N-Terminal 2,3-diaminopropionic acid (Dap) peptides as efficient methylglyoxal scavengers to inhibit advanced glycation endproduct (AGE) formation

Article abstract of DOI:10.1016/j.bmc.2009.02.018

2,3-Diaminopropionic acid (Dap) and N-terminal Dap peptides have been found to inhibit in vitro protein-modifications by methylglyoxal (MG), one of the highly reactive α-dicarbonyl compounds. MG scavenging potency of the newly synthesized N-terminal Dap p

Full text of DOI:10.1016/j.bmc.2009.02.018

Bioorganic & Medicinal Chemistry 17 (2009) 2310–2320
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
N-Terminal 2,3-diaminopropionic acid (Dap) peptides as efficient
methylglyoxal scavengers to inhibit advanced glycation
endproduct (AGE) formation
a
a
a
N. André Sasaki a,b, , Maria Concepcion Garcia-Alvarez , Qian Wang , Ludmila Ermolenko ,
*
Gisèle Franck a, Naïma Nhiri a, Marie-Thérèse Martin a, Nicolas Audic b, Pierre Potier z
a Institut de Chimie des Substances Naturelles, CNRS, 91198 Gif-sur-Yvette Cedex, France
b Pharmamens, Laboratoire de Chimie Thérapeutique, Faculté de Pharmacie, 80037 Amiens Cedex 1, France
a r t i c l e i n f o
a b s t r a c t
Article history:
2,3-Diaminopropionic acid (Dap) and N-terminal Dap peptides have been found to inhibit in vitro pro-
Received 23 June 2008
Revised 6 February 2009
Accepted 10 February 2009
Available online 20 February 2009
tein-modifications by methylglyoxal (MG), one of the highly reactive a-dicarbonyl compounds. MG scav-
enging potency of the newly synthesized N-terminal Dap peptides is demonstrated by RP-HPLC, SDS–
PAGE and non-denaturing PAGE analysis, assays for enzymatic activity and cell viability study and was
compared with that of known AGE inhibitors, such as aminoguanidine, pyridoxamine, metformin and
carnosine. Two addition products of MG and
tures are characterized by 1H and 13C NMR spectroscopy to indicate that both of them are pyrazines
derived from 2 molecules of MG and 1 molecule of -Dap- -Leu. Mutagenic activities of -Dap- -Leu
and -Dap- -Val and their metabolites according to the Ames assay are found to be negative.
Ó 2009 Elsevier Ltd. All rights reserved.
L-Dap-L-Leu are separated by HPLC and their chemical struc-
This paper is dedicated to the memory of
Professor Pierre Potier, an outstanding
international figure in medicinal chemistry
L
L
L
L
L
L
Keywords:
AGEs inhibitors
a-Dicarbonyl compounds
Methylglyoxal
N-Terminal 2,3-diaminopropionic acid
(Dap) dipeptides
1. Introduction
These a-dicarbonyl compounds lead to the non-enzymatic for-
mation of toxic and immunogenic AGEs that are cross-linking and
non-cross-linking of proteins. Many of these cross-links include
heterocyclic structures such as MOLD (methylglyoxal lysine di-
mer), GOLD (glyoxal lysine dimer), GODIC (glyoxal lysine arginine
dimer), MODIC (methylglyoxal lysine arginine dimer), pentosidine
and glucosepane. Examples of non-cross-linking AGEs are G-H1,
MG-H1, 3DG-H1 (hydroimidazolone adducts of arginine with GO,
Non-enzymatic glycation of proteins, known as the Maillard
reaction, is initiated by the condensation of amino groups present
in proteins with compounds containing reactive carbonyl groups.
Early-stage of this reaction leads to the formation of the early gly-
cation adduct, fructosyl-lysine, via Schiff base and concomitant
Amadori rearrangement. Later stage Maillard reaction is the forma-
tion of advanced glycation endproducts (AGEs) by the reaction of
e
MG and 3-DG, respectively), CML (N -carboxymethyl lysine), CEL
e
amino, guanido and thiol groups of proteins with
compounds derived from the degradation of glycated proteins.
The most notable highly reactive -dicarbonyls are glyoxal (GO),
a
-dicarbonyl
(N -carboxyethyl lysine) and Argpyrimidine.1–4 Glycation reactions
take place endogenously in all tissues and body fluids under phys-
iological conditions.
a
methylglyoxal (MG) and 3-deoxyglucosone (3-DG) that are origi-
nated from non-enzymatic dephosphorylation of triosedihy-
droxyacetone phosphate and glyceraldehyde-3-phosphate as well
as the lipid peroxidation, the glucose auto-oxidation and degrada-
tion of glycated proteins.
The consequence of AGE formation is the reticulation of pro-
teins. This phenomenon has been observed in long-lived proteins
such as tissue collagen, lens crystallin, fibronectin, tubulin, lami-
nin, actin, hemoglobin, albumin and lipids associated on low-den-
sity lipoproteins. Protein modification in the form of AGEs
progressively increases with aging and it is considered to contrib-
ute to the modification of normal tissues. The formation and accu-
mulation of increased AGEs has been implicated in the
development of cataracts, uremia, atherosclerosis, Alzheimer’s dis-
ease, Parkinson’s disease and above all, clinical complications of
* Corresponding author. Tel.: +33 1 69823101; fax: +33 1 69077247.
E-mail address: andre.sasaki@icsn.cnrs-gif.fr (N.A. Sasaki).
z
Deceased on February 3rd 2006.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.02.018

N. A. Sasaki et al. / Bioorg. Med. Chem. 17 (2009) 2310–2320
2311
type 2 diabetes, such as micro- and macroangiopathy, retinopathy,
nephropathy, neuropathy and ulcer.5–13 It is to be noted that the
concentration of MG was found to be 2–6-fold higher in patients
with both insulin-dependent and non-dependent diabetes.14 Nor-
mally, the glutathione-dependent glyoxylase system (glyoxalase I
were in the range of 80–98%, those of the following two-step reac-
tion were almost quantitative.
2.2. Dicarbonyl-scavenging activity of Dap and N-terminal Dap
dipeptides
and glyoxalase II) catalyzes the detoxification of the
a-dicarbonyls
by transforming them into
D
-lactate, glycolate and acetol.15,16
2.2.1. Evaluation by RP-HPLC
However, dysfunction of this metabolism of detoxification stimu-
lates the accumulation of the AGEs in the body leading to protein
damage during general aging and several pathological
conditions.17
In view of the wide occurrence and their deleterious conse-
quences of AGEs, numerous agents have been reported as potent
On the assumption that the nucleophilic trapping of a-dicar-
bonyl compounds by a scavenger is much more rapid than that
of the basic functional groups of polypeptide or protein, monitor-
ing the quantity or quality of the unmodified polypeptide or pro-
tein can serve as a means to evaluate the sequestering capacity
of a tested scavenger.
scavengers of the reactive
dine (PimagedineÒ), pyridoxamine (PyridorinÒ),
min, carnosine, tenilsetam, thiamine, OPB-9195,
a
-dicarbonyls. These are aminoguani-
-arginine, metfor-
-penicillamine,
Efficacy of each scavenger in this study was first assessed on RP-
HPLC by comparative measurement of the remaining intact insulin
vis-à-vis the control when it is incubated with MG in phosphate
buffer of pH 7.4 at 37 °C for 24 h in the presence of a scavenger
compound (1.1 mol equiv with respect to MG). In this work, we
have chosen insulin (51 amino acids with 1 Arg and 1 Lys) as model
polypeptide because of its easy access and well documented HPLC
studies thereupon.32 Krause and his colleagues have reported that
the retention times of native bovine insulin and those of GO- and
MG-modified insulin differ from each other on HPLC analysis.33
L
D
and LR-90.18–28 Although aminoguanidine has shown to be an
excellent anti-AGE drug, it has been abandoned due to adverse side
effects that were observed in phase three clinical trials in patients
with diabetes. On the other hand, thiamine and pyridoxamine are
considered as highly promising AGE inhibitors. Despite interesting
scavenging effects, studies on the chemical mechanisms of many of
these scavengers with respect to the reactive
yet to be confirmed except for aminoguanidine, metformin, and
-penicillamine. Taking into account of the 1,2-vicinal carbonyl
groups in the molecule, it seems rational to postulate 1,2-diamino
compounds for trapping -dicarbonyls by forming six-membered
a-dicarbonyls have
As a scavenger, we have arbitrarily tested D-Dap-L-Leu. When incu-
D
bated with MG alone, insulin peak on HPLC chromatogram dimin-
ishes in proportion to the increasing amount of MG in
concentration dependent manner until its complete disappearance
at 100 equiv level as shown in Figure 1a. However, under the same
a
ring piperazine and/or pyrazine derivatives. Validity of this ap-
proach has been exploited in the use of o-phenylenediamine or
1,2-diamino-4,5-dimethoxybenzene for the quantification of plas-
mic dicarbonyls.29,30 However, surprisingly, no work has been
conditions, incubation of insulin with MG in the presence of
D-Dap-
L-Leu (dipeptide/MG = 1.1 equiv) significantly reduces this trend
(Fig. 1b). In case insulin is incubated with 10 equiv of MG, it takes
about 7 days to observe complete degradation (Fig. 2b). In order to
shorten the incubation time to 24 h and thus facilitate the analysis,
100 and 110 M equiv of MG and MG-scavenger, respectively, with
respect to insulin, were used throughout this study.
reported on the therapeutic use of 1,2-diamino systems as a-dicar-
bonyl scavengers presumably due to toxic properties of the aro-
matic diamines. We deemed that it would be worthy to evaluate
therapeutic potential of 1,2-vicinal diaminoalkyl compounds as
sequestrating agents of the highly reactive
a
-dicarbonyls. To this
Quantification of the protected insulin is performed by com-
paring the insulin incubated with MG in the presence of a scaven-
ger with that of the control. When insulin is incubated in 10 mM
phosphate buffer of pH 7.4 (containing 0.1 M NaCl) with MG in
end, introduction of an acidic functional group such as CO2H or
SO3H into the 1,2-diamino containing molecule is considered a pri-
mary requirement for facilitating the ready excretion of the resul-
tant condensation products by kidney. In this context, dipeptides
with 2,3-diaminopropionic acid (Dap) moiety in its N-terminal ap-
pears to be an interesting model since side chain functional group
of the C-terminal amino acid can serve as a site for enhancing bio-
availability or therapeutic effects of the molecule. We report here
our studies on Dap and N-terminal Dap dipeptides to demonstrate
the presence of D-Dap-L-Leu at 37 °C and pH 7.4 for 24 h, 83% of
insulin is found to be protected from the MG-modification
(Fig. 1b). Among the products formed under these reaction condi-
tions, the one with retention time of 10 min in Figure 1b corre-
sponds to Adduct I or II formed by 2 mol of MG and 1 mol of
D-
Dap- -Leu (vide post). From co-injection and mass spectrometry
L
their potential as reactive
a
-dicarbonyl compound scavengers.
analysis, we confirmed that the peak with retention time of
14.8 min in Figure 1b belongs to that of unmodified human insu-
lin. Then, we investigated the scavenging potency of the different
N-terminal Dap-dipeptides that were prepared for this study.
Thus, insulin was incubated in the same conditions as described
above. Figure 3 illustrates that N-terminal Dap-dipeptides, such
as Dap-Leu, Dap-Nle, Dap-Pro and Dap-Ile possess excellent
capacity to prevent the degradation of insulin by MG. For the
comparison, efficacy of other known scavengers such as amino-
guanidine, pyridoxamine, penicillamine, cysteine, metformin and
carnosine was also investigated under the same conditions.
Whereas aminoguanidine, penicillamine and cysteine are potent
2. Results and discussion
2.1. Synthesis
N-Terminal Dap dipeptides were prepared by coupling N,N0-di-
Boc-2,3-Dap-OH31 with the methyl ester of amino acids in the
presence of EDC and HOBt. After alkaline hydrolysis of the methyl
ester with LiOH, N,N0-di-Boc groups were removed by the treat-
ment with 3 M HCl–dioxane (or THF) to obtain dipeptides as dihy-
drochloride salts (Scheme 1). While yields of the coupling reaction
Scheme 1. Synthesis of Dap dipeptides.

2312
N. A. Sasaki et al. / Bioorg. Med. Chem. 17 (2009) 2310–2320
Figure 1. (a) Degradation of insulin incubated with different quantity of MG in
0.1 M NaCl and 10 mM phosphate buffer of pH 7.4 at 37 °C for 24 h. (b) Protection of
insulin by
D
-Dap-
L
-Leu against different quantity of MG under the same conditions
as described above;
D
-Dap- -Leu/MG = 1.1. RP-HPLC conditions: Column: Grace-
L
Vydac Protein and Peptides C18 218TP54 (4.6 ꢀ 250 mm); injection volume 20
lL
of the reaction mixture; flow rate: 1 mL/min; temperature 25 °C; eluent A:
H2O + 0.1% TFA; eluent B: CH3CN/H2O 60/40 + 0.1% TFA; linear gradient: 40% B to
100% B in 35 min; detection: PDA (chromatograms extracted at 220 nm).
scavengers of MG, the sequestration capacity of pyridoxamine
and metformin was shown to be significantly low and that of car-
nosine was undetectable under the studied conditions. It is
noticeable that the a-dicarbonyl scavenging potency of 1,2-diami-
no moiety of Dap is much higher than that of 1,3-analogue of 2,
Figure 2. (a) Stability of insulin in a 0.1 M NaCl and 10 mM phosphate buffer of pH
7.4 at 37 °C for 24 h. (b) Degradation of insulin in the presence of 10 equiv of MG
under the same conditions as described for Figure 1a. (c) Protection of insulin by
D-Dap-L-Leu against 10 equiv of MG (D-Dap-L-Leu/MG = 1.1) under the same
conditions as described for Figure 1a. RP-HPLC conditions: the same as those
described for Figure 1.
4-diaminobutyric acid.
2.2.2. Evaluation by electrophoresis
mer and trimer as shown in Figure 5. On the contrary, the same
2.2.2.1. Test on RNase A. We next turned our attention to evaluate
the scavenging potency of N-terminal Dap-dipeptides by standard
SDS–PAGE electrophoresis. Bovine pancreatic ribonuclease A (RNase
A, 124 amino acids with 10 Lys and 4 Arg) has been extensively used
treatment in the presence of L-Dap-L-Leu or L-L-Dap-L-Val inhibits
the protein modification. The test was carried out under the same
conditions as those for RNase A. It is to be noted that in both cases
of RNase A and lysozyme, N-terminal Dap peptides are much more
efficient MG scavenger than AG that leads to the formation of a
dimer.
as a model protein for cross-linking studies.3,34 We have selected
L-
Dap-L-Leu and L-Dap-L-Val as cross-linking inhibitors for the similar
study. The ratio of MG to RNase A is 1:1 since incubation with equi-
molar amount of MG is found enough to observe the formation of
RNase A dimer and trimer. Consequently, 1.1:1 mole ratio of scaven-
ger with respect to MG was employed. After 48 h of incubation of
RNase A with MG at 37 °C, protein cross-linking is observed. How-
2.2.2.3. Test on glyoxalase I. A third electrophoresis study was
carried on human glyoxalase I (184 amino acids with 18 Lys and
5 Arg), a key protein for the detoxification system of the reactive
a
-dicarbonyl compounds.16 Since the basic functional groups in
ever, addition of
cess. Similarly,
structural modification by MG (Fig. 4).
L
-Dap-
-Dap-
L
-Leu to the co-incubation inhibits this pro-
glyoxalase I are much more abundant than in RNase and in lyso-
zyme, formation of dimer or trimer might not be observed under
previous SDS–PAGE conditions when glyoxalase I was incubated
with equimolar amount of MG. PAGE of glyoxalase I was then per-
formed under native conditions to demonstrate the change in the
electrophoretic mobility of the MG-treated protein. Figure 6 dem-
onstrates that exposure to 10 mM of MG for 24 h markedly in-
creases the mobility of the protein toward the anode, a change
L
L-Val protects the protein against the
2.2.2.2. Test on chicken lysozyme. SDS–PAGE electrophoresis
was also used for demonstrating the inhibitory efficacy of tenisle-
tam against lysozyme modification by 3-DG.24 In our study, it is
found that incubation of chicken lysozyme (129 amino acids with
6 Lys and 11 Arg) with MG modifies the protein by forming the di-
consistent with the loss of positive charges from
e-amino and

N. A. Sasaki et al. / Bioorg. Med. Chem. 17 (2009) 2310–2320
2313
Figure 3. Efficiency of Dap and N-terminal Dap-dipeptides to prevent the insulin modification by MG in comparison with that of known a-dicarbonyl scavengers.
Figure 6. Non-denaturing PAGE of glyoxalase I incubated with MG in the presence
and absence of -Dap- -Leu at 37 °C and pH 7.4 for 24 h: (1) incubated control
glyoxalase I, (2) glyoxalase I + MG (glyoxalase I/MG = 1/1), (3) glyoxalase I + MG +
-Dap- -Leu (1/1/1.1), (4) glyoxalase I + MG + -Dap- -Leu (1/1/2.2), (5) molecular
weight standard.
L
L
Figure 4. SDS–PAGE of RNase A incubated with MG in the presence and absence of
N-terminal Dap-dipeptides at 37 °C and pH 7.4 for 48 h: (1) incubated control
L
L
L
L
RNase A, (2) RNase A + MG (RNase A/MG = 1/1), (3) RNase A + MG +
L-Dap-L-Leu (1/
1/1.1), (4) RNase A + MG + -Dap- -Val (1/1/1.1), (5) RNase A + MG + AG (1/1/1.1).
L
L
2.2.3.1. Test on RNase A. The enzymatic activity of RNase A was
measured according to the method developed by Greiner–Stoeff-
elle and his colleagues.35 When incubated with equimolar amount
of MG at 37 °C for 24 h, RNase A loses 90% of its enzymatic activity
under the studied conditions. On the other hand, it retains its activ-
ity from 70% to 94% upon incubation with MG in the presence of
Dap peptides. Although AG is also a good inhibitor, the superiority
of the dipeptides with alkyl side chains, L-L-Dap-L-Leu, in particular,
is significant as shown in Figure 7. To our surprise, Dap alone
turned out to be much less effective agent for the conservation of
the enzymatic activity.
Figure 5. SDS–PAGE of lysozyme incubated with MG in the presence and absence
of N-terminal Dap-dipeptides at 37 °C and pH 7.4 for 48 h: (1) incubated control
2.2.3.2. Test on glyoxalase I. The enzymatic activity of glyoxalase
I was assayed by measuring the rate of formation of S-D-lactoylglu-
tathione from the hemithioacetal that is in turn formed non-enzy-
matically from MG and reduced glutathione. Thus, the
hemithioacetal formed from 1/1 mixture of MG and reduced gluta-
thione, after allowing 15 min for the non-enzymatic reaction, was
lysozyme, (2) lysozyme + MG (lysozyme/MG = 1/1), (3) lysozyme + MG +
-Leu (1/1/1.1), (4) lysozyme + MG + -Dap- -Val (1/1/1.1), 5) lysozyme + MG + AG
(1/1/1.1).
L-Dap-
L
L
L
guanidine groups and gain of negative charges, presumably via for-
mation of CEL and MG-H1 moieties. Incubation of glyoxalase I with
incubated with glyoxalase I in the absence or presence of
L-Dap-L-
MG in the presence of L-Dap-L-Leu inhibits this process.
Leu and the formation of S- -lactoylglutathione was followed by
D
measuring the absorbance at 240 nm. Incubation of human glyox-
alase I with equimolar amount of MG modifies the protein. This
modification induces a diminution of the enzymatic activity to
50% in comparison with the control. Simultaneous incubation of
2.2.3. Evaluation by enzymatic activity
Measurement of a remaining enzymatic activity of an enzyme
incubated with equimolar amount of MG in the presence of a
MG-trapping agent is one way to evaluate its scavenging potency.
the enzyme with MG in the presence of the dipeptide L-Dap-L-

2314
N. A. Sasaki et al. / Bioorg. Med. Chem. 17 (2009) 2310–2320
mine, to a lesser extent. On the contrary, the N-terminal Dap-
dipeptides with alkyl side chains on the C-terminal amino acids
such as -Dap- -Ile, -Dap- -Leu and -Dap- -Nle, and -Dap- -Val,
L
L
L
L
L
L
L
L
in particular, are excellent cell protectors to counter the MG in-
duced effects. We are intrigued to find out that Dap-containing
dipeptide ester with 1-hexadecanol is a cytotoxic compound.
2.3. NMR studies on addition products of MG with dipeptide
scavenger D-Dap-D-Leu
It is easily conceivable that nucleophilic addition of 1,2-dia-
mines to MG can lead to piperazine and/or pyrazine derivatives. In-
deed, the addition reaction of MG and
D-Dap-D-Leu under
incubation conditions (pH 7.4 at 37 °C) provides many products
as depicted in Figure 9. In order to find out the nature of some ma-
jor addition products, we have separated two of them, Adduct I and
Adduct II, by preparative HPLC for the NMR studies. From 1H NMR,
13C NMR (Table 1) and mass spectroscopic analyses, we tentatively
postulate the structures of Adduct I and Adduct II as pyrazine
derivatives. For Adduct I, it is speculated that the monohydrate
form of MG37 is involved in the formation of piperazine ring sys-
tem. As for Adduct II, the formation of dihydropyrazine ring by
the condensation of MG and 1,2-diamine functional groups of
dipeptide followed by the addition–condensation reactions. It is
clear that 1 mol of 1,2-diamine-containing peptide scavenges
2 mol of MG in both cases.
Figure 7. Effect of conserving RNase A enzymatic activity by N-terminal Dap-
dipeptides after incubation with MG at 37 °C and pH 7.4 for 48 h.
Leu protects the polypeptide from the structural modifications and
preserves the enzymatic activity up to 96%.
2.2.4. Evaluation by cell viability
2.4. Evaluation of the mutagenic activity of
L-Dap-L-Leu
It is suggested that carbonyl stress-induced loss of mitochon-
drial integrity could contribute to the cytotoxicity of MG. The
MG-induced effects such as ATP depletion and mitochondrial dys-
function could be prevented by pre-incubation of the cells with the
carbonyl scavengers.36
Mutagenic activity of -Dap- -Leu and its metabolites (produced
L
L
by S9 human liver fraction) was evaluated on the Salmonella
typhimurium TA7001, TA7002, TA7003, TA7004, TA7005, TA7006
(these six strains are mixed in the assay) and TA98 according to
the Ames assay. The test substances and positive controls (2-ami-
noanthracene, 4-nitroquinoline N-oxide and 2-nitrofluorene) were
incubated with different strains S9 human fraction at 37 °C for
90 min and then incubated with Indicator medium for 60 h before
the analysis. The experiments were performed in duplicate and
each point in 48 wells. The results are expressed as mutagenicity
ratio in comparison to solvent control (sterile water).
As evident from Figure 8, MG suppresses cell growth up to
about 33% under the conditions of this study. D-Dap alone has
turned out to be slightly cytotoxic. The known AGE inhibitors such
as aminoguanidine, metformin, pyridoxamine, carnosine and pen-
icillamine can be considered non-toxic. However, the viability of
the cell incubated with MG in the presence of these compounds
considerably diminishes except in the case of AG and penicilla-
Figure 8. MG scavenging effect of N-terminal-Dap-dipeptides and known AGE inhibitors in the protection of EAhy926 endothelial cells.

N. A. Sasaki et al. / Bioorg. Med. Chem. 17 (2009) 2310–2320
2315
Figure 9. RP-HPLC chromatograms of reaction products of MG and
L
-Dap-
L
-Leu. Incubation conditions:
L
-Dap-
L
-Leuꢁ2HCl (3.7 mM) and MG (3.4 mM) in 0.1 M NaCl and
10 mM phosphate buffer of pH 7.4 at 37 °C for 24 h. RP-HPLC conditions: column: symmetry 300 C-18 (4.6 ꢀ 250 mm); injection volume: 100
lL of the reaction mixture; flow
rate: 1 mL/min; temperature 40 °C; eluent A: H2O + 0.1% TFA, eluent B: CH3CN/H2O 60/40 + 0.1% TFA; linear gradient: 10% B to 50% B in 15 min; detection: PDA
(chromatograms extracted at 220, 280 and 360 nm).
Concentrations of the Ames test were chosen as follows:
-Leu alone: 10 M, 1 M and 0.1 M; mixture of -Dap- -Leu/MG:
10 M, 1 M and 0.1 M; MG: 10 M.
As seen from Table 2, the test substance alone ( -Dap-L-Leu) or
L
-Dap-
tive diseases.46–48 In this regard, it is interesting to know the Cu(II)
chelating property of the N-terminal Dap containing peptides. Our
preliminary results obtained from the measurement of Cu(II) in
ultrafiltrate of human serum by atomic absorption spectrophotom-
L
l
l
l
l
l
L
L
l
l
L
in combination with MG is neither mutagenic against the 6 mixed
and TA98 strains nor on the S9 fraction of human liver in the tested
concentrations.
etry indicates that
excellent Cu(II) chelators, as potent as trientine and carnosine, sig-
D-Dap-L-Nle and D-Dap-L-Leu are found to be
nificantly better than AG and metformin but less efficient than
penicillamine in the 1-15 mM range.49
Throughout the present in vitro studies, we have demonstrated
that the N-terminal Dap is the essential moiety for the MG-trap-
ping capacity regardless of its stereochemistry. As shown in Figure
3, insulin is equally well protected against MG modification both
D-
Although
MG,
-Dap has been reported to induce renal tubular necrosis.38
- and -Dap undergo transamination to form pyruvate and ammo-
nia by widely occurring 1,2-diaminopropionate ammonia-lyase in
D-Dap and L-Dap alone are excellent scavengers of
D
D
L
bacteria, notably in S. typhimurium.39 Moreover,
D-Dap showed a
mutagenic activity with S9 mix, in S. typhimurium TA 100 strain.40
For these reasons, we chose the N-terminal Dap-containing dipep-
tides for further assessments of the therapeutic potency of the new
MG scavengers.
by D-Dap-L-Leu and L-Dap-L-Leu. While the former is an excellent
insulin protector, the latter significantly contributes to conserve
enzymatic activities of RNase A and glyoxalase 1 in confrontation
with MG. With respect to the stereochemistry of the C-terminal
amino acid, it seems to play little role for the scavenging potency
of the dipeptides as evidenced in Figure 3 for the Dap-Leu series.
As to the functional group on the side chain of the C-terminal ami-
no acid, longer alkyl groups in general give satisfactory results
compared with the polar or aromatic ones (Fig. 3).
It has been generally accepted that transition metal ions may
play an important role during AGE formation in vivo by catalyzing
the generation of superoxide and
search groups have reported the reduction of AGE formation by
a
-dicarbonyls.41,42 Several re-
trapping Cu(II) with chelating agents such as trientine,43
D-penicil-
lamine27 pyridoxamine, 6-diaminopyridoxamine, trolox, amino-
guanidine,44 carnosine, tenilsetam, OPB-9195.45 Recent progress
in the so-called metal-chelation therapy in Alzheimer’s and other
CNS diseases may further promote the development of rational
strategies for the treatment of these AGE related neurodegenera-
3. Conclusions
In vitro efficacy of N-terminal Dap containing dipeptides as
scavengers of reactive
a-dicarbonyl compounds such as MG is

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N. A. Sasaki et al. / Bioorg. Med. Chem. 17 (2009) 2310–2320
Table 1
1H and 13C NMR chemical shifts of Adduct I and Adduct II. Hydrogens and carbons were attributed from detailed NMR spectroscopic analyses
Atom numbering
Adduct I
dH/ppm (600 MHz, (CD3)2SO)
dC/ppm (500 MHz, CD3OD)
Pyrazine- moiety
11
1
9
168.21
163.68
122.00
OH
6
7
8
3
11.64 (s, 1H)
7.26 (d, J = 1.5 Hz, 1H]
124.40
150.20
110.10
55.97
6.79 (d. J = 1.5 Hz, 1H)
4.87 (t, J = 5.4 Hz, 1H)
4.45 (dd, J = 5.4 Hz, J = 13.5, 1H)
4.37 (dd, J = 5.4 Hz, J = 13.5, 1H)
2.55 (s. 3H)
4
47.02
10
18.14
Leucine-moiety
NH
12
13
14
15
16
17
8.76 (d, J = 8 0 Hz, 1H)
175.31
52.64
41.23
26.14
23.34
21.60
4,25–422 (m, 1H)
1.57–1.52 (m, 3H)
0.87 (d, J = 5.9 Hz, 3H)
0.80 (d, J = 5.9 Hz, 3H)
Adduct II
dH/ppm (600 MHz, (CD3)2SO)
dC/ppm (500 MHz, CD3DO)
Pyrazine-moiety
8
11
5
6
2
3
7
9
10
206.52
165.29
158.55
150.56
142.85
141.95
50.10
8.92 (s, 1H)
4 24 (s, 2H)
2.29 (s, 3H)
2.46 (s, 3H)
30.16
22.06
Leucine-moiety
12
13
14
15
16
17
175.90
52.30
42.06
26.23
23.38
22.06
4.33 (br s, 1H)
1.64–1.62 (m, 3H)
0.88 (br s, 6H)
For atom numbering, see Figure 9.
Table 2
Mutagenicity of L-Dap-L-Leu alone and in combination with MG against 7 Salmonella strains
Test substance
Concentrations (lM)
Human S9
6 Mixed strains
TA98 Strain
L
L
L
L
-Dap-
-Dap-
-Dap-
-Dap-
L
L
L
L
-Leu
10/1/0.1
10/1/0.1
10/1/0.1
10/1/0.1
10
10
2.6
51.7
–
No
Yes
No
Yes
No
Yes
No
Yes
No
Yes
ꢂ
ꢂ
ꢂ
-Leu (metabolites)
-Leu + MG
-Leu + MG (metabolites)
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
+
MG
MG (metabolites)
ꢂ
+
+
ꢂ
Positive control (4NQO)
Positive control (2AA) (metabolites)
Negative control (solvent)
Negative control (solvent)
+
ꢂ
demonstrated. In a physiological solution, insulin, RNase A, lyso-
zyme and glyoxalase I are highly susceptible to modifications by
addition of MG. However, the presence of the new N-terminal
Dap containing dipeptides in the tested medium efficiently
inhibits this trend. Among the MG scavengers known in the lit-
erature, only AG matches to the new type of compounds pre-
sented here in terms of the efficacy to inhibit the modification
of proteins. Despite the seemingly excellent bifunctional charac-
with D-(+)-glucose in the absence or presence of the peptides
and metal ions. For the inhibition of intracellular AGE formation,
these scavengers must possess high physiological stability and
excellent bioavailability. Indeed, our preliminary results suggest
that selection of D-configuration in one or both of the constitut-
ing amino acids in dipeptides seems to be very important factor
to circumvent the problem arising from proteolysis. Pharmacoki-
netic studies of these dipeptides are currently under investiga-
tion in our laboratory. In view of the increasing evidences that
protein-AGE formation is closely associated with aging and dia-
betes complications, some of N-terminal Dap dipeptides de-
ters of Dap-dipeptides as
a-dicarbonyl scavenger and Cu(II) che-
lator, further studies are needed to confirm their efficiency by
measuring, for example, the formation of AGEs of BSA incubated

N. A. Sasaki et al. / Bioorg. Med. Chem. 17 (2009) 2310–2320
2317
scribed in this paper may provide an effective means to deceler-
ate and treat these insidious processes.
complex is taken as control (100%). The subsequent cleavage of
RNA with glycated RNase A was measured at 688 nm. The scav-
enging potency of AG, D
Phe, -Dap- -Leu, -Dap- -Ile,
study.
L
-Dap,
D
-Dap-
D-Ala, L-Dap-Gly, D-Dap-D-
L
L
L
L
L
-Dap-L
-Val were assessed for this
4. Experimental
4.1. Materials and methods
4.1.3.2. Glyoxalase I. The activity of glyoxalase I was assayed by
measuring the rate of formation of S- -lactoylglutathione from
D
Methylglyoxal, aminoguanidine hydrochloride, 1,1-dimethyl-
biguanide hydrochloride (metformin), pyridoxamine dihydrochlo-
ride, 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl-2H-tetrazolium
bromide (MTT), 2-aminoanthracene, 4-nitroquinoline N-oxide
and 2-nitrofluorene, human insulin, bovine pancreatic RNase A,
chicken lysozyme, yeast RNA were purchased from Sigma–Aldrich.
the hemithioacetal formed non-enzymatically from MG and re-
duced glutathione. Hemithioacetal was prepared by incubating 2
mM MG with 2 mM reduced glutathione in 50 mM sodium phos-
phate, pH 6.6, for 20 min at 37 °C. The normal hemimercaptal con-
centration was 600 lM, assuming the equilibrium constant for the
hemithioacetal formation was 3.33 Mꢂ1. The reaction mixture was
allowed to stand for 15 min to ensure the equilibration of hemi-
thioacetal formation. An aliquot of enzyme of control or that of
L
-carnosine and DL-penicillamine were purchased from Fluka.
DL-Diaminobutyric acid, 2,3-diaminopropionic acid ( -, - and DL-),
methyl esters of amino acids, N-Cbz- -Leu, 1-hexadecanol, 1-[3-
D
L
L
co-incubation with MG in the absence or presence of
was added to the hemithioacetal solution and the formation of
S- -lactoylglutathione was monitored by following absorbance at
240 nm;
240 = 2.86 mMꢂ1 cmꢂ1
L-Dap-L-Leu
(dimethylamino)propy]-3-ethylcarbodiimide hydrochloride (EDC)
and 1-hydroxybenzotriazole (HOBt) were from Acros Organics.
Tris-glycine SDS–PAGE gel and prestained SDS–PAGE standards
were obtained from Bio-Rad (Marnes-la-Coquette, France). Thymi-
dine was purchased from Invitrogen (Cergy-Pontoise, France).
Trypsin/EDTA was from Invitrogen (France). Human glyoxalase I
was obtained from Protein’eXpert SA (Grenoble, France). All the
S. typhimurium strains are obtained from Biogenic (France) and
the S9 human liver fraction was purchased from BD Biosciences
(France).
D
D
e
.
The activity of glyoxalase I was calculated in unit per ml where
unit is the amount of enzyme required to catalyze the formation of
1
l
mol of S-
D-lactoylglutathione per minute under assay con-
ditions.
4.1.4. Cell culture
A continuous cell line of immortalized EAhy926 endothelial
cells derived from fusion of human umbilical vein endothelial cells
(HUVECs) with A549 lung carcinoma cells was routinely cultured
in 75 mL flasks in moist air containing 5% CO2 at 37 °C using Dul-
becco minimal essential medium (DMEM) containing 4.5 g/L glu-
cose and supplemented with 10% fetal calf serum, 1% glutamine,
4.1.1. RP-HPLC
Control experiments were realized by incubating insulin
(34 lM) in phosphate buffer (10 mM, pH 7.4 + 0.1 M NaCl) at 37
°C for 24 h in absence or presence of MG (3.4 mM). Efficacy of
each scavenger in this study was assessed by comparative mea-
surement of the remaining intact insulin vis-à-vis the control
(insulin alone) when it is incubated under the same conditions
with MG (3.4 mM) in the presence of a scavenger compound
(3.75 mM, 1.1 mol equiv with respect to MG). Each compound
was assessed for three times and RP-HPLC analyses were realized
under the following conditions: column: Symmetry 300 C-18
HAT (100 lM hypoxantine, 0.4 lM aminopterin, 16 lM thymidine.
The cultured cell line was routinely harvested by trypsin treatment
(0.5 g/L trypsin/EDTA from Invitrogen) when cells EAhy926 at-
tained 90% confluence. All experiments were carried out on 12-
well dishes where cells EAhy926 were seeded at 1 ꢀ 105 cells per
well in 2 mL of DMEM. Cells were left overnight to attach to the
plate, and the appropriate carbonyl scavenger (1 mM) was then
(4.6 ꢀ 250 mm); injection volume: 100
lL of the reaction mix-
ture; flow rate: mL/min; temperature 40 °C; eluent A:
added 30 min prior to the addition of MG (600
lM). Following a
1
48 h exposure to carbonyl scavenger and/or MG, cell viability
was evaluated on the reduction of 3-(4,5-dimethylthiazol-2-yl)
2,5-diphenyl-tetrazoliumbromide (MTT).50 Results are expressed
as the ratio (%) of the number of cells with treatment to the num-
ber of cells without treatment (control) and is the mean S.E (n = 3
triplicates). The detection is carried out by UV spectrometry at
570 nm.
H2O + 0.1% TFA, eluent B: CH3CN/H2O 60/40 + 0.1% TFA; linear gra-
dient: 50% B to 55% B in 15 min; detection: PDA (chromatograms
extracted at 220 nm).
4.1.2. SDS–PAGE and non-denaturing PAGE analysis
Stock solution of a model protein was prepared by solubilizing
the protein in phosphate buffer to give a final concentration of 10–
15 mg/mL. The resultant solution was transferred into Eppendorf
tubes and incubated with MG in the presence or absence of
4.2. Chemical synthesis
4.2.1. N,N0-Di-Boc-2,3-
To a solution of N,N0-Di-Boc-2,3-
and -Leu-OMe hydrochloride (6.17 g, 33.97 mmol) in CH2Cl2 (150
L
-Dap-L
-Leu-OMe
-Dap-OH (9.39 g, 30.88 mmol)
a
-dicarbonyl scavenger for 24 h (glyoxalase I) or 48 h (RNase A
and lysozyme) at 37 °C then stored at ꢂ20 °C prior to analysis.
PAGE electrophoresis was performed by loading 10 L of the incu-
L
l
L
bated protein on a 8–16% gradient Tris-glycine SDS–PAGE gel. The
glyoxalase I samples were analyzed on 12% Tris-glycine PAGE gel.
mL) were added Et3N (4.74 mL, 33.97 mmol), EDC (7.10 g, 37.05
mmol) and HOBt (4.59 g, 33.97 mmol) and the reaction mixture
was stirred at room temperature overnight. Water was added
and the aqueous phase was extracted three times with EtOAc.
The combined organic layers were washed successively with 1 N
HCl, H2O, saturated NaHCO3 and brine, dried over Na2SO4 and fil-
tered. The solvent was evaporated under reduced pressure and
the residue was purified by flash column chromatography on silica
gel with heptane–EtOAc (3/1 then 1/1) to give 13.30 g of N,N0-Di-
L-Dap-L-Leu, L-Dap-L-Val and AG have been used as scavengers.
4.1.3. Assay for enzymatic activity
4.1.3.1. RNase A. Enzymatic activity of RNase A was assayed as
described in the literature.35 Thus, 600
employed as working concentration. 10
l
l
g/mL of yeast RNA was
L of RNAse (concentra-
tion of 10
lg/mL) that has been subjected to glycation by MG
in the absence or presence of MG-scavenger was added to 1 mL
of methylene blue-RNA solution. The difference between the
absorbance value of the methylene blue-RNA complex at 688
nm and the one obtained after the enzymatic digestion of the
Boc-L-2,3-Dap-L-Leu-OMe (quantitative yield).
[a
]
D
ꢂ44.6 (c 2.0, CHCl3).
1H NMR (300 MHz, CD3CN) d 7.05 (d, J = 7.9 Hz, 1H), 5.94 (br s,
1H), 5.57 (br s, 1H), 4.42 (ddd, J = 8.6, 8.3, 5.9 Hz, 1H), 4.11–4.09

2318
N. A. Sasaki et al. / Bioorg. Med. Chem. 17 (2009) 2310–2320
(m, 1H), 3.66 (s, 3H), 3.41–3.22 (m, 2H), 1.66–1.55 (m, 3H), 1.42,
1.41 (two s, 18H), 0.92 (d, J = 6.4 Hz, 3H), 0.89 (d, J = 6.3 Hz, 3H);
13C NMR (75 MHz, CD3CN) d 174.0, 171.6, 157.8, 156.7, 80.3,
80.0, 56.5, 52.9, 51.7, 43.0, 41.2, 28.7, 25.5, 23.3, 21.8;
MS (ESI) m/z 454 [M+Na]+.
4.2.7. -Dap-
L
-Proꢁ2HCl
[a]
L ꢂ72.2 (c 1.3, H2O).
D
1H NMR (300 MHz, D2O) d 4.62 (m, 1H), 4.39 (m, 1H), 3.62 (d,
J = 7.6 Hz, 1H), 3.57 (m, 2H), 3.42 (dd, J = 13.6, 6.1 Hz, 1H), 2.05
(m, 2H), 1.95 (m, 2H); 13C NMR (62.5 MHz, D2O) d 173.1, 164.9,
59.6, 52.6, 46.0, 39.2, 28.2, 22.3; MS (ESI) m/z 202 [M+H]+; HRMS
calcd for C9H20N3O3 (M+H) 202.1192, found 202.1196.
4.2.2. N,N0-Di-Boc-
To a solution of N,N0-Di-Boc-
L
-2,3-Dap-
L
-Leu
-2,3-Dap-L-Leu-OMe (12.05 g,
L
27.96 mmol) in THF/MeOH/H2O (1/1/1, 140 mL) at 0 °C was added
LiOHꢁH2O (1.17 g, 27.96 mmol) and the reaction mixture was stir-
red at room temperature until all of the starting methyl ester dis-
appeared (about overnight, followed by TLC). The reaction mixture
was acidified with aqueous KHSO4 solution to pH 4, and then ex-
tracted five times with CH2Cl2. The organic extracts were dried
over Na2SO4, filtered through Celite and evaporated under reduced
4.2.8.
L
-Dap-
[a] +22.4 (c 1.2, H2O).
D
L
-Ileꢁ2HCl
1H NMR (300 MHz, D2O) d 4.52 (t, J = 5.9 Hz, 1H), 4.46 (d,
J = 4.9 Hz, 1H), 3.60 (d, J = 5.9 Hz, 2H), 2.05 (m, 1H), 1.82 (m, 2H),
1.45 (m, 1H), 1.27 (m, 1H), 0.97 (d, J = 6.9 Hz, 3H), 0.90 (t,
J = 7.3 Hz, 3H); 13C NMR (62.5 MHz, D2O) d 175.5, 166.8, 58.8,
51.1, 40.3, 37.0, 25.3, 15.7, 11.6; MS (ESI) m/z 218 [M+H]+; HRMS
calcd for C9H20N3O3 (M+H) 218.1505, found 218.1537.
pressure to give 11.70 g (100%) of the crude 2,3-di-Boc-L-Dap-L-Leu
that was used directly to the next reaction without further
purification.
4.2.9. N-Cbz-L-Leu-OC16H33
[
a
]
ꢂ19.7 (c 2.0, CHCl3).
To a solution of L
-Cbz-Leu-OH (2.85 g, 10.75 mmol) and 1-hex-
D
1H NMR (300 MHz, CD3OD) d 4.37 (dd, J = 8.7, 5.6 Hz, 1H), 4.21
(br t, J = 6.2 Hz, 1H), 3.44 (dd, J = 14.1, 4.8 Hz, 1H) , 3.27 (dd,
J = 14.1, 8.3 Hz, 1H), 1.78–1.59 (m, 3H), 1.44, 1.44 (two s, 18H),
0.95 (d, J = 6.5 Hz, 3H), 0.93 (d, J = 6.9 Hz, 3H); 13C NMR (75 MHz,
CD3OD) d 178.1, 172.8, 158.8, 157.8, 80.8, 80.6, 56.4, 53.5, 43.2,
42.2, 28.9, 26.1, 23.8, 22.2; MS (ESI) m/z 440 [M+Na]+; HRMS calcd
for C19H35N3O7Na (M+Na) 440.2373; Found: 440.2380.
adecanol (2.60 g, 10.75 mmol) in CH2Cl2 (100 mL) were added EDC
(2.27 g, 11.83 mmol) and DMAP (131 mg, 1.075 mmol) and the
reaction mixture was stirred at room temperature overnight.
Water was added and the aqueous phase was extracted three times
with EtOAc. The combined organic layers were washed succes-
sively with 1 N HCl, H2O, saturated NaHCO3 and brine, dried over
Na2CO3 and filtered. The solvent was evaporated under reduced
pressure and the residue was purified by flash column chromatog-
raphy on silica gel with heptane–EtOAc (30/1 then 20/1) to give
4.2.3.
L
-Dap-
L-Leuꢁ2HCl
2.69 g of N-Cbz-L-Leu-OC16H33 (51% unoptimized yield).
A solution of 2,3-di-Boc-
M HCl–dioxane (130 mL) was stirred at room temperature for 6 h.
The volatile was removed under reduced pressure to give 7.65 g of
L
-Dap-L-Leu (11.00 g, 26.38 mmol) in 3
1H NMR (300 MHz, CDCl3) d 7.34–7.25 (m 5H), 5.31 (br s, 1H),
4.38 (dt, J = 5.3, 8.7 Hz, 1H), 4.11 (t, J = 6.7 Hz, 2H), 1.74–1.47 (m,
5H), 1.35–1.26 (m, 26H), 0.96-0.86 (m, 9H); 13C NMR (75 MHz,
CDCl3) d 173.3, 156.1, 136.5, 128.2, 128.2, 67.0, 65.6, 52.7, 41.0,
32.1, 29.8, 29.7, 29.7, 29.5, 29.3, 28.6, 26.0, 24.9, 22.9, 22.8, 22.1,
14.3; MS (ESI) m/z 512 [M+Na]+.
L
-Dap-
L
-Leuꢁ2HCl in quantitative yield.
[a
]
D
+3.4 (c 1.0, 6 N HCl).
1H NMR (300 MHz, CD3OD) d 4.37 (t, J = 5.8 Hz, 1H), 4.30 (dd,
J = 9.4, 5.6 Hz, 1H), 3.45 (dd, J = 13.9, 6.0 Hz Hz, 1H), 3.34 (dd,
J = 13.9, 5.3 Hz, 1H), 1.64–1.49 (m, 3H), 0.78 (d, J = 6.4, 3H), 0.74
(d, J = 6.4 Hz, 3H); 13C NMR (75 MHz, CD3OD) d 176.6, 167.3,
53.0, 51.7, 41.4, 40.6, 26.1, 23.4, 21.5; MS (ESI) m/z 218 [M+H]+;
HRMS calcd for C9H20N3O3 (M+H) 218.1505, found 218.1512.
4.2.10.
L
-Leu-OC16H33ꢁHCl
A suspension of N-Cbz-
L-Leu-OC16H33 (2.4 g, 4.91 mmol) and
10% Pd–C (240 mg) in EtOAc was hydrogenated under atmospheric
pressure for 4 h. The catalyst was removed by filtration through
Celite. The filtrate was evaporated. The residue was treated with
0.1 M HCl in dioxane and evaporated to dryness to give 1.83 g of
4.2.4.
L
-Dap-
[a] +7.8. (c 1, H2O).
D
D-Leuꢁ2HCl
L
-Leu-OC16H33ꢁHCl (95% yield).
1H NMR (300 MHz, CD3OD) d 4.38 (dd, J = 6.7, 5.0 Hz, 1H), 4.30
(t, J = 7.8Hz, 1H), 3.60-3.43 (m, 2H), 1.67–1.56 (m, 3H), 0.86 (d,
J = 6.0 Hz, 3H), 0.83 (d, J = 6.0 Hz, 3H); 13C NMR (75 MHz, D2O) d
175.9, 166.0, 52.3, 50.7, 39.6, 39.1, 24.5, 22.0, 20.9; MS (ESI) m/z
218 [M+H]+; HRMS calcd for C9H20N3O3 (M+H) 218.1505, found
218.1552.
1H NMR (300 MHz, CD3OD) d 4.87 (br s, 1H), 4.31–4.19 (m, 2H),
4.03 (t, J = 7.0 Hz, IH), 1.86–1.66 (m, 5H), 1.43-1.29 (m, 26H), 1.11
(d, J = 6.2 Hz, 3H), 1.02 (d, J = 6.0 Hz, 3H), 1.00 (t, J = 6.8 Hz, 3H); 13C
NMR (75 MHz, CD3OD) d 171.2, 67.7, 52.6, 40.9, 33.2, 30.9, 30.8,
30.8, 30.6, 30.4, 29.7, 27.0, 25.8, 23.9, 22.7, 22.6, 14.5; MS (ESI)
m/z 356 [M+H]+.
4.2.5. -Dap-
D
-Leuꢁ2HCl
Dꢂ11.7 (c 1.0, H2O).
4.2.11. N,N0-Di-Boc-
L-Dap-L-Leu-OC16H33
1H NMR (300 MHz, CDCl3) d 7.18 (br s, 1H), 5.85 (br s, 1H), 5.36
(br s, 1H), 4.58–4.51 (m, 1H), 4.24–4.22 (m, 1H), 4.11 (t, J = 6.8 Hz,
2H), 3.52–3.43 (m, 2H), 1.67–1.54 (m, 5H), 1.45–1.44 (two s, 18H),
1.35–1.26 (m, 26H), 0.93 (br d, J = 4.9 Hz, 6H), 0.88 (t, J = 6.6 Hz,
3H); 13C NMR (75 MHz, CDCl3) d 172.7, 170.7, 157.2, 156.3, 80.3,
80.0, 65.6, 55.5, 51.1, 42.6, 41.4, 32.1, 29.8, 29.7, 29.6, 29.5, 29.3,
28.6, 28.5, 28.4, 26.0, 24.9, 23.0, 22.8, 22.0, 14.3; MS (ESI) m/z
664 [M+Na]+.
[a]
D
1H NMR (300 MHz, D2O) d 4.46 (m, 2H), 3.58 (dd, J = 6.1,
0.9 Hz, 2H), 1.68 (m, 3H), 0.87 (m, 6H); 13C NMR (62.5 MHz,
D2O) d 176.0, 166.1, 52.0, 50.4, 39.6, 38.9, 24.4, 22.1; MS (ESI)
m/z 218 [M+H]+; HRMS calcd for C9H20N3O3 (M+H) 218.1505,
found 218.1537.
4.2.6. -Dap-
L-Nleꢁ2HCl
[a
]
D
Dꢂ43.0 (c 1.4, H2O).
1H NMR (300 MHz, D2O) d 4.43 (dd, J = 6.6, 5.1 Hz, 1H), 4.33 (dd,
J = 8.1, 5.7 Hz, 1H), 3.60 (dd, J = 14.5, 5.1 Hz, 1H), 3.53 (dd, J = 14.5,
6.6 Hz, 1H), 1.82 (m, 2H), 1.34 (m, 4H), 0.86 (t, J = 7.2 Hz, 3H); 13C
NMR (75 MHz, D2O) d 175.6, 166.0, 53.8, 50.6, 39.4, 29.9, 27.1, 21.6,
13.0; MS (ESI) m/z 218 [M+H]+; HRMS calcd for C9H20N3O3 (M+H)
218.1505, found 218.1522.
4.2.12.
L
-Dap-
[a] +13.1 (c 2.0, MeOH).
D
L
-Ile-OC16H33ꢁ2HCl
1H NMR (300 MHz, CD3OD d4.34–4.28 (m 2H), 4.01–3.86 (m, 2H),
3.41–3.29 (m, 2H), 1.61–1.39 (m, 5H), 1.16–1.05 (m, 26H), 0.76 (d,
J = 6.4 Hz, 3H), 0.72 (d, J = 6.4 Hz, 3H), 0.66 (t, J = 6.7 Hz, 3H), 0.90
(t, J = 7.3 Hz, 3H); 13C NMR (75 MHz, CD3OD) d 174.6, 167.2, 58.8,

N. A. Sasaki et al. / Bioorg. Med. Chem. 17 (2009) 2310–2320
2319
67.2, 53.0, 51.8, 41.4, 40.9, 33.2, 30.9, 30.6, 30.6, 30.6, 30.3, 29.7, 27.0,
26.1, 23.8, 23.4, 21.8, 14.6; MS (ESI) m/z 442 [M+H]+; HRMS calcd for
C25H52N3O3 (M+H) 442.4009, found 442.3983.
NMR (62.5 MHz, D2O) d 71.6, 167.6, 51.8, 51.5, 40.2, 40.1; MS
(ESI) m/z 191 [M+H]+; HRMS calcd for C6H15N4O3 191.1144,
found 191.1146.
4.2.13.
L
-Dap-
L
-p-CF3-Pheꢁ2HCl
4.2.21.
D
-Dap-Glyꢁ2HCl
[a] +38 (c 0.2, MeOH).
D
1H NMR (300 MHz, D2O) d 7.58 (d, J = 7.9 Hz, 2H), 7.35 (d,
J = 8.1 Hz, 2H), 4.69 (m, 1H), 4.25 (t, J = 6.0 Hz 1H), 3.27 (dd,
J = 13.9, 5.8 Hz, 1H), 3.17 (d, J = 6.0 Hz, 2H), 3.04 (dd, J = 14.0,
9.3 Hz, 1H); 13C NMR (75 MHz, D2O) d 174.1, 165.7, 140.7, 130.0,
129.6 (q, J = 32.9 Hz), 125.5, 125.0 (q, J = 271.0 Hz) 54.3, 50.7,
39.5, 36.3; MS (ESI) m/z 320 [M+H]+; HRMS calcd for C13H17F3N3O3
(M+H) 320.1222, found 320.1236.
1H NMR (300 MHz, D2O) d 4.53 (t, J = 5.9 Hz, 1H), 4.08 (d, J = 2.6
Hz, 2H), 3.63 (d, J = 5.9 Hz, 2H); 13C NMR (75 MHz, D2O) d 176.3,
167.6, 51.1, 42.6, 39.9; MS (ESI) m/z 161 [M+H]+; HRMS calcd for
C5H13N3O3 161.1039, found 161.1042.
4.2.22.
D-Dap-
D
-Aspꢁ2HCl
[a]
D
ꢂ32.4 (c 0.7, H2O).
4.2.14.
L
-Dap-D
L-p-F-Pheꢁ2HCl
1H NMR (300 MHz, D2O) d 4.75 (m, 1H), 4.38 (dd, J = 6.3, 5.3 Hz,
1H), 3.50 (dd, J = 14.4, 5.3 Hz, 1H), 3.42 (dd, J = 14.4, 6.4 Hz, 1H),
2.93 (d, J = 6.3 Hz, 2H); 13C NMR (75 MHz, D2O) d 174.2, 173.2,
165.9, 50.6, 49.4, 39.4, 35.1; MS (ESI) m/z 220 [M+H]+; HRMS calcd
for C7H14N3O5 220.0933, found 220.0903.
1H NMR (300 MHz, D2O) d 7.20–7.15 (m, 2H), 7.03–6.95 (m, 2H),
4.66–4.59 (m, 1H), 4.27 (t, J = 5.7 Hz, 0.5H), 4.25 (t, J = 5.8 Hz, 0.5H),
3.43 (d, J = 6.2 Hz, 0.5H), 3.42 (d, J = 5.7 Hz, 0.5H), 3.20–3.10 (m,
1H), 3.17 (d, J = 6.0 Hz, 1H), 2.97 (dd, J = 14.1, 8.6 Hz, 0.5H), 2.94
(dd, J = 14.1, 9.2 Hz, 0.5H); 13C NMR (75 MHz, D2O) d 174.3,
174.2, 165.8, 165.7, 162.8 (d, J = 243.7 Hz), 133.0, 132.8, 131.6,
116.3, 116.0, 54.7, 54.5, 50.7, 50.4, 39.5, 35.7, 35.5; MS (ESI) m/z
270 [M+H]+; HRMS calcd for C12H17FN3O3 (M+H) 270.1254, found
270.1255.
4.2.23.
D-Dap-
L
-Lysꢁ3HCl
1H NMR (300 MHz, D2O) d 4.42 (dd, J = 6.6, 5.1 Hz, 1H), 4.28 (dd,
J = 8.0, 5.8 Hz, 1H), 3.52 (m, 2H), 2.90 (t, J = 7.6 Hz, 2H), 1.82 (m,
2H), 1.60 (m, 2H), 1.37 (m, 2H); 13C NMR (75 MHz, D2O) d 174.9,
166.1, 53.5, 50.7, 39.5, 39.2, 29.7, 26.3, 22.1; MS (ESI) m/z 233
[M+H]+.
4.2.15.
L
-Dap-
[a] +22.2 (c 2.0, H2O).
D
L
-Valꢁ2HCl
1H NMR (300 MHz, D2O) d 4.54 (t, J = 5.9 Hz, 1H), 4.42 (d,
J = 5.0 Hz, 1H), 3.60 (d, J = 5.9 Hz, 2H), 2.29 (m, 1H), 0.97 (t,
J = 6.7 Hz, 6H); 13C NMR (62.5 MHz, D2O) d 175.5, 166.9, 59.5,
51.1, 40.3, 30.4, 19.0, 17.6; MS (ESI) m/z 204 [M+H]+; HRMS calcd
for C8H18N3O3 (M+H) 204.1348, found 204.1365.
Acknowledgments
We thank Vincent Hesry of CELLIS PHARMA (Saint-Malo,
France) for performing the mutagenicity assays and Sophie Da
Nascimento (Université de Picardie Jules Verne, Amiens, France)
for her technical assistance.
4.2.16.
D
-Dap-
D
-Alaꢁ2HCl
[
a]
D
ꢂ21.9 (c 1.0, MeOH).
References and notes
1H NMR (300 MHz, D2O) d 4.53 (q, J = 7.4 Hz, 1H), 4.48 (t,
J = 6.0 Hz, 1H), 3.64 (d, J = 6.0 Hz, 2H), 1.50 (d, J = 7.4 Hz, 3H); 13C
NMR (75 MHz, D2O) d 176.7, 166.5, 51.2, 49.9, 40.3, 16.6; MS
(ESI) m/z 176 [M+H]+; HRMS calcd for C6H14N3O3 (M+H)
176.1035, found 176.1037.
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L
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L
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D
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D
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[a]
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1H NMR (300 MHz, CD3OD) d 7.32 (m, 5H), 4.79 (dd, J = 9.9, 4.4
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L
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D
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[
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