Synthesis and intramolecular reactions of Tyr-Gly and Tyr-Gly-Gly related 6-O-glucopyranose esters

Article abstract of DOI:10.1016/j.carres.2003.07.011

6-O-(L-Tyrosylglycyl)- and 6-O-(L-tyrosylglycylglycyl)-D-glucopyranose were synthesized by condensation of the pentachlorophenyl esters of the respective di- and tripeptide with fully unprotected D-glucose. The intramolecular reactivity of the sugar conjugates was studied in pyridine-acetic acid and in dry methanol, at various temperatures and for various incubation times. The composition of the incubation mixtures was monitored by a reversed-phase HPLC method that permits simultaneous analysis of the disappearance of the starting material and the appearance of rearrangement and degradation products. To determine the influence of esterification of the peptide carboxy group on its amino group reactivity, parallel experiments were done in which free peptides were, under identical reaction conditions, incubated with D-glucose (molar ratios 1:1 and 1:5). Depending on the starting compound, different types of Amadori products (cyclic and bicyclic form), methyl ester of peptides, and Tyr-Gly-diketopiperazine were obtained.

Full text of DOI:10.1016/j.carres.2003.07.011

Carbohydrate
RESEARCH
Carbohydrate Research 339 (2004) 67–75
Synthesis and intramolecular reactions of Tyr-Gly and Tyr-Gly-Gly
related 6-O-glucopyranose esters
ꢀ*
Lidija Varga-Defterdarovic and Gorana Hrlec
-
Department of Organic Chemistry and Biochemistry, Ruder Boꢁskovicꢀ Institute, P.O. Box 180, HR-10002 Zagreb, Croatia
Received 20 March 2003; accepted 20 July 2003
Abstract—6-O-(
L
-Tyrosylglycyl)- and 6-O-(
L
-tyrosylglycylglycyl)-
tachlorophenyl esters of the respective di- and tripeptide with fully unprotected
D
-glucopyranose were synthesized by condensation of the pen-
-glucose. The intramolecular reactivity of the sugar
D
conjugates was studied in pyridine–acetic acid and in dry methanol, at various temperatures and for various incubation times. The
composition of the incubation mixtures was monitored by a reversed-phase HPLC method that permits simultaneous analysis of the
disappearance of the starting material and the appearance of rearrangement and degradation products. To determine the influence
of esterification of the peptide carboxy group on its amino group reactivity, parallel experiments were done in which free peptides
were, under identical reaction conditions, incubated with D-glucose (molar ratios 1:1 and 1:5). Depending on the starting com-
pound, different types of Amadori products (cyclic and bicyclic form), methyl ester of peptides, and Tyr-Gly-diketopiperazine were
obtained.
ꢀ 2003 Elsevier Ltd. All rights reserved.
Keywords: 6-O-(L-Tyrosylglycyl)-D-glucopyranose; 6-O-(L-Tyrosylglycylglycyl)-D-glucopyranose; D-Glucose; Peptide; Amadori rearrangement
1. Introduction
volved in numerous physiological functions.8 In vivo,
leucine enkephalin is rapidly hydrolyzed, forming three
tyrosine containing fragments: Tyr, Tyr-Gly, and Tyr-
Gly-Gly.9;10
In our earlier studies on the synthesis of leucine en-
kephalin related 6-O-glycoconjugates, it was for the first
time found that, depending on the employed solvent
these glycoconjugates undergo different intramolecular
rearrangement reactions. Thus, in pyridine–acetic acid
(1:1) bicyclic Amadori compounds prevailed,11 while in
dry methanol solution cyclic sugar-related imidazolidi-
nones were obtained (Fig. 1).12–14
To determine the influence of the peptide length on
intramolecular reactivity, in the present paper we report
the synthesis of Tyr-Gly and Tyr-Gly-Gly related 6-O-
glucopyranose esters, on the first plane as model systems
for glycoproteins, compounds involved in the innate and
adaptive immune response15 but also as compounds,
which mimic the reactivity of sugar 6-phosphate esters.16
Their behavior and reactivity were examined in pyridine–
acetic acid and in dry methanol solution, at various
temperatures and times of incubation and compared with
Glucose and other reducing sugars, during the early
stage of the Maillard reaction, react in vivo nonenzy-
matically with free amino groups of proteins, glyco-
proteins, lipids, and nucleic acids to form labile Schiff
bases which are then, by Amadori rearrangement, con-
verted to more stable keto-amines.1 Formed keto-
amines, or Amadori products, undergo over time, a
series of complex reactions leading to a class of per-
manently modified proteins implicated in the patho-
genesis of diabetes mellitus,2;3 normal processes of
aging,4;5 and AlzheimerÕs disease.6 These reactions also
occur during food processing or cooking, affecting fla-
vors, color, and organoleptic properties of foods.7
In addition to numerous endogenous biologically
active peptides, our recent interest has been focused on
enkephalins, particularly leucine enkephalin (Tyr-Gly-
Gly-Phe-Leu), an endogenous opioid pentapeptide in-
* Corresponding author. Fax: +385-1-4680-195; e-mail: lidija@irb.hr
0008-6215/$ - see front matter ꢀ 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2003.07.011

68
L. Varga-Defterdaroviꢀc, G. Hrlec / Carbohydrate Research 339 (2004) 67–75
containing glucoconjugate fractions were separated by
column chromatography. 13C NMR shift data for each
of them, showed six signals at positions indicative for
the C-1 atom and three signals in the region corre-
sponding to the C-6 atom of the sugar moiety, thus
suggesting a low primary hydroxyl group selectivity to-
ward esterification with those peptides. Repeated chro-
matography gave compounds 3 and 4 (Fig. 2), as well as
a mixture of glucopeptides in which the corresponding
peptide molecule is located at C-3 or at C-2of the sugar
moiety. Attempts to separate those compounds were so
far unsuccessful. Removal of the Boc-protecting groups
(with TFA) from peptide conjugates 3 and 4, resulted in
the corresponding trifluoroacetate salts of 5 and 6.
Desalting on a Dowex (Ac) column and lyophilization
afforded pure 5 and 6, which were used in the subse-
quent incubation experiments.
R1
O
R2
O
R3
O
CH
C O CH2
H2N
C NH CH C NH CH
n
O
OH
O
OH
n = 3
H
OH
glucopyranose ester
Py-AcOH
MeOH
R1
CH
NH
O
O R3
CH
O
C
C
R2
CH
NH
R2
C
NH
CH N
O
n
CH
O C
HN
H2C
O
OH
n
O
HOCH
HOCH
HO
C
CH2
NH
CH
R3
CH
R1
HCOH
HCOH
CH2
OH
C
O
O
bicyclic Amadori compound
cyclic sugar-related
imidazolidinone
O
HO
OR1
R1
OH
O
R2
O
R3
O
C OH
CH
CH2 HN
C NH CH C NH CH
O
H
n
HO
R1
CH
NH
O
CH
CH
O
C
2O
C
cyclic Amadori compound
R2
C(NH
)
OR2
R NH
CH2
n
CH N
n
CH
O C
HN
Comp.
R
R1
R2
n
HOCH
HOCH
__________________________________
CH
R3
1
2
H
H
H
H
H
H
a
1
2
1
2
1
2
1
2
1
2
HCOH
HCOH
C
O
OH
3
Boc
Boc
H
Boc
Boc
H
4
a
CH2OH
sugar-related
5
a
6
H
H
a
10
11
12
13
H
H
Me
Me
H
H
imidazolidinone
H
H
b-d
b-d
H
HO
H
R1
O
R2
O
R3
O
C OH
O
OH
+
OH
CH
H2N
C NH CH C NH CH
n
HO
O
HO
O
H
O
OH
OH
HO
HO
OH
O
O
D-glucose
OH
peptide
O
H
HO
OH
OH
OH
d
OH
O
H
Figure 1. Possible routes of an intramolecular reactions of 6-O-
pentapeptidyl- -glucopyranose as a function of employed solvent.
b
OH
OH
D
a
c
those obtained when free Tyr-Gly and Tyr-Gly-Gly were
incubated with -glucose, under the same conditions.
D
OH
O
C
O
(
)
CH2 NH C
O
C
n
H2
CH
CH2
OH
O
HO
OH
CH2
2. Results and discussion
2.1. Synthesis of starting materials
NH
O
HN
CH2
NH
O
OH
7 (n = 1)
8 (n = 2)
9
6-O-(
-glucopyranose 3 and 4 were synthesized following a
previously described procedure.17 Di- and tripeptide
L-Tyrosylglycyl)- and 6-O-(L-tyrosylglycylglycyl)-
D
Figure 2. Structures of compounds 1–13.

L. Varga-Defterdaroviꢀc, G. Hrlec / Carbohydrate Research 339 (2004) 67–75
69
(A) 1+Glc(1:1)/Py-AcOH
(B) 1+Glc(1:5)/Py-AcOH
2.2. Incubation experiments
100
%
90
Tyr-Gly and Tyr-Gly-Gly related 6-O-glucopyranose
esters 5 and 6 were incubated, at various temperatures
and times of incubations. For that purpose two solvent
systems were chosen pyridine–acetic acid (1:1) and dry
methanol. Pyridine–acetic acid possesses the balance of
acidity and basicity necessary for acid–base catalyzed
reactions such as the Amadori rearrangement,18 while
methanol has been shown to be an ideal solvent for in-
tramolecular rearrangement of enkephalin-related 6-O-
glycoconjugates to the corresponding 4-imidazolidinone
derivatives.12–14
50 °C
37 °C
50 °C
37 °C
80
70
60
50
40
30
20
10
0
1
2
1
2
1
2
1
2
9
days
1
12
9
1
12
(C) 2+Glc(1:1)/Py-AcOH
(D) 2+Glc(1:5)/Py-AcOH
Reversed-phase HPLC assay was used that permits
simultaneous monitoring of the disappearance of the
starting material and the appearance of rearrangement
and degradation products. The structures of isolated
incubation products 7–13 are presented in Figure 2.
Figures 3–6 show their distribution and relative con-
centrations, depending on the starting compound and
the incubation conditions, as estimated from peak areas
in HPLC chromatograms.
100
90
80
70
60
50
40
30
20
10
0
%
37 °C
50 °C
37 °C
50 °C
1
2
1
2
1
2
1
2
days
The incubation of 6-O-(Tyr-Gly)-D-glucopyranose 5
2
13
9
2
13
9
in pyridine–acetic acid (1:1) gave bicyclic Amadori
compound 7 and cyclo-(Tyr-Gly) 9, which prevailed
(Fig. 3A). The yield of 9 was positively correlated with
temperature, not as a result of 7 instability, but as a
result of intramolecular aminolysis of 5. The optimum
for 7 formation was a temperature of 37 ꢁC and one day
Figure 4. Relative amounts of products formed by incubations of
D
-glucose with Tyr-Gly (A, B), and
D
-glucose with Tyr-Gly-Gly (C, D)
in pyridine–acetic acid (1:1).
(A) 5/MeOH
100
(B) 6/MeOH
%
90
(A) 5/Py-AcOH
37 °C
50 °C
37 °C
50 °C
80
70
60
50
40
30
20
10
0
100
%
90
80
70
60
50
40
30
20
10
0
5
7
9
22 °C
4 °C
37 °C
50 °C
1
2
1
2
1
2
1
2
days
5
7
9
10
6
11
1
2
1
2
1
2
1
2
days
Figure 5. Relative amounts of products formed by incubations of 6-O-
esters 5 and 6 in dry methanol.
(B) 6/Py-AcOH
incubation, while longer periods and higher temperature
tend to favor its degradation.
It was earlier determined that intramolecular reaction
100
90
80
70
60
50
40
30
20
10
0
%
6
22 °C
37 °C
50 °C
4 °C
8
of the
L-tyrosine-related 6-O-glucopyranose ester gave
less than 1% of an unexpected cyclic Amadori com-
pound (although a bicyclic compound would be ex-
pected, Fig. 1) indicating that, in the mentioned
molecule, rearrangement and ester bond hydrolysis oc-
curred simultaneously.19 Our result shows that addition
of only one simple amino acid, such as glycine (com-
pound 5), stabilized Amadori product 7 to such an ex-
tent that it could easily be isolated, and no ester bond
hydrolysis was detected.
1
2
1
2
1
2
1
2
days
Figure 3. Relative amounts of products formed by incubations of 6-O-
esters 5 (A) and 6 (B) in pyridine–acetic acid (1:1).

70
L. Varga-Defterdaroviꢀc, G. Hrlec / Carbohydrate Research 339 (2004) 67–75
free amino groups. While Amadori compound 12 was
(A) 1+Glc/MeOH
(B) 2+Glc/MeOH
the main product obtained from 1 (exception: two days
at 50 ꢁC) (Fig. 4A), esterification of 1 (compound 5)
(Fig. 3A) shifts the reaction pathway in the direction of
intramolecular aminolysis giving diketopiperazine 9 as
the main product. At the same time, esterification of
tripeptide (compound 6) (Fig. 3B) completely obstructed
intramolecular aminolysis. Reaction of free peptides 1
100
%
90
80
70
60
50
40
30
20
10
0
37 °C
50 °C
37 °C 50 °C
and 2 with D-glucose afforded slightly larger amount of
Amadori compounds 12 and 13 (Fig. 4A and C), than
were those of 7 and 8 obtained from 6-O-glucopyranose
esters 5 and 6 (Fig. 3A and B). That may be explained by
the fact that an unbonded small peptide can more easily
1
2
1
2
1
2
1
2
days
9
1
12
9
2
13
Figure 6. Relative amounts of products formed by incubation of Tyr-
Gly and Tyr-Gly-Gly with -glucose in dry methanol.
D
approach the C-1 atom of free D-glucose, than the same
peptide esterified to C-6 of the same sugar molecule.
The tautomer equilibrium composition of Amadori
compounds was determined from relative peak intensi-
ties in 13C NMR (Me2SO-d6) spectra and is presented in
Section 3. Drawing a parallel between our results with
previously published ones,11 it was deduced that bicyclic
Amadori compound 7 was obtained exclusively in its
b-furanose form, while 8, on which the addition of one
more glycine residue facilitated twisting of the peptide
molecule, opened the possibility for some a-furanose
formation (a/b 14:86). 12 and 13, obtained by hydrolysis
of the ester linkage in 7 and 8, gave much more hetero-
geneous mixtures of tautomers in solution: both a,b-
furanose and b-pyranose forms were detected, but not a
trace of a-pyranose.
It is well known that, under appropriate conditions,
compounds containing an a-aminoamide moiety, such
as peptides, condense with aldehydes and ketones giving
4-imidazolidinone derivatives.22 Although sugars, in
their open form, are aldehydes or ketones, no studies
were done on the reaction of free sugars with peptides in
terms of peptide-related 4-imidazolidinone formation.
As our earlier studies showed that intramolecular cy-
clization of leucine enkephalin 6-O-monosaccharide es-
ters in methanol solution resulted in the formation of
novel glycation products containing a 4-imidazolidinone
ring12–14 (Fig. 1), we incubated esters 5 and 6 in meth-
anol at both 37 and 50 ꢁC (Fig. 5). The rates of disap-
pearance of starting compounds 5 and 6 were linearly
dependent on reaction time for both temperatures, but
slower than in pyridine–acetic acid (1:1). Moreover,
during these incubations alcoholysis took place giving
the corresponding peptide methyl esters 10 and 11, re-
spectively. While 11 was the only isolable product gen-
erated from 6-O-ester 6, the dipeptide moiety in 5 is
sufficiently activated to give, in addition to Tyr-Gly-
OMe 10, diketopiperazine 9 as a result of intramolecular
aminolysis. Although, in those incubations, not a trace
of the desired 4-imidazolidinone type of compounds was
obtained, the low yield of bicyclic Amadori compound 7
(Fig. 5A) suggested that, in methanol, the N-terminal
amino group of compound 5 possesses affinity to attach
Introduction of an additional glycine residue (com-
pound 6) further stabilized Amadori compound 8 which
was in that case, isolated as a main product. At the same
time esterification with D-glucose obstructed cyclization
of the peptide to diketopiperazine 9 (Fig. 3B).
The remaining levels of starting 5 and 6, after a one
day incubation period, demonstrate that although 5 re-
acts more readily than 6 do, the amounts of 7 formed
were lower than those of 8. Moreover, while the
amounts of 7 were, at different temperatures, after two
days, negligibly higher or even lower than after one day,
the amounts of 8 were several times larger. Incubation
of 5 and 6 at 50 ꢁC gave, in addition to the compounds
mentioned, a broad spectrum of degradation products.
Both dipeptide and tripeptide related 6-O-gluco-
conjugates 5 and 6 gave bicyclic Amadori compounds
7 and 8 in relatively good yields. Comparing that result
with the previous finding that intramolecular reaction of
Tyr-Pro related 6-O-glucopyranose ester gave cyclo-
(Tyr-Pro) as the only product, while the rearrangement
of Tyr-Pro-Phe related 6-O-glucopyranose ester was
stopped when a glycosylamine was formed,20 we de-
duced that the intramolecular rearrangement of the
peptide-related glycoconjugates depends, not only on
the length of the peptide, but also on the nature of the
incorporated amino acids.
Parallel experiments were done in which Tyr-Gly (1)
and Tyr-Gly-Gly (2) were incubated with D-glucose in
pyridine–acetic acid (1:1) (Fig. 4A and C). An amino
group of 1 and 2 has great affinity, not only for the C-1
atom of
and 13), but also toward the Gly2 carbonyl group (af-
fording diketopiperazine 9). An excess of -glucose
D-glucose (resulting in Amadori compounds 12
D
(Fig. 4B and D) caused the significant lowering of pep-
tides reactivity and thereby the yields of Amadori
products 12 and 13 were lower. That is in agreement
with the results of the Maillard reaction of
-glucose.21
From the reaction pathways of free (1, 2) and este-
L-leucine and
D
rified peptides (5, 6), it could be concluded that this type
of esterification influences the reactivity of the peptides

L. Varga-Defterdaroviꢀc, G. Hrlec / Carbohydrate Research 339 (2004) 67–75
71
to the C-1 sugar atom and, at the same time, the in-
tramolecular nucleophilic addition of the Gly2 nitrogen
to the already formed Schiff base was not possible.
Considerably low reactivity was observed in meth-
reagent, or heating with H2SO4. Column chromato-
graphy was performed on silica gel (Merck; 0.040–
0.063 mm). The following solvents were used: (A)
petroleum ether–ethyl acetate–acetic acid (14:10:1), (B)
ethyl acetate–acetone–water (4:5:0.25), (C) ethyl
acetate–ethanol–acetic acid–water (7:1:1:1). RP HPLC
was performed on a Varian 9010 HPLC system with
Eurospher 100 reversed-phase C-18, 5 lm, analytical
(250 · 4 mm) and semipreparative (250 · 8 mm) columns
(flow rate 0.7 mL minꢀ1 for analytical and 1.1 mL minꢀ1
for semipreparative separations) under isocratic condi-
tions using the following solvents: (D) 21% methanol in
0.1% aq TFA and (E) 15% methanol in 0.1% aq TFA.
RP HPLC column eluates were monitored by their UV
absorbance at 280 nm. The trifluoroacetate salts of the
obtained compounds were desalted on a Dowex 1X2200
(Ac) column (Aldrich, Milwaukee, WI; 10 · 0.8 cm,
eluent water), lyophilized and as such used in the incu-
bation experiments. The optical purity of all prepared
compounds was examined on Chiral Plates (Aldrich) in
methanol–water–acetonitrile (1:1:4) with ninhydrin de-
anolic solutions of
D-glucose and peptides 1 or 2, in
which even at 50 ꢁC more than 50% of the unreacted
peptide remained (Fig. 6A and B). From neither 1 nor 2
not a trace of a 4-imidazolidinone related rearrangement
product was obtained. Moreover, although 1 and 2
followed similar reaction pathways, giving diketopiper-
azine 9 and Amadori products 12 or 13, either of them
prefers a particular product. While 1 gave low yields of
12 and next to nothing of 9, tripeptide 2 as the main
product, particularly at higher temperatures, gave 9 and
only traces of Amadori product 13. The same reactivity
order (dipeptide > tripeptide) in terms of Amadori
product formation, was obtained when different di- and
tripeptides were incubated with
D-glucose in phosphate
buffer.23 A very good example how solvent and the
aldehyde employed influence the reaction pathway is the
incubation of Tyr-Gly-Gly (2) with D-glucose in meth-
1
anol in which case not a trace of a 4-imidazolidinone
related product was detected, while just Tyr-Gly-Gly is
the shortest N-terminal amino acid sequence of en-
kephalin, which with acetaldehyde leads to rapid 4-im-
idazolidinone formation.24
It could be summarized that, under precisely con-
trolled incubation conditions, either Tyr-Gly or Tyr-
Gly-Gly related 6-O-glucopyranose esters 5 and 6, or
tection. The H and 13C NMR spectra (one- and two-
dimensional), in Me2SO-d6 solution (at 20 ꢁC in 5 mm
NMR tubes) were recorded with Varian Gemini 300
spectrometer, operating at 75.5 MHz (13C) and
300.1 MHz (1H). Chemical shifts, in ppm, are referred to
Me4Si. The tautomers equilibrium composition (%) was
determined from the relative peak intensities in 13C
NMR spectra. Elemental analyses were carried out at
ꢁ
ꢀ
the Microanalytical Laboratory, Rudꢂer Boskovic Insti-
model systems with free peptides 1 or 2 and
D
-glucose
gave Amadori rearrangement products. It is obvious
that esterification through the primary hydroxyl group
tute.
of
D
-glucose influences the reactivity of the peptide
3.2. Synthesis of starting materials
amino group. While esterification of Tyr-Gly, in pyri-
dine–acetic acid, accelerated aminolysis, esterification of
Tyr-Gly-Gly made it impossible. In methanol solution
esterification of both peptides opened a new reaction
pathway giving their methyl esters as main products.
The results reported here should facilitate understanding
of the in vivo behavior of those small endogenous opioid
metabolites.
3.2.1. N,O-Di-tert-butyloxycarbonyl-
To an ice-cold solution of N,O-di-tert-butyloxycar-
bonyl-
-tyrosine25 (11.60 g, 30.4 mmol) and N,N0-di-
L-tyrosylglycine.
L
cyclohexylcarbodiimide (DCC) (6.27 g, 30.4 mmol) in
N,N-dimethylformamide (160 mL), N-hydroxysuccin-
imide (3.50 g, 30.4 mmol) was added. The reaction
mixture was stirred for 1 h at 0 ꢁC and 2h at room
temperature, and left overnight at +4 ꢁC. The precipi-
tated DCHU was filtered off and the filtrate added
dropwise to the stirred slurry of glycine (2.28 g,
30.4 mmol), 1 M NaOH (60 mL), and NaHCO3 (5.46 g,
65 mmol). The mixture was stirred for 1 h at room
temperature and filtered. The ice-cold filtrate was acid-
ified to pH 3.5% with 10% citric acid solution and ex-
tracted with chloroform (3 · 100 mL). Combined
extracts were washed with water (100 mL), dried
(Na2SO4), and evaporated. The residue was crystallized
(ethanol–water) to give product (8.15 g), which was
purified by stirring overnight with benzene (200 mL).
Filtration afforded pure title compound (5.56 g, 42%); Rf
(A) 0.34; mp 152–154 ꢁC. Anal. calcd for C21H30N2O8:
C, 57.52; H, 6.90; N, 6.39. Found: C, 57.40; H, 6.95; N,
3. Experimental
3.1. General methods
Trifluoroacetic acid (TFA) was of spectroscopic grade
(Uvasol, Merck, Darmstadt, Germany). All other sol-
vents were distilled at the appropriate pressure. Mps
were determined in open capillaries and are uncorrected.
Optical rotations were measured at room temperature
using an Optical Activity LTD automatic AA-10 pola-
rimeter. Reactions were monitored by TLC on glass
plates (Silica Gel 60 F254, Merck, Darmstadt, Germany)
using detection with ninhydrin, the chlorine–iodine

72
L. Varga-Defterdaroviꢀc, G. Hrlec / Carbohydrate Research 339 (2004) 67–75
1
6.38. H NMR: d 1.29 and 1.48 (Boc CH3), 2.73/3.02
(Tyr b/b0CH2), 3.81 (Gly CH2), 4.20 (Tyr aCH), 6.95
(Tyr NH), 7.07–7.33 (Tyr arom.), 8.29 (Gly NH), 12.65
(Gly COOH). 13C NMR: d 27.33 and 28.22 (Boc CH3),
36.89 (Tyr bCH2), 40.80 (Gly CH2), 55.65 (Tyr aCH),
78.21 and 83.15 (Boc Cq), 121.12 (Tyr eCH), 130.47 (Tyr
dCH), 136.19 (Tyr cC), 149.44 and 151.64 (Boc CO),
155.59 (Tyr fC), 171.53 and 172.37 (Tyr, Gly CO).
phenyl ester (2.06 g, 3 mmol) and imidazole (1.02 g,
15 mmol) were added. The resulting mixture was stirred
overnight at room temperature. After evaporation of the
solvent ethyl acetate (100 mL) was added, extracted with
10% citric acid (2 · 100 mL) and water (100 mL), dried
(Na2SO4), and evaporated. The residue (1.85 g) was
purified by column chromatography (solvent B) to yield
esterificated D-glucose (mixture of 6-O-, 3-O-, and 2-O-
esters) (860 mg, 48%) [Rf (B) 0.53 and 0.58]. Repeated
column chromatography (solvent B) gave the pure title
3.2.2.
pentachlorophenyl ester. To a solution of N,O-di-tert-
butyloxycarbonyl- -tyrosylglycine (4.38 g, 10 mmol) in
N,O-Di-tert-butyloxycarbonyl-
L
-tyrosylglycine
22
D
compound 3 (405 mg, 22%); Rf (B) 0.53; mp 129 ꢁC; ½aŠ
L
+21 (c 1.0, MeOH). Anal. calcd for C27H40N2O13: C,
53.99; H, 6.71; N, 4.66. Found: C, 53.95; H, 6.48; N,
4.76. H NMR: d 1.29 and 1.48 (Boc CH3), 2.74/3.02
dichloromethane (100 mL), DCC–pentachlorophenol
complex26 (10.05 g, 10 mmol) was added, stirred for 2h
at room temperature and left overnight at +4 ꢁC. The
precipitated DCHU was filtered off and the filtrate
evaporated. The oily residue was precipitated with
diisopropyl ether. After two recrystallizations from
dichloromethane–diisopropyl ether pure title compound
was obtained (6.60 g, 96%); Rf (A) 0.79; mp 156–157 ꢁC.
Anal. calcd for C27H29N2O8Cl5: C, 47.22; H, 4.25; N,
4.08. Found: C, 47.36; H, 4.11; N, 4.15.
1
(Tyr b/b0CH2), 2.94 (bGlc H-2), 3.00 (aGlc H-4), 3.05
(bGlc H-4), 3.12( bGlc H-3), 3.15 (aGlc H-2), 3.34 (bGlc
H-5), 3.44 (aGlc H-3), 3.82( aGlc H-5), 3.93 (Gly CH2),
4.00/4.21 (a,bGlc H-6/60), 4.08 (Tyr aCH), 4.34 (bGlc
H-1), 4.58 (aGlc 2-OH), 4.80 (aGlc 3-OH), 4.92( aGlc
H-1), 4.97 (bGlc 2-OH), 5.00 (bGlc 4-OH), 5.10 (aGlc
4-OH), 5.16 (bGlc 3-OH), 6.41 (aGlc 1-OH), 6.73 (bGlc
1-OH), 6.99 (Tyr NH), 7.08 (Tyr eCH), 7.34 (Tyr dCH),
8.44 (Gly NH). 13C NMR: d 27.33 and 28.22 (Boc CH3),
36.85 (Tyr bCH2), 40.65 (Gly CH2), 55.67 (Tyr aCH),
64.88 (a,bGlc C-6), 69.25 (aGlc C-5), 70.26 (bGlc C-4),
70.66 (aGlc C-4), 72.31 (aGlc C-2), 73.02 (aGlc
C-3), 73.61 (bGlc C-5), 78.21 and 83.18 (Boc Cq), 92.51
(aGlc C-1), 97.13 (bGlc C-1), 121.16 (Tyr eCH), 130.45
(Tyr dCH), 136.21 (Tyr cC), 151.62(Tyr fC), 149.43 and
151.64 (Boc CO), 170.23 and 172.66 (Tyr, Gly CO);
anomer ratio a-p/b-p 58:42.
3.2.3.
bonyl-
L
-Tyrosylglycine (1). N,O-Di-tert-butyloxycar-
L-tyrosylglycine (200 mg, 0.46 mmol) was dis-
solved in 90% trifluoroacetic acid solution (1 mL)
containing anisole (0.2mL). The solution was stirred at
room temperature for 30 min. Addition of dry diiso-
propyl ether (0 ꢁC) and subsequent centrifugation gave
the pure TFA · 1 (144 mg, 89%); RP HPLC tR (D)
15.65 min. 1H NMR: d 2.83/3.01 (Tyr b/b0CH2), 3.61
(Tyr aCH), 3.91 (Gly CH2), 6.70 (Tyr dCH), 7.08 (Tyr
eCH), 8.79 (Gly NH), 9.23 (Tyr OH). 13C NMR: d 36.52
(Tyr bCH2), 41.12(Gly CH2), 53.82(Tyr aCH), 115.60
(Tyr eCH), 125.13 (Tyr cC), 130.87 (Tyr dCH), 156.93
(Tyr fC), 169.08 and 171.14 (Tyr, Gly CO).
3.2.6. 6-O-(N,O-Di-tert-butyloxycarbonyl-
cylglycyl)- -glucopyranose (4). Compound
obtained starting from -glucose (1.62g, 9 mmol), N,O-
di-tert-butyloxycarbonyl- -tyrosylglycylglycine penta-
L
-tyrosylgly-
D
4
was
D
L
3.2.4.
L-Tyrosylglycylglycine (2). This compound was
L
obtained starting from N,O-di-tert-butyloxycarbonyl-
-
tyrosylglycylglycine27 (496 mg, 1 mmol), dissolved in
90% trifluoroacetic acid solution (2mL) containing
anisole (0.3 mL) and using the same procedure as de-
scribed for 1. Yield: 377 mg (92%) of TFA · 2; RP HPLC
tR (E) 15.47 min. H NMR: d 2.50/2.66 (Tyr b/b0CH2),
1
3.39/3.50 (Gly3 CH2), 3.50/3.56 (Gly2 CH2), 3.66 (Tyr
aCH), 6.36 (Tyr eCH), 6.71 (Tyr dCH), 7.82(Tyr N H),
7.92(Gly NH), 8.48 (Gly2 NH), 9.15 (Tyr OH). 13C
3
2
NMR: d 36.32(Tyr bCH2), 40.72(Gly CH2), 41.89
(Gly3 CH2), 53.87 (Tyr aCH), 115.47 (Tyr eCH), 124.91
(Tyr cC), 130.61 (Tyr dCH), 156.72(Tyr fC), 168.66 and
171.14 (Tyr, Gly2, Gly3 CO).
3.2.5. 6-O-(N,O-Di-tert-butyloxycarbonyl-
cyl)- -glucopyranose (3). To a stirred solution of
-glucose (1.62g, 9 mmol) in dry pyridine (60 mL), N,O-
di-tert-butyloxycarbonyl- -tyrosylglycine pentachloro-
L-tyrosylgly-
D
D
L

L. Varga-Defterdaroviꢀc, G. Hrlec / Carbohydrate Research 339 (2004) 67–75
73
42.0 (Gly3 CH2), 55.9 (Tyr aCH), 64.9 (a,bGlc C-6),
69.3 (aGlc C-5), 70.3 (bGlc C-4), 70.7 (aGlc C-4), 72.3
(aGlc C-2), 73.1 (aGlc C-3), 73.6 (bGlc C-5), 74.9 (bGlc
C-2), 76.6 (bGlc C-3), 78.4 and 83.3 (Boc Cq), 92.6 (aGlc
C-1), 97.1 (bGlc C-1), 121.2 (Tyr eCH), 130.5 (Tyr cC),
136.2(Tyr dCH), 149.5 and 155.8 (Boc CO), 151.7 (Tyr
fC), 169.7, 170.1, and 172.4 (Tyr, Gly2, Gly3 CO);
anomer ratio a-p/b-p 51:49.
156.90 (Tyr fC), 168.86, 169.06, and 170.11 (Tyr, Gly2,
Gly3 CO); anomer ratio a-p/b-p 55:45.
3.3. Incubation experiments and reversed-phase HPLC
analysis
6-O-(
1.9 lmol) or 6-O-(
L
-Tyrosylglycyl)-
D
-glucopyranose (5) (0.8 mg,
-glucopyra-
L
-tyrosylglycylglycyl)-
D
nose (6) (0.9 mg, 1.9 lmol) were incubated in pyridine–
acetic acid (1:1, 1 mL) at 4, 22, 37, and 50 ꢁC and in dry
methanol (1 mL) at both 37 and 50 ꢁC. For comparison,
3.2.7. 6-O-(L-Tyrosylglycyl)-D-glucopyranose (5). Com-
pound 3 (200 mg, 0.34 mmol) was dissolved in 90% tri-
fluoroacetic acid solution (2mL) containing anisole
(0.3 mL). The solution was stirred at room temperature
for 30 min. Addition of dry diethyl ether (0 ꢁC) and
subsequent centrifugation gave the pure TFA · 5
the reactivity of free peptides and
D-glucose, in the
same solvents, were examined at both 37 and 50 ꢁC.
In experiments with peptide–sugar molar ratio 1:1,
L
-tyrosylglycine (1) (0.7 mg, 2.8 lmol) or
glycylglycine (2) (0.8 mg, 2.8 lmol) were incubated sep-
arately with -glucose (0.5 mg, 2.8 lmol) either in
L-tyrosyl-
(174 mg, 99%); Rf (C) 0.16; RP HPLC tR (D) 12.73 and
22
D
13.29 (5a and 5b); mp 106–125 ꢁC (dec); ½aŠ +45.3
D
(c 2.0, EtOH). Anal. calcd for C17H24N2O9 ·
CF3COOH · 2H2O: C, 41.46; H, 5.31; N, 5.09. Found:
pyridine–acetic acid (1:1, 1.5 mL) or in dry methanol
(1.5 mL). When the molar ratio peptide–sugar was 1:5,
1
C, 41.71; H, 5.79; N, 5.07. H NMR: d 2.85/3.02 (Tyr
the peptide (2.8 lmol) and
D
-glucose (2.5 mg, 14 lmol)
b/b0CH2), 2.93 (bGlc H-2), 3.01 (bGlc H-3), 3.04 (bGlc
H-4), 3.07 (aGlc H-4), 3.13 (aGlc H-2), 3.17 (bGlc H-5),
3.35 (aGlc H-5), 3.45 (aGlc H-3), 3.96/3.98 (Gly CH2),
4.05 (Tyr aCH), 4.09/4.37 (a,bGlc H-6/60), 4.31 (bGlc
H-1), 4.91 (aGlc H-1), 6.71 (Tyr eCH), 7.08 (Tyr dCH),
8.14 (Tyr NH), 9.00 (Gly NH), 9.50 (Tyr OH). 13C
NMR: d 38.48 (Tyr bCH2), 40.43 (Gly CH2), 53.52(Tyr
aCH), 64.77 (a,bGlc C-6), 68.99 (bGlc C-4), 70.02( aGlc
C-4), 70.42( aGlc C-5), 72.08 (aGlc C-2), 72.77 (aGlc
C-3), 73.34 (bGlc C-2), 74.60 (bGlc C-3), 76.31 (bGlc
C-5), 92.28 (aGlc C-1), 96.88 (bGlc C-1), 115.38 (Tyr
eCH), 124.67 (Tyr cC), 130.59 (Tyr dCH), 156.70 (Tyr
fC), 168.94 and 169.53 (Tyr, Gly CO); anomer ratio a-p/
b-p 51:49.
were dissolved in pyridine–acetic acid (1:1, 4.5 mL). The
progress of the reaction was monitored by RP HPLC.
Aliquots were withdrawn from the incubation mixtures,
immediately frozen, and lyophilized. The respective
samples were directly analyzed by RP HPLC (for details
see Section 3.1).
3.4. Isolation of products formed during incubations
3.4.1.
-tyrosylglycyl-(1 ! O} (7).
-glucopyranose (5) (50 mg, 0.12mmol) was dissolved in
cyclo-{N-[-6)-1-Deoxy-b-
D
-fructofuranos-1-yl]-
L
6-O-(
L
-Tyrosylglycyl)-
D
pyridine–acetic acid (1:1, 50 mL) and the solution was
stirred for 17 h at 37 ꢁC. The solvent was evaporated off,
the residue was dissolved in water (1 mL), applied to a
short (10 · 0.8 cm) Dowex 1X2200 (Ac) column, and
eluted with water. Fractions containing title compound 7
were pooled, evaporated, and the residue purified by
semipreparative RP HPLC (solvent D). Lyophilization
and crystallization from methanol–diethyl ether gave
pure title compound 7 (7.8 mg, 17%); Rf (C) 0.41; RP
3.2.8. 6-O-(L-Tyrosylglycylglycyl)-D-glucopyranose (6).
Compound 4 (150 mg, 0.22 mmol) was deprotected by
using 90% trifluoroacetic acid solution as described in
the preparation of 5, affording TFA · 6 (134 mg, 98%);
Rf (C) 0.13; RP HPLC tR (E) 12.27 and 12.95 (6a and
22
D
6b); mp 127–135 ꢁC (dec); ½aŠ +72( c 1.0, EtOH). Anal.
calcd for C19H27N3O10 · CF3COOH · 3H2O: C, 40.32;
HPLC tR (D) 15.19 min; mp 130–132 ꢁC (dec); ½aŠ +33
D
1
H, 5.48; N, 6.72. Found: C, 40.93; H, 5.51; N, 6.83. H
(c 1.0, MeOH). Anal. calcd for C17H22N2O8 ·
CF3COOH · H2O: C, 44.36; H, 4.90; N, 5.44. Found: C,
44.95; H, 5.00; N, 4.92. 13C NMR for 7 (b-furanose): d
34.0 (Tyr bCH2), 43.0 (Gly CH2), 51.0 (Fru C-1), 61.8
(Tyr aCH), 62.9 (Fru C-6), 72.6 (Fru C-4), 77.0 (Fru
C-3), 80.8 (Fru C-5), 101.6 (Fru C-2), 115.6 (Tyr eCH),
125.5 (Tyr cC), 130.2(Tyr dCH), 156.6 (Tyr fC), 168.2
and 169.2(Tyr, Gly CO); only b-f.
NMR: d 2.84/2.99 (Tyr b/b0CH2), 2.92 (bGlc H-2), 3.04
(aGlc H-4), 3.08 (Glc H-4), 3.13 (bGlc H-3), 3.15 (aGlc
H-2), 3.36 (bGlc H-5), 3.46 (aGlc H-3), 3.77 (aGlc H-5),
3.82(Gly 3 CH2), 3.88 (Gly2 CH2), 4.01 (Tyr aCH), 4.06/
4.30 (a/bGlc H-6/60), 4.33 (bGlc H-1), 4.91 (aGlc H-1),
6.70 (Tyr eCH), 7.05 (Tyr dCH), 8.11 (Tyr NH), 8.41
(Gly2 NH), 8.81 (Gly3 NH), 9.42(Tyr O H). 13C NMR: d
36.31 (Tyr bCH2), 40.55 (Gly2 CH2), 41.80 (Gly3 CH2),
53.85 (Tyr aCH), 64.88 (a,bGlc C-6), 69.26 (aGlc C-5),
70.25 (bGlc C-4), 70.65 (aGlc C-4), 72.31 (aGlc C-2),
73.02( aGlc C-3), 73.60 (bGlc C-5), 74.83 (bGlc C-2),
76.55 (bGlc C-3), 92.51 (aGlc C-1), 97.11 (bGlc C-1),
115.61 (Tyr eCH), 125.05 (Tyr cC), 130.77 (Tyr dCH),
3.4.2. cyclo-{N-[-6)-1-Deoxy-a,b-
D
-fructofuranos-1-yl]-
-Tyrosylgly-
D-glucopyranose (6) (87 mg, 0.19 mmol) was
L
-tyrosylglycylglycyl-(1 ! O} (8). 6-O-(
L
cylglycyl)-
dissolved in pyridine–acetic acid (1:1, 87 mL) and the
solution was stirred for 48 h at room temperature. The

74
L. Varga-Defterdaroviꢀc, G. Hrlec / Carbohydrate Research 339 (2004) 67–75
solvent was evaporated off, and following the isolation
procedure described for compound 7, but using solvent
E, the title compound 8 was obtained (15 mg, 19%); Rf
HPLC (solvent D) to give the pure title compound 12
(20 mg, 27%); Rf (C) 0.14; RP HPLC tR (D) 15.59 min;
mp 116–120 ꢁC (dec); ½aŠ +9 (c 1.0, MeOH). Anal.
22
D
(C) 0.34; RP HPLC tR (E) 14.32min; mp 128–139 ꢁC
calcd for C17H24N2O9 · CF3COOH: C, 44.36; H, 4.90;
N, 5.40. Found: C, 44.58; H, 4.71; N, 5.37. 13C NMR for
12 (a-furanose): d 35.2(Tyr bCH2), 41.0 (Gly CH2), 50.5
(Fru C-1), 60.7 (Fru C-6), 61.6 (Tyr aCH), 75.2(Fru
C-4), 81.7 (Fru C-5), 83.1 (Fru C-3), 101.0 (Fru C-2),
115.5 (Tyr eCH), 124.9 (Tyr cC), 130.6 (Tyr dCH), 156.6
(Tyr fC); for 12 (b-furanose): d 35.0 (Tyr bCH2), 41.0
(Gly CH2), 50.9 (Fru C-1), 61.5 (Tyr aCH), 62.5 (Fru
C-6), 74.6 (Fru C-4), 78.2(Fru C-3), 82.4 (Fru C-5), 99.5
(Fru C-2), 115.4 (Tyr eCH), 124.7 (Tyr cC), 130.5 (Tyr
dC), 156.6 (Tyr fC); for 12 (b-pyranose): d 35.1 (Tyr
bCH), 41.0 (Gly CH2), 52.3 (Fru C-1), 61.6 (Tyr aCH),
64.0 (Fru C-6), 68.7 (Fru C-5), 69.1 (Fru C-4), 70.3 (Fru
C-3), 95.4 (Fru C-2), 115.4 (Tyr eCH), 124.7 (Tyr cC),
130.5 (Tyr dCH), 156.6 (Tyr fC). For all of them 167.4,
167.5, 167.6, and 170.6 (Tyr, Gly2, Gly3 CO); anomer
ratio b-p/a-f/b-f 40:40:20.
22
(dec); ½aŠ +54.7 (c 0.7, MeOH). Anal. calcd for
D
C19H25N3O9 · 1/2CF COOH · H2O: C, 46.69; H, 5.39;
3
N, 8.17. Found: C, 46.34; H, 5.29; N, 8.89. 13C NMR for
8 (a-furanose): d 34.8 (Tyr b), 42.3 (Gly3 CH2), 43.8
(Gly2 CH2), 48.4 (Fru C-1), 61.9 (Tyr aCH2), 62.2 (Fru
C-6), 72.4 (Fru C-4), 78.2(Fru C-5), 83.5 (Fru C-3),
103.6 (Fru C-2), 115.5 (Tyr eCH), 124.5 (Tyr cC), 130.7
(Tyr dCH), 156.7 (Tyr fC); for 8 (b-furanose): d 34.6
(Tyr bCH2), 41.6 (Gly3 CH2), 43.2(Gly 2 CH2), 51.8 (Fru
C-1), 61.3 (Fru C-6), 61.9 (Tyr aCH2), 71.8 (Fru C-4),
77.6 (Fru C-3), 82.5 (Fru C-5), 100.7 (Fru C-2), 115.5
(Tyr eCH), 125.1 (Tyr cC), 130.2(Tyr dCH), 156.5 (Tyr
fC). For both of them 167.3, 167.5, 169.0, and 169.3
(Tyr, Gly2, Gly3 CO); anomer ratio a-f/b-f 14:86.
3.4.3. cyclo-(L-Tyrosylglycyl) (9). 6-O-(L-Tyrosylgly-
cyl)-D-glucopyranose (5) (50 mg, 0.12mmol) was dis-
solved in pyridine–acetic acid (1:1, 50 mL) and the
solution was incubated for 17 h at 50 ꢁC. Following the
procedure described for the isolation of compound 7,
the title compound 9, being identical with formerly pre-
pared one,28 was obtained (8 mg, 33%); Rf (C) 0.75; RP
HPLC tR (D) 18.80 and tR (E) 22.11 min. 13C NMR: d
38.28 (Tyr bCH2), 43.75 (Gly CH2), 55.88 (Tyr aCH),
115.20 (Tyr eCH), 125.98 (Tyr cC), 131.30 (Tyr dCH),
156.58 (Tyr fC), 166.04 and 167.68 (Tyr, Gly CO).
3.4.6.2. Method b: Incubation of
L
-tyrosylglycine (1)
-Tyrosylglycine (1) (120 mg, 0.5 mmol)
D-glucose (68 mg, 0.38 mmol) were dissolved in
and
and
D-glucose. L
pyridine–acetic acid (1:1, 170 mL) and the solution was
incubated for two days at 37 ꢁC. The solvent was
evaporated off and the residue was purified by semi-
preparative RP HPLC (solvent D). Crystallization from
methanol–diethyl ether gave the title compound 12
(23 mg, 15%), which was identical (mp, optical rotation,
RP HPLC retention times, and NMR data) with com-
pound 12 obtained by Method a.
3.4.4.
sylglycyl)-
L
-Tyrosylglycine methyl ester (10). 6-O-(
D
L-Tyro-
-glucopyranose (5) (75 mg, 0.19 mmol) was
dissolved in dry methanol (90 mL) and the solution was
incubated for three days at 50 ꢁC. The solvent
was evaporated off and the residue was purified by
semipreparative RP HPLC (solvent D) giving the title
compound 10 (15 mg, 37%); Rf (C) 0.46; RP HPLC tR (D)
21.97 min. 13C NMR: d 36.29 (Tyr bCH2), 40.71 (Gly
CH2), 51.99 (CH3), 53.67 (Tyr aCH), 115.43 (Tyr eCH),
124.67 (Tyr cC), 130.62(Tyr dCH), 156.63 (Tyr fC),
168.83 and 169.86 (Tyr, Gly CO).
3.4.7. N-(1-Deoxy-D-fructos-1-yl)-L-tyrosylglycylglycine
(13). Bicyclic Amadori compound 8 (42mg, 0.1 mmol)
was dissolved in 0.1 M NH4OH (21 mL) and the solu-
tion was stirred at room temperature for 1 h. The solvent
was removed and the residue was purified by semipre-
parative RP HPLC, using solvent E, to give the pure
title compound 13 (20 mg, 44%), which was found to be
identical (comparison of mp, optical rotation, RP
HPLC retention time, and NMR data) with formerly
prepared Amadori compound 13;29 Rf (C) 0.11; RP
HPLC tR (E) 14.47 min. 13C NMR for 13 (a-furanose): d
35.2(Tyr bCH2), 41.1 (Gly2 CH2), 42.0 (Gly3 CH2), 50.3
(Fru C-1), 60.7 (Fru C-6), 61.5 (Tyr aCH2), 75.2(Fru C-
4), 81.8 (Fru C-5), 83.1 (Fru C-3), 101.0 (Fru C-2), 115.5
(Tyr eCH), 124.7 (Tyr cC), 130.5 (Tyr dC H), 156.6 (Tyr
fC); for 13 (b-furanose): d 34.9 (Tyr bCH2), 41.1 (Gly2
CH2), 42.0 (Gly3 CH2), 50.8 (Fru C-1), 61.3 (Tyr aCH2),
62.6 (Fru C-6), 74.6 (Fru C-4), 78.2(Fru C-3), 82.6 (Fru
C-5), 99.5 (Fru C-2), 115.5 (Tyr eCH), 124.7 (Tyr cC),
130.5 (Tyr dCH), 156.6 (Tyr fC); for 13 (b-pyranose): d
35.1 (Tyr bCH2), 41.1 (Gly2 CH2), 42.0 (Gly3 CH2), 51.9
(Fru C-1), 61.5 (Tyr aCH2), 64.0 (Fru C-6), 68.7 (Fru
C-5), 69.1 (Fru C-4), 70.1 (Fru C-3), 95.6 (Fru C-2),
3.4.5.
(
L
-Tyrosylglycylglycine methyl ester (11). 6-O-
-glucopyranose (6) (100 mg,
L
-Tyrosylglycylglycyl)-D
0.22 mmol) was dissolved in dry methanol (80 mL). In-
cubation and purification was performed as described
for 10, but using solvent E, yielding the title compound
11 (23 mg, 41%); Rf (C) 0.36; RP HPLC tR (E) 23.05 min.
3.4.6. N-(1-Deoxy-D-fructos-1-yl)-L-tyrosylglycine (12).
3.4.6.1. Method a: Hydrolysis of compound 7. Bicyclic
Amadori compound 7 (68 mg, 0.18 mmol) was dissolved
in 0.1 M NH4OH (28 mL) and the solution was stirred at
room temperature for 15 min. The solvent was removed
and the residue was purified by semipreparative RP

L. Varga-Defterdaroviꢀc, G. Hrlec / Carbohydrate Research 339 (2004) 67–75
75
ꢁ
13. Varga-Defterdarovic, L.; Vikic-Topic, D.; Horvat, S.
ꢀ
J. Chem. Soc., Perkin Trans. 1 1999, 2829–2834.
ꢀ
ꢀ
115.6 (Tyr eCH), 124.9 (Tyr cC), 130.5 (Tyr dCH), 156.6
(Tyr fC). For all of them 167.2, 167.3, 168.4, and 171.2
(Tyr, Gly2, Gly3 CO); anomer ratio b-p/a-f/b-f 44:42:14.
ꢁ
ꢁꢁ ꢀ
14. Horvat, S.; Roscic, M.; Horvat, J. Glycoconjugate J. 1999,
16, 391–398.
15. Rudd, P. M.; Elliott, T.; Crosswell, P.; Wilson, I. A.;
Dwek, R. A. Science 2001, 291, 2370–2376.
16. Sobal, G.; Menzel, J.; Sinzinger, H. Prostag. Leukotr.
Essential Fatty Acids 2000, 63, 177–186.
Acknowledgements
ꢁ
ꢀ
17. Horvat, S.; Varga-Defterdarovic, L.; Horvat, J.; Modric-
Zganjar, S.; Chung, N. N.; Schiller, P. W. Lett. Pept. Sci.
ꢀ
This work was supported by the Ministry of Science and
Technology of Croatia grant no. 0098054. The authors
thank Mrs. Milica Perc for excellent technical assistance.
ꢁ
1995, 2, 363–368.
18. Yaylayan, V. A.; Huyghues-Despointes, A. Crit. Rev.
Food Sci. Nutr. 1994, 34, 321–369.
ꢁ
19. Jeric, I.; Simicic, L.; Stipetic, M.; Horvat, S. Glycoconju-
ꢁ
ꢀ
gate J. 2000, 17, 273–282.
ꢁ ꢀ
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