The OMNI kit is the ultimate system for

synthesizing long peptides. It utilizes an

enzyme to efficiently couple peptide

fragments of various lengths to

assemble your desired product.

OMNI kit peptide synthesis kit

EnzyPep B.V.

1

Contents

OMNI Kit Components .................................................................................................................................. 2

Storage .......................................................................................................................................................... 2

Introduction .................................................................................................................................................. 3

Advantages of the OMNI Kit: ........................................................................................................................ 4

Description: ................................................................................................................................................... 4

Summarized Substrate Profile for OMNI Reaction: .................................................................................. 7

Substrates ................................................................................................................................................. 8

Reaction .................................................................................................................................................... 9

Recommended Procedure ...................................................................................................................... 10

Additional Materials Needed ...................................................................................................................... 12

OMNI Reaction Guidelines .......................................................................................................................... 13

Troubleshooting .......................................................................................................................................... 14

CEPS information ........................................................................................................................................ 15

Other Products - Specific Ligases ................................................................................................................ 15

Ordering Information .................................................................................................................................. 16

Appendix I ................................................................................................................................................... 17

Compatible Reagents .............................................................................................................................. 17

Incompatible Reagents ........................................................................................................................... 17

References: ................................................................................................................................................. 17

Contact us ................................................................................................................................................... 18

Notice to Purchaser ................................................................................................................................ 18

2

OMNI Kit components

Omniligase-1 stock solution:

Omniligase-1 in 25 mM Tricine buffer, pH 7.5 (1.0 mg/mL) …….…………………...….110 µL

Coupling reaction stock solution:

Acyl donor (Ac-Asp-Phe-Ser-Lys-Leu-OCam-Leu-OH, 10 mM) and

nucleophile (H-Ala-Leu-Arg-NH2, 15 mM) in water……………………………………….50 µL

Storage

Store at -20°C. Avoid freezing re-thawing steps.

Recommendation: Aliquot (e.g. 5 µL) the omniligase-1 enzyme immediately and store at -20°C.

3

Introduction

Chemo-enzymatic peptide synthesis (CEPS), the enzymatic ligation of chemically synthesized peptide fragments, is potentially one of the most cost-efficient technologies for the synthesis of medium-sized and long peptides (e.g. 20-100 amino acids) and peptide-to-protein couplings. When compared to chemical fragment coupling, CEPS offers several notable advantages: protection of the side-chain functionalities is not necessary, racemization is absent, and it uses less solvents. The OMNI kit is the ultimate system for assembly of long peptides and peptide-to-protein couplings. It utilizes a peptide ligating enzyme to efficiently couple peptide fragments of various lengths into your desired product. The coupling enzyme peptiligase1 was developed by EnzyPep B.V. (Geleen, the Netherlands) for application in the chemo-enzymatic synthesis of peptides (CEPS). Omniligase-1 is a fourth generation peptiligase variant that has been selected for its exceptionally broad substrate specificity. Typically, omniligase-1 catalyzes the reaction between an ester (acyl donor) and amine (acyl acceptor, nucleophile) fragment (Figures 1, 2). The enzyme can be used to perform linear peptide synthesis, but also catalyzes peptide macrocyclization very efficiently (Figure 1B). Figure 1: Schematic illustration of the OMNI reaction. Panel A: Linear peptide coupling reaction.

Panel B: Head-to-tail peptide cyclization.

4

Advantages of the OMNI Kit:

• Fast peptide-peptide, peptide-to-protein coupling reactions within minutes, under mild

aqueous conditions.

• No racemization.

• Protection of substrate side residues is not required.

• Broad substrate scope ligase, not needing recognition tags, results in traceless coupling.

• Although intrinsically traceless, the use of peptide linkers between peptides, peptide

fragments or proteins is possible (e.g. for conjugation).

• Simple, efficient peptide cyclization.

Description:

Enzyme

Omniligase-1 is a highly engineered ligase developed by EnzyPep BV. It combines the properties

of the hyperstable, cation-independent subtilisin BPN’ variant Sbt1492 and of thiolsubtilisins,

such as subtiligase3. The enzyme is exceptionally robust and displays activity in the presence of

organic co-solvents (e.g. up to 50% DMF or 50% DMSO), chaotropic agents (up to 4M GnCl, 2M

urea), disrupting agents (SDS, EDTA, Triton X-100) and reducing agents (DTT and TCEP). It is stable

at temperatures up to 55°C (unfolds at 70°C).

Substrate scope of omniligase-1. Similar to peptiligase1, omniligase-1 has six substrate

recognition subsites; four at the acyl donor binding site; S1-S4, and two at the amine site; S1' and

S2' (Figures 2, 3; nomenclature of Schechter & Berger). The full substrate scope of the enzyme

has been determined (Figure 3). The substrate scope of omniligase-1 is very broad.

5

Figure 2. Substrate recognition sites of omniligase-1. S1-S4 are substrate pockets at the acyl

donor site. At the acyl acceptor side, S1'and S2' substrate binding pockets are present.

Y-(Z)m-P4P3P2P1-Ester + H2N-P1'P2'-(Z)n-OH (or -NH2 or Y) Acyl donor (ester) fragment Nucleophilic (amine) fragment

where the fragments are water-soluble, and

Z = unprotected or protected amino acid or non-peptidic moiety Y= terminal protecting group (optional)

P4 = hydrophobic L-amino acid or protected L-amino acid P3 = any L-amino acid

P2 = any L-amino acid (preferably not Glu, Asp and Trp) P1 = any L-amino acid except Pro (preferably not Gly and Asp)

P1' and P2‘= any L-amino acid except Pro m, n = any number Apart from P4-P1, P1' and P2', all other components (X,Y,Z) can be of non-peptidic nature, e.g. polymer.

6

Figure 3. The substrate profile of omniligase-1. Acyl donor site, the P1-P4 pocket preference are

depicted.

The acyl donor pockets S4-S1 determine the reaction rate (product yield after 15 minutes is depicted in Figure 3), the amine pockets S1' and S2' determine the reaction yield. The S4 pocket prefers hydrophobic P4 amino acids. If appropriate, hydrophobicity of P4 can be achieved by using side-chain protection. The P1-P4, P1' and P2' pocket preference are depicted in Figure 3.

P1 P2

P3 P4

3

7

Figure 4. The substrate profile of omniligase-1. For the acyl acceptor site, S1' and S2' pocket were

screened. All combinations of first two amino acid in the substrate positions P1' and P2' (H-Xxx-

Yyy-Peptide) were tested and the yields are reported in the table below.

The above mentioned yields are obtained using 15 mM of acyl acceptor. Higher yields are obtained when the reactions are performed more concentrated. For obtaining maximal yield of your reaction, it is strongly advised that you consult Figure 4. or EnzyPep directly when selecting your ligation positions. If a suboptimal coupling sequence is desired (orange-red part of the table) EnzyPep has specific ligases available that are better suited for these specific ligations.

8

Summarized substrate profile for OMNI reaction:

Y-(Z)m-P4P3P2P1-Ester + H2N-P1'P2‘-(Z)n-OH (or -NH2 or Y) Acyl donor (ester) fragment Nucleophilic (amine) fragment where the fragments are water-soluble, and

Z = amino acid Y= terminal protecting group (optional)

P4 = hydrophobic L-amino acid or protected L-amino acid P3 = any L-amino acid

P2 = any L-amino acid except Pro (preferably not Glu, Asp and Trp) P1 = any L-amino acid except Pro (preferably not Gly and Asp)

P1' and P2'= any L-amino acid except Pro (preferably not Asp and Glu) m, n = any number Apart from P4-P1, P1' and P2', all other components (X,Y) can be of non-peptidic nature, e.g. polymer

Substrates

Acyl donor

Alkyl, aryl and thioesters can be utilized, but carboxyamidomethyl (Cam) esters or substituted Cam esters (e.g. –Cam-Leu-OH or –Cam-Leu-NH2) are recommended. The substitution of the Cam ester can be used to alter the polarity, and thus the solubility of the ester fragment. Cam esters and substituted Cam esters can be generated on resins commonly used for solid phase peptide synthesis4.

Because omniligase-1 has such a broad specificity, the acyl donor (ester) fragment normally requires N-terminal protection to prevent formation of cyclic peptides or polymers. The use of a phenylacetyl protection group is recommended; it can be removed easily with commercially available acylases (e.g. immobilized PenG acylase, available from Iris Biotech, order no.EZ60040). Acyl acceptor

The nucleophilic (amine) peptide fragment requires no protection at all. All amino acids except prolines in the P1' and P2' are accepted.

9

Reaction

The reaction is exceptionally fast (typically 5-10 min at an enzyme:product ratio of 1:100 for a 2-

fragment coupling). For convenience, most coupling reactions can be performed at ambient

temperature. Conversion is usually above 80% (hydrolysis ≤ 20%) at a 1.1 to 1.5 molar excess of

the amine fragment. Conversion generally improves with the length of the fragments. Details can

be found in the following section.

Important considerations:

Substrate concentration

The amine fragment concentration is crucial for the reaction yield. A higher concentration correlates to a better coupling yield. pH

A pH between pH 8.0 - 8.5 is optimal and required. At lower pH, the amine fragment is protonated, and thus unreactive; at higher pH, the Cam-ester is chemically hydrolyzed. Because peptides are usually acidic after TFA (trifluoroacetic acid) cleavage and a preparative HPLC step, it is critical to check the pH (and adjust accordingly) before starting the enzymatic coupling reaction. Temperature

A temperature range of 4 to 50 °C can be used, however ambient temperature is preferred.

Additives

Do not use any reagents that react with thiols such as maleimides or iodoacetamides, since they will inhibit the active site cysteine.

Head-to-tail cyclizations

The use of omniligase-1 with an unprotected ester fragment can be employed to manufacture the corresponding head-to-tail macrocyclic peptide without significant generation of dimers or polymers. Because of the broad substrate specificity, most macrocycles offer several coupling options with no restriction to Gly or Pro in order to prevent racemization. The reaction is intrinsically traceless, but can also be used with peptide linkers between other peptides, peptide fragments or proteins.

10

Recommended Procedure

Omniligase-1 is provided with a test reaction kit containing:

-110µL omniligase-1 solution at a concentration of 1000 µg/mL

-50 µL peptide test coupling solution (pH 2.5) containing 10 mM Ac-Asp-Phe-Ser-Lys-Leu-OCam-

Leu-OH (Ac-DFSKL-OCam-L-OH) and 15 mM H-Ala-Leu-Arg-NH2 (H-ALR-NH2) in MilliQ water.

These solutions should be stored at -20°C. To perform the omniligase-1 test reaction, please

follow the protocol below:

Before starting:

5 glass HPLC vials

1. Prepare fresh 1M pH 8.5 potassium phosphate buffer (Solution A).

2. Prepare a TCEP stock solution (100 mg/mL) in MilliQ water; this can be stored at -20°C

(Solution B).

3. Prepare a quenching solution of water/acetonitrile/methanesulfonic acid (48/48/4, v/v/v) to

stop the enzymatic reaction (Solution C).

Reaction:

1. Thaw the frozen acyl donor and amine fragment stock solution as well as omniligase-1 stock

solution (it is advised to immediately make aliquots and store at -20°C, e.g. of 5 µL). Keep

working solution on ice.

2. Add 475 µL quenching solution (Solution C) to four of the HPLC vials (1, 2, 3 and 4)

3. Add 75 µL phosphate buffer (Solution A) to the fifth, empty “reaction” HPLC vial. Add 2.5 µL

TCEP (Solution B) and 2.0 µL omniligase-1 solution to the reaction vial.

4. Add 25 µL of the peptide Cam-ester and amine stock solution to the reaction vial, mix well

and adjust pH if required1 (final pH ~ 8.0 to 8.5).

5. Monitor the reaction over time. After 1, 15, 30 and 60 minutes quench the reaction by

adding 25 µL of the reaction mixture to the quenching HLPC vials 1, 2, 3 and 4, respectively.

6. Analyze the samples by HPLC or LC-MS. See below an overlay of the different HPLC spectra

and corresponding masses.

1It is critical to measure and adjust the pH value for the omniligase reaction to reach high conversions and yields.

11

Figure 5. Reaction profile monitored in time.

Analysis Conditions

Analytical HPLC was performed on an Agilent 1260 Liquid Chromatograph, using a reversed-

phase column (Dr. Maisch, C18, 5 µm particle size, 250 × 4.6 mm) at 40°C. UV detection was

performed at 220 nm using a UV-VIS 204 Linear spectrometer. The gradient program was: 0-12

min linear gradient ramp from 30% to 50% eluent B (eluent A: 0.5 mL/L methane sulfonic acid in

H2O; eluent B: 0.5 mL/L MSA in acetonitrile). The flow rate was 1 mL/min and injection volumes

were 25 μL.

Ester (M = 821.4)

Ac-DFSKL-OCam-L-OH

Amine (M = 357.3)

H-ALR-NH2

Product (M = 989.6)

Ac-DFSKLALR-NH2

Hydrolyzed ester (M = 650.3)

Ac-DFSKL-OH

12

Additional Materials Needed

Materials:

Glass HPLC vials

Solutions:

Solution A: 1M K-phosphate buffer, pH 8.5

Solution B: TCEP [tris-(2-carboxyethyl)phosphine]stock solution (100 mg/mL; available from Iris

Biotech, order code: LS-3405) in MilliQ water

Solution C: MilliQ water/acetonitrile/methanesulfonic acid (48/48/4, v/v/v)

1-5 M NaOH or HCl, for pH adjustments

13

OMNI Reaction Guidelines

Identification of ideal coupling positions

To identify suitable fragments for coupling, find a coupling position with a hydrophobic P4

residue and favorable amino acids in P1' and P2' pockets. Please note that a combination of sub-

optimal amino acids can lead to a low yield.

There should be no prolines near the coupling site.

High yield and conversion

Use as high a concentration of the amine fragment as possible.

Always check (and adjust) the pH before starting the reaction.

Addition of TCEP (or optionally DTT) is preferred as it activates the enzyme; oxidative

conditions, disulfides or maleimides can inhibit the enzyme.

Analysis

Analyze the coupling reaction by HPLC-MS, use acidic conditions (<pH 3, see above) to quench

the reaction.

14

Troubleshooting

(Almost) no conversion at all:

Check the enzyme activity and sequence of acyl donor fragment (is the enzyme still frozen or

been thawed too many times?).

Are all substrates in solution? Precipitated/aggregated peptides can inhibit the enzyme.

Are reductive conditions being used? (oxidative conditions can inhibit omniligase-1)

Only (or excessive) hydrolysis:

Is it possible to increase the amine concentration?

Check pH.

Are the two amino acids at the P1'and P2' positions sub-optimal? Try to choose a different

coupling position.

Are the two amino acids at the P1' and P2' pocket positions accessible (e.g. when coupling to

specific proteins)? Try using disrupting agents (urea, GnCl) or a peptide spacer.

If none of the above works, or you are in need of an enzyme for a specific coupling reaction,

please contact EnzyPep (contact details at last page).

15

CEPS information

Peptiligases are the patented and proprietary products of EnzyPep (Brightlands Campus, 6167 RD Geleen, Netherlands). Omniligase-1 is available under license on request for using CEPS technology to manufacture pharmaceutical grade peptides and peptide conjugates.

Other products-Restriction ligases

Other peptiligases are available in both research and pharmaceutical grades. At the present time, over 100,000 unique peptiligases can be expressed, of which about 250 are well-characterized. In contrast to omniligase-1, many other designer peptiligases show very tight specificity, only permitting the CEPS coupling of closely defined sequences. This tight specificity:

• avoids the necessity of N-terminal protection of the ester fragment,

• assists purification by restricting coupling only to fragments with recognized amino acids in P4-P1, P1' and P2' positions,

• allows chain-specific coupling to heterodipeptides and branched chain peptides. EnzyPep also offers feasibility study services to help identify (a) whether CEPS can be used to assemble a particular polypeptide, and (b) provide or design peptiligases to optimize a CEPS procedure. If you are interested in any of these additional products or services, please contact EnzyPep at info@enzypep.com or tel: +31 (0)46 47 60 675

16

Ordering information

OMNI Kit

Omniligase-1 from B. subtilis, research grade, E.C. 3.4.21.62, molecular weight 27394 Da

including model peptides

Kit containing 110µg of enzyme; sufficient for 150 reaction, or a total of 1g of peptide; including

50µL acyl donor (Ac-Asp-Phe-Ser-Lys-Leu-OCam-Leu-OH, 10 µM) and nucleophile (H-Ala-Leu-

Arg-NH2, 15 µM) as model peptides for learning, how to work successfully with this technology.

Store

Code: EZK2020 EUR 850,-

17

Appendix I

Compatible reagents

The table below shows the maximum concentration of each reagent tested at EnzyPep. Higher

levels may be acceptable, but they should be tested before use. Please note that some of these

reagents may partially or completely denature omniligase-1.

Reagent Acceptable Concentration

DTT (dithiothreitol) available from Iris Biotech GmbH: order code RL-1020

1-5 mg/mL

TCEP tris-(2-carboxyethyl)phosphine hydrochloride available from Iris Biotech GmbH: order code LS-3405

1-5 mg/mL

EDTA 10 mM

SDS 5% (w/v)

Triton X-100 5% (w/v)

Urea 6 M

Guanidinium chloride 2 M

Incompatible reagents

These reagent are incompatible with the ligation reaction.

Reagent

Maleimide

Iodoacetamide

Epoxides

High concentrations of alcohols (transesterification)

High concentrations of thiols (thioester synthesis)

Ammonia or other primary amines (amide synthesis)

18

Contact us

Contact us

Customer Service/Ordering Technical support

Iris Biotech GmbH EnzyPep B.V.

Waldershofer Str. 49-51 D-95615 Marktredwitz Germany

Urmonderbaan 22 6167 RD Geleen The Netherlands

Phone: +49 9231 9619 73 Phone +31 (0)46 47 60 675

Web: www.iris-biotech.de Web: www.enzypep.com

e-mail: info@iris-biotech.de e-mail: info@enzypep.com

Notice to clients

Omniligase-1 is patented technology by EnzyPep B.V.

After purchase of OMNI Kit its components may be used for research purposes, only, without any

further payments of license fees and may not be used for any other purpose, including, but not

limited to, use in drugs, in vitro diagnostics purposes, therapeutics or in humans.

EnzyPep products are available from Iris Biotech GmbH and may not be transferred to third

parties, sold, resold, modified for resale, or used to manufacture commercial products or to

provide a service to third parties without written approval of EnzyPep B.V.

The use of Omniligase-1 to manufacture commercial products is possible with license agreement

with EnzyPep B.V., only.

19

References:

1. Toplak, A., Wu, B., Nuijens, T., Quaedflieg, P.L.J.M. & Janssen, D.B. (2016) Adv. Synth.

Catal.,358, 2140. 2. Jackson, D. Y., Burnier, J., Quan, C., Stanley, M.;Tom, J., Wells, J. A. (1994) Science, 266, 243. 3. Braisted, A.C., Judice, J.K.; Wells, J.A. (1994) Methods in Enzymology, 289, 300. 4. Nuijens T., Toplak, A., van de Meulenreek, M. B. A. C., Schmidt M.,Goldbach M., Quaedflieg

P. J. L. M. (2016) Tetrahedron Lett., 57, 3635.