Unprotonated Ligand Research Articles

Computational Study of Fluorinated Diglyoxime-Iron Complexes: Tuning the Electrocatalytic Pathways for Hydrogen Evolution.

The ability to tune the properties of hydrogen-evolving molecular electrocatalysts is important for developing alternative energy sources. Fluorinated diglyoxime-iron complexes have been shown to evolve hydrogen at moderate overpotentials. Herein two such complexes, [(dAr(F)gBF2)2Fe(py)2], denoted A, and [(dAr(F)g2H-BF2)Fe(py)2], denoted B [dAr(F)g = bis(pentafluorophenyl-glyoximato); py = pyridine], are investigated with density functional theory calculations. B differs from A in that one BF2 bridge is replaced by a proton bridge of the form O-H-O. According to the calculations, the catalytic pathway for A involves two consecutive reduction steps, followed by protonation of an Fe(0) species to generate the active Fe(II)-hydride species. B is found to proceed via two parallel pathways, where one pathway is similar to that for A, and the additional pathway arises from protonation of the O-H-O bridge, followed by spontaneous reduction to an Fe(0) intermediate and intramolecular proton transfer from the ligand to the metal center or protonation by external acid to form the same active Fe(II)-hydride species. Simulated cyclic voltammograms (CVs) based on these mechanisms are in qualitative agreement with experimental CVs. The two parallel pathways identified for B arise from an equilibrium between the protonated and unprotonated ligand and result in two catalytic peaks in the CVs. The calculations predict that the relative probabilities for the two pathways, and therefore the relative magnitudes of the catalytic peaks, could be tuned by altering the pK(a) of the acid or the substituents on the ligands of the electrocatalyst. The ability to control the catalytic pathways through acid strength or ligand substituents is critical for designing more effective catalysts for energy conversion processes.

Binding of H+ and Zn(ii) ions with a new fluorescent macrocyclic phenanthrolinophane

The new macrocyclic ligand 1,9(4,7)-diphenanthroline-3,7,11,15-tetraazacyclohexadecaphane (L) was synthesized by a 2 : 2 reaction of 1,10-phenanthroline-4,7-dialdehyde with 1,3-diaminopropane, followed by reduction with NaBH(4). L contains two phenanthroline groups linked together by two 1,3-diaminopropane chains in such a way that the heteroaromatic nitrogen atoms point outside the ligand cavity. The ligand structure defines two pairs of identical compartments displaying a specific ability in the binding of protons (1,3-diaminopropane) and metal ions (phenanthroline). Protonation and Zn(II) coordination were studied by means of potentiometric and spectroscopic ((1)H NMR, UV-vis, fluorescence) techniques. Both protonation and Zn(II) coordination consistently affect the fluorescence emission properties of L, giving rise to enhancement or quenching of the emission, depending on the species involved. L becomes emissive upon protonation, but the formation of the highly protonated species, in particular the fully protonated [H(6)L](6+), quenches the emission. The mono- and dinuclear Zn(II) complexes of the unprotonated ligand are non-emissive, like free L, while Zn(II) binding to [HL](+) activates the emission. The most interesting aspect, however, is the chelation enhancement of quenching (CHEQ) observed upon Zn(II) binding to [H(2)L](2+) and [H(4)L](4+), being among the few examples of CHEQ effect observed for Zn(II) complexes. Hydrogen bonding between a metal coordinated water molecule and a phenanthroline group seems to be responsible for the CHEQ observed for [ZnH(2)L](4+).

Impact of Ligand Protonation on Eigen-Type Metal Complexation Kinetics in Aqueous Systems

The impact of ligand protonation on metal speciation dynamics is quantitatively described. Starting from the usual situation for metal complex formation reactions in aqueous systems, i.e., exchange of water for the ligand in the inner coordination sphere as the rate-determining step (Eigen mechanism), expressions are derived for the lability of metal complexes with protonated and unprotonated ligand species being involved in formation of the precursor outer-sphere complex. A differentiated approach is developed whereby the contributions from all outer-sphere complexes are included in the rate of complex formation, to an extent weighted by their respective stabilities. The stability of the ion pair type outer-sphere complex is given particular attention, especially for the case of multidentate ligands containing several charged sites. It turns out that in such cases, the effective ligand charge can be considerably different from the formal charge. The lability of Cd(II) complexes with 1,2-diaminoethane-N,N'-diethanoic acid at a microelectrode is reasonably well predicted by the new approach.

β-[H2N(CH2)2NH2]0.5[ZnHPO3], a second modification of ethylenediamine zinc hydrogen phosphite

The title compound, poly[dizinc(II)-mu-ethylenediamine-di-mu-(hydrogen phosphito)], beta-[H(2)N(CH(2))(2)NH(2)](0.5)[ZnHPO(3)] or [Zn(2)(HPO(3))(2)(C(2)H(8)N(2))](n), is a hybrid organic/inorganic solid built up from ethylenediamine molecules (which lie about inversion centres), Zn(2+) cations (coordinated by three O atoms and one N atom) and HPO(3)(2-) hydrogen phosphite groups. The organic species bond to the Zn atom as unprotonated ligands, acting as bridges between infinite ZnHPO(3) layers that propagate as very buckled (001) sheets. The zincophosphite sheets contain polyhedral four- and eight-membered rings in a 4.8(2) topology. beta-[H(2)N(CH(2))(2)NH(2)](0.5).ZnHPO(3) complements the previously described alpha modification of the same stoichiometry [Rodgers & Harrison (2000). Chem. Commun. pp. 2385-2386].

Butane-1,4-diamine zinc(II) hydrogen phosphite

The title compound, poly[zinc(II)-mu-butane-1,4-diamine-mu-(hydrogen phosphito)] (C(4)H(12)N(2))(0.5)[ZnHPO(3)], is a hybrid organic-inorganic solid built up from 1,4-diaminobutane molecules, Zn(2+) cations (coordinated by three O atoms and one N atom) and HPO(3)(2-) hydrogen phosphite groups. The organic species bonds to the Zn atom as an unprotonated ligand, resulting in it acting as a bridge between infinite ZnHPO(3) layers, which propagate in (100). The complete butane-1,4-diamine species is generated from a H(2)N(CH(2))(2)- half molecule by inversion symmetry. The zincophosphite sheets contain polyhedral four- and eight-membered rings in a 4.8(2) topology.

A new complex of HgBr2 and pyrimidine-2-thione: (pyrimidine-2-thionato-kappaS)(pyrimidinium-2-thionato-kappaS)mercury(II) tetrabromomercury(II).

The title compound, [Hg(C(4)H(4)N(2)S)(C(4)H(3)N(2)S)](2)[HgBr(4)], consists of [Hg(pymt)(pymtH)](+) complex cations (pymtH is pyrimidine-2-thione) lying across twofold rotation axes in space group Fddd, with linearly coordinated mercury at an Hg-S distance of 2.357 (3) A, and [HgBr(4)](2-) anions lying at sites of 222 symmetry. The Hg atom is additionally coordinated by two N and two Br atoms, forming a 2+4 effective coordination sphere. The protonated ligand is connected via N-H.N hydrogen bonds to the neighbouring unprotonated ligand, thus forming infinite chains of cations.

Di-organocobalt complexes of macrocyclic ligands

Three new di-metallorganic cobalt complexes of the type trans-(Bz) 2Co(chel), where Bz is a benzyl group σ-bonded to cobalt atom and chel is an equatorial chelating system constituted by an amino-oximic ligand and its conjugated base, were synthesised. The protonated and the unprotonated ligands interact through an O–H ⋯ O bridge stabilising the entire structure. The complexes differ in the equatorial moiety which is derived from the following ligands: HLN-py=3-[(2-pyridyl)ethylimino]-butan-2-one oxime), HLN-Ph=3-[(2-phenyl)ethylimino]-butan-2-one oxime and the analogous HLN-PhCl=3-[(2-chlorophenyl)ethylimino]-butan-2-one oxime. Two of these compounds, namely those derived from HLN-py and HLN-PhCl were structurally characterised by means X-ray diffractometry. Data reveal that each complex is characterised by the presence of two unusually long cobalt–carbon bonds which are 2.120(4) Å (mean value) in complex with HLN-py ligand and 2.119(4) Å (mean value) in complex with HLN-PhCl. These data are consistent with a strong mutual trans-influence exerted by one ligand on the other.

Complexation Kinetics of N‐Alkyl‐Substituted Diamino Diamides with Nickel (II) in Aqueous Solution

AbstractEquilibrium constants are deter mined for the protonation and metal complexation of the nickel(II) complexes with 4‐methyl‐4,7‐diazadecanediamide (4‐Me‐L‐2,2,2), 4,7‐dimethyl‐4,7‐diazadecanediamide (4,7‐N,N′‐Me2‐L‐2,2,2), 4‐ethyl‐4,7‐diazadecanediamide (4‐Et‐L‐2,2,2), and 4‐methyl‐4,8‐ diazaundecane diamide (4‐Me‐L‐2,3,2), in 0.10 M KCl at 25.0°C. The formation kinetics of these nickel (II) complexes have been studied under the same conditions with use of the stopped‐flow technique. The possible path ways for the complexation reaction of nickel (II) with these ligands are discussed. The first metal‐nitrogen bond formation is proposed as the rate‐determining step for the reactions of nickel (II) with the unprotonated ligands; proton loss is the rate‐limiting step in the reactions of nickel (II) with the monoprotonated ligands. Similarly, in dissociation reactions of these nickel (II) complexes, the rate‐determining step for the water dissociation pathway is the break age of the second nickel‐nitrogen bond; the rate‐determining step for the proton‐assisted path way is the protonation of the released amino group. The important factors determining the reactivity of these complexes are considered. The kinetic results of the formation and dissociation reactions of these complexes are consistent with dissociative mechanism.

Kinetics and mechanism of copper(II) complex formation with tripodal aminopolythiaether and aminopolypyridyl ligands in aqueous solution.

The complex formation kinetics of aquated copper(II) ion reacting with 12 related tripodal ligands have been studied in aqueous solution at 25 degrees C, mu = 0.10 M (NaClO4). For most of the ligands studied, specific formation rate constants have been resolved for both the unprotonated and monoprotonated ligand species. All of the tripodal ligands included in this study contain a bridgehead amine nitrogen with the three legs consisting of 2-methylthioethyl or 2-ethylthioethyl and/or 2-pyridylethyl or 2-pyridylmethyl. Since the bridgehead nitrogen is too sterically hindered to participate in initial coordinate bond formation, the first bond must involve a thiaether sulfur or a pyridine nitrogen on one of the pendant legs followed by coordination to the bridgehead nitrogen to complete the first chelate ring. All kinetic data are interpreted in terms of this presumed sequence in the bond formation steps. For the two ligands in which all three pendant legs contain thiaether sulfur donor atoms, the rate-determining step appears to be at the point of second bond formation (chelate ring closure), although the distinction is not well defined. For all other unprotonated ligands, the kinetic behavior is consistent with the first-bond formation being rate-determining. Upon protonation, the rate-determining step appears to shift to the point of proton loss associated with second-bond formation in several cases. A particularly interesting observation is that the tripodal ligand tris(ethylthioethyl)amine (TEMEA) exhibits specific Cu(II) complex formation rate constants that are virtually identical to those for a closely related macrocyclic ligand, 1,4,8-trithia-11-azacyclotetradecane ([14]aneNS3), but the calculated CuIIL dissociation rate constants differ by a factor of 1000. A further comparison of the calculated dissociation rate constants for Cu(II)-tripodal ligand complexes indicates that a Cu(II)-N(pyridine) bond is approximately 10(4) times stronger than a Cu(II)-SR2 bond. This leads to the conclusion that a 1:1 Cu(II)-SR2 complex would have a predicted stability constant of about 0.04 M-1 in aqueous solution--the first estimate obtained for the strength of a single Cu(II)-S(thiaether) bond.

Kinetics and mechanism of the complexation reaction of copper(II) with diazadioximes

The protonation constants of four diazadioximes, 3,3,8,8-tetramethyl-4,7-diazadecane-2,9-dione dioxime (L 1), 3,3,9,9-tetramethyl-4,8-diazaundecane-2,10-dione dioxime (L 2), 3,3,6,6,9,9-hexamethyl-4,8-diazaundecane-2,10-dione dioxime (L 3), and 3,3,10,10-tetramethyl-4,9-diazadodecane-2,11-dione dioxime (L 4), have been determined potentiometrically in 0.10 mol dm −3 NaClO 4 at 25.0±0.1°C. The formation of their copper(II) complexes and the deprotonation reactions at the two oxime sites of these complexes have been investigated quantitatively by the potentiometric technique and by the measurement of their electronic spectra. The kinetics of complexation obtained by using the stopped-flow technique indicate that the first copper(II)–amino nitrogen bond formation between copper(II) and the unprotonated ligands is the rate-determining step and that the proton loss is the rate-limiting step in the reaction of copper(II) with the monoprotonated ligand. The thermodynamic, spectroscopic, and kinetic data are discussed in relation to a combination of steric, inductive, and ring-strain effects.

Ligands with radical character for high oxidation states in manganese and iron complexes

Hybrid density functional calculations have been performed to study the ligand radical character for highly oxidized iron and manganese complexes. This investigation was undertaken because amino acid radicals are found to play key roles in the mechanisms for substrate conversion in many proteins that contain iron or manganese complexes. Large basis sets were used and dielectric cavity methods were employed to estimate the effects of the protein surrounding. Two enzymes are of particular interest in this study. For ribonucleotide reductase the possibility that the tryptophane Trp48 obtains radical character in the course of the radical transfer between Tyr122 and Cys439 was studied. Possibilities of modelling this effect by a directly coordinated unprotonated histidine or tyrosine ligand were investigated. For photosystem II, the main interest is in the possibility that a histidine ligand might affect the stability of oxygen radical states, which could be important for OO bond formation.

Metal complexes of propane-1,2,3-triamine. Potentiometric and calorimetric investigations

The complexes formed by the ions Co2+, Ni2+, Cu2+, and Zn2+ with the ligand propane-1,2,3-triamine (L1) in 0.15 mol dm−3 aqueous NaCl at 25 °C have been studied by potentiometry and calorimetry (ligand to metal molar ratio, 1–3; pH range from 3.0 to 6.5–11.0). Stability constants have been obtained from potentiometric data by means of the least-squares program HYPERQUAD for the following species: [M(L1)]2+ and [MH(L1)]3+ for all four metal cations; [M(L1)2]2+ and [MH(L1)2]3+ for Ni2+, Cu2+, and Zn2+; [MH2(L1)2]4+ for Ni2+ and Cu2+; and [M(OH)(L1)]+ for Cu2+ only. Stabilities increase in the order Zn < Co < Ni < Cu. The complexes of L1 have approximately the same stability as the corresponding ones formed by the related triamines 2-methyl (2-methylamine) propane-1,3-diamine (L3) and 2-ethyl (2-methylamine)propane-1,3-diamine (L4). The stability constants have been used to obtain, from the calorimetric titration experiments, the formation enthalpies of all the metal complexes except those for cobalt(II) as well as the corresponding entropy changes. The thermodynamic quantities are compared with the corresponding values previously found for L3, in order to derive information about the coordination ability of the ligand. The analysis suggests that the unprotonated ligand forms a fac-tridentate complex in most cases, but for some species, such as [Cu(L1)2]2+, [CuH(L1)2]3+, and [Zn(L1)]2+, the thermodynamic quantities indicate that at least one of the nitrogen atoms of the triamine is either loosely bonded or not coordinated to the metal ion. A gas-phase molecular mechanics analysis shows that the strain energies of the fac-[Co(L1)2]2+ and -[Ni(L1)2]2+ symmetrical (s) and unsymmetrical (u) isomers are very similar. If the same solvation energy is assumed for the two isomers, each isomer will be present in roughly equal amounts in solution. Keywords: propane-1,2,3-triamine, potentiometry, calorimetry, complexation, cobalt, nickel, copper, zinc.

Ionization state and pKa of pterin-analogue ligands bound to dihydrofolate reductase.

The ionization state and pKa of the inhibitor 6,8-dimethyl-N5-deazapterin bound to the recombinant human dihydrofolate reductase (rhH2folate reductase) complex with NADPH was determined by a spectrofluorimetric method. The excitation spectra for bound ligand as a function of pH from 6.1 to 9.7 indicated it was the same cationic form as for unbound ligand, which is protonated on N3. However, the lower limit for the pKa of the bound form was determined to be 9.1, a value about pH 2.5 higher than that for free ligand, indicating that ligand bound to the enzyme is protonated at neutral pH. The excitation spectra for bound ligand as a function of pH were generated by computer simulation by employing corrections for the pH dependence of the proportion of bound ligand (variable Kd; ligand-dissociation constant) and taking account of the different pKa values for bound and unbound forms. A plot of Kd values against pH showed a bell-shaped curve indicating that 6,8-dimethyl-N5-deazapterin bound to rhH2folate reductase.NADPH to form a ternary complex of ionised enzyme with protonated ligand and/or protonated enzyme with unprotonated ligand; the spectrofluorimetric results are consistent with the first alternative.

Binary and ternary complexes of nickel(II) with 2-aminomethylbenzimidazole and salicylaldehyde: kinetic and equilibrium studies

The complexation of NiII with 2-aminomethylbenzimidazole (L) has been investigated at 20–40 °C, I= 0.30 mol dm–3. Both monoprotonated and unprotonated ligands bind the metal ion to form [NiL]2+. and the rate and activation parameters for the formation and acid-catalysed dissociation of this chelate are calculated. In the presence of Salicylaldehyde (Hsal), a mixed-ligand complex. [NiL(sal)]+, is also formed as an intermediate which further condenses to the Schiff-base complex, [NiL′]+[HL′=N-(benzimidazol-2-ylmethyl)salicylideneimine]. The kinetics of the fast ternary complex formation and its slow intramolecular transformation to the Schifi base complex have been investigated at 25 °C. The presence of L in the co-ordination sphere of Ni2+ enhances the dissociation of [NiL(sal)]+ to [NiL]2+ and sal– with respect to [Ni(Sal)]+, as evidenced by the stability constants of [NiL(sal)]+ and [Ni(sal)]+. Calculations based on the values of ΔS° for the ionisation of H2L2+ and the formation of [NiL]2+combined with Saq°(H+) and Saq°(Ni2+) data yielded the values Saq°(H2L2+)–Saq°(L)= 110, Saq°(L)–Saq°(HL+)=–116 and Saq°([NiL]2+)–Saq°(L)=–294 J K–1 mol–1, which presumably reflect the varying solvent-ordering effects of L, HL+, H2L2+ and [NiL]2+.

Voltammetric and spectroscopic study of 2-mercapto-3-pyridinol and its complexes with iron in dimethylsulphoxide

The redox behaviour of the ligand 2-mercapto-3-pyridinol (PSHOH) and its complexes with iron have been studied in dimethylsulphoxide. Depending on the metal-to-ligand mole ratio and the amount of base in the system, iron(II) forms three complexes which are dark-green [Fe II(PSHO) 2], dark-blue [Fe II(PSO) 2(PSHO)] 3− and red-brick [Fe II(PSO) 3] 4−, with stability constants of 2.5 × 10 7, 4.0 × 1O 9 and 5.0 × 10 8, respectively. The complexes have been characterized by cyclic voltammetry, controlled-potential electrolysis, optical spectroscopy and magnetic susceptibility measurements. The interaction of Fe III with the totally unprotonated ligand produces Fe II and the corresponding dimer, without the formation of a stable complex between the thiol radical and the metal ion.

Copper(II) complexation by 6-(alkylamino)methyl-2-hydroxymethylpyridines with varying alkyl chain length in aqueous solutions. Kinetics and thermodynamics

Kinetic parameters have been obtained from stopped-flow experiments for the complexation of copper(II) by a series of 6-(alkylamino)methyl-2-hydroxymethylpyridines with alkyl chains varying from C1 to C14, in aqueous solutions (T= 25 °C, pH 2.0–5.0). The apparent rate constants for complex formation at pH 3.5 were found to be weakly dependent on the alkyl chain length. A kinetic model has been proposed which is in excellent agreement with the experimental data, especially the change of the reciprocal relaxation time (or observed rate constant) with pH. Even in a fairly acidic pH range, the contribution of the unprotonated ligand species cannot be neglected. Equilibrium measurements were carried out in parallel using UV–visible spectrophotometry and potentiometric titrations in order to determine the values of thermodynamic parameters such as ionization and stability constants.

Effects of ring size on thermodynamic and kinetic properties of copper(II)-diaminodiamide complexes in aqueous solution

In order to study the effects of ring size on the thermodynamic and kinetic properties of copper(II)–diaminodiamide complexes, a new quadridentate ligand, N,N′- bis(carbamoylmethyl)-trimethylenediamine (L131), was synthesized. The log K values for the protonation constants of the ligand at 25.0 °C and 0.1 mol dm–3 NaClO4 are 8.22 and 6.33. The log K value for the formation constant of [Cu(L131)]2+ is 13.68, and the log K values for the deprotonation constants of [Cu(L131)]2+ and [Cu(H–1L131)]+ are –6.94 and –8.25, respectively. The complexation kinetics of copper(II) with this ligand has been studied under the same conditions using the stopped-flow technique. The resolved formation rate constants (in 0.10 mol dm–3 NaClO4 at 25.0 °C) for copper(II) reacting with the unprotonated and monoprotonated ligand, L131 and [HL131]+, are 1.88 × 109 and 1.79 × 106 dm3 mol–1 s–1 respectively. Two possible pathways for the complexation reaction are discussed.

Thermodynamic and kinetic studies on the complexation reactions of copper(II) with NN′-bis(β-carbamoylethyl)-trans-cyclohexane-1,2-diamine

The interactions of NN′-bis(β-carbamoylethyl)-trans-cyclohexane-1,2-diamine (L) and copper(II) in aqueous solution have been investigated by potentiometric and spectrophotometric techniques. The log K values for the protonation constants of the ligand at 25.0 °C and 0.1 mol dm–3 NaClO4 are 9.34 and 4.49. The log K value for the formation constant of [Cu(L)]2+ is 12.92, and the log K values for the deprotonation constants of [Cu(L)]2+ and [Cu(H–1L)]+ are –7.72 and –9.02 respectively. Electronic spectra of [Cu(L)]2+, [Cu(H–1L)]+, and [Cu(H–2L)] have been recorded and are discussed. The complexation kinetics of copper(II) with this ligand have been studied under the conditions using the stopped-flow technique. The resolved formation rate constants (in 0.10 mol dm–3 NaClO4 at 25.0 °C) for copper(II) reacting with the unprotonated and monoprotonated ligand, L. and HL+, are 4.83 × 107 and 4.10 × 102 dm3 mol–1 s–1 respectively. Two possible pathways for the complexation reaction are discussed.

The palladium(II) promoted hydrolysis of the methyl esters of glycyl-L-leucine, glycyl-L-alanine and L-alanylglycine

The palladium(II)-promoted hydrolysis of the methyl esters of glycyl-L-leucine, glycyl-L-alanine and L-alanylglycine have been studied at 25 °C and I=0.1 M in the pH range 4–5. At a 1:1 metal to ligand ratio the peptide esters act as tridentate ligands, donation occurring via the terminal amino group, the deprotonated amide nitrogen, and the carbonyl group of the ester. Due to the high Lewis acidity of Pd(II) rapid hydrolysis of the ester function by water and hydroxide ion occurs. Rate constants k OH and k H 2O have been obtained for base hydrolysis and water hydrolysis of the coordinated peptide esters at 25 °C. The rate constants for base hydrolysis are 3.4 X 10 6 M −1 s −1 (L-alaglyOMe), 6.4 X 10 6 M −1 s −1 (gly-L-alaOMe) and 2.3 X 10 7 M −1 s −1 (gly-L-leuOMe). Base hydrolysis of the coordinated peptide esters is at least 10 6 times that of the free unprotonated ligand. Activation parameters have been obtained for both water and base hydrolysis of the Pd(II) complex of methyl L-alanylglycinate and possible mechanisms for the hydrolyses are considered.

The pH-dependence of the binding of dihydrofolate and substrate analogues to dihydrofolate reductase from Escherichia coli

The interaction of dihydrofolate reductase (EC 1.5.1.3) from Escherichia coli with dihydrofolate and folate analogues has been studied by means of binding and spectroscopic experiments. The aim of the investigation was to determine the number and identity of the binary complexes that can form, as well as p K a values for groups on the ligand and enzyme that are involved with complex formation. The results obtained by ultraviolet difference spectroscopy indicate that, when bound to the enzyme, methotrexate and 2,4-diamino-6,7-dimethylpteridine exist in their protonated forms and exhibit p K a values for their N-1 nitrogens of above 10.0. These values are about five pH units higher than those for the compounds in free solution. The binding data suggest that both folate analogues interact with the enzyme to yield a protonated complex which may be formed by reaction of ionized enzyme with protonated ligand and/or protonated enzyme with unprotonated ligand. The protonated complex formed with 2,4-diamino-6,7-dimethylpteridine can undergo further protonation to form a protonated enzyme-protonated ligand complex, while that formed with methotrexate can ionize to give an unprotonated complex. A group on the enzyme with a p K a value of about 6.3 is involved with the interactions. However, the ionization state of this group has little effect on the binding of dihydrofolate to the enzyme. For the formation of an enzyme-dihydrofolate complex it is essential that the N-3/C-4 amide of the pteridine ring of the substrate be in its neutral form. It appears that dihydrofolate is not protonated in the binary complex.