Advantages of the OMNI Kit

Contents

OMNI Kit Components

Storage

Introduction

Advantages of the OMNI Kit:

Description:

Summarized Substrate Profile for OMNI Reaction:

Substrates

Reaction

Recommended Procedure

Additional Materials Needed

OMNI Reaction Guidelines

Troubleshooting

CEPS information

Other Products-Specific Ligases

Ordering Information

Appendix I

Compatible Reagents

Incompatible Reagents

References:

Contact us

Notice to Purchaser

Certificate of Analysis

OMNI Kit components

Omniligase-1 stock solution:

Omniligase-1 in 25 mM Tricine buffer, pH 7.5 (0.33 mg/mL)…….…………………….334µL

Coupling reaction stock solution:

Acyl donor (Ac-Asp-Phe-Ser-Lys-Leu-OCam-Leu-OH, 10 µM) and nucleophile (H-Ala-Leu-Arg-NH2, 15 µM) 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.

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. 20-100amino 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 peptiligase1was 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.

A

B

Advantages of the OMNI Kit:

• Fastpeptide-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 enzymeis 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-1has six substrate recognition subsites; four atthe acyl donor binding site; S1-S4, and two atthe 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.

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 -NH2or Y)

Acyl donor (ester) fragmentNucleophilic (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= anyl-amino acid except Pro(preferably not Glu, Asp and Trp)

P1= anyl-amino acid except Pro (preferably not Gly and Asp)

P1'and P2‘= anyl-amino acid except Pro

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

Figure 3.The substrate profile of omniligase-1.

The acyl donor pocketsS4-S1determine the reaction rate (product yieldafter 15 minutes is depicted above, maximum conversion after 60 min or longer), the amine pocketsS1' and S2' determine the reaction yield. The S4 pocket prefers hydrophobic P4amino 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.

Omniligase-1 accepts all amino acids except proline in the S1'and S2' substrate binding pockets (Figure 3).

Summarized substrate profile for OMNI reaction:

Y-(Z)m-P4P3P2P1-Ester + H2N-P1'P2‘-(Z)n-OH (or -NH2or Y)

Acyl donor (ester) fragmentNucleophilic (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= anyl-amino acid except Pro(preferably not Glu, Asp and Trp)

P1= anyl-amino acid except Pro (preferably not Gly and Asp)

P1'and P2‘= anyl-amino acid except Pro

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 from Iris Biotech 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.

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 thereaction 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 rangeof 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-1with 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.

Recommended Procedure

Omniligase-1is provided with a test reaction kit containing:
-334 µL omniligase-1 solution ata concentration of 330 µg/mL
-55 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.

Thesesolutions should be stored at -20°C. To perform the omniligase-1test reaction, please follow the protocol below:

Before starting:

5 glass HPLC vials

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

2. Prepare a TCEP stock solution (100 mg/mL) in MilliQwater; 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 B) to fourof 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 welland adjust pH if required[1](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 vials1, 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.

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.

Additional Materials Needed

Materials:

Glass HPLC vials

Solutions:

Solution A: 1MK-phosphate buffer, pH 8.5

Solution B:TCEP [tris-(2-carboxyethyl)phosphine]stock solution (100 mg/mL) in MilliQwater

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

1-5 M NaOH or HCl, for pH adjustments

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.

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).

CEPS information

Peptiligases are the patented and proprietary products of EnzyPep (Brightlands Campus, 6167 RD Geleen, Netherlands).

Omniligase-1is 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 canbe expressed, of which about 250are well-characterized.

In contrast to omniligase-1, many other 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 not coupling fragments with false amino acids in the 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 r tel: +31 (0)46 47 60 675

Ordering information

Please contact IRIS Biotech GmbH.

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) / 1-5 mg/mL; 6.5-30 mM
TCEP tris-(2-carboxyethyl)phosphine / 1-5 mg/mL; 3.5-17.5 mM
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)

References:

1. Toplak, A., Wu, B., Nuijens, T., Quaedflieg, P.L.J.M. & Janssen, D.B. (2016) Adv. Synth. Catal. (in press).
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. de Beer R. J, Nuijens T., Wiermans L., Quaedflieg P. J. L. M., Rutjes F. P. (2012) Org. Biomol. Chem., 10, 6767-6775.

Contact us

Contact us
Customer Service/Ordering / Technical support
Tel: +31 (0)46 47 60 675
Web:
e-mail:

Notice to purchaser

OMNI Kitcomponents may be used for research purposes only. They 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 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 BV.

1

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