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Fig. 5. Western blot of K-Ras antigens from AsPCl cells treated with KRASSS or KRASSA for 72 h. Lane 1: untreated cells; lane 2: lipofection alone; lanes 3-6: 0.5 yM, 1.0 yM, 1.5 yM, and 2.0 yM KRASSS; lanes 7-10: 0.5 yM, 1.0 yM, 1.5 yM, and 2.0 yM KRASSA.

Fig. 5. Western blot of K-Ras antigens from AsPCl cells treated with KRASSS or KRASSA for 72 h. Lane 1: untreated cells; lane 2: lipofection alone; lanes 3-6: 0.5 yM, 1.0 yM, 1.5 yM, and 2.0 yM KRASSS; lanes 7-10: 0.5 yM, 1.0 yM, 1.5 yM, and 2.0 yM KRASSA.

Fig. 6. Preparative C18 HPLC of N-Gly-Aba-Ado conjugate on 10 x 250 mm Alltima column eluted over 25 min from 5 to 69% CH3CN in 0.1% aqueous CF3CO2H at 1 mL/ min, and 50°C monitored at 260 nm.

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Fig. 7. MALDI-TOF mass spectrum of N-Gly-Aba-Ado chimera. Experimental mass: 435.5 Daltons; calculated mass: 434.4 Daltons.

C3UJ2triNwrAfr 21J VirJI tTM A<Mu f^MMV MOM! -'[ ' "J.".

Fig. 7. MALDI-TOF mass spectrum of N-Gly-Aba-Ado chimera. Experimental mass: 435.5 Daltons; calculated mass: 434.4 Daltons.

For the challenge of preparing a labeled probe, we first synthesized N-Gly-d-Ala-Gly-Gly-Aba-GCATCGTCGCGG (WT3613), a chelator-PNA specific for MYC mRNA on a 2 ^mol Fmoc-XAL-PEG-PS column in a single automated run. The overall yield following preparative RP-HPLC was 35.4%. The purified peak (Fig. 8) displayed a MALDI-TOF mass spectrum with a main peak at 3613.8 Daltons, in agreement with the predicted mass, 3614.5 Daltons (Fig. 9) (176).

The IGF-1 analog d-(Cys-Ser-Lys-Cys), which enables PNA uptake by cells bearing receptors for IGF-1, includes a disulfide bridge between terminating cysteines. The cyclization and purification of such peptides typically limits overall yield of an oligonucleotide-peptide conjugate. Following conjugation and purification, a peptide with a pair of cysteines to be cyclized is usually assembled on solid phase, deprotected, cleaved, and chromatographically purified. The reduced cysteine thiols are then oxidized nonspecifically by air in a basic solution (pH 8.5) at high dilution to minimize dimerization and oligo-merization (142).

Fig. 8. Analytical C18 HPLC of N-Gly-D-Ala-Gly-Gly-Aba-GCATCGTCGCGG PNA chimera as in Fig. 6.

Unfortunately, PNAs are poorly soluble when the pH is higher than 4, which also lowers yield. Deprotection/oxidation of the cysteines while the peptide chain is still on the support provides an alternative to the dilute aqueous route. Solid-phase cyclization is based on the assumption that for polymer-bound reac-tants intramolecular processes are preferred owing to pseudodilution (177). Under the latter conditions, deprotection and cyclization of cysteine residues are achieved simultaneously. Thus, the solubility problem of PNAs can be avoided, and the preparative steps can be simplified, improving overall yield. We therefore tested different conditions of cyclization on solid phase in order to find a simple and efficient cyclization method for peptide-PNA-peptide chimeras that would obviate the need for postsynthetic cyclization. We protected the two cysteine side chains with the S-acetamidomethyl (Acm) instead of S-triphenylmethyl (Trt) because Acm gives a higher yield of cyclization in (Me)2NCHO (178).

We optimized cyclization methods in a PNA dimer/peptide nonamer control sequence, N-Ado-Ado-Gly-Gly-Gly-Gly-d(Cys-Asn-Gly-Arg-Cys). Solution and solid-phase (Scheme 1) methods were compared. The maximum yield, 75%, was obtained on solid phase (Fig. 10). MS of the purified peak agreed with the predicted mass of the cyclized peptide, 1328.2 Daltons, which was x :>

3X0 Xffi Uffi i".*'IL0<2lt>\'a jnc t}ia X>S' -TJCJ, cm <l\Jit rKtjri I :pr »JC^I 3a 2001

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Fig. 9. MALDI-TOF mass spectrum of N-Gly-D-Ala-Gly-Gly-Aba-GCATCGT CGCGG PNA chimera. Experimental mass: 3613.8 Daltons; calculated mass: 3614.5 Daltons.

144 Daltons less than the linear, Acm-protected peptide (Fig. 11) (176). Solidphase cyclization (178) followed by PNA extension, cleavage, deprotection, and purification improves yield and saves significant effort, compared with postsynthetic aqueous cyclization and a second chromatographic purification (142).

The final synthetic milestone was the synthesis and cyclization of N-Gly-d-Ala-Gly-Gly-Aba-GCATCGTCGCGG-(Gly)4-d(Cys-Ser-Lys-Cys) (WT4261), a chelator-PNA-peptide specific for MYC mRNA and the IGF-1R. We used Fmoc-PAL-PEG-PS (0.2-0.3 mmol/g) for assembling the targeting peptides because it is compatible with PNA synthesis. Our experimental results suggested that if the loading of resin is higher than 0.3 mmol/g, cyclization on resin would lead to complicated products due to crossreaction between peptide chains. Three different reagents have been utilized for deprotection/oxidation of resin-bound peptide: (1) Tl(tfa)3 (179), (2) I2 under acidic conditions (180); (3) I2 in (Me)2NCHO E (181). Tl(tfa)3 is expensive, highly toxic, and requires

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Fig. 10. Preparative C18 HPLC of cyclized N-Ado-Ado-(Gly)4-D(Cys-Asn-Gly-Arg-Cys) conjugate as in Fig. 6.

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Fig. 10. Preparative C18 HPLC of cyclized N-Ado-Ado-(Gly)4-D(Cys-Asn-Gly-Arg-Cys) conjugate as in Fig. 6.

anhydrous conditions. In addition, it is possible that Tl(tfa)3 would react with amino or hydroxyl groups on nucleobases during the cyclization of cysteine residues. CF3CO2H or HOAc could not be used as the solvent owing to the acid lability of the PAL anchor. Hence, we tested two deprotection/oxidation conditions with I2/(Me)2NCHO: cyclization of cysteine residues (1) prior to addition of the Gly-d-Ala-Gly-Gly-Aba-PNA residues, and (2) after assembly of the complete peptide-PNA-peptide sequence.

For route 1, (Gly)4-d(Cys-Ser-Lys-Cys)-resin was suspended in (Me)2NCHO. Oxidation was carried out with I2 (10 eq) for 4 h at room temperature (Scheme 2). The resin was washed with (Me)2NCHO to remove excess iodine and dried under vacuum. Two micromole equivalents of dry resin were placed in an empty column for PNA extension and chelator coupling. Cleaved and deprotected sequence 4 was purified by preparative RP-HPLC at 50°C as above and gave an overall yield of 19.5%. Over 26 coupling cycles and one deprotection/oxidation step, the average yield per cycle was therefore 94%. MALDI-TOF-MS indicated a mass of 4261.8 Daltons, consistent with the predicted 4261.0 Daltons. The result indicated that the intramolecular disulfide

Fig. 11. MALDI-TOF mass spectrum of N-Ado-Ado-(Gly)4-D(Cys-Asn-Gly-Arg-Cys) conjugate. Experimental mass: 1327.3 Daltons; calculated mass: 1328.2 Daltons.

S-Acm S-Acm

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