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© 1995 Oxford University Press 2868-2876

Footnote

Phosphorus 31 solid state NMR characterization of oligonucleotides covalently bound to a solid support

Phosphorus 31 solid state NMR characterization of oligonucleotides covalently bound to a solid support P. M. Macdonald* , M. J. Damha 1 , K. Ganeshan , R. Braich 1 and S. V. Zabarylo

Department of Chemistry and Erindale College, University of Toronto, Toronto , Ontario M5S 1A2, Canada and 1 Department of Chemistry, McGill University, Montreal , Quebec H3A 2K6, Canada

Received June 3, 1996; Accepted June 22, 1996

ABSTRACT

31P cross polarization (CP) magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectra were acquired for various linear and branched di- and tri-nucleotides attached to a controlled pore glass (CPG) solid support. The technique readily distinguishes the oxidation state of the phosphorus atom (phosphate versus phosphite), the presence or absence of a protecting group attached directly to phosphorus (cyanoethyl), and other large changes in the phosphorus chemistry (phosphate versus phosphorothioate). However, differences in configurational details remote from the phosphorus atom, such as the attachment position of the ribose sugar (2'5' versus 3'5'), or the particulars of the nucleotide bases (adenine versus uridine versus thymine), could not be resolved. When different stages of the oligonucleotide synthetic cycle were examined, 31 P CPMAS NMR revealed that the cyanoethyl protecting group is removed during the course of chain assembly.

INTRODUCTION

Solid-phase synthesis has become the method of choice for producing oligonucleotides of defined sequence ( 1 , 2 ). The most popular solid-phase supports consist of long-chain alkylamine-modified controlled-pore glass (CPG) ( 3 , 4 ) and highly crossed-linked polystyrene ( 5 ). In the case of CPG, the long-chain alkyl spacer renders the terminal nucleoside highly accessible to coupling reagents while the pore size favours the synthesis of very long DNA and RNA oligomers ( 6 ). A typical synthesis involves successive rounds of coupling of an activated protected nucleoside precursor to the terminal pentose of the growing oligonucleotide, followed by capping of non-coupled sites and oxidation of phosphite triester linkages in preparation for the next round of coupling ( 1 ). When the nascent oligonucleotide has been completely assembled, it is deprotected and cleaved from the solid support.

It is undoubtedly true that such synthetic strategies as described above have been enormously successful, and that their commercialization has placed oligonucleotide synthesis within the reach of the non-specialist. Multiple gram solid-phase synthesis of DNA and RNA and analogs for therapeutic-based investigations ( 7 ), and for X-ray crystallography, NMR and other physical studies, has recently become a reality ( 8 , 9 ). It is equally true that, because the chemistry involved in the individual steps is difficult to study in situ , the ultimate success for any particular oligonucleotide sequence can only be determined upon its release from the solid support. Perhaps the most common means for monitoring synthesis efficiency is spectroscopic quantification of trityl cations released during chain elongation. The major limitation of this technique is that, other than 5'-detritylation, it does not provide any information about structural/chemical modifications occurring at other positions in the oligonucleotide chain.

Solid state NMR spectroscopy affords the capability of characterizing chemical structure, conformation and dynamics in solid-phase supported syntheses. It has been used, for instance, to analyze surface modifications of silica particles in a variety of situations ( 10 , 11 ). In this report we describe the first cross-polarization (CP) magic angle spinning (MAS) phosphorus-31 ( 31 P) NMR investigations of oligonucleotides covalently bound to a solid support. 31 P was chosen as the nucleus of interest because, first, it is 100% naturally abundant and has good sensitivity in the NMR experiment, so that no isotopic enrichment is necessary. Second, since many of the chemical manipulations performed during the course of a typical solid phase oligonucleotide synthesis result in changes directly affecting the phosphorus atom, the 31 P NMR spectrum should provide a sensitive monitor of the course of the synthesis. In this study we explore the capabilities and limitations of 31 P CPMAS NMR for reporting on the detailed chemistry of the phosphorus atom in oligonucleotides bound to controlled-pore glass, and for resolving different oligonucleotide structural motifs.

EXPERIMENTAL

Synthetic procedures

Synthesis of ApU 1a and 1b . Dinucleoside monophosphate ApU 1a , shown in Figure 1 A, was synthesized by the phosphodichloridite procedure ( 12 , 13 ). To a cold solution (0oC) of 2,4,6-collidine (3.63 mmol, 490 [mu]l) in dry THF (0.8 ml) was added 2-cyanoethylphosphodichloridite (CNEOPCl 2 , 0.55 mmol, 68 [mu]l). A THF solution of N 6 -benzoyl-5'- O -monomethoxytrityl-2'- O - t -butyldimethylsilyladenosine (0.50 mmol, 379 mg, 1.2 ml THF) was added dropwise over a period of 15 min. After 30 min of stirring at room temperature, a THF solution of 2',3'- O -di- t -butyldimethylsilyluridine (0.40 mmol, 189 mg, 1.5 ml THF) was added and the reaction mixture stirred for 1 h at room temperature. Finally, a solution of iodine (0.1 M) in THF/water (3:1.5; without any pyridine) was added to oxidize the phosphite triester intermediate. After 5 min, the reaction mixture was dissolved in chloroform (50 ml) and washed with 5% sodium bisulfite aqueous solution (5 ml). The organic phase was washed with water (3 * 50 ml) to remove collidine, dried (anhydrous sodium sulfate), filtered and concentrated. The crude product was purified on a silica gel column by elution with ethyl acetate/chloroform, gradient 25-50% ethyl acetate. The pure product ApU 1a was obtained in 84% yield (454 mg). ApU 1a was stirred with triethylamine/acetonitrile (4:6 v/v) for 90 min at room temperature to afford, after evaporation under reduced pressure, ApU 1b (the reaction was monitored by tlc which indicated quantitative conversion of 1a into the more polar product 1b ) ( 14 ).


Figure 1 . Structures of the various linear and branched, di- and trinucleotides synthesized specifically for this study.

Physical properties of 1a . TLC, ethyl acetate, R f = 0.80; methanol/chloroform 0.5:9.5 (two developments), R f = 0.35. UV (EtOH, nm): [lambda] max, 236, 262sh, 278; [lambda] min, 248 31 P NMR (CDCl 3 ): -1.15, -1.28 p.p.m. (upfield relative to 85% H 3 PO 4 , external capillary). 1 H NMR (CDCl 3 , 500 MHz, [delta] p.p.m.), anomeric protons: 6.095 (isomer 1), 6.128 (isomer 2), 1:3 ratio.

Physical properties of 1b . TLC, methanol/methylene chloride 1:9, R f = 0.15. UV (EtOH, nm): [lambda] max, 231,275; [lambda] min, 228,249. 31 P NMR (CDCl 3 ): -0.43 p.p.m. (upfield relative to 85% H 3 PO 4 , external capillary). 1 H NMR (CDCl 3 , 500 MHz, [delta] p.p.m.), anomeric proton: 6.220. Synthesis of ApU 2 , ApUpU 3 and branched A(pU)pU 4 attached to CPG . The structures of 2 , 3 and 4 are illustrated in Figure 1 A. The required 5'-MMT-3'-silylated uridine-CPG support was prepared as previously described ( 15 ). Syntheses of linear and branched oligomers were conducted via the silyl-phosphoramidite method as described ( 16 , 17 ). Ancillary reagents for automated synthesis (Applied Biosystem DNA synthesizer, Model 381A) were prepared as follows: (a) coupling reagent: 0.5 M solution of tetrazole in acetonitrile; (b) capping: cap A, 10% acetic anhydride/10% 2,6-lutidine/THF and cap B, 16% N -methylimidazole/THF; (c) oxidation: 0.1 M iodine in THF/pyridine/water (75:20:2 v/v/v); de-methoxytritylation: 5% trichloroacetic acid/ dichloromethane. Syntheses were conducted on a 1 [mu]mol scale and repeated (4*) to obtain ~150 mg of each solid support (total: 4 [mu]mol of bound oligomer). Average coupling efficiencies of ribo-U and ribo-A3'- amidites were circa 98.3% and 95.1%, respectively. Coupling of ribo-A2',3'-bisphosphoramidite solution (0.03 M) onto CPG-ribo-U (30 [mu]mol/g) yielded ~70% of the expected branched trinucleoside diphosphate A(pU)pU, 4 ( 18 ). A small portion of each support was deprotected ( 16 , 17 ) and analyzed by HPLC and gel electrophoresis (UV shadowing) to confirm the efficiency of synthesis and the purity (~90% in each case) (HPLC conditions: reverse-phase Whatman Partisil ODS-2 column; solvent A: 20 mM KH 2 PO 4 , with a linear gradient 0-50% solvent B: methanol, over 25 min, 1.5 ml/min flow, room temperature). Synthesis of TpoTpoT 5 , TpsTpoT 6 and TpsTpsT 7 attached to CPG. These oligomers, the structures of which are illustrated in Figure 1 B, were synthesized on an Applied Biosystem DNA synthesizer (Model 381A) via the phosphoramidite method (see above). Coupling yields were 96-98% (trityl assay method). For the preparation of TpsTpsT, the sulfurizing reagent tetraethylthiuram disulfide (TETD) ( 19 ), obtained from Applied Biosystems, was used in place of the iodine oxidizing reagent. The CPG beads were allowed to react with TEDT for 10 min at room temperature and then washed extensively with acetonitrile. In the case of TpsTpoT, the iodine solution was replaced by TETD reagent after the introduction of the first internucleotide (`po') linkage. Four micromoles (~150 mg) of oligomer-bound CPG were obtained in each case for 31 P NMR analysis. HPLC and gel electrophoresis of the products cleaved from the support showed that, as expected, the deprotected, crude phosphorothioate samples were composed of a mixture of diastereomers, of ~90% purity. Synthesis of TpoT 8 via t-butyl hydroperoxide oxidation. TpoT-CPG cyanoethylphosphite triester was prepared by coupling DMT-T cyanoethylphosphoramidite reagent/tetrazole in acetonitrile to T-CPG (5 * 1 [mu]mol). The solid support was then capped with acetic anhydride/2,6-lutidine/ N -Me imidazole/THF (5 min, room temperature). A solution containing t -butyl hydroperoxide (0.5 M, 6 ml) was drawn into a synthesis column containing TpT-CPG (5 [mu]mol) phosphite triester ( 20 ). After 3 min the solution was expelled and the solid washed with dichloromethane followed by ether, and dried under vacuum. A small portion of the support was detritylated to determine the extent of coupling (~100%).

NMR spectroscopy

Solid state NMR spectra were acquired using a Chemagnetics CMX300 spectrometer equipped with a Chemagnetics MAS probe doubly tuned to the resonance frequencies of phosphorus-31 (121.25 MHz) and hydrogen-1 (299.53 MHz). Samples consisting of 100-125 mg of controlled-pore glass bearing 4-5 [mu]mol oligomer were spun in a 7.5 mm o.d. zirconium rotor at a spinning rate of 6000 Hz. A single contact cross polarization technique was employed using a contact time of 2 ms with proton decoupling during the acquisition period. The proton radio field strength was 50 000 Hz. The magic angle was set using the resonance signal from zinc(II) bis( O , O '-diethyldithiophosphate) ( 21 ). Spectra were acquired using a sweep width of 100 kHz, a data size of 2 kHz, and a recycle delay of 2 s. Adequate signal-to-noise was generally achieved upon averaging ~32 000 transients (~18 h acquisition time). All chemical shifts were referenced to external 85% H 3 PO 4 .

RESULTS AND DISCUSSION

Solid state 31 P NMR of free oligonucleotides

Before we describe the results of 31 P CPMAS NMR studies of nucleotides bound to CPG, it is informative to examine the spectra of similar compounds free of CPG, but likewise in the solid state. Since large quantities of such nucleotides are readily available, the spinning rotor of the MAS probe may be filled to contain upwards of a millimole of phosphorus, provided no CPG is present. Such compounds serve, therefore, the dual functions of acting as convenient references for the optimization of cross-polarization and signal acquisition conditions, and providing a perspective on the 31 P NMR spectra of nucleotides bound to CPG. The cross-polarization contact time (2 ms) and the relaxation delay (2 s) used throughout these measurements were chosen by optimizing such spectra.

Figure 2 shows cross-polarization 31 P NMR spectra of a dry powder of the dinucleotides ApU 1a and ApU 1b acquired under both static and MAS conditions. In this instance the 3' position of the ribose sugar of adenosine is linked via a phosphate bridge to the 5' position of the ribose sugar of uridine. Results are shown for the phosphate with ( 1a ) and without ( 1b ) its cyanoethyl (CNE) protecting group. During the course of a solid-phase oligonucleotide synthesis, the CNE-protecting group blocks the non-bridging phosphate oxygen from undergoing chemical modification, and is removed just prior to the release of the completed oligonucleotide from the solid support.


Figure 2 . 31 P CP NMR spectra of a dry powder of the dinucleotide ApU 1 not attached to controlled pore glass. Top two spectra: CNE-protecting group attached 1a , static and MAS conditions. Bottom two spectra: CNE-protecting group removed 1b , static and MAS conditions. The static spectra were acquired using cross-polarization as described in the text, but with a spin echo (tau = 30 [mu]s) technique to avoid ring-down problems. The principal components of the chemical shift tensor, [sigma] 11 , [sigma] 22 and [sigma] 33 , are indicated. The MAS spectra ([omega] r = 4500 Hz) were also acquired using cross-polarization, but without any spin echo refocusing. The spinning side bands (SSBs) are indicated with asterisks while the isotropic resonances are indicated as [delta] 0 .

The broad 31 P NMR spectra obtained for dry powdered samples of 1a and 1b under static conditions arise from the fact that the chemical shielding experienced by a nucleus in a magnetic field is a function of both isotropic (i.e. orientation-independent) and anisotropic (i.e. orientation-dependent) interactions between the nuclear spin magnetic moment and the magnetic field. In liquids the rapid isotropic molecular motions average the anisotropic interactions to zero, leaving only the isotropic interactions. Consequently, in the NMR spectrum of a liquid sample the resonance lines are narrow and the frequency at which a resonance line is observed is a function of the isotropic chemical shift only. In solids both the isotropic and the anisotropic interactions come into play. Consequently, in the NMR spectrum of a solid sample the frequency at which a resonance line is observed is a function of the orientation of that nucleus with respect to the magnetic field, according to

[omega] = [omega] 0 (1 - [delta] 0 ) + [omega] 0 (1/2)([sigma] 33 - [delta] 0 )(3 cos 2 [theta] - 1 - [eta]sin 2 [theta] cos 2 [Phi] ) 1

where [omega] = 2'[pi][nu] is the observed frequency, [omega] 0 is the Larmor frequency, and [delta] 0 is the isotropic chemical shielding. The anisotropic chemical shielding is described in terms of a second-order tensor having principal components [sigma] 11 , [sigma] 22 and [sigma] 33 . The coordinate system for this tensor is fixed with respect to the molecular geometry. The angles [theta] and [Phi] are the colatitude and azimuthal polar angles, respectively, describing the orientation of the chemical shift tensor with respect to the magnetic field direction. The asymmetry parameter [eta] is defined such that [eta] = ([sigma] 22 - [sigma] 11 ) /[sigma] 33 . The anisotropic chemical shift tensor elements are defined such that [brvbar][sigma] 33 [brvbar] >= [brvbar][sigma] 22 [brvbar] >= [brvbar][sigma] 11 [brvbar].

In a solid powder sample all orientations of the chemical shift tensor with respect to the magnetic field are possible and the NMR spectrum consist of a superposition of resonance lines from individual spins at individual orientations. The result is a so-called powder pattern lineshape in which the values of the principal components of the chemical shift tensor appear as inflection points. For instance, in Figure 2 the static 31 P NMR spectrum (i.e. in the absence of MAS but employing CP) for both 1a and 1b consists of a powder pattern lineshape from which the values of the principal chemical shift tensor components are readily measured, as shown by the arrows indicating the frequencies of [sigma] 11 , [sigma] 22 and [sigma] 33 . Table 1 lists the values of the principal chemical shift tensor components measured for these and all other compounds studied here. The overall shape of such powder spectra is defined in terms of two quantities: the chemical shift anisotropy and the asymmetry parameter. The chemical shift anisotropy corresponds to the frequency spread in the spectrum, [brvbar][sigma] 33 - [sigma] 11 [brvbar], and the values measured for compounds 1a and 1b are typical of phosphate di- and triesters in the solid state ( 22 , 23 ). Note that removing the CNE-protecting group has little effect on the size of the chemical shift anisotropy. The asymmetry parameter, [eta], was defined above and one observes that a major effect of removing the CNE-protecting group is to increase the asymmetry parameter, as is readily apparent if one compares the values of [sigma] 11 versus [sigma] 22 in the static 31 P NMR spectra of 1a versus 1b in Figure 2 . Converting a neutral CNE-protected phosphotriester to an anionic phosphodiester decreases the symmetry of the electronic charge distribution about the phosphorus nucleus. This will cause the asymmetry parameter to increase. Any deeper interpretation of the spectral changes observed upon removal of the CNE-protecting group requires information regarding the orientation of the principal tensor components relative to the molecular geometry. This information cannot be obtained from powder NMR spectra alone. Instead, one needs to investigate the orientation dependence of the NMR spectra of single crystals of the species of interest.

Table 1 31 P NMR isotropic chemical shifts and anisotropic chemical shift tensor elements for free oligonucleotides and oligonucleotides bound to CPG
Oligonucleotide

[delta] 0

[sigma] 11

[sigma] 22

[sigma] 33

[brvbar][sigma] 33 - [sigma] 11 [brvbar]

[eta]

CPG Free

1 ApU static ( 1a )

-2.0

71

52

-122

193

0.16

+TEA ( 1b )

-2.0

86

26

-112

198

0.53

ApU MAS ( 1a )

-2.0

71

53

-122

193

0.16

+TEA ( 1b )

-2.0

86

25

-112

198

0.53

CPG Bound

2 ApU

-2.0

84

26

-110

194

0.53

+TEA

-2.2

85

25

-111

196

0.54

3 ApUpU

-2.0

90

25

-115

205

0.56

+TEA

-2.1

88

25

-114

202

0.55

4 A(2'pU)3'pU

-2.0

89

27

-116

205

0.53

+TEA

-2.2

88

26

-116

204

0.53

5 TpoTpoT

-2.0

93

24

-117

210

0.59

+TEA

-2.1

87

25

-113

200

0.55

6 TpsTpsT (i)

66.0

102

54

-157

259

0.31

(ii)

56.0

95

38

-132

227.0

0.43

+TEA

56.0

92

38

-130

222

0.41

7 TpsTpoT (po)

-2.0

85

30

-115

200

0.48

+TEA

-2.0

90

25

-116

206

0.56

(ps)

66.0

104

55

-160

264

0.30

+TEA

56.0

91

32

-123

214

0.48

8 TpT I 2 oxidized

-2.0

80

27

-106

186

0.50

t -BuOOH oxidized

-2.0

81

30

-110

191

0.46

All chemical shifts are in parts per million (p.p.m.) referenced to 85% H 3 PO 4 . Anisotropic chemical shift tensor elements are defined such that [brvbar][sigma] 33 [brvbar] >= [brvbar][sigma] 22 [brvbar] >= [brvbar][sigma] 11 [brvbar] where [sigma] ii = ([delta] ii - [delta] 0 ) and [delta] 0 and [delta] ii are the observed isotropic and anisotropic chemical shifts. The asymmetry parameter is defined as [eta] = ([sigma] 22 - [sigma] 11 )/[sigma] 33 .

When such solid powder samples are spun rapidly at the so-called magic angle, the powder pattern spectrum breaks up into a series of spinning side bands (SSBs) plus the isotropic chemical shift resonance line. The SSBs are separated from the isotropic resonance line by multiples of the spinning rate [omega] r . If the spinning rate exceeds the size of the interaction ([sigma] 33 - [delta] 0 ) then only the isotropic chemical shift resonance line remains visible. MAS produces a large enhancement in signal-to-noise because the entire spectral intensity is now focussed under a few narrow resonance lines rather than spread out over a range of frequencies. In the 31 P NMR spectra of solid powders of the same two dinucleotides, 1a and 1b , again in Figure 2 , under MAS conditions ([omega] r = 4500 Hz) one observes an isotropic resonance line at [delta] 0 = -2.0 p.p.m. accompanied by a series of SSBs. The isotropic resonance line is identified using the fact that its frequency remains constant at different sample spinning rates, while the positions of the SSBs (indicated by asterisks in the figure) are directly a function of the rate of sample spinning.

The isotropic chemical shifts, [delta] 0 , for 1a and 1b (see Table 1 for details) are typical of phosphate di- and triesters in general and oligonucleotides in particular ( 22 - 24 ). The presence or absence of the CNE-protecting group has little impact on [delta] 0 , consistent with the observed [Delta][delta] 0 in solution of ~0.6-0.8 p.p.m. (see Experimental). The isotropic chemical shift of phosphorus is known to be sensitive to the phosphorus-oxygen bond angles, and distortions occurring in the solid state can lead to differences in the observed isotropic chemical shift positions for the identical compound in the solid versus the solution state 31 P NMR spectrum ( 25 ). Since the isotropic chemical shifts observed here in the solid state are similar to those reported for identical compounds in solution, we conclude that any bond angle distortions in the solid are slight.

It is also possible to extract the anisotropic chemical shift information (i.e. the values of the individual phosphorus chemical shift tensor components) from such 31 P CPMAS NMR spectra using the graphical method of Herzfeld and Berger ( 26 ). This method relies on measuring the intensities and frequencies of the SSBs versus the isotropic resonance line. For example, in the 31 P CPMAS NMR spectra of 1a versus 1b in Figure 2 , it is evident that the ratio of the intensity of the (n +1) SSB intensity to that of the isotropic resonance line is very different in the two compounds. (The n +1 SSB is immediately downfield of the isotropic resonance line.) This is a manifestation of the different asymmetry parameters characterizing the chemical shift tensor in the two cases. When the intensities of all the SSBs are taken into account for the 31 P CPMAS NMR spectra of ApU 1a and 1b , and the analysis of Herzfeld and Berger ( 26 ) is applied, one obtains an excellent agreement between the values of the chemical shift tensor components obtained under static versus MAS conditions (see Table 1 ). Therefore, both isotropic and anisotropic chemical shift parameters can be obtained from 31 P CPMAS NMR spectra. This is an important point because, as will be described below, for di- and tri-nucleotides attached to a solid support it is not possible to acquire static 31 P NMR spectra in a reasonable length of time. Thus, one has no choice but to employ MAS.

Solid state 31 P NMR of oligonucleotides bound to CPG

When attempting to observe the 31 P NMR spectrum of short oligonucleotides bound to CPG, the bulk of the sample volume consists of the solid-phase support itself. Consequently, the maximum phosphorus content of any one sample is only 5 [mu]mol for a monophosphate dinucleoside, and MAS becomes an absolute necessity for obtaining reasonable signal-to-noise spectra in reasonable lengths of time. Figure 3 shows representative 31 P CPMAS NMR spectra of selected di- and tri-nucleotides bound to CPG, after removal of the 5'-trityl protecting group. The top spectrum was obtained with the dinucleotide ApU 2 . As with compound 1a , the internucleotide linkage is 3'-5', but the 2' position of the uridine ribose sugar is now linked via a long-chain alkylamine spacer to the surface of the CPG support (Fig. 1 ). The middle and bottom spectra in Figure 3 were obtained with the linear trinucleoside diphosphate (`trinucleotide') ApUpU 3 and the branched `trinucleotide' A(2'pU)3'pU 4 , respectively, both attached to CPG via a long-chain alkylamine linkage to the 2' position of the ribose ring of uridine. In the linear trinucleotide, both phosphodiesters are involved in 3'-5' links to the adjoining ribose sugars. In the branched trinucleotide, one phosphate bridge links the 3' position of the adenosine ribose ring to the 5' position of the CPG-attached uridine ribose ring, while another links the 2' position of the adenosine ribose to the 5' position of a second uridine ribose (see Fig. 1 for structural details).


Figure 3 . 31 P CPMAS NMR spectra of linear and branched di- and trinucleotides attached to a CPG solid-phase support. Top spectrum: ApU 2 . Middle spectrum: ApUpU 3 . Bottom spectrum: A(2'pU)3'pU 4 . All cases [omega] r = 6200 Hz. The SSBs are indicated with asterisks while the isotropic resonances are indicated with arrows.

In Figure 3 the 31 P CPMAS NMR spectra of 2 , 3 and 4 each consist of an isotropic phosphorus resonance line ([delta] 0 = -2.0 p.p.m.) plus a series of SSBs ([omega] r = 6200 Hz). The isotropic resonances are indicated with an arrow while the SSBs are indicated with asterisks. The chemical shift of the isotropic resonance line in all three cases is typical of a phosphate phosphorus (Table 1 ), and is virtually identical to that of the dinucleotides ApU 1a or 1b not bound to CPG. It follows that the oxidation step in the oligonucleotide synthetic cycle, from the phosphite to the phosphate triester, occurs to completion in situ , since a phosphite triester is expected to manifest an isotropic chemical shift in the region of 140 p.p.m. ( 22 , 23 ). Moreover, it appears that the presence ( 1a and 1b ) or absence ( 2 , 3 and 4 ) of the monomethoxytrityl protecting group on the ribose ring has no influence on the observed isotropic chemical shift.

In each case in Figure 3 , only a single isotropic 31 P NMR resonance line is resolved, so that it is not possible to differentiate between di- and trinucleotides, nor between linear and branched trinucleotides, on the basis of their 31 P NMR isotropic chemical shifts when bound to CPG. Any difference in chemical shift between the two 3'-5' linked phosphodiesters in the linear trinucleotide is expected to be quite small ( 24 ). As for the branched trinucleotide, it is known from 31 P NMR measurements in solution that the isotropic chemical shift of 2'-5' linked phosphodiesters occurs about 0.94 p.p.m. towards higher field than that of 3'-5' linked phosphodiesters ( 24 ). The width-at-half-height ([Delta][nu] 1/2 ) of the isotropic resonance lines in the solid state spectra in Figure 3 is typically ~75 Hz. This rather broad linewidth probably reflects a distribution of bond angles amongst the individual phosphoesters-a distribution which cannot average out due to the large energy barriers to rotation in the solid state. When the differences in two isotropic chemical shifts are small relative to the linewidth, they will not be resolved. It may also occur that bond angle distortions in the solid state, relative to the solution state, render the isotropic chemical shifts of 2'5' and 3'5' phosphodiesters into similar values. In either case, it would appear that 31 P CPMAS NMR spectroscopy has limited utility for differentiating such structural details in the solid state.

The chemical shift anisotropy and the asymmetry parameters for the dinucleotide, 2 , and the linear, 3 , and branched, 4 , trinucleotides (Table 1 ) all correspond closely with the values measured for ApU lacking its CNE-protecting group (i.e. 1b ). In fact, no change in spectral features was observed after the supports were treated with triethylamine solution for 90 min, conditions known to selectively cleave CNE groups ( 14 ). Note that the principal components of the chemical shift tensor, and the asymmetry parameter in particular, are quite sensitive to the presence or absence of the CNE-protecting group, as shown in Figure 2 and detailed in Table 1 . Thus, 31 P CPMAS NMR indicates that CNE-protecting groups are removed from the phosphate during the course of chain assembly.

The size of the anisotropic chemical shifts indicate further that the phosphate group experiences no large amplitude motions when the nucleotides are bound to CPG and there is no solvent present. As for any difference in the mobilities of the two phosphate diesters in ApUpU 3 , proximal and distal to the CPG, these would have to be rather large before they could be differentiated, given the MAS technique employed and the levels of phosphorus available in these samples. The fact is that the isotropic chemical shifts of these two phosphorus atoms are similar, and the isotropic chemical shift will not be affected by motional averaging. The chemical shift anisotropy would be reduced by motional averaging, and this would affect the intensities of the SSBs, but not their frequencies. Small differences in motional averaging will affect primarily the intensities of those SSBs furthest in frequency from the isotropic chemical shift position. However, these very intensities are the lowest to begin with and have the highest uncertainty in quantification.

The spectra in Figure 4 demonstrate that 31 P CPMAS NMR spectroscopy is well suited for differentiating large changes in phosphorus chemistry, such as the substitution of a phosphorothioate for a phosphate moiety. Here we compare three linear thymidine trinucleotides: one containing only phosphate linkages TpoTpoT, 5 , a second containing only phosphorothioate linkages TpsTpsT, 6 , and a third containing both phosphate and phosphorothioate linkages TpsTpoT, 7 . In the preparation of TpsTpsT, 6 , the sulfurizing reagent tetraethylthiuram disulfide (TETD/acetonitrile) ( 19 ) was used in place of the iodine oxidizing reagent (see Experimental). In the synthesis of TpsTpoT, 7 , the iodine solution was replaced by TETD reagent after the introduction of the first phosphate `po' linkage. Spectra were recorded before (Fig. 4 A) and after (Fig. 4 B) treating the supports with triethylamine.


Figure 4 . 31 P CPMAS NMR spectra of phosphate and phosphorothioate thymidine trinucleotides attached to a CPG solid-phase support. ( A ) Before TEA treatment, ( B ) after TEA treatment. Top spectrum: TpoTpoT 5 . Middle spectrum: TpsTpsT 6 . Bottom spectrum: TpsTpoT 7 . All cases [omega] r = 6400 Hz, except 7 after TEA treatment where [omega] r = 5200 Hz. The SSBs are indicated with asterisks while the isotropic resonance lines are indicated with arrows.

The spectrum of the trinucleotide containing only phosphate linkages, TpoTpoT, 5 , is virtually identical before and after treatment with triethylamine (top spectra in parts A and B of Fig. 4 ). In both cases one observes a single isotropic resonance line (denoted by an arrow in the figure) accompanied by SSBs (denoted by asterisks in the figure). The isotropic resonance line displays a chemical shift consistent with phosphate phosphorus ( 22 , 23 ). Analysis of the relative intensities and positions of the isotropic resonance and the SSBs by the Herzfeld and Berger method ( 26 ) reveals that the asymmetry parameter of TpoTpoT, 5 , both before and after treatment of triethylamine, is rather similar to that of the dinucleotide ApU, 1b , lacking a CNE group as discussed above. This indicates again that the CNE-protecting group can be removed prior to any treatment with triethylamine. The fact that the 31 P isotropic chemical shifts of the thymidine nucleotides are nearly identical to those of the adenosine and uridine nucleotides above, is a further indication that in the solid state chemical details at points distant from the phosphorus atom itself are not manifested in the spectrum.

The spectrum of the trinucleotide containing only phosphorothioate linkages, TpsTpsT, 6 , prior to treatment with triethylamine (middle spectrum in Fig. 4 A), consists of two distinct isotropic resonance lines (66.0 and 56.0 p.p.m., respectively) and two associated patterns of SSBs. Summing the intensities of the isotropic resonance line plus SSBs yields an intensity ratio of approximately 3:2 for the 66.0 p.p.m. versus 56.0 p.p.m. component. Herzfeld and Berger analysis ( 26 ), shows that the 66.0 p.p.m. component has an asymmetry parameter of 0.31 while the 56.00 p.p.m. component has an asymmetry parameter of 0.43. Again, since no resonance line is observed in the phosphite region of the spectrum, one concludes that the oxidation step, this time with tetraethylthiuram disulfide, has occurred to completion. When one treats the CPG-bound TpsTpsT, 6 , with triethylamine one obtains the middle spectrum in Figure 4 B, which shows only a single isotropic resonance line at 56.0 p.p.m. and an asymmetry parameter of 0.41.

We suggest that the two components in the 31 P CPMAS NMR spectrum of TpsTpsT, 6 , prior to triethylamine treatment correspond to populations of 6 with and without the CNE- protecting group. First, evidence in the literature indicates that converting a triester to a diester results in a large upfield shift in the isotropic chemical shift of phosphorothioates, but has little effect on phosphates. For instance, (EtO) 3 P(S) has an isotropic chemical shift of 68.1 p.p.m. ( 27 ), but loss of one ethyl ester to produce (EtO) 2 P(S)O - yields an isotropic chemical shift of 48.2 p.p.m. ( 28 ). Other examples abound and have been compiled by Tebby ( 23 ). In contrast, the isotropic chemical shift of the phosphotriester (EtO) 3 P(O) equals -1.2 p.p.m. ( 29 ) which shifts to only -1.8 p.p.m. upon loss of one ethyl ester to produce (EtO) 2 P(O)O - ( 30 ). Second, the asymmetry parameter for the 56.0 p.p.m. isotropic chemical shift phosphorothioate component is significantly larger than that of the 66.0 p.p.m. isotropic chemical shift phosphorothioate component. The same trend of an increase in the asymmetry parameter of the chemical shift tensor components was observed upon removal of the CNE-protecting group for the case of 1a versus 1b as discussed above. Third, upon treatment of TpsTpsT, 6 , with triethylamine to effect CNE-protecting group removal, the only spectral component remaining was that with the isotropic chemical shift of 56.0 p.p.m., while its asymmetry parameter was nearly identical to that of the 56.0 p.p.m. component present prior to triethylamine treatment.

In the spectrum of the mixed phosphate-phosphorothioate trinucleotide TpsTpoT, 7 , prior to treatment with triethylamine, as shown at the bottom of Figure 4 A, one observes two isotropic resonance lines and two overlapping sets of SSBs. One isotropic resonance line occurs at -2.0 p.p.m. and Herzfeld and Berger analysis of its SSBs ( 26 ) yields an asymmetry parameter equal to 0.48, consistent with a phosphate phosphorus lacking its CNE-protecting group. The second isotropic resonance occurs at 66.0 p.p.m., and Herzfeld and Berger analysis of its SSBs ( 26 ) yields an asymmetry parameter equal to 0.30, consistent with a phosphorothioate phosphorus having its CNE-protecting group intact. Thus, oxidation of the first internucleotide linker to `po' with the iodine reagent apparently removes CNE-protecting groups, while oxidation of the second internucleotide linker to `ps' with TETD reagent leaves the CNE-protecting groups at least partially intact.

In the spectra of TpsTpoT 7 prior to TEA treatment (bottom spectrum in Fig. 4 A), it appears that the total summed intensity of the isotropic chemical shift line plus SSBs is lower for the phosphorothioate versus phosphate spectral component. We note that caution must be exercised before relying on intensities from CPMAS NMR spectra for quantification. First, the cross-polarization conditions employed here were optimized for phosphate esters, and are not necessarily optimal for phosphorothioate esters. Thus, any disagreement between the relative intensities of phosphate versus phosphorothioate resonances might be purely fortuitous. That being said, the similarities in the proton populations surrounding the two phosphorus types provides little reason to anticipate large differences in their rates of cross-polarization, or relaxation, or intensity enhancements under cross-polarization conditions. Second, the rate of sample spinning in this particular instance places the first spinning side band from the phosphate resonance at ~56.0 p.p.m., i.e. virtually overlapping the expected resonance frequency of any phosphorothioates lacking a CNE-protecting group. Third, it is likely that a portion of the phosphorothioates have lost their CNE-protecting groups, as above for TpsTpsT, 6 , prior to TEA treatment. This will split each resonance into two components and complicate quantification of intensities. In summary, the phosphorothioate versus phosphate levels cannot be stated unequivocally from this particular spectrum.

The bottom spectrum of Figure 4 B show the results of triethylamine treatment of the trinucleotide TpsTpoT, 7 on its 31 P CPMAS NMR spectrum. After TEA treatment, two spectral components are again evident. One displays an isotropic chemical shift equal to -2.0 p.p.m. and an asymmetry parameter of 0.56, consistent with a phosphate phosphorus lacking any CNE-protecting group. The second displays an isotropic chemical shift equal to 56.0 p.p.m. and an asymmetry parameter of 0.48, consistent with a phosphorothioate phosphorus lacking any CNE-protecting group. Note that in this spectrum the spinning frequency was slowed to 5200 Hz (versus 6400 Hz for the others in this figure) in order to avoid overlap of SSBs from the phosphate and phosphorothioate powder patterns. The sum of the integrated areas of the SSBs plus the isotropic resonance line is now approximately the same for the phosphate and phosphorothioate resonances. This suggests, but does not prove (see qualifying statements above), that virtually all available sites (5'-hydroxyls) were coupled during the second round of synthesis which added the terminal (Tps) residue. This conclusion is supported by the coupling yield efficiencies (~98%) measured by the trityl assay method (see Experimental).

Concerning removal of the CNE-protecting group

The 31 P CPMAS results obtained here indicate that this technique is particularly sensitive to the presence or absence of the CNE-protecting group. For phosphate phosphorus, removal of the CNE-protecting group produces a large increase in the spectral asymmetry parameter [eta], but only small changes in the isotropic chemical shift. For phosphorothioate phosphorus, removal of the CNE-protecting group produces a relatively small increase in the value of [eta], but a large upfield shift in the isotropic chemical shift. The results suggest further that removal of CNE-protecting groups can occur during the course of oligonucleotide synthesis prior to their deliberate removal. This is somewhat surprising since it has generally been assumed that oligomers prepared on CPG supports retain CNE-protecting groups prior to deprotection with a base (usually concentrated aqueous ammonia). In principle, CNE groups can be removed in the course of the capping reaction, or subsequent to capping during the iodine/pyridine/water oxidation reaction ( 31 ). We conducted model experiments in solution which show that ApU phosphate-triester ( 1a ) is stable to the oxidizing iodine/pyridine/water reagent (24 h) and to the capping acetic anhydride/ N -methylimidazole solution (30 min) under conditions used in the solid-phase synthesis. Also, a sample of TpU at the phosphite triester stage was stable, in solution, to the capping reagent as judged by liquid state 31 P NMR ([delta] 140.1 and 140.3 p.p.m., CDCl 3 ). Addition of iodine/pyridine/water to this sample produced a TpU phosphate triester, demonstrating that, when free in solution, a nucleoside cyanoethyl phosphite or phosphate triester is resistant to the reagents present in the capping or oxidation solutions. This suggests that some surface property of the support itself contributes to the removal of CNE groups.

Letsinger and co-workers ( 20 ) observed that partial loss of a P-O methyl protecting group occurred during iodine oxidation of an oligonucleotide phosphite bond to CPG, and that the loss of the protecting group could be avoided by substituting tertiary butyl peroxide ( t -BuOOH) as the oxidizing agent. We tested whether this strategy could be employed to avoid removal of the CNE-protecting group in our syntheses by comparing the 31 P CPMAS NMR spectra for TpT bound to CPG 8 for the case of iodine versus t -BuOOH oxidation. The results of the spectral analysis are shown in Table 1 and they indicate that the CNE-protecting group is lost during the course of the solid phase synthesis regardless of the method of oxidation. Again, this suggests that some property of the CPG surface environment encourages removal of the CNE-protecting group. Further investigations of these effects are clearly warranted. It would be interesting to monitor the 13 C CPMAS NMR spectrum of 13 C-labelled cyanoethyl versus methyl protecting groups to directly observe the point at which they are removed, rather than indirectly through 31 P NMR.

SUMMARY AND CONCLUSIONS

Solid-state CPMAS 31 P NMR spectroscopy of oligonucleotides attached to a solid-phase support provides specific information on the chemistry of the phosphorus atom located within the oligonucleotide backbone. The phosphorus oxidation state is readily differentiated, but the detailed configuration of the phosphorus-sugar linkages (2'5' versus 3'5') are beyond the present limits of resolution. Other large changes in the phosphorus chemistry, such as removal of the CNE-protecting group during chain assembly, or replacement of a phosphate with a phosphorothioate moiety, are readily accessible to characterization. It seems likely that, in the future, one will be able to employ CPMAS 31 P NMR to monitor individual steps during a solid phase oligonucleotide synthetic cycle in situ, and to assess the efficiency of sulfuration, and other modifications at phosphorus, such as those that are relevant in therapeutic (antisense) applications [for a recent review see ( 32 )].

ACKNOWLEDGEMENTS

This work was supported by operating grants from the National Science and Engineering Research Council (NSERC) of Canada to PMM and MJD.

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