Peptide synthesis utilizing
micro-flow technology
Literature Seminar #1
2020.11.19
B4 Kazuki Oikawa

Contents
1. Introduction
2. Micro-flow technology for peptide synthesis
ー Solid-phase α-peptide synthesis

ー Solution-phase α-peptide synthesis
ー Cyclic peptide synthesis

3. Application and latest development
ー Total synthesis of Feglymycin
ー Rapid total synthesis of protein
ー Optimization by Deep learning

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 merits of both small molecular drugs andmacromolecular drugs!!
J. L. Lau, M. K. Dunn, Bioorg. Med. Chem. 2018, 26, 2700–2707.

3

Introduction〇 Peptide drugs
Since 1980, the number ofapproved peptide drugs
gradually increased andover 60 drugs are approved.

155 peptide drugs arein active clinicaldevelopment(2017)

Total sales of peptide drugs is$50 billion and 5% of total
pharmaceutical sales.
This percentage is estimated togrow 9-10% annually!
J. L. Lau, M. K. Dunn, Bioorg. Med. Chem. 2018, 26, 2700–2707.

4

Introduction〇 Peptide drugs

The number of long-chain, complex peptide drugs are increasing due toimprovements in peptide synthesis and manufacture technology

More scaled-up, high-yielding, cost-effective, and less-wasteful
method of peptide synthesis are needed…
J. L. Lau, M. K. Dunn, Bioorg. Med. Chem. 2018, 26, 2700–2707.

5

Introduction〇 What is Flow reaction?
Batch reaction

・Large reactor
・Charging, reaction, recovery is
conducted one by one

Flow reaction

・Small channel・Continuous reaction
6

Introduction〇 Equipment for micro-flow reaction
Micro reactor

Components

J. Britton, C. L. Raston, Chem. Soc. Rev. 2017, 46, 1250–1271

7

Introduction〇 Features of micro-flow reaction✓ Short diffusion length⇒ Precise control of short reaction time
✓ Large surface-to-volume⇒ Rapid and precise temperature control✓ Thin nature of microreactor⇒ Light penetration efficiency✓ Small reaction space⇒ Minimized risk in handling dangerous compounds✓ Continuous operation or increasing the number of reactors⇒ Easy scale-up

Micro-flow technology enables the use of highly reactive,
unstable, explosive or toxic compounds
S. Fuse, Y. Otake, H. Nakamura, Chem. Asian J. 2018, 13, 3818-3832

8

Introduction〇 Features especially in peptide syntheses

Problems of conventional syntheses
・Liquid-phase approach⇒ 〇 Large scale

× Complicated purification Long reaction time・Solid-phase approach⇒ 〇 Rapid reaction Easy purification

× Low atom economy Small scale

・Both approaches⇒ × Epimerization Aggregation

9

Contents
1. Introduction
2. Micro-flow technology for peptide synthesis
ー Solid-phase α-peptide synthesis

ー Solution-phase α-peptide synthesis
ー Cyclic peptide synthesis

3. Application and latest development
ー Total synthesis of Feglymycin
ー Rapid total synthesis of protein
ー Optimization by Deep learning

4. Summary
10

Solid-phase α-Peptide synthesis〇 Synthesis on a solid-phase column reactor

Rapid preheat⇒ Rapid reaction without degradation

M. D. Simon, B. L. Pentelute et al, ChemBioChem 2014, 15, 713–720.

11

Solid-phase α-Peptide synthesis〇 Synthesis on a solid-phase column reactor
Achieved rapid peptide synthesisthat took only 3 minutes toextend 1 residue!

M. D. Simon, B. L. Pentelute et al, ChemBioChem 2014, 15, 713–720.

12

Solution-phase α-Peptide synthesis〇 Synthesis via rapid and strong activation using Triphosgene

S. Fuse, Y. Mifune, T. Takahashi, Angew. Chem. Int. Ed. 2014, 53, 851–855

13

Solution-phase α-Peptide synthesis〇 Synthesis via rapid and strong activation using Triphosgene✓ Short reaction time
✓ High yield and Low epimerizationlevel (compared to batch reaction)

S. Fuse, Y. Mifune, T. Takahashi, Angew. Chem. Int. Ed. 2014, 53, 851–855

14

Cyclic peptide synthesis〇 Synthesis of CODs
Cyclooligomeric depsipeptides (CODs)
・Structures derived from repeated oligopeptidol monomer units
・Exhibit a wide variety of biological activities

D. Lücke, T. Dalton, S. V. Ley, Z. E. Wilson, Chem. Eur. J. 2016, 22, 4206–4217.

15

Cyclic peptide synthesis〇 Synthesis of CODs

D. Lücke, T. Dalton, S. V. Ley, Z. E. Wilson, Chem. Eur. J. 2016, 22, 4206–4217.

16

Cyclic peptide synthesis〇 Synthesis of CODs

Optimized flow conditions allowed inter- andintramolecular couplings in high yields and with
reduction in effort compared to batch method.
D. Lücke, T. Dalton, S. V. Ley, Z. E. Wilson, Chem. Eur. J. 2016, 22, 4206–4217.

17

Contents
1. Introduction
2. Micro-flow technology for peptide synthesis
ー Solid-phase α-peptide synthesis

ー Solution-phase α-peptide synthesis
ー Cyclic peptide synthesis

3. Application and latest development
ー Total synthesis of Feglymycin
ー Rapid total synthesis of protein
ー Optimization by Deep learning

4. Summary18

Total synthesis of Feglymycin〇 Feglymycin
・Oligopeptide composed of 13 amino acids
・Strong anti-HIV activity and moderate antimicrobial activity
・A lot of highly epimerization-prone residues (Hpg, D-Dpg)⇒ Despite the importance, synthetic methodology is very limited…

S. Fuse, Y. Mifune, H. Nakamura, H. Tanaka, Nat. Commun. 2016, 7, 13491

19

Total synthesis of Feglymycin〇 Synthetic protocol
Linear/Convergent hybrid approach

S. Fuse, Y. Mifune, H. Nakamura, H. Tanaka, Nat. Commun. 2016, 7, 13491

20

α-Peptide synthesis (redisplay)〇 Synthesis via rapid and strong activation using Triphosgene✓ Short reaction time✓ High yield and Low epimerizationlevel (compared to batch reaction)

S. Fuse, Y. Mifune, T. Takahashi, Angew. Chem. Int. Ed. 2014, 53, 851–855

21

Total synthesis of Feglymycin〇 Optimization

S. Fuse, Y. Mifune, H. Nakamura, H. Tanaka, Nat. Commun. 2016, 7, 13491

22

Total synthesis of Feglymycin〇 Linear synthesis

S. Fuse, Y. Mifune, H. Nakamura, H. Tanaka, Nat. Commun. 2016, 7, 13491

23

Total synthesis of Feglymycin〇 Convergent synthesis

S. Fuse, Y. Mifune, H. Nakamura, H. Tanaka, Nat. Commun. 2016, 7, 13491

24

Rapid total synthesis of protein〇 DARPin pE59 & Barnase

・ 130-residue protein
・ Established versatility as aprotein-binding scaffold (DARPin)・ Nanomolar affinity for pERK 2 (pE59)

・ 113-residue protein
・ Potent RNase with endonuclease activity
・ Not require cofactors or metal ionsfor folding or catalytic activity.
25

S. K. Mong, A. A. Vinogradov, M. D. Simon, B. L. Pentelute, ChemBioChem 2014, 15, 721–733.

Rapid total synthesis of protein〇 Synthetic protocol
Combination of micro-flow technology and NCL

26

S. K. Mong, A. A. Vinogradov, M. D. Simon, B. L. Pentelute, ChemBioChem 2014, 15, 721–733.

α-Peptide synthesis (redisplay)〇 Synthesis on a solid-phase column reactor

Achieved rapid peptide synthesisthat took only 3 minutes toextend 1 residue!
M. D. Simon, et al, ChemBioChem 2014, 15, 713–720.

27

Rapid total synthesis of protein〇 Typical synthetic problems

28

S. K. Mong, A. A. Vinogradov, M. D. Simon, B. L. Pentelute, ChemBioChem 2014, 15, 721–733.

Rapid total synthesis of protein〇 Typical synthetic problems

29

S. K. Mong, A. A. Vinogradov, M. D. Simon, B. L. Pentelute, ChemBioChem 2014, 15, 721–733.

Rapid total synthesis of protein〇 Native chemical ligation (NCL)

30

S. K. Mong, A. A. Vinogradov, M. D. Simon, B. L. Pentelute, ChemBioChem 2014, 15, 721–733.

Rapid total synthesis of protein〇 Native chemical ligation (NCL)

31

S. K. Mong, A. A. Vinogradov, M. D. Simon, B. L. Pentelute, ChemBioChem 2014, 15, 721–733.

Optimization by Deep learning〇 Fully automated synthesizer✓ High fidelity✓ Rapid✓ Minimum amount of biproducts✓ Incorporation of
non-canonical amino acids

A. J. Mijalis, B. L. Pentelute, et al, Nature chem bio 2017, 13(5), 464-466

32

Optimization by Deep learning〇 Access to highly reproducible data

S. Mohapatra, B. L. Pentelute, et al, ACS Cent. Sci. 2020 https://doi.org/10.1021/acscentsci.0c00979

33

Optimization by Deep learning〇 Prediction of Fmoc deprotection traces

S. Mohapatra, B. L. Pentelute, et al, ACS Cent. Sci. 2020 https://doi.org/10.1021/acscentsci.0c00979

34

Optimization by Deep learning〇 Supplement about fingerprint

S. Mohapatra, B. L. Pentelute, et al, ACS Cent. Sci. 2020 https://doi.org/10.1021/acscentsci.0c00979

35

Optimization by Deep learning〇 Prediction for GLP-1 synthesis

Predictions from the model match the experimental values⇒ However, what is width and height useful for…?
S. Mohapatra, B. L. Pentelute, et al, ACS Cent. Sci. 2020 https://doi.org/10.1021/acscentsci.0c00979

36

Optimization by Deep learning〇Interpretation of aggregation
Aggregation is a sequence-dependent event that results in poor synthetic outcome

S. Mohapatra, B. L. Pentelute, et al, ACS Cent. Sci. 2020 https://doi.org/10.1021/acscentsci.0c00979

37

Optimization by Deep learning〇 Sequence optimization using single-point mutations

Mutation of amino acids which wereactivated for aggregation led to adecrease of aggregation in most cases

S. Mohapatra, B. L. Pentelute, et al, ACS Cent. Sci. 2020 https://doi.org/10.1021/acscentsci.0c00979

38

Optimization by Deep learning〇 Future optimization
Reaction conditions also have room for improvement

In prediction, use of PyAOP and extended coupling time will improve the yields

The difference is small, but these minor effects add
up to have a detrimental impact in long peptide
synthesis>99% coupling efficiency per incorporated aminoacid is crucial
S. Mohapatra, B. L. Pentelute, et al, ACS Cent. Sci. 2020 https://doi.org/10.1021/acscentsci.0c00979

39

Contents
1. Introduction
2. Micro-flow technology for peptide synthesis
ー Solid-phase α-peptide synthesis

ー Solution-phase α-peptide synthesis
ー Cyclic peptide synthesis

3. Application and latest development
ー Total synthesis of Feglymycin
ー Rapid total synthesis of protein
ー Optimization by Deep learning

4. Summary
40

Summary
・Peptide drugs have been attracting attention in recent years
and more efficient synthesis method is needed
・Micro-flow technology has great possibility to develop peptidesynthesis in terms of precise reaction control and ease of scaleup
・In solution-phase, rapid and low-epimerization level synthesis
was achieved.
・In solid-phase, rapid and automated continuous synthesis wasachieved.
41

Appendix

β-Peptide synthesis〇 β-amino acids
Amino acids which have amino group bonded to the β carbon
・Many of β-amino acids lack chiral center at α-position→ Epimerization doesn’t occur

・Interesting properties from structural and biological viewpoint(ex: stable against proteolytic degradation)43

β-Peptide synthesis〇 First report of micro-flow synthesis✓High yield✓Short reaction time✓Molecule efficient

P. Watts, C. Wiles, S. J. Haswell, E. Pombo-Villar, Tetrahedron 2002, 58, 5427–5439

44β-Peptide synthesis〇 First report of micro-flow synthesis

EntryPG-AA1

AA2

Yield(Flow) Yield(Batch)1

quant.

46%

2quant.

57%

3quant.

35%

4quant.

36%

5quant.

35%

P. Watts, C. Wiles, S. J. Haswell, E. Pombo-Villar, Tetrahedron 2002, 58, 5427–5439

45β-Peptide synthesis〇 First report of micro-flow synthesis✓Deprotection using only 1 equiv. of base✓Achieved multi-step synthesis✓Separation using electrophoresis

V. George, P. Watts, S. J. Haswell, E. Pombo-Villar, Chem. Commun. 2003, 2886–2887.

46β-Peptide synthesis〇 EOF (Electroosmotic flow)

The motion of liquid induced by an applied potential acrossmicrochannel

An electric field causes layer to move towards the negative electrode→ Causes bulk liquid to move within the channel

Positive counter ions form a double layer

Negatively charged material on the surface

P. Watts, C. Wiles, S. J. Haswell, E. Pombo-Villar, Tetrahedron 2002, 58, 5427–543947β-Peptide synthesis〇 Dmab
A protecting group which is orthogonal to both acid(TFA) andpiperidine-labile protecting groups(PG).
This PG is removed under mild conditions(NH2NH2). It is crucial
because EOF is retarded if the pH is less than 3.
〇 PFP (pentafluorophenyl)
PFP esters are active esters which are useful for laboratory
peptide synthesis.
They are less susceptible to spontaneous hydrolysis during
conjugation reaction.

48

β-Peptide synthesis〇 Preparation of PFP ester〇 Preparation of Dmab-protected amino acids49

β-Peptide synthesis〇 Synthesis using fluorous tag

Fluorous compounds show high affinity for
fluorous solvents⇒ ・Easy purification (using FSPE)
・Overcome poor solubility

O. Flögel, J. D. C. Codée, D. Seebach, P. H. Seeberger, Angew. Chem. Int. Ed. 2006, 45, 7000–700350β-Peptide synthesis〇 Fluorous solid-phase extraction (FSPE)✓ High separability utilizing “fluorousaffinity”✓ High compatibility with mass
spectrometry(MS)51

Zhang, C. et al. Anal. Chem. 2017, 89, 8, 4566–4572

Solid-phase α-Peptide synthesis〇 Synthesis on a solid-phase column reactor
Automated peptide synthesis in a short time was achieved

M. D. Simon, B. L. Pentelute et al, ChemBioChem 2014, 15, 713–720.

52

Solid-phase α-Peptide synthesis〇 Synthesis on a solid-phase column reactor
Comparison of (ALF)7 to manually produced (ALF)7

M. D. Simon, B. L. Pentelute et al, ChemBioChem 2014, 15, 713–720.

53

Solution-phase α-Peptide synthesis〇 α-Peptide synthesis via photochemical reaction

Rearrangement of nitrone to amide

Substrate scope

Y. Zhang, et al, Angew. Chem. Int. Ed. 2013, 52, 4251–425554

Solution-phase α-Peptide synthesis〇 Synthesis via rapid and strong activation using Triphosgene

S. Fuse, Y. Mifune, T. Takahashi, Angew. Chem. Int. Ed. 2014, 53, 851–855

55

Cyclic peptide synthesis〇 Optimization

D. Lücke, T. Dalton, S. V. Ley, Z. E. Wilson, Chem. Eur. J. 2016, 22, 4206–4217.

56

Total synthesis of Feglymycin〇 Deprotection of Alloc group〇 Isomerization of C-terminal Glu

57

Total synthesis of Feglymycin〇 Detail of linear synthesis

S. Fuse, Y. Mifune, H. Nakamura, H. Tanaka, Nat. Commun. 2016, 7, 13491

58

Total synthesis of Feglymycin〇 Detail of linear synthesis

S. Fuse, Y. Mifune, H. Nakamura, H. Tanaka, Nat. Commun. 2016, 7, 13491

59

Total synthesis of protein〇 LC-MS data of DARPin pE59 fragments

60

S. K. Mong, A. A. Vinogradov, M. D. Simon, B. L. Pentelute, ChemBioChem 2014, 15, 721–733.

Total synthesis of protein〇 LC-MS data of DARPin pE59 fragments

61

S. K. Mong, A. A. Vinogradov, M. D. Simon, B. L. Pentelute, ChemBioChem 2014, 15, 721–733.

Total synthesis of protein〇 LC-MS data of Barnase fragments

62

S. K. Mong, A. A. Vinogradov, M. D. Simon, B. L. Pentelute, ChemBioChem 2014, 15, 721–733.

Total synthesis of protein〇 LC-MS data of Barnase fragments

63

S. K. Mong, A. A. Vinogradov, M. D. Simon, B. L. Pentelute, ChemBioChem 2014, 15, 721–733.

Total synthesis of protein〇 Resin64