Volume 6 Number 3 March 1979
Nucleic A c i d s Research
A conformational study of nucleic acid phosphate ester bonds using phosphorus-31 nuclear
magnetic resonance'
Cornells 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 C6-N-(dimBthyl)adenylyl-C3',5')-uridina and some
nucleotide methyl esters) i s presented. The temperature dependent phosphorus31 chemical s h i f t s were analyzed by standard thBrmodynamic procedures. I t i s
shown that gt conformations about the P-0 ester bonds have a lower free energy
content r e l a t i v e to gg conformers.
INTRODUCTION
Phosphorus-31 n u c l e a r magnetic resonance (NMR) h a s been used
t o study a wide v a r i e t y of b i o l o g i c a l systems, such a s t h e s t r u c t u r e of n u c l e i c acid c o n s t i t u e n t s ' , polynucleic acids ' ' ' ,
7 8
9 10
11
drug-nucleic acid i n t e r a c t i o n s ' , tRNA's ' , DNA unwinding ,
12 13
and metabolism in intact tissue and cells '
Phosphorus generally occurs as a phosphate group in biological molecules. Unlike other spectroscopic methods,
P-NMR provides
direct information about phosphate residues, due to i t s high
sensitivity to chemical (solvent, pH, metal ions) and structural
(valence bond and torsion angles) environment. Especially the
l a t t e r 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 H- and
C-NMR techniques . The two remaining torsion angles u' and co
(nomenclature as proposed by Sundaralingam ) about the P-0 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
P chemical shift of
© Information Retrieval Limited 1 Falconberg Court London W1V5FG England
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Nucleic Acids Research
phosphate esters with the O-P-0 valency angle was established
both on empirical *
and theoretical grounds
. Furthermore
theoretical calculations, in agreement with experimental data,
showed this bond angle to be intimately related to the torsion
19 20
angles about the phosphodiester bonds * . On these grounds
P-NMR was proposed as a useful probe for the geometric arrangement about the phosphate group. From both the semi-empirical MO
calculations
and the experimental model studies '
it was in-
ferred 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
31
in nucleic acids 2,6 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-(dimethyDadenylyl-(3',5')-uridine (nuApU, Fig. 1 ) , was dictated by the
following considerations:
1. The thermodynamics of the stack-destack equilibrium were
recently determined 21 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 °C to 100 °C).
CH,
CH,
FigUTB 1 .
Structure of 6-N-(dimethyl)adBnylyl-(3',5')•
uridine, m_ApU.
(U)
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In this paper we studied the temperature dependence of the chemical shift of m 2 ApU and of the corresponding mononucleotides,
guided by independent information as mentioned above.
EXPERIMENTAL
All mononucleotides and dinucleotides were synthesized via
22 2 3
an improved phosphotriester approach ' ; 3',5'-cyclic adenosine
monophosphate (A>p) was obtained from Aldrich Co., Belgium.
Tetramethylphosphoniumbromide (TMPB) was prepared as described by
24
Maier . All nucleic acid constituents were treated with Dowex
cation-exchange resin (Na -form), lyophilized and dissolved in a
10~
M EDTA solution in 99.9% D.O. The NMR reference compound
(either trimethylphosphate (TMP) or TMPB or both) was added and
the pH adjusted by adding a concentrated solution of DC1 or NaOD.
The
P-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 D_0 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
31
P-NMR.
External 85% H,POU is a very popular reference standard in
ot
P-NMR, 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
25
. Especially in temperature dependent studies, where
shielding
the magnetic susceptibility difference between sample and reference is not constant, one has to take recourse to an internal
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standard.
In analogy to tetramethylammoniumchloride (TMA), which is a
well-known and satisfactory internal reference for nucleotide
proton-NMR
, we have selected TMPB as internal standard. TMPB
has the following characteristics:
a. Its resonance position is 22.9 ppm downfield of external 85%
H 3 PO 4 at 25 JC.
_
i r
The compound is water-soluble and the o31
P chemical shift is
independent of pH and nucleotide concentration.
£. The compound is magnetically isotropic; furthermore no hydrogen bonding is expected.
31
Figure 2 shows the temperature dependence of the
P chemical
shift of A>p and of another frequently used reference compound
TMP. In the temperature range from 0 •*• 100 °C the change in chemical shift of A>p is about 0.60 ppra. 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
-2A.75-I
Figure 2.
P chemical shifts vs. temperature
for reference compoundsi top: A>p,
pH°7.0, 0.02 fli bottom: TUP, phW.O,
0.02 n (Shifts relative to TnPB).
-24.50-
-2A.25fi •
IPPM)
-20.00-
-1975-
-iaso20
1138
40
60
80
TI°C)
100
Nucleic Acids Research
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-0 bond angle is anharraonic, 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 Krugh that the
addition of methanol to a dinucleoside diphosphate sample results
in a significant effect on the
P 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-0 ester bonds and/or differences in (solvatation) behaviour of phosphodiesters vs. phosphotriesters.
Whatever the origin of these shift, it is clear that analysis
31
P chemical shift of dinucleotides in terms of
of the changes in
rotations about the ID' and to torsions demands a correction for
this phenomenon. Therefore, in the remainder of this report the
31
P chemical shift data will be expressed relative to A>p at the
same temperature.
B.
31
P
Chemical shift of 6-N-(dimethyl)adenylyl-(3',5')-uridine.
The temperature profile of the
P chemical shift of m,ApU
2 6
(Fig.
1) is shown in Figure 3. Following Gorenstein et al. ' we
31
ascribe the downfield shift of the
P signal to the increased
amount of gt (tg) conformations about the P-0 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. a 210° implies t etc. Furthermore the shorthand gt describes the
blend of conformers g t, g t, tg and tg about the a' and u
bonds respectively. This applies as well to the gg notation (im-
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1.25-
6
I PPM)
1.00-
0.75-
0.50-
60
20
80
100
T(°C)
p
nA
Figure 3.
P chemical shift V8. temperature for m2ApU, phW.O,
0.02 (1 (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 n^ApU 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-0 ester bonds
IS 21 27
*
*
. The unstacked molecules exist
in a large number of states characterized by main degrees of
conformational freedom about the P-0 ester bonds (gg and gt).
The fully extended phosphodiester tt conformation has been argued
to be considerably less favored because of eclipsed interactions
14
between the lone pair electrons on the ester oxygen atoms . 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
accompanied by a shift of the equilibrium g g
~Z (ff*» gg) to the
righthand side. As the conforEational transitions are fast on the
NMR time-scale, the recorded
1140
P chemical shift will be a weighted
Nucleic Acids Research
time-average of the chemical shifts in the gg and gt conformations,
giving rise to a net downfield shift of the
P signal.
O u
Powell et al. have described the treatment of temperaturedependent CD.-spectra of dinucleotides in terms of a two-state
stack-unstack equilibrium:
a —a
In K - In —
= AS°/R - A H°/RT
a - ax
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 (<5 ) , {idem for
ag
all gt conformations S . ) and bearing in mind that the unstacked
9z
state (at the infinite high temperature limit) will be a 50-50
mixture of gg and gt conformations, the equation becomes:
i(<5
+6 .) - 6
&Si
In K = In
&
Si
**E = A S°/R - AH°/RT
5 exp - gg
S
That is, at any chosen temperature, the
P chemical shift is
described by:
S
+ }(<5
+ S t) exp(AH°/RT - AS°/R)
exp
1 + exp(AH°/RT - AS°/R)
An iterative least-squares procedure was devised to fit the unknown independent parameters S , S , AH°, AS° to the experiment31
&0
9t
c
al
P shift us. temperature profile of nuApU. The iteration
converged and resulted in AH° = -11.3 kJ/mole and AS° = -40.2 J/
/mole.deg, i.e. a "melting temperature" T (= AH°/AS°) of 286 K.
The corresponding thermodynamic parameters obtained from CD and
H-NMR , which are mutually consistent, are AH = -25.8 kJ/mole,
AS° = -84.1 J/mole.deg and Tm = 307 K. Comparison of the two sets
leads to an obvious conclusion: The 31 P chemical shifts cannot be
described by a simple two state equilibrium in this form.
In order to rule out the possibility of intermolecular ass31
ociation effects on the
P chemical shifts, the sample was diluted 10 times and measured at three temperatures: The recorded
shifts were identical within the experimental error (aa. 0.5 Hz).
In Figure 4 the
P chemical shift of nuApU is plotted ve.
the stacking percentage as calculated from the thermodynamic
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Nucleic Acids Research
Figure 4.
1.50-
P chemical shift ve. percentage of
stacking of m^ApU.
1.25-
e
(PPMI
1J0O-
0.75-
0.50-
10
20
30
to
50
50
70
% STACK
80
21
parameters derived from CD and proton-NMR
. The figure indicates
31
why the iteration of the
P shift data converges on the low
values for &H° 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
P chemical shift
and the percentage of stacking (as determined from CD and H-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:
( D 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
about the P-0 ester bonds.
Close inspection of the
P 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.
£. Estimation of the thermodynamio parameters in a three-state
equilibrium.
The experimental chemical shift (<5
) in a three-state equi-
librium is given by:
S
exp
=
x - - S- g g g g
+x.(5.+x
4
gt gt
gg gg
(x = mole fraction), or:
S
gt
^
where K
+
K
ggSgg
1 + K . + K
gt
gg
= exp(AH°/RT - AS°/R) denotes the equilibrium constant
of the equilibrium
Stacked (g~g~) Z Unstacked (.gt)
(K
is defined analogously).
The problem boils down to the determination of seven indepen-
dent parameters: Sg-g-, 6gt,
igg,
A H ° t , A S ° t , AH°^ and AS° g . Our
program ANALYS (written in Fortran IV) is used to fit these parameters to the experimental temperature profile of nuApU by means
of a standard Newton-Raphson procedure. In order to obtain meaningful results the number of parameters to be extracted from the
31
P shift data must be reduced, therefore the following assumptions are made:
\CL /
o
™
~~ —
(b)
&
o
QQ
Off
- 2.5 ppm (relative to A>p)
These estimates are based on the observation that the reported
ambient temperature
P-NMR spectrum of yeast tRNA
e
displays
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 aa. 0 ppm, relative weight
•v76 (all shifts recalculated relative to A>p using 4 ^ p
- *A>D
=
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4.7 ppm). The crystal structure of yeast tRNA
Phe
shows that most
of the phosphate groups have a. g g orientation about the u' and
29
u P-0 ester bonds . Only a few gt conformations are observed.
Therefore we assign the cluster of
P 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
P-NMR spectrum of the trinucleotide 4-N-(dimethyl)cytidylyl-(3',5')-4-N-(dimethyl)cytidylyl-(3',5')-6-N-(dimethyD4
4
6
adenosine (m,C-m9C-m,A) also renders support for our estimate of
4
6
6 t- It is deduced from proton-NMR that the -nuC-nuA part of this
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 S .
Two model calculations are now presented, each having one
extra constraint in addition to those mentioned above:
fft "
gg
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
H-NMR a AS°-value of -84.1 J/mole.deg
R
21
is found for the two-state stack-destack equilibrium of m-ApU
Splitting the two-state destacked state into two entities (i.e.
unstacked gt and gg conformers) reduces this AS° value by approximately R In 2 = 5.9 J/mole.deg: i.e. A S°
gt
= A S°
= -78.2 J/mole.deg.
gg
The
thermodynamic a n a l y s i s by means of program A N A L Y S {vide
was
carried o u t , y i e l d i n g & - - = & =
0.15 p p m , A H ° t
supra)
= -25.4
kJ/mole and A H ° = -27.0 k J / m o l e .
gg
11
K
' Ktuo-atate
(CD) = the
gt two
* K gequilibria
g
Constraining
to yield the same "averaged"
thermodynamic parameters as obtained from CD and
1144
H-NMR resulted
Nucleic Acids Research
in: S - - - S
- 0.15 ppm; AH° t = -25.8 kJ/mole; AH°
= -25.8 kJ/
/mole; AS° = -80.0 J/mole.deg; AS° = -75.8 J/mole.deg (Note:
o
o
AH . and AH
were refined independently). The difference in free
gt
9g
^o
energy content (AGr) of the two destacked blends of conformers is
now a consequence of the destacked gt conformers having a larger
entropy content with respect to the destacked gg conformers.
Valence force field calculations , 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^-value
chosen. Pilot calculations in which 5 . was varied from 2.0 to
3.0 ppm showed changes in AH° and AS 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
P shift
data are thus found to be contradictory to quantum mechanical
19 32 3 3 34
model calculations ' ' ' •> which predicted the enthalpy term
of gg conformers to be oa. 12 kJ/mole lower than gt conformers.
D.
P Chemical shifts of mononualeotideB.
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 (m 2 Ap0Me). Within the experimental temperature range the 5'-O-methylphosphates resonate
about 0.4 2 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
interraolecular associattion effects as nuApOMe and ApOMe display
the same chemical shift (within the experimental error), whilst
the self-associating tendency of 6-N-(dimethyl)adenosine is known
1145
Nucleic Acids Research
350-1
3.256
(PPM)
3.00-
2.75-
20
60
80
100
T(°C)
FigurG 5.
P chsmical shift i>a. temperature profile of nucleoside
methylphosphatea: circles - rieOpA, filled circles - MeOpU, squares - ApOMe,
triangles - m^ApOMe, croeeas - UpOMs.
to be larger than that of adenosine
. The same small differences
have been observed for the corresponding 3'- and 5'-monophosphate
nucleosides and were ascribed to a subtle influence of the ribose31
3
phosphate backbone upon the
P chemical shift .
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-0 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 ve.
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
31
2
a
P resonance at 1.9 5 ppm lower field than diethylphosphate ,
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 field . Thus, if an estimate
1146
Nucleic Acids Research
for the rotamer population about the P-0 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" (<5
= 0.15 ppm;
S
gt - 2 * 5 PP m ) The differences in chemical shift between DMP and the nucleoside 5'-O-methylphosphates at 25 °C is about 1.21 ppm; the
= 1 > 6 3 ppm
difference « D M p - 63,_methylphosphates
" 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 2 5 C
(roughly 60 % ) . From the thermodynamic parameters derived in
Section C {vide supra) for m-ApU an assembly consisting of about
60 % g~g~ (stacked state), 25 % gt and 15 % gg is found at 25 °C,
i.e. 1-60 % 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.
CONCLUSION
In summary,
31
P 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-0 ester bonds, gg and gt, have similar enthalpy contents, the
2
latter being slightly favored. In contrast to Gorenstein et at.
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
P-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 t 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
1147
Nucleic Acids Research
f o r Chemical Research (S.O.N.) with f i n a n c i a l a i d from t h e Netherl a n d s O r g a n i z a t i o n f o r t h e Advancement of Pure Research (Z.W.O.).
We wish t o thank Dr. J . H . van Boom and h i s group f o r generous
a d v i c e i n t h e f i e l d of s y n t h e s i s and p u r i f i c a t i o n . We a r e i n d e b t ed t o Mr. C. Erkelens f o r h i s t e c h n i c a l a s s i s t a n c e . The s t i m u l a t ing and e n l i g h t i n g d i s c u s s i o n s with Drs. A . J . H a r t e l , H.P.M. de
Leeuw and C.S.M. Olsthoorn a r e g r a t e f u l l y acknowledged.
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