RESULTS AND DISCUSSION

In document Betaine analogues and related compounds for biomedical applications (Page 63-66)

CHAPTER 3 SYNTHESES OF COMPENSATORY SOLUTES

3.3 RESULTS AND DISCUSSION

The majority of the synthetic compensatory solutes were easy to synthesize and the yield was usually more than 75%. Applying the principles of a few existing methods, new solutes were synthesized. The general method of synthesis of these solutes is, the starting materials were mixed and the mixture left in the fume cupboard for a few days till the solids precipitated. To remove any starting materials or impurities in the product, all the solutes were washed several times with n-propanol, isopropanol and dichloromethane, and sucked dry, till a clear white crystalline powder was obtained. The structure and the composition of the products were confirmed by 1H NMR and elemental analysis. Results of elemental analysis from Campbell Microanalytical Laboratory are as shown in Table 3.1. The laboratory claims that ± 0.3 % is the standard error of their results. Hydration state of each solute was estimated from the results of their elemental analysis.

Only two solutes, dimethylthetin and deanol betaine, were obtained as hydrohalides and rest of the solutes were obtained in a free base form. Solutes obtained as a hydrohalide were converted to free base by applying to a Dowex 50 column (H+ form) as described in section 3.2.1. The product was eluted from the column using 5.1 M ammonia solution. The final product was isolated from the eluant by evaporation under reduced pressure using a rotary evaporator.

Though the yield of most of the syntheses was good, low yields were observed for a few solutes such as DEHB, TEHB and HPHB. Different methods of synthesis were tried to increase the yield of the product. To prevent polymerization products being formed during syntheses of DEHB and TEHB, we tried adding hydroquinone in order to inhibit the polymerization reaction. Though adding hydroquinone resulted in better yields, the final product was brownish in colour and several attempts to remove this colour from the solutes were unsuccessful. It was later realised that these brownish solutes were not suitable for spectrophotometric methods of measuring enzyme activity, which involved following the change in absorbance at wavelengths in the near UV. Thus, hydroquinone was not added for further syntheses.

All the three cyclic betaines, CB-1, 2 & 3, were not difficult to synthesize and the products were obtained easily by just mixing the starting materials. However, impurities in the product being the main problem, all the three cyclic betaines had to be washed several times with solvents to remove the starting materials. Furthermore, to recover the final product, solids were suspended in isopropanol overnight and filtered. For unknown reasons, aqueous solutions of CB-2 and CB-3 were acidic and were destabilizing the enzymes during analytical measurements. As CB-2 and CB-3 were synthesized during the finishing stages of this project and due to the time constraints, attempts were not made to remove those acidic impurities. To prevent this problem in future syntheses, we suggest that the solutions of CB-2 and CB-3 should be passed through an anion exchanger and then recrystallized using a rotary evaporator, to get the final product. SB-1 was synthesized using a method described by Barnhurst (1961) and the synthesis was complicated compared to other sulfobetaines. One of the starting materials, vinylsulfonic acid is available only in the form of 25% w/v vinylsulfonic acid sodium salt in water and to covert it into a free acid, concentrated HCl had to be added along with the starting materials. Concentrated HCl was again added during the later stages of the SB-1 synthesis to separate inorganic salts from the product. Due to these additions, the product contained a lot of acidic impurities and had to be resuspended in isopropanol several times to get the pure product. Even though SB-1 went through several cleaning steps, we still suspect that the final product contained a small percentage of impurities and this was evident by the inconsistent results obtained during different experiments using SB-1 (discussed in later chapters).

CSB-1 and CSB-2 were also synthesized during the later stages of the project, so were only used for DNA melting studies and not in enzyme stability experiments. Due to the difficulties faced during synthesis and very low yields, HPHB and TEHB have not been used in any of the experiments in this thesis. Also, CB-2 and CB-3, due to their strong acidic impurities, were not used in any of the experiments.

Table 3.2 lists the estimated costs of synthetic solutes described in this chapter. For comparison purposes, the cost estimation has been based on the raw materials required for the synthesis, using the prices of raw materials in the Sigma and Aldrich catalogues. The listed costs are an estimation based on a laboratory scale synthesis and do not take into account the cost of solvents required for the synthesis, which would be recovered in an industrial process. If an industrial process is designed for the syntheses of these solutes, the cost of the solutes would reduce as the raw materials would be purchased in bulk and syntheses would be in a large scale.

Table 3.2: Estimated cost of 500 g of synthetic compensatory solutes compared with price of 500 g of natural solutes based on the prices in the Sigma catalogue.

Synthetic Solutes Natural Solutes – Sigma, MO, USA Solute Estimated cost of

500g (A $) Solute Price of 500 g (A $)

Propio betaine 66.40 Sorbitol 27.20

DB 678.40 Glycine betaine 164.00 HDB 45.90 Trehalose 959.00 HGB 1173.20 DEHB 56.90 TEHB 32.80 HPHB 88.80 DMT 88.10 CB-1 385.20 CB-2 123.40 CB-3 165.70 Sulfobetaines SB-1 189.10 SB-2 1565.40 SB-3 709.10 SB-4 1381.70 CSB-1 1642.10 CSB-2 1575.10

Comparing the estimated cost of synthetic solutes with the price of natural solutes (Table 3.2), we observe that a few solutes such as propio betaine, HDB, DEHB and DMT are inexpensive and are a fraction of the price of natural solutes. Solutes DB and HGB are slightly expensive compared to other carboxylic acid solutes.

The sulfonic acid analogues or sulfobetaines are expensive as well due to the high cost of their raw materials. However, the costs listed in Table 3.2 are based only on a laboratory scale synthesis, and designing a proper large scale process for the syntheses of these expensive solutes could reduce their cost substantially.

In document Betaine analogues and related compounds for biomedical applications (Page 63-66)