Design Strategies for Developing Chiral Macrocyclic Chelators as a Diagnostic Tool

Conspectus

Macrocyclic chelators have become an indispensable part of the field of molecular imaging due to their ability to form highly stable complexes with various metal ions for diverse diagnostic and therapeutic applications. Among them, DOTA (1,4,7,10-tetra­azacyclo­dodecane-1,4,7,10-tetraacetic acid), which serves as a gold standard in both stability and versatility, is extensively utilized in clinical contexts as contrast agents for a series of imaging modalities, including MRI (magnetic resonance imaging), PET (positron emission tomography), SPECT (single-photon emission computed tomography), and optical imaging. Despite its advantageous properties, in certain cases, DOTA still has limitations to be solved, such as slow radiolabeling kinetics and possible in vivo decomplexation, which have led to increased research interest in structural modifications of the DOTA ligand.

To date, the most popular design strategy for DOTA analogues focuses on the modification of pendant arms, which can be simply achieved through the N-derivatization of the tetraaza macrocycle. Another strategy is to modify the macrocyclic ring structure, with the most successful example being the macrocycle pyclen, which contains a pyridine moiety. In 2022, pyclen-based Gd³⁺ complex gadopiclenol was approved by the U.S. FDA (United States Food and Drug Administration) as a next-generation MRI contrast agent for clinical use. However, reported studies on similar structural modifications of macrocyclic rings are relatively rare, as this requires the redesign of synthetic procedures that can be highly complicated.

In this Account, we discuss our design strategies of various macrocyclic chelators with chiral substituents on the macrocyclic ring for different biomedical imaging applications. We start with structural modifications to the frequently used DOTA chelator through the introduction of four symmetrical chiral substituents on its macrocyclic backbone. Results of NMR studies confirmed that the incorporated chiral substituents can prevent the interconversion between two TSAP/SAP regioisomers formed in the complexation process of DOTA with lanthanides. The chiral substituents also provide increased ligand rigidity and can enhance the coordination geometry for better encapsulation of the metal, preventing possible dechelation and transmetalation, thereby achieving higher coordination stability. A series of DOTA analogues with different chiral substituents were studied, and we have revealed that the TSAP/SAP isomeric ratios and pharmacokinetics of chiral DOTA varied with the steric hindrance and polarity of attached chiral groups. In particular, the Gd³⁺ complexes of chiral DOTAs with symmetric aliphatic groups exhibit enhanced relaxivity over that of achiral DOTA, making them ideal contrast agents for MR imaging. Through the subsequent optimization of introduced chiral groups, various improved chelators were then developed for photoluminescence or for MRI.

Next, we focused on further pendant arm modifications of these chiral DOTA platforms. Multiple functionalized pendant arms were attached to the chiral macrocycle as chromophores or targeting vectors, resulting in versatile chiral bifunctional chelators for targeted circularly polarized luminescence (CPL), PET/SPECT, and radiotherapy applications. Moreover, our chiral modifications are not limited to the 12-membered DOTA macrocycle. Recently, we broadened our design strategy to alter the macrocyclic ring sizes, an approach with relatively few literature examples. The chiral analogues of 9-membered NOTA and 10-membered DETA systems were synthesized for ⁶⁸Ga radiolabeling, with the influence of cavity sizes and positions of chiral substitution investigated. Follow-up studies will go deeper into the metal ion selectivity effects of differently sized chiral macrocycles to pave the way for future designs on novel macrocyclic chelators.

Key References

• Dai, L. ; Jones, C. M. ; Chan, W. T. K. ; Pham, T. A. ; Ling, X. ; Gale, E. M. ; Rotile, N. J. ; Tai, W. C.-S. ; Anderson, C. J. ; Caravan, P. ; Law, G.-L. . Chiral DOTA chelators as an improved platform for biomedical imaging and therapy applications. Nat. Commun. 2018, 9­(1), 857 10.1038/s41467-018-03315-8 . This work reported the synthesis of a series of chiral DOTA chelators with modified macrocyclic backbones. The introduced chiral substituents enhanced the rigidity and stability of these chelators, enabling control of structural isomerism of their lanthanide complexes.

• Zhang, J. ; Dai, L. ; Webster, A. M. ; Chan, W. T. K. ; Mackenzie, L. E. ; Pal, R. ; Cobb, S. L. ; Law, G. L. . Unusual magnetic field responsive circularly polarized luminescence probes with highly emissive chiral europium (III) complexes. Angew. Chem., Int. Ed. 2021, 60­(2), 1004–1010 10.1002/anie.202012133 . In this work, functionalized pendant arms were attached to various chiral DOTA chelators to create targeted CPL luminescence probes. The Eu³⁺ complexes of these modified chiral DOTAs exhibited improved photophysical properties and magnetic CPL responses.

• Zhang, J. ; Dai, L. ; He, L. ; Bhattarai, A. ; Chan, C.-M. ; Tai, W. C.-S. ; Vardhanabhuti, V. ; Law, G.-L. . Design and synthesis of chiral DOTA-based MRI contrast agents with remarkable relaxivities. Commun. Chem. 2023, 6­(1), 251 10.1038/s42004-023-01050-w . Two chiral DOTAs with hydrophilic functional groups were developed as Gd-based contrast agents, showing high water solubility and improved secondary-sphere relaxivity, suitable for potential MRI applications.

• Zhu, Y. ; Cheng, Z. ; Zhang, J. ; Feng, L. ; Mishra, A. ; de Rosales, R. T. M. ; Brown, R. K. ; Long, N. ; Law, G.-L. . Influences of Ring Sizes and Chiral Substituents on Macrocyclic Chelators for Improving ⁶⁸Ga Radiolabeling. JACS Au 2026, 6­(1), 113–123 10.1021/jacsau.5c00946 . Chiral analogues of 9-membered NOTA, 10-membered DETA, and 12-membered DOTA chelators were synthesized with detailed radiolabeling, equilibrium, and computational studies to reveal the influence of macrocyclic cavity size and chiral substitution positions on Ga³⁺ coordination properties.

1. Introduction

Macrocycles are ubiquitous in nature and are extensively used as chelators to form metal complexes. Classic examples are hemes and chlorophylls featuring porphyrin complex-based structures which display key functional roles such as for oxygen transportation in animals and energy transfer in plants, respectively. , Within these natural macrocycles, the central metal ion is firmly encapsulated within the ligand cavity, thereby conserving the coordination chemistry, metal stability, and functions of the biomolecules against harsh physiological environments. Due to their robustness, this has created a niche for scientists to investigate and mimic the structure and function of these natural macrocyclic systems. It has been proven that macrocyclic ligands are able to form metal complexes with enhanced thermodynamic stability and kinetic inertness compared to acyclic ligands, a phenomenon commonly known as the macrocyclic effect. , In past decades, numerous synthetic macrocyclic ligands, especially macrocyclic polyamines, have been developed for clinical, industrial, and laboratory applications such as molecular imaging, radiotherapy, biosensing, and metal ion separations. −

The tetraazamacrocycle DOTA (1,4,7,10-tetra­azacyclo­dodecane-1,4,7,10-tetra­acetic acid) and its derivatives stand out as “gold standard” macrocyclic polyamines that form stable lanthanide and transition-metal complexes with high kinetic inertness and unique properties that are well suited for in vivo applications including magnetic resonance imaging (MRI), positron emission tomography (PET), single-photon emission computed tomography (SPECT), and optical imaging. ,− Several metal complexes of DOTA derivatives have been approved by the U.S. Food and Drug Administration (FDA) as diagnostic probes for clinical use despite limitations such as slow radiolabeling kinetics and possible decomplexation in vivo, ,− leading to the design of next-generation ligands with higher inertness, improved coordination properties, or ligand versatility. − In this regard, there are three mainstream design strategies for improving macrocyclic polyamines: (1) modifying the pendant arms, (2) modifying the macrocyclic backbone, and (3) altering the macrocyclic ring size (Figure ).

1.

Three strategies for developing improved macrocyclic polyamines for biological applications with examples from the literature − or approved by the FDA.

At present, the most common and easiest structural modification for DOTA-based macrocyclic chelators is changing the N-substituent pendant arms. This strategy is prevalent among MRI contrast agents as it improves the water relaxivity of Gd-DOTA complexes by altering the water-exchange mechanics around the Gd³⁺ center, , such as FDA-approved gadobutrol and gadoteridol that have hydroxyl-based arms. Or another example by Gianolio’s group, showed by introducing a trisulfonated pyrene moiety on the DOTA, this provided enhanced relaxivity via increased molecular weight. Functionalizing pendant arms with a vector could also give rise to bifunctional chelators (BFCs) where the macrocyclic complex is responsible for the imaging and the functional arm imparts specific targeting which is crucial in many imaging modalities. In a typical BFC design, one carboxylic arm is generally covalently conjugated to a cancer-binding antibody, peptide or protein via a linker to enable specific targeting of cancer cells. In general, the convenience of pendant arm modifications offers extraordinary versatility to the DOTA platform for various imaging applications, and this strategy has been extensively studied. ,,−

Modifying the DOTA macrocycle backbone is much more challenging from a synthetic perspective; it also changes the ligand’s preorganization to afford distinct coordination geometry. In earlier reports, bulky alkyne groups or chiral substituents were incorporated on the macrocyclic ring to create new MRI contrast agents with improved relaxivity. , More recently, a chiral DOTA analogue with two sets of hydroxyl-methylene substituents on the adjacent macrocyclic carbons was also reported, exhibiting high structural rigidity and hydrophilicity. The most significant improvement is FDA-approved MRI contrast agent gadopiclenol: the pyridine moiety on the macrocycle backbone gives it significant stability and a great increase in the water relaxivity of its Gd³⁺ complexes. , However, similar structural engineering of macrocyclic ring is still limited by synthetic challenges, with fewer literature examples than for the pendant arm modifications.

Hancock et al. reported in the 1980s that the cavity size and coordination geometry of macrocyclic ligands play a crucial role in the formation kinetics and thermodynamic stability of their complexes. This triggered researchers to investigate macrocyclic chelators with different ring sizes to seek better metal ion selectivity. A representative example is the 9-membered chelator NOTA (1,4,7-triaza­cyclononane-1,4,7-triacetic acid). With a smaller cavity size than DOTA, NOTA exhibits ideal binding affinity to smaller transition and post-transition metal ions like Ga³⁺ for ⁶⁸Ga-labeled PET imaging. − A larger macrocyclic ring, the 14-membered TETA (1,4,8,11-tetra­azacyclotetra­decane-1,4,8,11-tetraacetic acid), has also demonstrated excellent compatibility with Cu²⁺ ions due to its favorable coordination geometry. − Other examples, like the 18-membered macropa, have emerged as novel chelators for large trivalent radiometals like ²²⁵Ac, ¹³³La, ¹⁷⁷Lu, ²¹³Bi, ²¹²Pb, ¹³¹Ba, and ^223/224Ra. − However, it is worth noting that, to date, DOTA is the only macrocyclic ring adopted in the FDA-approved clinical contrast agents. The research on macrocyclic chelators of different sizes is still a diamond in the rough that requires further development to fulfill their full potential for biological applications.

When designing DOTA-based macrocyclic chelators for lanthanide coordination, an important phenomenon that should be taken into consideration is the stereoisomerism of the resulting complexes. It is well documented that there are four conformational isomers present in aqueous solution: two pairs of enantiomers with square antiprismatic (SAP, corner/side) and twisted square antiprismatic (TSAP, corner/side) geometries (Figure ). The SAP and TSAP conformations arise through the rotation of carboxylate pendant arms, , whereas the side and corner conformations are categorized by the geometry of the tetraaza macrocycle. In nonchiral Ln-DOTA systems, the four conformational isomers are interconvertible on a time scale of 10–100 ms through rotation of the acetate arms or inversion of the macrocyclic ring. − With different steric crowding and activation energy for conformation exchange, SAP and TSAP isomers exhibit water-exchange rates that can differ by up to 2 orders of magnitude, resulting in distinct MRI contrast performances. ,,− They can also generate multiple resonances in paramagnetic NMR spectroscopy, hindering the accurate determination of protein structures. , Therefore, researchers are devoted to controlling the ratio of conformational isomers in Ln-DOTA complexes. , Similar structural isomerism was also found in various metal complexes of the other macrocyclic ligands, such as the NOTA systems. −

2.

Illustration of the geometric isomerism in Ln-DOTA complexes. (a) SAP and TSAP isomerism and (b) “side” and “corner” isomerism.

The selective formation of SAP and TSAP isomers can be achieved by enhanced ligand rigidification of DOTA analogues. ,,− When introducing bulky or chiral substituents on the pendant arms or the macrocyclic ring of DOTA chelator, the resulting Ln³⁺ complexes can be locked into either SAP or TSAP geometry. However, compared to the widely studied chiral modifications on acetate pendant arms, literature examples of chiral substitution on the DOTA macrocycle are relatively limited, likely due to synthetic challenges. ,, In our original design of chiral DOTA systems, we employed an optimized synthetic route with simplified steps, convenient purification, and high overall yields. It enabled the gram-scale preparation of various rigid chiral DOTA chelators bearing symmetric macrocyclic backbones for in-depth studies of their coordination chemistry with different metal ions. We then explored the other modification strategies, including pendant arm modifications and variations in macrocyclic ring sizes. − These efforts led to the development of a series of chiral macrocyclic chelators for biological applications, such as sensors and imaging probes.

2. Modification of Macrocyclic Backbones.

In our first work to improve DOTA, we focused on its macrocyclic ring framework for an enhanced ligand preorganization (Figure ). With our optimized synthetic route featuring a tetrameric cyclization reaction, chiral DOTA ligands L1–L5 were synthesized with methyl, ethyl, isobutyl, benzyl, and aminobutyl groups, respectively (Figure ). The chiral substituents with varied steric hindrance gave rise to different properties such as polarity, ligand rigidity, and hence different influences on the coordination isomers of its metal complexes. This led to further designs of ligands L6–L8 to explore controlling the coordination isomers.

3.

Design of chiral DOTA analogues with modified macrocyclic backbones in order of increased steric hindrance. ,,

4.

Structures of the lanthanide (Ln) complexes with chiral DOTA ligands L1–L8 bearing diverse chiral substituents. ,

According to the crystal structure of one of the resulting chiral macrocyclic complexes, GdL4, while the carboxylic pendant arms were pulled toward the metal center for coordination, all four chiral substituents on the macrocycle pointed in the opposite direction, creating a more rigid framework and simultaneously forming a shield to protect the metal surface.

We then studied the structural isomerism of our chiral DOTA complexes with Eu³⁺ and Yb³⁺, which showed that the extra steric hindrance from the chiral substituents restricts ring inversion, thereby preventing the interconversion between the corner and side pairs (Figure ). As a result, the conformation of TSAP and SAP isomers was “locked” after complexation, enabling chromatographic isolation. According to our ¹H NMR studies, the isomeric ratio of our chiral DOTA complexes correlated strongly with the steric hindrance of chiral substituents (Figure ): the TSAP isomers normally prefer sterically crowded environments at the metal center. The only exception is EuL5, where only the SAP isomers were observed, which might be attributed to the change in the solvation environment around the polar amine substituent.

5.

Restricted isomerism of our chiral DOTA complexes with an enlarged corner view of each macrocyclic ring in the dashed boxes. (Planes formed by the four nitrogen atoms are highlighted in blue; some atoms are omitted for clarity.)

6.

SAP/TSAP isomer ratios in the Eu³⁺ complexes of chiral DOTA L1–L8 according to the integration of ¹H NMR signals. ,

When MRI contrast agents are evaluated, one of the most important parameters is relaxivity, which can be influenced by the coordination isomers formed in Gd³⁺ complexation. For DOTA-based MRI contrast agents, the TSAP isomers exhibit faster water exchange kinetics, hence accounting for more relaxivity enhancement than the SAP isomers as they fall in the optimal window for water exchange. , Our chiral GdL1–GdL5 showed very fast water exchange rates with a much shorter water residence time (τ_M ^310 K) than that of achiral Gd-DOTA. The r ₁ and r ₂ values of achiral Gd-DOTA, GdL1, GdL2, and GdL5 were measured at 1.4 T, 37 °C in water, 4.5% (w/v) human serum albumin (HSA) solution, and human plasma (Table ). Notably, we synthesized and separated four isomers of GdL2, namely, GdL2A (SAP), GdL2B (TSAP), Gd­( R )­L2A (SAP), and Gd­( R )­L2B (TSAP), to investigate their influences. Our chiral DOTA complexes demonstrate moderately higher r ₁ relaxivities than Gd-DOTA, showing potential to bring about improved imaging performance in a clinical setting.

1. Relaxivities of Gd³⁺ Complexes in Water, HSA, and Human Plasma Measured at 1.4 T, 37 °C .

	Water		4.5% w/v HSA		Human Plasma
Sample	r 1	r 2	r 1	r 2	r 1	r 2
GdL1	4.1	5.1	4.5	5.3	4.6	5.5
GdL2A	4.2	5.5	4.7	5.5	4.7	5.6
GdL2B	3.9	4.9	4.8	6.0	4.9	6.1
Gd( R )L2A	3.9	5.0	4.5	4.9	4.6	4.8
Gd( R )L2B	3.9	4.9	4.8	6.1	4.9	6.1
GdL5	4.1	5.9	5.1	6.3	5.1	6.3
GdL8	6.8	/	/	/	/	/
Gd-DOTA	3.2	3.2	4.1	4.8	4.0	4.0

^aMeasured in water at 1.5 T, 37 °C from ref .

The kinetic inertness of GBCAs is an important parameter that can imply in vivo stability. The stabilities of the SAP and TSAP isomers of GdL2 were evaluated separately. Gd biodistribution studies in BALB/c mice revealed that, different from the rapid and exclusive elimination of normal Gd-DOTA via the renal pathway as previously reported, the biodistribution profiles of our chiral DOTA complexes varied with the bulkiness of their chiral macrocyclic backbones (Figure ).

7.

Biodistribution profile of different Gd³⁺ complexes in mice over time. Reproduced from ref . Available under a CC BY 4.0 license. Copyright 2018 The Authors.

Although L3 and L4, with bulky chiral substituents, result in a high abundance of TSAP isomers that are beneficial as GBCAs, they are not water-soluble. Therefore, we modified the four chiral benzyl groups on ligand L4 to give L6–L8 with improved water solubility. GdL8, with four hydrophilic sulfonyl groups, demonstrated the highest TSAP/SAP ratios and r ₁ relaxivity, making it ideal for GBCA applications (Table ).

Moreover, the aromatic chiral substituents on L7 and L8 which yielded one dominant isomer (TSAP) in their lanthanide complexes make them suitable for circularly polarized luminescence (CPL) applications. CPL is a photophysical phenomenon of chiral molecular systems characterized by a high luminescence dissymmetry factor (g_lum) which reflects the difference in emission intensities between right and left circularly polarized light. It is useful for probing chiral information in biological systems. , Chiral organic compounds normally have g_lum values ranging from 10^–4 to 10^–3, while our chiral lanthanide complexes can yield g_lum > 10^–1 as they enable CPL emission from the magnetic-dipole-allowed f–f transitions. , The TSAP isomer of TbL7 showed a high g_lum value of 0.285 at 542.5 nm (Figure a) from its magnetic-dipole-allowed transition (⁵D₄ → ⁷F₅, ΔJ = 1). Similarly, TbL8 also displays comparable CPL properties, with a g_lum value of 0.241 at 542.5 nm (Figure b). The measured g_lum values for both compounds are among the highest reported for Tb³⁺ complexes.

8.

Total emission (green) and CPL (red) spectra for the TSAP isomers of (a) TbL7 and (b) TbL8. Reprinted with permission from ref . Copyright 2019 American Chemical Society.

In our follow up studies, hydrophilic chiral groups were selected as functional substituents to enhance the hydrophilicity as MRI contrast agents. The overall relaxivity of GBCAs is contributed by inner-sphere, secondary-sphere, and outer-sphere relaxivities. In practice, the mainstream approach is to enhance the inner-sphere relaxivity by increasing the rotational correlation time τ_R. However, the contribution from second-sphere relaxivity is generally considered minor unless there are hydrophilic phosphate or hydroxyl groups around the Gd³⁺ ion. Our L9 was optimized through conjugating L5 with aromatic sulfonic acid substitution (Figure ). Besides improving water solubility, the aromatic substituents also bring about higher molecular weight and accelerated secondary-sphere water exchange. L10 features four hydroxy-functionalized chiral groups around the macrocycle for enhancing both inner-sphere and second-sphere relaxivities. All of the measured compounds have much higher relaxivity than Gd-DOTA in both water and 4.5% HSA solution (Table ).

9.

Gd³⁺ complexes of ligand L9 and L10 with backbone modifications for enhanced water solubility and relaxivity with their water coordination spheres.

2. Relaxivities of the Gd³⁺ Complexes of L9 and L10 under 1.4 T, 37 °C, pH 7 .

Complex	r 1 in H2O	r 2 in H2O	r 1 in 4.5% HSA	r 2 in 4.5% HSA
(TSAP) GdL9	7.4 (0.1)	8.4 (0.1)	12.4 (0.8)	16.3 (1.5)
(SAP) GdL9	14.5 (0.3)	20.0 (0.2)	17.5 (1.0)	51.9 (2.8)
GdL10	5.2 (0.2)	6.1 (0.2)	5.7 (0.4)	7.5 (0.6)
Gd-DOTA	3.2	3.2	4.1	4.8

^aData recorded in ref . The standard deviation is shown in parentheses.

Subsequently, pH-dependent r ₁ relaxivity measurements of GdL9 and GdL10 were conducted. The r ₁ relaxivity profiles obtained over pH 2 to 11 had no significant changes, suggesting that no catalyzed proton exchange took place. This result indicates that the considerable improvement of relaxivity for GdL9 and GdL10 with hydrophilic functional chiral groups resulted from the enhanced contribution of the secondary and outer-sphere relaxivity. In further kinetic and biodistribution studies, GdL9 and GdL10 also demonstrated better kinetic inertness and longer retention in plasma compared with Gd-DOTA.

3. Pendant Arm Modifications

Next, we revisited pendant arm functionalization on our chiral DOTA platforms. Many functionalized pendant arms have been developed to improve the properties of DOTA analogues, including chromophores in luminescent applications, − linkers for vector conjugation in targeted radiopharmaceuticals, − and molecular switches as responsive probes for sensor and imaging applications − (Figure ). Our designs of functionalized chiral DOTA focus on two directions: (1) incorporating a sensitizing unit into our pendant arm to develop luminescent probes or (2) adding functional groups to form a conjugation handle to create BFCs with conjugated targeting vectors.

10.

Design of chiral DOTA with functional pendant arms for different applications. ,

On the basis of this strategy, we introduced a chelating chromophore on the modified pendant arm to enhance the luminescence and also water solubility of our complexes to improve upon the properties observed for TbL7 and TbL8 complexes which were poorly emissive although displayed CPL properties. Various water-soluble aliphatic chiral DOTA chelators, namely L12–L16 (Figure a), were synthesized for luminescence studies; the extra carboxylic linker also provides a site for bioconjugation. As a proof of concept for developing target-specific bioprobes, we further designed ligand L17 that is conjugated to a mitochondria-specific, cell-penetrating peptoid (CPPo), Npmb-NLys-Npcb-NLys-NH₂. Due to the ease of synthetic incorporation of bioconjugation handles and initiated by our earlier studies, which revealed our chiral DOTAs to exhibit improved radiolabeling efficacy with ⁶⁴Cu and ¹⁷⁷Lu¹, we further developed BFC designs for targeting vectors that are necessary for radiopharmaceutical applications. This led to our designs of L18 and L19 (Figure b), where L18 features a cyclic Arg-Gly-Asp (RGD) peptide conjugated on the pendant arm for binding integrin receptors on cell surfaces, − while L19 has a pendant arm containing a tosyl group which interacts with bovine serum albumin protein and also serves as a bioconjugation site.

11.

Structures of (a) ligands L11–L17 with functional sensitizing units for luminescent probes and (b) ligands L18 and L19 with targeting vectors as BFCs for PET/SPECT applications.

The luminescence properties of Eu³⁺ complexes of L12–L17 were studied (Table ). All of the chiral compounds listed in Table are in the SAP form with (TSAP)­EuL13 being the only exception, which is selected to investigate the influence of coordination geometries. (SAP)­EuL11–EuL17 with varied chiral backbones did not exhibit significantly different quantum yields, while (TSAP)­EuL13 has a much lower quantum yield. This difference can be explained by the higher sensitization efficiency (η_sens) of the SAP isomers (80–90%). In general, the luminescent quantum yields of the chiral EuL12–EuL17 are much higher than that of nonchiral EuL11. This is attributed to the lack of luminescence quenching from coordinating water molecules in the first coordination sphere, as calculated from Parker’s and Horrocks’ equations , and also due to the chiral groups further shielding the Eu³⁺ metal center. EuL12–EuL17 not only have high quantum yields but also comparable g_lum values. The highest g_lum observed was 0.240 of EuL15, belonging to its ⁵D₀ → ⁷F₁ transition.

3. Photophysical Properties of EuL11–EuL17 Measured in 0.1 M HEPES, pH 7.3, at 298 K, Excited at 350 nm, with a 380 nm Long-Pass Filter .

	EuL11	EuL12	(SAP) EuL13	(TSAP) vEuL13	(R)EuL13	EuL14	EuL15	EuL16	(R)EuL17
Φ(%)	18.4 ± 0.4	27.0 ± 0.3	28.0 ± 1.5	19.8 ± 1.8	26.3 ± 0.6	25.6 ± 0.8	24.1 ± 0.7	26.7 ± 0.2	31.3 ± 1.1
τ H2O (ms)	0.976 ± 0.09	1.09 ± 0.08	1.30 ± 0.02	1.06 ± 0.02	1.21 ± 0.05	1.35 ± 0.006	1.33 ± 0.006	1.26 ± 0.02	1.39 ± 0.004
τ D2O (ms)	1.71 ± 0.08	1.93 ± 0.007	2.14 ± 0.01	1.65 ± 0.09	2.14 ± 0.004	2.21 ± 0.005	2.15 ± 0.002	2.10 ± 0.003	2.06 ± 0.003
Q	0.23	0.18	0.06	0.10	0.13	0.05	0.04	0.08	–0.02
Q	0.14	0.10	–0.01	0.03	0.05	–0.02	–0.03	0.01	–0.08
Φ Eu Eu (%)	24.3 ± 1.8	29.2 ± 1.4	30.9 ± 1.5	26.6 ± 1.9	32.2 ± 0.7	32.5 ± 0.7	32.4 ± 0.6	31.9 ± 0.2	33.2 ± 0.2
η sens (%)	74.6 ± 3.4	92.4 ± 4.1	90.3 ± 1.9	74.4 ± 3.1	81.6 ± 3.4	78.8 ± 1.3	74.2 ± 3.4	83.7 ± 1.5	94.5 ± 2.5
R	2.61	2.53	2.54	2.50	2.48	2.54	2.52	2.45	2.55
B	3312	4860	5040	3564	4734	4608	4338	4806	5634

^a EuL11–EuL17 are all in the SAP form, except (TSAP)­EuL13.

^bRelative to quinine sulfate in 0.1 M H₂SO₄ (λ_ex = 350 nm, Φ = 0.577). Estimated errors in the quantum yield and lifetime are ±15% and ±10%, respectively.

^cMeasuring the ⁵D₀ → ⁷F₂ transition.

^dCalculated with Parker’s equation.

^eCalculated with Horrocks’ equation.

^fI­(⁵D₀ → ⁷F₂)/I­(⁵D₀ → ⁷F₁).

^gB = ε_350 nmΦ.

The magnetic circularly polarized luminescence (MCPL) responses of EuL12–EuL17 were also examined under static external magnetic fields. The results showed an obvious linear increase in g_lum values from the magnetic dipole-allowed ⁵D₀ → ⁷F₁ transition with increasing magnetic field strength (Figure ). In particular, for the ( S )- and ( R )-SAP-EuL13, a 20% enhancement in g_lum values was obtained when the magnetic field increased from 0 to 1.4 T at room temperature. It is also worth noting that a linear trend between Δg_lum and the applied magnetic field strengths was observed. The high sensitivity and enhancement to the magnetic field enable these complexes to be suitable as magneto-optical probes. ,

12.

(a) Magnetic-field-dependent g_lum spectra of (S)- and (R)-SAP-EuL13 in 0.1 M HEPES buffer, pH 7.3, excited at 350 nm at room temperature. Arrows indicate the trends in g_lum changes; inserted diagrams show the direction of the applied magnetic field. (b) g_lum and (c) Δg_lum as functions of magnetic field strength for the ⁵D₀ → ⁷F₁ transition. Reproduced from ref . Available under a CC BY 4.0 license. Copyright 2020 The Auhtors.

The potential of L18 and L19 as radiopharmaceutical chelators was evaluated through cold and hot radiolabeling studies with frequently used metal ions in diagnostic and therapeutic radiopharmaceuticals, along with the chiral ethyl ligand L13 that can also be considered as BFCs due to its extra bioconjugation site. Conducted for our chiral BFCs. L18 was selected for ⁶⁴Cu²⁺ radiolabeling because it showed outstanding Cu²⁺ complexation kinetics. An extremely high radiolabeling yield of >99% was obtained at pH 6.05 after heating at 40 °C for 30 min. Nonadentate ligand L13 offers a saturated coordination sphere for lanthanide ions with improved stability, thus it was used to examine the radiolabeling efficacy with ¹⁷⁷Lu³⁺. As expected, effective ¹⁷⁷Lu labeling was achieved with a yield of 96% after incubating at 45 °C for 15 min (pH 6.0). This highlights the potential of L13 as a convenient platform for biological applications, as antibodies and peptides conjugated to L13 will not be affected by the mild conditions.

4. Modification of Macrocyclic Sizes

In our latest work, we explored chiral modifications on other macrocyclic chelators with different cavity sizes (Figure ). The 9-membered NOTA is the most promising and well-studied macrocyclic ligand after DOTA, thus we designed chiral NOTA L20 with ethyl substituents for comparison with our benchmark chiral DOTA L2 (Figure ). The 10-membered 1,4,7-triazacyclodecane-1,4,7-triacetate acid (DETA) chelator was first developed in 1971 with several follow-up investigations of its structure and basic coordination properties. − According to Hancock et al.’s metal ion selectivity theory, − DETA’s 6-membered coordination ring might be beneficial for chelating small metal ions like Ga³⁺; nevertheless, studies on DETA complexes are rather limited. Based on DFT calculations, we selected a combination of chiral methyl and ethyl groups for our chiral DETA design,s and both the α- and β-versions of chiral DETA were synthesized, namely, L21 and L22.

13.

Design of differently sized chiral macrocyclic chelators for ⁶⁸Ga radiolabeling.

14.

Structures of the Ga³⁺ complexes with the four differently sized chiral macrocyclic ligands and their parent achiral chelators. Adapted from ref . Available under a CC BY-NC-ND 4.0 license. Copyright 2025 The Authors.

Unlike chiral DOTAs, chiral NOTA and DETA cannot be synthesized via cyclization of aziridines; new synthetic routes employing chiral amino acid derivatives as building blocks to construct linear intermediates for cyclization were designed. ⁶⁸Ga radiolabeling studies suggested a general RCY (radiochemical yield) trend of L20 (9) > L2 (12) > L21 (α-10) > L22 (β-10). Chiral NOTA L20 gave nearly quantitative RCYs under all of the investigated conditions, displaying the best ⁶⁸Ga radiolabeling kinetics among the four chelators. In comparison, the RCYs for the 10- and 12- membered chelators are inferior at room temperature, reaching only >90% conversion at increased temperature. This conveys that fast kinetics is desired for radiolabeling, contrary to properties favoring good complex inertness. To gain deeper insight into the influences of ring sizes and chiral substituents on Ga³⁺ coordination stability, equilibrium studies, DFT calculations, and kinetic stability tests were conducted.

It is well known that the stability of a metal chelate is defined by both kinetic and thermodynamic stability. We first investigated the basicity of our chiral chelators and the thermodynamic stability of their Ga³⁺ complexes through potentiometric titrations (Table ). According to the overall protonation constants (∑ log K _i ^H), the basicity of these chiral macrocycles is similar to that of their parent achiral ligands. NMR titration results of L20 correlate well with its speciation diagram produced from potentiometric titration data, suggesting that the chiral substituents do not affect the protonation sequence of a macrocyclic ligand. In terms of Ga³⁺ stability constants (log K _GaL), a trend of L20 > L21 > L22 > NOTA > L2 > DOTA was observed, which is further supported by our DFT studies. Subsequent in vitro serum stability tests and kinetic studies under acid and alkaline conditions confirmed that the kinetic inertness of GaL2 and GaL20–GaL22 is favorable for PET imaging applications.

4. Results of Equilibrium Studies and DFT (Density Functional Theory) Calculations for L2 and L20–L22 .

	L20	L21	L22	L2	NOTA	DETA	DOTA
log K 1 H	11.16 ± 0.02	11.42 ± 0.19	10.55 ± 0.16	11.95 ± 0.21	12.00 ± 0.01	14.8 ± 0.18	11.14
log K 2 H	6.47 ± 0.16	7.57 ± 0.19	6.61 ± 0.01	9.53 ± 0.11	5.65 ± 0.02	6.12 ± 0.02	9.69
log K 3 H	3.49 ± 0.18	3.77 ± 0.20	4.12 ± 0.11	5.26 ± 0.08	3.19 ± 0.03	3.69 ± 0.02	4.84
log K 4 H	/	/	/	4.00 ± 0.07	/	/	3.95
∑ log K i H	21.12	22.76	21.28	30.74	20.84	24.61	29.62
log K GaL	31.20	31.02	30.45	27.26	29.60	/	26.05
ΔG°B of GaL (kcal mol–1)	–271.8	–272.0	–270.8	–229.4	–269.5	–268.4	–229.5
Calc. log K GaL	31.28	31.46	29.79	26.02	/	28.85	/

^aI = 0.1 M NMe₄Cl, 25 °C, where K _i ^H = [H_iL]/[H_i‑1L]­[H⁺]. The stability constant (log K _GaL) values are the averages of two sets of data from two independent measurements.

^bI = 0.1 M NMe₄Cl, 25 °C (from ref ).

^cFrom ref .

^dI = 0.1 M KCl, 25 °C (from ref ).

^eI = 0.1 M KCl, 25 °C (from ref ).

^fFrom ref .

Our studies yielded three conclusions: (1) Introducing chiral substituents onto the macrocyclic backbones of NOTA, DETA, and DOTA improved their Ga³⁺ coordination stability. (2) Introducing an S-methyl group at the α-position of DETA resulted in more ideal overall ligand basicity and Ga³⁺ chelating affinity than at the β-position. (3) Consistent with Hancock et al.’s theory, the 6-membered ring of chiral DETA can counteract the reduction in Ga³⁺ binding affinity caused by increased macrocyclic cavity size, rendering the stability constants of L21 and L22 close to that of L20 with a smaller macrocycle. As a proof-of-concept study, this work provides insights for the future design of next-generation chiral macrocycles with varied cavity sizes.

5. Summary and Outlook

We report a class of novel chiral macrocyclic chelators with different chiral substituents on their backbones as versatile biological probes. We employed three design strategies for developing our chiral ligands: (1) modification of the macrocyclic backbone, (2) modification of the pendant arms, and (3) alteration of the macrocyclic ring size. We started with the less common but most effective approach of macrocyclic backbone modification by introducing chiral groups into the macrocycle framework of DOTA. With enhanced rigidity on the macrocyclic ring, these chiral DOTA ligands prevent the interconversion between geometric isomers, thereby controlling the isomeric ratio or reducing the number of isomers in the resulting lanthanide complexes. The introduced chiral substituents resulted in improved coordination geometry, which endowed our chiral DOTA complexes with significantly higher stability compared to that of the achiral analogues. Proof-of-concept studies revealed their advantageous radiochemical, luminescence, and biocompatibility, highlighting their great potential for different biological applications. We then modified the pendant arm structures of these chiral DOTAs to create various bifunctional chelators (BFCs) for targeted diagnostic and therapeutic applications. Our recent study explored the effect of macrocyclic ring sizes by developing chiral analogues of the 9-membered NOTA and 10-membered DETA chelators for ⁶⁸Ga³⁺ radiolabeling. The observed radiolabeling efficacy and thermodynamic stability trends provide valuable information about the effect of macrocyclic cavity sizes and chiral modifications on the ligand coordination chemistry.

In addition to NOTA, DETA, and DOTA, there are still many differently sized macrocyclic chelators with limited studies, such as the 11-membered 1,4,8-triazacycloundecane-1,4,8-triacetic acid (UNTA) , and the 13-membered 1,4,7,10-tetraaza­cyclotridecane-1,4,7,10-tetraacetic acid (TRITA) ,− (Figure ). The lack of reports on these macrocycles arises from the difficult synthesis and purification and has confined the study of their metal coordination chemistry. Although DOTA and NOTA have been regarded as the benchmark or “gold standard” chelator, studies on the less-explored macrocycles should not be neglected. The fundamental design principal of macrocyclic chelators needs to be taken into consideration, such as ligand denticity, rigidity, coordination geometry, and their match with the size of metal ions. − Therefore, understanding the metal coordination chemistry in differently sized macrocyclic chelators is of immense importance to gain insight into the structure–property relationships. Our future work will focus on expanding our class of chiral macrocycles by developing other pendant arms as well as exploring other rings such as the 11- and 13-membered macrocycles. We are convinced that these fundamental studies on metal-size selectivity of macrocyclic chelation will pave the way for the future design of next-generation macrocyclic ligands which will serve as a tool to drive applications in metal sequestration and in advanced diagnostic applications.

15.

Current research gap of the 11- and 13-membered chiral macrocyclic chelators. ,−

Biographies

Yifan Zhu obtained his B.Eng. degree in pharmaceutical engineering from Changzhou University in China in 2022. He is currently a Ph.D. candidate in the Department of Chemistry, The Hong Kong Polytechnic University. His research focuses on the development of chiral macrocyclic chelators for molecular imaging.

Ga-Lai Law is currently a full professor at The Hong Kong Polytechnic University. Her current research interest focuses on lanthanide coordination chemistry in the development of new multifunctional compounds, including chiral supramolecular assemblies with unique topologies as well as chiral macrocyclic chelators for luminescence, diagnostic imaging, and theranostics.

The reported research was funded by The Hong Kong Polytechnic University (4BCC8), the Hong Kong Research Grants Council (PolyU253002/14P, PolyU153009/19P, PolyU153013/17P, 15300919, and 15301120) and also supported by and the National Natural Science Foundation of China (NSFC, 21875201).

The authors declare no competing financial interest.