A Salmochelin S4-Inspired Cipro ﬂ oxacin Trojan Horse Conjugate

: A novel cipro ﬂ oxacin − siderophore Trojan Horse antimicrobial was prepared by incorporating key design features of salmochelin, a stealth siderophore that evades mammalian siderocalin capture via its glycosylated catechol units. Assessment of the antimicrobial activity of the conjugate revealed that attachment of the salmochelin mimic resulted in decreased potency, compared to cipro ﬂ oxacin, against two Escherichia coli strains, K12 and Nissle 1917, in both iron replete and deplete conditions. This observation could be attributed to a combination of reduced DNA gyrase inhibition, as con ﬁ rmed by in vitro DNA gyrase assays, and reduced bacterial uptake. Uptake was monitored using radiolabeling with iron-mimetic 67 Ga 3+ , which revealed limited cellular uptake in E. coli K12. In contrast, previously reported staphyloferrin-based conjugates displayed a measurable uptake in analogous 67 Ga 3+ labeling studies. These results suggest that, in the design of Trojan Horse antimicrobials, the choice of siderophore and the nature and length of the linker remain a signi ﬁ cant challenge.

T he problem of antimicrobial resistance has reached a critical level, and authorities now believe humankind is entering a post-antibiotic era where minor infections and routine medical procedures will become major morbidity threats. 1−4 New strategies in the form of new antibiotic targets or modification of current antibiotics to bypass resistance mechanisms are urgently needed. 4−8 One approach is a Trojan Horse delivery strategy that targets outer membrane barrier permeability resistance 9−13 by utilizing bacterial iron transport, mediated by siderophores, to promote active antibiotic uptake; a number of examples can be found in the literature. 14−20 This strategy has been met with some success, with a number of siderophore−drug conjugates entering clinical trials. 4,21−23 One example, cefiderocol (Fetroja, Figure 1), was recently approved by the U.S. Food and Drug Administration (FDA) for treatment of complicated urinary tract infections (cUTIs). 24 Salmochelin siderophores, identified in 2003, 25 are a family of siderophores consisting of glycosylated variants of the high-Fe 3+ -affinity tris-catecholate siderophore enterobactin produced by many of the Enterobacteriaceae. 26 The family derives from the diglycosylated hexadentate salmochelin S4 ( Figure 2) and includes a series of di-and monoglycosylated hydrolysis products. 27 Salmochelins are biosynthesized by a range of bacteria, including Salmonella spp. and various pathogenic E. coli strains. 25,28,29 The salmochelins belong to a class of stealth siderophores, which maintain their role in bacterial iron acquisition while evading the host innate immune response. 30 As a response to an invading pathogen, the mammalian host secretes immunoprotein siderocalin to capture Fe 3+ -loaded catecholate-based siderophores. 31,32 The glycosyl units in the salmochelins prevent siderocalin binding, thus allowing the siderophores to avoid capture. The ability to produce and utilize salmochelins relies on the iroA gene cluster, 30 which encodes the enzymes responsible for the glycosylation of catechol units, the secretion of the biosynthesized salmochelins, the uptake of their Fe 3+ complexes, and finally, the hydrolysis of both the apo-and Fe 3+ -bound siderophores. 33 While the salmochelin uptake mechanism 34,35 is thought to share similarities with that of enterobactin, 18,36−38 the enterobactin receptor protein FepA, found in all E. coli,i s unable to mediate significant uptake of salmochelin S4, whereas the outer membrane receptor protein IroN, which is expressed only in strains that harbor iroA, allows uptake of both salmochelin S4 and enterobactin (see Figure 3). 25 This key difference means that the presence of the iroA gene cluster is considered a virulence factor.
The production of siderocalin by the host may limit the application of catecholate siderophores in Trojan Horse antimicrobials, unless the siderophore component is structur-ally modified to evade the immune response, while maintaining high affinity for Fe 3+ . The use of salmochelin-based siderophore components offers the opportunity of specifically targeting bacteria that express the salmochelin transport machinery, while minimizing disruptions to the host microbiome. This approach has been successfully demonstrated by Nolan et al. with the chemoenzymatic synthesis of glucosylated enterobactin−β-lactam conjugates ( Figure 4). 39 As part of our wider investigation into fluoroquinolone siderophore conjugates, 40−42 we herein report the design and synthesis of a first generation ciprofloxacin−salmochelin S4 inspired Trojan Horse antimicrobial 1 ( Figure 5), designed to evade the mammalian siderocalin immune response and to selectively target bacteria expressing salmochelin transport machinery.

■ RESULTS AND DISCUSSION
Design and Synthesis. In our approach to the design of a first generation salmochelin S4-inspired ciprofloxacin conjugate, the salmochelin S4 structure was simplified to a synthetically accessible structure, comprising an aliphatic link based on L-lysine between the catechol units and a reduction of siderophore denticity from hexadentate to tetradentate. L-Lysine was utilized to mimic the chirality and exact length of the backbone between the catechol moieties in salmochelin S4    ACS Infectious Diseases pubs.acs.org/journal/aidcbc Article in a hydrolytically stable manner; the ester backbone in salmochelin is prone to hydrolysis under physiological conditions. The tetradentate siderophore mimic allows for improved synthetic tractability compared to when the full triserine scaffold of salmochelin S4 is used. The carboxylic acid group of the lysine moiety provides an attachment point for the antimicrobial. It was shown previously that tetradentate, diamine-linked bis(catecholates) can function as siderophore components in Trojan Horse antimicrobials, which were shown to penetrate at least the outer membrane of Gramnegative bacteria, including E. coli. 43,44 We anticipated compound 1 to possess similar iron-binding properties to the tetradentate linear enterobactin dimer 45 ( Figure 2) and a previously described salmochelin S1 mimic, 46 as both feature similar 2,3-dihydroxybenzamide iron-chelating moieties attached to a 5-atomic backbone. We have previously investigated the Fe 3+ coordination chemistry of these two tetradentate bis(catecholates) by using both UV−vis and CD spectroscopy and observed rapidly equilibrating mixtures of 1:1 and 2:3 complexes, both monomers and dimers. In compound 1, however, the deprotonated carboxylic acid and adjacent carbonyl donor of the ciprofloxacin moiety could act as an additional third iron chelating unit and hence affect the iron-binding properties. 47,48 The C5-β-glycosyl-2,3-dihydroxybenzoyl units of salmochelin S4 (highlighted in Figure 2) were amalgamated into the design but connected via N α ,N ε of L-lysine, with the Cterminus providing an appropriate handle for attachment of the parent antibiotic, ciprofloxacin. Suitably functionalized catechol units were synthesized using previously described methodologies. Commercially available methyl 3-methoxysalicylate 2 was iodinated with iodine monochloride 49 and then demethylated. This was followed by benzylation of the free phenolic hydroxyl groups to give 3. Acetyl-protected β-glucose was installed via nickel-promoted Negishi coupling using an adapted literature procedure 50,51 to give aryl-C-glucoside 4. The glucosyl acetyl protecting groups of 4 were substituted with benzyl ethers, resulting in the formation of two compounds 5 and 6, which were combined and then hydrolyzed to give the free carboxylic acid 7 (Scheme 1). 50 In order to furnish a suitably functionalized antimicrobial to allow the salmochelin analogue to be constructed, L-lysineappended ciprofloxacin 10 was synthesized as shown in Scheme 2. Commercially available ciprofloxacin was converted into its benzoyl ester 8 via a transient t-butyloxycarbonyl nitrogen protection/deprotection strategy. It was then coupled to N α ,N ε -diboc-L-lysine via EDC-mediated amide formation to give 9.
Deprotection of the lysine-associated protecting groups yielded 10. Lysine functionalized ciprofloxacin was coupled with glucosylated catechol 7 to give benzyl-protected salmochelin-inspired conjugate 11. Global debenzylation with Pearlman's catalyst 52 furnished salmochelin-inspired ciprofloxacin conjugate 1 in a moderate yield. Experimental details are provided in the Supporting Information.
Fe 3+ Complex Formation. The Fe 3+ complex of 1 (1:1 ratio) was prepared by combining equimolar amounts of 1, dissolved in DMSO, and FeCl 3 hexahydrate, dissolved in water. The solvents were removed in vacuo, and the resulting residue was taken up in a volatile buffer (ammonium acetate) to enable the species formed at pH 7.4 to be examined by native ion mass spectrometry (experimental details are provided in the Supporting Information). Complexation was confirmed by UV−vis spectroscopy ( Figure S1). The observed λ max of the ligand-to-metal charge transfer band at 544 nm is consistent w i t ht h ep r e v a l e n c eo fac h r o m o p h o r ei nw h i c ht w o catecholate units are coordinated to Fe 3+ , i.e., an equimolar Fe 3+ -to-1 ratio. 46 Interestingly, the negative ion ESI mass spectrum showed evidence for the formation of both 1:1 and 2:2 species, the latter consisting of two Fe 3+ cations and two 1 5− ligands (Scheme S1). The most abundant ions were observed at m/z 553.1336, 583.1437, 737.8462, and 1107.2695. On the basis of their isotope patterns and charges, these peaks could be assigned to the acetate adduct of the monomeric species The composition of these ions suggests that the acetate in the 1:1 species and the carboxylate and keto O-donors of ciprofloxacin in the 2:2 species may be recruited to complete the 6-fold coordination sphere of the iron center. Due to the rigid nature of ciprofloxacin, its O-donor atoms can only coordinate to an adjacent iron center, and this may trigger dimer formation. In addition, H-bonding interactions with the glucose unit on the siderophore may support dimer formation.
In addition, a solution containing Fe 3+ and conjugate 1 in a 2:3 ratio was prepared in an analogous way. The λ max of the ligand-to-metal charge transfer band in the UV−vis spectrum did not shift significantly ( Figure S1), and the most abundant ions observed in the negative ion ESI mass spectrum were those assigned to 1:1 and 2:2 species, as above, plus free ligand (conjugate 1). The formation of a 2:3 complex (Scheme S1) was not apparent ( Figure S3). This suggests that the coordination of a third ligand is not favored in this case, potentially due to steric hindrance or competitive binding of the acetate or keto and/or carboxylate O-donors of the ciprofloxacin unit.
ACS Infectious Diseases pubs.acs.org/journal/aidcbc Article two bacterial strains: (1) E. coli K12 (BW25113), a common laboratory strain that does not express IroN, the outer membrane receptor protein required for active salmochelin uptake, 25 and (2) E. coli Nissle 1917, a probiotic strain that expresses IroN and is able to utilize salmochelin. 29 In addition to serving as a negative control, the K12 strain was investigated to confirm that nonpathogenic bacterial strains that are unable to utilize salmochelin remain unaffected. The size of 1 renders it unlikely to be taken up passively via porins, OmpF or OmpC. 53−57 OmpF is considered the main uptake pathway for ciprofloxacin but has a molecular weight limit of around 600 Da. 58 In the absence of active transport or porin-mediated uptake, passive diffusion across the outer membrane is a possibility; this has been suggested to occur for ciprofloxacin, although to a much lesser degree than uptake via porins. 59 However, it is unlikely that 1, with a projected polar surface area of 401 Å, 60 would display a similar capacity as ciprofloxacin for passive diffusion, with a corresponding approximated polar surface area of 82 Å, in its zwitterionic form; a high polar surface area correlates strongly with decreased membrane permeability. 61 It was hoped that the presence of the salmochelin transport machinery in Nissle 1917 would support active uptake of the Fe 3+ complex of 1 and hence increase its antibacterial activity, thereby allowing the selective targeting of this strain.
The antibacterial activity assays were carried out in MOPS acetate minimal media, 62 either in the presence of 100 μM Fe 3+ (iron replete conditions) or with no added Fe 3+ (iron deplete conditions, <18 pM Fe). Details of the composition and iron content of the media are provided in the Supporting Information.
As expected, in E. coli K12 (BW25113), which lacks the IroN transporter, 1 demonstrated a much lower antibacterial activity than ciprofloxacin under both iron replete and iron deplete conditions. Disappointingly, when the activity was tested against Nissle 1917, capable of expressing IroN, a similar lack of activity of 1 was observed, suggesting that active uptake of 1 is not taking place (Table 1). This observation led to a further investigation of factors limiting uptake/activity (vide infra).
Antibacterial Activity vs E. coli K12 (BW25113). Conjugate 1 was added to the growth media at varying concentrations (0−100 μM, Figure 6). At higher concentrations of 1 in iron replete conditions, immediate Fe 3+chelation was evident by the emergence of a characteristic purple color due to Fe 3+ −siderophore complex formation. 45 Due to the significant absorbance of these complexes at 600 nm, the optical density of the cell cultures is displayed at 800 nm (OD 800 ). As expected, at plateaued bacterial growth (after 48 h) and in the absence of antimicrobial agents, the bacterial cell density obtained in Fe 3+ replete media was more than twice as high as that obtained under Fe 3+ limitation (Figures 6  and S6).
In iron replete media, ciprofloxacin, the parent antimicrobial of 1, was active at low concentrations between 0.1 and 1 μM (Figure 7a), while 1 only showed growth suppression once the concentration started to approach that of Fe 3+ in the growth medium (>75 μM). In iron deplete conditions, a clear growth  While the latter might be an indication of salmochelin-mediated uptake of 1, the attenuation of bacterial growth may also be attributed to the competition of 1 with native siderophores for the more limited Fe 3+ resource, starving the cells of the vital nutrient. Again, the antibacterial activity of 1 was much lower than that of the parent antibiotic ciprofloxacin (∼75×, Figure 7b). Hence, to further explore the relationship between siderophore-mediated Fe 3+ uptake and the antibacterial activity of 1, a radiolabeling study with the Fe 3+ -mimetic 67 Ga 3+ was undertaken. 67 Ga-Radiolabeling and Bacterial Uptake. Hexadentate siderophores exhibit exceptionally high affinity for both Fe 3+ and the nonredox active, Fe 3+ -mimetic Ga 3+ (K D >10 −30 M). 63 Functionalized Ga 3+ and Fe 3+ siderophore complexes are both efficiently recognized by bacterial siderophore membrane transporters, indicating that bacteria cannot distinguish the trivalent ions at the time of siderophore-mediated entry. Ga 3+ represents an ideal surrogate to study the behavior of Fe 3+ complex species. 64,65 Commercial availability of two radioactive imaging isotopes of Ga 3+ , 68 Ga (t 1/2 = 1.1 h), a positron emission tomography (PET) imaging isotope, and 67 Ga (t 1/2 = 3.3 d), a longer lived isotope utilized for single photon emission computed tomography (SPECT), provides opportunities to study the stability and pharmacokinetics of Ga− siderophore complexes in vitro and in vivo. Indeed, this approach has already been explored for the imaging of fungal infections in rats, taking advantage of the siderophoremediated uptake of 68 Ga in fungi such as A. fumigatus, 66 as well as the assessment of a novel desferrichrome-based conjugate that exhibited enhanced potency in Gram-positive and Gram-negative strains. 19 Here, we elected to use 67 Ga, as the longer half-life of this isotope permits extensive long-term stability and internal-ization studies. It has been shown that 67 Ga-labeled deferoxamine (DFO) retains active uptake via bacterial Fe 3+ -transport in S. aureus with DFO acting as a xenosiderophore. 67,68 In order to synthesize 67 Ga-1,wefirst transformed 67 Ga-citrate to GaCl 3 and monitored the radiolabeling of 1 by radio-HPLC. 19 Complexation proceeds quickly with quantitative yield, producing an apparent molar activity of 90 nmol/MBq. In order to probe if the 67 Ga-1 was sufficiently inert for subsequent uptake experiments, we monitored complex inertness in LB broth over the course of 2 h, during which no significant dechelation was observed. Next, we assessed the time-dependent bacterial uptake of 67 Ga-1 in E. coli K12 (MG1655) in iron replete LB over 2 h. The percentage of internalized 67 Ga-1 was <10% of the total 67 Ga and comparable to the uptake of a 67 Ga-citrate control. The results in iron deplete LB media were similar, with <10% internalization, comparable to that seen with 67 Ga-citrate (Figure 8).
To confirm if this measured uptake mirrored low transport into bacteria, with the resultant absence of activity, the study was expanded to assess three compounds previously reported by us, staphyloferrin A-inspired conjugates 12, 13, and 14 ( Figure 9). 40 These conjugates retain and mirror the original siderophore structure more closely and provide a 6-coordinate ligand environment for corresponding Fe 3+ and Ga 3+ complexes. These compounds showed activity against E. coli NCTC10418 and some inhibition of DNA gyrase in vitro, albeit at a lower level than the parent ciprofloxacin. 41 All three compounds have an estimated polar surface area of 329 Å when fully protonated. In analogy to 67 Ga-1, 67 Ga-12, 67 Ga-13, and 67 Ga-14 were synthesized under identical conditions.     The radiolabeled complexes were less stable than 67 Ga-1 in both iron replete and iron deplete LB broth, exhibiting the percentage of intact radiochemical complexes ranging from 67 Ga-14 (70%) to 67 Ga-12 (60%) and 67  . No further trans-chelation was observed after 9 h, indicating that a relative equilibrium state is reached. In iron deplete LB broth, complex stability was marginally lower but again was maintained over 9 h.
To assess relative complex inertness in comparison with the 6-coordinate chelator ethylenediamine-tetraacetate (EDTA), the radiochemical gallium complexes were challenged with a 10-fold excess with respect to conjugate concentration. Complex inertness was monitored using radio-HPLC. After 2 h, the relative inertness can be ranked as follows: 67 Ga-14 > 67 Ga-12 > 67 Ga-13 > 67 Ga-1 ( Figure S11). This result indicates that, in the presence of a six-coordinate, competing chelator, the trans-chelation from a 4-coordinate donor such as 1 occurs rapidly; the 6-coordinate, staphyloferrin-based conjugates provide a more inert coordination environment, with the lysine-derived structure, 67 Ga-14, showing the best stability in this challenge assay.
Uptake experiments in E. coli K12 (MG1655) in both iron replete and deplete LB broth were assessed in comparison with 67 Ga-1. Although still low (up to 14.7% after 0.33 h for 67 Ga-13, Figure 10), the uptake of all three conjugates was higher than that observed with the 67 Ga-citrate control and marginally higher than that observed with 67 Ga-1, suggesting a very modest degree of translocation through bacterial cell membranes, corroborating antibacterial activity results obtained in previous work. 40 −42 Antibacterial Activity vs E. coli Nissle 1917. The antibacterial activities of 1 and ciprofloxacin were also assessed against a strain of E. coli capable of producing and transporting salmochelin, E. coli Nissle 1917. The probiotic Nissle 1917 shares many characteristics with uropathogenic E. coli strains, including the ability to produce several siderophores, specifically salmochelin, enterobactin, yersiniabactin, and aerobactin, to be able to adapt to environmental challenges. 29 It was hoped that the presence of the salmochelin transport machinery, including the outer membrane transporter IroN, 25 would increase the bacterial uptake and hence antibacterial activity of 1 in Nissle 1917.
However, the antimicrobial activity of 1 was found to be similar to that observed with the K12 strain. In iron replete media, clear growth inhibition was only seen at 100 μM concentrations and above, when the concentration of 1 reached the 100 μM concentration of Fe 3+ (Figure 11a). This suggests that, under these conditions, the sequestration of Fe 3+ by the siderophore unit of 1 becomes so significant that it deprives the growth medium of this vital nutrient. The fact that ciprofloxacin already suppresses growth at ∼1000× lower concentrations (0.1 μM, Figure 12a) is consistent with a lack of bacterial uptake of 1.
In iron deplete media, a slight growth inhibitory effect is already evident at a 1 μM concentration of 1 (Figure 11b). This observation is consistent with both a fiercer competition between the siderophore unit of 1 and native siderophores for the small amount of Fe 3+ available in the deplete medium and the siderophore-mediated uptake of 1. However, if active uptake is occurring, the lower activity of 1 compared to that of ciprofloxacin (Figure 12b) suggests that it is insufficient to compensate for the decrease in uptake of the antimicrobial via porins and passive diffusion caused by attachment of the salmochelin unit in 1, even though the expression of highaffinity siderophore transport proteins, such as IroN, should be upregulated under Fe 3+ limited conditions. 69 The observation that the activity of 1 vs E. coli Nissle 1917 is significantly lower than that of its parent drug ciprofloxacin led us to consider poor binding to the drug target DNA gyrase as an additional possible reason for the observed reduction in potency.
Gyrase Inhibition Assay. A key factor in the antimicrobial activity of ciprofloxacin conjugates is their continued ability to inhibit DNA gyrase, the cytoplasmic drug target of ciprofloxacin. In order to investigate the impact of   ACS Infectious Diseases pubs.acs.org/journal/aidcbc Article salmochelin-inspired siderophore conjugation on the ability of ciprofloxacin to inhibit gyrase, 1 was evaluated in an in vitro assay using a commercial DNA gyrase supercoiling assay. Initially, 1 was studied over a range of concentrations (0.5−20 μM) and no inhibition of gyrase activity was observed, as indicated by no reduction in the presence of supercoiled DNA plasmids on agarose gels ( Figure 13). Inhibition of the intracellular drug target DNA gyrase by 1 was detectable at concentrations of 30 μM and above, with complete inhibition at 75 μM ( Figure 14), indicating a decrease in gyrase inhibitory activity in comparison with the parent drug (10 μM). However, this moderate decrease in gyrase inhibitory activity does not explain the drastic drop in the antibacterial potency of 1. This, plus the lack of higher activity vs the Nissle 1917 strain, suggests that poor active transport of 1 is a major factor in the observed lack of antimicrobial activity against both strains of E. coli.

■ SUMMARY AND CONCLUSIONS
A ciprofloxacin siderophore conjugate 1 has been synthesized by linking glycosylated catechol units 7, found in salmochelin S4, to L-lysine modified ciprofloxacin 10. The resulting siderophore−ciprofloxacin conjugate was designed to evade the mammalian immune response and to selectively target pathogenic bacteria that express the salmochelin receptor IroN. Initial screening of 1 against a common laboratory strain of E. coli lacking the IroN transporter demonstrated much reduced antibacterial activity compared to the parent antibiotic ciprofloxacin, with concentrations ca. 250−1000× higher than required to obtain similar activity in identical conditions. Increased antibacterial activity was observed in iron deplete media. Probing cellular uptake via 67 Ga labeling suggested the absence of significant bacterial cell uptake of 67 Ga-1, consistent with the absence of IroN.
When the activity was examined vs Nissle 1917, a strain capable of expressing IroN, a similar concentration dependence was observed, with 1 again requiring concentrations 250− 1000× higher than ciprofloxacin to obtain similar growth inhibition, suggesting the presence of IroN did not lead to significantly greater uptake. Again, higher activity was observed in iron deplete media.
While the DNA gyrase inhibitory activity of 1 was significantly lower than that of the parent drug, the relatively moderate decrease in gyrase inhibitory activity does not explain the drastic drop in the antibacterial potency of 1.
These results suggest that one major obstacle in the successful application of salmochelin-based Trojan Horse antibiotics with an intracellular drug target is the delivery of the conjugate into the bacterial cell via Fe 3+ -siderophore transporters.
The observed formation of dimeric 2:2 species by native mass spectrometry provides a potential explanation for the observed lack of cellular uptake of conjugate 1. If the tendency to form dimers also applies to biological media, it is unlikely that these dimers would be recognized by the outer membrane receptor IroN and fit through the iron−salmochelin transporter.
Hence, our first generation conjugate, 1, the tetradentate mimic of the hexadentate siderophore salmochelin S4, appears poorly suited to target the salmochelin-mediated uptake pathway. A similar relationship was observed for enterobactin−ciprofloxacin Trojan Horse conjugates by Nolan and co-workers, 18 who observed that an intact hexadentate enterobactin unit was required for good antibacterial activity, whereas tetradentate or bidentate equivalents displayed poor activity, possibly due to reduced recognition by the corresponding outer membrane receptor. 18 It is also conceivable that the lack of a flexible linker between the siderophore component and ciprofloxacin renders 1 too rigid and bulky to be able to pass through the IroN transporter or the closely linked ciprofloxacin directly impedes siderophore binding.
The poor cellular uptake shown in the case of 1, along with its increase in activity in iron deplete media, may point to extracellular Fe 3+ sequestration as an additional mechanism of action, alongside DNA gyrase inhibition.
While the lack of significant uptake and activity of 1 is disappointing, it offers some direction to a future second generation design. The size and polar surface area of the conjugate are sufficient to prevent passive uptake via porin channels or passive diffusion through the cell membrane, suggesting that optimization of the siderophore component and linker to boost uptake in salmochelin-utilizing strains could be sufficient to transform similar conjugates into narrowspectrum antimicrobials. In addition, while the application of a biolabile linker designed to cleave the ciprofloxacin from the siderophore inside the cell could retrieve the DNA inhibitory activity, a biolabile link will not increase antibiotic efficacy unless the conjugate is delivered, at an appropriate level, into the bacterial cytoplasm. Future studies will be focused on optimizing active conjugate transport into bacterial cells using a combination of targeted chemical synthesis and radiolabeled 67 Ga complex uptake studies.