Practical modification of peptides using ligand-free copper-catalyzed azide–alkyne cycloaddition

Accepted Manuscript Practical modification of peptides using ligand-free copper-catalyzed azide–alkyne cycloaddition Yoshiaki Kitamura, Ryuto Sakamoto, Takao Shiraishi, Haruka Oguri, Satoshi Ohno, Yukio Kitade PII:

S0040-4020(16)30414-8

DOI:

10.1016/j.tet.2016.05.029

Reference:

TET 27756

To appear in:

Tetrahedron

Received Date: 9 March 2016 Revised Date:

10 May 2016

Accepted Date: 11 May 2016

Please cite this article as: Kitamura Y, Sakamoto R, Shiraishi T, Oguri H, Ohno S, Kitade Y, Practical modification of peptides using ligand-free copper-catalyzed azide–alkyne cycloaddition, Tetrahedron (2016), doi: 10.1016/j.tet.2016.05.029. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT

Practical modification of peptides using ligand-free copper-catalyzed azide–alkyne cycloaddition

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Yoshiaki Kitamuraa,b, Ryuto Sakamotoa, Takao Shiraishic, Haruka Oguria, Satoshi Ohnoa,b and

Department of Biomolecular Science, Graduate School of Engineering, Gifu University, 1-1

Yanagido, Gifu 501-1193, Japan b

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a

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Yukio Kitadea,b,c,d,*

Department of Chemistry and Biomolecular Science, Faculty of Engineering, Gifu

University, 1-1 Yanagido, Gifu 501-1193, Japan

United Graduate School of Drug Discovery and Medical Information Sciences, Gifu

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c

University, 1-1 Yanagido, Gifu 501-1193, Japan Department of Applied Chemistry, Faculty of Engineering, Aichi Institute of Technology,

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d

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1247 Yachigusa, Yakusa-cho, Toyota, Aichi 470-0392, Japan

∗ To whom correspondence should be addressed: Department of Chemistry and Biomolecular Science, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan Tel.: +81-58-293-2640; Fax: +81-58-293-2640; E-mail: [email protected] 1

ACCEPTED MANUSCRIPT Keywords

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Peptides; Post-modification; Orthogonal techniques; Click chemistry; CuAAC

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ACCEPTED MANUSCRIPT Abstract A convenient method for the post-modification of any peptide bearing a disulfide bond via

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rapid ligand-free copper-catalyzed azide–alkyne cycloaddition (CuAAC) was developed. N-hydroxysuccinimide (NHS) esters and maleimides bearing an aryl acetylene residue to allow installation of a terminal alkyne moiety into peptides were efficiently synthesized. The

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ligation of glutathione (GSH) using the maleimides and disulfide-linked dimer of GSH

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(GSSG) using the NHS esters proceeded smoothly to afford the corresponding terminal alkyne-labeled glutathione derivatives, respectively. The terminal alkyne-labeled GSH and GSSG efficiently coupled with 4-fluorobenzylazide to provide the corresponding glutathione analogues in quantitative yield. In any case, side reactions including cleavage of disulfide

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bonds were not observed. The application of somatostatin, disulfide-linked cyclic

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tetradecapeptide, to this method was achieved.

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ACCEPTED MANUSCRIPT Introduction Practical methods for labeling peptides and proteins with functional molecules are important

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in various fields ranging from chemical biology to medicine.1 Oligopeptides are typically labeled using orthogonal techniques such as the installation of non-natural functional groups on natural amino acid residues (including the N-terminal residue), followed by a reaction

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such as condensation,2 Staudinger ligation,3 olefin metathesis,4 Diels-Alder reaction5 or

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1,3-dipolar cycloaddition.6 A conjugation technique involving copper-catalyzed azide–alkyne 1,3-dipolar cycloaddition (CuAAC) was developed independently in the Sharpless and Meldal laboratories in 2001.7 This is one of the most common conjugation methods for orthogonal peptide modification due to its selectivity, versatility, and insensitivity to changes

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in pH and temperature. Many applications of CuAAC for peptide labeling have been reported over the past decade, employing Cu(I) species as the catalyst. However, Cu(I) is

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thermodynamically unstable and easily oxidized to catalytically inactive Cu(II), or quickly to

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disproportionation to Cu(II) and Cu(0).8 These drawbacks are circumvented by using a combination of Cu(II) salts and a reducing agent (e.g., copper sulfate (CuSO4) and sodium ascorbate (NaAsc)) to generate Cu(I) species in situ. NaAsc is a representative reductant for Cu(II) in CuAAC reactions in organic and materials synthesis, but cleaves disulfide bonds9 which often stabilize the secondary and/or tertiary structures of peptides and proteins. Moreover, dehydroascorbate and other ascorbate byproducts formed in CuAAC could react 4

ACCEPTED MANUSCRIPT with the ε-amino group of lysine and the guanidino group of arginine, leading to covalent modification and potential aggregation of the protein.10 These side reactions can be prevented

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when using sodium ascorbate as a reducing agent through the concomitant use of expensive Cu(I)-stabilizing ligands such as tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA), tris[(1-(3-hydroxypropyl)-1H-1,2,3-triazol-4-yl)methyl]-amine

(THPTA),

and

bidentate

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chelators such as bathophenanthroline disulfonic acid disodium salt (BPS, BPDS). A

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ligand-free CuAAC between terminal alkyne-modified peptide containing a disulfide bond and azide compound was previously reported,11 but the reaction required heating conditions (80 ºC) which can cause denaturation of peptides on occasions. Including this case, most of the existing methods for labeling peptides using CuAAC have been utilized non-natural

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peptides bearing pre-installed terminal alkynes. We here report a convenient method for the post-modification of disulfide-containing natural peptides via a rapid and mild ligand-free

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CuAAC at room temperature.

Scheme 1. Post-modification of peptides using a rapid ligand-free CuAAC.

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ACCEPTED MANUSCRIPT We recently developed a rapid ligand-free CuAAC for the post-labeling of oligonucleotides.12 Under optimized Cu(II)-NaAsc conditions, oligonucleotides bearing aryl

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acetylene derivatives rapidly reacted at room temperature with azide compounds in the absence of copper ligands and the corresponding labeled oligonucleotides were obtained quantitatively. We thus anticipated that our Cu(II)-NaAsc protocol would be applicable to the

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labeling of peptides and proteins.

Results and discussion

Synthesis of NHS ester and maleimide derivatives

In our previous study, it was found that terminal alkynes conjugated aromatic groups had

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higher reactivity with azide in CuAAC than that on aliphatic groups.13 Based on the knowledge, N-hydroxysuccinimide (NHS) esters 1 and 2, and maleimide 3 bearing an aryl

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acetylene residue, were designed to allow installation of a terminal alkyne moiety into

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peptides. NHS esters and maleimides easily react with amino groups and mercapto groups, respectively, and various reagents containing maleimides and/or an NHS ester have been developed and used to conjugate functional molecules onto biomolecules such as peptides and oligonucleotides.14 The synthesis of 1, 2 and 3 is shown in Scheme 2. 4-Ethynylbenzoic acid (4) is easily prepared from 4-iodobenzoic acid15 and was reacted with NHS to give the NHS ester 1. In 6

ACCEPTED MANUSCRIPT addition, 4 was treated with 5-aminopentan-1-ol in the presence of HOBt and EDC·HCl to afford amide 5 in moderate yield. Subsequent oxidation of the hydroxyl group of 5 to a

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carboxyl group using the TEMPO/NaClO/NaClO2 system, followed by treatment with NHS, gave the corresponding NHS ester 2. Compound 5 was converted to the corresponding azide 6 via the diphenylphosphate using diphenylphosphorylazide (DPPA). Reduction of the azide

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group in 6 using PPh3 gave the corresponding amine in quantitative yield; this amine was

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reacted with maleic anhydride in acetic acid to afford the desired maleimide 3 in 81% yield.

Scheme 2. Reagents and conditions: (a) NHS, EDC·HCl, DMAP, DMF, rt, 73%; (b) 5-aminopentan-1-ol, HOBt, EDC·HCl, DMF, rt, 72%; (c) i) TEMPO, NaClO, NaClO2, 0.1 M PB (pH 6.5), rt; ii) NHS, EDC·HCl, DMAP, DMF, rt, 45%; (d) i) DPPA, DBU, toluene, 0 ºC then rt,; ii) NaN3, DMF, 60 ºC, 78%; (e) i) PPh3, MeOH, rt; ii) maleic anhydride, AcOH, reflux, 81%.

Modification of peptides 7

ACCEPTED MANUSCRIPT To investigate the feasibility of peptide labeling via CuAAC, an initial study was undertaken using glutathione (GSH) and its oxidized disulfide-linked dimer (GSSG). GSH is a tripeptide

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comprising three amino acids (cysteine, glutamic acid and glycine; γ-GluCysGly) and is present in most mammalian tissues. GSH and GSSG play vital roles in defending cells against oxidative stress and toxins.16 GSH and GSSG are small natural peptides and ease of

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handling. The aryl acetylene moiety was introduced into GSSG and GSH using NHS esters 1

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or 2, or maleimide 3. The ligation of GSSG using 1 (1.0 equiv) in 0.1 M phosphate buffer (pH 7.0)/DMSO (1:3, v/v) for 1 h at room temperature afforded the corresponding mono-modified GSSG 7 in 51% yield along with a small amount of di-modified GSSG.

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Similarly, the reaction between GSSG and 2 gave the mono-modified product 8 in 56% yield. The system was also used to modify GSH using 3. GSH smoothly reacted with 3 within 15

yield.

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min, and the corresponding terminal alkyne-labeled GSH 9 was obtained in quantitative

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F-labeled synthon for positron emission

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The synthesis of 4-[18F]-fluorobenzylazide, a

tomography imaging (PET), reported by a few researchers17 prompted us to conduct CuAAC between the terminal alkyne-labeled glutathione analogues and 4-fluorobenzylazide (10) under our optimized conditions18 using CuSO4·5H2O and NaAsc in 0.1 M phosphate buffer (pH 7.0)/DMSO/MeCN at room temperature. The coupling between 7 and 10 (10 equiv) was incomplete (70% yield as judged by HPLC analysis) after 15 min, whereas the use of 20 8

ACCEPTED MANUSCRIPT equiv of 4-fluorobenzylazide accelerated the reaction and provided the corresponding GSSG analogue 11 quantitatively within 15 min. Coupling of 8 and 10 (10 equiv) was complete after

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15 min to afford 12 quantitative yield. It should be noted that no cleavage of disulfide bonds was observed in both cases. Under the present conditions, terminal alkyne-labeled GSH 9 efficiently coupled with 10 (10 equiv) to provide the corresponding GSH analogue 13 in

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quantitative yield.

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ACCEPTED MANUSCRIPT O N H O

HO

OH O

S S

O

O

NH2

H N

1 or 2 0.1 M PB/DMSO rt, 1 h

O H N

N H NH2

O

OH

O

O HO

GSSG

N H O

N3

O

F 10 CuSO4—5H2O, NaAsc 0.1 M PB/DMSO/MeCN rt, 15 min

O

S

O

O

O H N

N H HN

OH

S

O

HO

NH2

H N

OH

O

O

O

H N

N H

HO

O

S

O H N

N H HN

O

7: X =

O 51%

S

O

OH

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HO

NH2

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X O

8: X =

OH

O

H N

56%

Y

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HO

F

N N

O

F

N

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11: Y = N

quant 12: Y =

N

H N quant

HO

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O

SH

O

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O

N

H N

N H

NH2

O N O

3

O

O

0.1 M PB/DMSO rt, 15 min HO OH

N H

OH

O 9 quant

GSH

O

N N

N N O

10 CuSO4—5H2O, NaAsc 0.1 M PB/DMSO/MeCN rt, 15 min

O H N

NH2

O

F

S

O

O

S

O

HO

N H NH2

O H N

OH

O 13 quant

10

ACCEPTED MANUSCRIPT Scheme 3. Introduction of an aryl acetylene moiety into GSSG and GSH, followed by CuAAC with 4-fluorobenzylazide (10).

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We next applied this method to the modification of larger oligopeptides. Somatostatin is a disulfide-linked cyclic tetradecapeptide and is an important hormone that inhibits the secretion of several other hormones, including growth hormone, insulin and glucagon.

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Somatostatin has cysteine residues at positions 3 and 14 that form an internal disulfide bond,

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and lysine residues at positions 4 and 9. Somatostatin was modified with NHS-ester 1 (1.0 equiv) in 0.1 M phosphate buffer (pH 7.0)/DMSO (1:3, v/v) at room temperature for 15 min. On the basis of HPLC and MALDI-TOF mass analysis, two mono-modified somatostatin analogues were mainly produced along with trace amounts of another mono-modified

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somatostatin analogue and a di-modified somatostatin analogue. UPLC-QTOF tandem mass spectrometry (MS/MS) analysis and the Edman degradation19 showed that the major

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mono-modified somatostatin analogues were 4-modified somatostatin 14 (41% yield) and

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9-modified somatostatin 15 (33% yield), and that a trace amount of 1-modified somatostatin 16 was generated. CuAAC between the terminal alkyne-labeled somatostatin 14 and 4-fluorobenzylazide 10 (10 equiv) in 0.1 M phosphate buffer (pH 7.0)/DMSO (1:3, v/v) for 15 min at room temperature proceeded smoothly to provide the desired product 17 in 81% yield. Also in this case, cleavage of the disulfide bond was not observed on the basis of HPLC analysis. 11

ACCEPTED MANUSCRIPT NH2 Gly

H2N Ala

Cys

Asn Phe Phe

Lys

Trp

S

Lys NH2

S HO2C Cys

Ser

Thr

Phe

Thr

somatostatin

O HN Gly

Cys

NH2 Asn Phe

Lys

Phe

Cys

Trp

S S HO2C Cys

Gly

H2N Ala

Thr

O

Phe

S HO2C Cys

Thr

H N Ala

Asn Phe Phe Trp

S

Lys NH2 Ser 14 41%

Lys

Ser 15 33%

Thr

NH2 Gly

Cys

Lys

Asn Phe Phe Trp

S

Lys NH2

Ser Thr 16 trace

Phe

N

Lys

Asn Phe

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S HO2C Cys

F

Phe Trp

S

14 0.1 M PB/DMSO/MeCN rt, 15 min

Cys

N N

HN

Gly

H2N Ala

O

Thr

O

10 CuSO4—5H2O, NaAsc

Lys N H

Thr

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S HO2C Cys

Phe

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H2N Ala

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1 0.1 M PB/DMSO rt, 15 min

Ser 17 81%

Lys NH2 Thr

Phe

Thr

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Scheme 4. Introduction of an aryl acetylene moiety into somatostatin, followed by CuAAC

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with 4-fluorobenzylazide (10).

Conclusions

We have developed a facile peptide modification method utilizing rapid ligand-free CuAAC. This method should be applicable to any peptide bearing a disulfide bond. We anticipate that this approach will offer an alternative strategy for the practical functionalization of peptides and proteins, including the functionalization of peptides and proteins for use in PET imaging. 12

ACCEPTED MANUSCRIPT Experimental section General

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All reactions were carried out under an argon atmosphere, unless otherwise noted. All reagents and solvents were purchased from commercial vendors and used without further purification, unless indicated otherwise. 1H and

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C NMR spectra were recorded on a JEOL

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C NMR). All NMR samples were prepared as CDCl3 solutions. Chemical shifts

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MHz for

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JNM AL-400 spectrometer or JNM ECS-400 spectrometer (400 MHz for 1H NMR and 100

(δ) are expressed in ppm and are internally referenced (0.00 ppm for TMS-CDCl3 for 1H NMR and 77.0 ppm for

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C NMR). EI mass spectra were taken on a JEOL JMS SX102A

instrument. DART-TOF mass spectra were taken on a JMS T100TD instrument.

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MALDI-TOF mass spectra were taken on a Shimazu AXIMA-CFR plus instrument. UPLC-QTOF mass spectra were taken on a Waters Xevo Q-Tof mass spectrometer. Flash

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column chromatography was performed using silica gel 60N [spherical neutral (63-210 µm)]

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from Kanto Chemical Co., Inc.

Synthesis of NHS ester and maleimide derivatives 2,5-Dioxopyrrolidin-1-yl 4-ethynylbenzoate (1)20 NHS (176 mg, 1.53 mmol), EDC·HCl (310 mg, 1.62 mmol) and DMAP (33.2 mg, 270 µmol) were added to a solution of 4-ethynylbenzoic acid (4) (186 mg, 1.27 mmol) in DMF (4 mL) 13

ACCEPTED MANUSCRIPT at 60 ºC, and then the mixture was kept for 30 min at 60 ºC. The resulting mixture was stirred at room temperature for 12 h. H2O was added and the resulting precipitate was collected by filtration and dried to give 1 as pale yellow solid (227 mg, 73 %); 1H NMR (CDCl3) δ 8.11 (d,

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J = 7.6 Hz, 2H), 7.63 (d, J = 7.6 Hz, 2H), 3.34 (s, 1H), 2.90 (s, 4H); 13C NMR (CDCl3) δ 169.1, 161.3, 132.5, 130.4, 128.9, 125.0, 82.3, 81.5, 25.7; MS (EI) m/z 243 (M+), HRMS (EI)

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H, 3.73; N, 5.76. Found: C, 63.86; H, 3.99; N, 5.77.

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Calcd for C13H9NO4 (M+): 243.0532. Found: 243.0524; Anal. Calcd for C13H9O4N: C, 64.20;

4-Ethynyl-N-(5-hydroxypentyl)benzamide (5)

A mixture of 4-ethynylbenzoic acid (4) (1.02 g, 7.0 mmol), HOBt (1.61 g, 10.5 mmol) and

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EDC·HCl (2.02 g, 10.5 mmol) in DMF (35 mL) was stirred at room temperature for 6 h. 5-Aminopentan-1-ol (1.46 mL, 1.42 mmol) was added and stirring was continued for 22 h.

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The mixture was partitioned between EtOAc and H2O. The organic layer was washed with

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brine, dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (n-hexane/EtOAc, 10:1~2:3) to give 5 as a colourless solid (1.17 g, 72 %); 1H NMR (CDCl3) δ 7.71 (d, J = 8.6 Hz, 2H), 7.54 (d, J = 8.6 Hz, 2H), 6.23 (s, 1H), 3.67 (d, J = 6.3 Hz, 2H), 3.47 (dd, J = 13.2 Hz, 7.1 Hz, 2H), 3.19 (s, 1H), 1.70–1.59 (m, 4H), 1.51–1.43 (m, 2H); 13C NMR (CDCl3) δ 169.2, 168.6, 166.9, 134.7, 132.3, 127.0, 125.3, 82.9, 79.5, 39.4, 30.8, 28.5, 25.7, 22.1; MS (DART) m/z 231 (M+), 14

ACCEPTED MANUSCRIPT HRMS (DART) Calcd for C14H17NO2 (M+): 231.1259. Found: 231.1210; Anal. Calcd for

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C14H17NO2: C, 72.70; H, 7.14; N, 6.06. Found: C, 72.43; H, 7.37; N, 6.06.

2,5-Dioxopyrrolidin-1-yl N-(4-ethynylbenzoyl)aminopentanoate (2)

A mixture of 5 (693 mg, 3.0 mmol), TEMPO (95.8 mg, 613 µmol), NaClO2 (80%, 693 mg,

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613 µmol) and NaOCl (5% in H2O, 1.5 mL) in 0.1 M phosphate buffer (pH6.5) (15 mL) and

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MeCN (15 mL) was stirred at room temperature for 13 h. The reaction mixture was quenched with cold H2O, and partitioned between EtOAc and H2O. The aqueous layer was acidified by 1 M HCl. The organic layer was washed with brine, dried over Na2SO4, and concentrated under reduced pressure to give the corresponding acid. NHS (414 mg, 3.60 mmol), EDC·HCl

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(690 mg, 3.60 mmol) and DMAP (73.3 mg, 600 µmol) were added to a solution of the acid in DMF (30 mL) with stirring at 60 ºC, and then the mixture was kept at 60 ºC for 30 min. The

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resulting mixture was stirred at room temperature for 14 h. The reaction mixture was

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partitioned between EtOAc and H2O. The organic layer was washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (n-hexane/EtOAc, 5:1~2:3) to give 2 as a colourless solid (464 mg, 45 %); 1H NMR (CDCl3) δ 7.73 (d, J = 8.2 Hz, 2H), 7.54 (d, J = 8.2 Hz, 2H), 6.32 (s, 1H), 3.53–3.48 (m, 2H), 3.19 (s, 1H), 2.85 (s, 4H), 2.69 (d, J = 6.9 Hz, 2H), 1.91–1.84 (m, 2H), 1.80–1.73 (m, 2H); 13C NMR (CDCl3) δ 169.3, 168.6, 167.0, 134.7, 132.4, 127.1, 125.4, 15

ACCEPTED MANUSCRIPT 82.9, 79.5, 39.5, 30.8, 28.6, 25.8, 22.1; MS (DART) m/z 343 (M+), HRMS (DART) Calcd for C18H19N2O5 (M+): 343.1294. Found: 343.1284; Anal. Calcd for C18H18N2O5: C, 63.15; H,

N-(5-Azidopentyl)-4-ethynylbenzamide (6)

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5.30; N, 8.18. Found: C, 63.31; H, 5.35; N, 7.90.

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DBU (180 µL, 1.2 mmol) and DPPA (260 µL, 1.2 mmol) were added a solution of 5 (231 mg,

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1.0 mmol) in DMF (10 mL) at 0 ºC, and then the mixture was kept for 4 h at 0 ºC. The resulting mixture was stirred for 30 min at room temperature. The reaction mixture was partitioned between EtOAc and H2O. The organic layer was washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by column

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chromatography on silica gel (n-hexane/EtOAc, 5:1~1:1) to give the corresponding diphenyl phosphate. NaN3 (300 mg, 4.62 mmol) was added a solution of the diphenyl phosphate in

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DMF (40 mL), and then the mixture was heated at 60 oC for 16 h. The reaction mixture was

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partitioned EtOAc and H2O. The organic layer was washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (n-hexane/EtOAc, 50:1~1:1) to give 6 as a colourless solid (200 mg, 78 %); 1H NMR (CDCl3) δ 7.71 (d, J = 8.2 Hz, 2H), 7.54 (d, J = 8.2 Hz, 2H), 6.20 (s, 1H), 3.46 (dd, J = 13.3 Hz, 7.3 Hz, 2H), 3.29 (t, J = 6.9 Hz, 2H), 3.19 (s, 1H), 1.69–1.61 (m, 4H), 1.50–1.43 (m, 2H);

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C NMR (CDCl3) δ 166.9, 134.8, 132.4, 127.0, 125.4, 82.9, 79.6, 16

ACCEPTED MANUSCRIPT 51.4, 40.0, 29.4, 28.7, 24.3; MS (DART) m/z 257 [M+H]+, HRMS (DART) Calcd for C14H17N4O [M+H]+: 257.1402. Found: 257.1403; Anal. Calcd for C14H16N4O: C, 65.61; H,

N-(5-Maleimidopentyl)-4-ethynylbenzamide (3)

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6.29; N, 21.86. Found: C, 65.47; H, 6.16; N, 21.69.

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A mixture of 6 (38.4 mg, 150 µmol) and PPh3 (59.3 mg, 226 µmol) in MeOH (3 mL) was

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refluxed for 4 h. The reaction mixture was diluted with EtOAc, and the resulting mixture was extracted with 1 M HCl three times. The combined aqueous extracts were concentrated under reduced pressure to give the corresponding amine as an ammonium chloride salt. NaOMe (10.2 mg, 150 µmol) was added to a mixture of the ammonium chloride salt and AcOH (4

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mL), and then maleic anhydride (29.4 mg, 300 µmol) was added and the resulting mixture was refluxed for 6 h. Maleic anhydride (64.4 mg, 657 mmol) was added to the mixture and

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refluxing was continued for 4 h. The mixture was partitioned between EtOAc and H2O. The

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organic layer was washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (n-hexane/EtOAc, 50:1~0:1) to give 3 as a colourless solid (37.5 mg, 81 %); 1H NMR (CDCl3) δ 7.73 (d, J = 8.1 Hz, 2H), 7.55 (d, J = 8.1 Hz, 2H), 6.66 (s, 2H), 6.15 (s, 1H), 3.56–3.53 (m, 2H), 3.47–3.42 (m, 2H), 3.19 (s, 1H), 1.73–1.61 (m, 4H), 1.40–1.33 (m, 2H); 13C NMR (CDCl3) δ 171.1, 166.8, 134.8, 134.8, 132.4, 127.0, 82.9, 74.5, 40.1, 37.5, 29.0, 28.3, 24.1; MS (DART) m/z 17

ACCEPTED MANUSCRIPT 311 (M+), HRMS (DART) Calcd for C18H19N2O3 (M+): 311.1395. Found: 311.1364; Anal.

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Calcd for C18H18N2O5: C, 69.66; H, 5.85; N, 9.03. Found: C, 69.66; H, 5.75; N, 8.96.

General procedure for introduction of aryl acetylene moiety to peptides

To an eppendorf tube was added peptide (1.0 mg) and 0.1 M phosphate buffer (pH 7.0) (100

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µL). Subsequently, a solution of 1, 2 or 3 (1.0 equiv) in DMSO (33.5 µL) was added to the

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mixture. The resulting mixture was vortexed for approximately 1 sec, and then left for 15 min–1 h. The mixture was passed through a membrane filter (ADVANTEC, DISMIC@-13CP, 0.45 µm) and washed with Miili-Q water (300 µL). The filtrate was purified by reverse-phase HPLC to give the corresponding peptide possessing aryl acetylene residue. Product yields

Flow

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Rate: 1 mL/min.

A : B = 100 : 0 (0 min) → 50 : 50 (25 min) → 100 : 0 (30 min);

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MeCN; Gradient:

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were determined from HPLC peak areas. A: 0.1% TFA in Milli-Q water, B: 0.1% TFA in

General procedure for rapid CuCAAC reaction To an eppendorf tube was added peptide contained aryl acetylene residue (10 mM solution in Milli-Q water, 2 µL, 20 nmol), 4-fluorobenzylazide (10) (10 mM solution in DMSO, 20 µL, 200 nmol), CuSO4· 5H2O (10 mM solution in Milli-Q water, 2 µL, 20 nmol), NaAsc (10 mM solution in Milli-Q water, 2 µL, 20 nmol), MeCN (22 µL), and 0.1 M phosphate buffer (pH 18

ACCEPTED MANUSCRIPT 7.0) (116 µL). The mixture was vortexed for approximately 1 sec, and then left for 15 min. The mixture was passed through a membrane filter (ADVANTEC, DISMIC@-13CP, 0.45

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µm) and washed with Milli-Q water (300 µL). The filtrate was purified by reverse-phase HPLC to give the corresponding peptide possessing 1,4-triazole unit. Product yields were determined from HPLC peak areas. A: 0.1% TFA in Milli-Q water, B: 0.1% TFA in MeCN; A : B = 100 : 0 (0 min) → 50 : 50 (25 min) → 100 : 0 (30 min);

Flow Rate: 1

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Gradient:

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mL/min.

Mass spectrometric analyses of peptides

Spectra were obtained by MALDI-TOF/MS and UPLC-QTOF/MS. 7: Calcd for

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C29H35N6O13S2 [M-H]-: 739.2. Found: 737.9. 8: Calcd for C34H44N7O14S2 [M-H]-: 838.2. Found: 837.9. 9: Calcd for C23H25N4O8S [M-H]-: 517.1. Found: 516.2. 11: Calcd for

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C36H41FN9O13S2 [M-H]-: 890.2. Found: 890.2. 12: Calcd for C41H50FN10O14S2 [M-H]-: 989.3.

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Found: 989.7. 13: Calcd for C30H31FN7O8S [M-H]-: 668.2. Found: 668.2. 14: Calcd for C85H109N18O20S2 [M+H]-: 1765.8. Found: 1765.8. 15: Calcd for C85H109N18O20S2 [M+H]-: 1765.8. Found: 1765.8. 16: Calcd for C85H109N18O20S2 [M+H]-: 1765.8. Found: 1764.7. 17: Calcd for C92H115FN21O20S2 [M+H]-: 1916.8. Found: 1916.6.

Edman’s degradation procedure for confirmation of the modification site 19

ACCEPTED MANUSCRIPT To an eppendorf tube was added 14 (20 nmol) and a PITC (phenylisothiocyanate) solution (10 µL, PITC/Et3N/Milli-Q water/DMF, 1:1:1:7, v/v). The mixture was heated at 50 ºC for 5

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min. The mixture was dried in vacuo at 50 ºC for 10 min. The residue was washed with n-Hexane/EtOAc (15:1) containing 0.02%EtSH (200 µL), EtOAc containing 0.02%EtSH (200 µL), acetone (3 × 200 µL) and dried in vacuo at 50 ºC for 10 min. TFA (10 µL) was

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added to the resulting residue, and then left for 7 min. The reaction mixture was dried in

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vacuo at 50 ºC for 10 min. Product analysis by MALDI-TOF/MS revealed a peak at 1691.1, which could be assigned to Edman degradation compound of 14.

Acknowledgments

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This work was supported by the Molecular Imaging Research Program and Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology

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(MEXT) of Japan. We acknowledge the Division of Instrumental Analysis, Life Science

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Research Center, Gifu University for maintenance of the instruments and kind support.

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ACCEPTED MANUSCRIPT References and notes 1.

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ACCEPTED MANUSCRIPT 16. Hirrlinger, J.; Dringen, R. Brain Res. Rev. 2010, 63, 177–188. 17. (a) Thonon, D.; Kech, C.; Paris, J.; Lemaire, C.; Luxen, A. Bioconjugate Chem. 2009,

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18. The optimized reaction conditions: peptide contained aryl acetylene residue (10 mM solution in Milli-Q water, 2 µL, 20 nmol), 4-fluorobenzylazide (10) (10 mM solution in DMSO, 20 µL, 200 nmol), CuSO4·5H2O (10 mM solution in Milli-Q water, 2 µL, 20 nmol), NaAsc (10 mM solution in Milli-Q water, 2 µL, 20 nmol), MeCN (22 µL), and

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0.1 M phosphate buffer (pH 7.0) (116 µL), rt, 15 min.

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19. Edman, P.; Hogfeldt, E.; Sillen, L. G.; Kinell, P. O. Acta Chem. Scand. 1950, 4, 283–

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20. Ravnsbæk, J. B.; Jacobsen, M. F.; Rosen, C. B.; Voigt, N. V.; Gothelf, K. V. Angew. Chem. Int. Ed. 2011, 50, 10851–10854.

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