A tetramethyl rhodamine (Tamra) phosphoramidite facilitates solid-phase-supported synthesis of 5′-Tamra DNA

Article abstract of DOI:10.1021/jo0011134

5-Carboxy Tamra 1 was conjugated to 4-hydroxypiperidine with BOP and N-methylmorpholine, and the resulting 5-(N-pipyridyl-4-hydroxy)-Tamra carboxamide 2 was treated with 2-cyanoethyl tetraisopropylphosphorodiamidite to give 5-[N-pipyridyl-4-O-(2-cyanoethyl diisopropylphosphoramidite)]-Tamra carboxamide 3. Solutions of 3 were coupled onto the 5′-hydroxyl of solid-phasesupported DNA fragments with standard amidite coupling techniques. Cleavage and deprotection with aqueous tert-butylamine cocktail gave 5-Tamra-functionalized DNA as well as an additional compound without the Tamra chromophore. A mass spectrum of this product showed the incorporation of tert-butylamine. The extra product was completely suppressed by including a 5 min acetylation step after coupling. A model study of 3 coupled onto thymidine-functionalized CPG showed similar results. NMR and mass spectra of cleaved products confirmed the addition of tertbutylamine to the minor product. Coupling a Tamra active ester onto T CPG which was previously coupled with N-(4-methoxytrityl)piperidyl-4-O-(2-cyanoethyl diisopropylphosphoramidite) 4 produced the same major Tamra-bearing product, which coeluted on reverse phase HPLC with the major product generated with 3.

Full text of DOI:10.1021/jo0011134

J . Org. Chem. 2000, 65, 9033-9038
9033
A Tetr a m eth yl Rh od a m in e (Ta m r a ) P h osp h or a m id ite F a cilita tes
Solid -P h a se-Su p p or ted Syn th esis of 5′-Ta m r a DNA
Matthew H. Lyttle,* Timothy G. Carter, Daren J . Dick, and Ronald M. Cook
Biosearch Technologies, Inc., 81 Digital Drive, Novato, California 94949
matt@biosearchtech.com
Received J uly 24, 2000
5-Carboxy Tamra 1 was conjugated to 4-hydroxypiperidine with BOP and N-methylmorpholine,
and the resulting 5-(N-pipyridyl-4-hydroxy)-Tamra carboxamide 2 was treated with 2-cyanoethyl
tetraisopropylphosphorodiamidite to give 5-[N-pipyridyl-4-O-(2-cyanoethyl diisopropylphosphora-
midite)]-Tamra carboxamide 3. Solutions of 3 were coupled onto the 5′-hydroxyl of solid-phase-
supported DNA fragments with standard amidite coupling techniques. Cleavage and deprotection
with aqueous tert-butylamine cocktail gave 5-Tamra-functionalized DNA as well as an additional
compound without the Tamra chromophore. A mass spectrum of this product showed the
incorporation of tert-butylamine. The extra product was completely suppressed by including a 5
min acetylation step after coupling. A model study of 3 coupled onto thymidine-functionalized CPG
showed similar results. NMR and mass spectra of cleaved products confirmed the addition of tert-
butylamine to the minor product. Coupling a Tamra active ester onto T CPG which was previously
coupled with N-(4-methoxytrityl)piperidyl-4-O-(2-cyanoethyl diisopropylphosphoramidite) 4 produced
the same major Tamra-bearing product, which coeluted on reverse phase HPLC with the major
product generated with 3.
In tr od u ction
compounds was divulged. More recently, a paper from
this same group13 gainsays the utility of these compounds
because of stability problems, and presents a technique
for Tamra free acid coupling to DNA, again functionalized
first with an amine phosphoramidite, on solid supports.
We present the synthesis and techniques for use of a
stable Tamra phosphoramidite, which is compatible with
modern automated oligonucleotide synthesis methods.
The material is as easy to make and use as the analogous
fluorescein amidite.10
Development of high performance oligonucleotide
labeling methods which are robust enough to work well
on automated synthesizers still presents a significant
challenge. Attachment of labels to the 5′ terminus of DNA
has been traditionally done in solution1-3 with nitrogen
or sulfur nucleophiles pendant on the DNA previously
incorporated as 5′ modifications.4-6 A variety of activation
chemistries have been developed to couple labels to these
nucleophiles.7-9 The reactions are generally slow and
require chromatography to remove excess labeling re-
agents from the desired products. The added ease of
fluorophore coupling to DNA still anchored on a solid
support, as well as obviation of the 5′-amine amidite
coupling, has been accomplished in the case of fluores-
cein10,11 by the use of fluorescein amidites. Although these
compounds have become widely used, the analogous
rhodamine compounds have yet to become commercially
available. The concept of rhodamine amidites has been
put forth in the patent literature;12 however, little
information regarding the synthesis and use of the
Exp er im en ta l Section
Ma ter ia ls a n d Meth od s. Pyridine, methanol (MeOH),
ethyl acetate (EtOAc), dimethylformamide (DMF), dichlo-
romethane (CH2Cl2), and acetonitrile (CH3CN) were Omnisolve
grade from VWR. Tetrahydrofuran (THF), sodium bicarbonate
(NaHCO3), N-methylimidazole, N-methylmorpholine, 4-hy-
droxypiperidine, ammonium acetate, chromatographic alu-
mina, acetic anhydride (Ac2O), tert-butylamine, concd HCl,
4-methoxytrityl chloride, diisopropylamine, and magnesium
sulfate (MgSO4) were from Aldrich; 2-cyanoethyl tetraisopro-
pylphosphorodiamidite was obtained from Chemgenes. Ben-
zotriazol-1-yl-oxy-tris(dimethylamino)-phosphonium hexa-
fluorophosphate (BOP) was obtained from Chem Impex. 5- and
6-Carboxy Tamra and 5-carboxy Tamra-OSu active ester were
made and purified in house and can also be obtained from
Molecular Probes. Tetrazole and S-ethyltetrazole were ob-
tained from AIC. DNA syntheses were performed on a Biose-
arch 8750 synthesizer with Cruachem DNA amidites. All other
DNA synthesis reagents were the same as those previously
reported.14 NMR was performed by Acorn NMR (Livermore,
CA). Elemental analyses were performed by Desert Analytics
(Tucson, AZ). High-resolution mass spectra were performed
at the UC Berkeley Department of Chemistry MS lab. MALDI
mass spectra were performed in-house on a Finnigan Laser-
Mat.
(1) Khanna, P. L.; Ullman, E. F. Anal. Biochem. 1980, 108, 156.
(2) Curtis, S. K.; Cowden, R. R. Histochemistry 1980, 68, 23.
(3) Urdea, M. S.; Warner, B. D.; Running, J . A.; Stempien, M.; Clyne,
J .; Horn, T. Nucleic Acids Res. 1988, 16, 4937.
(4) Sinha, N. D.; Cook, R. M. Nucleic Acids Res. 1988, 16, 2659.
(5) Connolly, B. A.; Rider, P. Nucleic Acids Res. 1985, 13, 4485.
(6) Chollet, A.; Kawashima, E. H. Nucleic Acids Res. 1985, 13, 1541.
(7) van der Laan, A. C.; Brill, R.; Kuimelis, R. G.; Kuyl-Yeheskiely,
E.; van Boom, J . H.; Andrus, A.; Vinayak, R. Tetrahedron Lett. 1997,
38, 2252.
(8) Adamczyk, M.; Fishpaugh, J . R.; Heuser, K. J . Bioconjugate
Chem. 1997, 8, 255.
(9) Mujumdar, S. R.; Mujumdar, R. B.; Grant, C. M.; Waggoner, A.
S. Bioconjugate Chem. 1996, 7, 356.
(10) Brush, C. US Patent 5,371,241, 1994.
(11) Adamczyk, M.; Chan, C.; Fino, J .; Mattingly, P. J . Org. Chem.
2000, 65, 596.
(12) Woo, S. L.; Menchen S. M.; Fung, S. US Patent 5,231,191, 1993.
(13) Vinayar, R. Tetrahedron Lett. 1999, 40, 7611.
(14) Wang, W. W.; Lyttle, M. H.; Borer, P. N. Nucleic Acids Res.
1990, 18, 3347.
10.1021/jo0011134 CCC: $19.00 © 2000 American Chemical Society
Published on Web 12/07/2000

9034 J . Org. Chem., Vol. 65, No. 26, 2000
Lyttle et al.
5-(N-P ipyr idyl-4-h ydr oxy)-Tam r a car boxam ide 2. 5-Car-
boxy Tamra 1, 11 g (26 mmol), was dissolved in 300 mL of
DMF. BOP (13 g, 29 mmol) was added, along with 4 mL of
N-methylmorpholine. The solution was magnetically stirred
for 15 min, and 4-hydroxypiperidine (11 g, 109 mmol) was
added. The deep red solution was stirred for 3 h, and 20 g of
NH4OAc dissolved in 50 mL of water was added. The DMF
was removed by rotary evaporation, with a bath temp < 40
°C. The residue was dissolved in 700 mL of CH2Cl2 and washed
with 700 mL of 1 N HCl. The organic phase was dried over
MgSO4, filtered, and evaporated. A column 7 × 30 cm of
alumina, previously wetted with 7 wt % water, was packed in
5% methanol in CH2Cl2. The crude product was dissolved in
200 mL of this solvent mixture and loaded onto the column. A
gradient to 15% MeOH was run over 6 L of mobile phase, and
deeply colored fractions were checked by TLC. Those contain-
ing pure product (0.5 Rf, 20% MeOH/2% pyridine/CH2Cl2) were
pooled and evaporated to give 6.2 g (46% yield) of 2 as a dark
solution was washed twice with 600 mL of satd NaHCO3 and
once with 600 mL of brine. The organic phase was dried over
MgSO4 and evaporated to a foam. Column chromatography
was performed on a 25 × 5 cm bed of silica initially packed
with 5:5:90 TEA:EtOAc:petroleum ether, and then eluted with
1 L of 5:5:90 TEA:EtOAc:petroleum ether, with 1 L of 5:25:70
TEA:EtOAc:petroleum ether, with 1L 5:50:45 TEA:EtOAc:
petroleum ether, then finally with 5:70:25 TEA:EtOAc:petro-
leum ether. Pure fractions were pooled and evaporated to give
1
26.5 g (77% yield) of 4. H NMR (CDCl3, δ): 7.5 (d, 4H), 7.35
(d, 2H), 7.2 (m, 4H), 7.1 (dd, 2H), 6.8 (d, 2H), 3.8 (s, 3H), 3.7
(m, 1H), 3.5 (m, 1H), 2.5 (m, 1H), 2.0-1.8 (m, 6H), 1.2-1.0
(dd, 12H). 31P NMR (PPM, CDCl3:) 146.2. MALDI m/e (fluo-
rescein matrix) calcd 573.5; found 573.5. Anal. Calcd for
C
39H49N5O6P: C, 71.18; H, 7.73; N, 7.32. Found: C, 71.22; H,
7.79; N, 7.30.
P r ep a r a tion of Mod el Com p ou n d s
1
solid. H NMR (DMSO-d6, δ): 8.0 (d, 1H), 7.7 (d, 1H), 7.2 (s,
Gen er a tion of T Mon on u cleosid e Ta m r a Ad d u ct
6 Usin g 3 (See F igu r e 4). T CPG, 10 g, was placed in a
150 mL coarse frit sintered glass funnel atop a 1 L
sidearm flask. Dichloroacetic acid (3%) in CH2Cl2 (100
mL) was poured in, and the support was agitated briefly
with a spatula. The solution became brightly orange.
After 2 min, the solution was drained, and the step was
repeated twice with 100 mL of fresh 3% dichloroacetic
acid in CH2Cl2. The support was then washed three times
with 100 mL of CH2Cl2, twice with 100 mL of CH3CN,
and once with 100 mL of pyridine. The support was
transferred into a 250 mL round-bottom flask, and 1 g
of 3 was added. Dry pyridine (40 mL) was added, and
the solvent was removed by rotary evaporation. A plug
of glass wool was used to keep the CPG from leaving the
flask. Once dry, high vacuum was applied to the flask
for 18 h. A solution of 100 mL of 0.4 M s-ethyltetrazole
in dry acetonitrile was prepared, and enough of this was
added to the flask containing the CPG to make a slurry.
The slurry was allowed to stand for 15 min and was
poured into a sintered glass funnel. The support was
washed three times with 100 mL of CH3CN, and then
60 mL of amidite oxidizer solution14 was added. After 5
min, the support was washed three times with 100 mL
of CH3CN and dried. For cleavage of the T mononucle-
otide conjugate from the support, the support was placed
in a 500 mL round-bottom flask fitted with a condenser,
and a mixture of 20 mL water and 60 mL of tert-
butylamine was added. The mixture was heated to a
gentle reflux overnight. The solution was cooled and
filtered, and the dark purple solution was concentrated
by rotary evaporation. The two component products were
separated by reverse phase column chromatography as
below. tert-Butylamine phosphate counterions were re-
moved by treatment with Dowex 50WX8-400 anion
exchanger which had been previously treated with aque-
ous ammonia. High-resolution mass spectrum of early,
major product 6 calcd for MH+ C40H45N5O12P ) 818.28036;
found 818.280730. The later product 6, containing tert-
butylamine, lost an OH- upon ionization, calcd for M+
(-OH-) C44H54N6O11P ) 873.358812; found 873.358970.
Gen er a tion of T Mon on u cleosid e Ta m r a Ad d u ct
6 Usin g 4 F ollow ed by 5-Ta m r a OSu (see F igu r e 4).
T CPG (10 g) was detritylated and washed as above, and
1 g of 4 was coupled onto the support in the same fashion
as 3. The detritylation procedure was repeated to remove
the MMT group, and the support was washed with two
100 mL washes of 10% triethylamine in CH2Cl2 to
generate the unprotonated amine. 5-Tamra OSu (1 g) was
mixed with 80 mL of DMF and added to the CPG in a
1H), 6.5 (m, 6H), 4.75 (s, 1H), 3.9 (m, 1H), 3.7 (m, 1H), 3.3 (s,
12H), 3.2-3.0 (m, 2H), 1.75 (m, 1H), 1.55 (m, 1H), 1.35 (M,
1H), 1.2 (m, 1H). MALDI m/e calcd 513.6, found 512.1. Anal.
Calcd for C22H31N3O5‚H2O: C, 67.78; H, 6.25; N, 7.90. Found:
C, 68.18; H, 5.85; N, 8.07.
5-[N-P ip yr id yl-4-O-(2-cya n oeth yld iisop r op ylp h osp h o-
r a m id ite)]-Ta m r a Ca r boxa m id e 3. Compound 2 (5.3 g, 10.3
mmol) was dried by rotary evaporation with 100 mL of dry
pyridine. A high vacuum was then applied to the material for
1 h. A solution of 3 g (10 mmol) of 2-cyanoethyl tetraisopro-
pylphosphorodiamidite and 180 mg of tetrazole were mixed
in 40 mL of dry acetonitrile, and the solution was added to
the flask containing 2. After swirling briefly to dissolve solids,
the deep red solution was allowed to stand 3 h. An aliquot for
TLC was prepared with about 1 mL of CH2Cl2 and 1 mL of
aqueous NaHCO3. A few drops of the reaction mixture were
added, and the lower layer was spotted on a TLC plate and
eluted with 20% MeOH/CH2Cl2/2% pyridine. Complete conver-
sion was observed (product Rf ) 0.7). The solution was stripped
and dissolved in 200 mL of CH2Cl2 and then washed with 100
mL of satd aqueous NaHCO3. The organic phase was dried
over MgSO4, filtered, and evaporated. The solid was further
dried by coevaporation with 100 mL of dry pyridine followed
by 100 mL of dry acetonitrile. The yield was 5.3 g, 72%. An
analytical sample was prepared by chromatography with the
same conditions as those used to purify 2, except that the
mobile phase included 2% pyridine. 31P NMR (PPM, CDCl3:)
147.4, 147.1. MALDI m/e calcd: 713.6, Found: 713.7. Anal.
Calcd for C39H49N5O6P: C, 65.53; H, 6.91; N, 9.80. Found: C,
65.83; H, 6.60; N, 10.19.
N-(4-Meth oxytr ityl)p ip er id yl-4-O-(2-cya n oeth yld iiso-
p r op ylp h osp h or a m id ite) 4. 4-Hydroxypiperidine (8 g, 79
mmol) was coevaporated twice with 100 mL of dry pyridine
and redissolved in 150 mL of dry CH2Cl2 under argon.
Diisopropylamine (26.5 mL) was added, followed by dropwise
addition of 4-methoxytrityl chloride (25 g, 80 mmol) dissolved
in 150 mL of dry CH2Cl2. After overnight stirring, the solution
was checked by TLC (1:2 acetone/petroleum ether, product Rf
0.75 with 10% H2SO4 spray and heat), and the conversion was
found to be complete. The mixture was washed three times
with 300 mL of satd aqueous NaHCO3 and then 400 mL of
brine. The organic phase was dried over MgSO4 and reduced
to a foam by rotary evaporation. Column chromatography was
performed on a 20 × 5 cm bed of silica initially packed with 2
L of 1:2 EtOAc:petroleum ether with 1% TEA, and then eluted
with 2 L of 1:2 EtOAc:petroleum ether with 1% TEA, and
finally 2:1 EtOAc:petroleum ether with 1% TEA. Pure fractions
were pooled and evaporated to give 22.5 g (76% yield) of N-(4-
methoxytrityl)-4-hydroxypiperidine. To all of this was added
a premixed solution of 18 g (60 mmol) of 2-cyanoethyl tetra-
isopropylphosphorodiamidite and 1.08 g of tetrazole in 300 mL
of dry acetonitrile. The solution was allowed to stand for 3 h,
whereupon TLC (5:25:70 TEA:petroleum ether:EtOAc, Rf
product 0.66, starting alcohol 0.5) showed complete conversion.
The solution was reduced to a tar by rotary evaporation, and
then the residue was redissolved in 700 mL of CH2Cl2. The

Synthesis of 5′-Tamra DNA
J . Org. Chem., Vol. 65, No. 26, 2000 9035
F igu r e 1. 5-Tamra isomers with 3-carboxylate in lactone form (1A) or as carboxylate (1B). Schematic for reaction of tert-butylamine
with 1A and possible mechanism for prevention by acetylation step. R ) N-piperidyl-4-O-phosphoryl 5′ DNA or thymidine.
250 mL Erlenmeyer flask with stopper. After 18 h, the
slurry was poured into 150 mL coarse sintered glass
funnel atop a 1 L sidearm flask. The support was washed
three times with 100 mL of CH3CN and dried. Cleavage
proceeded as above.
byproduct. For the mononucleotide model compounds, a
5 × 20 cm column of Toso-Haas Amberchrom 50-100
µm packing was used with the same sequence of mobile
phase elutions. MALDI Mass spectra of the T-10 products
were performed according to Ball and Packmann.15
DNA Syn th esis. DNA was made on Biosearch 8750
DNA synthesizers with standard conditions as previously
described.14 dGDMF was used to ensure complete depro-
tection of dG during deprotection with the Tamra depro-
tection cocktail, which was 1:3 water:tert-butylamine.
Test sequences 5′-TTTTTTTTTT-3′, 5′-CGATCTGAAT-
AGCTT-3′ and 5′-TCCAGCTCATCGAGGTCATA-3′ were
made at 0.2, 1.0, and 1.0 µmol synthesis scales, respec-
tively, on standard CPG supports. The final DMT was
removed, and after thorough washing a solution of 200
µL of 50 mg/mL of 3 in dry acetonitrile and 200 µL of 0.4
M S-ethyltetrazole in dry acetonitrile were introduced
onto the column by the DNA synthesizer. After 10 min,
the coupling solution was washed off the column with
acetonitrile, and oxidation was performed. Next, a mix-
ture of acetic anhydride, N-methylimidazole and THF
(1:1:8) was automatically introduced to the column. After
5 min exposure, the acetylation solution was rinsed with
acetonitrile and the CPG containing the 5′-Tamra-labeled
DNA was washed thoroughly with acetonitrile. The CPG
was placed into screw cap eppendorf tubes and 1 mL of
the Tamra deprotection cocktail added to each. The tubes
were heated at 55° C for 18 h and cooled and the purple
solutions evaporated.
Anion-exchange HPLC analyses were performed as
follows: 2-20 µL of the aqueous samples, depending on
the concentration, was injected onto a Dionex anion-
exchange column (4.6 × 250 mm); samples were eluted
at 2 mL/min with aqueous buffers of (A) 0.025 M Tris‚
HCl and 0.01 M Tris, and (B) 0.025 M Tris‚HCl, 0.01 M
Tris, and 1.0 M NaCl using a linear gradient of 1:0 to
0:1 over 20 min, with UV detection at 260 nm. The
detector was a Waters 996 photodiode array. Samples for
base composition analysis were treated as previously
described,16 with analysis by reverse phase HPLC as
follows: 20 µL of the aqueous sample was injected onto
a HAISIL HL C18 5 µm column (4.6 × 150 mm); samples
were eluted at 1 mL/min with buffers of (A) 0.1M TEAA,
5% acetonitrile, (B) acetonitrile, with a linear gradient
of 1:0 to 0:1 over 20 min. UV detection at 260 nm.
Resu lts a n d Discu ssion
The key to understanding the complex chemistry
exhibited by the rhodamine dyes lies in the disposition
of the 3-carboxylate. The various species present at
various pHs probably follow the scheme established for
fluorescein17 and is either strongly violet colored or not
depending on the disposition of the 3-carboxylate18 (see
Figure 1, 1A and 1B). The two forms also exhibit marked
differences in solubility characteristics, in our hands. The
Reverse phase separation of 5′-Tamra-TTTTTTTTTT-
3′ and additional products made without the post oxida-
tion acetylation step was accomplished by first equili-
brating a Biosearch Micropure II cartridge with aceto-
nitrile and then water and then loading a solution of 30
O.D. units crude DNA onto the column in 0.5 mL of
water. Acetonitrile (20%) in water eluted the first product
as a bright pink solution, and then 50% acetonitrile in
water gave the second, less-colored band containing the
(15) Ball, R. W.; Packman, L. C. Anal. Biochem. 1997, 246, 185.
(16) Wheatly, J . B.; Lyttle, M. H.; Hocker, M. D.; Schmidt, D. E. J .
Chromatogr. A. 1996, 77.
(17) Sun, W.; Gee, K. R.; Klaubert, D. H.; Haugland, R. P. J . Org.
Chem. 1997, 62, 6469.
(18) Ramette, R. W.; Sandell, E. B. J . Am. Chem. Soc. 1956, 78, 4872.

9036 J . Org. Chem., Vol. 65, No. 26, 2000
Lyttle et al.
F igu r e 2. Synthesis of Tamra amidite 3.
reactivity of the 3-carboxylate toward reagents used to
functionalize the 5- or 6-carboxylates and deprotect
Tamra-tagged DNA probably determines the quantity
and character of side reaction products formed. We
believe that the synthesis and use of the Tamra deriva-
tive below demonstrate these principles in action.
Syn th esis of Ta m r a Am id ite 3. The first reaction
with Tamra was the formation of an amide with a
bifunctional amine containing a hydroxyl for later at-
tachment of the phosphoramidite group. Linear variants,
6-amino-1-hexanol and 2-(2-aminoethoxy)ethanol, both
gave many products, according to TLC. 4-Hydroxypip-
eridine gave much better results; mostly a single product
was formed. Extraction of the Tamra amide 2 proved
tricky; usually one extracts organic solvent solutions of
crude BOP amide formation products19 with mild base
such as satd NaHCO3 to remove the hydroxybenzotri-
azole and other unwanted aqueous base soluble materi-
als. In this case, the product 2 was quite soluble in the
aqueous layer. Extraction instead with 1 N HCl gave
satisfactory results; very little Tamra amide 2 was
observed in the aqueous layer. Column chromatography
on damp alumina gave 46% yield of 2, which was quite
pure by NMR and had a good elemental analysis. After
thorough drying, the Tamra amide alcohol 2 was con-
verted into the amidite 3 by reaction with a stoichiomet-
ric amount of 2-cyanoethyl tetraisopropylphosphorodia-
midite and a catalytic amount of tetrazole. Unreacted
compound 2 was easily removed by washing an ethyl
acetate solution of the product with aqueous NaHCO3.
The product amidite 3 was dried by stripping from dry
pyridine and then acetonitrile and high vacuum. The
yield was over 70% for this step. Chromatography of the
final product was unnecessary; no increase in activity was
seen upon purification (see Figure 2).
F igu r e 3. Anion exchange (AX) HPLC of: A, 5′-Tamra-
TTTTTTTTTT-3′ without post-coupling acetylation step; B,
same sequence with 5 min with post-coupling acetylation step.
product peak with the major product present in about
90% purity (see Figure 3, lower panel).
The unacetylated DNA sample containing the two
products was subjected to reverse phase chromatography
(see Figure S1), and mass spectral analyses were per-
formed on each separated component. The earlier, major
peak had the correct mass for the desired conjugate (calcd
3553.6, found 3555.6 amu), while the later, minor peak
had a molecular weight of the desired product plus 73.1
amu (calcd 3626.8, found 3628.3), within experimental
error ((0.2%) for the addition of tert-butylamine, the
amine present in the deprotection cocktail, as demon-
strated in Figure S2.
To more accurately explore the tert-butylamine addi-
tion phenomena, 3 was coupled onto T CPG and the
resulting mononucleotide 5′-Tamra product 5 cleaved,
purified, and analyzed by reverse phase HPLC (see
Figure 4). As with the 5′-Tamra T-10 study discussed
above, two products were observed when the coupled
CPG was treated with aqueous tert-butylamine and heat.
When the CPG bearing the oxidized Tamra T mono-
5′-Ta m r a DNA Syn th esis. Satisfactory (over 95%)
coupling efficiencies were obtained either by manual
syringe coupling methods20 or with automated coupling.
Ten milligrams of 3 dissolved in 200 µL of acetonitrile
was used. When the CPG immobilized sequence 5′-
TTTTTTTTTT-3′ was treated with an acetonitrile solu-
tion of 3 and activator, followed by oxidation and then
cleavage from the CPG and deprotection in tert-butyl-
amine and water, two products were observed by AX
HPLC, as shown in Figure 3 (upper panel). When a 5
min acetylation step (Ac2O, base) was included after the
amidite oxidation, cleavage, deprotection, and AX HPLC
analysis revealed complete suppression of the minor
(19) Hudson, D. J . Org. Chem. 1988, 53, 617.
(20) Lyttle, M. H.; Adams, H.; Hudson, D.; Cook, R. M. Bioconjugate
Chem. 1997, 8, 193.

Synthesis of 5′-Tamra DNA
J . Org. Chem., Vol. 65, No. 26, 2000 9037
F igu r e 4. Solid-phase-supported synthesis of 5′-Tamra-linked Thymidine mononucleotide model compounds.
nucleotide 5 was subjected to a 5 min acetylation step,
as above, only the major product was observed. The two
components generated by the unacetylated synthesis
were separated by reverse phase chromatography and
subjected to NMR analysis. A large singlet resonance at
δ 1.2 with an area of 9 protons was seen in the minor
product whereas only minimal absorbances were seen in
the major mononucleotide adduct in that region of the
NMR spectrum, given in Figure S3. High-resolution mass
spectra of each product confirm the molecular formulas,
although the minor product appeared to lose an OH group
during ionization (see Figure 1, where R ) 5′-(4-O-
piperidyl phosphate)thymidine). The NMR and mass
spectra confirmed the addition of tert-butylamine to the
minor product. The aromatic region of each NMR spec-
trum of both the major and minor Tamra mononucleotide
model compounds was similar, with no loss of relative
area, which dispels the possibility of aromatic substitu-
tion by tert-butylamine. As well, the minor byproduct
containing the extra mass did not show the characteristic
(λmax 556 nm) Tamra spectrum in either the T-10 DNA
or the model studies. An added tert-butylamine at the
3-carboxylate would force the lower ring out of the plane
of the xanthene nucleus and disrupt the full conjugation
needed to show the Tamra spectrum. The λmax of
the minor tert-butylated byproducts was ∼240 nm, close
to that of unsubstituted N,N-dimethylanaline. On the
F igu r e 5. AX HPLC of unpurified 5′ Tamra DNA made with
3. A ) 5′-Tamra-CGATCTGAATAGCTT-3′, B ) 5′-Tamra-
TCCAGCTCATCGAGGTCATA-3′.
basis of the evidence above, we believe that tert-buty-
lamine is adding to the lactone form of the 3-carboxylate
during the cleavage and deprotection step as shown in
Figure 1.
specifically by an amine nucleophile.21 Further support
for the structure of 6, and also a comparison of the
products generated through Tamra amidite coupling
versus Tamra active ester addition, was given by the
alternate synthesis shown in Figure 4. In this case N-(4-
methoxytrityl)piperidinyl-4-O-(2-cyanoethyl diisopropy-
lphosporamidite) 4 was first added onto T CPG. After
oxidation and washing, the MMT group was removed,
and then Tamra-OSu was added to the amine. The major
Tamra-containing products from both syntheses were
A possible mechanism for the curtailment of byproduct
formation by acetylation is that the two configurations
of the Tamra 3-carboxylate are in rapid equilibrium, and
that the free 3-carboxylate (structure 1B, Figure 1) is
acetylated, forming a mixed anhydride. The equilibrium
is thus shifted until no more of the vulnerable lactone
form is present. The mixed anhydride can then only
interact with tert-butylamine to regenerate the unreac-
tive 3 carboxylate anion; recall mixed anhydride amide-
forming reactions in peptide chemistry where the least
hindered side of a mixed anhydride is functionalized
(21) Vaughn, J . R.; Osato, R. L. J . Am. Chem. Soc. 1951, 73, 5553.

9038 J . Org. Chem., Vol. 65, No. 26, 2000
Lyttle et al.
isolated by chromatography, and HPLC analysis showed
identical compounds by coelution (see Figure S4). This
should allay any fears about dissimilar Tamra DNA
conjugates being generated by amidite or active ester
couplings.
Con clu sion
Results have been presented regarding the preparation
and use of a new Tamra amidite. Improved product DNA
conjugate quality has been demonstrated by the use of a
post-coupling acetylation step. Detailed spectral studies
of DNA conjugates and model compounds show that tert-
butylamine can add to the Tamra to generate a stable
byproduct, and that the position of addition is most likely
across the lactone ring. The studies also show that
identical compounds can be generated from Tamra amid-
ite and Tamra-OSu active ester couplings to the ap-
propriate substrates.
Two heterogeneous sequences, 5′-CGATCTGAAT-
AGCTT-3′ and 5′-TCCAGCTCATCGAGGTCATA-3′ were
5′ labeled on the CPG support with 3, followed by
oxidation and acetylation as described above. All ma-
nipulations were automatically executed by the DNA
synthesizer. Figure 5 shows AX HPLC of these unpurified
5′-Tamra 15 and 20 mers. PDA (photodiode array) spectra
of each major product peak showed the presence of Tamra
in the products (See Figure S5). Tamra amidite solutions
on the DNA synthesizer retained equivalent to fresh
activity after 7 days. Following 18 h of heating (55 °C)
in tert-butylamine/water and evaporation, a digest of each
sequence with phosphodiesterase II, followed by alkaline
phosphatase, showed the absence of any modified bases
which could be due to the added treatments (data not
shown). GDMF was used to allow effective G deprotection22
in the milder (than aqueous ammonia) Tamra cocktail.
Tamra amidite coupling to 5′-OH DNA immobilized on
a solid support will be more efficient and result in cleaner
products than those for 5′ labeling methods previously
reported, i.e., that of using first a 5′ amine functional-
ization amidite followed by active ester coupling. We hope
that these methods will facilitate the development and
use of other rhodamine fluorophores in DNA labeling.
Su p p or tin g In for m a tion Ava ila ble: Figures S1-S5 are
available free of charge via the Internet at http://pubs.acs.org.
(22) Vu, H.; McCollum, C.; J acobsen, K.; Theilsen, P.; Vinayak, R.;
Spiess, E.; Andrus, A. Tetrahedron Lett. 1990, 31, 7269.
J O0011134