A Versatile Approach to Noncanonical, Dynamic Covalent Single- and

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A Versatile Approach to Noncanonical, Dynamic Covalent Single and Multi-Loop Peptide Macrocycles for Enhancing Antimicrobial Activity James F. Reuther, Andrew C. Goodrich, P. Rogelio Escamilla, Tiffany A. Lu, Valarie Del Rio, Bryan W. Davies, and Eric Van Anslyn J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 21 Feb 2018 Downloaded from http://pubs.acs.org on February 21, 2018

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Journal of the American Chemical Society

A Versatile Approach to Noncanonical, Dynamic Covalent Single and Multi-Loop Peptide Macrocycles for Enhancing Antimicrobial Activity James F. Reuther,a Andrew C. Goodrich,b P. Rogelio Escamilla,a Tiffany A. Lu,a Valarie Del Rio,a Bryan W. Davies,b* and Eric V. Anslyna* a

Department of Chemistry, University of Texas at Austin, Austin, TX 78712

b

LaMontange Center for Infectious Diseases, Department of Molecular Biosciences, University of Texas at Austin, Austin, TX 78712 KEYWORDS: Peptide Macrocycles, Dynamic Covalent Chemistry, Cationic Antimicrobial Peptides, Solid-Phase Peptide functionalization, Drug-Resistant Bacteria ABSTRACT: Peptide oligomers offer versatile scaffolds for the formation of potent antimicrobial agents due to their high sequence versatility, inherent biocompatibility, and chemical tunability. Though many methods exist for the formation of peptide based macrocycles, increasingly pervasive in commercial antimicrobial therapeutics, the introduction of multiple looped structures into a single peptide oligomer remains a significant challenge. Herein, we report the utilization of dynamic hydrazone condensation for the versatile formation of single, double, and triple-loop peptide macrocycles using simple dialdehyde or dihydrazide small molecule crosslinkers, as confirmed by MALDI-TOF MS, HPLC, and SDS-PAGE. Furthermore, incorporation of aldehyde containing side-chains onto peptides synthesized from hydrazide C-terminal resins (HyRes) resulted in tunable peptide macrocycle assemblies formed directly upon resin cleavage post-SPPS. Both of these type of dynamic covalent (DC) assemblies produced significant enhancements to overall antimicrobial properties when introduced into a known antimicrobial peptide, Buforin II, when compared to the original unassembled sequence.

INTRODUCTION The recent and rapid evolution of antibioticresistant bacterial strains has led to the unfortunate, undeniable need for novel therapeutics to tackle this systemic problem.1-3 One approach takes inspiration from cationic antimicrobial peptides (CAMPs), integral components of the innate human immune system that ward off infections. Peptides offer a promising alternative to small molecule antibiotics because their large sequence diversity and chemical tunability make it challenging for bacterial strains to develop resistance.4-5 However, developing resistance is still possible and potential ramifications of utilizing CAMPs as broad-spectrum antibiotics stems from the possibility of compromising essential immune system responses that utilize natural CAMPs.6 One alternative is to incorporate unnatural functionalities into amino acid side-chains as a means of disguising the CAMPs while still retaining their potent antimicrobial properties.7-8 This tactic complements those that utilize unnatural backbone structures to mimic CAMPs such as β-peptides,9-10 peptoids,11-12 and oligothioetheramides,13-15 to name a few. In contrast, dynamic combinatorial chemistry provides a highly convergent approach for the high-throughput screening of a large library of drug candidates through dynamic exchange of reversible covalent bonding moieties.16-20 The incorporation of dynamic covalent (DC) functionalities into biologically active CAMPs and CAMP mimics could provide a marriage of these two principles offering a simple approach for incorporation of noncanonical functionalities as well as induction of topologi-

cal transitions. DC functionalities have been incorporated into peptide oligomers leading to interesting applications, such as templated assembly of fluorophores,21 α-helix formation,22 macrocyclization,23 peptide-peptide 24 ligation, self-replication,25 formation of catenanes,26 and formation of β-hairpin mimics.26 Very recently, our group reported the facile incorporation of aldehyde and hydrazide functionalities using solid-phase copper-catalyzed azide-alkyne cycloaddition (SP-CuAAC) “click” reactions (Scheme 1).27 Alkyne derivatives of these functionalities

Scheme 1. Reversible condensation of hydrazides onto aldehydes (a) and solid-phase copper-catalyzed azidealkyne cycloaddition (SP-CuAAC) incorporation of unnatural, dynamic covalent (DC) aldehyde and hydrazide functionalities into peptide oligomers containing unnatural azidolysine (AzK) amino acid residues (b).

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Figure 1. MALDI-TOF MS of macrocycles (MC; a and b) and multi-loop macrocycles (MLC; c) with two loops formed upon reaction of DiAl/Hy and TetAl/Hy peptides, respectively, with small molecule crosslinkers. These small molecules include flexible, aliphatic dihydrazides and rigid aromatic dialdehydes all showing efficient cyclization. All reactions were conducted at c = 1.0 mM in either 1:1 water: acetonitrile (a and c) or 1:1 PBS buffer (pH = 7.4): acetonitrile (b).

are “clicked” onto azidolysine (AzK) amino acid residues incorporated via solid-phase peptide synthesis (SPPS). When mixing these complementary peptides, the aldehyde and hydrazide side-groups rapidly react in aqueous environments to form unnatural peptide quaternary structures linked by dynamic hydrazone bonds. These unique topological quaternary structures include simple macrocycles, multi-loop macrocycles, zipper-like assemblies, and ladder polymers. Interestingly, upon DCassembly, these abiotic peptide quaternary structures were found to increase overall antimicrobial efficiency versus Staphylococcus Aureus when compared to their linear components. Of these various topological structures the templated ladder polymers and single-loop macrocycles (MCs) showed the most dramatic enhancements in activity. These enhancements led to overall modest antimicrobial effectiveness, as the peptide oligomers were not specifically designed to perform as antibiotics. Nonetheless, the observed trend motivated us to explore this phenomenon further. Herein, we demonstrate an expansion of our previous work showing the ability to induce DC-assembly of aldehyde and hydrazide containing peptide oligomers using simple small molecule crosslinkers. This approach allows for the formation of MCs when combined with dialdehyde (DiAl) or dihydrazide (DiHy) peptides or multi-loop macrocycles (MLCs) when mixed with tetra- or hexa-functionalized (TetAl/Hy and HexAl/Hy, respectively) oligomers. Additionally, we utilized hydrazide Cterminal resins (HyRes) as a means to synthesize peptide oligomers containing both hydrazide (on C-terminus post-resin cleavage) and aldehyde (through SP-CuAAC)

functionalities. Upon resin cleavage, these peptides spontaneously cyclize/oligomerize into various structures depending on the overall spacing of the DC-functionalities. Lastly, the effect of these various cyclization strategies on the antimicrobial properties of Buforin II, a naturally occurring CAMP, was tested versus various bacterial strains, including two antibiotic resistant strains, showing dramatically enhanced antimicrobial effectiveness when cyclized.

Table 1. Model DC-Peptides utilized for cyclization studies including peptides with 2, 4, and 6 aldehyde or hydrazide functionalities (X = Al or Hy, respectively). Peptide

Sequence

DiX1

N-RTRXFTXGRYF-C

DiX2

N-RTXRFTGXRY-C

DiX3

N-RXTRFTGRXY-C

DiX4

N-XRTRFTGRYXG-C

TetX1

N-RTXVXTXRXY-C

TetX2

N-XGXTDHRLDSXGXE-C

TetX3

N-XIHEXQNRXSARXVTW-C

HexX

N-XRLTXTFSXSFTXGTRXSGTXLRTXTRE-C

RESULTS AND DISCUSSION Peptide-Small Molecule Macrocyclizations


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Journal of the American Chemical Society DiAl1 and DiAl4 were also monitored by HPLC (Figure 2ad) showing complete conversion to the cyclic product as evidenced by the shift in retention time from 18.9 and 18.1 min to 18.7 and 16.6 min, respectively (All reported analytical HPLCs are 30 min gradient runs from 5% to 95% acetonitrile in water with 0.1 v% formic acid background). Additionally, the presence of one sharp peak in the crude reaction mixture after 2 hrs of reaction nicely portrays the fast, quantitative conversion of this cyclization strategy. Further evidence of the quantitative nature of this macrocyclization strategy is supported by 1H NMR where we observe the complete disappearance of the chemical shifts associated with the aldehyde protons at ~10.6 ppm upon cyclization of DiAl4 with ADH (Figure S1). Peptide reaction solutions remained stable over the course of monitoring for 10 days in 1:1 water: acetonitrile demonstrating the overall usefulness of this cyclization strategy.

Figure 2. Analytical HPLC chromatograms monitored at 254 nm absorbance of linear DiAl1 (a), DiAl4 (c), TetAl1 (e), TetAl2 (g) along with the peptides macrocyclized with ADH (b and h) or SDH (c and f) showing quantitative conversion to the corresponding DC-MC or DC-MLC when reacting for 2 hrs at c = 1.0 mM in 1:1 water: acetonitrile.

Several of the peptides synthesized in our previous report were used as models to examine the effect of using small molecule crosslinkers to induce cyclization (Table 1). DiAl and DiHy peptides were found to rapidly cyclize (ca. 1-3 hrs) when introduced to small molecule dialdehydes or dihydrazides at 0.5 to 3.0 mM concentrations. This is indicated by the presence of one dominant mass ion in the MALDI-TOF MS of these various peptide macrocyclization reactions analyzed without purification (Figure 1a and b). Four DiAl and DiHy peptides were studied with different amino acid (AA) spacing between the DC functionalities (ca. 2, 4, 6, and 8 AAs spaced for DiAl/Hy1-4, respectively). The tabulated observed and theoretical mass ions for all reported MALDI-TOF MS analysis can be found the supporting information. For cyclizations of DiAl2, 3, and 4 with flexible dihydrazide linkers, i.e. succinic dihydrazide (SDH) and adipic dihydrazide (ADH), complete conversion was observed in the MALDI-TOF MS as evidenced by the complete disappearance of starting material. DiAl1, the smallest of the macrocycles formed with only 2 AAs spaced between aldehydes, showed a small amount of starting material which is believed to be consequence of ionization. The conversion of the peptide macrocyclizations for

Mixing TetAl DC-peptides with the same crosslinker molecules showed high efficacy formation of double-loop MLCs (Figure 1c) with quantitative conversions. DC-macrocyclization of TetAl derivative TetAl1, with equally spaced aldehyde units every other AA, as well as TetAl2, with two aldehyde units at either termini spaced by one AA, with SDH or ADH occurred very efficiently as evidenced by MALDI-TOF MS. Further evidence of quantitative conversion was demonstrated using HPLC (Figure 2e-h) where the DC-macrocyclization of TeAl1 and TetAl2 causes a shift in retention time when allowed to react with SDH or ADH. The presence of only one distinct peak for these reactions is again indicative of the formation of one distinct product, even though the possibility of different cyclization products is present for the formation of double-loop MLCs. For example, the presence of four aldehyde moieties on the peptide oligomer results in the potential for several different crosslinking possibilities but the dynamic nature of the hydrazone linkage is hypothesized to allow for the most thermodynamically favored product to form through reversible exchange. DiHy1-4, TetHy2, and TetHy3 were found to macrocyclize when mixed with rigid aromatic dialdehyde crosslinkers but with some loss of efficacy (See Supporting Figure S2). The DiHy peptides cyclizations often display the presence of other mass ions in the MALDI-TOF MS associated with reaction of the N-terminus with the dialdehyde crosslinkers to form an imine. Switching the solvent system to 1:1 PBS buffer (pH = 7.4): acetonitrile reduced the abundance of side reactions for macrocyclization of DiHy peptides but did not eliminate them in all cases, as evidenced by HPLC. Further discussion of these cyclizations can be found in the supporting information (Supporting Section VIII).These macrocyclizations displayed one peak in the MALDI-TOF MS associated with product formation (Figure 1b and c), but due to the potential for side reactions to occur we mainly focused on cyclizations of aldehyde containing DC-peptides. Reactions between HexAl and small molecule dihydrazide crosslinkers resulted in gel formation due to the propensity to crosslink peptides intermolecularly. This property may hold interesting application potential



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in the formation of self-healable peptide hydrogels, but this is not the focal point of this study. Again, when employing buffered systems, the HexHy macrocyclizations precipitated completely from solution. Figure 3. DC-peptides with aldehyde side-chains synthesized on hydrazide C-terminal resins (HyRes) assemble into varDialdehyde ious macrocyclic and oligomeric structures (shown experiments were from c = 3.3 mM solutions), as characterized by crosslinkers, MALDI-TOF MS, based on the number of amino acids between DC functionalities. The four peptide synthesized, however, reHyRes1-4, are ordered by increasing number of AA spacing (2, 4, 6, and 10, respectively). The adopted DC-assemblies acted more include intramolecular macrocycles (green sphere), linear/macrocyclic dimers (red cube), linear/macrocyclic trimers selectively (blue triangle), and macrocyclic tetramers (purple hexagon). forming triple-loop clic/oligomeric assemblies, as demonstrated by MALDIMLCs when mixed with HexHy in 1:1 water: acetonitrile TOF and SDS-PAGE, which decreased in abundance as a (Figure S4). The analytical HPLC and MALDI-TOF MS of function of concentration. Transitioning from HyRes2 to HexHy support the quantitative formation of HyRes4 showed a continuous increase in intramolecular MLC(HexHy+TDA) and MLC(HexHy+ThDA), similar to macrocyclization of the aldehyde side group with the hyaforementioned examples. This approach provides a facile drazide terminus, with this DC-assembly predominating method for forming controlled macrocyclic topologies for HyRes4. Furthermore, for HyRes1-3, mass ions correwith multiple ring structures built into macromolecular sponding to macrocyclic and linear dimers/trimers were architectures. clearly observable whereas HyRes4 was found to adopt only macrocyclic DC-assemblies. Hydrazide C-Terminal Resin (HyRes) Peptides To study the effect of incorporating more rigid or As a means to incorporate both aldehyde and hydrazide DC-functionalities into a single peptide, we utilized a commercially available 2-chlorotrityl resin with an appended acyl hydrazide that becomes accessible postacid cleavage (HyRes) in place of the C-terminal carboxylate. Peptides were synthesized using this SPPS resin with aldehydes, incorporated using SP-CuAAC, spaced from the hydrazide terminus to varying degrees. HyRes1 to 4 increase the spacing between DC-functionalities in ascending order, with 2, 4, 6, and 10 AAs spacing, respectively (Figure 3). This degree of spacing greatly altered the assembly process for these HyRes peptides as revealed by MALDI-TOF MS. All HyRes peptides, were dissolved at various concentrations (ca. c = 0.05 mM – 25.0 mM) in 1:1 water: acetonitrile to observe the effect of concentration on the adopted DC-assemblies. Surprisingly, the concentration played little role on the assembly process with only small changes observed via MALDI-TOF MS or SDSPAGE (Figure S5 and S6, respectively). For HyRes1, the short relative spacing for aldehyde and hydrazide groups highly disfavor intramolecular macrocyclization with the peptide almost completely adopting dimeric macrocyclic/oligomeric DC-assemblies. Additionally, HyRes1 assembled into trimeric macrocy-

more flexible amino acids into the HyRes sequence, two additional model HyRes peptides were synthesized with incorporated proline or glycine AA residues, respectively (Figure S8). HyRes2Flex was synthesized with the same sequence as HyRes2 except with presumably the most flexible and freely rotating AA (glycine) incorporated in between DC-functionalities. Additionally, five adjacent proline AAs were incorporated into a HyRes peptide 12mer (HyResPro5), similar length as HyRes4, to see if rigidifying the backbone would favor linear oligomerization. Surprisingly, neither sequence manipulation had much effect on the overall adopted DC-assemblies with HyRes2flex predominantly adopting dimer macrocyclic/oligomeric assemblies, similar to HyRes2, and HyResPro5 assembling into mostly intramolecular macrocycles, similar to HyRes4. Antimicrobial DC-Buforin Assemblies As previously mentioned, the DC-assembly process was found to enhance antimicrobial properties of model peptides in our previous report.25 Because these previously studied peptides were not specifically designed for their use as antimicrobial agents, their assemblies displayed modest activities. Still, this discovery prompted us


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Journal of the American Chemical Society to test the effect of DC-assembly on a naturally-occurring CAMP sequence, buforin II. This 21mer peptide was first isolated from an Asian toad species, Bufo bufo gargarizans, and has been shown to inhibit cellular function of bacterial organisms upon cell membrane penetration.28-29 Furthermore, buforin II exhibits good selectivity towards bacterial organisms over human red blood cells, as reported by Kumar and coworkers.8 These are desirable properties from a therapeutic stand point which contributed to our choice of this sequence to screen. Table 2. Designed antimicrobial buforin II peptides with 2, 3, or 4 DC-functionalities incorporated into the sequence (X = Al or Hy). Peptide

Sequence

DiX Buforin

N-XTRSSRAGLQFPVGRVHRLLRXK-C

TriX Buforin

N-XTRSSRAGLQFPXVGRVHRLLRXK-C

TetAl Buforin

N-AlTRSSRAAlGLQFPVGRAlVHRLLRAlK-C

HyRes Buforin

N-AlTRSSRAGLQFPVGRVHRLLRKF-Hy

Five DC-buforin II peptides were synthesized using SPPS and SP-CuAAC with two, three and four DCfunctionalities incorporated. Additionally, one HyRes buforin peptide was synthesized with the aldehyde incorporated at the N-terminus (Table 2). One of the true benefits to this method for cyclization is the versatile ability to screen a variety of different preformed peptide tertiary structures by utilizing various crosslinking agents. This versatility should mesh nicely with high-throughput screening of novel therapeutic agents using peptide scaffolds. For this study, we screened the various MC and MLC assemblies described as well as two DC-quaternary assemblies from our previous report (vide infra). The DC-buforin assemblies were first characterized using the typical techniques: MALDI-TOF MS and HPLC (Figure 4). Overall, a similar trend was observed as

in the model DC-peptide assemblies with the aldehyde containing buforin peptides (DiAl and TetAl) cyclizing very efficiently when reacting with either SDH and ADH, as evidenced by the presence of one distinct mass ion in the MALDI-TOF and one sharp peak in the HPLC chromatogram. This also marks the largest peptide macrocycle formed in this study with 20 AA residues between the two DC- functionalities further demonstrating the overall versatility of this cyclization strategy. The macrocyclizations of DiHy buforin with TDA IDA, or ThDA in 1:1 PBS buffer (pH = 7.4): acetonitrile displayed additional mass ions in the MALDI-TOF MS again associated with incomplete cyclizations as well as N-terminus imine condensation, even after 3 days. Also, the HPLC traces displayed several peaks, especially when reacted with TDA, again believed to be associated with different byproducts formed. Furthermore, some observed masses corresponded to hydrolyzed hydrazide functionalities after 3 days in solution suggesting decreased stability in comparison to the cyclizations of the DiAl peptides. This would decrease their overall usefulness as antimicrobial therapeutics. Finally, two quaternary topologies were also chosen for our antimicrobial studies, formed in an analogous manner as from our previous report, including the intermolecular MC and zipper (Z) assemblies formed upon DC-reaction between DiAl+DiHy buforins and TriAl+TriHy buforins, respectively. The MALDI-TOF MS displayed predominant mass ions associated with the desired products, with some observed starting material believed to be formed upon ionization. The analytical HPLC displayed no observable starting material, shifting the retention time in between each starting material retention time in both cases. The slightly broadened chromatographic peaks may be indicative of formation of other topological structures although only the MC and zipper assemblies were observed in the MALDI-TOF MS. To further confirm the formation of buforin DCassemblies, SDS-PAGE was performed using tricine-SDS buffer systems for the efficient separation of peptides based on molecular weight (MW) from 3 to 30 kDa (Fig-

Figure 4. MALDI-TOF MS (a) and analytical HPLC traces (b; monitored at 254 nm) of DC-assemblies for antimicrobial buforin II peptides with two, three, and four aldehydes or hydrazides incorporated including MC assemblies with DiAl buforin (1-2), MLC assemblies with TetAl buforin (3,4), DiAl + DiHy buforin intermolecular MC (5), and TriAl + TriHy buforin zipper-assembly (6).



MS. This is not too surprising, however, because the ablation efficiencies are much higher for lower MW species in MALDI-TOF causing low MW products to predominate. The sequence-defined oligomerization reached a degree of polymerization of x = 6, but the predominant band still appeared to be the lowest MW intramolecular MC product. Again, it is hard to delineate whether the observed bands are associated with linear oligomers or larger MC products. Similarly, the reaction between TriAl+TriHy buforin also displayed higher MW bands which again are believed to be associated with oligomerization. These buforin peptide assemblies are the longest and highest MW structures attempted for this DC-assembly process, and the large overall spacing between DC-functionalities is believed to be the cause of the oligomerization. Figure 5. Tricine SDS-PAGE of antimicrobial buforin DC-peptides and assemblies after reacting for 3 days at 3.0 mM including DiAl (A), DiHy (B), MC(DiAl+SDH) (C), MC(DiAl+ADH) (D), MC(DiHy+TDA) (E), MC(DiHy+ThDA) (F), DiAl+DiHy (G; oligomer and MC), TriAl (H), TriHy (I), TetAl (J), MLC(TetAl+SDH) (K), Z(TriAl+TriHy) (L), HyRes buforin (M; oligomers and MC).

ure 5).30 For the DiAl, TriAl, and TetAl buforin peptides alone (lanes A, H, and J), some dimerization can be observed consequence of imine condensation of the Nterminus onto the aldehyde side groups. This is consistent with the results reported previously. The macrocyclization reactions DiAl buforin with SDH and ADH (lanes C and D) conducted in 1:1 acetonitrile: water showed a single narrow band eliminating the presence of the dimerized DiAl byproduct. The macrocyclization reactions of DiHy buforin with TDA and ThDA (lanes E and F) run in 1:1 PBS buffer: acetonitrile displayed some Nterminus dimerization via SDS-PAGE as evidenced by the higher MW band, consistent with our results observed previously. The formation of MLC-buforin assemblies through reaction of TetAl buforin with SDH was confirmed using SDS-PAGE (lane K) as well, showing one distinct, narrow band associated with the cyclized product. The buforin DC-quaternary structures and HyRes buforin displayed interesting patterns in the SDS-PAGE suggesting oligomerization occurring. For the reaction between DiAl+DiHy buforin, the desired macrocyclic product (lane G; theoretical MW = 6030 Da) clearly predominates as evident by the dark band slightly below the 6 kDa peptide reference marker. However, three other bands were observed in the higher MW region believed to be either linear oligomers or larger macrocyclic products. This is different than what we have observed previously suggesting that increasing the spacing between DCfunctionalities begins to favor oligomerization over cyclization. In a similar manner, the HyRes buforin peptide displayed even higher MW oligomerization, despite only observing intramolecular MC assemblies via MALDI-TOF

Enhancement of Antimicrobial Activity through DCAssembly The minimal inhibitory concentration (MIC) of each linear DC-peptide, DC-assembly, and positive control buforin II unaltered was determined for three Gramnegative strains: E. coli W3110 (wild-type), the isogenic cationic antimicrobial peptide (CAMP) resistant mutant WD101,31 and multi-drug resistant A. baumannii (Ab) 507532 (Figure 6). Additionally, these samples were screened against Gram-positive multi-drug resistant S. aureus (MRSA) but were found to have no activity. MIC determination was conducted using standard methods.33 For all Gram-negative bacteria studied, the DCassemblies displayed vastly enhanced antimicrobial properties when compared to the uncyclized DC-peptides and the unaltered control buforin II. Buforin II activity was previously reported using an agar trypticase soy broth with MIC = 16 µM versus E. coli W3110.28-29 However, we found buforin II had no antimicrobial activity (ca. MIC > 128 µM) versus the aforementioned Gram-negative bacteria when tested by standard Mueller-Hinton broth assay. Similarly, the linear DC-peptides DiAl/Hy and TriAl/Hy displayed little to no activity in Mueller-Hinton broth with the lowest MIC = 64 µM versus Ab5075 for DiHy, TriAl, and TriHy buforin (For more plotted MIC values of linear DC-peptides and cyclizing agents, see Figure S10). All linear DC-peptides showed no activity (ca. MIC > 128 µM) versus either E. coli strain. Remarkably, upon DC-assembly, many of the peptide samples became highly active versus the bacterial strains tested, in particular the multi-drug resistant A. baumannii strain Ab5075. Collectively, the macrocyclizations of DiAl buforin with SDH or ADH (ca. MIC = 32 µM versus all strains) outperformed the MICs formed from DiHy buforin peptides with dialdehyde crosslinkers. This is not too surprising considering the lower overall stability of the DiHy buforin cyclizations which may be contributing to the lower activities. When cyclized with IDA, DiHy buforin showed the highest turn-on in antimicrobial effectiveness but still had relatively modest activities (ca. MIC = 64, 32, and 64 µM for E. coli W3110, Ab5075, and E. coli WD101, respectively; see supporting infor-


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Figure 6. MIC values (µM) of buforin DC-assemblies, DC-peptides, and buforin II versus wild-type E. coli W3110, drug-resistant A. baumannii 5075, and colistinresistant E. coli WD101 conducted in Mueller-Hinton broth showing a turn-on in antimicrobial activity upon DC-assembly with the crude reactions of TriAl+TriHy buforin, DiAl+DiHy buforin, and HyRes buforin performing the best (Arrows indicated activity greater than 128 µM which were not measured).

mation). The TetAl buforin MLC derivatives displayed low antimicrobial efficacy versus both E. coli strains, but were effective against Ab5075 with MIC = 16 and 32 µM when cyclized with SDH or ADH, respectively. The most efficient antimicrobial agents of the DC-assemblies tested were the two DC-peptide quaternary assemblies (i.e. DiAl+DiHy and TriAl+TriHy buforin) and HyRes buforin showing large increases in antimicrobial activity versus all strains tested. Interestingly, these three samples were found to oligomerize in solution forming higher MW sequence-defined polymers mixed with macrocyclic/zipper-like DC-assemblies. The crude reaction mixture of DiAl+DiHy buforin displayed the most consistently effective antimicrobial properties with MIC = 8 µM for all Gram-negative strains tested. The DC-assembly with TriAl+TriHy buforin also showed efficient antimicrobial activity versus Ab5075 with MIC = 8 µM and decent activity versus E. coli strains with MIC = 32 µM. The lowest MIC value measured for the DCbuforin assemblies was the HyRes buforin peptide with MIC = 8, 4, and 16 µM effectiveness versus E. coli W3110, Ab5075, and E. coli WD101, respectively. The overall effectiveness of these DC-buforin assembles versus E. coli WD101 portrays the impact of this strategy for developing antimicrobial agents. This E. coli strain is resistant to CAMPs including polymixins,31 which are used as last line antibiotic treatments.34 DC-assembly of DiAl+DiHy buforin proved to be very effective versus this resistant strain of E. coli with MIC = 8 µM with HyRes buforin coming in second (ca. MIC = 16 µM). This suggests that the DC-assembly process can enhance CAMP activity to overcome traditional resistance mechanisms.

The antimicrobial activity for buforin II is achieved through the unusual mechanism of penetration through the cell membrane of bacterial cells without membrane disruption followed by interacting with specific cellular factors.35 The key components of the peptide sequence responsible for cell permeation include the hinged proline region as well as the α-helical segment on the C-terminal end of the peptide sequence. The DCcrosslinking of buforin II is hypothesized to stabilize these specific tertiary structure enabling motifs allowing for enhanced cell permeability and leading to overall increases in antimicrobial efficiencies. In addition to this hypothesis, we can also not rule out potential changes to the mechanistic pathways for antimicrobial activity. Clearly, this hypothesis will require further studies for conformation, but the dramatic increase in antibiotic activity found for our DC-crosslinking warrants such future work. CONCLUSION Herein, we report the use of small molecule crosslinkers to induce topological transitions in peptides containing complementary DC-functionalities (i.e. aldehydes and hydrazides). We also utilized commercially available resins for the design and synthesis of HyRes peptides with aldehydes and hydrazides. With these various peptides, we were able to construct peptide-based MCs and MLCs with one, two, or three loops per peptide. Furthermore, peptides with large spacing between complementary DC-functionalities were shown to form sequence-defined polymers with MWs reaching ~14 kDa. This DC-assembly process interestingly showed an enhancement of the overall antimicrobial activity of peptide systems when employing the well-known CAMP buforin II. These assemblies were found to be very efficient at inhibiting the growth of Gram-negative, drugresistant bacterial strains leading to potential applications as peptide-based therapeutics and broad-spectrum antibiotics. Future work includes the further development of DiAl, TetAl, and HyRes CAMPs along with the synthesis of novel crosslinkers to further screen the potential impact of ring size, amphiphilicity, and CAMP sequence on the antimicrobial effectiveness of peptide structures. Furthermore, we are exploring the combination of peptides with different properties to monitor the effect of this combination on the overall biological activity of the assembly.

ASSOCIATED CONTENT For synthetic procedures, characterization of peptide oligomers, additional characterization of DC-assemblies and procedures for antimicrobial screening, see the supporting information. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * [email protected] * [email protected]



Funding Sources The presented work was supported financially under the DAPRA Fold-Fx program (N66001-14-2-4051), NIH (AI25337), and the Welch Regents Chair (F-0046 and F-1870).

ACKNOWLEDGMENT We would also like to thank Maria Persons of the University of Texas at Austin Proteomics Facility and Ian Riddington of the Mass Spectrometry Facility at UT Austin for their aid in MALDI-TOF MS and HRMS acquisition.

ABBREVIATIONS CAMP, Cationic Antimicrobial Peptides; DC, dynamic covalent; SP-CuAAC, solid-phase copper catalyzed azide-alkyne cycloaddition; SPPS, solid-phase peptide synthesis; MC, macrocycles; MLC, multi-loop macrocycles; SDH, succinic dihydrazide; ADH, adipic dihydrazide; TDA, terephthaldehyde; IDA; isophthaldehyde; ThDA, 2,5thiophenedicarboxaldehyde; HyRes, hydrazide C-terminal resin; MIC, minimum inhibitory concentration

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Insert Table of Contents artwork here Dynamic Covalent Macrocyclization

Etc…

Gram-Negative Bacteria

Triggered inhibition of bacterial growth upon DC-assembly Active versus drugresistant strains


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A Versatile Approach to Noncanonical, Dynamic Covalent Single- and

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