Solution phase synthesis of ISIS 2922 (Vitravene) by the modified H-phosphonate approach

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Solution phase synthesis of ISIS 2922 (Vitravene) by the modiﬁed

H-phosphonate approach

Colin B. Reese* and Hongbin Yan

1

Department of Chemistry, King’s College London, Strand, London, UK WC2R 2LS.

E-mail: colin.reese@kcl.ac.uk; Fax: ϩ44 (0)207 8481771

Received (in Cambridge, UK) 9th September 2002, Accepted 15th October 2002

First published as an Advance Article on the web 13th November 2002

ThesynthesisofVitravene,a21-meroligonucleotidephosphorothioate(d[Gp(s)Cp(s)Gp(s)Tp(s)Tp(s)Tp(s)Gp(s)Cp(s)-

Tp(s)Cp(s)Tp(s)Tp(s)Cp(s)Tp(s)Tp(s)Cp(s)Tp(s)Tp(s)Gp(s)Cp(s)G]) by the modiﬁed H-phosphonate approach in

solution is reported. The synthetic strategy adopted involves the use of only four diﬀerent trimer building blocks. The

dimer and trimer blocks are prepared by a novel four component procedure in which the coupling agent (diphenyl

phosphorochloridate 5b) and the sulfur-transfer reagent (N-[(2-cyanoethylsulfanyl)succinimide] 19) are added at the

same time. Yields both of the building blocks and in the block coupling reactions are very satisfactory. The fully-

protected 21-mer 38 is unblocked by a ﬁve-step procedure to give Vitravene, which is isolated as its sodium salt.

6a was added in situ and the products were then worked up

to give the S-(4-chlorophenyl) phosphorothioate triester 3a.

Following ‘detritylation’ and acetylation (steps iii and iv), the

Introduction

The antisense^1,2and antigene²approaches to chemotherapy

could well revolutionise the treatment of cancer, certain viral

infections and a number of other diseases in years to come.

products were treated with E-2-nitrobenzaldoxime 7 and

N ¹,N ¹,N ³,N ³-tetramethylguanidine (TMG) in acetonitrile

One antisense oligonucleotide analogue, Vitravene has already

solution to give the partially-protected dinucleoside phosphate

4a. The protecting groups were then removed from the base

been approved³by U.S. Food and Drug Administration for the

topical treatment of cytomegalovirus retinitis in AIDS patients

residues and terminal hydroxy functions to give the completely

and a number of other oligonucleotide sequences are presently

unblocked dinucleoside phosphate. If a phosphorothioate

undergoing advanced human clinical trials. For this reason, the

large-scale synthesis of oligonucleotides and particularly of

their phosphorothioate analogues has become a matter of con-

siderable importance and indeed of urgency. So far, the demand

diester internucleotide linkage (as in 4b) was required, the

S-(4-chlorophenyl) sulfur transfer reagent 6a was replaced by

4-[(2-cyanoethyl)sulfanyl]morpholine-3,5-dione¹⁰8. Following

the ‘detritylation’ and acetylation steps, the 2-cyanoethyl group

for the relatively large (say, up to 1.0 kg) quantities of material

was removed by treatment with 1,8-diazabicyclo[5.4.0]undec-7-

required for clinical trials has been met⁴by scaling-up

ene (DBU) in the presence of chlorotrimethylsilane (to ensure

phosphoramidite-based solid phase synthesis. However, should

anhydrous conditions) to give the partially-protected dinucleo-

an oligonucleotide drug be approved for a systemic treatment,

then most probably hundreds of kilograms or perhaps even

tonnes of that speciﬁc sequence will be required. With this in

side phosphorothioate 4b. The protecting groups were then

removed from the base residues and the terminal hydroxy

functions in the same way.

mind, we have recently turned our attention to the development

Phosphoramidite-based solid phase synthesis, irrespective of

of a solution phase oligonucleotide synthesis that has the

the target sequence, almost always involves the stepwise addi-

potential to be scaled-up to the level likely to be required.

tion of monomeric building blocks. Thus no particular syn-

thetic strategy is possible. However, this need not be the case

Until fairly recently, the phosphotriester approach⁵appeared

to be the method of choice for the synthesis of oligonucleo-

both in the phosphotriester and the modiﬁed H-phosphonate

tides in solution. However, we then developed an alternative

approaches. It can be seen from Fig. 1 that the Vitravene

approach,^6,7which is based on H-phosphonate coupling fol-

lowed by the in situ addition of a sulfur transfer reagent. We

now believe that this modiﬁed H-phosphonate approach, which

could also be considered to be a modiﬁed phosphotriester

approach, is the preferred method of solution phase oligo-

nucleotide synthesis and that it is particularly suitable for the

preparation of phosphorothioate analogues of oligonucleo-

tides. The protocol that we followed in our initial studies⁶on

the modiﬁed H-phosphonate approach is illustrated for the

preparation of a dinucleoside phosphate and a dinucleoside

phosphorothioate in Scheme 1. Coupling between a protected

nucleoside 3ЈH-phosphonate 1 and a partially-protected nucleo-

side 2 with a free 5Ј-hydroxy function was rapidly eﬀected with

bis(2-chlorophenyl) phosphorochloridate 5a in pyridine–

dichloromethane solution at Ϫ40 ЊC. No attempt was made to

isolate the resulting relatively sensitive⁸H-phosphonate diester.

When a standard phosphodiester internucleotide linkage was

required, 2-[(4-chlorophenyl)sulfanyl]isoindole-1,3(2H )-dione⁹

Fig. 1 Trimer components of Vitravene.

sequence (d[Gp(s)Cp(s)Gp(s)Tp(s)Tp(s)Tp(s)Gp(s)Cp(s)Tp(s)-

Cp(s)Tp(s)Tp(s)Cp(s)Tp(s)Tp(s)Cp(s)Tp(s)Tp(s)Gp(s)Cp(s)-

G])¹¹can be divided into seven trimers of which one (CTT)

occurs three times and another (GCG) occurs twice. Therefore

it is possible to base the synthesis of Vitravene on only four

trimer blocks. This is clearly advantageous and would particu-

larly be so if a really large scale synthesis were to be under-

taken. Another obvious advantage of basing a synthesis on

trimer building blocks is that contamination of the product

with n Ϫ 1 and n Ϫ 2 sequences is avoided. This is very unlikely

DOI: 10.1039/b208802a

J. Chem. Soc., Perkin Trans. 1, 2002, 2619–2633

This journal is © The Royal Society of Chemistry 2002

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Scheme 1 Reagents and conditions: i, 5a, C₅H₅N, CH₂Cl₂, Ϫ40 ЊC, 5–10 min; ii, a, 6a or 8, C₅H₅N, CH₂Cl₂, Ϫ40 ЊC, 15 min, b, C₅H₅N–water

(1 : 1 v/v), Ϫ40 ЊC to room temp.; iii, HCl, CH₂Cl₂, Ϫ50 ЊC, 5 min; iv, Ac₂O, C₅H₅N, room temp., 15 h; v, 7, TMG, MeCN, room temp., 12 h;

vi, DBU, Me₃SiCl, CH₂Cl₂, room temp., 30 min.

to be the case in a synthesis based on monomeric building

blocks. We now report the synthesis, by the modiﬁed H-

phosphonate procedure in solution of Vitravene in which this

21-mer oligonucleotide phosphorothioate is assembled in six

coupling steps, each involving a trimer H-phosphonate building

block.

steps (Scheme 1). First, a solution containing both the 3ЈH-

phosphonate 1 and the component 2 with the free 5Ј-hydroxy

function was treated with the coupling reagent 5a at Ϫ40 ЊC

(Scheme 1, step i) and then, after 5–10 min, the sulfur-transfer

reagent (e.g. 8) was added (step ii). This procedure is perfectly

satisfactory for small scale synthesis. However, coupling reac-

tions (step i) will most probably be exothermic and therefore,

in a large scale synthesis, it may well be necessary to mix the

reactants much more slowly, perhaps by the controlled addi-

tion of the coupling reagent to a solution containing the

other two components. This could lead to the intermediate

H-phosphonate diester being exposed to the coupling reagent

for a considerable time and therefore to the possible occurrence

of undesired side-reactions.¹⁵Furthermore, it is inconvenient to

carry out reactions on a very large scale at as low a temperature

as Ϫ40 ЊC.

Results and discussion

1

Monomeric building blocks

Vitravene is derived from 2Ј-deoxycytidine, 2Ј-deoxyguanosine

and thymidine, and it is necessary to prepare two monomeric

building blocks from each nucleoside. These building blocks

were obtained from 4-N-benzoyl-5Ј-O-(4,4Ј-dimethoxytrityl)-

2Ј-deoxycytidine 9; B = 10, 5Ј-O-(4,4Ј-dimethoxytrityl)-2-N-

In order to address the last point, it was decided to look into

the possibility of carrying out coupling reactions at room tem-

perature. As both coupling and sulfur-transfer reagents are

electrophilic, it seemed that it might be possible to carry out

four component reactions by adding the two reagents simul-

taneously to a solution containing a mixture of the 3ЈH-

phosphonate 1 and the component 2 with the free 5Ј-hydroxy

function. This would allow the intermediate H-phosphonate

diester to react with the sulfur-transfer reagent as soon as it was

formed and its exposure to the excess of coupling reagent would

thereby be minimized. A possible problem that could then arise

is that the H-phosphonate monoester 1 might react with the

sulfur-transfer reagent (RS–X) to give the corresponding phos-

phorothioate diester 17 before coupling occurred. This reaction

could be promoted by the coupling reagent in that the sulfur-

transfer reagent might be expected to react more readily with a

mixed anhydride (e.g. 18), formed between the H-phosphonate

monoester 1 and the coupling reagent (e.g. 5b), than with the H-

phosphonate monoester 1 itself.

isobutyryl-2Ј-deoxyguanosine 9;

B = 11 and 5Ј-O-(4,4Ј-

dimethoxytrityl)thymidine 9; B = 13, all of which were com-

mercially available. In order to minimize side-reactions and to

increase the solubility of the protected oligonucleotide inter-

mediates in organic solvents, it was decided to protect guanine

residues also on O-6 with the 2,5-dichlorophenyl group¹²and

thymine residues on O-4 with the phenyl group.¹³The 2Ј-

deoxyguanosine derivative 9; B = 11 was converted in high yield

into its 6-O-(2,5-dichlorophenyl) derivative 9; B = 12 by a previ-

ously reported procedure¹²and the thymidine derivative 9;

B = 13 was converted (Scheme 2a and Experimental) into its

4-O-phenyl derivative 9; B = 14 in 82% isolated yield. All three

5Ј-O-DMTr derivatives 9; B = 10, 12 and 14 were converted

(Scheme 2b, step iii) into the triethylammonium salts of their

3ЈH-phosphonates 1, B = 10, 12 and 14, respectively, in high

yields by a previously reported procedure¹⁴and into the corre-

sponding 3Ј-O-levulinyl derivatives 15, B = 10, 12 and 14 in

good yields. With the exception of the 4-O-phenylthymidine

derivatives 1 and 15, B = 14 (see Experimental), these prepar-

ations have already been reported.⁷

The reaction between 5Ј-O-(4,4Ј-dimethoxytrityl)thymidine

3ЈH-phosphonate 1; B = 13 and the morpholine-3,5-dione

reagent¹⁴8 in pyridine-d₅was monitored at the ambient tem-

perature by ³¹P NMR spectroscopy and found to be very slow

indeed. It is noteworthy that the corresponding reaction with

N-[(2-cyanoethyl)sulfanyl]succinimide 19 was found to proceed

2

Four component synthesis of dimer and trimer blocks

In the initial work^6,7on the modiﬁed H-phosphonate approach,

fully-protected blocks (e.g. 3b) were prepared in two separate

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Scheme 2 Reagents and conditions: i, 1-methylpyrrolidine, Me₃SiCl, MeCN, room temp., 1 h; ii, a, POCl₃, 0 ЊC, 20 min, b, PhOH, 0 ЊC, 3 h, c, H₂O,

0 ЊC to room temp.; iii, a, 16, Me₃CؒCOCl, C₅H₅N, Ϫ20 ЊC, 1 h, b, H₂O, C₅H₅N, Ϫ20 ЊC to room temp.; iv, (CH₃COCH₂CH₂CO)₂O, Et₃N, DMAP,

CH₂Cl₂, room temp.; v, HCl, CH₂Cl₂, Ϫ50 ЊC, 5 min; vi, pyrrole, Cl₂CHCO₂H, CH₂Cl₂, room temp., 15 min.

and N-[(2-cyanoethyl)sulfanyl]succinimide 19 as the sulfur-

transfer reagent. The four component reactions were all carried

out in pyridine solution at room temperature. Diphenyl phos-

phorochloridate 5b was used instead of bis(2-chlorophenyl)

phosphorochloridate 5a because it is commercially avail-

able and, as far as can be judged, equally eﬀective. N-[(2-

Cyanoethyl)sulfanyl]succinimide 19 has two advantages over

the morpholine-3,5-dione sulfur-transfer reagent 8. First, it is

more soluble in pyridine and secondly, it appeared to react

more selectively with the intermediate H-phosphonate diester

in the presence of excess both of an H-phosphonate monoester

1 and the coupling reagent (e.g. 5a or 5b). It is convenient at this

stage to use a system of abbreviations for protected oligo-

nucleotide phosphorothioates^6,7in which nucleoside residues

and internucleotide linkages are italicized if they are protected

in a deﬁned way. In the present context, C, G, and T represent

at a rate approximately an order of magnitude slower. However,

the reaction involving the morpholine-3,5-dione reagent 8 pro-

ceeded much more rapidly in the presence of bis(2-chloro-

phenyl) phosphorochloridate 5a. Fortunately, the four

component coupling reactions appeared to proceed still more

rapidly and high isolated yields of S-(2-cyanoethyl)-protected

dinucleoside phosphorothioates 21 (Scheme 3a) were obtained

(Table 1, entries 1–3) when only a 20% excess of the 3ЈH-

phosphonate building block 1 was used in the presence of

diphenyl phosphorochloridate 5b as the coupling reagent

2Ј-deoxycytidine protected with a benzoyl group on N-4 of its

cytosine residue (as in 10), 2Ј-deoxyguanosine protected with

an isobutyryl group on N-2 and a 2,5-dichlorophenyl group on

O-6 of its guanine residue (as in 12) and thymidine protected

with a phenyl group on O-4 of its thymine residue (as in 14),

respectively; p(s) represents an S-(2-cyanoethyl)-protected

phosphorothioate and p(H) (which is unprotected and there-

fore not italicized) represents an H-phosphonate monoester

if it is attached to a monomer or if it is placed at the end

of a sequence. Thus DMTr-Cp(H) and HO-Cp(s)G-Lev

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Table 1 Four component solution phase oligonucleotide synthesis^a

H-Phosphonate

monomer

(mol equiv.)

Coupling

reagent 5b

(mol equiv.)

Sulfur-transfer

reagent 19

(mol equiv.)

Total

reaction

time/min

Entry

no.

5Ј-OH

Component

Isolated

yield (%)

Product^b

1

2

3

4

5

6

7

8

DMTr-Cp(H) (1.20)

DMTr-T p(H) (1.20)

DMTr-Cp(H) (1.20)

DMTr-Gp(H) (1.40)

DMTr-Cp(H) (1.40)

DMTr-Gp(H) (1.40)

DMTr-T p(H) (1.40)

HO-G-Lev

HO-T -Lev

HO-Cp(s)G-Lev

HO-Tp(s)T-Lev

HO-Cp(s)T-Lev

HO-Tp(s)T-Lev

2.5

25

HO-Cp(s)G-Lev

HO-Tp(s)T-Lev

HO-Cp(s)T-Lev

HO-Gp(s)Cp(s)G-Lev

DMTr-Gp(s)Cp(s)G-OH

DMTr-Cp(s)Tp(s)T-OH

DMTr-Gp(s)Cp(s)T-OH

DMTr-Tp(s)Tp(s)T-OH

95.6

93.8

96.7

94.4

94.8

93.1

94.0

94.2

^aReactions were carried out by adding a solution of diphenyl phosphorochloridate 5b and N-[(2-cyanoethyl)sulfanyl]succinimide 19 in anhydrous

pyridine dropwise over a period of 5 min to a stirred solution of the H-phosphonate monomer and the 5Ј-OH component in anhydrous pyridine at

room temperature. ^bThe products indicated in entries nos. 1–4 were obtained by treating the initially-obtained four component reaction products

with dichloroacetic acid and pyrrole in dichloromethane solution (see Scheme 3a and Experimental); the products indicated in entries nos. 5–8 were

obtained by treating the initially-obtained four component reaction products with hydrazine hydrate in pyridine–acetic acid–water (see Scheme 3b

and Experimental).

Scheme 3 Reagents and conditions: i, 5b, 19, C₅H₅N, room temp.; ii, pyrrole, Cl₂CHCO₂H, CH₂Cl₂, 0 ЊC, 10 min; iii, a, N₂H₄ؒH₂O, C₅H₅N, AcOH,

H₂O, b, CH₂Ac₂; iv, 16, Me₃CؒCOCl, Ϫ20 ЊC, 1 h, b, H₂O, C₅H₅N, Ϫ20 ЊC to room temp.

(Table 1, entry no. 1) represent the triethylammonium salt of

5Ј-O-(4,4Ј-dimethoxytrityl)-4-N-benzoyl-2Ј-deoxycytidine 3ЈH-

phosphonate 1; B = 10 and the partially-protected dinucleoside

phosphorothioate 21; B = 10, BЈ = 12, respectively.

Three partially-protected dinucleoside phosphorothioates,

HO-Cp(s)G-Lev, HO-Tp(s)T-Lev and HO-Cp(s)T-Lev (Table

1, 1–3) were required for the synthesis of Vitravene. These

dimers 21 (Scheme 3a) were prepared in two steps and the

average overall yield was ca. 95.4%. Following the four com-

ponent coupling and sulfur-transfer step (step i), the 5Ј-

terminal DMTr protecting groups were removed by treatment

with dichloroacetic acid in the presence of pyrrole¹⁶(step ii).

The required trimer blocks [HO-Gp(s)Cp(s)G-Lev (and

DMTr-Gp(s)Cp(s)G-OH), DMTr-Cp(s)Tp(s)T-OH, DMTr-

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Gp(s)Cp(s)T-OH and DMTr-Tp(s)Tp(s)T-OH; Table 1,

entries 4–8] were also prepared by four component synthesis

(Scheme 3a). However, a larger excess (1.40 mol equiv.) of the

3ЈH-phosphonate building block 20 was required in order to

force the coupling process to completion. This is presumably

because the 1 ϩ 2

3 coupling reactions are slower than those

involving two monomers (1 and 2), and therefore an increased

proportion of the H-phosphonate monoester 20 will get con-

verted¹²into phosphorothioate diester (corresponding to 17).

The partially-protected trimer HO- Gp(s)Cp(s)G-Lev (entry

no. 4) was required (see below) as the 3Ј-terminal building block

in the Vitravene synthesis. The other four partially-protected

trimers 24 [DMTr-Gp(s)Cp(s)G-OH, DMTr-Cp(s)Tp(s)-

T-OH, DMTr-Gp(s)Cp(s)T-OH and DMTr-Tp(s)Tp(s)-

T-OH; entries 5–8] were prepared from the corresponding

four component coupling products 22 [DMTr-Gp(s)Cp(s)-

G-Lev, DMTr-Cp(s)Tp(s)T-Lev, DMTr-Gp(s)Cp(s)T-Lev

and DMTr-Tp(s)Tp(s)T-Lev, respectively] by treatment with

hydrazine hydrate in pyridine–acetic acid–water in the usual

way¹⁷(Scheme 3b, step iii). These trimers 24 were isolated in an

average yield of ca. 94%; they were then phosphonylated by the

triethylammonium p-tolyl H-phosphonate–pivaloyl chloride

procedure¹⁴(step iv) to give the corresponding H-phosphonate

building blocks 25 in an average yield of ca. 92%.

3

Assembly of the fully-protected Vitravene sequence

As indicated above, the Vitravene sequence was prepared in six

coupling steps, each of which involved a trimer H-phosphonate

building block 25. Four component synthesis was not

attempted as it seemed possible that relatively large excesses of

trimer H-phosphonates would then be required to drive the

coupling reactions to completion, especially in the later

stages. The procedure adopted for the ﬁrst coupling reaction

(i.e. 3 ϩ 3

6; Table 2, entry no. 1) is illustrated in Scheme 4

(see also Table 2, footnote a). The trimer H-phosphonate 26

(1.25 mol equiv.) was ﬁrst allowed to react with the 5Ј-OH com-

ponent 27 (2.0 mmol, 1.0 mol equiv.) and pivaloyl chloride¹⁸

(2.5 mol equiv.) in anhydrous pyridine solution at 0 ЊC. After 30

min, in order to eﬀect sulfur-transfer as rapidly as possible,

suﬃcient N-[(2-cyanoethyl)sulfanyl]succinimide 19 was added

to saturate the reaction solution, which was then allowed to

warm up to room temperature. After 30 min, the products were

worked up and chromatographed to give the fully-protected

hexamer 28 in 94% isolated yield (Table 2, entry 1). In addition,

a small quantity (ca. 3%) of a less polar impurity, believed to be

the symmetrical hexamer 30, was obtained. The formation of

this impurity, which was easily removed by chromatography,

was probably mainly due to the H-phosphonate 26 being

contaminated with a small quantity of the corresponding

dephosphonylated material (i.e. DMTr-Cp(s)Tp(s)T-OH).

The 5Ј-O-DMTr protecting group was then removed from the

fully-protected hexamer 28 by treatment with dichloroacetic

acid in the presence of pyrrole in dichloromethane solution to

give the 5Ј-OH component 29, required for the next coupling

step (Table 2, entry no. 2), in 91.5% overall yield for the three

steps (Scheme 4, steps i–iii).

A

small proportion of each 5Ј-OH component was

retained for complete deprotection (see below). The remainder

of the material was coupled with the appropriate trimer

H-phosphonate 26 and the resulting product was ‘detritylated’,

as in the above preparation of the partially-protected hexamer

29 (Scheme 4), by treatment with dichloroacetic acid and

pyrrole in dichloromethane solution. It can be seen from

Table 2 that the excess of trimer H-phosphonate was increased

by ca. 6–10% in each successive coupling step. Nevertheless

the whole synthesis was relatively economical in trimer

H-phosphonates in that the average excess used in the six coup-

ling steps (Table 2, entries nos. 1–6) was only ca. 50%. This

compares favourably with large scale phosphoramidite-based

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Scheme 4 Reagents and conditions: i, Me₃CؒCOCl, C₅H₅N, 0 ЊC, 30 min; ii, 19, C₅H₅N, 0 ЊC to room temp., 30 min; iii, Cl₂CHCO₂H, pyrrole,

CH₂Cl₂, 0 ЊC, 30 min.

solid phase synthesis. The pivaloyl chloride/trimer H-phosphon-

ate molar ratio was ca. 2.0 in each coupling step. As indicated

above, suﬃcient sulfur-transfer reagent 19 was added to satur-

ate the reaction solution. Thus the actual quantity used

depended on the volume of pyridine used, which in turn,

depended on the total weight of nucleotide material (i.e. the

combined weight of the trimer H-phosphonate and the 5Ј-OH

component). It can be seen from the penultimate column of

Table 2 that, in order as far as possible to maintain the molar

concentrations in each coupling reaction, the total nucleo-

tide concentration by weight was increased as the synthesis

progressed. Small quantities of by-products, believed to be

symmetrical hexamers (e.g. 30) were also isolated (see Experi-

mental) from the products of the reactions leading to the

fully-protected nonamer and pentadecamer (entries 2 and 4).

The scale of the coupling reactions with respect to the

quantity of 5Ј-OH component used ranged from 3.0 mmol in

the case of HO-Cp(s)Tp(s)Tp(s)Gp(s)Cp(s)G-Lev 29

(molecular weight, 3017.5 daltons; entry 2) to 1.25 mmol in the

case of the partially-protected octadecamer 37 (molecular

weight, 8616.5 daltons; entry 6). Following the latter coupling

reaction (entry 6) and subsequent treatment with N-[(2-

cyanoethyl)sulfanyl]succinimide 19, 11.86 g (1.12 mmol) of

fully-protectedVitravene,DMTr-Gp(s)Cp(s)Gp(s)Tp(s)Tp(s)-

Tp(s)Gp(s)Cp(s)Tp(s)[Cp(s)Tp(s)Tp(s)]₃Gp(s)Cp(s)G–Lev

38 (molecular weight, 10,608 daltons) was isolated from the

products. The isolated yields of the fully-protected oligonucleo-

tide phosphorothioates (Table 2, entries 1–6) averaged over 91%

and the isolated overall yields of the 5Ј-OH components

(entries 1–5) averaged over 88%. It should be emphasized that

none of these reactions has yet been optimised.

guanine NH protons (column 2), and the relative integrals of

the two sets of signals are in good accordance with the theor-

etical values. The dC : dG ratios can also be determined from

the integrals of the H-8 (dG) and H-6 (dC) resonance signals

(columns 3 and 4, respectively). It seems likely from the data

in column 4 that the H-6 proton of a 5Ј-terminal dC residue

resonates ca. 0.17 ppm downﬁeld from that of a non-terminal

dC residue. It is also noteworthy that the H-1Ј and H-3Ј protons

of dG residues (columns 6 and 8, respectively) resonate ca.

0.25–0.3 ppm downﬁeld from the H-1Ј and H-3Ј protons of dC

and dT residues (columns 7 and 10, respectively). Thus the dG :

(dC ϩ dT) ratios may be determined. Despite the presence of

large numbers of diastereoisomers, the 3Ј-terminal levulinyl

methyl protons (column 12) invariably resonate as a sharp-

singlet at δ 2.12. The dT : dG ratios may be determined by

dividing the integrals of the thymine 5-methyl protons reson-

ance signals (at ca. δ 2.06; column 13) by one-half of the

integrals of the guanine isobutyryl methyl protons resonance

signals (at δ 0.93–0.94; column 14). Assignments have not been

made in the relatively high ﬁeld regions of the spectra where the

resonance signals of the 2-cyanoethyl and levulinyl methylene

protons are found. The ¹H NMR spectra of the fully-protected

sequences (i.e. the 5Ј-O-DMTr and the 5Ј-O-acetyl derivatives

of the 5Ј-OH components) are also relatively uncomplicated.

5

Unblocking of protected oligonucleotide phosphorothioates

The unblocking of the 5Ј-OH components involves four steps.

This process, as exempliﬁed by the unblocking of the hexamer,

HO-Cp(s)Tp(s)Tp(s)Gp(s)Cp(s)G-Lev 29 is illustrated in

Scheme 5. The 5Ј-hydroxy function is ﬁrst acetylated (step i).

This is a precaution to avoid the possible attack of the hydroxy

function¹⁹on the neighbouring phosphorothioate triester

internucleotide linkage. Step ii involves the DBU-promoted

removal of the S-(2-cyanoethyl) protecting groups from the

internucleotide linkages. Chlorotrimethylsilane is added as a

water scavenger in this step. Treatment with the conjugate base

of E-2-nitrobenzaldoxime 7 (step iii) then leads¹³to the

removal of the 6-O-(2,5-dichlorophenyl) and 4-O-phenyl pro-

tecting groups from the guanine and thymine residues, respect-

ively. The remaining acyl protecting groups are then removed

(step iv) from the cytosine and guanine base residues and the

3Ј- and 5Ј- terminal hydroxy functions by treatment with con-

centrated aqueous ammonia at 55 ЊC. 2-Mercaptoethanol is

added²⁰to the reaction solution in order to minimize the possi-

bility of desulfurization. Finally, an aqueous solution of the

fully-unblocked hexamer, d[Cp(s)Tp(s)Tp(s)Gp(s)Cp(s)G] 41

was passed through a column of Dowex 50 (Na^ϩform) to con-

vert it into its sodium salt. The other four 5Ј-OH components

31, 33, 35 and 37 were converted by the same four step process

4

¹H NMR spectra of the 5Ј-OH components

As they are complex mixtures of diastereoisomers (theoretically

32 for the hexamer 29, 256 for the nonamer 31, 2048 for the

dodecamer 33, 16384 for the pentadecamer 35 and 131072 for

the octadecamer 37), it is perhaps surprising that the ¹H NMR

spectra of the 5Ј-OH components 29, 31, 33, 35, 37 (abbreviated

to HO-6-Lev, HO-9-Lev, HO-12-Lev, HO-15-Lev and HO-18-

Lev, respectively, in Table 3) are relatively simple, and indeed

that their complexity does not appear to increase signiﬁcantly

with increasing molecular weight. Furthermore, the chemical

shifts of the resonance signals assigned to speciﬁc protons in

the base and sugar residues (Table 3, columns 1–14) vary only

within very narrow bands for all ﬁve sequences. Although no

attempt has been made to conﬁrm the base sequences by NMR

spectroscopy, it is very easy to conﬁrm the base ratios. Thus, for

all ﬁve sequences, the cytidine NH protons (Table 3, column 1)

resonate at δ 11.25–11.27, almost 1 ppm downﬁeld from the

Scheme 5 Reagents and conditions: i, Ac₂O, C₅H₅N, room temp., 16 h; ii, DBU, Me₃SiCl, room temp., 30 min; iii, E-2-nitrobenzaldoxime 7, DBU,

MeCN, room temp., 12 h; iv, a, conc. aq. NH₃(d 0.88), HSCH₂CH₂OH, 55 ЊC, 15 h, b, Dowex 50 (Na^ϩform).

2624

J. Chem. Soc., Perkin Trans. 1, 2002, 2619–2633

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(Scheme 5) into the sodium salts of d[Cp(s)Tp(s)Tp(s)Cp-

(s)Tp(s)Tp(s)Gp(s)Cp(s)G] 42, d[Cp(s)Tp(s)Tp(s)Cp(s)Tp(s)-

Tp(s)Cp(s)Tp(s)Tp(s)Gp(s)Cp(s)G]43,d[Gp(s)Cp(s)Tp(s)Cp(s)-

Tp(s)Tp(s)Cp(s)Tp(s)Tp(s)Cp(s)Tp(s)Tp(s)Gp(s)Cp(s)G]

44

and d[Tp(s)Tp(s)Tp(s)Gp(s)Cp(s)Tp(s)Cp(s)Tp(s)Tp(s)Cp(s)-

Tp(s)Tp(s)Cp(s)Tp(s)Tp(s)Gp(s)Cp(s)G] 45. Relatively small

quantities (ca. 0.01–0.02 mmol) of these ﬁve 5Ј-OH com-

ponents were unblocked in this way and the overall yields of the

isolated sodium salts all exceeded 80% (see Experimental). The

capillary gel electrophoresis (CGE) proﬁles and the ³¹P NMR

spectra of the unblocked sequences are illustrated in Figs. 2 and

3 (see below), respectively; the molecular weights of these

unblocked oligonucleotide phosphorothioates, as determined

by electrospray ionization mass spectrometry, are listed in Table

4. Finally, the unblocking of fully-protected Vitravene (DMTr-

Gp(s)Cp(s)Gp(s)Tp(s)Tp(s)Tp(s)Gp(s)Cp(s)Tp(s)[Cp(s)-

Tp(s)Tp(s)]₃- Gp(s)Cp(s)G-Lev 38 was carried out on a larger

(ca. 0.1 mmol) scale. The 5Ј-O-DMTr group was ﬁrst removed

by the procedure described above (Scheme 4, step iii). The four-

step unblocking process, outlined in Scheme 5, was then fol-

lowed to give completely unblocked Vitravene (d[Gp(s)Cp(s)-

Gp(s)Tp(s)Tp(s)Tp(s)Gp(s)Cp(s)Tp(s)Cp(s)Tp(s)Tp(s)Cp(s)-

Tp(s)Tp(s)Cp(s)Tp(s)Tp(s)Gp(s)Cp(s)G]) as its sodium salt in

76% overall isolated yield. The CGE proﬁle and ³¹P NMR spec-

trum of Vitravene are illustrated in Figs. 2f and 3f (see below),

respectively, and its molecular weight, as determined by eletro-

spray ionization mass spectrometry, is included in Table 4. In

1

addition, the H NMR spectrum of Vitravene is illustrated in

Fig. 4.

6

Conclusions

In our opinion, it may be concluded from the results of this

study that the solution phase synthesis of oligonucleotide

phosphorothioates by the modiﬁed H-phosphonate approach

shows much promise. In the ﬁrst place, it seems likely that it will

be possible to carry out the four component synthesis of dimer

and trimer (and perhaps tetramer) blocks on a very much larger

scale. This could prove to be of considerable practical import-

ance in solid phase as well as in solution phase synthesis. Thus

building blocks such as trimer H-phosphonates 25 or the

corresponding phosphoramidites 46 could be used instead

of monomer H-phosphonates¹⁸1 or monomer phosphor-

amidites²¹47 in solid phase synthesis. This could introduce a

strategic advantage when short (e.g. dimer or trimer) repeating

sequences were present and would also avoid separation prob-

lems due to contamination with n Ϫ 1 and n Ϫ 2 sequences.

Although yields were, on the whole, good in the block coup-

ling reactions (Table 2), we do not believe that this procedure

has been optimized. Nevertheless, the CGE data (Fig. 2) sug-

gest that the ﬁrst four coupling steps (Table 2, entries 1–4)

were very successful indeed. The least successful coupling

step appears to be 3 ϩ 15

18 (Fig. 2e and Table 2, entry 5);

J. Chem. Soc., Perkin Trans. 1, 2002, 2619–2633

2625

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Table 4 Electrospray ionization mass spectrometrically determined molecular weights (in daltons) of completely unblocked oligonucleotide

phosphorothioates

Oligonucleotide

Molecular formula

Measured M.W.

Theoretical M.W.

6-mer 41

9-mer 42

12-mer 43

15-mer 44

18-mer 45

Vitravene

C₅₈H₇₅N₂₀O₃₁P₅S₅

1863.38

2809.33

3755.44

4725.89

5686.75

6683.13

1863.55

2809.33

3755.10

4725.89

5686.68

6682.48

C₈₇H₁₁₃N₂₇O₄₈P₈S₈

C₁₁₆H₁₅₁N₃₄O₆₅P₁₁S₁₁

C₁₄₅H₁₈₈N₄₄O₈₁P₁₄S₁₄

C₁₇₅H₂₂₇N₅₀O₉₉P₁₇S₁₇

C₂₀₄H₂₆₃N₆₃O₁₁₄P₂₀S₂₀

Fig. 2 Capillary gel electrophoretic proﬁles of the fully-unblocked (a) 6-mer 41, (b) 9-mer 42, (c) 12-mer 43, (d) 15-mer 44, (e) 18-mer 45, (f ) 21-mer

(Vitravene), (g) mixture of all six sequences.

the fact that this coupling appears to be less successful than

3 ϩ 18 21 (Fig. 2f and Table 2, entry 6) suggests that the

diesters, resonance signals at ca. δ 56 ppm. In all six spectra,

there are also very weak resonance signals at ca. δ 0, which

indicate that not more than ca. 0.5% of the total internucleotide

linkages are phosphodiester rather than phosphorothioate

diesters. Although care was taken during the unblocking steps,

it is believed that even this extent of phosphodiester contamin-

ation can be decreased. Finally, the ¹H NMR spectrum (in

D₂O) of Vitravene is illustrated in Fig. 4. As Vitravene is theor-

etically a mixture of over 10⁶(i.e. 2²⁰) diastereoisomers and

contains six diﬀerently situated 2Ј-deoxycytidine, ﬁve diﬀer-

ently situated 2Ј-deoxyguanosine and ten diﬀerently situated

thymidine residues, it is hardly surprising that the resonance

signals are rather broad. The most downﬁeld group of signals

(δ 7.4–8.15, 21 H) is assigned to the H-6 resonances of the

coupling eﬃciency does not necessarily fall oﬀ with increasing

size of the 5Ј-hydroxy component. The block coupling reac-

tions were carried out on a relatively large scale inasmuch as

11.86 g (ca. 1.12 mmol) of fully-protected Vitravene 38 was

obtained. It must be emphasized that, following unblock-

ing, none of the oligonucleotide phosphorothioates was further

puriﬁed at the phosphorothioate diester stage.

Electrospray ionisation mass spectrometry provided satisfac-

tory molecular weight values (Table 4) for all of the completely

unblocked sequences. The ³¹P NMR spectra (in D₂O) of all

six completely unblocked oligonucleotide phosphorothioate

sequences (Fig. 3) display, as expected for phosphorothioate

2626

J. Chem. Soc., Perkin Trans. 1, 2002, 2619–2633

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Experimental

¹H NMR spectra were measured at 360 MHz with a Bruker AV

360 spectrometer; ¹³C NMR spectra were measured at 90.6

MHz with the same spectrometer. Tetramethylsilane was used

as an internal standard, and J values are given in Hz. ³¹P NMR

spectra were measured at 145.8 MHz with the same spec-

trometer; 85% orthophosphoric acid was used as an internal

standard. Reverse phase high-performance liquid chromato-

graphy (HPLC) was carried out on a 250 × 4.6 mm Hypersil

ODS 5µ column: the column was eluted with water–acetonitrile

mixtures [programme A: linear gradient of water–acetonitrile

(70 : 30 v/v to 20 : 80 v/v) over 10 min and then isocratic elution;

programme B: linear gradient of water–acetonitrile (70 : 30 v/v

to 10 : 90 v/v) over 10 min and then isocratic elution]. Capillary

gel electrophoresis (CGE) was carried out by Mrs Joan Bar-

rington at the Avecia Research Centre, Grangemouth with a

Hewlett-Packard 3D CE instrument. Electrospray ionisation

mass spectra were measured by Mr Brian Powell at the Avecia

Research Centre, Manchester with a Micromass Quattro

Ultima mass spectrometer. Merck silica gel 60 F₂₅₄TLC plates

were developed in solvent systems A [dichloromethane–

methanol (95 : 5 v/v) and B [dichloromethane–methanol (93 : 7

v/v)]. Merck silica gel 60 (Art 7729 and 9385) were used for

short column chromatography. Acetonitrile, 1-methylpyrrol-

idine, pyridine and triethylamine were dried by heating, under

reﬂux, over calcium hydride and were then distilled. Dichlo-

romethane and 1,2-dichloroethane were dried by heating, under

reﬂux, over phosphorus pentaoxide and were then distilled. 1,8-

diazacyclo[5.4.0]undec-7-ene (DBU) and N ¹, N ¹, N ³, N ³-

tetramethylguanidine (TMG) were dried by heating with cal-

cium hydride at 60 ЊC and were then distilled under reduced

pressure. 5Ј-O-(4,4Ј-Dimethoxytrityl)-2Ј-deoxyribonucleoside

derivatives were purchased from Cruachem Ltd., Glasgow.

Fig. 3 ³¹P NMR spectra (in D₂O) of the sodium salts of fully-

unblocked (a) 6-mer 41, (b) 9-mer 42, (c) 12-mer 43, (d) 15-mer 44,

(e) 18-mer 45, (f ) 21-mer (Vitravene).

N-[(2-Cyanoethyl)sulfanyl]succinimide 19

N-Bromosuccinimide (17.80 g, 0.10 mol) and bis(2-cyanoethyl)

disulﬁde¹⁰(17.20 g, 0.10 mol) were heated, under reﬂux, in dry

1,2-dichloroethane (30 cm³) in an atmosphere of argon for 2 h.

After the reaction mixture had been cooled down to room tem-

perature, hexane (300 cm³) was added and the mixture was

stirred for 10 min. The upper layer was decanted and the oily

residue was triturated with ethyl acetate (20 cm³) for 10 min.

The solid obtained was collected by ﬁltration and washed with

diethyl ether (70 cm³) to give the title compound as an oﬀ-white

solid (12.4 g, 67.3%) (found, in material recrystallized from

absolute ethanol: C, 45.8; H, 4.3; N, 15.2. C₇H₈N₂O₂S requires:

C, 45.64; H, 4.38; N, 15.21%), mp 110–112 ЊC; δ_H[(CD₃)₂SO]

2.71 (4 H, s), 2.75 (2 H, t, J 6.9), 3.02 (2 H, t, J 6.9);

δ_C[(CD₃)₂SO] 18.30, 29.07, 33.18, 119.62, 178.31.

5Ј-O-(4,4Ј-Dimethoxytrityl)-4-O-phenylthymidine 9; B

؍

14

1-Methylpyrrolidine (52.0 cm³, 0.50 mol) and chlorotrimethylsi-

lane (12.7 cm³, 0.10 mol) were added to a stirred suspension of

5Ј-O-(4,4Ј-dimethoxytrityl)thymidine²²9; B = 13 (21.78 g, 40.0

mmol) in dry acetonitrile (200 cm³) at room temperature. After

1 h, the reactants were cooled to 0 ЊC (ice–water bath) and

stirred for 10 min. Phosphorus oxychloride (5.59 cm³, 60 mmol)

was then added, followed after 20 min by phenol (22.59 g, 0.24

mol). After 3 h, water (10 cm³) was added and the stirred prod-

ucts were allowed to warm up to room temperature. After 16 h,

the products were concentrated under reduced pressure. The

residue was dissolved in dichloromethane (200 cm³) and the

solution was washed with saturated aqueous sodium hydrogen

carbonate (2 × 150 cm³). The aqueous layer was back-extracted

with dichloromethane (2 × 30 cm³). The combined organic

layers were dried (MgSO₄) and evaporated under reduced pres-

sure. The residue was fractionated by short column chrom-

atography on silica gel. Appropriate fractions, which were

Fig. 4 ¹H NMR spectrum (in D₂O) of the sodium salt of fully-

unblocked 21-mer (Vitravene).

cytosine, the H-8 resonances of the guanine and the H-6 reson-

ances of the thymine residues. The next nearest group of signals

(δ 5.8–6.25, 26.18 H) is assigned to all twenty-one H-1Ј reson-

ances of the nucleoside residues and the H-5 resonances of

the six cytosine residues; the integral is therefore 3% below

the theoretical value (i.e. 27 H). However, the integral of the

highest ﬁeld group of signals (δ 1.65–2.85, 72 H), assigned to all

forty-two H-2Ј resonances of the nucleoside residues and the

5-methyl proton resonances of the ten thymine residues is in

accordance with the theoretical value.

Studies directed towards the further development of this

methodology and its application to the synthesis of RNA

sequences are now being undertaken in this laboratory.

J. Chem. Soc., Perkin Trans. 1, 2002, 2619–2633

2627

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eluted with dichloromethane–methanol (99 : 1 v/v), were com-

bined and evaporated under reduced pressure to give the title

compound 9; B = 14 (20.36 g, 82%) as a colourless glass; R_f0.35

(system A) δ_H[(CD₃)₂SO] 1.73 (3 H, s), 2.18 (1 H, m), 2.30 (1 H,

m), 3.26 (2 H, m), 3.75 (6 H, s), 3.97 (1 H, m), 4.34 (1 H, m),

5.37 (1 H, d, J 4.6), 6.15 (1 H, t, J 6.3), 6.92 (4 H, d, J 8.9), 7.13–

7.48 (14 H, m), 7.99 (1 H, s); δ_C[(CD₃)₂SO] 11.89, 41.08, 55.26,

55.42, 63.72, 70.41, 86.07, 86.19, 86.27, 103.45, 113.65, 122.43,

126.08, 127.18, 128.02, 128.31, 129.85, 130.10, 135.60, 135.78,

142.24, 145.06, 152.18, 154.49, 158.52, 158.54, 170.14.

organic layers were then concentrated under reduced pressure

to ca. 40 cm³and toluene (50 cm³) was added. The resulting

solution was applied to a short column of silica gel. Elution of

the column and concentration of the combined appropriate

fractions [eluted with dichloromethane–methanol (95 : 5 v/v)]

gave the title compound 1; B = 14 as a colourless glass (7.50 g,

95.4%); δ_H[(CD₃)₂SO] 1.14 (9 H, t, J 7.3), 1.68 (3 H, s), 2.25

(1 H, m), 2.44 (1 H, m), 2.98 (6 H, quart, J 7.2), 3.28 (2 H, m),

3.75 (6 H, s), 4.12 (1 H, s), 4.75 (1 H, s), 5.79 (0.5 H, s), 6.12

(1 H, t, J 6.3), 6.92 (2 H, d, J 8.9), 6.93 (2 H, d, J 8.9), 7.14–7.48

(14.5 H, m), 7.99 (1 H, s), 10.76 (1 H, br); δ_P[(CD₃)₂SO] 1.07

(d, J 585.9).

3Ј-O-Levulinyl-4-O-phenylthymidine 15; B

؍

14

Levulinic anhydride¹²(2.14 g, 10.0 mmol) was added to a

stirred solution of 5Ј-O-(4,4Ј-dimethoxytrityl)-4-O-phenyl-

thymidine 9; B = 14 (3.16 g, 5.09 mmol), triethylamine (1.75

cm³, 12.6 mmol) and 4-(dimethylamino)pyridine (0.050 g, 0.41

mmol) in dry dichloromethane (25 cm³) at room temperature.

After 1 h, the products were poured into saturated aqueous

sodium hydrogen carbonate (30 cm³). The layers were separated

and the aqueous layer was back-extracted with dichloro-

methane (3 × 20 cm³). The combined organic layers were dried

(MgSO₄) and evaporated to dryness under reduced pressure.

The residue was dissolved in dichloromethane (50 cm³), and

pyrrole (3.2 cm³, 46 mmol) and then dichloroacetic acid (2.5

cm³, 30 mmol) were added to the stirred solution at room tem-

perature. After 15 min, the products were poured into saturated

aqueous sodium hydrogen carbonate (30 cm³). The organic

layer was separated and was again washed with saturated aque-

ous sodium hydrogen carbonate (30 cm³). The combined aque-

ous layers were back-extracted with dichloromethane (4 × 30

cm³). The combined organic layers were dried (MgSO₄) and

evaporated to dryness under reduced pressure. The residue was

triturated with ethyl acetate (20 cm³). The crystalline precipitate

was collected by ﬁltration and washed with cold ethyl acetate

(3 cm³) to give the title compound 15; B = 14 (1.74 g, 82%)

(found, in material crystallized from dichloromethane–ethyl

acetate: C, 60.3; H, 5.9; N 6.7. C₂₁H₂₄N₂O₇requires: C, 60.57;

H, 5.81; N, 6.73%), mp 184–185 ЊC; R_f0.33 (system A);

δ_H[(CD₃)₂SO] 2.09 (3 H, d, J 0.8), 2.12 (3 H, s), 2.24 (1 H, m),

2.37 (1 H, m), 2.51 (2 H, t, J 6.4), 2.75 (2 H, t, J 6.4), 3.67 (2 H,

m), 4.05 (1 H, m), 5.22 (1 H, m), 5.28 (1 H, t, J 5.2), 6.15 (1 H,

dd, J 5.8 and 8.2), 7.19 (2 H, m), 7.30 (1 H, m), 7.46 (2 H, m),

8.20 (1 H, s); δ_C[(CD₃)₂SO] 12.08, 27.69, 29.51, 37.40, 37.77,

61.12, 74.68, 85.20, 85.72, 103.32, 122.11, 125.75, 129.52,

142.25, 151.84, 154.26, 169.94, 172.01, 206.88.

Four-component synthesis of partially-protected dinucleoside

phosphorothioates

(a) HO-Cp(s)G-Lev 21; B

؍

10, BЈ

؍

12. A solution of

triethylammonium 5Ј-O-(4,4Ј-dimethoxytrityl)-4-N-benzoyl-2Ј-

deoxycytidine 3ЈH-phosphonate⁷1; B = 10 (5.753 g, 7.2 mmol)

and 2Ј-deoxy-6-O-(2,5-dichlorophenyl)-2-N-isobutyryl-3Ј-O-

levulinylguanosine¹²2; BЈ = 12 (3.482 g, 6.0 mmol) in dry pyrid-

ine (15 cm³) was evaporated to dryness under reduced pressure

and the residue was redissolved in dry pyridine (30 cm³). To this

stirred solution at room temperature, a solution of N-[(2-cyano-

ethyl)sulfanyl]succinimide 19 (2.763 g, 15.0 mmol) and diphenyl

phosphorochloridate 5b (3.1 cm³, 14.96 mmol) in dry pyridine

(40 cm³) was added dropwise over a period of 10 min. After a

further period of 10 min, water (5 cm³) was added. The react-

ants were stirred for 15 min more, and the products were then

partitioned between dichloromethane (250 cm³) and saturated

aqueous sodium hydrogen carbonate (250 cm³). The organic

layer was separated and the aqueous layer was back-extracted

with dichloromethane (2 × 20 cm³). The combined organic

layers were dried (MgSO₄) and evaporated under reduced

pressure. The residue was co-evaporated with dry toluene

(3 × 50 cm³) and then dissolved in dichloromethane (80 cm³).

The stirred solution was cooled to 0 ЊC (ice–water bath) and

ﬁrst pyrrole (4.16 cm³, 60.0 mmol) and then a solution of

dichloroacetic acid (4.95 cm³, 60.0 mmol) in dichloromethane

(40 cm³) were added. After 10 min, the products were cautiously

poured into saturated aqueous sodium hydrogen carbonate

(250 cm³). The organic layer was separated and was washed

again with saturated aqueous sodium hydrogen carbonate

(250 cm³); it was then dried (MgSO₄) and evaporated under

reduced pressure. The residue was fractionated by short column

chromatography on silica gel: the appropriate fractions, which

were eluted with dichloromethane–methanol (97 : 3 v/v), were

combined and evaporated under reduced pressure to give

HO-Cp(s)G-Lev 21; B = 10, BЈ = 12 (5.98 g, 95.6%) as a colour-

less glass; R_f0.22 (system A); t_R11.22 min (programme A);

δ_H[(CD₃)₂SO] includes the following signals: 0.95 (6 H, m), 2.13

(3 H, s), 3.60 (2 H, m), 4.19 (1 H, m), 4.35 (1 H, m), 4.44 (2 H,

m), 5.07 (1 H, m), 5.24 (1 H, m), 5.51 (1 H, m), 6.16 (1 H, t,

J 6.7), 6.46 (1 H, t, J 7.0), 7.37–7.75 (7 H, m), 8.01 (2 H, d,

J 7.4), 8.33 (1 H, m), 8.57 (1 H, m), 10.37 (1 H, br s), 11.29 (1 H,

br); δ_P[(CD₃)₂SO] 27.20, 27.46.

Triethylammonium 5Ј-O-(4,4Ј-dimethoxytrityl)-4-O-phenyl-

thymidine 3ЈH-phosphonate 1; B

؍

14

A solution of ammonium 4-methylphenyl H-phosphonate¹⁴

(5.64 g, 29.8 mmol) and triethylamine (8.36 cm³, 60 mmol) in

methanol (15 cm³) was evaporated under reduced pressure. The

residue and 5Ј-O-(4,4Ј-dimethoxytrityl)-4-O-phenylthymidine

9; B = 14 (6.21 g, 10.0 mmol) were dissolved in dry pyridine

(20 cm³), and the solution was concentrated under reduced

pressure. After the residue had been co-evaporated with dry

pyridine (20 cm³), it was redissolved in pyridine (60 cm³) and

the solution was cooled to Ϫ20 ЊC (industrial methylated spirits

(IMS)–dry ice bath). Pivaloyl chloride (3.67 cm³, 29.8 mmol)

was then added dropwise over a period of 5 min to the stirred

solution. After a further period of 1 h, water (60 cm³) was

added and the reaction mixture was allowed to warm up to

room temperature. After 1 h, the products were partitioned

between dichloromethane (300 cm³) and saturated aqueous

sodium hydrogen carbonate (2 × 200 cm³). The layers were

separated and the aqueous layer was back-extracted with

dichloromethane (2 × 40 cm³). The combined organic layers

were washed with 0.5 M triethylammonium phosphate buﬀer

(pH 7.0; 2 × 100 cm³). The combined aqueous layers were back-

extracted with dichloromethane (2 × 40 cm³). The combined

(b) HO-Tp(s)T-Lev 21; B

؍

BЈ

؍

14. This partially-

protected dinucleoside phosphorothioate was prepared from

triethylammonium

5Ј-O-(4,4Ј-dimethoxytrityl)-4-O-phenyl-

thymidine 3ЈH-phosphonate 1; B = 14 (7.08 g, 9.01 mmol) and

3Ј-O-levulinyl-4-O-phenylthymidine 2; BЈ = 14 (3.126 g, 7.51

mmol), following the procedure and stoichiometry described in

(a) above for the preparation of HO-Cp(s)G-Lev 21; B = 10, BЈ

= 12. HO-Tp(s)T-Lev 21; B = BЈ = 14 (6.10 g, 93.8%) was

isolated as a colourless glass following short column chrom-

atography; R_f0.26 (system A); t_R11.35 min (programme A);

δ_H[(CD₃)₂SO] includes the following signals: 2.10 (9 H, m), 2.74

(2 H, m), 2.94 (2 H, m), 3.15 (2 H, m), 3.69 (2 H, m), 4.22 (1 H,

m), 4.28 (1 H, m), 4.36 (2 H, m), 5.13 (1 H, m), 5.24 (1 H, m),

2628

J. Chem. Soc., Perkin Trans. 1, 2002, 2619–2633

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5.32 (1 H, m), 6.17 (2 H, m), 7.18 (4 H, m), 7.29 (2 H, m), 7.44

(4 H, m), 8.01 (1 H, m), 8.15 (1 H, m); δ_P[(CD₃)₂SO] 27.78,

27.83.

To this stirred solution at room temperature, a solution of

N-[(2-cyanoethyl)sulfanyl]succinimide 19 (2.872 g, 15.59 mmol)

and diphenyl phosphorochloridate 5b (3.23 cm³, 15.58 mmol) in

pyridine (40 cm³) was added dropwise over a period of 15 min.

After a further period of 15 min, water (5 cm³) was added. The

reactants were stirred for 15 min more, and the products were

then partitioned between dichloromethane (250 cm³) and satur-

ated aqueous sodium hydrogen carbonate (250 cm³). The layers

were separated and the aqueous layer was back-extracted with

dichloromethane (2 × 30 cm³). The combined organic layers

were dried (MgSO₄) and then concentrated under reduced pres-

sure to ca. 20 cm³. The resulting solution was cooled to 0 ЊC

(ice–water bath) and a mixture of hydrazine monohydrate (3.05

cm³, 62.9 mmol), water (6.2 cm³), glacial acetic acid (31 cm³)

and pyridine (40 cm³) was added with stirring. After 15 min,

pentane-2,4-dione (10 cm³, 97 mmol) was added and the react-

ants were allowed to warm up to room temperature. After 15

min, the products were cautiously poured into saturated aque-

ous sodium hydrogen carbonate (250 cm³). The layers were sep-

arated and the organic layer was again washed with saturated

aqueous sodium hydrogen carbonate (2 × 200 cm³). The com-

bined aqueous layers were back-extracted with dichloro-

methane (2 × 50 cm³). The combined organic layers were dried

(MgSO₄) and evaporated under reduced pressure. Toluene

(20 cm³) was added and the resulting solution was fractionated

by short column chromatography on silica gel: the appropriate

fractions, which were eluted with dichloromethane–methanol

(96 : 4 v/v), were combined and evaporated under reduced pres-

sure to give DMTr-Cp(s)Tp(s)T-OH 24; B = BЈ = 14, BЉ =

10 (8.90 g, 93.1%) as a colourless foam; R_f0.17 (system A); t_R

16.5 min (programme B); δ_H[(CD₃)₂SO] includes the following

signals: 2.10 (7 H, m), 2.25 (1 H, m), 2.63 (1 H, m), 2.78 (1 H,

m), 2.92 (4 H, m), 3.12 (4 H, m), 3.72 (6 H, m), 4.05 (1 H, m),

4.33 (7 H, m), 5.16 (2 H, m), 5.49 (1 H, d, J 4.4), 6.17 (3 H, m),

6.89 (4 H, m), 7.13–7.66 (23 H, m), 7.98 (4 H, m), 8.17 (1 H, m),

11.29 (1 H, br); δ_P[(CD₃)₂SO] 27.94, 27.99, 28.05, 28.12, 28.27,

28.30.

(c) HO-Cp(s)T-Lev 21; B

؍

10, BЈ

؍

14. This partially-

protected dinucleoside phosphorothioate was prepared from

2Ј-deoxy-(4,4Ј-dimethoxytrityl)-4-N-benzoylcytidine 3ЈH-phos-

phonate 1; B = 10 (4.793 g, 6.00 mmol) and 3Ј-O-levulinyl-4-O-

phenylthymidine 2; BЈ = 14 (2.082 g, 5.00 mmol), following the

procedure and stoichiometry described in (a) above for the

preparation of HO-Cp(s)G-Lev 21; B = 10, BЈ = 12. HO-

Cp(s)T-Lev 21; B = 10, BЈ = 14 (4.25 g, 96.7%) was isolated as a

colourless glass following short column chromatography; R_f

0.24 (system A); t_R9.21 min (programme A); δ_H[(CD₃)₂SO]

2.11 (6 H, m), 2.73 (3 H, m), 2.96 (2 H, m), 3.17 (2 H, m), 3.67

(2 H, m), 4.28 (2 H, m), 4.39 (2 H, m), 5.15 (1 H, m), 5.27 (2 H,

m), 6.19 (2 H, m), 7.18 (2 H, d, J 8.0), 7.28 (1 H, m), 7.38–7.54

(5 H, m), 7.63 (1 H, m), 8.01 (3 H, m), 8.35 (1 H, m), 11.29 (1 H,

br); δ_P[(CD₃)₂SO] 27.77, 27.84.

Four-component synthesis of partially-protected trinucleoside

diphosphorothioates

(a) HO-Gp(s)Cp(s)G-Lev 23; BЈ

؍

BЉ

؍

12; B

؍

10. A

solution of triethylammonium 2Ј-deoxy-6-O-(2,5-dichloro-

phenyl)-5Ј-O-(4,4Ј-dimethoxytrityl)-2-N-isobutyrylguanosine-

3ЈH-phosphonate 20; BЉ = 12 (5.23 g, 5.51 mmol) and HO-

Cp(s)G-Lev 21; B = 10, BЈ = 12 (4.101 g, 3.93 mmol) in dry

pyridine (10 cm³) was evaporated to dryness under reduced

pressure and the residue was redissolved in dry pyridine (19

cm³). To this stirred solution at room temperature, a solution of

N-[(2-cyanoethyl)sulfanyl]succinimide 19 (1.811 g, 9.83 mmol)

and diphenyl phosphorochloridate (2.04 cm³, 9.84 mmol) in

pyridine (27 cm³) was added dropwise over a period of 15 min.

After a further period of 15 min, water (5 cm³) was added. The

reactants were stirred for 15 min more, and the products were

then partitioned between dichloromethane (150 cm³) and satur-

ated aqueous sodium hydrogen carbonate (150 cm³). The

organic layer was separated and the aqueous layer was back-

extracted with dichloromethane (2 × 20 cm³). The combined

organic layers were dried (MgSO₄) and evaporated under

reduced pressure. The residue was co-evaporated with dry tolu-

ene (2 × 50 cm³) and was then redissolved in dichloromethane

(60 cm³). The solution was cooled to 0 ЊC (ice–water bath) and

ﬁrst pyrrole (2.73 cm³, 39.3 mmol) and then a solution of

dichloroacetic acid (3.24 cm³, 39.3 mmol) in dichloromethane

(20 cm³) were added. After 15 min, the products were cautiously

poured into saturated aqueous sodium hydrogen carbonate

(150 cm³). The organic layer was separated and was washed

again with saturated aqueous sodium hydrogen carbonate

(150 cm³); it was then dried (MgSO₄) and evaporated under

reduced pressure. The residue was fractionated by short column

chromatography on silica gel: the appropriate fractions, which

were eluted with dichloromethane–methanol (97 : 3 v/v) were

combined and evaporated under reduced pressure to give HO-

Gp(s)Cp(s)G-Lev 23; BЈ = BЉ = 12, B = 10 (6.15 g, 94.4%) as a

colourless foam; R_f0.17 (system A); t_R13.7 min (programme

A); δ_H[(CD₃)₂SO] includes the following signals: 0.95 (12 H, m),

2.13 (3 H, m), 3.61 (2 H, m), 4.23 (1 H, m), 4.41 (6 H, m), 5.10

(1 H, m), 5.18 (1 H, m), 5.33 (1 H, m), 5.51 (1 H, m), 6.19 (1 H,

t, J 6.6), 6.44 (2 H, m), 7.36–7.51 (10 H, m), 7.98 (2 H, d, J 7.3),

8.21 (1 H, m), 8.57 (2 H, m), 10.32 (1 H, br s), 10.37 (1 H, br. s),

11.29 (1 H, br); δ_P[(CD₃)₂SO] 27.85, 27.91, 27.98, 28.12.

(c) DMTr-Gp(s)Cp(s)T-OH 24; B

؍

10, BЈ

؍

14, BЉ

؍

12.

This partially-protected trinucleoside diphosphorothioate was

prepared from triethylammonium 2Ј-deoxy-6-O-(2,5-dichloro-

phenyl)-5Ј-O-(4,4Ј-dimethoxytrityl)-2-N-isobutyrylguanosine-

3ЈH-phosphonate 20; BЉ = 12 (6.204 g, 6.53 mmol) and HO-

Cp(s)T-Lev 21; B = 10, BЈ = 14 (4.10 g, 4.66 mmol), following

the procedure and stoichiometry described in (b) above for the

preparation of DMTr-Cp(s)Tp(s)T-OH 24; B = BЈ = 14, BЉ =

10. DMTr-Gp(s)Cp(s)T-OH 24; B = 10, BЈ = 14, BЉ = 12

(7.45 g, 94%) was isolated as a colourless foam following short

column chromatography; R_f0.19 (system A); t_R16.25 min

(programme A); δ_H[(CD₃)₂SO] includes the following signals:

0.89 (6 H, m), 2.09 (3 H, s), 2.11 (1 H, m), 2.26 (1 H, m), 2.76

(3 H, m), 2.91 (4 H, m), 3.47 (1 H, m), 3.70 (6 H, s), 4.06 (1 H,

m), 4.23–4.47 (7 H, m), 5.20 (1 H, m), 5.45 (1 H, m), 5.51 (1 H,

d, J 4.2), 6.19 (2 H, m), 6.47 (1 H, t, J 6.6), 6.72 (4 H, m), 7.13–

7.71 (21 H, m), 7.98 (3 H, m), 8.18 (1 H, m), 8.48 (1 H, s), 10.21

(1 H, br s), 11.29 (1 H, br); δ_P[(CD₃)₂SO] 27.67, 27.71, 27.73,

28.15, 28.27, 28.29.

(d) DMTr-Tp(s)Tp(s)T-OH 24; B

؍

BЈ

؍

BЉ

؍

14. This

partially-protected trinucleoside diphosphorothioate was pre-

pared from triethylammonium 5Ј-O-(4,4Ј-dimethoxytrityl)-4-

O-phenylthymidine-3ЈH-phosphonate 20; BЉ = 14 (4.57 g, 5.82

mmol) and HO-Tp(s)T-Lev 21; B = BЈ = 14 (3.60 g, 4.16

mmol), following the procedure and stoichiometry described in

(b) above for the preparation of DMTr-Cp(s)Tp(s)T-OH 24; B

= BЈ = 14, BЉ = 10. DMTr-Tp(s)Tp(s)T-OH 24; B = BЈ = BЉ =

14 (5.95 g, 94.2%) was isolated as a colourless foam following

short column chromatography; R_f0.27 (system A); t_R16.5 min

(programme B); δ_H[(CD₃)₂SO] includes the following signals:

1.77 (3 H, s), 2.04 (3 H, s), 2.06 (3 H, m), 2.11 (2 H, m), 2.24

(b) DMTr-Cp(s)Tp(s)T-OH 24; B

؍

BЈ

؍

14, BЉ

؍

10. A

solution of triethylammonium 2Ј-deoxy-5Ј-O-(4,4Ј-dimethoxy-

trityl)-4-N-benzoylcytidine-3ЈH-phosphonate 20; BЉ = 10 and

HO-Tp(s)T-Lev 21; B = BЈ = 14 (5.40 g, 6.236 mmol) in dry

pyridine (10 cm³) was evaporated to dryness under reduced

pressure and the residue was redissolved in pyridine (30 cm³).

J. Chem. Soc., Perkin Trans. 1, 2002, 2619–2633

2629

View Article Online

(1 H, m), 2.65 (2 H, m), 2.91 (4 H, m), 3.12 (4 H, m), 3.74 (6 H,

s), 4.04 (1 H, m), 4.25–4.40 (7 H, m), 5.17 (2 H, m), 5.48 (1 H, d,

J 4.4), 6.17 (3 H, m), 6.91 (4 H, m), 7.13–7.48 (24 H, m), 7.96

(3 H, m); δ_P[(CD₃)₂SO] 27.97, 28.03, 28.11, 28.26, 28.28.

triethylammonium salt of DMTr-Cp(s)Tp(s)Tp(H) 26. The

triethylammonium salt of DMTr-Gp(s)Cp(s)Tp(H) 32 (7.62 g,

91.0%) was isolated as a colourless glass following short column

chromatography of the products; t_R9.4, 9.8 min (programme

B); δ_P[(CD₃)₂SO] 1.04 (1 P, d, J 584.0), 27.53, 27.60, 27.66,

28.01, 28.15 (2 P, m).

(e) DMTr-Gp(s)Cp(s)G-OH 24; B

؍

10, BЈ

؍

BЉ

؍

12.

This partially-protected trinucleoside diphosphorothioate was

prepared from triethylammonium 2Ј-deoxy-6-O-(2,5-dichloro-

phenyl)-5Ј-O-(4,4Ј-dimethoxytrityl)-2-N-isobutyrylguanosine

3ЈH-phosphonate 20; BЉ = 12 (5.92 g, 6.23 mmol) and HO-

Cp(s)G-Lev 21; B = 10; BЈ = 12 (4.64 g, 4.45 mmol), following

the procedure and stoichiometry described in (b) above for the

preparation of DMTr-Cp(s)Tp(s)T-OH 24; B = BЈ = 14, BЉ =

10. DMTr-Gp(s)Cp(s)G-OH 24; B = 10, BЈ = BЉ = 12 (7.85 g,

94.8%) was isolated as a colourless foam following short

column chromatography; R_f0.22 (system A); t_R17.1 min (pro-

gramme B); δ_H[(CD₃)₂SO] includes the following signals: 0.90

(12 H, m), 3.69 (6 H, s), 4.11 (1 H, m), 4.22–4.40 (7 H, m), 4.65

(1 H, m), 5.14 (1 H, m), 5.49 (1 H, m), 5.56 (1 H, m), 6.16 (1 H,

m), 6.45 (2 H, m), 6.71 (4 H, m), 7.13–7.74 (19 H, m), 7.98 (2 H,

d, J 7.6), 8.15 (1 H, m), 8.48 (1 H, s), 8.53 (1 H, s), 10.20 (1 H, br

s), 10.34 (1 H, br s), 11.30 (1 H, br); δ_P[(CD₃)₂SO] 27.65, 27.71,

27.83, 27.86.

(c) DMTr-Tp(s)Tp(s)Tp(H) 34. This trimer H-phosphon-

ate was prepared from DMTr-Tp(s)Tp(s)T-OH 24; B = BЈ = BЉ

= 14, (5.536 g, 3.64 mmol), following the procedure and

stoichiometry described in (a) above for the preparation of the

triethylammonium salt of DMTr-Cp(s)Tp(s)Tp(H) 26. The

triethylammonium salt DMTr-Tp(s)Tp(s)Tp(H) 34 (5.60 g,

91.2%) was isolated as a colourless glass following short column

chromatography of the products; t_R11.6 min (programme B);

δ_P[(CD₃)₂SO] 1.23 (1 P, d, J 582.5), 27.91, 27.98, 28.01, 28.08

(2 P, m).

(d) DMTr-Gp(s)Cp(s)Gp(H) 36. This trimer H-phosphon-

ate was prepared from DMTr-Gp(s)Cp(s)G-OH 24; B = 10; BЈ

= BЉ = 12 (7.44 g, 4.0 mmol), following the procedure and

stoichiometry described in (a) above for the preparation of the

triethylammonium salt of DMTr-Cp(s)Tp(s)Tp(H) 26. The

triethylammonium salt DMTr-Gp(s)Cp(s)Gp(H) 36 (7.36 g,

90.8%) was isolated as a colourless glass following short column

chromatography of the products; t_R9.5, 10.2 min (programme

B); δ_P[(CD₃)₂SO] 1.12 (1 P, d, J 590.0), 27.48, 27.63, 27.76 (2 P,

m).

Preparation of triethylammonium salts of trimer H-phosphonate

building blocks

(a) DMTr-Cp(s)Tp(s)Tp(H) 26. A solution of ammonium

4-methylphenyl H-phosphonate (1.702 g, 9.0 mmol) and tri-

ethylamine (2.5 cm³, 17.9 mmol) in dry methanol (10 cm³)

was evaporated under reduced pressure, and the residue was

redissolved in dry pyridine (10 cm³). This solution was re-

evaporated. The residue was dissolved in dry pyridine (10 cm³)

and DMTr-Cp(s)Tp(s)T-OH 24; B = BЈ = 14, BЉ = 10 (4.598 g,

3.0 mmol) was added. After the resulting solution had been

concentrated under reduced pressure, the residue was redis-

solved in dry pyridine (10 cm³) and the solution was again

evaporated. Finally, the residue was redissolved in dry pyridine

(30 cm³) and the resulting solution was cooled to Ϫ20 ЊC (IMS–

dry ice bath). Pivaloyl chloride (1.11 cm³, 9.0 mmol) was added

over a period of 5 min to the stirred solution. After 1 h, water

(10 cm³) was added and the products were allowed to warm up

to room temperature over a period of 1 h. The products were

partitioned between dichloromethane (150 cm³) and 1.0 M

potassium phosphate buﬀer (pH 7.0, 80 cm³). The organic layer

was separated and the aqueous layer was back-extracted with

dichloromethane (2 × 15 cm³). The combined organic layers

were washed with 0.5 M tetramethylguanidinium (TMGH^ϩ)

phosphate buﬀer (pH 7.0, 2 × 60 cm³), and the combined aque-

ous layers were back-extracted with dichloromethane (2 × 15

cm³). Finally, the combined organic layers were extracted with

0.5 M triethylammonium phosphate buﬀer (pH 7.0, 80 cm³).

After it had been separated and dried (MgSO₄), toluene (100

cm³) was added to the organic layer. The resulting solution was

applied to a short column of silica gel. The column was eluted

with dichloromethane–methanol mixtures: the appropriate

fractions, which were eluted with dichloromethane–methanol

(85 : 15 v/v), were combined and concentrated under reduced

pressure to ca. 150 cm³. This solution was extracted with 0.5 M

triethylammonium phosphate buﬀer (pH 7.0, 80 cm³). The

organic layer was separated, dried (MgSO₄) and evaporated

under reduced pressure to give triethylammonium DMTr-

Cp(s)Tp(s)Tp(H) 26 (4.86 g, 95.4%) as a colourless glass; t_R

9.0 min (programme B); δ_P[(CD₃)₂SO] 1.48 (1 P, d, J 589.1),

27.99 (2 P, s).

HO-Cp(s)Tp(s)Tp(s)Gp(s)Cp(s)G-Lev 29

A solution of the triethylammonium salt of DMTr-Cp(s)-

Tp(s)Tp(H) 26 (4.24 g, 2.50 mmol, 1.25 mol equiv.) and HO-

Gp(s)Cp(s)G-Lev 27 (3.31 g, 2.00 mmol, 1.00 mol equiv.) in

dry pyridine (5 cm³) was evaporated under reduced pressure.

The residue was redissolved in dry pyridine (23 cm³) and the

solution was cooled to 0 ЊC (ice–water bath). Pivaloyl chloride

(0.62 cm³, 5.0 mmol, 2.5 mol equiv.) was added dropwise over a

period of 30 s to the stirred solution. After a further period of

30 min, ﬁnely powdered N-[(2-cyanoethyl)sulfanyl]succinimide

19 (1.513 g, 8.2 mmol, 4.1 mol equiv.) was added. The reactants

were allowed to warm up to room temperature over a period of

30 min. The products were then partitioned between dichloro-

methane (100 cm³) and saturated aqueous sodium hydrogen

carbonate (100 cm³). The organic layer was separated, and the

aqueous layer was back-extracted with dichloromethane (20

cm³). The combined organic layers were dried (MgSO₄) and

concentrated under reduced pressure. The products were frac-

tionated by short column chromatography on silica gel: elution

of the column with dichloromethane–methanol (96 : 4 v/v)

yielded an impurity [0.20 g, R_f0.42 (system B)]; elution with

dichloromethane–methanol (94 : 6 v/v) and evaporation of the

combined appropriate fractions gave a colourless glass (6.25 g),

R_f0.37 (system B). This material (6.20 g) was dissolved in

dichloromethane (19 cm³) and the stirred solution was cooled

to 0 ЊC (ice–water bath). Pyrrole (2.6 cm³, 37.5 mmol)

and dichloroacetic acid (2.0 cm³, 24.2 mmol) were then added.

After 30 min, the products were partitioned between dichloro-

methane (50 cm³) and saturated aqueous sodium hydrogen

carbonate (80 cm³). The layers were separated and the aqueous

layer was back-extracted with dichloromethane (10 cm³). The

combined organic layers were dried (MgSO₄) and evaporated

under reduced pressure. The residue was fractionated by short

column chromatography on silica gel: the appropriate fractions,

which were eluted with dichloromethane–methanol (91 : 9 v/v)

were combined and evaporated under reduced pressure to give

HO-Cp(s)Tp(s)Tp(s)Gp(s)Cp(s)G-Lev 29 (5.48 g, 91.5%) as

a colourless glass; R_f0.33 (system B); δ_H[(CD₃)₂SO] includes

the following signals: 0.94 (12 H, m), 2.07 (6 H, m), 2.12 (3 H,

s), 3.66 (2 H, m), 4.27–4.45 (17 H, m), 5.15 (4 H, m), 5.27 (1 H,

(b) DMTr-Gp(s)Cp(s)Tp(H) 32. This trimer H-phosphon-

ate was prepared from DMTr-Gp(s)Cp(s)T-OH 24; B = 10, BЈ

= 14, BЉ = 12 (7.648 g, 4.50 mmol), following the procedure and

stoichiometry described in (a) above for the preparation of the

2630

J. Chem. Soc., Perkin Trans. 1, 2002, 2619–2633

View Article Online

m), 5.51 (2 H, m), 6.18 (4 H, m), 6.45 (2 H, m), 7.13–7.74 (24 H,

m), 7.97 (6 H, m), 8.17 (1 H, d, J 7.6), 8.35 (1 H, m), 8.50 (1 H,

m), 8.56 (1 H, m), 10.32 (1 H, br), 10.36 (1 H, br s), 11.27 (2 H,

br); δ_P[(CD₃)₂SO] 27.76, 27.81, 27.88, 27.96, 27.98, 28.04,

28.09, 28.16, 28.27, 28.35.

following signals: 0.94 (12 H, m), 2.06 (18 H, m), 2.12 (3 H, s),

3.66 (2 H, m), 4.26–4.43 (34 H, m), 5.19 (10 H, m), 5.28 (1 H,

m), 5.51 (2 H, m), 6.19 (10 H, m), 6.45 (2 H, m), 7.11–7.74 (51

H, m), 7.98 (14 H, m), 8.17 (3 H, m), 8.34 (1 H, m), 8.49 (1 H,

m), 8.56 (1 H, m), 10.32 (1 H, br), 10.36 (1 H, br s), 11.26 (4 H,

br); δ_P[(CD₃)₂SO] 27.76, 27.83, 27.89, 27.96, 28.02, 28.10,

28.18, 28.25, 28.29, 28.35.

HO-[Cp(s)Tp(s)Tp(s)]₂Gp(s)Cp(s)G-Lev 31

The triethylammonium salt of DMTr-Cp(s)Tp(s)Tp(H) 26

(6.77 g, 3.99 mmol, 1.33 mol equiv.) and HO-Cp(s)Tp(s)-

Tp(s)Gp(s)Cp(s)G-Lev 29 (9.05 g, 3.00 mmol, 1.00 mol

equiv.) were coupled together in the presence of pivaloyl chlor-

ide (0.98 cm³, 7.96 mmol, 2.65 mol equiv.) in pyridine (45 cm³)

at 0 ЊC as in the above preparation of the hexamer 29. After

30 min, ﬁnely powdered N-[(2-cyanoethyl)sulfanyl]succinimide

19 (2.96 g, 16.1 mmol, 5.36 mol equiv.) was added. The reaction

was allowed to proceed for 1 h and the products were worked up

as in the above preparation of the hexamer 29. The products

were then fractionated by short column chromatography on

silica gel: elution of the column with dichloromethane–

methanol (96 : 4 v/v) yielded an impurity [0.25 g, R_f0.42 (sys-

tem B)]; elution with dichloromethane–methanol (94 : 6 v/v)

and evaporation of the combined appropriate fractions gave a

colourless glass (12.90 g), R_f0.36 (system B). Pyrrole (3.76 cm³,

54.2 mmol) and dichloroacetic acid (3.58 cm³, 43.4 mmol) were

added to a stirred solution of this material (12.70 g) in dichloro-

methane (27 cm³) at 0 ЊC (ice–water bath) and the reaction was

allowed to proceed for 40 min before it was worked up accord-

ing to the procedure described above in the preparation of the

partially-protected hexamer 29. The products were fractionated

by short column chromatography on silica gel: the appropriate

fractions, which were eluted with dichloromethane–methanol

(90 : 10 v/v), were combined and evaporated under reduced

pressure to give HO-[Cp(s)Tp(s)Tp(s)]₂Gp(s)Cp(s)G-Lev 31

(11.70 g, 90.4%) as a colourless glass; R_f0.30 (system B); δ_H

[(CD₃)₂SO] includes the following signals: 0.94 (12 H, m), 2.07

(12 H, m), 2.12 (3 H, s), 4.11–4.43 (27 H, m), 5.23 (8 H, m), 5.51

(2 H, m), 6.18 (7 H, m), 6.45 (2 H, m), 7.13–7.74 (38 H, m),

7.97 (10 H, m), 8.17 (2 H, m), 8.34 (1 H, m), 8.49 (1 H, m), 8.56

(1 H, m), 10.33 (1 H, br), 10.37 (1 H, br. s), 11.27 (3 H, br);

δ_P[(CD₃)₂SO] 27.77, 27.83, 27.89, 27.97, 28.03, 28.10, 28.18,

28.24, 28.30, 28.35.

HO-Gp(s)Cp(s)Tp(s)[Cp(s)Tp(s)Tp(s)]₃Gp(s)Cp(s)G-Lev

35

The triethylammonium salt of DMTr-Gp(s)Cp(s)Tp(H) 32

(5.457 g, 2.93 mmol, 1.53 mol equiv.) and HO-[Cp(s)Tp(s)-

Tp(s)]₃Gp(s)Cp(s)G-Lev 33 (11.00 g, 1.92 mmol, 1.00 mol

equiv.) were coupled together in the presence of pivaloyl

chloride (0.72 cm³, 5.84 mmol, 3.05 mol equiv.) in pyridine (42

cm³) solution at 0 ЊC as in the above preparation of the hexamer

29. After 30 min, ﬁnely powdered N-[(2-cyanoethyl)sulfanyl)-

succinimide 19 (2.763 g, 15.0 mmol, 7.83 mol equiv.) was added.

The reaction was allowed to proceed for 1 h and the products

were worked up as in the above preparation of the hexamer 29.

The products were then fractionated by short column chromato-

graphy on silica gel: elution of the column with dichloro-

methane–methanol (96 : 4 v/v) yielded an impurity [0.35 g, R_f

0.43 (system B)]; elution with dichloromethane–methanol (93 :

7 v/v) and evaporation of the combined appropriate fractions

gave a colourless glass (13.24 g), R_f0.26 (system B). Pyrrole

(2.38 cm³, 34.3 mmol) and dichloroacetic acid (2.86 cm³, 34.7

mmol) were added to a stirred solution of this material (13.00

g) in dichloromethane (17 cm³) at 0 ЊC (ice–water bath) and the

reaction was allowed to proceed for 1 h before it was worked up

according to the procedure described above in the preparation

of the partially-protected hexamer 29. The products were

fractionated by short column chromatography on silica gel:

the appropriate fractions, which were eluted with dichloro-

methane–methanol (90 : 10 v/v) were combined and evaporated

under reduced pressure to give HO-Gp(s)Cp(s)Tp(s)[Cp(s)-

Tp(s)Tp(s)]₃Gp(s)Cp(s)G-Lev 35 (11.817 g, 86.5%) as a

colourless glass; R_f0.25 (system B); δ_H[(CD₃)₂SO] includes the

following signals: 0.93 (18 H, m), 2.05 (21 H, m), 2.12 (3 H, s),

3.63 (2 H, m), 4.20–4.43 (43 H, m), 5.09–5.20 (12 H, m), 5.32

(1 H, m), 5.51 (2 H, m), 6.18 (12 H, m), 6.43 (3 H, m), 7.10–7.74

(64 H, m), 7.96 (17 H, m), 8.17 (5 H, m), 8.49 (1 H, m), 8.56

(2 H, m), 10.31 (2 H, br), 10.36 (1 H, s), 11.25 (5 H, br);

δ_P[(CD₃)₂SO] 27.76, 27.86, 27.97, 28.02, 28.10, 28.26, 28.33,

28.36.

HO-[Cp(s)Tp(s)Tp(s)]₃Gp(s)Cp(s)G-Lev 33

The triethylammonium salt of DMTr-Cp(s)Tp(s)Tp(H) 26

(6.26 g, 3.69 mmol, 1.42 mol equiv.) and HO-[Cp(s)Tp(s)-

Tp(s)]₂Gp(s)Cp(s)G-Lev 31 (11.37 g, 2.60 mmol, 1.00 mol

equiv.) were coupled together in the presence of pivaloyl

chloride (0.91 cm³, 7.4 mmol, 2.85 mol equiv.) in pyridine (47

cm³) solution at 0 ЊC as in the above preparation of the hexamer

29. After 30 min, ﬁnely powdered N-[(2-cyanoethyl)sulfanyl)-

succinimide 19 (3.09 g, 16.8 mmol, 6.47 mol equiv.) was added.

The reaction was allowed to proceed for 1 h and the products

were worked up as in the above preparation of the hexamer 29.

The products were then fractionated by short column chromato-

graphy on silica gel: elution with dichloromethane–methanol

(93 : 7 v/v) and evaporation of the combined appropriate

fractions gave a colourless glass (14.37 g), R_f0.29 (system B).

Pyrrole (3.17 cm³, 45.7 mmol) and dichloroacetic acid (3.80

cm³, 46.1 mmol) were added to a stirred solution of this

material (13.80 g) in dichloromethane (23 cm³) at 0 ЊC (ice–

water bath), and the reaction was allowed to proceed for 1 h

before it was worked up according to the procedure described

above in the preparation of the partially-protected hexamer 29.

The products were fractionated by short column chromato-

graphy on silica gel: the appropriate fractions, which were

eluted with dichloromethane–methanol (90 : 10 v/v) were com-

bined and evaporated under reduced pressure to give HO-

[Cp(s)Tp(s)Tp(s)]₃Gp(s)Cp(s)G-Lev 33 (12.58 g, 88%) as a

colourless glass; R_f0.26 (system B); δ_H[(CD₃)₂SO] includes the

HO-Tp(s)Tp(s)Tp(s)Gp(s)Cp(s)Tp(s)[Cp(s)Tp(s)Tp(s)]₃-

Gp(s)Cp(s)G-Lev 37

The triethylammonium salt of DMTr-Tp(s)Tp(s)Tp(H) 34

(4.45 g, 2.64 mmol, 1.67 mol equiv.) and HO-Gp(s)Cp(s)-

Tp(s)[Cp(s)Tp(s)Tp(s)]₃Gp(s)Cp(s)G-Lev 35 (11.50 g, 1.58

mmol, 1.00 mol equiv.) were coupled together in the presence of

pivaloyl chloride (0.65 cm³, 5.28 mmol, 3.34 mol equiv.) in

pyridine (38 cm³) solution at 0 ЊC as in the above preparation of

the hexamer 29. After 30 min, ﬁnely powdered N-[(2-cyano-

ethyl)sulfanyl)succinimide 19 (2.502 g, 13.58 mmol, 8.59 mol

equiv.) was added. The reaction was allowed to proceed for 1 h

and the products were worked up as in the above preparation of

the hexamer 29. The products were then fractionated by short

column chromatography on silica gel: elution of the column

with dichloromethane–methanol (93 : 7 v/v) and evaporation of

the combined appropriate fractions gave a colourless glass

(12.50 g), R_f0.26 (system B). Pyrrole (1.83 cm³, 26.4 mmol) and

dichloroacetic acid (2.20 cm³, 26.7 mmol) were added to a

stirred solution of this material (12.00 g) in dichloromethane

(14 cm³) at 0 ЊC (ice–water bath) and the reaction was allowed

to proceed for 1 h before it was worked up according to the

procedure described above in the preparation of the partially-

protected hexamer 29. The products were fractionated by short

J. Chem. Soc., Perkin Trans. 1, 2002, 2619–2633

2631

View Article Online

column chromatography on silica gel: the appropriate fractions,

which were eluted with dichloromethane–methanol (90 : 10 v/v)

were combined and evaporated under reduced pressure to give

HO-Tp(s)Tp(s)Tp(s)Gp(s)Cp(s)Tp(s)[Cp(s)Tp(s)Tp(s)]₃

Gp(s)Cp(s)G-Lev 37 (11.01 g, 84%) as a colourless glass; R_f

0.26 (system B); δ_H[(CD₃)₂SO] includes the following signals:

0.93 (18 H, m), 2.05 (30 H, m), 2.12 (3 H, s), 3.66 (2 H, m), 4.20–

4.50 (52 H, m), 5.17 (15 H, m), 5.32 (1 H, m), 5.51 (3 H, m), 6.18

(15 H, m), 6.45 (3 H, m), 7.10–7.74 (80 H, m), 7.94 (19 H, m),

8.16 (5 H, m), 8.49 (2 H, m), 8.56 (1 H, m), 10.31 (2 H, br),

10.36 (1 H, br. s), 11.25 (5 H, br); δ_P[(CD₃)₂SO] 27.77, 27.87,

27.93, 27.97, 28.02, 28.09, 28.26, 28.32.

mmol) to give Ac-[Cp(s)Tp(s)Tp(s)]₃Gp(s)Cp(s)G-Lev

(0.189 g, 94%) as a colourless froth.

(d) HO-Gp(s)Cp(s)Tp(s)[Cp(s)Tp(s)Tp(s)]₃Gp(s)Cp(s)-

G-Lev 35 (0.200 g, 0.028 mmol) was treated with acetic

anhydride (0.052 cm³, 0.55 mmol) to give Ac-Gp(s)-

Cp(s)Tp(s)[Cp(s)Tp(s)Tp(s)]₃Gp(s)Cp(s)G-Lev (0.188 g,

93.5%) as a colourless froth.

(e) HO-Tp(s)Tp(s)Tp(s)Gp(s)Cp(s)Tp(s)[Cp(s)Tp(s)-

Tp(s)]₃Gp(s)Cp(s)G-Lev 37 (0.200 g, 0.023 mmol) was treated

with acetic anhydride (0.044 cm³, 0.47 mmol) to give Ac-

Tp(s)Tp(s)Tp(s)Gp(s)Cp(s)Tp(s)[Cp(s)Tp(s)Tp(s)]₃Gp(s)-

Cp(s)G-Lev (0.185 g, 92%) as a colourless froth.

DMTr-Gp(s)Cp(s)Gp(s)Tp(s)Tp(s)Tp(s)Gp(s)Cp(s)Tp(s)-

[Cp(s)Tp(s)Tp(s)]₃Gp(s)Cp(s)-G-Lev 38

Complete unblocking of 5Ј-acetylated fully-protected oligo-

nucleotide phosphorothioates

The triethylammonium salt of DMTr-Gp(s)Cp(s)Gp(H) 36

(4.65 g, 2.30 mmol, 1.84 mol equiv.) and HO-Tp(s)Tp(s)Tp(s)-

Gp(s)Cp(s)Tp(s)[Cp(s)Tp(s)Tp(s)]₃Gp(s)Cp(s)G-Lev 37

(10.75 g, 1.25 mmol, 1.00 mol equiv.) were coupled together in

the presence of pivaloyl chloride (0.57 cm³, 4.63 mmol, 3.71

mol equiv.) in pyridine (35 cm³) solution at 0 ЊC as in the above

preparation of the hexamer 29. After 30 min, ﬁnely powdered

N-[(2-cyanoethyl)sulfanyl)succinimide 19 (2.303 g, 12.50 mmol,

10.0 mol equiv.) was added. The reaction was allowed to pro-

ceed for 1 h and the products were worked up as in the above

preparation of the hexamer 29. The products were then frac-

tionated by short column chromatography on silica gel: elution

of the column with dichloromethane–methanol (92 : 8 v/v) and

evaporation of the combined appropriate fractions gave DMTr-

Gp(s)Cp(s)Gp(s)Tp(s)Tp(s)Tp(s)Gp(s)Cp(s)Tp(s)[Cp(s)-

Tp(s)Tp(s)]₃Gp(s)Cp(s)G-Lev 38 as a colourless glass (11.86

g, 89.6%), R_f0.27 (system B). δ_H[(CD₃)₂SO] includes the follow-

ing signals: 0.85–0.95 (30 H, m), 2.05 (30 H, m), 2.12 (3 H, s),

3.68 (6 H, s), 4.2–4.6 (61 H, m), 5.19 (16 H, m), 5.5 (5 H, m),

6.18 (16 H, m), 6.46 (5 H, m), 6.69 (4 H, m), 7.12–7.74 (98 H,

m), 7.96 (22 H, m), 8.17 (6 H, m), 8.48 (4 H, m), 8.56 (1 H, m),

10.18 (1 H, br. s), 10.31 (2 H, br), 10.36 (2 H, br), 11.25 (6 H,

br); δ_P[(CD₃)₂SO] 27.60, 27.66, 27.76, 27.82, 27.97, 28.02,

28.10, 28.26, 28.31.

Substrate (e.g. 39; ca. 0.005–0.02 mmol) was dissolved in dry

pyridine (1 cm³) and the solution was evaporated under reduced

pressure. After this procedure had been repeated once more,

dry acetonitrile (1.0 cm³) was added to the residue. Chloro-

trimethylsilane (ca. 0.025 cm³) and DBU (9 mol equiv. per

phosphorothioate triester linkage) were then added and the

resulting solution was stirred at room temperature. After 30

min, the reaction solution was concentrated under reduced

pressure. The residue was dissolved in acetonitrile (1.5–2.0 cm³)

at room temperature and E-2-nitrobenzaldoxime 7 (ca. 5 mol

equiv. per phosphorothioate triester linkage) was added to the

stirred solution. After 12 h, the products were evaporated under

reduced pressure. Concentrated aqueous ammonia (d 0.88, 1.1–

1.2 cm³) and 2-mercaptoethanol (0.10 cm³) were added and

the resulting mixture was heated at 55 ЊC for 15 h. The cooled

products were evaporated under reduced pressure and the

residue was co-evaporated with ethanol (2 × 2 cm³). The residue

was then dissolved in methanol (2 cm³) and ethyl acetate

(30 cm³) was added. After centrifugation, the supernatant was

discarded. This process was repeated once more and the pre-

cipitate was kept in vacuo to remove the retained solvent. The

precipitate was dissolved in water (2 cm³) and the solution was

applied to a column (5 cm × 2 cm diameter) of Dowex 50 HCR

W2 (Na^ϩform) cation-exchange resin. The column was eluted

with water, and the appropriate fractions were combined and

concentrated (to ca. 3 cm³) under reduced pressure. The con-

centrated solution was freeze-dried to give the sodium salt of

the fully-unblocked oligonucleotide phosphorothioate.

Acetylation of partially-protected oligonucleotide phosphoro-

thioates with free 5Ј-hydroxy functions

Substrate (e.g. 29; ca. 0.02–0.06 mmol) was dissolved in dry

pyridine (1 cm³) and the solution was evaporated under reduced

pressure. After this procedure had been repeated, the residue

was dissolved in pyridine (2 cm³) and acetic anhydride (10–

25 mol equiv. with respect to substrate) was added to the stirred

solution at room temperature. After 16 h, methanol (0.3 cm³)

was added and, after 10 min, the products were partitioned

between dichloromethane (15 cm³) and saturated aqueous

sodium hydrogen carbonate (15 cm³). The layers were separated

and the aqueous layer was back-extracted with dichloro-

methane (5 cm³). The combined organic layers were dried

(MgSO₄) and evaporated under reduced pressure. The residue

was fractionated by short column chromatography on silica gel:

the appropriate fractions, which were eluted with dichloro-

methane–methanol (93 : 7 to 92 : 8 v/v) were combined and

evaporated under reduced pressure.

(a) Ac-Cp(s)Tp(s)Tp(s)Gp(s)Cp(s)G-Lev 39 (0.050 g,

0.016 mmol) was converted into the sodium salt of d[Cp(s)-

Tp(s)Tp(s)Gp(s)Cp(s)G] 41 (0.027 g, 84%); δ_H[D₂O] includes

the following signals: 1.7–2.7 (18 H, m), 3.65–4.3 (18 H, m),

5.75–6.2 (8 H, m), 7.45–7.95 (6 H, m); δ_P[D₂O] 56.70.

(b) Ac-[Cp(s)Tp(s)Tp(s)]₂Gp(s)Cp(s)G-Lev (0.050 g, 0.011

mmol) was converted into the sodium salt of d[Cp(s)Tp(s)-

Tp(s)Cp(s)Tp(s)Tp(s)Gp(s)Cp(s)G] 42 (0.029 g, 86%); δ_H[D₂O]

includes the following signals: 1.75–2.8 (30 H, m), 3.7–4.3

(27 H, m), 5.85–6.2 (12 H, m), 7.45–7.95 (9 H, m); δ_P[D₂O]

56.30, 56.39, 56.47, 56.64, 56.77, 56.89.

(c) Ac-[Cp(s)Tp(s)Tp(s)]₃Gp(s)Cp(s)G-Lev (0.060 g, 0.010

mmol) was converted into the sodium salt of d[Cp(s)Tp(s)-

Tp(s)Cp(s)Tp(s)Tp(s)Cp(s)Tp(s)Tp(s)Gp(s)Cp(s)G] 43 (0.037

g, 89%); δ_H[D₂O] includes the following signals: 1.7–2.8 (42 H,

m), 3.7–4.4 (36 H, m), 5.8–6.3 (16 H, m), 7.45–8.0 (12 H, m);

δ_P[D₂O] 56.58, 56.83.

(a) HO-Cp(s)Tp(s)Tp(s)Gp(s)Cp(s)G-Lev 29 (0.150 g,

0.05 mmol) was treated with acetic anhydride (0.047 cm³, 0.50

mmol) to give Ac-Cp(s)Tp(s)Tp(s)Gp(s)Cp(s)G-Lev 39

(0.141 g, 93%) as a colourless froth.

(d) Ac-Gp(s)Cp(s)Tp(s)[Cp(s)Tp(s)Tp(s)]₃Gp(s)Cp(s)G-

Lev (0.050 g, 0.0068 mmol) was converted into the sodium salt

of

d[Gp(s)Cp(s)Tp(s)Cp(s)Tp(s)Tp(s)Cp(s)Tp(s)Tp(s)Cp(s)-

(b) HO-[Cp(s)Tp(s)Tp(s)]₂Gp(s)Cp(s)G-Lev 31 (0.250 g,

0.057 mmol) was treated with acetic anhydride (0.054 cm³, 0.57

mmol) to give Ac-[Cp(s)Tp(s)Tp(s)]₂Gp(s)Cp(s)G-Lev

(0.240 g, 95%) as a colourless froth.

Tp(s)Tp(s)Gp(s)Cp(s)G] 44 (0.029 g, 84%); δ_H[D₂O] includes

the following signals: 1.7–2.8 (51 H, m), 3.7–4.4 (45 H, m),

5.75–6.25 (20 H, m), 7.45–8.0 (15 H, m); δ_P[D₂O] 56.50, 56.56,

56.58, 56.71, 56.80.

(c) HO-[Cp(s)Tp(s)Tp(s)]₃Gp(s)Cp(s)G-Lev 33 (0.200 g,

(e)

Ac-Tp(s)Tp(s)Tp(s)Gp(s)Cp(s)Tp(s)[Cp(s)Tp(s)-

0.035 mmol) was treated with acetic anhydride (0.066 cm³, 0.70

Tp(s)]₃Gp(s)Cp(s)G-Lev (0.050 g, 0.0058 mmol) was

2632

J. Chem. Soc., Perkin Trans. 1, 2002, 2619–2633

View Article Online

converted into the sodium salt of d[Tp(s)Tp(s)Tp(s)Gp(s)Cp-

(s)Tp(s)Cp(s)Tp(s)Tp(s)Cp(s)Tp(s)Tp(s)Cp(s)Tp(s)Tp(s)Gp(s)-

Cp(s)G] 45 (0.029 g, 83%); δ_H[D₂O] includes the following

signals: 1.65–2.8 (66 H, m), 3.65–4.4 (54 H, m), 5.7–6.25 (23 H,

m), 7.45–8.05 (18 H, m); δ_P[D₂O] 56.30, 56.39, 56.51, 56.61.

and ethyl acetate (200 cm³) was added to the solution. The

resulting precipitate was collected by centrifugation. This pre-

cipitation procedure was repeated once more and the solid

obtained was dried in vacuo. A solution of this material in water

(15 cm³) was passed through a column (6 cm × 4 cm diameter)

of Dowex 50 HCR W2 (Na^ϩform) cation exchange resin. The

appropriate fractions were pooled and concentrated under

reduced pressure to ca. 15 cm³. The resulting solution was

freeze-dried to give the sodium salt of d[Gp(s)Cp(s)Gp(s)Tp(s)-

Tp(s)Tp(s)Gp(s)Cp(s)Tp(s)Cp(s)Tp(s)Tp(s)Cp(s)Tp(s)- Tp(s)-

Cp(s)Tp(s)Tp(s)Gp(s)Cp(s)G] (Vitravene) as an oﬀ-white froth

(0.61 g, 76% overall yield); δ_H[D₂O] includes the following

signals: 1.65–2.85 (72 H, m), 3.65–4.2 (61 H, m), 5.8–6.25

(26 H, m), 7.4–8.15 (21 H, m); δ_P[D₂O] 55.99, 56.44, 56.69.

Complete unblocking of fully-protected Vitravene 38

Pyrrole (0.29 cm³, 4.18 mmol) and dichloroacetic acid (0.18

cm³, 2.18 mmol) were added to a cooled (ice–water bath),

stirred solution of DMTr-Gp(s)Cp(s)Gp(s)Tp(s) Tp(s)Tp(s)-

Gp(s)Cp(s)Tp(s)[Cp(s)Tp(s)Tp(s)]₃Gp(s)Cp(s)G-Lev 38

(1.500 g, 0.141 mmol) in dichloromethane (5 cm³). After 1 h, the

products were partitioned between dichloromethane (35 cm³)

and saturated aqueous sodium hydrogen carbonate (30 cm³).

The layers were separated and the aqueous layer was back-

extracted with dichloromethane (10 cm³). The organic layers

were combined, dried (MgSO₄) and evaporated under reduced

pressure. The residue was fractionated by short column

chromatography on silica gel: the appropriate fractions, which

were eluted with dichloromethane–methanol (91 : 9 v/v), were

combined and evaporated under reduced pressure to give a

colourless glass (1.383 g). A solution of this material (1.30 g) in

dry pyridine (3 cm³) was evaporated under reduced pressure.

After this procedure had been repeated once more, the residue

was dissolved in dry pyridine (5 cm³). Acetic anhydride (0.12

cm³, 1.3 mmol) was then added to the stirred solution at room

temperature. After 16 h, methanol (2 cm³) was added and, after

a further period of 10 min, the products were partitioned

between dichloromethane (40 cm³) and saturated aqueous

sodium hydrogen carbonate (40 cm³). The layers were separated

and the aqueous layer was back-extracted with dichlorometh-

ane (10 cm³). The combined organic layers were dried (MgSO₄)

and evaporated under reduced pressure. The residue was frac-

tionated by short column chromatography on silica gel: the

appropriate fractions, which were eluted with dichloro-

methane–methanol (91 : 9 v/v) were pooled and concentrated

under reduced pressure to give a colourless froth (1.186 g).

A solution of this material (1.00 g) in dry pyridine (5 cm³)

was evaporated under reduced pressure. After this procedure

had been repeated once more, dry acetonitrile (15 cm³) was

added to the residue. Chlorotrimethylsilane (0.52 cm³, 4.1

mmol) and then DBU (2.60 cm³, 17.4 mmol) were added to the

stirred mixture at room temperature. After 30 min, the products

were concentrated under reduced pressure. The residue was dis-

solved in dry acetonitrile (15 cm³) at room temperature and

E-2-nitrobenzaldoxime 7 (3.211 g, 19.3 mmol) was added to the

stirred solution. After 15 h, the products were evaporated under

reduced pressure. A mixture of the residue, 2-mercaptoethanol

(2.0 cm³) and concentrated aqueous ammonia (d 0.88, 18 cm³)

was heated at 55 ЊC. After 15 h, the products were cooled and

evaporated under reduced pressure. Absolute ethanol (5 cm³)

was added and the resulting mixture was evaporated to dryness.

After this procedure had been repeated twice more, the residue

was dissolved with the aid of soniﬁcation in methanol (20 cm³)

Acknowledgements

We thank Avecia Ltd. for generous ﬁnancial support. We

also thank Mrs Joan Barrington (Avecia Research Centre,

Grangemouth) and Mr Brian Powell (Avecia Research Centre,

Manchester) for carrying out capillary gel electrophoresis

and mass spectrometric molecular weight determinations,

respectively.

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J. Chem. Soc., Perkin Trans. 1, 2002, 2619–2633

2633

Article Doi

Solution phase synthesis of ISIS 2922 (Vitravene) by the modified H-phosphonate approach

DOI: 10.1039/b208802a

Source and publish data:

Authors:

Article abstract of DOI:10.1039/b208802a

Full text of DOI:10.1039/b208802a

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