**2.2 Development of d-block metal ions probes based on the cleavage of C–B bonds in B-containing probes**

It is well established that macrocyclic polyamine ligands such as 1,4,7-triazacyclononane ([9]aneN3) **1**, 1,4,7,10-tetraazacyclododecane ([12]aneN4, cyclen) **2**, and *Design, Synthesis, and Biological Applications of Boron-Containing Polyamine and Sugar… DOI: http://dx.doi.org/10.5772/intechopen.105998*

1,4,7,10,13-pentaazacyclopentadecane ([15]aneN5) **3** are able to form more stable complexes **4**–**6** with metal ions such as Cu2+, Ni2+, and Zn2+ in aqueous solution (**Figure 1**) than metal complexes of linear polyamine types [16, 17]. In addition, metal ions in these complexes, especially the Zn2+ ion in Zn2+-cyclen complex (**5**), possess strong Lewis acidity and the deprotonated Zn2+-bound H2O (HO− ) functions as a nucleophile and a base in aqueous solution at neutral pH [18–23].

Bendel and coworkers reported that 11B NMR/MRI would be a potential technique for the imaging of boron agents in the body [24, 25]. However, a functional system for achieving this has not been established yet. In this context, we hypothesized that the sp2 boron in **7** and **8** would be changed to the sp3 boron due to the formation of metal complexes **9a** and **10a** and the following interaction of metal-bound H2O (OH− ) with boron at neutral pH, resulting in change in the 11B NMR signals (**Figure 2**) [26]. However, the products obtained after the addition of Zn2+ to **7** (L1 ) (**Figure 3a**) were **11a** (ZnL3 ) and boric acid (B(OH)3), as confirmed by an X-ray structure analysis (**Figure 3b**). The findings strongly indicated that the Zn2+-bound H2O (**9a** and **10a**) is efficiently deprotonated

#### **Figure 2.**

*The C–B bond hydrolysis of phenylboronic acid-pendant 12-membered tetraamine (cyclen) to produce inorganic boric acid.*

#### **Figure 3.**

*X-ray crystal structures of (a)* **7** *(L1 ) and (b)* **11a** *(ZnL3 ) with B(OH)3.*

due to the double activation by Zn2+ and B to produce the Zn2+-bound HO− (**9b** and **10b**), which hydrolyzes the C–B bond. The hydrolytic cleavage of the C–B bond of **7** (L1 ) was also observed by the measurement of 11B NMR upon the addition of Zn2+, in which the 11B NMR signal of **7** (L1 ) at 31.1 ppm was shifted to 19.4 ppm that corresponds to B(OH)3.

The 11B NMR spectral change of **7** (L1 ) was promoted by Cu2+, Fe2+, Co2+, and Ni2+ but not by Ca2+ and Mg2+ (**Table 1**). Hydrolysis of the C–B bond of **7** (L1 ) with Cd2+ was faster than that with Zn2+, possibly due to the strong nucleophilicity of the Cd2+-bound HO– [27]. Meanwhile, the C–B bond cleavage of **7** (L1 ) by Mn2+ and Fe3+ was slow.

The intracellular uptake of boron in **7** and **8** into Jurkat T cells was determined by ICP-AES, and the results indicated that the uptake of **8** was higher than that of **7**, possibly due to the hydrophobicity of the boronic ester group. The Zn2+-induced C–B bond cleavage of **8** (L2 ) by intracellular Zn2+ was observed in living cells. The Jurkat T cells were sequentially treated with **8** (L2 ) and Zn2+ complex of pyrithione (Zn2+ ionophore to transfer Zn2+ into cells) for 20 min and 1 h, respectively. The cells were washed with CS-RPMI and PBS and then transferred to a quartz NMR tube, whose


*a All data are referenced to external BF3·Et2O in CDCl3 (δ = 0 ppm). <sup>b</sup>*

*Δδ = δ (***7** *(L1 ) with metal ions) – δ (***7** *(L1 )).*

*c Approximate reaction time for the completion of C–B bond cleavage.*

#### **Table 1.**

*11B NMR spectral change of* **7** *(L<sup>1</sup> ) (20 mM) upon the addition of d-block metal ions (20 mM) in 1 M HEPES buffer at pD 7.4 and 25 °C [26].a*

*Design, Synthesis, and Biological Applications of Boron-Containing Polyamine and Sugar… DOI: http://dx.doi.org/10.5772/intechopen.105998*

#### **Figure 4.**

*In-cell 11B NMR spectra of* **8** *(L2 ) in the absence of Zn2+–pyrithione (ionophore) and in the presence of Zn2+–pyrithione (BF3·Et2O was used as an external references). The Jurkat T cells (4 × 108 cells) were incubated with 33 μM* **8** *(L2 ) in culture medium at 37 °C for 1 h, and then (a) DMSO (as negative control), (b) 2.5 μM Zn2+–pyrithione, and (c) 10 μM Zn2+–pyrithione at 37 °C for 20 min.*

11B NMR spectra were measured in D2O containing PBS. As shown in **Figure 4**, the 11B signal for B(OH)3 (ca. 19 ppm) in Jurkat T cells was observed with a positive correlation to the concentrations of Zn2+-pyrithione complex, indicating the successful detection of the intracellular Zn2+ ions. It should be noted that the 11B signal for **8** (ca. 31 ppm) in the absence of Zn2+ was observed as a broad signal.
