Synthesis of pyrrole carboxamide nucleotide triphosphates - Putative labelled nucleotide analogues

Article abstract of DOI:10.1016/S0040-4039(02)00225-3

The syntheses of pyrrole carboxamide nucleotide triphosphates and the initial results of their incorporation by Klenow fragment are reported.

Full text of DOI:10.1016/S0040-4039(02)00225-3

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 43 (2002) 2289–2291
Synthesis of pyrrole carboxamide nucleotide
triphosphates—putative labelled nucleotide analogues
R. James D. Nairne,* Lea Pickering and Clifford L. Smith
Amersham Pharmacia Biotech UK Limited, Amersham Place, Little Chalfont, Buckinghamshire HP7 9NA, UK
Received 1 November 2001; revised 22 January 2002; accepted 30 January 2002
Abstract—The syntheses of pyrrole carboxamide nucleotide triphosphates and the initial results of their incorporation by Klenow
fragment are reported. © 2002 Elsevier Science Ltd. All rights reserved.
Nucleoside analogues in which the natural pyrimidine
or purine base moiety has been substituted by a pyrrole
nucleus have been shown to be good universal bases.
The 3-nitropyrrole nucleoside 1 (Fig. 1) has been incor-
porated into oligonucleotides using phosphoramidite
chemistry and has been shown to be effective in the
preparation of universal sequencing primers.1 However,
enzymatic incorporation of this class of analogue from
the triphosphate level has been found to be less
efficient, possibly due to the poor hydrogen bonding
ability of the nitro group. With this limitation in mind,
interest in pyrrole nucleosides that have more effective
hydrogen bonding donor–acceptor groups has been
growing. Pyrroles with one 2 or two carboxamide 3
substituents have recently been prepared.2 The triphos-
phates of these analogues are also better substrates for
polymerase enzymes and oligonucleotides that contain
these base analogues have been synthesised via
phosphoramidites.
In connection with our ongoing programme of research
into the properties of nucleic acids and their analogues,
we wished to prepare analogues of the pyrrole dicar-
boxamides which are capable of being labelled, either as
the triphosphate or after incorporation. In this commu-
nication, we report the synthesis of two related carbox-
amide pyrrole nucleotide analogues. We have prepared
a 3,4-dicarboxamide pyrrole nucleotide, in which both
amide groups are directly attached to the pyrrole
nucleus, and a 3-carboxamide pyrrole nucleotide in
which only one amide is attached to the pyrrole
nucleus. Both analogues have additional functionalisa-
tion to allow the attachment of a reporter group (such
as a fluorophore) to the molecule. Labelled nucleoside
analogues of this type have a possible role in nucleic
acid analysis.
The pyrrole monocarboxamide (Scheme 1) was pre-
pared starting from methyl 4-chloroacetoacetate 4.3 The
Figure 1.
Keywords: amides; nucleic acid analogues; nucleotides; pyrroles.
* Corresponding author. Fax: +44 (0) 1494 543627; e-mail: james.nairne@uk.amershambiosciences.com
0040-4039/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(02)00225-3

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R. J. D. Nairne et al. / Tetrahedron Letters 43 (2002) 2289–2291
Scheme 1. Reagents and conditions: (i) (EtO)3CH, Ac2O, reflux, 100%; (ii) glycine, KOH, MeOH, reflux, 58%; (iii) Ac2O, reflux,
36%; (iv) NaN3, DMF then NaHCO3, H2O, 56%; (v) PPh3, pyridine, H2O, not isolated; (vi) O-succinimidyl-N-(tBOC)
aminocaproate, MeCN, 68%; (vii) a-chloro-(3,5-di-O-p-toluoyl)-2-deoxy-
D-ribose, NaH, MeCN, 68%; (viii) HCONH2, MeONa,
MeOH, THF, reflux, 54%; (ix) POCl3, (MeO)3PO, (EtO)3PO then Bu3N·H3P2O7, DMF.
3,4-bis(ethoxycarbonyl) pyrrole. This was converted in
b-keto ester was reacted with triethyl orthoformate, and
then glycine to give the vinyl amine 5. Conversion to
the aminomethyl pyrrole 6 was effected in three steps;
cyclisation to form the pyrrole ring with concomitant
decarboxylation was accomplished using acetic anhy-
dride in 36% yield. Displacement of the chloride with
sodium azide and subsequent reduction using
triphenylphosphine in pyridine gave the amine 6. The
amine was reacted directly with the NHS ester of
BOC-protected 6-aminocaproic acid to give the func-
tionalised pyrrole 7. Glycosylation was effected by
alkylation of the sodium salt of the pyrrole with a-
two steps to the mono(amide) 11 by monosaponifica-
tion according to the procedure of Nicolaus and
Mangoni6 followed by EDC coupling with mono-BOC-
1,3-propanediamine. This pyrrole was glycosylated
under identical conditions as 7 (Scheme 2). Once again,
1H NMR showed that only one isomer, 12, was formed
and that this gave spectra consistent with the formation
of the b-anomer.7 The toluoyl groups were removed
using sodium methoxide in methanol resulting in trans-
esterification of the 4-ethoxycarbonyl moiety, and the
sugar was then silylated 13 with TBDMS-Cl. This latter
step proved necessary for purification of the nucleoside
analogue 14. Amidation was carried out under identical
conditions to the formation of 95 and the synthesis of
the triphosphate was completed by desilylation using
ammonium fluoride and phosphosphorylation. The
triphosphate 15 was treated with TFA to remove the
BOC protecting group, and the resultant amine 16 was
reacted with the NHS ester of 5(6)-fluoresceinyl-6-
aminocaproic acid in DMSO to give the fluorescently
labelled triphosphate 17.
chloro-(3,5-di-O-p-toluoyl)-2-deoxy-D-ribose in aceto-
nitrile. A single product 8 was obtained. 1H NMR
spectra of the product showed features that were
entirely consistent with the product having the b
configuration.4 This was followed by amidation of the
methyl ester function using formamide and sodium
methoxide in refluxing methanol5 with concomitant
removal of the toluoyl protecting groups to give the
nucleoside 9. The nucleoside analogue was converted to
the nucleotide analogue 10 in a solvent mixture of
trimethyl and triethyl phosphate using phosphorous
oxychloride, followed by addition of tributylammonium
pyrophosphate in DMF.
The triphosphates were screened for incorporation
using Klenow fragment in a primer extension assay.
The pyrrole dicarboxamides 15, 16 and 17 were incor-
porated as dA and dC, the monocarboxamide 10 was
incorporated as dT.
Preparation of the functionalised pyrrole-3,4-dicarbox-
amide 17 was initiated from the commercially available

R. J. D. Nairne et al. / Tetrahedron Letters 43 (2002) 2289–2291
2291
Scheme 2. Reagents and conditions: (i) 10% K2CO3(aq.), 52%; (ii) BOCNH(CH2)3NH2, EDC, HOBt, THF, 77%; (iii) NaH, MeCN,
then a-chloro-(3,5-di-O-p-toluoyl)-2-deoxy- -ribose, 90%; (iv) MeONa, MeOH, 77%; (v) TBDMS-Cl, imidazole, DMF, 90%; (vi)
D
HCONH2, MeONa, MeOH, THF, reflux; (vii) NH4F, MeOH, 72% from 13; (viii) POCl3, P(O)(OMe)3, P(O)(OEt)3, then
Bu3NH·P2O7, DMF; (ix) TFA; (x) 6-fluorescein-5-(and 6-)carboxamidohexanoic acid N-hydroxysuccinimidyl ester, DIPEA,
DMSO.
References
5-Ar-H
J=8 Hz, 2, 6-Ar-H
5. Jagdmann, G. E.; Munson, H. R.; Gero, T. W. Synth.
6
), 7.46 (1H, J=2.5 Hz, pyrrole-H
6
), 7.89 (2H, d,
).
6
), 7.94 (2H, d, J=8.4 Hz, 3, 5-Ar-H
6
1. Bergstrom, D. E.; Zhang, P.; Toma, P. H.; Andrews, P. C.;
Nicholls, R. J. Am. Chem. Soc. 1995, 117, 1201.
Commun. 1990, 20, 1203.
6. Nicolaus, R. A.; Mangoni, L. Gazz. Chim. Ital. 1956, 86,
358.
2. Lakes, D.; Guo, M. J.; Brown, D. M.; Salisbury, S. A.;
Smith, C. L.; Felix, I.; Kumar, S.; Nampalli, S.
Nucleosides, Nucleotides Nucleic Acids 2000, 19, 1599.
3. Japanese Patent JP 7-157466.
7. 1H NMR data for the nucleoside 12 (300 MHz, CDCl3): l
1.14 (3H, t, J=7 Hz, CH2CH3
(2H, quintet, J=6.6 Hz, NCH2CH2
ArCH3), 2.36 (3H, s, ArCH3), 2.62 (2H, br m, 2%-CH2
3.11 (2H, app q, J=6.2 Hz, CH2CO), 3.40 (2H, app q,
2NHCO2 Bu), 4.10 (2H, m, CO2CH2CH3),
4.52 (1H, m, 4%-CH), 4.54 (2H, m, 5%-CH2), 5.27 (1H, br s,
CH2NHCO), 5.55 (1H, m, 3%-CH), 5.95 (1H, dd, J=7.5
and 6.3 Hz, 1%-CH), 7.15 (2H, d, J=8.1 Hz, 3,5-ArH), 7.20
(2H, d, J=8.1 Hz, 3,5-ArH), 7.52 (1H, d, J=2.6 Hz,
pyrrole-H), 7.61 (1H, d, J=2.6 Hz, pyrrole-H), 7.82 (2H,
d, J=8.1 Hz, 2,6-Ar-H), 7.85 (2H, d, J=8.1 Hz, 2,6-Ar-
), and 9.71 (1H, br t, NH
6
), 1.36 (9H, s, C(CH
CH2N), 2.33 (3H, s,
),
6 )3), 1.68
4. 1H NMR data for the nucleoside analogue 8 (300 MHz,
6
6
6
6
CDCl3): l 1.20–1.26 (2H, m, CH2
1.45–1.55 (2H, m, CH2), 1.91–1.94 (2H, m, CH2
(2H, app t, CH2NHBOC, J=7 Hz), 2.22 (3H, s, ArCH3
2.39 (3H, s, ArCH3), 2.64 (2H, m, 2%-CH2), 2.95 (2H, dd,
J=6 and 13 Hz, CH2CONH), 3.68 (3H, s, CO2CH3), 4.29
(2H, d, J=6 Hz, NHCH2-pyrrole), 4.47–4.59 (3H, m,
4%-CH2, 5%-CH2), 5.28 (1H, s, br, amide NH), 5.60 (1H, m,
3%-CH), 6.03 (1H, dd, J=7.7 and 6.2 Hz, 1%-CH), 6.67 (1H,
br, amide NH), 6.81 (1H, d, J=2.5 Hz, pyrrole-H), 7.30
(2H, d, J=8 Hz, 3, 5-Ar-H), 7.31 (2H, d, J=8.4 Hz, 3,
6
), 1.33 (9H, s, C(CH3 3
), 2.07
),
6 ) ),
6
6
6
t
J=7 Hz, CH
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
t
H6
6 CO2 Bu).
6