Covalent labelling at 5'-end of synthetic oligonucleotides by one step generation of alkylamino group

Indian Journal of Chemistry Vol. 41B, June 2002, pp. 1246-1250

Covalent labelling at 5'-end of synthetic oligonucleotides by one step generation of alky lamino group

Krishna K Dubey, Yashveer Singh, Sanjay Kumar, Ramendra K Singh & Krishna Misra*

Nucleic Acids Research Laboratory, Department of Chemistry, University of Allahabad, Allahabad 211 002 Email: krishnamisra@hotmail.com

Received 3 October 2000; accepted (revised) 22 August 2001

Labelling of oligonucleotides can be carried out at 5'- and / or 3'-positions, phosphate backbone or nucleobases. The widely used labelling strategies, however, involve labelling of 5'- position where the label is covalently attached through a linker. Usually -NH2 or -SH functionalities are generated for coupling of the reporter groups. In the present work, a rapid one step procedure for generating 5'-alkylamino function has been developed. 5'-Butylamino thymidine and 5'-butylamino- d(TGCT) have been synthesised using solid phase synthesis. The efficacy of the method reported herein has been evaluated by labelling 5'- position with a tluorophore, 4-acetylamino-I, 8-naphthalimido-N-caproic acid (NCA).

In recent years, the detection, quantification and identification of nucleic acids have achieved importance due to the advent of Human Genome Mapping Project (HGMP) and DNA based clinical diagnostics. Hood's group in late 1980's developed automated nucleic acid sequencing system using laser based fluorescence detection ^I. This made researchers aware of the benefits of fluorescence detection, which were not accessible with traditional method of radioisotopic detection. Using fluorescence detection means that problems like instability, storage, disposal problems and associated health hazards can be avoided^2• Moreover, introduction of new hardware and detection system, ensures that fluorescence detection can be performed with almost same level of sensitivity as radioisotopic detection. Furthermore, fluorescence process itself is dependent on local environment and hence can be explored to probe molecular interactions, biochemical processes and cellular functions. Fluorescence detection is useful in relevant techniques of fluorescence in situ hybridisation (FISH)3 and the technology of chip based DNA arrays prepared using combinatorial chemistry methods⁴. The advent of fluorescence resonance energy transfer (FRET)5-7 technique has made possible real time monitoring of oligonucleotide hybridisation with enhanced sesitivity of detection.

This can be of great use in antisense technology.

Application of technologies described above re- quires easy access to appropriately labelled nucleic acids and oligonucleotides. Fluorescent labelling at 5'- OH, is the most widely used labelling strategl. This

usually involves the generation of either thiol or amino functionality at 5'_ position with latter being predomi- nantly used. The most common method employed for derivatisation of 5'- position involves generation of 5'- alkylamino function. A number of methods are avail- able like introducing 5'-arruno-5'-deoxythymidine monomer^9, N-trityl-6-amino hexanol^lO, 2-(bio- tinylamide)" or by reaction of diamines with 5'- phosphorimidazolide. However, the best method avail- able for derivatisation of 5'-position involves the reac- tion of 5'-OH with carbonyl diimidazole (CDI) fol- lowed by coupling with hexamethylene diamine^'2.

In the present work, a simple one step procedure for generating 5'-alkylamino functionality has been developed. This was achieved by llsing suitably protected amine, viz. N- (4 -bromobutyl) phthalimide, which on reaction with 5' -OH of thymidine and d- TGCT, followed by subsequent deprotection gives 5'- aminobutyl thymidine and 5'-aminobutyl-d(TGCT) respectively. The procedure is simple and can effectively be applied for derivatisation of 5'-OH of oligonucleotides in solid phase synthesis.

Results and Discussion

The present method is a simple one step procedure for generating alkylamino function at 5'-OH of synthetic oligonucleotides in solid phase. This was carried out by using momomer thymidine and a tetramer, d(TGCT) attached to a long chain alkylamino-controlled pore glass (LCAA-CPG) solid support. The tetra mer used in the present work was

MISRA et al. : COY ALENT LABELLING AT 5' ·END OF SYNTHETIC OLIGONUCLEOTIDES 1247

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B = ^base

n = ^{1 (T)}

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Scheme I-Synthesis of 5'-aminobutyl thymidine / d (TGCT) and covalent attachment with NCA; (a) trichloroacetic acid (3%) in dichloromethane; (b) N-(4-bromobutyl)phthalimideITEA IDMF/pyridine; (c) aq. ammonia (25%), 55°C, 12 h; (d) p-nitrophenyl ester of NCA.

synthesised in our laboratory on Omnifit DNA bench synthesiser at 0.2 IlM scale using phosphotriester approach of solid phase oligonucleotide synthesis¹³.

The method utilises the reaction of an alkyl halide with suitably protected amine function, viz., N- (4- bromobutyl) phthalimide with 5'-OR of monomer and tetramers, respectively. Since the method involves the generation of ethereal linkage, strongly basic conditions were employed by adding triethyl amine (TEA) in pyridine / DMF as th'e last step in the synthesis (Scheme I), The deprotection of base was done by incubation on water bath at 55°C. Subsequent detachment from resin and hydrolysis of phthaloyl group could be done with 25 % ammonia thereby producing 5' -aminobutyl thymidine and 5'- aminobutyl tetramer, respectively. Since stringently pure fraction of oligonucleotides are required, purification of monomer and tetramer was carried out by RPLC using reverse phase CIS column (Figure 1).

The ethereal linkage so generated was found to withstand usual deprotection employed in oligonucleotide synthesis.

The method has certain advantage over other methods, for instance usual derivatisation 12 requires activation of 5'-OR with COl followed by coupling with hexamethylene diamine whereas present method does not require any activation and usual deprotection with ammonia used to cleave the oligonucleotide from the solid support also removes the phthaloyl group thereby generating the required 5'-aminoalkyl functionality. Besides it has other advantages like (i) the length of alkyl chain can be adjusted as required,

1.0

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I

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R<.:lclIliulI Tim<.: (mill)

Figure I-HPLC profile of 5'-aminobulyl tetra mer (peak I, r. t.

3.6 min) and NCA labelled tetramer (peak 2, r.t. 3.9 min) on CIS analytical column, 125 x 5 mm. Solvent A fo,r 2 min, then a linear gradient of solvent A and 60 % Baver 6 min at a flow rate of 0.5 mL / min; Solvent A: aqueous ammonium acetate buffer (0. 1M, pH 6.93); solvent B: acetonitrile.

for appropriate spacing; (ii) the end amino group can be kept intact with phthaloyl group for storage purposes; (iii) coupling can be effected with any reporter group having functionality reactive towards amino group.

To evaluate the efficacy of the method reported in the present work,S' -covalent tagging was carried out with a fluorescent reporter group, 4-acetylamino-l, 8- naphthalimido-N-caproic acid (NCA). NCA is a highly fluorescent moiety developed in our laboratory and has been used for covalent tagging of antisense

1248 ^{INDIAN J.} CHEM., SEC B, JUNE 2002

oligonucleotides. This when covalently tagged to an antisense 8-, 33- and 41-mer, shows a minimum detec- tion of 10'9 MIL and as such serves as useful reporter group for antisense 0ligonucleotides^l4. The core system of NCA is 4-acetylamino-l, 8-naphthalic anhydride that has been condensed to a. spacer, viz. 6-amino caproic acid (Figure 2). Thus NCA contains free car- boxylic group which can be coupled with alkylamino functionality generated in monomer and the tetramer.

The 5'-butylamino thymidine and d (TGCT) were con- densed with p-nitrophenyl ester of NCA in presence of a base. The labelled monomer and tetramer were puri- fied by HPLC. The comparative emission spectra of NCA labelled thymidine and d(TGCT) was studied in methanol and ammonium acetate buffer, respectively.

A gradual decrease in relative fluorescence emission by 5 % was observed in labelled thymidine and 18 % in

Figure 2---4-Acetylamino-l, 8-naphthalimido-N-caproic acid (NCA).

100

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case of the labelled tetramer vis-a-vis NCA itself (Figure 3). This observation is compatible with in- creasing length of oligonucleotide. The minimum de- tection level was however 10-⁹ M / L in both the cases similar to the NCA itself.

The method thus, can be used for generating 5'- alkylamino functionality in synthetic oligonucleotides in solid phase synthesis. Since the method involves only one step procedure, it is simple to adopt for covalent labelling of synthetic oligonucleotides.

Experimental Sedion

Thymidine, phosphotriesters of thymidine, guanosine, cytidine and N-(4-bromobutyl) phthalimide were purchased from Sigma Chemical Company, USA.

I-Methylimidazole, mesitylene-sulphonyl nitrotriazole (MSNT) and dicyclohexyl carbodimide (DCC) were obtained from Fluka, Switzerland. p-Nitrophenol, am- monium acetate, hpIc grade water, methanol, acetoni- trile were purchased from E. Merck Company, India.

HPLC was carried out on Pharmacia LKB DBF using a reverse phase CI8 column and fluorescence spectra were recorded on Kontron SFM 25 spectrofluorometer.

DMTr-d (TGCT): It was synthesised using phosphotriester chemistry- at 0.2 ~ scale on Omnifit DNA bench synthesiser by a reported procedure^l2• The 5' -DMTr group was kept intact on oligonucleotide.

Figure 3-Relative fluorescence intensities ofNCA (----) in methanol; S'-NCA labelled thymidine (--0--) in methanol; S'-NCA labelled tetramer (--X--) in aq. ammonium acetate buffer at a concentration of 0.04 00; Ac. ^{360 nm.}

MISRA et al. : COY ALENT LABELLING AT 5' -END OF SYNTHETIC OLIGONUCLEOTIDES 1249

5' -Aminobutyl d(TGCT): The tetramer attached to solid support (LCAA-CPG), was taken in small reaction vessel having a medium porosity frit and stop-cock with a controlled inlet for nitrogen. This was washed well with anhydrous dioxane (HPLC grade, 3 x 3 mL) and dimethoxytrityl group was deprotected with trichloroacetic acid (3 % in dichloromethane). Resin was washed well with dichloromethane followed by dimethyl formamide (3 x 3 mL) and pyridine (3 x 3 mL). The resin now containing tetramer with free 5'-OH was then suspended in anhydrous pyridine (1.0 mL). N-(4- bromobutyl) phthalimide (0.014 mM; 4.0 mg) was added followed by 0.4 mL TEA in pyridine / DMF.

The reaction was allowed to proceed with occasional shaking and monitored on TLC. The reaction was complete in about 4 hr . After this time the resin was washed well with pyridine (5 x 3 mL). Resin was taken in eppendorf tube and treated with conc.

ammonia (25 %, 4 mL) for 2 hr at r.t. to cleave the support and phthaloyl group. Aqueous phase was filtered using sintered funnel to collect the derivatized tetramer in round bottom flask. Flask was sealed carefully and immersed in a shaking water bath for 12 hr at 55°C to remove amino protecting groups.

After this the flask was taken out, cooled and evaporated in vacuo to a gum and taken in ammonium acetate buffer (0.1 M) for purification on reverse phase HPLC. A gradient HPLC system was set up using 0.1 M ammonium acetate as solvent A and acetonitrile as B. Fractions were monitored at 260 nm and pooled accordingly. These fractions were Iyophilised to dryness.

5'-Aminobutyl thymidine: 5'-Dimethoxytrityl thymidine (25 mg; loading 1.71 J..lmol) linked with CPG solid support was derivatised using N-(4- bromobutyl)phthalimide (4.0 mg; 0.014 mmol) by the procedure discussed above. Crude reaction mixture containing 5'-aminobutyl thymidine was purified by column chromatography using a linear gradient of 5- 10 % methanol in dichloromethane as eluent; Rr 0.6 (dichloromethane: methanol:: 9 : 1, v/v).

p-Nitrophenyl ester of NCA: p-Nitrophenyl ester of NCA was prepared by a reported procedure^l4.

Labelling of 5' -aminobutyl thymidine with NCA. 5'-Aminobutyl thymidine was suspended in dry DMF (1.5 mL). p-Nitrophenyl ester of 4-acetylamino- 1, 8-naphthalimido-N-caproic acid (NCA), dissolved in dioxane (0.5 mL) was added to above suspension followed by triethylamine (0.2 mL) and pyridine (0.2

mL). The reaction was allowed to proceed for 6 hr at r.t. with constant stirring. The labelled thymidine was purified by column chromatography using a linear gradient of 10 - 20 % methanol in dichloromethane as eluent. Rr 0.75 (dichloromethane : ethylacetate :: 8 : 2, v/v).

Labelling of 5'-aminobutyl d(TGCT) with NCA.

5'-Aminobutyl tetramer was dissolved in a mixture of 1.0 M Na2C03/ NaHC03 buffer (PH 9.0, 250 J..lL). To this p-nitrophenyl ester of NCA (500 J..lL) dissolved in a mixture of 1.0 M Na2C03/ NaHC03 buffer (PH 9.0):

DMF:H20 (5:2:3, v/v) was added. The reaction mixture was vortexed and wrapped with aluminium foil to prevent light exposure. This was stirred for 8 hr at r.t. in dark. The labelled tetra mer was then passed through Sephadex G 25 using ethanol: water (8:2, v/v) as eluent. The filtrate containing NCA labelled tetramer was collected, concentrated and resuspended in aqueous ammonium acetate buffer (1.0 mL) and purified by HPLC in the same way as described for aminobutyl thymidine. The NCA labelled fractions were monitored at 260 nm. Pure labelled sample showing absorption at 260 nm were pooled and Iyophilised.

Fluorescence measurement of NCA labelled thymidine / d(TGCT): The NCA labelled thymidine / tetramer were dissolved in methanol and aqueous ammonium acetate buffer (0.1 M; pH 7.0). Fluorescence emission spectra of labelled thymidine and tetra mer were recorded at excitation wavelength of 360 nm at 0.04 OD concentration (minimum detectable concentration). The emission was scanned in the range of 200 - 600 nm. The maximum emission was observed at 460 nm accompanied with a 5 % decrease in labelled thymidine and 18 % decrease in labelled tetra mer vis-a-vis NCA itself.

Acknowledgement

Financial assistance from CSIR, New Delhi (KKD) and NVS Chemicals Pvt. Ltd. (YS) is gratefully acknowledged.

References

1 Smith L M, Sanders J Z, Kaiser R J, Hughes P, Dodd C, Con- nel C R, Heiner C, Kent C E & Hood L E, Nature 321,1986, 674.

2 Mansfield E S, Worley J M, Mackenzie S E, Surrey S, Rap- paport E & Fortina P, Mol Cell Probes, 9, 1995, 145.

3 Yang M J & Cao J, Prog Biochem Biophys, 25, 1998, 333.

4 Lipshutz R J, Fodor SPA, Gingeras T R & Lockhart D J, Na- ture Genet, 21,1999,21.

5 Stryer L, Annu. Rev Biochem, 47, 1978, 819.

1250 ^{INDIAN J.} CHEM., SEC B, JUNE 2002

6 Mergny J, Garestier T, Rougee M, Lebedev A V, Chas- signol M, Thuong, N T & Helene C, Biochemistry, 33, 1994, 15321.

7 Singh Y, Pandey A, Dubey K K, Watal G & Misra K, Curr Sci, 78, 2000, 478.

8 Davies M J, Shah A & Bruce I J, Chem Soc Rev, 29, 2000, 97.

9 Chollet A & Kawashima E H, Nucleic Acids Res, 13, 1985, 1529.

10 Connolly B A & Rider P, Nucleic Acids Res, 13,1985,3131.

II Kemp T, Sundquist W I, Chou F & Hu H L, Nucleic Acids Res, 13, 1985,45.

12 Dubey K K, Singh R K & Misra K, Nucleic Acids Symp Ser 34,1995, 177.

13 Gait M J, Matthes W D, SinghM, Sproat B S & Titmas R C, Nucleic Acids Res, 10, 1992, 6243.

14 Dubey K K, Singh R K & Misra K, Neurochem Int, 31/3, 1997,405.