Tag-assisted Liquid-phase
Peptide Synthesis
Literature Seminar #2
2022.01.19
M1 Kazuki Oikawa

Contents
1. Introduction
2. Benzyl-type tag
3. AJIPHASE®
4. Summary2

Introduction〇 Peptide drugs
Small molecule drugs

Peptide drugs

Macromolecular drugs

Molecular weight300-500

1000-10000

50000-150000

Oral administration〇〇 or ××

Cell penetration◎〇

×Target selectivity×〇◎

Synthetic approach

ChemicalChemical or Biological

Biological

Manufacturing cost

Low

Relatively Low

High

Possibility to launch

Low

Relatively High

Relatively High

Peptide drugs have the advantages of both small moleculardrugs and macromolecular drugs!!
Lau, J. L.; Dunn, M. K. Bioorg. Med. Chem., 2018, 26, 2700–2707.

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Introduction〇 Peptide drugs

There is a great need for the development ofpeptide synthesis methods.
Lee, J.; Kim, C. Macromol. Res., 2021, 29(1), 2-14.

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Introduction〇 Peptide synthesis
Liquid phase (LPPS)

Solid phase (SPPS)

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Introduction〇 Peptide synthesis
Solid phase (SPPS)

Liquid phase (LPPS)

Time〇×Solubility〇 (Biphasic)× (As peptide chain grows)

Purification〇×Cost×〇Reagent

× (Excess)〇Scale-up△〇

New synthetic method of peptide that combines theadvantages of SPPS and LPPS is needed.

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Introduction〇 Tag chemistry

Requirements
・C-terminal protection (Orthogonal to N-terminal protecting group)・Improve solubility in organic solvent
・Purification without complicated process7

Introduction〇 Classical PEG tag

Bayer, E.; Mutter, M. Nature, 1972, 237, 2-14.

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Introduction〇 Classical PEG tagProblems
・ Difficulty of reaction analysis
・ Complexity of purification (want it even easier!)・ Loading efficiency of amino acid9Contents
1. Introduction
2. Benzyl-type tag
3. AJIPHASE®
4. Summary
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Benzyl-type tag〇 3,4,5-Tris(octadecyloxy)benzyl alcohol
Concept・Application to Fmoc strategy
・Well-defined molecular structure
・Easy purification (size-exclusion chromatography)

Tamiaki, H.; Obata, T.; Azefu, Y.; Toma, K. Bull. Chem. Soc. Jpn., 2001, 74, 733-738.

11Benzyl-type tag〇 3,4,5-Tris(octadecyloxy)benzyl alcohol

Purification
Coupling : Gel-filtration chromatography
Deprotection : Washing with methanol
Tamiaki, H.; Obata, T.; Azefu, Y.; Toma, K. Bull. Chem. Soc. Jpn., 2001, 74, 733-738.

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Benzyl-type tag〇 Didocosyloxybenzyl alcohol
Concept
・Easier purification (precipitation)・Tolerance of protecting group (Fmoc and Boc)

Tana, G.; Kitada, S.; Fujita, S.; Okada, Y.; Kim, S.; Chiba, K. Chem. Commun., 2002, 237, 2-14.

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Benzyl-type tag〇 Didocosyloxybenzyl alcohol
C-terminal proline ⇨ Risk of diketopiperazine formation

Benzyl type protection is weak to acid condition⇒ Optimization of substituent group

Tana, G.; Kitada, S.; Fujita, S.; Okada, Y.; Kim, S.; Chiba, K. Chem. Commun., 2002, 237, 2-14.

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Benzyl-type tag〇 Didocosyloxybenzyl alcohol
Synthesis of A-TNF-α (Tana, G.; Kitada, S.; Fujita, S.; Okada, Y.; Kim, S.; Chiba, K. Chem. Commun., 2002, 237, 2-14.

)

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Benzyl-type tag〇 Disulfide formation

S-S bond was formed efficiently even at high concentrations!
Kitada, S.; Fujita, S.; Okada, Y.; Kim, S.; Chiba, K. Bioorg. Med. Chem. Lett., 2011, 21, 4476-4479.

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Benzyl-type tag〇 Disulfide formation
Synthesis of Somatostatin

Kitada, S.; Fujita, S.; Okada, Y.; Kim, S.; Chiba, K. Bioorg. Med. Chem. Lett., 2011, 21, 4476-4479.

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Benzyl-type tag〇 Head-to-Tail cyclization
Retrosynthetic strategy for Mahafacyclin B

Fujita, Y.; Fujita, S.; Okada, Y.; Chiba, K. Org. Lett., 2013, 15(6), 1155-1157.

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Benzyl-type tag〇 Head-to-Tail cyclization
Synthesis of tagged amino acid

Synthesis of linear peptide

Fujita, Y.; Fujita, S.; Okada, Y.; Chiba, K. Org. Lett., 2013, 15(6), 1155-1157.

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Benzyl-type tag〇 Head-to-Tail cyclization
Cyclization

Various peptides can be synthesized with high efficiency, purity, and yieldby controlling solubility and reactivity with hydrophobic tags.
Fujita, Y.; Fujita, S.; Okada, Y.; Chiba, K. Org. Lett., 2013, 15(6), 1155-1157.

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Benzyl-type tag〇 Improved process
Precipitation at every step ⇨ ○ Prevent ‘‘Double Hit’’✕ Excessive use of solvents

Okada, Y. et al. Org. Process. Res. Dev. 2019, 23, 2576-2581.

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Benzyl-type tag〇 Improved process

Trapping remaining Fmoc-AA-OH using
sacrificial primary amine⇒ Reduce the number of precipitation steps

Okada, Y. et al. Org. Process. Res. Dev. 2019, 23, 2576-2581.

22Benzyl-type tag〇 Improved process
Large scale synthesis of Icatibant acetate

Okada, Y. et al. Org. Process. Res. Dev. 2019, 23, 2576-2581.

23Benzyl-type tag〇 Short summary

A variety of benzyl-type tags with different properties havegreatly expanded the scope of peptide compound synthesis.

Okada, Y. et al. Org. Process. Res. Dev. 2019, 23, 2576-2581.

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Contents
1. Introduction
2. Benzyl-type tag
3. AJIPHASE®
4. Summary
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AJIPHASE®
〇 Fluorene-derived tag
Concept・Fmoc strategy
・Prevent diketopiperazine formation
・Productization

Takahashi, D.; Yamamoto, T. Tetrahedron. Lett. 2012, 53, 1936-1939.

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AJIPHASE®
〇 Fluorene-derived tag
Development of trityl type tag

Achieving a balance between the stability of the ester bond and a highloading yield using the trityl-type tag was difficult…
Takahashi, D.; Yamamoto, T. Tetrahedron. Lett. 2012, 53, 1936-1939.

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AJIPHASE®
〇 Fluorene-derived tag
Development of fluorene-type tag

Fluorene-derived tag (10f) could have the performance for use as a tag!
Takahashi, D.; Yamamoto, T. Tetrahedron. Lett. 2012, 53, 1936-1939.

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AJIPHASE®
〇 Fluorene-derived tag
Synthesis of peptide without diketopiperazine formation

Takahashi, D.; Yamamoto, T. Tetrahedron. Lett. 2012, 53, 1936-1939.

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AJIPHASE®
〇 Diphenylmethyl-derived tagConcept・Fmoc strategy
・Synthesis of C-terminal amide peptide

Takahashi, D.; Yamamoto, T. Org. Lett. 2012, 14(17), 4514-4517.

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AJIPHASE®
〇 Diphenylmethyl-derived tag
Synthesis of Eptifibatide

Takahashi, D.; Yamamoto, T. Org. Lett. 2012, 14(17), 4514-4517.

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AJIPHASE®
〇 Further development
Concept
・Higher solubility in organic solvents
・More efficient washing process (Extraction)・Longer peptide and hydrophobic peptide synthesis

Takahashi, D.; Inomata, T.; Fukui, T. Angew. Chem. Int. Ed. 2017, 56, 7803-7807.

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AJIPHASE®
〇 Further developmentTakahashi, D.; Inomata, T.; Fukui, T. Angew. Chem. Int. Ed. 2017, 56, 7803-7807.

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AJIPHASE®
〇 Further development
Synthesis of Degarelix

Takahashi, D.; Inomata, T.; Fukui, T. Angew. Chem. Int. Ed. 2017, 56, 7803-7807.

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AJIPHASE®
〇 Further development
Synthesis of Bivalirudin

Takahashi, D.; Inomata, T.; Fukui, T. Angew. Chem. Int. Ed. 2017, 56, 7803-7807.

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AJIPHASE®
〇 Further development

Takahashi, D.; Inomata, T.; Fukui, T. Angew. Chem. Int. Ed. 2017, 56, 7803-7807.

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Contents
1. Introduction
2. Benzyl-type tag
3. AJIPHASE®
4. Summary
37

Summary
・ Peptide drugs have been attracting attention in recent years
and more efficient synthetic method is needed.
・ Hydrophobic tagging can streamline the synthetic process ofpeptide with a simpler purification method.
・ Hydrophobic tags can also control the solubility andreactivity of peptide compounds.
・ Large-scale peptide synthesis and commercialization of tags
have been achieved through structural optimization of tags.
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