**6. Supramolecular structures formed by self-organization of** *N-***lipidated peptides anchored to cellulose**

Cellulose is a polysaccharide with two different types of hydroxyl groups i.e. primary and secondary. The primary hydroxyl groups are significantly more reactive then the secondary. Since the chains of polyanhydroglucose interacts with each other in the precisely defined way these functional groups are positioned within the reasonably regular fashion on he surface of cellulose. In the crystalline region of cellulose [102] the every second primary hydroxyl groups are exposed and accessible for interaction with reagents making after the transformation relatively regular pattern of anchored molecules separated by the distance of one anhydroglucose residue. Due to this advantageous feature of the cellulose the space available in between molecules anchored on the cellulose surface is sufficient for docking another molecules. Based on this assumption Kaminski and co-workers proposed entirely new approach for designing artificial receptors. According to the proposed concept, appropriate structure of molecules anchored on cellulose creates precisely defined and functionalized space for trapping ligands as presented on Figure 4.

The relatively weak bonding forces and conformational flexibility of both partners make docking of ligands to receptors difficult to study, to categorize by any kind of empirical rules, or to predict based on molecular modeling. Even in the case of interactions between relatively simple molecules, the possible bonding and repulsive forces of mutual hostguest interactions are multifaceted, very numerous, and difficult in terms of molecular modeling [103]. For the more advanced models involving flexible ligands and complex flexible receptor structures the rational construction plan of the host structure still exceeds our capabilities [104]. Thus, design of the molecular trap was done intuitively by mimicking structural features occurring in natural receptors, synthesis of the library of them by methods of combinatorial chemistry and selection of the most efficient representatives.

Strong, yet reversible binding force for the most of potential guest molecules were achieved by introducing into binding pockets most of the structural attributes responsible for weak intermolecular interactions [105]. These include hydrogen-bond donors and acceptors, lipophilic and hydrophilic fragments supplemented with π-donors and πacceptors as depicted on Figure 5.

All these elements were allocated inside the linear structure forming the matrix of podands in such a way as to separate the flexible *N-*lipopeptide fragment from the solid support by relatively rigid, aromatic rings. Thus, a bonding "pocket" was composed from the tethered fragments of "walls" constructed from aromatic rings, expanded with a diversity of

interactions offered by flexible peptide fragments, and finally closed with a "zipper" of hydrophobic chains of lipidic fragments. Due to the conformational flexibility of interacting partners, the relative direction of the functional groups of a ligand as well as that of the binding pockets could be readjusted to the most energetically favored orientation of both counterparts [106]. Thus structures immobilized on cellulose support *via* triazine linker created the mosaic of binding holes, mimicking the behavior of receptor formed from the neighboring, identical lipidated peptides.

Cellulose Functionalysed with Grafted Oligopeptides 265

**Carbohydrate**

**Carbohydrate**

O O O HO OH O N N N O NH

NH

n

Ala-Glu-NH-CO-(CH2)16CH3

**acceptor**

**Peptide**

**donor**

**L i p i d**

**acceptor**

**Peptide**

**donor**

**L i p i d**

deprotection of *N-*terminus and completing the synthetic procedure by binding of carboxylic acid (Scheme 8). In the synthesis of the peptide fragment, DMT/NMM/BF4- was used as a coupling reagent. For the final acylation of immobilized tripeptides with carboxylic acid more lipophilic DMT/NMM/TosO- was found more suitable as a coupling

**Carbohydrate**

**Carbohydrate**

**acceptor**

**Peptide**

**donor**

**L i p i d**

**Carbohydrate**

n

O O O OH

> N N N O NH

> > NH Ser-Glu-NH2

n

**4 5**

12345

O HO

<sup>O</sup> <sup>O</sup> HO OH OH

NH2

*Rubus laciniatus Beta vulgaris convar. Crassa (E 162)* 

**Figure 6.** Influence of the structure of podands on binding of antocyane dyes from *Rubus laciniatus* and

**Figure 5.** The concept of binding pockets with most of the structural attributes responsible for weak

**acceptor**

**Peptide**

**donor**

**L i p i d**

**acceptor**

**Peptide**

**donor**

**L i p i d**

**Carbohydrate**

**Carbohydrate**

n

O O HO O OH

> N H

**3**

O

O O

<sup>O</sup> <sup>O</sup> HO OH OH

**acceptor**

**Peptide**

**donor**

**L i p i d**

**Carbohydrate**

N O O HO O OH

**2**

12345

O N <sup>O</sup> <sup>N</sup> Me Cl

n

O

**Cellulose**

<sup>O</sup> <sup>O</sup> <sup>O</sup> OH

*Beta vulgaris* extracts.

OH HO

HO O OH OH

**1**

intermolecular interactions.

**acceptor**

**Peptide**

**donor**

**L i p i d**

**acceptor**

**Peptide**

**donor**

**L i p i d**

**acceptor**

**Peptide**

**donor**

**L i p i d**

reagent [107].

**Figure 4.** "Molecular traps" formed by podands regularly positioned on the support.

In the absence of some elements (Figure 6. **1-4**) binding process was substantially deteriorated compared to the binding ability of complete receptor structure (Figure 6, **5**).

Thus, the fully serviceable monolayer immobilized on cellulose was prepared in the stepwise process involving functionalization of cellulose with 1,3,5-triazine derivative followed by reaction with *m*-fenylenediamine, attachment of *N-*Fmoc amino acids, deprotection of *N-*terminus and completing the synthetic procedure by binding of carboxylic acid (Scheme 8). In the synthesis of the peptide fragment, DMT/NMM/BF4- was used as a coupling reagent. For the final acylation of immobilized tripeptides with carboxylic acid more lipophilic DMT/NMM/TosO- was found more suitable as a coupling reagent [107].

264 Cellulose – Medical, Pharmaceutical and Electronic Applications

neighboring, identical lipidated peptides.

interactions offered by flexible peptide fragments, and finally closed with a "zipper" of hydrophobic chains of lipidic fragments. Due to the conformational flexibility of interacting partners, the relative direction of the functional groups of a ligand as well as that of the binding pockets could be readjusted to the most energetically favored orientation of both counterparts [106]. Thus structures immobilized on cellulose support *via* triazine linker created the mosaic of binding holes, mimicking the behavior of receptor formed from the

**Figure 4.** "Molecular traps" formed by podands regularly positioned on the support.

In the absence of some elements (Figure 6. **1-4**) binding process was substantially deteriorated compared to the binding ability of complete receptor structure (Figure 6, **5**).

Thus, the fully serviceable monolayer immobilized on cellulose was prepared in the stepwise process involving functionalization of cellulose with 1,3,5-triazine derivative followed by reaction with *m*-fenylenediamine, attachment of *N-*Fmoc amino acids,

**Figure 5.** The concept of binding pockets with most of the structural attributes responsible for weak intermolecular interactions.

**Figure 6.** Influence of the structure of podands on binding of antocyane dyes from *Rubus laciniatus* and *Beta vulgaris* extracts.

Cellulose Functionalysed with Grafted Oligopeptides 267

**Figure 7.** Dependence of water permeability through *N-*lipidated amino acids layer.

elaidic

oleic

0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18

cinnamic

10-undecenic

Rate of water permeability

[1/min]

inside the receptor pocket [109].

solution of S-(þ)-naproxen (right).

competitiveness of process (Scheme 10).

Process of binding triphenylmethane dyes with an array of *N-*lipidated dipeptides peptides immobilized on cellulose according to the manner described above was not dependent on initial concentration of ligand reaching equilibrium within 20-30 min. The selectivity and rate of binding depends on the structure of the peptide fragment as well as *N-*lipidic moiety. Even tiny structural changes in guest molecules were detected by monitoring the alteration of the binding pattern [108]. Measurements of fluorescence of fluorescein docked inside the receptor pocket revealed difference in λmax, curvature and intensity of fluorescence depended on the structure of the peptide motif and lipidic fragment of binding pocket. This strongly suggests an alternation of charge distribution

erucic

palmitic

ricinic

alanine

phenyloalanine serine arginine

Binding of colorless ligand was monitored by replacement of the reporter dye due to

**Figure 8.** The library of *N-*lipidated dipeptides immobilized on cellulose disks treated with 10 mM/L solution of bromochlorophenol blue (1) (left); the same disks after subsequent treatment with 20 mM/L

**Scheme 9.**Synthesis of *N-*lipidated peptides (amino acids) immobilized on cellulose *via* aromatic linker.

Loading of cellulose support was calculated on the basis of N and Cl content determined by elemental analysis 9-10 μmol/cm2 with the anticipated ratio of molecular fragment triazine/*m*-phenylenediamine/amino acid/carboxylic acid.

The studies of water permeability through the monolayer achieved with 28-element library of *N-*acylated aminoacid prepared from Ala, Phe, Ser and Arg, and cinnamic, 10-undecenic, elaidic, oleic, erucic, palmitic, and ricinic acids confirmed that penetration of water is possible only in the presence of hydrophlilic functional groups incorporated into the monolayer. In their absence the podands were allocated sufficiently dense on cellulose fibers to inhibit penetration of water. This means that lipidic "zip" separate the interior of binding pocket, but the lipidic barrier remains still penetrable at least to the small, highly polar molecules of water.

**Figure 7.** Dependence of water permeability through *N-*lipidated amino acids layer.

O

O

<sup>O</sup> <sup>O</sup> HO OH

<sup>O</sup> <sup>O</sup> HO OH

triazine/*m*-phenylenediamine/amino acid/carboxylic acid.

n

1)

1) NaOH 2) DCMT

OH

NH2

<sup>O</sup> <sup>O</sup> <sup>O</sup> OH

HO

N H R

**DMT/NMM/BF4**

2) Piperidine

**Scheme 9.**Synthesis of *N-*lipidated peptides (amino acids) immobilized on cellulose *via* aromatic linker.

Loading of cellulose support was calculated on the basis of N and Cl content determined by elemental analysis 9-10 μmol/cm2 with the anticipated ratio of molecular fragment

The studies of water permeability through the monolayer achieved with 28-element library of *N-*acylated aminoacid prepared from Ala, Phe, Ser and Arg, and cinnamic, 10-undecenic, elaidic, oleic, erucic, palmitic, and ricinic acids confirmed that penetration of water is possible only in the presence of hydrophlilic functional groups incorporated into the monolayer. In their absence the podands were allocated sufficiently dense on cellulose fibers to inhibit penetration of water. This means that lipidic "zip" separate the interior of binding pocket, but the lipidic barrier remains still penetrable at least to the small, highly polar

Fmoc

O OH

OH

HO

N N N <sup>O</sup> Cl Me

HO

O

<sup>O</sup> <sup>O</sup> HO OH

> N H

> > N H

O OH

O

O

<sup>O</sup> <sup>O</sup> HO OH

> O N H

N H

R'

n

O

OH

O OH

O

n

O

<sup>O</sup> <sup>O</sup> HO OH

n

O N H

R

**DMT/NMM/Tos**

OH

H2N

NH2

OH

O OH

<sup>O</sup> <sup>O</sup> <sup>O</sup> OH

> R' O

1)

<sup>O</sup> <sup>O</sup> <sup>O</sup> OH

HO

OH

OH

HO

N N N <sup>O</sup> Me NH2

OH

HO

2) TFA, TIS, H2O

N N N <sup>O</sup> Me <sup>R</sup>

O

OH

O OH

HO <sup>n</sup>

N H

O OH

O

OH

<sup>O</sup> <sup>O</sup> <sup>O</sup> OH

<sup>O</sup> <sup>O</sup> <sup>O</sup> OH

HO

OH

OH

HO

HO

N N N

<sup>O</sup> Me

molecules of water.

Process of binding triphenylmethane dyes with an array of *N-*lipidated dipeptides peptides immobilized on cellulose according to the manner described above was not dependent on initial concentration of ligand reaching equilibrium within 20-30 min. The selectivity and rate of binding depends on the structure of the peptide fragment as well as *N-*lipidic moiety. Even tiny structural changes in guest molecules were detected by monitoring the alteration of the binding pattern [108]. Measurements of fluorescence of fluorescein docked inside the receptor pocket revealed difference in λmax, curvature and intensity of fluorescence depended on the structure of the peptide motif and lipidic fragment of binding pocket. This strongly suggests an alternation of charge distribution inside the receptor pocket [109].

Binding of colorless ligand was monitored by replacement of the reporter dye due to competitiveness of process (Scheme 10).

**Figure 8.** The library of *N-*lipidated dipeptides immobilized on cellulose disks treated with 10 mM/L solution of bromochlorophenol blue (1) (left); the same disks after subsequent treatment with 20 mM/L solution of S-(þ)-naproxen (right).

Cellulose Functionalysed with Grafted Oligopeptides 269

n

An array of *N-*lipidated peptides immobilized on cellulose was also used in studies of tissue homogenates for early diagnosing thyroid gland cancer [112], which is the most common malignancy of the endocrine system. There were found different binding patterns of healthy

By incorporation into the peptide fragment of receptor amino acid residues characteristic of catalytic triade of the hydrolytic enzymes the binding pockets demonstrated catalytic activity [113]. These were able to catalyse hydrolysis of esters bond [114]. All members of the library of 36 structures formed by permutations of Ser, Glu, His acylated with 6 long chain carboxylic acids were active as esterase and effectively catalyzed hydrolysis of p-nitrophenyl ester of Z-L-Leu-L-Leu-OH at pH 7-7.5 and temp not exceeding 20oC. The postulated mechanism of

O

**P R O D U C T**

<sup>O</sup> <sup>O</sup> <sup>O</sup> <sup>O</sup>

<sup>O</sup> <sup>H</sup> <sup>O</sup> O

<sup>O</sup> <sup>O</sup> <sup>O</sup>

**Figure 9.** The postulated mechanism of catalytic activity of *N-*lipidated tripeptides of catalytic triade.

In this case the progress of hydrolysis was so fast that under the conditions of the experiment it was difficult to identify the most catalytically active structure. The extinction at 405 nm increased from the initial value of 0.300 for the substrate to more than 0.800 before the first cycle of measurements was completed. The rate of reaction diversified enough for identification of the most active catalytic structures was afforded by using significantly less

As the final effect of catalytic activity is the transformation of relatively non-polar organic molecule into ionic species, one can expect application of this phenomena for the construction of sensors [115]. There are also many other interesting area of application of catalytically active *N-*lipidated peptides immobilized on cellulose. Most expected are effects stereochemical results of such transformations, expecting open access to essentially

OMe

O H O <sup>H</sup> <sup>O</sup> O H O H

H N

R <sup>O</sup> <sup>H</sup>

N N N MeO O

O R 1 <sup>H</sup> <sup>N</sup>

H N O

N H

R 3

N H <sup>O</sup> R 2

> H O H

> > O H O

<sup>O</sup> <sup>O</sup> <sup>O</sup>

O O H

**S U B S T R A T**

<sup>H</sup> <sup>O</sup> O H

N N N

H

N

R

H N O

OMe

N H O

R 2

R 3 O

N H

H N

O

H O H

O H

H <sup>O</sup> <sup>H</sup>

and cancer tissue for the various types of cancer.

O

H N O

N H

N H

R 3

N N N MeO O

N H <sup>O</sup> R 2

<sup>H</sup> <sup>O</sup> O H

catalytic activity of *N-*lipidated oligopeptides is presented on Figure 9.

R 1 N H

R <sup>O</sup> <sup>H</sup>

O O H

reactive, sterically hindered substrate Z-Aib-Aib-ONp.

O H

<sup>O</sup> <sup>O</sup> <sup>H</sup> <sup>O</sup> O H

N <sup>N</sup> C H2 N N N

H N

O

R 1, R 2, R 3 = , C H <sup>2</sup>C H <sup>2</sup>COOH , CH <sup>2</sup>O H

N H

R 3

R 2

H <sup>O</sup> <sup>H</sup>

H N O

> H N O

H N R 1

**I N T E R M E D I A T E**

O H O

<sup>H</sup> <sup>O</sup> H

<sup>H</sup> <sup>O</sup> H

**Scheme 10.** Proposed mechanism of competitive binding colorless analyte and reporter dye.

Docking colorless *N-*phenylpiperazines pairs of analogues with or without a fluorine atom in the phenyl ring [110] revealed that using an array of artificial receptors it is possible to verify the presence of such ligand modification.

Analysis of the binding pattern of *N-*phenylpiperazine derivatives showed two characteristic binding patterns dependent on the structure of amino acid residues interacting with ligands. For most amino acid residues weaker binding of fluorinated analogues and stronger binding of native phenyl substituted analogues was observed with an exception of the receptors bearing tryptophane residue inside the binding pocket [111].

An array of *N-*lipidated peptides immobilized on cellulose was also used in studies of tissue homogenates for early diagnosing thyroid gland cancer [112], which is the most common malignancy of the endocrine system. There were found different binding patterns of healthy and cancer tissue for the various types of cancer.

268 Cellulose – Medical, Pharmaceutical and Electronic Applications

**Scheme 10.** Proposed mechanism of competitive binding colorless analyte and reporter dye.

verify the presence of such ligand modification.

Docking colorless *N-*phenylpiperazines pairs of analogues with or without a fluorine atom in the phenyl ring [110] revealed that using an array of artificial receptors it is possible to

Analysis of the binding pattern of *N-*phenylpiperazine derivatives showed two characteristic binding patterns dependent on the structure of amino acid residues interacting with ligands. For most amino acid residues weaker binding of fluorinated analogues and stronger binding of native phenyl substituted analogues was observed with an exception of the receptors bearing tryptophane residue inside the binding pocket [111].

By incorporation into the peptide fragment of receptor amino acid residues characteristic of catalytic triade of the hydrolytic enzymes the binding pockets demonstrated catalytic activity [113]. These were able to catalyse hydrolysis of esters bond [114]. All members of the library of 36 structures formed by permutations of Ser, Glu, His acylated with 6 long chain carboxylic acids were active as esterase and effectively catalyzed hydrolysis of p-nitrophenyl ester of Z-L-Leu-L-Leu-OH at pH 7-7.5 and temp not exceeding 20oC. The postulated mechanism of catalytic activity of *N-*lipidated oligopeptides is presented on Figure 9.

**Figure 9.** The postulated mechanism of catalytic activity of *N-*lipidated tripeptides of catalytic triade.

In this case the progress of hydrolysis was so fast that under the conditions of the experiment it was difficult to identify the most catalytically active structure. The extinction at 405 nm increased from the initial value of 0.300 for the substrate to more than 0.800 before the first cycle of measurements was completed. The rate of reaction diversified enough for identification of the most active catalytic structures was afforded by using significantly less reactive, sterically hindered substrate Z-Aib-Aib-ONp.

As the final effect of catalytic activity is the transformation of relatively non-polar organic molecule into ionic species, one can expect application of this phenomena for the construction of sensors [115]. There are also many other interesting area of application of catalytically active *N-*lipidated peptides immobilized on cellulose. Most expected are effects stereochemical results of such transformations, expecting open access to essentially unlimited access to configurational arrangement of stereogenic centers of polypeptide fragment [116].

Cellulose Functionalysed with Grafted Oligopeptides 271

[10] Rosenau, T.; Renfrew, A.H.M.; Adelwoehrer, C.; Potthast, A.; Kosma, P. Cellulosics modified with slow-release reagents. Part I. Synthesis of triazine-anchored reagents for slow release of active substances from cellulosic materials. *Polymer* 2005, *46*, 1453–1458. [11] Nemati, F.; Kiani, H.; Hayeniaz, Y.S. Cellulose-Supported Ni(NO3)2×6H2O/2,4,6- Trichloro-1,3,5-Triazine (Tct) as a Mild, Selective, and Biodegradable System for

[12] Blackwell, H.E. Hitting the SPOT: small-molecule macroarrays advance combinatorial

[13] Benes, M.J.; Adamkova, K.; Turkova, J. Activation of beaded cellulose with 2,4,6-

[14] Lenfeld, J.; Benes, M.J.; Kucerova Z., 3,5-Diiodo-L-tyrosine immobilized on bead

[15] Danner, J.; Lenhoff, H.M.; Heagy, W. Glutathione-bound celluloses: preparation with the linking reagent s-triazine trichloride and use in chromatography*. J. Solid-Phase* 

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[17] Gardner, Thomas S. Cellulose esters of amino acids. (Eastman Kodak Co.). (1949), US

[18] Volkmer, R. Synthesis and application of peptide arrays: Quo vadis SPOT technology.

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