Transport of Free and Peptide-Bound Glycated Amino Acids: Synthesis, Transepithelial Flux at Caco-2 Cell Monolayers, and Interaction with Apical Membrane Transport Proteins

Article abstract of DOI:10.1002/cbic.201000759

In glycation reactions, the side chains of protein-bound nucleophilic amino acids such as lysine and arginine are post-translationally modified to a variety of derivatives also known as Maillard reaction products (MRPs). Considerable amounts of MRPs are taken up in food. Here we have studied the interactions of free and dipeptide-bound MRPs with intestinal transport systems. Free and dipeptide-bound derivatives of N⁶-(1-fructosyl)lysine (FL), N⁶-(carboxymethyl)lysine (CML), N⁶-(1-carboxyethyl)lysine (CEL), formyline, argpyrimidine, and methylglyoxal-derived hydroimidazolone 1 (MG-H1) were synthesized. The inhibition of L-[³H]lysine and [¹⁴C]glycylsarcosine uptakes was measured in Caco-2 cells which express the H⁺/peptide transporter PEPT1 and lysine transport system(s). Glycated amino acids always displayed lower affinities than their unmodified analogues towards the L-[³H]lysine transporter(s). In contrast, all glycated dipeptides except Ala-FL were medium- to high-affinity inhibitors of [¹⁴C]Gly-Sar uptake. The transepithelial flux of the derivatives across Caco-2 cell monolayers was determined. Free amino acids and intact peptides derived from CML and CEL were translocated to very small extents. Application of peptide-bound MRPs, however, led to elevation (up to 80-fold) of the net flux and intracellular accumulation of glycated amino acids, which were hydrolyzed from the dipeptides inside the cells. We conclude 1) that free MRPs are not substrates for the intestinal lysine transporter(s), and 2) that dietary MRPs are absorbed into intestinal cells in the form of dipeptides, most likely by the peptide transporter PEPT1. After hydrolysis, hydrophobic glycated amino acids such as pyrraline, formyline, maltosine, and argpyrimidine undergo basolateral efflux, most likely by simple diffusion down their concentration gradients.

Full text of DOI:10.1002/cbic.201000759

DOI: 10.1002/cbic.201000759
Transport of Free and Peptide-Bound Glycated Amino
Acids: Synthesis, Transepithelial Flux at Caco-2 Cell
Monolayers, and Interaction with Apical Membrane
Transport Proteins
Michael Hellwig,[a] Stefanie Geissler,[b] Renꢀ Matthes,[a] Anett Peto,[a] Christoph Silow,[a]
Matthias Brandsch,[b] and Thomas Henle*[a]
In glycation reactions, the side chains of protein-bound nucleo-
philic amino acids such as lysine and arginine are post-transla-
tionally modified to a variety of derivatives also known as Mail-
lard reaction products (MRPs). Considerable amounts of MRPs
are taken up in food. Here we have studied the interactions of
free and dipeptide-bound MRPs with intestinal transport sys-
tems. Free and dipeptide-bound derivatives of N6-(1-fructosyl)-
lysine (FL), N6-(carboxymethyl)lysine (CML), N6-(1-carboxyethyl)-
lysine (CEL), formyline, argpyrimidine, and methylglyoxal-de-
rived hydroimidazolone 1 (MG-H1) were synthesized. The inhib-
ition of l-[3H]lysine and [14C]glycylsarcosine uptakes was mea-
sured in Caco-2 cells which express the H+/peptide transporter
PEPT1 and lysine transport system(s). Glycated amino acids
always displayed lower affinities than their unmodified ana-
logues towards the l-[3H]lysine transporter(s). In contrast, all
glycated dipeptides except Ala-FL were medium- to high-affini-
ty inhibitors of [14C]Gly-Sar uptake. The transepithelial flux of
the derivatives across Caco-2 cell monolayers was determined.
Free amino acids and intact peptides derived from CML and
CEL were translocated to very small extents. Application of
peptide-bound MRPs, however, led to elevation (up to 80-fold)
of the net flux and intracellular accumulation of glycated
amino acids, which were hydrolyzed from the dipeptides
inside the cells. We conclude 1) that free MRPs are not sub-
strates for the intestinal lysine transporter(s), and 2) that diet-
ary MRPs are absorbed into intestinal cells in the form of di-
peptides, most likely by the peptide transporter PEPT1. After
hydrolysis, hydrophobic glycated amino acids such as pyrraline,
formyline, maltosine, and argpyrimidine undergo basolateral
efflux, most likely by simple diffusion down their concentration
gradients.
Introduction
Amino acids are targets for a variety of non-enzymatic chemi-
cal processes during food processing, such as oxidations or re-
actions with reducing sugars and their degradation products.
This process is generally referred to as the “Maillard reaction”
or “glycation”.[1]
droimidazolone 1, MG-H1, 9)[8] and N5-(5-hydroxy-4,6-dimethyl-
2-pyrimidinyl)-l-ornithine (argpyrimidine, Apy, 10) as a fluores-
cent minor product.[9] Pentosidine (11) is an amino acid con-
taining a lysine and an arginine residue.[10] In the late stages of
the Maillard reaction, AGEs, proteins, sugars, and their degra-
dation products react with one another to form colored high-
molecular-weight networks, which is why the Maillard reaction
is also termed “nonenzymatic browning”.
In the first stage of this process, lysine e-amino groups react
with reducing sugars such as glucose, lactose, and galactose to
form the Amadori products N6-(1-deoxy-1-fructosyl)lysine (fruc-
toselysine, FL, 1; Scheme 1), N6-(1-deoxy-1-lactulosyl)lysine (lac-
tuloselysine, 2), and N6-(1-deoxy-1-tagatosyl)lysine (tagatosely-
sine, 3), respectively. Amadori products can degrade to highly
reactive 1,2-dicarbonyl compounds, which again react with
lysine e-amino groups and arginine guanidino groups to form
“advanced glycation end products” (AGEs). Lysine, for example,
can be modified to N6-(carboxymethyl)lysine (CML, 4),[2] N6-(1-
carboxyethyl)lysine (CEL, 5),[3] 6-(2-formyl-1-pyrrolyl)norleucine
(formyline, Fom, 6),[4] and 6-(2-formyl-5-hydroxymethyl-1-pyrro-
lyl)norleucine (pyrraline, Pyrr, 7).[5,6] 6-(3-Hydroxy-2-methyl-4-
oxo-4(1H)-1-pyridinyl)-l-norleucine (maltosine, Mal, 8) is
formed during disaccharide degradation through reactions be-
tween lysine residues and isomaltol.[7] Reactions between argi-
nine residues and methylglyoxal lead to N5-(5-methyl-4-oxo-5-
hydro-2-imidazolonyl)-l-ornithine (methylglyoxal-derived hy-
Humans are exposed to these substances from heat-treated
foods such as bread, cereals, cookies, and dairy products. The
Amadori products, mainly those derived from glucose and di-
[a] M. Hellwig,+ R. Matthes, A. Peto, C. Silow, Prof. Dr. T. Henle
Institute of Food Chemistry, Technische Universitꢀt Dresden
01062 Dresden (Germany)
Fax: (+49)351-463-34138
E-mail: thomas.henle@chemie.tu-dresden.de
[b] S. Geissler,+ Dr. M. Brandsch
Membrane Transport Group, Biozentrum
Martin-Luther-Universitꢀt Halle-Wittenberg
Weinbergweg 22, 06120 Halle (Germany)
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201000759.
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Transport of Free and Peptide-Bound Glycated Amino Acids
enhance oxidative stress, to induce low-grade inflammation,
and to promote atherosclerosis.[15] Many AGEs are strongly re-
tained in end-stage renal disease.[16] Patients are encouraged
to reduce their dietary AGE intakes in order to optimize their
inflammatory status[17] and to prevent diseases. However,
whether the accumulation of (dietary) AGEs in physiological
fluids of patients detrimentally aggravates the diseases or
whether it simply represents a side effect of uncontrolled
sugar and carbonyl stress has not yet been shown.[18]
Food-borne AGEs can only affect physiological functions if
they are absorbed from the diet. If it is assumed that MRPs,
like other amino acids, are liberated from proteins, they arrive
at the intestinal epithelial barrier bound in small peptides or,
to a lesser extent, as the free amino acids.[19] Hypothetically,
these products can cross the intestinal epithelium paracellular-
ly by simple diffusion or transcellularly by diffusion, by endocy-
totic processes, or mediated by transport proteins. For the
translocation of lysine and arginine through the apical mem-
brane, enterocytes possess at least three different amino acid
transporters: namely the systems B0,+, b0,+, and y+.[20] Di- and
tripeptides are transported by the proton-coupled peptide
transporter 1 (PEPT1), which is driven by a transmembrane H+
gradient and catalyzes the cotransport of its substrates with
H+ into intestinal and other cells (for a review, see ref. [21]).
PEPT1 accepts many modified amino acids and dipeptides as
substrates as long as the structural requirements for substrate
binding and translocation are met.[22]
From the transit of immunoreactivity, Koschinsky et al.[13] es-
timated the absorption of AGEs in general to be about 10%,
even though AGEs are a structurally quite heterogeneous
group of substances (Scheme 1). We hypothesize that MRPs,
due to their structural diversity, must interact quite differently
with intestinal and renal amino acid and peptide transporters.
Circumstantial evidence is provided by balance studies show-
ing that less than 5% of ingested protein-bound fructoselysine
(1) and pentosidine (11), but 50–100% of protein-bound pyrra-
line (7), appear in the urine.[23] In infants, the urinary excretion
of CML (4) is dependent on the dietary intake.[24] The question
that therefore arises is of how these compounds cross the in-
testinal barrier. Pyrraline (7) and maltosine (8) are actively
transported by human PEPT1 in peptide form, but leave the
cells as free 7 and 8 after peptidolysis.[25–27] Like fructoselysine
(1) and CML (4),[28] free 7 and 8 are not transported through
Caco-2 cells.
Scheme 1. Chemical structures of the investigated Maillard reaction prod-
ucts fructoselysine (1), lactuloselysine (2), tagatoselysine (3), CML (4), CEL (5),
formyline (6), pyrraline (7), maltosine (8), MG-H1 (9), argpyrimidine (10), and
pentosidine (11).
This study was directed towards characterizing the interac-
tion of a broader range of glycated amino acids and dipeptides
with membrane transport systems that might accept these
types of compounds as substrates. Furthermore, the net trans-
epithelial transport of these compounds across Caco-2 cell
monolayers cultured on permeable filters in Transwell cham-
bers was determined. Taken together, these techniques pro-
vide new information on the degree and mechanism of intesti-
nal AGE absorption in vitro.
and oligosaccharides, are quantitatively the dominant Maillard
reaction products (MRPs) in food. Amounts of between 0.5 and
1.2 g are ingested daily, together with 25 to 75 mg of AGEs
(mainly 4 and 7).[11] Recently, the daily CML intake from two
standard diets used for clinical studies was determined to be
2.2–5.4 mg.[12] Because concentrations of other AGEs in most
foods are not known, it is impossible to assess their daily
intake.
The question of whether or not dietary AGEs play a causa-
tive role in the etiology of diseases such as diabetes and
uremia is intensely debated.[13,14] Dietary AGEs are reported to
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T. Henle et al.
acids or dipeptides, were absent, as verified by amino acid
analysis (AAA).
Results and Discussion
The fluxes of UV-absorbing amino acids (6, 7, 8, 10, 11) and
the corresponding dipeptides were measured by RP-HPLC after
optimization of the gradients in terms of the best possible sep-
aration of the dipeptides from their constituent glycated
amino acids. Other fluxes were measured by AAA with use of a
lithium-based system with different gradient programs. The
fluxes of 1a and 1b, however, additionally had to be measured
with a sodium system, because fructoselysine (1) was not sepa-
rated from the parent dipeptides in the lithium system.
Synthesis and analysis of free and peptide-bound glycated
amino acids
The Maillard reaction products investigated in this study are
not commercially available, so sufficient amounts (30–200 mg)
of ligands had to be synthesized. Dipeptides with the glycated
amino acids both in the N-terminal and in the C-terminal posi-
tions were prepared, because the sequence of a peptide is cru-
cial for its inhibitory and transport characteristics.[22,29] In this
study, the synthesis of 12 glycated peptides is described for
the first time. Moreover, the synthesis of MG-H1 (9, Scheme 2)
Inhibition of l-[3H]lysine uptake
The human intestinal cell line Caco-2 is a commonly used
model for study of the intestinal transport of di- and tripep-
tides and amino acids.[32] Caco-2 cells express the PEPT1 and
different amino acid transporters in their apical membranes.
The three systems B0,+, b0,+, and y+,[20] responsible for the
transport of lysine and arginine, are also possible candidates
for the transport of MRPs of lysine and arginine, and so the in-
teraction of free and peptide-bound MRPs with the l-lysine
transporter(s) was examined. Radiolabeled l-[3H]lysine was
used as the reference substrate for the lysine transporter(s). In
competition assays, inhibition of the uptake of l-[3H]lysine by
each compound was first investigated with a concentration of
10 mm to determine the substrate specificity. If the com-
pounds were able to inhibit the transport by at least 40%, in-
creasing concentrations of them were applied for inhibition to
allow calculation of their IC50 values. These values were con-
verted into inhibition constants (Ki) as described earlier.[33]
At a concentration of 10 mm, free and dipeptide-bound
MRPs inhibited the transport of l-[3H]lysine by 20–75% and 0–
38%, respectively (Table 1). For strongly inhibiting compounds,
Ki values between 0.32 mm for 7[25] and 4.6 mm for 10 were
calculated. The unlabeled reference amino acid l-lysine itself
inhibited l-[3H]lysine transport with a Ki value of 0.11ꢁ
0.01 mm (Table 1). Gly-Sar and the other tested unlabeled un-
modified dipeptides showed no affinity towards the lysine
transporter(s).
Scheme 2. Syntheses of N5-(5-methyl-4-oxo-5-hydroimidazolon-2-yl)-l-orni-
thine (MG-H1, 9) and N5-(5-hydroxy-4,6-dimethylpyrimidin-2-yl)-l-ornithine
(argpyrimidine, Apy, 10). a) l-Arg, HCl (12n), RT, 8 h. b) DMSO, NaOAc, RT,
3 h. c) l-Arg, HCl (12n), RT, 20 h.
was significantly improved in terms of yield and ease of prepa-
ration by allowing unprotected arginine and the dimethylace-
tal 14 of methylglyoxal (which forms methylglyoxal in situ) to
react in 12m HCl. At arginine concentrations of about
10 mgmLꢀ1, compound 9 was obtained as the main product in
one step in more than 40% molar yield. The synthesis could
also be adapted for peptides incorporating 9, but the incuba-
tion times were shortened in order to prevent peptide bond
cleavage. We are currently investigating whether this can be
applied as a general method for the synthesis of hydroimid-
azolones.
In conclusion, a minor part of the free MRPs, but not the di-
peptide-bound derivatives, interacts with the transporter(s) for
the amino acids l-lysine and l-arginine, either as substrates or
as inhibitors. Pyrraline (7) was the only compound to inhibit l-
[3H]lysine transport similarly to l-lysine, and it can be consid-
ered a high-affinity ligand (substrate or inhibitor).
Another new strategy[30] was applied for the synthesis of arg-
pyrimidine (10, Scheme 2). The 3-acetoxypentane-2,4-dione
(16) precursor, however, was not synthesized from lead(IV) ace-
tate,[30] but from 3-chloropentane-2,4-dione[31] (15), and was
then incubated in 12m HCl with unprotected arginine. Argpy-
rimidine (10) could be utilized as a “building block” for the syn-
thesis of Ala-Apy and, after introduction of the Boc protecting
group, for the synthesis of Apy-Ala. Taken together, the synthe-
sis operations afforded all compounds in sufficient and partly
unexpectedly high yields and purities as their formates, ace-
tates, or hydrochlorides. Impurities, especially other amino
Inhibition of [14C]Gly-Sar uptake
Unlike amino acid transport, the uptake of peptides from the
gut lumen is mediated by a single transport protein—PEPT1—
that tolerates many side-chain-modified peptides without
losing affinity.[22] Therefore, the interactions of glycated amino
acids and dipeptides with PEPT1 in Caco-2 cells were studied.
The radiolabeled dipeptide [14C]Gly-Sar, which is relatively re-
sistant to intra- or extracellular enzymatic hydrolysis, served as
the reference substrate. Competition experiments were first
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Transport of Free and Peptide-Bound Glycated Amino Acids
wards PEPT1 were strongly modulated, relative to
those of the parent dipeptides, by the modifications.
Peptides with pyrrole-modified side chains (6a/6b,
7a/7b) displayed the highest affinities (34–190 mm).
By Brandsch’s classification,[32] Ala-CML, Ala-CEL, Mal-
Ala, and both dipeptide derivatives of pyrraline, of
formyline, and of argpyrimidine can be classified as
high-affinity ligands (Ki <0.5 mm). The other tested
dipeptides can be considered medium-affinity li-
gands (0.5 mm<Ki <5 mm), except for Ala-FL, which
is a low-affinity compound. Gly-Sar represents a
medium-affinity PEPT1 substrate with a Ki value of
0.74ꢁ0.01 mm (Table 1), whereas the unmodified
lysine and arginine dipeptides are high-affinity sub-
strates with Ki values between 0.18 and 0.34 mm. It
is the hydrophobic peptides that show high affinity,
which agrees with recent findings about the affinity-
enhancing effects of hydrophobic side chain modifi-
cations.[29,34] Interestingly, the alanyl peptides 4a
and 5a, which each have a negative charge in the
side chain, bind to the carrier with affinities five and
13 times higher, respectively, than the correspond-
ing lysyl dipeptides 4b and 5b. This could possibly
be the result of unequal distribution of charges in
the binding region of PEPT1. In contrast, the alanyl
dipeptides of all other tested compounds had lower
affinities towards PEPT1 (1.6 to 5.6 times) than the
corresponding lysyl dipeptides. This is also consis-
tent with recent studies on model compounds in
which bulky hydrophobic groups were introduced
onto the lysyl side chains of Ala-Lys and Lys-Ala,
thereby strongly enhancing the affinities of lysyl
peptides.[29]
Table 1. Inhibition of l-[3H]lysine (2 nm) and [14C]Gly-Sar (10 mm) uptake in Caco-2 cells
by Maillard reaction products and control substances. Uptakes were measured at
pH 6.0 either for 5 min (l-[3H]lysine) or for 10 min ([14C]Gly-Sar) in the absence (control)
or in the presence of the compounds, either at fixed concentrations (10 mm) for
% uptake or at increasing concentrations (0.01–10 mm) of unlabeled compounds. Ki
values were derived from competition curves. Data are meansꢁSEM. n=3, 4.
Compound
l-[3H]Lysine uptake
uptake Ki [mm]
[% of
[14C]Gly-Sar uptake
uptake [% of Ki [mm]
control]
control]
fructoselysine
Ala-FL
FL-Ala
lactuloselysine
tagatoselysine
carboxymethyllysine
Ala-CML
CML-Ala
carboxyethyllysine
Ala-CEL
CEL-Ala
formyline
Ala-Fom
Fom-Ala
pyrraline
Ala-Pyrr
Pyrr-Ala
maltosine
Ala-Mal
Mal-Ala
MG-H1
Ala-(MG-H1)
(MG-H1)-Ala
argpyrimidine
Ala-Apy
1
41ꢁ1
2.7ꢁ0.1
32ꢁ2[a]
60ꢁ1
36 ꢁ2
49ꢁ2
33ꢁ3
93ꢁ4
9.9ꢁ0.4
14ꢁ1
90ꢁ4
11 ꢁ1
16ꢁ1
8.7ꢁ2.2[a]
1a 100ꢁ6
–
>10 (ꢂ13)[b]
4.5ꢁ0.2
8.7ꢁ1.1
3.5ꢁ0.1
–
1b
2
69ꢁ11
80ꢁ6
40ꢁ1
61ꢁ1
87ꢁ3
94ꢁ3
70ꢁ4
97ꢁ11
96ꢁ8
42ꢁ1
82ꢁ5
96ꢁ7
25ꢁ1[c]
–
–
3
4
3.1ꢁ0.6
–
–
–
–
–
–
4a
4b
5
5a
5b
6
6a
6b
7
7a
7b
8
8a
8b
9
9a
9b
10
0.07ꢁ0.01
0.9ꢁ0.1
–
0.22ꢁ0.01
1.1ꢁ0.1
–
2.2ꢁ0.3
82ꢁ2
–
–
10ꢁ1
9ꢁ1
0.09ꢁ0.01
0.05ꢁ0.01
>10[c]
0.19ꢁ0.01[c]
0.03ꢁ0.01[c]
>10[d]
0.32ꢁ0.04[c]
77ꢁ2[c]
5.5ꢁ0.4[c]
9.4ꢁ0.2[c]
85ꢁ2
66ꢁ5[c] >10[c]
78ꢁ7[c] >10[c]
48ꢁ5
3.5ꢁ0.4[d]
95ꢁ1 >10[d]
92ꢁ5 >10[d]
17ꢁ1
0.73ꢁ0.05[d]
0.25ꢁ0.02[d]
–
0.95ꢁ0.02
0.59ꢁ0.05
6.7ꢁ0.6
0.37ꢁ0.04
0.19ꢁ0.03[e]
0.74ꢁ0.01[c]
–
10ꢁ1
39ꢁ4
62ꢁ11
n.det.
1.6ꢁ0.2
89ꢁ1
–
–
17 ꢁ1
n.det.
55ꢁ5
4.6ꢁ1.1
39ꢁ3
10a 74ꢁ2
–
–
–
13ꢁ1
Apy-Ala
Gly-Sar
Lys
Arg
Ala-Lys
Lys-Ala
Ala-Arg
Arg-Ala
10b
n.det.
105ꢁ5[c]
17 ꢁ2[c]
18ꢁ1
n.det.
15ꢁ1[c]
101ꢁ4[c]
96ꢁ3
0.11ꢁ0.01[c]
0.027ꢁ0.003
–
85ꢁ4
–
–
–
–
8.6ꢁ0.2
13ꢁ1
0.23ꢁ0.02
0.34ꢁ0.02[f]
0.18ꢁ0.01
0.29ꢁ0.02
84ꢁ6
12
13
74ꢁ3
12ꢁ1
Transepithelial transport across intestinal epithe-
lial cell monolayers
65ꢁ8
12ꢁ1
[a] Value from ref. [28]. [b] Extrapolated beyond measurement range. [c] Value from
ref. [25]. [d] Value from ref. [27]. [e] n=2. [f] Value from ref. [29]. n.det.: not deter-
mined.
Interactions of compounds with transport systems
do not necessarily mean that they are indeed trans-
ported. The molecular features necessary for uptake
inhibition need not be the same as for translocation,
performed at 10 mm concentrations of each compound to de-
termine the substrate specificity. For ligands that inhibited
[14C]Gly-Sar uptake by at least 40%, competition assays were
subsequently carried out with increasing concentrations of the
compounds, to allow calculation of their Ki values. At a concen-
tration of 10 mm, most of the free modified amino acids inhib-
ited [14C]Gly-Sar uptake only weakly, with the exceptions of
fructoselysine (1), lactuloselysine (2), tagatoselysine (3), and
argpyrimidine (10), for which Ki values between 3.5 and
8.7 mm were calculated (Table 1). According to Brandsch,[32]
these compounds can be classified as medium- (3) and low-af-
finity ligands (1, 2, 10) for PEPT1. For purposes of comparison,
l-lysine and l-arginine showed no affinity towards PEPT1.
The glycated dipeptides, however, inhibited [14C]Gly-Sar
uptake by 40 to 94%. Ki values between 34 mm (Pyrr-Ala) and
>10 mm (Ala-FL) were determined (Table 1). The affinities to-
so the inhibitors might be nontransported compounds with
certain affinities towards the transporters. On the other hand,
several other carriers at the intestinal epithelium can be re-
sponsible for the translocation of substances. Flux measure-
ments were performed as in our previous studies;[25,27] [14C]Gly-
Sar and l-[3H]lysine were again used as the reference sub-
strates. The space marker [14C]mannitol served as a reference
compound for paracellular transport processes. Despite their
relatively low inhibitions of l-[3H]lysine uptake, all free amino
acids were subjected to the transport experiment. No CML or
CEL was found in the receiver compartment after 120 min
(Table 2). The flux rates of other glycated amino acids ranged
between 0.008 and 0.09% cmꢀ2 hꢀ1 and were always lower
than that of the space marker [14C]mannitol (0.13ꢁ
0.03%cmꢀ2 hꢀ1) and far lower than the flux of l-[3H]lysine
(6.86ꢁ0.15%cmꢀ2 hꢀ1). This argues against active transport of
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T. Henle et al.
to the free glycated amino acids (e.g., formyline 6,
0.04%cmꢀ2 hꢀ1; 6 from 6a, 3.37%cmꢀ2 hꢀ1). Most flux
rates were higher than the flux of the [14C]mannitol
space marker. This means that glycated dipeptides are
absorbed into the cells, most probably by PEPT1, and
hydrolyzed to the free modified amino acids and ala-
nine by intracellular peptidases. The amino acids reach
the basolateral compartment either through the action
of different amino acid transporters or possibly by
simple diffusion.
Table 2. Transepithelial flux of Maillard reaction products across Caco-2 cells and
cellular uptake after 120 min. Fluxes were determined in the presence of the
compound in question (1 mm), [14C]Gly-Sar (10 mm), l-[3H]lysine (2 nm), and
[14C]mannitol (10 mm), at pH 6.0 (apical) and pH 7.5 (basolateral) over 2 h. Data are
meansꢁSEM. n=3.
Compound
Flux of the Cellular
dipeptide
uptake[a]
[%cmꢀ2 hꢀ1] [%]
Flux of the
amino acid
[%cmꢀ2 hꢀ1
Cellular upta-
ke[a]
[%]
]
fructoselysine
Ala-FL
FL-Ala
lactuloselysine
tagatoselysine
carboxymethyllysine
Ala-CML
CML-Ala
carboxyethyllysine
Ala-CEL
CEL-Ala
formyline
Ala-Fom
Fom-Ala
pyrraline
Ala-Pyrr
Pyrr-Ala
maltosine
Ala-Mal
Mal-Ala
1
–
–
0.008ꢁ0.003[b] 0.03ꢁ0.01[b]
1a n.d.
1b n.d.
2
3
4
n.d.
n.d.
–
–
–
n.d.
n.d.
n.d.
n.d.
n.d.[b]
n.d.
n.d.
n.d.
n.d.
Pronounced differences with regard to the tenden-
cies of the glycated amino acids to leave the cells were
found. The total proportions of glycated dipeptides and
amino acids inside the cells and in the receiver com-
partment after 120 min are shown in Figure 1. More
than 50% of the glycated amino acids hydrolyzed from
the dipeptides of CML, CEL, maltosine, and MG-H1 after
absorption remain in the Caco-2 cells. This underlines
the transport capacity of PEPT1 as the likely responsible
carrier, capable even of transporting its substrates
uphill against a concentration gradient.[21] The MRPs
face the next barrier, the basolateral cell membrane,
not as dipeptides, but as amino acids. Hydrophobic gly-
cated amino acids such as 6, 7, and 10 can pass
through the basolateral membrane more quickly than
4, 5, 8, or 9, which are strongly retained, if not trapped,
inside the cells. We assume that the strong retention,
especially of hydrophilic amino acids, inside the cells is
due to the fact that they have to diffuse through the
basolateral membrane, a process that is easier for hy-
drophobic amino acids. The hydrophobic amino acids
permeate to the basolateral cell side to a greater extent
if their side chain modifications lack the capacity to
donate hydrogen bonds, as is the case for formyline 6,
but not for 7, 8, and 10. No correlation between the af-
finity of a dipeptide towards PEPT1 and the actual
transport was found.
–
–
–
0.62ꢁ0.02[b]
2.46ꢁ0.08
13ꢁ2
4a 0.02ꢁ0.01 0.05ꢁ0.01 0.11ꢁ0.01
4b 0.02ꢁ0.01 0.05ꢁ0.01 0.17ꢁ0.07
5
–
–
n.d.
1.36ꢁ0.07
3.1ꢁ1.5
12ꢁ1
5a 0.03ꢁ0.01 0.04ꢁ0.01 0.06ꢁ0.01
5b 0.03ꢁ0.01 0.17ꢁ0.01 0.10ꢁ0.05
6
–
–
0.04ꢁ0.01
3.37ꢁ0.13
1.09ꢁ0.16
0.07ꢁ0.02[c]
1.06ꢁ0.27[c]
0.28ꢁ0.08[c]
0.02ꢁ0.01[d]
0.27ꢁ0.08[d]
0.16ꢁ0.06[d]
0.09ꢁ0.08
0.24ꢁ0.11
0.22ꢁ0.03
0.01ꢁ0.01
0.30ꢁ0.02
0.14ꢁ0.04
0.03ꢁ0.01
0.08ꢁ0.01
2.39ꢁ0.08
0.86ꢁ0.08
0.6ꢁ0.1[c]
8.8ꢁ0.7[c]
2.6ꢁ0.2[c]
0.07ꢁ0.01[d]
16ꢁ2[d]
6a n.d.
6b n.d.
n.d.
n.d.
–
n.d.[c]
n.d.[c]
–
n.d.[d]
n.d.[d]
–
n.d.
n.d.
–
7
–
7a n.d.[c]
7b n.d.[c]
8
–
8a n.d.[d]
8b n.d.[d]
2.7ꢁ0.2[d]
0.32ꢁ0.04
10ꢁ2
MG-H1
9
–
Ala-(MG-H1)
(MG-H1)-Ala
argpyrimidine
Ala-Apy
9a n.d.
9b n.d.
10
10a n.d.
10b n.d.
11
3.12ꢁ0.17
0.05ꢁ0.02
1.79ꢁ0.14
0.38ꢁ0.03
0.01ꢁ0.01
–
0.23ꢁ0.02
4.01ꢁ0.24
–
n.d.
n.d.
–
Apy-Ala
pentosidine
[14C]Gly-Sar
[14C]mannitol
l-[3H]Lys
–
2.79ꢁ0.47 7.67ꢁ0.16 –
–
–
–
–
0.13ꢁ0.03[c]
6.86ꢁ0.15
[a] Total cellular uptake at the end of the experiment (120 min). [b] Value from
ref. [28]. [c] Value from ref. [25]. [d] Value from ref. [27]. n.d.: not detectable.
glycated amino acids by any of the Caco-2 amino acid trans-
port systems.
Conclusions
The transport studies were then performed with glycated di-
peptides. Caco-2 cells express membrane-bound peptidases,
so the dipeptides were partly hydrolyzed in the donor com-
partment during the flux measurement. The fluxes and cellular
uptakes reported in Table 2 therefore have to be regarded as
minimum values. All MRPs except for fructoselysine appeared
inside the cells and in the receiver compartment, but only the
dipeptides of CML and CEL could be recovered, to small ex-
tents, in intact form. When calculated for the intact dipeptides,
the flux rates of 4a/4b and 5a/5b are lower than that of the
space marker. However, all dipeptides were hydrolyzed very
quickly inside the cells, and the MRPs passed into the receiver
compartment in the form of amino acids. Therefore, the fluxes
are also calculated for the amino acids cleaved from the dipep-
tides (Table 2). Amino acids from the donor compartment
cannot interfere with this calculation because they are not
transported. The net flux rates of glycated amino acids, when
applied as dipeptides, are increased by up to 80-fold relative
Free glycated amino acids are not inhibitors of the lysine trans-
port system(s), nor are they transported in significant amounts
across cell monolayers. In contrast, several glycated dipeptides
are high-affinity inhibitors of [14C]Gly-Sar uptake. In particular,
the results for the carboxyalkylated peptides (4a/4b and 5a/
5b) show that not only side chain hydrophobization but also
the introduction of hydrophilic and charged carboxyl groups
can lead to strong inhibitors of [14C]Gly-Sar uptake. Those de-
rivatives that show high rates of [14C]Gly-Sar uptake inhibition
and high flux rates across Caco-2 monolayers are most likely
substrates of the intestinal proton-coupled peptide transporter
PEPT1. Depending on the kind of modification and the peptide
sequence, glycated peptides can be transported by PEPT1 into
the cells, where they are rapidly hydrolyzed. After addition of
glycated dipeptides to the apical compartment, no dipeptide
derivatives were detected in the basolateral compartment, but
free glycated amino acids were found. In particular, hydropho-
1274
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ChemBioChem 2011, 12, 1270 – 1279

Transport of Free and Peptide-Bound Glycated Amino Acids
more hydrophobic they are, the higher is the chance of their
appearance in the circulation.
Experimental Section
Materials: Boc-Ala-OSu, l-arginine, di-tert-butyl dicarbonate, glyox-
ylic acid monohydrate, and 1,1-dimethoxyacetone were obtained
from Fluka. Pd on activated charcoal (Pd/C, 10%, w/w), 3-chloro-
pentane-2,4-dione, glycylsarcosine (Gly-Sar), and Ala-Lys were pur-
chased from Sigma–Aldrich. Boc-Ala-Lys, Boc-Lys-Ala, Boc-Ala-Arg,
and Fmoc-Arg-Ala were from IRIS Biotech (Martinsried, Germany).
Microcrystalline cellulose (particle size 20–160 mm), pyruvic acid,
and N,N-diisopropyethylamine (DIPEA) from Merck were used. Ala-
OtBu and Lys-Ala were purchased from Bachem (Bubendorf, Swit-
zerland) and O-(N-succinimidyl)-N,N,N’,N’-tetramethyluronium tetra-
fluoroborate (TSTU) from Molekula (Taufkirchen, Germany). The fol-
lowing substances were synthesized by literature methods: 3-deoxy-
glucosone[6] (3-DG), fructoselysine[35] (1), 3-deoxypentosone[4] (3-
DPs), lactuloselysine[4] (2), formyline[4] (6), and pentosidine[36] (11).
The water used for the preparation of buffers and solutions was
obtained by use of a Purelab plus purification system (USFilter,
Ransbach-Baumbach, Germany). All other chemicals were pur-
chased from standard suppliers and were of the highest purity
available.
&
&
Figure 1. Total basolateral ( ) and intracellular ( ) proportions of glycated
peptides and amino acids at 120 min after apical application of glycated
peptides (1 mm). Molar amounts of glycated peptides and the correspond-
ing glycated amino acids were summed, divided by the initial peptide
amount, and expressed in %.
The human colon carcinoma cell line Caco-2 was obtained from
the German Collection of Microorganisms and Cell Cultures
(Braunschweig, Germany). Cell culture media, supplements, and
trypsin solution were purchased from Invitrogen or PAA (Cçlbe,
Germany). Fetal bovine serum was from Biochrom (Berlin, Germa-
ny). [Glycine-1-14C]Gly-Sar (specific radioactivity 56 mCimmolꢀ1
)
and l-[4,5-3H]lysine monohydrochloride (specific radioactivity
99 Cimmolꢀ1) were synthesized by GE Healthcare. [14C]Mannitol
(specific radioactivity 53 mCimmolꢀ1) was obtained from Hartmann
Analytic (Braunschweig, Germany).
Thin layer chromatography: TLC was performed on Merck silica
gel 60 plates with dichloromethane/methanol/25% aqueous NH3
(2:2:1, v/v/v) as the mobile phase. Visualization was achieved by
spraying the plates with a solution of ninhydrin in ethanol (0.1%)
acidified with glacial acetic acid (3%, v/v) followed by heating until
the appearance of spots. TLC plates were also used for the spot-
ting test to identify target fractions after chromatographic separa-
tions. Each fraction (1 mL) was spotted onto the TLC plate and
sprayed either with the ninhydrin reagent or with a solution of tri-
phenyltetrazolium chloride (TTC, 1%) in NaOH (1n).[4,35]
High-pressure liquid chromatography: All analytical HPLC analy-
ses were performed with an ꢂkta 10XT high-pressure gradient
system (Amersham Pharmacia Biotech, Uppsala, Sweden), consist-
ing of a P-900 pump with an online degasser (Knauer, Berlin, Ger-
many), a column oven, and a UV-900 UV detector. All separations
were performed with a stainless steel column (150ꢃ4.6 mm) filled
with Eurospher-100 RP-18 material of 5 mm particle size with an in-
tegrated guard column (5ꢃ4 mm) of the same material at a
column temperature of 308C. The injection volume was 50 mL. A
previously published solvent and gradient system permitted the
separation of 6 from both its peptides; the measurements were
performed at a wavelength of 293 nm.[4]
For the analyses of 10, 11, and the peptides 10a/10b, the eluent
was ammonium formate (pH 4.0, 10 mm), to which heptafluorobu-
tyric acid (HFBA, 650 mLLꢀ1) was added (solvent A, final pH: 3.5).
Solvent B consisted of a mixture of ammonium formate (pH 4.0,
50 mm, 200 mL) and methanol (800 mL), to which HFBA
(650 mLLꢀ1) was added. A linear gradient from 3 to 40% B in
20 min and then to 80% B in 3 min was applied at a flow rate of
bic glycated amino acids formed in the later stages of the Mail-
lard reaction can quickly permeate the basolateral cell mem-
brane either by simple diffusion or through the action of
amino acid transporters, whereas hydrophilic amino acids are
released much more slowly. Uptake rates of different dipep-
tides of the same MRP differ by factors of up to 4. It is highly
relevant whether MRPs are in the N- or the C-terminal position.
These findings are of nutritional and physiological relevance
and should be discussed as part of “risk assessment”. The data
show that free and dipeptide-bound Amadori products, which
represent more than 90% of the MRPs detected in foods, are
not taken up into cells in vitro. Others such as CML (4), CEL (5),
and MG-H1 (9) are strongly retained inside the cells. Further
studies should show whether these products are released very
slowly or are irretrievably trapped inside epithelial cells until
desquamation. In particular, few hydrophobic MRPs such as
pyrraline (7), maltosine (8), formyline (6), or argpyrimidine (10)
are transported through the cells. Because dietary pyrraline (7),
but not fructoselysine (1), is to a large extent excreted in the
urine, and because 7, but not 1, is absorbable in its peptide
form,[23] it can be assumed that maltosine (8), formyline (6),
and argpyrimidine (10) can also be absorbed after the diges-
tion of glycated food proteins. Further research should focus
on hepatic metabolism and renal handling. Digestibility studies
with proteins modified by the Maillard reaction are required in
order to provide information as to what extent MRPs appear in
absorbable peptide forms, and whether they are hydrolyzed
by luminal or membrane-bound peptidases. The longer these
MRPs are peptide-bound during intestinal digestion and the
ChemBioChem 2011, 12, 1270 – 1279
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1275

T. Henle et al.
1.0 mLminꢀ1. Argpyrimidine and its dipeptides were quantified
with use of a fluorescence detector (F-1050, Merck Hitachi) at exci-
tation and emission wavelengths of 320 and 380 nm, respectively.
For pentosidine, the wavelengths were set at 335 and 385 nm, re-
spectively. External calibration was performed with the synthesized
standards.
with a fraction collector (RediFrac, Amersham Pharmacia Biotech)
and the presence of the product was monitored by spotting of the
fractions (1 mL) on TLC plates and spraying with the appropriate re-
agent(s). Fractions containing the target product were repeatedly
concentrated to dryness and taken up in water until the smell of
pyridine or HCl had become imperceptible. Combined fractions
containing sodium citrate buffer were first desalted as described
earlier.[27] All products were stored at ꢀ208C.
Amino acid analysis (AAA): Flux measurements were performed
with an amino acid analyzer (S 433, Sykam, Fꢄrstenfeldbruck, Ger-
many) and a PEEK column (150ꢃ4.6 mm) filled with the cation ex-
change resin LCA K07/Li (particle size, 7 mm). Lithium buffers were
purchased ready for use from Sykam and employed for different
gradient programs according to the manufacturer’s instructions.
The lithium system was also used for the determination of the pu-
rities of synthesized substances, which were injected at a concen-
tration of 40 mgmLꢀ1. Flux samples of fructoselysine dipeptides ad-
ditionally had to be analyzed with the Alpha Plus amino acid ana-
lyzer (LKB Biochrom, Cambridge, UK) and a PEEK column (190ꢃ
4.6 mm) filled with a cation-exchange resin (particle size, 5 mm,
K. Grꢄning, Olching, Germany). The conditions are available from
the literature.[37] With both systems, post-column derivatization
with ninhydrin was applied, and VIS detection was performed with
integrated two-channel photometers working simultaneously at
440 nm and 570 nm. External calibration was performed with the
synthesized standards. The injection volume was 50 mL.
Ala-FL (1a) and FL-Ala (1b): Boc-Ala-Lys (507.3 mg, 1.6 mmol), and
d-glucose (1.73 g, 9.6 mmol) were dissolved in dry methanol
(90 mL) and heated under reflux for 4 h. The methanol was then
evaporated in vacuo and the residue was taken up in water
(150 mL). The pH was adjusted to 2.0 and the solution was trans-
ferred to a column (2.5ꢃ20 cm) filled with the cation exchanger
Lewatit S100, previously equilibrated with HCl (6m) and water
(each 250 mL). The column was washed with water (300 mL) to
remove the sugar, and the product was eluted with NH3 (2m ,
300 mL) after overnight incubation.[35] Ammonia was removed with
the aid of a rotary evaporator and the residue was subjected to
IEC on a column (1.5ꢃ50 cm) in the Py+ form. Compound 1a was
eluted with pyridinium acetate buffer (pH 4.35, 0.4n, 230–340 mL),
as revealed by the spotting test (TTC and ninhydrin). The product
was dissolved in methanol (2–3 mL) and precipitated in ice-cold
butanone as described in the literature[4,35] to yield 1a as a white
solid (384.7 mg, 53.2%).
For compound 1b, Boc-Lys-Ala (506 mg, 1.59 mmol) and d-glucose
(1.72 g, 9.6 mmol) were dissolved in water (50 mL). After freeze-
drying, the lyophilizate was incubated for 4 h at 708C in a sand
bath in a drying oven. The synthesis mixture was worked up as de-
scribed for 1a. Compound 1b was eluted with pyridinium acetate
buffer (pH 4.35, 0.4n, 350–550 mL) and was isolated as a white
solid after precipitation in ice-cold butanone (309.3 mg, 49.4%).
Tagatoselysine (3): Boc-Lys-OH (621.7 mg, 2.5 mmol) and d-galac-
tose (2.71 g, 15.1 mmol) were dissolved in N,N-dimethylformamide
(DMF)/methanol (3:7, v/v, 100 mL) and heated under reflux for 2 h.
The solvents were then evaporated in vacuo with repeated addi-
tion of water. Removal of the Boc protecting group and the excess
of the sugar were performed by IEC as described for 1a. Ammonia
was removed with the aid of a rotary evaporator and the residue
was subjected to IEC on a column (1.5ꢃ50 cm) in the Py+ form.
The spotting test (ninhydrin, TTC) revealed that 3 eluted with pyri-
dinium acetate buffer (pH 4.35, 0.4n, 160–250 mL). The product
was precipitated in ice-cold butanone as described above, to yield
3 as a white solid (420.6 mg, 43.4%).
Nuclear magnetic resonance spectroscopy, mass spectrometry,
and elemental analysis (EA): Proton spectra were recorded with a
Bruker DRX 500 instrument (Rheinstetten, Germany) at 500 MHz in
D2O as the solvent. Chemical shifts are given in parts per million
(ppm) relative to the internal HOD signal (4.70 ppm). For ESI-MS, a
PerSeptive Biosystems Mariner time-of-flight mass spectrometer
fitted with an electrospray ionization source (ESI-TOF-MS, Applied
Biosystems) working in the positive mode was used. Calibration of
the mass scale was established with a mixture of bradykinin, angio-
tensin I, and neurotensin. After appropriate dilution with formic
acid (1%) in aqueous acetonitrile (50%), the samples were injected
into the ESI source by syringe pump at a flow rate of 5 mLminꢀ1
.
EA data were obtained with a Euro EA 3000 elemental analyzer (Eu-
rovector, Milano, Italy). Elemental analysis was used to calculate
the product contents of the preparations. The percentage of nitro-
gen in the preparation was divided by the theoretical percentage
of nitrogen of the target substance and the content was expressed
in per cent by weight. All data relating to the characterization of
synthesis products are given in the Supporting Information.
Synthesis of ligands—general procedures: Purification of ligands
was performed by ion-exchange chromatography (IEC) with the
strongly acidic cation exchange resin DOWEX 50 WX-8 (200–400
mesh) unless otherwise stated. Before use, the resin was activated
with three times its volume both of HCl (6n) and of water. When
the resin was to be used in the H+ form, the material was placed
in a suitable Econo glass column (BioRad, Munich, Germany) and
equilibrated with three times its volume of HCl (0.01n). When it
was to be used in the Na+ form, the activated resin was rinsed
with three times its volume both of NaOH (1n) and of water,
placed in a suitable column, and equilibrated with three times its
volume of sodium citrate buffer (pH 3.00, 0.1n). When the resin
was required in its pyridinium (Py+) form, it was rinsed with three
times its volume both of aqueous pyridine (2m) and of water,
placed in a suitable column, and equilibrated with three times its
volume of pyridinium formate buffer (pH 3.00, 0.1n). The synthesis
mixtures were dissolved in equilibration buffer (30 mL) and applied
to the column after adjustment of the pH to 3.0. After rinsing of
the resin with a small volume of equilibration buffer, the products
were eluted by gravity with the elution buffers stated below at
flow rates of 0.2–0.4 mLminꢀ1. Fractions (10 mL) were collected
CML (4), Ala-CML (4a), CML-Ala (4b), CEL (5), Ala-CEL (5a), and
CEL-Ala (5b): These syntheses were performed by reductive alkyla-
tion of lysine derivatives with a-keto acids.[28] The amounts of reac-
tants given below were dissolved in water (30 mL) and the pH of
the solutions was adjusted to 8.75 with NaOH (1m) prior to the ad-
dition of Pd catalyst. The mixture was hydrogenated at room tem-
perature (RT) and atmospheric pressure for 24 h. During the syn-
theses of 5, 5a, and 5b, the H2 was renewed and hydrogenation
was continued for further 24 h. The catalyst was then filtered off.
Boc-CML and Boc-CEL were dissolved in HCl (3m) to concentra-
tions of 10–20 mgmLꢀ1 and heated under reflux for 3 h to remove
the Boc protecting group. The protected peptides were dissolved
in aqueous acetic acid (10%, 1000 mL) and heated under reflux for
4 h at 708C.[6] After the removal of the acids with the aid of a
rotary evaporator, the products were subjected to IEC on a column
(1.5ꢃ50 cm) in the H+ form. For the elution, a step gradient of as-
cending HCl concentrations (1, 1.5, 2m HCl, each 300 to 600 mL)
was applied. The spotting test (ninhydrin) showed that the prod-
ucts were well resolved from their educts and usually eluted with
1.5–2m HCl. After the evaporation of HCl, the products were
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ChemBioChem 2011, 12, 1270 – 1279

Transport of Free and Peptide-Bound Glycated Amino Acids
lyophilized to yield the hydrochlorides as slightly yellow amor-
phous powders.
the buffer, the residue was lyophilized, to yield 9a as a light
brownish powder (24.7 mg, 27.2%).
CML (4): Boc-Lys (1000 mg, 4.1 mmol), glyoxylic acid (480 mg,
5.2 mmol), Pd/C (50 mg). Yield 517 mg (46.4%).
Ala-CML (4a): Boc-Ala-Lys (500 mg, 1.6 mmol), glyoxylic acid
(187 mg, 2.0 mmol), Pd/C (38 mg). Yield 369 mg (60.7%).
CML-Ala (4b): Boc-Lys-Ala (500.3 mg, 1.6 mmol), glyoxylic acid
(710.8 mg, 7.5 mmol), Pd/C (50.6 mg). Yield 183.4 mg (31.2%).
CEL (5): Boc-Lys (1243 mg, 5.1 mmol), pyruvic acid (586 mg,
6.5 mmol), Pd/C (84.0 mg). Yield 980 mg (61.5%).
Ala-CEL (5a): Boc-Ala-Lys (500.0 mg, 1.6 mmol), pyruvic acid
(357 mg, 4.0 mmol), Pd/C (34 mg). Yield 430.0 mg (66.9%).
CEL-Ala (5b): Boc-Ala-Lys (500 mg, 1.6 mmol), pyruvic acid
(695 mg, 7.7 mmol), Pd/C (35 mg). Yield 377 mg (58.4%).
Ala-Fom (6a) and Fom-Ala (6b): For 6a, Boc-Ala-Lys (486 mg,
1.5 mmol) and 3-DPs (1000 mg) were dissolved in water (5.6 mL).
The solution was mixed with cellulose (4.5 g), and after lyophiliza-
tion the mixture was incubated at 708C in a drying oven. The
brown cake was extracted with water (3ꢃ100 mL). The pH of the
combined aqueous phases was adjusted to 1.0, and the solution
was extracted with ethyl acetate (3ꢃ100 mL). The pH of the solu-
tion was adjusted to 4.5, and the extraction was repeated (3ꢃ
100 mL ethyl acetate). The combined organic layers were evaporat-
ed to dryness with the aid of a rotary evaporator. Boc-protected in-
termediates were deprotected as described above for carboxyalky-
lated peptides. After evaporation of acetic acid, the residue was
subjected to IEC on a column (1.5ꢃ20 cm) in the Py+ form. Elution
was performed first with pyridinium formate buffer (pH 3.75, 0.3n,
250 mL) and then with pyridinium formate buffer (pH 4.05, 0.3n).
Compound 6a eluted between 20 and 140 mL of the second
buffer. After evaporation, the residue was precipitated in butanone
as described above, to yield 6a as a light yellow powder (73.4 mg,
15.2%).
(MG-H1)-Ala (9b) was prepared accordingly, from a mixture of 13
(81.6 mg, 0.23 mmol) and 14 (26.6 mL, 0.22 mmol) in HCl (12m,
9 mL), which was stirred at RT for 4 h. During IEC, 9b eluted with
the second elution buffer (270–400 mL). After the removal of the
buffer, the product was lyophilized, to yield 9b as a light yellow
powder (27.1 mg, 31.9%).
Argpyrimidine (10): 3-Chloropentane-2,4-dione (15, 1.68 mL,
14.9 mmol) was dissolved in DMSO (25 mL), and anhydrous sodium
acetate (2.44 g, 29.7 mmol) was added.[31] After the mixture had
been stirred for 3 h at RT, water was added (200 mL). The mixture
was extracted with diethyl ether (5ꢃ100 mL) after cooling. The ex-
tracts were dried (MgSO4) and concentrated to dryness. The re-
maining brownish oil was subjected to flash chromatography (FC)
on silica gel (20 g) with petroleum ether (40–608C)/ethyl acetate
(8:2, v/v). Target fractions of 3-acetoxypentane-2,4-dione 16 eluted
between 80–170 mL as revealed by the spotting test (TTC). The
slightly red oil that remained after evaporation of the solvents was
immediately added to a solution of l-arginine (1.047 g, 6.0 mmol)
in HCl (12m, 35 mL).[30] A second portion of 16 was added after 3 h
and the mixture was stirred at RT for 20 h. The solution was then
diluted with water (200 mL) and extracted with diethyl ether (3ꢃ
100 mL). The aqueous phase was concentrated to dryness and the
residue was subjected to FC on silica gel (30 g) with methanol/
ethyl acetate (2:1, v/v).[9] TLC of the fractions revealed that 10
eluted between 60–180 mL (Rf =0.77). After evaporation of the sol-
vents, argpyrimidine was isolated by IEC on a column (1.5ꢃ50 cm)
in the Py+ form. Elution was performed first with pyridinium for-
mate buffer (pH 4.05, 0.3n, 200 mL), and then with pyridinium ace-
tate buffer (pH 4.35, 0.4n). Compound 10 eluted with the second
buffer (380–600 mL). After buffer removal, the residue was precipi-
tated in butanone as described for 1a, to yield 10 as a white
powder (468.0 mg, 29.7%).
Ala-Apy (10a) and Apy-Ala (10b): Boc-Ala-OSu (840.9 mg,
2.94 mmol), compound 10 (199.1 mg, 0.78 mmol), and DIPEA
(330 mL, 1.9 mmol) were dissolved in a mixture of DCM (20 mL) and
methanol (10 mL). After overnight stirring at RT, the solvents were
removed under reduced pressure. The residue was dissolved in
NaHCO3 solution (5%, 50 mL) and extracted with ethyl acetate (3ꢃ
50 mL). The pH was adjusted to 1.0 with HCl (6m), and the aque-
ous phase was extracted with ethyl acetate (3ꢃ100 mL). The com-
bined organic layers were dried (Na2SO4) and concentrated to dry-
ness. The protected derivative was dissolved in HCl (6m)/tetrahy-
drofuran (1:1, v/v, 30 mL) and stirred at RT for 1 h.[38] The solvents
were removed with the aid of a rotary evaporator. Compound 10a
was then isolated by IEC on a column (1.5ꢃ50 cm) in the Py+
form. Elution was performed first with pyridinium acetate buffer
(pH 4.35, 0.4n, 300 mL) and then with pyridinium acetate buffer
(pH 5.00, 0.4n). Compound 10a eluted with the second buffer
(440–650 mL). After the removal of the buffer, the residue was
lyophilized, to yield 10a as a white powder (53.3 mg, 17.9%).
For 10b, compound 10 (162.7 mg, 0.64 mmol) was dissolved at
28C in a mixture of tetrahydrofuran (10 mL) and Na2CO3 solution
(0.5m, 10 mL). Di-tert-butyl dicarbonate (560 mg, 1.28 mmol) was
then added slowly. The cooling bath was removed and the mixture
was stirred for 2 h at RT. After removal of the solvents, the residue
was partitioned between water (30 mL) and ethyl acetate (10 mL).
The pH was adjusted to 2.4 and the aqueous phase was extracted
with ethyl acetate (3ꢃ30 mL). The combined organic layers were
extracted with water (2ꢃ20 mL), dried (Na2SO4), and concentrated
under reduced pressure. The residue, consisting of Boc-argpyrimi-
dine, was taken up in DCM (5 mL), and DIPEA (329 mL, 1.9 mmol)
and TSTU (232 mg, 0.77 mmol) were added. The mixture was
The synthesis of Fom-Ala (6b) was performed in the same way,
starting from Boc-Lys-Ala (452.7 mg, 1.4 mmol) and 3-DPs (750 mg)
dissolved in water (4.8 mL) and deposited on cellulose (3.8 g).
During IEC, compound 6b eluted with 15–200 mL of the second
elution buffer. Precipitation in butanone provided 6b as a light
yellow powder (56.8 mg, 12.4%).
MG-H1 (9), Ala-(MG-H1) (9a), and (MG-H1)-Ala (9b): l-Arginine
(1.009 g, 5.8 mmol) and 1,1-dimethoxyacetone (14, 678 mL,
5.7 mmol) were dissolved in HCl (12m, 110 mL). After the system
had been stirred at RT for 8 h, water (200 mL) was added and the
solution was concentrated to dryness in vacuo. The dried residue
was taken up in water (150 mL), and the pH was adjusted to 2.0.
The solution was transferred to a column (2.5ꢃ20 cm) filled with
the cation exchanger Lewatit S100, previously equilibrated with
HCl (6m) and water (each 250 mL). The column was washed with
water (300 mL) to remove uncharged byproducts, and the product
was eluted immediately with HCl (4m, 300 mL). After evaporation
of the acid, the residue was subjected to IEC on a column (1.5ꢃ
50 cm) in the Na+ form. Elution was first performed with sodium
citrate buffer (pH 4.50, 0.2n, 300 mL) and then with sodium citrate
buffer (pH 5.28, 0.3n). MG-H1 (9) eluted with the second buffer
(180–330 mL). After desalting and lyophilization, off-white amor-
phous 9 was obtained (794.4 mg, 41.5%).
Ala-(MG-H1) (9a) was prepared accordingly, from a mixture of 12
(78.5 mg, 0.23 mmol) and 14 (28.0 mL, 0.23 mmol) in HCl (12m,
9 mL), which was stirred at RT for 4 h. After the removal of byprod-
ucts as described for 9, the residue was subjected to IEC on a
column (1.5ꢃ50 cm) in the Py+ form. Elution was performed first
with pyridinium acetate buffer (pH 4.35, 0.4n, 400 mL), and then
with pyridinium acetate buffer (pH 5.00, 0.4n). Compound 9a
eluted with the second buffer (140–200 mL). After evaporation of
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T. Henle et al.
stirred at RT for 20 min. A portion of Ala-OtBu hydrochloride
(261.0 mg, 1.4 mmol) in DCM (5 mL) was then added, and the solu-
tion was stirred for 30 min at RT. DCM was removed and the resi-
due was dissolved in diethyl ether (100 mL) and extracted with HCl
(1m, 2ꢃ50 mL), NaHCO3 solution (5%, 2ꢃ50 mL), and water (2ꢃ
50 mL). The organic layer was concentrated under reduced pres-
sure and the residue was deprotected and isolated as described
for 10a. Compound 10b eluted with the second elution buffer
(630–880 mL). After the removal of the buffer, the residue was
lyophilized, to yield 10b as a white powder (32.7 mg, 11.9%).
Ala-Arg (12) and Arg-Ala (13): Boc-Ala-Arg (509.7 mg, 1.4 mmol)
was deprotected as described for 10a. The dried residue was sub-
jected to IEC on a column (1.5ꢃ50 cm) in the Na+ form. The
column was first rinsed with sodium citrate buffer (pH 5.35, 0.3n,
400 mL), and then with sodium citrate buffer (pH 6.00, 0.3n,
400 mL). The product was then eluted with sodium citrate buffer
(pH 6.00, 0.5n, 300 mL). After desalting, off-white amorphous 12
was obtained (452.6 mg, 92.7%).
For 13, Fmoc-Arg-Ala (704.8 mg, 1.5 mmol) was dissolved in DMF/
methanol/morpholine (72:8:20, v/v/v, 100 mL). The mixture was
stirred at RT for 1 h and then concentrated to dryness. The residue
was partitioned between water (100 mL) and diethyl ether (50 mL).
The aqueous phase was extracted with diethyl ether (2ꢃ50 mL)
and then concentrated to dryness. The residue was subjected to
IEC on a column (1.5ꢃ50 cm) in the Na+ form. After rinsing of the
column with sodium citrate buffer (pH 6.00, 0.3n, 500 mL), the
product was eluted with sodium citrate buffer (pH 6.00, 0.5n). TLC
showed a chromatographically pure fraction eluting between 180–
310 mL (Rf =0.27). After desalting, off-white amorphous 13 was ob-
tained (270.4 mg, 49.4%).
Cell culture: Caco-2 cells were routinely cultured in culture flasks
(75 cm2) with minimum essential medium supplemented with fetal
bovine serum (10%), gentamicin (50 mgmLꢀ1), and nonessential
amino acid solution (1%) at 378C under a humidified atmosphere
[CO2 (5%), O2 (95%)].[25,28,33] Cultures with a confluence of 80%
were treated for 5 min with Dulbecco’s phosphate-buffered saline
followed by a 2 min incubation with trypsin solution. For uptake
experiments, the cells were seeded in 35 mm disposable Petri
dishes (Sarstedt, Nꢄmbrecht, Germany) at a density of 0.8ꢃ106
cells per dish. The uptake measurements were performed on the
seventh day after seeding. Protein content per dish was deter-
mined by a Pierce Protein Assay (660 nm, Thermo Fisher Scientific)
by the manufacturer’s protocol.
For the flux measurements, Caco-2 cells were cultured on permea-
ble polycarbonate Transwell cell culture inserts (diameter 24 mm,
pore size 3 mm, Costar, Bodenheim, Germany) with a cell density of
0.2ꢃ106 cells per filter for 21 days.[25,28] The lower (receiver) com-
partment contained medium (2.6 mL) and the upper (donor) com-
partment medium (1.5 mL). The transepithelial electrical resistance
was measured at day 21 with a Millicell ERS (Millipore Intertech).
Transport studies: Uptake of [14C]Gly-Sar and l-[3H]lysine into
Caco-2 cells cultured on plastic dishes was measured at RT as de-
scribed earlier.[25,28,33] The uptake buffer contained Mes/Tris (pH 6.0,
25 mm), NaCl (140 mm), KCl (5.4 mm), CaCl2 (1.8 mm), MgSO4
(0.8 mm), glucose (5 mm), [14C]Gly-Sar (10 mm), or l-[3H]lysine
(2 nm), together with unlabeled compounds at increasing concen-
trations (0–10 mm). After incubation either for 10 min ([14C]Gly-Sar
uptake) or for 5 min (l-[3H]lysine uptake), the cells were quickly
washed four times, dissolved in Igepal CA-630 buffer, and prepared
for liquid scintillation spectrometry. The nonsaturable component
of [14C]Gly-Sar and l-[3H]lysine uptake (diffusion, adherent radioac-
tivity) determined by measuring the uptake of the labeled com-
pound in the presence of the unlabeled compound [Gly-Sar
(50 mm) or l-lysine (20 mm)] represented 8% and 21% of the total
uptake, respectively. This value was used during nonlinear regres-
sion analysis of inhibition constants.[25]
Transepithelial flux experiments at Caco-2 cell monolayers were
performed at day 21 after seeding at 378C in a shaking table incu-
bator.[25,28,33] In brief, after washing of the inserts with buffer
[Hepes/Tris (pH 7.5, 25 mm), NaCl (140 mm), KCl (5.4 mm), CaCl2
(1.8 mm), MgSO4 (0.8 mm), glucose (5 mm)], uptake was started by
addition of uptake buffer (pH 6.0, 1.5 mL) containing compounds
(1 mm) to the donor side. After 10, 30, 60, and 120 min, samples
(200 mL) were taken from the receiver compartment (2.6 mL) and
replaced with fresh buffer (pH 7.5). Samples were stored at ꢀ208C
until analysis by HPLC. After 2 h, the filters were quickly washed
four times with ice-cold uptake buffer, cut out of the plastic inserts
and stored in TCA solution (10%, 1 mL), which was frozen and de-
frosted three times. Before HPLC and AAA, the samples were dilut-
ed appropriately with the solvent A and loading buffers, respec-
tively.
Data analysis: Results are given as means ꢁSEM (n=4 to 9). IC50
values (that is, concentration of unlabeled compounds necessary
to inhibit 50% of [14C]Gly-Sar or l-[3H]lysine carrier-mediated
uptake) were determined by nonlinear regression by using the lo-
gistic Equation (1) for an asymmetric sigmoid (allosteric Hill kinet-
ics):
MaxꢀMin
Y ¼ Min þ
ð1Þ
P
1 þ ðX=IC50Þ
where Max is the initial Y value, Min the final Y value, and the
power P represents Hill’s coefficient (SigmaPlot program, Systat,
Erkrath, Germany), and converted into inhibition constants (Ki).[33]
Flux data were calculated after correction for the removed
amounts by linear regression of appearance in the receiver well
versus time.
Acknowledgements
We thank Karla Schlosser and Dr. Uwe Schwarzenbolz, Institute
of Food Chemistry, TU Dresden, for carrying out the amino acid
analyses and the acquisition of the mass spectra, respectively. We
are grateful to members of the Institute of Organic Chemistry, TU
Dresden, namely Dr. Margit Gruner and Anett Rudolph for record-
ing the NMR spectra and Anke Peritz for performing the elemen-
tal analyses. This work was supported by the Deutsche For-
schungsgemeinschaft grants HE 2306/9–1 and BR 2430/2–1.
Keywords: glycation · Maillard reaction · membranes · pept1 ·
peptides
[1] T. Henle, Amino Acids 2005, 29, 313–322; b) F. Ledl, E. Schleicher,
Angew. Chem. 1990, 102, 597–626; Angew. Chem. Int. Ed. Engl. 1990, 29,
565–594.
[2] M. U. Ahmed, S. R. Thorpe, J. W. Baynes, J. Biol. Chem. 1986, 261, 4889–
4894.
[3] M. U. Ahmed, E. Brinkmann Frye, T. P. Degenhardt, S. R. Thorpe, J. W.
Baynes, Biochem. J. 1997, 324, 565–570.
[4] M. Hellwig, T. Henle, Eur. Food Res. Technol. 2010, 230, 903–914.
[5] T. Nakayama, F. Hayase, H. Kato, Agric. Biol. Chem. 1980, 44, 1201–1202.
[6] T. Henle, A. Bachmann, Z. Lebensm.-Unters. Forsch. 1996, 202, 72–74.
[7] F. Ledl, H. Osiander, O. Pachmayr, T. Severin, Z. Lebensm.-Unters. Forsch.
1989, 188, 207–211.
[8] T. Henle, A. W. Walter, R. Haeßner, H. Klostermeyer, Z. Lebensm.-Unters.
Forsch. 1994, 199, 55–58.
1278
www.chembiochem.org
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2011, 12, 1270 – 1279

Transport of Free and Peptide-Bound Glycated Amino Acids
[9] I. N. Shipanova, M. A. Glomb, R. H. Nagaraj, Arch. Biochem. Biophys.
1997, 344, 29–36.
[23] A. Fçrster, Y. Kꢄhne, T. Henle, Ann. N.Y. Acad. Sci. 2005, 1043, 474–481.
ˇ
[24] K. Sebekovꢅ, G. Saavedra, C. Zumpe, V. Somoza, K. Klenoviscovꢅ, I. Bir-
[10] a) D. R. Sell, V. M. Monnier, J. Biol. Chem. 1989, 264, 21597–21602;
b) S. J. Cho, G. Roman, F. Yeboah, Y. Konishi, Curr. Med. Chem. 2007, 14,
1653–1671.
louez-Aragon, Ann. N.Y. Acad. Sci. 2008, 1126, 177–180.
[25] M. Hellwig, S. Geissler, A. Peto, I. Knꢄtter, M. Brandsch, T. Henle, J. Agric.
Food Chem. 2009, 57, 6474–6480.
[11] T. Henle, Kidney Int. 2003, 63, S145-S147.
[26] S. Geissler, M. Hellwig, M. Zwarg, F. Markwardt, T. Henle, M. Brandsch, J.
Agric. Food Chem. 2010, 58, 2543–2547.
[27] S. Geissler, M. Hellwig, F. Markwardt, T. Henle, M. Brandsch, Eur. J.
Pharm. Biopharm. 2011, 78, 75–82.
[12] I. Birlouez-Aragon, G. Saavedra, F. J. Tessier, A. Galinier, L. Ait-Aimeur, F.
Lacoste, C. N. Niamba, N. Alt, V. Somoza, J. M. Lecerf, Am. J. Clin. Nutr.
2010, 91, 1220–1226.
[13] T. Koschinsky, C.-J. He, T. Mitsuhashi, R. Bucala, C. Liu, C. Bꢄnting, K.
Heitmann, H. Vlassara, Proc. Natl. Acad. Sci. USA 1997, 94, 6474–6479.
[14] a) T. Henle, Mol. Nutr. Food Res. 2007, 51, 1075–1078; b) N. Ahmed, P. J.
Thornalley, Diabetes Obes. Metab. 2007, 9, 233–245.
[28] S. Grunwald, R. Krause, M. Bruch, T. Henle, M. Brandsch, Br. J. Nutr.
2006, 95, 1221–1228.
[29] I. Knꢄtter, B. Hartrodt, S. Theis, M. Foltz, M. Rastetter, H. Daniel, K. Neu-
bert, M. Brandsch, Eur. J. Pharm. Sci. 2004, 21, 61–67.
[30] N. Sreejayan, X. P. Yang, K. Palanichamy, K. Dolence, J. Ren, Eur. J. Phar-
macol. 2008, 593, 30–35.
[31] L. Valgimigli, G. Brigati, G. F. Pedulli, G. A. DiLabio, M. Mastragostino, C.
Arbizzani, D. A. Pratt, Chem. Eur. J. 2003, 9, 4997–5010.
[32] M. Brandsch, Expert Opin. Drug Metab. Toxicol. 2009, 5, 887–905.
[33] I. Knꢄtter, G. Kottra, W. Fischer, H. Daniel, M. Brandsch, Drug Metab.
Dispos. 2009, 37, 143–149.
[34] R. Tateoka, H. Abe, S. Miyauchi, S. Shuto, A. Matsuda, M. Kobayashi, K.
Miyazaki, N. Kamo, Bioconjugate Chem. 2001, 12, 485–492.
[35] R. Krause, K. Knoll, T. Henle, Eur. Food Res. Technol. 2003, 216, 277–283.
[36] T. Henle, U. Schwarzenbolz, H. Klostermeyer, Z. Lebensm.-Unters. Forsch.
1997, 204, 95–98.
ˇ
[15] K. Sebekovꢅ, V. Somoza, Mol. Nutr. Food Res. 2007, 51, 1079–1084.
[16] G. Cohen, G. Glorieux, P. Thornalley, E. Schepers, N. Meert, J. Jankowski,
V. Jankowski, A. Argiles, B. Anderstam, P. Brunet, C. Cerini, L. Dou, R.
Deppisch, B. Marescau, Z. Massy, A. Perna, J. Raupachova, M. Rodriguez,
B. Stegmayr, R. Vanholder, W. H. Hçrl, Nephrol. Dial. Transplant. 2007, 22,
3381–3390.
[17] J. Uribarri, S. Woodruff, S. Goodman, W. Cai, X. Chen, R. Pyzik, A. Yong,
G. E. Striker, H. Vlassara, J. Am. Diet. Assoc. 2010, 110, 911–916.
[18] J. M. Ames, Mol. Nutr. Food Res. 2007, 51, 1085–1090.
[19] S. A. Adibi, D. W. Mercer, J. Clin. Invest. 1973, 52, 1586–1594.
[20] R. Devꢀs, C. A. R. Boyd, Physiol. Rev. 1998, 78, 487–545.
[21] H. Daniel, Annu. Rev. Physiol. 2004, 66, 361–384.
[22] a) P. D. Bailey, C. A. Boyd, J. R. Bronk, I. D. Collier, D. Meredith, K. M.
Morgan, C. S. Temple, Angew. Chem. 2000, 112, 515–518; Angew. Chem.
Int. Ed. 2000, 39, 505–508; b) C. U. Nielsen, R. Andersen, B. Brodin, S.
Frokjaer, M. E. Taub, B. Steffansen, J. Controlled Release 2001, 76, 129–
138; c) T. Terada, K. Inui, Curr. Drug Metab. 2004, 5, 85–94; d) M.
Brandsch, I. Knꢄtter, F. H. Leibach, Eur. J. Pharm. Sci. 2004, 21, 53–60;
e) I. Rubio-Aliaga, H. Daniel, Xenobiotica 2008, 38, 1022–1042; f) B. S.
Vig, T. R. Stouch, J. K. Timoszyk, Y. Quan, D. A. Wall, R. L. Smith, T. N.
Faria, J. Med. Chem. 2006, 49, 3636–3644.
[37] T. Henle, H. Walter, I. Krause, H. Klostermeyer, Int. Dairy J. 1991, 1, 125–
135.
[38] M. A. Glomb, C. Pfahler, J. Biol. Chem. 2001, 276, 41638–41647.
Received: December 16, 2010
Published online on April 28, 2011
ChemBioChem 2011, 12, 1270 – 1279
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1279