430-480 nm1200 mW cm-2

Modeled from PDB ID: 1RKK

HPU Department of Chemistry

R&D Department, Ameritox

Society for Redox Biology and Medicine

HPU Office of Undergraduate Research and Creative Works

WFU Center for Redox Biology and Medicine

WFU Center for Molecular Communication and Signaling

Synthesis and Evaluation of the Unique Antimicrobial Peptide Polyphemusin-1William A. LeFever and Andrew J. Wommack*

Department of Chemistry, High Point University

Background:

Acknowledgements:

1) Miyata, T.; et al. Antimicrobial Peptides, Isolated from Horseshoe Crab Hemocytes, Tachyplesin II, and Polyphemusins I and II: Chemical Structures and Biological Activity. J. Biochem. 1989, 106, 663–668. 2) Hancock R. E. W.; The antimicrobial peptide polyphemusin localizes to the cytoplasm of Escherichia coli following treatment. Antimicrob. Agents

Chemother. 2006, 50, 1522–1524. 3) Simon, M. D.; et al. Rapid flow-based pepetide synthesis, Chembiochem. 2014, 15, 713-720 4) Tam, J. P.; et al. J. Am. Chem. Soc.1991,113,6657–6662. . 5) Hoyle, C. E.; Bowman, C. N. Thiol–Ene Click Chemistry. Angew. Chem. Int. Ed. 2010, 49, 1540–1573.

◆ Polyphemusin-1 (PM1) is a cationic antimicrobial peptide fromLimulus polyphemus, the American horseshoe crab.1

◆ The main structural motif of native PM1 is a β-hairpin turnproduced by two intramolecular disulfide bonds, granting PM1high antimicrobial activity against Gram-negative bacteria. 2

◆ The importance of reversible disulfide bridging to PM1 activitycan be determined by synthetically replacing disulfide bondsthat participate in the β-hairpin turn on native PM1.

◆ Permanently reduced derivatives of PM1 were synthesized byreplacing each cysteine in the sequence with alanine to preventdisulfide bridging and the β-hairpin turn.

◆ Photoredox-catalyzed TEC was performed on a truncated PM1derivative containing a free thiol and a vinyl carbon to form aninert thioether coupling mimicking the native disulfide bridge.

Figure 1: Native PM1 oxidized (left) and reduced (right) structures

Solid Phase Peptide Synthesis:

Figure 3: General description of the preparation of each PM1 derivative through Fmoc-SPPS 3

Disulfide bridges were induced on synthetic native PM1 analogs by dissolving pure peptide in .1% acetic acid with 5% DMSO.4

FLOW ChemistrySPPS Reactor Body

3-min/coupling!!!

Characterization TEC Coupling Reaction

(A) crude native PM1 pre-oxidation. (B) pure native PM1 pre-oxidation.(C) crude oxidized PM1. (D) pure oxidized native PM1. (E) crudetruncated native PM1 pre-oxidation. (F) pure truncated native PM1 pre-oxidation. (G) crude oxidized truncated native PM1. (H) pure oxidizedtruncated native PM1. (I) crude PM1-alanine. (J) pure PM1-alanine.(K) crude truncated PM1-alanine. (L) pure truncated PM1-alanine.

Ru2+ photoredox

catalysis, followed by

alkylative quench

1) Ru(bpy)3Cl2 (0.50 mol%)p-toluidine (0.50 equiv)

20 mM NaOAc, pH 5.1, (0.10 mM)

452 nm, Blue LEDs

HSS

I

O

NH2

(>50 equiv)

5 min, then pH <4.0

S

NH2

O

NOT OBSERVEDby LC-MS

RCFGRYvinylGlyV RCFGRYvinylGlyV

RCFGRYvinylGlyV

Starting b-turn model peptidefrom polyphemusin-1

91% Yield

2) 75 mM HEPES, pH 8.0,

23 °C, 30 min

entry yielda,bsubstrate sequence

1 91 (2, 3, 0)RCFGRYvinylGlyV

2 84 (6, 3, 0)

3 51 (12, 15, 20)

4 78 (5, 4, 9)

5 72 (8, 6, 11)

6 74 (6, 5, 10)

KCFGRYvinylGlyV

7 71 (2, 3, 8)

ECFGRYvinylGlyV

ACFGRYvinylGlyV

ICFGRYvinylGlyV

VCFGRYvinylGlyV

VCWGRYvinylGlyV

Figure 6: Example installation of the redox-inert disulfide on vG-PM1

Conclusions and Future Work

Native-PM1-red crudeObserved m/z[M+3H] = 817.3[M+4H] = 613.1Expected: 820.7/615.5

Native PM1-red pureObserved m/z[M+3H] = 818.5[M+4H] = 613.9Expected: 820.7/615.5

Native PM1-ox pureObserved m/z[M+3H] = 817.1[M+4H] = 613.1Expected: 819.7/614.5

PM1-red-frag crudeObserved m/z[M+2H] = 582.9Expected: 584.2

PM1-red-frag pureObserved m/z[M+2H] = 582.9Expected: 584.2

PM1-ox-frag pureObserved m/z[M+2H] = 581.6Expected: 583.2

Ala-PM1 crudeObserved m/z[M+3H] = 776.1[M+4H] = 582.4Expected: 777.9/583.7

Ala-PM1 pureObserved m/z[M+3H] = 776.1[M+4H] = 582.4Expected: 777.9/583.7

Ala-PM1-frag crudeObserved m/z[M+2H] = 551.0Expected: 552.13

Ala-PM1-frag pureObserved m/z[M+2H] = 550.7Expected: 552.13

vG-PM1 frag pureObserved m/z[M+2H] = 659.3[M+3H] = 440.0

Oxidized PM1 frag pure

11.427

11.109

10.433

A

Native PM1

B

C

D 10.422

E

F

G

H

10.219

10.261

10.640

Figure 7: MS and HPLC characterization of purified vinylglycinesubstituted PM1 derivative after photocatalyzed TEC coupling.

RRWCFRVCYRGFCYRKCRVCYRGFCYR

RRWAFRVAYRGFAYRKARVAYRGFAYR

RRWvGFRVvGYRGFCYRKCRVvGYRGFCYR

Figure 2: PM1 sequences and cysteine replacements

10.849

I10.800

J10.638

K 9.786

9.816L

◆ Photoredox-catalyzed TEC will be performed on a full length PM1derivative containing vinylglycine residues and tested forantimicrobial activity4. RRWvGFRVvGYRGFCYRKCR

Figure 4: HPLC traces and MS of crude and purified native PM1.

Figure 5: Synthetic route to Fmoc-vinylglycine from Fmoc-Met-OH5

FmocHN

O

OCH3

H

SCH3

FmocHN

O

OHH

SCH3

HOBt (1.1 equiv)HBTU (1.05 equiv)DIPEA (2.0 equiv)

DMF (0.40 M)MeOH (3.0 M)

23 °C, 1 h O

OCH3

H

SCH3

FmocHN

O

MeOH/MeCN(1:1, 0.50 M)0 °C ® 23 °C

12 h

0.25 M KIO4 (1.1 equiv)

18 h, refluxxylenes

FmocHN

O

OCH3

H

81% isolated yield

1 M HCl,HOAc,

reflux, 1 h

Fmoc-VinylGly-OH

FmocHN

O

OH

H

Fmoc-Met-OH

O

OCH3

H

SCH3

FmocHN

O

H

◆ Antimicrobial assays will berun in collaboration with theBlackledge Lab at HPU todetermine minimum inhibitoryconcentration (MIC) values foreach PM1 derivative.