Synthesis of modified oligonucleotides containing 4-guanidino-2-pyrimidinone nucleobases

TETRAHEDRON

Pergamon

Tetrahedron 57 (2001) 179±194

Synthesis of modi®ed oligonucleotides containing

4-guanidino-2-pyrimidinone nucleobases

*

Jordi Robles, Anna Grandas and Enrique Pedroso

Â Á Â Â Á

Departament de Quõmica Organica, Facultat de Quõmica, Universitat de Barcelona, Martõ i Franques 1-11, E-08028 Barcelona, Spain

Received 24 July 2000; revised 23 October 2000; accepted 24 October 2000

AbstractÐOligonucleotides incorporating 4-guanidino-2-pyrimidinone nucleobases have been prepared. These nucleobase analogues were

designed to mimic the double hydrogen bond donor pattern of protonated cytosines in parallel triple helices. Guanidine-, N-methyl-, N,N-

dimethyl-, and N,N⁰-dimethylguanidine-containing nucleoside H-phosphonates were used for the synthesis of oligonucleotide analogues

1. Introduction

because of the increased electrostatic repulsion between

negatively charged chains, and their stability depends on

the pH and ion concentration (Mg²¹, K¹) of the medium.

Finally, unmodi®ed oligonucleotides are not stable to

nucleases, and their permeation across cellular membranes

is quite limited.

Oligonucleotides are currently used in molecular biology

for the detection, sequencing, ampli®cation and mutation

of gene segments.¹More recently, therapeutic applications

have also been designed based on their hybridization poten-

tial, which would permit the recognition of cellular targets

such as RNA or DNA.²Interaction with proteins has also

been devised. The formation of speci®c complexes could

either block, enhance or simply regulate the function of

the corresponding gene or protein, thus correcting the

genetic disorder previously produced.

In order to increase the af®nity of the third strand for

dsDNA, several options have been studied. On the one

hand, triple-helix formation can be facilitated by the

addition of DNA ligands⁸or polyamines.⁹On the other

hand, chemical modi®cation of the third strand either at

the internucleoside phosphate groups,¹⁰or at the sugar¹¹or

nucleobase moieties¹²often increases the stability to

In particular, segments of nuclear double stranded (ds) DNA

could be targeted by the formation of triple helices.³In such

complexes, the third oligonucleotide chain binds to DNA

polypurine tracks establishing Hoogsteen-type hydrogen

bonds in the major groove. Different triple helix motifs⁴

may be formed depending on the sequence of the third

chain and its relative orientation with respect to the poly-

purine-containing strand of the duplex, which becomes the

central strand of the triplex. Pyrimidine-containing oligo-

nucleotides form T(A´T) and C¹(G´C) triads⁵and are

oriented parallel to the polypurine strand, while in the

purine motif the alignment of the third chain is antiparallel

and A(A´T) and G(G´C) triplets are formed.

Although triple helix-forming oligonucleotides produce

regulatory effects in biological media,⁶their possible

pharmacological application is hampered by several limi-

tations.⁷First, they only recognize polypurine tracks.

Second, triple helices are less stable than double helices,

Figure 1. (a) Canonical C¹(G´C) triad in triple helices (relative orientation

of strands is represented by white and black circles, respectively); (b) 4-

Guanidino-2-pyrimidinone nucleobase as an analogue of protonated cyto-

sine in the C¹(G´C) triplet.

Keywords: guanidine; H-phosphonate; nucleobase; triple helix; oligonu-

cleotide analogues.

* Corresponding author. Tel.: 134-93-4021263; fax: 134-93-3397878;

e-mail: pedroso@qo.ub.es

PII: S0040-4020(00)00991-1

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J. Robles et al. / Tetrahedron 57 (2001) 179±194

Figure 2. Alternatives for the preparation of modi®ed oligonucleotides containing 4-guanidino-2-pyrimidinone nucleobases.

nucleases, reduces the electrostatic repulsion between

negatively charged chains and provides additional inter-

actions that favour triplex formation.

group (pK_a13) is more basic than the imino nitrogen of

cytosine, the guanidine-modi®ed nucleobase would be

protonated at neutral pH.

Much more speci®c problems have been also addressed. The

most important is the pH dependence for parallel triple helix

formation, which often precludes the formation of this type

of complex in physiological media.¹³The reason is that

cytosine needs to be protonated to form two Hoogsteen

hydrogen bonds with the central guanine in the C¹(G´C)

triad (Fig. 1). Although several studies indicate that the

pK_aof cytosine (pK_a4.5) increases when it is immersed

in a polyanionic oligonucleotide strand,¹⁴triplex stability

decreases as the pH increases.¹⁵In order to overcome this

pH dependence, several analogues have been designed.

Some are neutral analogues that reproduce the hydrogen

bond scheme of protonated cytosine,^16±23others are either

more basic than cytosine or have better stacking proper-

ties.^24±26Most stabilize the triple helix at neutral pH when

introduced in the third strand, and even suppress the pH

dependence, but they are dif®cult to synthesize. Only

8-oxoadenine and 5-methylcytosine derivatives are

commercially available.

In comparison with protonated cytosine, the guanidine

group introduces additional bonds between the third and

the central strands, which probably displaces the backbone

of the third strand and leads to non-isomorphic triplets (Fig.

1). However, lack of isomorphism with standard triplets

does not seem to signi®cantly lower the af®nity of the

third strand, since protonated cytosine analogues in which

the sugar was also displaced away from the major groove do

form triple helices.^16±18Behr and cowokers had also

proposed the use of oligonucleotides containing guanidine

nucleobases analogues for triple helix formation,²⁸and they

reported on the synthesis of a 4-guanidino-2-pyrimidinone

nucleoside.²⁹They also described the synthesis of closely-

modi®ed oligonucleotides^30,31but, to our knowledge, the

introduction of 4-guanidino-2-pyrimidinone nucleoside in

oligonucleotides has not been accomplished yet.

Here we report on the development of a synthetic method-

ology to obtain oligonucleotides containing this protonated

cytosine analogue.

In order to retain the hybridization properties of protonated

cytosine, we reasoned that an amidinium group could act as

the double hydrogen bond donor. Pyrimidine nucleobases

modi®ed at the 4-position are easy to obtain,²⁷so we con-

sidered the 4-guanidino-2-pyrimidinone analogue shown in

Fig. 1 a good candidate. Moreover, since the guanidine

2. Results

We assayed three strategies to obtain modi®ed oligonucleo-

tides incorporating 4-guanidino-2-pyrimidinone nucleobases

J. Robles et al. / Tetrahedron 57 (2001) 179±194

181

Table 1. Conversion yields for the transformation of 4-triazolyl- and 4-

phenoxy-2-pyrimidinone nucleosides into 4-guanidino derivatives (speci®c

details for each treatment are shown in the Experimental)

facilitate the reaction on a solid matrix, addition of a crown

ether or use of Li₂CO₃, which would procure a more homo-

geneous reaction medium, were also evaluated, but in no

case did results improve.

These results were applied to preliminary experiments on a

solid support. The synthetic precursor of the modi®ed

nucleoside was the 3⁰-H-phosphonate derivative of 5⁰-O-

DMT-5-methyl-4-triazolyl-2-pyrimidinon-1-yl-2⁰-deoxyribo-

side (Fig. 2, strategy A), which was obtained from 5⁰-O-

DMT-thymidine in a single step.³⁶In our hands, this reac-

tion gave much better results than substitution of guanidine

for the triazolyl group on the phosphoramidite derivative.

Trimer TXT (where X represents the triazolyl-modi®ed

nucleoside) was assembled using standard H-phosphonate

chemistry,³⁷and tuning of the modi®cation and cleavage/

deprotection conditions was assayed. Best results were

achieved when a 4 h-treatment of the oligonucleotide-

resin with guanidine (608C) was followed by reaction with

conc. aq. ammonia at room temperature. Analysis of the

crude by reversed phase HPLC and MALDI-TOF mass

spectrometry showed TT^gT (where T^gis the guanidine-

modi®ed nucleoside) as the major product (86%). Two

minor side-products, TC^mT (where C^maccounts for

5-methylcytosine) and TTT, were also observed, and their

relative amount increased signi®cantly when the treatment

with aqueous ammonia was carried out at 508C.

Z

Base^a

K₂CO₃DBU N(C₂H₅)₃N(C₂H₅)₃/NMI

Triazolyl (1)

pppp

ppp

pp

ppp

p

ppp

3-Nitrotriazolyl (2)

3-Bromotriazolyl (3)

4-Nitrophenoxy (4)

2-Nitrophenoxy (5)

2,4-Dichlorophenoxy (6)

p

nr

p

nr

ppp

2,4,5-Trichlorophenoxy (7) pp

pp

2,3,4,5,6-

Pentachlorophenoxy (8)

^aFor conversion yields, the following convention is used: pppp, 100% after

6±8 h; ppp, 100% after 8±16 h; pp, 50±75% after 16 h; p, 25±50% after

16 h; nr, no reaction.

by solid phase synthesis (Fig. 2). The ®rst alternative (A)

uses a convertible nucleobase derivative,³²which can be

transformed into the desired guanidine derivative in a single

step before the ®nal deprotection and cleavage of the oligo-

nucleotide from the resin. In both the second and third

strategies (B and C), guanidino-derivatized nucleosides

are the synthons used for oligonucleotide assembly. The

main difference between B and C is the protection of the

guanidine group, and different methods for the formation of

internucleoside phosphate linkages have also been used.

We then proceeded with the synthesis of a longer chain,

0

⁵TTTTT^gTTT^gTT^gTTTTT³. The oligomer was solid-

phase assembled using the same methodology as above,

and the resin was treated with the guanidine´HCl/K₂CO₃

mixture in DMF at 608C for 6 h to introduce the guanidine

groups. The modi®ed oligonucleotide was then cleaved

from the resin by treatment with ammonia at room tempera-

ture for 2 h, and the resulting crude was puri®ed by reversed

phase HPLC. Detailed HPLC and PAGE analysis of the

puri®ed strand under more stringent conditions showed

that it was a mixture of products. Analysis by MALDI-

TOF mass spectrometry con®rmed that the mixture was

constituted by at least three products, containing one, two

or three guanidino-modi®ed nucleobases, respectively.

Assuming that a longer oligomer might require more time

for the substitution of triazole by guanidine, increased times

for the modi®cation step were also assayed (8, 12 or 48 h),

but results did not improve. Further attempts to isolate the

desired product from the mixture also failed. Consequently,

preparation of oligonucleotides modi®ed in several posi-

tions using convertible nucleosides appeared not to be the

method of choice. Even with the relatively strong conditions

used for the substitution of triazolyl groups by guanidine,

mixtures of products were obtained from which the target

oligonucleotide could not be isolated.

2.1. Synthesis using strategy A

Strategy A required reaction conditions to replace a leaving

group by a guanidine moiety without damaging the oligo-

nucleotide chain. Several triazolyl^33,34and phenoxy-

based^34,35leaving groups were assayed, since they are acces-

sible from pyrimidine nucleosides and labile to oxygen and

nitrogen nucleophiles to yield the corresponding substi-

tution products. In particular, we synthesized several 3⁰-O-

acetyl-5⁰-O-dimethoxytrityl-5-methyl-2-pyrimidinone deriva-

tives (see below) as model nucleosides to test the reaction

conditions for their conversion into guanidine-containing

derivatives by reaction with guanidine hydrochloride in

the presence of a base. The results of these experiments

are summarized in Table 1.

As can be inferred from Table 1, the best combination was

using triazolyl or 3-nitrotriazolyl convertible groups, and

K₂CO₃as the base for their transformation into guanidine

derivatives. Poorer results were always obtained with phen-

oxy derivatives, even those activated towards nucleophile

attack by electron-withdrawing groups. No improvements

(data not shown) were made by changing the solvent (aceto-

nitrile, tetrahydrofuran, dioxane or water). Since we antici-

pated that the low solubility of K₂CO₃in DMF would not

2.2. Synthesis using strategy B

A second approach used a preformed guanidino-modi®ed

nucleoside for the synthesis of the oligonucleotide (Fig. 2,

strategy B), which could thus be obtained avoiding the

reaction with guanidine on the solid support. We protected

the guanidine group for the solid-phase assembly of the

oligonucleotide by the phosphite triester approach. As stated

below, this is unnecessary if the synthesis is performed

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J. Robles et al. / Tetrahedron 57 (2001) 179±194

Figure 3. Synthesis of the protected guanidine nucleoside synthon.

using H-phosphonate chemistry. Thus, our ®rst goal was to

®nd a suitable protection for the guanidine moiety. Since the

permanent protection scheme in oligonucleotide synthesis is

based on groups labile to bases, we chose the same type of

protection for guanidines to allow all protecting groups to be

removed in a single step.

ditions.⁴⁰Unfortunately, HPLC analysis of the crude

showed that in neither case was a major product obtained,

even after long reaction times (ca. 16 h). The crude from the

ammonia treatment was formed by a complex mixture, in

which the protected trimer was the main component, and a

second, longer treatment with ammonia at 508C was not

effective either. The crude obtained after the treatment

with LiOH contained TTT as the major product. As we

had checked in a separate experiment that the guanidino-

modi®ed nucleobase was stable to the LiOH treatment at

room temperature, transformation into thymine was attri-

buted to partial hydrolysis of the protected guanidine

nucleobase, which is probably more labile than the unpro-

tected one because of the withdrawing effect of the nitro-

benzoyl protecting group.

The ®rst protecting group that we assayed was 9-¯uorenyl-

methoxycarbonyl (Fmoc), which is suitable for the protec-

tion of the arginine side chain.³⁸However, the nucleobase-

linked guanidine reacted poorly with the protecting chloro-

formate reagent, even in harsh conditions. The 4-nitro-

benzoyl group was found to be a good alternative, since it

could be easily introduced onto guanidine and was labile

enough to aqueous ammonia at room temperature. The

protected intermediate for oligonucleotide synthesis was

obtained as shown in Fig. 3.

2.3. Synthesis using strategy C

First, triazolyl-modi®ed nucleoside 11 was prepared from

5⁰-O-DMT-3⁰-O-TBDMS-thymidine by reaction with

phosphotriazolide.³⁹Subsequently, the guanidine group

was introduced using the optimized conditions shown in

Table 1 (guanidine´HCl1K₂CO₃in DMF, 608C) to yield

modi®ed nucleoside 12a. Guanidine protection was then

performed by reaction with excess 4-nitrobenzoyl chloride

in pyridine at room temperature, yielding the acylated

derivative 13 as inferred from ¹H NMR and FAB-MS analy-

sis. In the following step, the secondary hydroxyl group

was deprotected by treatment with tetrabutylammonium

¯uoride. Finally, nucleoside 14 was functionalized as the

phosphoramidite derivative 15 required for oligonucleotide

synthesis.

From the two previous strategies assayed, three conclusions

could be drawn. First, because of the dif®culty of intro-

ducing guanidine at the pyrimidinone ring of a resin-linked

oligonucleotide, it seemed advisable to use a preformed

guanidino-nucleoside to assemble the modi®ed oligomer.

Second, the nucleophilicity of guanidine was diminished

by the electron-withdrawing character of the pyrimidinone

ring, since the nucleobase-linked guanidine reacted only

with strongly electrophilic reagents such as nitrobenzoyl

chloride. Third, the electron-withdrawing character of the

nitrobenzoyl protecting group also rendered the pyrimidi-

none-guanidine bond more labile to the nucleophile

reagents commonly used for the oligonucleotide deprotec-

tion. Thus, a third alternative was designed (Fig. 2, strategy

C) that relied on the use of unprotected guanidine deriva-

tives for the synthesis of oligonucleotides.

Trimer TYT (where Y represents the guanidino-protected

nucleoside) was assembled on the resin using standard

phosphoramidite chemistry. During the synthesis, the

coupling ef®ciency of the modi®ed nucleoside phosphor-

amidite was similar to that of thymidine phosphoramidite.

Two reagents to cleave and deprotect the trimer were

assayed: concentrated aqueous ammonia, and a solution of

LiOH in methanol, which affords mild deprotection con-

We then turned our attention into the H-phosphonate

chemistry. Because of the rather speci®c reactivity with

O-nucleophiles of the intermediate generated in the

coupling step, a mixed acyl-phosphonyl anhydride, oligo-

nucleotides can be synthesized with unprotected nucleo-

bases without interference.⁴¹In our case, N-reaction of

J. Robles et al. / Tetrahedron 57 (2001) 179±194

183

Figure 4. Synthesis of the guanidine nucleosides 3⁰-H-phosphonate derivatives.

guanidine groups during oligonucleotide synthesis should

also be disfavoured because of their lower nucleophilicity

(see above). Nevertheless, in order to assess that secondary

reactions would not take place during the oligonucleotide

assembly, the guanidine unprotected nucleoside 12a was

treated with adamantoyl chloride in the presence of pyri-

dine. No acylation of the nucleoside was observed even after

6 h-reaction at room temperature.

deprotected derivatives 16, from which the desired H-phos-

phonate derivatives 17 were obtained by selective O-phos-

phonylation with diphenylphosphite in pyridine⁴²and basic

hydrolysis of the intermediate phosphite diester. All

products were characterized by NMR and mass spectro-

metry.

In order to evaluate the synthetic suitability of the new

analogues, a series of trimers TZT (where Z represents the

modi®ed nucleosides, namely guanidino-, N-methylguani-

dino-, N,N⁰-dimethylguanidino- and N,N-dimethylguani-

dino-derivatives) were synthesized and characterized.

Standard H-phosphonate methodology was used, with the

only exception that the amount of pyridine in the coupling

reaction mixture was increased because of the low solubility

of the H-phosphonate derivatives 17 in 1:1 acetonitrile/

pyridine mixtures. As a result, pyridine solutions of

both these nucleosides and the thymidine H-phosphonate

Hence, the guanidino-nucleoside H-phosphonate building

blocks were obtained as summarized in Fig. 4. 5⁰-O-

DMT-3⁰-O-TBDMS-4-guanidino-3-methyl-2-pyrimidinone

derivative (12a) was ®rst obtained as described above (see

Fig. 3), and the same reaction was used to synthesize other

derivatives such as N-methylguanidine (12b), N,N-

dimethylguanidine (12c) and N,N⁰-dimethylguanidine

nucleosides (12d). Treatment of nucleosides 12 with

tetrabutylammonium ¯uoride rendered the 3⁰-hydroxyl-

Figure 5. Reversed phase HPLC traces of TZT crudes: (a) Z: guanidine derivative; (b) Z: N-methylguanidine derivative; (c) Z: N,N⁰-dimethylguanidine

derivative; (d) Z: N,N-dimethylguanidine derivative.

184

J. Robles et al. / Tetrahedron 57 (2001) 179±194

0

Figure 6. Reversed phase HPLC traces of crudes ⁵TTTTZTTZTZTTTTT³(19), where Z is: (a) guanidine derivative; (b) N-methylguanidine derivative; (c)

N,N⁰-dimethylguanidine derivative; (d) N,N-dimethylguanidine derivative.

building blocks, and an acetonitrile solution of adamantoyl

chloride were used. In these experiments, the coupling

yields of the modi®ed H-phosphonate derivatives 17 were

identical to that of thymidine (90±95%), except for the

guanidine derivative 17a which coupled less ef®ciently

(87%). Once assembled, trimers were cleaved from the

resin by treatment with either conc. aqueous ammonia or

0.05 M LiOH in methanol at room temperature for 3 h, and

the corresponding crudes were analysed by reversed phase

HPLC (Fig. 5). HPLC pro®les showed, for all of the four

derivatives and any cleavage treatment, a single major

product, which was identi®ed by MALDI-TOF mass spec-

trometry as the target oligonucleotide. Yields after synthesis

and puri®cation were also comparable to those obtained

with unmodifed nucleosides (45±60%).

and N,N-dimethylguanidine-containing oligonucleotides

were purer than those incorporating guanidine or N-methyl-

guanidine (Fig. 6). The major products were puri®ed and

obtained homogeneous, as shown by reversed phase HPLC

or PAGE analysis, and unambiguously identi®ed as the

desired oligonucleotides by MALDI-TOF mass spectro-

metry, except the guanidine-containing oligomers. These

particularly modi®ed oligonucleotides were identi®ed by

MALDI-TOF and electrospray mass spectrometry to be

mixtures of products with different numbers of guanidine

groups, resulting from the cleavage of the guanidine-nucleo-

side linkage and formation of either thymine or 5-methyl-

cytosine. Consequently, only short chains with a single

guanidine modi®cation can be obtained with this method.

Since no side products due to either N-acylation of

guanidine or partial substitution of guanidine by water or

ammonia were found, we undertook the synthesis of

longer strands. In particular, we synthesized the modi®ed

3. Discussion

Three strategies have been explored for the solid phase

synthesis of modi®ed oligonucleotides containing 4-guani-

dino-2-pyrimidinone nucleobases, which could act as new

analogues of protonated cytosine and increase the stability

of triple helices at neutral pH. This is an important goal to

allow oligonucleotides to be used in antigene strategies for

the control of gene expression.

0

oligonucleotides ⁵TTTTCTTZTCTTTTT³(18) and

0

⁵TTTTZTTZTZTTTTT³(19), where Z represents either

the guanidino-, N-methylguanidino-, N,N⁰-dimethyl-

guanidino- or N,N-dimethylguanidino-modi®ed nucleoside,

respectively, by the same procedure described for the

assembly of trimers TZT. The 3⁰-H-phosphonate of 4-N-

benzoyl-5⁰-O-DMT-2⁰-deoxycytidine was used to introduce

cytidine in 15-mer 18. Again, no signi®cant differences

were observed in the coupling ef®ciency of the modi®ed

nucleosides. The corresponding 5⁰-O-DMT-oligonucleo-

tide-resins were then treated with conc. aqueous ammonia

at room temperature to produce the deprotection and

cleavage from the resins, for 3 h in the case of 15-mers

19, and for 15 h to completely deblock cytidines in

15-mers 18. Reversed phase HPLC analysis of the crudes

showed that in all cases major products had been obtained,

although the degree of homogeneity was different between

the various oligonucleotides. Independently of the number

of modi®ed nucleosides present, N,N⁰-dimethylguanidine-

Our ®rst choice was to use the `convertible-nucleoside'

approach. Setting up the best conditions for the substitution

reaction required testing different compounds as well as

different bases to liberate guanidine from its hydrochloride

(Table 1). Of the different leaving groups tested, triazolyl

and 3-nitrotriazolyl were more easily substituted by guani-

dine than phenoxy-functionalized 2-pyrimidinones, and

best results were achieved when K₂CO₃was added to the

reaction mixture.

Oligonucleotides were synthesized using the H-phospho-

nate method and the 4-triazolyl-2-pyrimidinone nucleoside

derivative. Two conclusions were inferred from these

J. Robles et al. / Tetrahedron 57 (2001) 179±194

185

experiments. In the ®rst place, complete conversion of

triazolyl- into guanidino-nucleobases is much more dif®cult

to achieve on a solid support than in a homogeneous

reaction medium. TC^mT and TTT were found to accompany

the target trinucleotide TT^gT, which could neverthe-

dimethyl-, or N,N⁰-dimethylguanidine. They were all

readily obtained by reaction of a protected triazolyl-deriva-

tized nucleoside with the corresponding guanidine deriva-

tive, using essentially the same conditions previously

optimized for the `convertible-nucleoside' approach.

less be isolated, and ₀the deprotection crude of

0

⁵TTTTT^gTTT^gTT^gTTTTT³contained, among others,

mono-, di-, and trisubstituted guanidine-containing

products. Thus, incomplete replacement of triazolyl groups

by guanidine was followed by reaction with aqueous

ammonia to yield either thymine or 5-methylcytosine.

The syntheses of the four different trinucleotides TZT

(Zguanidino-nucleoside) proceeded smoothly, and

homogeneous crudes were obtained after the deprotection

step. However, the syntheses of more complex oligo-

0

nucleotides, such as ⁵TTTTCTTZTCTTTTT³(18) and

0

⁵TTTTZTTZTZTTTTT³(19), indicated that dimethyl-

guanidine-containing oligonucleotides are more easily

accessible than the others. The N-methylguanidine-contain-

ing oligomers could also be isolated and characterized as

the target products, but not the guanidine-containing oligo-

nucleotides.

Secondly, when the guanidine-treated oligonucleotide-resin

was deprotected with ammonia at a higher temperature, the

relative proportion of TC^mT and TTT with respect to TT^gT

increased. This indicated a certain lability of the guanidine-

pyrimidinone linkage to nucleophiles, which increased with

temperature.

Even though it is generally accepted that guanidine groups

are dif®cult to acylate because they generally remain proto-

nated, we cannot rule out that the byproducts accompanying

the target oligonucleotides were branched oligomers formed

by incorporation of nucleotide synthons onto the unpro-

tected guanidine group (or, to a lesser extent, onto the

N-methyl substituted derivative). Nevertheless, the lability

of the guanidine-pyrimidinone linkage to the oligonucleo-

tide deprotection conditions, which is enhanced when the

guanidine moiety is linked to electron-withdrawing groups,

may also explain the low homogeneity of guanidine- and

As a result, we decided to evaluate a different alternative

using nucleoside synthons containing the guanidine group,

which was protected to prevent side reactions during oligo-

nucleotide elongation.

Guanidine protection was found not to be straightforward,

since its nucleophilicity was reduced by covalent linkage to

the 2-pyrimidinone nucleobase. It reacted only with strongly

electrophilic carbonyl derivatives such as 4-nitrobenzoyl

chloride, but not with 9-¯uorenylmethyl chloroformate.

The 4-nitrobenzoyl group was labile to ®nal deprotecting

reagents such as concentrated aqueous ammonia or

methanolic LiOH solutions, using conditions in which the

guanidine-pyrimidinone linkage was stable.

N-methylguanidine-containing

oligonucleotides.

An

increase in the number of methyl substituents, either by

steric hindrance or by electronic factors, yields more homo-

geneous crudes and thus allows the target products to be

obtained more easily.

Our ®rst synthetic target was, again, modi®ed trimer TT^gT.

Oligonucleotide assembly by the phosphite triester

approach proceeded smoothly, but the crude obtained after

ammonia deprotection was a complex mixture of products

containing the guanidine-protected trinucleotide. Deprotec-

tion with LiOH afforded TTT as the main product. Two

conclusions were deduced from these ®ndings. On the one

hand, reactions on resin-linked oligonucleotides may be

more dif®cult than on nucleosides in solution. Quantitative

removal of the 4-nitrobenzoyl group from the guanidine-

modi®ed nucleoside had been achieved, whereas not all

protecting groups could be eliminated from the insolubi-

lized trimer. On the other hand, the guanidine-pyrimidinone

linkage was labile to nucleophiles, especially to hydroxide

anions. This lability was most probably enhanced because

of the additional electron-withdrawing effect of the nitro-

benzoyl group.

In summary, oligonucleotide analogues incorporating

4-alkylguanidino-2-pyrimidinones have been prepared

using the H-phosphonate methodology. We are currently

studying their hybridization to single- or double-stranded

DNA fragments.

4. Experimental

Unless otherwise indicated, all chemicals were purchased

from commercial suppliers (reagent grade) and used without

puri®cation. Dry CH₃CN was obtained by distillation over

CaH₂and storage over CaH₂lumps. CH₂Cl₂was neutralized

and dried by passing through basic Al₂O₃and storage over

CaH₂. Dry tetrahydrofuran was obtained by distillation over

sodium metal in the presence of benzophenone. N,N-

dimethylformamide was bubbled with nitrogen to remove

volatile contaminants and dried by storage over CaH₂.

Amine-free pyridine was obtained by distillation over

ninhydrin and further dried by storage over CaH₂lumps.

Since it was dif®cult to ®nd a suitable protecting group for

the guanidine moiety, we worked with guanidine-containing

nucleoside analogues without protecting the guanidine

group. In order to minimize side reactions at this function,

H-phosphonate derivatives were used for the oligonucleo-

tide elongation, but the combination of solvents for the

nucleoside synthons and the coupling reagent had to be

optimized.

NMR spectra were recorded with either a Varian Gemini or

a Varian Unity operating at 200 and 300 MHz, respectively.

(d values are relative to internal Si(CH₃)₄(¹H, ¹³C) or

external H₃PO₄(³¹P). IR-FT spectra were recorded in a

Nicolet 510 apparatus. UV analyses were performed using

a Varian Cary 5E spectrophotometer. Low resolution mass

spectra of nucleosides were recorded in a Hewlett±Packard

We prepared four guanidine-modi®ed nucleosides, contain-

ing either the naked guanidine moiety or N-methyl-, N,N-

186

J. Robles et al. / Tetrahedron 57 (2001) 179±194

5988 A (FAB) apparatus and oligonucleotides were

analysed using VG-Quattro (ES) or Perseptive Biosystems

Voyager DEe-RP (MALDI-TOF) instruments. High

resolution mass analyses were carried out in the facilities

of the University of Vigo (Spain). Calculated values for

neutral compounds (M) are indicated in all cases. Oligo-

nucleotides were quanti®ed by their UV absorption at

260 nm in OD₂₆₀units.

4.2.1. 3-Nitrotriazolyl derivative 2. Mp 102±1048C; TLC

(AcOEt/hexane 2:1): R_f0.25; ¹H NMR (200 MHz,

CDCl₃): d9.38 (s, 1H; H-triazolyl), 8.44 (s, 1H; H±C6),

7.40±7.20 (m, 9H; H-phenyl), 6.85 (d, ³J(H, H)9 Hz, 4H;

H-phenyl), 6.38 (m, 1H; H±C1⁰), 5.45 (m, 1H; H±C3⁰), 4.17

(m, 1H; H±C4⁰), 3.79 (s, 6H; CH₃O), 3.46 (m, 2H; H±C5⁰),

2.92 (m, 1H; H±C2⁰), 2.45 (m, 1H; H⁰±C2⁰), 2.10 (s, 3H;

CH₃CO), 2.00 (s, 3H, CH₃±C5); ¹³C NMR (50 MHz,

CDCl₃) d170.2 (OCOCH₃), 158.7 (Cphenyl), 156.9

(C4), 153.1 (C2) 148.2 (Ctriazolyl), 146.2 (Ctriazolyl),

144.2 (C6), 143.9 (Cphenyl), 135.0, 134.8 (Cphenyl),

129.9, 128.0, 127.9, 127.3, 113.2 (Cphenyl±H), 105.6

(C5), 87.8, 87.3, 85.3 (Cq, C1⁰, C4⁰), 74.8 (C3⁰), 63.3

(C5⁰), 55.3 (CH₃O), 39.7 (C2⁰), 21.0 (CH₃CO), 16.0

(CH₃±C5); LRMS (FAB, magic bullet, Xe, positive

mode): m/z: 683.1 [M1H]¹; calcd for C₃₅H₃₄O₉N₆[M]:

682.6795 (average).

4.1. 3⁰-O-Acetyl-5⁰-O-dimethoxytrityl-1-[5-methyl-4-

triazolylpyrimidin-2(1H)-onyl]-b-d-2⁰-deoxyriboside (1)

Triazole was suspended in dry CH₃CN (10 mL/mmol azole)

under argon atmosphere, and the mixture was cooled in an

ice bath. POCl₃(2 equiv.) and dry triethylamine (15 equiv.)

were subsequently added, and left to react for 30 min. Then,

a solution of 3⁰-O-acetyl-5⁰-O-dimethoxytritylthymidine in

CH₃CN (10 mL/mmol azole) was added dropwise through a

cannula. When the addition was ®nished, the ice bath was

removed and the mixture left to react overnight at room

temperature. The dark mixture was cooled in an ice bath,

and treated with 1 M aq. triethylammonium hydrogencarbo-

nate (5 mL). After 10 min, the suspension was concentrated

by partial elimination of the solvent (approx. 50%). AcOEt

(100 mL) was added, and the organic phase washed twice

with both 1 M aq. triethylammonium hydrogencarbonate

and brine. The organic solution was dried over Na₂SO₄,

®ltered to remove the dessicant and evaporated to dryness.

Triazolyl derivative 1 was obtained as a brown solid

(quantitative yield, 5±8 mmol scale). No further puri®cation

was needed.

4.2.2. 3-Bromotriazolyl derivative 3. Mp 94±968C; TLC

(CH₂Cl₂/MeOH 10:1): R_f0.75; ¹H NMR (200 MHz,

CDCl₃): d9.18 (s, 1H; H-triazolyl), 8.37 (s, 1H; H±C6),

7.40±7.20 (m, 9H; H-phenyl), 6.83 (d, ³J(H, H)9 Hz, 4H;

H-phenyl), 6.39 (m, 1H; H±C1⁰), 5.44 (m, 1H; H±C3⁰), 4.18

(m, 1H; H±C4⁰), 3.79 (s, 6H; CH₃O), 3.44 (m, 2H; H±C5⁰),

2.90 (m, 1H; H±C2⁰), 2.42 (m, 1H; H⁰±C2⁰), 2.10 (s, 3H;

CH₃CO), 2.00 (s, 3H; CH₃±C5); ¹³C NMR (50 MHz,

CDCl₃) d170.3 (OCOCH₃), 158.7 (Cphenyl), 157.1

(C4), 153.6 (C2), 146.9 (Ctriazolyl), 146.1 (Ctriazolyl),

143.9 (Cphenyl), 142.8 (C6), 135.0, 134.7 (Cphenyl),

129.9, 128.0, 127.9, 127.2, 113.3 (Cphenyl±H), 105.8

(C5), 87.5, 87.2, 85.1 (Cq, C1⁰, C4⁰), 74.9 (C3⁰), 63.4

(C5⁰), 55.3 (CH₃O), 39.6 (C2⁰), 21.0 (CH₃CO), 16.4

(CH₃±C5); LRMS (FAB, magic bullet, positive mode):

m/z: 850.4, 848.4 [M1Xe]¹, 756.4 [M1K]¹, 740.4, 738.4

[M1H]¹, 718.5, 716.4, 715.4 [M1H]¹; calcd for

C₃₅H₃₄O₇N₅Br [M]: 716.5780 (average).

Mp 103±1058C; TLC (CH₂Cl₂/MeOH 10:1): R_f0.75 (blue

1

¯uorescent spot); H NMR (200 MHz, CDCl₃): d9.30 (s,

1H; H-triazolyl), 8.32 (2, 1H; H-6), 8.09 (s, 1H; H-triazo-

lyl); 7.40±7.20 (m, 9H; H-phenyl), 6.83 (d, ³J(H, H)9 Hz,

4H; H-phenyl), 6.42 (m, 1H; H±C1⁰), 5.41 (m, 1H; H±C3⁰),

4.15 (m, 1H; H±C4⁰), 3.78 (s, 6H; CH₃O), 3.47 (m, 2H;

H±C5⁰), 2.92 (m, 1H; H±C2⁰), 2.43 (m, 1H; H⁰±C2⁰),

2.10 (s, 3H; CH₃CO), 2.00 (s, 3H, CH₃±C5); ¹³C NMR

(50 MHz, CDCl₃) d170.2 (OCOCH₃), 158.6 (Cphenyl),

158.1 (C4), 153.6 (C2), 153.3 (Ctriazolyl), 146.4 (Ctriazo-

lyl), 144.9 (Cphenyl), 144.0 (C6), 135.0, 134.9 (Cphenyl),

129.9, 127.9, 127.8, 127.1, 113.2 (Cphenyl±H), 106.0 (C5),

87.3, 87.1, 84.9 (Cq, C1⁰, C4⁰), 74.9 (C3⁰), 63.6 (C5⁰), 55.2

(CH₃O), 39.6 (C2⁰), 21.0 (CH₃CO), 16.5 (CH₃±C5); LRMS

(FAB, magic bullet, Xe, positive mode): m/z: 661.2, 660.2

[M1Na]¹, 639.2, 638.2 [M1H]¹; calcd for C₃₅H₃₅O₇N₅

[M]: 637.6819 (average).

4.3. 3⁰-O-Acetyl-5⁰-O-dimethoxytrityl-1-[5-methyl-4-

phenoxy-pyrimidin-2(1H)-onyl]-b-d-2⁰-deoxyribosides

(4-nitrophenoxy derivative 4, 2-nitrophenoxy derivative

5,2,4-dichlorophenoxyderivative6,2,4,5-trichlorophenoxy

derivative7and2,3,4,5,6-pentachlorophenoxyderivative8)

4.3.1. General procedure. 3⁰-O-Acetyl-5⁰-O-dimethoxy-

tritylthymidine (0.6 g, 1.0 mmol) was dissolved in dry

CH₂Cl₂(10 mL) under inert atmosphere, and the ¯ask was

cooled in an ice bath. Triethylamine (0.7 mL, 5.0 mmol),

mesitylene chloride (0.3 g, 1.5 mmol) and dimethylamino-

pyridine (60 mg, 0.5 mmol) was added. After 10 min, the

bath was removed and the mixture left to react for 6 h at

room temperature. The reaction ¯ask was cooled again in an

ice bath, and triethylamine (0.85 mL, 6.0 mmol), DBU

(0.1 mL, 0.8 mmol) and the corresponding phenol (4-nitro-

phenol, 2-nitrophenol, 2,4-dichlorophenol, 2,4,5-trichloro-

phenol or 2,3,4,5,6-pentachlorophenol, 5 mmol) was added.

After 5 min, the ice bath was removed and the mixture left

to react for 3 h, after which time the reaction ¯ask was

cooled in an ice bath and 10% aqueous NaHCO₃was

added to quench the reaction. After 10 min, the solvents

were removed by evaporation under vacuum, and the corre-

sponding crude puri®ed by silica gel column chromato-

graphy using AcOEt/hexane/triethylamine 50:50:1 as

4.2. 3⁰-O-Acetyl-5⁰-O-dimethoxytrityl-1-[4-azolyl-5-

methylpyrimidin-2(1H)-onyl]-b-d-2⁰-deoxyribosides (3-

nitrotriazolyl derivative 2, 3-bromotriazolyl derivative 3)

A similar procedure was used to obtain derivative 1, except

for carrying out the reaction with 2.5 equiv. of azole (3-

nitrotriazole⁴³or 3-bromotriazole⁴⁴), 5 equiv. of triethyl-

amine, and diphenylphosphochloridate (2 equiv.) instead

of POCl₃. 3-Nitrotriazolyl derivative 1 was obtained as

dark brown solid (85% yield, 3 mmol scale), and 3-bromo-

triazole derivative 2 as a light brown solid (65% yield,

3 mmol scale). No further puri®cation was necessary.

J. Robles et al. / Tetrahedron 57 (2001) 179±194

187

eluent. The desired fractions were pooled and the solvent

removed under vacuum to yield the target product as a solid.

4.3.5. Trichlorophenoxy derivative 7. White solid, 70%

yield; mp 112±1148C; TLC (AcOEt/hexane 2:1): R_f

1

0.50; H NMR (200 MHz, CDCl₃): d8.10 (s, 1H; H±

4.3.2. 4-Nitrophenoxy derivative 4. Yellow solid, 60%

yield; mp 105±1078C; TLC (AcOEt/hexane 2:1):

C6), 7.50, 7.08 (s, s, 2H; H-chlorophenyl), 7.40±7.20 (m,

3

9H; H-phenyl), 6.85 (d, J(H, H)9 Hz, 4H; H-phenyl),

1

3

R_f0.45; H NMR (200 MHz, CDCl₃): d8.28 (d, J(H,

H)9 Hz; H-nitrophenyl), 8.11 (s, 1H; H±C6), 7.40±7.20

(m, 11H; H-phenyl, H-nitrophenyl), 6.85 (d, ³J(H,

H)9 Hz, 4H; H-phenyl), 6.40 (m, 1H; H±C1⁰), 5.42 (m,

1H; H±C3⁰), 4.11 (m, 1H; H±C4⁰), 3.81 (s, 6H; CH₃O), 3.43

(m, 2H; H±C5⁰), 2.70 (m, 1H; H±C2⁰), 2.34 (m, 1H; H⁰±

C2⁰), 2.08 (s, 3H; CH₃CO), 1.67 (s, 3H, CH₃±C5); ¹³C NMR

(50 MHz, CDCl₃) d170.3 (OCOCH₃), 169.4 (C4), 158.6

(Cphenyl), 156.4 (Cnitrophenyl), 154.8 (C2), 145.3 (C6),

144.1 (Cphenyl), 142.0 (Cnitrophenyl), 135.2, 135.1 (Cphe-

nyl), 130.0, 128.0, 127.9, 127.1, 113.3 (Cphenyl±H), 125.3,

122.7 (Cnitrophenyl±H), 104.4 (C5), 87.1, 85.5, 84.2 (Cq,

C1⁰, C4⁰), 75.0 (C3⁰), 63.5 (C5⁰), 55.3 (CH₃O), 39.3 (C2⁰),

21.0 (CH₃CO), 11.8 (CH₃±C5); LRMS (FAB, magic bullet,

Xe, positive mode): m/z: 841.4, 840.4 [M1Xe]¹, 731.5,

730.5 [M1Na]¹, 707.5, 708.6 [M1H]¹, calcd for

C₃₉H₃₇O₁₀N₃[M]: 707.7253 (average).

6.40 (m, 1H; H±C1⁰), 5.40 (m, 1H; H±C3⁰), 4.10 (m, 1H;

H±C4⁰), 3.81 (s, 6H; CH₃O), 3.46 (m, 2H; H±C5⁰), 2.70 (m,

1H; H±C2⁰), 2.33 (m, 1H; H⁰±C2⁰), 2.08 (s, 3H; CH₃CO),

1.68 (s, 3H; CH₃±C5); ¹³C NMR (50 MHz, CDCl₃)

d170.4 (OCOCH₃), 169.3 (C4), 158.6 (Cphenyl), 155.0

(C2), 146.7 (C6), 144.1 (Cphenyl), 142.0 (Cchlorophenyl),

135.2, 135.1 (Cphenyl), 131.0, 129.9, 128.0, 127.2, 125.5,

117.8, 113.3 (Cphenyl±H, Cchlorophenyl±H), 104.3 (C5),

87.1, 86.5, 84.8 (Cq, C1⁰, C4⁰), 75.0 (C3⁰), 63.5 (C5⁰), 55.3

(CH₃O), 39.3 (C2⁰), 21.0 (CH₃CO), 11.7 (CH₃±C5); LRMS

(FAB, magic bullet, positive mode): m/z: 899.2, 897.3

[M1Xe]¹, 791.3, 789.4, 787.4 [M1Na]¹, 766.3, 764.2

[M1H]¹, calcd for C₃₉H₃₅O₈N₂Cl₃[M]: 766.0620 (average).

4.3.6. Pentachlorophenoxy derivative 8. White solid, 55%

yield; mp 87±898C; TLC (AcOEt/hexane 2:1): R_f0.60; ¹H

NMR (200 MHz, CDCl₃): d8.13 (s, 1H; H±C6), 7.40±

3

7.20 (m, 9H, H-phenyl), 6.85 (d, J(H, H)9 Hz, 4H; H-

4.3.3. 2-Nitrophenoxy derivative 5. Yellow solid, 40%

yield; mp 98±1008C; TLC (AcOEt/hexane 2:1): R_f0.30;

phenyl), 6.40 (m, 1H; H±C1⁰), 5.42 (m, 1H; H±C3⁰), 4.10

(m, 1H; H±C4⁰), 3.81 (s, 6H; CH₃O), 3.46 (m, 2H; H±C5⁰),

2.75 (m, 1H; H±C2⁰), 2.38 (m, 1H; H⁰±C2⁰), 2.08 (s, 3H;

CH₃CO), 1.71 (s, 3H; CH₃±C5); ¹³C NMR (50 MHz,

CDCl₃) d170.3 (OCOCH₃), 168.9 (C4), 158.6 (Cphenyl),

154.6 (C2), 144.1 (C2), 142.4 (Cchlorophenyl), 135.2, 135.1

(Cphenyl), 129.9, 128.0, 127.2, 113.3 (Cphenyl±H), 103.7

(C5), 87.1, 85.6, 84.5 (Cq, C1⁰, C4⁰), 75.0 (C3⁰), 63.5 (C5⁰),

55.3 (CH₃O), 39.3 (C2⁰), 21.0 (CH₃CO), 11.6 (CH₃±C5);

LRMS (FAB, magic bullet, positive mode): m/z: 969.0,

967.1, 965.2 [M1Xe]¹, 861.3, 859.3, 857.3, 855.2

[M1Na]¹, 834.3 [M1H]¹, calcd for C₃₉H₃₃O₈N₂Cl₅[M]:

834.9515 (average).

3

¹H NMR (200 MHz, CDCl₃): d8.18 (dd, J(H, H)7 Hz,

1 Hz; H-nitrophenyl), 8.12 (s, 1H; H±C6), 7.65 (m, 1H;

nitrophenyl), 7.40±7.20 (m, 11H; H-phenyl, H-nitrophe-

3

nyl), 6.87 (d, J(H, H)9 Hz, 4H; H-phenyl), 6.40 (m,

1H; H±C1⁰), 5.40 (m, 1H; H±C3⁰), 4.20 (m, 1H; H±C4⁰),

3.81 (s, 6H; CH₃O), 3.46 (m, 2H; H±C5⁰), 2.70 (m, 1H; H±

C2⁰), 2.37 (m, 1H; H⁰±C2⁰), 2.10 (s, 3H; CH₃CO), 1.70 (s,

3H; CH₃±C5); ¹³C NMR (50 MHz, CDCl₃) d170.3

(OCOCH₃), 169.8 (C4), 158.6 (Cphenyl), 154.8 (C2),

145.3 (C6), 144.1 (Cphenyl), 141.7 (Cnitrophenyl), 135.2,

134.9 (Cphenyl, Cnitrophenyl), 130.0, 128.0, 127.9, 127.1,

113.3 (Cphenyl±H), 126.5, 126.1, 125.7 (Cnitrophenyl±H),

104.6 (C5), 87.1, 86.5, 84.4 (Cq, C1⁰, C4⁰), 75.2 (C3⁰), 63.5

(C5⁰), 55.3 (CH₃O), 39.2 (C2⁰), 21.0 (CH₃CO), 11.8

(CH₃±C5); LRMS (FAB, magic bullet, positive mode):

m/z: 841.4, 840.4 [M1Xe]¹, 731.6, 730.5 [M1Na]¹,

708.7, 707.5 [M1H]¹, calcd for C₃₉H₃₇O₁₀N₃[M]:

707.7253 (average).

4.4. 3⁰-O-Acetyl-5⁰-O-dimethoxytrityl-1-[4-guanidine-5-

methyl-pyrimidin-2(1H)-onyl]-b-d-2⁰-deoxyriboside (9)

4.4.1. Experiments of Table 1. Approx. 0.03 mmol of deri-

vatives 1±8 were dissolved in a small vial in 300 mL of dry

dimethylformamide (or dimethylformamide/triethylamine)

and treated with guanidine hydrochloride (15 mg) and the

corresponding mixture of reagents (see Table 1 for details).

The reaction was monitored by TLC analysis (CH₂Cl₂/

MeOH 10:1).

4.3.4. Dichlorophenoxy derivative 6. White solid, 65%

yield; mp 88±908C; TLC (AcOEt/hexane 2:1): R_f0.45;

¹H NMR (200 MHz, CDCl₃): d8.12 (s, 1H; H±C6), 7.42

(dd, ³J(H, H)7 Hz, 1 Hz; H-chlorophenyl), 7.40±7.20 (m,

3

11H; H-phenyl, H-chlorophenyl), 6.85 (d, J(H, H)9 Hz,

4.4.2. From nucleoside 1. Nucleoside (0.50 g, 0.8 mmol)

was dissolved in dry dimethylformamide (10 mL). K₂CO₃

(0.32 g, 2.4 mmol) and guanidine hydrochloride (0.15 g,

1.6 mmol) were added, and the corresponding mixture was

heated in an external bath at 1008C. The reaction was

completed in 3 h according to TLC analysis. The mixture

was left to cool at ambient temperature and ®ltered to

remove the insoluble residue. The solid was further washed

with CHCl₃and all the ®ltrates were pooled. The solvent

was removed by evaporation in vacuo to obtain a residue

which was puri®ed by silica gel column chromatography

eluting with CH₂Cl₂/MeOH/Et₃N (100:5:1). The desired

fractions were pooled and evaporated to dryness to yield a

white solid (0.35 g, 70% yield). Mp 1808C (decomp.); TLC

(CH₂Cl₂/MeOH 100:10): R_f0.3 (blue ¯uorescent spot); ¹H

4H; H-phenyl), 6.42 (m, 1H; H±C1⁰), 5.42 (m, 1H; H±C3⁰),

4.11 (m, 1H; H±C4⁰), 3.81 (s, 6H; CH₃O), 3.44 (m, 2H;

H±C5⁰), 2.70 (m, 1H; H±C2⁰), 2.37 (m, 1H; H⁰±C2⁰),

2.08 (s, 3H; CH₃CO), 1.71 (s, 3H; CH₃±C5); ¹³C NMR

(50 MHz, CDCl₃) d170.3 (OCOCH₃), 169.3 (C4), 158.6

(Cphenyl), 155.0 (C2), 149.6 (C6), 144.1 (Cphenyl), 141.5

(Cchlorophenyl), 135.2, 135.1 (Cphenyl), 131.7, 129.9,

128.0, 124.8, 113.3 (Cphenyl±H, Cchlorophenyl±H),

104.3 (C5), 87.1, 85.4, 84.4 (Cq, C1⁰, C4⁰), 75.1 (C3⁰),

63.5 (C5⁰), 55.3 (CH₃O), 39.2 (C2⁰), 21.0 (CH₃CO), 11.8

(CH₃±C5); LRMS (FAB, magic bullet, positive mode): m/z:

865.4, 863.4 [M1Xe]¹, 755.4, 753.4, [M1Na]¹, 732.8,

731.5 [M1H]¹, calcd for C₃₉H₃₆O₈N₂Cl₂[M]: 731.6172

(average).

188

J. Robles et al. / Tetrahedron 57 (2001) 179±194

NMR (200 MHz, CDCl₃): d7.60 (s, 1H; H±C6), 7.40±

7.20 (m, 9H; H-phenyl), 6.82 (d, ³J(H, H)9 Hz, 4H;

H-phenyl), 6.46 (m, 1H; H±C1⁰), 5.38 (m, 1H; H±C3⁰),

4.17 (m, 1H; H±C4⁰), 3.78 (s, 6H; CH₃O), 3.45 (m, 2H;

H±C5⁰), 2.45 (m, 2H; H±C2⁰), 2.07 (s, 3H; CH₃CO), 1.55

(s, 3H; CH₃±C5); ¹³C NMR (50 MHz, CDCl₃) d170.3

(OCOCH₃), 169.0 (Cguanid), 160.3 (C4), 158.5 (Cphenyl),

156.0 (C2), 144.2 (Cphenyl), 135.5 (C6), 134.3 (Cphenyl),

130.0, 128.0, 127.9 127.0, 113.4 (Cphenyl±H), 110.5 (C5),

86.9, 85.5, 83.8 (Cq, C1⁰, C4⁰), 75.3 (C3⁰), 63.6 (C5⁰), 55.2

(CH₃O), 38.6 (C2⁰), 21.0 (CH₃CO), 13.0 (CH₃±C5); IR

mode): m/z (%): calcd for C₃₃H₃₅O₈N₅P [M1H]¹:

660.2223, found 660.2217 (47).

4.6. 3⁰-O-t-Butyldimethylsilyl-5⁰-O-dimethoxytrityl-1-[5-

methyl-4-triazolylpyrimidin-2(1H)-onyl]-b-d-2⁰-deoxy-

riboside (11)

5⁰-Dimethoxytritylthymidine (5.7 g, 10.4 mmol) was

dissolved in dry pyridine (50 mL) under inert atmosphere.

To this solution, imidazole (2.4 g, 15.6 mmol) and t-butyl-

dimethylsilyl chloride (1.1 g, 15.6 g) was added. When the

initial nucleoside was completely consumed according to

TLC analysis (approx. 6 h), the excess of silylating agent

was hydrolysed by addition of water (5 mL). After 10 min,

the solvents were removed by evaporation. The residue was

partitioned between AcOEt (200 mL) and 10% aq. tartaric

acid (200 mL). The organic phase was separated and

washed with 10% aq. tartaric acid (200 mL), 10% aq.

NaHCO₃and brine. The organic solution was dried over

Na₂SO₄and the solvent was removed by evaporation in

vacuo to yield a white solid (6.4 g, 97% yield), which was

further used without puri®cation. Mp 101±1028C; TLC

(CH₂Cl₂/MeOH 10:1): R_f0.65; ¹H NMR (200 MHz,

CDCl₃): d8.85 (s, 1H; H±N3), 7.64 (s, 1H; H±C6),

(KBr; only most signi®cant bands): (n3500±3200 (NH

Å

st), 1730 (OCvO st), 1675 (CvO st), 1605 (C±NH guan.

st) cm²¹; HRMS (FAB, magic bullet, positive mode): m/z

(%): calcd for C₃₄H₃₈O₇N₅[M1H]¹: 628.2771, found

628.2753 (100).

4.5. Triethylammonium5⁰-O-dimethoxytrityl-1-[5-methyl-

4-triazolylpyrimidin-2(1H)-onyl]-b-d-2⁰-deoxyriboside

3⁰-H-phosphonate (10)

Triazole (2.0 g, 29.3 mmol) was suspended in dry CH₃CN

(30 mL) under argon atmosphere, and the mixture was

cooled in an ice bath. PCl₃(0.80 mL, 9.0 mmol) and dry

triethylamine (6.3 mL, 45.0 mmol) were subsequently

added, and the mixture left to react for 30 min. Then, a

solution of 5⁰-dimethoxytritylthymidine (1.0 g, 1.8 mmol)

in CH₂Cl₂/CH₃CN (2:1, 30 mL) was added dropwise

through a canula. When the addition was ®nished, the ice

bath was removed and the mixture left to react overnight at

room temperature. The reaction ¯ask was cooled in an ice

bath and 1 M aq. triethylammonium hydrogencarbonate was

added (5 mL). After 10 min, the suspension was concen-

trated by partial elimination of solvent (approx. 50%).

AcOEt (100 mL) was added, and the corresponding organic

phase was washed twice with 1 M aq. triethylammonium

hydrogencarbonate (100 mL). The organic solution was

dried over Na₂SO₄and the solvent was removed in vacuo.

The residue was puri®ed by column chromatography (SiO₂)

using CH₂Cl₂/MeOH/Et₃N 100:5:1 as eluent. The desired

fractions were pooled, and after evaporation of the solvent

and coevaporation with dry CH₂Cl₂, a white solid was

obtained (0.87 g, 62% yield). Mp 168±1698C (decomp.);

TLC (CH₂Cl₂/MeOH/Et₃N 100:10:1): R_f0.45 (blue ¯uor-

3

7.45±7.25 (m, 9H; H-phenyl), 6.80 (d, J(H, H)9 Hz,

4H; H-phenyl), 6.33 (m, 1H; H±C1⁰), 4.54 (m, 1H;

H±C3⁰), 3.93 (m, 1H; H±C4⁰), 3.76 (s, 6H; CH₃O), 3.32

(m, 2H; H±C5⁰), 2.24 (m, 2H; H±C2⁰), 1.45 (s, 3H, CH₃±

C5), 0.80 (s, 9H, CH₃±CSi), 0.04, 0.06 (ss, 6H, CH₃±Si);

¹³C NMR (50 MHz, CDCl₃) d163.8 (C4), 158.6

(Cphenyl), 150.2 (C2), 144.2 (Cphenyl), 135.6, 135.4,

135.3 (C6, Cphenyl), 130.0, 128.0, 127.9 127.0, 115.2

(Cphenyl±H), 110.0 (C5), 86.7, 84.8 (Cq, C1⁰, C4⁰), 72.0

(C3⁰), 62.8 (C5⁰), 55.2 (CH₃O), 41.6 (C2⁰), 25.7 (CH₃±CSi),

18.0 (CqSi), 11.9 (CH₃±C5), 24.6, 24.8 (CH₃Si).

For the following reaction, the same procedure described for

the preparation of derivative 1 was used. After the reaction

and the corresponding work-up, the product was redissolved

in CH₂Cl₂and precipitated with diethylether/hexane (1:1,

200 mL). A light brown solid was obtained after ®ltration

and drying (5.4 g, 76% overall yield). TLC and NMR

analyses showed that the product was pure enough not to

be further puri®ed. Mp 96±988C; TLC (CH₂Cl₂/MeOH

10:1): R_f0.40 (blue ¯uorescent spot); ¹H NMR

(200 MHz, CDCl₃): d9.38 (s, 1H; H-triazolyl), 8.48 (s,

1H; H±C6), 8.23 (s, 1H; H-triazolyl), 7.45±7.22 (m, 9H;

H-phenyl), 6.85 (d, ³J(H, H)9 Hz, 4H; H-phenyl), 6.37 (m,

1H; H±C1⁰), 4.58 (m, 1H; H±C3⁰), 4.15 (m, 1H; H±C4⁰),

3.85 (s, 6H; CH₃O), 3.48 (m, 2H; H±C5⁰), 2.65 (m, 1H;

H±C2⁰), 2.38 (m, 1H; H⁰-C2⁰), 2.00 (s, 3H; H₃C±C5),

0.87 (s, 9H, CH₃±CSi), 0.15, 0.13 (ss, 6H, CH₃±Si); ¹³C

NMR (50 MHz, CDCl₃) d158.6 (Cphenyl), 158.0 (C4),

154.0 (C2), 153.2 (Ctriazolyl), 145.5 (Ctriazolyl), 145.0

(Cphenyl), 144.6 (C6), 135.1 (Cphenyl), 130.0, 128.0,

127.9 127.1, 113.2 (Cphenyl±H), 105.7 (C5), 87.5, 87.1,

86.8 (Cq, C1⁰, C4⁰), 70.6 (C3⁰), 62.0 (C5⁰), 55.2 (CH₃O),

42.3 (C2⁰), 26.7 (CH₃±CSi), 17.9 (CqSi), 16.5 (CH₃±C5),

24.6, 24.9 (CH₃Si); LRMS (FAB, magic bullet, Xe,

negative mode): m/z: 847.4 [M22H1Xe]², 788.1

[M23H12K]², 747.9, 747.1 [M22H1K]²; calcd for

C₃₉H₄₇O₆N₅Si [M]: 709.9061 (average).

1

escent spot); H NMR (200 MHz, CDCl₃): d9.30 (s, 1H;

H-triazolyl), 8.38 (s, 1H; H±C6), 8.07 (s, 1H, H-triazolyl),

2

7.45±7.20 (m, 9H; H-phenyl), 6.88 (d, J(H, P)620 Hz,

3

1H; H±P), 6.84 (d, J(H, H)9 Hz, 4H; H-phenyl), 6.39

(m, 1H; H±C1⁰), 5.05 (m, 1H; H±C3⁰), 4.18 (m, 1H; H±

C4⁰), 3.78 (s, 6H; CH₃O), 3.45 (m, 2H; H±C5⁰), 3.03 (q,

³J(H, H)7 Hz, 6H; CH₂ammonium), 2.43 (m, 1H; H±

3

C2⁰), 1.92 (s, 3H; CH₃±C5), 1.33 (t, J(H, H)7 Hz, 9H;

CH₃ammonium); ¹³C NMR (50 MHz, CDCl₃) d158.5

(Cphenyl), 158.0 (C4), 153.9 (C2), 153.2 (Ctriazolyl),

146.7 (Ctriazolyl), 144.9 (Cphenyl), 144.1 (C6), 135.2

(Cphenyl), 130.0, 128.0, 127.9 127.0, 113.2 (Cphenyl±H),

3

105.7 (C5), 87.3, 86.9, 85.9 (Cq, C1⁰, C4⁰), 72.8 (d, J(C,

P)4 Hz; C3⁰), 62.7 (C5⁰), 55.2 (CH₃O), 45.6 (CH₂ammo-

nium), 41.1 (C2⁰), 16.4 (CH₃±C5), 8.8 (CH₃ammonium);

³¹P NMR (121 MHz, CDCl₃) d3.54; IR (KBr; only most

signi®cant bands): n2630±2600, 2500 (PO±H st), 1065,

Å

1030 (PvO st) cm²¹; HRMS (FAB, magic bullet, positive

J. Robles et al. / Tetrahedron 57 (2001) 179±194

189

3

4.7. 3⁰-O-t-Butyldimetylsilyl-5⁰-O-dimethoxytrityl-1-[4-

guanidine-5-methylpyrimidin-2(1H)-onyl]-b-d-2⁰-deoxy-

ribosides (12a, 12b, 12c, 12d)

phenyl), 6.84 (d, J(H, H)9 Hz, 4H; H-phenyl), 6.44 (m,

1H; H±C1⁰), 4.52 (m, 1H; H±C3⁰), 4.02 (m, 1H; H±C4⁰),

3.86 (s, 6H; CH₃O), 3.40 (m, 2H; H±C5⁰), 3.17 (bs, 6H,

CH₃±N), 2.40, 2.25 (m, 2H; H±C2⁰), 1.73 (s, 3H; CH₃±

C5), 0.88 (s, 9H, CH₃±CSi), 0.07, 0.06 (bs, 6H, CH₃±Si);

¹³C NMR (50 MHz, CDCl₃) d169.1 (Cguanid), 159.0

(C4), 158.4 (Cphenyl), 156.1 (C2), 144.5 (Cphenyl), 137.7

(C6), 137.1 (Cphenyl), 130.0, 128.1, 127.8, 126.8, 113.1

(Cphenyl±H), 112.0 (C5), 86.4, 86.0, 85.5 (Cq, C1⁰, C4⁰),

71.6 (C3⁰), 62.6 (C5⁰), 55.2 (CH₃O), 41.9 (C2⁰), 29.7 (CH₃±

N), 25.7 (CH₃±CSi), 18.0 (CqSi), 14.5 (CH₃±C5), 24.7,

24.9 (CH₃Si); LRMS (FAB, magic bullet, Xe, negative

mode): m/z: 728.1, 727.0 [M2H]²; calcd for

C₄₀H₅₃O₆N₅Si [M]: 727.9644 (average).

Derivatives 12 were obtained using the same general proce-

dure described for derivative 9. Triazolyl compound 11 (2±

5 mmol) was dissolved in dry dimethylformamide (10 mL/

mmol nucleoside) under inert atmosphere. K₂CO₃(3 equiv.)

and the guanidine derivative (2 equiv.; guanidine´HCl for

12a, methylguanidine´HCl for 12b, 1,1-dimethylguani-

dine´H₂SO₄for 12c and 1,2-dimethylguanidine´HBr⁴⁵for

12d) was then added. The mixture was stirred in a bath at

1008C until the initial nucleoside was completely consumed

according to TLC analysis (6±12 h depending on the analo-

gue). The mixture was left to cool, and ®ltered to remove the

insoluble residue. The solid was further washed with CHCl₃

and all the ®ltrates were pooled. The solvents were ®nally

removed by evaporation in vacuo to obtain a residue which

was puri®ed by column chromatography (SiO₂) using

CH₂Cl₂/MeOH/Et₃N as eluent: 12a and 12d eluted with a

100:10:1 mixture, 12b with 100:4:1 and 12c with 100:3.1.

The corresponding product was obtained by evaporation of

solvents.

4.7.4. N,N⁰-Dimethylguanidine derivative 12d. White

solid, 70% yield; mp 85±888C; TLC (CH₂Cl₂/MeOH

1

10:1): R_f0.60; H NMR (200 MHz, CDCl₃): d8.30 (s,

1H; H±N3), 7.75 (s, 1H; H±C6), 7.50±7.32 (m, 9H; H-

3

phenyl), 6.85 (d, J(H, H)9 Hz, 4H; H-phenyl), 6.41 (m,

1H; H±C1⁰), 4.55 (m, 1H; H±C3⁰), 4.07 (m, 1H; H±C4⁰),

3.83 (s, 6H; CH₃O), 3.43 (m, 2H; H±C5⁰), 2.93 (bs, 6H;

CH₃±N), 2.42, 2.25 (m, 2H; H±C2⁰), 1.65 (s, 3H; CH₃±C5),

0.87 (s, 9H, CH₃±CSi), 0.06, 0.05 (bs, 6H, CH₃±Si); ¹³C

NMR (50 MHz, CDCl₃) d169.1 (Cguanid), 158.4 (Cphe-

nyl), 147.0 (C2), 142.2 (Cphenyl), 135.3 (C6, Cphenyl),

129.9, 127.9, 127.8, 126.8, 113.0 (Cphenyl±H), 112.0

(C5), 86.5, 86.4, 85.3 (Cq, C1⁰, C4⁰), 71.5 (C3⁰), 62.6

(C5⁰), 55.1 (CH₃O), 41.7 (C2⁰), 28.2 (CH₃±N), 25.6

(CH₃±CSi), 16.4 (CqSi), 14.5 (CH₃±C5), 24.7, 25.0

(CH₃Si); LRMS (FAB, magic bullet, Xe, negative mode):

m/z: 808.5, 806.5 [M2 3H12K]², 728.1, 727.0 [M2H]²;

calcd for C₄₀H₅₃O₆N₅Si [M]: 727.9644 (average).

4.7.1. Guanidine derivative 12a. White solid, 70% yield;

1

mp 128±1308C; TLC (CH₂Cl₂/MeOH 10:1): R_f0.25; H

NMR (200 MHz, CDCl₃): d7.61 (s, 1H; H±C6), 7.40±

3

7.15 (m, 9H; H-phenyl), 6.75 (d, J(H, H)9 Hz, 4H; H-

phenyl), 6.31 (m, 1H; H±C1⁰), 4.28 (m, 1H; H±C3⁰), 3.90

(m, 1H; H±C4⁰), 3.71 (s, 6H; CH₃O), 3.32 (m, 2H; H±C5⁰),

2.32, 2.15 (m, 2H; H±C2⁰), 1.55 (s, 3H, CH₃±C5), 0.75 (s,

9H, CH₃±CSi), 20.07, 20.13 (ss, 6H, CH₃±Si); ¹³C NMR

(50 MHz, CDCl₃) d170.4 (Cguanid), 161.0 (C4), 158.6

(Cphenyl), 155.6 (C2), 144.5 (Cphenyl), 136.0 (C6), 135.7

(Cphenyl), 130.1, 128.2, 127.8, 126.9, 113.2 (Cphenyl±H),

112.1 (C5), 86.6, 86.3, 85.6 (Cq, C1⁰, C4⁰), 71.9 (C3⁰), 62.8

(C5⁰), 55.3 (CH₃O), 41.9 (C2⁰), 25.7 (CH₃±CSi), 18.0

(CqSi), 11.6 (CH₃±C5), 24.7, 24.9 (CH₃Si); HRMS

(FAB, magic bullet, positive mode): m/z (%): calcd for

C₃₈H₅₀O₆N₅Si [M1H]¹: 700.3530, found 700.3554 (100).

4.8. 3⁰-O-t-Butyldimetylsilyl-5⁰-O-dimethoxytrityl-1-[5-

methyl-4-(4-nitrobenzoyl)guanidine-pyrimidin-2(1H)-

onyl]-b-d-2⁰-deoxyriboside (13)

Guanidine derivative 12a (0.7 g, 1 mmol), previously dried

by coevaporation with dry CH₃CN, was dissolved in dry

pyridine (10 mL) under inert atmosphere. 4-Nitrobenzoyl

chloride (0.4 g, 2.2 equiv.) was added to the solution. The

initial nucleoside was completely consumed in 2 h accord-

ing to TLC analysis. The solution was cooled in an ice bath,

and 10% aq. NaHCO₃(2 mL) was added to hydrolyse the

excess of acid chloride. After 10 min, solvent was removed

by evaporation. The residue was partitioned between AcOEt

(100 mL) and 10% aq. NaHCO₃(100 mL). The organic

phase was separated and washed again with 10% aq.

NaHCO₃(100 mL), 10% aq. tartaric acid (100 mL) and

brine. The organic solution was dried over Na₂SO₄and

the solvent removed. The resulting residue was puri®ed by

silica gel column chromatography using CH₂Cl₂/MeOH/

Et₃N 100:1:1 as eluent. Solvent elimination rendered the

product as a bright yellow solid (0.45 g, 75% yield). Mp

4.7.2. N-Methylguanidine derivative 12b. White and

hygroscopic solid, 60% yield; mp 125±1278C; TLC

(CH₂Cl₂/MeOH 10:1): R_f0.30; ¹H NMR (200 MHz,

CDCl₃): d7.68 (s, 1H; H±C6), 7.46±7.25 (m, 9H; H-phen-

3

yl), 6.80 (d, J(H, H)9 Hz, 4H; H-phenyl), 6.43 (m, 1H;

H±C1⁰), 4.45 (m, 1H; H±C3⁰), 4.12 (m, 1H; H±C4⁰), 3.85

(s, 6H; CH₃O), 3.42 (m, 2H; H±C5⁰), 2.98 (bs, 3H; CH₃±N),

2.45, 2.13 (m, 2H; H±C2⁰), 1.70 (s, 3H; CH₃±C5), 0.88 (s,

9H, CH₃±CSi), 0.07 (bs, 6H, CH₃±Si); ¹³C NMR (50 MHz,

CDCl₃) d170.0 (Cguanid), 160.5 (C4), 158.3 (Cphenyl),

156.2 (C2), 143.6 (Cphenyl), 135.5 (C6, Cphenyl), 129.9,

128.0, 127.7, 127.0, 113.0 (Cphenyl±H), 112.0 (C5), 86.4,

86.1, 85.6 (Cq, C1⁰, C4⁰), 71.5 (C3⁰), 62.8 (C5⁰), 55.1

(CH₃O), 42.0 (C2⁰), 28.2 (CH₃±N), 25.6 (CH₃±CSi), 18.1

(CqSi), 11.4 (CH₃±C5), 24.5, 24.8 (CH₃Si); LRMS (FAB,

3-amino-1,2-propanodiol, Xe, negative mode): m/z: 712.6

[M2H]²; calcd for C₃₉H₅₁O₆N₅Si [M]: 713.9378 (average).

1

126±1288C; TLC (CH₂Cl₂/MeOH 100:10): R_f0.80; H

NMR (300 MHz, CDCl₃): d8.45±8.20 (m, 5H; H-nitro-

phenyl, H±C6), 7.50±7.28 (m, 9H; H-phenyl), 6.90 (d, ²J(H,

H)9 Hz, 4H; H-phenyl), 6.38 (m, 1H; H±C1⁰), 4.60 (m,

1H; H±C3⁰), 4.05 (m, 1H; H±C4⁰), 3.85 (s, 6H; CH₃O), 3.45

(m, 2H; H±C5⁰), 2.60, 2.36 (m, 2H; H±C2⁰), 1.67 (s, 3H,

CH₃±C5), 0.87 (s, 9H, CH₃±CSi), 0.07, 0.05 (bs, 6H, CH₃±

Si); ¹³C NMR (75 MHz, CDCl₃) d158.8 (Cphenyl), 148.9

4.7.3. N,N-Dimethylguanidine derivative 12c. White

solid, 55% yield; mp 126±1288C; TLC (CH₂Cl₂/MeOH

1

10:1): R_f0.60; H NMR (200 MHz, CDCl₃): d8.08 (s,

1H; H±N3), 7.65 (s, 1H; H±C6), 7.48±7.25 (m, 9H; H-

190

J. Robles et al. / Tetrahedron 57 (2001) 179±194

(C6), 144.3 (Cphenyl), 135.3 (Cphenyl), 130.1, 128.2,

128.0, 127.2, 113.3 (Cphenyl±H), 129.7, 123.7, 123.2

(Cnitrophenyl±H), 86.8, 86.7 (Cq, C1⁰, C4⁰), 71.8 (C3⁰),

63.2 (C5⁰), 55.2 (CH₃O), 42.2 (C2⁰), 25.6 (CH₃±CSi),

17.9 (CqSi), 11.6 (CH₃±C5), 24.7, 25.0 (CH₃Si); LRMS

(FAB, magic bullet, Xe, negative mode): m/z: 848.6, 847.6

[M2H]²; calcd for C₄₅H₅₂O₉N₆Si [M]: 849.0149 (average).

H±C3⁰), 4.20, 4.17 (m, 1H; H±C4⁰), 3.76, 3.75 (s, s, 6H;

CH₃O), 3.60±3.45, 3.35 (m, 5H; H±C5⁰, H±C iPr, CH₂OP),

2.70 (m, 1H; H±C2⁰), 2.59 (m, 1H; H±CHCN), 2.42±2.25

(m, 2H; H±CHCN, H±C2⁰), 1.58 (bs, 3H; H₃C±C5), 1.15±

1.10 (m, 12H; CH₃iPr); ³¹P NMR (121 MHz, CDCl₃):

d149.3, 148.7; HRMS (FAB, magic bullet, positive

mode): m/z (%): calcd for C₄₈H₅₆O₁₀N₈P [M1H]¹:

935.3857, found 935.3813 (100).

4.9. 5⁰-O-Dimethoxytrityl-1-[5-methyl-4-(4-nitrobenzoyl)

guanidine-pyrimidin-2(1H)-onyl]-b-d-2⁰-deoxyriboside

(14)

4.11. 5⁰-O-Dimethoxytrityl-1-[4-guanidine-5-methyl-

pyrimidin-2(1H)-onyl]-b-d-2⁰-deoxyribosides (16a, 16b,

16c, 16d).

Nucleoside 13 (0.55 g, 0.6 mmol) was dissolved in dry tetra-

hydrofuran (10 mL) under inert atmosphere. Solid tetrabu-

tylammonium ¯uoride trihydrate (0.37 g, 1.2 mmol) was

added. When the initial nucleoside was completely

consumed according to TLC analysis (approx. 6 h),

MeOH was added (2 mL), and 10 min later the solvent

was removed by evaporation. The crude was directly puri-

®ed by column chromatography (SiO₂) using CH₂Cl₂/

MeOH/Et₃N 100:1:1 as eluent. After evaporation of the

solvent, a bright yellow solid was obtained (0.30 g, 68%

yield). Mp 146±1488C; TLC (CH₂Cl₂/MeOH 100:10):

4.11.1. General procedure to obtain derivatives 16.

Nucleoside 12 (1±4 mmol) was dissolved in dry tetra-

hydrofuran (10 mL/mmol nucleoside) under inert atmos-

phere, and solid tetrabutylammonium ¯uoride trihydrate

was added (2 equiv.). When the initial nucleoside was

completely consumed (TLC analysis, normally after 4±

6 h), MeOH was added (2 mL) and, after 10 min, the

solvent was removed by evaporation. The crude was

directly puri®ed by silica gel column chromatography

using CH₂Cl₂/MeOH/Et₃N as eluent: 16a eluted with a

100:10:1 mixture, 16b and 16c with 100:5:1, and 16d

with 100:7.1. The product was obtained after elimination

of the solvent.

1

R_f0.45; H NMR (300 MHz, CDCl₃): d8.35±8.05 (m,

5H; H-nitrophenyl, H±C6), 7.40±7.25 (m, 9H; H-phenyl),

6.85 (d, ³J(H, H)9 Hz, 4H; H-phenyl), 6.38 (m, 1H;

H±C1⁰), 4.60 (m, 1H; H±C3⁰), 4.18 (m, 1H; H±C4⁰), 3.70

(s, 6H; CH₃O), 3.42 (m, 2H; H±C5⁰), 2.70, 2.55 (m, 2H;

H±C2⁰), 1.68 (s, 3H; CH₃±C5); ¹³C NMR (75 MHz, CDCl₃)

d159.3 (Cnitrophenyl), 158.7 (Cphenyl), 149.8 (C6),

144.3 (Cphenyl), 135.3 (Cphenyl), 130.0, 128.1, 128.0,

127.2, 113.3 (Cphenyl±H), 123.2 (Cnitrophenyl±H), 86.9,

86.7 (Cq, C1⁰, C4⁰), 71.8 (C3⁰), 63.3 (C5⁰), 55.2 (CH₃O),

42.1 (C2⁰), 12.5 (CH₃±C5); HRMS (FAB, magic bullet,

positive mode): m/z (%): calcd for C₃₉H₃₉O₉N₆[M1H]¹:

735.2778, found 735.2772 (72).

4.11.2. Guanidine derivative 16a. White solid, 85% yield;

1

mp 160±1628C; TLC (CH₂Cl₂/MeOH 10:1): R_f0.20; H

NMR (300 MHz, CDCl₃): d7.65 (s, 1H; H±C6), 7.47±

3

7.20 (m, 9H; H-phenyl), 6.83 (d, J(H, H)9 Hz, 4H; H-

phenyl), 6.38 (m, 1H; H±C1⁰), 4.46 (m, 1H; H±C3⁰), 4.18

(m, 1H; H±C4⁰), 3.79 (s, 6H; CH₃O), 3.40 (m, 2H; H±C5⁰),

2.46 (m, 1H; H±C2⁰), 2.25 (m, 1H; H±C2⁰), 1.59 (s, 3H;

CH₃±C5); ¹³C NMR (50 MHz, CDCl₃) d170.6 (Cguanid),

160.9 (C4), 158.3 (Cphenyl), 156.6 (C2), 144.3 (Cphenyl),

136.2, (C6), 135.5, 135.4 (Cphenyl), 129.9, 127.9, 127.8,

126.6, 112.9 (Cphenyl±H), 112.1 (C5), 86.6, 86.1, 85.7

(Cq, C1⁰, C4⁰), 71.8 (C3⁰), 63.4 (C5⁰), 55.1 (CH₃O), 41.6

(C2⁰), 14.0 (CH₃±C5); IR (KBr; only most signi®cant

4.10. 5⁰-O-Dimethoxytrityl-1-[5-methyl-4-(4-nitrobenzoyl)

guanidine-pyrimidin-2(1H)-onyl]-b-d-2⁰-deoxyriboside

3⁰-O-cyanoethyl-N,N-diisopropylphosphoramidite (15)

bands): n3500±3200 (NH, OH st), 1685 (CvO st), 1610

Å

(C±NH guan. st) cm²¹; HRMS (FAB, magic bullet, positive

mode): m/z (%): calcd for C₃₂H₃₆O₆N₅[M1H]¹: 586.2665,

found 586.2692 (100).

Nucleoside 14 (0.20 g, 0.27 mmol), previously dried

by coevaporation with dry CH₃CN, was dissolved in

dry CH₂Cl₂(5 mL) under inert atmosphere. Then,

2-cyanoethoxy-bis(N,N-diisopropylamino)phosphine (100

mL, 0.32 mmol) and tetrazole (10 mg, 0.13 mmol) were

added. When the initial nucleoside (R_f0.15) was com-

pletely consumed, as shown by TLC analysis (approx.

1 h), MeOH (1 mL) was added to destroy the excess of

phosphine. After 10 min, the solvent was removed by

evaporation. The residue was dissolved in CH₂Cl₂

(50 mL), and washed with 10% aq. NaHCO₃(2£50 mL)

and brine. The solution was dried over Na₂SO₄, and the

solvent removed by evaporation. The product was puri®ed

by column chromatography (SiO₂) using CH₂Cl₂/hexane/

Et₃N (47.5:47.5:5) as eluent. Evaporation of solvents and

precipitation with cold hexane rendered the product

(mixture of two diastereomers) as a yellowish solid

(125 mg, 50% yield). Mp 129±1308C (decomp.); TLC

(CH₂Cl₂/AcOEt/triethylamine 45:45:10): R_f0.45, 0.40;

¹H NMR (300 MHz, CDCl₃): d8.35±8.05 (m, 5H;

H-nitrophenyl, H±C6), 7.40±7.20 (m, 9H; H-phenyl), 6.82

(m, 4H; H-phenyl), 6.35 (m, 1H; H±C1⁰), 4.63 (m, 1H;

4.11.3. N-Methylguanidine derivative 16b. White solid,

90% yield; mp 121±1228C; TLC (CH₂Cl₂/MeOH 10:1):

1

R_f0.20; H NMR (300 MHz, CDCl₃): d7.60 (s, 1H;

H±C6), 7.45±7.20 (m, 9H; H-phenyl), 6.82 (d, ³J(H,

H)9 Hz, 4H; H-phenyl), 6.44 (m, 1H; H±C1⁰), 4.55 (m,

1H; H±C3⁰), 4.08 (m, 1H; H±C4⁰), 3.77 (s, 6H; CH₃O), 3.40

(m, 2H; H±C5⁰), 2.87 (bs, 3H; CH₃±N), 2.44 (m, 1H; H±

C2⁰), 2.23 (m, 1H; H±C2⁰), 1.61 (bs, 3H; CH₃±C5); ¹³C

NMR (75 MHz, CDCl₃) d169.8 (Cguanid), 160.3 (C4),

158.6 (Cphenyl), 156.6 (C2), 144.6 (Cphenyl), 135.8,

135.7 (C6, Cphenyl), 130.1, 128.2, 127.9, 126.9, 113.3

(Cphenyl±H), 113.2 (C5), 86.7, 85.8, 85.6 (Cq, C1⁰, C4⁰),

72.3 (C3⁰), 63.9 (C5⁰), 55.2 (CH₃O), 41.8 (C2⁰), 29.8 (CH₃±

N), 14.1 (CH₃±C5); IR (KBr; only most signi®cant

bands): n3500±3200 (NH, OH st), 1680 (CvO st),

Å

1610 (C±NH guan. st) cm²¹; HRMS (FAB, magic bullet,

positive mode): m/z (%): calcd for C₃₃H₃₈O₆N₅[M1H]¹:

600.2822, found 600.2850 (45).

J. Robles et al. / Tetrahedron 57 (2001) 179±194

191

4.11.4. N,N-Dimethylguanidine derivative 16c. Yellowish

solid, 70% yield; mp 137±1398C; TLC (CH₂Cl₂/MeOH

4.12.2. Guanidine derivative 17a. White solid, 80% yield;

mp 118±1208C (decomp.); TLC (CH₂Cl₂/MeOH/Et₃N

1

10:1): R_f0.30; H NMR (300 MHz, CDCl₃): d7.60 (s,

100:10:1): R_f0.20; H NMR (300 MHz, CDCl₃): d7.71

3

1H; H±C6), 7.45±7.20 (m, 9H; H-phenyl), 6.82 (d, J(H,

(s, 1H; H±C6), 7.45±7.20 (m, 9H; H-phenyl), 6.88 (d, ²J(H,

3

H)9 Hz, 4H; H-phenyl), 6.56 (m, 1H; H±C1⁰), 4.56 (m,

1H; H±C3⁰), 4.10 (m, 1H; H±C4⁰), 3.78 (s, 6H; CH₃O), 3.38

(m, 2H; H±C5⁰), 3.07 (bs, 6H; CH₃±N), 2.52 (m, 1H;

H±C2⁰), 2.25 (m, 1H; H±C2⁰), 1.61 (bs, 3H; CH₃±C5);

¹³C NMR (75 MHz, CDCl₃) d168.4 (Cguanid), 159.3

(C4), 158.4 (Cphenyl), 156.2 (C2), 144.6 (Cphenyl), 135.6

(Cphenyl), 135.3 (C6), 130.0, 128.1, 127.9, 126.8, 113.1

(Cphenyl±H), 113.0 (C5), 86.8, 85.6, 85.5 (Cq, C1⁰, C4⁰),

72.4 (C3⁰), 64.1 (C5⁰), 55.2 (CH₃O), 41.8 (C2⁰), 26.9 (CH₃±

N), 14.2 (CH₃±C5); IR (KBr; only most signi®cant bands):

P)620 Hz, 1H; H±P), 6.83 (d, J(H, H)9 Hz, 4H; H-

phenyl), 6.42 (m, 1H; H±C1⁰), 4.93 (m, 1H; H±C3⁰), 4.25

(m, 1H; H±C4⁰), 3.78 (s, 6H; CH₃O), 3.41 (m, 2H; H±C5⁰),

2.83 (q, ³J(H, H)7 Hz, 6H; CH₂ammonium), 2.63 (m, 1H;

H±C2⁰), 2.35 (m, 1H; H±C2⁰), 1.54 (s, 3H; CH₃±C5), 1.05

(t, ³J(H, H)7 Hz, 9H; CH₃ammonium); ¹³C NMR

(75 MHz, CDCl₃) d167.0 (Cguanid), 159.1 (C4), 158.4

(Cphenyl), 155.2 (C2), 144.2 (Cphenyl), 138.1, (C6),

135.3 (Cphenyl), 129.8, 127.9, 127.7, 126.7, 112.7

(Cphenyl±H), 109.1 (C5), 86.6, 85.7, 85.1 (Cq, C1⁰, C4⁰),

73.4 (C3⁰), 63.1 (C5⁰), 55.0 (CH₃O), 45.3 (CH₂ammonium),

40.3 (C2⁰), 19.4 (CH₃±C5), 9.8 (CH₃ammonium); ³¹P NMR

(121 MHz, CDCl₃) d3.3; IR (KBr; only most signi®cant

n3500±3200 (NH, OH st), 1700 (CvO st), 1665, 1610

Å

(C±NH guan. st) cm²¹; HRMS (FAB, magic bullet, positive

mode): m/z (%): calcd for C₃₄H₄₀O₆N₅[M1H]¹: 614.2978,

found 614.2950 (100).

bands): n3400±3100 (NH, OH st), 2340 (PO±H st), 1680

Å

(CvO st), 1630 (C±NH guan. st), 1065 (PvO st) cm²¹

;

HRMS (FAB, magic bullet, positive mode): m/z (%): calcd

4.11.5. N,N⁰-Dimethylguanidine derivative 16d. White

solid, 70% yield; mp 149±1508C; TLC (CH₂Cl₂/MeOH

for C₃₂H₃₇O₈N₅P [M1H]¹: 650.2380, found 650.2385 (56).

1

10:1): R_f0.25; H NMR (300 MHz, CDCl₃): d7.60 (s,

3

1H; H±C6), 7.45±7.20 (m, 9H; H-phenyl), 6.82 (d, J(H,

4.12.3. N-Methylguanidine derivative 17b. White solid,

85% yield; mp 160±1628C (decomp.); TLC (CH₂Cl₂/

MeOH/Et₃N 100:10:1): R_f0.20; ¹H NMR (300 MHz,

CDCl₃): d7.85 (s, 1H; H±C6), 7.40±7.20 (m, 9H; H-phen-

H)9 Hz, 4H; H-phenyl), 6.43 (m, 1H; H±C1⁰), 4.51 (m,

1H; H±C3⁰), 4.13 (m, 1H; H±C4⁰), 3.78 (s, 6H; CH₃O), 3.42

(m, 2H; H±C5⁰), 2.92, 2.90 (s, s, 6H; CH₃±N), 2.52 (m, 1H;

H±C2⁰), 2.20 (m, 1H; H±C2⁰), 1.65 (bs, 3H; CH₃±C5); ¹³C

NMR (75 MHz, CDCl₃) d169.1 (Cguanid), 159.1 (C4),

158.3 (Cphenyl), 156.4 (C2), 144.5 (Cphenyl), 135.6

(Cphenyl), 135.3 (C6), 129.9, 128.0, 127.8, 126.8, 113.0

(Cphenyl±H), 112.6 (C5), 86.6, 85.7, 85.6 (Cq, C1⁰, C4⁰),

72.0 (C3⁰), 63.4 (C5⁰), 55.2 (CH₃O), 41.6 (C2⁰), 27.8

(CH₃±N), 14.2 (CH₃±C5); IR (KBr; only most signi®cant

2

3

yl), 6.84 (d, J(H, P)620 Hz, 1H; H±P), 6.80 (d, J(H,

H)9 Hz, 4H; H-phenyl), 6.28 (m, 1H; H±C1⁰), 4.95 (m,

1H; H±C3⁰), 4.23 (m, 1H; H±C4⁰), 3.75 (s, 6H; CH₃O), 3.42

(m, 2H; H±C5⁰), 3.01 (q, ³J(H, H)7 Hz, 6H; CH₂ammo-

nium), 2.95 (bs, 3H; CH₃±N), 2.66 (m, 1H; H±C2⁰), 2.37

(m, 1H; H±C2⁰), 1.60 (s, 3H; CH₃±C5), 1.27 (t, ³J(H, H)

7 Hz, 9H; CH₃ammonium); ¹³C NMR (75 MHz, CDCl₃)

d163.7 (Cguanid), 158.6 (Cphenyl), 156.5 (C2), 144.4

(Cphenyl), 135.8, 135.4 (C6, Cphenyl), 130.1, 128.2,

127.9, 127.1, 113.2 (Cphenyl±H), 111.1 (C5), 86.7, 85.6,

85.0 (Cq, C1⁰, C4⁰), 73.9 (C3⁰), 63.2 (C5⁰), 55.2 (CH₃O),

45.7 (CH₂ammonium), 40.5 (C2⁰), 28.3 (CH₃±N), 12.3

(CH₃±C5), 9.0 (CH₃ammonium); ³¹P NMR (121 MHz,

CDCl₃) d3.2; IR (KBr; only most signi®cant bands):

bands): n3500±3200 (NH, OH st), 1680 (CvO st), 1620

Å

(C±NH guan. st) cm²¹; HRMS (FAB, magic bullet, positive

mode): m/z (%): calcd for C₃₄H₄₀O₆N₅[M1H]¹: 614.2978,

found 614.2983 (100).

4.12. Triethylammonium 5⁰-O-dimethoxytrityl-1-[4-

guanidine-5-methylpyrimidin-2(1H)-onyl]-b-d-2⁰-deoxy-

riboside 3⁰-H-phosphonate (17a, 17b, 17c, 17d)

n3500±2900 (NH, OH st), 2365 (PO±H st), 1700 (CvO

Å

st), 1620 (C±NH guan. st), 1080 (PvO st) cm²¹; HRMS

(FAB, magic bullet, positive mode): m/z (%): calcd for

C₃₃H₃₉O₈N₅P [M1H]¹: 664.2536, found 664.2507 (100).

4.12.1. General procedure to obtain derivatives 17.

Nucleoside 16 (0.5±1 mmol), previously dried by coeva-

poration with dry CH₃CN, was dissolved in dry pyridine

(10 mL/mmol) under inert atmosphere. Diphenylphosphite

(10 equiv.) was then added. The reaction, according to TLC

analysis, was normally completed in 45±60 min. The reac-

tion mixture was then cooled in an ice bath, and 1 M aq.

triethylammonium hydrogencarbonate was added (2±5 mL).

After 30 min, the solvents were removed by evaporation.

The residue was redissolved in CHCl₃(50 mL) and washed

with 1 M aq. triethylammonium hydrogencarbonate (50 mL).

The organic phase was set apart, and the aqueous phase was

reextracted with CHCl₃(50 mL) until TLC analysis showed

no nucleoside in the aqueous solution. The organic phases

were pooled, dried over Na₂SO₄and the solvent was

removed by evaporation. The crude was puri®ed by column

chromatography (SiO₂) using CH₂Cl₂/MeOH/triethylamine

100:20:2 as eluent. The desired products were obtained after

evaporation of the solvent and precipitation with diethy-

lether/hexane 1:1.

4.12.4. N,N-Dimethylguanidine derivative 17c. White solid,

85% yield; mp 150±1528C (decomp.); TLC (CH₂Cl₂/MeOH/

Et₃N 100:10:1): R_f0.25; ¹H NMR (300 MHz, CDCl₃):

d7.55 (s, 1H; H±C6), 7.55±7.25 (m, 9H; H-phenyl), 6.83

(d, ²J(H, P)620 Hz, 1H; H±P), 6.82 (d, ³J(H, H) 9 Hz, 4H;

H-phenyl), 6.28 (m, 1H; H±C1⁰), 4.92 (m, 1H; H±C3⁰), 4.22

(m, 1H; H±C4⁰), 3.78 (s, 6H; CH₃O), 3.38 (m, 2H; H±C5⁰),

3

3.09 (bs, 6H; CH₃-N), 3.04 (q, J(H, H)7 Hz, 6H; CH₂

ammonium), 2.63 (m, 1H; H±C2⁰), 2.35 (m, 1H; H±C2⁰),

3

1.50 (s, 3H; CH₃±C5), 1.32 (t, J(H, H)7 Hz, 9H; CH₃

ammonium); ¹³C NMR (75 MHz, CDCl₃) d168.7(Cguanid),

158.6 (C4), 158.4 (Cphenyl), 156.5 (C2), 144.4 (Cphenyl),

135.6, 135.4 (C6, Cphenyl), 130.1, 128.1, 127.8, 126.8,

113.1 (Cphenyl±H), 111.8 (C5), 86.7, 85.2, 85.1 (Cq, C1⁰,

C4⁰), 73.8 (C3⁰), 63.6 (C5⁰), 55.2 (CH₃O), 45.5 (CH₂

ammonium), 40.3 (C2⁰), 27.0 (CH₃±N), 14.2 (CH₃±C5),

8.6 (CH₃ammonium); ³¹P NMR (121 MHz, CDCl₃)

d3.7; IR (KBr; only most signi®cant bands): n3500±

Å

192

J. Robles et al. / Tetrahedron 57 (2001) 179±194

3000 (NH, OH st), 2380 (PO±H st), 1675 (CvO st), 1605

(C±NH guan. st), 1060 (PvO st) cm²¹; HRMS (FAB,

magic bullet, positive mode): m/z (%): calcd for

C₃₄H₄₁O₈N₅P [M1H]¹: 678.2693, found 678.2660 (100).

and treated with the corresponding solution (2 mL was

generally used) for 2 h. The mixture was ®ltered through

glass wool to remove the glass beads, which were washed

with water (2 mL). The ®ltrates were then concentrated by

evaporation in vacuo to 1±2 mL. If these solutions were not

clear, ®ltering through 0.45 mm nylon ®lters was carried

out.

4.12.5. N,N⁰-Dimethylguanidine derivative 17d. White

solid, 76% yield; mp 118±1208C (decomp.); TLC (CH₂Cl₂/

MeOH/Et₃N 100:10:1): R_f0.25; ¹H NMR (300 MHz,

CDCl₃): d7.58(s, 1H; H±C6), 7.55±7.20 (m, 9H; H-phenyl),

6.89 (d, ²J(H, P)620 Hz, 1H; H±P), 6.82 (d, ³J(H,

H)9 Hz, 4H; H-phenyl), 6.48 (m, 1H; H±C1⁰), 4.92 (m,

1H; H±C3⁰), 4.27 (m, 1H; H±C4⁰), 3.78 (s, 6H; CH₃O), 3.40

4.16. Analysis, puri®cation and characterization

Reversed-phase HPLC analyses of oligonucleotides were

carried out on Nucleosil C18 columns (25£0.5 cm,

10 mm), using linear gradients of 0.01 M aq. triethylammo-

nium acetate (solvent A) and 1:1 CH₃CN/H₂O (solvent B),

(¯ow: 1 mL/min, detection wavelength: 260 nm). Trimers

TXT were analysed using a linear gradient from 15 to 35%

of B in 20 min (gradient A), and 15-mers with either

gradient A or a gradient from 15 to 75% of B in 30 min

(gradient B). Analyses of 15-mers by gel electrophoresis

were run under denaturing conditions (7 M urea), on 20%

polyacrylamide gels, at 500±750 V for 3±4 h). Reaction

with `stain-all' dye (Sigma) was used to detect the products

in the gel.

3

(m, 2H; H±C5⁰), 2.97 (q, J(H, H)7 Hz, 6H; CH₂ammo-

nium), 2.90 (bs, 6H; CH₃±N), 2.65 (m, 1H; H±C2⁰), 2.30

3

(m, 1H; H±C2⁰), 1.58 (s, 3H; CH₃±C5), 1.28 (t, J(H,

H)7 Hz, 9H; CH₃ammonium); ¹³C NMR (75 MHz,

CDCl₃) d166.4 (Cguanid), 159.8 (C4), 158.4 (Cphenyl),

144.4 (Cphenyl), 135.6 (Cphenyl), 135.2 (C6), 130.1, 128.1,

127.8, 126.8, 113.1 (Cphenyl±H), 111.8 (C5), 86.6, 85.4,

85.1 (Cq, C1⁰, C4⁰), 74.0 (C3⁰), 63.5 (C5⁰), 55.2 (CH₃O),

45.6 (CH₂ammonium), 40.2 (C2⁰), 27.9 (CH₃±N), 14.0

(CH₃±C5), 9.0 (CH₃ammonium); ³¹P NMR (121 MHz,

CDCl₃) d3.6; IR (KBr; only most signi®cant bands):

n3500±3000 (NH, OH st), 2340 (PO±H st), 1700 (CvO

Å

st), 1615 (C±NH guan. st), 1070 (PvO st) cm²¹; HRMS

(FAB, magic bullet, positive mode): m/z (%): calcd for

C₃₄H₄₁O₈N₅P [M1H]¹: 678.2693, found 678.2665 (100).

Oligonucleotides (protected with the DMT group at the

5⁰-hydroxyl) were puri®ed on a semipreparative PRP-1

column (Hamilton), using the same gradient as for the

analysis of the crude (see above), but with 0.05 M aq.

triethylammonium acetate as eluent A (¯ow: 2 mL/min,

detection Wavelength: 280 nm). The desired fractions

were pooled and lyophilized to dryness. Oligonucleotides

were dissolved in water/HAcO 3:1 and left to react for

30 min at 48C in order to eliminate the DMT group, and

lyophilized again. Oligonucleotides were redissolved in

water and eluted through a Sephadex G-10 exclusion

column to remove organic impurities, and the oligo-

nucleotide-containing fractions pooled and lyophilized.

4.13. Oligonucleotide synthesis

Solid-phase syntheses were performed using a 380B

Applied Biosystems synthesizer. All reagents, nucleoside

derivatives or resins not described in this Experimental

Section or prepared according to published procedures

were of commercial origin (Glen Research). Standard

procedures were used for the assembly of oligonucleotides

using phosphite triester chemistry⁴⁶(DMT-on).

4.14. Oligonucleotide synthesis using H-phosphonate

chemistry

Oligonucleotides were analysed MALDI-TOF MS (nega-

tive mode), using a trihydroxyacetophenone/ammonium

citrate matrix mixture, and either a re¯ector (trimers)

or a linear detector (15-mers). The following convention

for the nucleoside analogues is used: T^g4-guanidine-,

Oligonucleotide syntheses were performed on DMT-T-CPG

at the 2±3 mmol scale. Steps of the synthesis cycle (adapted

from standard H-phosphonate chemistry³⁷): (1) depro-

tection: 3% trichloroacetic acid in dichloromethane; (2)

coupling (5£): 0.035 M nucleoside 3⁰-H-phosphonate1

0.17 M adamantoyl chloride, both in either pyridine/

CH₃CN 1:3 (synthesis with triazolyl nucleoside (10) or

pyridine/CH₃CN 1:1 (syntheses of oligonucleotides con-

taining the guanidine analogues (17), 1 min; (3) capping:

0.1 M isopropyl phosphite10.17 M adamantoyl chloride

in pyridine/CH₃CN 1:3, 2 min. To simplify the puri®cation

step, the dimethoxytrityl group was kept on the 5⁰-hydroxyl

group of the last nucleoside. After the chain assembly,

oxidation of H-phosphonate diester bonds to phosphates

was carried out by reaction with 3% iodine in lutidine/tetra-

hydrofuran/water 75:23:2 for 30 min, followed by a second

treatment with 1.5% iodine in lutidine/triethylamine/tetra-

hydrofuran/water 38:25:36:2 for an additional 30 min.

T^mg4-N-methylguanidine-,

T^dmg4-N,N-dimethylguani-

dine- and T^dmg24-N,N⁰-dimethylguanidine-2-pyrimidinone

nucleoside.

0

4.16.1. ⁵TXT³. XT^g: 45% yield (synthesis1puri®cation);

reversed-phase HPLC t_R(gradient A): 13.4 min; MS

(MALDI-TOF, negative mode): 890.8 [M2H]²; calcd for

C₃₁H₄₃O₁₈N₉P₂[M]: 891.67 (average); XT^mg: 55% yield

(synthesis1puri®cation); reversed-phase HPLC t_R(gradient

A): 13.3 min; MS (MALDI-TOF, negative mode): 903.74

[M2H]²; calcd for C₃₂H₄₅O₁₈N₉P₂[M]: 905.70 (average);

XT^dmg: 55% yield (synthesis1puri®cation); reversed-

phase HPLC t_R(gradient A): 13.8 min; MS (MALDI-

TOF, negative mode): 917.65 [M2H]²; calcd for

C₃₃H₄₇O₁₈N₉P₂[M]: 919.72 (average); XT^dmg2: 60%

yield (synthesis1puri®cation); reversed-phase HPLC t_R

(gradient A): 13.5 min; MS (MALDI-TOF, negative

mode): 917.89 [M2H]²; calcd for C₃₃H₄₇O₁₈N₉P₂[M]:

919.72 (average).

4.15. Deprotection and cleavage

Oligonucleotide-resins (10±25 mg) were placed in a vial

J. Robles et al. / Tetrahedron 57 (2001) 179±194

193

0

5⁰

4.16.2. TTTTCTTXTCTTTTT³(18). XT^g: after puri-

®cation by HPLC, mixture of two products; MS (MALDI-

TOF, negative mode): 4507.98 [M2H]², 4466.11 [M2

CN₂H₃]², 2554.67 [M22H]²²; calcd for C₁₄₉H₁₉₇O₁₀₀N₃₅P₁₄

[M]: 4511.97 (average); XT^mg: 12% yield (synthesis1

puri®cation); reversed-phase HPLC t_R(gradient A): 12.9

min; MS (MALDI-TOF, negative mode): 4523.41 [M2

H]², 2261.99 [M22H]²²; calcd for C₁₅₀H₁₉₉O₁₀₀N₃₅P₁₄

[M]: 4525.99, (average); XT^dmg: 25% yield (synthesis1

puri®cation); reversed-phase HPLC t_R(gradient A): 12.9

min; MS (MALDI-TOF, negative mode): 4537.18 [M2

H]², 2269.20 [M22H]²²; calcd for C₁₅₁H₂₀₁O₁₀₀N₃₅P₁₄

[M]: 4540.02 (average); XT^dmg2: 22% yield (synthesis1

puri®cation); reversed-phase HPLC t_R(gradient A): 13.0

min; MS (MALDI-TOF, negative mode): 4536.43 [M2

H]²2269.09 [M22H]²²; calcd for C₁₅₁H₂₀₁O₁₀₀N₃₅P₁₄

[M]: 4540.02, (average).

Â Á

6. Giovannangeli, C.; Helene, C. Antisense Nucleic Acids Drug

Dev. 1997, 7, 413±421.

7. Chan, P. P.; Glazer, P. M. J. Mol. Med. 1997, 75, 267±282.

8. Mergny, J. L.; Duval-Valentin, G.; Nguyen, C. H.; Perrouault,

Â

L.; Faucon, B.; Rougee, M.; Montenay-Garestier, T.; Nisagni,

Â Á

E.; Helene, C. Science 1992, 256, 1681±1684.

9. Musso, M.; Thomas, T.; Shirahata, A.; Sigal, L. H.; van Dyke,

M. W.; Thomas, T. J. Biochemistry 1997, 36, 1441±1449.

10. Nielsen, P. E. Ann. Rev. Biophys. Biomol. Struct. 1995, 24,

167±183.

11. Freier, S. M.; Altmann, K.-H. Nucleic Acids Res. 1997, 25,

4429±4443.

12. Luyten, I.; Herdewijn, P.; Eur J. Med. Chem. 1998, 33, 515±

576.

13. Plum, G. E.; Park, Y. W.; Singleton, S. F.; Dervan, P. B.;

Breslauer, K. J. Proc. Natl. Acad. Sci. USA 1990, 87, 9436±

9440.

14. Asensio, J. L.; Lane, A. N.; Dhesi, J.; Bergqvist, S.; Brown, T.

J. Mol. Biol. 1998, 275, 811±822.

0

5⁰

4.16.3. TTTTXTTXTXTTTTT³(19). XT^g: after puri-

®cation by HPLC, mixture of products; MS (MALDI-TOF,

negative mode): 4618.97 [M2H]², 4576.94 [M2CN₂H₃]²,

4536.76 [M2C₂N₄H₅]², 4492.95 [M2C₃N₆H₁₄]²; calcd for

C₁₅₃H₂₀₅O₁₀₀N₃₉P₁₄[M]: 4624.10 (average); XT^mg: 8%

yield (synthesis1puri®cation); reversed-phase HPLC t_R

(gradient A): 12.8 min; MS (MALDI-TOF, negative

mode): 4661.98 [M2H]²2334.13 [M22H]²²; calcd for

C₁₅₆H₂₁₁O₁₀₀N₃₉P₁₄[M]: 4666.18 (average); XT^dmg: 16%

yield (synthesis1puri®cation); reversed-phase HPLC t_R

(gradient A): 13.2 min; MS (MALDI-TOF, negative

mode): 4705.14 [M2H]², 2354.38 [M22H]²²; calcd for

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C₁₅₉H₂₁₇O₁₀₀N₃₉P₁₄[M]: 4708.26 (average); XT^dmg2

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14% yield (synthesis1puri®cation); reversed-phase HPLC

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This work was supported by funds from the Spanish MEC

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Article Doi

Synthesis of modified oligonucleotides containing 4-guanidino-2-pyrimidinone nucleobases

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