New tripodal metal-chelating agents derived from nitrilotriacetic acid (NTA) and extended by three unnatural amino acids D-penicillamine (D-Pen) are presented. ID-Pen is actually the drug most extensively used to treat copper (Cu) overload in Wilson's disease and as such is a very attractive building block for the design of chelating agents. D-Pen is also a bulkier analogue of cysteine, with the beta-methylene hydrogen atoms replaced by larger methyl groups. The hindrance of the gem-dimethyl group close to the thiol functions is demonstrated to influence the speciation and stability of the metal complexes. The ligands L-4 (ester) and L-5 (amide) were obtained from NTA and commercial D-Pen synthons in four and five steps with overall yields of 14 and 24%, respectively. Their ability to bind Cu(I), thanks to their three thiolate functions, has been investigated using both spectroscopic and analytical methods. UV, CD, and NMR spectroscopy and mass spectrometry evidence the formation of two Cu(I) complexes with L-5: the mononuclear complex CuL5 and one cluster (Cu2L5)(2). In contrast, the bulkier ethyl ester derivative L-4 cannot accommodate the mononuclear complex in solution and thus forms exclusively the cluster (Cu2L4)(2). Cu K-edge X-ray absorption spectroscopy (XAS and EXAFS) confirms that Cu(I) is bound in trigonal-planar sulfur-only environments in all of these complexes with Cu- - -S distances ranging from 2.22 to 2.23 angstrom. Such C3-symmetric CuS3 cores are coordination modes frequently adopted in Cu(I) proteins such as metallothioneins. These two ligands bind Cu(I) tightly and selectively, which makes them promising chelators for intracellular copper detoxification in vivo.