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

Shasad Sharif, Monique Chan Huot, Peter M. Tolstoy, Michael D. Toney, K. Hanna M Jonsson, Hans Heinrich Limbach

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Abstract

By use of 15N NMR spectroscopy, we have measured the pK a 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.

Original languageEnglish (US)
Pages (from-to)3869-3876
Number of pages8
JournalJournal of Physical Chemistry B
Volume111
Issue number15
DOIs
StatePublished - Apr 19 2007

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Pyridoxal Phosphate
phosphates
Pyridine
Phosphates
pyridines
Nuclear magnetic resonance
Nitrogen
aqueous solutions
Schiff Bases
nuclear magnetic resonance
acids
Acids
nitrogen
imines
Hydrogen bonds
rings
Deprotonation
Protonation
Chemical shift
Phenol

ASJC Scopus subject areas

  • Physical and Theoretical Chemistry

Cite this

Sharif, S., Huot, M. C., Tolstoy, P. M., Toney, M. D., Jonsson, K. H. M., & Limbach, H. H. (2007). 15N nuclear magnetic resonance studies of acid-base properties of pyridoxal-5′-phosphate aldimines in aqueous solution. Journal of Physical Chemistry B, 111(15), 3869-3876. https://doi.org/10.1021/jp067334g

15N nuclear magnetic resonance studies of acid-base properties of pyridoxal-5′-phosphate aldimines in aqueous solution. / Sharif, Shasad; Huot, Monique Chan; Tolstoy, Peter M.; Toney, Michael D.; Jonsson, K. Hanna M; Limbach, Hans Heinrich.

In: Journal of Physical Chemistry B, Vol. 111, No. 15, 19.04.2007, p. 3869-3876.

Research output: Contribution to journalArticle

Sharif, Shasad ; Huot, Monique Chan ; Tolstoy, Peter M. ; Toney, Michael D. ; Jonsson, K. Hanna M ; Limbach, Hans Heinrich. / 15N nuclear magnetic resonance studies of acid-base properties of pyridoxal-5′-phosphate aldimines in aqueous solution. In: Journal of Physical Chemistry B. 2007 ; Vol. 111, No. 15. pp. 3869-3876.
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abstract = "By use of 15N NMR spectroscopy, we have measured the pK a 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.",
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T1 - 15N nuclear magnetic resonance studies of acid-base properties of pyridoxal-5′-phosphate aldimines in aqueous solution

AU - Sharif, Shasad

AU - Huot, Monique Chan

AU - Tolstoy, Peter M.

AU - Toney, Michael D.

AU - Jonsson, K. Hanna M

AU - Limbach, Hans Heinrich

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N2 - By use of 15N NMR spectroscopy, we have measured the pK a 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.

AB - By use of 15N NMR spectroscopy, we have measured the pK a 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.

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