Tocopherol (Vitamin E) Modified Oligonucleotides

Sheena Aitken, Ricky Archer, Grant McGeoch, Catherine McKeen and Douglas Picken; Link Technologies Ltd, Bellshill, UK. catherine@linktech.co.uk

Introduction
As with cholesterol modifications, tocopherol has been shown to have potential use in the delivery of oligonucleotides into cells. Vitamins such as tocopherol are not produced by the target cells, but are used by the latter and therefore vitamins are recognised. They are thought to be internalised by cells only after interaction with a binding protein and therefore have the potential for specific targeting of a cell type.1,2,3 We have looked at the use of tocopherol CEP (1) and octyltocopherol CEP (2) in oligonucleotide synthesis with the aim to define the optimum deprotection conditions. The synthesis conditions were not optimised since these are reported elsewhere.
4

Results & Discussion

Deprotection Optimisation
A set of tocopherol (1) modified T6 oligonucleotides were synthesised using the conditions described above. A poly T sequence was chosen to prevent any misleading results arising from incomplete deprotection of amino nucleobases. After synthesis, the resin was emptied from the column into a sample tube and the appropriate deprotection solution added. Cleavage and deprotection were carried out simultaneously. The oligonucleotides were then deprotected as shown in Table 1. Analysis by MALDI MS is shown in Table 2. All cases showed that the full-length product was present (one example is shown in Figure 1). In all but method D this was the main product. In this case the main 1 product was observed as M+158 (Figure 2). This is thought to be due to incomplete removal of the TBDMS deprotection solution prior to analysis since after further desalting only the full-length product was observed. The results show 1 is compatible with all deprotection methods A-E. The deprotection tests were repeated with comparable 2 results using the following sequence TTC GGC TTG TCC GTG GAA TCT CAC AGC TTA T, where the 5-end was modified by either 1 or 2.
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and the resulting thiol-modified oligonucleotide was eluted from the column with MeCN/water (1:1). In all cases the desired thiolmodified oligonucleotide was obtained. Examples of the HPLC and MS data of the tocopherol-O-C6-S-S-C6-O-oligonucleotide before cartridge purification and the HS-C6-O-oligonucleotide after purification are shown in Figures 4-7. HPLC of the latter shows two peaks since the dimerised oligonucleotide (oligoS-S-oligo) is observed. The MS data is summarised in Table 4.
500 450 400 350 300 250 200 150 100 50 0 0 5 10 15 20 25 0 5 10 15 20 25 350 300

mAU

250 200 150 100 50 0

ml

mAU

ml

Figure 4. HPLC data of tocopherol-thiol-modified oligonucleotide.

MALDI-TOF Mass Spectrum
Intens. [a.u.] 10279.9 Intens. [a.u.]

Figure 5. HPLC data of thiolmodified oligonucleotide.

MALDI-TOF Mass Spectrum

2000

%
9657.1

300

100

100

90

90

250
80

1500

80

70

70

200

The hydrophobic nature of tocopherol has also been utilised as a means of improving the purification of ribozymes. We have also investigated the use of tocopherol as a means of allowing an initial purification of thiol-modified oligos with a view to improving the efficiency of a second ion-exchange purification. Thiol-modified oligonucleotides are often used as a means of incorporating labels such as dyes by reaction with maleimides or acetamides. There is often a need to carry out multiple purifications in order to ensure the purity of the thiol-modified oligonucleotide prior to conjugation and a simple, efficient method of removing DMT-containing N-1 failures would be advantageous.

60

60

Table 2. Summary of MS data for toc-modified poly-T oligos. Method A B C D E

MALDI-TOF Mass Spectrum
Intens. [a.u.]

150

50

1000

50

100
9535.8

10418.2

9699.2

2248.6072 2248.6072 2248.6072

Intens. [a.u.]

2251 2414, 2256 2255

%
MALDI-TOF Mass Spectrum
120

0
6000 7000 8000 9000 10000 11000 12000 13000
m/z

9222.5

2248.6072

2252

9986.5

10

9361.3

50

10162.4

20

9845.4

2248.6072

2255

30

9795.0

Calculated MW

Observed MW

40

40

30

500

20

10

0
6000 7000 8000 9000 10000 11000 12000 13000
m/z

Acquired: 2011-03-18T17:05:24.000 Comment 1: LMS Comment 2: 10712 G:\MS Data\MS externe\13630\0_A17\1

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Acquired: 2011-03-18T17:05:32.000 Comment 1: LMS Comment 2: 10712 G:\MS Data\MS externe\13630B\0_A18\1

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Figure 6. MS data of tocopherolInstrument: ultraflex s/n: 25002.00093 D:\Methods\flexControlMethods\Oligos 13000 Da.par

Figure 7. MS data of thiol-modified

Instrument: ultraflex s/n: 25002.00093 D:\Methods\flexControlMethods\Oligos 13000 Da.par

thiol-modified oligonucleotide.

oligonucleotide.

%
100

100

Initially, the elution step was only carried out at the end of the purification after the reduction step, but this has since been repeated using an elution step to remove any failures which retained a DMT group at the end of the synthesis prior to carrying out the disulphide reduction. Also, to show that all the DMT-containing oligo sequences are efficiently removed, a sample of a tocopherol-thiololigonucleotide (Figure 3) mixed with a sample of DMT-O-C6-S-S-Ooligonucleotide (Figure 8) has been purified using the same method. The results are pending. This purification technique will also be carried out with octyltocopherol and other hydrophobic groups.

2252.4

2413.9

90

90

200
80

100

80

2274.2

70

80

70

150
60 60

Experimental
Synthesis Conditions
Synthesis was carried out on an ABI 394 DNA/RNA automated synthesiser using the following reagent specifications: standard CEPs and spacers (Bz-dA, dmf-dG, Ac-dC, dT, SP18, SP9, SPC12 and SPC3) - 0.1M in acetonitrile, coupling time = 30s; tocopherol CEPs (1 and 2) - 0.1M anhydrous, alcohol-free, dichloromethane, coupling time = 900s; solid support - dT 1000 SynBase 1mol column; activator - 0.25M ETT6; cap A - THF/pyridine/acetic anhydride (8:1:1); cap B - 10% methylimidazole in THF; oxidiser 0.02M iodine in THF/pyridine/water (7:2:1); deblock - 3% TCA/DCM.

50

60

50

100
40 40

40
2256.4 2456.8

30

30

20

2527.2

50

2296.3

20

20

2602.5

10

10

0
1800 1900 2000 2100 2200 2300 2400 2500 2600 2700
m/z

0
2000 2100 2200 2300 2400 2500 2600 2700 2800 2900
m/z

Acquired: 2010-07-12T23:01:42.000 Comment 1: Comment 2: G:\MS Data\MS externe\11290\0_A24\2

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Acquired: 2010-07-12T21:29:12.000 Comment 1: HCN Comment 2: 2248 G:\MS Data\MS externe\11292\0_A22\1 Instrument: autoflex

Printed: 15:44:40

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Figure 1. MALDI-MS of tocopherol

Instrument: autoflex s/n: 238120.00054

Figure 2. MALDI-MS of tocopherol

s/n: 238120.00054

modified T6 oligonucleotide with method B.

D:\Methods\flexControlMethods\Oligos 6000 Da.par

D:\Methods\flexControlMethods\Oligos 6000 Da.par

modified T6 oligonucleotide with method D. Figure 8. Thiol-modified oligonucleotide.

Versatility of Tocopherol (1)

While 2 already encompasses a C8 spacer, 1 has the versatility to be used with either a hydrophobic spacer such as spacer C12 or a hydrophilic spacer such as spacer 18 (HEG). To show this a series of tocopherol (1) oligonucleotides were synthesised with a spacer between the oligonucleotide and tocopherol. In all cases the sequence used was TTC GGC TTG TCC GTG GAA TCT CAC AGC TTA T. The MS data is shown in Table 3.

Conclusions
Both tocopherol (1) and octyltocopherol (2) are compatible with all deprotection methods A-E as shown in Table 1. Tocopherol (1) has the versatility to be used in conjunction with any number of hydrophobic or hydrophilic spacers. Hydrophobic groups such as tocopherol can be used to modify oligonucleotides with a view to a simple and efficient means of removing DMT-containing N-1 failures from thiol modified oligonucleotides.

Deprotection Conditions
The deprotection conditions tested are shown in Table 1. Thereafter, all deprotections were carried out using AMA at room temperature for 2h.
Table 1. Deprotection conditions used on toc-modified poly-T oligos. Method A B C D E Solution AMA AMA Ammonium hydroxide i. AMA ii. Et3N/Et3N:3HF/NMP (1.5:2:3) EDA/toluene Time 10min 2h 16h i. 10min ii. 2.5h 4h Temp. 65C 65C 55C i. 65C ii. 65C RT

Table 3. Summary of MS data for toc-spacer-modified oligos. Spacer None 18 C12 C3 9 Calculated MW 9954.8966 10298.0117 10218.0372 10091.8968 10165.9334 Observed MW 9965 10309 10229 10103 10177

Further Information
Any queries regarding this work should be directed to Dr Catherine McKeen, Product Manager, Link Technologies Ltd. www.linktech.co.uk

Analysis Conditions
RP-HPLC on all oligos was carried out using a Waters X-Bridge OST C18, 2.5m, 4.6x50mm column, Buffer A: 0.1M TEAA, Buffer B: MeCN, with a gradient of 0-50% B over 15min (X-Bridge oligos gradient) at a flow rate of 1ml/min. MALDI-TOF was carried out on a Bruker Ultraflex. This data was provided by EGT-SA. LCMS was carried out on an Agilent 6220 Accurate Mass TOF LCMS with 1260 pumps and DAD, using a Zorbax C18, 3.5m, 2.1x30mm column, Buffer A: 190mM HFIP, 7mM TEAA, 5% MeOH in water, Buffer B: MeOH, with a gradient of 0-100% B over 12min. All samples were at a concentration of 2OD/ml.
1 Delivery of Oligonucleotides and Analogues: The Oligonucleotide Conjugate-Based Approach, F. Marlin, P. Simon, T. Saison-Behmoaras and C. Giovannangeli, ChemBioChem, 11, 1493-1500, 2010. 2 Efficient in vivo delivery of siRNA to the liver by conjugation to alpha-tocopherol, K. Nishina, T. Unno, Y. Uno, T. Kubodera, T. Kanouchi, H. Mizusawa and T. Yokota, Mol. Ther., 16, 734-740, 2008. 3 Resolution of liver cirrhosis using vitamin-A coupled liposomes to deliver siRNA against a collagen-specific chaperone, Y. Sato, K. Murase, J. Kato, M. Kobune, T. Sato, Y. Kawano, R. Takimoto, K. Takada, K. Miyanishi, T. Matsunaga, T. Takayama and Y. Niitsu, Nat. Biotechnol., 26, 431-442, 2008. 4 Attachment of Vitamin E Derivatives to Oligonucleotides during Solid-Phase Synthesis, D. Will and T. Brown, Tet. Letts ., 33, 2729-2732, 1992. 5 Fast and simple purification of chemically modified hammerhead ribozymes using a lipophilic capture tag, B.S. Sproat, T. Rupp, N. Menhardt, D. Keane, and B. Beijer, Nucleic Acids Res., 27, 1950-1955, 1999. 6 While this was the only activator used for this work, 0.5M ETT,

In all cases the desired product was obtained indicating that 1 gives the option of incorporating either a hydrophilic or hydrophobic spacer between the tocopherol and the oligonucleotide.

Use of Tocopherol Modifications to Aid Purification

The possibility of using hydrophobic modifications such as tocopherol or cholesterol derivatives was investigated using 1. The same 31mer mixed DNA sequence was used as before where the thiol-modifier (3) was used to incorporate O-C6-S-S-C6-O between the oligonucleotide and 1 (Figure 3). As a control the same oligonucleotide sequence was modified with 3 where the terminal DMT group was retained. A 25s coupling time for the thiol modification was used, rather than the recommended 300s, to enhance failures in the crude.

Figure 3. Tocopherol-thiol-modified oligonucleotide.

After synthesis each of the oligonucleotides were cleaved and deprotected using AMA at room temperature for 2h then immediately loaded onto a Varian DNA TOPS column. The standard protocol for purifying DMT-ON DNA oligonucleotides was carried out. For the tocopherol oligonucleotides an extra step of addition of 100mM TCEP in water to each of the columns (leaving for 5min before being removed) was carried out. This step was repeated

0.3M BTT and 0.25M DCI have all been used to successfully couple both tocopherol derivatives 1 & 2 to oligonucleotides.

Table 4. Summary of MS data of tocopherol-thiol-modified oligonucleotides before and after TOPS purification. Oligo before TOPS Toc-O-C6-SS-C6-oligo Toc-O-C6-SS-C6-oligo DMT-O-C6-SS-C6-oligo Calculated MW 10281.8914 10281.8914 9791.6614 Observed MW 10314 10314 9799.56 Oligo after TOPS HS-C6-O-oligo HS-C6-O-oligo HS-C6-O-oligo Calculated MW 9667 9667 9667 Observed MW 9661 9657 9665