Photoamplification and Multiple Tag Release in a Linear Peptide-Based Array of Dithiane Adducts

Externally sensitized photoinduced fragmentation in dithiane adducts of carbonyl compounds[1] requires the presence of an electron transfer (ET) sensitizer, and therefore can be made contingent on a molecular recognition event. This event “arms” the binary photoactive system by bringing the adduct and the sensitizer into the immediate proximity of each other.[2] Such conditional photorelease of dithianes, which are readily detectable at subpicomolar levels, presents new opportunities for useful bioanalytical applications. We have recently developed this concept into a fundamentally novel methodology for direct screening of solution phase combinatorial libraries, in which various 2-alkyl substituted dithianes are used as encoding digits.[3] By immobilizing the dithiane-masked benzophenones on polymeric beads or dendrimers we have further shown that one molecular recognition event can trigger the release of multiple copies of the encoding dithiane tags, amounting to amplification on surface.[4] Such photoamplification is possible because each externally sensitized fragmentation of the benzophenone-dithiane adducts unmasks more sensitizer which, in turn, unmasks its neighbors carrying the amplification chain and therefore boosting the sensitivity. The next logical question becomes whether this photoamplification methodology can be implemented in a linear array of masked sensitizers, and – if such amplification is possible – whether the propagation of the effect and the release of dithiane tags occurs sequentially (i.e. not unlike the one-dimensional propagation in the Bickford Fuse), randomly, or in some other unusual order? In this Communication we report on photoamplification in linear polypeptide scaffolds. Synthesis of masked benzophenone sensitizers tethered to Fmoc-protected lysine is outlined in Scheme 1. We chose four 2-substituted dithianes, ethyl- (a), propyl- (b), pentyl- (c), and octyl-(d) to positionally encode the lysine residues in the polypeptide chain. The lithiated dithianes were reacted with 3-benzoyl benzoic acid 1, furnishing adducts 2a–d, which were converted into N-hydroxysuccinimide (NHS) esters 3a–d and tethered to Fmoc protected lysine. The modified lysines 4a–d, in a form of their NHS esters 5a–d, were utilized in solid state peptide synthesis on 0.48 mmol/g TentaGel beads. The peptides were capped with lysine-tethered benzophenone as the photoinitiator (Figure 1). As linear amplification in a single strand was the focus of our study we chose the low loading beads as a scaffold mimicking infinite dilution in solvent (i.e. preventing the inter-strand sensitization – see supporting information). Figure 1 Dithianes release after 10 min irradiation as a function of their position in polypeptides 6a–d (sensitizer is at position 0). Scheme 1 Synthesis of photoactive lysine conjugates. The following tetra-, hepta-, and decapeptides, containing photoactive lysine residues and spacers, were synthesized: LysBP-LysDT1-LysDT2-LysDT3-BEAD (6a) LysBP-Gly-LysDT1-Gly-LysDT2-Gly-LysDT3-BEAD (6b) LysBP-Met-LysDT1-Met-LysDT2-Met-LysDT3-BEAD (6c) LysBP-Gly-Gly-LysDT1-Gly-Gly-LysDT2-Gly-Gly -LysDT3-BEAD (6d) where LysDTX represents lysine residue outfitted with benzophenone masked with 2-alkyldithiane, and LysBP is the lysine carrying “free” tethered sensitizer. The standard synthetic Fmoc-piperidine protocol was used, except that in order to assure the completion at each step of the peptide synthesis, the time for coupling of NHS esters 5a–d was increased to 3 days per residue. Mono and bis-glycyl spacers were introduced to probe the distance dependence, whereas methionine was tested as a potential co-sensitizer or quencher. The beads were suspended in acetonitrile, degassed, and irradiated using a 330 nm long pass filter to selectively excite the sensitizer. Subsequent release of dithianes into solution was monitored by GCMS. After 10 min of irradiation all three dithianes were found in the solution, indicating that the linear amplification and the binary encoding in a linear array of dithiane tags are possible. This finding is critical to the development of our methodology for encoding and screening of solution phase combinatorial libraries. A single library member can now be readily encoded using a complete stringed set of tags which are necessary for its subsequent identification. Beyond this, the unusual ordering of the initial relative efficiencies presented an interesting opportunity to probe the mechanism of the release. The bar graph in Figure 1 shows relative dithiane peak intensities after 10 min irradiation as a function of their position in the polypeptide chain. It illustrates that the overall efficiency of release from the position most distal to the sensitizer is generally higher. A more detailed analysis of the release is shown in Figure 2. Due to the secondary photooxidation of the released dithiane, its concentration over the course of photolysis reaches a maximum and then decreases. We approximate this release by a trinomial function A = at3 + bt2 + ct, where term c gives us ∂A/∂t at time zero, allowing for an accurate comparison of the initial rates of release (values c are underlined in Figure 2). Again, as a rule, the most distal dithianes were released with higher initial efficiency. Figure 2 Dithianes release from peptides 6a–d. Position from the sensitizer: ● proximal, ∎ middle, ▴ distal. We suggest that the outcome of photoamplification in peptides is governed by the migration of the formed radical cation from one dithianyl moiety to the next in the chain. Such charge transfer between photoactive residues should be unbiased and random, because the oxidation potentials of alkyl dithianes are nearly degenerate and the system does not have thermodynamic sinks, other than the wasteful back electron transfer (BET) trap. The probability of BET from benzophenone anion radical is the lowest for the most distal dithiane, accounting for the experimental observation where the most efficient release is from the distal position in 6b–d (Figure 3). Because of BET, the proximal radical cation does not live long enough to fragment efficiently. This stochastic approach provides rationale for most of the experimental observations, if one recalls that as soon as the fragmentation occurs a fresh copy of the sensitizer is unmasked. The unmasked sensitizer is in turn excited, initiating similar radical cation hopping away from the radical anion, with the most distal position this time being the one closest to the original terminal benzophenone. Figure 3 Radical cation hopping in the polypeptide chain. One peculiar experimental observation is the release from tetrapeptide 6a (Figure 2), which has no spacers between the photoactive lysines. In this peptide the release of the two distal dithianes seems to occur simultaneously. This result may point to a specific mode of radical cation propagation in a system where dithiane adducts are spatially close to each other and, therefore, are capable of forming interdithiane two center three electron (2c3e) S-S bonds. Such 2c3e bonds are well precedented in the literature.[5] Previously, we have also shown that additional stabilization in bis-dithianyl radical cations considerably increases their quantum yields of fragmentation.[6] We therefore suggest that in tight systems such as 6a, propagation of the radical cation may occur through the direct formation of 2c3e bonds, so that the charge density migrates in a form of a dimeric dithiane radical cation (Figure 4). Release of either dithiane from these pairs has near equal probability, which explains the comparable initial efficiencies of release from the second (1203) and the third (997) adduct. The dimer formed by the first and the second dithiane is again at a disadvantage of BET. Figure 4 Charge density migration via dimeric cation-radicals. Introduction of methionine as a spacer (cf. 6b and 6c) had produced an overall effect of competitive quencher, even though Met+• is known to form the 2c3e bonds with various heteroatoms.[7] To assess the effect of polymeric matrix on the amplified release, the photoactive dithiane adducts were immobilized in a reversed order of the glycine separated heptapeptide 6b: LysDT3-Gly-LysDT2-Gly-LysDT1-Gly-LysBP-BEAD (7b) i.e. the lysine carrying the sensitizer was immobilized first. Irradiation of 7b produced the same preferential release of the distal dithiane (from the terminal LysDT3) attesting to the generality of the described phenomenon. The second control was to utilize the dithiane adducts of 4-formylbenzoic acid not capable of amplification. While upon irradiation of 8b (Figure 5) we see the most distal dithiane eventually released into solution, the combined relative quantum efficiency of the release from the aldehyde-based linear array is an order of magnitude smaller. Most likely this is due to the premature reduction of the only sensitizer in the strand during the extended photolysis needed to fragment all three dithiane adducts. In the amplified release from 6a–d and 7b every fragmentation event replenishes the sensitizer, which carries the photoamplification chain. Figure 5 Peptide 8b based on benzaldehyde adducts, not capable of amplification. In conclusion, we have demonstrated that binary dithiane encoding and amplification can be implemented in linear polypeptide-based arrays of photoactive externally sensitized dithiane adducts. Variable efficiency of dithiane release also opens up an exciting opportunity for developing a positionally encoded molecular barcode system beyond simple binary encoding.