Volume
6 Number 3 March 1979
Volume 6 Number 3 March 1979
Nucleic Acids Research
Nucleic Acids Research
A conformational study of nucleic acid phosphate ester bonds using phosphorus-31 nuclear
mgnetic resonanceI
Comelis A.G. Haasnoot and Cornelis Altona
Gorlaeus Laboratories, State University, P.O. Box 9502, 2300 RA Leiden, Netherlands
Received 20 November 1978
ABSTRACT
A systematic phosphorus-31 nuclear magnetic resonance study of some
nucleic acid constituents (6-N-(dimethyl)adenylyl-(3',5')-uridine and some
nucleotide methyl esters) is presented. The temperature dependent phosphorus31 chemical shifts were analyzed by standard thermodynamic procedures. It is
shown that gt conformations about the P-O ester bonds have a lower free energy
content relative to gg conformers.
INTRODUCTION
Phosphorus-31 nuclear magnetic resonance (NMR) has been used
to study a wide variety of biological systems, such as the structure of nucleic acid constituents 2'3 , polynucleic acids ' '. 5.' 6
drug-nucleic acid interactions7'8 tRNA's9' 10, DNA unwinding 11
and metabolism in intact tissue and cells12'13
Phosphorus generally occurs as a phosphate group in biological molecules. Unlike other spectroscopic methods, 31P-NMR provides
direct information about phosphate residues, due to its high
sensitivity to chemical (solvent, pH, metal ions) and structural
(valence bond and torsion angles) environment. Especially the
latter dependency is regarded as a promising tool in the study
of nucleic acid constituents. Of the six torsion angles that
define the sugar-phosphate backbone of nucleic acids 14, only four
(involving the ribose moiety) are amenable to analysis by IH- and
15
13
C-NMR techniques . The two remaining torsion angles w' and w
(nomenclature as proposed by Sundaralingam 16) about the P-O ester
bonds cannot be monitored directly via coupling constants; the
conformational behaviour of these bonds in solution must be inferred from indirect evidence.
Recently, a correlation between the 31P chemical shift of
C Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England
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phosphate esters with the O-P-O valency angle was established
both on empirical 3s' 1 7 and theoretical grounds 18 Furthermore
theoretical calculations, in agreement with experimental data,
showed this bond angle to be intimately related to the torsion
angles about the phosphodiester bonds 19 ',20 On these grounds
31P-NMR was proposed as a useful probe for the geometric arrangement about the phosphate group. From both the semi-empirical MO
calculations 8 and the experimental model studies 2'6 it was inferred that trans conformations about the phosphodiester bonds
would cause a substantial deshielding relative to the corresponding gauche conformations.
Up till now the above findings have been used in a qualitative manner to provide information on the helix-coil transition
in nucleic acids
A quantitative analysis of the
p shift vs.
temperature in nucleotides in terms of conformational behaviour
is called for. In our opinion such an analysis should first of
all be carried out on a model compound with well-known characteristics, determined by independent techniques.
Our choice of model compound for this study, 6-N-(dimethyl)adenylyl-(3t,5t)-uridine (m6ApU, Fig. 1), was dictated by the
following considerations:
1. The thermodynamics of the stack-destack equilibrium were
recently determined21 in this laboratory by two independent
physical methods (circular dichroism (CD) and proton NMR).
2. The compound shows a large shift of the stack-destack equilibrium over the accessible temperature range (78% + 15%
stack going from 0 0C to 100 °C).
.
.
CH311 1CH3
N6
HN
HA
)N9Ns
Figure 1
Structure of
OH
N3
H2
uridine,
m2
1136
0
°
H
P 2i
HS
H5<
0,H
H
(U)
0
0
HI~
H3
6-N-(dimethyl1)adenyy1-(3 ',5')-
m6ApU.
Nucleic Acids Research
In this paper we studied the temperature dependence of the chemical shift of
and of the corresponding mononucleotides,
guided by independent information as mentioned above.
m6ApU
EXPERIMENTAL
All mononucleotides and dinucleotides were synthesized via
an improved phosphotriester approach22'23; 3',5'-cyclic adenosine
monophosphate (A>p) was obtained from Aldrich Co., Belgium.
Tetramethylphosphoniumbromide (TMPB) was prepared as described by
24 All
Maier2.
nucleic acid constituents were treated with Dowex
cation-exchange resin (Na +-form), lyophilized and dissolved in a
M EDTA solution in 99.9% D20. The NMR reference compound
10
(either trimethylphosphate (TMP) or TMPB or both) was added and
the pH adjusted by adding a concentrated solution of DOl or NaOD.
The 31P-NMR spectra were recorded on a JEOL PFT-100 Fourier
Transform spectrometer operating at 40.48 MHz, interfaced with
a JEOL EC-6 (8k datapoints) or a JEOL EC-100 (16k datapoints)
computer for data accumulation. The spectrometer was field-frequency locked on the deuterium resonance of D20 used as solvent,
heteronuclear proton noise decoupling was used throughout. The
sample temperature was regulated with a JEOL VT-3C temperature
controller; the temperature was measured before and after each
experiment with a Pt-resistance thermometer (type 204, ThermoElectric, Leiden, Holland), differences were less than 0.2 C.
All reported shifts are denoted using the 6-convention,
i.e. positive values represent shifts to lower field.
RESULTS AND DISCUSSION
A. Choice of reference standard in 3 P-NMR.
External 85% H3PO4 is a very popular reference standard in
but the values of the line positions obtained by this
external reference technique will depend on the bulk susceptibility of the sample and will not be a true measure of intramolecular
shielding 25. Especially in temperature dependent studies, where
the magnetic susceptibility difference between sample and reference is not constant, one has to take recourse to an internal
31P-NMR,
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standard.
In analogy to tetramethylammoniumchloride (TMA), which is a
well-known and satisfactory internal reference for nucleotide
proton-NMR 26, we have selected TMPB as internal standard. TMPB
has the following characteristics:
a. Its resonance position is 22.9 ppm downfield of external 85%
H3P04 at 25 0C.
b. The compound is water-soluble and the 3lP chemical shift is
independent of pH and nucleotide concentration.
c. The compound is magnetically isotropic; furthermore no hydrogen bonding is expected.
Figure 2 shows the temperature dependence of the 3lP chemical
shift of A>p and of another frequently used reference compound
TMP. In the temperature range from 0 + 100 0C the change in chemical shift of A>p is about 0.60 ppm. This shift cannot be due to
changes in torsion angles about the P-0 ester bonds, as the phosphate group is incorporated into a "rigid" six-membered ring.
Neither can changes in the glycosidic base torsion angle X account
for this shift as 3',5'-cyclic uridine monophosphate (which has a
small magnetic anisotropy compared to the adenine base) displays
-24.75-
Figure 2.
3
P chemical shifts vs. temperature
for reference compounds; top: A>p,
pH=7.0, 0.02 M; bottom: TMP, pH=7.0,
0.02 M (Shifts relative to TMPB).
-24.50-
-24.25-
(PPM)
-20.00-x
-19.75X
TM-P
-115O
0
1138
20
40
60
80
T (C)
100
Nucleic Acids Research
3
virtually the same shift as A>p . Two possible explanations are
conceivable: Due to the increase in temperature higher vibrational
energy levels are populated. If the energy potential well for the
O-P-O bond angle is anharmonic, the effect will be a considerable
change in chemical shift of the phosphorus atom. More likely,
however, the shift is induced by changes in solvatation and/or
hydrogen bonding of the phosphate group. The latter mechanism is
supported by the observation of Reinhardt and Krugh8 that the
addition of methanol to a dinucleoside diphosphate sample results
in a significant effect on the 31p chemical shift, which cannot
be due to an unstacking process, being in upfield direction (vide
infra).
The temperature dependence of TMP apparently is due to the
same mechanism, for TMP displays a temperature profile very similar to that of A>p (Fig. 2). The small difference (< 0.07 ppm)
between the temperature profiles of the shifts of the two compounds
may be due to the extra degree of freedom in TMP, i.e. possible
rotations about the P-O ester bonds and/or differences in (solvatation) behaviour of phosphodiesters vs. phosphotriesters.
Whatever the origin of these shift, it is clear that analysis
of the changes in 3lP chemical shift of dinucleotides in terms of
rotations about the w' and w torsions demands a correction for
this phenomenon. Therefore, in the remainder of this report the
31 chemical shift data will be expressed relative to A>p at the
same temperature.
B.
31P Chemical shift of 6-N-(dimethyl)adenylyl-(3',5')-uridine.
The temperature profile of the 3lp chemical shift of m6ApU
(Fig. 1) is shown in Figure 3. Following Gorenstein et at. 22, we
ascribe the downfield shift of the 3lP signal to the increased
amount of gt (tg) conformations about the P-O ester bonds at
elevated temperature.
A side-step on nomenclature seems now appropriate: The
shorthand notations g (gauche) and t (trans) are operational
definitions, i.e. they include non-classical rotamers, e.g. w =
2100 implies t etc. Furthermore the shorthand gt describes the
blend of conformers g t, g t, tg and tg about the wt and w
bonds respectively. This applies as well to the gg notation (im1139
Nucleic Acids Research
1.25-
6
(PPM)
1.00
0.75-
0.50I
0
I
20
40
60
I
I
80
I
100
T (°C)
Figure 3.
P chemical shift vs. temperature for m2ApU, pH=7.0,
0.02 M (shifts relative to A>p).
plying g g , g g , g g and g g). When a particular conformer
is depicted, this will be indicated by using superscripts (vide
supra).
It has been shown unequivocally that m6ApU is engaged in an
equilibrium between destacked forms and a single righthanded stacked conformation, in which the latter must have g g orientations
about the P-O ester bonds1' 21'27. The unstacked molecules exist
in a large number of states characterized by main degrees of
conformational freedom about the P-O ester bonds (gg and gt).
The fully extended phosphodiester tt conformation has been argued
to be considerably less favored because of eclipsed interactions
between the lone pair electrons on the ester oxygen atoms14. In
addition, quantum mechanical calculations have shown that this
conformation is in fact a high energy conformation, therefore the
tt form is left out of further consideration. Thus the shift in
the stack-destack equilibrium on increasing temperature will be
(gt, gg) to the
accompanied by a shift of the equilibrium 9g g
righthand side. As the conformational transitions are fast on the
NMR time-scale, the recorded 31P chemical shift will be a weighted
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Nucleic Acids Research
time-average of the chemical shifts in the gg and gt conformations,
giving rise to a net downfield shift of the 31 signal.
Powell et al.28 have described the treatment of temperaturedependent C.D.-spectra of dinucleotides in terms of a two-state
stack-unstack equilibrium:
a
ln K = ln
u
-a
AS0/R
=
-a
a
-
AH0/RT
x
where a refers to the physical property monitored, the subscripts
u and x denote the unstacked state and stacked state respectively. Introducing the simplifying assumption that all gg conformers
give rise to almost identical chemical shifts (6Og ), (idem for
all gt conformations 6 gt ) and bearing in mind that the unstacked
state (at the infinite high temperature limit) will be a 50-50
mixture of gg and gt conformations, the equation becomes:
ln K = ln
AS /R - AH /RT
6
That
is,
at any chosen
exp
-6
gg
temperature, the
31P
chemical shift is
described by:
6
6
exp
+
gg
(6
+ 6
1 +
exp(AH
gt
) exp(AH0/RT /RT - AS /R)
AS0/R)
An iterative least-squares procedure was devised to fit the unknown independent parameters 6 gg' gt' AH0, AS° to the experimental
P shift vs. temperature profile of m2ApU. The iteration
converged and resulted in AH= -11.3 kJ/mole and AS0 = -40.2 JI
/mole.deg, i.e. a "melting temperature" T m (= AH0/AS0) of 286 K.
The corresponding thermodynamic parameters obtained from CD and
1H-NMR which are mutually consistent, are AH° = -25.8 kJ/mole,
AS0 = -84.1 J/mole.deg and T = 307 K. Comparison of the two sets
leads
to
n obviousm
31
P chemical shifts cannot be
leads to an obvious conclusion: The
described by a simple two state equilibrium in this form.
In order to rule out the possibility of intermolecular association effects on the 31P chemical shifts, the sample was diluted 10 times and measured at three temperatures: The recorded
shifts were identical within the experimental error (ca. 0.5 Hz).
In Figure 4 the 31P chemical shift of m2ApU is plotted vs.
the stacking percentage as calculated from the thermodynamic
21,
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Figure 4.
1.50
P chemical shift vs. percentage of
stacking of m6ApU.
1.256
(PPM)
1.0-
0.75-
0.5010
20
30
0o
go
60 70
% STACK
parameters derived from CD and
80
proton-NMR21.
The figure indicates
31P shift data converges on the low
values for AH and AS
Still working under the simplifying assumption that all gg conformations (including the stacked g g)
give rise to approximately the same chemical shift, the change
in chemical shift of the dinucleotide with increasing temperature
is solely due to the increasing amount of gt conformations. If
upon destacking the gt and gg conformations were equally populated
(i.e. equienergetic destacked gt and gg conformers) the plot
should reveal a linear dependence between the 31P chemical shift
and the percentage of stacking (as determined from CD and 1H-NMR).
However, as the plot shows an S-shaped curvature, it is concluded
that the two destacked states are not equienergetic. A qualitative
analysis of the data in terms of a three-state equilibrium is now
in order. The states envisaged are:
(1) a stacked conformer having a g g conformation,
(2) a blend of unstacked conformers having gt conformations,
(3) a blend of unstacked conformers having gg orientations
why the iteration of the
0
0
.
about the P-O ester bonds.
Close inspection of the 31P shift data (Fig.3) reveals that the
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displacement with temperature (i.e. the derivative of the curve)
is larger at lower than at higher temperatures. This implies that
upon destacking at lower temperatures the gt conformations are
populated to a larger extent than the gg conformations, in other
words: the destacked gt conformation has a lower free energy
content with respect to the destacked gg conformation.
C.
Estimation of the thermodynamic
equilibrium.
parameters in
The experimental chemical shift (
librium is given by:
xg
exp
(x
=
mole fraction),
6
e,V
=
+
g
xgt
gt
exp)
+
in
a
a
three-state
three-state equi-
xgg
or:
6--
+
g g
+
K
+ K
gt gt +gggg
+ K
I + K
gt
exp(AH0/RT
- AS /R) denotes the equilibrium constant
where Kgt
of the equilibrium
Stacked (g g ) ' Unstacked (gt)
(K9 is defined analogously).
The problem boils down to the determination of seven indepenOur
dent parameters: 8g~q~ 6gt' 6qgg AHt0 ,St, AH 0 and A S0
program ANALYS (written in Fortran IV) is used to fit these parameters to the experimental temperature profile of m26ApU by means
of a standard Newton-Raphson procedure. In order to obtain meaningful results the number of parameters to be extracted from the
31 shift data must be reduced, therefore the following assumptions are made:
(a) 6 -g- = 6
gg
gqg
(b) 6gt = 2.5 ppm (relative to A>p)
These estimates are based on the observation that the reported
ambient temperature 31P-NMR spectrum of yeast tRNAPhe 10displays
four signals: (1) at 3.7 ppm, relative weight 1, assigned to the
free terminal phosphate; (2) at 2.4 ppm, relative weight 1; (3)
at 2.1 ppm, relative weight 1; (4) at ca. 0 ppm, relative weight
6
"v76 (all shifts recalculated relative to A>p using 6TMP
A>p =
-
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4.7 ppm). The crystal structure of yeast tRNAPhe shows that most
of the phosphate groups have a g g orientation about the w' and
29
. Only a few gt conformations are observed.
w P-O ester bonds
Therefore we assign the cluster of
signals around 0 ppm to
the phosphate groups having a g g conformation and the signals
at 2.1 and 2.4 ppm to phosphate groups having a gt conformation.
This assignment also backs assumption (a) as the cluster (g g)
resonates at about the same frequency as A>p (g g ) does.
The 31P-NMR spectrum of the trinucleotide 4-N-(dimethyl)-
31P
cytidylyl-(3',5')-4-N-(dimethyl)cytidylyl-(3',5')-6-N-(dimethyl)-
(m2C-m4C-m6A)
adenosine
also renders support for our estimate of
2
2
2
6 part of this
It
is
deduced
from
proton-NMR
that the -m2C-m2A
6gt.qt
~~~~~~~~~30
trinucleotide is completely destacked . The phosphate group of
this part gives rise to a signal at 1.3 ppm. As this resonance
is almost temperature independent, it is concluded that the gt
and gg conformations in this particular case are about equienergetic and contribute almost equally to the observed shift. Thus an
upper limit of 2.6 ppm is found for 6gt.
Two model calculations are now presented, each having one
extra constraint in addition to those mentioned above:
I. AS0
AS0
~~~~~~4
gt
The entropy of the "unstacked state" consists of a solvatation and a mixing-of-states term. The forementioned constraint
thus implies the two blends of destacked states to have approximately the same solvatation entropy and the same number of destacked conformers. From CD and 1H-NMR aAS -value of -84.1 J/mole.deg
6
21
is found for the two-state stack-destack equilibrium of m2ApU
Splitting the two-state destacked state into two entities (i.e.
unstacked gt and gg conformers) reduces this AS0 value by approxim0
ASgq
-78.2 J/mole.deg.
ately R ln 2 = 5.9 J/mole.deg: i.e. AS 0qt
The thermodynamic analysis by means of program ANALYS (vide supra)
= 6
was carried out, yielding
= 0.15 ppm, AH0
= -25.4
kJ/mole and AH0 = -27.0 kJ/mole.
qq
II. K two-state (CD) =K gt + Kgg
-
6g-g-
Constraining the two equilibria to yield the same "averaged"
thermodynamic parameters as obtained from CD and 1H-NMR resulted
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-25.8 kJ/
= 0.15 ppm; AH t = -25.8 kJ/mole; AH0
in: g -g- = 6
999
gg (Note:
qg
0
/mole; AS0 = -80.0 J/mole.deg; AS 0 = -75.8 J/mole.deg
AHRt and AHR0 were refined independently). The difference in free
°) of the tWo destacked blends of conformers is
energy content AG
now a consequence of the destacked gt conformers having a larger
entropy content with respect to the destacked gg conformers.
Valence force field calculations 31, indicating three favored gt
conformers (tg , tg and g t) against two favored gg conformers
(g g and g g ) point to the same effect.
It should be stressed that the thermodynamic results presented above are to a surprising degree insensitive to the 6 gt-value
chosen. Pilot calculations in which 60t
g was varied from 2.0 to
3.0 ppm showed changes in AH0 and AS0 of less than four percent.
The question whether the difference in AG is caused by a
by a change in the enthalpy or in the entropy term cannot be settled at present. It seems reasonable to hold both terms responsible
for this phenomenon, the calculations presented above mark the
boundaries for the changes in each term. Be this as it may, both
calculations purport to show the free energy content of destacked
gt conformers to be lower indeed compared to the free energy
content of the destacked gg conformers. The experimental 3lP shift
data are thus found to be contradictory to quantum mechanical
model calculations19 3233,34 which predicted the enthalpy term
of gg conformers to be ca. 12 kJ/mole lower than gt conformers.
D.
31P ChemicaZ shifta of mononucZeotides.
Figure 5 shows the temperature dependent shifts of the nucleoside methylphosphates: uridine 5'-O-methylphosphate (MeOpU),
uridine 3'-O-methylphosphate (UpOMe), adenosine 5'-O-methylphosphate (MeOpA), adenosine 3'-O-methylphosphate (ApOMe) and 6-Ndimethyladenosine 3'-O-methylphosphate (m26ApOMe). Within the experimental temperature range the 5'-O-methylphosphates resonate
about 0.42 ppm to lower field than the 3'-O-methylphosphates.
Small, but consistent differences are noted between the adenosine
and uridine derivatives. The latter differences cannot be due to
intermolecular associattion effects as m2ApOMe and ApOMe display
the same chemical shift (within the experimental error), whilst
the self-associating tendency of 6-N-(dimethyl)adenosine is known
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Nucleic Acids Research
3.50-
3.25-
6
(PPM)
3.00-
,
2.75-
l
I
20
140
I
60
I
80
100
T(OC)
P chemical shift i's. temperature profile of nucleoside
methylphosphates: circles - MeOpA, filled circles - MeOpU, squares - ApOMe,
triangles - m6ApOMe, crosses - UpOMe.
Figure 5.
2
to be largerthan that of adenosine5. The same small differences
have been observed for the corresponding 3'- and 5'-monophosphate
nucleosides and were ascribed to a subtle influence of the ribosephosphate backbone upon the 3i P chemical shift 3
The large shift differences observed between the 3'- and 5'mononucleotide methylesters cannot be ascribed to differences in
conformational behaviour between the two types of residues. For
differences in rotamer distributions about the P-O ester bonds
(gg versus gt) in the two groups of compounds are expected to
show up as differences in the slopes of the chemical shift vs.
temperature profiles. This not being the case, the differences
between the chemical shifts of 3'- and 5'-esters must be due to
intrinsic differences in shielding related to the nature of the
ester groups attached. Such intrinsic differences have indeed
been observed. For instance dimethylphosphate (DMP) gives rise to
P resonance at 1.95 ppm lower field than diethylphosphate2,
a
another, even more subtle example is set by 3',5'-cyclic ribonucleotides and 3',5'-cyclic thymidine monophosphate, where the deoxy
compound resonates to 0.5 ppm higher field3. Thus, if an estimate
1146
Nucleic Acids Research
for the rotamer population about the P-O ester bonds is to be
made, a correction for the intrinsic deshielding of the methyl
group is needed, in order to relate the methyl esters to the
previously established "dinucleotide-scale" (6 g = 0.15 ppm;
= 2.5 ppm).
The differences in chemical shift between DMP and the nucleoside 5'-0-methylphosphates at 25 0C is about 1.21 ppm; the
difference
3'-methylphosphates 1.63 ppm. Using these
values as intrinsic deshielding corrections, subtraction of the
respective differences from the 3'0- and 5'-O-methylphosphate
nucleosides yields a "corrected" chemical shift of ca. 1.56 ppm
relative to A>p, thus indicating a substantial amount of gt conformers being present in the methylphosphate derivatives at 25 °C
(roughly 60 %). From the thermodynamic parameters derived in
Section C (vide supra) for m6ApU an assembly consisting of about
60 % g g (stacked state), 25 % gt and 15 % gg is found at 25 °C,
i.e. s60 % of the unstacked molecules pertain to gt conformers.
The close resemblance of the unstacked dinucleotide to the methylphosphates is indicative for the similarities in the behaviour of
phosphodiester bonds.
6gt
6DMP
CONCLUSION
In summary, 3lP NMR studies of nucleic acid constituents are
clearly capable to provide direct information about the phosphodiester geometry. It is shown that the conformations about the
P-O ester bonds, gg and gt, have similar enthalpy contents, the
latter being slightly favored. In contrast to Gorenstein et al.2
we conclude therefore that the major driving force for formation
of a single-stranded helical structure in dinucleotides remains
the base-base interaction. Moreover, it is clear that 31P-shift
vs. temperature profiles by themselves do not yield quantitative
information on the thermodynamics of the stack-destack equilibrium
in polynucleotides, because these shift effects monitor the equilibrium gg Z gt in which the relative contribution of g g (stacked) cannot be separated from the total gg blend.
ACKNOWLEDGEMENT
This research was supported by the Netherlands Foundation
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Nucleic Acids Research
for Chemical Research (S.O.N.) with financial aid from the Netherlands Organization for the Advancement of Pure Research (Z.W.O.).
We wish to thank Dr. J.H. van Boom and his group for generous
advice in the field of synthesis and purification. We are indebted to Mr. C. Erkelens for his technical assistance. The stimulating and enlighting discussions with Drs. A.J. Hartel, H.P.M. de
Leeuw and C.S.M. Olsthoorn are gratefully acknowledged.
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