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Secondary Amino Alcohols: Traceless Cleavable Linkers for Use in Affinity Capture and Release

: Capture and release of peptides is often a critical operation in the pathway to discovering materials with novel functions. However, the best methods for efficient capture impede facile release. To overcome this challenge, we report secondary amino alcohol-based linkers for release of peptides after capture. These amino alcohols are based on serine (seramox) or isoserine (isoseramox) and can be incorporated into peptides during solid-phase peptide synthesis via reductive amination. Both linkers are quantitatively cleaved within minutes under NaIO 4 treatment. Cleavage of isoseramox produced a native peptide N -terminus. This linker also showed broad substrate compatibility; incorporation into a synthetic peptide library resulted in the identification of all sequences by nanoLC-MS/MS. The linkers are cell compatible; a cell-penetrating peptide that contained this linker was efficiently captured and identified after uptake into cells. These findings suggest secondary amino alcohol-based linkers might be suitable tools for peptide discovery platforms.


Introduction
Synthetic peptide libraries are used to discover sequences that bind to protein targets, penetrate cells, or facilitate site-selective bioconjugations. [1][2][3][4][5][6] These experiments require the selective capture of hits from chemical or biological samples and subsequent release. After release, the isolated peptides are analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to determine their sequences. The best approaches for efficient capture impede quantitative release. Indeed, the widely adopted streptavidin-biotin affinity capture and release approach suffers from this hurdle (Figure 1a). To release biotinylated peptides denaturing conditions [e.g., boiling in sodium dodecyl sulfate (SDS) buffer] 7 are required, which can lead to degradation and contamination. Therefore, there is a need for new approaches for efficient retrieval of peptides after capturing with streptavidin.
Cleavable linkers are attractive tools that enable the release of peptides after affinity capture. [8][9][10] Linkers consist of chemical moieties that can be 1) incorporated into peptides and 2) cleaved under specific conditions. Various cleavable linkers have been described, and their cleavages can be induced with enzymes, 8 photoirradiation, 11 metals, [12][13][14] nucleophiles, 15,16 electrophiles, 8 acids, 17 reductants, 18 and oxidants. [19][20][21] Vicinal diol-based linkers are compatible with standard solidphase peptide synthesis (SPPS) (Figure 1b). The 1,2-diol motif is stable toward basic and acidic conditions. Treatment of linkerembedded peptides with sodium periodate facilitates cleavage. These oxidative conditions are compatible with folded proteins 20 and with cells. 22 Amino alcohols are an alternative periodate-labile motif and can be cleaved 1000-times faster than diols. 21,23 An SPPS compatible linker based on this structure has been reported. 19 However, this linker requires a nine-step synthesis, presenting challenges for its widespread applicability. Here, we report secondary amino alcohols as a new class of oxidatively cleavable linkers. Secondary amino alcohols exhibit the same reactivity as primary amino alcohols towards NaIO4 oxidation in small molecule studies. 23 We designed two cleavable linkers with this motif based on serine (seramox) and isoserine (isoseramox). These structures can be inserted into a peptide backbone during SPPS via reductive amination. Both linkers are quantitatively cleaved by periodate within minutes (Figure 1c). The isoseramox linker releases a native peptide N-terminus upon cleavage; this is the first example of a traceless periodate cleavable linker. Isoseramox was incorporated into a 216membered peptide library, showing broad substrate tolerance for synthesis and cleavage. Isoseramox was also compatible with cell assays. A cell-penetrating peptide containing isoseramox entered the cell cytosol before it was captured and selectively released by periodate. Secondary amino alcohol linkers are thus enabling tools for experiments requiring the capture and release of synthetic peptides.

Results and Discussion
Secondary amino alcohols can be incorporated into peptides during standard SPPS. Peptide 1 (SLAGV) was prepared by Fmoc-based SPPS. Gly-derived aldehyde 2 was attached to the N-terminal serine residue via reductive amination on-resin (Scheme 1a). The resulting secondary amine was then Bocprotected. Removal of the Fmoc group enabled the elongation of the peptide chain, generating peptide 3 (Y-seramox-SLAGV). Simultaneous cleavage of the peptide from the resin and side chain deprotection provided seramox-containing peptide 4 in 90% crude yield ( Figure S1a). 24 The cleavable secondary amino alcohol moiety in the peptide backbone is based on a natural Gly-Ser dipeptide, wherein a single carbonyl is replaced with a methylene group. This Ψ(CH2NH) functionality is considered a bioisostere of an amide. 25 A seramox-containing peptide was quantitively cleaved in 5 min, faster than a diol-containing peptide (Figure 2). The peptides [500 µM in 1X phosphate buffered saline (PBS), pH 7.5] were treated with two equivalents of NaIO4 (1 mM). Under these conditions, less than 30% of diol 4a was cleaved after 60 min. Seramoxcontaining peptide 4b was cleaved to full conversion after 5 min. Quantitative cleavage of 4b was also accomplished at lower concentrations of peptide and periodate (10 µM and 30 µM respectively) in three hours ( Figures S3, S4).
[a] H2N-OH•HCl was not added for cleavage of the isoseramox peptide (4c), and the quenching was performed for 5 min.
The cleavage of seramox generates an N-terminal aldehyde product (Scheme 1b). Coordination of seramox to the periodate (A) and cleavage of the Cα-Cβ bond generates an imine (B) in the peptide backbone. This imine is spontaneously hydrolyzed, affording a primary amine on the C-terminal fragment (C), and an aldehyde on the N-terminal fragment (D). Aldehydes could represent a handle for further functionalization. [26][27][28] However, aldehydes suppress MS signals, especially hindering MS/MS sequencing. This issue is not only present with seramox, but also with known diol and amino alcohol linkers (Figure 1b) and was circumvented by transforming the intermediate aldehydes into oximes with H2N-OH•HCl (Figure 2). 29 Secondary amino alcohol linkers based on isoserine (isoseramox) enable traceless release of N-terminal peptides. The generation of amines instead of aldehydes at the N-terminus upon periodate cleavage overcomes the MS sequencing challenges of known linkers. Furthermore, applications requiring the release of peptides for biological purposes may benefit from the regeneration of the native peptide structure. 30 In designing the isoseramox linker, we hypothesized that replacing the seramox moiety with an isoserine derivative would reposition the cleavable bond (C1-C2), thus generating a native N-terminal peptide upon periodate treatment (D, Scheme 2a).
Isoseramox-containing peptide 4c was quantitatively converted to N-terminal peptide 5c (LAGV) in 5 min with NaIO4 ( Figure 2). Despite the change from a primary alcohol (seramox) to a more sterically hindered secondary alcohol (isoseramox), reaction times were not affected. To our knowledge, this example is the first reported periodate cleavage that generates native peptides at the N-terminus. Both the amine and the alcohol functionality of the (iso)seramox linkers appear to be essential in enhancing cleavage rates. Since changing from a diol to an amino alcohol resulted in faster periodate cleavage, we tested a 1,2-diamine linker. A 2,3diaminopropionic acid (Dap)-derived linker was incorporated into peptides and its cleavage efficiency was studied ( Figure S5). Cleavage of the diamine required 18 hours to proceed to full conversion, slower than (iso)seramox linkers. Protonation of both amines at pH 7.5 may slow their rate of coordination to the periodate. We hypothesize that the (iso)seramox alcohol facilitates coordination to the periodate (Scheme 2a, 4c to A), while the amine accelerates the cleavage step (Scheme 2a, A to B). 31 Figure 3. Isoseramox linkers have a broad substrate tolerance for both incorporation and cleavage. Synthesis of a diverse peptide library without (library A) and with (library B) the isoseramox linker. Library A was cleaved from resin and isolated. Library B was conjugated to biotin, cleaved from resin, pulled down with streptavidin magnetic beads, and cleaved with NaIO4. Both libraries were sequenced using nanoLC-MS with >90% recovery of sequences in both cases. This highlights that the incorporation and the cleavage of isoseramox tolerates most classes of amino acids. N-terminal Ser and Thr were excluded to avoid formation of N-terminal aldehydes. N-terminal Cys was excluded because it is known to chelate periodate. Secondary amino alcohol linkers have a broad substrate tolerance. We incorporated isoseramox into a combinatorial peptide library to test (1) the efficiency of its addition on several N-terminal residues and (2) the tolerance of its oxidative cleavage in the presence of diverse amino acid side chains. A combinatorial library of 216 members was prepared by split-and-pool SPPS. The library contained 17 canonical amino acids in 8 positions (1234LAYK), four of which were variable positions (Figure 3). 32 Position 1 could be Asp, Met, His, Trp, Tyr, Val, Lys and Gln (containing basic, acidic, amidic, aromatic and aliphatic residues), position 2: Ser, Ala and Leu, position 3: Glu, Asn and Gly, and position 4 Phe, Pro and Thr. At this stage, part of the library was cleaved for use as a reference pool (library A). The second part was alkylated with isoseramox and then biotinylated (library B). Library B was immobilized onto magnetic streptavidin beads and were separately desalted by micro solid-phase extraction and analyzed by nanoLC-MS/MS on an Orbitrap Fusion Lumos spectrometer. The MS/MS spectra were analyzed with the sequencing software PEAKS 8.5 and the complete list of sequences obtained was additionally filtered for peptides fitting the library design (correct length and correct monomers in positions 1, 2, 3 and 4). 33 Efficient and complete cleavage would result in a product distribution identical to reference library A. Indeed, the sequences identified from the reference library A and the cleaved library B were almost identical: 90% (194 out of 216) and 94% (202 out of 216) of peptides were identified, respectively. Sequences with all eight possible residues in position 1 were identified. This demonstrates that incorporation and cleavage of the isoseramox linker are efficient regardless of the neighboring residues. Oxidized methionine residues were observed in both library A (89%) and library B (96%), though this heterogeneity did not impact sequencing. Oxidized Met is recognized by the PEAKS sequencing software as a post-translational modification. No oxidations or modifications of other residues, such as Trp or His, were observed. Furthermore, sodium periodate cleavage has previously been shown to be compatible in proteomics-based workflows, as the oxidation was found not to interfere with sequencing. 10 These results indicate that secondary amino alcohol linkers can be used for capture and release workflows.
Protein secondary structure and folding is not disrupted by sodium periodate treatment. After studying (iso)seramox in the context of short peptides, we investigated whether periodate-based amino alcohol cleavage could be extended to more complex scaffolds. The miniprotein Z33 was synthesized by SPPS. Z33 adopts a 33residue helix-helix bundle. The −helical structure of the synthetic Z33 was confirmed by circular dichroism (CD) spectroscopy. After treating Z33 with 4 mM of sodium periodate (i.e., four times more concentrated than the harshest cleavage condition) for 30 minutes, the CD spectrum showed no alteration ( Figure S6). This result indicates that the −helical structure was not disrupted by periodate treatment. The compatibility of Z33 with NaIO4 is in agreement with several previously published reports using periodate cleavages on folded proteins, antibodies, protein complexes and whole cells. [34][35][36] (Iso)seramox-based recovery of biotinylated peptides from streptavidin beads is more efficient than SDS denaturation strategies ( Figure 4). After capture of biotinylated peptides with streptavidin, it is currently difficult to release the peptides for analysis. A frequently used method to disrupt the strong biotinstreptavidin interaction relies on heating the complex in SDS containing buffer. 7 We compared this strategy to our (iso)seramox based recovery workflow. Two sets of peptides were prepared, containing seramox-Lys(Biotin) (10a-c) or Gly-Ser-Lys(Biotin) (12a-c), at their C-termini, respectively. To assess the effect of peptide recovery conditions on sensitive moieties, one peptide in each set contained a phosphoserine (pSer) and one peptide contained a TAMRA fluorophore. All peptides were individually immobilized on magnetic streptavidin beads. For 10a-c, periodate treatment enabled the recovery of cleaved peptides 11a-c (Method A; 0.5 mM, 10 min). Peptides 12a-c were recovered according to described denaturing procedures by heating in SDS buffer with excess biotin (Method B; 0.4% SDS, 20 mM biotin, 95 °C, 15 min). While the recovery solutions of 11ac were homogeneous, suspensions with precipitated material were observed for 12a-c. LC-MS analysis showed that the seramox-based strategy enabled the recovery of pure material. Alternatively, SDS denaturation caused release of several side products along with the desired peptides ( Figure 4). TAMRA and pSer were intact after both seramox and denaturation recovery strategies.
Isoseramox linkers are also compatible with cell-based assays. When screening for peptide binders to whole cells or for cellpenetrating peptides (CPPs) it is often necessary to recover hit peptides from cells. 3 Here we show that the isoseramox linker can be used to recover a CPP from HeLa cells.
We synthesized a variant of the known CPP D-penetratin, containing both biotin for capture and isoseramox for release (10, Figure 5a). Reactions of 10 (50 µM) with NaIO4 (3 equiv) provided quantitative conversion to cleaved product in 10 min ( Figure S7). Peptide 10 contains an oxidizable Met residue; only 30% Met oxidation was observed, indicating that oxidative cleavage of the linker is faster than oxidation of the Met sidechain. However, when performing peptide library assays at the MS detection limit, heterogeneity in the sample could cause loss of sequencing information.
D-Penetratin-containing isoseramox retains its cell-penetrating activity (Figure 5b). A derivative containing TAMRA (12) was synthesized and incubated with HeLa cells. Confocal imaging of the cells displayed diffuse fluorescence in the cytosol and nucleus, in addition to endosomes. These images suggest that the linker does not disrupt the peptide's cell-penetrating activity and access to the cytosol ( Figure S8).
We then captured and released D-Penetratin derivative 10 after delivery into HeLa cells (Figure 5a). HeLa cells were incubated with 10 µM peptide 10 for 1.5 h, washed extensively to remove membrane-bound peptide, and lysed. The lysate containing internalized biotinylated peptide was then incubated with streptavidin coated magnetic beads. Treatment of the magnetic beads with NaIO4 enabled the recovery of 3 ng of D-penetratin (11), 37 enough material for characterization by LC-MS (see Supporting Information). By comparison, results in our laboratory utilizing a protease cleavable linker did not result in the recovery of any peptide. 38 These results demonstrate the potential of the isoseramox linker for future applications in cell-based peptide library screening.

Conclusion
We have described secondary amino alcohols as a new class of cleavable linkers. (Iso)seramox exhibits broad applicability for peptide library screening. The linkers can be incorporated into peptides during on-resin synthesis. Cleavage of these secondary amino alcohols was shown to be faster than known diol linkers. Furthermore, isoseramox is the first example of a traceless, periodate-labile linker, where cleavage liberates native N-terminal peptides. The broad substrate compatibility of isoseramox was demonstrated in the synthesis, capture, and release of a peptide library. The recovery of 94% of sequences demonstrates 1) the efficiency of reductive amination of isoseramox onto several Nterminal residues and (2) the tolerance of its oxidative cleavage with various peptides. Additionally, isoseramox facilitated the recovery of peptides after uptake into cells. Taken together, these experiments highlight the applicability of (iso)seramox toward the release of peptides from streptavidin beads after screening assays and affinity capture. The tolerance of cells to NaIO4 39 could expand the potential of (iso)seramox linkers to controlled release of cargo in living systems. 13,40 We are optimistic that these easy-to-use linkers will be valuable tools not only for the discovery of functional peptides and peptidomimetics, but also in small molecule and polymer applications.