**4. Chiral SAMs and enantioselective crystallization on chiral SAMs**

As mentioned above, SAMs are organic assemblies formed by the adsorption of molecules from solution or the gas phase onto the surface of solids. If the adsorbed molecules are chiral, the self assembled monolayer is also rendered chiral. The chirality of the molecule can be distributed within the monolayer interior or located at the terminus of the molecule. However, the chirality is only expressed when the chiral constituent is exposed at the monolayer surface. Chiral SAMs are used in chiral systems. The SAMs can be used to specifically interact with chiral species, such as proteins or amino acids. Chiral SAMs have been used in enantioselective crystallization. In this case, a racemic solution of a chiral molecule is crystallized on a chiral SAM. The chiral SAM serves as a nucleating surface for one of the enantiomers, thereby increasing its crystallization on the SAM. Thus, enantioselective crystallization is achieved.

Dressler and Mastai studied the enantioselective crystallization of glutamic acid on self assembled monolayers of cysteine (Dressler and Mastai, 2007). The chiral SAMs were prepared by immersing gold covered glass slides in aqueous solutions of cysteine (10mM) for 2 hours. These SAMs were then characterized by X-ray diffraction, micro-Raman measurements, X-ray photoelectron spectroscopy, contact angle and ellipsometry.

on small metallic islands, in contrast to large islands, where the α-form is favoured. The increased frequency of the high energy form (β-glycine) with decreasing feature sizes is a result of the high supersaturation that is generated from fast solvent evaporation. (Lee, A.Y.

The crystallization of racemic molecules is very similar to the crystallization of achiral molecules. However, because of their chirality, racemic molecules can form different types of crystals with different compositions. If the crystal lattice contains equal left and right handed molecules arranged in an ordered manner, the crystal is heterochiral and referred to as a racemic compound. In the case where the crystal lattice is composed of only one enantiomer (left or right), the crystal is homochiral and referred to as a conglomerate. In

Chiral resolution is the process of the separation of racemates into their enantiomers. Many methods have been developed for this purpose. The most common is the use of chiral stationary phases in HPLC. However, large scale chiral separation is based on the classical crystallization method, which incorporates diasteriomeric transformation and chiral seeding. Even today, new methods of chiral crystallization are being designed. Lahav et al. developed a method, called "tailor-made" additives, which has been exploited for the kinetic resolution of conglomerates (Addadi, 1982). These additives have molecular structures which are very similar to one of the enantiomorphs of the substrate crystals. Consequentially, the inhibitors stereoselectively adsorb to the surfaces of one of the enantiomorphs, delaying or preventing its growth. As a result, enantiomeric excess is achieved. It is important to mention that in order to acheive chiral resolution by crystallization, the racemate system must spontaneously resolve upon crystallization to a

**4. Chiral SAMs and enantioselective crystallization on chiral SAMs** 

As mentioned above, SAMs are organic assemblies formed by the adsorption of molecules from solution or the gas phase onto the surface of solids. If the adsorbed molecules are chiral, the self assembled monolayer is also rendered chiral. The chirality of the molecule can be distributed within the monolayer interior or located at the terminus of the molecule. However, the chirality is only expressed when the chiral constituent is exposed at the monolayer surface. Chiral SAMs are used in chiral systems. The SAMs can be used to specifically interact with chiral species, such as proteins or amino acids. Chiral SAMs have been used in enantioselective crystallization. In this case, a racemic solution of a chiral molecule is crystallized on a chiral SAM. The chiral SAM serves as a nucleating surface for one of the enantiomers, thereby increasing its crystallization on the SAM. Thus,

Dressler and Mastai studied the enantioselective crystallization of glutamic acid on self assembled monolayers of cysteine (Dressler and Mastai, 2007). The chiral SAMs were prepared by immersing gold covered glass slides in aqueous solutions of cysteine (10mM) for 2 hours. These SAMs were then characterized by X-ray diffraction, micro-Raman

measurements, X-ray photoelectron spectroscopy, contact angle and ellipsometry.

et al., 2006)

conglomerate.

**3. Enantioselective crystallization** 

enantioselective crystallization is achieved.

nature, racemic compounds greatly outnumber conglomerates.

In order to study the interactions between the chiral crystals and the chiral SAMs, pure enantiomers of glutamic acid were first crystallized on the L and D cysteine films. Interestingly, enantiomers of glutamic acid with identical chirality to that of the cysteine SAMs did not grow on the SAMs in a specific orientation and were unchanged from crystals grown in solution (Table 4). On the other hand, enantiomers with chirality opposite to that of the cysteine films grew in a preffered orientation. In either case, morphological differences between crystals grown on the SAMs and those grown in solution were not very significant.


Table 4. Crystal habits and crystal orientation of DL, D and L glutamic acid crystallized on cysteine films and in pure solution. (Dressler and Mastai, 2007)

The chiral cysteine SAMs were then utilized for the enantioselective crystallization of glutamic acid. For this purpose, L and D cysteine SAMs were immersed in supersaturated solutions of DL-glutamic acid for ca. 2 hours. The crystals were then characterized using various techniques. It is important to note here that glutamic acid grows as a monohydrate crystal from water and therefore has a different structure from its enantiomers which were discussed previously. Figure 14 presents SEM images of glutamic acid monohydrate crystals grown on L and D cysteine surfaces and grown from solution. Crystals grown from solution have a needle-like morphology with an average size of 300 μm whereas crystals grown on the cysteine surfaces grow in well ordered arrays of rectangular/plate like shaped crystals with a typical size of 10-30 μm. X-ray diffraction measurements of the crystals showed preffered orientation of the glutamic acid crystals (Table 4).

Fig. 14. SEM images featuring the crystal morphology of rac-glutamic acid crystallized (A) from solution, (B) on a chiral D-cysteine surface and (C) on a chiral L-cysteine surface. (Dressler and Mastai, 2007)

The enantiomeric excess of the crystals grown on the chiral SAMs was investigated using polarimetry. The crystals were gently removed from the chiral films, dissolved in 5N HCl

Crystallization on Self Assembled Monolayers 55

Banno *et al.* studied the enantioselective crystal growth of leucine on self assembled monolayers with covalently attached D or L-leucine molecules (Banno *et al.*, 2004). The monolayers were formed in the following way: gold substrates were prepared by vapour deposition of gold on quartz crystals. The substrates were flame annealed and subsequently immersed in 2 mM ethanolic solutions of 11-mercaptoundecanoic acid (MAU). Leucine was then covalently attached to the MAU monolayer. The crystals on the SAM were grown by

In order to identify the leucine crystals grown on the monolayers, X-ray diffraction and QCM (quartz crystal microbalance) measurements were utilized. Figure 16 shows XRD patterns of the leucine SAMs after immersion in a 175 mM D or L-leucine solution for 3 hrs. For the D-leucine SAM, the diffraction peak was observed only after it was immersed in the D-leucine solution, whereas no peak appeared when it was immersed in the L-leucine solution. With the L-leucine SAM, exactly the opposite results were obtained. The diffraction angles of the two observed peaks were identical to eachother and equal to 6.07°. The above mentioned cross inversion between D and L-leucine strongly suggests that the crystallization on the Leucine SAM is highly enantioselective. The enantioselectivity was also confirmed by the increase in surface mass resulting from the grown leucine crystals on

Fig. 16. XRD patterns of (a) D and (b) L-leucine SAMs after immersion in pure enantiomeric

The leucine monolayers were also immersed in saturated DL-leucine solutions. As shown in Figure 17, a diffraction peak was observed at 6.34° for both D and L-leucine SAMs. This happens because in the presence of equimolar amounts of D and L enantiomers of leucine, the racemic crystal tends to form instead of either one of the two pure enantiomeric crystals.

Fig. 17. XRD patterns of (a) D and (b) L-leucine SAMs after immersion in racemic leucine

immersing the substrates in saturated solutions of D, L or DL-leucine.

the modified SAM, detected by QCM.

leucine solutions. (Banno *et al.*, 2004)

solution. (Banno *et al.*, 2004)

solution and their optical activity was measured. The results show that the enantiomeric excess of glutamic acid crystals collected from the L-cysteine films is about 31% e.e. enriched with D-glutamic acid (Table 5). The crystallization on the D-cysteine films resulted in 27% e.e. enriched with L-glutamic acid. The optical activity of the crystallization solution was also measured (at different intervals). The optical activity results measured from the solution and from the crystals are in good agreement with each other.


Table 5. Enantiomeric excess of crystals crystallized in solutions and on chiral cysteine surfaces. (Dressler and Mastai, 2007)

Dressler and Mastai also studied the crystallization of histidine on the chiral cysteine films. They demonstrated that the cysteine films induce a modification in histidine morphology and lead to specific orientation of the histidine crystals. They also found that the L and D cysteine surfaces were active in chiral discrimination. Again, the enantiomeric excess was ca. 30%.

Singh *et al.* used chiral self assembled monolayers (Figure 15) as resolving auxiliaries in the crystallization of valine (Singh *et al.*, 2010). When starting with racemic solutions, the crystals obtained on the chiral SAMs contained one of the valine enantiomers in excess. The enantiomer obtained in excess was always of opposite chirality to the monolayer used. The chiral resolution was enhanced as a result of decreasing supersaturation. In addition, Singh et. al also monitored the crystallization of valine on chiral SAMs beginning with initial solutions containing one of the enantiomers in excess. When starting with a solution containing 50% ee, crystals of one pure enantiomer were obtained on the SAMs whereas mixtures of the pure enantiomer and the racemic compound were obtained in control experiments, not containing the chiral SAMs. The enantiomer obtained on the chiral SAMs was the enantiomer initially present in excess, regardless of the chirality of the monolayer being used.

Fig. 15. Chemical structures of the chiral self assembled monolayers. (Singh *et al.*, 2010)

solution and their optical activity was measured. The results show that the enantiomeric excess of glutamic acid crystals collected from the L-cysteine films is about 31% e.e. enriched with D-glutamic acid (Table 5). The crystallization on the D-cysteine films resulted in 27% e.e. enriched with L-glutamic acid. The optical activity of the crystallization solution was also measured (at different intervals). The optical activity results measured from the

Table 5. Enantiomeric excess of crystals crystallized in solutions and on chiral cysteine

Dressler and Mastai also studied the crystallization of histidine on the chiral cysteine films. They demonstrated that the cysteine films induce a modification in histidine morphology and lead to specific orientation of the histidine crystals. They also found that the L and D cysteine surfaces were active in chiral discrimination. Again, the enantiomeric excess was ca. 30%.

Singh *et al.* used chiral self assembled monolayers (Figure 15) as resolving auxiliaries in the crystallization of valine (Singh *et al.*, 2010). When starting with racemic solutions, the crystals obtained on the chiral SAMs contained one of the valine enantiomers in excess. The enantiomer obtained in excess was always of opposite chirality to the monolayer used. The chiral resolution was enhanced as a result of decreasing supersaturation. In addition, Singh et. al also monitored the crystallization of valine on chiral SAMs beginning with initial solutions containing one of the enantiomers in excess. When starting with a solution containing 50% ee, crystals of one pure enantiomer were obtained on the SAMs whereas mixtures of the pure enantiomer and the racemic compound were obtained in control experiments, not containing the chiral SAMs. The enantiomer obtained on the chiral SAMs was the enantiomer initially

solution and from the crystals are in good agreement with each other.

present in excess, regardless of the chirality of the monolayer being used.

Fig. 15. Chemical structures of the chiral self assembled monolayers. (Singh *et al.*, 2010)

surfaces. (Dressler and Mastai, 2007)

Banno *et al.* studied the enantioselective crystal growth of leucine on self assembled monolayers with covalently attached D or L-leucine molecules (Banno *et al.*, 2004). The monolayers were formed in the following way: gold substrates were prepared by vapour deposition of gold on quartz crystals. The substrates were flame annealed and subsequently immersed in 2 mM ethanolic solutions of 11-mercaptoundecanoic acid (MAU). Leucine was then covalently attached to the MAU monolayer. The crystals on the SAM were grown by immersing the substrates in saturated solutions of D, L or DL-leucine.

In order to identify the leucine crystals grown on the monolayers, X-ray diffraction and QCM (quartz crystal microbalance) measurements were utilized. Figure 16 shows XRD patterns of the leucine SAMs after immersion in a 175 mM D or L-leucine solution for 3 hrs. For the D-leucine SAM, the diffraction peak was observed only after it was immersed in the D-leucine solution, whereas no peak appeared when it was immersed in the L-leucine solution. With the L-leucine SAM, exactly the opposite results were obtained. The diffraction angles of the two observed peaks were identical to eachother and equal to 6.07°. The above mentioned cross inversion between D and L-leucine strongly suggests that the crystallization on the Leucine SAM is highly enantioselective. The enantioselectivity was also confirmed by the increase in surface mass resulting from the grown leucine crystals on the modified SAM, detected by QCM.

Fig. 16. XRD patterns of (a) D and (b) L-leucine SAMs after immersion in pure enantiomeric leucine solutions. (Banno *et al.*, 2004)

The leucine monolayers were also immersed in saturated DL-leucine solutions. As shown in Figure 17, a diffraction peak was observed at 6.34° for both D and L-leucine SAMs. This happens because in the presence of equimolar amounts of D and L enantiomers of leucine, the racemic crystal tends to form instead of either one of the two pure enantiomeric crystals.

Fig. 17. XRD patterns of (a) D and (b) L-leucine SAMs after immersion in racemic leucine solution. (Banno *et al.*, 2004)

Crystallization on Self Assembled Monolayers 57

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It should be noted that the primary interaction between the enantiomers attached on the SAMs and prenucleation aggregates formed in solution is due to hydrophobic bonding or Van der Waals interaction. It is assumed that crystal growth proceeds only when the chirality of the prenucleation aggregate of pure enantiomers formed in solution is the same as that of the attached enantiomer. In racemic solution, on the contrary, most of the prenucleation aggregates are considered to be in DL-form, resulting in the growth of the racemic crystalline phase on both D and L-leucine attached SAMs.
