Peptide Oligomers from Ultra-Short Peptides using

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Author’s Accepted Manuscript Peptide Oligomers from Ultra-Short Peptides using Sortase Natalya Voloshchuk, Long Chen, Qiang Li, Jun F. Liang www.elsevier.com/locate/bbrep

PII: DOI: Reference:

S2405-5808(17)30050-X http://dx.doi.org/10.1016/j.bbrep.2017.02.005 BBREP396

To appear in: Biochemistry and Biophysics Reports Received date: 27 July 2016 Revised date: 25 January 2017 Accepted date: 3 February 2017 Cite this article as: Natalya Voloshchuk, Long Chen, Qiang Li and Jun F. Liang, Peptide Oligomers from Ultra-Short Peptides using Sortase, Biochemistry and Biophysics Reports, http://dx.doi.org/10.1016/j.bbrep.2017.02.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Peptide Oligomers from Ultra-Short Peptides using Sortase

Natalya Voloshchuk, Long Chen, Qiang Li, Jun F. Liang* Department of Chemistry, Chemical Biology, and Biomedical Engineering, Charles V. Schaefer School of Engineering and Sciences, Stevens Institute of Technology, Hoboken, NJ 07030, USA

*Correspondence author Dr. Jun F. (James) Liang Department of Chemistry, Chemical Biology, and Biomedical Engineering, Stevens Institute of Technology, Castle Point on Hudson, Hoboken, NJ 07030, USA. Tel.: 201-216-5640; Fax: 201-216-8240. Email: [email protected]

ABSTRACT Sortase A catalyzed ligation of ultra-short peptides leads to inter/intra-molecular transpeptidation to form either linear or cyclic oligomers dependent upon the peptide length. Cyclic peptides were the main products for peptides with more than 15aa. However, for ultra-short ( 5 hrs) reaction. There were good correlations between the formation of cyclic peptides and the production of corresponded linear peptides in the hydrolyzed forms, indicating the presence of SrtAStaph-mediated ring opening reactions in cyclic oligomers. Oligomers containing more peptide units were especially prone to SrtAStaph mediated hydrolysis, and all GGGLPRT tetramers (both cyclic and linear) formed at the beginning of reactions disappeared at the end of 24hour reaction (Fig. 1).

90

90

Linear

Peptide distribution (%)

Peptide distribution (%)

Hydrolysis

75

60 45 30 15

Trimer

Cyclic

Hydrolysis

75

Linear

Dimer

Cyclic

0

60 45 30 15 0

1

3

5

24

1

Reaction time (hrs)

3

5

24

Reaction time (hrs)

Figure 2. Kinetics of dimer and trimer product formation in SrtAStaph catalyzed GGGLPRT-OMe ligation.

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It is known that the polyglycine sequence at the N-terminus of the peptide can greatly affect SrtAStaph mediated protein ligation. Good reactive activity was usually observed for proteins/peptides with three or more glycine residues at N-termini22. SrtAStaph mediated transpeptidation would not happen for proteins or peptides with only one N-terminal glycine

22,23

.

To test the reactivity of SrtAStaph to small peptides with less than three N-terminal glycines, we extended our study to peptide GLPRT-OMe and GGLPRT-OMe. Both GLPRT-OMe and GGLPRTOMe gave oligomer products (Fig. 2), indicating that a single aminoglycine was sufficient for nucleophilic attack at the small LPXT containing substrates. These results were in consistent with findings from recent studies.26, 27 However, significant differences in reaction kinetics and product distribution were found for GGGLPRT-OMe, GGLPRT-OMe, and GLPRT-OMe (Fig. 2): first, reactions of GLPRT-OMe and GGLPRT-OMe were associated with fewer amounts of linear oligomers. The sum of cyclic oligomers in the final products of GLPRT-OMe and GGLPRT-OMe reached 85%, which was higher than the 65% in GGGLPRT-OMe; second, SrtAStaph-mediated ringopen (hydrolysis) reaction was only observed for the cyclic products from GGGLPRT-OMe but not these from GLPRT-OMe or GGLPRT-OMe. Cyclic oligomers from GLPRT-OMe and GGLPRTOMe increased steadily during the course of reaction (Fig. 3A & 3B). Obviously, LPRT-G or LPRTGG linkages in cyclic oligomers from GLPRT-OMe and GGLPRT-OMe were not good substrates for SrtAStaph because the longer glycine linker limited the steric accessibility of the substrate to the active site, supporting the finding that LPXTG sequences containing three or more N-terminal glycines are good nucleophile binding sites of SrtAStaph22, 23; third, the main products for GLPRTOMe and GGLPRT-OMe at the end of 24 hour reaction were cyclic trimers and tetramers. Neither GLPRT-OMe nor GGLPRT-OMe formed a cyclic dimer, a main cyclic product of GGGLPRTOMe. In contrast, cyclic pentamer was only found for GLPRT-OMe. Because GGGLPRT-OMe, 9

GGLPRT-OMe, and GLPRT-OMe existed as random coils in solutions, potential contributions of peptide secondary structures to SrtAStaph-mediated small peptide ligation could be excluded. Kinetic results suggested that linear dimers did exist at the early phase of GLPRT-OMe and GGLPRT-OMe reactions (Fig. 3C & 3D) but they failed to precede intramolecular transpeptidation to form cyclic products. We know that side chain interactions among amino acid residues in cyclic peptides will be much stronger than those in linear ones. Therefore, the strong ring stain might explain why dimers were the main cyclic oligomer for GGGLPRT-OMe but not for GLPRT-OMe and GGLPRT-OMe. Data from GLPRT-OMe, GGLPRT-OMe and GGGLPRT-OMe (Fig.1) suggested that peptide lengths had dramatic effects on the final products of SrtAStaph-mediated small peptide ligations. 100 Cyclic Linear

100

Cyclic Linear

A

Oligomer distribution (%)

Oligomer distribution (%)

120

80 60 40 20

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0 1

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Cyclic Hydrolyzed

C

100

Oligomer distribution (%)

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Reaction time (hrs)

Reaction time (hrs)

Oligomer distribution (%)

B

80 60 40 20

Cyclic Hydrolyzed

D

80 60 40 20 0

0 5 hr

24 hr

Trimer

--

5 hr

5 hr

24 hr

24 hr

Trimer

Tetramer

10

--

5 hr

24 hr

Tetramer

Figure 3. A & B) Kinetics of SrtAStaph-catalyzed transpeptidation of GLPRT-OMe (A) and GGLPRTOMe(B); C & D) Comparison of cyclic and hydrolyzed products in the reaction mixtures of GLPRT-OMe (C) and GGLPRT-OMe (D) at end of 5 and 24 hour reactions. Data represent relative amount of a product in the reaction mixture at the specific time point.

The important role of peptide length in SrtAStaph-catalyzed small peptide cyclization was confirmed by using small peptides with increased peptide lengths (7-10 amino acids) but containing only one N-terminal glycine (GHKLPRT-OMe, GHHPHLPRT-OMe, and GVPGVGLPRT-OMe) (Fig. 4). Like other small peptides, all three selected peptides do not have specific secondary structures and exist as random coils in solutions (Fig. 4, top). Cyclic peptides were also the main products (yield>81%) for all three peptides. Peptide lengths had the same effect on SrtAStaph-catalyzed peptide ligation: cyclic dimers were major products of GHHPHLPRT-OMe and GVPGVGLPRTOMe but were not found for GHKLPRT-OMe (Fig. 4, bottom).

11

Ellipticity (millidegree)

2 0 -2 -4 -6

GLPRT-OMe GGLPRT-OMe GGGLPRT-OMe

-8 -10 -12 -14 -16 190

200

210

220

230

240

250

Wavelength (nm) Peptides GHKLPRT GHHPHLPRT GVPGVGLPRT GGGWLGALFKALSKLLPRT

Monomer 83

Yield (%) Dimer Trimer 54 38 52 60 24 -

Tetramer 27 -

Figure 4. SrtAStaph mediated ligation and cyclization of peptides of different lengths. Top, CD spectra of peptides; Bottom, calculated yields of products from SrtAStaph-catalyzed transpeptidation.

SrtAStaph-mediated small peptide ligations were summarized in Figure 5. SrtAStaph catalyzed small peptides to form linear peptide oligomers efficiently (yield >85%) and rapidly ( 14 amino acids) for cyclization but even 13

dimers of these peptides would exceed the length limit (< 30 amino acids) to generate stable ligation products.

We

tested

SrtAStaph

catalyzed

peptide

ligation

on

a

19-aa

peptide

(GGGWLGALFKALSKLLPRT-OMe). As we had expected, oligomers from this peptide were not found. However, high yield (~83 %) and single cyclic product from the direct cyclization of GGGWLGALFKALSKLLPRT-OMe was produced (Fig. 4, Bottom).

There has been an increased interest and rapid expansion in the study of cyclic peptides over the last decade. Strategies for synthesizing cyclic peptides include side chain to side chain, side chain to terminal group, and terminal group to terminal group (head-to-tail) cyclization. These methods, however, are inefficient for cyclization of large peptides (>10 residues) due to the large entropic barriers for such reactions and competing intermolecular oligomerization. Findings from this study provide useful information to the synthesis of linear and cyclic oligomers from ultra-short peptides for wide pharmaceutical and biomedical applications. We know that cyclic peptides may have other advantages over their linear counterparts due to their unusual biological activity, improved thermodynamic stability, and increased resistance to protease digestion.24,28,29, In addition, nanostructured surfaces and scaffolds are essential for assisting bone and neuron cell growth and have a wide range of biomedical applications30,31. Peptides or peptide oligomers with specific sequences can also self-assemble to form materials with regular nanostructures and demonstrate unusual stability7,8. Because of good biocompatibility, biomaterials using self-assembled peptides instead of synthetic

polymers

have

been

generated

recently32,

33

.

Taking

peptide

GGGWLGALFKALSKLLPRT-OMe as an example: although both linear and cyclic peptide selfassembled in water, they behaved differently (Fig. 6A) and formed peptide aggregates with distinguished super-molecular structures (Fig. 6B). 14

700

A

Fluorescence Intensity

600

Linear Cyclic

500 400 300 200 100 0 400

450

500

550

Wavelength (nm)

B

Figure 6. Changed self-assembling properties of linear and cyclic GGGWLGALFKALSKLLPRT peptides. A) Self-assembly of linear and cyclic peptides in solutions as confirmed using fluorescence probe 1,8-ANS; B) Typical structures of self-assembled linear (left) and cyclic (right) peptides in DI water as visualized using SEM . Scale bar = 1.0 µm.

ACKNOWLEDGEMENTS This work was partially supported by NIH grant GM081874.

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HIGHLIGHTS 

Sortase A catalyzed ultra-short peptide to form high yield (75-93%) oligomers



Peptides with one aminoglycine form stable oligomers



Cyclic oligomers are predominant when oligomers become large (>15aa)

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