Pergamon

0040-4039(95)01405-5

Tetrahedron Letters, Vol. 36, No. 38, pp. 6823-6826, 1995 Elsevier Science Ltd

Printed in Great Britain 0040-4039/95 $9.50+0.00

SOLID PHASE SYNTHESIS OF DIRECTLY LINKED PNA-DNA-HYBRIDS

Frank Bergmann, Willi Bannwarth # and Steve Tam*

Roche Research Center. ttoffmann-La Roche Inc., Nutley. New Jersey 07110

Abstract: The synthesis of directly linked PNA-DNA-hybrids and the results of thermal melting studies are described.

The extraordinary binding affinity of Peptide Nucleic Acid (PNA) to DNA and RNA as demonstrated by

Egholm et al. 1,2 has stirred considerable interest in antisense research. Biological application of this new series

of agents, however, is limited by its undesirable self-aggregation properties and/or its inability to activate RNase

H. As a possible solution to these problems, we have studied several PNA-DNA hybrid molecules.

In this study, hybrid molecules with direct linkage of a PNA and an oligodeoxyribonucleotide sequence, in

both orientations, have been synthesized. In 5'-PNA-DNA-3' hybrids, the DNA is linked through an amide

bond to the PNA, and in 5'-DNA-PNA-3' hybrids, the two component units are connected via a phosphamide

bond. The synthetic strategy of the former type of hybrids, as outlined in Scheme 1, is similar to our recently

reported method on the preparation of peptide-DNA hybrids. 3 The DNA part is synthesized first with allyl

phosphoramidites on a sarcosine modified LCAA-CPG 8 support terminating in a 5'-amino-5'-deoxynucleoside.

The Boc/Z-protected PNA building blocks (commercially available from Millipore) can then be coupled directly

to the amino-terminus applying the recommended conditions. 2 The final cleavage procedure is carried out with:

a) triflic acid/TFA/dimethylsulfide/m-cresol 1:10:6:2 (lh at r.t.), 4 b) conc. NH3 (2h at 70°C). However, since

purine deoxyribonucleotides would be depurinated under these strongly acidic deprotection conditions, this

procedure can only be used for hybrids containing pyrimidine bases in the DNA part. For mixed sequences thai

also contain purine bases, Fmoc/acyl-protected PNA building blocks 5 have to be employed. In these cases, the

final deprotection can be easily carried out in a single treatment with concentrated ammonia (2h at 70"C).

For the synthesis of 5'-DNA-PNA-3' hybrids another support modification has to be used (Scheme 2).

Therefore, LCAA-CPG 8 was functionalized with 12-hydroxydodecanoic acid dimethoxytrityl-ether6/HBTU/

Hiinigs Base (1:1:2) in dry DMF. After capping with 5% Ac20 in DMF and removal of the DMTr group with

2% TCA, the first PNA building block was coupled to the alcohol function with HATU/Hfinigs Base/DMAP in

DMF to form an ester bond which can be cleaved later in the final deprotection step with anhydrous ethanolic

ammonia. The PNA sequence was then assembled with Fmoc/acyl-protected monomers, or in the thymine case

with Boc-protected PNA monomer, using standard conditions. 5 Continuation of the synthesis with oligo

deoxyribonucleotides was carried out with the standard 2-cyanoethyl-phosphoramidite methodology. Treatment

with anhydrous ethanolic ammonia overnight then gave the fully deprotected carboxamide product. Aqueous

ammonia, on the other hand, yielded a mixture of carboxamide and carboxylic acid products. Since the

phosphamide PNA-DNA linkage is labile under acidic conditions for removal of the Z-protecting groups, Z-

protected PNA monomers are not suitable for this synthesis.

# Present address: PRPC, F. Hoffmann-La Roche Ltd., CH-4002 Basel, Switzerland

6823

6824

B 0 CH3 . ( ~ R 1 - CPG - ~ NFI-C-CH2-N-C-CH2-CH2- ~-

DMTr OR 1 O O

B

l,I " -ooM,,

! H,~'~ ~. O- ]]5-~Q [-- O. ]R 1 R 2 =

" ~ [~R2~ J n R' = H H ( N H ~ ) v

B I ( R = Boc B = Thy Cyt z Ade z Gua z J O ' ' ' [ ii) r O R = Fmoc B = Thy, Cyt Bz, Ads Bz, Gua iBu

R-HN % ~ COOH

E 1 O 0 B ,B R 1 - C P G R 2-a l l y l \ ,

O R " L J m ~ J n B = Thy, Cyt, Ade, Gua

Scheme 1 ; Synthesis o! 5'-PNA-DNA,3' hybrids i) Standard DNA-synthesis using allyl phosphorarnidites; 5'-amino-derivative for the last coupling, ii) PNA-Synthasis (Boo- cleavage: FA//m-creso195:5 (2x3'); Fmoc--cleavage: 20% piperidine/DMF (1 '+2x7'); coupling: PNA (0.05M, 5eq)/HATU/ HiJn~gs Base 1:0.9:2 in DMF (20'); Capping: 5% Ac20/DMF (5'). using Boc in addition 5% piperidine/DMF (2'); Wash: DMF/ CH2CI2 1:1, pyridine before coupling step. iii) conc. NH 3 2h 70"C. iv) conc. NH3 2h 70"C or by applying of Z-protected building blocks (homopyrimidine-sequences): a) CF3SO3HICF3COOH/CH3SCH3/m-cresol 1:10:6:2, b) conc, NH 3 2h 70"C.

As a n odel sequence a homopyrimidine 13-mer 5'-TC'I'I'CCTCTCTCT-3' targeting a complementary

homopurine sequence within the HIV gag mRNA was selected. DNA sequences 1, 2 and 14 and RNA

sequence 15 were synthesized using standard procedures. Phosphamide containing DNA sequences were

prepared according to Bannwarth's procedure. 7 The aI1-PNA sequence 6 was synthesized by the recommended

conditions. 2 Hybrid molecules 5 and 7-13 were prepared according to procedures in Scheme 1 and/or Scheme

2. All compounds were purified by preparative PAGE except PNA 6 which was purified by RP18-HPLC. All

structures were confirmed by MALDI-TOF- and/or ESI-MS. The results of thermal melting studies are shown

in the Table.

The i,lcorporation of a phosphamide linkage at an interior position of the DNA fragment results only in a

small Tm drop of about 2-3"C using the 5'-amino-5'-deoxythymidine phosphoramidite building block (sequence

3) and only about I 'C drop using the corresponding 5'-amino-2',5'-dideoxycytidine phosphoramidite building

block (sequence 4). However, introduction of a single PNA building block at an interior position of the

sequence (5) leads to a substantial drop of Tm (ca. 16"C for DNA and 12.5"C for RNA). The all-PNA sequence

6 shows a strong binding to DNA and particularly to RNA. However, it exhibits a large hysteresis effect

between melting and association curve. The ramp-up curve reveals an unspecific melting behavior probably due

to higher aggregations, whereas the ramp-down curve reflects the true duplex transition. Replacement of 1, 2 or

3 PNA building blocks in 6 by deoxyribonucleotides at the 3'-end (7-9) leads to a lower decrease of Tin and

maintains the typical hysteresis effect for the PNA. Interestingly, PNA-DNA hybrids 10 and 11 consisting

approximately half of DNA and half of PNA show a big Tm drop when targeting DNA 14 but only a marginal

6825

drop when targeting RNA 15 (compared to the DNA-DNA and DNA-RNA duplexes) and they do not exhibit

the hysteresis effect. Both the DNA and the PNA part must have contributed to the binding with RNA since the

individual 6- or 7-mers show only very low Tm's. The DNA sequence 12 capped with PNA at the 3'- and 5'-

end shows a slightly higher Tm compared to a duplex of 13-mer 14 or 15 with the 11-mer 5'-CTCTCTCCTTC-

3' (16), suggesting only a small contribution of the 2 terminal PNA residues to the binding. This type of

capped DNA sequences and 5'-DNA-PNA-3' hybrids are no longer a substrate for 3'-exonuclease and their

stability in human serum is about 25 times higher than of the corresponding DNA sequence or 5'-PNA-DNA-3'

hybrids (data not shown). Additional studies to confirm in vivo nuclease protection will be carried out. 9

O OMTr-R 1 RI= CPG - ~ N H C ( C H 2 ) 1 1 0 - -

1. 2°/. TCA 2. i) O R = Boc B - Thy, (Cyt z, Ade z, Gua z)

R-HN ~ ~ COOH R = Fmoc B = Thy, Cyt Bz, Ade Bz, Gua iBu

Boc.HN ~ N v " l L R~ R 1 = NH 2

I * 1. TFA/m-cresot 95:5 2. ii) O R - Boc B = Thy, (Cyt z', Ade z, Gua z)

R-HN ~ ' / ~ COOH R - Fmoc B = Thy, Cyt Bz, Ade Bz, Gua iBu

~, ,~1 iv) O O R ~ N H 2 ~ H N ~ . n H N ~ t ' ~ R '

B

iii) DMTr-O~ O-p-Ey ",.,CN

1 B r B l Thy , 1 . CPG R2. cyacoethy,, ° / .Oo/ ,Oo . , . . ,

C J R ] ~ (~ R~ LHN*"*"x~ b~"g"~J Hn N ~ I'~K" R "i B = Thy, Cyt, Ade, Goa

Scheme 2: Synthesis of 5'-DNA-PNA-3* hybrids

i) PNA (10eq, 0.1M)/HATU/Hiinigs Base/DMAP 1:1:3:1 in DMF (2h), then capping with 5% Ac20/DMF (15'). ii) PNA- Synthesis: Coupling: PNA (5eq, 0.05M)/HATU/Hiinigs Base 1:0.9:2 in DMF (20'); Capping: 5% Ac20/DMF (5'), if Bo¢ is used,one wash with 5% piperidine/DMF (2'); Boc-cleavaga: TFA/m-cresol 95:5 (2x3'); Fmoc..cleavage: 20% piperidine/ DMF (1'+2x7'); Wash: DMF/CH2CI 2 1:1, pyridine before coupling step. Jii) Standard DNA-synthesis using i3-cyanoethyl phosphoramidites (1 coupling 30'). iv) NH3/EtOH (70"C overnight).

In summary, we have developed procedures to directly link PNA and DNA together. For application to

antisense research the 5'-DNA-PNA-3' hybrids and DNA sequences capped with PNA could be of great interest

since they have both the advantages of PNA and DNA molecules, namely, enhanced exonuclease stability,

ability to induce RNase H activity, improved cell permeability and good RNA binding properties. A series of

these hybrid molecules are currently under biological investigation and will be reported in due course.

6826

5'-AGAAGGAGAGAGA-3' 5'-AGAAGGAGAGAGA-3' DNA 14 RNA 15 ramp DNA 14 RNA 16

5'-TCTCTCTCCTTCT-3' 1

43.3 54.8 5'- NH'~FCTCTC TCCTTCT-3 ' 2 43.5 54.2

5'-TCTCTCTCNHTccTrc T-3 ' 3

5'-TCTCTCTNHCCTTCT-3' 4

5'-TCTCT~a+TCCTTCT-3 ' S

Tm Tm AH AS AG AH AS AG

43.9 54.9 43.7 55.1 wr 84.8 240,3 13.2 102.3 284.8 17.4

41.5 62.0 41.3 51.6 42.9 54.0 42.7 53.6

27.7 42.5 27.6 42.3

(50.7) 67.9 72.1 85.1 46.6 67.7

H- TCTCTCTCCTrCT-Lys-NH 2 6

H.TCTCTCT~TT~HT_ 3, ? 53.2 66.0 69.9 83.2 43.4 64.1

H-TCTCTCTCC'FIr~CT-3' 6 52,1 58.9 69.4 36.3 59.5

51.7 55.3 69.3 H'TCTCTCTCc7NHTCT-3' g 32.8 56.3

27.6 51.5 H-TCTCTC~HTccTTCT-3 ' 1C 27.3 50.6

5'.TCTCTC TGCTTCT-NH 2 11 34.5 51.8 33.9 51.7

H._,I, UTCTCTC CTTC_T_N H 2 .~ 42.7 53.4 423 53.0

5'- NH2C TCTCTCTCTTC T-N H2 1Q 41.3 52.6 41.2 52.3

5'- CTCTCTCGTTC-3' 16 41.1 52.6 41.1 52.9

I-able: Tm and thermodynamic constants

up 80.5 227.2 12.9 100.9 280.3 17.4 dowr

up 69.9 257.9 13.0 95.4 266.3 16.0 dow¢

up 84.4 239.6 13.0 105.3 294.5 17.0 down

up 60.3 173.8 8.5 99.0 285.7 13.9 down

up 93.4 263.2 15.0 161.6 445.6 28.8 down

up 84.8 239.2 13.5 148.9 412.4 26.0 down

up 57.8 159.1 10.4 119.0 329.8 20.7 down

JP 45.0 1201 9.2 1089 302.5 18.8 :Jown

JP 46.4 128.5 8.1 94.9 264.8 16.0 ~wn

up 56.9 158.9 9.5 93.6 260.1 16.1 dowr~

up 83.4 236.9 12.6 98.1 273.5 16.6 down

up 87.0 248.7 12.9 95.4 265.8 16.2 down

~k~wv 79.8 226.4 12.4 94.4 262.7 16.1

PNAs are in bold, underlined and italicized; NH/NH2- superscript = 5'-amino-5'-deoxynucleotide; NH2 = carbox-amide- terminus; H = amino-terminus; buffer: 100raM NaCI, 10raM Na-phosphate, 0.1 mM EDTA, pH 7; total strand concentration: 3~tM; ramp rate: 0.5"C/rain; Trn in "C; AH in kcal/mol; AS in cal/moI-K; ~G in kcal/mol at 25"C, calculated with a deviation of :1:5%; BH, AS and &G have negative sign for duplex formation and positive sign for duplex melting.

Acknowledgments: We would like to thank Dr. W.-R. Li and Dr. R. Goodnow for providing the Fmoc-protected PNA building

blocks, Dr. H. Michel and Dr. M. Sochacki for measurement of MALDI-TOF- and ESI-MS, Dr. D. Pruess for helpful discussions

regarding thermal melting studies and Mr. E. Kttng and Mr. P. Iaiza for excellent technical assistance.

References and Notes:

1. Egholm, M.; Buchardt, O.; Nielsen, P.E.; Berg, R.H.J. Am. Chem. Soc. 1992, 114, 1895. 2. PNA Information Package, Millipore Corp., Bedford, MA, USA, and references therein. 3. Bergmann, F.; Bannwarth, W. Tetrahedron Lett. 1995, 36, 1839. 4. Tam, J.P.; Heath, W.F.; Merrifield, R.B.J. Am. Chem. Soc. 1986, 108, 5242. 5. The synthesis of monomeric Fmoc-protected PNA building blocks will be published elsewhere. 6. Preparation of 12-hydroxydodecanoic acid 4,4'-dimethoxytrityl ether: 1 g (4.62 retool) 12-Hydroxydodecanoic acid was twice co-

evaporated with 20 ml of dry pyridine, then dissolved in dry pyridine (20 ml). 1.72 g (5.08 retool) 4,4'-dimethoxytrityl chloride were added. The reaction mixture was stirred overnight, then quenched with MeOH (5 ml), evaporated, diluted with CH2CI2 (50 ml) and washed with sat. NaHCO3 soln. (50 ml). The layers were separated, and the aqueous phase was back-extracted with CH2CI2 (2x 25 ml). The combined organ, layers were dried over Na2SO 4 and evaporated. Purification by flash-chromatography (40 g SiO'2) applying a CH2CI2 / 0-4% MeOH gradient containing 1% Et3 N yielded 2.16 g (79%) of an orange-yellow oil. The structure was confirmed by NMR, UV and MS.

7. Bannwarth, W. Helv. Chim. Acta 1988, 7l, 1517. 8. Abbreviations: LCAA-CPG: Long chain alkylamine-controlled pored glass; BOC: t-butyloxycarbonyl; Z: benzyloxycarbonyl;

Fmoc: 9-fluorenylmethoxycarbonyl; HATU: [O-(7-azabenzotriazol-l-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; HBTU: 2-(1H-henzotriazol-l-yl)-l,l,3,3-tetramethyluronium hexafluorophosphate; DMAP: 4-dimethylaminopyridine; HIV: human immunodeficiency virus; PAGE: polyacrylamide-gcl electrophoresis; MALDI-TOF-MS: malrix-assisted laser desorption ionization-time of flight mass spectrometry; ESI-MS: electro-spray ionization mass spectrometry; Tin; melting temperature.

9. Sands, H. et al. Molecular Pharmacology 1995, 47, 636.

(Received in USA 15 June 1995; revised 19 July 1995; accepted 21 July 1995)