Click Peptides, Design and Applications of

Yoshiaki Kiso and Atsuhiko Taniguchi Youhei Sohma

Department of Medicinal Chemistry, Kyoto Pharmaceutical University, Yamashina-ku, Kyoto, Japan Department of Physical Chemistry, Kyoto Pharmaceutical University, Yamashina-ku, Kyoto, Japan
doi: 10.1002/9780470048672.wecb632

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Article Contents
Biologic Background Chemistry Chemical Tools and Techniques

A click peptide is a chemically modied peptide analog used to understand the biologic function of peptides. A click peptide does not exhibit the inherent biologic activity of the original peptide because of a simple chemical modication of the peptide backbone and; by adding an exogenous action (click) such as pH-change or photo-irradiation, easily affords the native peptide in situ with a quick and easy one-way conversion via a native amide bond-forming reaction. For example, the designed click peptides of Alzheimers amyloid peptide (A) 142, which did not exhibit a self-assembling nature because of a backbone isomerization from a native Gly25 -Ser26 bond to a -ester bond, could migrate to the intact A142 under physiologic conditions by pH- or photo-triggered click via an ON intramolecular acyl migration reaction. This click peptide overcomes the difculties in handling A142 in syntheses and biologic experiments, which claries the currently unexplained processes of Alzheimers disease.

The use of intact peptides in an in vitro experiment sometimes causes considerable discrepancies in the biologic data because of the difculties in handling and controlling the properties of peptides such as low water-solubility and highly aggregative nature. A click peptide is a chemically modied peptide analog used to understand the biologic function of peptides while overcoming the problems associated with the peptide properties (Fig. 1) (13). A click peptide has the ability to:

Additionally, in the design of the click peptide, a simple modication of the peptide backbone is desired to minimize difculties in chemical synthesis of the click peptide and the conversion process to the native peptide.

Biologic Background
The highly aggregative feature of peptides is a signicant obstacle against establishing a reliable in vitro biologic experimental system to investigate the major causative agents of diseases. Recent amyloid peptide (A)-related studies have encountered such problems because of their highly aggregative nature. A more clear understanding of the pathologic mechanism of A would be of great value to discover novel drug targets against Alzheimers disease (AD). A is the main proteinaceous component of amyloid plaques found in the brain as a pathognomonic feature of AD (4), and it has have been found to be neurotoxic in vitro and in vivo (5). Several studies have supported the hypothesis that neurotoxicity and the kinetics of A142 aggregation are related directly to the assembly state in solution. However, the pathologic self-assembly of A142 in amyloid plaque formation, a process currently unexplained, is very difcult to demonstrate in vitro because of its uncontrolled self-assembly. The synthesized A142 in 1

Control the following natures of the original peptide. Physicochemical property (e.g., water-solubility, self-assembly, aggregation, or folding) Biologic activity (e.g., ligand-receptor binding afnity, or enzyme-substrate binding afnity) Convert to the native peptide in situ by an exogenous action (click). Examples of such an action include pH-change and photo-irradiation. Because a rapid amide bond formation is required, an intramolecular reaction is desired. An atom-economical reaction should be selected as the conversion reaction to avoid a side effect derived from coreleased products in biologic experimental conditions.

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Click Peptides, Design and Applications of

Click peptide does not exhibit the nature of the original peptide.

Native peptide is easily prepared in situ.

chemical modification peptide A peptide B

click peptide A peptide B

click peptide

quick and one-way conversion

native peptide

An exogenous action triggers the native amide bond formation under physiological conditions.
Figure 1 A concept of chemical biology-oriented click peptide.

To understand the biological function.

itself contains various oligomeric forms (6), and A142 undergoes time- and concentration-dependent aggregation in an aqueous TFA(triuoroacetic acid)acetonitrile solution used in high performance liquid chromatography (HPLC) purication (7). Moreover, the A142 monomer forms aggregates easily even in a standard storage solution such as dimethylsulfoxide (8). This uncontrolled self-assembly in an in vitro experiment causes considerable discrepancies in the biologic data. As a result of its highly aggregative nature, difculties in handling A142 have hampered the progress of A142-related AD research such that constructs have been used instead of the native peptide. Recently, it has been ascertained that the pathologic self-assembly nature of inherent peptides or proteins is one major event that leads to the development of many diseases such as prion protein in prion disease, -synuclein in Parkinsons disease, and islet amyloid polypeptide in type 2 diabetes, as well as A142 in AD (9).

O-Acyl isopeptide method

In 2003, we discovered that the presence of an O-acyl instead of N-acyl residue within the peptide backbone changed the secondary structure of the native peptide signicantly (10). The coupling and deprotection efcacies improved during solid-phase peptide synthesis of peptide derivatives that possess difcult sequences, most likely because the O-acyl isopeptide prevented the formation of secondary structures that disfavor coupling. Generally, the difcult sequences are hydrophobic and are prone to aggregate in solvent during synthesis and purication. This aggregation is attributed to inter-/intra-molecular hydrophobic interactions and the hydrogen bond network among resin-bound peptide chains, which results in the formation of extended secondary structures such as -sheets (11). In addition, the target peptide was generated subsequently by an ON intramolecular acyl migration reaction. This nding marked the beginning of the development of the O-acyl isopeptide method for peptide synthesis. The method has begun to be used by several other groups, especially Mimna et al. (12), Coin et al. (13) and B orner et al. (14), which indicates that the O-acyl isopeptide method is widely advantageous for peptide preparation.

Chemistry
We designed and synthesized the click peptide based on the O-acyl isopeptide (Fig. 2) (13). The click peptide has an O-acyl isopeptide structure of the corresponding native peptide. Hydrogen bond interactions between peptide chains often play a crucial role in the onset of physicochemical and biologic actions through their contributions to the conformational stability of the higher order structure. In the click peptide, a newly formed ester bond results in the inhibition of the ordered hydrogen bond interactions, which lead to conformational changes, and mask the activity of the native peptide. Additionally, the target peptide was generated via an ON intramolecular acyl migration reaction. The ON intramolecular acyl migration is a very attractive chemical reaction for the click peptide. The ON intramolecular acyl migration is capable of rapid amide bond formation under physiologic conditions (pH 7.4) because of the energetically favorable intramolecular ve-membered ring intermediate. It is an atom-economical reaction; thus, no byproduct is released during conversion to the parent peptide, which is a great advantage in toxicology in biologic experimental systems. 2

Synthesis of O-acyl isopeptide

The synthetic procedure of O-acyl isopeptides has been well established (10). O-Acyl isopeptides can be prepared easily using Fmoc-based solid-phase peptide synthesis. After Boc-Ser/Thr-OH is coupled to a peptide-resin to obtain Boc-Ser/ Thr-peptide-resin, Fmoc-Xaa-OH is esteried to the -hydroxy group of Ser/Thr residue using a DIC (1,3-diisopropylcarbodiimide)-DMAP (4-dimethylaminopyridine) method. Although esterication on the resin might induce epimerization at the esteried amino acid residue, the use of an O-acyl isodipeptide unit, which can be synthesized easily in solution, could avoid this problem (15). The protected O-acyl isopeptide-resin is obtained through coupling of additional amino acid residues using conventional techniques. During Fmoc removal of the second amino acid residue at the N-terminal side next to the ester bond, caution should be used to prevent the formation of diketopiperazines. Finally, the desired O-acyl isopeptide is obtained by

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Click Peptides, Design and Applications of

e.g.) pH- / photo-sensitive protection protective group H N R2 H N O peptide B

R O H 1 O O -acyl isopeptide structure

peptide A

click peptide based on the O -acyl isopeptide

click e.g.) pH-change photo-irradiation protective group

to A142 via ON intramolecular acyl migration in phosphate buffered saline (pH 7.4) at 37 C with a half-life of 1 minute (Fig. 3), whereas the TFA salt of the isopeptide was stable at 4 C in both solid state and dimethylsulfoxide solution. The A142 isopeptide proved to be easier to synthesize and purify than A142; moreover, it provided an opportunity to prepare A142 in situ rapidly under physiologic conditions. The spontaneous self-assembly of A142 could thus be more studied effectively to elucidate the inherent pathologic functions of the aggregative A142 in AD. Moreover, slower migration (t1/2 = 3 hours) was observed at pH 4.9, and no migration at pH 3.5 after incubating for 3 hours. These results suggest that this pH-dependent ON intramolecular acyl migration enables pH-triggered click for controlled in situ production of an intact A142 from the click peptide.

Photo-triggered click peptide of A142

Furthermore, we have synthesized a photo-triggered click peptide of A142, in which a photocleavable 6-nitroveratryloxycarbonyl group (16) was introduced at the -amino group of Ser26 in the A142 isopeptide (Fig. 4) (2, 3). The photo-triggered system had been employed to provide well-dened, time-controllable, and position-selective activation by light irradiation when studying biologic systems, because light is the fastest medium and can be focused to a restricted area. In size-exclusion chromatography, oligomers of A142 increased in quantity with incubation time (pH 7.4, 37 C) at the expense of the monomer. On the other hand, the photo-triggered click peptide remained in the monomeric form after 24 hours of incubation. Similarly, thioavin-T uorescence intensity (17), which corresponds to the extent of bril formation, increased with incubation time in the case of A142, yet unchanged in the case of the photo-triggered click peptide after 24 hours of incubation. Our results indicated that the click peptide was nonaggregative. The isopeptide structure resulted in complete inhibition of the aggregative nature of A142. Photo-irradiation of the click peptide and subsequent ON intramolecular acyl migration afforded intact A142 rapidly in situ . In the absence of light, the click peptide was stable during storage. This method may provide a useful system to investigate the biologic dynamics of A142 in AD with high spatial and temporal resolution by photochemically inducible activation of peptide self-assembly.

O H2N H N R O H 1 O R2 peptide B

peptide A

ON intramolecular acyl migration

pH 7.4

native amide bond R2 peptide A N H H N O HO O peptide B R1 H

native peptide
Figure 2 A click peptide based on the O-acyl isopeptide method.

TFA treatment followed by HPLC purication. Because O-acyl isopeptides generally exhibit high solubility in various media and are eluted as a sharp single peak in HPLC purication, they are puried easily to give pure isopeptides (1, 3, 10).

Chemical Tools and Techniques

An experimental system that can prepare a bioactive peptide easily in situ is advantageous to investigate biologic functions. The click peptide would be a useful tool to establish such an experimental system. As a tool with a similar concept, a caged compound is a synthetic molecule whose biologic activity is masked by a covalently attached photocleavable protecting group, and affords the bioactive molecule by photo-irradiation. Generally, such a photo-triggered system is considered to be advantageous for studying the dynamic processes of peptides and proteins, because upon photoactivation, only a short duration of time is required to control the spatiotemporal dynamics 3

pH-Triggered click peptide of A142

An application of the method to Alzheimers A142 revealed that the O-acyl isopeptide of A142 (in which the Gly25 -Ser26 sequence of A142 was isomerized to a -ester bond) could be synthesized effectively and stored without spontaneous self-assembly (13). Intact monomer A142 could then be obtained from the isopeptide under physiologic experimental conditions. The water solubility of the TFA salt of the isopeptide was 100-fold higher than that of A142 (0.14 mg mL1 ). Puried isopeptides could be converted quantitatively

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Click Peptides, Design and Applications of

1 H

10

20

Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys Leu Val Phe Phe Ala Glu Asp Val

NH

(Gly25) O O (Ser26)

40 HO
Ala lle Val Val Gly Gly Val Met Leu Gly lle lle

TFA-H2N 30
Ala Gly Lys Asn

pH-triggered click peptide

pH-triggered click pH 7.4

NH

(Gly25) O

O (Ser26)
pH 7.4

t1/2 = 1 min 1 H 10 20
Glu Asp Val Gly

H2N

Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys Leu Val Phe Phe Ala

25 26

40 HO
Ala lle Val Val Gly Gly Val Met Leu Gly lle lle

30 A1-42

Ser

Ala Gly Lys Asn

Figure 3 The production of A 142 from the pH-triggered click peptide via a pH-dependent ON intramolecular acyl migration reaction.

of the native compounds (18, 19). A fundamental drawback of the caged strategy on large peptides and proteins is that a small photocleavable group does not always mask biologic activity, because high potency may be attributed to large sections of the peptide structure (19, 20). This drawback can be overcome by the click peptide strategy because the native properties are masked in the isopeptide and released by O-acyl to N-acyl migration. This advantage should open new doors for the development of novel and useful photo-triggered tools to probe systems in chemical biology and medical science. Because difculties in handling A142 in syntheses and biologic experiments would hamper the progress of A142-related AD research, we expect that the click peptide method will help to clarify the currently unexplained processes of AD. Moreover, many amyloidogenic diseases, as well as A142 in AD, have recently attracted much attention and are being studied to discover novel drug targets (9). Thus, we hope that the click peptide strategy would be applied widely to these amyloid-related peptides or proteins. Click peptide can serve as a tool to study peptide folding and aggregation in chemical biology-oriented research, because acyl migration can be induced chemically or photochemically to release the native peptide. 4

References
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2.

3.

4. 5.

6.

7.

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Click Peptides, Design and Applications of

Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys Leu Val Phe Phe Ala Glu

Asp

Val

spontaneous rearrangement

Leu Gly lle Ala lle Val Val Gly Gly Val Met

lle Ala

Asn Gly Lys

intact A142

Understanding of inherent pathological function of A142

Figure 4 A photo-triggered click peptide. The production of A 142 by photo-triggered click followed by ON intramolecular acyl migration reaction.

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14. 15.

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Adams SR, Tsien RY. Controlling cell chemistry with caged compounds. Annu. Rev. Physiol. 1993;55:755784. Shimizu M, Yumoto N, Tatsu Y. Preparation of caged compounds using an antibody against the photocleavable protecting group. Anal. Biochem. 2006;348:318320. Bosques CJ, Imperiali B. Photolytic control of peptide self-assembly. J. Am. Chem. Soc. 2003;125:75307531.

See Also
Peptides, Chemistry of; Peptide Synthesis; Proteins, Chemical Modications of; Protein Misfolding; Synthetic Proteins to Elucidate Biologic Function

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