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As previously mentioned, P2Naq is based upon the P2X peptide family used by the Makhatadze group to measure the enthalpic contributions of various amino acids at a guest site in the formation of a helix.8 _{Its synthetic sequence is Ac-} DKDGDGYISAAEAϘ(OMe/OMe)AQ-NH2, where Ϙ once again represents Naq. Fmoc- solid phase peptide synthesis was once again straightforward; an increased yield was obtained by synthesizing the isoleucine-serine sequence into the growing peptide chain using a Fmoc- protected pseudo-proline derivative, where the serine residue side chain hydroxyl is reversibly bound to its own amine with an acid labile dimethylacetal group thereby reducing the likelihood of premature termination of chain extension. Additionally, when synthesized on a CEM Liberty microwave synthesizer, it was necessary to switch from the common piperidine Fmoc deprotection reagent to the less basic piperazine to circumvent succinimide formation between Asp5 and Gly6, increasing the overall yield of the peptide substantially. Peptide identity was confirmed via MALDI-TOF-MS. It is of note that purified and lyophilized P2Naq(OMe/OMe) is more or less completely insoluble in any solution besides at least 4 M GdnHCl or neat TFA, or mixtures of the two (high concentrations of urea is

possibly effective, but it was not tested). When added to aqueous solutions that do not contain high denaturant concentrations, the peptide forms hard, millimeter scale gel-like aggregates that are transparent with no apparent scattering from the bulk solution. They can, however, be recovered quickly using neat TFA or slowly adding >4 M GdnHCl solutions. In addition to P2Naq(OMe/OMe), peptides P1 and P2A, as described above, were synthesized under the same conditions.

3.3.1 Deprotection/Activation of P2Naq(OMe/OMe)

Despite the success of using the hypervalent iodide(III) oxidant PIFA to activate Naq in Ac-Naq(OMe/OMe)- OMe, all attempts to similarly deprotect P2Naq(OMe/OMe) failed. Indeed, while initial experiments were performed with blind excess of the PIFA reagent resulting in indistinct products, none of which had an obvious, non-aggregate naphthoquinone spectrum, even careful titrations of PIFA into a solution of P2Naq(OMe/OMe) showed an unexpected one equivalent lag of in the formation of an activated Naq product, Figure 3.4. The best explanation was that another species in solution, presumably the Tyr₇ of P2Naq(OMe/OMe), was reacting significantly faster with PIFA than Naq(OMe/OMe) since

Figure 3.4. Titration of P2Naq(OMe/OMe) with substoichiometric aliquots of PIFA. One equivalent is added before oxidation of Naq(OMe/OMe) is evident as observed at 340 nm. The lines are drawn to guide the eye.

oxidation is effected through a covalent adduct between the oxidant and its target and tyrosine is less sterically occluded than Naq(OMe/OMe).

Since it was unknown at the time whether the observed phenomena was going to be true for all oxidants commonly used to activate methyl-ether protected quinones suggesting that the tyrosine is far more reductive than Naq(OMe/OMe) is, other common methods of removing methyl-ether protecting groups were explored, all of which can be described as push-pull nucleophilic displacement reagents, where an oxophilic Lewis acid activates the methoxy sufficiently to allow a nucleophile to displace the methyl group freeing the hydroxyl. The vast majority of the reagents attempted, from BF3-methylsulfide or AlCl₃/EDT in CH₂Cl₂, both of which only partially deprotected Naq(OMe/OMe) even though they were in vast excess, to TMSBr/EDT/thioanisole/m-cresol, which showed no appreciable deprotection, were completely unsatisfactory. There were two exceptions to this observation. In the case of triflic acid/thioanisole in TFA, there was clear removal of the methyl protecting groups from Naq(OMe/OMe), but it was met with a mixture of backbone hydrolysis products around Naq; as if Naq, once deprotected, was involved in the pseudo- catalytic hydrolysis of peptide bonds before and after itself. On the other hand, using TMSI/EDT/thioanisole in TFA to deprotect P2Naq(OMe/OMe) gave a single predominant product with a very clear reduced spectrum not unlike that of the protected Naq(OMe/OMe). Mass spectroscopy, both MALDI-TOF-MS and ESI-MS, revealed a dehydration reaction had occurred, but this was not all together unexpected given the number of amides in the peptide. However, the product was not redox active as purified, and only displayed redox activity after being accidentally exposed to a highly acidic aqueous solution. Given the observations, it is believed that the dehydration product was actually a

cyclization of reduced Naq to the peptide backbone, Scheme 3.1. The heptaNaq project described in Chapter 2 was conceived to untangle the various reactions that were complicating the selective deprotection of Naq in P2Naq. Subsequent attempts to further fine-tune the reactivity of the push-pull systems proved difficult; the use of TMS-triflate in TFA, for instance, generated both presumptive backbone cyclization and hydrolysis products, while using B(OTFA)₃ in TFA in a large excess seemingly hydrolyzed the backbone of an extended helix from the N- and C- terminals and, though interpretation of the MALDI-TOF spectra of the HPLC purified products was difficulty, did not appear to deprotect Naq(OMe/OMe).

The success of using stoichiometric amounts of cerium ammonium nitrate (CAN) to deprotect heptaNaq(OMe/OMe) in the presence of heptaTyr (Chapter 2), provided the basis for successfully deprotecting P2Naq(OMe/OMe). Treatment of P2Naq(OMe/OMe) in 8 M GdnHCl with 100 mM TFA, see discussion above concerning the solubility of P2Naq(OMe/OMe), with 2-3 equivalents of CAN at room temperature for twenty minutes yields deprotected P2Naqox in a ~60% overall HPLC yield; the remaining 40% is made up of