Synthesis and study of the fluorescein conjugate of the nucleotide dPTP

Article abstract of DOI:10.1081/NCN-100002489

The synthesis of a fluorescein conjugate on the non-natural base P is described. The ability of the newly synthesised fluorescein-dPTP and of a fluorescein-11-dUTP to compete with the natural nucleotide TTP was also studied. Overall the efficiency of labe

Full text of DOI:10.1081/NCN-100002489

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SYNTHESIS AND STUDY OF THE FLUORESCEIN
CONJUGATE OF THE NUCLEOTIDE dPTP
W. J. Cummins a , A. L. Hamilton a , C. L. Smith a & M. S. J. Briggs a
a Amersham Pharmacia Biotech UK Limited , Amersham Place, Little Chalfont,
Buckinghamshire, HP7 9NA, England
Published online: 07 Feb 2007.
To cite this article: W. J. Cummins , A. L. Hamilton , C. L. Smith & M. S. J. Briggs (2001) SYNTHESIS AND STUDY OF THE
FLUORESCEIN CONJUGATE OF THE NUCLEOTIDE dPTP, Nucleosides, Nucleotides and Nucleic Acids, 20:4-7, 1049-1051, DOI:
10.1081/NCN-100002489
To link to this article: http://dx.doi.org/10.1081/NCN-100002489
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS, 20(4–7), 1049–1051 (2001)
SYNTHESIS AND STUDY OF THE FLUORESCEIN
CONJUGATE OF THE NUCLEOTIDE dPTP
W. J. Cummins,∗ A. L. Hamilton, C. L. Smith, and M. S. J. Briggs
Amersham Pharmacia Biotech UK Limited, Amersham Place,
Little Chalfont, Buckinghamshire HP7 9NA, England
ABSTRACT
The synthesis of a fluorescein conjugate on the non-natural base P is described.
The ability of the newly synthesised fluorescein - dPTP and of a fluorescein-
11-dUTP to compete with the natural nucleotide TTP was also studied. Overall
the efficiency of labelling a nucleic acid with a fluorescein moiety was found
to be approximately equal.
The bicyclic nucleobase analogue 6H,8H-3-4dihydropyrimido[4,5-c]-[1,2]-
oxazin-7-one (P) is a mimic of both cytosine and thymidine, since it can form
stable hydrogen bonded pairs with either guanine or adenine. This property arises
because the base analogue is able to exist in both the imino and amino tautomer.
The corresponding deoxyribonucleoside of P (dP) has been made and incorporated
into oligonucleotides via chemical synthesis as its phosphoramidite derivative (1,2)
and via enzymatic incorporation as its triphosphate derivative (3) (dPTP). In the
latter case dPTP was shown to be an excellent substrate for the Taq polymerase with
incorporation in place of TTP, at only a fourfold lower efficiency, although substi-
tution for dCTP gave much reduced efficiency (3). This highlighted the potential
for the dP to be used in probes and primers where the base analogue can be placed
into positions of degeneracy to reduce the number of probes and primers required.
The synthesis of functionalised derivative of dPTP would allow the introduction of
a fluorescent label such as fluorescien via enzymatic means.
∗Corresponding author.
1049
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Copyright 2001 by Marcel Dekker, Inc.
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ORDER
REPRINTS
1050
CUMMINS ET AL.
Figure 1. Reagents a) TIPDSCl2, py, N2, 0◦C, 68% b) K2OsO42H2O, N-methylmorpholino-N-
oxide, acetone, 72% c) NalO4, THF, H2O, 93% d) 2,2,5,5-tetramethyl-1-aza-2,5-disilacyclopentane-
1-propyl magnesium bromide, Et2O, THF, N2, not isolated then e) MMTCl, Et3N, DCM, 61% the two
steps f) N-hydroxyphthalimide, Ph3P, DEAD, THF, N2g) 1,2,4, triazole, POCl3, Et3N, MeCN 55%
over the two steps h) NH3, dioxane i) Bu4NF, THF 68% over two steps j) AcOH, MeCN, H2O not
isolated then k) NHS of N-(trifluoroacetyl)caproic acid, Et3N, DMF, 60% over two steps l) POCl3,
(MeO)3PO, (EtO)3PO then Bu3NH H3P2O7, DMF, 40% m) NH3(aq) then NHS ester of 5-(6) carboxyl
fluorescein, DMF, Na2CO3 buffer pH 8.5.
Amulti-stepsyntheticroute, asshowninFigure1, startingfrom5ꢁ-allyluridine
achieved the synthesis of a dPTP derivative with a functionalised arm. The triphos-
1
phate derivative showed consistent 31P and H NMR spectra. The diastereomers
were not separated during the course of the synthesis. Subsequent conjugation of
the amino side chain with the NHS ester of 5-(6)-carboxy fluorescein provided the
fluorescein conjugated nucleotide 6 (denoted as fluorescein-dPTP).

ORDER
REPRINTS
FLUORESCEIN CONJUGATE OF THE NUCLEOTIDE dPTP
1051
Incorporation Assays
The ability of fluorescein-dPTP and fluorescein-11-dUTP (11 atom linker
between C5 on the base and fluorescein) to compete with the natural dNTP’s
as substrates for exonuclease free Klenow fragment DNA polymerase I (EFK)
was examined using the extension of a 33P-labelled 15 mer primer hybridised to
a 24 mer template.
The primer (1 pmol) and template (2 pmol) were hybridised in x2 Klenow
buffer (100 mM Tris-HCl pH 7.5, 10 mM MgCl2, 10 mM β-mercaptoethanol) by
heating at 75◦C for 3 minutes then allowing to cool to 30◦C over at least 30 minutes.
To this was added EFK polymerase (0.1 U) and inorganic pyrophosphatase (20 mU)
plus a mix of either fluorescein-dPTP and dTTP or fluorescein-11-dUTP and dTTP.
The total nucleotide concentration in all the reactions was fixed at 20 µM, but the
ratio of the dNTP to the labelled nucleotide was varied. Reactions were incubated
at 37◦C for 2 minutes before adding formamide/EDTA stop solution. The reactions
were then heated at 95◦C for 2 minutes before loading samples (4 µl) onto a 20%
acrylamide/7M urea gel. After running at 1.8 kV for 2 hours the gel was fixed and
dried before exposing to a phosphor screen for analysis and quantification.
The results from the above gel analysis clearly show that the fluorescent
nucleotide analogue (fluorescein dPTP) is incorporated approximately 10–15%
more efficiently than the corresponding dUTP derivative. However, we have shown
that the subsequent extension of primers with fluorescein - dPTP at the 3ꢁ end is
approximately two-fold less efficient than the corresponding primer with a dUTP
derivative at its 3ꢁ end (unpublished results). Thus as a means of introducing a
fluorescein group into an oligo enzymatically with modified nucleotides then the
dP derivative is approximately equal to uridine.
REFERENCES
1. Kong Loo Lin, P.; Brown, D.M. Nucleic Acids Research, 1989, 17, 10373–10383.
2. Kong Loo Lin, P.; Brown, D.M. Nucleic Acids Research, 1992, 20, 5149–5142.
3. Zaccolo, M.; Williams, D.M.; Brown, D.M.; Gherardi, E. J. Mol. Biol., 1996, 255,
589–603.

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