**1. Introduction**

240 Cellulose – Medical, Pharmaceutical and Electronic Applications

Chinese J Anal Chem 2006;4(34):525P-528.

2009;26(9):1649-1652.

University; 2008.

biosynthesis. Pestic Sci 1987;21:253-267.

HPLC. Journal of Instrumental Analysis 2007;26:55-58.

Chromatography. Asian J Chem 2009;21(8): 6095-6101.

methamidophos. J Agric Food Chem 2006;54:8134−8138.

[100] Chen SW, Lin KD, Liu WP. Determination of metalaxyl enantiomeric purity by nonchiral high performance liquid chromatography with circular dichroism detector.

[101] Han XQ, Li J, Yun CL, Sun YY, Wang HS. Chiral Separation of Compounds on Covalently Bonded Cellulose Chiral Stationary Phase by NO1Tflal and Reversed Phase

[102] Qiu J, Wang QX, Zhou ZQ, Yang SM. Enantiomeric separation and circular dichroism detection of metalaxyl acid metabolite by Chiral High Performance Liquid

[103] Lin K, Zhou SS, Xu C, Liu WP. Enantiomeric resolution and biotoxicity of

[104] Polcaro CM, Berti A, Mannina L, Marra C, Sinibaldi M, Viel S. Chiral HPLC resolution of neutral pesticides. J Liq Chromatogr Relat Technol 2004;27:49-61. [105] Cai XJ, Li Z, Xu XZ. Study on Enantioseparation of several herbicides on chiral stationary phases with HPLC. Journal of Instrumental Analysis 2007;26(6):891-894. [106] Lin CM, Liu JY, Zhang DT. Enantioseparation of naproanilide on cellulose

[107] Chen SW, Cai XY, Liu WP. Characterization of napropamide enantiomers by CD and determination of the enantiomeric ratios in water. Spectrosc Spect Anal

[108] Burden RS, Carter GA, Clark T, Cooke DT, Croker SJ, Deas AHB, Hedden P, James CS, Lenton JR. Comparative activity of the enantiomers of triadimenol and paclobutrazol as inhibitors of fungal growth and plant sterol and gibberellin

[109] Yang HY. Study on environmental behavior of insecticide pyraclofos. Zhejiang

[110] Pan H, Duan R, Liao Y, Xu BM. Chromatographic separation of chiral pesticides

[111] Liu WP, Lin K, Gan JY. Separation and aquatic toxicity of enantiomers of the

derivatives chiral stationary phases. Agrochemicals 2007;46(8):526-528.

and their intermediates. Chemistry & Bioengineering 2007;24(8):76-78.

organophosphorus insecticide trichloronate. Chirality 2006;18:713-716.

Advantages offered by immobilization of any component of the reacting system are rewarding all additional efforts and the cost of the support. The majority of the methods reported have been based on the principles of solid phase organic synthesis (SPOS) in which the substrate is attached to the polymer support and excesses of reactants and reagents used to drive each synthetic step to completion. Then simple filtration affords a polymer bound product. While this approach is undoubtedly effective, there are a number of drawbacks which include the requirement for additional chemical steps to attach starting material, to develop synthetic methodology for the solid phase and to cleave products. More recently, solution phase methods, which circumvent these difficulties, have been introduced as alternatives to SPOS. These allow the use of excess of reagents followed by sequestrating either the product or excess reagents and byproducts from the reaction mixture using an insoluble functionalized polymer. Isolation and purification can then be achieved by simple filtration and evaporation.

Usually polystyrene and PEG based resins are commonly used as matrixes in SPOS, but nowadays there is observed also increased application of various beaded cellulose supports [1]. These show different solvent swelling profiles relative to those exhibited by the standard organic polymers and, being biomolecules, are biodegradable. Cellulose framework is attracting growing attention due to favorable biophysical properties, biocompatibility, low immunogenicity, relatively high resistance to temperature, inertness under broad range of reaction conditions and solvents and many other unique properties. Moreover, native cellulose microfribrils are abundant within slightly diversified properties dependent on the origin within relatively low cost. These properties make cellulose very useful for biochemical and biological investigations of interactions in aqueous as well as organic media.

© 2013 Kaminski et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Kaminski et al., licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Solid supported reagents were found exceedingly useful in all syntheses involving excessive amounts of substrates [2]. Inexpensive cellulose is offering high loading potential but concurrently attended by the threat of side reaction of nucleophilic hydroxyl groups. Using relatively inert towards hydroxylic group under ambient conditions triazine coupling reagents it was possible to obtain monofunctional triazine condensing reagents **1** and bifunctional reagent **2** by the treatment of cellulose with 2,4-dichloro-6-methoxy-1,3,5 triazine or cyanuric chloride respectively [3]. An independent approach towards immobilization of cyanuric chloride was confirmed the general utility of this procedure [4]. The loading of the cellulose carrier has been established by determination of Cl and N contents. For the standard laboratory Whatman filter paper, typical anchoring of triazine condensing reagent gave density of loading 0.6 – 1.0\*10-6 mmole/cm2.

Cellulose Functionalysed with Grafted Oligopeptides 243

**Figure 1.** Chromatograms of selected dipeptides or amides obtained by using triazine coupling reagents immobilized on cellulose matrix divided chess-wise into separate "square flat reactors" by

> N N N N+

cellulose

**Scheme 2.** Tracelss chiral coupling reagents **2a-e** prepared on cellulose.

OMe

Cl

**<sup>1</sup> 3a-e 2a-e**

In the more advanced approach, chiral coupling reagents immobilized on the cellulose were prepared and then used for enantioselective activation of racemic substrates [7]. Traceless enantiodifferentiating reagents [8] were obtained by using the cellulose membrane loaded

2 eq.

NH 3CH H Cbz COOH

THF/0oC/0.5h

N N N

OMe

+

2NH R

THF/r.t./14h

N N N

cellulose **6**

OH

**5**

OMe

O

+

Cbz NH 3CH H

COOH

O

O

cellulose

Cbz O NH 3CH <sup>H</sup>

Cbz NH R NH 3CH H

O

**4**

separation lines imprinted with polysiloxane.

with 2,4-dichloro-6-methoxy-1,3,5-triazine.

THF/0oC/0.5h

tertiary amine

N N N

Cl

OMe

O

cellulose

**Scheme 1.** Peptide **4** synthesis using monofunctional **1a** and bifunctional **1b** triazine condensing reagents reagents immobilized on cellulose.

An expedient matrix for the preparation of indexed library of amides and oligopeptides has been obtained by the demarcation of the surface of the cellulose plates chess-wise by the thin lines imprinted by polysilane, allocated separated, squared area for parallel synthesis of each individual compound (flat reactors) [5]. Application of carboxylic components into compartments of the matrix afforded "superactive" [6] triazine esters **3** linked to the support. Applying of amino components afforded the indexed library of amides and oligopeptides. The extraction of the final products from the solid support gave chromatographically homogeneous amides and oligopeptides in 60-99% yield. Chromatograms of the crude extracts from the diagonal fields of the part of the 8x12 library of amides and dipeptides are presented on Figure 1. When the size of matrix compartment corresponds to the size of typical ELISA plate, the amount of product recovered by extraction from the single "square flat reactor" was sufficient for elucidation of the structure of product by ES-MS or FAB-MS, for determination of their purity by HPLC, and even for studies involving 1H-NMR.

Increasing the dimensions of the matrix field or application of powdered cellulose enabled "bulk" (1-10 mmole) synthesis of amides and oligopeptides. The further modification of this synthetic procedure as well as the "shape" of cellulose support, opened possibility to design of tailor made system of immobilized triazine coupling reagents the best suited to the given synthetic goal.

N N N X Cl

NMM

reagents reagents immobilized on cellulose.

O

**1a,b**

**1a** X=OCH3 **1b** X=Cl

synthetic goal.

condensing reagent gave density of loading 0.6 – 1.0\*10-6 mmole/cm2.

Z-NH-CHR-COOH

**Scheme 1.** Peptide **4** synthesis using monofunctional **1a** and bifunctional **1b** triazine condensing

An expedient matrix for the preparation of indexed library of amides and oligopeptides has been obtained by the demarcation of the surface of the cellulose plates chess-wise by the thin lines imprinted by polysilane, allocated separated, squared area for parallel synthesis of each individual compound (flat reactors) [5]. Application of carboxylic components into compartments of the matrix afforded "superactive" [6] triazine esters **3** linked to the support. Applying of amino components afforded the indexed library of amides and oligopeptides. The extraction of the final products from the solid support gave chromatographically homogeneous amides and oligopeptides in 60-99% yield. Chromatograms of the crude extracts from the diagonal fields of the part of the 8x12 library of amides and dipeptides are presented on Figure 1. When the size of matrix compartment corresponds to the size of typical ELISA plate, the amount of product recovered by extraction from the single "square flat reactor" was sufficient for elucidation of the structure of product by ES-MS or FAB-MS, for determination of

Increasing the dimensions of the matrix field or application of powdered cellulose enabled "bulk" (1-10 mmole) synthesis of amides and oligopeptides. The further modification of this synthetic procedure as well as the "shape" of cellulose support, opened possibility to design of tailor made system of immobilized triazine coupling reagents the best suited to the given

N+ O

Cellulose Cellulose

their purity by HPLC, and even for studies involving 1H-NMR.

N N N X

O

Cellulose

Solid supported reagents were found exceedingly useful in all syntheses involving excessive amounts of substrates [2]. Inexpensive cellulose is offering high loading potential but concurrently attended by the threat of side reaction of nucleophilic hydroxyl groups. Using relatively inert towards hydroxylic group under ambient conditions triazine coupling reagents it was possible to obtain monofunctional triazine condensing reagents **1** and bifunctional reagent **2** by the treatment of cellulose with 2,4-dichloro-6-methoxy-1,3,5 triazine or cyanuric chloride respectively [3]. An independent approach towards immobilization of cyanuric chloride was confirmed the general utility of this procedure [4]. The loading of the cellulose carrier has been established by determination of Cl and N contents. For the standard laboratory Whatman filter paper, typical anchoring of triazine

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

N N N O

O

**2 3 4**

Z

R' O

NH2

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

R

N H

Z

H

O R'

O

**Figure 1.** Chromatograms of selected dipeptides or amides obtained by using triazine coupling reagents immobilized on cellulose matrix divided chess-wise into separate "square flat reactors" by separation lines imprinted with polysiloxane.

In the more advanced approach, chiral coupling reagents immobilized on the cellulose were prepared and then used for enantioselective activation of racemic substrates [7]. Traceless enantiodifferentiating reagents [8] were obtained by using the cellulose membrane loaded with 2,4-dichloro-6-methoxy-1,3,5-triazine.

**Scheme 2.** Tracelss chiral coupling reagents **2a-e** prepared on cellulose.

Chiral quaternary *N-*triazinylammonium derivatives **2a-e** immobilized on the membrane were obtained *in situ* by treatment of **1** with appropriate tertiary amines (*N*methylmorpholine, column 1; strychnine, column 2, brucine column 3; quinine column 4; and sparteine, column 5). Chirality of cellulose support (column 1) was found sufficient for enantiospecific activation of L enantiomer of racemic Z-Ala-OH with L/D ratio exceeding 90/10.

L-enantiomer content D-enantiomer content

Cellulose Functionalysed with Grafted Oligopeptides 245

O O

n

toluenesulfonates in the presence of sodium bicarbonate or DIPEA. Activation of carboxylic components proceeded under conditions similar to the standard synthesis in solution yielding "superactive" esters of *N-*protected amino acids anchored to the support *via*

> <sup>O</sup> OH O

> > O

loading: 9.2 \*10-6 mol/cm2

A synthetic value of triazine reagents immobilized on cellulose was confirmed by dipeptide synthesis. The reagents were found efficient in the synthesis of Z-, Boc, or Fmoc protected chromatographically homogenous dipeptides in 72-91%. Moreover, experiments involving activation of sterically demanding 2-aminoisobutyric acid (Aib) confirmed that an access to the reactive centers of immobilized reagents remains principally unrestricted, although

The other modification of cellulosic fibers with tri-functional triazines was applied as control release system. The compounds employed were immobilized on cellulose substituted with monochlorotriazinyl (MCT) anchor group for fixation of an active substance and tuning the reactivity to facilitate release control. While the compounds were completely stable under dry conditions, the active substances were released simply by surrounding humidity. The reagents offered intriguing perspectives for the preparation of modified cellulosic material for single-use application in fields such as healthcare, cosmetics,

Cellulose was found also useful support for efficient control of selectivity of chemical reactions. In the classic procedure for the nitration of phenols, use of nitric and sulfuric acid mixtures results in the formation of *ortho* and *para* products with a ratio of about 2:1. Nitration of phenols and naphthols in the presence of biodegradable cellulose-supported Ni(NO3)2×6H2O/2,4,6-trichloro-1,3,5-triazine system proceeded in acetonitrile at room temperature regioselectively. *Ortho*- nitrated phenols were obtained within a short reaction time with good yields. The reaction conditions were mild, and the employed cellulose could be recovered several times for further use [11]. The suggested mechanism proposes that cellulose acts as a template by forming hydrogen bonds between OH groups, phenol, and nitrate anions. This complex would transform substrate into the *ortho*-substituted

intermediate followed by regioselective rearrangement to *o*-nitro phenol.

slightly lower yield and purity of respective peptides were noticed in this case [9].

N N N

MeO N

OH

O

+

OH OH

O S O <sup>O</sup> -

OH

O O O

**Scheme 3.** Stable triazine coupling reagents immobilized on cellulose plate.

OH

OH

OH

triazine ring (see Scheme 3).

or personal hygiene [10].

Further structure modification of the immobilized triazine **1** proceeded directly on the membrane using chiral tertiary amines yielding spatially addressed five sub-libraries of enantiodifferentiating condensing reagents (Figure 2, 1-5). In all cases enantiodifferentiating activation of *rac*-Z-Ala-OH afforded triazine "superactive" ester **3a-e** with different enantiomeric composition. It has been found that the effect of chiral amine used as additional chiral selector predominate an effect of cellulose. Enantiomerically enriched esters **3a-e** in reaction with L-Phe-OMe (S1); D-Phe-OMe (S2), and H-Gly-OMe (S3) gave a library of alanine dipeptides of divergent configuration and enantiomeric purity (not linked to the support) and side-products (still immobilized on the cellulose membrane). The method opened an access to L and D alanine derivatives directly from racemic substrates. The best results (ee 92-99%) in the synthesis of L-alanine peptides were obtained in condensations mediated by *N-*methylmorpholine (column 1) or sparteine (column 5) when matching effects of cellulose and chiral selector were cooperated. The best results in the synthesis of D-alanine peptides (ee 91-98%) were obtained in condensations mediated by strychnine (column 2).

The disadvantage of procedure described above is caused by limited stability of *N*triazinylammonium chlorides **2** prepared on cellulose. Stable immobilized triazine coupling reagents were obtained in reactions of cellulose with *N-*methylmorpholinium ptoluenesulfonates in the presence of sodium bicarbonate or DIPEA. Activation of carboxylic components proceeded under conditions similar to the standard synthesis in solution yielding "superactive" esters of *N-*protected amino acids anchored to the support *via* triazine ring (see Scheme 3).

loading: 9.2 \*10-6 mol/cm2

**Scheme 3.** Stable triazine coupling reagents immobilized on cellulose plate.

244 Cellulose – Medical, Pharmaceutical and Electronic Applications

90/10.

strychnine (column 2).

Chiral quaternary *N-*triazinylammonium derivatives **2a-e** immobilized on the membrane were obtained *in situ* by treatment of **1** with appropriate tertiary amines (*N*methylmorpholine, column 1; strychnine, column 2, brucine column 3; quinine column 4; and sparteine, column 5). Chirality of cellulose support (column 1) was found sufficient for enantiospecific activation of L enantiomer of racemic Z-Ala-OH with L/D ratio exceeding

**Figure 2.** Enantiomeric composition of the products of enantioselective activation of *rac*-Z-Ala-OH and

L-enantiomer content D-enantiomer content

Further structure modification of the immobilized triazine **1** proceeded directly on the membrane using chiral tertiary amines yielding spatially addressed five sub-libraries of enantiodifferentiating condensing reagents (Figure 2, 1-5). In all cases enantiodifferentiating activation of *rac*-Z-Ala-OH afforded triazine "superactive" ester **3a-e** with different enantiomeric composition. It has been found that the effect of chiral amine used as additional chiral selector predominate an effect of cellulose. Enantiomerically enriched esters **3a-e** in reaction with L-Phe-OMe (S1); D-Phe-OMe (S2), and H-Gly-OMe (S3) gave a library of alanine dipeptides of divergent configuration and enantiomeric purity (not linked to the support) and side-products (still immobilized on the cellulose membrane). The method opened an access to L and D alanine derivatives directly from racemic substrates. The best results (ee 92-99%) in the synthesis of L-alanine peptides were obtained in condensations mediated by *N-*methylmorpholine (column 1) or sparteine (column 5) when matching effects of cellulose and chiral selector were cooperated. The best results in the synthesis of D-alanine peptides (ee 91-98%) were obtained in condensations mediated by

The disadvantage of procedure described above is caused by limited stability of *N*triazinylammonium chlorides **2** prepared on cellulose. Stable immobilized triazine coupling reagents were obtained in reactions of cellulose with *N-*methylmorpholinium p-

coupling with L-Phe-OMe (S1); D-Phe-OMe (S2), and H-Gly-OMe (S3).

A synthetic value of triazine reagents immobilized on cellulose was confirmed by dipeptide synthesis. The reagents were found efficient in the synthesis of Z-, Boc, or Fmoc protected chromatographically homogenous dipeptides in 72-91%. Moreover, experiments involving activation of sterically demanding 2-aminoisobutyric acid (Aib) confirmed that an access to the reactive centers of immobilized reagents remains principally unrestricted, although slightly lower yield and purity of respective peptides were noticed in this case [9].

The other modification of cellulosic fibers with tri-functional triazines was applied as control release system. The compounds employed were immobilized on cellulose substituted with monochlorotriazinyl (MCT) anchor group for fixation of an active substance and tuning the reactivity to facilitate release control. While the compounds were completely stable under dry conditions, the active substances were released simply by surrounding humidity. The reagents offered intriguing perspectives for the preparation of modified cellulosic material for single-use application in fields such as healthcare, cosmetics, or personal hygiene [10].

Cellulose was found also useful support for efficient control of selectivity of chemical reactions. In the classic procedure for the nitration of phenols, use of nitric and sulfuric acid mixtures results in the formation of *ortho* and *para* products with a ratio of about 2:1. Nitration of phenols and naphthols in the presence of biodegradable cellulose-supported Ni(NO3)2×6H2O/2,4,6-trichloro-1,3,5-triazine system proceeded in acetonitrile at room temperature regioselectively. *Ortho*- nitrated phenols were obtained within a short reaction time with good yields. The reaction conditions were mild, and the employed cellulose could be recovered several times for further use [11]. The suggested mechanism proposes that cellulose acts as a template by forming hydrogen bonds between OH groups, phenol, and nitrate anions. This complex would transform substrate into the *ortho*-substituted intermediate followed by regioselective rearrangement to *o*-nitro phenol.

## **2. Cellulose acylated (grafted) with amino acids or peptides**

Designing of new materials based on renewable natural resources is one of the most important scientific and technological challenges. The aim of these efforts is to open an access to materials which will have to replace toxic or non-biodegradable materials derived from fossil resources, while offering similar mechanical, thermal, or optical properties. In contrast to polymer membranes, cellulose shows high thermostability up to temperatures of about 180 °C, making it possible to use cellulose for reactions at elevated temperatures [12]. To date filter papers have been mostly used as the solid support.

Cellulose Functionalysed with Grafted Oligopeptides 247

Since the hydroxyl groups are moderately reactive, the process of functionalization of cellulose is often preceding with more complex modification procedures. A reactive intermediate containing isocyanate groups was prepared by treatment of cellulose with 2,4 tolylene diisocyanate. The reactions of the intermediate with amino acids and their esters gave cellulose derivatives containing amino acid residues. The isocyanate groups reacted with amino acid esters in DMSO at low temp. under nitrogen to give high conversions. The amounts of amino acid esters bound to the cellulose through urea linkage were evaluated as 0.35-1.07 mmol/g. The selective adsorption and chelation of metal ions indicated that celluloses containing lysine and cysteine residues adsorbed 0.051 and 0.056 mmol Cu2+/g,

An essential drawback of the ester linkage applied for anchoring peptides is instability towards aqueous media of pH > 7, not uncommon for bio-assay and stripping conditions. Moreover, cellulose and cellulose membranes show only a limited acid stability. This acid sensitivity severely restricts palette of reagents and reaction conditions that can be applied, even for the most stable commercially available cellulose materials. Therefore, besides the direct esterification of cellulose membranes with amino acids, many publications describe

Cellulose undergoes facile alkaline etherification which, given the availability of up to three hydroxyl groups per glucopyranose residue, offers the potential to provide very high loading supports. Several companies already offer already modified cellulose membranes. Specially prepared cellulose membranes with a stably attached aminated spacer of 8 to 12 PEG units (PEG300-500) are available, which in contrast to common cellulose membranes is

The materials, on which polypeptides were immobilized on different shaped cellulose products *via* chemically stable ether linkage have antimicrobial or anticancer activities. These were used as wound dressings, sutures, artificial blood vessel, catheters, dialysis membranes, clothing, and stents. Thus, material, on which beetle defensin analogue Arg-Leu-Leu-Leu-Arg-Ile-Gly-Arg-Arg was immobilized on cotton fabric showed high antimicrobial activity even after repeated washing and autoclave sterilization [28]. Also other nonapeptide Arg-Leu-Tyr-Leu-Arg-Ile-Gly-Arg-Arg immobilized to aminofunctionalized cotton fibers by a modification of the SPOT synthesis technique was active against *S. aureus,* methicillin-resistant *S. aureus* and mouse myeloma cells and human leukemia cells. The assays revealed that these fibers maintained inhibition activity against

Cellulose with intrinsic osteoinductive property useful for the preparation of the bone substitutes was obtained by immobilization of peptides containing Arg-Gly-Asp (RGD) fragment [30]. Biomaterials from bacterial-derived cellulose modified with cell adhesion peptide became a promising material as a replacement for blood vessels in vascular surgery

bacteria and cancer cells after washing and sterilization by autoclaving [29].

respectively [27].

[31].

the use of more stable ether or amide linkers.

stable under strong acidic and basic conditions.

The classic immobilization procedure involved the use of cyanuric chloride [13] as linker for anchoring broad range of amino acids and peptides on cellulose. Lenfeld and coworkers [14] immobilized 3,5-diiodo-tyrosine (DIT) on cellulose beads activated by the reaction with 2,4,6-trichloro-1,3,5-triazine and used prepared materials as sorbents in affinity chromatography of proteases. Also glutathione-bound cellulose for use in chromatography was prepared with cyanuric chloride as linking agent [15].

The library of p-nitrophenyl esters of oligopeptides anchored with *N-*terminal amino-acids *via* triazine linkage to the cellulose were synthesized step by step and after digestion with tissue homogenate were used for colorimetric differentiation of hydrolytic activity of primary subcutaneously growing tumor of Lewis lung carcinoma (LLC) bearing mice, lung metastatic colonies of LLC, blood serum of LLC bearing mice, and appropriate tissue homogenate of the healthy mice [16].

Cellulose is a polysaccharide containing free hydroxyl groups. In the first report cellulose free OH groups were esterified with amino acids activated previously by the transformation into appropriate acid halide or anhydride, in the presence of a catalyst, such as Mg(ClO4)2, H2SO4, H3PO4, or ZnCl2 [17]. Recently, the more convenient procedure involved the coupling method of Fmoc protected amino acids such as Fmoc-β-Ala-OH or Fmoc-Gly-OH [18] by using activating reagents such as *N*,*N'*-diisopropylcarbodiimide (DIC), 1,1' carbonyldiimidazole (CDI) in presence of a base, e.g. *N-*methyl-imidazole (NMI) [19]. There are also recommendations suggesting the use of 1,1'-carbonyl-di-(1,2,4-triazole) (CDT) instead of CDI in order to reduce the risk of the deprotection of Fmoc-amino acids during the coupling reaction [20]. Since the early reports by Frank, cellulose has found widespread application as a support in the synthesis of peptides and oligonucleotides. This involved the use of cellulose in the form of sheets, membranes, disks [21] or cotton thread used as supporting material [22]. Despite these precedents, alternative cellulose supports, notably beads which can offer considerably higher loading levels than that obtained with planar supports [23]. Beaded cellulose can be easily prepared by the coagulation-regeneration technique involving the addition of a solution of a soluble cellulose derivative, commonly the xanthate [24,25] or acetate [26], to a rapidly stirred, inert, immiscible solvent. The beads, thus formed, are precipitated either by a sol-gel process or by a reduction in reaction temperature. Chemical regeneration of the hydroxyl groups and sieving produces the active beads with the desired size distribution.

Since the hydroxyl groups are moderately reactive, the process of functionalization of cellulose is often preceding with more complex modification procedures. A reactive intermediate containing isocyanate groups was prepared by treatment of cellulose with 2,4 tolylene diisocyanate. The reactions of the intermediate with amino acids and their esters gave cellulose derivatives containing amino acid residues. The isocyanate groups reacted with amino acid esters in DMSO at low temp. under nitrogen to give high conversions. The amounts of amino acid esters bound to the cellulose through urea linkage were evaluated as 0.35-1.07 mmol/g. The selective adsorption and chelation of metal ions indicated that celluloses containing lysine and cysteine residues adsorbed 0.051 and 0.056 mmol Cu2+/g, respectively [27].

246 Cellulose – Medical, Pharmaceutical and Electronic Applications

**2. Cellulose acylated (grafted) with amino acids or peptides** 

To date filter papers have been mostly used as the solid support.

was prepared with cyanuric chloride as linking agent [15].

homogenate of the healthy mice [16].

beads with the desired size distribution.

Designing of new materials based on renewable natural resources is one of the most important scientific and technological challenges. The aim of these efforts is to open an access to materials which will have to replace toxic or non-biodegradable materials derived from fossil resources, while offering similar mechanical, thermal, or optical properties. In contrast to polymer membranes, cellulose shows high thermostability up to temperatures of about 180 °C, making it possible to use cellulose for reactions at elevated temperatures [12].

The classic immobilization procedure involved the use of cyanuric chloride [13] as linker for anchoring broad range of amino acids and peptides on cellulose. Lenfeld and coworkers [14] immobilized 3,5-diiodo-tyrosine (DIT) on cellulose beads activated by the reaction with 2,4,6-trichloro-1,3,5-triazine and used prepared materials as sorbents in affinity chromatography of proteases. Also glutathione-bound cellulose for use in chromatography

The library of p-nitrophenyl esters of oligopeptides anchored with *N-*terminal amino-acids *via* triazine linkage to the cellulose were synthesized step by step and after digestion with tissue homogenate were used for colorimetric differentiation of hydrolytic activity of primary subcutaneously growing tumor of Lewis lung carcinoma (LLC) bearing mice, lung metastatic colonies of LLC, blood serum of LLC bearing mice, and appropriate tissue

Cellulose is a polysaccharide containing free hydroxyl groups. In the first report cellulose free OH groups were esterified with amino acids activated previously by the transformation into appropriate acid halide or anhydride, in the presence of a catalyst, such as Mg(ClO4)2, H2SO4, H3PO4, or ZnCl2 [17]. Recently, the more convenient procedure involved the coupling method of Fmoc protected amino acids such as Fmoc-β-Ala-OH or Fmoc-Gly-OH [18] by using activating reagents such as *N*,*N'*-diisopropylcarbodiimide (DIC), 1,1' carbonyldiimidazole (CDI) in presence of a base, e.g. *N-*methyl-imidazole (NMI) [19]. There are also recommendations suggesting the use of 1,1'-carbonyl-di-(1,2,4-triazole) (CDT) instead of CDI in order to reduce the risk of the deprotection of Fmoc-amino acids during the coupling reaction [20]. Since the early reports by Frank, cellulose has found widespread application as a support in the synthesis of peptides and oligonucleotides. This involved the use of cellulose in the form of sheets, membranes, disks [21] or cotton thread used as supporting material [22]. Despite these precedents, alternative cellulose supports, notably beads which can offer considerably higher loading levels than that obtained with planar supports [23]. Beaded cellulose can be easily prepared by the coagulation-regeneration technique involving the addition of a solution of a soluble cellulose derivative, commonly the xanthate [24,25] or acetate [26], to a rapidly stirred, inert, immiscible solvent. The beads, thus formed, are precipitated either by a sol-gel process or by a reduction in reaction temperature. Chemical regeneration of the hydroxyl groups and sieving produces the active An essential drawback of the ester linkage applied for anchoring peptides is instability towards aqueous media of pH > 7, not uncommon for bio-assay and stripping conditions. Moreover, cellulose and cellulose membranes show only a limited acid stability. This acid sensitivity severely restricts palette of reagents and reaction conditions that can be applied, even for the most stable commercially available cellulose materials. Therefore, besides the direct esterification of cellulose membranes with amino acids, many publications describe the use of more stable ether or amide linkers.

Cellulose undergoes facile alkaline etherification which, given the availability of up to three hydroxyl groups per glucopyranose residue, offers the potential to provide very high loading supports. Several companies already offer already modified cellulose membranes. Specially prepared cellulose membranes with a stably attached aminated spacer of 8 to 12 PEG units (PEG300-500) are available, which in contrast to common cellulose membranes is stable under strong acidic and basic conditions.

The materials, on which polypeptides were immobilized on different shaped cellulose products *via* chemically stable ether linkage have antimicrobial or anticancer activities. These were used as wound dressings, sutures, artificial blood vessel, catheters, dialysis membranes, clothing, and stents. Thus, material, on which beetle defensin analogue Arg-Leu-Leu-Leu-Arg-Ile-Gly-Arg-Arg was immobilized on cotton fabric showed high antimicrobial activity even after repeated washing and autoclave sterilization [28]. Also other nonapeptide Arg-Leu-Tyr-Leu-Arg-Ile-Gly-Arg-Arg immobilized to aminofunctionalized cotton fibers by a modification of the SPOT synthesis technique was active against *S. aureus,* methicillin-resistant *S. aureus* and mouse myeloma cells and human leukemia cells. The assays revealed that these fibers maintained inhibition activity against bacteria and cancer cells after washing and sterilization by autoclaving [29].

Cellulose with intrinsic osteoinductive property useful for the preparation of the bone substitutes was obtained by immobilization of peptides containing Arg-Gly-Asp (RGD) fragment [30]. Biomaterials from bacterial-derived cellulose modified with cell adhesion peptide became a promising material as a replacement for blood vessels in vascular surgery [31].

Application of cellulose as a support for synthesis of complex template-assembled synthetic proteins (TASP) by orthogonal assembly of small libraries of purified peptide building blocks has been reviewed [32]. In most cases the linear template precursor was prepared by standard solid phase peptide synthesis (SPPS) on synthetic resin with orthogonal protecting groups followed by head-to-tail cyclisation of the linear precursor peptide and anchoring the template structure on cellulose. The strategy involving cleavable linker allowed control of the progress of synthesis on polystyrene resin. Final assembly of peptides prepared under standard SPPS conditions proceeded by successive cleavage of orthogonal protecting groups followed by coupling of predefined peptides.

Cellulose Functionalysed with Grafted Oligopeptides 249

matrices orients the fusion partner away from the solid support [41] reducing steric hindrance; and their high-affinity binding to cellulose is considered nearly irreversible. [42] At present, many CBD-tagged affinity ligands are purified before attachment to their solid support matrix. [43] For large-scale applications, it could be beneficial to directly immobilize the affinity ligand

Horseradish peroxidase (HRP) was immobilized to cellulose with cellulose-binding domain (CBD) as a mediator, using a ligand selected from a phage-displayed random peptide library. A 15-mer random peptide library was panned on cellulose-coated plates covered with CBD in order to find a peptide that binds to CBD in its bound form. The sequence LHS, which was found to be an efficient binder of CBD, was fused to a synthetic gene of HRP as an affinity tag. The tagged enzyme (tHRP) was then immobilized on microcrystalline cellulose coated with CBD, thereby demonstrating the indirect immobilization of a protein to cellulose *via* three amino acids selected by phage display

As a model system, it has been developed a fusion protein, which consisted of antibody-binding proteins L and G fused to a cellulose-binding domain (LG-CBD) tethered directly onto cellulose. Direct immobilization of affinity purification ligands, such as LG-CBD, onto inexpensive support matrices such as cellulose is an effective method for the generation of functional, singleuse antibody purification system. This straightforward preparation of purification reagents make antibody purification from genetically modified crop plants feasible and address one of

In several cases it could be beneficial to directly immobilize the affinity ligand at the source of production, thus avoiding the cost and time required for purification. A potential use of cellulose-supported affinity ligands for purification of other bioproducts from homogenates from genetically modified plants expressing recombinant proteins is under intensive studies. To examine the potential of immobilizing affinity purification ligands onto cellulose matrices in a single step, the yeast *P. pastoris* were engineered to express and secrete a chimeric protein consisting of antibody-binding proteins L and G[45] fused to a cellulosebinding domain. A similar fusion was recently reported for cell capture in hollow-fiber bioreactors. There are reports on the direct immobilization of chimeric LG-CBD proteins onto cellulosic resins for antibody purification. Both protein L and protein G domains retained dual functionality demonstrated by the specific binding and purification of scFv and IgG antibodies from complex feed stocks of yeast supernatants and tobacco plant homogenates. This is a step towards the rapid generation of inexpensive affinity purification reagents and systems, to reduce the costs associated with downstream processing of pharmaceutical products, including antibodies, from complex production systems such as

Copolymers having polypeptide side chains grafted on cellulose main chain were used for adhesion of fibroblasts. The factor likely to play a key role in determining the binding ability was the balance between the hydrophilicity and hydrophobicity of the main- and side-chain

the major bottlenecks facing commercialization of plant-derived pharmaceuticals [45].

at the source of production, thus avoiding the cost and time required for purification.

library and CBD [44].

genetically modified crop plants.

components [46].
