Nucleotides and Nucleic Acids

1 Introduction

A decade has passed

BY J. B. HOBBS

since this Reporter began to contribute this chapter to this series of volumes, and the progress in certain areas of nucleotide chemistry has been astonishing. In a recent paper in which 96 different oligodeoxyribonucleotide heptadecamers were utilized for isolation of cDNA clones, the chemical content is des- cribed in the sentence "Oligonucleotides were synthesised by a solid phase phosphotriester method". A decade ago, the synthesis of a single 17-mer would have merited a full paper. The widespread de- mand for chemically and enzymically synthesised gene fragmentation has prompted the publication of a book on the methodol~gy,~ and among the considerable synthetic feats reported in this area are the syn- theses of sequences containing the biologically active genes for human interferon aZ3 and immune interferon y14 of 511 and 453 base

pairs length , respectively. Once again several symposia, includ- ing a Tetrahedron "Symposium-in-Print", must be commended for their interesting content. However, in a year of burgeoning activity in the field of nucleotide chemistry and biochemistry, two reports must be singled out as presaging possibly the shape of things to come. One is the demonstration6 that a molecule of RNA, a unit of the ribo- nucleoprotein Ribonuclease P, possesses catalytic activity, cleaving a tRNA precursor molecule to yield the correct 5'-terminal sequence of the tRNA: the activities normally ascribed to enzymes may thus be performed by a polynucleotide in the absence of protein, and the single-strandedness and consequent flexibility of RNA may endow it with a whole range of functional activity which is just beginning to be recognized. The other suggests that recombinant RNA technology, in which the enzyme Q B replicase is used to amplify RNA sequences engineered into its natural RNA substrates, is close to being real ised.

1

7

2 Mononucleotides

2.1 Chemical Synthesis. - Among novel nucleotides reported which

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198 Organophosphorus Chemistry

have been prepared by phosphorylation of the unprotected nucleosides with phosphoryl chloride in trialkyl phosphates (Yoshikawa's method) are a series of 2-alkyl- and 2-arylinosine 5'-phosphates and 2-(sub- stituted) benzylthioinosine 5 ' -phosphates , some seventeen nucleo- tides in all, which were investigated as inhibitors of IMP dehydro- genase, 5-f luoro-2 I , 3 I - d ideoxy-3 ' -f luorouridine 5 ' -phosphate9 and 5- (2,5-dimethoxyphenyl) -2 ' -deoxyur idine 5 ' -phosphate , lo which were probable mechanism-based inhibitors of thymidylate kinase, and 6- phenylthiouridine 5 '-phosphate, which in turn afforded 6-mercapto- uridine 5'-phosphate on treatment with sodium hydrosulphide, and thence 6-methylthiouridine 5'-phosphate on alkylation with methyl iodide. The 5'-phosphate of "Tiazofurin" (2-B-D-ribofuranosyl- thiazole-4-carboxamide) was obtained in better yield using the Sowa- Ouchi procedure (phosphoryl chloride-acetonitrile-pyridine-water) and displayed antitumour activity against mouse leukzemia. l2 The 5'-triphosphate and 3',5'-cyclic phosphate were also prepared from tiazofurin 5'-phosphate by standard methods, the latter being less effective as an antitumour agent. uridine 5'-phosphate has been prepared from the nucleoside by in- cubation with thlpidine kinase (fromEscherichia coli) and ATP, and acts as a mechanism-based inhibitor of thymidylate synthetase, thus accounting for the cytotoxic activity of the nucleoside: cells lacking thymidine kinase are far more resistant to the drug.13 L- Riboguanosine has been prepared by the usual procedures of nucleo- side synthesis, derivatised to afford 9-(2,3-di-g-acetyl-@-L-ribo- furanosyll-E -palmitoylguanine, and treated with g,g-bis(lH-benzo- triazolyl) phosphoromorpholidate to give (11, which upon successive

2',5-Difluoro-2'-deoxy-~-

2

0

<'r,p-;- OAc OAc

2 (2) R' = H, R = P03H2

( 3 ) R' = P03H2, R 2 = H

deblocking with triethylamine, ammonia,and weak acid affords the enantiomer of 5'-GMP. l4 It is intended to compare the oligomeri-

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7: Nucleotides and Nucleic Acids 199

sation of activated GMP on a poly(C) template with that of its mirror image. The 2'-phosphate, 3'-phosphate, 2',3'-phosphate,and 5 ' - phosphate of 3'-C-methyluridine have been prepared by treatingthe 5'-

g-benzoyl- or 2',3'-gfg-ethoxymethylidene derivativesof the nucleo- side, as appropriate, with 6-cyanoethyl phosphate and DCC, followed by deblocking and (for the 2',3'-phosphate) further cyclisation. Allo-uridine 5I-phosphate (2) has been prepared from 2',3'-g,g-iso- propylidene-6'-~-pivaloyl-allo-uridine using the same reagents, and - allo-uridine 6'-phosphate (3) by incubating the nucleoside with 4- nitrophenyl phosphate and malt sprouts (or carrot) phosphotrans- f erase. While (2) is a substrate for 5'-nucleotidase, ( 3 ) is not, although (3) is hydrolysed by unspecific phosphatases. Allo-uridine 5',6'-monophosphate was readily formed by treating (3) with DCC, and was also formed each time (3) was activated chemically with a view to preparing 6'-allo-UDP. Incubation of cytidine 2',3'-monophos- phate with allo-uridine and pancreatic ribonuclease afforded (3'+6')-

Cp(al1o-U), which was hydrolysed to the expected products by ribo- nucleases A and T2.

15

2',3'-Dideoxy-3'-~-(phosphonomethyl~adenosine ( 4 ) has been

0 I I I

X(CH,),O-P-O-(AdO- 5 ')

OH

y 2

O=P-OH I

OH (4)

prepared by condensing N_'-benzoylzdenine with 1,2-di-g-acetyl-5-2- benzoyl-3-deoxy-3-(diethoxyphosphosphonomethyl)-~-D-ribofuranose, de- acetylation at the 2'-position with hydroxylamine, reduction to the 2'-deoxy species using phenoxythiocarbonyl chloride and tributyltin hydride, and deb10cking.l~ methylsilyl bromide.

The ethyl groups were removed using tri-

A number of w-haloalkyl esters of 5'-AMP (5,6) have been pre- pared by esterifying 5'-AMP with the appropriate u-haloalkan-1-01 using DCC, as potential active-site-directed reagents for dehydro- genases. l8 The bromohexyl species (6,"=6) inactivated yeast alco- hol dehydrogenase rapidly, but the other compounds, while competent

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200 C)rganophosphorus Chemistry

alkylating agents, did not exhibit active-site-directed affinity labelling. phosphates by trimethyl phosphate at 37-60° and over the pH range 7-10 shows that the rate of methyl ester formation is practically independent of the nature of the base, and also of pH, being typi-

19 cally s. 9% after 24 hours at 37O, but rises with temperature. Not surprisingly, the methylation of the bases is both pH- and temperature-dependent, reflecting the ionised forms predominant at different pH values.

A study of the methylation of deoxynucleoside 5'-

Renewed interest has been shown in developing phosphotriesters which are labile in vivo but which, being comparatively lipophilic, may serve to carry nucleotides across biological membranes. Treat- ment of 3'-~-acetyl-2',5'-dideoxy-5'-iodo-thymidine (or-5-fluoro- uridine) with the silver salt of bis(pivaloyloxymethy1) phosphate affords phosphotriesters (7). 2o The fluoro compound prevented

0 A c

( 7 ) R = Me or F

growth of CHO cells in culture. When 5-fluoro-2'-deoxyuridine was treated firstly with damp phosphoryl chloride in triethyl phosphate and then with 3-aminopropan-1-01 or propane-1,3-diol, ( 8 ) and (9) were formed, respectively. 21 of esterifying the nucleoside with trimethylene phosphate using di- ethyl azodicarboxylate and triphenyl phosphine. The corresponding 2'-deoxythymidine species were also prepared. While (81 , rather than (91, was effective in prolonging the lifespan of leukzemic mice, it was less effective than 5-fluorouracil. In designing (8) it was hoped that it would be degraded in a similar way to cyclophosphamide, to afford the nucleoside 5'-phosphoramidate and acrolein, but no evidence for this could be obtained. In another study, not only (91 , but also (10) and (11) were prepared by treating 2'-deoxy-5- fluorouridine with the correspondingly substituted 2-chloro-2-0x0-

A better synthesis of ( 9 ) consisted

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7: Nucleotides and Nucleic Acids 20 1

0

A HN

I I

4 F X = NH, R1 = R2 = H

X = 0, R ' = R 2 = H

X = 0, R' = PhCH,O, R = H

X = 0, R' = R 2 = F 1

X =0, R = O H , R 2 = H

2

I OH

1,3,2-dioxaphosphorinanes (prepared by treating 2-benzylglycerol and 2,2-difluoropropan-l,3-diol, respectively, with phosphoryl chloride), and (12) was also derived by hydrogenolysis of Again, the analogous 2Ldeoxythymidine species were made similarly. lytic studies, compound (11) was readily hydrolysed at room temper- ature, particularly at higher pH, apparently to the 2,2-difluoro-3- hydroxypropyl ester of 5-fluorouridylate, and (11) was almost as effective as 5-fluorouridine in inhibiting the growth of murine leukaemia L1210 cells. Treatment of 5'-chloro-2',5'-dideoxy- thymidine first with 2-chloro-1,3,2-dioxaphospholane and then with elemental sulphur has been used to prepare (13), and similar treat- ment of E4 , 2 ' , 3 '-triacetyl-=-cytidine affords (14) .

In hydro-

23

0 I

0-P=S LA OAc

(1 4)

Another way to render nucleotides lipid-soluble lies in syn- thesising actual liponucleotides. When 3'-~-l~vulinoyl-2'ileoxy- thymidine was treated firstly with 2-chlorophenyl-~,~-bis-(l-benzo- triazolyl) phosphate, and then with 1-hexadecanol, and then de- blocked using the standard methods, the hexadecyl ester of 5'-dTMP was formed in high yield. 24

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202 Organophosphorus Chemistry

Similarly, treatment of 1,2-di-~-palmitoyl-~-glycerol with the same phosphorylating agent, and then with 3'-~-lae~ulinoyl-2~-decixy- thymidine, gave 5'-O-(1,2-di-~-palmitoyl-~-glycero-3-phosph0)-2~- - deoxythymidine, after deblocking.

When 9-(l-g-monomethoxytrityl-lI3-dihydroxy-2-propoxymethyl~- adenine is treated with trichloroethylphosphorodichloridite in THF at -78O, then oxidised with aqueous pyridine and unblocked, the phosphodiester ( 15) is obtained. 25 phates" corresponding to (15) but with 2'-deoxyadenosine as the 5'- residue or as the 3'-residue are obtained similarly. Studies with spleen and snake venom phosphodiesterases show that neither is able to recognise the "ring-open" structure as a normal substrate,

The two "dinucleoside phos-

OH (1 6 )

though spleen phosphodiesterase can digest the phosphodiester with 2'-deoxyadenosine as the 5I-residue (sic) and snake venom phospho- diesterase degrades the isomer with 2'-deoxyadenosine as the 3 ' - residue. The guanine derivative (16) is currently of considerable interest as an antiviral agent, since it is readily converted to its monophosphate by virally specified thymidine kinase in herpesvirus- 1-infected cells, and subsequently to its triphosphate which in- hibits the viral DNA polymerase and can become incorporated into DNA.26r27

Mercuriation of dUMP with mercuric acetate followed by treat- ment with styrene or 3-nitrostyrene in the presence of lithiumtetra- chloropalladate gives the corresponding 5-styryl-dUMP species, which may be oxidised to 5-formyl-dUMP by osmium tetroxide and sodium periodate in the presence of sulphuric acid. If C-8 of the styrene

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7: Nucleotides and Nucleic Acids 203

is first labelled with a carbon isotope, this represents a conven- ient method of introducing the label as a substituent at C-5 of the uracil ring,with reduction nucleotides. 28 ribonucleotides and polyribonucleotides. While the bromine atom of (~-5-(2-bromovinyl)-2'-deoxyuridylate is fairly inert, as befits a vinyl halide, on incubation with thymidylate synthetase from Lactobacillus casei it becomes highly reactive, and species such as (17) and (18) are formed.

offering a route to labelled thymine This reaction is reportedly also effective with

29

0

dRib-5'- P dRib-5'- P

(17) R = OH (19) R = Br

(18) R = SCH,CH,OH (20) R = SCH,CH,OH

These are thought to arise by attack of the enzyme at C-6 convert- ing the bromovinyl species to the ally1 bromide (19) in which the bromine atom is readily replaced to give (201, and Michael addition to the enone then generates (17) and (181, and the 1,4-oxathiane resulting from cyclisation of (20).

A Russian review on the synthesis, properties and applications of nucleotidyl peptides of both phosphoester and phosphoamide types has been published. 30 A number of analogues of tryptophanyl aden- ylate have been prepared by treating N-benzyloxycarbonyl-tryptophan with isobutylchloroformate and triethylamine, and then with various AMP analogues (8-bromo-,2-chloro-,7-deaza-, es.). 31 blocking, the products were tested as substrates in the amino- acylation reaction catalysed by tryptophanyl-tRNA synthetase, and the 2-chloro-,7-deaza-,and 2'-deoxy- analogues proved competent.

After de-

A novel procedure for the chemoselective phosphorylation of N- unprotected nucleosides begins by converting the hydroxy-group to be phosphorylated to the aluminium alkoxide. 32 For instance, treat- ment of a 5'-g-t-butyldimethylsilyl (TBDMS)-2'-deoxynucleoside first- ly with lithium aluminium tetra(dimethylamide1 and then with three equivalents of a phenol derivative (ed. 4-nitrophenol) affords a

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204 Organophosphorus Chemistry

lithium triaryloxyaluminate, LiA1(OAr)30R, in which the 3I-hydroxy group of the nucleoside (ROH) is activated as its alkoxide. This reacts readily with dialkyl phosphorochloridates to form the cor- responding nucleoside dialkyl phosphotriesters. If an aryl phos- phorodichloridateis used instead, and the product then treated with (say) the lithium 5I-alkoxide of 2'-deoxy-3'-g-TBDMS-adenosine, the corresponding aryl dinucleosidyl phosphate (21) is formed. While yields are good, it seems unlikely that this procedure will replace the currently more widely-used methods, especially when simple rapid routes to nucleotide monomer units for use in "phosphotri- ester" oligonucleotide synthesis are available. For instance, treatment of 2'-deoxycytidine successively in the same pot with 2,4,5-trichlorophenylbenzoate, dimethoxytrityl chloride, and 4- chlorophenyl-2-cyanoethylphosphorochloridate with N-methylimidazole affords (2 2 1 in 60% yield without isolation of intermediates. 33

0 RO JH! -0- P-0 JR DMTr 0 4: 0-P-OC6H4CI-4 II I

OAr OCH,CH,CN

( 2 1 ) R = TBDMS ( 2 2 )

When - N6, 5I-g-bis (dimethoxytrityll-adenosine and N6,3'-pbis- (dimethoxytrity1)-adenosine are condensed using 2-chlorophenyl- phosphorobis(l~-1,2,4-triazolide) and TPS- or MS-5-(pyridin-2-yl)- tetrazoles, a single diastereoisomer of the resulting phospho- triester (23) is obtained. 34 Evidence obtained from 31P and 'H & JDMTr ..\i-;R, ~ OR

DMTrO -P-0 ODMTr 0- P- 0 OR

OMe c6 H4 c 1-2

(23) ( 2 4 ) R =TBDMS

n.m.r. and CD spectroscopy suggests that the Sp diastereoisomer is formed, with specific ligand behaviour by the nitrogen atom of the pyridine ring dictating the path of the incoming group during con-

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7: Nucleotides and Nucleic Acids 205

densation.

Oxidation of the protected dinucleoside phosphite (24) using iodine in [ I 7 O , 1803-H20 affords a mixture of diastereoisomers , which is separable by column chromatography to give, after deblocking, the oxygen chiral isotopomers of [170,1801-UpA. 35 Hydrolysis of the major isotopomer in [170,1803-H20 using nuclease S1 (a process known to proceed with inversionat phosphorus) yieldschiral AMP which on analysis by methods previously reported is found to have - Sp stereochemistry, showing that its parent isotopomer possessed - Rp chirality and the original major triester product was Sp. Methylation of (gp) - [170,180]UpA using methyl iodide in DMSO then gives two diastereoisomeric triesters, (25) and (261 , whose ab- solute configurations have been established by inspecting the l80- isotopic shifts in the 31P n.m.r. spectra, in comparison with the same compounds lacking the l80 isotope. triester resonating at lower field in the 31P n.m.r. spectrum shows

The methyl phospho-

HoY2ra 0 OH

0:. /

OH OH

Howra 0 OH

MeO.. / dP\

OH OH

the larger isotopic shift on replacement of 16 0 by l8O, indicating that the isotopically substituted oxygen is doubly bonded to oxygen, and is thus the gp diastereoisomer (26); the high-field resonance belongs to the Rp diastereoisomer (25). study performed using oxygen chiral isotopomers of d(GpA) reached essentially the same conclusions, with one important qualification: the positions of resonances in the 31P n.m.r. spectra in the methyl phosphotriesters of protected 1 1801 d (GpA) are solvent-dependent , with the Sp diastereoisomer resonating upfield of Ep in deuterio-

An essentially similar

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206 C)rganophosphorus Chemistry

chloroform, and downfield of Ep in i6-DMSO?6 Thus for configura- tional assignments based on chemical shifts, care must be taken that the same solvents are used. With this prerequisite, the chemical shift differences can be used for unequivocal assignment of stereochemistry in the 160-phosphotriesters. isotopomers of d(GpA) were obtained by deprotection of the sepa- rated diastereoisomers of ( 2 7 ) , which was synthesised using stan-

The oxygen chiral

0 I

MeO-y=@

~ C O C H ~ O M ~

(27)

Rovhy 0 I I

PhS - P =O

NHPh

(28) R = DMTr

(29) R = (PhSI2P(O)

(30) R = P0,H2

dard "phosphite" phosphotriester methodology in order to prepare stereospecif ically l80-labelled deoxydinucleoside phosphate blocks for incorporation into oligonucleotides.

Treatment of phosphoranilidodichloridate with thiophenol and triethylamine affords S,S-diphenylphosphoranilidodithioate, which on limited hydrolysis with lithium hydroxide followed by treatment with cyclohexylamine gives cyclohexylammonium S-phenylphosphor- anilidothioate. This may be coupled to 5'-G-dimethoxytrityl-2'- deoxythynidinc using TPS-C1 to give ( 2 8 ) . When this is deblocked with acetic acid and coupled to S,S-diphenyl phosphorodithioate using mesitylenedisulphonyl chloride (MDS-C1) , ( 2 9 ) is obtained. Treatment of ( 2 9 ) with hypophosphite removes a single thiophenyl group from the 5'-position, and brief oxidation with aqueous io- dine removes the other, to give (30). Conversely, treatment of ( 2 9 ) with isoamyl nitrite and acetic acid removes the anilidate group, after which similar treatment with aqueous iodine removes predominantly the thiophenyl group on the 3'-phosphate to give S,S- diphenyl -2 ' -deoxythyid ine 3'-phosphate 5'-phosphorodithioate.Thus each phosphate group of ( 2 9 ) can be unblocked selectively to allow

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7: Nucleotides and Nucleic Acids 207

further manipulation - such as nucleoside 3',5'-polyphosphate syn- thesis.37.

Several aroylphosphonic acids have been coupled to 5I-g-dimeth- oxytrityl-2'-deoxythymidine using MDS-C1 to afford the correspond- ing 3'-aroylphosphonates (31), which may be further coupled to 3'-

(31) R = H, X =H,CI,OMe

(32) R = 3'-Ac-dThd-5:

X = H , C I , O M e

0 I

Me-P- R

( 3 3 ) R = NMe2

( 3 4 ) R = 3I-62-dThd-5'

- O-acetyl-2'-deoxythymidine using MDS-C1 and 3-nitro-lg-1,2,4-tria- zole to give (32). 38 and a catalytic amount of DBU, after which standard deprotection affords the dithymidine phosphonate, which may be silylated and then oxidised with aqueous iodine, or with sulphur, to afford the cor- responding phosphate and thiophosphate respectively, if required. The aroyl group thus acts as an effective mask of the P-H function, Treatment of 5'-g-trity1-2'-deoxythymidine with methyl bis(N,N- dimethy1amino)phosphine in chloroform under nitrogen affords the phosphonamidite (33) which when treated with 3'-g-benzoyl-2*-deoxy- thymidine and 1g-benzotriazole in acetonitrile for a short time gives the dinucleosidyl phosphonite (341, which is readily oxidised to the corresponding methylphosphonate using t-butyl hydroperoxidtz?? The lg-benzotriazole apparently functions by protonating the dimethyl- amino group of (33), and reaction times must be kept short to pre- vent acid-catalysed ligand exchange leading to(3'-39- and(5'-59- linked phosphonites being formed. phosphonate d[Ap(Me)T] was prepared similarly.

The aroyl group is removed using n-butylamine

The corresponding methyl-

Several adenosine 5'-phosphorodiamidates [(35), (36)] have been prepared and subjected to conformational study using CD and n.m.r. in an effort to rationalise solubility differences between (35)and

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208 Organophosphorus Chemistry

(36) .40 (36), and to a lesser extent in (35), compared with AMP, and it is

A marked shift towards syn-conformation is evident in

0 II I

I R- P- O-(AdO-5 )

R (35) R = NH,

R = NHMe, NMe2,

N(CH , CH, 1, 0

T h v 8

(37) X = 0, Y = NH,, B = T h y

(38) X = NH, Y = OH, B = Ade, Cyt, Gua

thought that the adenine ring may stack with the 5'-phosphoro- diamidate group in (36). Treatment of d(TpT)withtwoequivalents of TPS-C1, and then with ammonia, affords the phosphoramidate (37), and the corresponding(3'-39- and (5 ' -53- linked isomers have been prepared s imi lar ly . 41 Alkaline hydrolysis of these species seems to proceed without intramolecular assistance from the 3'- or 5'- hydroxy groups : the hydrolysis of (3 '-3 ' ) d [Tp (hH2 1 TI gives dT and dTp ( N H 2 ) in equal quantity, that of (5 '-5 ' ) d [Tp (NH2 1 TI gives dT and d[(NH2)pT] in equal quantity, and that of d[Tp(NH2)T] gives dT and a 1:l mixture of 2'-deoxythymidine 3 ' - and 5'-phosphoramidate, in equal quantity. Dinucleoside phosphoramidates containing 3I-E- phosphoramidate links (38) have been prepared by condensing mixed anhydrides of unprotected 5-deoxynucleotides and mesitoic acid with 3 Lamino-2 ' ,3 ' -dideoxythymidine in aqueous solution. * oxy)-butyl esters of dideoxynucleoside phosphates ( 3 9 ) have been prepared by transesterification of the 4-chlorophenyl dideoxy- nucleoside phosphotriesters with 4-(aminooxy)-l-butanol in the presence of caesium fluoride. 43

4-(Amino-

The aminooxybutyl triesters were

81

(CH, ),ONH, 2 (39) B' = T h y , B = T h y or Cyt

0

E t 0- P=O

1 or B = Ade or Gua, 8' = T h y

developed for use as affinity mutagens.

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7: Nucleotides and Nucleic Acids 209

Upon prolonged incubation of neutral mixtures of arsenate and inosine or adenosine (or their 2l-deoxy congeners), arsenic mono- nucleotides are formed spontaneously, with the nucleoside 5 ' -

arsenates predominating over the 2 ' - and 3l-arsenates. 44 the 5'-arsenate could substitute for 5 I - W as a substrate for myo- kinase, adenosine 5'-arsenophosphate could not be detected as a product, and may thus be highly unstable if it is formed at all.

While

The cyanophosphonate (40) has been prepared and used in a Horner-Emmons condensation during synthesis of the nucleoside anti- biotic sinefungin. 45

2.2 Cyclic Nuc1eotides.- Treatment of 5'-g-monomethoxytrityl- uridine with tris(1-imidazoly1)phosphine at low temperature gives a high yield of the nucleoside 2',3'-phosphorimidazolidite (41) which on oxidation with aqueous iodine, followed by deblocking, affords uridine 2',3'-monophosphate. 46 This cyclic phosphity-

NHCO(CH2) *COOR I

(CH,),

HN Hy*"t

MMTrO

0 0

'P' I

0

(42) R = CAMP-2'

lation reaction seems general for diols with hydroxy groups sep- arated by 2.7-3.0:. ed similarly, gave a polymeric product.

Unprotected 2'-deoxythymidine, on being treat-

2'-Fluoro-2'-deoxyadenosine 3',5'-monophosphate has been pre- pared from the nucleoside using standard methods in order to solve a problem of assignments in l3C n.m.r. spectra in related species. 2'-g-Succinyladenosine 3',5'-monophosphate has been condensed with ~-(4-aminobutyl)-J-ethylisoluminol using isobutyl chloroformate to give (421, a chemiluminescent, stable, immunologically active derivative of CAMP which has been used to develop a homogenous immunoassay for cyclic nucleotides. 48 Treatment of CAMP with

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Organophosphorus Chemistry 2 10

allyl bromide in methanolic DMF affords N1-allyl-cAMP, which after Dimroth rearrangement and ring closure with N-iodosuccinimide yields (43). 49 If CAMP is exposed to allyl bromide in aqueous sodium hydroxide, 2'-G-allyl-cAMP is formed, and treatment with potassium iodide and titanium (111) chloride affords 2'-0-(3- hydroxy-2-iodopropyl)-cA!!P. Both iodopropyl derivatives were designed as potential affinity labels of CAMP-binding sites in enzymes.

( 4 3 ) (44) R' = Me, R 2 = EtJPri ,BunJF, B r , I R 1 = R 2 = Et

R' = Bzl , R2= Pr i , I

The alkyl esters of a number of 5-substituted-2'-deoxyuridinr 3',5'-monophosphate species (44) have been prepared by treating the silver salts of the cyclic nucleotides with the appropriate alkyl iodides. 50 and characterised using 31P n.m.r., the shift of the phosphorus atom bearing the alkoxy group axial being ca. 1.3 p.p.m. downfield of that of the equatorial isomer.

Where possible, the diastereoisomers were separated

In a review "Wadsworth-Emmons Reaction Revisited", the utility of the reaction for preparing chiral nucleoside phosphorothioates, dinucleoside phosphorothioates, and P-chiral oxygen-labelled phos- phates has been considered in detail. 51 For instance, protected uridine and cytidine 3',5'-monophosphates, on treatment with ani- line in the presence of triphenylphosphine and carbon tetrachloride, afford mixtures of the diastereoisomers of the corresponding nu- cleoside cyclic 3',5'-phosphoranilidates (451, which may be sep- arated chromatographically and converted to the corresponding cyclic phosphorothioates using sodium hydride and carbon disulphide, with

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7: Nucleotides and Nucleic Acids 211

PhNH, P

0 0 OH II \

( 4 5 ) B = Ura, Cyt

retention of configuration. 52

( 4 6 ) X = S

(47) x = '80

(48) X = MeS

If [15N] -aniline is used, the ab-

solute configuration may be assigned to the individual diastereo- isomers of (45) by measuring the coupling constants between-31P and I5N nuclei.

Two methods have been reported for the stereoretentive conver- sion of cyclic phosphorothioates into oxygen-labelled phosphates. In one, a diastereoisomer of 2'-deoxythymidine 3',5'-phosphoro- thioate [e2. Sp-(46)] is heated under reflux with [1801-chloral to afford (sp) -2 ' -deoxythymidine 3 ' , 5 I - [180] phosphate (47 ) , with high stereoretention. 53 In the other, (46) is first methylated with methyl iodide to give (481, which upon hydrolysis with [1801-sodium hydroxide in [1801- H 0 gives (47) quantitatively. 54 of the oxygen isotope was determined by methylation of (47) and analysis using 31P n.m.r, spectroscopy. When the procedure was repeated on an unequally proportioned mixture of (46) and its Ep diastereoisomer, similar analysis of the products showed that l80

was distributed accordingly between the axial and equatorial positions in the same unequal ratio. The same observation of stereoretentive replacement and gp (85:15) 5'-pthymidyl 3'-g-thymidyl phosphorothioate was subjected to the same treatment. It is thought that single pseudo- rotation of the pentacoordinate intermediate formed during hydro- lysis must occur.

The position 2

by l80 was made when a mixture of Rp

The stereochemical course of base-catalysed hydrolysis of 2-

The Ep diastereoisomer (49) was hydrolysed in 160-aqueous barium hydroxide at looo to afford 2 I-deoxyadenosine 5 ' - [160,170,1801- phosphate and 2'-deoxyadenosine 3 I - [l60, 170, 1801 - phosphate, and the unseparated mixture of products incubated with ATP, PEP, adenylate

deoxyadenosine 3 I , 5 '- [170, l801 - phosphate has been established. 55

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2 12 Organophosphorus Chemistry

cyclase,and pyriivate kinase to convert selectively the 5'-mOnO- phosphate to the 5'-triphosphate which was chiral at P,. This

was then used as a substrate for adenylate cyclase from Brevi- bacterium liquifaciens, and the cdAMP formed analysed by methy-

OH

(50)

lation and examination of the "P n.m.r. spectrum (the preparation of chiral [l60, 170 ,180]- phosphate esters and their stereochemical analysis have recently been usefully summarised) ? 6

2 '-deoxyadenosine 3 I - [l6O, 170,180]-phosphate was cyclised using diphenyl phosphorochloridate and t-butoxide, and analysed similar- ly. Both products of hydrolysis were found to have been formed with inversion of stereochemistry at phosphorus. The possibility of C-0 bond cleavage during hydrolysis was excluded by 31P n.m.r. examination of the products of hydrolysis of cdAMP in [l801-H2O. Thus hydrolysis of the cyclic phosphodiester proceeds with inver- sion, and pseudorotatory processes which might lead to retention are not observed to a significant extent.

The residual

When ribonucleoside 3',5'-monophosphates are treated with one- half equivalent of TPS-C1, and then with dimethylamine, the Sp diastereomers (50) of the 3',5'-N,N-dimethylphosphoramidate products are formed stereoselectively, almost to the exclusion of the gp diastereoisomers, as revealed by 31P n.m.r. analysis. While yields are modest, this represents a remarkably direct and rapid route to the chirally defined species. When 2'-deoxy- thymidine is treated with tris(N,N-dimethy1amino)phosphine the 3 ' , - 5'-N,N-dimethylphosphoramidite (51) is obtained, which on treat- ment with methanol or phenol at low temperature gives the methyl and phenyl 3',5'-phosphites of 2'-deoxythymidine, (52) and (53) respectively,as diastereoisomeric mixtures. It has proven possible to separate the cis isomers ( 5 4 ) of (52) and (53), and thus to analyse the conformations of these, and of their trans isomers (55) by n.m.r. 58 dioxaphosphorinane ring predominantly in a twist conformation, rather than the chair form illustrated, with the proportion being

57

The latter are in fact found to have the 1,3,2-

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7: Nucleotides and Nucleic Acids 213

(51) R = Me,N

(52) R = Me0

(53) R = PhO

( 5 4 ) X = Me0 or PhO, Y = lone pair

( 5 5 ) X = lone pair, Y = Me0 or PhO

particularly high for the phenyl phosphite. The reasons for this are not clear.

The diastereoisomeric 5'-amino-5'-deoxyadenosine 3',5'-phosphoro- thioates have been prepared by cyclising bis(4-nitrophenyl) 5'- amino-5'-deoxyadenosine 5'-phosphorothioate with tetramethyl- ammonium hydroxide and separating the resultant diastereoisomers on DEAE-Sephadex. These, with a large number of other CAMP analogues, were tested for their ability to stimulate transcription - vivo in E.coli. 59 phosphorothioate (CAMPS) proved particularly effective, suggesting that the catabolite gene activator protein may interact stereo- selectively with the axial oxygen attached to phosphorus. diastereoisomer of CAMPS stimulated CAMP-dependent protein kinase in rat liver cells, thus leading to increased glycogenolysis, while the Rp diastereoisomer antagonised this activity. 6o

CAMP and cGMP analogues have been used to probe interactions between the cyclic nucleotides and the catalytic sites in mammalian phos- phodiesterases.

The gp diastereoisomer of adenosine 3',5'-

The Sp

A number of

61

3 Nucleoside Polyphosphates

In a variant on the carbonyl bis(imidazo1e)method for nucleo- side polyphosphate synthesis, AMP has been treated with N,N'-acyl- dibenzotriazoles to afford high yields of adenosine 5'-phosphoro- (El-benzotriazolidate), which upon treatment with orthophosphate gives a moderate yield of ADP. 62

vantage over the classical method. 5-Amino-4-imidazolecarbox- amide riboside 5'-triphosphate, the proposed alarmone for 10-formyl- tetrahydrofolate deficiency, has been synthesised by phosphorylation

There seems to be no clear ad-

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214 Organophosphorus Chemistry

of the nucleoside using Yoshikawa's method, and conversion of the 5'-monophosphate to the triphosphate using Michelson's method (treat-

63 ment with diphenylphosphorochloridate, followed by pyrophosphate). Treatment of amino-group-containing antigens with adenosine 5'-tri- metaphosphate permits the antigen to be "tagged" with ATP, attached via a y-phosphoramidate linkage. 64 species be used in competition with non-labelled antigens to bind to immobilised antibodies in a novel immunoassay. Acidic treat- ment of the ATP-antigen-antibody complex formed releases ATP for quantitative estimation using luciferase. Mixed anhydrides of AMP, ADP and ATP with mesitoic acid are formed by treating the respective nucleotides with mesitoyl chloride. 65 were treated with chloroacetaldehyde to afford the corresponding 1,-

It is proposed that these

The resultant species (56)

Me 0 0

. II II B U ~ 0-P-0-P-0- R M e a!! - { O - i { OH n 0- (Ado- 5 ' )

OH I OH I 'Me

( 5 6 ) n = 1-3 (57) R = Guo-5', dThd-5'

- N6-etheno-derivatives , and were also oxidised with periodate and then condensed with 4-azidobenzoyl hydrazide in order to link a photoactivatable group to the nucleoside moiety. The compounds were prepared as potential affinity reagents for ATP-dependent enzymes.

If isobutyl tributylstannyl 2-phenyl phosphorothioate is oxid- ised with iodine in the presence of the bis(tributylstanny1) esters of GMP or dTMP, the unsymmetrical alkyl nucleosidyl diphosphates (57) are formed. 6 6

ponent is oxidised by iodine via a 6-membered transition state ( 5 8 )

to afford monomeric isobutyl metaphosphate, which then attacks the bis(tributylstanny1) nucleotide. This procedure thus affords alkyl dinucleos idyld iphosphates under homogeneous neutral conditions.

It is proposed that the phosphorothioate com-

The 5'-(B,y-peroxytriphosphate) analogues of all the common ribo- nucleosides and 2~deoxyribonucleosides ( 5 9 ) have been prepared by treating the nucleoside 5t-phosphoromorpholidates with peroxy- diphosphate. 67 Carbonyl bis(imidazole1 could also be used to effect condensation in most cases. The species (59) are sensitive

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7: Nucleotides and Nucleic Acids 215

Ph

B"3

(58)

0 0 0 II II II

HO-P-0-0-P-0-P-0-R I I OH OH

I OH

(59) R Nucleosidyl-5'

to acids and to reducing agents, such as hydrazine and low-valency transition metals. Thermal degradation of the adenosine derivative affords ADP, AMP,and some ATP, and it has been suggested that a species such as (60), which might fragment to form AMP or adenosine

0- 0-0 0- 0 \ I II

// \o/i-T- 0 0- 0-

(60 1

0- (Ado-5')

O X 0 0 II I II I I I l l I

HO-P-C-P-0- P-0-R

OH Y OH OH

(61) X = Y = CI

(62) X = Y = F (63) X = F; Y = H

R = Ado-5' I or Guo-5 /

5'-(y-peroxytriphosphate) could be formed. Isotopic labelling should permit this to be resolved. Of many enzymes tested, only NAD' pyrophosphorylase appeared able to utilise adenosine 5 ' - ( 6, y-

peroxytriphosphate) as a substrate, though it was a potent inhibitor in a number of cases. The selectivity of enzymes for a single stereoisomer of a six-membered metal-nucleotide chelate, which species (59) cannot form, may account for the lack of substrate activity.

Adenosine and guanosine 5'-phosphoromorpholidates have been condensed with dichloromethylene-,difluoromethylene-,sndmonofluoro- methylene-bis(phosphonic) acids to give isopolar isosteric ana- logues (61-63) of ATP and GTP, and with acetylene-1,2-bis(phosphonic) acid to give ( 6 4 1 , anisopolar but non-isosteric analogue of ATP. 68

The changes in the downfield shift between P n.m.r. spectra of the analogues were taken to reflect the differ- ences between electronegativity and dn bonding effects at P the Pa resonance value is expected to be invariant), and increased with decreasing electronegativity of the 6,y-bridging groups. Ion-

and Pa in the 31P Y

(since Y

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216 Organophosphorus Chemistry

0 0 0 0 0 0 II II I I II I I I I

I I I I I I HO-P-Cr C-P-0 - P-0-(Ado- 5 ' )

OH OH

HO-P-0- P- 0 -P-0 - (dfhd - 5 1 I

OH OH NH, OH

( 6 4 ) ( 6 5 )

selective electrode measurements and 31P n.m.r. data were used to evaluate the binding of divalent metal cations to the analogues, which refled steric and electronic features. While (64) readily formed a complex with barium ions, its complex with calcium ions was weak compared with the other analogues, and it failed to bind magnesium ions significantly. The changes in chemical shift at Pa betweenuncmplexed and metal-complexed ATP were compared with the equivalent changes in the analogues as an index of "goodness of analogue quality", and it was concluded that the B, y-dif luoro- methylene species afforded the closest analogue of ATP.

- pl- (Thymidine-5' ) -P1-aminotriphosphate - (65) has been prepared by treating the 2'-deoxythymidine analogue of (35) with pyro- phosphate. 69 On alkaline hydrolysis, (65) affords a mixture of 2'-deoxythymidine 3 ' - and 5'-phosphoramidates, 2'-deoxythymidine, and inorganic phosphates, while on reaction with cytidine in aqueous ammonia it gives (2'-5')- and (3'-5')-Cp(c!T), cytidine 2',3'-phos- phate, and thymidine. The 5'-g-phosphonomethyl' analogues of ATP and GTP reported last year7' function as phosphate donor substrates for uridine kinase fromL1210 cells, albeit with a lower ymax than ATP . 71 transition-state complex required may account €or the difference in rates.

A reduced ability of the magnesium chelates to form the

In an enzymatic synthesis of (Sp)-adenosine 5'-g-(l-thiotri- phosphate) (ATPaS), adenosine 5'-phosphorothioate is incubated with adenylate kinase and pyruvate kinase immobilised on a copolymer of acrylamide and N-acryloxysuccinimide ,together with ATF and phosphcenol pyruvate (PEP). 72

(Sp)-ADPaS formed by stereoselective phosphorylation of the pro-R oxygen of AMPS by adenylate kinase, to give the desired product, but also regenerates the ATP consumed by the adenylate kinase. The (gp)-ATPcrS can thus be obtained on a 2 0 millimole scale. Adenosine 5'-(3-thiotriphosphate) (ATPyS) can be prepared on a 50 millimole

The pyrnvate kinase not only phosphorylates the

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7: Nucleotides and Nucleic Acids 217

scale using glycerokinase, pyruvate kinase, triose phosphate isomer- ase, glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate kinase,and lactate dehydrogenase immobilised on the same polymer as above, together with dihydroxyacetone, thiophosphate, PEP,and ADP as substrates, 73 Essentially, thiophosphate is incorporated into 3-phosphoglyceroyl thiophosphate by the action of glyceraldehyde 3-phosphate dehydrogenase, and the thiophosphoryl group then transferred to ADP to yield ATPyS in a process catalysed by phos- phoglycerate kinase.

In a novel method for synthesis of ATPBS and ADPBS, adenosine is treated with phosphoryl chloride to give adenosine 5'-phosphoro- dichloridate, and then with 2',3'-g,g-methoxymethylideneadenosine 5'- (2-thiodiphosphate) to give El- (5'-adenosyl)-p2- 5'- (2' ,3'-meth- oxymethy1idene)adenosyl 2-thiocyclotriphosphate (66). 7 4 On suc- cessive treatment with sodium periodate (to cleave the adenosine ring),2-mercaptoethanol (to quench excess periodate), and buffers of pH2 (to deblock the protected ribose ring) and 10.5 (for B-eli- mination of the oxidised adenosine moiety), ATPBS is formed in good yield. The formation of the cyclotriphosphate (66) was indicated

by the observation that if work-up was performed in [1801-H20, the ATPBS obtained was labelled with l80 at Pa and P . treated successively with diphenylphosphorochloridate followed by 2 , 3 -2,g-methoxymethylideneadenosine 5 '-phosphate ,El - (5 -adenosyl) - - P2- 15'- (2' ,3'-methoxymethylidene) adenosyl] l-thiopyrophosphate (67) is formed. ( 6 6 ) above then affords ADPBS in good yield.74 at pH2 is omitted, the product is the protected ADPBS used above to synthesise ATPBS. reaction sequence, and the resulting [ B-1801-ADPBS (and unlabelled

If AMPS is

Use of the same deblocking sequence as described for If the treatment

(Ep)- [C~-~~O]-AMPS has been subjected to this

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218 Organophosphorus Chemistry

S 0 II I I

(Ad 0- 5' 1 -0 - P-0 - P- 0 OH I OH I - y 0 \ y e

ADPBS)isolated, treated with PEP and pyruvate kinase {to give (2~)- [B-180]-ATPBS} and acetyl phosphate and acetate kinase {to give (9)- [B-180]-ATPBS} and the isotopic labelling of the thiotriphosphate chain then investigated by degradation, methylation,and mass spec- troscopic analysis. 75 had occurred during the coupling step, so that both (gp)- and (?PI- [ B - 01-ADPBS had been formed in equal quantity. As a check, (5~)- [cr-l8OI- AMPS was treated successively with diphenylphosphorochlori- date and AMP, and the resultant Rp and Sp epimers of the diadenosyl monothiopyrophosphate formed found to contain equal quantities of l80. Thus phosphoanhydride formation by Michelson' s procedure in- volves neither retention nor inversion, but epimerisation at phos- phorus. It is likely that pyridine, which is present in the coup- ling step, displaces diphenyl phosphate forming a pyridinium phos- phate intermediate in which the stereochemistry is randomised either by exchange with other pyridine molecules or by reversible dissociation to pyridine and the nucleoside metaphosphate within a solvent cage. The pyridine is then displaced when the phospho- anhydride is formed.

The results indicated that epimerisation

18

(Rp) - [ B y-l80, Y - ~ ~ O I I - GTPyS has been prepared as described pre- viously for the corresponding ATPyS species in another Report7' and used to investigate the stereochemical course of thiophosphoryl group transfer catalysed by mitochondria1 PEP carboxykinase. 76 thiophosphoryl group is transferred by the enzyme to oxaloacetate giving chiral [ 01-thiophospho-en01 pyruvate, and the stereo- chemistry of this step analysed by further transfer of the thiophos- phoryl group to ADP using pyruvate kinase (inversion) to give chiral [ y - l 8 0 1 -ATPyS, and thence to AMP using adenylate kinase (inversion) to give chiral [B-1801-ADPt3S, which was analysed for isotopic labell-

The

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7: Nucleotides and Nucleic Acids 219

ing as described above (Ref. 75). Thiophosphoryl transfer was found to have proceeded with inversion, probably by a single dis-

17 17 placement mechanism. (Sp)-[ By- 0,y- 0,180]-GTPyS (68) has been used as a substrate to determine the stereochemical course of phos- phoryl transfer in the reaction catalysed by adenylosuccinate synthetase. 77 into ATPyS enzlmically (see above, ref. 73) and transferred

The chiral thiophosphate released is incorporated

S 0 0 \ II II

8 -P-O- P-OR I I OH OH

8 ( 6 8 ) R = Guo-5 ' 1 8 "0

(691 R = Ado-5' 1 0 = "0

thence to form ATPBS using the methods already previously described. inversion at phosphorus had occurred during thiophosphoryl trans- fer. inorganic pyrophosphatase, and analysis of the chiral thiophos- phate released, as above, indicates that the hydrolysis occurs with inversion of configuration at P Y ? ~ formation of an enzymic acyl phosphate intermediate which is then hydrolysed with C-0 cleavage was excluded by a single-turnover ex- periment in which inorganic pyrophosphate was hydrolysed by the enzyme in [1801-H20, when equimolar proportions of [ 041- and [l80, 1603]-phosphate were formed. single-step direct phosphoryl transfer to water takes place. Sim- ilar studies using (Rp) - [BY-~~O,~-~*O~-ATPYS have been undertaken to determine the stereochemical course of hydrolysis catalysed by adenosine triphosphatase from a thermophilic bacterium. 79 hydrolysis was performed in [1701-H20, and the thiophosphate re- leased analysed as above to show that inversion at phosphorus had taken place, probably by direct in-line displacement of ADP. Hydrolysis of ATPyS in['801H20 was also performed, and the thio- phosphate released contained only a single l8O atom, excluding the possibility of oxygen scrambling between thiophosphate and water.

Analysis by 31P n.m.r. spectroscopy showed that net

(Sp)-[ f3y-170,y-170,1801-ATPyS (69) is a substrate for yeast

The possibility of initial

16

It appears, therefore, that

The

The stereochemical course of the reaction catalysed by yeast phenylalanyl-tRNA synthetase has been investigated using firstly (sp) - a-l80, 3 B -180]-ATPaS and unlabelled phenylalanine, and

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220 Organophosphorus Chembtry

secondly (sp) -ATPaS and [ ' * O ] -phenylalanine. 8o The phenylalanyl adenylate formed in each case is cleaved using hydroxylamine to afford chiral [l8O1- AMPS, which is converted to (Sp)-ATPctS using adenylate kinase and pyruvate kinase and analysed to determine the position of the l8O atom. It has been found that negative ion fast atom bombardment (FAB) - mass spectrometry can be used for direct analysis of the underivatised ATPctS.The principal mass ions obtained for the ATPclS derived from the first experiment are shown( 70) and

H O - ! - O t ~ - @ ~ / - O i 0 (Ado-5' 1

OH OH I OH

97J 179J 275J

(70) (M-HI- = 5 2 4

clearly indicate that180 is in the a B-bridge position. The comparable values obtained for ATPctS from the second experiment were 97, 177, 275, and 524, showing that l80 was in an ct non-bridge position. Both results were confirmed by 31P n.m.r. analysis, and demonstrate that inversion occurs at Pa upon formation of the phenylalanyl adenylate. The mass spectrometric analysis requires far less material than the 31P n.m.r. method, with 100 nanomoles of product sufficing to establish the position of l80 incorporation by normal as well as linked-scan mass spectrometry.

(Sp)-Adenosine - 5 I - c o(-'~O, 18021 -triphosphate has been prepared by phosphorylating adenosine using thiophosphoryl chloride, hydro- lysing the resultant 5 ' -thiophosphorodichloridate in [1803 -H20, enzymic conversion to (sp) - [ kinase, then hexokinase), displacement of the sulphur atom by oxi- dation with bromine in [170 l - H 2 0 , and finally re-phosphorylation with pyruvate kinase. 81 stereochemical course of formation of tryptophanyl adenylate by tryptophan-tRNA synthetase from beef pancreas , with the 1801 -AMP released on treatment with hydroxylamine being analysed using methods described elsewhere. 5 6

ADP with inversion at Pa, probably by an associative "in-line" mechanism, was found to have occurred.

18 02]-ADPaS (adenylate kinase, pyruvate

The product was used to investigate the

[ l60 , l7O,

Once again, displacement of

82 , 8 3 Adenosine 5 I - [ 6 , y - l 8 O 1 - triphosphate has been prepared.

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7: Nucleotides and Nucleic Acids 22 1

One synthetic method involves phosphorylation of (gp)-[ p180]-ADPBS using PEP and pyruvate kinase (which phosphorylates specifically the pro-S oxygen atom, in this case oxygen-18) to give (?p)-[By- 01-

ATPBS, which is then oxidised with bromine water. A better method involves treating 2,O-diethyl thiophosphorochloridate with [1801-H20

to form the bridge-oxygen-labelled tetraethyl dithiopyrophosphate(7Q. The ethyl groups are removed with TMS-iodide and after desilylation of (72) in bicarbonate buffer, oxidation with bromine water affords

18

180-bridge-labelled pyrophosphate (73 in good yield.

(71) R = Et, X S

(72) R = TMS,X = S

(73) R = H , X = O

I ; ; ; RO-pP-@-pP-0~

I I OR OR

On treatment with (73), adenosine 5'-phosphoromor~holidate gives the desired ATP analogue. 82

described and was also used to prepare the analogously 170-labelled corn pound^.^^ Both [ By-1801-ATP and [ By-1701-ATP were used to follow the bicarbonate-dependent By-bridge + B-non-bridge positional oxygen isotope exchange catalysed by carbamoyl phoaphate synthetase from E.coli.

A variant of this latter route has been

Investigation of the effects of 170 and l80 isotopes with regard to chemical shifts and line-broadening in the 31P n.m.r. spectra of a number of specifically oxygen-labelled nucleotides have led to the conclusion that there is an approximately linear relationship between the 31P n.m.r. chemical shifts and the spin-spin coupling constant be- tween 170 and 31P nuclei.84 The complexes formed between (2~)- [CX-~~OI-ADP and Mg2+ or Co3+ ions have been studied in order to measure the effects of the metal ions on the 31P chemical shifts in the u,B-bidentate A and A chelate isomers: in the A isomers the metal ion is bonded to l60, in the A isomers to l8O. Similar studies were performed using ( ~ ~ ) - [ C ~ - ~ ~ O ~ - A D P , and it was found that binding of the Co3+ ion to l 7 O in the A isomer produced a downfield signal ( 9 8 p.p.m.1 and binding to l 6 O in the A isomer an upfield signal (-82 p.p.m.) in the 170 n.m.r. spectra, while bind- ing of the Mg2+ ion gave an averaged signal due to rapid exchange of both species. "label" in macromolecular systems, by virtue OF its quadrupolar or dipolar line-broadening effects in 31P n.m.r. spectra. This pro- posal has been amply justified (see Section 6).

magnitude of the 180-isotope effect in

It was proposed that 170 would be useful as a

170 N.m.r.

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222 Organophosphorus Chemhtry

spectroscopy has been applied to a number of l70-labelled phos- phorus compounds, including thiophosphate and phosphonate analogues of ATP, in order to assign unambiguously the chemical shifts of all l 7 O resonances associated with bridging and non-bridging oxygen atoms in these species.85 found to occur on protonation of a 170-P function demonstrating further that l 7 O n.m.r. spectroscopy can be used to quantitate site and deqree of charge neutralisation in phosphate ester anions.

An upfield shift of some 50 p.p.m. was

Similar studies using I 5 N and l7O n.m.r. spectroscopy have been carried out on I 5 N - and 170-labelled imidodiphosphates , including adenylyl imidodiphosphate, which demonstrated inter alia that pro- tonation of the tetraanion of adenylyl imidodiphosphate occurs ex- clusively on the y-phosphoryl oxygen atoms. a 6 This suggests that the imino-tautomers of the imidodiphosphate moiety do not occur to any significant extent, since these, if present, would be likely sites for protonation.

Oxygen exchange studies in which the l8O isotopes of Mg.[0~-~~0, Y - ~ ' O ~ ] - ATP are exchanged with l 6 O of solvent water during hydro- lysis of ATP by actomyosin have led to the suggestion that, in addition to two spurious and artefactual pathways, two "legit- imate" pathways for the hydrolysis of ATP by actomyosin occur distinguishably. In one pathway, actin and myosin are dissociated in the exchange reactions: in the other they remain associated. Arising in part from this, it has been proposed that the two heads of myosin are functionally different, with one head able to cleave bound Mg.ATP reversibly (and thus support oxygen exchange) while it is free of actin, and the other able to do so only when actin is bound. This accounts for the experimental observation that the two different hydrolysis pathways differ considerably in the degree of oxygen exchange for which they are responsible. A model of obligatorily sequential interaction of the actin heads with the myosin has been proposed which, if established, will represent a considerable contribution to muscle biochemistry by studies of this type. Other significant studies on solvent oxygen exchange with 1 Y-~~OI-ATP have been reported in connection with the catalytic activities of mitochondria1 ATPase8' and of chloroplast ATPase and ATP synthase?'

a 7

Reverse-phase h.p.1.c. has been used to separate the A and A

diastereoisomers of u,B-bidentate Cr(III)-(H 0 ) ADP, using 2 4

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7: Nucleotides and Nucleic Acids 223

ethanesulphonic acid as ion-pairing reagent and isocratic buffer (a faster and more convenient method than the cycloheptaamylose columns used previously), and the interconversion of the two forms has been studied as a function of pH, temperature, buffer type and concentration. 91 The kinetic data suggest that

specific base catalysis occurs via an S N I C R mechanism, as indicated: loss of a proton from the A - diastereoisomer gives ( 7 4 ) , which opens to give the 8-monodentate chelate ( 7 5 ) which can rotate at the Pa-O-PB bridge, and thus form the (deprotonated) A-

diastereoisomer ( 7 6 ) . The A -a,$-bidentate diastereoisomers of a number of chromium (111) and cobalt (111) aquoammine complexes of ADP have been prepared and investigated as competitive inhibit- ors (with respect to ADP) of creatine kinase. 9 2 Generally the Cr (111) - nucleotide complexes bound more strongly to the enzyme than the equivalent Co(II1) complexes, and the binding affinity increased with the number of water molecules coordinated to the metal atom,due to stronger hydrogen-bonding interactions between the complex and the protein.

Investigation of the specificity of the ATPase from the therm- ophilic bacterium PS3 for the magnesium and cadmium complexes of (Ep)- and (Sp)-ATPaS and -ATPBS elicited no special preference for the ATPaS diastereoisomers,but a preference for (Sp)-Mg.ATPBS and (Rp)-Cd.ATPBS (A-stereochemistry) over their respective diastereo-

79 isomers, showing that the A-B,y-nucleotide chelate is favoured. The same chelate was found to be favoured by tryptophanyl-tRNA synthetase from yeast. 31 In studies on the geometry of the metal ion-GTP complex bound at the active site of EF-Tu from Bacillus stearothermophilus, the comparative abilities of the magnesium and cadmium complexes of (Ep)- and (SpI-GTPclS, GTPBS, and also of GTPyS

93 to inhibit an antibiotic-induced GTPase activity were observed.

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224 Organophosphorus Chemistry

It was concluded that the pro-R oxygen atoms at Pa and PB interacted with the protein and the pro-S oxygen at P dinated to the metal ion. In a related study, e.p.r.spectroscopy was used to observe the complexes formed when GTP was added to EF-Tu in the presence of manganese ions, and using data on the kinetics of these conditions permitted specific association of the individual e.p.r. signals with the complexes EF-Tu.Mn.GTP, EF-Tu.Mn.GDP.P and EF-Tu.Mn.GDP. 9 4

from superhyperfine coupling between the 170 nucleus and the manganese ion, it was possible to show, by u~ing[a-~~O$-GTP, B-170,]-GTP, and [ v-~'O~]-GTP , that the manganese ion was coordin-

ated to PB in the complexes EF-Tu.GDP.Pi and EF-Tu.GDP, and by working in 11701-H,0 it was possible to deduce the likely number of water molecules coordinated to the manganese ion in each com- plex, and the number of metal-protein interactions.

B was

32P]-orthophosphate release from [y_32Pl-GTP under

i' By observing the line broadening resulting

Diadenosine 5' , 5 ' I -g1,p4-tetraphosphate (Ap4A) ( 7 7 ) is presently arousing much interest. Its role in cellular meta- bolism has been reviewedg5 and has been the subject of at least one Meeting.96 It seems to arise, with Ap3Gpp, Ap4G, Ap3G,and Ap A in organisms such as E.coli and Salmonella typhimurium in response to stress, such as heat shock, exposure to ethanol, or cell oxidation, and it has been proposed that these molecules may be "alarmones" which mediate heat-shock response. 9 7

phosphohydrolase enzyme has been purified from E.coli which hydro- lyses ApnA (p=3-6) and also Gp4G, to give the corresponding nu- cleoside-5'-diphosphate as one product in each case. 98 platelets store considerable amounts of Ap3Ag9 which, on release in response to thrombin-induced aggregation, Slowly induces aggregation, apparently by heing hydrolysed to ADP. This effect is antagonised by A p 4 A . The syntheses of

3

A pyro-

Human

100

0 0 0 0

( A d o - 5 1-0-P-X - P-Y - P-X- P-0- (Ado- 5 ) I II II II II I

I I I I OH OH OH OH

(77) x = Y = 0

(78) X = 0, Y = CH,

( 7 9 ) X = CH,, Y = 0

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7: Nucleotides and Nucleic Acids 225

phosphonate analogues of Ap4A, (78) and ( 7 9 1 , have been realised by activating methylenediphosphonate with carbonyl bis(imidazo1e) and coupling it to the other appropriate compon- ents. lo' The U.V. spectra, hypochromicity,CD, and formation and properties of the Mg2+ and Zn2+ complexes of a large number of bisnucleoside oligophosphates, including (771, and (791, Ap3A,and a number of "cap" structures have been reported. lo2 complexes display base-stacking interactions at neutral pH and low temperature, the degree of stacking depending on the length of the polyphosphate chain (as does the stability of the complexes). CD studies also indicate that Ap4A binds to biogenic amines, basic proteins, and poly(dT) (with a stoicheiometry of 2 residues of dTMP per molecule of Ap4A)fo3 Most cations cause destacking of the adenine rings .

~ 1 1 metal

Treatment of 5'-g-TBDMS-2'-deoxythymidine successively with morpholino-g,g-bis(1-benzotriazolyl) phosphate (801 , and with 244- nitropheny1)ethanol in the presence of N-methylimidazole, followed by 5'-deprotection with 4-toluenesulphonic acid and further treat- ment with ( 8 0 ) , affords (81). This species is functionally akin to (29) in that either phosphate group may be selectively deprot-

-

I

0 N-P-0 O-P-N

0 W I o==';-NWo 0 0 I aN> N

I CH,CH,C,H,NO,- 4

( 8 0 ) (81)

ected for further elaboration. Treatment of (81) with triethyl- mine removes the benzotriazole group, and subsequent reaction with orthophosphate gives the 5'-diphosphate. Thereafter, the 4-nitrophenethyl group may be removed with DBN, and finally, treatment with pyrophosphate yields 2'-deoxythymidine 3'-triphos- phate 5 '-diphosphate. lo4 2 '-g-Acetyl-3 '-trif luoroacetamido-3 I-

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226 Organophosphorus Chemistry

deoxy-N6-benzoyladenosine has also been phosphorylated using (80) , and the product treated successively with triethylamine and pyro- phosphate, and then deprotected using ammonia to afford 3'-amino- 3 I-deoxyadenosine 5 '-triphosphatelo5 2 ' ,3 '-Dideoxy-3 '-aminonucleo- side 5'-triphosphates areeffective inhibitors of both bacterial and mammalian DNA polymerases, becoming incorporated at the 3'-termini of growing chains to terminate elongation. They may therefore represent a useful alternative to 2',3'-dideoxynucleotides for use in DNA gel sequencing methods. lo6 been prepared from the nucleoside using standard methods, and foundtobe apowerful and selective inhibitor of DNA polymerase a from HeLa cells, although without activity on DNA polymerases Band y . Modification of dGTP at the 2-amino group may thus be useful in designing polymerase-specif ic affinity probes.

- N2- (4-n-Butylphenyl)-dGTP has

lo8

The B2 subunit of ribonucleotide reductase from E.coli con- tains a tyrosine radical essential for enzymic activity. When 2'- azido-2'-deoxy-CDP or 2'-azido-2'-deoxy-UDP is supplied to the enzyme, the tyrosine radical decays and concomitantly a new trans- ient radical is formed, in which the characteristics of the e.p.r. spectrum suggest hyperfine coupling to one nitrogen nucleus and one hydrogen nucleus. 109'110 If 2'-2d -2'-azido-2'-deoxy-UDP is used instead, no significant change in the e.p.r. spectrum is seen, indicating that the radical is not localised on a carbon atom. Furthermore, deuteriation at H-1' or H-3' also does not al- terthe spectrum from that seen with 2'-azido-2'-deoxy-UDP. ever, when [ 2'-l5N1 -2'-azido-2'-deoxy-UDP is used the e.p.r. spectrum splitting pattern alters, and the radical is thus thought to be localised on the nitrogen atom, one possible structure being (82). Isolation of the enzyme from bacteria grown in

How-

H30"20wra OH * N - O O E n z -

(82 1 R i b-5-PPP- mRNA

(83)

[l7OI-H2O might help to resolve the structure further: the

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7: Nucleotides and Nucleic Acids 227

mechanism of action of this enzyme has long been an enigma.

5-Azacytidine 5'-monophosphate and 5-azacytidine 5'-triphosphate have been prepared from the nucleoside by standard methods. Both are more unstable than the nucleoside, possibly due to the phosphate groups acting as general base catalysts, promoting attack of water at C-6 of the triazine rinq with subsequent decomposition. However, the triphosphate is a substrate for AMP(CMP)tRNA nucleotidyltrans- ferase from yeast, permitting 5-azacytidine residues to replace one or both cytidine residues in the ..CpCpA terminus of tRNAPhe. The modified tRNA molecules were functionally active in the charging re- action and in protein synthesis, and the 5-azacytidine residues were far less susceptible to hydrolysis, possibly due to protection from attack of water by stacking interactions.

- PI- (7-Methyl-5 '-guanosyl) -p2- (5 I -adenosyl) pyrophosphate has been prepared in fair yield by treating 7-methylguanosine 5'-phosphate successively with carbonyl bis(imidazo1e) and AMP. However, much poorer yields were realised when attempting to apply this method to the synthesis of ''cap'' structures. phenyl) -P2- - (7-methyl-5 '-yuanosyl) -1-thiopyrophosphate is treated with iodine in the presence of pAmpG, or with silver nitrate in the presence of pAmpGpU (prepared by phosphotriester methods: Am is 2 ' -

- 0-methyladenosine) , the ''cap" structures m G (5' )pppAmpG and m G ( 5 ' ) -

pppAmpGpU are formed respectively. The presence of the 7-methyl- guanosine-containing terminus in eukaryotic mRNA stimulates its cell- free translation to a degree which is dependent on ionic strength, pH, and other parameters. The presence of added 7-methylquanosine- 5'-triphosphate causes little inhibition of globin mRNA translation at pH values below 7.2, but significant inhibition above this value. Since the p_Ka value of the N-1 proton of m GTP is ca. 7 . 4 under the conditions optimal for cell-free translation, it has been suggested that the presence of the enolate form ( 8 3 ) in the "cap" is necess- ary for the recognition of 7-methylguanosine by cap-binding protein

114 as a preliminary to translation.

If, however, pl-(S-

7 7

7

The reaction of N-methyl-3-nitrosourea (MNU) with dATP has been examined in vitro at room temperature at pH7. The major products are dATP and dADP methylated at P y and PB respectively, although products of base-methylation (at N1 and N 1 and bis-methylation (on base and Py of the triphosphate chain) also occur.'15

3

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228 Organophosphorus Chemistry

results thus suggest that significant alteration in the constitution of cellular deoxyribonucleotide pools can occur when cells are ex- posed to MNU.

Cytidine 5'-tetraphosphate has been found as a significant con- taminant (0.3-3.9%) in commercial samples of CTP. ''' contamination arises by dismutation of CTP or otherwise is not clear; Nucleoside 5'-triphosphates may conveniently be concentrated from aqueous solutions by coprecipitation with calcium fluoride, and ATP may be separated almost quantitatively from solutions as low as 5 nanomolar in concentration. The technique may also be used for nucleoside 5'-diphosphates, but not for monophosphates or cy- clic nucleotides. Dimethyl sulphoxide containing lithium chloride has been described as a good non-aqueous solvent for ATP, and per- mits derivatisation of ATP using such reagents as succinic an- hydride, terephthaloyl chloride and hexamethylene diisocyanate to be performed directly. The amphiphilic cyclosymmetric decapep- tide gramicidin S binds nucleotides in aqueous solution and the resulting complexes partition in organic solvents, the phase-trans- fer efficiencies increasing in the order monophosphate < diphosphate < triphosphate. The interaction between gramicidin S and nucleotide is thought to be primarily ionic in nature, between protonated &-amino groups of ornithine residues and the nucleotide phosphate groups.

Whether the

4 Oligo- and Poly-nucleotides

4.1 Chemical Synthesis.- A review on phosphorylation in the syn- thesis of mono- and oligonucleotides has appeared12* and several extensive reviews on the chemical synthesis of oligo- and poly- nucleotides have been published. 121

In the synthesis of oligodeoxyribonucleotides, species such as (84) have hitherto generally been prepared by treating the appro- priate base-protected 5'-~-dimethoxytrityl-2'-deoxynucleoside with an N,N-dialkylmethoxychlorophosphoramidite. However, these re- agents are difficult to prepare and very sensitive to moisture, and the use of bis-(dialky1amino)methoxyphosphines (85-87) as an al-

122,123 ternative method for forming (84) has now been described. These species are formed by reaction of methoxphosphorodichlori-

dite with the appropriate secondary amines, and treatment of the

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7: Nucleotides and Nucleic Acids 229

tH MeO-P- NR,

I NR,

DMTrO

(84 ) B* = Protected base

( 8 5 ) R Me,CH

(86) R,= (CH ,I4 (87) R,= (CH,CH,),O

protected deoxynucleoside with (851, (861, or (87) in the presence of a weak acid ( tetrazole122 or 4,5-dichloroimidazole 123) affords

the appropriate species (84) in high yield. Little or no(3'-3')- coupling occurs. Since this procedure generates no insoluble amine hydrochlorides, species (84) may be prepared in situ and used directly for solid-phase oligonucleotide synthesis without the necessities of filtration and purification, thus simplifying hand- ling and avoiding possible exposure to moisture of phosphity- lating reagents. 8-Cyanoethyl N,N-dialkylchlorophosphormidites

(88-90) have been prepared by treatment of 8-cyanoethylphosphoro- dichloridite with the appropriate secondary mines (or, better, with their N-trimethylsilylated derivatives) at low temperature, and when these were used to phosphitylate base-protected 5'-g-dimethoxy-

R ,N - P -0CH CH, CN I

Cl

(88) R = Me

(89) R = Me,CH

(90) R,= (CH2CH2120

trityl-2'-deoxynucleosides in the presence of N_-ethyldiisopropyl- amine, the appropriate species (91) were formed. stable species have been utilised for oligonucleotide synthesis on controlled-pore glass beads, and offer the useful property that removal of the cyanoethyl groups and base-protecting groups as well as the oligonucleotide from the base can all be accomplished by a single treatment with concentrated ammonia, without (it is claimed) internucleotide cleavage or isomerisation. The diiso- propylphosphoramidites formed from ( 8 9 ) were found to be particul- arly useful. Substituted 5-phenyltetrazoles have been found to catalyse the reaction of (84, NR2 = morpholino) and similar phos-

124, 125 These

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230 Organophosphorus Chemktry

phoramidites with 5'-hydroxy-group-bearing components in oligo- deoxyribonucleotide synthesis. 126 When the phosphoromorpholidite was mixed with these compounds in acetonitrile, no amidite signals other than those of the phosphoromorpholidite parent could be de- tected using 31P n.m.r. spectroscopy, indicating that the active coupling species were present in low concentration. Activation is thought to involve protonation of (84) as the rate-determining step: 5-(4-nitrophenyl)tetrazoleI a stronger acid than tetrazole, is also a better catalyst, and its use €or coupling reactions is there- fore recommended.

Hydroxybenzotriazole-activated phosphotriester intermediates in oligonucleotide synthesis have been formed and used both in polymer-supported oligodeoxyribonucleotide synthesis127 and in oligoribonucleotide synthesis. 128 In the former study,base-pro- tected 5'-g-dimethoxytrityl-2'-deoxynucleoxynucleosides were treated with 2-chlorophenyl g,g-bis(l-benzotriazolyl) phosphate to give products of general formula (92) which were used to elongate a growing

B*

0 II

0 C,H ,Cl- 2

f B

C H

,Me

( 9 2 ) Me

(93)

oliqodeoxyribonucleotide chain, attached via its 3'-terminus to a k i e s e l g u h r - - p o l y a c r y l a m i d e support, in the 5'-direction. The coupling reaction is performed in high yield by adding ( 9 2 ) in the presence of N-rnethylimidazole and N,N-diisopropylethylamine, and no other condensing agent is necessary. The latter study utilised base-protected 5'-~-laevulinoyl-2'-~-methoxytetrahydropyranyl ribonucleosides which were phosphorylated as above to give the functional equivalents of (921, which then reacted with a slight molar deficiency of base-protected 2'-g-methoxytetrahydropyranyl ribonucleosides to give essentially the (3 ' - 5 ')-linked products, G. coupling occurred selectively at the 5'-hydroxy group of the ribonucleosides, with only minor quantities of by-products. This useful observation was applied to prepare a pentamer.

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7: Nucleotides and Nucleic Acids 23 1

Oligodeoxyribonucleotides have also been prepared using di(1,2- dimethylethenylene) pyrophosphate to generate the reactive inter- mediates. Using this reagent, intermediates such as ( 9 3 ) are formed,and coupled without prior isolation with the 5'-hydroxy group of a base- and sugar-protected deoxyribonucleoside, to give a phosphotriester in which the phosphate group is esterified by 3-

hydroxybutan-2-one. When extending such a protected dinucleoside phosphotriester which had been deblocked at the 3'-hydroxy group in order to prepare a tetranucleotide, ~-(1,2-dimethylethenylenedioxy- phosphory1)imidazole (94) was found to be a better phosphorylating agent than the pyrophosphate. The pyrophosphate has, however, been used to prepare 2'-deoxythymidylyl-(3'-5~)-puromycin. 130

(94) I CH,CH,C,H,NO,-4

(95)

The synthesis and thorough characterisation of a number of base-protected guanosine 3 ' - and guanosine 2'-phosphotriesters, in which the sugar hydroxy functions were protected by TBDYS groups, have been reported, and these compounds used to prepare fully-pro- tected GpGp. 13' TBDMS groups have also been used to protect the sugar in adenosyl derivatives during the synthesis of homogeneous adenylate(3'-53-oligomers by the phosphotriester method. 132 this study the 4-nitrophenylethyl group was used, inter alia, to protect the phosphate group. The same protecting group was in- troduced in a solution synthesis of protected di-(2'-deoxy)nu- cleoside phosphotriesters by utilising 4-nitrophenylethyl morpho- lino phosphorochloridite as phosphitylating agent. 133 The inter- mediates of type ( 9 5 ) which were formed were coupled with the 5'- hydroxy group of a protected 2'-deoxynucleoside in the presence of 1g-tetrazole to obtain the desired products. The 4-nitrophenyl- ethyl group is easily removed by B-elimination using DBU; it has also been introduced as a protecting group at O4 of thymine and uracil bases by treating their sugar-protected nucleosides with 4-nitrophenylethyl iodide and silver carbonate, and the 04- (4-

In

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232 Organophosphorus Chemistry

nitrophenylethy1)pyrimidine species thus formed are stable during the processes involved in phosphotriester oligonucleotide synthesis and removal of the most commonly used phosphate- and sugar-protect- ing groups. 134

A number of new groups for protection of phosphate, base,and sugar functions during oligonucleotide synthesis have been described. The 2-methyl~ulphonylethyl~~~ and 2-cyano-1, l-dimethylethyl 13 6

groups have both been introduced, by use of their respective mor- pholino phosphorochloridites as phosphitylating reagents, as phos- phate-protecting groups in oligodeoxyribonucleotide "phosphite" triester syntheses. Both are removed rapidly by 0.2M sodium hydroxide in methanolic dioxan, conditions which do not disturb other common protecting groups, and both are suitable for use in solid-phase syntheses. 2-Chloro-4-tritylphenyl cyanoethyl phos- phate has been condensed with base-protected S'-g-dimethoxytrityl- 21-deoxynucleosides to give monomer units of type (96) for use in

B'lc

CH

oligodeoxyribonucleotide synthesis. 137 The lipophilic group facilitates purification of the products of coupling by extraction and flash gel chromatography, and is readily removed, at the con- clusion of synthesis, by treatment with 4-nitrobenzaldoximate. Conditions for removal of the (previously described) 5-chloro-8- guinolyl protecting group from phosphate in fully protected d(TpT) have been examined. 138 phosphoramidate, while, if the 3'-hydroxy group of the 3'-terminal deoxythymidine is free, use of aqueous alkali leads to formation of the (3'-3')-linked dinucleoside phosphate. However, zinc ace- tate in aqueous pyridine or pyridine 2-carbaldoxime in aqueous dioxan effected selective removal of the 5-chloro-8-quinolyl group. 31P N.m.r. spectroscopy has been used to compare the kinetics of removal of the cyanoethyl group from phosphate in fully-protected mono- and oligonucleotides by different organic bases.

Use of ammonia results in formation of the

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7: Nucleotides and Nucleic Acids 233

In an effort to circumvent acid-catalysed depurination of N6- benzoyladenosine residues during the acidic detritylation of 5 ' - hydroxy groups in oligonucleotide synthesis, the N6-amino group of the adenine ring has been protected as its N-methyl-2-pyrrolidine amidine derivative(f0rmed by treating 2'-deoxyadenosine with 1-metk ~1-2~2-diethoxypyrrolidine) l4O or as a dialkylformamidine deriva- tive (formed by treating 2'-deoxyadenosine with the dimethyl acetal of the corresponding N,N-dialkylformamide). suppressed in these species, probably because the amidine formation preserves the basicity of the N6-arnino function. The amidines are largely stable to the normal conditions of oligonucleotide coupling and deprotection, the di-n-butylformamidine derivative being the most stable and promising of the dialkylformamidine series. The - N-methylpyrrolidine amidine protection is readily removed using ethylenediamine in a phenol-water solvent. The 2,2,2-trichloro-t- butyloxycarbonyl group has been introduced to protect the amino groups of the nucleobases as their urethane derivatives during oligo- nucleotide synthesis. It is claimed that the urethane-pro- tection thus introduced is more stable than the acyl protection normally used. The 2-nitrophenylsulphenyl group has also been introduced as a protecting group at the amino functions of the nucleobases by treating the silylated nucleosides with 2-nitro- phenylsulphenyl chloride.

the normal processes of oligonucleotide phosphotriester synthesis, and is readily removed using triethylammonium thiocresolate in pyridine. yl-g6-diphenylcarbamoyl derivative, and this pattern of protection is compatible with the processes of oligonucleotide synthesis using - S,S-diphenylphosphorodithioate nucleoside triesters reported previously. 144 At the conclusion of synthesis, the protecting groups are removed with methanolic ammonia. The protection of uracil residues by alkylation or acylation at the N-3 position has been reported. In the former, uridine protected at the 3 ' - and 5'- hydroxy groups was treated with methoxyethoxymethyl chloride. The y3 -methoxyethoxymethy lur idine species remained stable through the processes of oligoribonucleotide synthesis, and was finally re- moved quantitatively using trityl fluoroborate in aqueous aceto- nitrile, a reagent which also removed dimethoxytrityl groups and 4-methoxybenzyl groups from sugar hydroxy-functions. The 2,2,2- trichloro-1,l-dimethylethoxycarbonyl, anisoyl, benzoy1,and chloro- benzoyl groups have been introduced at the N-3 position of uracil

Depurination is

This group is also compatible with 1 4 3

2 '-Deoxyguanosine has been protected as its N2-propion-

14 5

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by treating sugar-protected uridine with the appropriate acid chlorides. 146 in the conditions normally used during oligonucleotide synthesis, and its use led to an improved yield when UpUp was synthesised using the S,S-diphenyl nucleoside 3'-phosphorodithioate method, in comparison with that obtained using unprotected uracil. The anis- oyl group was removed using methanolic ammonia, 2'-Deoxythymidine has been converted to its 4-2-phenyl derivative? and 2'-deoxy- guanosine to its 6-g-(2-ni t rophenyl)-2-N-phenylacetyl and 6-g-(3,-

5-dichlorophenyl) -2-2-phenylacetyl derivatives. 147 to be promising groups for protection of the bases during oligo- deoxyribonucleotide synthesis.

The N3-anisoyl derivative proved to be most stable

These appear

The 4-methoxybenzyl group has been introduced as a protecting group at the 2'-OH of ribonucleosides by treating 3',5'-protected uridine with sodium hydride and 4-methoxybenzyl bromide, or by treating 2',3'-g,~-(dibutylstannylene)uridine with 4-methoxybenzyl bromide and separating the 2'- and 3'-g-methoxybenzyl uridines formed . 14* is removed, as indicated above, with trityl fluoroborate. 2'-4- (1,3-Benzodithiol-2-yl)uridine has been prepared by treating 3',5'- blocked uridine with 1,3-benzodithiolium tetrafluoroborate and utilised in a synthesis of UpU. 149 removed using 0.01 M hydrochloric acid. The use of pyridinium 4- toluenesulphonate as acid catalyst for introducing the tetrahydro- furanyl group at the 2'-position of 3',5'-protected guanosine has been found extremely effective? and the tetrahydrofuranyl group has been used to protect the 2'-hydroxy group in, for instance, the

ment of 5 -g-monomethoxytrityl-2 I -g-tetrahydrofuranyl-N4-benzoyl- cytidine with 2-chlorophenylphosphorobis ( lH-1 ,2 ,4- t r iazo l ida te~ , followed by 2'-2-tetrahydrofuranyl-N_ -benzoylcytidine affords

At the conclusion of oligoribonucleotide synthesis it

The benzodithiolyl group is

synthesis of the anticodon heptanucleotide of tRNA fMet . I5O Treat-

4 (97)

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7: Nucleotides and Nucleic Acids 235

in reasonable yield, indicating a good degree of selective 5'-phos- phorylation. 15' Some 3'-3'-linked by-product is also formed. Also, treatment of base-protected 2'-g-tetrahydrofuranylnucleosides with 2-chlorophenyl 4-anisido phosphorochloridate showed fair selectivity for the 5'-position in forming ( 9 8 ) , although some bisphosphory- lated product was also obtained. In an extensive study on the effects of substrate structure and reaction conditions on the rates of detritylation and depurination of 5'-g-dimethoxytrityl-3'-g- (3,5-dinitrobenzoyl) -2 '-deoxy-N6-benzoyladenosine , the use of tri- fluoroacetic acid in lI2-dichloroethane was recommended as afford- ing optimal detritylation without purination when deblocking during oligonucleotide synthesis. 152 In a similar study involving slightly different substrates, the use of 3% trichloroacetic acid in nitromethane-methanol(95:5) was reported to give rapid detri-

153 tylation without depurination.

Cytidine 2'-phosphate has been used as a protecting group for the 3'-terminus in the "triester" synthesis of oligodeoxyribo- nucleotides. 154 A short, protected oligonucleotide block contain- ing aryl phosphotriester links is Coupled at its 3I-terminal phos- phate to _N4 ,g3 ,g5 ' - tribenzoylcytidine to give a terminus of general structural type (99). Detritylation of (99) then permits the chain to be extended in a 5'-direction by coupling with monomer

or oligomer blocks in the usual manner. Unblocking using pyri- dinealdoximate and ammonia removes the 2-chlorophenyl groups and the acyl groups, and causes partial lossofphosphocytidine as cytidine 2',3'-monophosphate, a process which is completed by treatment with lead acetate. The cytidine 2I-phosphate not only performs as a convenient and easily removed protecting group for

6 the 3'-terminus, but also suppresses depurination at adjacent - benzoyl-2'-deoxyadenosine residues.

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236 Organophosphorus Chemistry

ArS0,X t Nu' (ArSO,N?? + X - I

(100)

R ' O 0 \II

2 /

P-0- + (101)

R O

(102)

1 1 R O 0 0 OR

I I /

R20

(105)

?-.. R ' O 0

'1- Nu2 2 /

R O (106)

+ (102) + H t

1

(101)

R1O 0 \ 11 1 - P-0SO2Ar + Nu

2 / R O

(103)

do 0 (102 1 \I1 + 1

2 /P-NU + ArS03- -

R O

(104)

/ R '0

+ N U ~ H or NU'

X = C l , tetrazole, 3-nitro-1,2, 4 -triazole

Nu' = pyridine, DMAP, N- methylimidazole

NuL = tetrazole, 3 -nitro-l,2,4-triazole

R = 2-chlorophenyl

R = 5 - O-trityl-2'-deoxythymidine

1

2 1

R 3 = 3'-0-acetyl-2'-deoxythyrnidine

Scheme 1

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7: Nucleotides and Nucleic Acids 237

In an effort to determine the likely nature of the intermediates formed during coupling reactions to form phosphotriesters in oligo- deoxyribonucleotide synthesis, the condensation effected between 5'- - O-trityl-2'-deoxythyidine 3'-(2-~hlorophenyl)phosphate and 3 I - p

acetyl-2'-deoxythymidine by TPS-C1 in the presence of pyridine, and also by TPS-nt and arylsulphonyltetrazolides, has been studied using 31P n.m.r. spectroscopy, and the nature of the catalysis of this process by organic bases has been investigated. 155 A summary of the course of reaction proposed is presented in Scheme 1. The arylsulphonyl chloride, tetrazolidgor nitrotriazolide (100) reacts with a nucleophile of type 1 (e.g. pyridine, N-methylimidazole,or DMAP) to form intermediate (101) which reacts rapidly with the phospho- diester (102) to form the mixed sulphonate-phosphate anhydride (103). Reaction of another molecule of nucleophile type 1 with the highly rezctive (103) (no 31P n.m. r. signal ascribable to this species could be observed) displaces arylsulphonate to form (104), which reac tswi thphosphodies te r (102) giving the tetrasubstituted pyro- phosphate (105). This completes the first step of phosphotriester formation, and (105) accumulates to over 80% in the 31P n.m.r. spectrum. The second step is catalysed by a nucleophile of type 2 (e.g. - 1H-tetrazole or 3-nitro-1,2,4-triazole), which attacks (105) to displace phosphodiester (102) and form (106). This step re- sults in release of a proton, and is thus itself catalysed by a nucleophile of type 1, acting as a base. Finally, attack of the alcoholic component on (106) affords the phosphotriester, a species which could also be attained by direct attack of the alcohol on (104). Nucleophiles of type 2 do not catalyse formation of the mixed anhydride (1031, but may be produced in the initial reaction of (100, x = tetrazole or nitrotriazole) with a nucleophile of type 1. Some evidence for the formation of species of type (101) was obtained by i.r. spectrometry: the formation of (104) and its subsequent courses of reaction were surmised, rather than establish- ed directly, in this study. However, the interactions of (101) with (102) and of (104) with (102) would probably be facilitated by electrostatic interaction leading to ion pair formation and thence to reaction. The finding that pyridine accelerates con- densation when arylsulphonyltetrazoles are used is accommodated nicely by this scheme, which makes an attractive rationale of a number of observations.

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238 Organophosphorus Chemistry

Reviews on solid-phase synthesis and biological applications of polydeoxyribonucl eotides l5 deoxyribonucleotide synthesis157 have been published. support, ~-acryloylpyrrol idine-N-acryloyloyl- l ,6-hexanediamine cross- linked with N,N'-diacryloyl-l , 2-ethanediamine, has been used for the synthesis of dT(pdT)6 by the phosphotriester method. 158 Con- trolled-pore glass functionalised with a long-chain alkylamine has been preferred to polydimethylacrylamide-kieselguhr as a support for oligodeoxyribonucleotide synthesis on the grounds that it does not swell or contract with changes of solvent, it is easily deri- vatised using a base-protected nucleoside 3'-g-succinate, and, since it is less polar, a weaker protic acid, carrying a smaller attendant risk of depurination, can be used for detrit~1ation.l~' The agent used was, in fact, 3% dichloroacetic acid in 1,2-dich- loroethane. 5'-O-Succinyluridine has been linked to long-chain alkylamine controlled-pore glass, and treated with trimethyl- orthobenzoate and 4-toluenesulphonic acid to give, after weak acid hydrolysis , immobilised uridine 2' (3 I ) -2-benzoate. 160 free nucleoside hydroxy group was then used to provide the 3'- terminus in a conventional oligodeoxyribonucleotide synthesis by phosphite or phosphotriester methods, and in analogy to the method described above using a cytidine 3'-phosphate terminus, the oligo- mer was finally cleaved from the support using lead (11) ions. Silica gel continues to be a widely used support, and good results have been reported on Fractosil 200 for a phosphotriester synthesis which used TPS-C1 and N_-methylimidazole in 1,2-dichloroethane as couplinq agent. The same combination has been used as coupling agent for oligodeoxyribonucleotide synthesis on porous glass, and on chloromethylated polystyrene. 162 support was functionalised by treatment with mercaptoethanol followed by oxidation with hydroqen peroxide, to give a polymer- attached 6-sulphonylethanol, to which the first protected nucleo- tide unit was coupled. At the completion of synthesis the oligo- mer was removed from the support by B-elimination using triethyl- mine. Cross-linked polystyrene functionalised with aminomethyl groups has been linked to the 5'-position of a 2'-deoxynucleoside- 3'-(2-chlorophenyl)phosphoro-4-anisidate y7a a succinyl linker(l07), and an oligodmxyribonucleotide synthesised by elgonation in the 3'- direction from this 5'-terminus, using protected dimer and trimer blocks bearing 3'-(2-chlorophenyl)phosphoro-4-anisidate termini. The procedure has certain advantaqes, in that if any of the 5 ' -

and on automated solid-phase ol igo- A new

The

In the latter case, the

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7: Nucleotides and Nucleic Acids 239

(107) @ = polymer support

hydroxy component used to lengthen the chain is sulphonated by the coupling agent employed (e.g. MS-NT), it is simply washed off the column and no sulphonated by-product remains attached to the poly- mer. Also, it is not necessary to apply acid or zinc bromide to the column to effect detritylation (with the attendant risk of de- purination) at any time. The disadvantage is that removal of the anisidate using isoamyl nitrite, the necessary preamble to each coupling step, is rather slow. Silica gel has been functionalised using 3-aminopropyltriethoxysilane, and the resultant aminopropyl- silica (108) treated with 11-bromoundecanoyl chloride, and then with phthalimide followed by hydrazine (Gabriel reaction) to give (109) , and this process repeated to afford (110). 164 The silica prepara-

~

(108) n = 0 (109) n = 1

(110) n = 2

tions bearing different lengths of alkyl spacers were then utilised as supports for automated solid-phase oligodeoxyribonucleotide synthesis of tetradeoxyadenylate using 5'-~-dimethoxytrityl-2'- deoxy-N6-phtha loyladenos ine 3 I - (2-phenyl ) phosphorothioate monomer units and isodurene disulphonyl chloride in the presence of 3-nit- 1,2,4-triazole as coupling agent. It was found that coupling yields improved with increasing length of the alkyl spacers. Vydak TP silica has been used as solid-phase support for the synthesis of a ribonucleotide nonadecamer using the phosphite procedure, 165

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240 Organophosphorus Chemistry

and of several ribonucleotide nonamers in an automated synthesis usingthesame method. The ribonucleotide monomer units used for elongation in the latter study were protected at the 2'-hydroxy group by TBDMS, except in the case of guanosine, where 2'-s-tri- isopropylsilyl protection was used, simply because the mixture of isomers of N2-benzoyl-5 I -2-monomethoxyguanosine silylated at the 2'- and 3'-positions by this group is more easily separated than the corresponding mixture of TBDMS derivatives.

Several papers have dealt with the matters of strategy and procedure in oligonucleotide synthesis. A set of oligodeoxy- ribonucleotide linker fragments has been constructed by the phos- photriester method from functionalised dimer blocks, using field desorption mass spectrometry (FD-rn.s.1 to identify and check the purity of the monomer and dimer units ~ynthesised.'~~ An im- proved syringe method for phosphite solid-phase synthesis uses two syringes in tandem: one contains the polymer support, en- trapped between two ceramic filters, and the second syringe is used to draw the reagents, wash solutions, etc. into the first syringe and over the polymer support from septum cap bottles, and to expel them at the end of the reaction or wash time required.168 This minimises exposure of the sensitive intermediates to moisture, and, since the size of the second syringe can be changed ad libi- - tum, it simplifies washing procedures. A practical and labour- saving idea involves the use of segmental solid supports. Suppose a number of different oligonucleotide sequences are re- quired to be synthesised at the same period in time. Whatman 3MM paper discs are functionalised by condensation with the appro- priate 5'-~-monomethoxytrityl-2'-deoxynucleoside 3'-succinate species required at the 3I-terminus of each of the desired se- quences using MS-nt, and the papers are marked accordingly, in pencil, to denote which of the required syntheses is to be per- formed on each individual disc. Then, all discs which require, say, a 2'-deoxycytidine unit to be added next are sorted together into a single vessel and the oligonucleotide chains addedtobythat unit. There then follows another re-sorting, depending on the next residue to be added, and so on. In fact, by having four elongation 'pots' on the go, one for each base, and a sufficient number of discs, one should be able to prepare 41° different decamers simultaneously using only 40 coupling reactions! At any rate, the originators of the idea claim that this renders automated

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7: Nucleotides and Nucleic Acids 24 1

synthesisers obsolete.

In addition to the synthetic feats mentioned in the intro- duction, the synthesis of a gene coding for the human epidermal growth factor urogastrone ,I7' and one coding for ribonuclease S

protein, 17' demand honourable mention. solid-phase methods using phosphoramidite reagents. The prize for the most unusual structure must go to the immobile cruciform junct ion. 172 structed by phosphotriester methods, each with the first half of its sequence complementary to the second half of the sequence of one of the others, in such a way that annealing should result in a cruciform junction, and electrophoretic and spectroscopic evi- dence indicated that this did, in fact, occur. Cruciform structures are presumed to occur transiently as intermediates during replication and recombination of DNA molecules but are normally mobile and difficult to study.

Both were prepared by

Four deoxyribonucleotide hexadecamers were con-

A number of oligonucleotides containing sugar- or base-modified nucleoside analogues have been prepared by the phosphotriester method. Thus, oligodeoxyribonucleotides containing riboadenosine, =-adenosine , 2 '-deoxy-2 -f luoroadenosine and -quanosine , 173 3 I - deoxy-3 I-f luoro- , chloro-, and azido-thymidine ,174 1- (2-deoxy-6-D- ribofuranosyl) pyrid-2-oneI 175 1- (2-deoxy-a-D-ribofuranosy1)-

1 7 7 pyrimidin-2-one , 17' and 2 1-deoxyguanosin-8-ylacetylaminof luorene have all been prepared. The protected derivatives of 5-methyl- aminomethyl-2-thiouridine (111) and (112) and of 5-carbomethoxy- methyl-2-thiouridine (113) have been prepared coupled with 2,2,2-trichloroethyl phosphate or 2-chlorophenyl phosphate using MS-triazolide, and the phosphodiesters thus derived from (111) and (113) used respectively in phosphotriester syntheses. At the conclusion of synthesis, the trichloroethyl group is removed from the (111)-containing oligonucleotide using zinc and acetylacetone or sodium hydroxide in aqueous dioxan, to afford the 5-methyl- aminomethyluridine-containing sequence found at the anticodon of tRNA Lys, from E.coli B; the (113)-containing sequence is that of the anticodon for tRNA3 Lys from rabbit liver.

Modified DNA duplexes containing 3',5'-pyrophosphate bonds have been synthesised. I 8 O

undergoes self-association in solution to give DNA-like duplexes, The decamer d ( pT-G-G-C-C-A-A-G-C-Tp)

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242 Organophosphorus Chemistry

""Jy,. 0

II I

0-P-R

DMTrO J Me 6 ( 1 1 4 ) R = CI

MMTrO

OH OMThp

(111) R = N(Me)COOCH2CCI,

(112) R =N(Me)COCF3

(113) R = COOMe

(115) R = IH- l ,2 ,3,4- tetrazole

(116) R = OH

and when these were treated with 1-ethyl-3 (3'-dimethylaminopropyl)- carbodiimide, the phosphate termini of contiguous decmer segments were coupled forming ( 3 '-5 9pyrophosphate links with near-quantita- tive efficiency. The presence of the pyrophosphate bonds was con- firmed by cleavage of the resultant polymers using trifluoroacetic anhydride. A protected deoxyribonucleotide hexadecamer with a central (3'-3 9-pyrophosphate bond was obtained accidentally as a by- product in a coupling reaction when d(T-T-G-G-T-T-A-Ap), fully protected except for a single phosphate dissociation at the 3 ' -

terminus, was treated with a large excess of TPS-C1 and !-methyl- imidazole (cf. Scheme l! 1. 181

Methylphosphonic dichloride and tetrazolemethylphosphonic chloride have been used to prepare oligo(deoxyribonuc1eoside methyl- phosphonate). Treatment of a base-protected 5'-g-dimethoxytrityl- 2'-deoxynucleoside with these reagents yields (114) or (115) which, when treated with a 2'-deoxynucleoside blocked at base and 3'- hydroxy goup, affords the protected 3',5'-dinucleosidyl methyl- phosphonate. 182 obtained when ( 1 1 5 ) was used. Oligo(deoxyribonuc1eoside methyl- phosphonate)synthesis has also been performed by solid-phase methods on derivatised silica or on cross-linked polystyrene. In the former case, species (114) in the presence of tetrazole was used as the lengthening unit in a procedure which otherwise re- sembled a conventional phosphotriester synthesis cycle. Coupling

Fewer (3'-3 4-linked dimeric by-products were

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yields were fair when thymidine units were involved, but markedly poorer when N2-isobutyrylguanosine units were involved. separation of oligo(2'-deoxythymidine methy1phosphonate)species on HPLC columns, less separation of the methylphosponate diastereo- isomers was observed as the chain length of the oligomer increased. In the synthesis on cross-linked polystyrene,coupling was performed using the simple 3'-methylphosphonates (116) using MS-nt as con- densing agent, and yields were higher, although the coupling re- action was very sensitive to moisture. The completed oligo- mers were removed from the support (succinyl linker) and base- protecting groups were removed using ethylenediamine in ethanol, with little or no cleavage of the methylphosphonate chains. Two series of oligomers were made, one containing all methylphosphonate linkages d [{Np(Me) InN1 , the other containing a single conventional 5'-nucleotide residue, d[Np{Np(Me) lnN1. The latter could be phos- phorylated at the 5I-terminus using-polynucleotide kinase and ATP, and the resulting species then separated by polyacrylamide gel electrophoresis according to chain length. Piperidine cleaves the methylphosphonate oligomers randomly to produce shorter oligomers, while treatment of the oligomers with acid produces apurinic sites, with the 4'-hydroxy groups liberated at these sites then attacking the adjacent 3'- or 5'- methylphosphonate groups to effect chain cleavage with 3 ' - or 5'- hydroxy group termini at the break points. These reactions may be used to characterise the oligomers. A single methylphosphonate link has been placed at two different sites in the lac operator sequence (which was prepare2 by phos- photriester synthesis), with each of the different methylphos- phonate diastereoisomers being introduced separately at each of the two sites.184 The binding of lac repressor protein was then studied with all four oligonucleotides, and binding was found to be affected by the presence of the methylphosphonate link, and sen- sitive to its stereochemistry, at one site (previously believed to be a repressor-phosphate contact site) but not at the other.

Upon

The protected dinucleosidyl phosphotriesters (117) and (118) have been prepared by conventional methods and their diastereo- isomers separated using silica gel chromatography, and used in turn to prepare tetrathymidylate ethyl esters. 185 Each tetranucleotide was obtained as a mixture of two diastereoisomers due to the pre- sence of an asymmetric phosphorus atom at the new internucleotidic link, and these were separated by reverse-phase chromatography.

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244 Organophosphorus Chemistry

(117) R = C c H 4 C 1 - 2

(118) R Et Tr 0 OAc

OR

Racemisation at phosphorus was observed to occur when, for instance, (118) was formed from a single diastereoisomer of (117) by trans- esterification with ethanol in the presence of caesium fluoride.

Once again (2 '-5 ')-linked oligoadenylates have been the subject of much investigation. 3'-O-Benzoyladenosine - derivatives have been utilised in an otherwise conventional phosphotriester synthesis of ''core tnmer'l (2'-5')ApApA. The phosphodiester linkage iso- mers of "2-5 A" [pppA(2')pA(2')pA] have been prepared by lead (11)- catalysed oligomerisation of adenosine 5'-phosphorimidazolidate, followed by separation of the 2'-5'/3'-5'-linked 5'-phosphorylated trimers and tetramers obtained, and treatment with carbonyl bis- (imidazole) followed by pyrophosphate. 187 (if any) formed on attack by nucleases, together with 31P n.m.r. spectroscopy, were used to identify the structures of the products. It was found that replacement of a (2 '-5')-internucleotidic link by its (3'-5')-isomer caused a loss in biological activity in inhibiting protein synthesis of at least one order of magnitude. In another extensive study on the influence of chain length, phosphorylation state,and heterocyclic base in "2-5A" on its ability to bind to and activate "2-5AVr-dependent endoribonuclease, a number of analogues of "2-5A" in which these parameters were altered were prepared similarly and examined. 188 and for three or more AMP residues in a (2'-5')-linked oligomer, were found to be stringent. However, both [A(2')p13A and the 5I-methyl ester of p [A (2 ) p] 2A (prepared by methylating p [A (2 ' 1 p] 2A using triphenylphosphine, 2,2'-dipyridyl disulphide, and methanol) both showed significant activity, far higher than core trimer, showing that the 5'-phosphoryl moiety of active species need not necess- arily be triphosphate, but may also be an alkyl phosphate. The llcaF.pedll species A(5')pn(5')A(2')~A(2')pA (n = 2-4) have also been prepared either by trea'fling the 5 ' -phosphorimidazolidate of core trimer with AMP or ADP (for = 2,3) or by treating pA(2')pA(2')pA

The cleavage products

The requirements €or adenine as base,

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7: Nucleotides and Nucleic Acids 245

with adenosine 5'-trimetaphosphate (for = 4) ,18' The correspond- ingly capped 5'-tetraphosphates of core tetramer and core dimer were also prepared similarly. While the capped tetraphosphates of core trimer and core tetramer were as active as "2-5A" in in- hibiting protein synthesis in L cells, the capped di- and triphos- phates were far less active, despite binding efficiently to "2-5A"- dependent endoribonuclease, while the capped tetraphosphate of core dimer was inactive, binding poorly to the enzyme. Degradation studies indicated that cytosolic enzymes degraded the capped tetra- phosphate of core trimer rapidly to give "2-5A", which then acti- vated the enzyme, while the capped di- and triphosphates were de- graded only slowly, without forming "2-5A". Capping of the 5'-di- or triphosphates of core trimer at PB or P thus leadstoloss of ability to activate the endonuclease, despite efficient binding. An antigen containing "2-5A" has been prepared by oxidation of "2-5A" at the 3'-terminus with periodate and conjungation with adipic dihydrazide linked at its other terminus to bovine serum albumin. The conjugate was used to immunise rabbits, and an antiserum capable of detecting only 20 femtomoles of (2'-5'blinked oligoadenylates was obtained. The tubercidin (7-deazaadenosine) analogue of "2-5A" (and the corresponding 5'-triphosphorylated tetramer) have also been prepared via phosphorimidazolidate oligo- merisation, and were found to bind to "2-5A8'-dependent endoribo- nuclease of mouse L cells without stimulating it, although the tetramer was as active as "2-5A" in inhibiting translation in a rabbit reticulocyte system. Thus there exist species-specif ic structural requirements of activation of the endoribonuclease. The "core trimer" analogue of tubercidin, as well as (2'-5')- and (3'-5')-tubercidylyl tubercidin, have been prepared by straight-

192 forward phosphotriester routes using the phosphite procedure. The (3'-5')-linked tubercidin analogue of ApA was hydrolysed faster than ApA by nuclease S1, suggesting that the enzyme is sensitive to base modification.

Y

3',5'-~,~-Tetraisopropyldisiloxanyladenosine has been treated with methyl phosphorodichloridite in the presence of 1H-lI2,4-tri- azole, then with N6 ,N6 , 2 , 3 I-2,g-tetrabenzoyladenosine, and the phosphite product oxidised with sulphur in pyridine to afford the

( 2 ' - 5 3 - linked diadenosyl methyl phosphorothioate (119)?93 The diastereoisomers of (119) were separable using t.l.c., and after deprotection the product obtained from the high gF isomer was

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S

Organophosphorus Chemistry

degraded by snake venom phosphodiesterase with a half-life of one day, tvhile that obtained from the low I& isomer had a half-life of 40 days. The former is thus almost certainly the sp diastereo- isomer. By comparison, A(2')pA was degraded with a half-life of 2% minutes! Treatment of the separate diastereoisomers of(119)with 0.2 M hydrochloric acid in dioxan resulted in desilylation only at the 5'-position, and the resulting material was used, as above, to prepare the diastereoisomeric phosphorothioate analogues of "core trimer", the diastereoisomer pair prepared from the Sp isomer of (119) being more stable to murine phosphodiesterase than the pair prepared from the Rp isomer. The stability of (2'-5')-linked oligoadenylates to enzymic hydrolysis is thus increased markedly by substitution of thiophosphate for phosphate. The preparation of G(5')p3ApA, G(5')p3A(2')pA, and G(5')p3ApA in which the tenninal adenosine was protected as its 2',3'-gIg-isopropylidene derivative, has been described. lg4 The pApA species were synthesised by phos- photriester methods or phosphoroimidazolidate oligomerisation, de- blocked, and coupled with GDP via treatment with carbonyl bis- (imidazole). The hydrolysis of these "cap" analogues by a di- nucleoside triphosphatase activity from rat liver nuclei was examined, and the species containing the (2'-5l)- internucleotidic link found to be hydrolysed much more slowly than the others.lg5 The polymerisation of unprotected ribonucleosides using phosphorus tris (azoles) affords a simple rapid route to (2l-5')- and (3'-59- linked oligoribonucleotides, with the tris(azo1es) formed from imidazole, 2-methylimidazole, and 2-ethyl-4-methylimidazole giving the best results. lg6 At low temperature in THF, phosphorimidazol- idites of type (120) are thought to be formed initially, and oligo- merise to give (121) which on oxidation with aqueous iodine affords (2'-5'1- and (3'-5')-linked oligomers. The formation of (3'-33- and (5'-5 I)- internucleotidic links was not observed. Oligomers of up to pentamer length were obtained, and linkage isomers were separable

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6

247

6

( 1 2 0 ) 8 =Ura, Ade, Cyt (121)

using reverse-phase h.p.1.c. In the diuridyl phosphate obtained, the linkage isomers were found to be formed in almost equal quan- tity with a slight preponderance of the (2'-5')-linked isomer, ir- respective of the imidazole species used.

4.2 Enzymatic Synthesis.- Poly(2-methylthio-7- deazainosinic acid) , lg7 poly (8-methyladenylic and poly (2-isopropyl- adenylic acid) lg9 have all been synthesised by polymerisation of their respective nucleoside 5'-diphosphates using polynucleotide phosphorylase from Micrococcus luteus. Poly(2-methylthio-7- deazainosinic acid) is unable to form normal Watson-Crick or Hoogsteen base pairs, but forms a rigid 2:l triple-stranded com- plex with poly(A). The homopolymer shows high thermal stability and is very resistant to hydrolysis by nuclease S1 and ribo- nuclease T2. Poly(8-methyladenylic acid) exhibits unusual spectroscopic properties, and is thought to exist as a single- stranded regular structure with alternating = and anti conform- ations. Poly-(2-isopropyladenylic acid) cannot form normal Watson-Crick base pairs due to steric hindrance, but forms a 1:l complex with poly(X) , presumably via coordination at N-7. Poly- (5-fluoro-2'-deoxyuridylic acid) has been prepared by polymeri- sation of 5-fluoro-2'-deoxyuridine 5'-triphosphate using terminal deoxynucleotidyl transf erase. 2oo structure and forms a duplex with poly(dA) of much lower thermal stability than poly (dA) .poly (dT) .

It exhibits little secondary

A number of spin-labelled nucleotides have been synthesised and incorporated into polynucleotides by enzymic methods. Thus, for instance, the dUTP derivatives (122) and (123) have been co- polymerised with other deoxynucleoside triphosphates using terminal deoxynucleotidyl transferase201 and the UDP derivatives (124) ,

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248 Organophosphorus Chemistry

(125), (124) and (126)lwith CDP using polyribonucleotide phosphorylase to give spin-labelled polyribonucleotides. 202t 203

(126), and (127) have been copolymerised with UDP or [for

The UDP deri-

R/' \(CH 2 ) n / fl \N H I @-.roo R,s\cH~q--. Me Me

Me Me

(122) R = dUTP-5, X =0, n = 1 ( 1 2 7 ) R = UDP-5

(123) R = dUTP-5, X = S, n = 1

(124) R = UDP-5, X = S, n = 1

(125) R =UDP-5 ,X = S , n = 2

(126) R = (4-deoxo-UDP)-4,X = S, n 1

vatives were prepared by alkylation of 5-mercapto-UDP [(124), (125), and (12711 or 4-thio-UDP (126) with an appropriate alkylating species. The incorporation of (126) into polymer was more effic- ient than that of (124). Poly(C) has also been spin-labelled by acylation with the anhydride of 3-carboxy-2,2,5,5-tetramethyl- pyrrolin-1-oxyl, but the acylated polymer was unstable to hydro- lysis, and lost the nitroxide label on standing.202 The spin- labelled polyribonucleotides have been used to examine internal motions in RNA duplexes203 and to measure the affinity of reverse transcriptase for some polynucleotide inhibitors in a competitive assay. 204

Oligoribonucleotides have been synthesised enzymatically by preparing trimer blocks bearing a phosphoranilidate 5'-terminus and a 2'-2-(2-nitrobenzyl) 3'-terminus. The nitrobenzyl and aniline blocking groups are removed from the termini of the blocks to be joined using light and acid respectively, and the blocks coupled using ATP and T4 REJA ligase. 205 The dinucleotide pCpA, prepared by a phosphotriester procedure, has been acylated at the 2 ' (3')-hydroxy groups of adenosine using carbonyl bis(imi- dazole) and various 2-acylamino acids, and the resultant species ligated to tRNAPhe from E.coli (which lacked its terminal C75-A76 residues) using T4 RNA ligase, in order to prepare "chemically misacylated" tRNAPhe. 206 1 ,N'-Ethenoadenosine 3 ' , 5 '-bis (phos-

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7: Nucleotides and Nucleic Acids 249

phate) and 1 ,g6-etheno-2-azaadenosine 3 ' , 5'-bis (phosphate) have been ligated to yeast tRNAPhe lacking its last four residues (tRNA enzyme. 72207 acceptor than the non-paired C75 in tRNA75. was performed to replace The fluorescent analogues were introduced as reporter groups to obtain information on mobility and stacking in the tRNA acceptor terminus. While replacement of by the fluorescent analogues gave a tRNA species inactive in aminoacylation, replacement of A73, followed by repair of the terminus using ATP(CTP) terminal nucleotidyl transferase gave functional tRNA molecules. Aminohex-1-yl) -p2- ( 5 -adenosyl)pyrophosphate has been derivatised by covalent attachment of biotin, fluorescein,or tetramethyl- rhodamine to the aminohexyl moiety, and the resultant species used as substrates for T4 RNA ligase, resulting in the attachment of the derivatised 6-aminohexyl 1-phosphate to the 3I-terminus of 5s RNA, yeast tRNAPhe, (Ap) C, and (Ap) 3A acceptors. 208 The biotin- labelled species thus obtained were bound quantitatively and selectively to avidin-agarose, while the fluorescein ahd tetra- methylrhodamine-labelled oligonucleotides were highly fluorescent, showing no quenching due to attachment to the receptor. In another method for fluorescent labelling, 2'-deoxycytidine 3'-phosphorothi- oate has been prepared by coupling 5 '-~-dimetho~ytrityl-~~-anisoyl- 2'-deoxycytidine with S-cyanoethyl phosphorothioate using TPS-C1 in pyridine, followed by deblocking. 209 On incubation with poly- nucleotide kinase and ATP, it affords 3'-~-(5'-phosphoryldeoxy- cytidyl) phosphorothioate [pdCp(S)], which may in turn be alkyl- ated with 3,6,7-trimethyl-4-bromomethyl-1,5-diazabicyc1o~3.3.0]- octa-3,6-diene-2,8-dione ("Monobromobimane") to give the fluores- cent 3'-~-(5'-phosphoryldeoxycytidyl) S-bimane phosphorothioate. Both pdCp(S) and its 5-bimane derivative may be ligated to the 3'- terminus of tRNA, and also to the 3'-terminus of oligodeoxyribo- nucleotides, so long as the latter have a ribonucleoside residue graftEd on as the 3 '-terminus.

1, or lacking its terminal residue (tRNA75) using the same

A similar procedure of tRNAfMet by 1 ,N'-ethenoadenosine.

The base-paired C72 in tRNA72 was a more efficient

El- (6-

3

In a number of studies, tRNA molecules have been cleaved in the anticodon loop region, the ends at the cleavage sites phos- phorylated or dephosphorylated, according to requirement, using polynucleotide kinase and alkaline phosphatase, to generate proper ends for ligation, and oligonucleotides grafted in, using T4 RNA

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250 Organophosphorus Chemistry

ligase, to produce tRNA molecules containing modified anticodon loops. This procedure has been performed to construct a UGA suppressor tRNA by modification of yeast tRNACYs , 210 to construct ten modified anticodon loops of different size and sequence in tRNAfMet in order to probe the requirements for its recognition by methionyl-tRNA synthetase, 211 molecules modified in the anticodon sequence in order to probe the dependence on sequence of the enzymatic conversion of adenosine in the "wobble" position to inosine. 212

ligase to ligate fragments of yeast tRNAPhe cleaved in the double- stranded regions by cobra venom ribonuclease has been examined, and strong discrimination in the ability to ligate nicks in different positions of the same helical array was found.

and to construct sixteen tRNAASP

The inability of T4 RNA

213

Under conventional assay conditions, T4 DNA ligase does not actively catalyse blunt-ended ligation of DNA molecules, but in the presence of high concentrations of macromoleculesfsuch as poly- ethylene glycol, or Ficoll 7 0 (a branched polysaccharide), or bovine plasma albumin, this process is stimulated up to a thousand- fold, a finding likely to be useful if it is wished to utilise blunt-end ligation to form linear or circular ligation products?14

A method has been described which permits "sticky ends" of different sequence generated by cleavage of DNA by restriction endonucleases to be joined in favourable cases. 215

duplex fragments are formed from digestion with different restric- tion endonucleases, with "sticky ends" 5'-C-T-A-G... and 5'-A-G-C- T... . These are non-complementary. The procedure involves con- trolled partial filling of the single-stranded ends using reverse transcriptase with dCTP and dTTP (for the first sequence) and dATP and dGTP (for the second sequence). The sticky ends are then reduced to 5'-C-T ... and 5'-A-G ... which are self-comple- mentary and may be joined using DNA ligase. In fact, sticky ends as short as one nucleotide long which are compl.ementary may be joined .

Suppose two

While most oligonucleotide syntheses reported have employed phosphotriester methods, enzymic methods utilising polynucleotide phosphorylaseand T4 RNA ligase have been used to synthesise 23 sequence variants of the 21-mer oligoribonucleotide which con- stitutes the binding site of bacteriophage R17 coat protein, to

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characterise the nature of the interaction of the protein with its binding site. 216

A number of studies on nucleotide analogues which, when incor- porated into DNA, would present potentially mutagenic sites, have appeared. dTTP and 0 -methyl-dTTP as partial substitutes for dTTP in primed DNA synthesis on a polyd(A-T) template, although little polymer is obtained in the complete absence of dTTP. 217 taining polymers formed (which contained up to 22% of the methyl- ated analogues) retained the secondary structure of poly d(A-T), as indicated by melting curve data. Since the analogues can form only a single hydrogen bond to adenine, this retention of secondary structure points to stacking interactions playing a major role in in the helix stabilisation. In contrast, poly d(A-T) alkylated to less than 4% total modification by methyl- or ethylnitrosourea showed distinct destabilisation of the secondary structure, possibly resulting from alkylation at the phosphodiester links. g- Methoxy-dCTP, formed by treating dCTP with methoxyamine hydro- chloride, can also substitute for dTTP in the above system, al- though it is not incorporated when poly d(G-C) is used as tem- plate primer. 218 Nearest-neighbour analysis of the analogue- containing product shows that N4-methoxy-2 ' -deoxycytidine sub- stitutes only for 2'-deoxythymidine. When used as template strand , the N4-methoxy-2 '-deoxycytidine-containing polymer incor- porated dATP opposite the analogue with little misincorporation, suggesting that the N-methoxycytosine moiety is in the imino form with the methoxy group anti to N3 of the ring, an arrangement which permits efficient base-pairing with adenine.

DNA Polymerase I from E.coli can utilise g2-methyl- 4

The analogue-con-

4

Random copolymers containing 2-aminopurine (2-AP) residues, poly d(2-APfG), and poly d(2-APfA) have been obtained by poly- merising mixtures of 2-aminopurinedeoxyriboside 5'-triphosphate with dGTP and dATP, respectively, on d(pGI8 and d(pA)* primers, using terminal deoxynucleotidyl transferase. 219 When poly d(2-APfG) was annealed to poly(dC1 , spectroscopic data suggested that hydrogen-bonding was occurring at N-1 of 2-aminopurine in the 2AP-C base mispair, and thus that either 2-aminopurine or cytosine was present as the disfavoured imino tautomer. The influence of local nucleotide sequence on the tendency to misincorporate 2- aminopurinedeoxyriboside 5'-phosphate in place of AMP during DNA

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252 Organophosphorus Chemistry

replication by DNA polymerases from various sources has been studied, using equimolar concentrations of these deoxynucleoside triphosphates as substrates (together with other dNTP), and se- quencing gels to determine the sites of misincorporation. 220 effect of base-pair stability of nearest-neighbour nucleotides on the fidelity of DNA synthesis has been studied using poly d(1-C) and poly d(G-C) as template primers, and dGTP, dITP and dCTP as substrates for DNA polymerase I (Klenow fragment): the G-C base pair is more stable than the I-C base pair,so comparison of the misincorporation rates reflects the influence of base-pairing at the terminus undergoing elongation. 221 The misincorporation was found to be higher adjacent to the stable G-C base pairs, and the ability of the exonuclease to edit the mistake was de- creased by the formation of stable base pairs after the mismatch, suggesting that clusters of G-C base pairs may represent mutation "hot-spots" in DNA. The ability of DNA polymerase ci to elongate mismatched primer termini has been examined using (dTIqdG, (dT) 8dC, and (dT) 10dGdT as primers annealed to poly (dA). 2 2 2 presence of the mismatch suppressed the elongation rate in all cases, and a minimum of five thymine residues needed to be insert- ed after the mismatch for the elongation rate to return to normal. Manganese (1I)ions lifted the attenuation of the elongation rate after a mismatch, a property which may be associated with their mutagenic potential. DNA containing apurinic and apyrimidinic sites (AP) has been prepared using a bacterial mutant, and its replication by a number of DNA polymerases was studied. 223 The results suggested that dAMP, in particular, tended to become in- serted opposite AP sites, a process which would result in trans- version mutations occurring at apurinic sites and transition mutations at apyrimidinic sites.

The

The

In vitro primed DNA synthesis on a single stranded bacterio- phage fd DNA template using three normal dNTP substrates and one deoxyribonucleoside triphosphate analogue modified in the base moiety has been used to effect complete substitution in one strand by the base analogue. 2 2 4

species were prepared, containing 2-aminopurineI 5-halogeno- pyrimidines, 8-azaquanineI es., as substrates for restriction endonucleases to study the influence of introduction of a base analogue at the cleavage sites. In general, structural alter- ations in the base pairs contiguous to the phosphodiester bonds

No less than twelve such modified DNA

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cleaver', vage.

by the enzymes substantially reduced the rates of clea-

5 Other Studies

5.1 Affinity Separation.- A review on the hybridization of nucleic acids immobilized on solid supports, which contains useful key protocols, has been published. 2 2 5

The 3'-(4-aminophenyl) phosphate derivatives of 2'-deoxy- cytidine, 2'-deoxyadeno~ine~and 2'-deoxyguanosine have been pre- pared by reduction of the corresponding nitrophenyl phosphodi- esters and coupled to succinylated aminoethyl-Sepharose using a water-soluble carbodiimide, to afford immobilised 2'-deoxynucleo- side 3'-phosphates as affinity adsorbents. 226 The affinity of nucleoside-metabolising enzymes for these adsorbents correlated reasonably well with their ability to be inhibited by the corres- ponding non-immobilised 4-aminophenyl phosphodiesters, and in some

Methylguanosine 5 ' - (3 - [ 4-aminophenyl] triphosphate) has been pre- pared by coupling 4-nitrophenol to 7-methyl-GTP using DCC and re- ducing the product, and has been coupled to Sepharose 4B using cyanogen bromide to afford an affinity adsorbent for mRNA cap- binding protein. 227 The adsorbed protein was eluted using 7- methyl-GTP. Cellulose has been treated successively with cyano- gen bromide, E-aminocaproic acid,and hydrazine to introduce a hydrazide group which was condensed with periodate-oxidised poly- (I) .poly(C) to afford a new poly(1) .poly(C) affinity matrix, which was effective in purifying (2 '-5 9-oligoadenylate synthetases. The complexes EF-Tu.GDP and EI-Tu.GTP (containing EF-Tu from E.coli or Thermus thermophilus) have been immobilised on cyanogen bromide-Sepharose, and immobilised EF-Tu.GDP could be converted to EF-Tu.GTP in situ enzymically, or by washing with a GTP-con- taining buffer . 229 The immobilised EF-Tu.GTP bound aminoacyl- tRNA specifically, and could thus be used to isolate tRNA iso- acceptors from bulk tRNA by aminoacylating the bulk tRNA using a single amino acid species and passing the mixture over the column.

cases the strength of binding was strongly pH-dependent. 7-

228

DNA from bacteriophage A has been coupled to epomxy-cellulose, and the accessibility of specific DNA sequences investigated by studying the digestion of the immobilised DNA by restriction

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254 0 rganop hospho rus Chemistry

endonucleases. 230 erated using Klenow fragment and the [ Q - ~ ~ P ] -labelled dNTP known to be required to fill in at the cleavage sites, was used to quantitate the cleavage. All sites were found to be randomly accessible, with variable site accessibility, and thus DNA-epoxy- cellulose should be a useful medium for the affinity isolation of sequence-specific DNA-binding proteins.

Radiolabelling of the recessed ends thus gen-

In a novel use of immobilised polynucleotides, poly(u)-cellu- lose has been utilised in an elegant experiment to test (andobtain evidence for) the classical two-tRNA-site model for the ribosomal elongation cycle. 2 3 1

5.2 Affinity Labelling.- A review on reactive derivatives of nucleoside 5'-mono-, di-, and tri-phosphates as affinity reagents has been published.

--

232

The majority of affinity labelling studies using reactive nucleotides have used the sarr,e species as previously reported, and well-established methodologies. These studies, in which no structural, chemica1,or procedural novelty is involved, will henceforth be omitted from this Report.

Periodate-oxidised nucleotides have once again been used for affinity labelling. In one study, 233 the labelling of succinyl- Casynthetase from E.coli by periodate-oxidised ADP was compared with labelling by ADP-2'-semialdehyde (I>. the species formed by selective reduction of periodate-oxidised ADP at the 3'-aldehydic function formed). Modification of the enzyme by the ADP-2I-semi- aldehyde occurred faster than that by periodate-oxidised ADP, and exhibited different concentration dependence and non-saturable kinetics, and the species thus did not appear to act as a proper affinity label for the active site, while periodate-oxidised ADP gave satisfactory results. In another study, ribonucleotide reductase from Corynebacterium nephridii has been labelled using periodate-oxidised CDP and ADP. 234 enzyme was associated with the elimination of pyrophosphate, and it was thought that the enzyme itself catalysed elimination of pyrophosphate, subsequently becoming labelled by the a,B-unsatur- ated nucleoside dialdehyde formed, or a morpholino-derivative thereof, as discussed in last year's Report. 70

Covalent attachment to the

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7: Nucleotides and Nucleic Acids 25 5

5'-Thiophosphoryladenylyl-(3'-5')-uridine may be prepared by incubating adenosine 5'-phosphorothioate with UTP and RNA polymer- ase in the presence of poly d(A-T), as the product of an abortive initiation reaction, and upon alkylation atthe sulphur atom using labelled 4-azidophenacyl bromide, (128) is obtained. 235 This has

0

N 3 ~ C O C H 2 S - 7- II 0- (ApU- 5' ) N3-@l-0 -1-0 - (U rd - 5 1

OH OH OH

( 1 28) 'NO,

(1 29)

been incubated with [cx-~~P~-CTP, RNA polymerase from E.coli, and a bacteriophage DNA transcription promotor of appropriate sequence, resulting in the formation of a transcription complex containing labelled ApUpC derivatised at the 5'-terminus with a photolabile group. After photolysis the labelled DNA and subunits of RNA polymerase were isolated, and the trinucleotide removed by cleavage at the thiophosphoryl group with phenylmercuric acetate. The DNA template and the (J and B subunits of RNA polymerase were found to have become labelled. 235 transcription by RNA polymerase in the presence of a mixture of ribonucleoside triphosphates containing chain terminators (3l-o- methyl-dCTP, 3'-Q-methyl-dGTPI 3'-dATP), resulting in the generation of an array of oligo- and poly-ribonucleotides of practically all lengths from 4 to 116 residues long, all of which contained a photolabel at the 5 I - t e r m i n ~ s . ~ ~ ~ Irradiation of the system then permitted the 5'-termini of these polymers to be attached to the nearest macromolecules, and the resulting labelled conjugates were separated on SDS gels, the RNA chains removed, as above, using phenylmercuric acetate, and the labelled components identified, together with the length and sequence of the RNA chains attached to them. the transcription complex could thus be followed. For RNA chains less than twelve residues long, the DNA template was the major com- ponent labelled, with the B and B ' subunits of the enzyme labelled as minor components, but with chains longer than twelve residues, only the B and 6 ' subunits were labelled. The u subunit was not labelled by RNA longer than the trinucleotide.

Further, (128) has been used to initiate

The path of the leading end of the nascent RNA through

Uridine 5'-phosphoromorpholidate has been treated with 4-azido-

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256 Organophosphorus Chemistry

2-nitrophenyl phosphate to give El- (4-azido-2-nitrophenyl) -P2- - (5'- uridy1)pyrophosphate (129), a photoaffinity analogue of UDP-galac- tose, which has been used as a photoaffinity label for lactose synthase and found to label the galactosyltransferase subunit.

237

Most photoaffinity labelling studies involving nucleotides have utilized 8-azidonucleotides, although 2-azido-ADP has been used to locate the tight ADP-binding site on membrane-bound coupling factor CF-1 of spinach chloroplasts, 238 phenyl)amino]propionyl-ATP has been used to label the ATP-binding site of the epidermal growth factor protein kinase. 239 benzophenone-type photoaffinity labels, 3'-g-(4-benzoyl)benzoyl- ADP has also been used for the labelling of chloroplast CF-l,240 and 3l-G- (4-benzoyl) benzoyl-ATP was used to lase1 the Ca2+, Mg2+- ATPase of sarcoplasmic reticulum, €or which purpose it was superior to both 8-azido-ATP and 3'-g-3-[lJ-(4-azido-2-nitrophenyl)amino]- propionyl-ATP.

and 3'-g- 3-"- (4-azido-2-nitro-

Of the

241

5.3 Post-synthetic Modification.- Unprotected oligo- and poly- nucleotides bearing a 5'-terminal phosphate group may be converted to their 5'-phosphoramidates by treatment with a water-soluble carbodiimide in imidazole buffer at pH6 to give the corresponding 5'-phosphorimidazolidate, and subsequent reaction with an amine [eLg. ethylenediamine, poly (L-lysine) , bovine serum albumin]. 24 2 The internucleotidic bonds are not ruptured by the carbodiimide, nor are the bases affected. The procedure offers a convenient method for attaching reporter groups to oligonucleotides. If nucleoside 5'-phosphorimidazolidates are reacted with oligo- or polynucleotides bearing unblocked 5I-phosphate termini, the cor- responding El- (5l-nucleosidyl) -P2- - (5'-oligonucleotidyl)-pyrophos- phate species are formed in good yield. 243 If adenosine 5'-phos- phorimidazolidate is used, €or instance, the products obtained are ligation intermediates and useful substrates for ligases.

The methylation of DNA may be followed by spectroscopic means using l 3 C and 31P n.m.r. if a I3C-enriched methylating agent such as methyl methanesulphonate is used. 244 spectra of model compounds permits the formation of methyl phos- phodiesters,methyl phosphotriesters,and a number of methylated base derivatives to be determined directly without degradation of the DNA and separation of the individual products. An enzyme activity

comparison with the

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7: Nucleotides and Nucleic Acids 257

has been identified in E.coli cells induced for adaptive response which can demethylate the phosphotriesters formed on exposure of DNA to methylating agents such as N-methyl-N'-nitro-N-nitroso- guanidine. 2 4 5

Oligodeoxyribonucleotides bearing a terminal 5'-phosphate group246 and a terminal 5'-thiophosphate group247 have been treated with (130) and (131), respectively, to obtain alkylating derivatives

(130) R = CH,CH,CI

(131) R = CH,

of polynucleotides. The derivatization by reagents (130) and (131) involves reaction of the aliphatic chloroethylamino groups with the terminal phosphate or phosphorothioate groups, to form the 0- or 5- alkylated phosphate esters. The "aromatic" chloroethyl group is comparatively unreactive, due to electron withdrawal by the formyl group on the aromatic ring, but is activated when the formyl group is reduced by borohydride. The alkylating polynucleotide deri- vatives were designed for use as affinity reagents. Other oligo- nucleotide species synthesised for the purposes of complementarily addressed alkylation include the 2 I , 3 I -g,g-[ 4- (N-2-chloroethyl-Ij- methylamino) benzylidene] derivative of the 3' -terminal adenosine residue of an oligoribonucleotide, 248 and the platinated product of treatment of a deoxyguanosine-containing oligodeoxyribonucleotide with [bromo-~,~,~',~'-tetrakis-(2-aminoethyl)-lI6-hexa~ethylene- diamine] diplatinum. 249 strands, apparently, by coordination at N - 7 of guanine.

The platinum complex links complementary

The thiation of cytidine residues using hydrogen sulphide in pyridine, followed by determination of the positions of the result- ing 4-thiouridine residues by fingerprinting or sequencing gel methods, has been used to determine the sites of cytidine residues accessible to the reagent, and hence to give information on the secondary structure of tRNAPhe from yeast250 and murine small nuclear RNA. 251

followed by hydrazine affords poly(C) containing a high proportion The reaction of polycytidylic acid with bisulphite

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258 Organophosphorus Chemistry

of N4-aminocytidine residues , which condense with bromopyruvic acid forming a Schiff base bearing a bromomethyl group ( ~ 3 2 1 . ~ ~ ~

This is highly reactive with thiol groups, permitting cross-linking

of poly(C) to glutathione and thiol-bearing proteins. Treatment of poly ( C ) with bisulphite and ethylenediamine results in near- quantitative conversion of the cytidine residues to IJ4-arninoethyl- cytidine. 253 The primary aliphatic amino group reacts quantitat- ively with N-hydroxy-succinimide esters, and the fluorescent re- porter group nitrobenzofurazan has been introduced in this way. Polyuridylic acid has been mercuriated at the 5-position with mercuric acetate, after which successive treatments with mercapto- ethylamine and 2,4-diriitro-5-azidofluorobenzene result in the introduction of the photoreactive aryl azide (133). 254

An elegant procedure has been described for determining, in a single step, the sites of cross-linking between oligoribonucleo- tides covalently linked by a cleavable bond. 255 linked duplexes are radioactively labelled at both 5'-termini using polynucleotide kinase, and then split randomly with alkali under conditions which e.ffect not more than one cut per duplex. The sample is separated into its components by gel electrophoresis in one dimension, the cross-link cleaved, and electrophoresis then applied in the second dimension, after which autoradiography is performed. The off-diagonal spots are those derived from the cross-linked duplexes. Two series of pairs of spots are seen, one member of each pair representing an undcgraded 5'-labelled chain, the other being a part-degraded 5I-labelled chain which had been linked to it. The number of pairs of spots in each series gives the number of nucleotide residues lying between the unlabel- led 3'-end of the degraded strand and the position of the cross- link. This method was used to elucidate the positions of nucleo-

Isolated cross-

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7: Nucleotides and Nucleic Acids 259

tide residues linked on photocrosslinking N-acetylvalyl-t-RNA bound in the ribosomal The precise site of cross-linking was identified by treating the cross-linked duplex with bisulphite, when all cytosine bases were converted to uracil except those involved in the cyclobutane dimer. Other RNA- RNA cross-linking studies have used reversible cross-linking agents [N-acetyl-5' - (4-glyoxylylbenzoyl)~ystarnine~~~ and 4 - (hydroxymethy1)- 4 , 5 ' , 8-trimethylp~oralen~~~ 3 and a similar two-dimensional electro- phoretic technique, and subsequently obtained the structures of the sequences linked by classical digestion and fingerprinting proced- ures. The reversible RNA-protein crosslinking agent bikethoxal {ethylene glycol bis [3- (2-ketobutyraldehyde) similarly in investigating crosslinking within 30s and 50s riboso- ma1 subunits.

g site to 16-18s ribosomal RNA. 256

ether]} has been used

2 59

5 . 4 Sequencing and Cleavage Studies.- Methods of rapid DNA sequence analysis, including gel sequencing,shotgun, cloning,and other methods, have been summarised. 260 quencer which uses the Maxam-Gilbert "chemical" procedure for gel sequencing has now been marketed. 261 Two marked improvements in the methodology of gel sequencing have been described:262 one involves using a tris-borate-EDTA buffer gradient along the length of the gel, with highest concentration at the lower end of the gel where the shorter oligonucleotides run. Since more buffer ions are available in this area to carry the current, the shorter oligo- nucleotides migrate less far than they would in a homogeneous buffer, while the larger oligonucleotides migrate comparatively further. The consequence is less spacing between the shorter oligonucleotides and more spacing between the longer ones, and hence better resolution. The second improvement involves using 2'-deoxyadenosine 5'- (1- [ 3 5 S ] thio) triphosphate instead of [C~-~~J?]~ATP as radiolabel in dideoxy sequencing reactions. The analogue is a good substrate for DNA polymerase I from E.coli, and the softer radiation from the sulphur radioisotope results in improved resolu- tion of the bands obtained on autoradiography.

An automatic DNA se-

If 3 * - [ 3 2 ~ 1 -end-labelled DNA is irradiated with U.V. light in the presence of spermine, and then heated briefly at 90° in buffer, thymine-specific cleavage of the chains occurs after breakdown of the thymine ring. cleavage occurs at both guanine and thymine positions, but

If spermine is replaced by methylamine,

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260 Organophosphorus Chemktry

predominantly at the former. These reactions are useful for confirmation of the results obtained from Maxarn-Gilbert sequen- cing. 2 6 3 Oxidation of DNA with N-bromosuccinimide, followed by treatment with piperidine, results in chain cleavage at guanine and cytosine residues, the rate of reaction with single-stranded DNA being much faster than with duplex DNA. 2 6 4

useful for confirming results from Maxam-Gilbert sequencing, or in "footprinting" studies (see below).

This may also be

A novel use for gel sequencing is to study misincorporation during DNA synthesis, a template DNA strand, is elongated in the presence of three highly purified oeoxynucleoside 5'-triphosphates, the rate of primer elongation measures the rate of misincorporation opposite template positions normally directing incorporation of the miss- ing base, and, if a chemically modified deoxyribonucleoside 5 ' -

triphosphate is added, any alteration in the rate of elongation reflects the propensity of the analogue to mispair and become in- corporated. 2 6 5

of substrate concentrations, on misincorporation can also be mon- itored. The technique is essentially of use for measuring the replication accuracy of DNA polymerases.

If a 5'-[32P'l -labelled primer , annealed to

The influence of local sequence, and of imbalance

Phosphotriester methods have been used to prepare d(G-G-G-A- A-T-T-C-T-T) and d(A-A-G-A-A-T-T-C-C-C), and the susceptibility to cleavage by restriction endonuclease Eco RI measured for each decamer alone,and for the duplex mixture of the two, to assess the influence of sequences adjacent to the recognition site on the cleavage reaction. 2 6 6 Each partly self-complementary decamer was cleaved by the enzyme, but slower than the duplex, which was cleaved preferentially in the adenine-rich strand. Oligomers of d(G-G-A-A-T-T-C-C) up to the pentamer, prepared using T4 DNA ligase, were found to be cleaved at the Eco RI site nearest the 5'-end which is flanked by only one dG.dC base pair, rather than three. The influence of adjacent-highly stabilised base pairs thus tends to suppress cleavage. A cunning use of restriction sequence methylase enzymes, which methylate only specific se- quences in DNA, together with that of a restriction endonuclease, Dpn1,which cuts only DNA methylated at adenine in both strands of its recognition sequence, effectively permits site-specific cleavage of DNA at 8- or 10- base pair sequences, rather than the

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7: Nucleotides and Nucleic Acids 26 1

4- to 6- base pair sequences recognized by most restriction endo- nucleases. 267 mapping of very long DNA molecules.

This is likely to prove useful in the physical

The identification of the potentially reactive sites in nucleic acids which are shielded from chemical or enzymic modification by intramolecular interaction, or intermolecular interaction with another macromolecule, using gel sequencing techniques, seems to have been dubbed "footprinting". Comparison of the patterns generated following chemical or enzymic cleavage under conditions where tertiary or intermolecular reactions cannot occur with those obtained under conditions where they do so permits identi- fication of the sequences involved in tertiary interactions. In some studies on tRNA, 2 6 8 r 2 6 9 ethylnitrosourea, which alkylates and thus labilizes phosphodiester bonds, has been used, but most other studies using this burgeoning technique have employed the chemical reagents used to effect chain cleavage via base modification in nucleic acid sequencing techniques, or specific endonucleases, and will not be catalogued here. However, the use of light for foot- printing DNA demands mention: exposure to U.V. light, followed by reduction of the modified DNA with borohydride (to reduce the carbonyl bonds in the photodimers, resulting in ring opening) and treatment with aniline acetate at pH 4.5 causes chain cleavage at the photodimer sites. Since a small distortion of the double helix is needed in order for the photodimers to form, any protein contacts which affect this distortion result in a modified cleav- age pattern. The use of light offers a number of interesting advantages: it eliminates the possibility of chemical or enzymic perturbation of the protein-nucleic acid interaction which the technique is meant to monitor, it can be performed very rapidly, in a wide variety of temperature and solvent conditions, and, most importantly, in vivo. 2 7 0

Many papers dealing with strand scission in DNA have appeared. 2-~-(5-~-Dimethylamino-l-naphthalenesulphonyl)- aminoethanol has been linked to the 5'-(4-chlorophenyl)phosphomonoester group of protected (pdTI9,using TPS-C1 and N-methylimidazole, and the resulting oligonucleotide deblocked to give the 5'-dansylaminoethyl ester of (pdTI9. When this is added to poly(A) and irradiated using a nitrogen laser, poly(A) is cleaved, while poly(C) and poly(U) are hardly affected in similar experiments. Derivatives of

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262 Organophosphorus Chemistry

this type are thus suitable for complementarily addressed laser scission. 271

been treated with bleomycin and ferrous ions in the presence of hydrogen peroxide, and the degradation products identified by spectroscopic and chromatographic techniques as the 3'-glycolic acid methyl cytosine and dGMP. 272 proved by total synthesis using triester methods. Cytosine was thought to arise via hydrolysis of cytosine propenal during work-

up, these products was the same as that suggested by another group and reported previously. 70 While the base sequence preference for light-induced strand scission in DNA by cobalt-bleomycins is simi- lar to that found for the iron-bleomycin-peroxide agent, namely cleavage at pyrimidine nucleoside residues on the 3'-side of guano- sine residues, the light-induced scission was unaffected by the concentration of dissolved oxygen, and little or no propenal product could be detected, although bases were released. 273 A different degradative mechanism may therefore be operating. The action of y-irradiation on DNA in vitro results in strand breakage with the formation of 5'-phosphate and 3'-phosphate and -phosphoglycolate termini. Neither type of 3'-terminus can be degraded by the 3 ' - 5 '

exonuclease activities of DNA polymerase I from E.coli or T4 DNA polymerase, and the3'-phosphoqlycolate terminus is unaffected by alkaline phosphatase (E.coli) and the 3'-phosphatase activity of T4 polynucleotide kinase, although the latter could remove 3'- terminal phosphate. 2 7 4 However, exonuclease I11 from E.coli can degrade both types of 3'-terminus, via initial removal of the 3'- phosphate or phosphoglycolate, and thus the repair of y-radiation- induced strand breaks could proceed via the sequence exonuclease I11 - DNA polymerase - DNA ligase. The damage caused in DNA by the neocarzinostatin chromophore results in strand breakage with 3'-phosphate and 5'-phosphate and 2'-deoxythymidine 5'-aldehyde termini. The last-named represent most of the drug-generated 5 ' -

termini (as quantitated by reduction with borohydride followed by labelling with in alkali with release of thymine. Neocarzinostatin thus generates strand breaks via selective oxidation at C-5' of deoxyribose. The sequence specificity of strand breakage in DNA by borohydride- reduced mitomycin C has been determined using sequencing gels, and the cleavage found to produce 5'-phosphate termini and more complex

The self-complementary hexamer d (C-G-C-G-C-G) has

ester derivatives of d (C1-Gp) and d (C-G-C-Gp) as well as The identity of the first product was

and the mechanism of degradation of DNA proposed to account for

Y-~'P I-ATP and polynucleotide kinase) and decompose

275

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7: Nucleotides and Nucleic Acids 263

3l-termini. 2 7 6

the sequence specificity of DNA cleavage by EDTA-distamycin-iron (11) , 277 278 distamycin-EDTA-iron (11) , 2 7 8

2 79 iron (11) , 277 and penta-N-methylpyrrolecarboxamide-EDTA-iron(I1) , four structurally closely-related agents. Careful examination of the cleavage patterns generated when methidiumpropyl-EDTA-iron (11) and deoxyribonuclease I, which show little specificity with regard to sequence, are used for footprinting show that the enzyme indicates larger binding site sizes than the iron-EDTA complex, possibly because it is a much larger molecule.

Sequencing gels have also been used to determine

EDTA-bis (distamycin) -

280

Tris (phenanthro1ine)-ruthenium (11) 281 and tris (4 , 7-diphenyl- phenanthro1ine)-ruthenium (11) 282 are chiral complexes which show stereoselectivity in binding to DNA, with the A enantiomers binding to the right-handed "B"-forrn DNA and the A enantiomers to left-handed "Z"-form DNA, and tris(4,7-diphenylphenanthroline)- cobalt (111) shows similar stereoselectivity, and cleaves the DNA chain on irradiation at 2 5 4 nm. 283

possibility of using specific cleavage by the A enantiomer as a probe for the occurrence of left-handed helical segments in DNA. The bis(1,lO-phenanthroline) - cuprous ion complex also shows sensitivity to DNA chain conformation, cleaving B-DNA rapidly, A-DNA[in, for instance, poly(rA) . poly(dT)] more slowly, and Z -

DNA not at all, in the presence of hydrogen peroxide. 284 This property may also be useful for detecting the presence of Z-DNA.

This seems to offer the

The agent believed responsible for inception of damage to DNA in these cleavage reactions by metal complexes is hydroxyl free radical, and e.pr. spin-trapping techniques have been used to show that iron complexes of nucleoside 5'-di- and -triphosphates catalyse hydroxyl radical formation from hydrogen peroxide, with the triphosphate complexes being most effective. 285

signifcant in mediating oxygen free radical damage in biological systems. It is thought that hydroxyl radicals are generated & vivo by a Fenton reaction between ferrous ions and hydrogen peroxide:

They may be

H202 + Fe2+ - Fe3+ + -OH +'OH

and the ferrous ion-nucleotide complexes promote this process. The ferric ion is then reduced by superoxide ion radical:

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264 Organophosphorus Chemi3try

and this reaction, together with the foregoing, constitutes the Ha ber -We i s s I' react ion : - -

H202 + '02 I__, O2 + OH + 'OH

and evidence has been presented that this occurs in vivo to cause single-strand breaks in DNA, 286 with the levels of intra-cellular (and, likely, chromatin-bound) iron and superoxide dictating the level of DNA damage and cell killing.

287,288

Sequencing studies have shown that sequence-selective modifi- cation in DNA with the mutagen 2-acetoxyamino-6-methyl-dipyrido[ 1 ,- 2-g: 3 I , 2 I - < ] imidazole , which occurs at C-8 of guanine bases , and oxidative damage by ozone to tRNA,290 which is thought to involve initial attack at C4-C5 of guanine, both occur in sequences rich in guanine, and this, together with results reported earlier, seem to indicate G-C cluster regions as mutational hot-spots.

289

As reported last year," the precursor to ribosomal RNA (rRNA) in Tetrahymena contains an intervening sequence (IVS) which is excised without the involvement of any enzyme: the rRNA precursor is "self-splicing". It now seems that a similar process occurs during the processing of the precursor to 35s rRNA in mitochondria of Neurospora. 291 hymena rRNA precursor with guanosine, which bemesincorporated at the 5'- end of the excised IVS, and with many other nucleosides and analogues, have been examined, and it appears that guanosine becomes bound at a specific site on the pre-rRNA, like a substrate to an enzyme, and the 3'-hydroxy group then attacks a specific UpA phos- phodiester link in the RNA. 292 been proposed. The structure of the linear excised IVS is thought to be one in which the two ends of the molecule are in close proxi- mity: structural homology has been found, also, with IVS'sfrom fungal mitochondria. 293 cyclisation, with formation of a circular RNA molecule which has lost 15 nucleotides from the 5I-end (L-15 IVS). This then under- goes slow reopening at a single phosphodiester bond which is the one forrneii in the cyclisationprocess, and the resultant species has, un- usually, 5'-phosphate and 3'-hydroxy group termini, and subsequently undergoes a further round of cyclisation and reopening to release pApCpCpU from the 5I-end and form (L-19 IVS). 2 9 4

of the linear IVS must permit the two termini to become adjacent to

The kinetics of the interaction of the Tetra-

A model for this interaction has

The excised linear IVS mediates its own

Thus the sequerce

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7: Nucleotides and Nucleic Acids 265

facilitate cyclisation by transesterification, and the resulting phosphodiester bond must be strained to lower the activation energy for hydrolysis. The principles familiar in enzyme chemistry seem to be emerging in RNA chemistry, and it is tempting to speculate as to how far the parallels may be drawn. Do metal ions act as effectors in RNA-mediated reactions? Would intercalators behave as in in- hibitors? How far might ionic strength regulate activity, possibly by stabilising duplex segments? The possibilities are enthralling.

5.5 Metal Complexes.- The adducts produced upon the binding to DNA, dinucleoside monophosphates, and various deoxyribonucleotide heteropolymers of cis-diamminedichloroplatinum (11) (DDP) and Cis- dichloro(ethy1enediamine)-platinum (11) (DEP) have been character- ised by enzymic digestion of the nucleic acid-drug complexes, sep- aration of the products using h.p.l.c., removal of the drug with thiourea,and analysis of the constituent nucleotides. 295

concentrations of DEP, intrastrand cross-linking occurs between neighbouring guanine bases, and the complex prevents enzymic re- moval of the intervening phosphate. At higher concentrations these sites become saturated, and two intrastrand guanine bases separated by a third base (removable by enzymic digestion) or a guanine base and an intrastrand adenine base separated by a third base (which was not removed during digestion) could be cross-linked. Small quantities of interstrand cross-links and some monofunctional adducts also occur. Similar results were obtained in a study of the binding of DDP to short oligonucleotides containing guanine and cytosine, and pH-dependent ‘H n.m.r. spectra showed that platinum invariably binds to N-7 of guanine.296 Studies on the effect of binding DDP to a single d(GpG)-containing strand of a deoxyribo- oligonucleotide duplex on the temperature for cooperative melting of the duplex show that while the melting temperature drops signi- ficantly from that for the underivatized duplex, indicating de- stabilization on platination, the degree of distortion induced in the duplex is probably not large. 297 ‘ 298

At low

A number of platinum (11) and magnesium (11) complexes of GMP have been prepared and characterised using FT-i.r. spectroscopy and other methods. *” The metal ions appeared, in general, to be co-

ordinated to N-7 of the base, and the platinum complexes, and mag- nesium complexes prepared from neutral solution, showed no direct metal ion-phosphate interaction. However, in magnesium complexes

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266 Organophosphorus Chemistry

prepared from acidic solution, direct coordination of the metal ion to phosphate was observed. Potentiometric p H titrations have been carried out to determine the pga values of the free acid forms of AMP, l,y6-etheno-AMP, and the monoanion of UMP, and the stability constants of their complexes with several divalent metal ions have been measured. 300

copper (II), and zinc ions with l,IJ'-etheno-AMP were much higher than those with AMP, due to an increased tendency for these metal ions to coordinate to the base as well as phosphate in the analogue. This tendency is even more marked in the ADP complexes, and a warn- ing has been given against drawing conclusions obtained from enzyme studies using metal ion-1 ,g6-ethenoadenosine derivatives on the assumption that they are effectively equivalent to the corresponding adenosine species. A similar study has been performed on the complexes of nucleoside 5'-diphosphates with divalent metal ions, using 'H n.m.r. spectroscopy 301 Macrochelate formation in the purine nucleotides at N-7 and phosphate occurs with several tran- sition-metal ions either intramolecularly at low dilution or intermolecularly with zinc and cadmium complexes of ADP and IDP, when dimeric stacks are formed. Upper limits have been listed for the concentrations of nucleoside 5'-diphosphates which should be used in studies meant to evaluate the properties of the monomeric species, or of their metal complexes. absorbance techniques have been used to measure magnesium ion chelation in a solution containing free ATP and Mg.ATP, in order to determine the apparent dissociation constant of Mg.ATP in physio- logical conditions and concentrations. 302

free magnesium ions was found to be less than millimolar in several different types of tissues, a value at which the concentration of the free ions would limit the rates of cellular reactions in which weak magnesium-nucleotide complexes, such as Mg.ADP, were involved. For glutamate dehydrogenases from bovine tissues, ATP and GTP are activator and inhibitor respectively, while their magnesium com- plexes lack these activities, a finding which emphasizes the critical importance of intracellular magnesium concentrations. 303

The stability constants of manganese (111,

31P N.m.r. and optical

The concentration of

The dissociation constants of a number of lanthanide-ATP com- plexes have been determined using a competition assay involving hexokinase, and the strength of interaction with ATP found to in- crease as the ionic radius of the lanthanide ion decreases.304 fluorescent lifetime of europium (111) ions in varying mole

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267 7: Nucleotides and Nucleic Acids

fractions of .deuterium oxide-water mixtures, and in the presence of ATP with or without added hexokinase or CF1-ATPase has been measured to determine the degree of hydration of the metal ion in these complexes, and the number of molecules of solvent displaced on binding to the protein. 30 5

Chelates of ATP and A(5')p4(5') with radioactive technetium have been prepared by treating the nucleotides with stannous chloride and [99mTc]-technetium pertechnetate. 306 Certain tumour

cell lines exhibit uptake of low levels of adenine nucleotides through their membranes, while untransformed cells do not, and [ 99mTc 1 Tc-Ap4A was found to accumulate preferentially in solid tumours in rats and rabbits to permit in vivo imaging. Such labelled nu- cleotides may therefore be useful for in vivo tumour detection.

A model for the transmembrane exchange reaction of ADP and ATP at the mitochondria1 membrane, which is critically dependent on the participation of a divalent metal ion - probably magnesium - and the differing stability constants of protonated and deprotonated metal-nucleotide species in ternary complexes with alternating 307 conformational states of the carrier protein, has been proposed.

6 Analytical Techniques and Physical Methods

The separation of nucleotides using h.p.1.c. has been re- 308 viewed.

DNA may be precipitated selectively from phosphate buffers by addition of cetylpyridinium bromide, thus facilitating its recovery, in high yield, following hydroxyapatite chromatography. 309 The DNA is subsequently separated from the detergent by washing with water and ethanol. DNA labelled by nick translation using 2'- deoxyadenosine 5 ' - (1- [35Sl -thio) triphosphate and used as a probe in Southern or colony-blot hybridisation has been found to have some advantages over [32Pl-labelled DNA : the half-life of the sulphur radiolabel is longer than that of phosphorus, and resolution on autoradiographs is improved. 3 10

Mechanisms for changes in the chemical shifts of phosphorus nuclei in 31P n.m.r. spectra of nucleotides have been reviewed, and a satisfactory correlation between 31P n.m.r. chemical shift

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268 Organophosphorus Chembtry

values in nucleoside and other organic phosphates with the v(P=O) stretching frequency in the i.r. spectrum has been established. 312

The introduction of oxygen-17 in place of oxygen-16 in the phos- phategroups of oligo- and polynucleotides has been used as a method for assigning the 31P n.m.r. spectra of these species. (with nuclear spin I= /2) is directly bonded to phcsphorus at a non- bridging site, its contribution to the dipolar transverszrelaxation of the phosphorus nucleus is substantially greater than that of the three flanking protons (3'-H;5'-H;5"-H) which are the normal con- tributors, and the line width of the phosphorus resonance is in- creased. Thus, comparison of a poly(I).poly(C) duplex, in which the poly(1) strand has been prepared by polymerisation of [ a -

IDP with polynucleotide phosphorylase , with one lacking the I7O isotope permits ready assignment of the signals due to poly(C) and to poly(1) .313 was compared with [1701poly(A) .poly(U) , the lack of change in the pattern of peaks despite introduction of the label suggested the occurrence of possible alternating conformations in the duplex. Using [ 0 1-phosphoryl chloride and the hydroxybenzotriazole phos- photriester method , d (CpGpC [ 01 pG) and d (C [ 01 pGpCpG) have been prepared. 314 Comparison of the signals of these species with that of the unlabelled oligonucleotide permits unequivocal assignment of the phosphorus signals, and repetition of the exercise in the pre- sence of actinomycin D allows characterisation of the shifts in re- sonances which ensue on binding of the drug. The spin relaxation in the 170 n.m.r. spectrum of [1701-poly(A) , due to the interaction of the nuclear quadrupole moment of 170 with bonding electrons measured over a range of temperatures, is dependent on P-0 bond orientation, and has been used to investigate internal motions in poly (A) . 315

When 170 5

1 7 - 021

In a similar exercise, in which poly(A) .poly(U)

1 7

17 17

A similar ploy for distinguishing resonances in the "P n.m.r. spectra of polynucleotides utilises phosphorothioate substitution. Copolymerisation of dATP with (gp,gp)-dTTPaS, or (SpI-dATPaS with dTTP, on a poly d(A-T) template using DNA polymerase I from Micro- coccus luteus , affords poly d( AzT) and poly d(TzA) , respectively, in which the deoxythymidine and deoxyadenosine units, respectively, bear phosphorothioate groups at their 5 '-positions. 316 Comparison of the 31P n.m.r. spectra of these species with that of poly d(A-T) permits unequivocal assignment of the phosphorus resonances, which are different in the d(TpA) and d(ApT) subunits, and suggest that

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7: Nucleotides and Nucleic Acids 269

a repeating dinucleotide structure is present, with carbonyl bis (imidazole) and [ l8O4] orthophosphate affords [ y - l 8 O ]-ATP, which when treated with yeast acetyl COA synthetase affords [ a-1804] pyrophosphate. 317 The resultant chemical shift of Pa generates an AB pattern in the 31P n.m.r. spectrum, allowing the 31F-31p coupling constant to be measured.

Treatment of ADP

4

The 31P shielding tensor of 5'-dCMP has been derived from a 318 single-crystal study of the free acid form of the nucleotide.

P { Hlheteronuclear two-dimensional nuclear Overhauser A 31 1

effect (n.0.e.) study of the effects of dipolar interactions between

319 phosphorus and intramolecular (and solvent) protons in ATP in deuterium oxide-water solutions on relaxation has been performed. All three phosphorus nuclei and particularly P showed dipolar interactions with solvent protons, while Pa and P sugar, and Pa with base study of 5'-GMP at different pH values and in the presence of lanthanum ions has been used to evaluate conformations in purine mononucleotides. 320 The coupling between 31P and l3C nuclei in the I3C n.m.r. spectrum of 2 I , 3'-cyclic mononucleotides has been used to evaluate a relationship between the conformational equili- bria of the sugar and cyclophosphate rings in these compounds, and in other studies the relationship between the conformational properties of the C3'-03' bond and the sugar rings in oligo- and polynucleotides in solution has been explored. 3 2 2 The magnetic shielding constant of the 31P nucleus has been measured for all 16 common dideoxynucleoside phosphates d(N1pN2) and found in general to be shifted to lower field for the compounds displaying large 31P-1H(31) and 31P-1H(5') (5") coupling constants. 323 mensional n.m.r. method employing a special pulse sequence has been developed in which only the protons which are scalar coupled to the 31P nucleus (H-31,€1-5'tH-5'') appear in the spectrum, and this, together with other data, has permitted assignment of the deoxyribose proton n.m.r. signals in d (ApGpCpT) . 324

Y interacted with

protons. Data from a ' 'P { 'H 1 n . 0 , e .

321

A two-di-

A number of studies employing 31P n.m.r. spectroscopy have been performed on oligomers containing cytosine and guanine residues, potentially capable of forming a left-handed conformation as in "Z"-DNA. In fact r(C-G-C-G) and r(C-G-C-G-C-G) seem to adopt a right-handed IrA1'-type double helical conformation, 3 2 5 while the

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270 Organophosphorus Chemistry

corresponding oligodeoxyribonucleotides form right-handed "B"-type double helices in dilute salt solutions,326 but tend to adopt the left-handed "2"-conformation in high salt solutions at low temper- ature. 327 The 5-methylcytosine-containing hexamer [ d (m C-G)] can adopt the "Z" conformation, and the B - Z transition has been studied over a range of temperatures and salt concentrations. 328 The 8-methyl- and 8-bromo-guanine-containing oligoribonucleotides

8 8 8 8 (C-br8G-C-br8G-C-br G) and (C-m G-C-m G-C-m G), prepared by phos- photriester methods, also appear to adopt a 'Z'-type left-handed duplex form. 329 Other sequences of interest examined using 31P n.m.r. spectroscopy include the covalently-linked RNA-DNA hybrid r(G-C-G)d(T-A-T-A-C-G-C) (representing the junction between an RNA

primer terminus and an Okazaki fragment), 330 the N-dimethylated analogue of the CpCpA terminus of tRNA: miC-m;C-mzA,331 and the triplet codon for phenylalanine, UP UP^?^

5

In studies on the effects of binding of intercalators to nucleic acids, evidence from 31P n.m.r. spectra suggests that ethidium bromide and bis(methidium1 spermine bind to poly d(G-C)

3 3 3 causing it to become converted from the ''Z" to the "B" form , while adriamycin elicits the same response in poly d (m5G-C) . 334 The 31P n.m.r. shifts observed when ethidium bromide becomes com- plexed to poly(A1 .oligo(U) and tRNAPhe have been studied as a function of temperature, and the results support the notion that 31P chemical shifts are sensitive indices of phosphate ester conformations. 335 phosphate backbone of double-stranded DNA fragments has been exam- ined using 31P n.m.r.

The effect of hydration on motions in the sugar-

336

In enzymic studies, 31P n.m.r. has been used to characterise the binding of nucleotide substrates to NAL)P+-specif ic isocitrate deh~drogenase~~~ and to examine the phosphorolytic activity of glycyl-tRNA synthetase from E.coli towards glycyl adenylate using a range of phosphorylated substrates, 338 and two-dimensional 31P n.m.r. has been used to determine the unidirectional flux between creatine phosphate and ATP in vivo in rat Also, the increased relaxation rates observed for the 31P nuclei when one of the two intrinsic zinc ions of RNA polymerase from E.coli is replaced by a paramagnetic manganese (11) or cobalt (11) ion have been used to determine the distances between the metal ion and the phosphorus atoms of bound ATP. 340

tissues. 339

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7: Nucleotides and Nucleic Acids 27 1

Pyrolysis electron-impact mass spectrometry and field desorp- tion mass spectrometry have been used to identify and characterise the protected nucleoside phosphites commonly used for "triester" oligonucleotide synthesis, 341 and the former technique has also been used to monitor directly the growth of the chain in solid- phase oligonucleotide synthesis in a procedure in which the polymer- bound oligonucleotide was pyrolysed directly in the mass spectro- meter at 280°. 3 4 2 triethylammonium salts of unprotected d(T-A-C-C) and d(G-G-T-A) r e veals a primary pattern of fragmentation apparently at the phosphe diester links, and sequence information can be derived from the pattern of primary and metastable ions, and also from the colli- sional activation secondary ions. 343 dissociation of a number of protonated dinucleoside phosphates produced by field desorption has been studied by linked scanning mass spectrometry, and isomeric sequences in both ribonucleotide and deoxyribonucleotide series could be distinguished. 344 Unde- rivatised dinucleoside monophosphates and other nucleotides have also been characterised by mass spectrometry using a fast atom

345 bombardment ionisation method.

Fast-atom-bombardment mass spectrometry of the

The collisionally activated

7 The Fourier transform-infra-red spectra of N -protonated and N7-methylated GMP, and also of GMP with various divalent metal ions bound at N-7, have been recorded. 346 phosphate vibrational frequencies were observed for direct metal- phosphate interactions, and N7-metal and proton-phosphate inter- actions were found to affect sugar-phosphate vibrational frequen- cies by inducing conformational changes around the sugar-phosphate bond. Raman spectroscopy has been used to characterise A-, B-, and Z-type structures in DNA, and certain intense Raman lines of the phosphodiester backbone in the region 750-850 cm-l have been identified as being particularly useful for qualitative identifi- cation of these structures. 347 Raman spectroscopy has also been used to monitor conformational changes in yeast tRNAASP by obser- ving changes in the ribose-phosphate backbone structural vibrations over a range of temperatures.

Large perturbations of

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