A New Generation of Peptide Conjugation Products

Tuesday, July 05, 2011
A New Generation of Peptide Conjugation Products

A New Generation of Peptide Conjugation Products

Conjugating with HyNic-peptides

Traditionally, two chemistries have been used to conjugate peptides to other biomolecules and surfaces; carbodiimide chemistry (EDC/NHS) and maleimido/thiol chemistry. Both chemistries have significant drawbacks. The EDC/NHS method conjugates peptides through lysine moieties on the peptide and as a consequence, chemically alters the epitope resulting in sub-optimal antibodies. Additionally, (EDC/NHS) chemistry tends to be very inefficient. Maleimido/thiol chemistry is currently the method of choice wherein a cysteine moiety is incorporated on the carboxy-terminus of the peptide and subsequently linked to a maleimido-modified protein carrier. Drawbacks to this method include the requirement to modify the protein carrier just prior to conjugation in order to prevent hydrolysis of the unstable maleimido moiety. Additionally, cysteine-containing peptides readily oxidize to disulfide linked peptides and become un-reactive. While at the same time, precluding the preparation of antibodies against cysteine-containing peptides since these will scramble or change the original epitope.

To overcome these drawbacks, SoluLinK has developed and introduced HydraLinK™ chemistry. HydraLinK chemistry is based on the conjugation of HyNic-modified peptides to 4FB-modified carriers. Figure 1 presents the chemistry of the conjugation of a HyNic-peptide (PepLink) to a 4FB-modified protein.

HyNic (hydrazinonicotinamide) was originally developed to bind technetium-99M to proteins for in vivo imaging.6 Subsequently, HyNic was incorporated on chemotactic peptides for imaging of focal sites of inflammation, on oxytocin and octreolide for tumor imaging and for imaging a wide variety of other targets (see PubMed search, keywords: HyNic, peptide).7-9

Incorporation of the HyNic moiety on a peptide is straightforward, high yielding and can be added at either N- or C-terminus using 6-Boc-HyNic (1) (SoluLinK catalog # S-3003) or FMOC-Lys-(ε-6-BocHyNic)OH (2) (SoluLinK catalog # 3034), respectively.

Cleavage from the resin is accomplished using TFA/acetone/water/triisopropylsilane (TIS)/water (92.5/2.5/2.5/2.5) for 2 h. The presence of the acetone forms a hydrazone with the deprotected hydrazine moiety in situ blocking any trifluoroacetamide formation from the reaction of TFA with the strongly nucleophilic hydrazine.10,11 Unlike cysteine peptides, HyNic-peptides are stable and do not scramble or dimerize.

The linking of HyNic-peptides (PepLink) to 4FB-modified proteins, oligonucleotides or surfaces is straightforward, stoichiometrically efficient and high yielding. In a standard conjugation, only a 2-5 mole excess of one of the HyNic-peptide (PepLink) is required to give >80% conjugate yield. Hydrazone bond formation can also be catalyzed and reaction kinetics improved 10-100-fold by the presence of aniline, often leading to conjugate yields > 95%. Optimal conjugation kinetics (formation of the hydrazone bond) is between pH 4.5-5.0. However, the reaction also proceeds at higher pH, albeit more slowly. When conjugating to an antibody, it is recommended that reactions be performed at pH 6.0, since antibody immunoreactivity can be deleteriously affected below pH 6.0. 12 A further advantage of using the HyNic/4FB pair is that the degree of conjugation can be conveniently monitored spectrophotometrically (in real-time). The formation of the bis-aryl hydrazone bond can be exploited to both trace and quantify the progress of the conjugation reaction using its known molar extinction coefficient (29,000 @ 354 nm).

Peptide Conjugation: Some Examples

HyNic-peptide to a 4FB-Protein

Conjugation of a HyNic-modified peptide to a 4FB-modified protein is a simple, two-step process wherein a 5-fold excess of HyNic-peptide is incubated with a 4FB-modified protein in buffer at pH 6.0 for 2 hours (Figure 2). The final protein-peptide conjugate is readily purified using a diafiltration device (MWCO:1000-3000).

HyNic-Peptide to a 4FB-Oligonucleotide

The preparation of peptide/oligonucleotide conjugates with SoluLinK’s PepLink technology is also straightforward. Simple addition of a 3-5 molar excess of a HyNic-peptide to a 4FB-modified oligonucleotide at pH 5.0-6.0 for 2 h @ RT, followed by diafiltration to remove excess peptide, yields the conjugate (see Figure 3). The 4FB-moiety can be incorporated on the oligonucleotide during solid phase synthesis using a 4FB-C6-phosphoramidite (SoluLinK catalog # S-1005). This is the first example of the preparation of a peptide/oligonucleotide conjugate wherein both moieties had their respective linkers incorporated during solid phase synthesis. To demonstrate the stability of the bis-arylhydrazone bond the oligo/peptide conjugates were heated at 94°C for 2h and subsequently analyzed by gel electrophoresis (Figure 3, lanes 6 and 7).

Peptide Conjugates using 4-HTA (4-hydrazidoterephthalamide)

4-BOC-HTA (3) (SoluLinK catalog # S-1014), is an aromatic hydrazide linker which is also available from SoluLinK for incorporation into peptides. The aromatic acylhydrazine (4-HTA), forms a hydrazide-based hydrazone that is more thermal labile than an aromatic hydrazine-based hydrazone. Conjugates formed with this linker can be cleaved at elevated temperatures in contrast to aromatic hydrazine-based hydrazone (HyNic) conjugates (Figure 4). A more thermal labile bond allows for the cleavage of solution phase conjugates or solid phase immobilized conjugates by simply elevating temperature.

HyNic-peptide to a 4FB-Modified Surface (Magnetic Microsphere)

Another application of HyNic-peptides (PepLink) is immobilization of peptides to 4FB-modified solid surfaces. For example, HyNic-peptides are easily immobilized on 4FB-modified magnetic beads (NanoLink; SoluLinK catalog # M-1001-010). Simple incubation of a HyNic-peptide with 4FB-modified magnetic beads at pH 5.0 for 30-60 minutes, followed by a brief wash, yields the bead immobilized peptide (Figure 5).

Solid Phase Synthesis of HyNic-Peptides

N-terminal HyNic incorporation using 6-Boc-HyNic

Method 1-1: Incorporation of HyNic to the N-terminus of a 10-mer peptide

  1. Dissolve 6-Boc-HyNic (5 eq), HBTU (5 eq) in a minimum amount of DMF.
  2. Add DiPEA (15 eq) to mixture and add immediately to Fmoc-deblocked peptide resin.
  3. Incubate for 1-2 h.
  4. Check completeness of reaction with Kaiser or TNBS test.
  5. Wash resin and repeat reaction cycle if required.
Application 1-1: Synthesis of HyNic-10-mer peptide

The peptide was assembled on a Rainin Symphony Peptide Synthesizer using Rink amide resin. All acylation reactions were carried out using a 5-fold excess of Fmoc-amino acid activated with HBTU/DiPEA. A coupling time of 1 hour was used for each cycle. The HyNic-peptidyl resin was cleaved using TFA/TIS/acetone/water (92.5/2.5/2.5/2.5) for 2 h. The crude peptide was analyzed by HPLC and ES-MS with an m/e of 1314.6 as expected for the desired peptide (Figure 6). The addition of acetone is critical as the in situ formation of the acetone hydrazone of the deprotected peptide blocks the formation of the trifluoroacetamide.13, 14 The product was isolated by RP-HPLC using a gradient of 90% A (0.1% TFA/water) to 100% B (0.1% TFA/80% ACN/20% water) in 15 min.

C-terminal HyNic incorporation using Fmoc-Lysine-ε-(6-Boc-HyNic) OH

Method 2-1: Incorporation of HyNic on the C-terminus of a peptide
  1. Dissolve Fmoc-Lysine-ε-(6-Boc-HyNic)OH (5 eq), HBTU (5 eq) in a minimum amount of DMF.
  2. Add DiPEA (15 eq) to mixture and add immediately to Fmoc-deblocked peptide resin.
  3. Incubate for 1-2 h.
  4. Check completeness of reaction with Kaiser of TNBS test.
  5. Wash resin and repeat reaction if required.
Application 2-1: Synthesis of C-terminal HyNic-14-mer peptide

The peptide was assembled on a Rainin Symphony Peptide Synthesizer using Rink amide resin. All acylation reactions were carried out using a 5-fold excess of Fmoc-amino acid activated with HBTU/DiPEA (5 eq). A coupling time of 1 hour was used for each cycle. The HyNic-peptidyl resin was cleaved using TFA/TIS/acetone/water (92.5/2.5/2.5/2.5) for 2 h. The crude peptide was analyzed by HPLC and ES-MS with an m/e 1612 as expected for the desired peptide (Figure 7). The addition of acetone is critical as the in situ formation of the acetone hydrazone of the deprotected peptide blocks the formation of the trifluoroacetamide.13, 14 The product was isolated by RP-HPLC using a gradient of 90% A (0.1% TFA/water) to 100% B (0.1% TFA/80% ACN/20% water) in 15 min.

N-Terminal Biotin incorporation using ChromaLink Biotin

Method 3-1: Incorporation of ChromaLink Biotin-COOH on N-terminus of a peptide
  1. Dissolve ChromaLink Biotin-COOH (5 eq), HBTU (5 eq) in a minimum amount of DMF.
  2. Add DiPEA (15 eq) to mixture and add immediately to Fmoc-deblocked peptide resin.
  3. Incubate for 1-2 h.
  4. Check completeness of reaction with Kaiser of TNBS test.
  5. Wash resin and repeat reaction if required.

Application 3-1: Synthesis of N-terminal biotinylated 10-mer peptide The peptide was assembled on a Rainin Symphony Peptide Synthesizer using Rink amide resin. All acylation reactions were carried out using a 5-fold excess of ChromaLink Biotin-COOH with HBTU/DiPEA (15 equiv.). A coupling time of 1 hour was used for each cycle. The ChromaLink Biotin- peptidyl resin was cleaved using TFA/TIS/water (95/2.5/2.5) for 2 h. The crude peptide was analyzed by ES-MS and had m/e 1869 as expected for the desired peptide. The product was isolated by RP-HPLC using a gradient of 90% A (0.1% TFA/water) to 100% B (0.1% TFA/80% ACN/20% water) over 15 min. RP-HPLC of the crude reaction product is illustrated in Figure 8.

Recommended HyNic-Peptide Cleavage Conditions

Cleavage

  1. Suspend resin in 92.5% TFA/2.5% acetone/2.5% triisopropylsilane/2.5% water
  2. Incubate for 2 hours
  3. Collect supernatant
  4. Precipitate with ether then place at -200°C for 15-20 min
  5. Centrifuge @ 3000 rpm
  6. Wash pellet with ether (1 eq.)
  7. Dissolve pellet in 1:1, acetonitrile:water
  8. Lyophilize
  9. Purify with HPLC if required; use a C18 column and detect at 214 nm.

Analytical

  1. The modified peptide will have a longer retention time in HPLC due to the addition of the hydrophobic linker.
  2. There may be two peaks in the final product, the HyNic acetone hydrazone along with the free hydrazine analog.

epLink™ Linker Reagents

Auxiliary Linker Reagents

Additional 4FB-activated reagents for conjugation to HyNic-peptides

4FB Linkers

References

  1. El-Andaloussi S., Holm T, and Langel, U., Cell-penetrating peptides: mechanisms and applications. Curr Pharm Des. 2005;11(28):3597-611. (Review)
  2. Torchilin VP., Recent approaches to intracellular delivery of drugs and DNA and organelle targeting. Annu Rev Biomed Eng. 2006;8:343-75 (Review)Zatsepin TS, Turner JJ, Oretskaya TS, and Gait MJ., Conjugates of oligonucleotides and analogues with cell penetrating peptides as gene silencing agents. Curr Pharm Des. 2005;11(28):3639-54
  3. Snyder EL, Saenz, CC, Denicourt, C, Meade BR, Cui,XS Kaplan, IM and Dowdy, SF, Enhanced targeting and killing of tumor cells expressing the CXC chemokine receptor 4 by transducible anticancer peptides. Cancer Res 2005 Dec 1;65(23):10646-50
  4. Debart F, Abes S, Deglane G, Moulton HM, Clair P, Gait MJ, Vasseur JJ, Lebleu B., Chemical modifications to improve the cellular uptake of oligonucleotides Curr Top Med Chem. 2007;7(7):727-37
  5. Abrams, M. J., Juweid, M., Tenkate, C. I., Schwartz, D. A., Hauser,M. M.; Gaul, F. E., Fuccello, A. J., Rubin, R. H., Strauss, H. W., Fischmann, A. J. Technetium-99m-human polyclonal IgG radiolabeled via the hydrazino nicotinamide derivative for imaging focalsites of infection in rats. J. Nucl. Med. 1990, 31, 2022-2028.
  6. Schwartz, D. A., Abrams, M. J.; Hauser, M. M.; Gaul, F. E., Larsen, S. K.; Rauh, D.;, Zubieta, J. A., Preparation of hydrazino-modified proteins and their use for the synthesis of technetium-99m-protein conjugates. Bioconjugate Chem. 1991, 2, 333.
  7. van der Laken, C. J., Boerman, O. C., Oyen, W. J. G., van der Ven, M. T. P., Edwards, D. S., Barrett, J. A., van der Meer, J. W. M.; Corstens, F. H. M., Technetium-99m labeled chemotactic peptides in acute infection and sterile inflammation. J. Nucl. Med. 1997, 38, 1310-1315.
  8. Miranda-Olvera AD, Ferro-Flores G, Pedraza-López M, de Murphy CA, De León-Rodríguez LM., Synthesis of oxytocin HYNIC derivatives as potential diagnostic agents for breast cancer. Bioconjug Chem. 2007 Sep- Oct;18(5):1560-7.
  9. Kos-Kudła B, Zemczak A, Foltyn W, Marek B, Strzelczyk J, Telega A, Zajecki W, Legaszewski T, Jurecka- Lubieniecka B. Octreotide suppression test in diagnosing and predicting the outcome of therapy in patients with neuroendocrine tumors. Preliminary report. Endokrynol Pol. 2007 Mar-Apr;58(2):123-9.
  10. Surfraz MB, King R, Mather SJ, Biagini SC, Blower PJ., Trifluoroacetyl-HYNIC peptides: synthesis and 99mTc radiolabeling., J Med Chem. 2007 Mar 22;50(6):1418-22.
  11. Greenland WE, Howland K, Hardy J, Fogelman I, Blower PJ., Solid-phase synthesis of peptide radiopharmaceuticals using Fmoc-N-epsilon-(hynic-Boc)-lysine, a technetium-binding amino acid: application to Tc-99m-labeled salmon calcitonin. J Med Chem. 2003 Apr 24;46(9):1751-7.
  12. Dirksen A, Dirksen S, Hackeng TM, Dawson PE., Nucleophilic catalysis of hydrazone formation and transimination: implications for dynamic covalent chemistry., J Am Chem Soc. 2006 Dec 13;128(49):15602-3.
  13. W. E. P. Greenland, K. Howland, J. Hardy, I. Fogelman, and P. J. Blower,Solid-Phase Synthesis of Peptide Radiopharmaceuticals Using Fmoc-N-E-(Hynic-Boc)-Lysine, a Technetium-Binding Amino Acid: Application to Tc- 99m-Labeled Salmon Calcitonin J. Med. Chem. 2003, 46, 1751-1757
  14. M. Bashir-Uddin Surfraz, Robert King, Stephen J. Mather, Stefano C. G. Biagini and Philip J. Blower, Trifluoroacetyl-HYNIC Peptides: Synthesis and 99mTc Radiolabeling, J. Med. Chem. 2007, 50, 1418-1422

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