Characterization of a novel hydroxypropyl methylcellulose (HPMC) direct compression grade excipient for pharmaceutical tablets.

Controlled release tablets are important dosage forms enabling a slower release of the drug and better pharmacokinetics for some drugs and hydrophilic matrix tablets utilizing hydroxypropyl methylcellulose (HPMC) are one of the most common types. One of the main challenges with using HPMC is its poor flow when implemented in a direct compression process or when utilized for continuous manufacturing for which novel grades of direct compression have been developed. In this work, three different direct compression (DC) grades of HPMC (K4M, K15M and K100M) were characterized and compared to their standard grade (CR) counterparts. These materials were compared in terms of density, particle size, morphology, surface area and powder flow using multiple techniques. Results showed that the materials were almost identical in terms of particle shape and although the DC grades had better flow, the particle size was slightly smaller with an unexpectedly higher surface area, which most likely resulted from the inclusion of co-processed silicon dioxide in the DC grades. The bulk, tapped and true densities were slightly higher for all of the DC grades. Of the eleven different parameters used to characterize the flow of the materials the DC grades showed better flow than their standard CR counterparts for nine of the parameters (Carr's Index, Erweka flow, FT4 Flow Rate Index, Mean Avalanche Time, Avalanche Scatter, Number of Avalanches, Shear Cell Uni-axial Compressive Strength and Shear Cell Flow Function Coefficient). Only the FT4 Basic Flowability Energy and Specific Energy showed the opposite trend which can be explained from the testing methodology. It is recommended to evaluate the DC grades of HPMC for processes where better flowing material would have an advantage, such as direct compression, continuous manufacturing, and roller compaction if the powder flow into the rolls is problematic.

Effect of Moisture Sorption on Free Volume and Relaxation of Spray Dried Dispersions: Relation to Drug Recrystallization.

The effect of vapor sorption on the free volume of drug-polymer spray-dried dispersions (SDDs) was investigated, along with the crystallization propensity of drug molecules in SDDs after exposure to humidity. Subsequently, the correlation of free volume change and relaxation time with drug recrystallization was examined. Four polymers, including polyvinylpyrrolidone, polyvinylpyrrolidone vinyl acetate copolymer, hydroxypropyl cellulose, and hydroxypropyl methylcellulose acetate succinate, and 2 drugs (indomethacin and ketoconazole) were selected for preparing SDDs. Free volume data of the exposed SDDs were obtained with positron annihilation lifetime spectroscopy, while the relaxation time was measured using a TA rheometer. Additionally, the crystallization propensity of active pharmaceutical ingredients (APIs) in the exposed SDDs was assessed using both polarized light microscopy and powder X-ray diffraction, followed by relating API crystallization inclination with expansion of holes and relaxation time. Finally, Cohen and Turnbull molecular transport model, along with its extensions by Vrentas and Duda, was qualitatively utilized for interpreting the recrystallization propensity of API molecules. In conclusion, API recrystallization is closely related to free volume change upon moisture sorption and relaxation time, but system dependent; overall, drug-hydroxypropyl methylcellulose acetate succinate SDDs appear physically stable against recrystallization due to less increase in free volume.

Enhancing tablet disintegration characteristics of a highly water-soluble high-drug-loading formulation by granulation process.

The objective of this study was to improve the disintegration and dissolution characteristics of a highly water-soluble tablet matrix by altering the manufacturing process. A high disintegration time along with high dependence of the disintegration time on tablet hardness was observed for a high drug loading (70% w/w) API when formulated using a high-shear wet granulation (HSWG) process. Keeping the formulation composition mostly constant, a fluid-bed granulation (FBG) process was explored as an alternate granulation method using a 2(4-1) fractional factorial design with two center points. FBG batches (10 batches) were manufactured using varying disingtegrant amount, spray rate, inlet temperature (T) and atomization air pressure. The resultant final blend particle size was affected significantly by spray rate (p = .0009), inlet T (p = .0062), atomization air pressure (p = .0134) and the interaction effect between inlet T*spray rate (p = .0241). The compactibility of the final blend was affected significantly by disintegrant amount (p < .0001), atomization air pressure (p = .0013) and spray rate (p = .05). It was observed that the fluid-bed batches gave significantly lower disintegration times than the HSWG batches, and mercury intrusion porosimetry data revealed that this was caused by the higher internal pore structure of tablets manufactured using the FBG batches.

Desleucyl-Oritavancin with a Damaged d-Ala-d-Ala Binding Site Inhibits the Transpeptidation Step of Cell-Wall Biosynthesis in Whole Cells of Staphylococcus aureus.

We have used solid-state nuclear magnetic resonance to characterize the exact nature of the dual mode of action of oritavancin in preventing cell-wall assembly in Staphylococcus aureus. Measurements performed on whole cells labeled selectively in vivo have established that des-N-methylleucyl-N-4-(4-fluorophenyl)benzyl-chloroeremomycin, an Edman degradation product of [19F]oritavancin, which has a damaged d-Ala-d-Ala binding aglycon, is a potent inhibitor of the transpeptidase activity of cell-wall biosynthesis. The desleucyl drug binds to partially cross-linked peptidoglycan by a cleft formed between the drug aglycon and its biphenyl hydrophobic side chain. This type of binding site is present in other oritavancin-like glycopeptides, which suggests that for these drugs a similar transpeptidase inhibition occurs.

Impact of moisture and magnesium stearate functionality on manufacturability of wet granulated metformin tablets.

During the development of a wet granulated 850 mg metformin hydrochloride tablet formulation, the tablets exhibited high friability (>3% w/w) irrespective of the source of extra-granular magnesium stearate (MgSt). High friability values indicated that an anti-bonding effect of MgSt was too high to be overcome by 3.3% w/w povidone as a binder in the formulation with 1.5% w/w residual granule moisture. Increasing the povidone concentration up to 7% w/w showed limited improvement in friability, with tablets showing variable friability depending on MgSt source. Characterization of MgSt indicated differences in crystallinity, surface area and particle morphology between different vendors. In addition, a new bulk yield strength test, which determines the MgSt fragmentation tendency, was found to be indicative of the MgSt performance in the tablet formulation. To improve bonding properties of granules, residual granule moisture was increased to 2% w/w at different povidone concentrations. At 2% w/w residual granule moisture content, regardless of MgSt source, the tablets showed significant improvement in friability (∼0.6% w/w) even at the lowest povidone concentration (3.3% w/w). The bonding power of higher residual granule moisture had a greater impact than higher povidone concentration in overcoming the anti-bonding effects of magnesium stearate.

REDOR constraints on the peptidoglycan lattice architecture of Staphylococcus aureus and its FemA mutant.

The peptidoglycan of Gram-positive bacteria consists of glycan chains with attached short peptide stems cross-linked to one another by glycyl bridges. The bridge of Staphylococcus aureus has five glycyl units and that of its FemA mutant has one. These long- and short-bridge cross-links create totally different cell-wall architectures. S. aureus and its FemA mutant grown in the presence of an alanine-racemase inhibitor were labeled with d-[1-¹³C]alanine, l-[3-¹³C]alanine, [2-¹³C]glycine, and l-[5-¹9F]lysine to characterize some details of the peptidoglycan tertiary structure. Rotational-echo double-resonance (REDOR) NMR of isolated cell walls was used to measure internuclear distances between ¹³C-labeled alanines and ¹9F-labeled lysine incorporated in the peptidoglycan. The alanyl ¹³C labels in the parent strain were preselected for C{F} and C{P} REDOR measurement by their proximity to the glycine label using ¹³C¹³C spin diffusion. The observed ¹³C¹³C and ¹³C³¹P distances are consistent with a tightly packed architecture containing only parallel stems in a repeating structural motif within the peptidoglycan. Dante selection of d-alanine and l-alanine frequencies followed by ¹³C¹³C spin diffusion rules out scrambling of carbon labels. Cell walls of FemA were also labeled by a combination of d-[1-¹³C]alanine and l-[¹5N]alanine. Proximity of chains was measured by C{N} and N{C} REDOR distances and asymptotic plateaus, and both were consistent with a mixed-geometry model. Binding of an ¹9F-labeled eremomycin analog in the FemA cell wall matches that of binding to the parent-strain cell wall and reveals the proximity of parallel stems in the alternating parallel-perpendicular mixed-geometry model for the FemA peptidoglycan lattice.

Cross-link formation and peptidoglycan lattice assembly in the FemA mutant of Staphylococcus aureus.

Staphylococcus aureus FemA mutant grown in the presence of an alanine-racemase inhibitor was labeled with d-[1-(13)C]alanine, l-[3-(13)C]alanine, [2-(13)C]glycine, and l-[5-(19)F]lysine to characterize some details of the peptidoglycan tertiary structure. Rotational-echo double-resonance (REDOR) NMR of isolated cell walls was used to measure internuclear distances between (13)C-labeled alanines and (19)F-labeled lysine incorporated in the peptidoglycan. The alanyl (13)C labels were preselected for REDOR measurement by their proximity to the glycine label using (13)C-(13)C spin diffusion. The observed (13)C-(13)C and (13)C-(19)F distances are consistent with a tightly packed, hybrid architecture containing both parallel and perpendicular stems in a repeating structural motif within the peptidoglycan.

NMR studies of protonation and hydrogen bond states of internal aldimines of pyridoxal 5'-phosphate acid-base in alanine racemase, aspartate aminotransferase, and poly-L-lysine.

Using (15)N solid-state NMR, we have studied protonation and H-bonded states of the cofactor pyridoxal 5'-phosphate (PLP) linked as an internal aldimine in alanine racemase (AlaR), aspartate aminotransferase (AspAT), and poly-L-lysine. Protonation of the pyridine nitrogen of PLP and the coupled proton transfer from the phenolic oxygen (enolimine form) to the aldimine nitrogen (ketoenamine form) is often considered to be a prerequisite to the initial step (transimination) of the enzyme-catalyzed reaction. Indeed, using (15)N NMR and H-bond correlations in AspAT, we observe a strong aspartate-pyridine nitrogen H-bond with H located on nitrogen. After hydration, this hydrogen bond is maintained. By contrast, in the case of solid lyophilized AlaR, we find that the pyridine nitrogen is neither protonated nor hydrogen bonded to the proximal arginine side chain. However, hydration establishes a weak hydrogen bond to pyridine. To clarify how AlaR is activated, we performed (13)C and (15)N solid-state NMR experiments on isotopically labeled PLP aldimines formed by lyophilization with poly-L-lysine. In the dry solid, only the enolimine tautomer is observed. However, a fast reversible proton transfer involving the ketoenamine tautomer is observed after treatment with either gaseous water or gaseous dry HCl. Hydrolysis requires the action of both water and HCl. The formation of an external aldimine with aspartic acid at pH 9 also produces the ketoenamine form stabilized by interaction with a second aspartic acid, probably via a H-bond to the phenolic oxygen. We postulate that O-protonation is an effectual mechanism for the activation of PLP, as is N-protonation, and that enzymes that are incapable of N-protonation employ this mechanism.

Uniformity of glycyl bridge lengths in the mature cell walls of fem mutants of methicillin-resistant Staphylococcus aureus.

Peptidoglycan (PG) composition in intact cells of methicillin-resistant Staphylococcus aureus (MRSA) and its isogenic Fem mutants has been characterized by measuring the glycine content of PG bridge structures by solid-state nuclear magnetic resonance (NMR). The glycine content estimated from integrated intensities (rather than peak heights) in the cell walls of whole cells was increased by approximately 30% for the FemA mutant and was reduced by 25% for the FemB mutant relative to expected values for homogeneous structures. In contrast, the expected compositions were observed in isolated cell walls of the same mutants. For FemA mutant whole cells, the increase was due to the presence of triglycyl bridge PG units (confirmed directly by mass spectrometric analysis), which constituted 10% of the total PG. These species were coalesced in some sort of a lattice or aggregate with spatial proximity to other PG bridges. This result suggests that the triglycyl-bridged PG units form a PG-like structure that is not incorporated into the mature cell wall.

Critical hydrogen bonds and protonation states of pyridoxal 5'-phosphate revealed by NMR.

In this contribution we review recent NMR studies of protonation and hydrogen bond states of pyridoxal 5'-phosphate (PLP) and PLP model Schiff bases in different environments, starting from aqueous solution, the organic solid state to polar organic solution and finally to enzyme environments. We have established hydrogen bond correlations that allow one to estimate hydrogen bond geometries from (15)N chemical shifts. It is shown that protonation of the pyridine ring of PLP in aspartate aminotransferase (AspAT) is achieved by (i) an intermolecular OHN hydrogen bond with an aspartate residue, assisted by the imidazole group of a histidine side chain and (ii) a local polarity as found for related model systems in a polar organic solvent exhibiting a dielectric constant of about 30. Model studies indicate that protonation of the pyridine ring of PLP leads to a dominance of the ketoenamine form, where the intramolecular OHN hydrogen bond of PLP exhibits a zwitterionic state. Thus, the PLP moiety in AspAT carries a net positive charge considered as a pre-requisite to initiate the enzyme reaction. However, it is shown that the ketoenamine form dominates in the absence of ring protonation when PLP is solvated by polar groups such as water. Finally, the differences between acid-base interactions in aqueous solution and in the interior of proteins are discussed. This article is part of a special issue entitled: Pyridoxal Phosphate Enzymology.

NMR studies of the stability, protonation States, and tautomerism of (13)C- AND (15)N-labeled aldimines of the coenzyme pyridoxal 5'-phosphate in water.

We have measured the pH-dependent (1)H, (13)C, and (15)N NMR spectra of pyridoxal 5'-phosphate ((13)C(2)-PLP) mixed with equal amounts of either doubly (15)N-labeled diaminopropane, (15)N(α)-labeled l-lysine, or (15)N(ε)-labeled l-lysine as model systems for various intermediates of the transimination reaction in PLP-dependent enzymes. At low pH, only the hydrate and aldehyde forms of PLP and the free protonated diamines are present. Above pH 4, the formation of single- and double-headed aldimines (Schiff bases) with the added diamines is observed, and their (13)C and (15)N NMR parameters have been characterized. For 1:1 mixtures the single-headed aldimines dominate. In a similar way, the NMR parameters of the geminal diamine formed with diaminopropane at high pH are measured. However, no geminal diamine is formed with l-lysine. In contrast to the aldimine formed with the ε-amino group of lysine, the aldimine formed with the α-amino group is unstable at moderately high pH but dominates slightly below pH 10. By analyzing the NMR data, both the mole fractions of the different PLP species and up to 6 different protonation states including their pK(a) values were obtained. Furthermore, the data show that all Schiff bases are subject to a proton tautomerism along the intramolecular OHN hydrogen bond, where the zwitterionic form is favored before deprotonation occurs at high pH. This observation, as well as the observation that around pH 7 the different PLP species are present in comparable amounts, sheds new light on the mechanism of the transimination reaction.

Staphylococcus aureus peptidoglycan tertiary structure from carbon-13 spin diffusion.

The cell-wall peptidoglycan of Staphylococcus aureus is a heterogeneous, highly cross-linked polymer of unknown tertiary structure. We have partially characterized this structure by measuring spin diffusion from (13)C labels in pentaglycyl cross-linking segments to natural-abundance (13)C in the surrounding intact cell walls. The measurements were performed using a version of centerband-only detection of exchange (CODEX). The cell walls were isolated from S. aureus grown in media containing [1-(13)C]glycine. The CODEX spin diffusion rates established that the pentaglycyl bridge of one peptidoglycan repeat unit of S. aureus is within 5 A of the glycan chain of another repeat unit. This surprising proximity is interpreted in terms of a model for the peptidoglycan lattice in which all peptide stems in a plane perpendicular to the glycan mainchain are parallel to one another.

Characterization of peptidoglycan in fem-deletion mutants of methicillin-resistant Staphylococcus aureus by solid-state NMR.

Compositional analysis of the peptidoglycan (PG) of a wild-type methicillin-resistant Staphylococcus aureus and its fem-deletion mutants has been performed on whole cells and cell walls using stable-isotope labeling and rotational-echo double-resonance NMR. The labels included [1-(13)C,(15)N]glycine and l-[epsilon-(15)N]lysine (for a direct measure of the number of glycyl residues in the bridging segment), [1-(13)C]glycine and l-[epsilon-(15)N]lysine (concentration of bridge links), and d-[1-(13)C]alanine and [(15)N]glycine (concentrations of cross-links and wall teichoic acids). The bridging segment length changed from 5.0 glycyl residues (wild-type strain) to 2.5 +/- 0.1 (FemB) with modest changes in cross-link and bridge-link concentrations. This accurate in situ measurement for the FemB mutant indicates a heterogeneous PG structure with 25% monoglycyl and 75% triglycyl bridges. When the bridging segment was reduced to a single glycyl residue 1.0 +/- 0.1 (FemA), the level of cross-linking decreased by more than 20%, resulting in a high concentration of open N-terminal glycyl segments.

Nuclear magnetic resonance and ab initio studies of small complexes formed between water and pyridine derivatives in solid and liquid phases.

The structure and geometry of hydrogen-bonded complexes formed between heterocyclic bases, namely, pyridine and 2,4,6-trimethylpyridine (collidine), and water were experimentally studied by NMR spectroscopy in frozen phase and in highly polar aprotic liquefied freon mixtures and theoretically modeled for gas phase. Hydrogen-bonded species in frozen heterocycle-water mixtures were characterized experimentally using 15N NMR. When base was in excess, one water molecule was symmetrically bonded to two heterocyclic molecules. This complex was characterized by the rHN distances of 1.82 Angstrom for pyridine and 1.92 Angstrom for collidine. The proton-donating ability of water in such complexes was affected by an anticooperative interaction between the two coupled hydrogen bonds and exhibited an apparent pK(a) value of about 6.0. When water was in excess, it formed water clusters hydrogen bonded to base. Theoretical analysis of binding energies of small model heterocycle-water clusters indicated that water in such clusters was oriented as a chain. The NMR estimated rHN distances in these species were 1.69 Angstrom for pyridine and 1.64 Angstrom for collidine. Here, the proton-donating ability of the hydroxyl group bonded to the heterocycle was affected by a mutual cooperative interaction with other water molecules in the chain and became comparable to the proton-donating ability of a fictitious acid, exhibiting an apparent pK(a) value of about 4.9. This value seems to depend only slightly on the length of the water chain and on the presence of another base at the other end of the chain if more than two water molecules are involved. Thus, the proton-donating ability of the outer hydroxyl groups of biologically relevant water bridges should be comparable to the proton-donating ability of a fictitious acid exhibiting a pK(a) value of about 4.9 in water. Driven by the mixing entropy, monomeric water presented in the aprotic freonic mixtures above 170 K but completely precipitated upon further cooling. Traces of water could be suspended in the mixtures down to 130 K in the presence of about 20-fold excess of heterocyclic bases. The obtained experimental data indicated that at these conditions water trended to form the symmetric 2:1 heterocycle-water complexes, whose bridge protons resonated around 6.7 ppm.

NMR studies of coupled low- and high-barrier hydrogen bonds in pyridoxal-5'-phosphate model systems in polar solution.

The 1H and 15N NMR spectra of several 15N-labeled pyridoxal-5'-phosphate model systems have been measured at low temperature in various aprotic and protic solvents of different polarity, i.e., dichloromethane-d2, acetonitrile-d3, tetrahydrofuran-d8, freon mixture CDF3/CDClF2, and methanol. In particular, the 15N-labeled 5'-triisopropyl-silyl ether of N-(pyridoxylidene)-tolylamine (1a), N-(pyridoxylidene)-methylamine (2a), and the Schiff base with 15N-2-methylaspartic acid (3a) and their complexes with proton donors such as triphenylmethanol, phenol, and carboxylic acids of increasing strength were studied. With the use of hydrogen bond correlation techniques, the 1H/15N chemical shift and scalar coupling data could be associated with the geometries of the intermolecular O1H1N1 (pyridine nitrogen) and the intramolecular O2H2N2 (Schiff base) hydrogen bonds. Whereas O1H1N1 is characterized by a series of asymmetric low-barrier hydrogen bonds, the proton in O2H2N2 faces a barrier for proton transfer of medium height. When the substituent on the Schiff base nitrogen is an aromatic ring, the shift of the proton in O1H1N1 from oxygen to nitrogen has little effect on the position of the proton in the O2H2N2 hydrogen bond. By contrast, when the substituent on the Schiff base nitrogen is a methyl group, a proton shift from O to N in O1H1N1 drives the tautomeric equilibrium in O2H2N2 from the neutral O2-H2...N2 to the zwitterionic O2-...H2-N(2+) form. This coupling is lost in aqueous solution where the intramolecular O2H2N2 hydrogen bond is broken by solute-solvent interactions. However, in methanol, which mimics hydrogen bonds to the Schiff base in the enzyme active site, the coupling is preserved. Therefore, the reactivity of Schiff base intermediates in pyridoxal-5'-phosphate enzymes can likely be tuned to the requirements of the reaction being catalyzed by differential protonation of the pyridine nitrogen.

15N nuclear magnetic resonance studies of acid-base properties of pyridoxal-5'-phosphate aldimines in aqueous solution.

By use of 15N NMR spectroscopy, we have measured the pKa values of the aldimines 15N-(pyridoxyl-5'-phosphate-idine)-methylamine (2a), N-(pyridoxyl-5'-phosphate-15N-idine)-methylamine (2b), and 15N-(pyridoxyl-idine)-methylamine (3). These aldimines model the cofactor pyridoxal-5'-phosphate (PLP, 1) in a variety of PLP-dependent enzymes. The acid-base properties of the aldimines differ substantially from those of the free cofactor in the aldehyde form 1a or in the hydrated form 1b, which were also investigated using 15N NMR for comparison. All compounds contain three protonation sites, the pyridine ring, the phenol group, and the side chain phosphate (1, 2) or hydroxyl group (3). In agreement with the literature, 1a exhibits one of several pKas at 2.9 and 1b at 4.2. The 15N chemical shifts indicate that the corresponding deprotonation occurs partially in the pyridine and partially in the phenolic site, which compete for the remaining proton. The equilibrium constant of this ring-phenolate tautomerism was measured to be 0.40 for 1a and 0.06 for 1b. The tautomerism is essentially unaltered above pH 6.1, where the phosphate group is deprotonated to the dianion. This means that the pyridine ring is more basic than the phenolate group. Pyridine nitrogen deprotonation occurs at 8.2 for 1a and at 8.7 for 1b. By contrast, above pH 4 the phosphate site of 2 is deprotonated, while the pyridine ring pKa is 5.8. The Schiff base nitrogen does not deprotonate below pH 11.4. When the phosphate group is removed, the pKa of the Schiff base nitrogen decreases to 10.5. The phenol site cannot compete for the proton of the Schiff base nitrogen and is present in the entire pH range as a phenolate, preferentially hydrogen bonded to the solvent. The intrinsic 15N chemical shifts provide information about the hydrogen bond structures of the protonated and unprotonated species involved. Evidence is presented that the intramolecular OHN hydrogen bond of PLP aldimines is broken in aqueous solution. The coupling between the inter- and intramolecular OHN hydrogen bonds is also lost in this environment. The pyridine ring of the PLP aldimines is not protonated in aqueous solution near neutral pH. The basicity of the aldimine nitrogens would be even lower without the doubly negatively charged phosphate group. Protonation of both the Schiff base and pyridine nitrogens has been discussed as a prerequisite for catalytic activity, and the implications of the present findings for PLP catalysis are discussed.

Coupling of functional hydrogen bonds in pyridoxal-5'-phosphate-enzyme model systems observed by solid-state NMR spectroscopy.

We present a novel series of hydrogen-bonded, polycrystalline 1:1 complexes of Schiff base models of the cofactor pyridoxal-5'-phosphate (PLP) with carboxylic acids that mimic the cofactor in a variety of enzyme active sites. These systems contain an intramolecular OHN hydrogen bond characterized by a fast proton tautomerism as well as a strong intermolecular OHN hydrogen bond between the pyridine ring of the cofactor and the carboxylic acid. In particular, the aldenamine and aldimine Schiff bases N-(pyridoxylidene)tolylamine and N-(pyridoxylidene)methylamine, as well as their adducts, were synthesized and studied using 15N CP and 1H NMR techniques under static and/or MAS conditions. The geometries of the hydrogen bonds were obtained from X-ray structures, 1H and 15N chemical shift correlations, secondary H/D isotope effects on the 15N chemical shifts, or directly by measuring the dipolar 2H-15N couplings of static samples of the deuterated compounds. An interesting coupling of the two "functional" OHN hydrogen bonds was observed. When the Schiff base nitrogen atoms of the adducts carry an aliphatic substituent such as in the internal and external aldimines of PLP in the enzymatic environment, protonation of the ring nitrogen shifts the proton in the intramolecular OHN hydrogen bond from the oxygen to the Schiff base nitrogen. This effect, which increases the positive charge on the nitrogen atom, has been discussed as a prerequisite for cofactor activity. This coupled proton transfer does not occur if the Schiff base nitrogen atom carries an aromatic substituent.

X-ray crystallographic structures of enamine and amine Schiff bases of pyridoxal and its 1:1 hydrogen-bonded complexes with benzoic acid derivatives: evidence for coupled inter- and intramolecular proton transfer.

Crystal structures of Schiff bases containing pyridoxal (PL), N-(pyridoxylidene)-tolylamine, C(15)H(16)N(2)O(2) (I), N-(pyridoxylidene)-methylamine, C(9)H(12)N(2)O(2) (III), and their 1:1 adduct with 2-nitrobenzoic acid, (I)(+) C(7)H(4)NO_4;- (II), and 4-nitrobenzoic acid, (III)(+) C(7)H(4)NO_4;- (IV), serve as models for the coenzyme pyridoxal-5'-phosphate (PLP) in its PLP-dependent enzymes. These models allow the study of the intramolecular OHN hydrogen bond of PL/PLP Schiff bases and the H-acceptor properties of their pyridine rings. The free base (I) forms hydrogen-bonded chains involving the hydroxyl side groups and the rings of adjacent molecules, whereas (III) forms related hydrogen-bonded cyclic dimers. The adducts (II)/(IV) consist of 1:1 hydrogen-bonded complexes, exhibiting strong intermolecular bonds between the carboxylic groups of the acids and the pyridine rings of (I)/(III). In conclusion, the proton in the intramolecular O-H...N hydrogen bond of (I)/(III) is located close to oxygen (enolamine form). The added acids protonate the pyridine ring in (II)/(IV), but only in the latter case does this protonation lead to a shift of the intramolecular proton towards the nitrogen (ketoimine form). All crystallographic structures were observed in the open form. In contrast, the formation of the pyridinium salt by dissolving (IV) leads to the cyclic aminal form.

NMR studies of solvent-assisted proton transfer in a biologically relevant Schiff base: toward a distinction of geometric and equilibrium H-bond isotope effects.

The tautomeric equilibrium in a Schiff base, N-(3,5-dibromosalicylidene)-methylamine 1, a model for the hydrogen bonded structure of the cofactor pyridoxal-5'-phosphate PLP which is located in the active site of the enzyme, was measured by means of 1H and 15N NMR and deuterium isotope effects on 15N chemical shifts at variable temperature and in different organic solvents. The position of the equilibrium was estimated using the one-bond 1J(OHN) and vicinal 3J(H(alpha)CNH) scalar coupling constants. Additionally, DFT calculations of a series of Schiff bases, N-(R1-salicylidene)-alkyl(R2)amines, were performed to obtain the hydrogen bond geometries. The latter made it possible to investigate a broad range of equilibrium positions. The increase of the polarity of the aprotic solvent shifts the proton in the intramolecular OHN hydrogen bond closer to the nitrogen. The addition of methanol and of hexafluoro-2-propanol to 1 in aprotic solvents models the PLP-water interaction in the enzymatic active site. The alcohols, which vary in acidity and change the polarity around the hydrogen bond, also stabilize the equilibrium, so that the proton is shifted to the nitrogen.