The Cross–linking of Animal Fibres : I–3: 4 – isoPropylidene 1: 2–5: 6–Dianhydromannitol

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<ul><li><p>402 CAPP &amp; SPEAKMAN-"CROSS-LINKING OF ANIMAL FIBRES- I" dug. 1949 </p><p>The Cross-linking of Animal Fibres 1-3 : 4 - isoPropylidene 1 : 2-5 : 6-Dianhydromannitol </p><p>C. W. CAPP and J. B. SPEAKMAN When animal fibres me treated with 3 : 4-iaopropylidene 1:: 2,- 5 : 6-.dianhydromannitol for 24]hr. at 6 0 " ~ . </p><p>in acid solution, cross-linking is brought about by reaction with the carboxyl groups of salt-linkages. The reaction proceeds more slowly at temperatures below 60c., and degradation is severe at high temperatures because of attack on the disulphide bonds. Under optimum conditions at 60%. the extent of cross- linking is smal1,'giving only about 7% increase in the resistance of the fibres to extension in water at 22*2"c., but even this limited amount of cross-linking causes an apprecisble reduction in the milling shrinkage of flannel. As the scaliness of the fibres is unaltered, the reduced shrinkage must be referred to the modified elastic properties of the.fibres, in agreement with the results of experiments with other crom-linking agents. </p><p>It is well known that epoxy compounds, such as ethylene oxide and propylene oxide, are capable of reaction with amines, alcohols, phenols, thiols, and carboxylic acids in the following manner- </p><p>R'.NH2:+ CHI--CH-R* + R'.NH.CHeCH(OH).R* '0/ </p><p>'0 ' R'.(!OOH + CH2-CH-R2 -+ R'TOOCHgCH(OH)*R2 The main peptide chains of proteins carry side- chains of all these types, and the conditions under which they react with epoxy compounds, especially propylene oxide, have been investigated by F'raenkel-Conrat and his colleagues1 in the case of soluble proteins, e.g. egg albumin and 1-lacto- globulin. Neutral, acidic, alkaline, and urea solu- tions of the proteins were used, and the optimum conditions for combination with basic, acidic, phenolic, and thiol side-chains established. In the light of these results, it seemed probable that di-epoxy compounds would be capable of cross- linking the main peptide chains of animal fibres, and a pure specimen of 3:4-isopropylidene 1 :2-5 :A-dianhydromannitol- </p><p>CHrCH-CH- CH-CH-CH, \O/ 6 \(/ </p><p>Y&lt; CHs CHs </p><p>was kindly provided by Professor W. N. Haworth for use in the following investigation*. As the amount of pure reagent (R 1) was very small, the preliminary experiments were carried out with a sample (R 2) of rather lower purity, obtained from another source, but the total quantity of reagent was so small that its action on animal fibres had to be examined exclusively by mechano-chemical methods4. </p><p>Experimental THE ACTION OF ~SOPROPYLIDENE DIANHYDRO- </p><p>A sample of non-medullated human hair was purified by extraction with alcohol and ether in a Soxhlet apparatus, followed by washing in distilled water, and 5-cm. lengths, taken from the intact root ends, were then attached to light glass hooks by means of dental cement. After the fibres had been calibrated by determining their load-xten- ion curves up to 30% extension in distilled water </p><p>MANNITOL ON UNTREATED FIBRES </p><p>Since the completion of this work in 194@ It has been reported* that Schlack has attempted to cross-link wool with dl-epoxy com- pounds. </p><p>a t 22*2"c., they were treated with isopropylidene dianhydromannitol (R 2) for 24 hr. a t the desired temperature and pH. In each case, two calibrated fibres and two unmounted fibres were treated in 6 C.C. of a solution made up by mixing 0.25 C.C. of R 2 with 2.76 C.C. of water and 3-00 C.C. of double- strength buffer solution. Owing to the limited amount of R 2 available, the initial pH values of the reagents were not measfired, the values given in sub- sequent tables being those of the double-strength buffers diluted with equal volumes of water. It should, however, be mentioned that the final pH values of the solutions, although measured rather inaccurately because only 6 c.5. of solution was availablefor use with the glass electrode in each case, were close to the nominal values. Phosphate buffers were used for pH 4.63-7-96, and potassium ohloride-boric acid-sodium hydroxide buffers for pH 8-00 and 9.20. </p><p>After treatment, the calibrated fibres were washed in running water for 20 hr. before redetermining their load-extension curves in distilled water a t 22.2"0., and the change in resistance to extension (30%) was then calculated. The unmounted fibres were used for supercontraction measurements. After each fibre had been mounted in a stainless steel setting frame4, its length wae measured with a travelling microscope. The fibre was then slackened, boiled in a 6% solution of sodium metabisulphite for 1 hr., rinsed, dried, drawn taut, and remeasured. In all cases the change in length was expressed as a percentage of the original length. </p><p>For reference purposes similar data were obtained for fibres which had been treated with double-strength buffer solutions diluted with an equal volume of water only. The results of both sets of experiments are given in Table I, each value being the mean of two closely agreeing observations on different fibres. </p><p>The change in resistance to extension of the ruagent-treated fibres is shown as B function of the temperature of treatment in Fig. 1. Maximum strengthening is realised a t about 5O0c., and since fibres treated a t this temperature show little or no supercontraction in a boiling solution of sodium bisulphite, it seems probable that strengthening is due to cross-linking of the main peptide chains. With rise of temperature above 500., however, the fibres are weakened progressively, to an extent which also increases with rise of pH. As the disulplude bonds of animal fibres are known to be </p></li><li><p>dug. 1949 CAPP &amp; SPEAKMAN-"CROSS-LlNKlNG OF ANIMAL FIBRES- I" 403 </p><p>Temperature of </p><p>Treatment ("C.) 25.0 </p><p>40.5 </p><p>50.0 </p><p>TABLE I Change in Itesistance </p><p>to Extension (yo) Reagent- Buffer- treated treated Fibres Fibres - 2.5 - 2.5 - 2.7 - 1.5 - 2.8 - 3.2 </p><p>0.3 - 1.5 0.5 - 2.0 2.1 - 1.3 5.3 0.9 4.a 1.1 4 .3 1.2 3.0 1.4 3.4 0.3 1.3 - 0.3 </p><p>- 0.9 - 0.8 0.7 - 0.7 </p><p>- 3.7 - 3.4 - 8.7 - 2.7 - 12.1 - 3.0 - 24.1 - 4.7 </p><p>- 51.7 - 25.8 - 77.4 - 31.8 </p><p>- 45.4 - 2 2 4 </p><p>Supercon traction (%) </p><p>Reagent- Buffer- treated treated Fibres Fibres </p><p>2 4 7 20.8 22.4 20.8 23.7 23.7 </p><p>6.0 20.9 2.9 19.9 1.7 14.0 0.0 18.6 </p><p>- 0.3 20.1 </p><p>2.4 23.5 - 0.2 1 7 4 </p><p>1.3 20.0 </p><p>1.5 25.5 4.2 20.9 1.4 24.0 </p><p>7.1 15.7 2.8 3.2 5.9 0.2 </p><p>0.1 28.2 </p><p>0.a 22.3 </p><p>1.0 21.6 </p><p>3.2 20.6 </p><p>10 30 40 50 60 70 80 90 100 Temperature of Treatment. ' C . </p><p>0 pH 4.53 X pH 5.93 + pH 6.94 </p><p>FIQ. 1 </p><p>attacked by water a t temperatures above about 55'12.5, it seems probable that the weakening brought about by the di-epoxy compound is due to reaction with the products of hydrolysis. </p><p>Some support for this view is provided by the failure of stretched fibres to acquire a permanent set when immersed in a solution of the reagent (R 2), in the usual concentration, for 24 hr. a t pH 4-53 and 50'12. After treatment, the stretched fibres were washed in distilled water and then released in boiling water. Data illustrating the rate of contraction of the fibres are given in Table 11, with corresponding data for fibres treated under similar conditions with the pH 4-53 buffer alone. </p><p>The failure of the reagent-treated fibres to acquire a set permanent to boiling water, in spite of the cross-linking which has been shown to occur under the above conditions, is not surprising if the strained disulphide bonds of stretched fibres are </p><p>TABLE I1 Percentage Extension after Release In Boiling </p><p>0 2 15 30 00min. Buffer alone ... 38.7 1.7 - 1.3 - 2.0 - 1 2 </p><p>46.8 6.6 0.3 - 0.1 - 2.0 Buffer + reagent 39.3 8 8 - 2.3 - 3.6 - 9.0 </p><p>4 3 7 8.7 - 2.0 - 4.7 - 7.7 </p><p>Treatment Water for- </p><p>hydrolysed at 50"c. and react with the di-epoxy compound without forming cross-linkages to m y marked extent. Few cross-linkages are formed when reduced fibres are treated with the di-epoky compound, as is shown in a later section, and it seems likely, therefore, that the above explanation why fibres are severely damaged by the reagent a t high temperatures, as well &amp;B why stretched fibres fail to acquire a permanent set in the reagent a t BO"o., is correct. It is, however, surprising that stretched fibres treated with the reagent a t B0"c. should supercontract when released in boiling water for 80 min., because unstretched fibres, treated under similar conditions, fail to contract in a boiling solution of sodium bisulphite. Some cross- linking does, however, occur when untreated fibres are boiled in sodium bisulphite solution, as is indicated by the fact that they supercontract only 30% compared with 60% for dmminated fibres. Cross-linking during boiling in sodium bisulphite solution wnnot occur with deamimted fibres, and the absence of supercontraction when fibres treated with the di-epoxy compound a t 6 0 C . are boiled in sodium bisulphite solution seems, therefore , t o be due to the stability conferred on the structure by the two types of linkage, viz. those formed by the di-epoxy compound and those formed by the bisulphite. </p><p>Further evidence that the strengthening of fibres treated with the di-epoxy compound a t 60C. is due to cross-linking of the main peptide chains was obtained in the following manner- Calibrated fibres were treated with isopropylidene dianhydro- mannitol (R 1) a t 60'0. under the same conditions as before, and restretched in distilled water to determine the increase in resistance to extension. The fibres were then immersed in 0.1 N. hydro- chloric acid for 24 hr. a t 22.2"c., and restretched in the solution with which they had reached equilibrium. Should either the mid or basic side- chains of salt-linkages react with the di-epoxy compound to form cross-linkages, the reduction in the resistance to extension in 0.1 N. hydrochloric acid, referred to the original resistance to extension in distilled water, should be leas than that given by untreated fibres in the same solution. The results are given in Table I11 with corresponding data for fibres treated with the buffer solutions alone under identical conditions. </p><p>TABLE I11 Temperature of Treatment 60'0. </p><p>(1) , (W (Ul) (W (il) - ( iv) 4.40 7.8 - 16.1 0.1 - 303 16.2 6.67 7 7 - 1 6 3 - 0 3 - 30.1 14.8 0.74 7.0 - 101 0.0 - 29.7 10.0 7.81 6.3 - 2 2 4 1.9 - 28.2 6.6 </p><p>- 24.2 - 3 3 - 43.2 10.0 8.70 4.1 </p></li><li><p>404 CAPP k SPEAKMAN-"CROSS-LINKING OF ANIMAL FIBRES- I" Aun. 1049 </p><p>Iy </p><p>fi all cases, the buffer-treated fibres show a greater fall in resistance to extension in 0.1 N. hydrochloric acid than the reagent-treated fibres, the difference being approximately the same a t pH 4.40 and 5.57 (column 6 of Table 111). The differences are shown as a function of pH in Fig. 2, and it is interesting that a minimum should be observed at pH 7.5, followed by a rapid rise in more alkaline media. It scema probable that the form of the curve is to be explained in the following terms- the cross-linking of intact fibres proceeds best in acid media, and diminishes with rise of pH until thiol groups are liberated by disulphide bond hydrolysis to take part in new cross-linking reactions. Although only a limited number of such cross-linkages can be formed, as shown in a later section, they prevent the severe swelling and weakening which buffer-treated fibres undergo in the acid medium. </p><p>Since the fall in resistance to extension of fibres treated with the di-epoxy compound at pH 4.40 and 5.57 is only about half that of corresponding huffer-treated fibres in 0.1 N . hydrochloric acid, it is obvious that either the acid or the basic side- chains of the salt-linkages which are normally broken by acid must have taken part in the cross- linking reaction responsible for the increased resistance to extension of the reagent-treated fibres. In order to discover whether acid or basic side-chains are involved, further experiments were carried out with deaminated fibres. </p><p>THE ACTION OF ~SOPROPYLIDENE DIANHYDRO- </p><p>After calibration, pairs of fibres were introduced into a 2-02. bottle containing 8.690 g. sodium nitrite dissolved in 34 C.C. water. Glacial acetic :tcid (7.2 c.c.) was then added, and the bottle closed with II stopper provided with a Bunsen valve. Deamination was allowed to proceed for 24 hr. a t 2 2 . 2 " ~ . The reagent was then renewed, and after a further 24 hr. the fibres were removed and washed overnight in running water. Their load-xtension curves were then redetermined in distilled water a t 22 .2"~ . In order to discover the extent of deamina- tion, the fibres were next immersed in 0.1 N. hydro- chloric acid for 24 hr. before determining their load-extension curves in the acid at 22.2%. Unlike untreated fibres, fully deaminated fibres show almost the same resistance to extension in 0.1 N. hydrochloric acid as in distilled water, and i t is obvious from the data of Table IV that the fibres used in these experiments were almost completely deaminated. The fibres were then washed in running water overnight before treatment for 24 hr. a t pH 6.05 and 50%. with the di-epoxy compound </p><p>MANNITOL ON DEAMINATED FIBRES </p><p>(R 1) in the usual concentrirtion. After treat,ment,, the fibres were :gain washed in runriing water before redetermining thcir load-extension curves in distilled water a t 2 2 . 2 " ~ Values for the clrange in resistance to extension a t the various stagrs of treatment are given in Table I V , with corrrspond- ing data for buffer-treated fibres. </p><p>1 Deaminated Bbrea in wtitt'r _.. ... ~ 2 b 7 - 23.5 Deaminated Rbrw i i t 0 . 1 N-HC'I ... - 27.7 - z'!.li Ueaniinatetl Rbrra. treated with </p><p>reagent and bntfer, in wHtw ... -- 1 9 4 - 16.0 2 Dearninated Bbres In wntrr ... ... - 26.3 -. 29.2 </p><p>Deaininated Bbrrs in 0.1 N-H(!I ... - 24.3 - 30.4 </p><p>The increase in rcsistarice to extension of the deaminated fibres (6.3 and 6.Si%), after treatment with the reagent, is so similar to that of untreated fibres as to leave no doubt that the carhoxyl groups of salt-linkages play an important part in the cross- linking reaction which is responsible for the strengthening of intact fibres by the di-epoxy coni- pound in acid media at 5 0 " c . Further support for this conclusion is provided by the fact that when fibres were treated for 24 hr. a t 50%. with the di-epoxy compound in presence of a phthalate buffer a t pH 4-99, the increase in resistance to extension in water, due to the treatment, was only 0.30/,, compared with 7.8% for treatment in presence of a phosphate buffer a t the same pH. Instead of combining with the fibre, the reagent combined with the carboxyl groups of phthalic acid, giving D buff-coloured, crystalline precipitate. Finally, since isopropylidene dianhytlro- mannitol appeared to be capable of cross-linking the acid side-chains of hair, it seemed likely that, alkali-resistant alginates might be obtained by cross-linking alginic acid with di-epoxy compounds. This work will form the subject of a later paper, but, in support of the above experiments with hair, it may be stated that highly successful results have been obtained. On nll these grounds, therefore, it seems...</p></li></ul>