Carbon protonation of 2,4,6-triaminopyrimidines: synthesis, NMR studies, and theoretical calculations

Several C5-substituted 2,4,6-triaminopyrimidine derivatives and their HBF4 salts were synthesized to study the carbon protonation of the pyrimidine ring. NMR investigations in DMSO-d6 prove experimentally that, in addition to the usual protonation at N1, the compounds can be protonated at C5 as well. We present several new stable cationic sigma-complexes in the pyrimidine series, where C5 protonation predominates over N1 protonation. Quantum chemical calculations using the B3LYP/cc-pVDZ method were utilized in the gas phase and also in DMSO solvent with the polarized continuum model (PCM) method to rationalize the observed protonation behavior. Results of the calculations accord with the experimental observations and prove that combined steric and electronic effects are responsible for the observed C5 protonation and for sigma-complex stability. We demonstrate that C5 protonation is a general feature of the 2,4,6-triaminopyrimidine system.     

Triethylenetetramine penta- and hexa-acetamide ligands and their ytterbium complexes as paraCEST contrast agents for MRI

The ligand triethylenetetramine-N,N,N',N',N',N'-hexaacetamide (ttham) was synthesized with the aim of forming lanthanide complexes suitable as contrast agents for magnetic resonance imaging applications utilizing the chemical exchange-dependent saturation transfer (CEST) effect. It was designed to exclude water molecules from the first coordination sphere and provide a high number of CEST active amide protons per lanthanide ion. The ligand was characterized by its protonation behavior and its complexation properties with ytterbium ions in aqueous solution. The basicity of the ttham backbone amine protons decreases in the order N(central(1)) > N(terminal(1)) > N(terminal(2)) > N(central(2)), as deduced from NMR titration experiments and from a comparison of its protonation constants with those of two ttham derivatives, in which either a terminal (N-benzyl-triethylenetetramine-N,N',N',N',N'-pentaacetamide, 1bttpam) or a central acetamide group (N'-benzyl-triethylenetetramine-N,N,N',N',N'-pentaacetamide, 4bttpam) is substituted with a benzyl group. This protonation sequence results from the combined influence of inductive effects, the intramolecular hydrogen bonding network, and the Coulomb repulsion between protonated ammonium groups. The ytterbium complex of ttham, [Yb(ttham)]Cl(3), is coordinatively frustrated. Due to steric constraints, in addition to the four backbone nitrogen atoms, only three of the four symmetry-equivalent terminal acetamide donors can coordinate simultaneously to the ytterbium ion, and the dangling fourth one exchanges quickly with the other three. The ytterbium complexes of a total of five ligands (ttham, 1bttpam, 4bttpam, 2,2',2'-triaminotriethylaminehexaacetamide (ttaham), and diethylenetriamine-N,N,N',N',N'-pentaacetamide (dtpam)) were studied with respect to their CEST properties. In solution, all of these complexes have a low symmetry. The presence of multiple magnetically different amide groups in each complex prevents the realization of very high CEST effects. These results nevertheless form an excellent basis for a further optimization of this class of ligands.     

Probing Protonation Sites of Isolated Flavins Using IR Spectroscopy: From Lumichrome to the Cofactor Flavin Mononucleotide

Infrared spectra of the isolated protonated flavin molecules lumichrome, lumiflavin, riboflavin (vitamin B₂), and the biologically important cofactor flavin mononucleotide are measured in the fingerprint region (600–1850 cm^?1) by means of IR multiple‐photon dissociation (IRMPD) spectroscopy. Using density functional theory calculations, the geometries, relative energies, and linear IR absorption spectra of several low‐energy isomers are calculated. Comparison of the calculated IR spectra with the measured IRMPD spectra reveals that the N10 substituent on the isoalloxazine ring influences the protonation site of the flavin. Lumichrome, with a hydrogen substituent, is only stable as the N1‐protonated tautomer and protonates at N5 of the pyrazine ring. The presence of the ribityl unit in riboflavin leads to protonation at N1 of the pyrimidinedione moiety, and methyl substitution in lumiflavin stabilizes the tautomer that is protonated at O2. In contrast, flavin mononucleotide exists as both the O2‐ and N1‐protonated tautomers. The frequencies and relative intensities of the two C?O stretch vibrations in protonated flavins serve as reliable indicators for their protonation site.     

强酸性环境中氧氟沙星的质子化模型研究

利用核磁共振波谱技术研究了不同浓度硫酸溶液中氧氟沙星(OFL)的1H,19F和13C核磁共振谱,对不同硫酸浓度引起的δH,δF,δC和JFC耦合常数的变化进行比较分析,由此推测其结构状态.综合1H,19F和13C核磁共振谱特点及其变化,提出OFL分子在强酸性环境中N1'被进一步质子化的结构模型.在浓硫酸溶液中,N1'被进一步质子化,并与F9形成氢键(N1'—H+┈F9),该结构使分子的共轭程度大幅降低,导致其荧光发射波长、荧光激发波长及紫外吸收波长均发生蓝移.硫酸溶液中氧氟酸和甲基氧氟沙星的荧光光谱行为进一步证明了OFL分子在浓硫酸溶液中质子化模型的合理性.     

Protonation of Nitramines: Where Does the Proton Go?

The reactions of nitramine, N ‐methyl nitramine, and N ,N ‐dimethyl nitramine with anhydrous HF and the superacids HF/MF₅ (M=As, Sb) were investigated at temperatures below −40 °C. In solution, exclusive O‐protonation was observed by multinuclear NMR spectroscopy. Whereas no solid product could be isolated from the neat HF solutions even at −78 °C, in the HF/MF₅ systems, protonated nitramine MF₆⁻ salts were isolated for the first time as moisture‐sensitive solids that decompose at temperatures above −40 °C. In the solid state, depending on the counterion, O‐protonated or N‐protonated cations can be formed, in accord with theoretical calculations which show that the energy differences between O‐protonation and N‐protonation are very small. The salts [H₂N‐NO₂H][AsF₆], [H₃N‐NO₂][SbF₆], [MeHNNO₂H][SbF₆], and [Me₂NNO₂H][SbF₆] were characterized by their X‐ray crystal structures.     

Study of the protonation (methylation) position and tautomeric structure of thiopyrimidine derivatives by 2D <Superscript>1</Superscript>H—<Superscript>15</Superscript>H NMR HSQC/HMBC. Experimental approach and theoretical modeling

Two-dimensional ₁H—₁₅N NMR HSQC/HMBC experiments enable the unambiguous determination of the protonation (methylation) position and tautomeric structure of nitrogen-containing heterocycles. In investigated thiopyrimidines protonation (or methylation) occurs at the N(1) atom of the pyrimidine ring. The tautomeric structures of these compounds were established based on the analysis of ₁H—₁₅N NMR spectra. Ab initio calculations of chemical shifts (GIAO B3LYP/6-31G(d)//HF/6-31G) are in full agreement with experimental values. The stability of various protonated (methylated) and tautomeric species is explained in terms of a thermodynamic approach.     

Encapsulation of halides within the cavity of a pentafluorophenyl-substituted tripodal amine receptor

Pentafluorophenyl-substituted tripodal amine L, tris[[(2,3,4,5,6-pentafluorobenzyl)amino]ethyl]amine, is becoming a potential receptor for encapsulation of Cl- and Br- within the pseudo-C3-symmetric tris(2-aminoethyl)amine (L1) cavity upon protonation of the secondary amines. 1H NMR titration results indicate that [H3L]3+ binds with Cl- and Br- strongly compared to the [H3L2]3+ receptor, where L2 is N,N',N' '-tris[(2-benzylamino)ethyl]amine.     

15N and 13C NMR study of protonated monoaminopyridines in CDCl3-DMSO

The 2-, 3- and 4-amino-pyridine and their protonated forms, obtained by reaction with pyridinium chloride, were investigated by 15N NMR spectroscopy. Exhaustive evidence has been found that the protonation occurs mainly on the annular nitrogen. Protonation of 4-aminopyridine by dehydrohalogenation of 1,1,2,2-tetrachloroethane (TCE) was also studied by 13C NMR spectroscopy, which indicated that the protonation occurs via the formation of adducts.     

Protonation of trimipramine salts of maleate, mesylate and hydrochloride observed by 1H, 13C and 15N NMR spectroscopy

Protonation of the tricyclic antidepressant drug trimipramine with maleic acid, methanesulfonic acid and hydrochloric acid was studied using 1H, 13C and 15N NMR spectroscopy at natural abundance. The effect of counter ions on the protonation was compared under identical conditions of solvent, concentration and temperature using homonuclear and heteronuclear one- and two-dimensional experiments. Differential protonation of the terminal tertiary amine nitrogen is determined from the indirect spin-spin couplings, chemical shifts, 13C relaxation data and variable-temperature experiments. In the maleate salt, only one of the acidic protons is involved in protonation, the other being associated with the anion moiety. 15N chemical shifts of the protonated nitrogens are nearly linearly related to the pK(a) of the constituent acid.     

Protolytic properties of polyamine wasp toxin analogues studied by 13C NMR spectroscopy

Acid-base properties of the natural polyamine wasp toxin PhTX-433 (1) and seven synthetic analogues [PhTX-343 (2), PhTX-334 (3), PhTX-443 (4), PhTX-434 (5), PhTX-344 (6), PhTX-444 (7), and PhTX-333 (8)], each having four protolytic sites, were characterized by 13C NMR spectroscopy. Nonlinear, multiparameter, simultaneous fit of all chemical shift data obtained from the NMR titration curves yielded macroscopic pKa values as well as intrinsic chemical shift data of all differently protonated macrospecies. Analyses of the chemical shift data demonstrated strong interactions between all four sites and provided information about complex relationships between chemical shift values and protonation state. Deprotonation of fully protonated forms starts at the central amino group of the polyamine moiety, and the extent of this trend depends on the distance to the flanking, protonated amino groups. The pKa1 values of 1-8 are in the range 8.2-9.4. Hence, some of the toxins are incompletely protonated at the pH and ionic strength conditions used for assessment of their interactions with ionotropic glutamate and nicotinic acetylcholine receptors, and the degree of protonation is expected to have pharmacological importance in the ion-channel binding event.     

1H NMR direct observation of enantiomeric exchange in palladium(II) and platinum(II) complexes containing N,N' bidentate aryl-pyridin-2-ylmethyl-amine ligands

The complexes [MCl(2)(kappa2-N approximately N')] (N approximately N' = 2-C(5)H(4)N-CH2-NHAr; Ar = 4-MeC(6)H(4), a; 2,6-Me(2)C(6)H(3), b; 4-MeOC(6)H(4), c; 4-CF(3)C(6)H(4), d; M = Pd, 1a-d; Pt, 2a-d) have been prepared and fully stereochemically characterized both in the solid state and in solution. Their behavior in DMSO-d6 solution is dependent on the substituents of the aryl group and on the metal. Complexes of palladium with substituents at the para position (1a, 1c, 1d) display a dynamic 1H NMR pattern when the solutions are heated. An enantiomeric exchange Slambda/Rdelta is suggested to explain such behavior. On the basis of the calculated negative DeltaS values, an associative mechanism involving the solvent is proposed. Under the same conditions, analogous complexes of platinum (2a, 2c, 2d) proved to be unstable, and release of the N approximately N' ligand was observed. Complexes 1b and 2b show temperature-variable 1H NMR spectra without any evidence accounting for enantiomeric exchange or decoordination. DFT calculations on models of 1a and 1b show that diastereomeric exchange Sdelta/Slambda is a process where the complex with the higher sterical hindrance, 1b, has a lower energy barrier.     

Protonation studies of modified adenine and adenine nucleotides by theoretical calculations and (15)N NMR

The acid/base character of nucleobases affects phenomena such as self-association, interaction with metal ions, molecular recognition by proteins, and nucleic acid base-pairing. Therefore, the investigation of proton-transfer equilibria of natural and synthetic nucleos(t)ides is of great importance to obtain a deeper understanding of these phenomena. For this purpose, a set of ATP prototypes was investigated using (15)N NMR spectroscopy, and the corresponding adenine bases were investigated by theoretical calculations. (15)N NMR measurements provided not only acidity constants but also information on the protonation site(s) on the adenine ring and regarding the ratio of the singly protonated species in equilibrium. Substituents of different nature and position on the adenine ring did not change the preferred protonation site, which remained N1. However, for 2-thioether-ATP derivatives a mixed population of N1 and N7 singly protonated species was observed. Reduction of basicity of 0.4-1 pK(a) units relative to ATP was also observed for all evaluated ATP derivatives, except for 2-Cl-ATP, for which K(a) was ca. 10,000-fold lower. To explain the substitution-dependent variations in the experimental pK(a) values of the ATP analogues, gas-phase proton affinities (PA), Delta Delta G(hyd), and pK(a) values of the corresponding adenine bases were calculated using quantum mechanical methods. The computed PA and Delta Delta G(hyd) values successfully explained the experimental pK(a) values. A computational procedure for the prediction of accurate pK(a) values was developed using density functional theory and polarizable continuum model calculations. In this procedure, we developed a set of parameters for the polarizable continuum model that was fitted to reproduce experimental pK(a) values of nitrogen heterocycles. This method is proposed for the prediction of pK(a) values and protonation site(s) of purine analogues that have not been synthesized or analyzed.     

Allenediazonium ions and their protonation chemistry: a DFT study

The parent allenediazonium monocation H2C[double bond]C[double bond]CH(N2+) and ten of its substituted derivatives XYC[double bond]C[double bond]C(Z)N2+ (with F, CF3, Me, OMe, and Me2N as substituents) were studied by DFT at the B3LYP/6-31++G** level. Except for the Me2N-substituted derivative that forms a monocation-N2 complex, structurally intact allenediazonium ions were obtained as minima in all cases. Protonation studies at various sites were performed on allenediazonium cations, and relative energies of the resulting minima were used to identify the energetically most favored dications. In the majority of cases, protonation at the central carbon of the allenic moiety (C2) is most favored, forming delocalized allyl cation-N2+ species. The same dication structure is formed via initial C3-protonation, followed by a formal hydride shift, in cases where a carbocation-stabilizing group is placed at C3. When a CF3 group is placed at C3, initial protonation at C1 resulted in a 1,3-fluorine shift, to generate a fluoroallyl cation linked to a CH2N2+ moiety. Structural features in the allenediazonium monocations and their protonated dications were examined, taking into account their geometrical features, computed charges, and the GIAO NMR shifts.     

The influence of protonation on molecular structure and physico-chemical properties of gossypol Schiff bases

Protonation of gossypol Schiff bases (S1 and S2), possessing different numbers of basic N-atoms, was studied using potentiometric, spectroscopic, ESI MS and PM5 methods. Titration of S1 and S2 with HClO(4), monitored by the FT-IR and (1)H NMR, indicated that the change from the enamine-enamine into the protonated imine-imine tautomeric form occurs at different Schiff base-H(+) ratio. The FT-IR and PM5 results show that for S1 the first protonation step occurs at Schiff base moiety whereas for S2 it is realised at N-atom of the morpholine. The formation of N(+)-HO hydrogen bond between morpholine moieties within S2 contributes to high pK(a(ACN)) = 22.65.     

Protonation behaviour of chiral tetradentate polypyridines derived from alpha-pinene

Detailed protonation experiments of the [5,6]-pinenebipyridine molecule and the unsubstituted [4,5]- and [5,6]-CHIRAGEN[0] ligands in various solvents indicate a variety of structures of the protonated species. UV-visible and NMR measurements (including (15)N chemical shifts) show the transition from trans to cis conformation of [5,6]-pinenebipyridine upon protonation. The [4,5]-CHIRAGEN[0] ligand, in which the protonation sites of the nitrogen atom donors are at opposite sides of the molecule, behave essentially like two independent bipyridine moieties; this behaviour was monitored by UV-visible, CD and NMR spectroscopy (including (15)N data). In the case of the [5,6]-CHIRAGEN[0], a pocket of donor atoms provides a chiral environment for two protons per ligand.     

Ab initio molecular dynamics-based assignment of the protonation state of pepstatin A/HIV-1 protease cleavage site

A recent 13C NMR experiment (Smith et al. Nature Struct. Biol. 1996, 3, 946-950) on the Asp 25-Asp25' dyad in pepstatin A/HIV-1 protease measured two separate resonance lines, which were interpreted as being a singly protonated dyad. We address this issue by performing ab initio molecular dynamics calculations on models for this site accompanied by calculations of 13C NMR chemical shifts and isotopic shifts. We find that already on the picosecond time-scale the model proposed by Smith et al. is not stable and evolves toward a different monoprotonated form whose NMR pattern differs from the experimental one. We suggest, instead, a different protonation state in which both aspartic groups are protonated. Despite the symmetric protonation state, the calculated 13C NMR properties are in good agreement with the experiment. We rationalize this result using a simple valence bond model, which explains the chemical inequality of the two C sites. The model calculations, together with our calculations on the complex, allow also the rationalization of 13C NMR properties on other HIV-1 PR/inhibitor complexes. Both putative binding of the substrate to the free enzyme, which has the dyad singly protonated (Piana, S.; Carloni, P. Proteins: Struct., Funct., Genet. 2000, 39, 26-36), and pepstatin A binding to the diprotonated form are consistent with the inverse solvent isotope effect on the onset of inhibition of pepsin by pepstatin and the kinetic iso-mechanism proposed for aspartic proteases (Cho, T.-K.; Rebholz, K.; Northrop, D.B. Biochemistry 1994, 33, 9637-9642).     

Proton-detected solid-state NMR spectroscopy of fully protonated proteins at 40 kHz magic-angle spinning

Remarkable progress in solid-state NMR has enabled complete structure determination of uniformly labeled proteins in the size range of 5-10 kDa. Expanding these applications to larger or mass-limited systems requires further improvements in spectral sensitivity, for which inverse detection of 13C and 15N signals with 1H is one promising approach. Proton detection has previously been demonstrated to offer sensitivity benefits in the limit of sparse protonation or with approximately 30 kHz magic-angle spinning (MAS). Here we focus on experimental schemes for proteins with approximately 100% protonation. Full protonation simplifies sample preparation and permits more complete chemical shift information to be obtained from a single sample. We demonstrate experimental schemes using the fully protonated, uniformly 13C,15N-labeled protein GB1 at 40 kHz MAS rate with 1.6-mm rotors. At 500 MHz proton frequency, 1-ppm proton line widths were observed (500 +/- 150 Hz), and the sensitivity was enhanced by 3 and 4 times, respectively, versus direct 13C and 15N detection. The enhanced sensitivity enabled a family of 3D experiments for spectral assignment to be performed in a time-efficient manner with less than a micromole of protein. CANH, CONH, and NCAH 3D spectra provided sufficient resolution and sensitivity to make full backbone and partial side-chain proton assignments. At 750 MHz proton frequency and 40 kHz MAS rate, proton line widths improve further in an absolute sense (360 +/- 115 Hz). Sensitivity and resolution increase in a better than linear manner with increasing magnetic field, resulting in 14 times greater sensitivity for 1H detection relative to that of 15N detection.     

Ion-pair interactions of lanthanide(III) complexes in aqueous solutions

The formation of ion-pair adducts between the cationic complex La(THP)3+ (THP = 1,4,7,10-tetrakis(2-hydroxypropyl)-1,4,7,10-tetraazacyclododecane) and the anionic complexes Tm(DOTA)- (DOTA = 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetate), Tm(DTPA)2- (DTPA = diethylenetriamine-N,N,N',N",N"-pentaacetate), Tm(TTHA)3- (TTHA = triethylenetetraamine-N,N,N',N",N"',N"'-hexaacetate), and Tm(DOTP)5- (DOTP = 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetrakis(methylenephosphonate)) is examined by 13C NMR spectroscopy. The induced 13C shifts of the La(THP)3+ complex are followed by titration of the Tm(III) complexes of DOTA, DTPA, and TTHA at pH 7. From these data, the stability constants are calculated to be beta 1 = 64 M-1 (1:1), beta 1 = 296 M-1 (1:1), and beta 2 = 26,000 M-2 (2:1) for the ion pairs of La(THP)3+, with Tm(DOTA)-, Tm(DTPA)2-, and Tm(TTHA)3-, respectively. The La(THP)3+,Tm(DOTP)5- system elicits chiral resolution of the rapidly interconverting Tm(DOTP)5- isomers.     

The conformation of biliverdin in dimethyl sulfoxide: implications for the coordination with copper

Biliverdin (BV) structure was analyzed by using NMR techniques and unrestricted density function theory simulations to explain the incapacity of BV to build coordination complex(es) with Cu²⁺ in dimethyl sulfoxide, which was confirmed by UV-Vis, EPR and NMR spectroscopy. NMR showed that N atoms are protonated in all four pyrrole rings. The structure is stabilized by two hydrogen bonds between NH moieties and carbonyl oxygens from opposite terminal pyrrole rings, and by the bending of propionyl chain with carboxyl group out of the plain toward central position of BV. The simulations of deprotonated BV, which builds copper complexes in water and chloroform as described previously, showed a different conformation and organization of hydrogen bonds. Taking into account that deprotonation represents a critical step in coordinate bonds formation, the protonation of an additional N atom may represent a key difference between the interactions of BV with copper in different solvents.

     

The mobility and ion structure of protonated aminoazoles

The reduced mobility of protonated pyrazole derivatives was measured by ion mobility spectrometry (IMS) in air, nitrogen, and carbon dioxide, at temperatures between 150 and 250°C. It was found that the mobility of protonated 5-amino-1-phenylpyrazole was higher than that of its 3- and 4-isomers. This was attributed to the fact that in the 5-isomer the preferred site of protonation is on the endocyclic nitrogen, which leads to delocalization of the ionic charge, and thus to a diminished interaction with the drift gas molecules. On the other hand, protonated 5-amino-1-methylpyrazole has a slightly lower mobility than its isomers, which is indicative of a different protonation mechanism.