Fluorescence resonance energy transfer dye nucleotide terminators: A new synthetic approach for high-throughout DNA sequencing

Article abstract of DOI:10.1081/NCN-100002307

Fluorescence resonance energy transfer (FRET) based dye-nucleotide terminators (10-13) were designed, synthesized, and formulated with Thermo Sequenase II DNA polymerase into a robust kit for high throughput DNA sequencing. The key energy transfer (ET) rigid and linear linker (2), required for the syntheses of energy transfer cassettes (6-9) was synthesized via Heck coupling reaction on t-Boc-L-4-iodo-phenylalanine (1) with N-TFA-propargylamine.

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FLUORESCENCE RESONANCE ENERGY TRANSFER
DYE NUCLEOTIDE TERMINATORS: A NEW SYNTHETIC
APPROACH FOR HIGH-THROUGHOUT DNA
SEQUENCING
Satyam Nampalli a , Mahesh Khot a , John R. Nelson a , Parke K. Flick a , Carl W. Fuller a
& Shiv Kumar b
a Amersham Pharmacia Biotech, Piscataway, New Jersey, 08855, U.S.A.
b Amersham Pharmacia Biotech, Piscataway, New Jersey, 08855, U.S.A.
Version of record first published: 07 Feb 2007.
To cite this article: Satyam Nampalli , Mahesh Khot , John R. Nelson , Parke K. Flick , Carl W. Fuller & Shiv Kumar (2001):
FLUORESCENCE RESONANCE ENERGY TRANSFER DYE NUCLEOTIDE TERMINATORS: A NEW SYNTHETIC APPROACH FOR HIGH-
THROUGHOUT DNA SEQUENCING, Nucleosides, Nucleotides and Nucleic Acids, 20:4-7, 361-367
To link to this article: http://dx.doi.org/10.1081/NCN-100002307
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS, 20(4–7), 361–367 (2001)
FLUORESCENCE RESONANCE ENERGY
TRANSFER DYE NUCLEOTIDE TERMINATORS:
A NEW SYNTHETIC APPROACH FOR
HIGH-THROUGHOUT DNA SEQUENCING
Satyam Nampalli, Mahesh Khot, John R. Nelson, Parke K. Flick,
Carl W. Fuller, and Shiv Kumar∗
Amersham Pharmacia Biotech, Piscataway, New Jersey 08855
ABSTRACT
Fluorescence resonance energy transfer (FRET) based dye-nucleotide
terminators (10–13) were designed, synthesized, and formulated with Thermo
SequenaseTM II DNA polymerase into a robust kit for high throughput DNA
sequencing. The key energy transfer (ET) rigid and linear linker (2), required
for the syntheses of energy transfer cassettes (6–9) was synthesized via
Heck coupling reaction on t-Boc-L-4-iodo-phenylalanine (1) with N-TFA-
propargylamine.
INTRODUCTION
Since the development of dideoxy terminator DNA sequencing (1), there
have been new developments and advances in DNA polymerases (2), fluorescence
DNA sequencing technology (3), and automated sequencers. Fluorescence reso-
nance energy transfer (FRET) based dye terminator DNA sequencing is the most
preferred method over the radio-isotope labeled one (poses severe problems in stor-
age, handling, and health risks) or the single/ET dye-primer sequencing (requires
four-lane sequencing). FRET dyes comprising a common donor dye and four differ-
ent acceptor dyes are superior to single dye labels in DNA sequencing by virtue of
∗ Corresponding author. Fax: 732-457-8353; E-mail: shiv.kumar@am.apbiotech.com
361
ꢀ
C
Copyright 2001 by Marcel Dekker, Inc.
www.dekker.com

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NAMPALLI ET AL.
generating, well separated, enhanced fluorescence signals for the detection/analysis
of four kinds of DNA sequencing fragments (4). To date, there are a number of ex-
amples of FRET DNA sequencing either by using FRET dye-labeled primers (5)
or terminators (6) with various ET linkers separating the donor and acceptor dyes.
As the Human Genome Project needs high-speed, high throughput sequencing for
deciphering the whole genome, further improvements in the sequencing technology
are necessary to help expedite the effort.
As part of the research program directed towards developing novel FRET
dideoxy nucleotide terminators for high-throughput DNA sequencing with im-
proved brightness, sequence read-length, accuracy, and reactivity with new DNA
polymerases, we have undertaken a new synthetic approach (7) to arrive at a four-
color set of fluorescent dye-labeled terminators.
NHTFA
NH2
I
DIEA, DMSO,rt
CuI, Et3N, (Ph3P)4Pd (0)
30% NH4OH, rt
TFAHN
CO2H
, DMF, rt
HO
O
O
H
H
H
_
t-BocHN
t-BocHN
CO2H
t-BocHN
CO2H
CO2
1
2
3
O
O
N
O
O
HO
O
O
HO
O
O
HO
O
O
_
CO2
_
CO2
_
CO2
H
N
5-ROX SE,
5-TAMRA SE,
H
H
N
O
1:1 aqueous TFA
O
N
O
5-REG SE,
5-R110 SE,DMSO
DIPEA, rt
H
H
H
RCOHN
CO2H
H2N
CO2H
t-BocHN
CO2H
4
6: R = 5-Rhodamine-X (ROX)
5
7: R = 5-Tetramethylrhodamine (TAMRA)
8: R = 5-Rhodamine-6-G (REG)
9: R = 5-Rhodamine 110 (R110)
Scheme 1.

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FRET DYE NUCLEOTIDE TERMINATORS
363
SYNTHESIS AND RESULTS
The primary goal was the design and synthesis of four FRET dye-labeled
cassettes, which could be coupled with reactive alkynylamino-dideoxynucleotides
or other biological molecules of interest. In order to achieve the set goal, the
FRET cassettes 6–9 (Scheme 1) with a rigid and yet linear linker were envi-
sioned to be synthesized from a common donor dye cassette 4, derived from a
tri-functionalmolecule, t-Boc-L-4-iodophenylalanine1. viaHeckcouplingreaction
with N-TFA-propargylamine. Thus, commercially available starting material 1 was
6, 7, 8, and 9
DSC, DMAP, DMF,-60 0C
11-ddNTPs, Na2CO3-NaHCO3 (pH 8.5), -30 0
C
HO
O
O
HO
O
O
_
_
CO2
CO2
H
H
N
N
O
O
+
N
O
NMe2
H
H
CO2
H
CO2
H
N
CONH(CH2)5CONH
N
CONH(CH2)5CONH
O
NH2
N
O
O
NH2
N
+
N
Me2N
O
N
N
N
3-HO9P3O
O
3-HO9P3O
O
10
HO
O
O
HO
O
O
11
_
_
CO2
CO2
H
H
N
N
O
O
+
+
NH2
NEtH
H
H
H
CO2
CO2
H
CONH(CH2)5CONH
CONH(CH2)5CONH
N
N
O
O
O
N
O
O
O
NH
NH
H2N
EtHN
O
N
N
NH2
3-HO9P3O
3-HO9P3O
O
O
12
13
Scheme 2.

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NAMPALLI ET AL.
conveniently subjected to the Heck (8) coupling reaction with N-TFA-propargy-
lamine using (Ph3P)4 Pd (0)/CuI/Et3N/DMF to provide the rigid-linear cassette
linker 2 in 87% yield. The base and acid sensitive protecting groups–TFA of para-
propargylamine, t-Boc of α-amine–and the free carboxylic acid group in 2 were
thought to be exploited in a sequential covalent attachment with a donor carboxy-
fluorescein, acceptor carboxy-rhodamine NHS esters, and alkynylamino-ddNTPs,
respectively. Accordingly, removal of the base sensitiveTFA group in 2 with 30%
NH4OH furnished 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) cas-
sette 4 in 88% yield. Removal of the acid sensitive t-Boc protecting group by treating
with ice-cold 1:1 aqueous TFA at rt afforded the TFA salt of 5 (88%), setting the
stage for preparing the energy transfer cassettes. The very first energy transfer cas-
sette synthesized was 5-FAM-PAPhe-ROX 6 (81%), resulting from the conjugation
of FAM-cassette 5 with 5-carboxy-X-rhodamine succinimidyl ester (5-ROX, SE)/
DIPEA in anhydrous DMSO. This ET cassette 6 when excited at 488 nm, showed
4 times enhanced ROX emission to that of single ROX dye. In order to have a four-
color set, FAM cassette 5 was conjugated independently with 5-TAMRA, 5-REG
and 5-R110 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.
According to the MM+/MO calculations, the relative spatial orientation of the
donor to acceptor dyes in these ET cassettes was shown to be parallel, which should
be optimal for FRET (9). 1H-NMR, 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.
Figure 1. Blue Argon-laser (448 nm) excited, normalized fluorescence emissions of the four-color
set 10–13 in 1 X TBE 8.0 M urea.

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365

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NAMPALLI ET AL.
Initial attempts to conjugate activated ET cassettes 6–9 (Scheme 2) with
alkynylamino 11- ddNTPs (11,12) using a large variety of activating reagents such
as TSTU/DIPEA, DCC/HOSu, HBTU, HATU, HOBt, PyBOP, PyBrOP, EDAC/
HOSu, and TFA-HOSu, at different temperatures produced low yields of the de-
sired ET terminators. However, employing DSC/DMAP/DMF/−60◦C conditions
to generate NHS esters of the acid functionalities in 6–9, and in situ conjugation
with 11-ddNTPs (11-ddCTP, 11-ddATP, 11-ddUTP, and 11-ddGTP) at −30◦C pro-
vided the desired ET dye terminators (13) 10, 11, 12, and 13, respectively, in yields
ranging from 15–20%. The normalized fluorescence emission spectra for these ET
terminators are given in Figure 1. The fluorescence emission enhancement rates of
these ET terminators compared to the corresponding single dye terminators were
found to be 18 (10), 6.5 (11), 5 (12), and 1.6 (13).
Of the many possible combinations of ET terminators, 6 different four-color
sets were synthesized and tested in the DNA sequencing experiments. DNA se-
quencing performed employing the four-color set of 10–13 with a variety of DNA
templates and Thermo SequenaseTM II DNA polymerase (14), found to be of high
quality with read-lengths in excess of 1000 base-pairs (Fig. 2). The four-color
FRET terminators’ kit has the demonstrated ability to be easily used on capillary
electrophoresis (APB’s MegaBACETM 1000 and ABI’s 310 and 3700) and slab gel
(ABI’s 373 and 377) based automated DNA sequencers. In terms of high throughput
sequencing results, using the capillary electrophoresis based MegaBACE (15), one
can sequence about 864 DNA samples to identify in excess of 0.5 million base-pairs
in 18 hrs.
CONCLUSION
A novel synthetic route for the four-color 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 DNA polymerase.
In addition, the FRET cassettes developed in this study have the potential of labeling
other reactive biological molecules, such as oligonucleotides and proteins.
ACKNOWLEDGMENTS
The authors wish to thank Inna Livshin, Jim Holecek for initial HPLC exper-
iments, Drs. Mark McDougall and C. Y. Chen for Mass and fluorescence spectral
analysis.
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