Oxidation of biotin during oligonucleotide synthesis

Article abstract of DOI:10.1081/NCN-200059277

A new "polystyrene biotin support" has been synthesized for the solid support synthesis of the 3′-biotinylated oligonucleotides. Several oligos were synthesized and were analyzed by the HPLC and Mass Spec. Oligo analysis revealed that the biotin gets oxidized to "biotin sulfoxide" during the synthesis. Copyright Taylor & Francis, Inc.

Full text of DOI:10.1081/NCN-200059277

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OXIDATION OF BIOTIN DURING OLIGONUCLEOTIDE
SYNTHESIS
Krishna Upadhya a , Iqbal K. Khattak a & Bashar Mullah a
a Applied Biosystemsu , Foster City, California, USA
Published online: 15 Nov 2011.
To cite this article: Krishna Upadhya , Iqbal K. Khattak & Bashar Mullah (2005) OXIDATION OF BIOTIN DURING
OLIGONUCLEOTIDE SYNTHESIS, Nucleosides, Nucleotides and Nucleic Acids, 24:5-7, 919-922, DOI: 10.1081/NCN-200059277
To link to this article: http://dx.doi.org/10.1081/NCN-200059277
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Nucleosides, Nucleotides, and Nucleic Acids, 24 (5–7):919–922, (2005)
Copyright D Taylor & Francis, Inc.
ISSN: 1525-7770 print/ 1532-2335 online
DOI: 10.1081/NCN-200059277
OXIDATION OF BIOTIN DURING OLIGONUCLEOTIDE SYNTHESIS
5
Krishna Upadhya, Iqbal K. Khattak, and Bashar Mullah
Applied Biosystems,
Foster City, California, USA
5
A new ‘‘polystyrene biotin support’’ has been synthesized for the solid support synthesis of the 3’-
biotinylated oligonucleotides. Several oligos were synthesized and were analyzed by the HPLC and Mass
Spec. Oligo analysis revealed that the biotin gets oxidized to ‘‘biotin sulfoxide’’ during the synthesis.
Keywords Biotin, Oligonucleotide Synthesis
INTRODUCTION
The strong affinity between biotin and streptavidin or avidin (Kd = 10À15/M) is
exploited in many molecular biology applications.[1] Biotin can be conjugated to
oligonucleotide at the 5’-end, at the 3’-end or internally depending on the
application. For the 3’-end biotin labeling of the oligo, commercially available,
biotin controlled pore glass (CPG) support is used.[2] Compared to CPG support,
polystyrene has special advantages.[3] Applied biosystems has developed a high-
throughput DNA synthesizer 3900TM, which exclusively uses the polystyrene
support (PS) for the high throughput DNA synthesis. To facilitate, the synthesis of a
high throughput 3’-biotin labeled oligonucleotides, there was a need for polystyrene
biotin support. Here in we report the synthesis of the ‘‘biotin polystyrene support,’’
and its use in the oligonucleotide synthesis. We also report here, for the first time,
the oxidation of biotin, to biotin sulfoxide, during the oligosynthesis.
SYNTHESIS OF BIOTIN SUPPORT
Biotin 1 was suspended in pyridine (Scheme 1) and was reacted with
t-butylchlorodiphenylsilane and t-butylbenzoylchloride sequentially to convert to
N-t-butylbenzoylbiotin 2.[4] The carboxyl group in compound 2 was activated and
coupled with 1-O-DMT-2-aminobutyl-1, 3-propane diol linker 3,[3] to yield
compound 4. The hydroxyl group in compound 4 was reacted with succinic
Address correspondence to Krishna Upadhya, Applied Biosystems, 850 Lincoln Centre Drive, Foster City,
CA 94404, USA; Fax: (650) 638-6343; E-mail: upadhykg@appliedbiosystems.com
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K. Upadhya, I. K. Khattak, and B. Mullah
SCHEME 1 Synthesis of biotin support.
anhydride to get compound 5. The carboxyl group in compound 5 was activated
and coupled to the amine functionalized support to yield biotin-support 6 and 7.
Any uncoupled, free amines were capped with acetyl groups.
3’-BIOTINYLATED OLIGONUCLEOTIDE SYNTHESIS
Standard columns were packed with 0.2 mmol biotin PS support or with
0.2 mmol biotin CPG support. Oligonucleotides were synthesized either on ABI
TABLE 1 Representative Biotinylated Oligonucleotides Synthesized
TTT TTT TTT TTT TTT TT-Biotin-3’
TGG ATAGCG ACG TGG-Biotin-3’
ATC ACT GGA TAG CGA GGT GG-Biotin-3’
CCC CAA TCA CTG GAT AGC GAC GTGG-Biotin-3’
CCC ACC CCC AAT CAC TGG ATA GCG ACG TGG-Biotin-3’
ATC AGC CCC ACC CCC AAT CAC TGG ATA GCG ACG TGG-Biotin-3’

K. Upadhya, I. K. Khattak, and B. Mullah
921
394TM or on ABI 3900TM DNA synthesizer, following standard cycles. On
completion of the synthesis, oligonucleotides were cleaved and deprotected
manually, by heating the ‘‘support’’ in concentrated ammonium hydroxide, at 65°C
for 3 h in a sealed vial. Oligos were analyzed and purified by HPLC and
characterized by MS (Table 1).
RESULTS AND DISCUSSION
HPLC analysis of the oligos (Figure 1) synthesized on ABI 3900TM from the
biotin-PS support showed two major products. They were separated and purified by
HPLC and analyzed by mass spectrometry. Interestingly, the mass difference
between the two products was 16 Dalton units, which accounts for one oxygen
atom. A plausible explanation is that the sulfur atom in the biotin gets oxidized to
sulfoxide by the oxidizing reagent, iodine/pyridine/water (bottle 15), used in each
cycle of the DNA synthesis. The oligos made on ABI 394TM by CPG support
yielded only the oxidized product. Commercial CPG support also yielded biotin
oxidized oligos. The amount of oxidized product depends on the length of the
oligo and the nature of the support used.
Reduced concentration of the iodine reagent did not have significant effect on
the oxidation. We also tried other oxidizing agents compatible with the DNA
synthesis protocols either to selectively curb the biotin oxidation or to oxidize
completely to the biotin sulfoxide. Replacing the iodine reagent with 10-camphor-
sulfonyl-oxaziridine (CSO) didn’t change the oxidation pattern. But the oxidizing
agent, t-butylhydroperoxide (TBHP) completely oxidizes the biotin made from PS
support to sulfoxide level, and the one made from biotin CPG support to a mixure
of sulfoxides and sulfone.
To our knowledge, this phenomenon of biotin oxidation during the oligo-
nucleotide synthesis mediated by the biotin support is not reported in the literature.
One reason would be that for many applications, biotin is labeled at the 5’-end of
FIGURE 1 HPLC of the oligo 1. A) oxidized, B) expected.

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K. Upadhya, I. K. Khattak, and B. Mullah
the oligo and therefore it is not subjected to the repeated oxidation cycles, yielding
the desired product. However in some other unrelated studies, the oxidation of
biotin to biotin sulfoxide, and it’s binding to avidin has been reported in the
literatures.[5,6] The study showed that oxidation did not significantly alter the affinity
toward avidin.
REFERENCES
1. Green, N.M. Biochem. J. 1963, 89, 585–591.
2. Nelson, P.S.; Kent, M.; Muthini, S. Oligonucleotide labeling methods 3. Direct labeling of oligonucleotides
employing a novel, non nucleosidic, 2-aminobutyl-1,3-propanediol backbone. Nucleic Acids Res. 1992, 20,
6253–6259.
3. Andrus, A. Evaluating and isolating ‘‘synthetic oligonucleotides.’’ 1992.
4. Fang, S.; Bergstrom, D. Fluoride cleavable biotinylation phosphoramidite for 5’-end–labeling and affinity
purification of synthetic oligonucleotides. Nucleic Acids Res. 2003, 31(2), 708–715.
5. Zhou, X.; Shearer, J.; Rokoita, S.E. A Ni(Salen)-biotin conjugate for rapid isolation of accessible DNA. J. Am.
Chem. Soc. 2000, 122, 9046–9047.
6. Garlick, R.K.; Giese, R.W. Dissociative binding of -and -sulfoxides of biotinylamidoethyl-3-(4-hydroxy-
3[125l]iodophenyl) propionamide to avidin. Biochem. J. 1990, 268, 611–613.