Preorganized PSP Ligands Yield Monomeric Cu(I) Complexes with Sub-zeptomolar Cu(I) Dissociation Constants

Abstract

Unraveling the function of biological copper requires tools that can selectively recognize and manipulate this trace nutrient within the complex chemical environment of biological systems. Increasing evidence suggest that cells maintain an exchangeable pool of Cu(I) that is buffered in the high zepto- to low attomolar range. While mixed amine-thioether donors have been commonly employed for the design of Cu(I)-selective ligands and probes, their dissociation constants are limited to the pico- to femtomolar range. To address this challenge, we combined our previously devised phosphine sulfide stabilized phosphine donor motifs with a rigid 1,2-phenylene or 1,8-naphthylene ligand backbone. The resulting ligands, phenPS and naphPS, bind Cu(I) with a 1:1 complex stoichiometry and offer dissociation constants of 0.6 and 0.8 zM, respectively. Concluding from the crystal structures of the free and Cu(I)-bound ligands, the 1,2-phenylene-bridged ligand phenPS provides a high degree of structural preorganization to accommodate the Cu(I) center without large conformational changes, while the 1,8-naphthylene bridged ligand revealed significant out-of-plane distortions in both the free and Cu(I)-bound states. Both ligands were accessed by palladium-catalyzed cross-coupling reactions from the corresponding arylhalides under mild conditions, an approach that could be readily expanded towards design of other ligands and probes.

Graphical Abstract

To design synthetic ligands that can compete with zeptomolar Cu(I) dissociation constants encountered in biology, we rigidified phosphine sulfide-stabilized phosphine (PSP-ligands) with aromatic backbones for conformational preorganization. The resulting chelators, phenPS and naphPS, are accessible through Pd-catalyzed coupling and form discrete 1:1 Cu(I) complexes with dissociation constants down to 0.6 zM. The outlined approach can be further expanded toward ultra-high affinity Cu(I) chelators and probes with tailored properties.

Introduction

Copper is an essential trace element for most living organisms, where it plays a vital role in a variety of processes such as cellular respiration, defense against reactive oxygen species, or connective tissue maturation.¹ As copper is stored and transported primarily as Cu(I) within the reducing intracellular milieu,² ligands that selectively recognize and manipulate Cu(I) under biologically relevant conditions are critical to elucidate the molecular mechanisms of intracellular copper trafficking and regulation. While free aquacopper(I) ions would be susceptible to disproportion or oxidation by dioxygen,³ increasing evidence suggest that the exchangeable pool of cellular copper is stabilized by bioligands, which buffer cytosolic Cu(I) at attomolar levels or below.^4–6 While dissociation constants below 1 fM are challenging to realize with commonly employed synthetic Cu(I) chelators based on thioether donors,^7–9 we recently demonstrated that dissociation constants down to 10 zM are attainable with aliphatic phosphine-based ligands containing auxiliary phosphine sulfide moieties.¹⁰ The latter not only participate in Cu(I) coordination but also exert an electron withdrawing effect on the phosphine sites, thus stabilizing the free ligand against protonation and oxidation. These phosphine sulfide-stabilized phosphines, typified by PSP-1 and PSP-2 (Chart 1), maintain strong Cu(I) coordinating ability despite dramatically reduced basicity compared to typical aliphatic phosphines. With a 1:1 Cu(I) complex stability constant of logK 20.0 (K_d 10 zM), PSP-2 offers an unprecedented Cu(I) affinity among synthetic ligands and is unaffected by Mn(II), Fe(II), or Zn(II), even at millimolar concentrations.¹⁰ The Cu(I) complexes of both PSP-1 and PSP-2, however, engage in additional dimerization equilibria at high micromolar concentrations, hampering the utility of these ligands for applications where Cu(I) availability must be precisely controlled. We previously obtained an X-ray crystal structure of the dimeric PSP-1-Cu(I) complex, which revealed only two fused 5-membered chelate rings per Cu(I) center as opposed to the triple ring fusion that would be expected in a C₃-symmetric 1:1 Cu(I) complex.¹⁰ This suggests that relief of steric strain imposed by triple ring fusion is the driving force for dimerization. A similar strain effect has been noted for tris(2-pyridylmethyl)phosphine, which coordinates to Fe(II), Ru(II) and Cr(III) only through a tridentate N₂P donor set with the third pyridine moiety left uncoordinated.¹¹ With two phosphine moieties linked through an ethylene bridge, PSP-2 has the potential to form a 1:1 Cu(I) complex via a linearly connected S-P-P-S donor set, circumventing fusion of three 5-membered rings across a single bond. Nevertheless, we still observed formation of a dimeric [(PSP-2)₂Cu(I)₂] complex with a dimer-to-monomer dissociation constant of 200 μM, which is only 5-fold less favorable than the corresponding value for the PSP-1-Cu(I) system. We suspected that the ability of the P-CH₂-CH₂-P motif to adopt an antiperiplanar conformation permits an alternative dimerization mode where the fusion of adjacent 5-membered rings is avoided entirely. Molecular modeling studies suggested that such a structure is indeed feasible (Figure S1, Supporting Information). While attempts to obtain diffraction-quality crystals of the dimeric species were unsuccessful, crystallization of PSP-2-Cu(I) at millimolar concentrations from an aqueous nitrate solution yielded a 1:1 coordination polymer, which indeed exhibits an antiperiplanar conformation of the diphosphinoethane backbone (Figure S2, Supporting Information).

We therefore reasoned that replacing the flexible ethylene bridge of PSP-2 with a rigid 1,2-phenylene or 1,8-naphthylene unit might prevent oligomerization, yielding exclusively monomeric Cu(I) complexes. As a potential added benefit, the phosphorus lone pairs of the free ligand would be held in close proximity, and the resulting preorganization could provide a further increase in binding affinity relative to PSP-2. Conformational restriction in cis-1,2-diaminocyclohexane-N,N,N’,N’-tetraacetic acid by the cyclohexane backbone resulted, for example, in an 14-fold increase of the Zn(II) stability constant compared to EDTA.¹²

Experimental Section

Synthesis.

Synthetic procedures and analytical data for phenPS, naphPS, Cu(I) complexes, and all intermediates are provided in the Supporting Information.

X-ray Structure Determination.

Suitable crystals were selected, mounted on a loop with ParatoneN oil, and placed in a cooled nitrogen gas stream at 173 K on a Bruker D8 APEX II CCD sealed-tube diffractometer with graphite monochromated Mo-Kα (0.71073 Å) radiation. Data collection, indexing, and initial cell refinements were carried out using APEX II software.¹³ Frame integration and final cell refinements were done using the SAINT software.¹⁴ All structures were solved using direct methods and difference Fourier techniques (SHELXTL, V6.12).¹⁵ Hydrogen atoms were placed in their expected chemical positions and were included in the final cycles of least-squares with isotropic U_ij’s related to the atom’s ridden upon. All nonhydrogen atoms were refined anisotropically except for the disordered solvent molecules CH₃CN and CH₂Cl₂. Additional details of data collection and structure refinement are given in Tables S2–11 (Supporting Information). Supplementary crystallographic data can be obtained free of charge from the Cambridge Crystallographic Data Centre via http://www.ccdc.cam.ac.uk/data_request.cif.

Cu(I) Binding Stoichiometry.

BCA (2,2’-bicinchoninic acid, 150 μM from a 50 mM stock solution in 0.1 M aqueous KOH), Cu(II)SO₄ (50 μM from a 30 mM aqueous stock solution) and sodium ascorbate (150 μM from a 60 mM aqueous stock solution) were sequentially added under stirring to aqueous buffer (10 mM PIPES, 0.1 M KCl) in a 1 cm path length quartz cuvette. A UV-vis spectrum was recorded and the solution was titrated in 1–10 μL aliquots with a 3 mM stock solution of phenPS in DMF or naphPS in DMSO. For phenPS, the endpoint occurred at 1.04 molar equivalents on the basis of the mass of ligand employed for stock solution preparation, consistent with incomplete removal of the solvent of crystallization as observed by ¹H NMR. Adjusting the assumed stock concentration from 3.00 to 2.88 mM resulted in saturation at exactly 1 molar equivalent, which was confirmed by an independent molar ratio titration with [Cu(I)MCL-2]PF₆ (Figure S5, Supporting Information).

Molar Ratio Titration with [Cu(I)MCL-2]PF₆.¹⁶

In a quartz cuvette with 1 cm pathlength, a 50 μM aqueous solution of phenPS was prepared by diluting the ligand from a stock solution (3 mM in DMF) into 2.95 mL of aqueous buffer (10 mM PIPES, 0.1 M KCl, pH 7) with rapid stirring. After recording a UV-vis absorption spectrum from 240 to 500 nm, [Cu(I)MCL-2]PF₆ (3 mM in water) was added in 5 μM aliquots up to 95 μM. An absorption spectrum was recorded after a 1 min mixing period following each aliquot. To derive an accurate reference spectrum of the Cu(I)-MCL-2 complex, a separate titration was conducted using pure DMF in place of the phenPS stock solution. The exact concentration of the phenPS stock solution was calibrated against [Cu(I)MCL-2]PF₆ by plotting the apparent fractional saturation of the ligand, calculated as f = (A −A₀) /(A_sat – A₀), where A corresponds to the absorbance at 290 nm and A₀ and A_sat refer to the spectra at 0 and 50 μM Cu(I), respectively, versus nominal molar equivalents of Cu(I) for the first 10 data points (0–0.9 molar equivalents). After concentration correction and subtraction of the DMF background absorption, non-linear least-squares fitting of the titration data over the wavelength range 250–400 nm was performed using a fixed logβ₁₁ of 21.2 for Cu(I)-phenPS to yield a logβ₁₂ = 31.86 ± 0.07 (or logK₂ = 10.66 ± 0.07) for Cu(I)₂phenPS.

Stability Constants of Cu(I) Complexes.

An aqueous stock solution (60 mM) of sodium bathocuproine disulfonate (BCS, Acros Organics) was prepared and calibrated by competition titration against MCL-1 as previously described.¹⁶ BCS (1.50 mM), sodium ascorbate (150 μM) and CuSO₄ (30 μM) were sequentially added via aqueous stock solutions to 3 mL of buffer (10 mM PIPES, 0.1 M KCl, pH 7.0) in a 1-cm path length cuvette equipped with magnetic stirring, and a UV-Vis absorption spectrum was recorded from 650–380 nm. PhenPS was added in 5 μM aliquots from a 2.88 mM stock solution in DMF and an absorption spectrum was acquired after a 2 min equilibration period following each aliquot. The titration was conducted in duplicate, and each dataset was analyzed by nonlinear least-squares fitting over the spectral range from 650–400 nm using the Specfit software package.¹⁷ The stability constant for naphPS was determined similarly, except that the ligand was delivered from a 3.0 mM stock solution in DMSO, and a 5 minute equilibration period was required to obtain a stable spectrum after each aliquot. The phenPS stability constant was further verified by direct competition with MCL-1 as previously described for PSP-2 (Figure S9, Supporting Information).¹⁰

Electrochemistry.

Redox potentials of phenPS and naphPS and the copper complexes were determined by cyclic voltammetry in MeOH containing 0.1 M Bu₄NPF₆ using a CH-Instruments potentiostat (model 600A). All measurements were carried out under an atmosphere of argon in a single compartment cell with a glassy carbon working electrode, a Pt counter electrode, and a non-aqueous Ag/AgNO₃ reference electrode (10 mM AgNO₃ in CH₃CN). The half-wave potentials (E_1/2) were referenced to ferrocenium (0.41 V vs. SHE)¹⁸ as external standard. All measurements were performed with a scan rate of 100 mV/s.

Results and Discussion

Synthesis.

While PSP-2 was previously obtained by direct substitution of lithiated trimethylphosphine sulfide with 1,2-bis(dichlorophosphino)ethane,¹⁰ dichlorophosphines are cumbersome to handle due to their high reactivity with atmospheric moisture, and only a few are commercially available. We therefore explored the utility of palladium-catalyzed cross coupling as a more versatile approach to install the aryl-P bond in the target ligands (Scheme 1).¹⁹ Although the secondary phosphine intermediates employed in such couplings are often air-sensitive liquids, we suspected that the presence of two phosphine sulfide moieties might confer air-stability and crystallinity as observed for the tertiary phosphines PSP-1 and PSP-2. Secondary phosphine 3 was accessible via selective substitution of the chloride leaving groups of methyl dichlorophosphite with Me₂PSCH₂Li followed by reduction of the phosphinite intermediate 2 with DIBAL. Isolated as a colorless crystalline solid, phosphine 3 indeed showed no evidence of decomposition after months of storage in air at room temperature.

Scheme 1.

While coupling of secondary phosphines with 1,2-dibromoarenes sometimes yields only the monosubstitution products,²⁰ phosphine 3 underwent the desired double coupling with 1,2-dibromobenzene 4 under the conditions described by Miki and Buchwald¹⁹ to furnish phenPS 5 in 45% yield after 18 hours. Likewise, reaction of 1,8-dibromonapthalene 6 with 3 produced the peri-substituted ligand naphPS 7 in 33% yield. Formation of these bis-PSP ligands, which might be expected to sequester palladium as unreactive tetracoordinate complexes, did not appear to inhibit the catalytic activity of the Pd/dippf system. Instead, the moderate reaction yields are due primarily to reductive dehalogenation, which is especially pronounced during the second coupling step.

X-ray Structure Determination.

To explore the degree of conformational preorganization towards Cu(I)-complexation, we determined the crystal structures of the free ligands, phenPS and naphPS, as well as the corresponding Cu(I) complexes 8 and 9, respectively. Single crystals suitable for crystallographic characterization were obtained by diffusing MTBE into a saturated solution of each ligand in dichloromethane. The Cu(I) complexes, [Cu(I)phenPS]BF₄ (8) and [Cu(I)naphPS]BF₄ (9) were isolated from a mixture of dichloromethane-acetonitrile (1:1) containing equimolar amounts of [Cu(CH₃CN)₄]BF₄ and ligand. Pertinent geometrical parameters of the crystal structures are compiled in Tables 1 and 2, and additional crystallographic data are provided in the Supporting Information. Structural representations, including atom numbering schemes, are shown in Figures 1 and 2.

TABLE 1.

Selected bond distances (Å) and angles (deg) describing the ligand geometry in the crystal structures of phenPS•CH₂Cl₂ (5) and naphPS• 3CH₂Cl₂ (7).

Parameter	5	7
P(1)-C(1)	1.8568(5)	1.8616(12)
P(1)-C(4)	1.8544(6)	1.8614(13)
P(1)-C(7)	1.8442(5)	1.8514(12)
P(1)•••P(1)’	3.0703(5)	2.9552(6)
P(1)-C(7)-C(12)	116.715(16)	121.84(9)
P(1)-C(1)-P(2)	108.20(3)	114.68(6)
C(1)-P(2)-S(1)	111.686(19)	114.74(4)
P(1)-C(7)•••C(7)’-P(1)’	3.66(9)	21.76(9)

TABLE 2.

Selected bond distances (Å) and angles (deg) describing the Cu(I)-coordination environment in the crystal structures of [Cu(I)phenPS]BF₄•CH₃CN (8) and [Cu(I)naphPS] BF₄•CH₃CN (9).

Parameter	8	9
Cu(1)-P(1)	2.2870(7)	2.2324(9)
Cu(1)-P(4)	2.2669(7)	2.2672(9)
Cu(1)-S(1)	2.3220(7)	2.3475(9)
Cu(1)-S(3)	2.3042(7)	2.3219(9)
P(1)•••P(4)	3.2343(10)	3.3426(9)
P(1)-Cu(1)-P(4)	90.51(3)	95.95(3)
S(1)-Cu(1)-S(3)	121.98(3)	109.72(3)
P(1)-C(7)-C(12)	119.16(19)	125.2(2)
P(1)-C(1)-P(2)	112.60(14)	114.45(16)
C(1)-P(2)-S(1)	110.48(9)	112.35(11)
P(1)-C(7)•••C(16)-P(4)	10.8(3)	22.21(17)

Figure 1.

ORTEP drawing and atomic numbering scheme for the crystal structures of (A) [Cu(I)phenPS]BF₄ 8 and (B) phenPS 5. Ellipsoids shown represent 50% probability. Hydrogen atoms and counter ions have been omitted for clarity.

Figure 2.

ORTEP drawing and atomic numbering scheme for the crystal structures of (A) [Cu(I)naphPS]BF₄ (9) and (B) naphPS (7). Ellipsoids shown represent 50% probability. Hydrogen atoms and counter ions have been omitted for clarity.

The tetrafluoroborate salt of Cu(I)-phenPS crystallized as a discrete monomeric complex in the orthorhombic space group Pbca. The Cu(I) center adopts a distorted tetrahedral coordination geometry with near C₂-symmetry, where only two of the four phosphine sulfide donors are bound to Cu, each forming a 5-membered chelate ring (Figure 1A). The P1-C7-C16-P4 torsion angle of 10.8 deg indicates a small distortion of the ligand scaffold; however, the Cu(I) center remains well aligned with the mean squares plane defined by the benzene ring. The Cu-P bond distances (2.287 and 2.267 Å ) compare well with those observed in [Cu(I)PSP-1]₂ and [Cu(I)PSP-2]_∞ (2.272(1) and 2.268(2) Å, respectively), indicating similar P-Cu bond strengths in all three complexes. In contrast to [Cu(I)PSP-1], the Cu-S bond lengths in [Cu(I)phenPS] show only small variations with an average of 2.323 ± 0.13 Å. Thus, compared to PSP-1, the phenPS ligand scaffold appears to accommodate better the bonding requirements imposed by Cu(I), resulting in overall reduced steric strain.

The free ligand phenPS (5), which crystallized in the orthorhombic space group Pccn, also adopts a C₂-symmetric geometry similar to its Cu(I) complex (Figure 1B). Thus, phenPS exhibits a high degree of conformational preorganization, such that simple rotations along the P1-C7 and P1’-C7’ bond axis are sufficient for Cu(I) complexation. Upon Cu(I) coordination, the P^•••P non-bonding distance increases from 3.07 to 3.23 Å. While the copper center remains aligned with the least-squares plane of the benzene ring, the P1-C7^•••C7’-P1’ torsion angle slightly increases from 3.7 to 10.8 deg, indicating some buildup of steric strain upon Cu(I) coordination. Likewise, upon formation of the 5-membered chelate ring the P1-C7-C16 and P1-C1-P2 bond angles increase from 116.7 to 119.2 deg and from 108.2 to 112.6 deg, respectively. Nevertheless, the overall geometrical changes of the ligand framework upon Cu(I) complexation are modest, suggesting only a small increase in strain energy.

The perisubstituted naphthalene ligand naphPS (7) crystallized in the trigonal space group P₃221 and also revealed a twofold symmetry, which coincides with the crystallographic C₂-axis (Figure 2B). The steric strain imposed by the phosphino substituents results in a displacement of the two P atoms by 0.47 Å above and below the naphthalene least-squares plane to yield a P1-C7^•••C7’-P1’ torsion angle of 21.8°. The out-of-plane shift of the phosphino substituents is also associated with a significant distortion of the naphthalene ring, which is twisted into a non-planar conformation. Compared to unsubstituted naphthalene,²¹ the ring deformation is indicated by a widening of the C7–C8-C9 bond angle from 120 deg from to 122.23(16) deg and lengthening of the C7–C8 and C8–C9 bonds 1.416(1) to 1.4352(1) Å and 1.3737(2) to 1.3864(15) Å, respectively. The resulting P^•••P non-bonding distance of 2.955 Å corresponds to approximately 76% of the sum of van der Waals radii of the phosphorous atoms (3.90 Å),²² indicating a forced overlap of the two phosphorous lone pairs. Similar P^•••P non-bonding distances and out-of-plane distortions have previously been reported for structurally related 1,8-bis(dialkylphosphino)naphthalenes.^23–24

Analogous to phenPS, the Cu(I) complex of naphPS adopts a distorted tetrahedral coordination geometry with a C₂-symmetric arrangement of the phosphine sulfide donors. The complex crystallized in the triclinic space group P-1 containing a single monomeric complex per asymmetric unit (Figure 2A). While Cu(I) coordination might be expected to alleviate the steric strain imposed by forced overlap of the two phosphorous lone pairs, the ligand framework of the Cu(I) complex remains surprisingly distorted as indicated by the P1-C7^•••C16-P4 torsion angle of 22.2 deg.

Similar to the free ligand, the two phosphorous donors are displaced above and below the least-squares plane of the naphthalene ring by 0.525(4) and 0.409(4) Å, respectively. Significant steric strain is also apparent from a comparison of the P^•••P non-bonding distances, which increased from 2.9552(6) to 3.3426(9) Å upon Cu(I) complexation. Presumably due to insufficient space between the peri-substituents, the copper center is pushed significantly above the mean squares plane of the naphthalene ring by 0.515(6)Å to maximize bonding interactions with the P and S donors.

A superimposition of the Cu(I)-coordination spheres of [Cu(I)phenPS]BF₄ (8) and [Cu(I)naphPS]BF₄ (9) revealed a remarkable similarity between the two complexes (Figure 3). Notably, the angle between the intersecting planes of the coordination tetrahedron, defined by P1-Cu1-P4 and S1-Cu1-S3, are essentially identical, with 71.818(19) deg for 8 and 71.17(3) deg for 9, and the positions of the P and S donor pairs are almost congruent.

Figure 3.

Superimposition of the Cu(I) coordination environment of [Cu(I)phenPS]BF₄ (8) and [Cu(I)naphPS]BF₄ (9). The alignment is based on minimizing the rms error for the positions of Cu1 and the surrounding donor atoms S1, S3, P1, and P4.

The overlay further underscores the favorable conformational preorganization of the phenylene-bridged ligand phenPS, whose geometry is well matched to accommodate the Cu(I) center. By comparison, the peri-substituted naphthalene ligand naphPS remains strained after Cu(I) coordination with significant out-of-plane distortion of the naphthalene backbone.

Cu(I) Complex Stoichiometry in Solution.

To evaluate the Cu(I) binding stoichiometry of phenPS and naphPS in solution, we conducted molar ratio titrations in PIPES buffer (pH 7.0, 0.1 M KCl) using 2,2’-bicinchoninic acid (BCA) as a colorimetric indicator. BCA forms with Cu(I) a purple 2:1 complex (λ_max = 562 nm, ε = 7,900 M^–1cm^–1)²⁵ with a Cu(I) stability constant of logβ₂ = 17.7 at pH 7.0 (0.1 M KClO₄, 25°C).¹⁶ Titration of 150 μM BCA and 50 μM Cu(I), prepared by in situ reduction of Cu(II)SO₄ with sodium ascorbate (150 μM), with either phenPS or naphPS yielded a linear decrease of the absorbance at 562 nm, thus indicating quantitative removal of Cu(I) by both ligands (Figure S3 and S4, Supporting Information). Consistent with a 1:1 complex stoichiometry, the equivalence point was reached at an equimolar concentration of Cu(I) and added ligand.

Additional insights into the coordination mode were obtained by comparing the ¹H NMR spectrum of the free ligand and the corresponding complex. As shown in Figure 4, the two dimethylphosphorothioylmethyl arms produce distinct pairs of resonances in the spectra of both ligands, with two doublets arising from the methyl groups and a pair of doublets of doublets from the bridging methylene units. Due to the high inversion barrier of the phosphorous center, each pair of methylene protons H^a and H^b is rendered diastereotopic with different chemical shifts. In addition to the geminal ^aH-C-H^b coupling, both protons would also be expected to couple with the two neighboring P nuclei (I = 1/2) to produce two doublets of doublets of doublets (ddd). The observed coupling pattern is simpler, however, with only two doublets of doublets of equal splitting, presumably due to negligible coupling with the trivalent P nucleus.

Figure 4.

¹H NMR (500 MHz) chemical shifts and coupling patterns for the dimethylphosphorothioylmethyl substituents of (A) phenPS (5) and (B) naphPS (7) before (top) and after (bottom) addition of 1 molar equivalent of [Cu(CH₃CN)₄]BF₄ in DMSO-d₆. Due to line broadening, the spectra in the presence of Cu(I) were recorded at 80°C. Signals due to solvent water are labeled with a star.

This interpretation is corroborated by the ¹H NMR spectrum of PSP-1,¹⁰ which features three chemically identical methylene groups with pairs of enantiotopic protons that should yield a doublet of doublets; however, only a simple doublet was observed with J_PH = 11.5 Hz. Likewise, the two methyl groups attached to each phosphine sulfide moiety are rendered diastereotopic and couple with the neighboring P nuclei to produce two doublets (J_PH = 13.1 Hz) at distinct chemical shifts. Upon addition of an equimolar amount of Cu(I), supplied from a stock solution of [Cu(CH₃CN)₄]PF₆ in CD₃CN, all signals are shifted downfield relative to free ligands. While we observed some line broadening at room temperature, heating to 80°C resulted in sharp spectra with similar qualitative coupling patterns compared to those of the ligands alone. Contrary to the crystal structure, which shows a C₂-symmetric coordination geometry involving only two out of the four phosphine sulfide substituents, the ¹H NMR experiments suggest a rapid dynamic exchange relative to the NMR timescale where all four phosphine sulfide substituents interact with the Cu(I) center, presumably in a pairwise alternating fashion. Altogether, the NMR studies and competition titrations with BCA paint a uniform picture in which both ligands bind Cu(I) with a 1:1 complex stoichiometry.

Stability Constants of Cu(I) Complexes.

Consistent with a strong Cu(I)-ligand interaction, the UV-vis titration of phenPS with [Cu(I)MCL-2]PF₆ in PIPES buffer (10 mM, pH 7.0, 0.1 M KCl, 25°C) produced a linear increase of the absorption maximum up to a 1:1 Cu(I)-phenPS ratio (Figure 5A). The resulting spectrum is identical to that of isolated [Cu(I)phenPS]BF₄ used for x-ray structural determination (Figure S5B, Supporting Information), thus confirming formation of the same species. Further addition of Cu(I) resulted in small but distinct spectral changes, indicating coordination of a second molar equivalent of Cu(I). Nonlinear least-squares fitting of the UV-vis traces past equimolar Cu(I)-ligand ratios converged with a simple 1:1 binding model and yielded a logK₂ of 10.66 ± 0.07 (K_d of 22.3 ± 3 pM) for binding of a second Cu(I) cation. Although the absorption changes appear linear in this titration range (Figure 5A, inset), the calculated species distribution diagram indicates that a significant portion of Cu(I) remains bound to MCL-2 (Figure S6, Supporting Information). As such, the affinity of MCL-2 is well-matched for determining the logK₂ of phenPS through a ligand competition titration.

Figure 5.

Determination of the Cu(I) binding affinities of phenPS (5) in PIPES buffer (pH 7.0, 10 mM, 0.1 M KCl, 25°C). A) Molar ratio titration of phenPS (50 μM) with [Cu(I)MCL-2]PF₆. Inset: Experimental (solid circles) and calculated (red trace) absorbances at 286 nm and fraction of Cu(I) remaining unbound to phenPS (hollow circles) from nonlinear least-squares fitting of the Cu(I)phenPS/Cu(I)₂phenPS coordination equilibrium. B) Competition titration with phenPS (5 μM aliquots) with Cu(I) (30 μM, produced by in situ reduction with sodium ascorbate) in the presence of 1.5 mM BCS. C) Change of absorbance at 483 nm for the titration in plot B and nonlinear least-squares fit based on data from 400–650 nm.

To determine the much higher stability constant of the 1:1 phenPS-Cu(I) complex, we performed a ligand competition titration in the presence of a large excess of bathocuproine disulfonate (BCS). The concentration of BCS-bound Cu(I) can be assessed by UV-vis spectroscopy based on the characteristic orange color of the [(BCS)₂Cu(I)]^– complex (λ_max = 483 nm, ε = 13,300 M^–1cm^–1).²⁶ Because commercially available BCS is supplied as an unspecified hydrate and contains a mixture of three regioisomers bearing the sulfonate groups at the meta or para positions of the 4- and 7-aryl rings, we determined the stability constant for the batch at hand through competition titration with MCL-1 as previously described.¹⁶ The measured logβ₂ value of 20.93±0.05 (pH 7.0, 0.1 M KCl, 25°C) was sufficiently different from that of the original batch (logβ₂ = 20.81 at pH 7.0, 0.1 M KClO₄, 25°C)¹⁶ to justify its use in the following competition titrations. Spectrophotometric titration of 30 μM Cu(I) with phenPS in the presence of a large excess of BCS resulted in a gradual decrease of the absorption band centered at 483 nm (Figure 5B). Nonlinear least-squares fitting from 400–650 nm yielded a logK of 21.21 ± 0.09 at pH 7.0 (10 mM PIPES, 0.1 M KCl, 25°C), corresponding to a dissociation constant of K_d = 0.6 zM (Figure 5C). To confirm this extraordinary binding affinity, we titrated phenPS directly into a solution containing MCL-1 in 2000-fold excess over Cu(I),¹⁰ which yielded a logK of 21.23 ± 0.05 (Figure S9, Supporting Information). Consistent with a favorable conformational preorganization, the Cu(I) affinity of phenPS is over an order of magnitude higher compared to PSP-2, while the propensity of the 1:1 Cu(I) complex to coordinate a second Cu(I) is decreased by a similar margin. Moreover, principal component analysis of the titration data indicated a clean Cu(I) exchange equilibrium between BCS and phenPS and provided no evidence for formation of a dimeric 2:2 complex.

A molar ratio titration of naphPS with [Cu(I)MCL-2]PF₆ in PIPES buffer (10 mM, pH 7.0, 0.1 M KCl, 25°C) yielded much smaller changes in the absorption spectrum compared to phenPS (Figure S7, Supporting Information). Based on these data, it was not possible to discern whether naphPS can bind a second molar equivalent of Cu(I) as observed for PSP-2 and phenPS. Nevertheless, the 1:1 Cu(I) complex stability constant was readily determined by Cu(I) competition titration against BCS using the same conditions as described for phenPS above (Figure S10, Supporting Information). Although naphPS might be expected to provide an even higher Cu(I) binding affinity than phenPS due to relief of lone pair repulsion between the two phosphino substituents upon Cu(I) coordination, the observed affinity remains essentially unchanged at logK = 21.10 ± 0.04 (10 mM PIPES, pH 7.0, 0.1 M KCl, 25°C). Apparently, the Cu(I) complex of naphPS retains a significant amount of strain energy as previously indicated by the out-of-plane distortion of the naphthalene ring in both the free ligand and the Cu(I) complex (Figure 2).

Electrochemistry.

To evaluate the redox properties of the Cu(I/II) couple bound to phenPS and naphPS, we conducted cyclic voltammetry studies in methanol using tetrabutylammonium hexafluorophosphate as the electrolyte. The Cu(I) complex of phenPS, which was formed in situ by addition of [Cu(I)(CH₃CN)₄]BF₄, showed a reversible redox process with a half-wave potential of E_1/2 = 0.152 V vs. Fc⁺/Fc and peak separation of 75 mV (Figure 6A). By comparison, the Cu(I) complex of naphPS produced a cathodically shifted redox wave with a half-wave potential of E_1/2 = –0.063 V vs. Fc⁺/Fc and 94 mV peak separation (Figure 6B). The dramatic shift by more than 200 mV indicates an increased stabilization of the Cu(II) state, presumably due to the shorter coordinative bond lengths, which might better accommodate the geometry of the naphPS ligand backbone and alleviate some of the steric strain.

Figure 6.

Cyclic voltammograms for (A) phenPS (0.4 mM) and (B) naphPS (0.4 mM) in the presence of [Cu(I)(CH₃CN)₄]BF₄ (0.2 mM) in methanol at 25°C (0.1 M Bu₄NPF₆, glassy carbon electrode, 100 mV/s scan rate, Ag⁺/Ag reference electrode with Fc⁺/Fc as external reference).

In the absence of copper, the cyclic voltammograms revealed irreversible oxidation processes for both ligands, with anodic peak potentials of 0.50 V vs. Fc⁺/Fc for phenPS and 0.31 V vs. Fc⁺/Fc for naphPS (Figure S11, Supporting Information). The significantly lower oxidation potential of naphPS is presumably due to a stabilizing effect of the naphthalene backbone as well as reduction in steric strain upon formation of the monocation, in which the phosphino substituent in the peri-position can exert now a stabilizing rather than destabilizing influence.²⁷

Conclusions

By combining our previously devised phosphine sulfide stabilized phosphine (PSP) donors with a rigid ligand backbone, we were able to achieve Cu(I) dissociation constants as low as 0.6 zM, a more than 15-fold affinity boost compared to the parent compound PSP-2. Concluding from the x-ray crystallographic studies, the phenylene-bridged ligand phenPS exhibits a high degree of structural preorganization, such that only small conformational changes are required to adopt the geometry of the Cu(I) complex. Although naphPS, which features a strained peri-substituted naphthalene ligand backbone, was expected to yield an even higher affinity, its Cu(I) dissociation constant was essentially identical with that of phenPS. Presumably due to the insufficient space between the perisubstituents in naphPS, the ligand cannot accommodate Cu(I) without significant out-of-plane distortion of the naphthalene ring. Both ligands bind Cu(I) with a 1:1 complex stoichiometry without evidence for the formation of Cu₂L₂ dimers or coordination polymers as observed with PSP-2. While our previous syntheses of aliphatic PSP-ligands relied on strongly nucleophilic organolithium reagents to introduce the final P-C bonds, the palladium-catalyzed cross-coupling approach utilized for producing phenPS and naphPS is compatible with a much broader range of functional groups and could be readily adopted for accessing functionalized ligands and probes with tailored properties for detecting and manipulating Cu(I) within complex biological environments.

Chart 1.