A new glycation product ‘norpronyl-lysine,’ and direct characterization of cross linking and other glycation adducts: NMR of model compounds and collagen

NMR is ideal for characterizing non-enzymatic protein glycation, including AGEs (advanced glycation endproducts) underlying tissue pathologies in diabetes and ageing. Ribose, R5P (ribose-5-phosphate) and ADPR (ADP-ribose), could be significant and underinvestigated biological glycating agents especially in chronic inflammation. Using [U-13C]ribose we have identified a novel glycoxidation adduct, 5-deoxy-5-desmethylpronyl-lysine, ‘norpronyl-lysine’, as well as numerous free ketones, acids and amino group reaction products. Glycation by R5P and ADPR proceeds rapidly with R5P generating a brown precipitate with PLL (poly-L-lysine) within hours. ssNMR (solid-state NMR) 13C–13C COSY identifies several crosslinking adducts such as the newly identified norpronyl-lysine, in situ, from the glycating reaction of 13C5-ribose with collagen. The same adducts are also identifiable after reaction of collagen with R5P. We also demonstrate for the first time bio-amine (spermidine, N-acetyl lysine, PLL) catalysed ribose 2-epimerization to arabinose at physiological pH. This work raises the prospect of advancing understanding of the mechanisms and consequences of glycation in actual tissues, in vitro or even ex vivo, using NMR isotope-labelled glycating agents, without analyses requiring chemical or enzymatic degradations, or prior assumptions about glycation products.

The second category involves fragmentation of the sugar carbon chain by retro-aldol reaction, hydrolytic cleavage or oxidative cleavage. The reactive sugar degradation products glyoxal and methylglyoxal in particular play a role in the formation of many AGEs. The two pathways to formation of CML [10,11] are a good example (Scheme S3), with oxidative cleavage of the Amadori product [3] competing with reaction of lysine with glyoxal [12].
Recently a third category of AGE has been discovered in vitro and in vivo: that of carboxylic amides, derived from β-dicarbonyl cleavage (Scheme S4) [13]. Of these N ε -acetyl lysine and N εformyl lysine are present in human plasma at comparable levels to CML [14].

Figure S1 Some important AGEs
Glucosepane is considered the most important known crosslink [10], being derived from glucose. Pentosidine and its precursor pentosinane are formed in the same manner as glucosepane from pentose sugars (which are formed by the autoxidation of glucose).
and DOPDIC are hydrolysed forms of glucosepane and pentosinane, respectively. GOLD and N ε -CML are the products formed by reaction of lysine with glyoxal (a product from the oxidation of sugars). MOLD (methylglyoxal lysine dimer) and N ε -CEL are the analogous products formed from methylglyoxal (a product from the fragmentation of sugars). Norfuraneol is formed from the amine-catalysed rearrangement and dehydration of pentose sugars.  The broad signal at 209 ppm, assigned to the carbonyl carbon of ribuloselysine, the Amadori product, reaches a maximum after 3 days before slowly decaying, although it is still present after 23 days suggesting the glycation process had not yet gone to completion. The broadness of the signal is presumably attributable to the polymeric nature of PLL: each ribuloselysine entity is in a slightly different chemical environment. The multiplet (doublet of doublets of doublet) at 202 ppm couples to 136 ppm and is from norfuraneol. The multiplet around 215 ppm is from relatively stable ketone intermediates; a corresponding feature is also present in reactions of R5P .

Figure S4
31 P solution-state NMR of ADPR before (blue) and after (red) reaction with PLL The ADPR signal loses intensity and is replaced with two doublets (asterisked) at − 7.3 and − 10.2 ppm (J = 20 Hz) which correspond to the βand α-phosphate groups of free ADP , released from ADPR, respectively.
Figure S5 13 C solution-state NMR of ADPR before (blue) and after (red) reaction with PLL ADPR assignment is given. The chemical shifts of C 1 -C 5 of the ribosyl phosphate moiety are dependent on the configuration of the anomeric carbon, C 1 , and accordingly signals from the α-anomer (hydroxyl group 'down') and the β-anomer (hydroxyl group 'up') are differentiated. After the generation of a precipitate (see text for details), the signals from the free ribose, asterisked, are significantly reduced, whereas the signals from ADP are unchanged in intensity. Taken with Figure S4, these results confirm that the ADP is released during the reaction that leads to crosslinking and precipitation. The same signal depletion over time was observed for glycation of PLL by R5P . Figure S6

Figure S7 13 C solution-state NMR spectrum of the supernatant phosphate buffer from [U-13 C]ribose glycated collagen (blue) and that of unreacted [U-13 C]ribose (red)
The dominant species in the supernatant is unreacted ribose, but significant quantities of glycolic and oxalic acids were also observed (signals indicated, see Section 5.1.2), along with a multitude of weaker signals from minor AGEs which require further assignment.

Figure S8
Comparison of the 13 C NMR spectrum of the supernatant from [U-13 C]ribose glycated collagen (blue) and the model system: [U-13 C]ribose glycated PLL (red) Several weak signals are observed in both spectra, such the signal due to CML at 52 ppm. However, many of the supernatant AGE signals are not observed in the model system, showing that PLL is an incomplete model for collagen glycation. The lack of norpronyl-lysine in the supernatant is expected as it is attached to the solid collagen. The absence of norfuraneol and acetic acid is surprising: either they are not generated in observable quantities, or bind strongly to collagen, or had degraded by the time the spectrum was obtained.
Table S1 Shifts of C 1 and C 2 in carboxylic acids are highly pH and concentration dependent, with carboxylate ion C 1 typically resonating 4-5 ppm to higher frequency than the corresponding acid [15] Values in the