N-terminal specific protein
modification
Literature seminar #1
2020.1.30
B4 Tamiko Nozaki

1

Contents
1. Introduction
2. Imidazolidinone formation of protein N-termini
3. Application of N-terminus selective imidazolidinone formation
4. Summary

2

Contents
1. Introduction
2. Imidazolidinone formation of protein N-termini
3. Application of N-terminus selective imidazolidinone formation
4. Summary3

1. Introduction

Protein Bioconjugation
ü Cysteine side chain modification

Inappropriate for proteins whose catalytic function or structural stability
rely on the Cys.
ü Lysine side chain modificationAkkapeddi, P., Azizi, S., Freedy, A. M., Cal, Pedro M. S. D, Gois, P. M.
P., & Bernardes, G. J. L. (2016).. Chemical Science, 7(5), 2954-2963.

Products are tend to heterogeneous mixture with altered activity.

4

Protein Bioconjugation

1. IntroductionüTryptophan side chain modification

Seki, Y., Ishiyama, T., Sasaki, D., Abe, J., Sohma, Y., Oisaki, K.,& Kanai, M. (2016). JACS, 138(34), 10798-10801.

Trp : The least abundant (∼1%)
Little surface exposureIncluded in the primary sequences of 90% of native proteins→Targeting Trp is the promising strategy to high selectivity while maintaining
structural integrity.
ü N-terminus is also the emerging target of of site-specific modification.

5

N-terminus as the target of protein modification

1. Introduction

α-amine group of the N-terminus is a uniquely reactive site for chemical
modification in single-chain protein.
N-terminal amine is charged as well as Lysine at physiological condition.
80.3 % of the N-terminal residues are solvent exposed .
N-terminal modification has minimal effect for the overall protein structure.

Rosen CB, Francis MB. (2017), Nature chemical biology. 7;13(7):697-705.

ü N-terminal residues are easily accessible, high selective modification target
with little disruption to protein function.
6

pKa of the α-amino group of N-terminal amino acid

1. Introduction

Electrostatic interactions have significant effect on the pKa of the α-amino group.
α-carbonyl groupN-terminal residue

Sereda TJ, Mant CT, Quinn AM, Hodges RS.,(1993),
Journal of Chromatography A,646(1):17-30.

ü pKa of α-amino group is around 6-8 (pKa of ε-amino group of Lysine : 10.3)

7

N-terminal modification for therapeutics

1. Introduction

Pegfilgrastim (Neulasta): N terminally PEGylated human recombinant granulocyte colony-simulating factor(rh-GCSF)

https://www.tga.gov.au/sites/default/files/auspar-pegfilgrastim-190328-pi.pdf

ü Increasing the stability and circulation half time of protein without
substantial effects on protein function and charge.
8

pH control method

1. Introduction

 Applied to any N-terminal amino acids
and the wide variety of attachment group.
 Complete site specificity is rarely achieved.

(Rosen CB, Francis MB., 2017)
9Using Specific side chain amino acid
Cysteine

Tryptophan

1. Introduction

Serine(Rosen CB, Francis MB., 2017) These methods need introduction of specific amino acid at N-terminus.

10

N-terminal transamination

Introduction

Gilmore, J. M., Scheck, R. A.,
Esser-Kahn, A. P., Joshi, N. S., &Francis, M. B. (2006). ACIE, 45(32),5307-5311.

Cassimjee, K. E., Manta, B., & Himo, F.
(2015). Organic & biomolecularchemistry, 13(31), 8453-8464.

(Rosen CB, Francis MB., 2017)

 AKT sequence at the N-terminus is optimal.

11

1. Introduction

Other one step chemical modification
Imidazolidinone formation

 Potentially general approach.
The absence of a proline in the second position is necessary.
Oxidative coupling(Rosen CB, Francis MB., 2017)

 Free Cys are reacted with orhto-aminophenol.

12N-terminal modification by enzymes

1. Introduction(Rosen CB, Francis MB., 2017) The reactions occur under mild condition.

13

1. Introduction

Summary of N-terminus selective protein modification methods
Independence ofamino acid sequence

The accessibility ofthe reagent

Site selectivity

High yield

pH control

/



Using Specific sidechain amino acid

/

/

-transamination







-Imidazolidinone
formation-Oxidative coupling

enzymes-→ Imidazoline formation method would be the general and selective method
for N-terminal modification.
14

Contents
1. Introduction
2. Imidazolidinone formation of protein N-termini
3. Application of N-terminus selective imidazolidinone formation
4. Summary

15

Identification of 2PCA

Imidazolidinone formation

2. Imidazolidinone formation of protein N-termini

Gilmore, J. M., Scheck, R. A., Esser-Kahn, A. P., Joshi, N. S., & Francis, M. B. (2006). ACIE, 45(32),5307-5311.

Imidazolidinone
Optimization of aldehyde

MacDonald JI, Munch HK, Moore T, Francis MB.(2015). Nature chemical biology;11(5):326-331.

16

2. Imidazolidinone formation of protein N-termini

Verification of N-terminus selective modification by 2PCA
ü Imidazoline formation is N-terminal selective.

Pyroglutamic acidü Modification does notoccur when N terminalis blocked or the second
amino acid is Pro.
LC-MS/MS analysis(MacDonald JI et al, 2015)17

2. Imidazolidinone formation of protein N-terminiReactivity of various N-terminal amino acids with 2PCA

(MacDonald JI et al, 2015)(d) The reaction showed good-to-excellent levels of conversion as quantified by LC/MS for a panel of XADSWAG peptides, where X represents a variable amino acid position. Conditions for b–d: 100 μM peptide,
10 mM 2PCA (or derivative), 10 mM phosphate buffer at pH 7.5 and 37 C. The samples were incubated for
1 h in b and for 4 h in c and d.
(e) The PADSWAG peptide was subjected to similar conditions as those in d but at pH 8.5. For d and e,experiments were run in triplicate, and error bars represent the s.d.

ü All of the N-terminal amino acid residues provided relatively good yields.

18

2. Imidazolidinone formation of protein N-termini

Bioconjugation of diverse functional group
Increasing water solubility

For functionalization(MacDonald JI et al, 2015)19

ü N-terminal specific attachment of various synthetic compounds to biomolecules.

2. Imidazolidinone formation of protein N-termini

N-terminal selective bioconjugation with 2PCASingle
PEGylatedprotein(MacDonald JI et al, 2015)→No PEGylation (negative control)→Single PEGylation→heterogeneous PEGylation

2PCA :
One-step selective N-terminal modification method under mild condition without
genetic engineering
20Identification of TA4C

2. Imidazolidinone formation of protein N-termini

2PCA
4 steps

TA4C

Onoda, A., Inoue, N., Sumiyoshi, E., & Hayashi, T. (2020).. ChemBioChem,21
ü TA4C would be the one-pot synthesized N-terminal selective modification reagent.

2. Imidazolidinone formation of protein N-termini

Verification of N-terminus selective modification by TA4C
ü N-terminal selective Imidazoline formation was confirmed.

ü Modification does not occur
when N-terminal is blocked or
the second amino acid is Pro.
(Onoda et al, 2019)

Figure S4. MS/MS spectrum of angiotensin I modified with 1.
Observed a-, b- and y-ions are shown. The parent ion peak islabeled with an asterisk. Conditions: peptide (100 M), 1 (10mM), phosphate buffer (10 mM, pH 7.5) at 37 C for 4 h.

22

2. Imidazolidinone formation of protein N-termini

Bioconjugation of diverse functional group
Optimized condition(Onoda et al, 2019)

Figure 3. (b) Chemicalstructures of functional
molecules attached to a TA4C
moiety and relative ratio ofmodification of RNase.
Conditions: protein (50 µM), 1,10, 11 (10 mM) or 12 (20 mM),
phosphate buffer (10 mM, pH7.5) at 37 C for 16 h.

ü N-terminal specific attachment of various functional molecules to protein

23

2. Imidazolidinone formation of protein N-termini

Facile process for generation of TA4C reagents

Dimroth rearrangement : 1-(4-nitrophenyl)-1H-1,2,,3-triazole-4-carbaldehyd

Optimized condition(Onoda et al, 2019)

ü Two step N-terminal modification24

2. Imidazolidinone formation of protein N-termini

TA4C reagent with azide/alkyne group for chemical modification(Onoda et al, 2019)

Dimroth rearrangement enables to prepare TA4C reagent with azide or alkyne group.
→ Strain-promoted azide-alkyne cycloaddition with functional moiety.
TA4C :One step N-terminal protein modification reagent synthesized in CuAAC reaction
or Dimroth rearrangement25

2. Imidazolidinone formation of protein N-termini

Summary of N-terminus selective imidazoline formation
2PCA(MacDonald JI et al, 2015)

TA4C(Onoda et al, 2019)26

Contents
1. Introduction
2. Imidazolidinone formation of protein N-termini
3. Application of N-terminus selective imidazolidinone formation
4. Summary27

Cytosolic Delivery of Proteins

3. Application of N-terminus selective
imidazolidinone formation methods

Most proteins taken up through the endocytosis
pathway are destroyed in lysosomes.
Kou, L., Sun, J., Zhai, Y., & He, Z. (2013). Asian Journal of Pharmaceutical Sciences, 8(1), 1-10.

Delivery strategy that can transport functional
proteins to intercellular targets using N-terminal
modification with 2PCA.

Sangsuwan, R., Tachachartvanich, P., & Francis, M. B. (2019). JACS, 141(6), 2376-2383.

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Amphiphilic polymer

3. Application of N-terminus selective
imidazolidinone formation methods

Lin, C. J., Sperling, R. A., Li, J. K., Yang, T., Li, P., Zanella, M., . . . Parak, W. J. (2008). Small, 4(3), 334-341.

Polymer backbone : poly(isobutylene-alt-maleic anhydride) (→hydrophilicity)→ Alkylamine chains are linked by the direct amidation (→hydrophobicity)→ Functional groups with an amino terminal group are also linked to the polymer292PCA containing polymer

3. Application of N-terminus selective
imidazolidinone formation methods

polymerProtein-polymer conjugation

Sangsuwan, R., Tachachartvanich, P., & Francis, M. B. (2019). JACS, 141(6), 2376-2383.

Remove free polymer
by chromatography
Inactivation of 2PCA
with NH2OH

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GFP-labeled polymers in living cell

3. Application of N-terminus selective
imidazolidinone formation methods

ü Polymer conjugated protein is
efficiently delivered into theliving cell.
(Sangsuwan, R. et al. (2019))

31

3. Application of N-terminus selective
imidazolidinone formation methods

Delivery of RNase A as a Cytotoxic Cargo Protein
RNase A

Leland, P. A., & Raines, R. T. (2001). Chemistry &Biology, 8(5), 405-413.

Figure 6. Delivery of RNase A to cells as a model cytotoxic cargo molecule. (a) Enzymatic activity of RNase A is unaffected by polymer
attachment. Absorbance at 300 nm was recorded at 1 min intervals. (b) HeLa cells were incubated alone (control), with polymer H, or with theconjugate of RNase A to polymer H for 6 h at 37 C. Viability was measured using an MTT assay and is reported as the percentage of surviving
cells compared to the untreated control. Error bars represent the standard error of three independent experiments. ****p < 0.0001 indicates asignificant difference between RNase A- polymer H-treated cells and unmodified RNase A-treated cells.
(Sangsuwan, R. et al. (2019))

ü Polymer conjugation did not affect the activity of RNase A.
ü RNase A-polymer exhibited cytotoxic effect.
→ Polymer conjugation did not effect the function of RNase A.

32

3. Application of N-terminus selective
imidazolidinone formation methods

2PCA method for ADC –Synthesis of 2PCA derivative
N terminal modification with 2PCA
→Site specific modification of antibody
with high stability and selectivity under mild condition

6-AM-2PCAFigure 1. Synthesis of 6-AM-2-PCA and its application for N-terminal modification of peptides or proteins. (A) 6-AM-2-PCA is synthesized
through azidization and subsequent oxidation of 6-(bromomethyl)-2-pyridinemethanol (1) to yield an azido intermediate (2) and ultimately,pyridinecarboxyaldehyde (3). (B) N-terminal modification of a protein with 6-AM-2-PCA results in an azide appended to the protein thatorthogonally reacts with a DBCO derivative through the aldehyde-amine reaction.

Li, D., Han, B., Wei, R., Yao, G., Chen, Z., Liu, J., . . . Zhao, Q. (2018).. mAbs, 10(5), 712-719.

6-AM-2PCA : Azide derivative of 2PCA → click reaction

33

3. Application of N-terminus selective
imidazolidinone formation methods

2PCA method for ADC –modification of antibody
ü Fluorescence-coupled anti HER2 Fab via 6-AM2PCA was distributed at plasma membranes.

https://www.takarabio.com/learningcenters/cdna-synthesis/cloning-antibodyvariable-regions

Figure 6. Immunostaining of SK-BR-3 breast
cancer cells by 6-AM-2-PCA-conjugatedanti-Her2 antibodies. (A) Three panels (leftto right) represent fluorescence of SK-BR- 3cells stained with modified anti-Her2 Fabfollowed by DBCO-ATTO 488, nuclei stainedwith DPAI, and the mergedimage. (B) Threepanels (left to right) represent fluorescence of SK-BR-3 cells stained with DBCOATTO 488 alone, nuclei stained with DPAI,and the merged image.
34(Li, D. et al., (2018))

Contents
1. Introduction
2. Imidazolidinone formation of protein N-termini
3. Application of N-terminus selective imidazoline formation methods
4. Summary

35

Summaryü N-terminal residues are easily accessible, high selective modification
target with little disruption to protein function.

ü Imidazoline formation by 2PCA or TA4C is the favorable for N-terminal
modification with high generality and selectivity under mild condition.

ü N-terminal selective imidazolidinone formation would be applied totherapeutic applications including material delivery and ADC.

36

Appendix

37

Endogenous N-terminus modification

Varland S, Osberg C, Arnesen T., PROTEOMICS. 2015;15(14):2385-2401.

ü Most proteins expressed in bacteria and some protein expressed in eukaryotes
including antibodies could be the target of artificial N-terminal modification. 38

tRAc-Xx-tRH-Xx

pKa(Sereda TJ, et al. (1993))

Model peptide
Ac-X-LGAKGAGVG-amide
H-X-LGAKGAGVG-amide

39Modification of Cysteine at protein N terminus
Native chemical ligation

Malins, L. R., & Payne, R. J. (2014). Current
Opinion in Chemical Biology, 22, 70-78.

Forming thiazolidine with aldehydeBandyopadhyay, A.; Cambray, S.; Gao, (2016) J. Chem. Sci. , 7, 4589.

 The reaction is relatively slow (48 h).
 It requires acidic condition.
 A large excess of the aldehyde is needed.
→ortho-boronic stabilizes the thiazolidinoboronate product through N→B coordination.
40

Modification of Cysteine at protein N terminus
Condensation with 2-cyanobenzothiazole derivatives

Zheng, Z., Chen, P., Li, G., Zhu, Y., Shi, Z., Luo, Y., . . . Liang, G. (2017). Chemical Science, 8(1), 214-222

 The reaction is rapid at neutral pH and generates stable product.
→CBT reacts with D-cysteine in the final step of D-luciferin synthesis.
41

Oxidative coupling

Obermeyer, A. C., Jarman, J. B., & Francis, M. B. (2014). JACS, 136(27), 9572-9579.

Proline still stood out as the most reactive species.
Free cysteine residues can be oxidized.

42

Synthesis of 2PCA intermediate(MacDonald JI et al, 2015)43

Protein scope for N-terminal biotunylation(MacDonald JI et al, 2015)44

Synthesis of TA4C precursor

Goud, G. L., Ramesh, S., Ashok, D., Reddy, V. P., Yogeeswari, P., Sriram, D., ... & Manga, V. (2017), MedChemComm, 8(3), 559-570.
(Search in SciFinder)45

Two step modification with TA4C

An amine precursor in DMSO (200 mM, 20 μL, final concentration: 100 mM) and 13 in DMSO (200 mM, 20 μL, finalconcentration: 100 mM) were mixed in a tube (1 mL). To the solution was added MOPS in H2O (400 mM, 1 μL, finalconcentration: 10 mM), and the mixture was incubated at 99 C for 30 min. The Dimroth rearrangement reaction wasassessed by 1H NMR measurement (400 MHz, DMSO-d6) (Figure S15~S18).
RNase in H2O (1 mM, 2.5 μL, final concentration: 50 μM) was diluted with potassium phosphate buffer (10 mM, pH 7.5, 42.5μL). To the resulting solution, a crude solution of TA4C 1, 10, 14 or 15 in DMSO (5.0 μL) was added, and the mixture wasincubated at 37 C for 16 h. The protein was then purified several times by centrifugal filtration in an Amicon Ultra-0.5
centrifugal filter device with a 10-kDa molecular weight cutoff (Millipore). Modification of proteins was analyzed by LC-MS.
46

Polymer variants

ü Polymer H showed the greatest
degree of conjugation (~68 %) andgreater cellar uptake.

47

Polymer immobilization of the ligand-binding domain of Erα’ (application)(MacDonald JI et al, 2015)

2PCA incorporates high-affinity binding proteins for endocrine-disrupting
compounds into polymer matrix that can be easily detected.
→Detection of those compounds in drinking water.

48

Capture and Recycling of SrtA (application)
Methods for the selective capture and subsequent recycling of enzyme were inhigh demand.
ü Lithocholic acid was attached to SrtA by
2PCA-based chemistry.
ü LA-SrtA is selectively captured with βCD
functionalized resin.

Rosen, C. B., Kwant, R. L., MacDonald, J. I., Rao, M., & Francis, M. B. (2016). ACIE, 55(30), 8585-8589.

49

N-terminal specific conjugation of ECM (application)

Vinculin
F-actin
DAPI
Lee, J. P., Kassianidou, E., MacDonald, J. I., Francis, M. B., & Kumar, S. 50(2016)., Biomaterials, 102, 268-276.

N-terminal modification for ADC

Thompson, P., Bezabeh, B., Fleming, R., Pruitt, M., Mao, S., Strout, P., . . .
Dimasi, N. (2015). Bioconjugate Chemistry, 26(10), 2085-2096.
51