Journal of Biochemical and Biophysical Methods, 26 (1993) 1-26 1 © 1993 Elsevier Science Publishers B.V. All rights reserved 0165-022X/93/$06.00

JBBM00972

NMR spectroscopic properties (1H at 500 MHz) of deuterated* ribonucleotide-dimers ApU*, GpC*, partially deuterated 2'-deoxyribonucleotide-dimers d( TpA* ), d(ApT * ), d( GpC * ) and their comparison

with natural counterparts (1H-NMR window)

A. F61desi, F.P.R. Nilson, C. Glemarec, C. Gioeli and J. Chattopadhyaya Department of Bioorganic Chemistry, Biomedical Center, UniL, ersity of Uppsala, Uppsala (Sweden)

(Received 28 July 1992) (Revised version received 17 August 1992)

(Accepted 1 September 1992)

Summary

Pure l '#,2',3' ,4'#,5',5"-2H6-ribonucleoside derivatives 10-14, 1'#,2',2",3',4'#,5',5',-2H7-2 '- deoxynucleoside blocks 15-18 and their natural-abundance counterparts were used to assemble partially deuterated ribonucleotide-dimers (* indicates deuteration at 1'#,2',3',4'#,5',5"(2H6)): ApU* 21, GpC* 22 and partially deuterated 2'-deoxyribonucleotide-dimers d(TpA*)23, d (ApT*)25 , d(GpC*) 26 (* indicates deuteration at 1 '#, 2',2",3',4'#,5',5"(2H7)) according to the procedure described by F61desi et al. (Tetrahedron, in press). These five partially deuterated oligonucleotides were subsequently compared with their corresponding natural-abundance counterparts by 500 MHz ~H-NMR spectroscopy to evaluate the actual NMR simplifications achieved in the non-deuterated part (1H_NMR window) as a result of specific deuterium incorporation. Detailed one-dimensional X H-NMR (500 MHz), two-dimensional correlation spectra (DQF-COSY and TOCSY) and deuterium isotope effect on the chemical shifts of oligonucleotides have been presented.

Key words: 1H-NMR spectroscopy; Deuterated ribonucleotide dimer; Deuterated 2'-deoxyribonucleo- tide dimer

RNA and DNA have a large number of biological function such as storage and propagation of genetic information or catalytic activity. These DNA and RNA molecules have different secondary (double strands, hairpin loops, internal loops,

Correspondence address: J. Chattopadhyaya, Department of Bioorganic Chemistry, Box 581, Biomedical Center, University of Uppsala, S-751 23 Uppsala, Sweden.

bulges) and tertiary (triple strands, helical junctions, pseudoknots) structural elements [1] that are connected to the specific biological functions. The study of the conformational behaviour of these macromolecules would most probably help us to understand how the conformational characteristics of DNA and RNA translate into interaction and recognition that culminate into specific biological function. Nuclear magnetic resonance (NMR) spectroscopy has emerged as one of the most powerful tools [2] in conjunction with various computational methods [2-6] which provide conformational information under biological condition. In this complex approach to solve the problem of solution geometry of DNA or RNA molecules, the NMR measurements provide the informations on bond torsion angles and interproton distances as inputs for the computational calculations for model building. The information on bond torsion can be extracted from homonu- clear two-dimensional (2-D) correlated spectroscopy (COSY) providing a direct proof of the existence of resolved scalar couplings (3JHH) , and correlates the chemical shifts of coupling partner through the single or multiple coherence transfer of nuclear spins as in DQF-COSY or by HOHAHA or TOCSY which visualize the structure of the spin system in a most direct and informative manner [2c,4]. The information on interproton distances are derived from 2-D nuclear Overhauser enhancement (NOESY) [5] results which indicate through-space inter- actions between two relaxing protons [5b-e]. In an effort to collect these primary conformational informations, it would be ideal that each resonance line and cross-peak due to two interacting nuclei is clearly separated in homonuclear proton-proton, heteronuclear proton-carbon, proton-phosphorus, carbon-phos- phorus, NOESY and ROESY experiments. Enormous developments both in hardware (increasing magnetic field, more powerful computers) and spectral editing methods [2f,4,6] allow us to partly tackle the problem of spectral complexity due to resonance overlap and allow the extraction of 3JHH and nOe volumes in an unambiguous manner from up to 14-16-mer duplex DNA and 8-12-mer single stranded RNA but it is simply impossible to collect all of these informations in a non-prejudicial manner from a larger molecule. Clearly, these problems are associated with spectral overlap which becomes more and more complex due to overcrowding of resonances particularly from the repeating pentose moieties with increasing chain length. The problem due to severe spectral overlap of proton resonances in absorption assignments and in measurements of 3JHH and nOe volume of a larger biologically functional DNA or RNA molecule could be solved by chemical means by substituting proton (1H) with deuteron (2H) in a chosen domain and extracting necessary information arising from the shorter NMR-visible non-deuterated part (1H-NMR window). By incremental shift of the ~H-NMR window in a series of oligo-DNA or RNA with identical sequence (Scheme 1), one should be able to put together the total structural information of a much larger oligonucleotide than what is possible today. The most important part in this concept is that two 1H-NMR windows in two oligomers of the series should have at least an overlap of a nucleotide residue with specific chemical shifts in order to be able to correlate protons from both windows with respect to the same nucleotide reference point (i.e. same proton resonances in both NMR-windows).

r.::.:3:j.:.%..~L~:3k3 ::.:2,:.~ .:2.::,7%'3;'31".:J':2".'.%'3:." I

I Succesive shift of "NMR-window" in a DNA or RNA molecule allows the incremental assemblage of conformational information such as torsional angles or nOes using the techniques of Multidimensional NMR spectroscopy

Scheme 1

OH OH

HO OH HO OH

t ' i "NMR-window" = Non-deuterated

DNA/RNA

r::...:...:.::.:~.l Deuterated DNA/RNA

Signifies shift of "NMR-window" across the whole length of the molecule. Note that the design of NMR-windows with a slight overlap enables one to walk on the molecule by a common nOe or a chemical shift on two adjacent "NMR-windows"

O-Tol O O-Tol O ~ 5 ~ O ~ cj.OCH:I ~sTO~ ~OAc H/D "~ ' I~ 2.1W" I'UD

J I i i Tol-O O-Tol Tol-O O-Tol

1 2 3 4

O-Tol OH

D D D

ToI-O O-Tol HO OH

OH t > ~ , / 5 . / O . ~ . B

H/Dr ~ ~ k ~ m ~ ' HID

HO ~ D

5: B = U 10: B = U 15: B = C Bz 6: B = C Bz 11: B = C Bz

16: B = A Bz 7: B = A Bz 12: B = A Bz 8: B - G DPc 13: B - G DPc 17: B-- ~Ac~OPC

- Ac -- Ae 18: B = T 9: B = T 14: B = T

I I 1' OH =

" 2L._ . ~ ~ / . ' X ' ~ ~.."/x / z ~

O~. .O OH O% dO "14 ..p.,~' D I ,.P~f ,, I -o io.. o li-0 fo.Lo s,s.o B I "r

e / HO -o i ~

19: B I = U ; B = A 20: B I = C ; B = G

21: B I = A ; B = U

22; B I = G ; B = C

2 3 : B~=T; B = A 24: B 1=C; B = G

25: B I = A ; B = T

26: B I = G ; B = C

H O --'~'~5- .O- A

~ t D - D ~ D 3 D H/ ' D 1 ~ . - - i ~ I r HID

HO 0 I

- O - P = O

H O . 5 H /5 + 0 A ~:

HO O I

- O - P - - O I O

N O OH

where: Tol = 4-toluoyl, Ac = acetyl, U = uracil-l-yl, C 8z = N4-benzoylcytosin-l-yl, Bz 6 DPC 2 b A = N -benzoyladenin-9-yl, G Ac = N -acetyl-O -diphenylcarbamoylguanin-9-yl,

1" = thymin-l-yl, A = adenin-9-yl, C = cytosin-l-yl, G = guanin-9-yl

S c h e m e 2

27

to

r~

i--S‘---T--‘-- -ii -7

5.6 5.4

H6 Hl’ I I IB5 H3'

I H5' H5"

Fig. 1. 500 MHz ‘II-NMR spectra of deuterated u-ribofuranose (> 97 atom % *H at C2, C3, C5/5’;

- 85 atom % 2H at C4; - 20 atom % ‘H at Cl), deuterated-~-o-nucieos~des and their natural-abun-

dance counterparts (99.985 atom % 1 HI. (A) shows 1 -O-methyl-2,3,5-tri-0-(4-toluoylj-(a /p: - l/10)-

o-ribofuranoside (3) and (B) shows its natural-abundance counterpart. (C) shows 1’#,2’,3’,4”,5’,5”-

2H,-uridine (10); (I31 shows natural-abundance counterpart; (G) shows the full height anomeric region of 1’*,2’,3’,4’#,5’,5”-*H,-uridine (10) from subspectrum (C). (E) shows 1’#,2’,3’,4’#,5’,5”-‘H,-N4-ben-

zoylcytidine (11); (F) shows natural-abundance counterpart.

_ --J ~_,I ~ A .......................

H2

B ) ~ HI" H2" H3" H4" H5" H5""

8.80 p p m 8.40 7.98 7.66 5.98 4.80 4.40 3.80

D)~ HI" t-I2" H4"

8.30 ppm 7.38 6.00 4.60 4.20 3.80

H6

t-12" . H5" H5""

(

7.60 ppm 5.84 4.20 3.80

Fig. 2. 500 MHz IH-NMR spectra of deuterated-fl-D-nucleosides ( > 97 atom % 2H at C2', C3', C5 ' / 5 " ; ~ 85 atom % 2H at C4' (C4'~); ~ 20 atom % ZH at CI ' (CY#)) and their natural-abundance counterparts (99.985 atom % 1H). (A) shows l '#,2' ,3' ,4 '#,5' ,5"-2H6-N6-benzoyladenosine (12); (B) shows natural-abundance counterpart. (C) shows l ' * , 2 ' , 3 ' , 4 ' # , 5 ' , 5 " - 2 H 6 - N 2 - a c e t y l - O 6 - d i p h e n y l -

carbamoylguanosine (13); (D) shows natural-abundance counterpart. (E) shows 1-(1'#,2',3',4'#,5',5 "- 2H6-/3-o-ribofuranosyl)-thymine (14): (F) shows natural-abundance counterpart.

(B jilL_. H2- H2"

rwyw~-~ ~, . . . . . I [ - w ' ' ' l . . . . I . . . . . . . . . I ' ' I I " ~ T ~ r ~ ' ' ' i ' - ' ' ' " ( " ' r ~ " ' l . . . . I . . . . I T ' ' ' l ' ' ~ r l ' ' 8.46 ppm 7.98 7.50 6.16 4.40 4.04 3.80 2.60 2.20

8.80 ppm 8.40 8.08 7.68 6.60 4.70 4.20 3.78 2.86 2.48

H8

(, _ A _

8.28 p p m 7.40 7.20 6,42 4.72 4.00 3.80 2.80 2.40

Fig. 3, 500 MHz 1H-NMR spectra of deuterated-fl-D-nucleosides ( > 97 atom % 2H at C2', C3', C5'/5"; ~ 85 atom % 2H at C4' (C4'#); ~ 20 atom % ZH at C1' (C1'#)) and their natural-abundance

f # p . t ¢ # ~ . 2 t 4 counterparts (99.985 atom % ~H). (A) shows 1 ,2 ,2 ,3 ,4 ,5,5 - H7-2-deoxy-N -benzoylcytidine (15); (B) shows natural-abundance counterpart. (C) shows l'#,2',2",3',4'#,5',5"-2HT-2'-deoxy-N6-ben - zoyladenosine (16); (D) shows natural-abundance counterpart. (E) shows 1'*,2',2",3',4'*,5',5"-2H7-2 '-

deoxy-N 2-acetyl-O6-diphenylcarbamoylguanosine (17); (F) shows natural-abundance counterpart.

H 6 H1 "

7.58 ppm 6.24

H 8 A H2A

I I

,cL .... ' ' ' ' ' ' ' I ' ' ' ' ' . . . . '

Dpm 8.0

H3" H4"

t . . . . I . . . . ~ - ' 7

4.40 4.00 T II "-r-r-r ' I . . . . 3.80 2.30

H6U HI'A

[ . . . . , , .

H5'/2'/3'/4'U

. tl ...ell' '° . ,c

I '

ppm ' 718 46 3.8

Fig. 4. 500 MHz I H - N M R spectra of deuterated-/3-D-nucleoside ( > 97 atom % 2H at C2' , C3', C 5 ' / 5 " ; 85 atom % 2H at C4 ' (C4'#); ~ 20 atom % 2H at CI ' (CI '#)) and its naturaL-abundance counterpart

(99.985 atom % IH) and natural and partially deuterated diribonucleoside-(3' ~ 5 ' ) -monophosphates in D 2 0 at 298 K. (A) shows l '# ,2 ' ,2" ,3 ' ,4 '# ,5 ' ,5"-2H7-thymidine (18); (B) shows natural-abundance counterpart . (C): natural ApU, (D): A p U * where the 1'#,2' ,3' ,4 '# and 5 ' / 5 " protons of the uridine (pU *) residue are exchanged with 2H. (E): naturaL GpC, (F): G p C * where the 1 '#,2 ' ,3 ' ,4 ' # and 5 ' / 5 " protons of the cytidine (pC *) residue are exchanged with 2H. The H1 '# appears as a singlet while the

H4 '# appears as a doublet due to its coupling to the phosphorus of the 3 ' ~ 5 ' phosphate linkage.

H2'/3'/4'C

H ~ C ....

' I ' ' I 0 4 4 4 !2 4.

Hlq2 1 HS'/HS"A

m'A . ]_ .

4.5 4.0

I HOC HI'G H5'/H5"C

H8G[ tl HI'C H5C H4'G H4'C HS'/H5"G

H3'G

8 0 45 4 DDm

H8A mA CH3T [ t H6T H5'/H5"A

H4'T [

HI'T H3'T H4'A | ~1 H2'A ,I, " ' I ' - l , / ~1 H2"A H2"T ] H2q"

oDm 5.0 4 2

H8A H2A H6T HI~I " H2'B-I2"T

H4'A H4"T H5'/HS" A H2'/H2"

' ' ' H3"I" i H5"F |

Dpm 6 2 4 75 4 50 4 25 4 00

Fig. 5. I H - N M R spectra of natural and partial ly deuterated di(2'-deoxyribonucleoside)-(3' ~ 5 ' ) - p # t t , , , # monophosphates in D 2 0 at 298 K. (A): natural d(GpC), (B): d (GpC* ) where the 1 ,2,2 ,3,4 and

5 ' /5" protons of the cytidine (pC*) residue are exchanged with 2H. (C): natural d(ApT), (D): d(ApT*) where the I'#,2',2",Y,4 '# and 5 ' / 5" protons of the thymidine (pT*) residue are exchanged with 2H. (E): natural d(TpA), (F): d(TpA*)where the 1'#,2',2",3',4 '# and 5 ' / 5" protons of the adenosine (pA*) residue are exchanged with ell. The H1 '# appears as a singlet while the H4 '# appears as a doublet due

to its coupfing to the phosphorus of the 3' ~ 5' phosphate linkages.

Although nucleosides have been selectively deuterated with very different amount of deuterium incorporation at both sugar [7] and nucleoside [8] levels using different chemical [7b-g,i-n,8a-g,10a,12] and enzymatic [7h,8h-m] methods, these monomers have not really found their application in conformational studies on

(A)

..... \5

0 H2'G

0

H3'C H2'C H4'C

I

H3'G ~ I..15 ,~G

E I ppm 4 4

H S " G

H5'C HS"C

et

~ 4 0 3e

5 8

ppm

• I I ' G - -

N 1 C

-1

H3C H2'C H4'C

I

HYC FI5"C

,0 58

Fig. 6. H O H A H A spectra of the natural and partially deuterated cytidylyl-(3' ~ 5')-guanosine (CpG) in D 2 0 at 298 K. Panel A represents the 2-D spectrum for the non-deuterated CpG. Panel B represents the 2-D spectrum of the partially deuterated C p G * where the 1'#,2',3',4 '# and 5 ' / 5 " protons of the guanosine residue ( p G * ) are exchanged. In the 1-D spectrum, the HI' appears as a singlet at 5.81 ppm while the H4' appears as a doublet at 4.2 ppm due to its coupling to the phosphorus of the 3' ~ 5'

phosphate linkage. In the 2-D spectrum, the J-network for the 3'-terminal residue has vanished.

i ~ i 2 I ! ppm 4 4 4 4 0 3 8

ppm

1

10

oligonucleotides. Examples of base deuteration [9] and its effect on the 2-D 1H-NMR and 2H-NMR studies on oligomers selectively deuterium labeled at given positions of the sugar moiety [10] are known. The most detailed studies on various dimers and trimers where one of the nucleotide units was fully enzymatically

HI'G HI'C

HI'C

J

(B)

HI'G

FI2'G 143'G H4'G H5'G H5"G

C)oO oo oOD H5'C H5"C

H2'C H3'C H4'C

-5~

ppm

ppm 4 44 42 40 3 B

H2'G H3'G H4'G H5'G H5"G

O 0 0 Oo 58

Dpm

0pm 4 .6 4 4 4 2 4 .0 3 8

Fig. 7. H O H A H A spectra of the natural and partially deuterated guanylyl-(3' --* 5')-cytidine (GpC) in D 2 0 at 298 K. Panel A represents the 2-D spectrum for the non-deuterated GpC. Panel B represents the 2-D spectrum of the partially deuterated GpC * where the 1'#,2' ,3 ' ,4 '# and 5 ' / 5 " protons of the cytidine (pC*) residue are exchanged. In the 1-D spectrum, the HI ' appears as a singlet at 5.84 ppm while the H4' appears as a doublet at 4.2 ppm due to its coupling to the phosphorus of the 3 ' -~ 5'

phosphate linkage. In the 2-D spectrum, the J-network for the 3'-terminal residue has vanished.

11

deuterated were carried out in the early seventies when the 2-D spectroscopy was not developed yet [8h-m]. This lack of data on the required level of deuteration necessary in the pentose sugar moieties in nucleotides for effective suppression of cross-peaks in 2-D NMR spectroscopy, in general, and how such deuteration may

(A) A

HIIU /

HI'A ~

115% ~ H2'U H3'U H4'U HS'U

oo H2'A H3'A H4'A H5'A H5"A

0 0 0 OG 0O i ' 1 '

ppm 4 8 4 6 4 4 4 2 4

i 6 0

ppm

HI 'U

HI'A

H2'A H3'A H4'A HS'A HS"A

Ipp- m ' I ' ' ~ ' 1 ' I ' I 4 . B 4 . 6 4 . 4 4 . 2 4 . 0

- 6 . 0

ppm

Fig. 8. H O H A H A spectra of the natural and partially deuterated adenylyl-(3' --* 5')-uridine (ApU) in D 2 0 at 298 K. Panel A represents the 2-D spectrum for the non-deuterated ApU. Panel B represents the 2-D spectrum of the partially deuterated A p U * where the 1'#,2',3',4 '# and 5 ' / 5 " protons of the uridine ( p U * ) residue are exchanged. In the 1-D spectrum, the HI' appears as a singlet at 5.75 ppm while the H4' appears as a doublet at 4.15 ppm due to its coupling to the phosphorus of the 3' --* 5' phosphate linkage. In the 2-D spectrum, the J-network for the 3'-terminal residue ( p U * ) has vanished.

12

(A)_~

HI'G ~ . _ . ~ _ _

HI'C

i

i H3'G H4'G I-LS'/H5 "G

01t1 H3'C HS'C H4'C

HS"C

H2'/H2"G

)Q)I H2'C H2"C

' • • l ' • i . . . . i . . . . i . . . . [ W

0 0 m 4 5 ' " ~ 0 3 5 3 0 2 5

boa

( B ) _ _ A ,

HI'C

HI'G

H2'/I~"G . ,G H.G - -

I

Dpm

pp~ 45 40 35 30 25

Fig. 9. HOHAHA spectra of the natural and partially deuterated 2'-deoxyguanylyl-(3' ~ 5 ' ) - 2 ' - deoxycytidine [d(GpC)] in D20 at 298 K. Panel A represents the 2-D spectrum for the non-deuterated d(GpC). Panel B represents the 2-D spectrum of the partially deuterated d(GpC*) where the 1'#,2',2",Y,4 '# and 5 ' / 5 " protons of the cytidine (pC*) residue are exchanged. In the 1-D spectrum, the HI ' appears as a singlet at 6.19 ppm while the H4' appears as a doublet at 4.1 ppm due to its coupling to the phosphorus of the 3' ~ 5' phosphate linkage. In the 2-D spectrum, the J-network for

the Y-terminal residue has vanished.

13

cause spectral simplification has promted us to take up this study on ~H-NMR window (Scheme 1) [11].

Our synthetic strategy [11] involved deuteration of methyl a/Cl-o-ribofuranoside 1 by Raney nickel-2H20 exchange reaction [7k-m,12] to give methyl le,2,3,4#,5, 5'-2H6-a/~-D-ribofuranoside 2 (> 97 atom % 2H at C2, C3, C5/5 ' ; ~ 85 atom %

(A) ~ L

HI'T

I

J HI'A "~i

H'YT H4'T HS'/HS"T H2q" H2"T ,

I I g , Ii H3'A H4'A HS'/HS"A H2'A H2"A

pgm ~ 3

j -6.0

J ppe

( B )

| t I H3"T H4'T HS'/I-B"T H2'T H2"T 6, o

HI'A ~ Ippm

[ ' . . . . . . . . I . . . . . . . ' ' I . . . . . . . . . [ ' ' ' ' ' ' ' ppm a 3 2 Fig. 10. H O H A H A spectra of the natural and partially deuterated thymidylyl-(3' ~ 5 ' ) - 2 ' - deoxyadenosine [d(TpA)] in D 2 0 at 298 K. Panel A represents the 2-D spectrum for the non-de- uterated d(Tpa). Panel B represents the 2-D spectrum of the partially deuterated d(TpA*) where the 1'#,2',2",3',4 '# and 5'/5" protons of the adenosine (pA*) residue are exchanged. In the 1-D spectrum, the HI ' appears as a singlet at 6.38 ppm while the H4' appears as a doublet at 4.10 ppm due to its coupling to the 3' ~ 5' phosporus of the phosphate linkage. In the 2-D spectrum, the J-network for the

3'-terminal residue has vanished.

14

(A)

Hl"r

H I ' A

( B ) _ _

H4"T H3"I" H5'T H5"T

I 0ee I-I2'/I-12"1"

I JllJ I H3'A H4 'A ladi'/H5"A

H2'/H2"A

ptm 4 ~ 4 0 ] 5 J o ~ 5

Hl'l"

HI'A ~

HYA H4'A HS'/H5"A H2'/I-12"A

~6 2

-ppm

I ' ' ' ' I ' ' ' ' I . . . . I ' ' ' ' 1 ' ' ' ' I ' ' ' '

opm 4.5 4.0 3 5 3 0 2 5

Fig. 1 l. H O H A H A spectra of the natural and partially deuterated 2'-deoxyadenylyl-(Y ~ 5')-thymidine (d(ApT)) in D20 at 298 K. Panel A represents the 2-D spectrum for the non-deuterated d(ApT). Panel B represents the 2-D spectrum of the partially deuterated d (ApT*) where the 1'#,2',3',4 '# and 5 ' / 5 " protons of the thymidine (pT*) residue are exchanged. In the 1-D spectrum, the HI ' appears as a singlet at 6.15 ppm while the H4' appears as a doublet at 4.00 ppm due to its coupling to the phosphorus of the 3' ~ 5' phosphate linkage. In the 2-D spectrum, the J-network for the 3'-terminal

residue has vanished.

Fig. 12. DQF-COSY spectra of the natural and partially deuterated adenylyl-(3' ~ 5')-uridine (ApU) in D~O at 298 K. Panel A: 2-D spectrum of the natural ApU. The cross peaks used for the determination of the vicinal 3JHH coupling constants are shown in the numbered boxes: (1) HI ' -H2'A, (2) H2'-H3'A, (3) H3'-H4'A, (4) H4'-H5'A, H4'-H5"A, (5) HI ' -H2 'U , (6) H2'-H3' , H3'-H4' , H4'-H5' and H4'-H5"U. Panel B: 2-D spectrum of A p U * where the I '#,2' ,3' ,4 '~ and 5 ' / 5 " of p U * have been exchanged with

deuterium. The empty boxes show that all cross peaks involving the pU* residue have vanished.

J

3

16

(A)

(B)

II

I III

' M I 2

t 4 0

I, 1

tl ° ppm 5 . 5 5 0 4 5

I I

III NI

-55

ppm

40

. . . . I ' ' ' ' I ' ' ' ' I . . . . I

pprn 5 . 5 5 . 0 4 .5 4 .0

Fig. 13. DQF-COSY spectra of the natural and partially deuterated guanylyl-(3' ~ 5')-cytidine (GpC) in DzO at 298 K. Panel A: 2-D spectrum of the natural GpC. The cross peaks used for the determination of the vicinal 3JHH coupling constants are shown in the numbered boxes: (1) HI'C-H2'G, (2) H3'C-H4'G, (3) H, (4) H4'-H5', H4'-H5"G, (5) HI'-H2'C, H2'-H3'C, H3'-H4'C, H4'-H5'C and H4'-H5"C. Panel B: 2-D spectrum of GpC* where the 1'#,2',3',4 '# and 5 ' / 5 " of pC* have been exchanged with deuterium. The empty boxes show that all cross peaks involving the pC* residue have

vanished.

,II ~ 4 . 0

5 4.,5

3

5 o

5.5

ppm

17

2H at C4 (C4'e); ~ 20 atom % 2H at CI (CI'*), ~ denotes partial deuteration) as main product, which was purified after toluoylation of the hydroxyl groups (see Scheme 2). The 500 MHz ~H-NMR spectrum of this deuterated sugar derivative 3 is shown in Fig. 1A to illustrate [11] the purity and deuteration level achieved. Subsequent 1-acetylation of this product resulted in 1-O-acetyl-2,3,5-tri-O-toluoyl- a//3-D-l#,2,3,4#,5,5'-2H6-ribofuranoside 4, which was condensed with silylated nucleobases uracil, N4-benzoylcytosine, N6-benzoyladenine, N2-acetyl-O6-diphen - ylcarbamoyl guanine and thymine [11] to afford the ribonucleoside blocks 5-9. The toluoyl groups from the fully protected nucleoside blocks were removed in a regioselectivc manner to give nucleobase protected nucleosides 10-14 [11]. The isomeric purities of these products were found to be excellent as evident through their 500 MHz 1H-NMR spectra (Figs. 1C-F and 2A-F) [11]. The deuterated ribonucleosides 11-14 were converted to the corresponding 2'-deoxynucleosides 15-18 [11]. The level of deuterium incorporation at the 2" was > 97 atom % as evidenced by integration of 1H-NMR spectra at 500 MHz (shown in Figs. 3A-F and 4A) of the residual proton resonance.

In order to investigate the effect of deuterium incorporation in the pentose moieties on the 1-D and 2-D 1H-NMR spectra such as DQF-COSY, HOHAHA (TOCSY) and NOESY, eight sets of deuterated dimers UpA* 19, CpG* 20, ApU* 21, GpC * 22, d(TpA*) 23, d(CpG * ) 24, d(ApT*) 25 and d(GpC *) 26 and their natural abundance counterparts and one trimer A*2'p5'A2'p5'A* 27 were synthesized [11] (* denotes the deuterated part in the molecule) using standard phosphotriester chemistry [13]. These partially deuterated dinucleotides and the trinucleotide were subsequently compared with the corresponding natural counter- parts to evaluate the actual NMR simplifications achieved in the ~ H-NMR window part (Scheme 1) as a result of specific deuterium incorporation. We have already reported [11] the synthesis of all of these deuterated dimers 19-26 and trimer 27, but we have only reported the NMR simplifications achieved in case of UpA* 19, CpG* 20, d(CpG*) 24 and the trimer A*2'p5'A2'p5'A* 27.

We herein report the 1-D and 2-D NMR spectra of ApU* 21, GpC* 22, d(TpA*) 23, d(ApT*) 25 and d(GpC*) 26 and their natural abundance counter- parts (Figs. 4C-17). Comparison of these deuterated and non-deuterated spectra (Figs. 4C-17) show, in conjunction with the partially deuterated dimers published in Ref. 11, that the complexities of the natural-abundance dimers are very different from case to case depending upon the aglycone and the nucleotide sequence. Such a comparison is useful to establish the varied degree of resonance overlap and simplifications achieved in them. These comparative studies for partially deuterated dinucleotides may be of interest in the future studies as reference spectra in the interpretation of XH-NMR window in larger oligo-DNA and oligo-RNA. In addition, we have examined the deuterium isotope effect on the chemical shifts of HI' and H4' of the deuterated residue and 1', 2'(2"), 3', 4', 5 ' /5" in the non-deuterated residue in all partially deuterated difiucleotides and 2,5A core and compared with their natural abundance counterpart. Such a comparison shows (Table 1) that the chemical shifts of the HI' and H4' of the deuterated residue in 19-27 shift only by 0.01-0.02 ppm while the chemical shifts

18

(A)

( B )

I.

1

p orn 5 5 5 0 4 5 4 0

6

5 1

5 0

- 5 5

LD~m

- 4 . 0

-4.5

5 0

-5.5

Dpm

ppm 5.5 5.0 45 4 0

Fig. 14. DQF-COSY spectra of the natural and partially deuterated cytidylyl-(3' ~ 5')-guanosine (CpO) in D20 at 298 K. Panel A: 2-D spectrum of the natural CpG. The cross peaks used for the determination of the vicinal 3JHH coupling constants are shown in the numbered boxes: (1) HI'C-H2'C, (2) H3'C-H4'C, (3) H2'C-H3'C, (4) H4'C-HS'C and H4'C-HS"C, (5) HI'G-H2'G, (6) H2'G-H3'G, (7) H3'G-H4'G, (8) H4'G-HS'G. Panel B: 2-D spectrum of CpG* where the 1'#,2',3',4 '# and 5'/5" of pG * have been exchanged with deuterium. The empty boxes show that all cross peaks involving the

pG * residue have vanished.

19

(A)

I I

I

i t

l ' 7

13

i i

i

lO

4 5 1 , , 2

1 2

6

ppm

ppr~ 6 a 2

I ! 8

I I

1o

(~) t , t I I. 4 5

;,'1 11 12 ~ I[ ~

13

1 2

o

' ' ' ' ' l ' ' , , ' ' ' ' , ' ' ' ' ' ' ' ' ' ' l ' ' ' ' ' " ' ' ' ' ' ' ' ' ' ' ' ' / ' . . . . . . ppm 6 4 2

Fig. 15. DQF-COSY spectra of natural and partially deuterated th~idylyl-(3' ~ 5')-2'-deoxyadenosine (d(TpA)) in D20 at 298 K. Panel A: 2-D spectrum of the natural d(TpA). The cross peaks used for the determination of the vicinal 3JHH coupling constants are shown in the numbered boxes: (1) HI'-H2"T, (2) HI'-H2'T, (3) H2'-H2"T, (4) H2"-H3'T, (5) H2'-H3'T, (6) H3'-H4'T, (7) H4'-H5'T, H4'-H5"T, (8) HI'-H2'A, (9) HI'-H2"A, (10) H2'-H2"A, (11) H2'-H3'A, (12) H2"-H3'A, (13) H3'-H4'A. Panel B: 2-D spectrum of d(TpA*) where the 1'#,2',2",3',4 '# and 5' /5" of pA* have been exchanged with

deuterium. The empty boxes show that all cross peaks involving the pA* residue have vanished.

20

(A)

( B )

I I

2

3

4

6 1

pDm 6 5 4 3

2

D IO

,!

3

4

6

Dl3m

I 2

3

4

r

L5

Dpm

pDm S 5 4 3

Fig. 16. DQF-COSY spectra of natural and partially deuterated 2'-deoxyadenylyl-(3' ~ 5')-thymidine (d(ApT)) in D20 at 298 K. Panel A: 2-D spectrum of the natural d(ApT). The cross peaks used for the determination of the vicinal 3Jmt coupling constants are shown in the numbered boxes: (1) HI'-H2'A, HI'-H2"A, (2) H2'-H2"A, (3) H2'-H3'A, H2"-H3'A, (4) H3'-H4'A, (5) H4'-H5'A, H4'-H5"A, (6) HI'-H2'T, HI'-H2"T, (7) H2'-H2"T, (8) H2'-H3'T, H2"-H3'T, (9) H3'-H4'T. Panel B: 2-D spectrum of d(ApT*)where the 1'#,2',2",3',4 '# and 5 ' / 5" of pT* have been exchanged with deuterium. The

empty boxes show that all cross peaks involving the pT* residue have vanished.

21

(A)

(B)

2

io L-i1 g 4

1

ppm 6 5 4 3

I 2

J !

! 4

I I

r

F

Di]m

L 2

3

I

Z4

pDm

ppm 6 5 4 3

Fig. 17. DQF-COSY spectra of natural and partially deuterated 2'-deoxyguanylyl-(3' ~ 5 ' ) - 2 ' - deoxycytidine (d(GpC)) in D20 at 298 K. Panel A: 2-D spectrum of the natural d(GpC). The cross peaks used for the determination of the vicinal 3JHH coupling constants are shown in the numbered boxes: (1) HI ' -H2'G, H2"-H3'G, (2) H2'-H2"G, (3) H2'-H3'G, H2"-HYG, (4) H3'-H4'G, (5) H4'-H5'G, H4'-H5"G, (6) HI'-H2'C, HI'-H2"C, (7) H2'-H2"C, (8) H2'-7H3'C, H2"-H3'C, (9) H3'-H4'C. Panel B: 2-D spectrum of d(GpC*) where the 1'#,2',2",3',4 '# and 5 ' / 5 " of pC* have been exchanged with

deuterium. The empty boxes show that all cross peaks involving the pC * residue have vanished.

TA

BL

E

1

~H

ch

emic

al s

hif

ts (

6

scal

e, r

efer

ence

: 8

(ac

eto

nit

rile

)= 2

.00

pp

m)

of

par

tial

ly d

eu

tera

ted

oli

go

mer

s an

d t

hei

r c

om

pa

riso

n (

de

ute

riu

m i

soto

pe

effe

ct)

wit

h

the

na

tura

l-a

bu

nd

an

ce

co

un

terp

art

s in

D2

0

at 2

98 K

(ex

cep

t fo

r th

e tr

ime

r w

hic

h i

s re

co

rde

d a

t 3

08

K)

at 3

-8

mM

co

nc

en

tra

tio

ns

Oli

go

me

r R

esi

du

e

HI'

H

2'

H2

" H

3'

H4

' H

5'

H5

" H

8/H

6

H2

/H5

/Me

5

Up

A

U

5.8

4

4.2

8

- 4.

51

4.2

0

3.8

0

3.75

7.

65

5.78

A

6.1

4

4.7

9

- 4

.57

4.

41

4.2

6

4.1

9

8.4

5

8.25

Up

A*

19

U

5

.86

4

.27

-

4.51

4

.19

3

.79

3.

75

7.6

2

5.7

8

A*

6

.14

-

- -

4.4

0

- -

8.4

6

8.2

6

Cp

G

C

5.8

8

4.21

-

4.31

4

.15

3.

79

3.7

0

7.5

9

5.75

G

5.9

7

4.5

0

- 4.

41

4.3

7

4.1

7

4.0

6

7.9

2

-

Cp

G*

2

0

C

5.8

8

4.21

-

4.31

4

.15

3

.79

3

.69

7

.59

5

.74

G *

5

.97

-

- -

4.3

7

- -

7.91

-

Ap

U

A

6.0

2

4.3

8

- 4

.63

4

.42

3

.94

3

.83

8

.29

8.

15

U

5.7

5

4.2

4

- 4

.20

4

.19

4

.26

4

.08

7

.67

5

.60

Ap

U*

2

1

A

6.03

4

.84

-

4.6

3

4.4

2

3.93

3

.83

8

.30

8.

17

U *

5

.75

-

- -

4.1

8

- -

7.6

7

5.6

0

Gp

C

G

5.8

0

4.7

4

- 4.

61

4.35

3

.89

3

.79

7

.89

-

C

5.8

4

4.2

2

- 4

.19

4

.16

4

.29

4

.07

7.

75

5.71

Gp

C*

2

2

G

5.81

4

.74

-

4.61

4.

35

3.9

0

3.7

9

7.91

-

C *

5

.84

-

- -

4.1

7

- -

7.75

5.

71

D(T

pA

) T

5

.9

2.11

1.

55

4.5

3

3.95

3

.60

3

.56

7

.27

1.

78

dA

6

.34

2

.82

2

.54

4

.73

4

.17

4

.0

4.0

8

.32

8

.07

d(T

pA*

123

24

of t he n o n - d e u t e r a t e d m o i e t y m o v e by < 0.01 p p m d u e to d e u t e r i u m i so tope ef fec t .

Such a c o m p a r i s o n shows tha t t h e d e u t e r i u m i so tope e f fec t on p r o t o n c h e m i c a l

shif ts is i n d e e d ve ry small . Th i s m e a n s tha t it w o u l d be poss ib le to ove r l ay t h e

s p e c t r u m of va r i ous ' N M R - w i n d o w ' c o n t a i n i n g a n a l o g u e s o f d e u t e r i u m - l a b e l e d

o l i g o - D N A o r R N A (as shown in S c h e m e 1) a n d c o m p a r e t he c r o s s - p e a k s in

m u l t i d i m e n s i o n a l c o r r e l a t i o n o r N O E S Y s p e c t r o s c o p y for t h e c o n f o r m a t i o n a l

s tud ies w i t h o u t any p r o b l e m d u e to t h e d i s t o r t i on o f t h e c h e m i c a l shif ts f r o m the

d e u t e r i u m i so tope e f fec t p r o v i d e d the c o n c e n t r a t i o n a n d the t e m p e r a t u r e a r e

iden t ica l .

Simplified description of the method

The IH-NMR experiments were performed [11] on a Bruker AMX-500 MHz spectrometer. The DQF-COSY and Hartmann-Hahn spectra were recorded in pure-phase absorption mode with the time proportional incrementation method (TPPI) and with low power preirradiation of the residual HDO peak during the relaxation delay. The DQF-COSY [15] spectra were acquired with 4096 complex data points in t 2 and 256 points in t v The data were zero filled to give a 4096×1024 point matrix and a sine-square bell window was applied in both directions before Fourier transformation. The Hartmann- Hahn [16] spectra were acquired with 2048 complex data points in t 2 and 256 points in t I. The data were zero filled to give a 2048 × 1024 point matrix and a sine-square bell window was applied in both directions before Fourier transformation.

Acknowledgements

T h e a u t h o r s t h a n k the S w e d i s h B o a r d for T e c h n i c a l D e v e l o p m e n t ( N U T E K )

a n d S w e d i s h N a t u r a l S c i e n c e R e s e a r c h C o u n c i l for g e n e r o u s f inanc ia l suppor t . W e

also t h a n k W a l l e n b e r g s t i f t e l s e n , U n i v e r s i t y o f U p p s a l a , F o r s k n i n g s r ~ d s n ~ i m n d e n

( F R N ) for f inanc ia l s u p p o r t s for p u r c h a s i n g the 500 M H z N M R s p e c t r o m e t e r .

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25

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26

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