Rotational correlation times of adenosine 5′-monophosphate in solution as deduced from 1H and 13C spin-lattice relaxation times

Vohimc’32. ..’

..

CHEMkAL

number 1 ‘.

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; .. . .

: QXR.ELATION

TFS

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OF.AD,ENOSLNE

AND 13C SPIN-EAti&

Chemkrry;Facdty

Received 25 November

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IMOTO, K&uyuki AK$%KA ..-

Departxen?,of

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AS DEDUCED-F>OM ‘; Toshiaki

:l April 1975

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&OT&TlONAL

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PHYSICS(LETTERS

,:

of Science,

5’-kNOPHOkPl&TE

RELAXATION

IN kOLUTrO&

TIMti

and Hiroyuki HATAN Kyoto

University, Kyoto

606,..Iapan

1974

..

Rotational correlation times (~~1of the 5’.AMPmolecule deduced from spin-lattice relaxation times (Ti) of different

protonsin

the molecule agree fairly WPU with each other in the tempernture range of 3.5-74°C.

values deduced from 13C T1 value+. tation of the 5’.A;MP molecule. . : _’

,..

These

“l-he same is ttie with or a~e,slow as compxcd to the overall IO-

r&Its indicate that the internal mctiws

.’Study.. of local m&ular motion in solution hae become fe2sibie using nucltiai spin relaxation time r,leas-

The purpose of the present report is to show how accurately one can elucidate molecular rotation in solution from .proton T1 measurements with-the FT method and to prove the above-mentioned hypothesis about the “rigidity” of a nucleoside or a nucleotide . in solutjon. For this purpose, adenosine S’-monophosphate (5’eAMP) has been chosen, and ‘H and 13C ’

obtained ,from the pulse Fourier-transform (F’T) NhlR,tec_h&que [l] , Spin-lattice relaxation times (?Q bf_the ,1%-Jnucleus of a C-H bond is particularly uJeful for this purpose, because it is prddon&ntIy deter$ned bi the dipolar interaction with the directly b;onbed proton [1] _ On the other hand, proton T1 s&n--lattice reJaxation~.times have been measured at -’ measut_ements have,not been used much for a quantita100 and 25.15 ME&, respectively, at various temperative’elu,cidation of molec’ular m&on in solution: This tures, and the rates of molecular rdtation i&different is largely due to the fact that the ielaxation of a pro.. pai-ts of the molecule deduced from both measureion in a molecule is often affecte_d by several neighborments are compared. ing protoni throughdipolar interactions that depend Fig. 1 shoti; the experimental r&ults for the temcrj$c,ally 0i-1the.inteiproton’djstarices which, usually, perature depende&of pr$o~‘~l’values of.a 0.5 M ._ ‘, ,’are utinqw~ qutitities. D20 solution of 5’-@!P (pH 8.1 j in thi temperature : .range c&3.5-.74”C. It is important to notice in fig. 1 rt ii apparant to us, however, particulariy in dealing ~~witi,biologic~_substances; thit it is necessary io dethat fhe T1 oi;;ll !he pToto& except H(2)show min.’ ..’ iqa and that th’ey occur at a same temperature, i:e., . rive‘iniormation conce$ing the molecular motion from $rofon‘ T1 @ties, -by .t*n’g advantage‘of ‘the.$igh 6-7’C. The unique behavior @‘Tl of H(Z), analogous urements

,q&itivity

of the proton resonkce.

Furthermore,

we

havi recently shown-‘that@ton Tl_measurements,

to tlie case of +oly(A)

[4], pr,ob.ably

indicate;‘that

it -.

is determ&d t y inte~moler;uler interaction rather

e+ & E? method, combined-with selective deuteri; II& Gbstiiutioti, ~,a? be used as.8 me’ans,& elucidate

.than the intramolecular dip?Iar jnteractior, which:is .. expected td be &mallti &is.ca+ On the’cther hand,. *i&e-interproton djstinces i? solution 131. This ,Tl:of the’other,profons should b&determined prkmeth,od:is ,based on the hypo’thes’is that the-molecule in ~ominafitly.by.iii&nolec@r &poiar_inieraction. rate. ‘,’ ‘:;c#i&ti& is :uffic$?tlc “rigidy’~‘$‘&a’~&e ro$a$or&l : ‘- In cases.whpie.the.sjjinl!attice.relaxation’ 11coiiolatipn .tinie bf any proton-protbn vector cln be,. (l/,Tl) of a ijrqton,is $$ei@Jned ijy.c!ipbl& iritera,c: rep&&ted j&as weli‘py tha’i of any C-H bond ob- ,. ,’ tiog, the fo!loning &xpr$ion is st&tly,v&d for an’ .’ i~.&i& frOti._l,?C Ti, m~asure’ &nts.-’ -I:. . _; isolated p&&f pi&ons that are: sep&t6d.l$ a &ad ., ‘. .I .,:

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Volume

32, number

1 ApriI 1975

CHEMICAL PHYSICS LETTERS

1

Table

1

Carbon-13 spin-lattice relaxation cYbons of 5’-AMI (0.5 M inDz0,

times (Tt) of theC-H pH 8-L) at 6.5’C and TV

values deduced therefrom Carbon -.---_..

2 8 1’ 2’ 3’ 4’ 5’

:,(Io-~ 1 (s) =) _- ._ ._...._.-----

0.080

0.8

0.079 0.078 0.078 0.079 0.083 0.044

0.8 0.9 0.9 0.9 0.8 0.7

s) br

3) The T! values include experimental 2.8

3.0

3.2

3.4

3.6

IO’/T(OKl’ Fig. 1. Temperature dependence of spin-lattice relaxation times (Tr) of non-exchangable protons in 5’-AMP (0.5 M in DzO, pH 8.1).Tr’s were measured with a JEbL PS-100 NMR spectrometer equipped with a Fourier tansform unit (P&CT100) and a spectrum computer(JEC-61, by applying 180”T-90” pulse sequences. About 10 mM of EDTA was added to the sample solution to eliminate the effect of contaminant paramagnctic metal ions on the relaxation times.

distance r and are undergoing

isotropic

a characteristic

rr,

-=‘r,1

time

I 1Or6 374HhG

constant

1

‘+.4

G + cd&,2

1 + 4w$,2

rotation

with

1 3

(1)

where rH and WH are the gyromagnetic ratio and Larmor angular frequency of a proton, respectively. Eq. (1) predicts, therefore, that at Tl (minimum) we will have U;T, = 0.62 (or 7, = 1 .O X 10mg s at 100 MHz). Eq. (,l) may also be applied to ,analyze relaxation data in a multi-proton system of a “rigid” molecule simply by replacing the right hand side with a .summation

of the.contributions

from

all neighboring

prcrtons, !s long as we$llow for errors due to deviations of the relwation curves from single exponential functions of time in a multi-proton system [S] . The fact .that T, (minimum) occurs at the same temperature for all the protons [except H(2)] of SI-AMP shows that the molecule takes one or more fmed.conformations in which a ve,ctor formed by any pair’of protons is tindergoing rotation at a,.rate nearly. equal to the rotation of the wh.ole molecule, i.e., = 1 X !Oeg So _‘.-, ‘. .‘. ,, ,y: .’ .’ .. .. ,. : :, ., ‘I.-.

ertoss of c 10% except C(5’), for which an error of J 20% may- bq include-d because of its fast relaxation. b! or values are calculated using the folkwing relationship [2] with the C-H distances(r) taken to be 1.085 A and 1.100 8, for the base and the ribose, respectivcty,

(1 1

F,

13

G&12’r

=-- lore C

_ 1 [ Ii- (“C--q+:

,

rt= and WC represent the gyromaanetic ratio and Larmor angular,frequency of the carbon nucleus, resp~crively.

where

For example, the possibility that the C(4’)-C(S) bond undergoes internal rotation faster than the overall rotation‘of the molecule can be excluded frcm the present results, since if this were the case, H(S) would have TI (minimum) at a temperature lower than that for the other protons. From the same reasoning, it may be concluded that the other internal motions, i.e., the rotational motion about the giycosidic bond and the rate of ribose puckering are also slow compared with the overall rotation of the molecule. To check this conclusion from the proton TL va!ues, carbon-13 TI’s have also been measured at the same temperature, i.e., 6YC. The results are given in table 1 together wiA& the rr values deduced from the Tl values. It is to be noted that the rr values obtained from different carbon atoms all agree with each other within the experimental error of z= 10% except for C(5’) for which an error of < 20% may be included because of its fast relaxation. Furthermore, the absolute values of ir (0.6-0.9 i: 10mg s) agree reasonably

1 .: CHEMICAL PHYSiCS LETlERS ‘. 1 April 1975 ‘: ...‘ .,;’ ._,. ‘. . ...:. -:~wolI with those obtained f&n the pro&r T, values -: ‘1 tioni equilibrium [6]: “:: : .: (+,:I..0 x. jcf9 s), in spite ; of the approximation inFig. 2 shows the Tr’versus l/?relation~obtained by ‘.‘.,volved in using’eq. (l).to analyze the proton TI data. the above proSdike from the proton TI values, and _Thcse results from’ 13C Tr measurements support the ,indicates clearly that all of the 2’-5’ protons have ‘-.‘.Coriclusion~from~ the proton T, measurements that. the identical rotational correlation times not only at ,,-‘~‘:AMP~molecule is sufficiently “rigid” in that the.in; .6,5”C but’also at all other temperatures studied. On terrral‘mptions amslow’as compared to the overall ro the other band,.fg. 2 shows small, but significant de“. taiion orthemolecule’at 6.5’C. viatipns from the or values for these protons for the ‘.., .’ _-Tos;e.if such a conclusion is also vaIid at ot,her .. protons H(8) end H(1’). We have found in a separate -temperatures, .the T1 versus’I/Trelationof fig. 1 isexperiment.that 5’-AMP is a mixture of a sj~n and an .’ ,‘.,_,Vqlurnc.32;nd.~ber

3..

converted ,.:

int,o a ~,,versuc

,1/T relation

by using

eq.

(1)

‘for a!l,the._protons.cxcept H(2) (see fig. 2). For this. bracedme to.be valid, it’must be established that no conformational change oc&s.with changing tempera‘-&e.that could cause a significant,change In the T1 of

the proton in question. This should obviously be true .,:.,for.2’-5’ protons whose Tt~‘values depend piedominantIy on.the conformation of tl-kribose moiety, which appear&remain unchanged at all temperatures stu-

‘tiled ‘as.verified by the constancy of the spin-spin -,-~.~--~_ .coupiing constants of these protons with temperature. ,. Op the other hkd,.the ,T1 Yiues of H(1’) and H(8j .&ill depend rather strongly on the rotational angle ‘. about the glycosidic bo,nd or the sy;z-arzti conforma‘.

-‘.

,. .

/ ,

I’

. .

form,

but

it pre-

ments at various temperatures are also plotted. As long as the mclecule is f‘rigid”, it is expected that the 7r values obtained from proton TI measurements by means ofeq. (1) will closely parallel those from 13C T, measurements. Fig. 2, shows, in fact, that this is true. 4 small but constani (20-30%) discrepancy is error of the T1 measurements.

This discrepan-

only for’an isolated pair of protons, and partly also from errors in the assumed C-H distances in the analysis of the 13C Ti data. A question that mny’also be asked is whether or not it is proper.to use the minimum values of.TI.in the dimer regi.on (approximately 50% of0.5 M AMP forms dimers at 6.5”C, as estimated from a separate shift measurement) to deduce 7r values in the’monomer r&on. It is important, and moreover useful, to note that the.value of T, at its minimum should rem& largely ‘the same regardless of.the de-

‘..

,. H12'144~5'1 -

-0. nc ! / 3.2 314

_

.”

monomer

cy cquld.‘arise from t,he application of eq. (1) to the multi-proton system, the equation being strictly valid

,.

Fig

in the

a change in the syn-nnti conformational equilibrium with temperature. A closer examination of this phenomenon will give useful inforrriation as to the dynamics of the~syn-anti conformational equilibrium. In_G&2 rr values deduced from 13C T, measure-

mental

(‘1.

.,

..

conformation

fers the a~rfr’conformation when it forms a stacked ,, dimer [7] .-It is probable in this connection that this unique behavi,lr of H(8) and H(l’) is associated with

noted, however, the values falling beyond the experi-

:.

‘.

.,

arrti

.,

2.6

3.6

grek of stacking as long as .the predominant source of.

IO’/TPKl,

.’ . ;_ :

2. Tempera&rc

1 3.6

dependence

of rotational

correlation

times.

_‘(7;) . of 5’-AMP deduced from ‘H and 13C.$in-lnttice relaxa-. : tion tim.? CT1 ;..r, values from proton 7’1’s are deduced from. : the,daia.tif fig. 1 by using eq. (I), while ‘i values from 13C .- r,‘i’-,ra ~btti+ by the procedure described in fo&no:d b) -‘:c,f.,~61~‘1.me v&&al bu tidicate;.the rmge of 7r v+lues O" .“tain~d,~~b_in_~]!heC-H,carbons

.;,...-:..1.,: ..:as..,-:‘.‘,:I.‘.,.‘.,-~‘:j .,,;. ‘,Y: -. :. ‘~.._.‘,._r_.‘,. ~_-,-,:. :

‘?., ; .,_. :,I

in themolcc+~.

.. .. ,: :..: ,.

,;:.:,

,‘.: :.

.: “:: .

.-,, ,_,

.

,rkltiation ccmes from intramolecular interactions.’ Using TL (minimum) in the dimer region for .the analysisin the monomer region-would, therefore, be corn-. pletely v&d for 2’-5’ protons, and approximately for., H(1’) and H(8):, ., ,’ ‘‘, ‘Bre& can’be seen in the, l& rr’ versus l/T cu_rves at “. 15’C in fig. 2. -The Straight-portion ab0.W z. 15’.C .._ obeys the relatio$ship .. ‘ . : 1’ : :. : : . . .., ._ .;. .- ,,.‘..‘:’ ,.. : : j, :.., y ,:.; ,,: .,,, -‘,....,~_,:., ,. .. ... ;_ ,._.. :. .: /‘_,, ._ ,, ,., -. .: ._’: _‘_.,.‘, ,., ,. ‘,

Volume 32, number I

CHEhlICAL

PHYSICS

L April 1975

LETTERS

slow (2 10Bg s) as compared to the overall rotatiorl in a purine nu’cleoside derivative with an activation enthalpy (4H) of 6-7 kcal/mole of the monomeric 5’for the rotation predominantly AMP. On the other hand, ihe increased slope below z 15°C indicates that the rotational motion becomes progressively harder in this tempeiature range with in&easing dimer formation. Carbon-l 3 T1 measurements have also been performed on some purine nucleosides, e.g., 2’;3’-isopropylideneadenosine,

2’,3’-isopropylideneguanosine,

and 2’,3’4sopropylidene.3,5’.cycloguanosine (all in DMSC!-d,).The results indicate that, within experimental errors of x 1O%,all the C-H groups in:a molecule, except the methyl groups and S’lmethylene groups, rotate at a common rate which is determined by the rotation of the whoIe molecule. It appears proper to anticipate that in general the rotation about the glycosidic bond and the ribose puckerin&rate are

(5 1OS9 s).

.’ References [l] T.C. Farrat and E.D. Becker, Pulse and Fourier transform NMR. Introduction to theory md methods (Acadcmic Press, New York, 19’11). [Z] K.F. Kuhlmann, D.M. Grant and RX. Hmis.1. Chem. Phys. 52 (1970) 3339. [3] K. Akzitia, T. Imoto and H. Hntanp. Chem. Fhys_ Lctters 21 (1973) 398. (41 K. Akasaka, Biopolymers-(197+), to b.e PubIishcd, [ 51 A. Abragam, The principlts of nuclear magnetism (Oxford Univ. Press, London, 1961) ch. 8, p_ 293. [6] T. Imoto, K. Akasaka and H. Hatano, Chem. Letters

(1974) 73.

[ 71 T. Imoto, K. Akasah

and H. Hatano, Bulletin of the 13th Annual Meeting of tic Biophysiti Society of lap;ln (1974) p. 26.