Covalent modification and surface immobilization of nucleic acids via the Diels-Alder bioconjugation method

Article abstract of DOI:10.1081/NCN-100002332

The importance of chemically modified and surface immobilized nucleic acids has inspired the development of a wide variety of complementary techniques for covalent oligonucleotide preparation and immobilization. We are developing technology based on the use of a Diels-Alder reaction for accomplishing the covalent modification of oligonucleotides. Reported herein is preliminary progress toward the establishment of robust reagents for introducing the reactive functionality, as well as studies employing the BIACORE system to demonstrate surface immobilization by the method.

Full text of DOI:10.1081/NCN-100002332

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COVALENT MODIFICATION AND SURFACE
IMMOBILIZATION OF NUCLEIC ACIDS VIA THE DIELS-
ALDER BIOCONJUGATION METHOD
Gregory M. Husar , Dominick J. Anziano , Michael Leuck & David P. Sebesta
Published online: 07 Feb 2007.
To cite this article: Gregory M. Husar , Dominick J. Anziano , Michael Leuck & David P. Sebesta (2001) COVALENT
MODIFICATION AND SURFACE IMMOBILIZATION OF NUCLEIC ACIDS VIA THE DIELS-ALDER BIOCONJUGATION METHOD,
Nucleosides, Nucleotides and Nucleic Acids, 20:4-7, 559-566, DOI: 10.1081/NCN-100002332
To link to this article: http://dx.doi.org/10.1081/NCN-100002332
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS, 20(4–7), 559–566 (2001)
COVALENT MODIFICATION AND SURFACE
IMMOBILIZATION OF NUCLEIC ACIDS VIA THE
DIELS-ALDER BIOCONJUGATION METHOD
Gregory M. Husar, Dominick J. Anziano, Michael Leuck,
and David P. Sebesta∗
Proligo, LLC, 2995 Wilderness Place, Suite 207, Boulder,
Colorado 80301
ABSTRACT
The importance of chemically modified and surface immobilized nucleic acids
has inspired the development of a wide variety of complementary techniques
for covalent oligonucleotide preparation and immobilization. We are develop-
ing technology based on the use of a Diels-Alder reaction for accomplishing
the covalent modification of oligonucleotides. Reported herein is preliminary
progress toward the establishment of robust reagents for introducing the re-
active functionality, as well as studies employing the BIACORE system to
demonstrate surface immobilization by the method.
Covalently modified nucleic acids are serving as increasingly important tools
in molecular biological, clinical and diagnostic applications (1). Commonly em-
ployed methods for constructing bioconjugates of oligonucleotides include the in-
troduction of the chemical modifier via automated synthesis (most typically via
specifically designed phosphoramidite reagents) or via post synthetic condensation
with reactive handles introduced during automated synthesis. While the former ap-
proach involves fewer steps and in principle requires only a single purification, the
preparation of labeled oligonucleotides by direct solid phase synthesis can be com-
plicated by reagent incompatibility with synthesis conditions as well as a relatively
limited selection of readily available specialty amidites.
∗Corresponding author.
559
ꢀ
C
Copyright 2001 by Marcel Dekker, Inc.
www.dekker.com

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HUSAR ET AL.
The second approach to oligonucleotide bioconjugates relies on introduction
of nucleophilic reactive handles during solid phase synthesis (generally N-alkyl
amines (2) or mercaptans (3–5)), followed by post-synthetic conjugation with elec-
trophilic reactive labels. While post-synthetic conjugation affords more flexibility
than relying on specialty amidite approaches, a number of complications can also
afflict this method. In our hands, phosphoramidite reagents, such as the commonly
employed MMT- or TFA-amino linkers, exhibit decreased stability and coupling
efficiency relative to standard nucleoside phosphoramidites. We have consistently
observed an increased lability for amidites derived from 1◦ vs 2◦ alcohols, along
with decreased coupling performance during solid-phase synthesis. Additionally,
in our hands, standard oligonucleotide deprotection conditions have led to de-
tectable percentages of N-cyanoethylation of amino linker moieties, often compli-
cating conjugate preparation by this method. Protected mercaptan reactive groups
require an additional downstream processing step as well; specifically, the reduc-
tion of the dimeric disulfide construct which often results from exposure to air,
consequently, amine condensation with electrophilic labeling reagents seems to
have seen more use. With respect to post-synthetic conjugate formations from
such functionally modified oligonucleotides, competing hydrolysis of labeling
reagent, incomplete conversion or multiple labeling of the oligo can all lead to
decreased yields and complicate isolation and purification of the desired conjugate
products.
We report herein preliminary progress toward a complementary method for
preparing covalently modified oligonucleotides by a post-solid phase synthesis
method relying on a cycloaddition reaction as the covalent binding event (6). Specif-
ically, the highly selective reaction between a diene and a dienophile (a Diels-Alder
cycloaddition) hasbeen exploitedincovalentbiomoleculemodifications (7,8). Con-
ceptually, the inertness and chemoselectivity of the Diels-Alder reactants offer
advantages in bioconjugation applications. For example, competing hydrolysis of
reactants under aqueous experimental conditions is less of a concern in a diene-
dienophile condensation and, in fact, the Diels-Alder process is actually accelerated
in aqueous solvents (9). Furthermore, due to the high selectivity dienes possess for
reactions with dienophiles, the Diels-Alder cycloaddition constitutes a previously
untapped dimension of chemical orthogonality for the covalent modification of
nucleic acids and other biopolymers (10).
RESULTS AND DISCUSSION
I. Reagents for Post-Synthetic Labeling of Oligonucleotides
An objective of our work is the direct comparison of the Diels-Alder bioconju-
gation method with other established techniques for biomolecule covalent modifica-
tion with respect to operational simplicity and efficiency of conjugate preparation.
Furthermore, comparable bioconjugate performance in applications is essential.

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MODIFICATION AND IMMOBILIZATION OF NUCLEIC ACIDS
561
1. LDA
2. oxalyl chloride
3. NaBH4
OH
OH
O
2
47% overall
1
1. CDI
2. 4-nitro-
phenethylamine
NO2
O
O
N
H
3
Scheme 1.
Initial efforts focussed on the design and synthesis of a reagent which could ulti-
mately be employed in high throughput solid-phase oligonucleotide synthesis to
reliably introduce the Diels-Alder reactive functionality. The decision at this stage
to incorporate the diene (vs the dienophile) via solid phase synthesis was driven by
two considerations: (1) the anticipated relative stability of unsubstituted 1,3-dienes
toward the variety of reaction conditions encountered during oligo synthesis and
(2) the abundance of relevant and structurally diverse dieneophile reactants avail-
able commercially as maleimide derivatives. A further design consideration was the
incorporation of a 2◦ alcohol amidite precursor to avoid the previously mentioned
shortcomings of primary alcohol derived amidites. Schemes 1 and 2 summarize
the synthesis of two 1,3-diene core structures employed in the current studies (11).
1. TBSCl, imid
1. KO-t-Bu
OTBS
2. Br2
OH
2. DOWEX H+
Br
Br
43% overall
4
5
NO2
1. CDI
2. 4-nitro-
phenethylamine
O
O
OH
N
H
6
7
Scheme 2.

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562
HUSAR ET AL.
1. LiAlH4, THF
2.ROCO(imid) (8)
3. Ac2O, TEA
1. K2CO3, MeOH
2. DMTCl, pyr
H
N
O
AcO
O
CN
R
O
OH
OAc
O
9
10a; ROH=2
10b; ROH=6
O
Cl
NC
P
H
H
N
DMTO
NC
N
O
N(iPr)2
DMTO
O
R
R
O
O
O
OH
11a-b
O
P
DIPEA, DCM
N(iPr)2
12a-b
Scheme 3.
Acyclic hexadiene 2 and hydroxymethylcyclohexadiene 6 were converted to their
respective 4-nitrophenethyl carbamates 3 and 7. We wanted to confirm the stability
of the diene functionality under various reaction conditions commonly employed
during automated solid phase oligonucleotide synthesis and deprotection and the
nitrophenyl moiety provided an inert chromaphore for UV detection. With the
exception of 1.0 M TBAF, the diene functionality proved completely stable to all
of the reaction conditions studied (12).
The next task was the conversion of diene alcohols 2 and 6 into suitable
phosphoramidite reagents. Previously, acyclic diene alcohol 2 was directly phos-
phitylated and this primary amidite was employed to 5ꢁ-end label a synthetic
oligonucleotide (7). Our interest was in designing a functional amidite which had
a DMT group (for in-process analysis of the coupling step), a 2◦-amidite and was
extendable, enabling incorporation at any position within an oligonucleotide chain.
Scheme 3 shows representative syntheses which afford diene amidites 12a and 12b.
Preliminary solid phase oligonucleotide syntheses with these reagents are under-
way and evaluation of Diels-Alder bioconjugations and surface immobilizations
with these diene oligo substrates will be reported in due course.
II. Surface Immobilization of Diene Modified Oligonucleotides
The BIACORE system offers the opportunity for the evaluation of surface
phenomena and we designed a set of experiments which would confirm the ap-
plication of the Diels-Alder bioconjugation as a new technique for surface im-
mobilization (10,13). While the BIACORE system has been a very powerful tool
for studying surface bound, non-covalent biomolecular associations (14), we were
curious to determine whether or not it could be exploited for the optimization of
key reaction parameters (e.g.; diene structure, linker structure, temperature, etc.)

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MODIFICATION AND IMMOBILIZATION OF NUCLEIC ACIDS
563
involved in covalent surface immobilization of biomolecules. A useful feature of
the SPR system is the lack of a requirement for a reporter group of any kind
on the molecule being immobilized. It is simply a change in mass density at the
liquid/ solid interface which affects the signal. Due to the ease of preparation (and
in some cases, commercial availability) of poly(ethylene glycol) (PEG) derivatives,
we performed some of our initial experimentation with examples from this class of
polymers.
We began by converting all four flow cells of the commercially available
carboxymethyl dextran coated gold chip (BIACORE CM5 chip) into a maleimide
(i.e.; dienophile) modified surface via sequential addition of EDC-NHS, ethylene
diamine and the bifunctional reagent sulfo-EMCS (15). A control experiment con-
firming the presence of surface bound maleimide was conducted by subjecting the
first flow cell to a 1.5 mM solution of methoxy poly(ethylene glycol) mercaptan
(MeO-PEG-SH; MW = 5000). At 25◦C, a change in the relative baseline response
signal of roughly 2000 response units (RU) was observed after 5 min exposure at
2 µL/min flow rate. For the purposes of studying covalent surface immobilization
chemistry, it is the magnitude of this change, and the rate of the change, in the base-
line response signal that are the key pieces of information. No additional increase
in the baseline response was observed upon continued exposure at this temperature
to the mercaptan PEG.
The second dieneophile flow cell was subjected to PEG cyclohexadiene sub-
strate 14b (Scheme 4) for 10 min under otherwise identical conditions, however, no
appreciable change in the baseline response signal was observed after that length of
time. In order to realize the same roughly 2000 RU increase in the baseline response
observed upon exposure to the solution of the thiol substrate, approximately 18 h
was required, indicating the Michael addition of the thiol group to the maleimide to
be much faster than the diene cycloaddition. Table 1 shows the results of a series of
experiments conducted at 25◦C and 37◦C with PEG dienes 14a–c, along with some
control PEG mercaptan data. As can be seen, higher degrees of surface functional-
ization occur at the higher temperature, and the degree of the increase is consistent
from diene to diene. It is interesting, however, that no appreciable difference in the
degree of surface substitution was observed when changing from the acyclic diene
1. CDI
2.
O
R
O
Me
ROH
O
N
H
O
n
O
Me
H2N
O
n
a; ROH=2
b; ROH=6
c; ROH=hydroxymethyl
13
14a-c
anthracene
Scheme 4.

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564
HUSAR ET AL.
Table 1.
RU 25◦C (time)b RU 37◦C (time)b
1727 (5 min) 3346 (5 min)
Entry
Substratea
1
2
3
4
MeO-PEG-SH (1.5 mM)
14a (1.5 mM)
14b (1.5 mM)
247 (3 h)
211 (3 h)
267 (3 h)
629 (3 h)
580 (3 h)
658 (3 h)
14c (1.5 mM)
aAll flow rates were 2 µL/min; all substrates were solutions in 100 µM
phosphate buffer (pH=6.5); bAll RU represent relative increases in the CM5
maleimide flow cell baseline response after adequate elution of running
buffer (100 µM phosphate, pH=6.5).
moiety to the cyclic diene examples. Based on Breslow’s original data on aqueous
acceleration of Diels-Alder reactions (9), we expected to see some difference in
going from the linear acyclic substrate 14a to cyclic diene 14b, and particularly
expected anthracene derivative 14c to display accelerated reaction rates. It is con-
ceivable that exposure of the diene functionality from the solution phase structure
of the PEG substrates is rate limiting, thus complicating the kinetic analysis of
the surface derivatization. Further investigations on the influence of diene struc-
ture in Diels-Alder bioconjugation applications are warranted and are currently
underway.
The immobilization of oligonucleotide substrates were addressed next. The
results of a variety of experiments are summarized in Table 2. Based on our ex-
perience in the PEG series, a thiol substrate immobilization was carried out as a
standard for comparison. Initially, 1.5 mM solutions of thiol oligo (16) (Entry 1) and
cyclohexadiene oligo 16 (Entry 2; for preparation of 16, see Scheme 5) (17) were
used, however, some later experiments were performed with more dilute solutions
(0.5 mM; Entries 4, 5). It is noteworthy that the increase in the relative response
observed in going from 25◦C to 37◦C was more dramatic in the oligo diene series
(4.4 fold increase at 37◦C) than in the PEG diene case (average 2.5 fold increase).
Table 2.
Entry
Substratea
RU 25◦C (time) RU 37◦C (time)
1
2
3
4
5
Oligo-SH (1.5 mM)
Oligo-cyclic diene (16; 1.5 mM)
5K-PEG-SH (1.5 mM)
Oligo-SH (0.5 mM)
Oligo-cyclic diene (16; 0.5 mM)
682 (10 min)
681 (10 h)
1814 (5 min)
325 (5 min)
241 (3 h)
−
−
3023 (5 min)
2618 (1 h)
1071 (3 h)
aAll flow rates were 2 µL/min; all substrates were solutions in 100 µM phosphate
b
buffer (pH=6.5); All RU represent relative increases in the CM5 maleimide
flow cell baseline response after adequate elution of running buffer (100 µM
phosphate, pH=6.5).

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MODIFICATION AND IMMOBILIZATION OF NUCLEIC ACIDS
565
NH2(CH2)6OPO2-5’-d(CTACCTACGATCTGACTAGC)
Diene NHS, DMF
15 (ODN99225)
O
O
H
N
O
O
N
H
O
N
H
ODN99225
2
O
16
Scheme 5.
CONCLUSION
We have prepared reagents for introducing Diels-Alder functionality to syn-
thetic oligonucleotides. We have demonstrated the surface immobilization of PEG
and oligonucleotide substrates by the Diels-Alder method and have begun a system-
atic optimization of the immobilization process employing the BIACORE system.
ACKNOWLEDGMENTS
The authors wish to thank Dr. Andreas Wolter, Dr. Jan Skouv, Dr. Stephen
Scaringe, Dr. John Pilon and Dr. Ken Hill for helpful discussions.
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HUSAR ET AL.
9. Rideout, D.C.; Breslow, R. J. Am. Chem. Soc. 1980, 102, 7816–7817.
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cleotide and was a generous gift from Dr. Brian Hicke of Gilead Sciences.
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lowing sequence: (a) CDI; (b) [NH2(CH2)3O(CH2)2OCH2]2; (c) succinic anhydride;
(d) DCC/NHS.

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