Volume 12 Number 14 1984

Nucleic Acids Research

Tendendes of 31p chemical shifts changes in NMR spectra of nucleotide derivatives

A.V.Lebedev Institute of Organic Chemistry, Siberian Division of the Academy of Sciences of USSR, Lavrent'ev Prospect 9, Novosibirsk 630090, USSR A.I.Rezvukhin Institute of Therapy, Siberian Division of the Academy of Medical Sciences of USSR, Vladimirovsky spusk 2a, Novosibirsk 630003, USSR

Received 2 May 1984; Revised 26 June 1984; Accepted 3 July 1984

ABSTRACT 31P MMR chemical shifts of the selected mono- and oligonucleotide derivatives, including the compounds with P-N, P-C, P-S bonds and phosphite nucleotide anal3+ues have been presented. The influence of substituents upon chemical shifts has been discussed. The concrete examples of P chemical shifts data application in the field of nucleotide chemistry have been considered.
,

INTRODUCTION 31p NMR spectroscopy is widely used at present in the chemistry of nucleotides. Some important aspects of 31P NMR biological applications e.g. studies of the conformations of nucleotide derivatives, metabolism of the latter in cells and tissues in vivo e.a. have been considered in reviews [1-5].However no attempts have been made to generalize and systematize the information about 31P chemical shifts of nucleotide derivatives. So the investigation carried out in the present work has been connected with the establishment of the tendencies in the changes of 31P chemical shifts of mono- and oligonucleotide derivatives. The considered data are included the typical examples of 31p chemical shifts and seemed to be useful for characterization of different nucleotide derivatives by their 31P NMR spectra [6]. The reactive intermediates and incompletely characterized compounds are indicated in the Tables with a sign (V).
RESULTS
All the compounds have been classified into groups according to the chemical environment of the phosphorus atom (see Tables 1-16). The 31P chemical shifts are referenced to 85% HP04 and
I RL Press Limited, Oxford, England.
5547

Nucleic Acids Research

positive values mean downfield shifts of 31p sifnals. Only 31p NM spectra recorded with spin-spin decoupling H - P have been considered in this review. The following factors seem to be necessary taken into account for the correct analysis and interpretation of 31p MR spectra of nucleotide derivatives. Ribo- and 2'-deoxyriboderivatives. The deshielding effect of 2'-hydroxy group (-0.5 ppm) has ',een found to exist for internucleotide phosphates, 3',5'-cyclophosphates and terminal 3'phosphates of mono- and oligonucleotides. On the contrary, the chemical shifts of terminal 5'-phosphates have been shown to be practically insensitive to presence or absence of 2'-hydroxy group. Phosphate group Position.In all 2'-deoxyderivatives the terminal 5'-phosphates are less shielded than 3'-phosphates. In riboderivatives the chemical shifts of 5'- and 3'-phosphate groups are depending on the medium effects [7]. The nature of bases. The substantial influence of heterocyclic bases on 31p chemical shifts has been observed for nucleoside 3' ,5'-cyclophosphates [7]and internucleotide phosphates in homopolyribonucleotides [7,8] and oligonucleotides [9,10]. The noticeable difference of chemical shifts of nucleoside 5'-phosphates (either ribo- or 2'-deoxyribo) has been found especially at low pH. On the contrary, such difference for nucleoside 3'phosphate and 2',3'-cyclophosphate has not been almost detected [7,8] . The influence of medium. The chemical shifts values of phosphate monoester group in aqueous solutions depend on pH in ionization region of these group and change in -4 ppm interval.The perturbations in pH regions corresponding to ionization of nucleotide bases can be observed simultaneously on the titration curves. The presence of bivalent metal ions in the solution can also lead to chemical shifts changes of nucleotide derivatives. These results have been discussed in details in[7,8,11-16]. The 31P chemical shifts of phosphate esters have been shown to be also sensitive to solvent effects and temperature[15,16]. We have investigated the influence of organic solvents on chemical shifts of phosphorus atoms in triethylammonium salt of
5548

Table 1 31P chemical shifts of phosphate monoesters (OH)2P(O)(OR) 6 Medilum Ref Nc 6 0 Medium Ref. R R -bzdA(Ac) 0.6 Py,PyH 6 4 -dT(Ac) 1.2 Py,PyH 17-22 21 6 5 (MeOTr)daT- O .8 2 -andC (Ac ) 0 .7 _,_ __ a) b$ -ibdG(iB) 0.4 1j7 6 pdTp 23

_~ ~ ~
3

Nucleic Acids Research

-.ppm r. _-.1pp

pdTpdT(Ac) [61. This influence has been established to be less than-1 ppm both for phosphomonoester and phosphodiester group. The lack of correlation connecting 31P chemical shifts with dipole moments or dielectric constants of solvents has been found. Probably these small changes in chemical shifts have been provided by conformational peculiarities of molecules in a concrete solvent [15] The counterion effect can be estimated from the comparison of chemical shifts for pyridinium (0.7 and -1.25ppm) and triethylammonium salt (-0.1 and -2.35ppm) of pdTpdT(Ac) in
pyridine as a solvent [6] All the factors mentioned above determine in more or less degree the values of 31 P chemical shifts of nucleotide derivatives. Therefore the comparison of chemical shifts must be necessary made under identical or at least similar conditions. Phosphate monoesters (PME: mononucleotides and terminal phosphates of oligonucleotides, Table 1). The signals 31P of NE groups in pyridine solutions are observed in 1.2+0.lppm interval depending on the nature of base and phosphate group position (3' or 5'); in DMF solutions from -0.1 to -1.Oppm (compound

Phosphate diesters (PDE: monoesters of terminal phosphates of mono- and oligonucleotides, internucleotide phosphates, Table 2). The upfield shift of terminal phosphate signal (for nonaqueous solutions) by 1.5+2.6ppm has been observed at the formation of alkyl esters of mono- and oligonucleotides and also of internucleotide bond (except methyl esters indicating rather small upfield shift: 0.1+0.3ppm, cp. chemical shifts of the compound 1.4 with those of 2.2 and2.8). In the case of trichloro)THe rirst position or trie number or compound corresponds to the number of the Table; the second after the point to the number of compound in that Table.
5549

1.6 *).

Nucleic Acids Research

31

P chemical shifts of phosphate diesters (HO)P(O) (OR1) (OR2)

MeCNEtPh4-ClPh4-NO2 Ph dTpdTR | d -dT(Aci 0.9 -dT (Ac) -1.4 -dT (Ac) -5.6

Table 2

Medium

iv 2 3V
4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Py,PyH
H 0,pH7
-It_,,_

-,,-

Py

Ref. 17

25, 29

(Tr)dT0NEtpdT-**) (MeOTr)U(2'-Ac)(MeOTr)U (2' -Ac) -

-dT(Ac) -dT(Ac) -dT -dT(Ac) -dT(Ac)

(MeOTr)Ut(2'-Ac)(MeO) -Tr dT-

-5.1 -6.0 -0.94 -1 . 2*) -1.0 -dT(Ac) -0.85 -U(Ac)2 -2.05 -CNEt -2.01 -CH CCl -3.64

30
6 6

Pi,PyH+ _,,_
-",
-,-",

23 18,29 31 32
32

U(St)_
A(3')dA-

4-HPh-U -A -dA

Poly(U)

Poly(A) Poly(G) Poly(C)

-5.7 -0.49 -0.72 -1.11 -0.55 -0.77 -0.96

-1.11

-", 1,4-Dioxane D20,pD7.4

_,,-,,-

32 33 6 6 6
7 7 8 8

H20,pH7
_,, -,-,,_

*)-2.5ppm in DMF,PyH+ [231 **).-1 .2ppm (for 5'-cyan othylphosphate ester group) ethyl esters the 31p chemical shift change has been found to be -4ppm (cp. chemical shifts of compounds 1.5 and 2.12). The signals of mononucleotide phenyl esters (compounds 2.32.5) in comparison with alkyl ones (except methyl and trichloroethyl esters) have been observed to be shifted upfield (4-5 ppm). The presence of substituents in phenyl ring leads to substantial changes in the chemical shifts (cp. chemical shifts of compounds 2.3-2.5). The nature of heterocyclic bases and substituents in hydroxy and amino groups has been found to affect the chemical shifts in PDE. E.g. the chemical shifts of 2-cyanoethyl esters of pandC(-1.3ppm[24]), pbzdA(-1.5ppm[24]), pdTE1.3ppm [24]), pdT(Ac) (-1.4ppm[25]) are noticeable different. The individual 31p signals of internucleotide phosphates of oligonucleotides dCpdTpd ApdG[26], dTpdTpdGpdG[27], dCpdCpdApdA[27] pUpUpU[6], dApdTpdGpd CpdApdT[28]and pdGpdCpdGpdCpdGpdC1281have been also observed. The reliableassignments of 31p signals have been performed only in works126,271.
5550

Nucleic Acids Research

Table 3 chemical shifts of phosphate triesters (R10)(R20)P(O)(OR3)

31p
N0 1 2 3 4 5

Et7 (Tr)dTPh8 (Tr)dT9 4-ClPh(Tr)dT10 Ph(Tr)dT11 PhSC H (Tr)dT12 MeOTr)U(2'-Ac)-, CH Cf 1 MeOTr)U(21-Ac)-|CI 2CC13

R1CN-EtCNEtCNEtPh(Tr)dT(Tr)dT-

R Cd ' CNEt- -dT(Ac) 4-ClPh- -dT(Ac) Ph- -dT(Ac) CNEt- -dT(Ac) Me- -dT(Ac)
GIVE

RO

-dT(Ac) -dT(Ac) -dT(Ac) Ph-dT(Ac)

-2.69 -7.8 -7.7 _ -2.3 CNEt-4.43 -U(Ac)2 -3.84 -3.89

-2.5 Py -7.70 -7.88 Py -12.3 Py -2.55 -2.70 Py -0.75 -1.03 Py

77 di | Solvent Ref. -2.24'P -

-2.67 -7.4 -7.4 -13.0 -2.2 -4.38

*) * Py CH2C12 py PY PY By

29 29 29 31 6 6 29 30 36 37 32 6

*) in CHCl -EtOH (4:1) **) _7.3 ana -7.6ppm in

Py1351

Phosphate triesters (PTE: diesters of mononucleotides and terminal phosphates of oligonucleotides, esters of internucleotide phosphates, Table 3). The signals of phosphorus atoms in alkyl PTE has been shifted upfield (0.9+1.8ppm) in respect to alkyl PDE, except methyl PTE (shift is near zero) and of trichloroethyl PTE (1.8+2.4ppm upfield). The signals of monophenyl PTE have been shifted upfield (4-5ppm) in respect to alkyl PTE (cp. chemical shifts of compounds 3.5 and 3.8) and diphenyl ones (compound 3.10) have been shifted to c.a. l0ppm,i.e. the additivity of substituents effect is observed. The internucleotide phosphate esterification leads to diastereomers formation. The individual signals of the latter are often observed in 31p NMR spectra (e.g. compounds 3.3,3.5-3.9,3.11-3.13). For oligonucleotide derivatives the formation of diastereomers is typical for any substitutions at internucleotide phosphorus (see below). The chemical shifts difference of PTE diastereomers i&fd2-d can reach 0.4ppm (compound 3.8). The change of medium and solvents has practically no influence on the phosphorus chemical shifts of PTE. E.g. the chemical shifts for compound 3.9 in pyridine and methylene chloride differs on 0.lppm. Monoamides of Phosphate monoesters (PME monoamides: amides of mononucleotides and terminal phosphates of oligonucleotides, Table 4). PME monoamides obtained with primary (secondary) ami5551

Nucleic Acids Research

Tableof4 monoamides of 31 P chemical shifts phosphate monoesters (HO) (R1 )P(O) (OR2)
Nc
1 2 3

R2
dT-dT -dT -dT -dT -dT -dT(Ac) -dT

Medium

Ref.

H2NH NC" H

4 mo!p.iline5 piperidine6 PhNH7 PhCH NH8

EH

8.85 9.32 5.0

5.0 5.5 -1.0 6.5 -10.3 -11.1 -5.1 -6.7

10 11

imi'daiole-

triazolsPy-

DMAP+*)

-dT(Ac) -dT(Ac)

-dT(Ac)

D20, pD13 D20, pD13 Py _,-,,_ _,, _,,-,,

_,,_

38 38 39 39 39 39 40 39

-,,_ _,,_

17,18,20,22 41

*) 4-N

NLdimethylaminopyridine

nes can be considered as PDE analogues esteric oxygen being substituted by NH(or NR) group. In comparison with PDE the downfield chemical shifts of phosphorus atom in these compounds are observed (by 6-7ppm; cp. chemical shifts of compounds 2.2 and 4.7, 2.3 and 4.6). The substitution of alkyl group at nitrogen by phenyl one leads as in PDE case to upfield shift of 31p signal (5-6ppm; cp. chemical shifts of compounds 4.3 and

4.6).
The introduction of CH2 group between phenyl and nitrogen should be noticed to destroy the conjugation including phosphorus, nitrogen atoms and i-system of benzene ring. That effect leads to downfield 31p chemical shift (cp. chemical shifts of compounds 4.6 and 4.7). The compounds with nitrogen atom of P-N bond being included in aromatic sr-system can be considered as another type of PME monoamides. E.g. imidazolides and triazolides of mononucleotides (compounds 4.8 and 4.9) have 31p signals at -(10-11ppm) region and phosphorus signal of N-phosphorylpyridinium group of mono- and oligonucleotides is observed at -(5-7ppm)(compounds

4.10,4.11). Monoaides of phosphate diesters (PDE monoamides: monoamides of terminal phosphate esters and internucleotide phosphates,
Table 5). The absence of ionized phosphate group leads to several peculiarities of PDE monoamides in respect to BME monoami5552

Nucleic Acids Research

Table 5 31 P chemical shifts of monoamides of phosphate diesters (R1 0)(R2)P(O)(OR3)

__

R1

R2

Rc3d'
-dT 12.08 -dT(Ac) 10.85 11.0 -dT(Ac) 9.05 9.20 -dT(Ac) 2.8 2.6 -dT(Ac) 5.1 4.8 -Et 8.6 8.4 2-ClPh- -2.25 -2.75 -Et -7.25 -7.45 -Et -10.95 -11.10 4-ClPh 15.51 -15.60

olve Ref

10 [MeO)2Tr]dTT

(Tr)dTH2N(Tr)dTC6H NIi'iH4 (Tr)dT(C H )NHC(O)C6H 1N 5V (Tr)dT6l 1 6 (Tr)dT6'H6_H BhiH7 (MeOTr)dT8 (Tr)dTimidazole99 (Tr)dTtriazoletriazole*) 1,4-Dioxane

1 2 3

dT-

H2N-

D 0 38
By 43 *) 6 By 44 *) 6 *) 6

Py 42 Py 37

iy

42

*) 33

des. The comparison of 31p chemical shifts of PDE monoamides with alkylamide and anilide groups with corresponding PTE let us to conclude that the influence of nitrogen atom (or NH group) leads to downfield shift of 31p signal (10-13ppm; cp. chemical shifts of compounds 3.5 and 5.3, 3.8 and 5.4). The PDE derivatives containing N-phosphorylurea fragment can be considered formally as PDE monoamides (compound 5.5). The comparison of their chemical shifts with those of PTE shows that the substitution of -OAlk groups by -NR-C(O)-NHR ones leads to downfield 31P signal shift (7-8ppm; cp, chemical shifts of compounds 3.5 and 5.5). The 31P resonance region of PDE imidazolides (compound 5.8) is shifted in whole downfield in respect to that for PME imidazolides (compound 4.8), but the resonance region of PDE triazolides (e.g. compound 5.9) coincides practically with that of PME triazolides (e.g. compound 4.9). In the last case the substitution of nucleoside group by 4-chlorophenyl one leads (as in the case of PDE and PTE) to upfield 31P signal shift (4-5ppm; cp.chemical shifts of compounds 5.9 and 5.10). The individual signals of diastereomers can be observed practically in all cases of PDE monoamides. Diamides of phosphate monoesters (PME diamides: terminal phosphate diamides of mono- and oligonucleotides, Table 6). The comparison of chemical shifts for diphenyl ester of pdT(Ac) (compound 3.4) with dianilide derivatives of mono- and oligonuc5553

Nucleic Acids Research

Table 6 P chemical shifts of diamides of phosphate monoesters (R10) (R2)P(0) (R3)
NO 1 2v

R1

R2

R3

cf1

62
-

Solvent Ref .

3? 4
6

5V

ThIHPhIWH-dT(Ac) 2. 9 (Tr)dT- imidazole- imidazole- -11.7 (Tr)dT- imidazole- PhCH2NH2.75 2.88 -dT(Ac) imidazole- C H NH2.33 2.49

(Tr) dT- triazole-

?Tr) dT-

triazole-

PhCH2NH-

tBillole-

-16.8

-0.1 -0.4

_,,_ _,,-,,-,,-

_,,_

Py

40 6

6 6 6 6

leotides (compound 6.1) shows that the influence of two nitrogen atoms leads to the downfield 31p signal shift (15ppm). The chemical shifts values of compounds 3.4, 5.7 and 6.1 (or 3.4, 3.8, 5.4 and 6.1) shows that the substitution of one oxygen atom by nitrogen one leads to the downfield shift (9-lOppm) and substitution of the second oxygen 5-6ppm only. Hence, non-additivity of the effect of nitrogen atoms in PME diamides has been observed. In the case of mixed diamides (compounds 6.3, 6.4 and 6.6) and diimidazolide of (Tr)dTp (compound 6.2) the substituent effects are almost additive, what follows from the chemical shifts values for compounds 3.5, 5.5, 5.9, 6.6 and also 3.5, 5.8, 6.2 and triimidazolidephosphate (6'= -16.3ppm, [45]). Mixed anhydrides of nucleotides with carbon acids (Table 7). The formation of one anhydride bond with carbon acid can be noted to be accompanied with upfield shifts of the signals of both terminal and internucleotide phosphorus atoms (8-1Oppm; cp. chemical shifts of compounds 7.2 and 1.4). The formation

Table 7 chemical shifts of mixed anhydrides of nucleotides with carbon acids (R1 )(R20)P(O)OC(O)(R3)

31p

2v HO3'(MeOTr)dTO4v MsC(0)05v PhNH6t (Tr)dTO5554

R1 Ho_

R d"| -Me-8.6

-dT(Ac) -dT

-dT(Ac) -dT(Ac)

-Ms -Ms

-dT(Ac)

-Ms -Ms Ph-

-7.5 -9.0 -9.3 -16.8 -3.20 -3.47 -8.85 -9.10

d _

Solvent

Ref.
46 6 47

-Py -1n-,,_
-,-tl-

-,,_

6 6

29

Nucleic Acids Research

8 ~~~~~Tabl1e con8tants 31 of prophosphate p chemical shifts and aoubling
of

derivatives

nuoleotides
| R2

(R1)P,,(O)(OR2)OBPo(O) OR3)(R
R

Ref. z3. 1 1 z -10.84 -8.54b) 22.6 12 HO-11.26 -7.02a) 23.6 12 H2 -dT H0. 17-20 HO10.3 -10.3c) -dT(Ac) -dT(Ac) 03 -11.2 -16.8c) 17.0 18 HO4-NO Ph -dT(Ac) 04 0-11.8 -19.7c) 18.1 48 -A HO5' SC(M)- -A 0-10.8 -17.3a) F49 6 H-+ HO-A 1.29 -11.59a)20.0 50 Nic+7* 1 -11.2 -6.8 17.5 25 -dT (Ac) T(Ac) 0ph_rH-dT (Ac) dT (Ac) O-12.4 -1.2c) 18.5 25 '9**O H11NH-11.5 -5.15c) 16.4 19 CEN HC(O) -dT(Ac) T(Ac) HO 6 -11.5 -5.60 20 H-OA -0.8d) 51 11.3 1 H6H112- -dT(Ac) -dT(Ac HO- -11.2 -12.0c) 17.0 52 12 (Tr)dT11.2 -12.4 17.0 6 -dT(Ac) -dT(Ac) HO- -12.0 -16.4c) 17.0 25 13 * Ph-12.0 -16.7 17.0 6 1i0 Phr17.0 -12.2c) 16.6 25 147 Tr d1-d(Ac 29,53 (Tr dTO- -dT(Ac) dT(Ac)(Tr)dTO0 -13.8 l5v 35 6v 4-ClPhO- -dT(Ac) -dT(Ac)4-ClPh (19.0,19.2,19.5) 35 7ir (Tr)dTO- 4-ClPh- 4-ClP- (Tr)dT (19.6t19.9,20.2) PhNH- -dT(Ac) -dT(Ac) PhNH40 -(7.0-7.4) 8'1 H_ -OA -10.14 -6.47d) 51 H19(H&)2PF(O)NHT) To diastereomers icotinaJide adenine dinucleotide; Tw) a) In H20, pH7.4; b) In TJU,-lgE[HJ7.4; o) In Py; d) In D20,pDlO

, -T

Nq R |

Rn

61H1N-

of two anhydride bonds leads to the 31p signal shift of terminal phosphate on 17-18ppm in the same direction (cp. chemical shifts of compounds 7.4 and 1.4), i.e. the effect can be considered as additive. From these data follows that the nature of carbon acid has no essential influence on chemical shifts values. The comparison of chemical shifts for compounds 7.5 and 7.3 shows the substitution of -OAlk group by -NHPh leads to downfield 31P signal shift (5-6ppm), i.e. the effect is the same as in the case of transition of PTE to PDE monoamides. Pyrophosphate derivatives of nucleotides (Table 8),31p NMR spectra of non-symetrical derivatives can be assigned as a rule to AB-type spin-systems, The difference of R.and P chemical shifts (and also values of coupling constants JPOP ) depends on the nature of substituents bonded with them, the acidity of medium and solvent effect. In some cases sufficiently resolved
5555

Nucleic Acids Research

Table 9 31 P chemical shifts and constants of triphosphate decoutni; rivatives of nuoleotides ( (R20)P(O)OP*(O)(OR2)M0PO)(OR2)(OR3)

T
2

Iffo_
HOleO-

R2 R3 IRR
-H
-H

1pR- d|PP dE | Solv. -11.43 -_22.83 -771 20. -dA -13.47 2 PM -23.46 -10.4 28.3 24.6 a) -A -12.7 -23.6 -11.2 DlKSO -11.45 -22.66 -7.33 19.8 19.8 H20 pH7
-12.25 -23.17 -10.6425.6 -12.7 -24.0 -11.5
23.6
a? b)

ef,
12 54 12 12 55 55

4 5 6 7 8 9
0 I

-H 4NO2h -H NH- -H C6 6 fiE-H PbNH- -H F-H

HO-

-A -A -A -A -A -A dT(Ac -H -H -A

-14.0 -24.5 -19.7 c) -11.8 -22.0 -3.6 22.5 22.5 DMSO:Py 6 -11.4 -23.2 -10.2 H20,pl1 56 -11.4 -22.1 -4.2 55 Py -10.11 -22.6 -17.5 J 935Hs H20,p11 49 -11.45 -21.6 -11.5 1 0 117.0 Py 21
-11.1 -22.8 -22.8J

H20p 857

a) In TMU, -lg[EC7.4; b) In DKF:KeOHm1:1; c) In DMF-:Py1:1

spectra cannot be obtained, because of small chemical shifts difference between phosphorus atoms of pyrophosphate bond. 31p NMR spectra trisubstituted pyrophosphates are similar to those of non-symmetrical mono- and disubstituted ones. The only difference consists in the possibility of existance of two diastereomers. The chemical shifts of asymmetrical phosphorus atoms for the latter often do not coincide (compounds 8.7-8.10). Symmetrically disubstituted pyrophosphates give a singletS in 31p MIR spectra being shifted upfield (11-12ppm) in respect to phosphomonoester signal (for non-aqueous solutions). Tetrasubstituted pyrophosphates can exist as three diastereomers in contrary to symmetrical disubstituted pyrophosphates. This circumstance complicates 31p IBR spectra, where several lines have been often observed (compounds 8.16, 8.17). All the regularities of the influence of substituents established for PDE and PTE can be used in the case of pyrophosphate derivatives. Triphosphate derivatives of nucleosides (nucleoside 5'-triphosphates, their i-derivatives,trinucleoside tripolyphosphates, Table 9). The 31P spectra of these compounds can be assigned usually to ABX-type of spin-systems (or AMX). Pj, signal (X nuc5556

31p

Nucleic Acids Research

Table 1 0 P chemical shifts and coupling constants of nucleotide analogues with P-S and P-Se bonds in aqueous solutions

No
1 2 3 4 5

Compound-

4L~

ef
58 59 60

7 8 9 10 11 12

43,5 (HO)P(S)(OH)(OA) 56.8 (AO)P(S) (OH) (OA) (MeOTr)dTOP(S) (OH) (2-ClPhO) 52.94 bzA2'-(2-NO Ph)pJP(S)(OCH )bzA(Bz) 71.2 (HO) (S)(8H)O(HO)P(O)(3A) 11.1 42.1 (HO)PO )(OH)O(HO)P(S)(OA) (HO) ( (OH)O(HO)4(6O(HO)P(O)(OA) 10.6 (HO) (O)(OH)O(HO)P!(S)O(HO)3aO)(OA) 11.4 11.4 (HO)P(O)(OH)O(HO)P(O)O(HO)P(S)(OA) 43.4 21.6 b (AO)P(O)(SCH. )(OH)

59

33.9 -6.3 -220 29.9 -30.0 -222

31.2 0.8 35. 19.6 29.0 -6. 6.5 273 -6. -t,- -,,-5. 27.5 205

58 58 49 58 58 61 62 62

(UO)P(Se)(0H3(OU)

KSiU(2'-Si)P(Se)P(Et)]pU(si)2p)

51.68**) 50.90 72.5 *) 70.4

*) Si means (CH3)3Si-group, **) Two diastereomers

leous) have been observed in highest field and medium change

effects substantially on the position of Piatom[6]. The Pchemical shift value varies slightly from compound to compound -(11-13ppm).The P6resonance position has been determined by substituent at P6 atom: from -3.6ppm (compound 9.5) to -19.7ppm (compound 9.4). The medium also effects substantially on P6chemical shift especially in the case of nucleoside 5'-triphosphates[6]. The Pand P6chemical shift difference can be interpreted in terms of general asymmetry of molecules, i.e. the diastereotopy of ,L_and P6atoms (compound 9.9). Sulphur and selenium containing analogues of nucleotides (Table 10). Sulphur containing derivatives of nucleotides have been widely used in studies of enzyme systems. The comparison of 31P chemical shifts of these compounds with oxygen analogues shows that the substitution of phosphoryl oxygen by sulphur in PME and in terminal phosphates of nucleoside di- and triphosphates leads to the downfield signal shift ('40-44ppm; cp. chemical shifts values of compounds 8.1 and 10.5, 1.1 and 10.1); in PDE, PLof nucleoside diphosphates, P.and P. of nucleoside triphosphates (52-58ppm; cp. chemical shifts of compounds 2.15 and 10.2 or 9.2 and 10.6); in PTE ('70ppm; cp. chemical shifts of
5557

Nucleic Acids Research

Table 11 31P pchemical shifts and coupling constants of nucleotide derivatives with P-C bond in aqueous solutions at variouspH

P0
1 2

Compound

dp ,) JI | J, iRef
R2
-H
pH

(R1O) (HO)3SO)CH2P;O) (OH) (OR2)

R-i
-

-A

6
11

(R10) (HO)-PPxr(O) (OH) YrV() (OH) (OR2

1

17.4 17.7 15.C 21.0 9.2 23.0 12.C

63 63

63

49

____

-A
-A -A -A -A -A

-H -H -H -H -CH3 -H

CH2
CC2 CHP CF2 CH2 0

4 5 6 7 8

0 4 -11.0 6 11.3 10 10.3 0 -10.2 0 -10.5 0 -10.2 -11.4 0 18.4 CH

8.4 14.8 8.5 13.7

63
26.0 8.5 64 30.5 17.7 64 28.0 16.0 64 32.0 57.0 64 26.0 8.5 63 63

14.412.1 2.4 8.6 2.4 8.6 2.2 4.2 6.8 17.7 5.1 6.8

compounds 3.6 and 10.4). If the sulphur substitutes the estertype oxygen the downfield 31p signal shifts -20ppm has been observed (cp. chemical shifts of compounds 2.1 and 10.10). These differences of chemical shifts values can be used for determination of the position of sulphur. The spectra analysis of sulphur containing nucleoside 5'-di(and tri)phosphates shows that the presence of sulphur leads to the changes in chemical shifts only for phosphorus atoms directly bonded with it; the other chemical shifts remain practically the same. The 31P chemical shifts of some nucleotide derivatives containing P=Se bond have been also known. The region of 31p resonance of these derivatives has been shifted to high field in respect to sulphur containing analogues ( cp.chemical shifts of compounds 10.11 and 10.2 ). Nucleotide derivatives with P-C bond (Table 11).The 31p Signals of nucleotide phosphonate derivatives have been shown to be shifted downfield (16-32ppm; depending on the medium influence and structure of compounds) in respect to corresponding phosphate derivatives (cp. chemical shifts of compounds 11.1
5558

Nucleic Acids Research

Table 1 2 chemical shifts of phosphite analogues of nucleotides (R1)P(OR2 (R_3_
)

31p

01

L2j[(DeTr]dT- -CH3 -OdT(Lev)

[(MeO)2Tr]dT- -CH3
-Cl

[CM0

N R2 t R3 )2Tr]dT- -CH3 1
1R

3)2

146.0

147.7148
140.8

145.4

139.6

167.2

(CD )3CO 138.9 CD 1 139,9 167.2 CDC13

Solvent Ref. (CD )2C T 65 CDl6

65

65
65

and 8.3, 11.2 and 8.1, 11.3 and 9.2). The substitution of hydaby chlorine or fluorine leads rogens in fragment Pp-CH2- Pto the upfield 31P signal shift (4-8ppm) and to changes in coufrom 8.5 to 57 Hz (cp. chemical shifts pling constants 2J of compounds 11.3-11.7). and (Table 12).Recently the daPen ta on 31P chemical shifts of some phosphite nucleotide analogues have been obtained with the development of "phosphite"l method of oligonucleotide synthesis. The 31P signals of these compounds have been shifted downfield in respect H3P04 being placed at 120-170ppm region[6].The data of the Table 12 shown qualitatively the 31P chemical shifts of nucleotide phosphite analogues to be determined by the regularities established for nucleotides - derivatives of orthophosphoric acid. However the reliable conclusions in that case cannot be made because of the lacking of additional data. Kucleoside 3' , 5' -cyclophosphate derivatives (Table 13) .These compounds have been considered separately because of upfield shift of 31P signals of their PDE (-1ppm) in respect to acyclic PDE (cp. chemical shifts of compounds 13.1 and 2.6, 13.2 and 2.15). The greater difference of chemical shifts values (up to 3-5ppm) has been observed between 3',51-cyclic and acyclic PTE, monoamides of PDE and others (cp. chemical shifts of compounds 13.5 and 3.6, 13.7 and 5.4, 13.8 and 10.2).The4dfor diastereomers of 3',5'-cyclic derivatives of PTE have been found to increase substantially in respect to acyclic ones (cp.d'1 and d2 of compounds 13.5 and 3.6, 13.7 and 5.4, 13.8 and 10.2, 13.10 and 12.2).

2i

5559

Nucleic Acids Research

Table 13 chemical shifts of nucleoside 31 ,5'-cyclophosphate derivatives
R

31

X , R

NO

R1

d'4

Medium
_,,_ DMSO-d -DMSO(1:1) DhSO-d6 -,(CD )2CO DMSO-d6 Py -,,DMSO (CD ) CO

Ref.

1
2 3

HOHOMeO-

4 5

EtOMeO6 (M&e) N7 PhNiHO8 Me9 0 MeO-

1 Me)2N- I*) Thy

0 Thy- H- -2.05 O Ade- HO- -1.58 0 Ade- HO- -4.02 -5.27 -2.47 -3.46 0 Ade- HO- -4.43 -6.14 0 Thy- H- -4.7 -6.7 7.53 6.91 O Thy- H0.64 -3.35 O Thy- HS Thy- H- 54.73 52.11 O Thy- H- 30.2 26.2 *) Thy- H- 129.5 123.2

D20, pD7

H- 144.9 139.6

ED13

44 44 70 68 68

7 7 66 67 67 .68 69

r *) Lone pair Nucleoside 2',3'-cyclophosphate derivatives (Table 14). The area of P chemical shifts for these compounds is quite different from those for nucleotide derivatives considered above. 2',3'-Cyclic PDE have been shifted downfield (-20ppm) in respect to acyclic PDE (cp. chemical shifts of compounds 14.1 and 2.11). The similar shifts have been observed for thiophosphate triesters (cp. chemical shifts of compounds 14.4 and 10.2).The treatment of (Tr)Up with N,Nt-dicyclohexylcarbodiimide in pyridine leads to the formation of 2',3'-cyclic derivative (compound 14.2) and further to compound 14.3, which characterizing by two individual signals in 31p NMR spectrum at 24.9 and 24.Oppm[6].The
Table 14

31p
NO
2

chemical shifts of nucleoside 2',3'-cyclophosphate derivatives

R1

R2

X/ sR2. Solvernt Ref.

6 6
72

-OH Tr- Ura3V Tr- Ura- -N(C6H11 )C(O)NHC6H11 -OH 4 H- Ura-OH 5 H- Cyt-

-OH~~~~~~~~~~~~~~~---O 1 T~~~~~~~~~UraTr-a= H=-_ 1706 - DM -b

Py 0 18.5 0 24.9 24.0 Py S 76.1 75.1 H 0 018.5 - M_eH

73

5560

Nucleic Acids Research

Table 15
chemical shifts and coupling constants of 3',5'-cyclopyrophosphate derivatives
31p

NO
1
H

R1
_-H

d- d4Ji.XMedium
-13.70 -14.45 27.6
DIF

23
74

2 H23.4 _13.06 -14.94 22.0 H2O,pH9

-dT(Ac)

-13.00

-14.40

Py(TPS)

23

latter correspond to two possible diastereomers. Nucleoside 3'.5'-cyclopyrophosphates and their derivatives (Table 15).These compounds form the special group, because of the substantial difference between their spectral data and those for corresponding substituted pyrophosphates. The main difference consists in upfield shift of P,and particularly P signals (u-. 2ppm, PF-2.5-4.0ppm, cp. chemical shifts of compounds 15.2 values has been also observed. and 8,12)oThe increase of The chemical shifts and coupling constants of these compounds depend substantially on the acidity of medium and solvent effect (see chemical shifts of compounds 15.1 and 15.2).The substitution of -dT(Ac) by phenyl group leads to upfield shift of corresponding phosphorus atom (C5ppm) as in the cases of PDE and PTE [61. Adenosine 5'-trimetaphosphate (Table 16).The 31p spectrum of this compound can be assigned to ABC-type of spin-systems in the region of 31P resonance of dianhydride phosphate fragments. Our experiments[6]have been shown that the small chemical shift and coupling constant differences being caused by medium effects substantially change the picture of 31p spectrum of adenosine 5'-trimetaphosphate. The analysis of 31p chemical shifts values in the Tables 1-16 let us to describe the influence of some substituents on 31p chemical shifts of nucleotide derivatives (Table 17). The group -OAlk (Alk A CH3, CH2CC13) has been chosen as the replaced one. chemical shifts changes can be The found tendencies of used for purpose of identification of structure of nucleotide

2Jp0p

31P

5561

Nucleic Acids Research

Table 16
0

p chemical shifts and coupling constants adenosine 5'-trimetaphosphate derivatives and their phosphonate analogues L N

H
n 0P 0'H

A
Ref. 75 6
76
76

X Y JPd I jB
O

-22.6 2 CR2 0 14.0 14.0 39 0 CR2 -24.4

1i Q

-22.9 -24.2 -24.6 23.5 23.7 -23.7 -24.0 24.7 21.9 19.5 -1.9 -24.8 10.0 20.0 20.0

J) 22.9

Solvent
Py DMS0 Py
-

-2.3 -23.8 4.3 4.9 20.0 18.0 4.5

Table 17 Substituent effects on 31P MIR chemical shifts of nucleotide derivatives (ppm)*) TI [CAlk [0A-T]Or ( Substituent o.=P-ONuc O=P-ONuc o=P-OITuc lI--OR OH xOAlK,

R=Alk,Ar,Nuc
~0

-OAlk', -OJuc** -OH, -OCH3 -OCH20Cl3 -OAr

~O

_(o.5-1.0)

(0.9-1.8)

(3-4)
-

-(1.5-2.5) -(0.5-1.0)

-0-g-RI(R'=Alk,Ar)
-NHER', NR&(R'=Alk)
-NHAr

-(4.0-4.7)

-(6-7)

-(4-5)

--10

-(6-7)

-14
-

(6-7)

|NR'--NR' (R =C 6H11

0
-

IX= _X7 \---h

-0-

R'(R'= H Alk) (RI= NtCH ),-(5-6)

-(7-8) () -(4-5)

(-1)

(10-12)

(-5)

(-5)
(--10)

CR,-"H 3) X= N

-(9-10) -(9-10)

-(2-3)_

-(4-5) -(8-9)

(--15)
-(20-24)

OR'(R'=HvAlk,Ar- -(10-12)
R"= -,-, NucC

-(10-12) A

jAElK65562

replaced group, Alk' Nuc - nucleoside

CH3, CH2CC13

Nucleic Acids Research

derivatives. E.g. earlier have been assumed that the during the study of mechanism of oligonucleotide synthesis by diester method let to suppose the mononucleotide (I) transform in presence of arylsulphonyl chloride in pyridine consequently to symmetrical dinucleosidepyrophosphate (II), trinucleosidetripolyphosphate (III), tetranucleosidetetrapolyphosphate (IV) and then trinucleosidetrimetaphosphate (V). The latter compound (V) has been considered to be the reactive agent phosphorylating the nucleoside component[77,78]. The detailed investigation of that process by 31P NMR spectroscopy has been shown the real formation of compounds (II(IV), together with the absence of compound (V) among products. The 31p chemical shifts difference has been found between the observed (-5ppm) and expected for the compound (V) C -21ppm;see Table 16). The 31P signal at -5ppm has been assigned to compound (VI)(cp. the chemical shift of compound 4.10). The latter compound (VI) has been determined to be the N-phosphorylpyridinium mononucleotide derivative as the reactive phosphorylating agent [17,19,79,80]. So the use of 31p MIR spectroscopy led to the establishment of the mechanism of oligonucleotide synthesis by diester method. The kinetic parameters of that process have been measured[20,31,81]. The similar investigation by 31P NMR spectroscopy let to determine .the structure of intermediates, the order of their transformations and kinetic parameters of reactions for triester method of oligonucleotide synthesis [30,31,33,35-371. The fonnation of the by-product in the reaction of synthesis of (CNEt)2pdT from pdT and 2-cyanoethanol in the presence of TPS have been noticed [811 ( the 31p signals at -5.6 and -7.9ppm) Taking into account the data of Tables3 and 13 these signals can be assigned to two diastereomers of 2-cyanoethyl ester of thymidine 3',5'-cyclophosphate (cp. chemical shifts of compounds 3.5,3.6 and 13.5). So 31P NMR spectroscopy being used in the field of nucleotide chemistry as follows from the present review of 31P chemical shifts data can be considered to be rather effective (and in some cases even unique) method carrying the information about structure and transformations of the investigated compounds.
5563

Nucleic Acids Research AcknowledRements We thank Academician D.G. Knorre and Dr. A.S. Levina for their critical readings of the manuscript and many helpful suggestions, Dr. V.F. Zarytova, E.M. Ivanova and V.P. Starostin for giving of some compounds, Mr. S.A. Krupoder and Mrs. E. Efirnova for the translation of manuscript into English. REFERENCES 1. Burt,C.T.,Cohen,S.M.and Barany,M.(1979) Armu. Rev. Biophys. and Bioeng., 8, 1-25 2. Mildvan,A.S (1977) Accounts Chem. Res., 10, 246-252 3. Radda, G.K., Seeley,P.J. (1979) Annu. Rev. Physiol., 41, 749-769 4. Ugurbil,K., Shulman,R.G., Brown,T,R. (1979) Biological Applications of Magnetic Resonance, pp. 537-589, Academic Press, New York 5. O'Neill,I.K., Richards,C.P. (1980) Annual Reports on MlAR Spectroscopy, 10A, pp. 134-236, Academic Press, New York 6. Lebedev,.V., Rezvukhin,A.I. (1983) Bioorgan. Khimiya, 9, 149-185 7. Cozzone,P.J., Jardetzky,O. (1976) Biochemistry, 15,48534859 8, Akasaka,K., Yamada.A., Hatano,H. (1975) FEBS Lett., 53,
9. Cohn,M., Hughes,T.R. (1960) J. Biol. Chem., 235,3250-3253 10. Tran-Dinh,S., Roux,M., Ellenberger,M. (1975) Nucl. Acids Res., 2, 1101-1110 11. Tran-Dinh,S., Neuman,J.M (1977) Nucl. Acids Res.,4,393-403 12. Labotka,R.J., Glonek,T., Myers,T.C. (1976) J. Amer. Chem. Soc., 98, 3699-3704 13. Cohn,M., Hughes,T.R. (1962) J, Biol. Chem., 237, 176-181 14. Avedikian,A.M., Besserre,D., Delepierre,M. (1982) J. Chim. Phys. Physicochim. Biol., 79, 73-79 15. Lerner,D.B., Kearns,D.P. (1980) J. Amer. Chem. Soc., 102,

339-341

7611-7612 16. Gorenstein,D.G., Findlay,J.B., Momii,R.K., Kar,D. (1976) Biochemistry, 15, 3796-3803 17. Zarytova,V.F, Knorre,D.G., Lebedev,A.V., Levina,A.S., Rezvukhin,A.I. (1974) Izv.Sib.Otd.Akad.Nauk SSSR,Ser.khim. nauk, iss.1, 85-96 18. Knorre,D.G., Lebedev,A.V., Levina,A.S., Rezvukhin,A.I., Zarytova,V.F. (1974) Tetrahedron, 30, 3073-3079 19. Zarytova,V.F., Ivanova,E.M,, Lebedev,A.V. (1976) Bioorgan. Khimiya 2, 189-198 20. Zarytova,V.F., Knorre,D.G., Khalimskaya,L.M. (1975) Zh. Fiz. Khimii 49, 2504-2512 21. Zarytova,V.F., Lebedev,A.V., Ratmanova K.I., Smirnov,V.D., Shabarova,Z.A., Khalimskaya,M. (19765 Izv.Sib.Otd.Akad. Nauk SSSR, ser.kbim.nauk, iss.6, 100-106 22. Zarytova,V.. , Knorre,D.G., Lebedev,A.V., Levina,A.S., Rezvukhin,A.I. (1973) Dokl. Akad. Nauk SSSR, 212, 630-633 23. Knorre,D.G., Lebedev,A.V., Zarytova,V.F. (1976) Nucl. Acids Res., 3, 1401-1418

5564

Nucleic Acids Research

24. Zarytova,V.F., Kuznetzova,L.M., Lomakina.T.S., Starostin, V.P. (1979) Izv.Sib.Otd.Akad.Nauk SSSR, ser.khim.nauk, iss.1, 93-100 25. Zarytova,V.F., Knorre,D.G., Lebedev,A.V., Levina,A.S., Rezvukhin,A.I. (1975) Izv.Sib.Otd.Akad.Nauk SSSR, ser.khim. nauk, iss.2, 139-149 26. Pardi A., Walker,R., Rapoport,H., Wider G., Wuitrich,K. (19835 J. Amer. Chem. Soc., 105, 1652-1653 27. Cheng,M, Kan,L.-S., Miller,P.S, L eutzinger,E.E., Ts'o, P.O.P. (1982) Biopolymers, 21, 697-701 28. Patel,D.J. (1974) Biochemistry, 13 2396-2402 29. Lebedev,A.V., Rezvukhin,A.I. (19755 Izv.Sib.Otd.Akad.Nauk SSSR, ser.khim.nauk, iss.2, 149-154 30. Zarytova,V.F., Shishkim,G.V., Khalimskaya,L.M. (1981) Bioorgan. Khimiya, 7, 900-906 31. Knorre,D.G., Zarytova V.F., Lebedev,A.V., Khalimskaya,L.,., Sheshegova,E.A. (19785 Nucl. Acids Res., 5, 1253-1272 32. Adamiak,R.W., Biala,E., Crzeskowiak,K., Kierzek,R., Kraszewski,A., Markiewicz,W.T., Stawinski,J., Wiewiorowski,M. (1977) Nucl. Acids Res., 4, 2321-2329 33. Dobrynin,V.N., Boldyreva,E.F., Bystrov,N.S., Severova,N.V., Chernov,B.K., Kolosov,M.N. (1978) Bioorgan. Khimiya, 4,

523-534

34. Knorre,D.G., Levina,A.S., Shubina,T.N. (1975) Izv.Sib.O*d. Akad.Nauk SSSR, ser.khim.nauk, iss. 3, 118-128 35. Drozdova,L.I., Zarytova,V.F., Khal'mskaya,L.M. (1981) Izv. Sib. Otd.Akad.Nauk SSSR, ser.khim.nauk, iss,1, 125-130 36. Zarytova,V.F., Khalimskaya,L.M., Yarmolinskaya,E.V. (1980) Izv.Sib.Otd.Akad.Nauk SSSR, ser.lkhim.nauk, iss.6, 78-86 37. Zarytova,V.F., Sheshegova,E.A. (1978) Bioorgan. Khimiya,

38. Tomasz,J., Simoncsits,A. (1981) Tetrahedron Lett., 22, 3905-3908 39. Zagrebelnyi,S.N., Zarytova,V.F., Levina,A.S., Semenova,.N. Yarmolinskaya,E.V., Yasnetzkaya,S.M. (1978) Bioorgan.Khimiya, 4, 729-734 40. Knorre, D.G., Mishenina,G.F., Sanukov,V,V., Shubina,T.N. (1977) Dokl. Akad. Nauk SSSR, 236, 613-616 41. Zarytova,V.F., Ivanova,E,M., Knorre,D.G., Forbruggen,H. (1980) Dokl. Akad. Nauk SSSR, 255, 1128-1131 42. Zarytova,V.F., Knorre,D.G., Lebedev,A.V., Levina,A.S., Rezvukhin,A.I. (1974) Izv.Sib.Otd.Akad.Nauk SSSRt, ser.khim. nauk, iss.3, 126-131 43. Knorre,D.G., Zarytova,V.F. (1976) Nucl. Acids Res., 3, 2709-2729 44. Zielinski,W.S., Stec,W.J. (1977) J. Amer. Chem. Soc., 99,
45. Sergeeva,N.F., Smirnov,V.D., Shabarova,Z.A., Prokof'fev,M.A. Zarytova,V.F., Lebedev,A.V., Knorre,D.G. (1976) Bioorgan. Khimiya, 2, 1056-1062 46. Eckstein,F. (1970) J. Amer.Chem.Soc., 92, 4718-4723 47. Drutsa,V.L., Zarytova,V.F., Knorre,D.G., Lebedev,A.V., Sokolova,N.I., Shabarova,Z.A. (1977) Dokl. Akad. Nauk SSSR, 233, 595-597 48. Trettyakova,S.S., Drutsa,V.L., Lebedev,A.V., Sokolova,N.I., Shabarova,Z.A. (1978) Bioorgan. Khimiya, 4, 910-914 49. Vogel,H.J., Bridger,W.A. (1982) Biochemistry, 21, 394-401
5565

4, 901-910

8365-8366

Nucleic Acids Research

50. Sarma,R.H., Mynott,R.J. (1972) Org. Magn. Res., 4, 577-584 51. Tran-Dinh,S., Roux,M. (1977) Eur. J. Biochem., 76, 245-249 52, Zarytova,V.F., Knorre,D.G., Lebedev,A.V., Levina,A.S., Rezvukhin,A.I. (1974) Izv.Sib.Otd.Akad.Nauk SSSR, ser.khim. nauk, iss.3, 121-125 53. Zarytova,V.F., Ivanova,E.M., Lebedev,A.V. (1976) Bioorgan. Khimiya, 2, 1196-1204 54. Zarytova,V.F., Knorre,D.G., Kurbatov,V.A., Lebedev,A.V., Samukoo,V.V.,Shishkin,G.V. (1975)Bioorgan.Thimiya, 1,793-799 55. Knorre,D.G.,EKurbatov,V.A. ,Samukov,.V.(1976) FEBS Lett.,70, 105-108 56. Babkina,G,T. Zarytova,V.P. ,Knorre,D.G.(1975) Bioorgan.

57. 58. 59. 60. 61.

62. 63.

64. 65. 66. 67. 68. 69. 70.

71. 72.

73. 74. 75.

76.

77.
78.

79. 80.
81.

Kbhmia, 1.b11-615 Nageswara Rao,B.D.,Cohn,M.(1977) Proc. Nat.Acad.Sci. USA,74, 5355-5359 Jaffe,E.R., Oohn,M. (1978) Biochemistry, 17, 652-657 Marlier,J.P.,Benkovic,S.J.(1980)Tetrahedron Lett.,21,11211124 Zielinaki,W.S. ,Lenikowski,Z.J. ,Stec,W.J. (1976) J.Chem.Soc. Chem. Communs.,, 772-773 Eckstein,F. ,Simonson,L.P. ,Bar,H.-P. (1974) Biochemistry, 13, 3806-3810 Nemer,LJ. ,Ogilvie,K.K. (1980)Tetrahedron Lett.21,4149-41 52 Crutchfield,Mo. ,Dungan,C.H. ,Letcher,J.H. ,VanWazer,J.R.(1967) Topics in Phosphorus Chemistry,vol.5,pp.35, 313-314, Interscience Publishers, New York-London-Sydney Blackburn,G.M. ,Kent,D.B. ,Kolkman,F.(1981) J. Chem. Soc. Chema Communs., 1188-1190 Beaucage,S.L. ,CaruthersN,.H. (1981 )Tetrahedron Lett. ,22, 1859-1862 Jarrest,R.L.,Lowe,G.,Potter,V.L.(1980)Chem.Communs.,1142-1145 Engels,J. ,Schleager,B-J.(1977)J.Med.Chem. ,20, 907-911 Bajwa,G.S. ,Bentrude,W.G.(1978)Tetrahedron Lett. ,19, 421-424 Stec,W.J. ,Zielinski,W.S. (1980) Tetrahedron Lett. ,21,1361-1365 Bajwa,G.S.,Bentrude,W.G. (1980) Tetrahedron Lett.,21,4683-4686 Zarytova,V.P. Rait V.K.,Chernikova,T.S.(1977) Bioorgan. Khimiya, 3, 1626-1 32 Usher,D.A. Ererich,E.S.,Eokstein,F.(1972) Proc. Nat. Acad. Sci. USA, 99, 115-118 Blackburn G.M.,Cohen,J.S.,Todd,A.R.(1964) Tetrahedron Lett., 39, 2873-i879 Zarytova,V.F. ,Lebedev,A.V,(1977) Bioorgan.Khilya,13,12111-1218 Glonek,T. ,Kleps,R.A. ,Myers,T.C.(1974) Soience,185,352-355 Glonek,T. ,Kleps,R.A. ,Myers,T.C.(1976) Bioinorg. Chem., 5, 295-304 Weimann,G. and Khorana,H.G.(1962)J.Amer.Chem.Soc. ,84, 43294341 Jacob,T.M. and Khoran ,H.G. (1964)J.Amer.Chem.Soc. ,86, 16301635 Zarytova,V.F. ,Graifer,D.M. ,Knorre,D,G. ,Lebedev,A.V. ,Rezvukhin A.I. (1978) Nucl. Acids Res., spec. publ., 209-212 Graifer,D.M.,Zarytova,V.F.,Ivanova,E.M.,,Lebedev,A.V Rezvukhin,A.I. (1978) Dokl. Akad. Nauk SSSR, 242, 61*-619 Zarytova,V2.P,LKnozo,D.G.,Starostin,V.P.,Khalimakaya,L.. (1979) Izv. Sib. Otd. Akad. Nauk SSSR, ser.khimi.nauk, iss.1,
88-93

5566