Synthesis of trifunctional cyclo-β-tripeptide templates

The concept of template-assembled synthetic proteins (TASP) describes a central scaffold that predefines the three dimensional structure for diverse molecules linked to this platform. Cyclic β-tripeptides are interesting candidates for use as templates due to their conformationally defined structure, stability to enzymatic degradation, and ability to form intermolecular stacked tubular structures. To validate the applicability of cyclic β-tripeptides within the TASP concept, an efficient synthesis of the cyclopeptide with orthogonal functionalization of the side chains is desired. A solid-phase-supported route with on-resin cyclization is described, employing the aryl hydrazide linker cleavable by oxidation. An orthogonal protection-group strategy allows functionalization of the central cyclic β-tripeptide with up to three different peptide fragments or fluorescent labels.


Introduction
Cyclic β-tripeptides form structurally well-defined secondary structures with the potential for alignment of the rings to form intermolecular aggregates [1]. Due to the unidirectional alignment of the carbonyl groups and the flattened-ring conformation, cyclic β-tripeptides form assemblies of stacked rings through hydrogen bonding [2]. Furthermore, they are exceedingly stable against proteolytic cleavage and enzymatic degrad-ation [3,4]. These properties make cyclic β-tripeptides interesting candidates for the concept of template-assembled synthetic proteins (TASP) [5,6]. The TASP concept describes a central scaffolding molecule directing all further attached molecules into a spatially predefined structure. Cyclic β-tripeptides are suitable for use as a central scaffold that carries different kinds of molecules and functionalities to direct them into a trigonal planar assembly. This idea was already used for the synthesis of a C 3 -symmetric ligand for the immune response receptor CD40 [7]. However, the synthetic route to these kinds of molecules is demanding due to solution-phase synthesis of the β-tripeptides and their final cyclization with low efficiency [8,9]. Moreover, only homofunctionalized cyclic β-tripeptides have been described so far [9,10]. To further investigate and exploit the potential of this class of circular peptides, a synthetic route should fulfil the requirements of (i) fast synthetic access and (ii) the possibility to attach different molecules on each side chain of the central scaffold. Here we report a new effective synthesis for cyclic β-tripeptides on a solid support, employing the oxidation-labile aryl hydrazide linker [11]. We also describe an orthogonal protection-group strategy to synthesize a trifunctional cyclic β-tripeptide that has the potential of forming intermolecular hydrogen-bonded stacks ( Figure 1).

General strategy
To equip the cyclic β-tripeptide with three different functional units, each amino acid residue needs to be specifically addressed. Furthermore, all three amino acids should have side chains long enough to avoid steric hindrance between the moieties and the core. Thus, homo-β-lysine was chosen as the underlying amino acid to build up the scaffold allowing sidechain functionalization by amide bond formation. To protect the lysine side chain for selective and orthogonal amide-bond formation following the solid-phase peptide synthesis (SPPS), the protection groups fluorenylmethoxycarbonyl (Fmoc) and carbobenzyloxy (Cbz) were applied. Alteration of the amine in the third β-homolysine side chain to an azide [12][13][14] employing the Wong azidation [15] enables Huisgen [3 + 2]-cycloaddition [16] as an orthogonal coupling method. In order to build up the peptide sequence in the presence of Fmoc, Cbz and the azide, an acid-labile protecting group was required for temporary protection of the primary α-amino group. Therefore, tert-butyloxycarbonyl (Boc) protection was used to mask all α-amino groups during solid-phase synthesis of the tripeptide.
In previous studies, synthesis of the cyclic β-tripeptide scaffold was provided by cyclization of the linear β-tripeptide obtained Scheme 1: Synthesis of cyclic peptides employing the oxidation-labile aryl hydrazide linker [11,24].
by solution-phase chemistry [8,9]. Purification by chromatography is required after each coupling step, and the final cyclization reaction often results in poor yields. Herein, an alternative approach is described based on SPPS followed by on-resin cyclization. Recently, Waldmann et al. discovered an on-resin head-to-tail cyclization based on a Boc protocol using the oxidation-labile aryl hydrazide linker [24] and cleavage from the resin under simultaneous cyclization [11]. This method was adapted for the synthesis of the cyclic β-tripeptide 4 ( Figure 2).
The synthesis of the cyclic β-tripeptide was performed according to Scheme 1 by using the commercially available 4-Fmoc-hydrazinobenzoyl AM NovaGel resin from Merck Biosciences. After coupling of the three β-amino acids by using the standard Boc-protocol, the hydrazide linker was oxidized to generate nitrogen as a good leaving group. Nucleophilic attack of the N-terminal amino group at the activated carbonyl group provided cleavage of the cyclized β-tripeptide from the resin. Purification did not require any chromatography since organization by intermolecular hydrogen bonding resulted in decreased solubility and precipitation of the β-tripeptide template 4 from methanol.

Functionalization of the β-tripeptide template
The cyclic β-tripeptide template 4 has the potential for orthogonal functionalization at the side chains with up to three different moieties, by successive amide-bond formation and by employing the Huisgen [3 + 2]-cycloaddition. Aggregation by stacking of the functionalized peptide rings will further provide a higher density of organized recognition motifs and labels. As a proof-of-concept, the successive coupling of a fluorophore 5(6)-tetramethylcarboxyrhodamine 5 (TAMRA-COOH) and a cell-penetrating peptide to the template 4 is reported, as well as the functionalization with the nucleobase recognition units thymine-1-yl acetic acid (6) and (N 4 -benzyloxycarbonyl)cytosine-1-yl acetic acid (7) (Figure 3). In all cases, the template contains a third option for functionalization by covalent attachment of molecules through [3+2]-cycloaddition under mild conditions.
The template approach allows for the preparation of constructs that, e.g., combine biological activity, fluorescence labelling and cell-penetrating or cell-directing units in one molecule to be used for in vivo studies. The defined spatial organization of recognition units such as nucleobases on the template provides  a specific hydrogen-bonding network orthogonal to the peptide ring aggregation, which will be of value for aggregate formation and molecular architectures defined by a three-dimensional network of hydrogen bonds.
Selective release of the first amino group was achieved by removal of the Cbz protecting group using TFA and m-cresol (95:5 v/v). The first functional unit of a recognition moiety was attached to the template as a carboxylic acid by amide-bond formation using PyBOP and DIEA, or PyBrOP and DIEA for coupling (Scheme 2). Exemplarily, TAMRA-COOH (5) and the nucleobase moieties thymine-1-yl acetic acid (6) and (N 4benzyloxycarbonyl)cytosine-1-yl acetic acid (7) were attached to the cyclo-β-peptide yielding monofunctionalized templates 14, 10 and 12 ( Figure 4).
The second amino group was Fmoc-deprotected with 20% piperidine in DMF. Coupling of the fully protected cell-pene-trating peptide penetratin 8 [26] with the homolysine side chain of the cyclic β-tripeptide 15 was accomplished by HOBt and HBTU activation in solution using a 10-fold access of penetratin. The β-peptide template 16 functionalized at two side chains was obtained, purified by HPLC, and characterized by mass spectrometry.

Conclusion
Cyclic β-tripeptides provide an interesting platform for the concurrent arrangement of side-chain bound functionalities in combination with the ability of intermolecular tubular aggregation of the rings. An easy and fast approach towards cyclic β-tripeptides was presented. The tripeptide scaffold was assembled completely on a solid support, thereby saving purification steps following amino acid coupling. Moreover, cleaving of the peptide from the resin and cyclization was performed in a single step employing the oxidation-labile aryl hydrazide linker [11]. Together with a convenient and efficient purification procedure, this method is beneficial with respect to yield and speed of the synthesis. Further, an orthogonal protection strategy was introduced to equip the β-peptide template with three different functional molecules allowing testing of the cyclic β-tripeptides as a scaffold for the defined orientation of functional units and the combination of up to three modules in one molecule. The advantages of using the β-peptide template within the TASP concept are the spatially defined and rigid structure, with a high stability against enzymatic degradation, and their ability to form intermolecular staples by backbone hydrogen bonding. This might be of special advantage to target or imitate multivalent and/or cooperative processes.
As an initial effort, we equipped the cyclic β-tripeptide with a fluorophore and a protected cell-penetrating peptide, which can be further functionalized with any biologically active molecule bearing an alkyne. Potentially, this generates a fluorescent and cell-permeable drug applicable for in vivo experiments and other biochemical assays. In addition, different nucleobases were linked to the central core in order to generate a template that can form a three dimensional network of hydrogen bonds.

Experimental General remarks
All technical solvents were distilled prior to use. The solvents of analytical and HPLC grade were used as supplied. The solvents used for the synthesis were obtained in quality puriss. abs. from Acros Organics, Sigma Aldrich, Merck, or VWR. All chemicals were of the highest grade available and used as supplied. All moisture-and oxygen-sensitive reactions were carried out under an inert gas atmosphere (nitrogen or argon). Analytical TLC was performed on Merck TLC aluminium sheets silica gel 60 F 254 . Detection was performed under UV light (254 nm) or by dipping into a solution of ninhydrin (3% in ethanol) followed by heating with a heat gun. Eluents and the appropriate R f values are indicated. The columns for flash chromatography were packed with silica gel 60 from Macherey-Nagel with a grit size of 0.063 to 0.2 mm and were run under a pressure of 1 to 1.5 bar. The substance was applied as a concentrated solution or adsorbed on silica gel. Eluents are indicated. Reverse-phase HPLC was performed on an Äkta Basic 900 from Pharmacia Biotech. UV detection was performed at 215, 254 and 280 nm wavelength. The solvents used for HPLC were of HPLC grade and degassed while being stirred in vacuo. Demineralized Water for HPLC use was preprocessed by the water treatment plant "Simplicity" from Millipore. All HPLC runs were performed by using linear gradients between A (0.1% aq TFA), B (0.1% TFA in methanol) or C (0.1% TFA in MeCN) and water (0.1% TFA) within 30 minutes. Flow rates were taken as 1 mL/min for the analyt-ical columns and 10 mL/min for preparative columns. All crude samples were dissolved in methanol or acetonitrile and filtered prior to use. Electrospray ionization (ESI) mass spectra were obtained on a Finnigan LCQ instrument. High-resolution mass spectra (HRMS) were obtained on a Bruker Apex IV FT-ICR-MS instrument. 1 H NMR and 13 C NMR spectra of samples dissolved in DMSO-d 6 , CDCl 3 or acetone-d 6 were recorded on a Varian Unity 300 (300 MHz) spectrometer. Residual solvent proton signals were used as an internal standard.