Diels-Alder bioconjugation of diene-modified oligonucleotides

Article abstract of DOI:10.1021/jo0100190

In an effort to offer complementary technology for covalent biomolecule modification (bioconjugation), we have developed a method that exploits the aqueous acceleration of Diels-Alder reactions for this purpose. Three different diene phosphoramidite reagents have been synthesized that enable diene modification of synthetic oligonucleotides prepared by the phosphoramidite method. Clean and efficient Diels-Alder cycloaddition of these diene oligonucleotides with maleimide dieneophiles was carried out, and the labeled oligonucleotide bioconjugates were characterized by HPLC and electrospray mass spectrometry. Dieneophile stoichiometry, temperature, and pH are all parameters that were shown to influence the efficiency of the process.

Full text of DOI:10.1021/jo0100190

5352
J . Org. Chem. 2001, 66, 5352-5358
Diels-Ald er Biocon ju ga tion of Dien e-Mod ified Oligon u cleotid es
Kenneth W. Hill,*,†,‡ J on Taunton-Rigby,† J effrey D. Carter,§ Eric Kropp,† Kurt Vagle,§
Wolfgang Pieken,§ Danny P. C. McGee,† Gregory M. Husar,§ Michael Leuck,§
Dominic J . Anziano,§ and David P. Sebesta*,§
NeXstar Pharmaceuticals, 2860 Wilderness Place, Boulder, Colorado 80301, and Proligo, LLC,
2995 Wilderness Place, Suite 207, Boulder, Colorado 80301
dsebesta@proligo.com
Received J anuary 5, 2001
In an effort to offer complementary technology for covalent biomolecule modification (bioconjugation),
we have developed a method that exploits the aqueous acceleration of Diels-Alder reactions for
this purpose. Three different diene phosphoramidite reagents have been synthesized that enable
diene modification of synthetic oligonucleotides prepared by the phosphoramidite method. Clean
and efficient Diels-Alder cycloaddition of these diene oligonucleotides with maleimide dieneophiles
was carried out, and the labeled oligonucleotide bioconjugates were characterized by HPLC and
electrospray mass spectrometry. Dieneophile stoichiometry, temperature, and pH are all parameters
that were shown to influence the efficiency of the process.
In tr od u ction
otide bioconjugates containing halide radioisotopes have
been employed in pharmacological and imaging studies.5
Commonly employed methods for oligonucleotide bio-
conjugate construction include the introduction of the
chemical modifier during solid-phase synthesis via the
phosphoramidite method or by post-solid-phase synthesis
condensation with reactive handles introduced during
automated synthesis. While the former approach involves
fewer steps and in principle requires only a single
purification, the preparation of labeled oligonucleotides
by direct solid-phase synthesis can be complicated by
molecular label incompatibility with synthesis conditions
as well as a relatively limited selection of readily avail-
able phosphoramidite reagents corresponding to the
desired conjugate moieties. Post-solid-phase synthesis
modification is one of the more commonplace methods
for the derivatization of oligonucleotides, and condensa-
tion of 5′- or 3′-primary alkylamine modified oligonucle-
otides with electrophilic derivatives of the molecular
labels has seen widespread use.6 Advantages of amino
oligonucleotide bioconjugation techniques include ready
availability of these derivatized oligomers and amine
reactive labels of interest; however, these methods suffer
from competing reagent hydrolysis and potential cross
reactivity with oligonucleotide functionality. Other fre-
quently employed oligonucleotide bioconjugation methods
employ the coupling of an alkyl mercaptan derivatized
oligonucleotide with an R-haloacetyl,7 a maleimide,8 or
an activated disulfide, such as a pyridyl disulfide.9
Michael addition of mercaptan oligonucleotides to male-
imides are particularly facile; however, the necessity of
The capacity of oligonucleotides to assemble into
complementary duplex structures with target nucleic acid
sequences via the formation of Watson-Crick base pairs
has led to a rich variety of technologies exploiting this
property and, in conjunction with dramatic advances in
information technologies, has contributed to unprece-
dented progress in the biotechnological arena.1 In a
general sense, covalently attached reporter groups on
oligonucleotide probes enable quantitative information
to be derived about the presence of a given nucleic acid
target sequence. In applications related to nucleic acid
sequencing, differential gene expression measurement,
and genotyping, oligonucleotides covalently modified with
one or more reporter groups (including haptens such as
biotin2 or organic fluorophores and/or fluorescence quench-
ers such as fluorescein and Cy3)3 enable the derivation
of genetic information in an ever more comprehensive
and operationally streamlined fashion.
Additionally, covalent attachment of poly(ethylene
glycol), peptides, or lipid modifiers to oligonucleotide
based active pharmaceutical ingredients has led to
improved activity and pharmacokinetic profiles for ex-
amples from this class of clinical candidates.4 Oligonucle-
* To whom correspondence should be addressed.
† NeXstar Pharmaceuticals.
‡ Current address: Somalogic, 1775 38th Street, Boulder, CO 80308.
§ Proligo.
(1) For examples, see: (a) Microarray Biochip Technology; Schena,
M., Ed.; Eaton Publishing, MA, 2000. (b) Collins, F. S. Nat. Genet. 1999,
23 (3), 249-252.
(2) Selinger, D. W.; Cheung, K. J .; Mei, R.; J ohansson, E. M;
Richmond, C. S.; Blattner, F. R.; Lockhart, D. J .; Church, G. M. Nat.
Biotechnol. 2000, 18, 1262-1268.
(5) Kuehnast, B.; Dolle, F.; Terrazzino, S.; Rousseau, B.; Loc’h, C.;
Vaufrey, F.; Hinnen, F.; Doignon, I.; Pillon, F.; David, C.; Crouzel, C.;
Tavitian, B. Bioconjugate Chem. 2000, 11, 627-636.
(6) Agrawal, S.; Christodoulou, C.; Gait, M. J . Nucleic Acids Res.
1986, 14, 6227-6245.
(3) Hessner, M. J .; Budish, M. A.; Friedman, K. D. Clin. Chem. 2000,
46(8), 1051-1056.
(4) (a) Corey, D. R. J . Am. Chem. Soc. 1995, 117, 9373-9374. (b)
Hicke, B. J .; Watson, S. R.; Koenig, A.; Lynott, C. K.; Bargatze, R. F.;
Chang, Y.-F.; Ringquist, S.; Moon-McDermott, L.; J ennings, S.; Fitz-
water, T.; Han, H.-L.; Varki, N.; Albinnana, I.; Willis, M. C.; Varki,
A.; Parma, D. J . Clin. Invest. 1996, 98, 2688-2692. (c) Rajur, S. B.;
Roth, C. M.; Morgan, J . R.; and Yarmush, M. L. Bioconjugate Chem.
1997, 8, 935-940. (d) Tung, C.-H.; Stein, S. Bioconjugate Chem. 2000,
11, 605-618.
(7) J ones, D. S.; Barstad, P. A.; Field, M. J .; Hachmann, J . P.; Hayag,
M. S.; Hill, K. W.; Iverson, G. M.; Livingston, D. A.; Palanke, M. S.;
Tibbetts, A. R.; Yu, L.; Coutts, S. M. J . Med. Chem. 1995, 38, 2138-
2144.
(8) Ghosh, S. S.; Kao, P. M.; McCue, A. W.; Chappele, H. L.
Bioconjugate Chem. 1990, 1, 71-76.
(9) Rajur, S. B.; Roth, C. M., Morgan, J . R.; Yarmush, M. L.
Bioconjugate Chem. 1997, 8, 935-940.
10.1021/jo0100190 CCC: $20.00 © 2001 American Chemical Society
Published on Web 07/12/2001

Diels-Alder Bioconjugation of Diene-Modified Oligonucleotides
J . Org. Chem., Vol. 66, No. 16, 2001 5353
Sch em e 1
mercaptan oligonucleotide disulfide reduction directly
prior to bioconjugation adds undesirable operational
complexity to all these related methods.
While amine- and mercaptan-based techniques will
always be mainstream methods for oligonucleotide con-
jugate formation, they are not without their problems,
and complementary techniques would serve a valuable
purpose in the field as well. In a particularly promising
recent account, Greenberg has described a conceptually
distinct variation on postsynthesis oligonucleotide bio-
conjugate formation in which a reactive 2′-amino uridine
nucleoside monomer (introduced during automated solid
phase synthesis) is selectively deprotected and condensed
with electrophilic labels prior to oligonucleotide depro-
tection and cleavage from synthesis support.10 Similarly,
Grinstaff describes Pd(0)-catalyzed covalent modification
of support-bound, 5-halouridine-containing oligonucle-
otides.11
The Diels-Alder [4 + 2] cycloaddition between a diene
and a dienophile remains among the more useful carbon-
carbon bond-forming reactions available to synthetic
organic chemists.12 Breslow’s discovery that aqueous
solvents accelerate the Diels-Alder reaction unveiled
further opportunities for exploitation of the methodology
in synthetic and physical organic chemistry applica-
tions.13-15 While the basis of the acceleration in water
remains the subject of much study, we recognized the
opportunity this phenomenon represented in the biotech-
nological arena. Specifically, the highly selective reaction
between a diene and a dienophile could be exploited in
covalent biomolecule modifications. The first example of
the application of the Diels-Alder reaction in a nucleic
acid context came from the work of Eaton and Tarasow.16
This group identified RNAs capable of catalyzing a
Diels-Alder reaction by in vitro selection from a library
of modified RNAs. Seelig and J a¨sche have recently
published a similar example of an RNA-catalyzed Diels-
Alder reaction.17
and standard reagents for oligonucleotide synthesis were
obtained from Proligo, Glen Research, Cruachem, Aldrich, and
Burdick and J ackson. Biotin maleimide (biotin-BMCC; 6b),
fluorescein maleimide 6c, and 1,6-bismaleimidohexane 8 were
obtained from Pierce. N-Ethylmaleimide (6a ) and coumarin
maleimide 6d were obtained from Aldrich. The 5K and 20K
PEG maleimides, 6e and 6f, respectively, were obtained from
Shearwater Polymers, Inc., and biotin maleimide 26 was
purchased from Molecular Biosciences. NMR spectra were
measured at 300 MHz. Electrospray mass spectral data were
recorded in the negative ion mode or from M-Scan. At least
three charge states were used to determine the reported
molecular weights. High-resolution FAB mass spectroscopy
data was obtained from the University of California, Berkeley,
mass spectroscopy laboratory.
The synthesis of all dienes, diene phosphoramidites and
oligonucleotides (shown in Schemes 1, 2, 5, and 6) are
described in the Supporting Information.
P r ep a r a tion of a Stock Solu tion of Acyclic Hexa d ien e-
Mod ified Oligon u cleotid e 5. Lyophilized 5′-diene oligo-
nucleotide5 was dissolved in 1 mL of 25 mM phosphate buffer
(pH ) 6.8) to give an oligonucleotide concentration of 2000
OD260nm/mL (approximately 54 mg/mL or 6 mM). The oligo-
nucleotide concentration was determined by measuring the
absorbance at 260 nm of the oligonucleotide solution and
converting to concentration using an extinction coefficient of
1 OD260nm ) 27ug/mL. The extinction coefficient was calculated
using the method published by Breslauer20 and using the
values from Sugimoto.21
Gen er a l P r oced u r e: F or m a tion of Diels-Ald er Bio-
con ju ga tes 7a -f. Generally, 2 equiv of maleimides 6a -f were
added to aliquots of the stock solution of 5, although for
reagent solubility reasons, as much as 10-12 equiv was
sometimes used. Samples of the reaction mixture were with-
drawn over time and analyzed by analytical anion-exchange
HPLC on a 4 × 250 mm Dionex Nucleopak PA-100 strong
anion-exchange column heated to 80 °C using the following
method: a linear gradient of 36% buffer B to 65% buffer B
over 30 min (buffer A: 25 mM Tris (pH ) 7.5), 1 mM EDTA,
10% acetonitrile; buffer B: buffer A + 1 M NaCl). The column
flow rate was 1 mL/min, and the chromatogram components
were observed by UV detection at 260 nm. After the reactions
appeared complete, the product was isolated by anion-
exchange chromatography, using a 9 × 250 mm Dionex
Nucleopak PA-100 column with a flow rate of 5 mL/min
employing the same gradient described above. The purified
conjugates were converted to the triethylammonium salt forms
in order to obtain good MS data. The products were loaded
onto a 4.6 × 250 mm PRP-1 column and washed with five
column volumes of 25 mM triethylammonium carbonate
followed by two column volumes of water. The conjugates were
then eluted from the column in 50% acetonitrile/water and
lyophilized. Anion-exchange HPLC and electrospray mass
spectrometry were performed on the final lyophilized conju-
gates.
Initial results in our laboratory18 have shown that the
Diels-Alder reaction is indeed amenable to oligonucle-
otide bioconjugation.19 The efficiency of the method
compares very favorably with established techniques for
oligonucleotide bioconjugation. We report herein the first
full account of this complementary technique for oligo-
nucleotide bioconjugation.
Exp er im en ta l Section
Gen er a l Meth od s. Derivatized controlled pore glass (CPG)
long-chain alkylamine-T solid support was obtained from
Prime Synthesis. Deoxynucleoside phosphoramidites, solvents,
(10) Hwang, J .-T.; Greenberg, M. M. Org. Lett. 1999, 1, 2021-2024.
(11) Khan, S. I.; Grinstaff, M. W. J . Am. Chem. Soc. 1999, 121,
4704-4705.
(12) Roush, W. R. Comprehensive Organic Synthesis; Paquette, L.,
Ed.; Pergamon Press: Oxford, 1991; Vol. 5, p 513.
(13) Rideout, D. C.; Breslow, R. J . Am. Chem. Soc. 1980, 102, 7816-
7817.
(14) Otto, S.; Blandamer, M. J .; Engberts, J . B. F. N. J . Am. Chem.
Soc. 1996, 118, 7702-7707.
Syn th esis of N-Eth yl Ma leim id e Oligon u cleotid e Con -
ju ga te 7a . Compound 7a was prepared from 200 µL of the
stock solution of 5 and 10 µL (2 equiv) of a stock solution of
6a (prepared by dissolving 6.4 mg of 6a in a solution of 150
µL of water and 50 µL of acetonitrile) according to the general
procedure. After 2 h at 35 °C, the reaction was complete. The
product was isolated as described above, and anion-exchange
(15) Blokzijl, W.; Engberts, J . B. F. N. J . Am. Chem. Soc. 1992, 114,
5440-5442.
(16) Tarasow, T. M.; Tarasow, S. L.; Eaton, B. E. Nature 1997, 389,
54-57.
(17) Seelig, B.; J a¨sche, A. Chem. Biol. 1999, 6, 167-176.
(18) Presented in part at the 214th National ACS meeting, 1997,
Las Vegas, NV.
(19) After the initiation of our studies, Seelig and J a¨sche reported
a conceptually similar biotinylation of a diene-modified RNA transcript
prepared from an anthracene modified guanosine phosphate diester
transcription initiator: Seelig, B.; J a¨sche, A. Tetrahedron Lett. 1997,
38, 7729-7732.
(20) Breslauer, K. J .; Frank, R.; Blocker, H.; Marky, L. A. Proc. Nat.
Acad. Sci. U.S.A. 1986, 83, 3746-3750.
(21) Sugimoto, N.; Nakano, S.; Yoneyama, M.; Honda, K. Nucl. Acids
Res. 1996, 24, 4501-4505

5354 J . Org. Chem., Vol. 66, No. 16, 2001
Hill et al.
Sch em e 2
HPLC analysis revealed a purity of 98%. Analysis by electro-
spray mass spectrometry gave a molecular weight of 8922.2
( 0.5 (calcd 8922.7).
Con tr ol Exp er im en t: Rea ction of 6a w ith 3. A mixture
of 3 and 6a under the conditions described above showed no
change by HPLC over time.
anion-exchange HPLC. Electrospray mass spectrometry gave
a molecular weight of 9096.9 ( 0.3 (calcd 9095.9).
Syn th esis of 5K P EG Oligon u cleotid e Con ju ga te 7e.
Compound 7e was prepared from 200 µL of the stock solution
of 5 and 11 mg (approximately 2 equiv) of 5K PEG maleimide
6e according to the general procedure. After 20 h at 25 °C,
the reaction appeared complete. The product was isolated as
described above and showed a purity of 95% by anion-exchange
HPLC analysis. Electrospray mass spectrometry gave a mo-
lecular weight centered at 13795.8 (0.8 (n ) 110) ( 44 (calcd
13797.6 (n ) 110)).
Effect of Aceton itr ile on th e F or m a tion of 7a . A 100
µL portion of the stock solution of 5 was placed into each of
two 1 mL microcentrifuge tubes. To the first tube was added
100 µL of 25 mM phosphate buffer (pH ) 6.8) and to the second
tube was added 100 µL of acetonitrile. A stock solution of 6a
was prepared by dissolving 46.25 mg of N-ethylmaleimide in
1 mL of acetonitrile. To each of these two mixtures was added
10 µL of the N-ethylmaleimide stock solution (10 equivalents
of 6a ). Both reactions were incubated at 25 °C and monitored
by analytical anion-exchange HPLC. After 15 min, the reaction
without acetonitrile was 50% complete while the reaction with
acetonitrile present took 240 min to reach 50% completion.
Syn th esis of Biotin yla ted Oligon u cleotid e 7b. Com-
pound 7b was prepared from 200 µL of the stock solution of 5
and 1.5 mg (approximately 2 equiv) of 6b according to the
general procedure. After 18 h at 35 °C, the reaction appeared
complete. This product was isolated as described above, and
anion-exchange HPLC analysis showed a purity of 97%.
Analysis by electrospray mass spectrometry gave a molecular
weight of 9333.4 ( 0.9 (calcd 9331.3).
Effect of Tem p er a tu r e on th e F or m a tion of 7b. Two 1
mL microcentrifuge tubes were each charged with 200 µL of
the stock solution of 5. To each was added 8 mg (0.015 mmol,
approximately 10 equiv) of 6b. The first tube was incubated
at 35 °C and the second at 60 °C. Both reactions were followed
by anion-exchange HPLC using the method described above.
The reaction run at 35 °C took 90 min to reach 50% completion.
The reaction at 60 °C took only 15 min to reach the 50%
completion mark and was complete after 2 h.
Syn th esis of F lu or escein -La beled Oligon u cleotid e 7c.
Compound 7c was prepared from 200 µL of the stock solution
of 5 and 3.8 mg (approximately 12 equiv) of fluorescein
maleimide 6c according to the general procedure. Most of the
fluorescein did not appear to dissolve, although the reaction
mixture did take on the expected yellow fluorescein color. The
reaction mixture was vortexed at room temperature and, after
18 h, appeared complete. The product was isolated as described
above and revealed to be 98% pure by anion-exchange HPLC.
Electrospray mass spectrometry gave a molecular weight of
9224.6 ( 0.9 (calcd 9224.1).
Syn th esis of Cou m a r in -La beled Oligon u cleotid e 7d .
Compound 7d was prepared from 230 µL of the stock solution
of 5 and 10 µL (ca. 1.2 equiv) of a stock solution of 6d (prepared
by dissolving 5.0 mg of 6d in 100 µL of dimethyl formamide)
according to the general procedure. After approximately 20 h
at 35 °C, the reaction appeared complete. The product was
isolated as described above and revealed to be 96% pure by
Effect of Tem p er a tu r e on th e F or m a tion of 7e. Under
the same conditions described above at 55 °C, the formation
of 7e was complete within 3 h.
Syn th esis of 20K P EG Oligon u cleotid e Con ju ga te 7f.
Prepared from 200 µL of the stock solution of 5 and 50 mg
(approximately 2 equiv) of 20K PEG 6f. After 20 h at 35 °C,
the reaction was complete. This material was not characterized
further.
F or m a tion of Oligon u cleotid e Dim er 9. Compound 9
was prepared from 200 µL of the stock solution of 5 and 22 µL
(ca. 1/3 equiv) of a stock solution of 8 (prepared by dissolving
5 mg (0.02 mmol) of 8 in 1 mL of 1,4 dioxane) according to the
general procedure. After 16 h at 25 °C, no further conversion
of 5 was observed by anion-exchange HPLC analysis. An
approximate yield of 75% was calculated on the basis of the
area of the peak due to 5 versus the peak area of the
oligonucleotide dimer 9 (no peak due to monoconjugated
oligonucleotide was observed, although this species could have
coeluted with 5). The product was isolated as before, and
anion-exchange analysis showed a purity of 98%. Electrospray
mass spectrometry gave a molecular weight of 17873.1 ( 0.5
(calcd 17872.3).
Syn t h esis of Biot in yla t ed Oligon u cleot id e 27 via
Diels-Ald er Biocon ju ga tion of 24 a n d Biotin Ma leim id e
26. Acyclic diene modified oligonucleotide 24 (prepared as
described in the Supporting Information; 27.5 OD) was dis-
solved in 1.0 mL of H2O. This solution was aliquoted into five
1.5 mL microcentrifuge tubes in equal 200 µL volumes (each
tube containing 5.5 OD260, 40.3 nmol). All samples were dried
under centrifugal conditions and reduced pressure at 85 °C.
One of the tubes containing oligonucleotide 24 (5.5 OD) was
preheated to 40 °C for 1 min and then treated with 26.9 µL
(10 equiv) of a stock solution of 26 (prepared from 3.94 mg of
26 in 0.500 mL of 100 mM phosphate buffer (pH ) 6.3 or 5.5)).
The solutions were prepared to afford a final diene oligonucle-
otide concentration of 1.5 mM. The solution was vortexed for
15 s, placed in a microcentrifuge and spun for 15 s, and then
transferred to a vibrating heating block (40 or 70 °C). Aliquots
(2 µL) were sampled at 15 min intervals. The 2 µL aliquots
were diluted to 22 µL with 0.1 M NaOH solution, diluted to
200 µL with water, and analyzed by analytical RP HPLC.
(Control experiments confirmed that this NaOH treatment

Diels-Alder Bioconjugation of Diene-Modified Oligonucleotides
J . Org. Chem., Vol. 66, No. 16, 2001 5355
Sch em e 3
effectively arrested the formation of the Diels-Alder adduct.)
All reverse-phase analyses were performed on a 4.1 × 250
Hamilton PRP-1 column with a 10 µm particle size using the
following methods: a linear gradient of 0% buffer B to 25%
buffer B over 25 min, or 0% buffer B to 40% buffer B over 40
min (buffer A: 100 mM triethylammonium acetate (pH 7.0);
buffer B: acetonitrile). The column flow rate was 1.5 mL/min.
At 40 °C, conversion to 27 was complete (product peak
confirmed by LC/MS analysis (Mscan)) within 90 min at pH
) 6.3 and within 30 min at pH ) 5.5. At 70 °C, 80% conversion
was observed within 20 min at pH ) 6.3. Electrospray MS
analysis of 27 showed a MW of 5554.1 (calcd 5555.0).
Syn t h esis of Biot in yla t ed Oligon u cleot id e 28 via
Diels-Ald er Biocon ju ga tion of 25 a n d Biotin Ma leim id e
26. Treatment of 25 under the conditions described above
afforded biotinylated oligonucleotide 28. Electrospray MS
analysis of 28 showed a MW of 5566.1 (calcd 5567.0). At 40
°C, complete conversion to 28 was observed within 30 min at
pH ) 5.5 (product peak confirmed by LC/MS analysis (Mscan)),
while at pH 6.3, 42% conversion was observed after 30 min.
At 70 °C, 72% conversion was observed within 20 min at pH
) 6.3 with complete conversion observed after 16 h (no time
points between 20 min and 16 h were sampled).
following sequence: 5′-d(CCA GTA CAA GGT GCT AAA
CGT AAT GG-[3′-3′]-T-[5′-5′]-T). This oligodeoxynucle-
otide is a truncated sequence of one of many DNA
aptamers identified by the SELEX process,22,23 having a
high binding affinity to L-selectin.4b The synthesis was
conducted on a Milligen 8800 oligonucleotide synthesizer
using standard solid-phase protocols at a 300 µmole
scale.24 After completion of the 5′ terminal solid-phase
addition cycle (trityl-off), the diene phosphoramidite 2
was coupled (0.2 M acetonitrile solution, 4 equiv, 30 min;
Scheme 2). Oxidation was carried out with 0.5 M NaIO4
in water for 10 min, and then the excess oxidant was
removed by washing with water followed by acetonitrile.
The oligonucleotide was then cleaved from the solid
support and deprotected under standard conditions using
ammonium hydroxide. Anion-exchange analysis of the
crude diene oligonucleotide 5 showed 45% full-length
material, and this was purified by reversed-phase HPLC.25
The product-containing fractions were pooled and con-
verted into the sodium salt form on a PRP-1 column. The
purified oligonucleotide was then lyophilized to a white
powder that was ca. 85% pure by analytical anion-
exchange HPLC. Mass spectrometric analysis of this
material gave the expected mass.
For bioconjugation experiments, a stock solution of 5
was prepared by dissolving a sample in 25 mM phos-
phate, pH ) 6.8, to give an approximate concentration
of 6 mM oligonucleotide (approximately 54 mg/mL).
Scheme 3 shows the conversion of diene oligonucleotide
5 into a number of Diels-Alder bioconjugates. Treatment
of an aliquot of the 6 mM stock solution of 5 with 2 equiv
of N-ethyl maleimide (6a ) at 35 °C resulted in complete
transformation within 2 h to a new product with a
slightly shorter retention time (as determined by HPLC
Resu lts a n d Discu ssion
F ir st-Gen er a tion Stu d ies. Our initial intentions
were to introduce a 5′-diene functional handle. The
decision at this stage to incorporate the diene (vs the
dienophile) via solid-phase synthesis was driven by the
anticipated relative stability of unsubstituted 1,3-dienes
toward the variety of reaction conditions encountered
during oligonucleotide synthesis as well as the abundance
of relevant and structurally diverse dieneophile reactants
available commercially as maleimide derivatives. Our
efforts began with the synthesis of acyclic diene 1, which
was prepared from ethyl sorbate as described by Choi
and co-workers.28 Direct phosphitylation of 1 was carried
out to afford the simple diene phosphoramidite 2 (Scheme
1).
(25) Bridonneau, P.; Bunch,S.; Tengler, R.; Hill, K.; Carter, J .;
Pieken, W.; Tinnermeier, D.; Lehrman, R.; Drolet, D. W. J . Chro-
matogr. B 1999, 726, 237-247.
(26) J a¨sche, A.; Bald, R.; Nordhoff, E.; Hillenkamp, F.; Cech, D.;
Erdmann, V. A.; Furste, J . P. Nucleosides Nucleotides 1996, 15, 1519-
1529.
Diene-modified oligonucleotide synthesis began with
the selection of the 28mer DNA fragment with the
(22) Tuerk, C.; Gold, L. Science 1990, 249, 505-510.
(27) Tarasow, T., M.; Tinnermeier, D.; Zysniewski, C. Bioconjugate
Chem. 1997, 8, 89-93.
(28) Choi, J .-K.; Ha, D.-C.; Hart, D.; Lee, C.-S.; Ramesh, S.; Wu, S.
J . Org. Chem. 1989, 54, 279-290.
(23) Ellingtion, A.; Szostak, J . Nature 1990, 346, 818-822.
(24) The large synthesis scale was employed to afford plenty of
material for process development and/or pharmacokinetics studies.

5356 J . Org. Chem., Vol. 66, No. 16, 2001
Hill et al.
Sch em e 4
Sch em e 5
analysis). Isolation and mass spectrometric characteriza-
tion of the product confirmed the formation of the
expected Diels-Alder cycloadduct 7a . As a control ex-
periment, excess N-ethylmaleimide was added to oligo-
nucleotide 3, which lacks the hexadiene functionality. No
conversion of 3 was observed under the reaction condi-
tions.
isolated products were colored due to the fluorescent
nature of the fluorescein and coumarin moieties.
The ability to generate poly(ethylene glycol) bioconju-
gates by the Diels-Alder bioconjugation method was
demonstrated. Treatment of the stock solution of 5 with
two equivalents of PEG maleimide 6e at 25 °C cleanly
afforded the expected PEG-oligonucleotide conjugate 7e
after 20 h. At 55 °C, complete conversion to 7e occurred
in only 3 h. Similarly, the 20 K PEG-oligonucleotide
conjugate 7f was prepared (2 equiv 6f, 35 °C; 20 h). The
PEG-oligonucleotide conjugates were isolated by reversed-
phase chromatography, and the molecular weights con-
firmed by mass spectrometry.26,27
A particularly compelling application of the Diels-
Alder conjugation methodology is in linking more than
one oligonucleotide to single platform. A solution of 5 was
treated with approximately 0.33 equiv of 1,6-bismale-
imidohexane (8) at 25 °C (Scheme 4). After 16 h the dimer
conjugate 9 had formed in approximately 80% yield
(based on 8). The dimer product was isolated by anion-
exchange chromatography and the molecular weight
confirmed by electrospray MS.
Similarly, oligonucleotide diene 5 was treated with 2
equiv of biotin maleimide 6b. This homogeneous solution
was warmed to 35 °C, and the course of the reaction was
followed by anion-exchange HPLC. After approximately
18 h, the peak corresponding to 5 had disappeared and
a new peak had grown in (95% yield by HPLC). This
product was isolated by anion exchange chromatography
and analyzed by electrospray mass spectrometry. The
mass obtained agreed with the expected mass of the
Diels-Alder adduct 7b . Much faster formation of bio-
tinylated oligonucleotide 7d was accomplished upon
treatment of 5 with 10 equiv 6b at 60 °C. Under these
conditions, complete conversion to 7b was observed in 2
h.
The diene oligonucleotide 5 was treated with 12 equiv
of fluorescein maleimide 6c or 1.2 equivalents of cou-
marin maleimide 6d . The reaction with the fluorescein
maleimide was conducted at room temperature (ap-
proximately 25 °C), while the coumarin maleimide cy-
cloaddition was run at 35 °C. The coumarin maleimide
6d was readily soluble in the solvent system used (5%
DMF in phosphate buffer); however, the fluorescein
maleimide solubility was limited and most of this solid
settled to the bottom of the reaction solution. Anion-
exchange analysis of the reaction mixtures showed the
diene oligonucleotide peak decreasing as a new peak grew
in with time. Both reactions took approximately 20 h to
go to completion, with HPLC yields of 95% and 93% for
the coumarin maleimide and fluorescein maleimide reac-
tions, respectively. Both products were isolated by anion
exchange chromatography and, in each case, the isolated
products gave the correct mass for the expected Diels-
Alder cycloaddition products 7c and 7d . Both of the
While we have not investigated the stereochemistry of
Diels-Alder reactions within the context of the oligo-
nucleotide conjugations, diene 1 forms the endo adduct
with N-methyl maleimide in toluene28 and in aqueous
buffer.29
Secon d -Gen er a tion Stu d ies. We were next inter-
ested in studying the influence of diene structure on
conjugation rate and establishing a reagent suitable for
the introduction of the diene functionality under high
throughput automated oligonucleotide synthesis condi-
tions. Cyclic 1,3-diene 14 was prepared as shown in
Scheme 5. Hydroxymethyl cyclohexene 10 was silylated
(TBSCl, imidazole; 95%), then treated with molecular
bromine to form a diastereomeric mixture of dibromides
12. Double elimination (potassium tert-butoxide, Aliquat
336) afforded silyl ether protected diene 13. Failure to
(29) Hill, K. W.; Taunton-Rigby, J .; Tarasow. T. Unpublished results.

Diels-Alder Bioconjugation of Diene-Modified Oligonucleotides
J . Org. Chem., Vol. 66, No. 16, 2001 5357
Sch em e 6
Sch em e 7
protect the hydroxyl group prior to elimination resulted
in intramolecular bromide displacement and the forma-
tion of bicyclic ether products. Finally, acidic silyl ether
cleavage and purification by distillation afforded hy-
droxymethyl cyclohexadiene substrate 14 in 75% overall
yield.
satile phosphoramidite reagent platform for diene incor-
poration during automated synthesis. Scheme 6 shows
the synthesis of diene amidite reagents 21a and 21b.
Chiral hydroxy nitrile 17 was reduced by treatment with
LiAlH4. Condensation of the unpurified amino diol with
diene carbonyl imidazolides 18a or 18b, followed by
peracetylation (for ease of purification and characteriza-
tion) gave diacetate dienes 19a and 19b. Acetate cleavage
(K2CO3/ MeOH), followed by selective protection of the
primary hydroxyl gave phosphoramidite precursors 20a
and 20b. Phosphitylation afforded the two diene amidites
Prior to proceeding further with diene oligonucleotide
synthesis, we performed a series of control experiments
establishing the compatibility of the diene functionality
under the various reaction conditions encountered during
automated solid-phase synthesis. To accomplish this,
dienes 1 and 14 were transformed into nitrophenethyl-
amine-derived carbamates 15 and 16 (Figure 1) by
sequential addition of carbonyldiimidazole and 4-nitro-
phenethylamine in DMF. The 4-nitrophenethyl moiety
provided a chemically inert chromophore for UV detec-
tion. These substrates were subjected to a number of
reaction conditions and evaluated by HPLC after 16 h.
The dienes both displayed complete stability to all
conditions studied except 1.0 M tetrabutylammonium
fluoride solution in THF. However, alternative desilyla-
tion conditions (TEA-HF) were suitable. Noteworthy is
the stability of the dienes toward the standard oxidizing
conditions (0.02 M I2, pyr/water/THF).
F igu r e 1. Reaction conditions evaluated include the follow-
ing: (a) A2O, pyr, THF; (b) N-Me-imidazole, THF; (c) 3% TCA/
CH2Cl2; (d) 10% DCA, PhMe; (e) 0.02 M I2, pyr/water/THF;
(f) t-Bu-OOH/PhMe; (g) 1.0 M TBAF/THF; (h) TEA-HF/ACN;
(i) 0.45 M tetrazole/ACN; (j) 0.25 M 4,5-dicyanoimidazole; (k)
NH4OH, 25 °C; (l) Nh4OH, 70 °C (4 h); (m) phenylacetyl
disulfide. *Except 1.0 M TBAF/THF. Significant decomposition
of 15 and 16 was onserved.
Comfortable with the requisite stability of the diene
functionality, we next designed and synthesized a ver-

5358 J . Org. Chem., Vol. 66, No. 16, 2001
Hill et al.
Sch em e 8
21a and 21b in reasonable overall yields. This amidite
platform features the DMT protected alcohol, which
facilitates colorimetric, in-process monitoring of the
coupling step and enables extension beyond the diene
functionality (vs exclusive 5′-end labeling).30
5.5 or 6.3), and the progress of the reactions was
monitored by HPLC analysis of aliquots sampled at
regular intervals. Under equivalent reaction conditions
(pH, temperature, concentration), the cyclic and linear
diene modified oligonucleotides displayed comparable
reaction rates. Diels-Alder bioconjugation reactions
conducted at pH 5.5 were observed to be consistently
faster than those at pH 6.3. For example, at 40 °C with
10 equiv of biotin maleimide 26, diene oligonucleotide 24
(1.5 mM) was completely transformed into conjugate 27
within 30 min at pH 5.5, while 90 min were required at
pH ) 6.3.
Incorporation of these reagents during automated
synthesis was carried out next. First, the support-bound,
fully protected oligodeoxynucleotide 23 was prepared.
Oligonucleotide 23 is a 15mer of the following sequence:
5′-d(GGG-AGG-ACG-ATG-CGG). The synthesis was car-
ried out on a controlled pore glass (CPG) synthesis
support using standard conditions on a Milligen 8800
DNA synthesizer at a 935 µmol scale.24 An analytical
portion was detritylated and subjected to ammonium
hydroxide deprotection. HPLC analysis of the resultant
oligonucleotide 22 showed the crude synthesis to be 63%
full-length material. Next, a number of 1 µmol portions
of the support-bound, trityl-on crude oligonucleotide were
separated and loaded into empty column casings for diene
phosphoramidite coupling studies on an ABI 394 syn-
thesizer. A coupling protocol employing 15 equiv of
amidite 21a or 21b for two 15 min coupling steps enabled
efficient incorporation of the acyclic and cyclic dienes
(Scheme 7). Anion-exchange HPLC analysis of depro-
tected crude oligonucleotides revealed clean incorporation
of the diene amidites; crude diene oligonucleotide purities
ranging from 62-68% full-length material were observed.
Electrospray MS analysis of the crude synthesis products
revealed the presence of the expected diene molecular
weights, and samples were purified to >80% purity by
anion-exchange HPLC.
Con clu sion
In conclusion we have shown that the Diels-Alder
reaction is a useful method of preparing oligonucleotide
conjugates in aqueous solution under mild conditions.
Reagents have been developed which facilitate ready
incorporation of the Diels-Alder reactive functionality
under standard solid-phase synthesis conditions. The
method provides a complementary alternative to amine-
and mercaptan-based oligonucleotide conjugations. Ap-
plications of the derived Diels-Alder bioconjugates and
surface immobilization by the method will be described
in due course.
Ack n ow led gm en t. We wish to thank Dr. Ted Tara-
sow for many insightful discussions. We also thank
Kevin J ohnson, David Tinnermeier, Kristi Vrkljan, and
Russ Lehrman for their expert technical assistance. The
authors also wish to thank Drs. Andreas Wolter and
Stephen Scaringe.
Our next objective was to determine the effect of the
diene structure on the rate of Diels-Alder bioconjugation.
A series of experiments was carried out in which 24 and
25 were each treated with either 2 or 10 equiv of biotin
maleimide 26 (Scheme 8). These reactions were con-
ducted at 25, 40 and 70 °C in phosphate buffers (pH )
Su p p or tin g In for m a tion Ava ila ble: Experimental pro-
cedures for synthesis and purification of all diene reagents and
oligonucleotides. HPLC chromatograms for crude and purified
oligonucleotides 5 and 7a ,f. Selected chromatograms for diene
stability studies described in Figure 1 and formation of
biotinylated oligonucleotide 28. This material is available free
of charge via the Internet at http://pubs.acs.org.
(30) Theisen, P.; McCollum, C.; Upadhya, K.; J acobson, K.; Vu, H.;
Andrus, A. Tetrahedron Lett., 1992, 33, 5033-5036.
J O0100190