Bicyclic Peptide Based Lectinomimic

Peptide based lectin mimetics represent an attractive approach for the development of artificial carbohydrate receptors that might find application in bio-analytical and medicinal fields. Taking into consideration the structure of typical lectin binding site, we have designed a novel artificial receptor molecule possessing a rigid three-dimensional structure, hydrogen-bonding site and lipophilic binding pocket to promote hydrophobic interaction and hydrogen-bonding. A new solid-phase synthetic approach that allows complete synthesis of desired bicyclic peptide 1 on the solid support was developed. CD spectra of peptides 1 and 2 indicate that the structure of 1 is rather rigid and preorganised for the three-dimensional monosaccharide substrates binding. The binding affinities of bicyclic peptide receptor 1 toward various carbohydrate substrates at physiologically relevant conditions were estimated by UV/vis and fluorimetric titration experiments, and the observed values are in the millimolar range. With these results we have demonstrated that the bicyclic peptide 1 represent a promising basis for the design of new and more efficient carbohydrate receptors that may have broader application in bio-analytical or medicinal field.


INTRODUCTION
ARBOHYDRATE recognition is one of the most sophisticated recognition processes in biological systems. [1]This process mediates many important aspects of cell-cell recognition, such as inflammation, cell differentiation, tumor cell colonization and metastasis.However, due to its complexity and weak binding affinities, carbohydrate recognition by natural systems is still poorly understood.On the other hand, studies on synthetic carbohydrate receptors could make significant contributions to a better understanding of this process and lead to the development of new agents for application in bio-analytical and medicinal fields.Considering the importance of the carbohydrate recognition, it is not surprising that design and synthesis of artificial receptors for these important biological substrates attracted a great deal of attention in biomimetic chemistry. [2]2a,3] This crude simplification has been based on the structural studies of the lectins, revealing that carbohydrate binding sites are typically shallow binding pockets where only binding of the terminal monosaccharide moieties can occur. [4]Binding of an individual lectin to monosaccharide substrate is extremely weak; the association constant (Ka) for these complexes are typically in the 10 -4 -10 -2 mol -1 dm 3 range.2a,3a] However, construction of artificial receptors for binding of carbohydrates in water represent particularly challenging task, mainly due to the weak hydrogen-bonding interactions.
Carbohydrate binding protein mimicking molecules using the peptide-based systems (lectinomimics) represent particularly attractive approach for the development of artificial carbohydrate receptors. [5]Given the number available L-and D-amino acid building blocks, even small peptides offer enormous diversity and potential for design and development of new, and more selective artificial carbohydrate receptors.4b,4c] Considering the structure of typical lectin binding site, we have designed a novel artificial receptor molecule possessing a rigid three-dimensional structure, hydrogen-bonding site and lipophilic binding pocket to promote hydrophobic interaction and hydrogen-bonding.As a model system we chose a cyclic cationic peptide antibiotic polymyxin B (PMB), [6] which is known to bind the lipid A disaccharide moiety of lipopolysaccharide (LPS) with the high affinity. [7]erein, we report the design, synthesis and binding properties of polymyxin based bicyclic peptide 1, Scheme 1, toward the various mono-and disaccharide substrates in aqueous media.

MATERIALS AND METHODS
Wang resin was purchased from Chem-Impex (Wood Dale, IL, USA) and Rink amide AM resin was purchased from Millipore-Sigma (Burlington, MA, USA).Fmoc-protected amino acids and coupling reagents (HOBt, HBTU, PyBOP) were purchased from Chem-Impex (Wood Dale, IL, USA).N-methylmorpholine, hydrazine, triethylamine and Pd(PPh3)4 was purchased from Sigma Aldrich (St. Louis, MO, USA).All solvents were purchased from Fisher-Scientific (Waltham, MA USA), and were analytical reagent grade or better.Individual peptides were synthesized on a PS3 automated peptide synthesizer (Gyros Protein Technologies, Tucson, AZ, USA).Mass spectrometry was performed on MALDI-TOF Voyager-DE STR (Applied Biosystems, Foster City, CA, USA) in reflector mode using α-cyano-4-hydroxycinnamic acid as a matrix and positive mode.Analytical RP-HPLC analyses and peptide purifications were performed on 1260 Infinity (Agilent Technologies, Santa Clara, CA, USA) liquid chromatography systems equipped with a UV/Vis detector.CD spectra were recorded on a Jasco-810 spectropolarimeter (Jasco, Easton, MD).Titration experiments were performed on Varian Cary 3 UV/vis spectrophotometer (Paolo Alto CA, USA) and Perkin Elmer LS50B Luminescence Spectrometer (Waltham, MA, USA).

Circular Dichroism Spectroscopy
CD spectra were recorded in water using a quartz cell of 1 mm optical path length.Spectra were measured over a wavelength range 180 -250 nm with an instrument scanning speed of 100 nm/min and a response time 1 s.The peptide concentrations were 1 mmol dm -3 for 1 and 1.2 mmol dm -3 for 2, and the CD spectra are the result of eight averaged scans taken at 25 °C.All CD spectra are baselinecorrected for signal contribution due to the water.CD spectra were modeled using the CDPro suite of programs employing SDP42 database.

Bicyclic Peptide Design and Solid-phase Synthesis
The conformationally constrained cage-like molecules, such as bicyclic peptide receptor 1, represent an interesting topology for designing artificial receptors capable of binding biologically important monosaccharides under physiological conditions.In this type of monosaccharide receptors, the three-dimensional lipophilic cavity is already preorganised for the three-dimensional monosaccharide substrates binding, meaning that there are less entropically unfavorable conformations formed in order to adopt the optimal complex geometry.In addition, incorporation of functionalities for hydrogen bonding interactions within receptor's three-dimensional structure will permit monosaccharide substrates not only to be complexed but to be encapsulated, allowing therefore monosaccharide binding in water.
Our synthetic receptor is modeled on cyclic decapeptide antibiotic polymyxin B. [6] Polymyxin B consists of a seven member ring containing four positively charged diaminobutyric acid (Dab) residues, one Thr residue, hydrophobic D-Phe and Leu residues, and the N-terminal lipophylic tail region composed of Dab-Thr-Dab-6-methyloctanoic acid segment.11c] Taking into consideration the structure of typical lectin binding site and above mentioned literature data, we based our peptide lectinomimic 1 on PMBN.In addition, since PMBN Orn analogue retains its binding affinity toward lipid A, and considering a greater selection of commercially available orthogonally protected Orn derivatives, we decided to incorporate Orn residues instead of Dab into the receptor's peptide sequence, Scheme 1.
Solid-phase synthesis of PMB [12] and its truncated analogue PMBN [11b,11c] has been reported previously.Described synthetic paths include solid-phase assembly of linear precursors using standard Fmoc-chemistry, followed by peptide cyclization in solution, or synthesis using a safety-catch sulfonamide linker and peptide cyclization with associated cleavage from the solid support.However, the same synthetic strategies cannot be applied for the synthesis of peptide receptor 1 due to its structural complexity (i.e.required bicyclic structure formed by incorporation of the bipyridine dicarboxylate moiety).Therefore, we have developed a new solid-phase synthetic approach that allows complete synthesis of desired bicyclic peptide 1 on the solid support.In general, our synthetic approach toward bicyclic peptide 1 was comprised of the resin attachment of the Asp residue via side chain, successful use of combination of six quasi-orthogonal removable protecting groups, stepwise Fmoc solid-phase synthesis of a linear precursor, on-resin head-to-tail cyclization, and in the final step on-resin attachment of the bipyridine dicarboxylate moiety.Anchoring of the first residue to the resin is one of the most critical steps in peptide synthesis.Choice of a wrong linker, loading method, or amino acid can have highly deleterious effects on the purity of synthesized peptides.In order to optimize this first step, two different types of resins were used: the hydroxymethyl-based Wang resins (substitution 0.95 mmol/g) yielding cyclic peptide with the C-terminal carboxylate moiety, and the aminomethyl-based Rink resins (substitution 0.43 mmol/g) yielding corresponding analogue with the C-terminal amide moiety.The level of the Asp residue attachment to the resins was determined by the spectroscopic (UV/vis) method as previously described in the literature. [8]The best results were obtained with the Rink amide resins (yields 75 % -80 %).The Fmoc chemistry was used throughout the linear peptidyl-resin precursor synthesis.After removal of Cterminal Allyl protecting group and N-terminal Fmoc protecting groups using standard deprotection methods, [9] linear peptide precursor was successfully cyclized on resin using PyBop/HOBt/NMM as we previously described. [13]ncorporation of bipyridine unit into peptide skeleton represented additional challenging synthetic task.For this purpose, Orn residues with ivDde protected side chain's amino groups were used.ivDde side chain protected Orn was chosen because ivDde can be removed by hydrazine reagent without affecting remaining protecting groups present on the peptide.In this way, we have generated two primary amino groups available for incorporation of the bipyridine dicarboxylate bridge.It was expected that coupling of bipyridine dicarboxylate under pseudo-dilution conditions [14] will result in the desired bicyclic peptide 1.However, after deprotection of Orn side chain amino groups with 2 % hydrazine in DMF and subsequential coupling of 1 eq of 2,2'-bipyridine-5,5'-dicarboxylic acid only undesired cyclic peptide 3 bearing two bipyridine dicarboxylate units was obtained (MALDI-TOF MS analysis: m/z = 1270 calculated; m/z = 1292 [M+Na] + observed), Scheme 1.This problem was solved by incorporating into the peptide precursor Orn residues with orthogonally protected side chain's amino groups.For this purpose, we chose Fmoc-Orn(Mtt)-OH and Fmoc-Orn(ivDde)-OH to incorporate in positions 2 and 5, respectively, of the cyclic peptidyl-resin precursor sequence.Selective removal of ivDde protecting group with 2 % hydrazine in DMF allowed incorporation of only one 2,2'bipyridine-5,5'-dicarboxylic acid moiety.In the next step, Mtt protecting group was removed with 1 % TFA in CH2Cl2, followed by on-resin macrocyclization using PyBop/NMM.Progress and completion of the coupling reaction and macrocyclization was monitored by the ninhydrin colorimetric test. [15]Cleavage of the resulting bicyclic peptide product from the resins was carried out in 95 % of trifluoroacetic acid (TFA)/CH2Cl2 mixture in the presence of triisopropylsilane (TIS) as a scavenger.This time desired peptide 1 was synthesized in satisfactory yield (85 %, MALDI-TOF MS analysis: m/z = 1026.15calculated; m/z = 1026.45observed.RP-HPLC analysis: Rt = 23.2min., linear gradient 100 % A → 100 % B in 30 min.at flow rate of 1 mL/min.As shown in Scheme 1, control cyclic peptide 2 was synthesized using the same solid-phase synthetic strategy (yield 90 %, MALDI-TOF MS analysis: m/z = 817,98, calculated; m/z = 818.41[M+H] + , 840.44 [M+Na] + observed.

Conformational Characterization by CD Spectroscopy
Since the receptor preorganization plays a key role in the complexation process, we investigated an effect of bipyridine dicarboxylate bridge on receptor's conformation by CD spectroscopy.The CD spectra were recorded in cacodylate buffer (pH = 6.5, 50 mmol dm -3 ).In both cases, the spectra minimums were obtained at λ = 196 nm characteristic of unordered peptide conformations, indicating absence of peptide conformational changes upon incorporation of the bipyridine bridge.In comparison to the control peptide 2, the observed more pronounced negative band at λ = 223 and positive band at λ = 212 nm for bicyclic peptide 1 can be interpreted as a contribution of aromatic bipyridine bridge to the CD spectrum rather than the consequence of conformational change. [16]11b,11c] However, previously reported 1 H NMR studies [17] suggested that conformation of free PMBN in aqueous solution is characterized by a type II' β-turn centered on the Dab 5 -D-Phe 6 -Leu 7 -Dab 8 sequence and an inverse γ-turn at Thr 10 .Based on our results and results of others, we can assume that receptor 1 has a rigid solution conformation and expect no significant conformational changes upon monosaccharide substrate binding, minimizing therefore the entropic penalty of binding.

Binding Studies
Our preliminary binding studies indicate that the bicyclic peptide lectinomimic 1 is capable of binding monosaccharides with millimolar affinities in aqueous media.The binding affinities of bicyclic peptide 1 toward various monosaccharide substrates in cacodylate buffer (pH = 6.5, Table 1.Association constants for 1 : 1 complexes between various carbohydrate substrates and 1 in aqueous media (pH = 6.5)  1.The association constants (log Ka) were calculated using the SPECFIT global analysis software. [8]In all cases, a good agreement between the two methods was obtained.The stoichiometry for complexes of 1 with monosaccharides was determined by the mole ratio method (Job plot) [10] to be 1:1, Figure 2. As shown in Table 1, the selectivity of the peptide based receptor 1 with respect to a specific monosaccharide is, at best, modest.On another hand, affinity toward larger substrate such as gentiobiose, a disaccharide composed of two D-glucose units, are much lower and thus not determinable by or UV/vis titration experiments under the same experimental conditions.The observed 1 : 1 stoichiometry for complexes of 1 with monosaccharide substrates and weak interaction with larger disaccharide substrate indicate that complexation occurs by insertion of the substrate molecule into receptor's lipophilic pocket, mimicking therefore similar process in lectins.The fact that the association constants for negatively charged mono-saccharides are identical, within an experimental error, to those found for the neutral substrates suggests a significant contribution of hydrophobic interactions to the overall complex stability.Importantly, estimated association constants are similar to those reported in the literature for lectin/monosaccharide complexes, proving further our designing principle for the bio-mimetic monosaccharide receptors containing the three-dimensional lipophilic cavity.

CONCLUSION
Despite the lack of bicyclic peptide 1 selectivity toward various monosaccharide substrates, our binding studies successfully demonstrated that it is possible to construct peptide-based artificial receptor molecules capable of binding biologically important carbohydrates at physiologically relevant conditions.The design of this biomimetic receptor is based on the premises that the receptor molecule should be soluble in water, should possess a three-dimensional intermolecular binding site into which the three-dimensional monosaccharide substrates may be inserted, and that the conformation of the receptor molecule should be restricted to reduce the entropic penalty of binding.
With these results, we have also demonstrated that the bicyclic peptide 1 represents a promising scaffold for the design of new and more efficient carbohydrate receptors that may have broader application in bioanalytical field.Further optimization of bicyclic peptide receptor 1 using combinatorial chemistry approach is currently underway in order to improve its binding properties and selectivity.

Figure 2 .
Figure 2. The Job plot indicating 1:1 stoichiometry for the complex between 1 and galactose.