Fluorescence resonance energy transfer terminators for DNA sequencing

Article abstract of DOI:10.1016/S0040-4039(00)01617-8

A four-colour set of fluorescence resonance energy transfer dideoxy nucleotide terminators (10-13), have been synthesised using a rigid and linear tri-functional molecule (2), synthesised via Heck coupling reaction of t-Boc-L-4-iodophenylalanine (1) with N-TFA-propargylamine. Evaluation of the terminators in DNA sequencing reactions, in combination with Thermo Sequenase(TM) II DNA polymerase, demonstrated them to be excellent reagents for high-throughput DNA sequencing. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4039(00)01617-8

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 41 (2000) 8867–8871
Fluorescence resonance energy transfer terminators for
DNA sequencing
Satyam Nampalli, Mahesh Khot and Shiv Kumar*
Nucleic Acid Chemistry, Amersham Pharmacia Biotech, 800 Centennial Avenue, Piscataway, NJ 08855, USA
Received 1 September 2000; accepted 19 September 2000
Abstract
A four-colour set of fluorescence resonance energy transfer dideoxy nucleotide terminators (10–13),
have been synthesised using a rigid and linear tri-functional molecule (2), synthesised via Heck coupling
reaction of t-Boc- -4-iodophenylalanine (1) with N-TFA-propargylamine. Evaluation of the terminators
L
in DNA sequencing reactions, in combination with Thermo Sequenase™ II DNA polymerase, demon-
strated them to be excellent reagents for high-throughput DNA sequencing. © 2000 Elsevier Science Ltd.
All rights reserved.
Development of new DNA polymerases1 and fluorescence resonance energy transfer (FRET)
dyes have helped improve dideoxy terminator DNA sequencing.2 FRET dyes comprised of a
common donor dye and different acceptor dyes are superior to single-dye-labels because they
generate well-separated, enhanced fluorescence signals for the four kinds of DNA sequencing
fragments to be detected using a single laser for analysis.3 To date, there are a number of
examples of energy transfer in DNA sequencing either by using fluorescence energy transfer
dye-labelled primers4 or terminators5 with various linkers separating the donor and acceptor
dyes. This work was directed towards finding FRET labelled terminators with improved
brightness, sequence read-length, accuracy, and reactivity with Thermo Sequenase™ II DNA
polymerase.
As part of the research program directed towards developing novel FRET dideoxy nucleotide
terminators and DNA polymerases for high-throughput DNA sequencing, we have undertaken
a novel synthetic approach to arrive at a four-colour set of fluorescent dye-labelled terminators.
The objective was the design and synthesis of four FRET dye-labelled cassettes, which could be
coupled with alkynylaminodideoxynucleoside-5%-triphosphates or other biological molecules of
interest. For this purpose, the FRET cassettes 6–9 (Scheme 1) with a rigid and yet linear linker
were envisaged to be synthesised from a common donor dye cassette 5, derived from a
* Corresponding author. Fax: 732-457-8353; e-mail: shiv.kumar@am.apbiotech.com
0040-4039/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)01617-8

8868
tri-functional molecule, t-Boc-
L
-4-iodophenylalanine 1 via a Heck coupling reaction with
N-TFA-propargylamine.
Scheme 1.
Thus, a convenient Heck6 coupling reaction of 1 with N-TFA-propargylamine using
(Ph3P)4Pd(0)/CuI/Et3N/DMF afforded the rigid-linear cassette linker 2 in 87% yield. The
para-propargylamino, t-Boc-a-amino, and the free carboxylic acid functional groups in 2 were
preconceived to be conjugated sequentially with a donor dye, acceptor rhodamine dyes, and
ddNTPs, respectively. Removal of the TFA protecting group with 30% NH4OH gave compound
3 (quantitative yield), which upon conjugation with 5-carboxyfluorescein succinimidylester
(5-FAM, SE) in anhydrous DMSO in the presence of DIPEA provided 5-FAM-PAPhe
(propargylamino phenylalanine) cassette 4 in 88% yield. Removal of t-Boc protecting group by
treating with ice-cold 1:1 aqueous TFA at rt afforded the TFA salt of 5 in 88% yield,7 ready for
preparing energy transfer cassettes. The very first energy transfer cassette synthesised was
5-FAM-PAPhe-ROX 6 (81%), resulting from the conjugation of FAM-cassette 5 with 5-car-
boxy-X-rhodamine succinimidyl ester (5-ROX, SE)/DIPEA in anhydrous DMSO. This ET

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cassette 6 when excited at 488 nm, showed four times enhanced ROX emission to that of single
ROX dye. In order to have a four-colour set, FAM cassette 5 was conjugated independently
with 5-TAMRA (5-carboxytetramethylrhodamine), 5-REG (5-carboxyrhodamine 6G) and 5-
R110 (5-rhodamine green carboxylic acid, trifluoroacetamide) NHS esters to provide 5-FAM–
PAPhe–TAMRA 7 (78%), 5-FAM–PAPhe–REG 8 (84%), and 5-FAM–PAPhe–R110 9 (69%)
ET cassettes, respectively.
Scheme 2.

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MM+/MO calculations showed that the donor and the acceptor dyes in these ET cassettes
have spatial orientations parallel to each other, which should be optimal for FRET.8 1H NMR,9
UV–vis, and TOF MS,10 data confirmed the molecular structures of these ET cassettes. The ET
linker, para-propargylamino phenylalanine 2, has the versatility to be conjugated not only with
widely used fluorescein and rhodamine dyes, but also with cyanines or any other fluorescent
tags. It is also worth mentioning that these ET cassettes have a great potential to label modified
primers, should one choose to do primer DNA sequencing.
Initial attempts to conjugate activated ET cassettes with alkynylamino 11-ddNTPs11,12
(Scheme 2) using a large variety of activating reagents13 such as TSTU/DIPEA, DCC/HOSu,
HBTU, HATU, HOBt, PyBOP, PyBrOP, EDAC/HOSu, and TFA–HOSu, at different temper-
atures produced low yields of the desired ET terminators. However, using the optimised
DSC/DMAP/DMF/−60°C conditions, we could convert 6, 7, 8, and 9 to the corresponding
NHS esters and in situ conjugate with 11-ddNTPs (11-ddCTP, 11-ddATP, 11-ddUTP, and
11-ddGTP) at −30°C to provide the desired ET dye terminators14,15 10, 11, 12, and 13,
respectively, in yields ranging from 15–20%. The normalised fluorescence emission spectra for
these ET terminators are given in Fig. 1. The fluorescence emission enhancement rates of these
ET terminators compared to the corresponding single dye terminators were found to be 18
(ROX), 6.5 (TAMRA), 5 (REG), and 1.6 (R110).
Figure 1. Argon-laser (488 nm) excited, normalised fluorescence emissions of the four-colour set: 10–13 in 1 X TBE
(tris-boric acid EDTA) 8.0 M urea
Of the 64 possible combinations of ET terminators (8 carboxyrhodamine 5/6 regio-isomers×2
caboxyfluorescein 5/6 regio-isomers×4 alkynylamino 11-ddNTPs), 12 were synthesised and
tested in the DNA sequencing experiments. DNA sequencing performed, employing the four-
colour set of 10–13 with a variety of DNA templates and Thermo Sequenase™ II DNA
polymerase, were found to be of high quality with read-lengths in excess of 800 bases.
In conclusion, a novel synthetic route for the four-colour set of FRET terminators has been
developed involving a rigid-linear ET cassette linker chemistry, and formulated the set into a
robust DNA sequencing kit with Thermo Sequenase II. Selection process for the best four-
colour set, energy transfer efficiencies of the ET cassettes, and DNA sequencing results will be
published elsewhere.

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Acknowledgements
The authors wish to thank Inna Livshin and Jim Holecek for initial HPLC experiments, Dr.
John Nelson for sequencing experiments, Drs. Mark McDougall and C. Y. Chen for Mass and
fluorescence spectral analysis, Drs. Carl Fuller and Parke Flick for many helpful discussions.
References
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3. Ju, J.; Ruan, C.; Fuller, C. W.; Glazer, A. N.; Mathies, R. A. Proc. Natl. Acad. Sci. USA 1995, 92, 4347–4351.
4. (a) Ju, J.; Kheterpal, I.; Scherer, J. R.; Ruan, C.; Fuller, C. W.; Glazer, A. N.; Mathies, R. A. Anal. Biochem.
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N.; Mathies, R. A. Nucleic Acids Research 1996, 24, 1144–1148. (d) Lee, L. G.; Spurgeon, S. L.; Heiner, C. R.;
Benson, S. C.; Rosenblum, B. B.; Menchen, S. M.; Graham, R. J.; Constantinescu, A.; Upadhya, K. G.; Cassel,
J. M. Nucleic Acids Research 1997, 25, 2816–2822.
5. Rosenblum, B. B.; Lee, L. G.; Spurgeon, S. L.; Khan, S. H.; Menchen, S. M.; Heiner, C. R.; Chen, S. M. Nucleic
Acids Research 1997, 25, 4500–4504.
6. Cortese, N. A.; Ziegler, C. B.; Hrnjez, B. J.; Heck, R. F. J. Org. Chem. 1978, 43, 2952.
7. 1H NMR of the common single dye cassette 5 (DMSO-d6): l 2.84 (2H, q, J=6, 15 Hz, benzylic), 3.11 (2H, dd,
J=3, 18 Hz, propargylic), 4.36 (1H, s, chiral), 6.50 (4H, m, aromatic), 6.66 (2H, s, aromatic), 7.25 (2H, d, J=9.0
Hz, aromatic), 7.34 (2H, d, J=9.0 Hz, aromatic), 7.37 (1H, d, J=6 Hz, aromatic), 8.25 (1H, d, J=6.0 Hz,
aromatic), 8.43 (1H, s, aromatic), 9.34 (1H, m, NH-).
8. Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 2nd ed. Kluwer Academic/Plenum: New York, 1999; pp.
367–390.
9. 1H NMR of 6 (DMSO-d6): l 1.72 (8H, br s, ROX-dye rings% homo-benzylic), 1.92 (8H, br s, ROX-dye rings%
benzylic), 2.83 (8H, br s, ROX-dye rings% methylenes adjacent N), 3.05 (2H, m, benzylic), 4.32 (2H, br s,
propargylic), 4.44 (1H, m, chiral), 6.49 (4H, m, aromatic), 6.62 (2H, s, aromatic), 7.16 (1H, d, J=9.0 Hz,
aromatic), 7.28 (5H, m, aromatic), 7.96 (1H, d, J=6.0 Hz, aromatic), 8.25 (1H, d, J=6.0, aromatic), 8.34 (1H,
1
s, aromatic), 8.46 (1H, s, aromatic). Compounds 7, 8 and 9 showed satisfactory H NMR data.
10. TOF MS ES m/z, cone 50v, 50% CH3CN/H2O: compound 6: 1089.66 (MH-3); 7: 985.83 (MH-3); 8: 1013.89
(MH-3); 9: 931.23 (MH-2).
11. Hobbs Jr., F. W.; Cocuzza, A. J. U.S. Patent 5 047 519, 1991.
12. The number 11 preceding the ddNTPs (dideoxynucleoside triphosphates), indicates the number of atoms in the
linker arm attached at 5-position in pyrimidines and 7 in 7-deazapurines.
13. Glossary of terms; TSTU (O-(N-succinimidyl)-1,1,3,3-tetramethyluronium tetrafluoroborate), DIPEA (N,N-di-
isopropylethylamine), DCC (N,N%-dicyclohexylcarbodiimide), HOSu (N-hydoxysuccinimide), HBTU (2-(1H-ben-
zotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate), HATU (2-(1H-azobenzotriazole-1-yl)-1,1,3,3-
tetramethyluronium hexafluorophosphate), HOBt (N-hydroxybenzotriazole), PyBOP (benzotraizole-1-yl-oxy-tris-
pyrrolidino-phosphonium hexafluorophosphate), PyBroP (bromo-tris-pyrrolidino-phosphonium hexafluorophos-
phate), EDAC (1-ethyl-3-(3%-dimethylaminopropyl)carbodiimide. HCl, TFA-HOSu (N-trifluoacetylhydroxysuc-
cinimide), DMAP (4-N,N-dimethylaminopyridine).
14. Typical experimental procedure: To a stirred mixture of ET cassette 8 (100 mg), 91 (mol) and N,N%-disuccinimidyl
carbonate (453 mmol, 5 equiv.) in anhydrous DMF (10 mL), was added anhydrous DMF solution of DMAP (454
mmol, 5 equiv.) at −60°C. TLC monitoring of the reaction within 5 min indicated complete conversion to the
NHS ester, which, without isolation was treated with 0.1 M Na2CO3ꢀNaHCO3 buffer solution of 11-ddATP (74
mmol, 0.8 equiv.) at −30°C and stirred at rt for 1 h. The reaction mixture was subjected to a silica gel column
(6 iso-PrOH:3 NH4OH:1 H2O) purification followed by Q-Sepharose FPLC (buffer A: 40% CH3CN/0.1 M TEAB
pH 7.5; buffer B: 40% CH3CN/1.0 M TEAB pH 7.5) and C18 reversed-phase HPLC (buffer C: 0.1 M TEAB, pH
7.0; buffer D: 100% CH3CN) to yield 11 (20%) as a pink fluffy solid. Similarly, compounds 10, 12 and 13 were
obtained in 15–20% yields.
15. TOF MS ES m/z, cone 100v, 20% CH3CN/H2O:ET terminator 10: 1687.79 (MH-4); 11: 1606.69 (MH-4); 12:
1612.49 (M-4); 13: 1569.23 (MH-1).