Volume 12 Number 15 1984
Nucleic Acids Research
Proton-coupled "N NMR spectra of nentral and protonated ethenoadenodw and ethenocytidlne*
H.Sierzputowska-Gracz1, M.Wiewidrowski1, L.Kozerski 2+ and W.von Philipsborn3
'Inst. Bioorganic Chemistry, Polish Academy of Sciences, 61704 Poznan, Noskowskiego 12, Inst.
Organic Chemistry, Polish Academy of Sciences, 00-961 Warszawa, Kasprzaka 44, Poland, and
Inst. Organic Chemistry, Univ. ZOrich, Winterthurerstrasse 190, CH-8057 ZOrich, Switzerland
Received 4 June 1984; Accepted 6 July 1984
ABSTRACT
The
N ohemical shifts and
N, H spin coupling constant*
were determined in the title compounds us ins the INEPT pulse
sequence and assigned with the aid of selective proton decoupling. The 5 / 1 5 N / and J/N,H/ values are discussed in terms of
involvement of the imidazole ring created by ethenobridging in
the electronic structure of the whole molecule. Both spectral
parameters indicate that the diligant nitrogen in this ring is
the primary site of protonation in these modified nuoleosldes.
It is concluded that 1 5 N KMR of nucleoside bases can be
largely a complementary method to 1 H and 13c NMR studies and,
in addition, oan serve as a direct probe for studies of nitrogen environment in oligomerio fragments of nucleic acids even
at moderately strong magnetic fields due to the higher speotral
dispersion compared with 'H and '-'C NMR speotra.
1. INTRODUCTION
The application 1-4 of multiple pulse sequences suoh as
INEPT^"*' in FT NMR spectrosoopy effectively removes the drawbacks in obtaining
N NMR spectra. Because of low natural
abundance of the N nucleus and its low sensitivity and negative gyromagnetio ratio, only a few studies of nucleoaldes and
8—1 •?
nuoleotides were published so far ~ , despite the vital role
played by the nitrogen atoms in biological activity of these
compounds. Concurrently 1E and 13C NMR •pectroaoopy have proven
powerful methods in studies of structure and conformation of
nucleosldes and nuoleotides . On the other hand,
N opeotroscopy, characterized by high chemical shift dispersion and
Bensitivlty to intra- and inter-molecular environment ' ' of
the nitrogen nucleus, can serve as a direot probe to study
these phenomena in heteroaromatlc bases. This oan be a valuable tool in our approaoh to chemical, synthesis of the oligo© IRL Press Limited, Oxford, England.
6247
Nucleic Acids Research
merle fragments of tRNA and studies of the tRNA structure 1 ft
function relationship .
In the present work, we report complementary results from
-"N NMR spectroscopy to the recent Investigations of a comparative structural analysis of parent and etheno-modified
cytidine and i t s hydrochloride in the crystal by means of
19
20
1
n
X-ray analysis
, IR spectroscopy
, and by H and JC NMR
21
15
in solution . Furthermore, similar
N NMR results for ethenoadenosine and i t s hydrochloride are presented.
2. EXPERIMENTAL
The
5
N NMR spectra were measured at 20.28 MHz on a Varian
XL—200 NMR spectrometer using a broadband variable temperature
probe. Chemical shifts were determined against external
CH_
NO. contained in a coaxial capillary. The pulse sequenoe
INEPT with or without refoousing pulses was used to acquire
fully
H-coupled spectra. Typioal parameters were: 50 ms delay
time T , a spectral width of 4000 Hz, 1 s acquisition time,
and a pulse sequenoe delay of 1 a. Routinely 6-9 h were required to obtain suffioient S/N ratios for the spectra of
O.k M solutions in DMSO-dg.
H frequencies for selective de-
coupling were determined in the same solutions and decoupling
power % H» was typically 20 Hz to avoid irradiation of other
closely lying proton resonances.
3. RESULTS AND DISCUSSION
3.1. Assignments of ohemioal shifts and coupling constants
Due to significant changeo introduced in purine and pyrimidine bases by ethenobridging also
N chemical shifts ohange
dramatically and, hence, should be assigned using unambiguous
arguments. These primarily stem from protan-ooupled speotra.
In Table 1 N,H spin coupling constants are presented for the
title compound* 2 and 2 a n d f o r adenosine /\J. In Table 2
chemical shifts for modified nucleosidea in neutral and protonated foraa are given.
Tn* assignment* of chamioal shifts are based on the magnitude
of resolvable ooupling observed, signal disappearance upon re—
6248
Nucleic Acids Research
tor
'ln i11 A I ^ ^ ^ I U J M
-WO
L ^ ^
-1VO.S
'l
«/"•*' •*• i
"^*r*n&
Figure 1i
In Figure 1, typical proton-coupled ^ N INEPT spectra are
illustrated which were used for spectral assignments.
15
N NMR INEPT opectra of ethenoadenooine /0.4 M in UMSO-dg/
referenced against CH-^HOj, in an external oapillary.
a/ Refooused spectrum of etnenoadenosine hydroohloride /2a/
irradiated at toe H-11 frequenoy in the proton spectrum.
Experimental conditions: 4000 Hz spectral width, 50 ma delay
tine, 0.8 s pulse sequenoe delay time, 1 s aoquialtion time,
13240 transients. The inset speotra show expansions for the
H-1 and N-9 resonanoaa. The N-1 resonance /upper inset/
spectrum shows the nonrefocused signal taken fron th# fully
ooupled speotrum, separation of outer lines 16.4 Hz. Tna
lower inset shows the expanded refooused signal at -182.0
ppm, separation of the outer lines 11.7 Hz. For the H-9
resonanoe the Inset Illustrates a refooused fully ooupled
signal and the nonrefocused signal after Irradiation of the
H-C/1'/ frequenoy, In upper and lower traoes respectively.
b/ Konrefooused fully ooupled speotrua of etnenoadenosine /Z/
after neutralization of the hydroohloride In DMSO-d^.
Experimental conditions as above /23,37O transients/. The
Inset shows expansion of the N-1 signal, separation of outer
lines 16.2 Ha, and of the N-9 signal.
6249
Nucleic Acids Research
Table 1 .
l
*H,H spin couplLng constant* in nucleosidea
'J
1
/1~^\
2
i"
bl
cl
.)
b)
C
88.2
90.1
15.8
14.0
12.4
(N1-H2I
17.1
15.5
14.8
(N3-H2I
11.0
10.5
11.8
(N7-H8I
8.4
IS9-II8I
d
* -X 3 'J
ibo.e
W
//
VI
J 3 )'
<
cl
0)
" 2>
fr^C'
'J
'"-3
Riboie
y
(aba. value*, - 0.2 H i ) .
'J
3el
7.0
(7.7)
(n-::2,
3.9
(3.9)
13.5
(13.0)
(N1-H11I
IN3-H2I
10.9
(7.7)
IN6-H10)
12.5
(12.7)
(N7-H8)
6.9
(7.7|
IN9-H8I
11.61
(N9-H1'1
6.2
16.0)
(N1-H6I
3.3
(3.31
12.9
(8.7|
(N3-H8I
(N4-H7I
3.2
5.3
(N1-NH2I
(4 . 3 ]
(S1-H10)
11 .61
(N9-H21)
4 . 5 f (4 . 5 1 ' (N3-H5)
(I13-H7)
3.3 f (3 .31
^N^^ 0
Rlboae
Valuei taken froo Ref. 14, 1 M solution in DMSO.
Valuei for 3'-MP taken
from Ref. 12, 0.08 M solution in water, p H " 7 .
Preaent work, 0.4 H aolution
in DMSO-d,.
0
For experimental conditions see notes d) and e) in Table 2. Values In parentheses refer to
protonated etheno*denosine (,2*) •
For experimental conditions see notes d) and e) In Table 2. Values in parentheses refer to
ethenocytidlne hydrochloride (3±).
Assignments may be interchanged.
^ Assignment confiraod by selective decoupling of H-5.
" • cb«siical shifts
protooatioo shifts.
EUiaaoadanoalD*
Protxoatloo «hlft c l
of athaooadtoosin* (21 and •th«nocytidln« (3) and th« oorraspoodloff
(2)
8-1
N-3
N-.
11-7
•-9
-178.5
-3.5
-152.2
• 4.6
-150.1
-40.4
-139. 1
-0. I
-207.3
• 2.0
K-3
dl
Eth»Bocytiain«
I3>
ProtOftatlon «hl(t*'
-III.I
-0.9
H-4
N-l
-1*4.0
-41.1
-232.0
i (pfa) with rtspvet to utarnal CBJIO2 in a capillary.
0.4 H solution of •tlwnoadtotMiM hydrochlorid* in DKSO-d, after Mutralliatloa by an «qnlvalcnt
oraoat of powdared K3CO3 " ^ flltarlnq.
0.4 K solatloo of th* hydrocfalorlda In DKfO-d(.
0.3 I solatloB of •tbsnocytldln* hydrochloride in DRfO-d( after aeotralltatlon by an equivalent
—ouot of powdered *2CO3 and filtering.
0.3 H solatloo of the hydrochlorld* In DMSO-dc.
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Nucleic Acids Research
aoval of the N,H coupling used in building up the INEPT signal
for a given nitrogen, and obemloal shift comparison with the
parent micleoaide. Sinoe ooupling oonstants through three bonds
oan be observed, signals are easily assigned by their oharaoteristio coupling pattern. Vhile ethenocytidine poses no problems the ambiguity arises in the oase of N-3 and N-7 atoms in
ethenoadenosine which are coupled to only one proton with much
the same coupling constants, see Table 1 and Fig. 1. The similarity of ooupling oonstants does not allow for unequivocal
assignments on this basis to either pyrimldlne or imidazole
I't 22
rings
' . Hence, the assignment of N-7 to the signal at
highest frequency is substantiated by oomparison with the 1-*N
KMR spectrum of parent adenosine J_ where this nitrogen, unambiguously assigned by deuteration of C/8/-H, absorbs at nearly
Ik
the same frequency .
Assignments of coupling oonstants are confirmed in ambiguous cases /N-1 in ethenoadenosine and N-3 in ethenocytidine/
by seleotlve decoupling of one of the three protons Involved
in coupling. The fully coupled spectrum /compare upper Inset
for the N-1 signal In Pig. la/ gives a oomplioated pattern for
whloh the sum of the Involved couplings can be evaluated from
the separation of the outer lines. It is worth noting that the
shape of the signal, the signal-to-noise ratio obtained, and
relative line Intensities depend drastioally upon the assumed
J values introduced into the multipulse INEPT sequence program
and, henoe, a good S/N ratio is required to deteot all lines
of an unknown signal pattern.
In the case of ethenoadenosine Irradiation one of the resonanoe frequencies of the protons Involved in coupling simplifies the N-1 signal pattern to a pair of doublets /refocused and nonrefooused, compare lower Inset In Pig. la/ so
that one coupling constant oan be evaluated and the sum of the
remaining two.
Using this teohnlque it was possible to assign three coupling constants, visible in the fine structure of the N-1 signal
of ethenoadenosine hydroohlorlde, by selective irradiation of
the H-10 and H-11 proton frequencies /Table 1/. Irradiation of
H-10 simultaneously gives an unambiguous assignment of the N-6
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Nucleic Acids Research
resonance by observing its reduced intensity due to removal of
J/N-6,H-1O/ coupling responsible for enhancement of the INEPT
signal in this oase. In ethanoadenoslne this experiment allows
for distinction between closely lying signals of the N-3 and
N-6 atoms.
In ethenooytidine, either neutral or protonated, the signal
of N-3 is a nonrefocused doublet /J = k.5 H z / of triplets
/J s 3.3 Hz/. This simplifies the assignments sinoe irradiation
of H-5 in the bydrochloride allows an assignment of the larger
coupling to this proton /Table 1/.
3.2. Chemioal shifts
The current rationale for an interpretation of
N ohemi21•"2h is based on a
cal shifts in heteroaromatic compounds 8 '9* <
general consensus that the dominant role in nitrogen screening
is played by the paramagnetic term expressed by equation:
i
where the
I.Q. term represents the distribution of the 2p
electrons in the ring system,
energy and
<r
> .
AE
an average excitation
the expectation value of the reciprocal
cube of the 2p orbital radius.
The interpretation of terms in the above equation Justifies8'9'23'
2k
et al.
' 2 5 the linear relationship found by Vitanovski
between nitroGen chemical shift and ir-electron den-
sity q "7J at the nitrogen atom in heterocyclic compounds which
shows a deorease of <3
with increasing TT-olectron density
.
In this respect it is useful to discuss the nitrogen shifts
in heterocyclio bases in terms of two distinct types of nitrogen valence states referred to as "pyridine like" /or diligant/
and "pyrrole like" /or triligant/ nitrogen atoms
. The latter
nucleus provides two electrons to the delocalized TT-orbital
system whereas the former only one. For this reason, q N values
from MO calculations are higher for the "pyrrole like" nucleus,
and its resonance is displaoed to much lower frequencies than
that of the "pyridine like" nucleus. In the latter, the lone
pair is not involved In the T-orbite.1 system and is, therefore,
accessible for oomplexation suoh as protonation. The protona6252
Nucleic Acids Research
tlon of "pyxidine like" nuclei can formally be considered as
a change to the "pyrrole like" valence atate, i.e. the nucleus
beoomes triligant. Such a change oauses an Increase in "JT—electron density at the protonated atom and consequently a shift
to lover frequencies. On the other hand, the possibility of
delocalization of an increased T-electron density may partially
counterbalance low-frequenoy shifts induced by protonation.
1-Methylimidazole k_ contains both types of nitrogen valence states discussed above and, therefore, its chemical
Oil
5 f\ 9ft
26 28
shifts
as
Sche
shifts '
' ~~
as shown
shown in
in the
the Scheme
can serve as reference
values for the nuoleosides studied:
CH3
-217-7 /ilgO/8
N/1/
-210.3 / H 2 0 /
-219.2 /DMSO/ 2
-221.7 /CDCI3/ 2 8
-13^.7 /fr-jO/6
K/3/
-119.1
/DMSO/
-209.8 / H 2 0 /
28
-12l*.1 /CDCI3/ 2 8
Scheme
Noteworthy is a fact that the average ohamical shift for 1methyl imidazole in water agrees with that found in imidazole
o
/-177 PPm / undergoing tautomerization. The protonation of N-3
shifts this characteristic value of the system to ca. -210 ppc
iiidicatinG that both nitrogens adopt nearly equally "pyrrole
like" valence states. Interestingly, formal protonation shifts
in water are 7«^ and -75.1 ppm for N-1 and N-3, respectively
/Scheme above/ whereas the protonation shift is negative for
N-1 and much smaller for N-3 in imidazoles undergoing tautomerizatlon 2 5 ' 2 7 ' 2 8 .
The possibility of simultaneous modification of purine and
pyrlmidine bases in oligomeric nuoleic acids poses the problem
of a differentiation between ethenocytidine and ethenoadenosine
6253
Nucleic Acids Research
moieties. This oan be judged from two regions In the
-'n NMR
spectrum. In the spectral region of the glyoosidlc nitrogens
this atom absorbs at distinctly lower frequencies in ethenooytldine than in ethenoadenosine /-2k ppo/. Even more specific
ohangea oan be figured out by comparison of the ohemioal shifts
of the two nitrogens in the imidazole rings created In both
nuoleosides. In ethenocytidine both of them absorb at much
lower frequency than corresponding atoms in ethenoadenosine
/Table 2 and Pig. 1/ and fall Into the spectral range free
from other resonances, i.e. -160 ppm to -190 ppm.
Hie individual
N chemioal shifts in both bases can also
be discussed in terms of a comparison of the imidazole rings
topologioally similar. Thus in the Imidazole ring present in
purine, Identified by the N-7 and N-9 atoms, the chemical
shifts are very close to those in the Isolated model imidazole
jt given in the Soheme above. On the other hand, In the imidazole ring formed by ethenobridglng /i.e. in 2/, the spread in
chemioal shifts between the two nitrogen atoms is only half
of that in the purine imidazole moiety, and distinct deahielding of N-1 vs. the formally similar N-9 is observed. These
observations may indicate closer similarity of the valence
states of these nitrogens than found in isolated imidazole
and atronger conjugation of this ring
with the purine moiety
than in purine itself. A similar situation is found in ethenocytidine / 2 / where the
N chemioal shifts in the Imidazole
ring are spread as much as in the corresponding imidazole ring
of •thenoadenosine, the resonances, however, being shifted by
12—14 ppm to lower frequency.
Protonation of both nuoleosides results in similar spectral
effects despite the structural differences between both systems. Thus, diligent nitrogens, N-6 in Z_ and N-*» in ^, are
shifted to lower frequenoy by about —h\ ppm whereas their trili&ant partners in the Imidazole ring show only small low-frequency effeots of -3.5 BtxuX -0.9 ppm in £ and %., respectively. The topologioally related N-3 and N-1 atoms show similar
deshieldlng effeots of +4.6 and +5-9 Ppm in £ and %.•
Tbe
ave-
rage resonance positions In imidazolium units are shifted to
higher frequency compared with the 1-aethyllmldazole model kg.
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Nucleic Acids Research
by 2k and 11 ppm In Z_ and 2.' respectively.
These results permit a rationalization regarding the site
of protonation and the •B"—electron distribution In the protonated molecule. The primary site of protonation In both oasea
Is the dlligant nitrogen In the Imldazole ring formed by ethenobridglng. However, the protonation shift in DMSO /-41 ppm/
is in both oases only about half of that found In the model
1-sethyllmidazole in H.O. The proton is In fast exchange as
confirmed by broadening of the N-6 signal /Fig. la/ and absenoe
of
J/X,H/ for this nitrogen in the INEPT speotrum. Although
these informations oould, In prinoiple, Indicate intermolecular
proton exchange or intramolecular tautomorization of a proton,
1 9—21
they also support the idea of charge transfer *
from the
lmldazolium moiety to the pyrimidine rings. This can be judged
from a high-frequency shift of the average lmidazolivm nitrogen
resonances, and a participation of N-3 in Z_ and N-1 in ^ In
stabilizing the lmidazolium oatlons is also evident from their
high-frequency shifts upon protonation of the respective nitrogen atoms N-6 and N-'t.
3.3. Coupling constants
N,H Coupling constants for oompounde 2 and 2.
and for
the
parent adenoslne /\J are collected in Table 1. A comparison
between parent adenosine and the modified nucleosides reveals
interesting trends. In adenosine, nitrogen spin coupling to
the H-2 proton in the pyrimldine ring ie larger than to the
H-8 proton In an imidazole ring -*•
. I n ethenoadenosine / 2 /
the coupling of N-1 with H-2 is reduced by about kCffj apparently
due to a change in hybridization of the nitrogen atom. Inter T
action of N-7 and N-9 with H-8 in the imidazole part are of the
same order of magnitude as in adenosine. N,H spin interactions
in the imidazole ring formed by etheno-bridging are quite different as compared with other inidazole rings. Thus, N-1 in
ethenoadenosine and K-3 in othenocytidine, fortally related to
K-9, reveal only weak ooupline of 3.9 and 3.3 Hz to 11-11 and
H-8 in ethenoadenosine and ethenocytidino, respectively, as
compared with S.k and 6.9 Hz fo^ N-9 in the purine moieties of
1 and 2_. Geminal coupling of the structurally related N-6 and
N-^* atoms reveal much stronger coupling through formal single
6265
Nucleic Acids Research
bonds /10.9 and 12.9 Hz, respectively/. These Interactions, of
the same order of magnitude as found for corresponding two-bond
N,H interactions, e.g. in thiazole /-10.6 H z / 2 8 ' 2 9 , may indicate enhanced double-bond character of the N-^,C-7 /and N-6,
C—10/ linkages due to conjugation of three double bonds as
1 9—21
suggested recently
. This idea is further supported by the
30^b drop in magnitude of these oouplings upon protonation of
the diligont nitrogen atom resulting in an inhibition of conjugation and effective isolation of the carbon-carbon double
bond in the imidazole moiety. Sinoe the above phenomenon is
the most significant change revealed upon protonation of the
two modified nuoleosides it can be of diagnostic value in recognizing the site of protonation. The observations discussed
above confirm oonolusions deduced from chemioal shift interpretation regarding the differences between both imidazole rings,
their distinctly different involvement in the electronic structure of the whole molecule, the site of protonation and proton
exchange.
In summary, the reported
N NMR spectra of modified nucleo-
sldes allow the conclusion that this technique gives results
1
11
complementary to the observations deduced from E or
C NMR
21
speotra as described recently . In addition, direct insight
into the structural environment of nitrogen nuclei in nucleoaide bases may be obtained. A distinctly larger spectral dispersion, even at moderately strong magnetic fields, in the
region of diligant and triligant nitrogen atoms as oompared
11
with the
C spectral range of double bonds, as well as large
protonation shifts seem to indicate useful applications in
studies of oligomeric fragments of nucleic acids. Further
experiments in this direotion are in progress.
ACKNOWLEDGEMENTS
The authors acknowledge the reoeipt of a scholarship to L.K.
from Varlan AG Zug during this research and finanoial support
from the Schweizerlsoher Nationalfonds zur Forderung der vissenschaftlicben Forschung and the Polish Academy of Sciences
/project MR-I 12/.
6256
Nucleic Acids Research
+
On leave to the Institute of Organic Chemistry, University of Zarich, 1981 -1982
•"N NMR Spectroscopy Part XII; Part XI see Ref. 1.
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Nucleic Acids Research
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