15N and 1H NMR study of ureido sugars, derivatives of 2-amino-2-deoxy-β-D-glucopyranosides

Article abstract of DOI:10.1002/(SICI)1097-458X(199810)36:10<727::AID-OMR359>3.0.CO;2-A

The ¹⁵N NMR spectra of a series of derivatives of 2-amino-2-deoxy-β-D-glucopyranose and dipeptides or secondary amines were recorded. In the dipeptide derivatives the chemical shift of nitrogen atom N-1 (linked to sugar) is essentially unchange

Full text of DOI:10.1002/(SICI)1097-458X(199810)36:10<727::AID-OMR359>3.0.CO;2-A

MAGNETIC RESONANCE IN CHEMISTRY
Magn. Reson. Chem. 36, 727È731 (1998)
15N and 1H NMR study of ureido sugars, derivatives of
2-amino-2-deoxy-b-D-glucopyranosides
M. Weychert,1 J. Klimkiewicz,1 I. Wawer,1* B.Piekarska-Bartoszewicz2 and A. Temeriusz2
1 Faculty of Pharmacy, Medical University of Warsaw, Banacha 1, 02097 Warsaw, Poland
2 Department of Chemistry, Warsaw University, Pasteura 1, 02093 Warsaw, Poland
Received 20 January 1998; revised 18 April 1998; accepted 18 April 1998
ABSTRACT: The 15N NMR spectra of a series of derivatives of 2-amino-2-deoxy-b-D-glucopyranose and dipep-
tides or secondary amines were recorded. In the dipeptide derivatives the chemical shift of nitrogen atom N-1
(linked to sugar) is essentially unchanged and the shifts of nitrogen atoms N-3 and N-6 are determined by the
nature of the R and R substituents at C-a carbon of the amino acid unit. The highest shielding of N-3 and N-6 is
1
2
observed for Gly units (R or R \ H) and decreases in the order Gly [ Val [ Phe [ Leu [ Ala. The deshielding
1
2
of the nitrogen and proton of the N-6ÈH group results from the intramolecular hydrogen bonding interaction with
the oxygen atom of the ester group. The experiment with HÈD isotopic exchange conÐrmed slow exchange of the
N-6ÈH protons whereas the N-1ÈH and N-3ÈH protons, not involved in intramolecular H-bond, exchange fast.
( 1998 John Wiley & Sons, Ltd.
KEYWORDS: NMR; 1H NMR; 15N NMR; ureido sugars
INTRODUCTION
given in Table 1. The data for other ureido sugars are
included for comparison.
In recent years, ureas with sugar substituents have
attracted considerable attention. Ureido sugars are
starting materials in the synthesis of nitrosoureido
Two signals were seen in the proton decoupled
natural abundance 15N NMR spectra of 2È4, but the
di†erence in chemical shifts was too small for reliable
assignment based on alkyl group increments and the
spectra were recorded using the INEPT technique. The
N-1ÈH signal appeared as a doublet with a 1J(NH) of
ca. [87 Hz; this resonance can be assigned to the
nitrogen linked to glucopyranose and the other reso-
nance is from N-3 nitrogen. The chemical shifts of N-1
are within the range [303.5 to [306.2 ppm and
nitrogen N-3 in compounds with dialkyl groups is
deshielded (16È26 ppm) when compared with N-1. The
nitrogen of a secondary or primary amine residue
incorporated in a ureido sugar is deshielded with
respect to that from the respective alkylamine. It is
worth comparing ureido sugars 1 and 2 with one and
two ethyl groups at N-3. The replacement of the
N-3ÈH hydrogen by a second ethyl group results in
a low-frequency shift of N-1 (of 4.7 ppm) and a high-
frequency shift of N-3 (of 7.6 ppm). A small substituent
e†ect (2 ppm) for N-1 and a high-frequency shift of
N-3 (of ca. 20 ppm) were observed7 for ureido sugars
with aromatic substituents (5È7), in agreement with
the known tendency9 that the nitrogen resonance in
arylamines is usually deshielded compared with that for
alkylamines.
sugars,
which
exhibit
antitumour
activity.
Streptozotocin1 and chlorozotocin2 are used in clinical
treatment and nitrosoureido sugars with other sub-
situents are being tested for this application. As part of
our continuing work on the synthesis3h5 and the deter-
mination of the structure of ureido sugars,5h8 we report
here the results of a 15N NMR study of a series of
derivatives of 2-amino-2-deoxy-b-D-glucopyranose and
secondary amines 1È7 and dipeptides 13È22 (Scheme 1).
For the sake of completeness, the results8 for amino
acid derivatives 8È12 are also included. The method of
synthesis of ureido sugars with dipeptide residues and
structure characterization by 1H and 13C NMR spec-
troscopy were reported recently.5 Compounds with an
amino acid or dipeptide contain two or three NH
groups and knowledge of their properties was required
prior to the next step in the synthesis, N-nitrosation,
which should be carried out selectively.
RESULTS AND DISCUSSION
The chemical shifts and coupling constants for 2È4 with
two alkyl groups and 13È22 with dipeptide residues are
In the spectra of ureido sugars with one L-amino acid
residue,8 the resonance of N-1 appeared at approx-
imately [300 ppm and the replacement of the amino
acid led to no change; the resonances of N-3 are within
the range [292 to [306 ppm and their chemical shifts
depend on the e†ects of the side-chain at the C-a carbon
of the amino acid unit.
* Correspondence to: I. Wawer, Department of Physical Chemistry,
Faculty of Pharmacy, Medical University of Warsaw, Banacha 1,
02-097 Warsaw, Poland. E-mail: wawer=farm.amwaw.edu.pl
Contract/grant sponsor: Medical University of Warsaw; Contract/
grant number: IIA-16 (1997).
Contract/grant sponsor: Warsaw University; Contract/grant number:
BST 562/14/97.
( 1998 John Wiley & Sons, Ltd.
CCC 0749-1581/98/100727È05 $17.50

728
M. WEYCHERT ET AL .
Scheme 1. Structures of the compounds studied.
High-resolution and solid-state 15N NMR techniques
remaining nitrogens with CH neighbours. The values of
2J(N-C,H) are 0.7È1.5 Hz11 and such small splittings
into triplets or doublets were not clearly seen in the
resonances. The intensity of the N-1 signal was usually
slightly smaller, probably because of the longer relax-
ation time of this nitrogen, bound to the bulky and rela-
tively rigid sugar moiety. The assignment of the 15N
shifts of the three NH groups was conÐrmed by the
correlation with the shifts of their attached proton in a
2D heteronuclear experiment (gs-HSQC). The most
deshielded 15N resonance assigned to N-6 is correlated,
as expected, with the most deshielded proton N-6ÈH
(Fig.1). The chemical shift of N-1, linked to glucose, is
ca. [300 ppm and is not responsive (as in ureido sugars
with one amino acid) to the changes of the substituent
at N-3. The chemical shifts of the N-3 nitrogens in
ureido sugars 13È22 are more variable. The highest
shielding is observed for nitrogen in Gly (R \ H) and
are being increasingly applied to studies of peptides;10
chemical shifts were found to depend on the conforma-
tion, nature of the amino acid, amino acid sequence and
the kind of hydrogen bonding. It seemed interesting to
measure 15N NMR spectra for ureido sugars 13È22
because these compounds are soluble in CDCl , and
3
the chemical shifts of dipeptide residues can be obtained
in a weakly interacting solvent whereas most available
data are for DMSO or water.
15N NMR spectra of dipeptide derivatives were
recorded with the INEPT technique and three doublets
appeared in the typical spectra in the narrow range
from [290 to [310 ppm. This di†erence in chemical
shifts of a few ppm is too small for unambiguous assign-
ment. Single-frequency 15NM1HN decoupling experi-
ments were Ðrst performed to di†erentiate the N-CH
nitrogen in Gly-containing compounds from the
2
( 1998 John Wiley & Sons, Ltd.
MAGNETIC RESONANCE IN CHEMISTRY, VOL. 36, 727È731 (1998)

15N AND 1H NMR OF UREIDO SUGARS
729
Table 1. 15N chemical shifts (in CDCl , d in ppm, reference CH NO ) and 1J(N,H)
3
3
2
coupling constants (Hz) (in parentheses) for peracetylated methyl b-D-glucopy-
ranosyl ureas with various substituents
Compound
N-1
N-3
N-6
Ref.
6
1
2
[301.2 (88.7)
[305.9 (87.7)
[306.2 (87.3)
[303.5 (87.3)
[297.9 (89.5)
[298.3 (89.3)
[299.2 (89.8)
[300.2 (89.4)
[300.2 (89.3)
[299.2 (88.2)
[300.1 (89.2)
[300.1 (89.1)
[300.7 (88.7)
[300.9 (91.5)
[300.2 (88.3)
[300.8 (89.2)
[300.5 (87.8)
[300.8 (89.7)
[300.8 (88.8)
[300.8 (90.1)
[300.6 (89.8)
[301.0 (88.0)
[294.1 (88.7)
[286.5
[279.8
[286.9
3
4
5
[277.4 (89.4)
[278.0 (89.2)
[280.9 (89.0)
[307.6 (90.1)
[292.2 (90.0)
[298.9 (89.1)
[294.1 (89.8)
[296.9 (89.9)
[306.4 (89.5)
[289.8 (90.6)
[290.7 (89.1)
[306.2 (90.6)
[296.1 (89.5)
[291.2 (91.0)
[294.4 (89.5)
[294.2 (89.5)
[306.4 (90.7)
[290.0 (90.5)
7
7
7
8
8
8
8
8
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
[263.7 (93.9)
[279.3 (91.8)
[261.9 (92.9)
[269.6 (93.3)
[273.3 (92.0)
[276.4 (94.0)
[276.0 (93.8)
[277.1 (93.7)
[267.3 (90.3)
[278.9 (93.9)
decreases in the order Gly [ Val [ Phe [ Leu [ Ala.
This order can be explained in terms of the b-e†ect of
the alkyl group R or R linked to the C-a carbon of
types of NH groups: (i) the chemical shift of N-1, linked
to sugar, is essentially unchanged; (ii) the chemical shift
of N-3 is determined by the e†ect of R , in similar way
4
5
4
the amino acid unit and is in agreement with the
sequence observed in N-acyl- and N-
as in other peptide compounds since the relationship is
linear; (iii) the chemical shift of N-6 is dependent on the
carbobenzyloxyamino acids11 and in ureido sugars 8È12
nature of R , but the proximity of the ester group
results in deshielding (approximately 20 ppm).
5
with L-amino acid residues.8
The plot of the nitrogen chemical shifts for ureido
sugars with dipeptides versus the above amino acid
sequence (Fig. 2) characterizes the behaviour of three
The plots shown in Fig. 2 give the possibility of pre-
dicting nitrogen chemical shifts for the ureido sugars
with other amino acid sequences such as those synthe-
sized by us (13È22, Scheme 1) for example, for ureido
sugar 23 with LeuÈLeu the values [300, [291 and
[264 ppm can be expected for N-1, N-3 and N-6,
respectively.
Since the d 15N measurements were carried out in
dilute CDCl solutions, the contribution of intermolec-
3
ular hydrogen bonding can be neglected; however,
Figure 2. Plot of 15N chemical shifts for ureido sugars
with dipeptides versus the amino acid sequence (from
Ref. 11).
Figure 1. 2D gs-HSQC 1H/15N NMR spectrum of 19.
( 1998 John Wiley & Sons, Ltd.
MAGNETIC RESONANCE IN CHEMISTRY, VOL. 36, 727È731 (1998)

730
M. WEYCHERT ET AL .
intramolecular hydrogen bonds are still present. IR and
1H NMR spectroscopy have frequently been applied to
characterize the inter- and intramolecular interactions
of amino acid derivatives in solution. A Fourier trans-
form IR study of ureido sugars with amino acid
residues12 indicated that the NH group is involved in
an intramolecular hydrogen bond, a pentagon (C type),
5
which is formed by association with the oxygen atom of
the ester group.
In ureido sugars with dipeptides, the 1H NMR
chemical shifts of N-1ÈH are in the range 4.95È5.61
ppm and those of N-3ÈH are 5.45È6.08 ppm, whereas
those of N-6ÈH are in the narrower range of 6.86È7.28
ppm; usually N-6ÈH is at least 1 ppm more deshielded
than N-3ÈH. The relationship between d 1H and d 15N
for the three NH groups is illustrated in Fig. 3. The
deshielding of N-6ÈH protons can be related to the
intramolecular hydrogen bonding interaction. In order
to conÐrm this supposition, an experiment with HÈD
isotopic exchange was carried out.
The NH protons are usually rapidly exchanged with
deuterium; however, in molecules where amide protons
are involved in intramolecular hydrogen bonds the rate
of the exchange reactions with the solvent will be con-
siderably slower. The HÈD exchange e†ect for labile
protons of biomolecules was used as an indicator of the
exposure of these protons to the solvent and yielded
information on the peptide conformations.13 Part of the
1H NMR spectrum of 19 is illustrated in Fig. 4; after
Figure 4. Part of 1H NMR spectrum of 19, (a) before and
(b) after addition of CD OD.
3
the addition of CD OD (50 ll) the signals of N-1ÈH
to the more pyramidal or trigonal geometry of the
3
and N-3ÈH lost 95% of their intensity whereas only a
nitrogen. The value of 1J(N,H) is linearly dependent on
slight decrease of the intensity of N-6ÈH was observed.
The 1J(N,H) coupling constants, given in Table 1, are
of interest because they could be related to the torsional
angles in conformations of peptides in solution. Accord-
ing to a general dependence, 1J(N,H) decreases from ca.
95 Hz for a planar trans peptide group to ca. 67 Hz for
an angle of 90¡ and increases again to ca. 90 Hz for a
planar cis arrangement, found in cyclic peptides. In the
linear dipeptides, 1J(N,H) is between 92.1 and 94.5
Hz.13 The extended conformation with an all-trans
orientations is far more probable than cis orientations
of NH and CxO groups for the dipeptide residue of
ureido sugars, hence the values of 1J(N,H) can be related
the percentage of
s
character of the nitrogen
orbitals,9,11 the higher the value of 1J(N,H), the higher
is the bond order of the nitrogen. Values of 87È90 Hz
were measured for 1J(N,H) for nitrogens N-1 and N-3
of the ureido bridge (Table 1), in agreement with those
usually observed for ureas. The shielding of [300 ppm
and the smaller 1J(N,H) of 87È88 Hz for N-1 suggest a
more pyramidal geometry of nitrogen linked to glucose.
The value of 1J(N,H) between 91.5 and 94 Hz and the
lowest shielding indicates that the geometry for N-6 is
more trigonal.
EXPERIMENTAL
The ureido sugars were synthesized from methyl 3,4,6-
tri-O-acetyl-2-deoxy-2-(4-nitrophenoxycarbonylamino)-b-
D-glucopyranoside and a secondary amine or respective
dipeptide (protected by an OMe, OEt or OBz group)
according to published procedures.3h5
15N NMR spectra were recorded on a Bruker
AM-500 spectrometer at 50.7 MHz, using the standard
INEPT Bruker sequence with following parameters:
15N pulse width 30 ls (90¡), relaxation delay 1.5 s,
acquisition time 2.95 s, spectral width 5 kHz and digital
resolution 0.17 Hz per point. The pulse sequence was
optimized for the average 1J(N,H) of ca. 90 Hz (t \ 1/4
1J(N,H)). Spectra were recorded using a 10 mm probe
Figure 3. Relationship between d 1H and d 15N for the
three NH groups in 13–22.
and the concentration was 0.5 M in CDCl . 15N chemi-
3
( 1998 John Wiley & Sons, Ltd.
MAGNETIC RESONANCE IN CHEMISTRY, VOL. 36, 727È731 (1998)

15N AND 1H NMR OF UREIDO SUGARS
731
cal shifts were referenced against neat external
).
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3
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This work was supported by the Medical University of Warsaw,
Grant IIA-16 (1997), and by Warsaw University, Grant BST 562/14/
97.
( 1998 John Wiley & Sons, Ltd.
MAGNETIC RESONANCE IN CHEMISTRY, VOL. 36, 727È731 (1998)