The multiple Maillard reactions of ribose and deoxyribose sugars and sugar phosphates

Abstract

Ribose 5-phosphate (R5P) undergoes the Maillard reaction with amines at significantly higher rates than most other sugars and sugar phosphates. The presence of an intramolecular phosphate group, which catalyzes the early stages of the Maillard reaction, provides the opportunity for the R5P molecule to undergo novel reaction paths creating unique Maillard products. The initial set of reactions leading to an Amadori product (phosphorylated) and to an alpha-dicarbonyl phosphate compound follows a typical Maillard reaction sequence, but an observed phosphate hydrolysis accompanying the reaction adds to the complexity of the products formed. The reaction rate for the loss of R5P is partially dependent on the pK(a) of the amine but also is correlated to the protonation of an early intermediate of the reaction sequence. In the presence of oxygen, a carboxymethyl group conjugated to the amine is a major product of the reaction of R5P with N-acetyllysine while little of this product is generated in the absence of oxygen. Despite lacking a critical hydroxyl group necessary for the Maillard reaction, 2-deoxyribose 5-phosphate (dR5P) still generates an Amadori-like product (with a carbonyl on the C-3 carbon) and undergoes phosphate cleavage. Two highly UV-absorbing products of dR5P were amine derivatives of 5-methylene-2-pyrrolone and 2-formylpyrrole. The reaction of dR5P with certain amines generates a set of products that exhibit an interesting absorbance at 340nm and a high fluorescence.

Figure 1

A comparison of the rates of disappearance of NAcLys (■) and R5P (▲) from solution at pH 7.5 and 37 °C. The loss of NAcLys over an approximately 70-h period was 0.015 M while 0.042 M of R5P disappeared from solution during this same timeframe.

Figure 2

The generation of N-glycoside/Amadori products for R5P (▲) and dR5P (■) in reaction with NAcLys at pH 7.4 and 37 °C. The relative amount of products, represented as peak areas, was obtained by integration of an extracted ion chromatogram performed at the appropriate m/z value. The concentrations of the sugar and NAcLys were 0.05 M and 0.10 M, respectively.

Figure 3

The percent phosphate (P_i) cleaved from various R5P/amine systems at pH 8 and 37 °C as determined by ³¹P NMR. The R5P concentration was 0.05 M, and the amine concentration was 0.10 M except for poly(lysine) where the amine concentration (of the R groups) was 0.050 M. Symbols: Poly(lysine) (+); Asn (◊); β-Ala (■); l-NAcLys (△); l-Ala (◆); Gly-gly (▲); Gly (□).

Figure 4

A series of three ³¹P NMR spectra following the reaction between NAcLys and R5P at pH 7.4 and 37 °C for five days. (Spectra are offset for clarity.) The reaction begins with a single R5P peak [represented by #] at approximately +4.5 ppm and, with time, P_i [represented by *] at approximately +3.2 ppm. Compounds giving downfield signals (+4.6 to +5.0 ppm) were generated.

Figure 5

(A) Rate of absorbance increase at 280 nm for R5P (▲), ribose + P_i (△), dR5P (■), and deoxyribose + P_i (□) with NAcLys at pH 7.4 and 37 °C. (B) Absorbance spectra for R5P (▲), ribose + P_i (△), dR5P (■), and deoxyribose + P_i (□) with NAcLys after 24 h (17.5 h for R5P) at pH 7.4 and 37 °C. (C) Absorbance spectra for three amino acids after a reaction with R5P at pH 8.0 and 37 °C. Reaction time was 10 h for Gly (▲) and Glu (◊), 7.3 h for Asn (■), and 6 h for Gly-Gly (◆).

Figure 6

The comparative absorbance spectra of reactions of NAcLys (0.10 M) and R5P (0.050 M) (pH 7.4) performed under the presence (■) and absence (▲) of oxygen. The incubations were held at 37 °C for five days.

Figure 7

The Total AGEs fluorescence (emission = 360 nm; excitation = 460 nm) generated in a reaction between a pentose sugar and NAcLys at pH 7.5 and 37 °C. The sugars employed are R5P (▲), dR5P (■), ribose and P_i (△), and deoxyribose and P_i (□). Inset: An expanded view of R5P, ribose and P_i, and deoxyribose and P_i.

Figure 8

Portions of ¹H NMR spectra of 0 h and 8 h reactions of dR5P with β-Ala (pH 7.5, 37 °C) performed in D₂O. Peaks at +5.5 ppm [represented by #] indicate the C-1 hydrogen does not lose significant intensity over this time period while peaks at +1.75 ppm, +2.05 ppm, and +2.35 ppm (shoulder) [represented by *] indicate the C-2 hydrogen signals (three sets stemming from the two ring hydrogens and the α/β forms) disappear as exchange for solvent deuterium occurs. Note the splitting pattern for the C-1 hydrogens moves from a multiplet (actually overlapping triplets) to a pair of singlets (for α and β forms).

Figure 9

Total ion chromatograms of the seven day reactions (37 °C; pH 7.4) of NAcLys with R5P (– – –) and dR5P (–––). Separation was performed on a 4.6 × 150 mm C₈ column using 80:20 H₂O:2-PrOH with 0.5% HCO₂H at 0.5 mL/min.

Scheme 1

Proposed early reactions of R5P (1) with amines stemming from the acyclic form in equilibrium with the furanose. A traditional series of Maillard reactions (path A) leads through an Amadori product (2) to an α-dicarbonyl product (3), while an isomerization series (path B) leading to ribulose 5-phosphate (4) may be encouraged by a general base catalysis (i.e., removal of the C-2 hydrogen) of the amine.

Scheme 2

Chemical equations of the early Maillard sequence of R5P and amines defining the rate constants. Methylglyoxal would be generated by a retro-aldolization along with glyceraldehyde phosphate.

Scheme 3

The general reaction mechanism for the formation of the Amadori product from a sugar and an amine.

Scheme 4

A proposed reaction scheme of Hauck et al. for the hydrolysis of an intramolecular 5-phosphate moiety from a pentose sugar containing a carbonyl group at the C-3 position.

Scheme 5

The reaction scheme for the exchange of hydrogen with solvent deuterium at the C-2 carbon of dR5P in a Maillard reaction with an amine. ¹H NMR analysis shows full loss of C-2 hydrogens over a period of approximately 10 h at 37 °C, thus indicating the sequence can occur twice to replace both hydrogens with deuterium.

Scheme 6

A proposed sequence of reactions between d-ribose and NAcLys leading to a m/z 115 [M + H⁺] product. The reaction is initiated (step a) by an attachment of the amine to form a Schiff base, followed by Amadori rearrangement (step b) and hydrolysis of the amine to yield a 1-deoxy-2,3-dicarbonyl compound (step c). The dicarbonyl compound can spontaneously rearrange to HMF by a previously reported mechanism.

Scheme 7

Tautomerization reaction of dR5P originating from an enamine (Scheme 5) leading to an Amadori-like compound.

Scheme 8

Structures of two products generated from a reaction at 37 °C and pH 7.4 between dR5P and β-Ala. A. Four-min retention-time compound, an amine derivative of 5-MP, with an absorbance maximum at 260 nm. B. Six-min retention-time compound, N-methyl-2-formylpyrrole, with an absorbance maximum at 290 nm.