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

Article abstract of DOI:10.1016/j.carres.2007.08.003

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 α-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 340 nm and a high fluorescence.

Full text of DOI:10.1016/j.carres.2007.08.003

Available online at www.sciencedirect.com
Carbohydrate Research 342 (2007) 2575–2592
The multiple Maillard reactions of ribose and deoxyribose sugars
and sugar phosphates
Admire Munanairi, Steven K. O’Banion, Ryan Gamble, Elizabeth Breuer,
Andrew W. Harris and Roger K. Sandwick*
Department of Chemistry and Biochemistry, Middlebury College, Middlebury, VT 05753, United States
Received 27 July 2007; accepted 10 August 2007
Available online 19 August 2007
Abstract—Ribose 5-phosphate (R5P) undergoes the Maillard reaction with amines at significantly higher rates than most other sug-
ars and sugar phosphates. The presence of an intramolecular phosphate group, which catalyzes the early stages of the Maillard reac-
tion, 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 a-dicarbonyl phosphate compound follows a typical
Maillard reaction sequence, but an observed phosphate hydrolysis accompanying the reaction adds to the complexity of the prod-
ucts formed. The reaction rate for the loss of R5P is partially dependent on the pKa of the amine but also is correlated to the pro-
tonation 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 gener-
ates a set of products that exhibit an interesting absorbance at 340 nm and a high fluorescence.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Keywords: Ribose 5-phosphate; Deoxyribose 5-phosphate; Maillard; Glycation
1. Introduction
(including glucose 6-phosphate and fructose 1,6-bis-
phosphate).1–3 Based on UV absorbance and browning
results, R5P reacts at rates that exceed 100-fold that of
glucose.1 A reaction with glycine at pH 8 and 37 ꢁC
monitored by 1H NMR spectroscopy gave a bimolecular
rate constant for the disappearance of R5P from solu-
tion of 0.22 Mꢀ1 hꢀ1.1 Considering this first step of the
Maillard reaction involves the attack of the deproto-
nated form of the amine, a pH-independent rate equa-
tion for this system can be rewritten as
A member of the pentose phosphate pathway, ribose
5-phosphate (R5P) exists at higher levels (5–20 lM) in
those cells requiring reduced nicotinamide adenine di-
nucleotide phosphate (NADPH) for reductive biosyn-
thesis or in dividing cells that require the molecule as a
precursor for purine and pyrimidine biosynthesis. As a
sugar, R5P is subject to spontaneous Maillard reactions
with cellular amines, including the N-terminal, lysine
(Lys) and arginine (Arg) groups of proteins. In fact,
the molecule has been shown to react at much higher
rates than most other common sugars (including
glucose, ribose, and fructose) and sugar phosphates
Rate ¼ k½sugarꢁ½R–NH2ꢁ
where [R–NH2] reflects the pH-dependent concentration
of the deprotonated amine. For the reported glycine
system,1 the rate constant would be recalculated as
approximately 7 Mꢀ1 hꢀ1. Given an amine of moder-
ately low pKa (e.g., 8.0) and 100 lM concentration, this
translates into an in vivo R5P glycation rate equaling
*
Corresponding author. Tel.: +1 802 443 3496; fax: +1 802 443
2072; e-mail: rsandwic@middlebury.edu
0008-6215/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2007.08.003

2576
A. Munanairi et al. / Carbohydrate Research 342 (2007) 2575–2592
approximately 0.05% of available amine per day at
physiologic R5P levels. Longer lived proteins would
thus experience 1% glycation in a 3-week period.
highly UV- and visible-absorbing advanced glycation
end-products (AGEs). The reaction of R5P in this se-
quence appears, however, to be more complex than
other sugars. It is possible that the presence of the amine
encourages isomerization of R5P to ribulose 5-phos-
phate (Ru5P) (see Scheme 1), and this reaction may or
may not be connected to an observed loss of phosphate
from the molecule.1,10 As in other Maillard systems, the
availability of O2 allows a different series of subsequent
reactions (compared to anaerobic conditions) and the
formation of reactive oxygen species superoxide and
hydrogen peroxide.11
Like R5P, 2-deoxyribose 5-phosphate (dR5P) has a
phosphate moiety perched over a reactive hemiacetal
group of a furanose ring. The sugar, however, lacks
the hydroxyl group at C-2 necessary for the enaminol
tautomerization step that occurs during the Amadori
rearrangement process. Thus, while it is likely that
dR5P undergoes some initial N-glycoside reaction with
amines in solution, it is impossible for dR5P to proceed
to melanoidin production and browning via a tradi-
tional Maillard series of reactions. However, as previ-
ously reported by Wondrak et al.12 and as we report
here, dR5P overcomes this ‘deoxy’ barrier to generate
a variety of Maillard-like compounds.
The relatively rapid glycation rate of R5P is clearly
related in some way to its attached phosphate group.1
Phosphates have been long employed as catalysts of
the Maillard browning reaction via a mechanism specu-
lated to be either general base catalysis4 or covalent
catalysis.5 In the case of R5P, a proximity effect of the
phosphate group perched over the reactive anomeric
center apparently promotes a greater reactivity in com-
parison to that observed by intermolecular phosphate.
(Sugar phosphates such as glucose 6-phosphate contain
intramolecular phosphate, but the location of the phos-
phate in an equatorial position is stereochemically dis-
tant from the C-1 carbon of the glucopyranoside.) The
proximity/orientation rate enhancements could also be
supplemented by steric effects of the phosphate on the
furanose ring structure leading to a higher level of the
acyclic structure, a form generally acknowledged as
the true initial reactant of the Maillard sequence. While
large discrepancies have been reported in the acyclic lev-
els of sugars as a result of the technique employed, the
acyclic level of R5P appears to be at least an order of
magnitude in excess of D-ribose and 2–3 orders of mag-
nitude greater than D-glucose.6–8
We report here the kinetics and major reaction pro-
ducts of R5P and dR5P (along with their corresponding
non-phosphorylated counterparts ribose and deoxyri-
bose) with simple amines. The molecules are capable
of producing a diverse set of products in a relatively
rapid timeframe in simulated physiological conditions
and, thus, need to be considered as potentially signifi-
cant glycation agents in cells.
The early steps of the reaction of R5P with amines
(Scheme 1) appear to be similar to the Maillard reaction
of other sugars. The initial attack of the nitrogen to pro-
duce an N-glycoside is followed by a rearrangement to
generate an Amadori compound.2,9 The formation of
a typical a-dicarbonyl compound has then been sug-
gested,1 which subsequently proceeds to a variety of
O-
O-
-O
O-
-O
-O
P
P
O
P
O
O
O
H2C
R
NH+
O
R
O
O-
OH
O-
OH
-O
H2C
-O
OH
H2C
NH
P
P
H2O
CH3
A.
O
O
O
O
RNH2
H2C
H2C
O
O
HO
OH
OH
OH
HO
O
O
O
RNH2
2
3
HO
OH
HO
OH
RNH2
-O
O-
O
O-
-O
1
P
P
B.
O
O
O
H2C
OH
H2C
OH
OH
OH
HO
O
HO
OH
4
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 a-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.

A. Munanairi et al. / Carbohydrate Research 342 (2007) 2575–2592
2577
2. Results
tants, but also by the transition of the N-glycoside to the
Amadori rearrangement product and beyond. The ini-
tial reaction of R5P and amine (deprotonated form) to
the N-glycoside is an equilibrium process as defined by
the equation and Keq below.
2.1. Rate of sugar consumption
The rate constants reflecting the loss of R5P with time in
reactions with glycine (Gly), L-alanine (Ala), glycylgly-
cine (Gly-gly), L-asparagine (Asn), b-alanine (b-Ala),
N-acetyl-L-lysine (NAcLys), and N-acetyl-L-arginine
(NAcArg) at pH 8.0 and 37 ꢁC are shown in Table 1.
(The NAcArg value listed is at best an estimate, given
the slow reaction time, that is, less than 20% reacted
over 18 days.) All determinations at this pH were per-
R5P þ NH2–XꢀPhosphoribosamino–X
½Phosphoribosamino–Xꢁ
Keq
¼
½R5Pꢁ½NH2–Xꢁ
Employing 1H NMR spectroscopy to determine the loss
of R5P from a NAcLys-containing solution at pH 10.5,
we observed rapid equilibrium (within minutes) with a
Keq = 7.6 Mꢀ1 (37 ꢁC). This value is comparable to
Keq = 2.5 Mꢀ1 determined by Schendel et al.16 for a
R5P/NH3 reaction and to Keq = 5.0 Mꢀ1 for a R5P/
tris(hydroxymethyl)aminomethane reaction obtained in
our laboratory (unpublished data). Given the neutral
pH of our reaction systems, only 0.3% of R5P would
be converted at equilibrium to the N-glycoside. Thus,
while the concentration of deprotonated amine is a com-
ponent of our rate equation, large-scale R5P and
NAcLys loss is primarily impacted by the rate of the
subsequent step to the Amadori product. The rate con-
stant (k*) listed in Table 1 is essentially a combined rate
constant for the first two steps, equivalent to k1k2/kꢀ1 or
Keqk2. This translates into a k2 value of 4.7 hꢀ1 for the
R5P/NAcLys system.
1
formed by measuring the intensities of the H peaks (a
and b) of the C-1 hydrogens of R5P. An LC/MS selec-
tive ion-monitoring (SIM) method following the m/z
97 fragment of R5P13 was also employed for some
amines, and the results (not shown) were in agreement
with the given NMR data. The molecular rate constants
(k) reflect the kinetics of the reaction between the R5P
and amine molecules while the adjusted k* corrects the
value for actual deprotonated amine concentrations.
The kinetic treatment of the early Maillard sequence is
based on the reaction series shown in Scheme 2. For
simplicity, the kinetics assumes irreversible steps to
generate the Amadori product14 and the a-dicarbonyl
compounds, an assumption that does not appear to be
completely true.15 (A slow reversal of these steps
occurs.)
It is important to note that the kinetics of the system
is not only impacted by the collision rate of the two reac-
Confirming the early steps are rate limiting to the
overall Maillard series, the trend in the ‘R5P-loss’ rate
Table 1. Kinetic data for the rate of sugar loss in a Maillard reaction with amine at 37 ꢁC
Sugara
Aminea
Technique
pKa (at 37 ꢁC)
pH
k (Mꢀ1 hꢀ1
)
kb (Mꢀ1 hꢀ1
)
b
R5P
R5P
R5P
R5P
R5P
R5P
R5P
R5P (2X)
R5P
Gly-gly
L-Asn
Gly
L-Ala
1H NMR
1H NMR
1H NMR
1H NMR
1H NMR
1H NMR
1H NMR
1H NMR
1H NMR
LC–MS
7.8
8.6
9.46
9.7
8.0
8.0
8.0
8.0
8.0
8.0
8.0
8.0
7.5
7.5
7.5
7.5
7.5
0.6
0.35
0.26
0.07
0.31
0.20
0.005
0.63
0.32
0.32
0.021
0.015
ꢂ0
1.0
1.8
7.8
3.6
28
b-Ala
9.95
L-NAcLys
L-NAcArg
L-NAcLys
L-NAcLys
L-NAcLys (0.5X)
L-NAcLys
L-NAcLys
L-NAcLys
10.25
12.25
10.25
10.25
10.25
10.25
10.25
10.25
36
89
113
180
180
11.8
10.7
ꢂ0
R5P
Ribose
dR5P
Deoxyribose
LC–MS
1H NMR
LC–MS
a Sugar concentrations were 0.050 M and amine concentrations were 0.10 M except where noted.
b Gly pKa at 37 ꢁC obtained from a literature value while L-NAcArg was estimated from a literature value at 25 ꢁC. pKa’s for Gly-gly, b-Ala, L-Ala,
L-Asn, and L-NAcLys at 37 ꢁC were determined by titration. The ribose and deoxyribose experiments contained equimolar amounts of inorganic
phosphate.
k1
k2
k3
α-Dicarbonyl
Phosphates &
Methylglyoxal
NAcLys + R5P
N-Glycoside
Amadori Product
k-1
NAcLys
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.15

2578
A. Munanairi et al. / Carbohydrate Research 342 (2007) 2575–2592
constants (either k or k*) with the various amino acids
correlates well with the corresponding rate constant ser-
ies calculated for the increase in 280 nm absorbance and
browning.1 As can be seen, there is variation in the k*
values for R5P loss ranging from the lower Gly-gly
value (1.0 Mꢀ1 hꢀ1) to higher b-Ala and NAcLys values
(ꢂ27 Mꢀ1 hꢀ1). An observed direct relationship between
the pKa of the amine and k* suggests that a reaction sub-
sequent to the initial deprotonated amine attack is
favored by a protonated form of the amine/imine.
Conforming to the general Maillard reaction (see
Scheme 3), this likely reflects the need for the Schiff base
to be protonated in order to proceed to the enaminol
and then on to the Amadori product. (This assumes
the pKa of the Schiff base parallels the pKa of the amine.)
This reaction would pull the initial equilibrium reaction
(most likely favoring the sugar and free amine at neutral
pH) toward the Amadori product and beyond. An alter-
native explanation for the variation observed in k* is
that a steric effect may play a role in the initial attack
with the long-tailed, less hindered NAcLys and b-Ala
having a greater effective collision frequency than the
more hindered amino acid reactants. This could be the
explanation for why L-Ala, with an a-carbon substitu-
ent, has a smaller k* than L-Gly and b-Ala.
Additional evidence supporting a second contributor
to the initial steps of the Maillard reaction is supplied
by pH studies. Given the accepted general pathway
states that a deprotonated nitrogen nucleophile is
required for attack, the reaction should proceed more
rapidly at higher pH. This appears to be the case for
Gly (data not shown), but not for NAcLys, which causes
more R5P loss at pH 7.4 than at pH 8. This supports
the suggestion that the initial formation of the Schiff
base is an equilibrium reaction favoring the free sugar
and amine, and that further progression in the path
(leading to the significant losses of sugar observed by
1H NMR spectroscopy or LC/MS) requires a low pH-
favoring reaction that drives the reaction through an
irreversible step(s).
R5P concentration, it is difficult to comprehend how
the steps contributing to the rate constant could be any-
thing other than first order (see below and Section 3).
One likely contributor to this effect is the higher levels
of potentially catalytic phosphate that accompany the
increase in R5P concentration. A comparison of the
rates of 280 nm absorbance increases for a reaction of
glycine in either a glyceraldehyde versus glyceralde-
hyde + Pi system or a ribose versus ribose + Pi system
indicates the catalytic effect of phosphate to be approx-
imately 10–12-fold per 1 mequiv of soluble phosphate
(data not shown). In our system where R5P is doubled
(to 100 mM), this would account for only about one-
third of the observed rate constant increase. Further
explanation may require a full elucidation of the mech-
anism of the inter- and intramolecular phosphate
catalysis.
A similar characterization of a reaction between
NAcLys and D-ribose and Pi (equimolar D-ribose and
Pi to simulate the concentrations of each in R5P) gave
a k = 0.021 Mꢀ1 hꢀ1 and a k* = 11.8 Mꢀ1 hꢀ1. When
interpreted in comparison to the R5P experiment, an
approximately 15-fold rate enhancement for the disap-
pearance of the sugar occurs when a phosphate group
is covalently attached to the ribose sugar. This is of
the range typically quoted for catalytic effects due to
proximity;17 however, a second explanation may be that
the phosphate group encourages higher levels of the acy-
clic R5P form, the form that is believed to be the true
initial reactant (along with the deprotonated amine) in
the Maillard reaction. A phosphate group existing on
C-5 would increase the level of the acyclic form due to
the inability of the phosphorylated sugar to take the
more stable pyranose form and possibly to other steric
factors (i.e., steric hindrance adding stress to the fura-
nose structure). The cited levels of acyclic forms of
D-ribose and R5P are 0.05%7 and 1%,6 respectively,
although it should be mentioned two different tech-
niques were used to obtain these values, and discrepan-
cies have frequently been seen among literature values
for acyclic form levels. In order to examine this in a dif-
ferent manner, the relative rates of color formation for
glyceraldehyde + Pi versus glyceraldehyde 3-phosphate
(G3P) with Gly were determined. Since both glyceralde-
hyde and G3P exist only in the acyclic forms, any rate
enhancement must be solely due to a proximity effect
of the intramolecular phosphate. In fact, G3P generated
280-nm products at a sixfold greater rate than glyceral-
An interesting effect on the rate constants is observed
when R5P concentrations are increased while holding
amine levels constant. While altering the initial amine
concentration does not result in an apparent change in
the rate constant, increasing the initial level of R5P in
solution yields significantly higher rate constants for
R5P loss (see Table 1). While this evidence may be inter-
preted as the reaction being of higher order relative to
O
H+
H+
OH
H
O
H
H
N
HO
HO
C
H
H
R
N+
N
R
C
H
N
C
C
H
C
H
C
C
H
R
R
C
H
H+
H+
H
H
Sugar
Amine
Schiff Base
Enaminol
Amadori Product
Scheme 3. The general reaction mechanism for the formation of the Amadori product from a sugar and an amine.

A. Munanairi et al. / Carbohydrate Research 342 (2007) 2575–2592
2579
0.12
0.1
dehyde in the presence of intermolecular phosphate. It is
likely that a proximity effect plays a similar role in the
intramolecular phosphate catalysis of the R5P Maillard
reaction; however, it is still unclear what portion it con-
tributes to the overall increase in rate.
0.08
0.06
0.04
0.02
0
With a k = 0.015 Mꢀ1 hꢀ1 in a reaction with NAcLys,
the Maillard activity of dR5P is considerably less (factor
of 10–20) than that of R5P. This is expected for two rea-
sons. First, replacement of the bulky –OH group with a
hydrogen at the C-2 position favors dR5P taking the
unreactive cyclic form versus the reactive acyclic form.
Second, the lack of the hydroxyl group on C-2 prevents
the N-glycoside from transitioning to an Amadori com-
pound by a normal Maillard reaction scheme. The N-
glycoside compound generated from dR5P is unable to
convert to the normal enaminol intermediate or the sub-
sequent C-2 carbonyl group, steps which are part of the
Maillard reaction sequence. The fact that dR5P does
generate Maillard-like reaction products (see below)
suggests that the molecule somehow overcomes the C-
2 ‘blockage’ to proceed in reactivity.
The loss of deoxyribose from a system containing
NAcLys and Pi at pH 7.4 and 37 ꢁC is at least an order
of magnitude less rapid than the comparable ribose/
NAcLys/Pi system. Over a 150 h incubation period at
37 ꢁC, no statistically significant loss of deoxyribose
was noted by analysis using LC/MS. Although not de-
tected, some deoxyribose indeed reacted as evidence of
the formation of an N-glycoside/Amadori product was
observed by liquid chromatography/mass spectroscopy
(LC/MS) (see below). Like dR5P, this slower rate is ex-
pected due to the combination of lower acyclic levels
(unlike dR5P, deoxyribose also can assume the more
stable pyranose form) and the lack of the hydroxyl
group on the C-2 needed for normal Maillard reaction
progression. The rate is considerable less than that ob-
served for dR5P, suggesting an enhanced reactivity pro-
moted by an attached phosphate group.
0
20
40
60
80
100
120
140
Time (h)
Figure 1. A comparison of the rates of disappearance of NAcLys (j)
and R5P (m) 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.
a-dicarbonyl forms. Under these conditions (pH 7.5;
37 ꢁC), a level of approximately 0.015 M NAcLys is tied
up in the N-glycoside/Amadori product as molecules
flow through the initial portion of the Maillard reaction.
Ultimately, there are some amine products that result in
the amine compound being consumed in the reaction;
however, it is not at a 1:1 ratio with R5P.
The overall amine (NAcLys or b-Ala) loss from solu-
tion was related to the initial level of R5P in solution. A
doubling of the R5P concentration resulted in an
approximately 50% increase in the amine loss. The ini-
tial concentration of amine appeared to play less of a
factor in the absolute amount of disappearance of amine
from solution, for example, a 0.050 M NAcLys solution
versus a 0.10 M NAcLys solution of the same R5P con-
centration exhibited approximately equal overall amine
losses from solution. Together these results suggest the
ultimate loss of amine from solution is related primarily
to the synthesis of amine-consuming, post-Amadori
R5P products. These products apparently are made in
limited amounts in comparison to non-amine-consum-
ing, post-Amadori R5P products. The data suggest less
than 30% of post-Amadori products involve an amine
attachment.
2.2. Rate of amine consumption
The disappearance of amine from solution does not stoi-
chiometrically couple with the loss of R5P. Figure 1
shows the loss of R5P versus the loss of NAcLys in a
reaction at 0.10 M NAcLys and 0.050 M R5P at pH
7.5 and 37 ꢁC using peak intensities and a SIMS method
of LC/MS13 to determine NAcLys and R5P concentra-
tions, respectively. (The fluorescamine method for deter-
mining free NAcLys amine groups yielded similar
values.) As can be seen, the loss of NAcLys from solu-
tion is approximately three times less than the absolute
decrease in R5P concentration. This effect is explained
by the reaction mechanism (see Scheme 1) where
NAcLys is consumed during the initial attack of the
amine onto the sugar but then is released after the Ama-
dori rearrangement as the sugar converts to one of the
The amine loss in an NAcArg/R5P system as mea-
sured by an LC/MS method or 9,10-phenanthrenequi-
none modification technique was insignificant over a
300 h incubation period at 37 ꢁC. This slow reaction rate
corresponds with the negligible loss of R5P over this
time frame and indicates a low reactivity of free Arg side
groups with initial free sugar. Any observable Arg mod-
ification in combined amine systems, therefore, appears
to be the result of a reaction between Arg and a product
generated by the reaction of R5P and an amine of lower
pKa.
The rate of loss of amine from an incubation of
NAcLys, D-ribose, and Pi is significantly less than that
seen for the R5P system. After 145 h of reaction, only
0.008 M of NAcLys was observed to be modified (data

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A. Munanairi et al. / Carbohydrate Research 342 (2007) 2575–2592
800
700
600
500
400
300
200
100
0
not shown) in comparison to approximately 0.021 M of
the amine when reacted with R5P. Although slower, the
relative ratio of D-ribose used to NAcLys modified
(1.5:1) at a point in the reaction where approximately
one-third of the sugar consumed is similar to the 2:1
ratio seen for R5P in the R5P/NAcLys reaction. This
suggests that the slower initial reaction of ribose to the
Amadori product is accompanied by a proportionally
slower reaction from the Amadori product (i.e., the step
recycling the amine). Thus, the catalytic effect of the
intramolecular phosphate in R5P is enhancing both
the pre-Amadori and post-Amadori reactions by rela-
tively similar levels.
Over a long period of time (ꢂ150 h), amine losses in
the dR5P/NAcLys system were approximately the same
as the amine consumption in the R5P/NAcLys system,
22% versus 21%, respectively. As opposed to the R5P
reaction, the loss of NAcLys is at a 3:1 stoichiometric
ratio to the loss of dR5P, implying that several NAcLys
residues ultimately become conjugated to a single dR5P
molecule. In a traditional Maillard reaction, the amine is
released upon the formation of one of two dicarbonyl
compounds via a rearrangement step. Lacking the C-2
hydroxyl group, dR5P cannot undergo this rearrange-
ment and thus appears to stay attached. Additional
amine molecules apparently must subsequently attach
to this N-glycoside (see below).
0
50
100
150
200
Time (h)
Figure 2. The generation of N-glycoside/Amadori products for R5P
(m) and dR5P (j) 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.
ide/Amadori compounds have been reached. This
likely represents the attachment of the amine onto sub-
sequently formed a-dicarbonyls and/or other products
(temporary and permanent) later in the sequence. Mod-
eling of the kinetics of the reaction series (see Section 3)
gave a k3 value of 0.03 hꢀ1 and a maximum Amadori
product level of 0.016 M.
A R5P/NAcLys reaction performed in the absence of
oxygen did not significantly alter the N-glycoside/Ama-
dori product ion peak pattern (data not shown) in com-
parison to the reaction shown in Figure 2 performed in
the presence of oxygen. The rates of loss of R5P and
NAcLys were also comparable. As expected, this indi-
cates the movement of molecules through the early por-
tion of the Maillard reaction, that is, up through and
including the transition of the Amadori product to the
next compound, is a series which proceeds without oxy-
gen involvement.
The loss of amine from the deoxyribose/Pi/NAcLys
system was insignificant over a 165-h incubation period,
confirming with the deoxyribose loss studies that the
Maillard reaction of deoxyribose is comparably slow.
In a manner similar to the ribose/R5P comparison, the
relative rates of the deoxyribose versus dR5P systems
point to a large catalytic effect of intramolecular phos-
phate on this series of reactions.
2.3. Rate of N-glycoside/Amadori product formation
The formation of N-glycoside/Amadori-like com-
pounds from dR5P is included in Figure 2. As can be
seen, there is a slower transition to a maximum level
in comparison to the R5P reaction and over the course
of the experiment, the ion peak areas of the chromato-
grams isolated on the selected Amadori/N-glycoside
mass (m/z 385 [M+H+] for dR5P) are not as intense.
A relatively slower initial reaction rate (with a similar
rate of transition away from this form) would account
for this rate pattern although it is important to note that
the current Maillard reaction mechanism would not
allow for an Amadori product to be formed in dR5P
where C-2 has lost a necessary hydroxyl participant of
the rearrangement. It is possible that the level seen is
reflective solely of the N-glycoside only, although it
is anticipated that equilibrium levels of the N-glycos-
ide would be established quickly, whereas our data indi-
cates a steady increase of this compound(s) over the first
24 h.
The relative concentration of the combined N-glycoside/
Amadori compounds generated by a NAcLys/R5P reac-
tion was determined by LC/MS via an extracted-ion
method measuring the peak areas under the chromato-
gram of the m/z 401 0.5 [M+H+] signal. (The N-gly-
coside and Amadori rearrangement compounds have
identical molecular masses. Although it is speculated
that the level of N-glycoside is of minor concentrations
(see Section 2.2), we do not have conclusive evidence of
this and thus will group the two species together for
these discussions.) The relative amounts of the N-glycos-
ide/Amadori compounds generated in the reaction with
time are shown in Figure 2. As can be seen, the concen-
tration maximum is reached at approximately 30–40 h
while the NAcLys levels continue to decline (see
Fig. 1) until about 5 days. This signifies that NAcLys
molecules continue to attach to R5P-derived com-
pounds even after the maximum level of the N-glycos-

A. Munanairi et al. / Carbohydrate Research 342 (2007) 2575–2592
2581
Table 2. Estimated time to reach 90% of maximum N-glycoside/
30
25
20
15
10
Amadori levela
System
Time (h)
R5P and NAcLys
dR5P and NAcLys
15
40
Ribose + Pi + NAcLys
Deoxyribose + Pi + NAcLys
105
>200
a The R5P, dR5P, and ribose values were determined graphically while
the deoxyribose level was estimated via a calculation from initial rate
patterns.
5
0
0
20
40
60
80
N-Glycoside/Amadori formation in the non-phos-
phorylated ribose or deoxyribose systems took longer
to reach a steady state/maximum level in comparison
to R5P or dR5P (see Table 2). N-Glycoside/Amadori
production in the ribose/Pi system took 105 h to reach
90% of a maximum value while the deoxyribose/Pi sys-
tem was only at 50% of an estimated peak level at this
same time point. The production of an N-glycoside/
Amadori-like compound in the deoxyribose system
was observed even when no statistically significant loss
of deoxyribose or NAcLys from solution was detected.
This implies the signals recorded for this compound rep-
resent relatively small concentration levels.
Time (h)
Figure 3. The percent phosphate (Pi) 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 (e); b-Ala (j);
L-NAcLys (n); L-Ala (r); Gly-gly (m); Gly (h).
to phosphate removal. The phosphate continues to be
cleaved from the pentose nucleus, even after all R5P
has disappeared from solution, again indicating that
R5P is converted to a second phosphate-containing
intermediate. The reaction of poly(lysine) with R5P
exhibits a greatly enhanced rate of phosphate cleavage
even at a lower molar level of amine. This is not ex-
plained solely by its relatively low pKa (9.5) as its phos-
phate hydrolysis rate is significantly greater than amines
of comparable pKa (such as Asn or Gly-gly). The close
proximity of several amines around the reaction site
must promote a higher rate of the cleavage reaction.
The loss of a C-5 phosphate from a ribose-like sugar
has been proposed by Hauck et al.10 to be the result
of a series of reactions initiated when a sugar intermedi-
ate contains a C-3 carbonyl group next to a C-4 hydr-
oxyl group, leading to the ability to form an enediol
between these two carbon centers (Scheme 4).
Along with the release of Pi from R5P into solution
during the Maillard reaction, 31P NMR analysis de-
tected the formation of several new compounds exhibit-
ing peaks 0–0.5 ppm downfield from the phosphate peak
of R5P (see Fig. 4). It is believed these represent the
phosphorylated forms of the Amadori product, the a-
dicarbonyl compounds, and other intermediates as the
R5P sugar core undergoes the various Maillard altera-
2.4. Hydrolysis of phosphate and formation of new
phosphate compounds
The reaction between R5P and amines eventually leads
to the cleavage of the phosphate group from the ribose
nucleus. Figure 3 shows the rate of phosphate hydrolysis
for several amines at 37 ꢁC and pH 8.0 as monitored by
31P NMR. There is less correlation of the rate of Pi for-
mation with the pKa of the attacking amine for this ser-
ies, although it is likely that the exceptionally slow
phosphate cleavage rates of some amines like NAcArg
(hydrolysis rate equivalent to the background rate) is
likely tied to their high pKa. (The rate of R5P hydrolysis
without amine at pH 8 was approximately 1.5% over a
70 h time period.) The rate of phosphate cleavage is di-
rectly proportional to both the initial concentration of
the amine and the initial concentration of R5P present
in the mixture. A lag time of 5–10 h is typically observed
for the first evidence of phosphate cleavage; this suggests
an initial transition of R5P to a second compound prior
O
OH
OH
O
OH
O
O-
C
CH
H
O
P
O-
C
C
H
CH
H
O
P
C
O-
O-
2-
HPO4
O
O
O
OH
C
CH2
C
C
C
CH3
Scheme 4. A proposed reaction scheme of Hauck et al.10 for the hydrolysis of an intramolecular 5-phosphate moiety from a pentose sugar containing
a carbonyl group at the C-3 position.

2582
A. Munanairi et al. / Carbohydrate Research 342 (2007) 2575–2592
metric ratio, suggesting that a reaction of dR5P is asso-
ciated with a simultaneous or nearly simultaneous
release of phosphate.
2.5. Absorbance changes
The relative rates of absorbance increases at 280 nm and
420 nm of R5P reacting with amino acids/dipeptides
have been previously reported1 to show a partial corre-
lation to the pKa of the attacking amine. The rate of UV
absorbance increase (at 280 nm) for the R5P/amine
(NAcLys) reaction is faster than other common sug-
ars1,2 and for the comparable dR5P/amine, ribose +
Pi/amine, and deoxyribose + Pi/amine systems (see
Fig. 5a). It is important to note that while the ribose/
Pi reaction with NAcLys is less than 15-fold less rapid
than the reaction of R5P in terms of sugar loss, the rate
of absorbance increase at 280 nm is only approximately
threefold slower. Thus, the comparative color develop-
ment for these two systems is not based solely on the ini-
tial rate of attack of the amine, but more likely to the
speed of unique products formed once the reaction pro-
gresses beyond the Amadori product.
Figure 4. A series of three 31P NMR spectra following the reaction
between NAcLys and R5P at pH 7.4 and 37 ꢁC for 5 days. (Spectra are
offset for clarity.) The reaction begins with a single R5P peak
[represented by #] at approximately +4.5 ppm and, with time, Pi
[represented by ] at approximately +3.2 ppm. Compounds giving
*
In addition, as can be seen in Figure 5b, R5P is unique
among this grouping in its production of an obvious
320 nm product along with a primary UV-absorbing
product in the 280–290 nm range. The reaction of R5P
with numerous amino acids/dipeptides all show the
320 nm shoulder to some degree, with some (like Gly)
having approximately similar absorbances at 320-nm
versus 280 nm, while others (like Asn) have a relatively
less intense 320-nm peak (see Fig. 5c). The 320-nm peak
appears to be dependent on the ratio of R5P to amine in
solution, a solution containing a R5P-to-NAcLys con-
centration ratio of 1.5:1 yields a dominant peak at
320 nm with a less intense 290-nm shoulder. Previous
reports have indicated that 2,3-a-dicarbonyls (non-phos-
phorylated) have absorbance maxima at 310 nm.10 In
comparison, ribulose 5-phosphate has an absorbance
maximum of 280 nm and hydroxymethylfuranone (4-hy-
droxyl-5-methyl-3(2H)-furanone, HMF), a spontaneous
product of ribulose 5-phosphate degradation, has a
reported absorbance maximum of 285 nm.10 The lack
of significant 310–320 nm absorbance indicates the reac-
tion series is deviating from the normal progression
through the a-dicarbonyl forms or that it is rapidly tran-
sitioning through these dicarbonyl intermediates. For
example, Asn has been reported to react with glucose
in an acrylamide-producing, decarboxylation reaction
that ultimately results in a fructosamine product.19,20
It is highly possible the same series can happen for the
reaction of Asn with R5P, forming a ribulosamine phos-
phate product with reduced conversion to an a-dicar-
bonyl form.
downfield signals (+4.6 to +5.0 ppm) were generated.
tions. In a reaction between R5P and ammonia, the
Maillard product phosphoribosamine exhibits a phos-
phate peak that is positioned as a shoulder (i.e., less than
0.05 ppm downfield) on the R5P phosphate peak. The
phosphate resonance of ribulose 5-phosphate, the C-2
ketose isomer of R5P and a possible product of our
reactions, is well differentiated from the R5P resonance
at 0.4 ppm downfield.18 It is likely that other com-
pounds existing in a C-2 ketose form (e.g., the phos-
phorylated forms of the Amadori product and the
a-dicarbonyl compounds) would be shifted downfield
in a similar fashion.
When the reaction was performed in the absence of
oxygen, the rate of phosphate cleavage was approxi-
mately 50% more rapid than when performed in the
presence of oxygen. This strengthens the evidence that
the hydrolysis of phosphate is a non-oxidative process.
(The formation of carboxymethyllysine (CML) via an
oxidative pathway (see Section 4.6) would generate 3-
phosphoglycerate (3PG) in place of Pi; 3PG yields a
phosphate peak in 31P NMR that is superimposable
upon the R5P phosphate resonance.18)
A similar analysis was performed using 31P NMR to
characterize the loss of phosphate from dR5P during a
Maillard-like reaction. Over a 120-h period, a solution
of dR5P and NAcLys at pH 7.5 and 37 ꢁC results in
the hydrolysis of 18% of its phosphate as Pi. (The com-
parable solution using R5P shows cleavage of 20% of its
intramolecular phosphate.) The loss of dR5P and the
formation of Pi appear over time to be at a 1:1 stoichio-
The availability of oxygen in the R5P-amine reactions
determines the development of the UV-absorbing com-

and L-Ala) exhibits this absorbance peak, while other
A. Munanairi et al. / Carbohydrate Research 342 (2007) 2575–2592
2583
50
40
30
20
10
0
a
25
20
15
10
5
250
275
300
325
350
375
400
0
Wavelength (nm)
0
10
20
30
40
50
60
Time (h)
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
(j) and absence (m) of oxygen. The incubations were held at 37 ꢁC for
5 days.
b
c
6
5
4
3
2
1
0
amines (e.g., glycine and L-lysine (non-acetylated)) fail
to produce significant amounts of the UV-absorbing
compound. The difference may be in the ability of some
amines to undergo additional rearrangement reactions
between the amine group and the a-carbon (such as
decarboxylation reactions of the a-carboxylate
group21,22) not possible with other amines including
NAcLys. This would thus reduce the transition of the
intermediate to the 350-nm-absorbing compound. A
4-day dR5P/b-Ala reaction performed in the absence
of oxygen (data not shown) gave similar 350-nm absor-
bance to that performed in the presence of oxygen, but
a decrease in the 280-nm absorbance was noted.
In a Maillard reaction with NAcLys and a phosphate
catalyst, ribose yielded relatively high absorbance in the
UV range with A280 rate increases of approximately
threefold less than the corresponding R5P system (see
Fig. 5). Analysis of the spectra generated by a ribose
reaction indicates compounds with absorbance maxima
of approximately 280 nm and 300 nm were generated. In
a fashion similar to other pentoses, D-ribose undergoes
the Maillard reaction to produce a-dicarbonyls23 and
thus should also exhibit an absorbance peak at
310 nm. Hauck et al.10 found that a 1-deoxy 2,3-dicar-
bonyl compound (a potential dicarbonyl formed from
ribose) converts through rearrangement to HMF. A rel-
atively rapid conversion of the dicarbonyl to HMF
would exhibit a lower 310-nm absorbance and a high
285-nm absorbance (of HMF). The presence of an
absorbance maximum for the ribose system in the
280–290 nm region suggests that this may be occurring
(see also Section 2.7).
250
270
290
310
330
350
370
390
Wavelength (nm)
4
3.5
3
2.5
2
1.5
1
0.5
0
250
300
350
400
Wavelength (nm)
Figure 5. (a) Rate of absorbance increase at 280 nm for R5P (m),
ribose + Pi (n), dR5P (j), and deoxyribose + Pi (h) with NAcLys at
pH 7.4 and 37 ꢁC. (b) Absorbance spectra for R5P (m), ribose + Pi
(n), dR5P (j), and deoxyribose + Pi (h) 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 (m) and Glu (e), 7.3 h for Asn (j), and 6 h for
Gly-Gly (r).
pounds. As shown in Figure 6, the presence of oxygen in
the system reduces the overall UV absorbance, creating
a spectrum with broad absorbance maxima of 280 nm
and 310 nm. The reaction under anaerobic conditions
favors the production of a compound of high absorptiv-
ity at 305 nm and a lower absorbing compound at
approximately 360 nm.
The absorbance maximum at 350 nm for dR5P in
reaction with NAcLys (see Fig. 5b) is interesting. The
reaction of this sugar with some amines (e.g., b-Ala
Comparatively, the deoxyribose/Pi/amine (NAcLys)
reaction generated a small rate of UV absorbance in-
crease (10-fold less than ribose/Pi/amine) with time over
a 150-h incubation period at 37 ꢁC. These data and our
results in the visible range are in agreement with the
study of Wondrak et al.,12 which evaluated the brown-

2584
A. Munanairi et al. / Carbohydrate Research 342 (2007) 2575–2592
ing (at 420 nm) of ribose versus deoxyribose solutions in
reaction with x-amino acids. The spectrum at 5 days
indicates the presence of a 260–280 nm absorbing com-
pound along with a 310–330 nm absorbing compound.
generator, exceeding the total AGE fluorescence capa-
bility of R5P by a factor of approximately fifteen (see
Fig. 7). When compared to a deoxyribose/Pi system,
dR5P generates fluorescent compounds at rates nearly
60-fold higher. A portion of this increased rate versus
that of the deoxyribose/Pi system is likely attributed to
the more rapid initial reaction between the sugar and
the amine; however, a second contributor could be the
presence of the phosphate in directing the molecule to
an end-point fluorophore.
2.6. Fluorescence
The general Maillard reaction has been reported to
produce glycation products that exhibit fluorescence
characteristics and R5P is no exception. As reported
previously, a spontaneous reaction of R5P with amines
or proteins rapidly produces a total AGEs fluorescence
(360 nm excitation; 460 nm emission) (see Fig. 7) at a
faster rate than most other sugars such as glucose, glu-
cose 6-phosphate, and fructose.2 While the presence of
the phosphate bound onto the ribose nucleus appears
to greatly speed up the original consumption of the su-
gar and the production of UV products, the functional
group does not add significant advantage over intermo-
lecular inorganic phosphate to the production of total
AGE fluorophores (Fig. 7). After a brief lag period, mix-
tures containing amines and D-ribose with equimolar
soluble phosphate exhibit a higher fluorescence than
R5P. (This greater fluorescence by ribose in comparison
to R5P was reported in short-term experiments using
bovine serum albumin at 55 ꢁC, but the opposite was
observed at 37 ꢁC.2) Total AGE fluorescence with ribose
was found to be directly correlated with phosphate con-
centration added to solution. Since the relatively lower
fluorescence shown by R5P versus ribose/Pi is not due
to the original rate of amine attack or to the formation
of the Amadori product (both which are more rapid for
R5P than D-ribose/Pi), it is likely that R5P is incapable
of generating highly fluorescent products until after
phosphate hydrolysis.
2.7. Characterization of major products by NMR and
LC/MS
The Maillard reaction of amines with sugars is initiated
by the attack of the amine nitrogen onto the carbonyl
carbon to generate an N-glycoside (Schiff base), which
subsequently has the opportunity to undergo rearrange-
ment to form the Amadori product (see Scheme 1). The
transition of the free amine and free sugar to the N-gly-
coside is a reversible process which, as stated previously,
slightly favors the N-glycoside for the R5P/amine sys-
tem. At neutral pH, where deprotonated amine concen-
trations are relatively low, the amount of N-glycoside in
comparison to free sugar and free amine will be compa-
1
rably low. An H NMR investigation of dR5P with b-
Ala confirmed this process. Lacking a hydroxyl group
on the C-2 carbon, dR5P cannot undergo conversion
to the Amadori product via traditional Maillard rear-
rangement. The overall loss of dR5P from solution
was relatively slow. However, as shown in Figure 8,
1H NMR spectroscopy indicates a relatively rapid loss
of the resonance of the hydrogen signals of the C-2 car-
While R5P is relatively quick in comparison to other
sugars to produce fluorescence products, dR5P in a
Maillard reaction is a significantly greater fluorescence
2500
20000
0
15000
0
140
10000
5000
0
Figure 8. Portions of 1H NMR spectra of 0 h and 8 h reactions of
dR5P with b-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,
0
20
40
60
80
100
Time (h)
+2.05 ppm, and +2.35 ppm (shoulder) [represented by ] indicate the
*
Figure 7. The total AGEs fluorescence (emission = 360 nm; excita-
tion = 460 nm) generated in a reaction between a pentose sugar and
NAcLys at pH 7.5 and 37 ꢁC. The sugars employed are R5P (m), dR5P
(j), ribose and Pi (n), and deoxyribose and Pi (h). Inset: An expanded
view of R5P, ribose and Pi, and deoxyribose and Pi.
C-2 hydrogen signals (three sets stemming from the two ring hydrogens
and the a/b 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 a and
b forms).

A. Munanairi et al. / Carbohydrate Research 342 (2007) 2575–2592
2585
bon during the initial 8-h period of the reaction (37 ꢁC),
a period where no significant loss of C-1 hydrogen inten-
sity was noted. Clearly, the loss of the C-2 hydrogen res-
onance is due to exchange of these hydrogens with
deuteriums of the D2O solvent. The exchange of C-2
hydrogens also occurs without amine12 but at a three-
fold slower rate. Thus, either the amine is promoting
the proton-to-deuterium exchange in a catalytic process
(i.e., the amine is acting as a general base for removal of
the proton) or, more likely, the exchange results from a
transient binding of the dR5P with the amine and a sub-
sequent temporary rearrangement of the double bond to
expel a hydrogen (Scheme 5).12
Being a dead-end product for dR5P, the enamine can
only reverse in direction, ultimately releasing the free
amine and generating the free acyclic sugar. The fact
we detected insignificant short-term losses of the C-1
hydrogen (a chemical shift of the resonance would be
expected to occur when the nitrogen is attached) while
at the same time observing the loss of C-2 hydrogens
agrees with our speculations that the equilibrium levels
of the N-glycoside at neutral pH are small. Thus, we
propose the bulk of the MS ion signal observed for
our combination of N-glycoside/Amadori product (see
above) represents the Amadori product.
which eluted at a longer retention time of 3.6 min in
comparison to the majority of reactants and other prod-
ucts. First noted of its 280–290 nm absorbance at 15 h,
the compound grew in amount until 96 h, when its level
reached a stable value. (At this point the compound ac-
counted for approximately 20% of the overall 280–
290 nm absorbance.) Comparison to a pure standard
confirmed this product as HMF. This compound is be-
lieved to be formed by a reaction series (Scheme 6)
involving a 1-deoxy-2,3-dicarbonyl intermediate derived
via a traditional Maillard sequence and a subsequent
rearrangement that includes cyclization and dehydra-
tion.10,23 After an initial lag period of about 10 h,
HMF is generated at a rate of approximately 5–10%
of that of D-ribose consumed. Reaching a steady level
of 0.35 mM at a point in the reaction sequence (at
ꢂ96 h) when less than 20% of the D-ribose has been con-
sumed suggests HMF may be reacting to form a subse-
quent product (e.g., polymerization).
LC/MS characterization of the D-ribose/Pi/NAcLys
reaction also clearly indicated the oxidative production
of a compound with a molecular mass compatible with
that of a CML product (actually in this case CM-
NAcLys). The compound’s identity was confirmed by
an MS2 fragmentation pattern that was in agreement
(for the NAcLys version of the molecule) with that re-
ported for CML by Treelink et al.24 Unlike the forma-
tion of HMF, the synthesis of CML appeared to be
LC/MS analysis of the reaction of D-ribose/Pi and
NAcLys at pH 7.4 and 37 ꢁC with extended times shows
the development of a compound mass equal to 114 Da,
OH
OH
H
H
C
OH
O
H
D
N
H2O
H
N
CH
C
C CH
C
H
C
C CH
N
H
H
H
H
H
in D2O
OH
H
OH
H
H
C
O
N
C
C CH
CH
C
H
N
D2O
D
H
D
H
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.
O
N
HN
HC
HC
CH3
C
H2C
C
OH
O
CH OH
CH OH
O
O
R-NH2
a
c
b
d
HC
HC
OH
OH
HC
HC
OH
OH
C
O
HC
HC
OH
OH
CH3
R-NH2
O
HC
OH
CH2OH
CH2OH
CH2OH
CH2OH
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.10

2586
A. Munanairi et al. / Carbohydrate Research 342 (2007) 2575–2592
initiated without any significant lag period. The ion sig-
nal for CML continued to rise in intensity throughout
the 165 h of the reaction. Also observed was the produc-
tion of a compound with an appropriate mass for a car-
boxyethyl (CEL) moiety onto the NAcLys. This
occurred with a significant lag time of over 72 h, sug-
gesting its formation was on a slower time scale than
the CML product. We did not observe a furfural prod-
uct (furfural itself or a Schiff base conjugate of furfural)
observed of pentoses in Maillard reactions;23,25,26 how-
ever, previous studies indicate higher temperatures
may be required for its formation.
LC/MS analysis of the reaction of R5P with NAcLys
at pH 7.4 and 37 ꢁC with extended time identified the
development of several new products, many of which
are awaiting further characterization. The appropriate
m/z peak representing the production of an N-glyco-
side/Amadori product was observed immediately in the
LC/MS spectrum, and the intensity of this signal rose
rapidly to peak levels in a 1-day period (for NAcLys
and b-Ala). Within relatively short times (10 h), the pro-
duction of a compound with mass = [R–NH2 + 58] was
noted, which matched the molecular mass and fragmen-
tation pattern of the CML product generated in the
D-ribose/Pi/NAcLys system. The CML product was
detected early in the reaction (at 7.5 h), and it continued
to be formed at a linear rate throughout the 150 h of
the experiment. Comparison of the intensity of the ion
peak areas for this signal in the R5P versus D-ribose/Pi
systems indicates CML is generated at approximately
the same rate despite R5P loss occurring at a signifi-
cantly faster pace. Again mirroring the comparative D-
ribose reaction, a CEL product was also generated after
a noted lag period (>48 h). It is believed that the synthe-
sis of a carboxymethyl moiety stemming from either
ribose or R5P onto NAcLys was via an oxidative
mechanism involving a C-2–C-3 enediol as proposed
by Smith and Thornalley.14 Confirmation of the mecha-
nism of synthesis of the CEL group awaits further anal-
ysis of a methylglyoxal intermediate.
presence of oxygen. Representing oxidized products of
R5P, the presence of these compounds suggests the con-
ditions of the incubation and analytical system had not
fully excluded oxygen. While there may have been other
products of minor ion intensity, the major MS signal no-
vel to the oxygen-free reaction was compatible with a
compound of a mass equivalent to [R–NH2 + 96]. The
compound had an absorbance in the 320 nm range
and little absorbance in the 280 nm region. As will be
discussed below, analogous compounds were discovered
in the dR5P/amine system (under aerobic conditions).
The [R–NH2 + 96] compound represents the attachment
of a C5H6O2 group onto the deprotonated form of the
amine. One candidate for this compound is an imine
derivative of HMF. This could result from the initial
isomerization of R5P (promoted by the general base ac-
tion of an amine) to ribulose 5-phosphate (Ru5P), which
can undergo spontaneous dephosphorylation, a rear-
rangement to generate a furanone ring, and then an at-
tack of the amine onto the ring carbonyl. We consider
this reaction series unlikely, however, as we fail to see
any evidence of HMF itself in a R5P-derived reaction.
(Also, a HMF-containing reaction system failed to rap-
idly react with an amine to generate this compound.) We
favor instead an imine conjugate of a triketone previ-
ously noted in Maillard reactions.27 The triketone is
speculated to result from spontaneous dephosphoryl-
ation of a 2,3-a-dicarbonyl compound.
In the dR5P reaction with NAcLys, LC/MS analysis
revealed the production of several new compounds,
implying that while the loss of dR5P from solution is
slow, the lack of the C-2 hydroxyl group does not fully
impede some conversion of the molecule to future prod-
ucts. (A practically identical set of products was
achieved in a reaction of dR5P with b-Ala; for clarity
the masses given will be only for the NAcLys reaction.)
This has been reported previously for solutions both
with and without amine.12 The likely explanation for
how the C-2 barrier is overcome is via a rearrange-
ment of the double bond initially formed between C-1
and C-2 (Scheme 5) to generate an Amadori-like C-3
ketose (Scheme 7). This transition to the C-3 ketose
can then, in the manner of R5P, lead to a variety of
other reactions including dephosphorylation. Occur-
ring in a manner similar to R5P (Scheme 4) by the estab-
lishment of the carbonyl at C-3, phosphate hydrolysis
A reaction of a R5P/NAcLys system performed in the
absence of oxygen gave some signals observed in the aer-
obic system including the CML and CEL products.
After a 5-day incubation, the CML signal was substan-
tially reduced (at only 15%) of that observed during a
comparable R5P/NAcLys reaction performed in the
HO
OH
H
H
H
C
H
O
C
H
C
N
C
H
N
H
N
C
C
C
H
C
H
C
H
H
H
H
Scheme 7. Tautomerization reaction of dR5P originating from an enamine (Scheme 5) leading to an Amadori-like compound.

A. Munanairi et al. / Carbohydrate Research 342 (2007) 2575–2592
2587
8
a
b
O
C
H
H2C
O
6
4
N
N
H2C
CH2
H2C
CH2
C
C
HO
O
HO
O
2
0
Scheme 8. Structures of two products generated from a reaction at
37 ꢁC and pH 7.4 between dR5P and b-Ala. (a) Four-minute retention-
time compound, an amine derivative of 5-MP, with an absorbance
maximum at 260 nm. (b) Six-minute retention-time compound, N-
methyl-2-formylpyrrole, with an absorbance maximum at 290 nm.
0
1
2
3
4
5
6
7
8
9
Time (min)
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.
age upon irradiation, has been shown to be formed
through an initial hydrogen abstraction from C-1 of a
deoxynucleotide (in DNA) by oxygen leading to super-
oxide.28,29 A scheme for the generation of the amine
form of 5-MP would not likely follow a similar path
as for the formation of 5-MF29,30 as the nitrogen
becomes incorporated in the ring rather than as a
double-bonded atom at C-1. It is more likely that the
amine ultimately attaches at a newly formed carbonyl
at C-4, cyclizes, and then rearranges to the 5-MP form.
The reaction mechanism is presently unclear. Another
product of DNA oxidative damage (via H2O2 and chro-
mium) is furfural.30 A scheme involving hydrogen
abstraction at C-5 leading to furfural generation has
been proposed by Mazzer et al.30 While we did not ob-
serve furfural in our b-Ala/amine reactions, the pyrrole
compound with a longer retention time (at ꢂ6 min) is
similar in structure to furfural. The mechanism for its
formation is speculated to be via a series of reactions
generating a pyrrole compound as described by Tressl
et al. for deoxyribose.31 Somewhere in the reaction
sequence an oxidation of C-5 would be required to gener-
ate the aldehydic group during phosphate cleavage; this
may occur in an analogous manner as for furfural for-
mation generated from DNA.30
While these compounds absorb in the 280-nm and
320-nm regions, their absorbance maxima are not at
350 nm, the unique wavelength observed in our UV–
vis studies. Instead, a short tR compound (2.8 min) of
an m/z 455 [M+H+] is compatible with a compound
with a 350-nm wavelength maximum. Similar to the
m/z 267 [M+H+] compound, this compound is an oxi-
dized product of the starting dR5P and NAcLys reac-
tants. The fragmentation pattern of the m/z 455
[M+H+] compound reveals a major peak at m/z 268,
indicating a loss of the molecular mass (187 amu) of a
NAcLys. While the identity of this compound needs fur-
ther investigation, it is likely that the molecule involves
two amine groups attached to a C5H6O moiety.
appears to be at a same rate as dR5P loss. Thus, the rate
of phosphate cleavage is relatively spontaneous com-
pared to the rate of transition of dR5P to the C-3 ketose
form.
Most obvious in the LC chromatogram of the dR5P/
NAcLys system at extended times was the generation of
two unique compounds with extended retention times
(tR 4.4 and 6.8 min) on a reversed phase column
(Fig. 9). Both compounds have an m/z 267 [M+H+],
which is compatible with a molecular formula repre-
sented by NAcLys attached through a double bond
(or two single bonds in a cyclic form) to a C5H4O group.
It is important to note that these products are oxidized
in respect to the starting reactants. The wavelengths of
maximum absorbance for the two compounds are
258 nm and 290 nm for the tR 4.4 min and tR 6.8 min
peaks, respectively. The two compounds account for
52% of the 280 nm absorbance and 20% of the 320-nm
absorbance, but none of the 350-nm and 420-nm
absorbance.
Employing b-Ala as the amine component, the two
1
compounds were characterized by H NMR spectros-
copy, and the structures (see Scheme 8) best fitting the
data were an amine derivative of 5-methylene-2-pyrro-
lone [5-MP, 3-(2-methylene-5-oxo-2,5-dihydro-1H-pyr-
rol-1-yl)propanoic acid (tR 4.0 min)], and a 2-formyl-
pyrrole compound [3-(2-formyl-1H-pyrrol-1-yl)pro-
panoic acid (tR 6.0 min)]. Judging from proton
spectra of an acetone-extracted solution, the two com-
pounds were synthesized in approximately equal
amounts. The reaction scheme in the generation of the
compounds requires oxidation, dehydration, and phos-
phate cleavage steps. A compound similar in structure
to the amine derivative of 5-MP is 5-methylene-2-fura-
none (5-MF). 5-MF, a marker of DNA oxidative dam-

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A. Munanairi et al. / Carbohydrate Research 342 (2007) 2575–2592
Additional related compounds observed with time
stants equaling approximately 5–10 Mꢀ1 were achieved
(data not shown). dR5P and non-phosphorylated ribose
forms would be anticipated to have equilibrium con-
stants of the same range. This implies very little N-
glycoside exists at typical reaction concentrations at
neutral pH and suggests any significant decrease in
observed sugar concentration occurs because of a
subsequent Amadori rearrangement step reported to be
largely irreversible.14 The lack of observable amounts
of deoxyribose or dR5P loss over extended times is con-
sistent with small equilibrium levels of the N-glycoside
and with the inability of these molecules to undergo a
traditional Amadori rearrangement process.
exhibited LC/MS signals at m/z 625 [M+H+] and 689
[M+H+]. These likely correspond to an NAcLys at-
tached to the m/z 455 compound and to an NAcLys
bridging two m/z 267 compounds, respectively.
The reaction of dR5P with amine performed in the ab-
sence of oxygen for 4 days at 37 ꢁC generated many of
the same products seen with the reaction performed in
the presence of oxygen. However, LC/MS analysis indi-
cated significantly lower amounts of signal from the
compounds of m/z 267 and 455 (see above) and instead
an enhanced signal of compounds two hydrogens great-
er in mass. The lack of oxygen likely prevents the syn-
thesis of the oxidized versions and instead the non-
oxidized forms predominate.
The kinetics of the early Maillard sequence is compli-
cated by the fact that free amine is consumed and then
regenerated in the early portions of the reaction produc-
ing a-dicarbonyls. Here, treatment of the rates in a sec-
ond-order manner for comparative purposes does not
depict the true kinetics as shown most clearly by systems
where sugars stoichiometrically in excess of amine con-
centrations continue to readily lose sugar concentrations
beyond the expected 1:1 ratio. A closer investigation of
the R5P/NAcLys system reveals additional kinetic in-
sights. Making an assumption of comparatively negligi-
ble equilibrium levels of the N-glycoside at neutral pH
and a recycling of the amine during its release from
the Amadori product, kinetics modeling was performed
using the reaction sequence of Scheme 2 to fit the pat-
tern of the Amadori product profile (i.e., a maximum
at approximately 40 h and a half maximum achieved
at 110 h, see Fig. 2) with the R5P and NAcLys data
(see Fig. 1 and kinetic data). The modeling resulted in
a k = 0.26 Mꢀ1 hꢀ1 and a k3 = 0.03 hꢀ1. Application
of our measured Keq = 7.6 Mꢀ1 for the deprotonated
NAcLys/R5P/N-glycoside equilibrium system yields a
k2 = 24.5 hꢀ1 for the system at pH 7.4. This theoretical
treatment of the data predicts that at its maximum, the
Amadori product at 0.016 M comprises approximately
33% of the sugar and 17% of the NAcLys. This is in
comparison to less than 0.3% of each existing as the
N-glycoside. In opposition to the predictions of the
modeling, the free amine level did not return to original
concentrations upon full consumption of the sugar and
subsequent movement of the molecules past the Amado-
ri compound. The progressive loss of amine after the
maximal level of Amadori product is achieved (see Figs.
1 and 2) implies a subsequent amine-consuming reaction
occurring post-Amadori rearrangement. It is likely that
reaction with the a-dicarbonyl or formation of subse-
quent Maillard product(s) (like CML) in a stable form
accounts for the long-term loss of amine from solution.
At 120 h, approximately 12% of the NAcLys in the R5P-
containing solution was lost to other compounds with
an estimated 40% of this contained as Amadori product
that had yet to be converted to future Maillard
compounds.
The sugar deoxyribose reacts with an amine in the
presence of a phosphate catalyst at extremely slow rates.
Over a period of 150 h, no significant deoxyribose or
NAcLys loss was detected by LC/MS analysis, and the
only major new product observed was a compound con-
sistent with a mass of an N-glycoside (m/z 305 [M+H+]
for the NAcLys conjugate). The intensity of the signal
for this compound grew linearly over the length of the
incubation, and it accounted for >90% of all new UV–
vis absorbance generated. Given that the equilibrium
level of N-glycoside is thought to be extremely low at neu-
tral pH, the linear rate of formation of this compound
implies its transformation into a secondary, similar mass
product. The possibility of a conversion to a pseudo-
Amadori product (a 3-ketose) in a similar fashion to
dR5P is likely. The lack of significant loss of deoxyribose
and NAcLys from solution along with the relatively
small intensity of the extracted-ion signal suggests the
formation of this compound to be relatively small.
3. Discussion
Ribose sugars and sugar phosphates undergo traditional
Maillard reactions leading to a diverse set of products.
As is common with most Maillard processes, the initial
reversible attack of amine onto the carbonyl group
yielding an N-glycoside (either in a straight-chain form
or in the presumably more stable cyclic form) is likely
an equilibrium process connected to the concentration
of the deprotonated form. Previous studies on the pen-
tose phosphate pathway metabolite phosphoribosamine
have determined an equilibrium constant (2.5 Mꢀ1 at
37 ꢁC) that slightly favors the N-glycoside over R5P
and ammonia.16 It is probable that other amines react-
ing with R5P exhibit N-glycosides of approximately
the same stability, although some factors may make
them slightly less favorable. In our attempts to establish
equilibrium constants of the R5P N-glycosides of
tris(hydroxymethyl)aminomethane (Tris) and NAcLys
employing systems of high pH (ꢂ10.5), equilibrium con-

A. Munanairi et al. / Carbohydrate Research 342 (2007) 2575–2592
2589
Treatment of the data in this manner with NAcLys
being recycled explains the discrepancy observed in the
rate constants of R5P loss when increasing levels of
R5P. When the kinetics are modeled employing the ac-
tual observed NAcLys concentrations (involving the
recycling of the amine), the solutions containing differ-
ent starting R5P levels yield rate constants that are more
equivalent. (A small increase in rate constant with pKa
was still observed.) When treated in this manner, rate
constants in the range of 0.3–0.6 Mꢀ1 hꢀ1 were
obtained.
Despite inaccuracies in the true values, the R5P sys-
tems with different amino acids exhibiting k* values
varying by more than an order of magnitude shows
the early Maillard system to be more complicated than
a simple attack of a deprotonated amine onto the sugar.
Higher rates at lower pH for some amines indicate a
protonation step may be involved, while some steric ef-
fects appear to be partially responsible for the differ-
ences in rates, for example, b-alanine versus L-alanine.
The similar kinetics of the early Maillard reaction of
amines with R5P in both the presence and absence of
oxygen suggests a common pathway to and just beyond
the Amadori rearrangement product. The observation
of the production of CML in reactions performed in
the presence of oxygen was expected as R5P is structur-
ally similar to other sugars at the reducing end of the
molecule. In contrast, the reaction of R5P in the absence
of oxygen generated little CML but instead produced a
compound compatible in mass with a triketopentane
conjugate. (The lack of evidence of the formation of
HMF suggests this is not a HMF conjugate.) Inorganic
phosphate was cleaved from the sugar over time at
appreciable rates in both the presence and absence of
oxygen. It is unknown whether this is due to a Mail-
lard-related reaction (e.g., phosphate cleavage from an
a-dicarbonyl) or to the isomerization of R5P to Ru5P,
perhaps promoted by the amine in solution.
cases likely leads to a 5-methyl group) discourages the
furfuryl alcohol and 2-(hydroxymethyl)pyrrole products
observed for deoxyribose12,31 in favor of unique pyrrole
monomeric and polymeric compounds. A pseudo-Ama-
dori product is formed from dR5P with amines at rela-
tively slow rates in comparison to R5P but at a fast pace
compared to deoxyribose with Pi. The difference in rates
of dR5P and deoxyribose/Pi clearly demonstrates the
impact of an intramolecular phosphate group at C-5
on the speed of the reaction. As in the R5P system, this
effect may be due to the phosphate causing an increased
tendency toward an acyclic form or to a catalytic role it
plays in rearrangement steps. The formation of the pseu-
do-Amadori product apparently is connected to the sub-
sequent loss of the phosphate and ultimately the
production of a pair of pyrrole isomers with absorbance
in the UV range. The reaction is an oxidative process;
the reaction run in the absence of oxygen instead gener-
ates a saturated form of this compound. As the O2-
exposed reaction progresses, additional amine molecules
link to this non-phosphorylated product, generating
other UV–vis-absorbing compounds, including (for
some amines) a unique 350-nm product. While the exact
identity of this product(s) is unknown, the compound(s)
bears similar UV–vis, fluorescence, and mass-building
patterns to a 2-(2-furoyl)-4(5)-(2-furanyl)-1H-imidazole
product formed by the condensation of glucoses with
two Lys followed by oxidation and dehydration.32,33
Surprisingly, no carboxyethyl groups (–CH2CH2-
COOH) were detected as a result of this reaction. These
hypothetically could be generated by the reaction of O2
with the dicarbonyl in the dR5P reaction series in a
similar manner as it occurs with the Amadori product
of any sugar/amine combination. The formation of this
compound may indicate the importance of the amine
being in a position such that a Schiff base ultimately lies
in conjugation with an enediol upon a removal of a
hydride by oxygen.
Previously reported to yield furfural products,25,26 the
most widely characterized of the studied group, D-
ribose, is believed to react in a traditional manner with
amines, generating an Amadori rearrangement product,
followed by a-dicarbonyls, and then to cyclization to the
furfural/furfurone product. The relatively rapid rate of
reaction (in comparison to common aldohexose sugars
such as glucose) is speculated to be partially tied to
the sugar’s higher level of acyclic form. The primary
products observed during the reaction at neutral pH
and 37 ꢁC are believed to be an HMF product and
CML.
The unique reactivity of pentose and deoxypentose 5-
phosphates before and after the hydrolysis of the intra-
molecular phosphate adds a new variation to a typical
Maillard reaction scheme. Further research is needed
to determine if these reactions have commercial applica-
tions or if they are of consequence physiologically.
4. Experimental
4.1. General methods
While the dR5P system lacks the C-2 hydroxyl group
necessary for the transition of the N-glycoside to the tra-
ditional Amadori product, the molecule is capable of
reacting in a Maillard fashion12 with some products ulti-
mately undergoing hydrolysis of phosphate. Like R5P,
the inclusion of a 5-phosphate moiety (which in some
D-Ribose 5-phosphate (disodium salt), D-deoxyribose
5-phosphate (sodium salt) (2-deoxy-D-erythropentose 5-
phosphate), D-ribose, D-deoxyribose (2-deoxy-D-erythro-
pentose), N-acetyllysine, b-alanine, N-acetylarginine,
4-hydroxyl-5-methyl-3(2H)-furanone (HMF), D2O, and
other L-amino acids and reagents were purchased of

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A. Munanairi et al. / Carbohydrate Research 342 (2007) 2575–2592
the highest purity from Sigma–Aldrich Chemical Co.
Solvents for liquid chromatography/mass spectrometry
(LC/MS) were purchased from Fisher Scientific Co.
and were of >99.9% purity.
LC/MS experiments (see below) using an extracted-ion
method (combined area of the m/z 215, 237, and 259
peaks).
Due to complications of the water peak and multiple
sugar forms (pyranose and furanose) in the NMR anal-
ysis, the determination of the loss of deoxyribose and ri-
bose from solution was performed by an extracted-ion
method using an electrospray injection (ESI) Agilent
Ion Trap LC–MS. The combined areas of the ex-
tracted-ion chromatogram (range = m/z 0.5) of the
[M+H+], [M+Na+], and [MꢀH++2Na+] signals were
measured for each sugar. This corresponds to the areas
of the m/z 135, 157, and 179 signals for deoxyribose and
the areas of the m/z 151, 173, and 195 signals for ribose.
Calculations of the kinetics of the sugar loss were per-
formed using a self-designed modeling program employ-
ing Microsoft Excelꢂ software. The second-order rate
constant was determined by fitting the actual disappear-
ance of the sugar with the best fit relationship using the
rate equation
Unless stated otherwise, all reactions involved the
preparation of solutions containing 0.10 M amine and
0.050 M sugar phosphate. For deoxyribose and ribose,
a reaction of 0.10 M amine, 0.050 M sugar, and
0.050 M sodium dihydrogen phosphate were prepared.
These solutions were brought quickly to the desired
pH (usually either pH 7.4 or 8.0) and then incubated
in a heating block at 37 ꢁC. Incubations for periods
extending to 7 days were performed for some experi-
ments. For extended-time experiments of greater than
3 days, the solutions were sterile-filtered prior to incuba-
tion to eliminate the possibility of microbial growth.
For reactions performed in the absence of oxygen,
component solutions were brought to the proper pH,
bubbled with nitrogen for several minutes, combined,
further bubbled with nitrogen, and then sealed tightly.
Samples were opened just prior to analysis; for studies
requiring a number of samples over a period of time,
multiple sealed tubes were prepared.
Rate ¼ k½sugarꢁ½amineꢁ;
where the sugar and amine concentrations were the ac-
tual concentrations prepared. While serving as a good
estimate of rate constants for comparative purposes,
the rate equation employed assumes a stoichiometric
loss of amine with the loss of the sugar, an assumption
shown by us and others not to be correct. (The amine
apparently is regenerated during the loss of sugar (see
Section 3).) Adjusted rate constants (k*) corresponding
to the actual deprotonated amine concentrations were
determined via the above equation employing deproto-
nated amine concentrations as calculated using the
amine’s pKa. The pKa for all amines except Gly were
determined at 37 ꢁC by titration of the amine (0.10 M)
with 1.0 M NaOH.
4.2. Rate of sugar consumption
Loss of R5P and dR5P from solution was determined by
1H nuclear magnetic resonance (NMR) spectroscopy
using 5-mm tubes and a 400 MHz Bruker Fourier-trans-
form (FT) NMR spectrometer. Typically, each 1H
experiment time point involved 16 scans (scan range
equaling 20 ppm), followed by an automatic baseline
correction and integration of the proton peaks. All
chemical shifts are relative to tetramethylsilane (TMS)
hydrogens. The concentrations of R5P or dR5P were
determined by comparing the integration of the com-
bined peaks of the a and b hydrogens of the anomeric
carbon at 5.2–5.5 ppm for R5P (two peaks) and at
5.5 ppm for dR5P (one combined peak for both the a
and b form) versus either an MeOH hydrogen peak at
approximately d 3.4 or an EtOH hydrogen triplet at
approximately d 1.2. (MeOH and EtOH are contained
in small amounts in the R5P and dR5P purchased from
Sigma Chemical Co.) The integration ratio of the initial
sample was set as equivalent to the initial R5P or dR5P
concentration (usually 0.05 M). This ratio was verified
by measuring this integration ratio on the appropriate
R5P or dR5P solution that lacked the amine. Measure-
ment of the R5P in solution by this manner was per-
formed until all R5P was consumed or for 7 days. The
results of the R5P analysis method was shown to be in
good agreement with results obtained via an LC–MS
technique of Huck et al.,13 which monitored the inten-
sity of the ion signal of the m/z 97 collision fragment
of the m/z 229 signal of the MS operating in a negative
mode. The dR5P results were in good agreement with
4.3. Rate of amine consumption and rate of N-glycoside/
Amadori product formation
The loss of NAcLys from solution was determined by an
extracted-ion method measuring the combined MS ion
areas (range = m/z 0.5) of the m/z 189, 211, and 233
signals. (Similar results of other amines (not shown)
were performed using the appropriate masses of the
mono-protonated and mono- and di-sodiated amine.)
The peak area of the extracted-ion peaks for the amine
at 0 h was set as the initial concentration of the amine
(typically 0.10 M). N-Glycoside/Amadori product for-
mation was determined in a similar manner by extract-
ing the ion signals corresponding to the masses of the
mono-protonated and mono- and di-sodiated forms of
the sugar–amine conjugate (mass = sugar plus amine
minus water). No direct translation of these values to
the corresponding concentrations could be made since
pure standards were not available. Comparative concen-

A. Munanairi et al. / Carbohydrate Research 342 (2007) 2575–2592
2591
trations over time are represented by the total MS peak
areas of these extracted ions.
an excitation wavelength of 360 nm ( 20 nm) filter with
an emission wavelength filter of 460 nm ( 20 nm). The
values given are not corrected for dilution or for
sensitivity.
In some cases lysine and arginine modifications were
assessed by a fluorescamine method34 and a 9,10-phe-
nanthrenequinone method,34 respectively. Measure-
ments were performed with excitation/emission
wavelengths (nm) of 380/485 (for Lys modification)
and 310/400 (for Arg modification). (Filters employed
had 20–40 nm of bandwidth to cover the optimum exci-
tation and emission wavelengths.)
4.6. Characterization of products by LC–MS and NMR
spectroscopy
All LC–MS chromatograms and spectra were obtained
using an Agilent Series 1100 LC/MSD Trap XCT Plus
Liquid Chromatogram–Mass Spectrometer with electro-
spray injection. Samples (5–25 lL) were injected, and
LC separation of the compounds was performed using
a 4.6 · 150 mm C8 Zorbex reversed phase column and
an 80:20 H2O–2-PrOH mobile phase (containing 0.5%
HCO2H). A photodiode array detector capable of
recording full spectra monitored the chromatographic
flow. ESI injection conditions employed a drying tem-
perature of 365 ꢁC, a nebulizer pressure of 50 psi, and
a dry gas flow of 9 L/min. Typically, an MS experiment
was performed in positive mode over a scan range of
m/z 50–2200 and a target mass of 250 Da. Use of an
automatic MSn mode allowed for fragmentation of
prominent MS signals. In some cases (such as for the
analysis of R5P loss), a negative-ion mode MS and/or
a manual mode was employed for supplemental infor-
mation. The instrument has the capability of generating
chromatograms via an ion-extraction method. This
proved useful in identifying unique signals and matching
the MS results with the UV–vis results of the LC–MS.
4.4. Hydrolysis of phosphate and formation of new
phosphate compounds
The cleavage of inorganic phosphate (Pi) from sugar
phosphates was monitored by 31P NMR. All solutions
were prepared in D2O rather than H2O at the appropri-
ate concentrations. FT 31P NMR was performed at a
frequency of 161.9 MHz with a scan range of 100 ppm
and a relaxation delay of 0.25 s. Typically, 200–500
scans were performed for each analysis. The amount
of phosphate hydrolyzed was determined by comparison
of the integration of the Pi peak at approximately
+3.4 ppm (vs a H3PO4 reference) versus the integration
of the total phosphate signals in solution. The phos-
phate peaks for R5P and dR5P were located at approx-
imately +4.8 ppm. With time, phosphate signals
typically developed that either overlapped the R5P or
dR5P or were downfield from the R5P or dR5P peak.
Our experience with phosphoribosamine (a reaction of
R5P with ammonia) suggests the phosphate signals of
N-glycosides overlap typically as a downfield shoulder
to the R5P peak. It is speculated that Amadori and
other rearrangement products (e.g., dicarbonyl phos-
phates) have chemical shifts slightly downfield from
the R5P phosphate signal (i.e., at approximately
+5.3 ppm). The signal for ribulose 5-phosphate
(Ru5P), a ketose isomer of R5P, is reported to be
0.4 ppm downfield from the R5P signal.18
1
HMF was identified by a combination of H NMR
and LC–MS. 1H NMR analysis was performed on reac-
tion samples in D2O. Standard solutions (0.0–1.0 mM)
of HMF were prepared in water, and 5 lL was injected
into the reversed phase column LC–MS system with
conditions listed previously. Analysis: UV–vis: kmax
285 nm;35 1H NMR (D2O): d 4.55 (s, 2H), d 2.2 (s,
3H);35 MS: m/z 115 [M+H+] (tR 3.6) with fragmenta-
tions of 88 (55) and 87 (100). Compounds in the Mail-
lard reactions, which yielded a match of retention
time, absorbance characteristics, m/z and fragmentation
4.5. Absorbance and fluorescence measurements
1
spectrum, and H NMR were identified as HMF. The
All ultraviolet/visible (UV–vis) absorbance spectra, sin-
gle-wavelength measurements, and fluorescence mea-
surements were performed with a BioTek Synergy HT
Microplate Reader using 200-lL samples. For UV–vis
characterization, samples were read without dilution
unless measurements greater than 2.0 A were obtained,
whereupon diluted samples were prepared and measured.
All absorbances are adjusted appropriately for any dilu-
tion and for a factor that corrects for the pathlength to
yield values in the commonly reported cmꢀ1 units.
Fluorescence measurements were performed using
black microwell plates, 100 lL of sample combined with
100 lL of water, and a sensitivity of 50. Total advanced
glycation end-products (AGEs) fluorescence employed
amount of HMF in the Maillard reaction solution was
quantified by a standard relationship generated for
A280 peak area versus HMF concentration. (A second
method employing the standard relationship between
the peak area of the m/z 168 signal versus HMF concen-
tration was not needed for this study as few interfering
compounds with UV absorbance exited the reversed
phase column at tR 3.6 min.)
The amine form of 5-MP generated by the dR5P/b-
Ala (or dR5P/NAcLys) reaction was likewise identified
by LC–MS and 1H NMR spectroscopy. (The b-Ala
results are presented here.) 1H NMR analysis was
performed on samples that were collected at 4.0 min,
acidified, extracted with EtOAc, dried, and resuspended

2592
A. Munanairi et al. / Carbohydrate Research 342 (2007) 2575–2592
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in CD2Cl2. Analysis: UV–vis: kmax 258 nm; H NMR
(CD2Cl2): d 2.7 (t, 2H), 3.95 (t, 2H), 4.95 (s, 1H), 5.05
(s, 1H), 6.25 (d, 1H), and 7.1 (d, 1H); MS: m/z 168
[M+H+] (tR 4.0 min) with fragmentations of m/z 150
(100) and 108 (5). COSY NMR analysis indicated the
d 2.7 and 3.95 peaks to be strongly coupled, as were also
the d 6.25 and 7.1 signals. Use of an 15N-labeled amine
resulted in a relatively small 15N–1H (ring hydrogens)
coupling value (ꢂ3 Hz) suggesting neither hydrogen is
directly adjacent to the ring nitrogen.
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the product of dR5P and b-Ala. H NMR analysis was
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This study was supported by a grant from the Vermont
Genetics Network, a five-year IDeA Networks of Bio-
medical Research Excellence (INBRE) project spon-
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MS instrument was funded by a grant from the Major
Research Instrumentation program of the National
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Magdalena Bokiej, Andrew Livermore, and Professor
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