp.) after structural and energy minimization.
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ported that some simple modifications of biotin derivatives block biotinidase activity. For example, amide cleavage appears to be sensitive to steric encumbrance near the biotin carboxylate. Biotin conjugated with the amine of p-aminobenzoic acid (PABA) is readily cleaved by biotinidase, but m-aminobenzoic acid is cleaved at only 10% of the rate of PABA, and o-aminobenzoic acid does not appear to be cleaved (22). Other parameters such as the distance from the ring structures of biotin to the carboxylate amide appear to also be important as shortening the aliphatic side chain decreases the activity to 2% of the unaltered rate when PABA conjugates are studied (22). Three of the new radioiodinated biotin derivatives were chemically modified near the biotin carboxylate to block the amidase action of biotinidase. Although it was known that alterations in the biotin moiety would decrease biotinidase activity, we chose to leave biotin unaltered with the exception of conjugation of the carboxylate with an amine containing moiety. It was felt that modification of biotin would likely result in a decreased binding with streptavidin. The early studies of Rosebrough indicated that an R-carboxylate group (from a cysteinyl linked biotin) was efficient at blocking cleavage of defero-acetylcysteinyl-biotin by biotinidase (26, 27). However, our desire to use the water solubilizing trioxatridecanediamine linker made the cysteinyl derivative unattractive for our studies. We felt that an R-methyl group from the biotin adduct with 3-aminobutyric acid might provide similar steric hindrance to that of an R-carboxyl group, so biotin derivative 25 was targeted for synthesis and evaluation. The results obtained suggest that the Rmethyl group does not provide enough steric hindrance to completely block biotinidase activity. For the purposes of these studies we chose to limit the number of biotin derivatives synthesized. However, based on the results with the R-methyl substituted biotin adduct 25, it would be of interest to synthesize and compare a biotinaspartic acid adduct, which would be equivalent to 25, except it would have an R-carboxylate group. The reports that the sarcosine (N-methylglycine) adduct of biotin completely blocked biotinidase activity (28, 29) led to targeting the radioiodinated biotin derivative 24 for synthesis and evaluation. Although 24 was prepared, the difficulties encountered in that synthesis led to the desire to prepare an additional N-methyl biotin derivative. From a limited survey of commercial sources, it appeared that there were very few commercially available N-methyl compounds that could be used as linkers. The N-methyl compounds that were available from commercial sources were not attractive for our studies as we felt that they would not provide enough water solubilization for the biotin derivatives, nor did they provide the spacer distance we were seeking. This led to consideration of synthesizing a N-methylamine or N,N′-dimethyldiamine derivative for use as a linker molecule. Interestingly, synthesis of a biotin-psoralen adduct containing a N,N′-dimethylhexanediamine linker had been previously reported (43), but it was thought that such an aliphatic linker would decrease water solubility of biotin derivatives. These considerations prompted us to synthesize N,N′-dimethyl-4,7,10-trioxa-1,13-tridecanediamine, 9, and to use it in the synthesis of radioiodinated biotin derivative 15. Biotinidase activity was measured utilizing centrifugation concentrators which retain molecules with a molecular mass over 10 kDa. Incubation of the radioiodinated biotin derivative [125I]13 at 37 °C in 10% mouse serum displayed nearly linear cleavage by biotinidase over a 2 h time period (Figure 1). Deactivation of biotinidase with
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HOHgBz prior to incubation showed that the cleavage was specific. To measure biotinidase stability of biotin derivatives [125I]13, [125I]15, [125I]24, and [125I]25, each was incubated in diluted mouse or human serum at 37 °C for 2 h. After incubation in serum, an excess of streptavidin was added to bind with biotin. The assay solutions containing serum, streptavidin, and radiolabeled biotin were then filtered through a size exclusion centrifugation membrane and rinsed with PBS and water. The radioactivity that went through the membrane represents that activity which was no longer attached to biotin. To demonstrate that the activity penetrating the membrane was due to biotinidase cleavage, pretreatment with HOHgBz was conducted. Additionally, to examine whether the biotin derivatives might bind nonspecifically in serum and/or on the membrane, centrifugation without added streptavidin was conducted. The results of the biotinidase activity assay are provided in Table 2. It is apparent that the radioiodinated biotin derivative which had no functional group for blocking serum biotinidase (i.e., 13) was rapidly degraded by biotinidase. It was equally apparent that the biotin derivatives containing N-methylbiotinamide functionalities (i.e., 15 and 24) were very stable to biotinidase degradation. Interestingly, the R-methyl biotinamide functionality (i.e., 25) was found to be relatively stable in mouse serum, but was cleaved at a intermediate rate in human serum. It is not known why there is such a difference in rate of cleavage in the two types of serum, but it may be due to differences in the way the serum was handled. It has been reported that mouse serum biotinidase is less active than human serum biotinidase (18), but perhaps more important is the fact that the mouse serum used was frozen and thawed twice whereas the human serum was fresh (refrigerated). Freeze-thawing is reported to decrease biotinidase activity (22). In addition to the biotin derivatives prepared and evaluated in this study, a large number of other biotin derivatives can be envisioned which are likely to block biotinidase activity in vivo. It seemed obvious that biotinidase activity could be completely blocked if the biotin amide were reduced to an amine. Therefore, as part of this investigation, the biotin-trioxadiamine adduct 10 was reduced with borane-methyl sulfide to give the secondary amino derivative. That compound was subsequently reacted with the TFP esters, iodobenzoate, 3, and stannylbenzoate, 4 (unreported results). Although these biotin derivatives had improved water solubility, they were difficult to purify and were found to be unstable upon storage, so this derivative was not investigated further. The literature also suggests that shortening the pentanoic acid side chain in biotin will cause a blockage of biotinidase activity (22). Therefore, compounds such as the norbiotinamine and its isothiocyanto derivative (44) might be used to prepare compounds that are stable to biotinidase activity. The goal of these studies was to obtain information with regards to biotin modifications and biotinidase stability, but it is also important to consider how modifications to biotin derivatives affect their binding with streptavidin or avidin. Since bulky substituents attached directly to the biotin carboxylate decrease binding with avidin, it might be expected that biotin derivatives containing bulky substituents adjacent to the carbonyl group, such as N-methyl derivatives and R-methyl groups, could result in decreased binding. Therefore, we are planning to evaluate the dissociation kinetics (44) of the compounds described herein to determine the effect on binding with r-streptavidin brought about by the modi-
Biotinidase Stabilized Biotin Derivatives
fications. If the binding is greatly decreased, then additional biotin derivatives designed to block biotinidase activity will be prepared and evaluated. Summary. In this study, four radioiodinated, water solubilized, biotin derivatives were evaluated for their stability toward degradation in serum by the enzyme biotinidase. The biotin derivative which had a simple amido bonded biotin, 13, was found to be unstable in both mouse and human serum when incubated at 37 °C. Two biotin derivatives, 15 and 24, which contained N-methylamido bonded biotin were found to be very stable toward degradation of biotinidase, similar to other previously reported biotin derivatives. In contrast to the N-methyl derivatives, a biotin derivative, 25, which contained a methyl group R to the biotin amido linkage was not stable to biotinidase, but was degraded at a slower rate than 13. The use of high specific activity radioiodinated biotin derivatives ([125I]13, [125I]15, [125I]24, and [125I]25) made the evaluation of their in vitro stability relatively easy. The radioiodinated biotin derivatives were readily prepared from p-tri-n-butylstannylbenzoyl biotin derivatives (12, 14, 22, and 23), but not without being highly purified on the HPLC. In addition to the biotinidase stabilizing functionalities in the biotin derivatives studied, a water solubilizing linker moiety was incorporated into each compound. ACKNOWLEDGMENT
We thank Dr. Robert Vessella and Kent Buhler for providing the mouse and human serum used in these studies. We thank Dr. Patrick Stayton and Lisa Klumb for providing r-streptavidin used in the studies and for their helpful comments on the manuscript. We are grateful for the generous financial support provided by the Department of Energy, Medical Applications and Biophysical Research Division, Office of Health and Environmental Research under Grant DE-FG0695ER62029. Supporting Information Available: HPLC chromatograms and 1H NMR spectra of the new compounds prepared in the research described in this manuscript (34 pages). Ordering information is given on any current masthead page. LITERATURE CITED (1) Green, N. M. (1975) Avidin. Adv. Protein Chem. 29, 85133. (2) Green, N. M. (1990) Avidin and Streptavidin. Methods Enzymol. 184, 51-67. (3) Diamandis, E. P., and Chrostopoulos, T. K. (1991) The Biotin-(Strept)Avidin System: Principles and Applications in Biotechnology. Clin. Chem. 37, 625-636. (4) Wilchek, M., and Bayer, E. A. (1988) The Avidin-Biotin Complex in Bioanalytical Applications. Anal. Biochem. 171, 1-32. (5) Hnatowich, D. J., Virzi, F., and Rusckowski, M. (1987) Investigations of Avidin and Biotin for Imaging Applications. J. Nucl. Med. 28, 1294-1302. (6) Goodwin, D. A. (1995) Tumor Pretargeting: Almost the Bottom Line. J. Nucl. Med. 36, 876-879. (7) Paganelli, G., Malcovati, M., and Fazio, F. (1991) Monoclonal antibody pretargetting techniques for tumor localization: the avidin-biotin system. Nucl. Med. Commun. 12, 211-234. (8) Wilbur, D. S., Hamlin, D. K., Vessella, R. L., Stray, J. E., Buhler, K. R., Stayton, P. S., Klumb, L. A., Pathare, P. M., and Weerawarna, S. A. (1996) Antibody Fragments in Tumor Pretargeting. Evaluation of Biotinylated Fab′ Colocalization with Recombinant Streptavidin and Avidin. Bioconjugate Chem. 7, 689-702. (9) Pathare, P. M., Wilbur, D. S., Heusser, S., Quadros, E. V., McLoughlin, P., and Morgan, A. C. (1996) Synthesis of
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