Volume 11 Number 4 1983
Nucleic Acids Research
On the conformation of 5-substituted uridines as studied by proton magnetic resonance
Wolfgang Uhl*, Josef Reiner + and Hans Gunter Gassen*
*lnstitut fUr Organische Chemie und Biochemie .Technische Hochschule Darmstadt, Petersenstrasse
22, D-6100 Darmstadt, and +Universitat Bayreuth/OCl, UniversitStsstrasse 30, D-8580 Bayreuth,
FRG
Received 29 November 1982; Accepted 18 January 1983
ABSTRACT
The proton magnetic resonance (pmr) spectra of 10 basemodified uridine derivatives xsUrd have been measured at 3°,
30°, and 60°C in order to correlate the electronic effects of
different substituents with the molecular conformation of the
respective nucleosides. The results presented demonstrate the
close relation between conformational parameters and the electron-affinity of the substituents as reflected by their Hammett
constants. Going from electron-donating to electron-acceptinq
groups, the portion of N-conformer in the ribose N;=SS equilibrium increases from 44% to about 90%. In addition the percentage
of gauche-gauche rotamer as measured for the exocyclic groups
changes from about 30% in nh5Urd to more than 80% in no 5 Urd.
INTRODUCTION
Minor nucleotides containing modifications in either the
base moiety or the ribose part occur in many nucleic acids. In
the case of tRNA, the proportion of modified nucleotides is much
higher than in the other polymers and a wide range of structural
variations has been established [1]. Many attempts have been
made to correlate their special molecular geometries with biochemical functions. Interest in these compounds stems from the
possibility that the monomers of ribonucleic and deoxyribonucleic acids may already exhibit some of the structural characteristics which exist in the natural occurring polynucleotides.
In particular, the influence of substituents in the heterocyclic
base on conformational parameters has been studied in great detail at the nucleoside level as well as in polynucleotides [2,3].
As a gross outcome it became clear that the effects of substitutions in the base moiety are not restricted to the respective pyrimidine or purine ring, but also exert strong effects
on the ribose and on the exocyclic group. However, a more sys-
© IRL Press Limited, Oxford, England.
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tematic approach is necessary to correlate the electronic properties of the substituents with their influence on the conformation of the nucleoside.
Egert et al. studied a series of 5-substituted uridines by
X-ray crystallography, pmr-spectroscopy, and quantum-chemical
methods revealing conformational changes as a function of the
electron-donor or -acceptor capability of the substituents [2].
Because of the packing forces among the molecules in the solid
state, the data obtained for the crystalline compounds cannot
be used without restrictions for nucleosides in solution. Therefore this type of information has to be supplemented by data
for nucleosides in the liquid phase. With the aid of a 250 MHz
spectrometer we could improve and enlarge nmr-data on x^Urd-type
nucleosides. The well resolved resonances of the ribose protons
allowed a detailed interpretation of the structural alterations
in the sugar moiety and the exocyclic group as induced by basemodifications. The selection of substituents was made according
to their electronic properties as reflected by their Hammett
constants, and not predominantly by biological significance.
MATERIALS AND METHODS
Uridine was purchased from Pharma Waldhof, DUsseldorf, and
was used without further purification. The preparation of the
5-substituted uridines followed known procedures [4,5]. They
were checked for homogeneity by thin layer chromatography and
paper chromatography in two different solvents each.
For pmr spectroscopy the samples were dissolved in 2.0 ml
5 mM Na 2 HPO 4 (pH 7.0) and lyophilized. Thereafter the residue
was evaporated twice from 500 til D_O (99.75% isotope purity)
and finally from 99.95% D O . Following this procedure the probe
was dissolved in 500 ul D 2 O (99.95%) and filled into an nmr
tube (d = 5 m m ) . The concentration of the nucleosides varied
from 10 to 15 mM.
The pmr spectra were recorded with the aid of a Bruker
250 MHz spectrometer in the FT mode. 300-500 interferograms
were accumulated with a digital resolution of 0.368 Hz/point.
The pulse angle used was 15°. The temperature was held constant
within ±1 degree. Chemical shifts are given in ppm downfield
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of the methyl resonance of 2,2, 3,3 -tetradeutero-3-(trimethylsilyl)-sodium-propionate.
RESULTS
Assignments of the nmr-signals. The general formula of the
modified nucleosides is presented in figure 1. The protons of
the base and the ribosyl moiety are numbered according to the
carbon atoms to which they are attached. The methylene protons
of the exocyclic group were labeled H_, and H.,_ and assigned
as proposed by Davies et al. [6] .
The hydrogen atom H, is located in the downfield region, shifting from about 7.3 to 9.7 ppm (figure 2 ) , depending on the
type of 5-substituent. The H^, doublet is centered at 5.9 ppm
and shows vicinal coupling with H-,. On both sides of the water
peak, a complex spectral region is found containing the multipletts of the 2' to 5' protons The identification of the individual signals could be achieved by their characteristic splitting patterns [7]. A computer program (LAOCOON II) was used as
an aid in this respect (figure 3 ) .
The conformation about the N-glycosllic bond. Many attempts
have been made to determine the conformation of the N-glycosilic
bond by different analytical methods. The numerous investigations include various nmr-techniques like J( C,H)-, NOE-, and
T--measurements, X-ray studies on crystalline compounds and
even theoretical approaches [2,8-10]. From the published data,
it can be derived that pyrimidine nucleosides substituted at
positions other than C^ and C g exist in predominantly anti-conformations. This is in accordance with the temperature dependence of the H, resonances obtained from the modified compounds
Figure 1
HO
General formula of the
uridine derivatives
x'Urd showing the numbering of the atoms and the
characteristic dihedral
angles -x = C6-N-|-C-| • —0-| • ,
ii)1 = 0 1 ,-C 4 ,-C 5 ,-O 5 , and
OH
^2
E C
3'- C 4'- C 5--°5[23].
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I.VJ..L
j
Figure 2
250 MHz pmr spectra of 5-aminouridine, uridine,
and 5-nitrouridine at 60°C
under investigation [10a]. Our data are also in aood agreement
with the linear correlation between I (J. ,., +J.,.,„) and
A6 (6H 5 , B ~6H 5 , c ) as published by Hruska and colleagues [11].
Obviously the substituents introduced into the 5-position of
the base moiety don't change the overall geometry of the nucleosides. Nevertheless there is some noticeable conformational difference between uridine itself and the 5-substituted derivatives,
accounting for the altered stacking behaviour and base pairing
patterns observed in modified oligo- or polynucleotides. Since
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V !
V
V 'i
Figure 3
The pmr spectrum at 250 MHz of 5-hydroxymethyluridine
in DjO (a). Computer simulated 250 MHz spectrum of
the ribose protons. LAOCOON II was used as computer
program (b).
the rules that govern these effects are still unknown, we tried
to find systematic correlations between the geometry of the
sugar portion and the type of substituent in the heterocyclic
base.
The ribose ring conformation. In solution the conformation of
the ribosyl moiety can be described as a rapidly equilibratina
mixture between the N-type (C^.-endo) and the S-type (C2,-endo)
rotational isomers. Whereas, the H
and K_, protons show a
nearly trans-diaxial (0 ^ 160
position in the C2,-endo ribosyl
conformation, they adopt an approximately diequatorial orientation (0 i> 90°) in the C,,-endo ribose (figure 4 ) .
Hence, a shift of the N ^ S equilibrium should become observable
by an increase or decrease of the J^,-, values [12}. Similar
correlations are valid for the H^, and H^, coupling constants.
Approximate methods have been developed to evaluate equilibrium compositions of the ribose ring from experimentally de-
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HOCH
HOCH
C ,-endo
conformation
(a)
(b)
Figure 4 Trans-diaxial and diequatorial orientation of H-, and
H 2 , in the C,,-endo (a) or C^.-endo (b) conformation
or the ribose
termined coupling constants with sufficient accuracy [8]. They
are based on the usual linear relationship between the time
average J and the J of each participating conformer:
N
obs
X- N J
S
X- S J
N
S
where X is the fraction of the N conformer and X represents
N
S
the fraction of the S isomer ( X + X = 1 ) . The data summarized
in table I were calculated from J-]!,! a n d J 3 ' 4 ' c o u P l i n i 3 s according to the method of [13].
Figure 5 shows the ribose conformation as a function of the
Hammett constants of the different 5-substituents.
From the graph, a close relation is found between the electron-affinity of *x an<3 the position of the H ^ S equilibrium.
Electron-attracting groups favour N-type conformations, electron-donating groups increase the amount of S-isomers.
Conformation of the exocyclic CH^OH-group. It is evident
from X-ray studies that in the crystalline state the infinite
number of possible conformations for the exocyclic group is restricted to a small range of rotational isomers.
Since similar limitations exist in solution as well, we attempted to identify the preferred conformation of the 5-substituted nucleosides by pmr spectroscopy. Details concerning the
population of the energetically favoured staggered conformers
by the exocyclic group (figure 6) are obtainable from an analysis
of the J, , J-B an<^ J4 1 cC coupling constants. Our data were derived
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Table I
Percentage of N-confonner for modified nucleosides
x^Urd as calculated from J
1 '2' and J 3 ' 4 'coupling
constants
no^Urd
3
30
60
1 .8
2.6
4.7
cn5Urd
3
30
60
2.5
2.6
3.6
fo Urd
3
30
60
2.6
2.6
3.3
br Urd
3
30
60
3.3
4.0
4.0
cl5Urd
3
30
60
3.7
4.0
4.0
hm5Urd
3
30
60
Urd
9.9
7.9
91
-1 .01
4.7
7.3
6.6
81
68
-0.63
-0.33
4.7
7.2
6.4
80
69
-0.60
-0.35
5.3
5.3
5.9
5.9
5.5
65
60
57
-0 .27
-0 . 18
-0 .12
5.1
5.5
5.9
6.0
5.5
62
61
57
-0.21
-0.19
-0.12
4.0
4.4
4.8
5. 1
5.5
5.5
5.5
57
54
51
-0.12
-0.07
-0.02
3
30
60
4.0
4.4
4.0
5.1
5.3
5.5
5.5
5.5
57
54
57
-0.12
-0.07
-0.12
mo 5 Urd
3
30
60
3.7
4.0
4.4
5.1
6.0
5.3
5.5
63
56
54
-0.23
-0.11
-0.07
m 5 Urd
3
30
60
4.8
4.8
5. 1
5. 1
5. 1
5. 1
5.5
5.4
5.5
51
50
49
-0.02
±0.00
+ 0.02
oh 5 Urd
3
30
60
4.8
5.1
4.8
5. 1
5.1
5.1
5.1
5.1
5.5
48
46
51
+ 0.03
+ 0.07
-0.02
nh^Urd
3
30
60
5.1
5.1
5. 1
5.5
5.5
4.8
4.8
4.8
44
44
44
+ 0.10
+0.10
+0.10
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50-
-0.2
Figure 5
0.2
0.4
0.8 [Op]
0,6
Population of the N-conformation in the ribose-moiety
as a function of the Hammett constants of the
5-substituents as 30°C
from the following equations
E JB =
4.5,c
P_
The observed coupling constants (a 4 ,„, B , J 4 I C I C ) are interpreted
in terms of values expected for each rotamer (J , J , J )
weighted according to the mole fractions (p+, p , p_) of each
isomer in the equilibrium. In accordance with previously published calculations the values of J used for
and
conformers were as follows [8]:
=
2.4
1. 3
=
2.6
J C = 10. 5
a
JC = 3.8
= 10.6
The assignment of the two methylene protons as originally suggested by R e m m and Shugar has been conformed by Davies and
Rabczenko [14,6]. The downfield signal, which usually shows a
smaller coupling to H.. is labeled Hc,_ (6HC, >6H.,_) whereas
H
D
hi
D
D
j
t,
H 5 , c is attributed to the upfield resonance.
In figure 7, the conformation of the exocyclic group is correlated with the Hammett constants of the substituents x. From
the diagram, it is obvious that the Y + rotamer is predominant in
every nucleoside examined. Furthermore, going from electron-donating to electron-withdrawing groups, there is a continous in1174
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(gauche-gaOcha)
(gaQcha-trans)
(trans- gaQcha)
Ya
Figure 6
Representation of the three classical staggered rotamers around the C. ,-Cr, bond of the exocyclic CH_OHgroup
crease in p . Measurements done at 60°C reveal considerable lower
values as compared to the 3°C series. This indicates that the
hindrance to free rotation around the C.,-C_,-bond due to the
potential barriers between the energetically favoured conformers
is markedly reduced at elevated temperatures.
DISCUSSION
The results obtained from this study demonstrate that each of
the two structural domains of the ribose moiety may be significantly influenced by the introduction of a substituent into the
heterocyclic base Altered electron distribution and geometric
properties induced in the pyrimidine ring are mediated to the
sugar portion and the exocyclic group by different mechanisms.
According to Egert et al. [2], alterations of the dihedral anale
•02
Figure 7
0B I opl
Correlation of the exocyclic group conformation with
the Hammett constants of the 5-substituents for
60°C (o) and 3°C (•)
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Table II
Nucleotide
Population of the rotational isomers around the
C^.-C,., bond of 5-substituted uridine derivatives
T
[°C]
J
4'5'B
[Hz]
J
4'5'C
[Hz]
P+
[%]
p
a
[%]
P_
log £
[%]
3
30
60
<1.0
2.4
2.6
<1 .0
2.8
3.3
116
84
77
1
16
21
_
0
2
-0.72
-0.52
3
30
60
2.4
2.4
2.8
2.8
3.7
4 .0
84
74
68
16
26
28
0
0
4
-0.72
-0.45
-0.33
fo Urd
3
30
60
2.5
2.6
2.9
3.0
3.7
4 .0
81
73
67
18
26
28
1
1
5
-0.63
-0.43
-0.31
br5Urd
3
30
60
2.6
2.9
2.9
3.3
3.7
4 .2
77
70
65
21
25
30
2
5
5
-0.52
-0.37
-0.27
cl5Urd
3
30
60
2.7
2.8
3. 1
3.5
4.0
4.2
74
68
63
23
28
29
3
4
8
-0.45
-0.33
-0.23
hm5Urd
3
30
60
2.6
2.9
3. 1
4 .0
4.0
4.4
69
67
61
29
28
32
2
5
7
-0.35
-0.31
-o. 19
Urd
3
30
60
2.6
3.1
3.1
4 .0
4.4
4.6
69
61
59
29
32
34
2
7
7
-0.35
-0.19
-0.16
mo 5 Urd
3
30
60
2.i
2.9
3.1
2.4
3.3
3.7
90
74
68
13
20
24
6
8
-0.45
-0.33
m 5 Urd
3
30
60
2.8
2.9
3. 1
4.0
4 .4
4.4
68
62
61
28
32
32
4
6
7
-0.33
-0.21
-0.19
oh5Urd
3
30
60
2.9
2.9
3. 1
4.2
4.4
4.6
65
62
59
30
32
34
5
6
7
-0.27
-0.21
-0.16
nh^Urd
3
30
60
2.2
3. 1
3.3
4.4
4.4
4.6
69
61
57
35
32
33
7
10
-0.19
-0.12
no^Urd
Z
C
en Urd
c
[p,
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X and the furanose N = S equilibrium are predominantly caused by
interactions between the C^-C, double bond and the ribose oxygen
0 1 . The considerable upfield shifts of the H & resonances in the
60° spectra reflect a change in the average rotational conformation of the base moiety about the glycosilic bond, indicating the
decreased deshielding effect of the CH_OH-group at elevated temperatures. Though there is no direct evidence from our data, the
orientation of the exocyclic group could be governed by a
C,-H
0., hydrogen bond. Depending on the type of substituent
in the 5-position of the base, both intramolecular interactions
may exhibit an increased or decreased bonding character as compared to uridine. Due to the different electron-affinities of
the donor and acceptor groups x, the electron distribution
within the Ti-system of the pyrimidine ring is altered. Since
electron-attracting groups strengthen the bonding interaction
between a lone pair at 0 1 and the TT* orbital of the C^-Cg double
bond, they should favour small dihedral angles x an d increased
populations of N-type conformers [2]. In addition, the electron
density at the Cg carbon atom is reduced which would result in
the stabilization of a C,-H
0., hydrogen bond and thereby increase the population of the gauche-gauche rotamer. Electrondonating groups are expected to exert an opposite influence. As
evident from the data presented in tables I and II, this has
been verified for the modified nucleosides. The nitro and amino
groups induce the most significant deviations from the molecular
geometry of uridine as would be expected from their Hanunett constants. In general, substituents with mesomeric effects exert a
stronger influence on the nucleoside structure as compared to
those that exhibit inductive forces exclusively. Thus, the conformational parameters of m^Urd and especially hm Urd show only
minor differences with regard to the unmodified compounds. The
temperature dependence of the data obtained from 3°, 30°, and
60°C measurements reflects the increasing compensation of the
intramolecular forces by molecular tumbling. The predominance
of N-type and p+-type- conformers observed in- the 3° spectra
is markedly reduced within the 60°C series. X-ray data obtained for 5-modified undines [2] reveal a number of unexpected conformations, though a general agreement with the pre-
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dieted geometrical properties could be established. Hence,
one must assume that in the crystalline state the strong
packing forces alter the conformational features of the
molecules as to be derived from the electron-affinities
of the various substitucnts. For the dissolved compounds,
the postulated graduation could be verified. The deviatinq D data obtained for 5-methoxyuridine must be explained by very
specific interactions that compete with the electronic effects
of the substituents as comprised by its individual Hammett
constant. Though a distinct mesomeric (+M) effect has been
established for the methoxy group based on experimental and
theoretical (CNDO/2) results, its electron-donating capability
seems to be reduced within the pyrimidine system [15,16]. This
may be due to a steric effect of the methyl grouo, forcing the
substituent to twist out of the base plane [2].
The efficiency of the different groups x
to pertubations
of electron distribution is sensitively monitored by the Hfi
hydrogen atoms since their chemical shifts closely parallel the
Hammett constants of the 5-substituents. Thus the H, resonance
5
of nh_Urd is located about 2.3 ppm upfield as compared to the
^
5
H- signal of no-Urd, whereas the 6 values for other protons remain nearly constant. Differences due to a modified deshielding
effect of the exocyclic groups are small and may be neglected in
this connection. Similar correlations have been described for a
number of related molecular systems [17,18]. The deviating upfield shift measured for the H, resonance of 5-cyanouridine is
caused by the strong diamagnetic anisotropy of the cyano group
located in the ortho position to the H, atom.
Since there is evidence that the conformational properties of
such base-modified nucleosides are maintained in larger nucleic
acid fragments, they should exert an influence on the structure
and dynamics of the corresponding polymers as well [19,20]. Uridine derivatives bearing electron-withdrawing 5-substituents are
expected to show an increased stacking tendency as compared to
those with electron-donating groups [21, 22] since intermolecular
stacking interactions invariably favour the N-conformation. In
addition, specific alterations in the geometry of base-base overlap may occur. Thus, 5-substituents are ideal candidates to mo1178
Nucleic Acids Research
dify~TThe dimerisation of nucleic acids though they do not directly interfere with the system of Watson-Crick hydrogen bonding.
Local changes in the conformation of one strand may impair the
complex formation with a second DNA or RNA.
ACKNOWLEDGEMENT
We are grateful to Mrs. E. R6nnfeldt for her help in preparing the manuscript and to Dr. S. Braun for his help in simulating the spectra. This work was supported by the Deutsche Forschungsgemeinschaft and Fond der Chemischen Industrie.
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