**4. Enzymatic synthesis and purification of sialyloligosaccharides**

#### **4.1 Synthesis of sialyloligosaccharides by recombinant sialyltransferases from marine bacteria**

Chemoenzymatic synthesis of various sialyloligosaccharides by mammalian sialyltransferases, and the purification of the product, has been reported (Sabesan & Paulson, 1986). However, mass-production of sialyloligosaccharides by using mammalianderived sialyltransferases remains problematic because the enzymes are unstable and difficult to produce as recombinant proteins in *Escherichia coli*. In comparison to mammalian sialyltransferases, bacterial sialyltransferases are generally more stable and productive in *E. coli* protein-expression systems (Tsukamoto et al., 2007, 2008; Yamamoto et al., 2006), and they show a broader acceptor substrate specificity (Izumi & Wong, 2001; Yu et al., 2005). Here, we report the methods that we developed to use recombinant sialyltransferases from marine bacteria to successfully produce large quantities of 6′-sialyllactose and synthesize various sialyloligosaccharides.

Purification of Marine Bacterial Sialyltransferases and Sialyloligosaccharides 79

(Drouillard et al., 2010). Our method was developed from a microbiological system for the large-scale production of 3′-sialyllactose that used high cell-density cultures of a genetically engineered *E. coli* strain expressing the *Neisseria meningitidis* gene for 2,3-sialyltransferase (Fierfort & Samain, 2008). To date, we have achieved the production of 6′-sialyllactose with a final concentration greater than 30 g L−1 of culture medium, by continuously feeding the culture with an excess of lactose. A detailed report of the production conditions is provided

**4.1.3 Synthesis of various sialyloligosaccharides by using purified recombinant 2,3-**

Using the procedure described in section 4.1.1, we could enzymatically produce 3′ sialyllactose (sialoside 6) by using 2,3-sialyltransferase instead of 2,6-sialyltransferase. While using a recombinant 2,3-sialyltransferase derived from *Photobacterium* sp. JT-ISH-224 to produce 3′-sialyllactose, we detected a by-product in the enzymatic reaction mixture and determined its structure to be 2,3′-disialyllactose (sialoside 7; Mine et al., 2010a). This recombinant 2,3-sialyltransferase can also transfer Neu5Ac from CMP-Neu5Ac to the anomeric hydroxyl groups of mannose and 6-mannobiose to produce sialosides 8 & 9, respectively (Mine et al., 2010b), and transfer Neu5Ac to inositols to produce sialosides 10 &

(A) 3′-sialyllactose (sialoside 6), (B) 2, 3′-disialyllactose (sialoside 7), (C) sialyl-6-mannobiose (sialoside 8), (D) sialyl-mannose (sialoside 9), (E) sialyl-1D-*chiro*-inositol (sialoside 10), (F) sialyl-*epi*-inositol

(E) (F)

11 (Mine et al., 2010c). The structures of sialosides 6–11 are shown in Figure 5.

OH HO OH

OH OH

OH

OH

OH NHAc

OH

HO O

(A) (B)

(C) (D)

HO HO

AcHNOH

HO OH

OH

OH AcHN OH

HO

O

HOOC

COOH

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

O

OH

HO OH

OH

HOOC

HO

HO OH

AcHNOH

OH OH

OH

HO OH

<sup>O</sup> OH OH

O

OH

O

OH NHAc

OH

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

OH HO O

O HOOC

OH

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

O

HO

OH

<sup>O</sup> OH

HO

**sialyltransferase from** *Photobacterium* **sp. JT-ISH-224** 

O

OH

HOOC

O

COOH

O

OH

O

OH

O

O HOOC

HO HO

> HO AcHN

HO OH HO

OH

O OH

OH

HO OH

OH

OH AcHN

O

HO

in Drouillard et al. (2010).

(sialoside 11).

Fig. 5. Structures of sialosides 6–11.

#### **4.1.1 Synthesis of 6′-sialyllactose from lactose and CMP-Neu5Ac by using purified recombinant 2,6-sialyltransferase from** *P. damselae* **JT0160**

Purified recombinant 2,6-sialyltransferase from *P. damselae* JT0160 shows broader acceptor substrate specificity than that of the mammalian enzymes. For example, it could transfer Neu5Ac to not only disaccharides but also mono- and tri-saccharides efficiently, and provided the corresponding sialosides (Fig. 4; Kajihara et al., 1996; Yamamoto et al., 1998).

Below, we describe an example of the enzymatic synthesis of 6′-sialyllactose (sialoside 1).


(A) 6′-sialyllactose (sialoside 1), (B) 2′-fucosyl-6′-sialyllactose (sialoside 2), (C) 3′, 6′-disialyllactose (sialoside 3), (D) 6-sialyl-*N*-acetylgalactosamine (sialoside 4), (E) 6-sialyl-methyl--D-galactopyranoside (sialoside 5). Fig. 4. Structures of sialosides 1–5.

#### **4.1.2** *In vivo* **synthesis of 6′-sialyllactose by using genetically engineered** *E. coli*

Recently, we succeeded in mass-producing 6′-sialyllactose by using a genetically engineered *E. coli* strain expressing the *Photobacterium* sp. JT-ISH-224 gene for 2,6-sialyltransferase

Purified recombinant 2,6-sialyltransferase from *P. damselae* JT0160 shows broader acceptor substrate specificity than that of the mammalian enzymes. For example, it could transfer Neu5Ac to not only disaccharides but also mono- and tri-saccharides efficiently, and provided the corresponding sialosides (Fig. 4; Kajihara et al., 1996; Yamamoto et al., 1998). Below, we describe an example of the enzymatic synthesis of 6′-sialyllactose (sialoside 1). 1. The reaction mixture was composed of 20 mg (55 mol) of lactose (Gal1-4Glc), 79 mg (110 mol) of CMP-Neu5Ac, and 0.6 U of the purified enzyme in 0.5 mL of 100 mM bis-

3. The product formed by the enzymatic reaction was analyzed by using thin layer chromatography (TLC) as follows: a small amount of the enzymatic reaction mixture was applied to a pre-coated silica gel plate (60 F254, Merck, Darmstadt, Germany), which was then developed with 2-propanol/acetic acid/water (3:2:1 v/v); for visualization of the organic compounds, the plate was dipped into a solution of 5% v/v

OH

OH

OH AcHN <sup>O</sup>

OH

(A) (B)

OH

HO

HO AcHN

HO OH

OH

HO HO

> HO AcHN

O

O HO OH

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

OH O

OH <sup>O</sup>

HO

NHAc

HO OH

OH

OH

OH

OH <sup>O</sup>

O HOOC

OH

O HOOC

OH

HO HO

O

OMe OH

OH

AcHNOH

OH

(A) 6′-sialyllactose (sialoside 1), (B) 2′-fucosyl-6′-sialyllactose (sialoside 2), (C) 3′, 6′-disialyllactose (sialoside 3), (D) 6-sialyl-*N*-acetylgalactosamine (sialoside 4), (E) 6-sialyl-methyl--D-galactopyranoside (sialoside 5).

(E)

HO

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

(C) (D)

Recently, we succeeded in mass-producing 6′-sialyllactose by using a genetically engineered *E. coli* strain expressing the *Photobacterium* sp. JT-ISH-224 gene for 2,6-sialyltransferase

**4.1.2** *In vivo* **synthesis of 6′-sialyllactose by using genetically engineered** *E. coli* 

**4.1.1 Synthesis of 6′-sialyllactose from lactose and CMP-Neu5Ac by using purified** 

**recombinant 2,6-sialyltransferase from** *P. damselae* **JT0160** 

2. The reaction mixture was incubated at 30°C for 2 h.

sulfuric acid in ethanol and then heated.

OH <sup>O</sup>

OH <sup>O</sup>

O HOOC

HO HO

> HO HO

> > AcHNOH

HO

AcHNOH

HO OH

OH

O HOOC

O HOOC

OH

O OH

O

OH

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

HO

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

HO

Tris buffer (pH 6.0).

Fig. 4. Structures of sialosides 1–5.

(Drouillard et al., 2010). Our method was developed from a microbiological system for the large-scale production of 3′-sialyllactose that used high cell-density cultures of a genetically engineered *E. coli* strain expressing the *Neisseria meningitidis* gene for 2,3-sialyltransferase (Fierfort & Samain, 2008). To date, we have achieved the production of 6′-sialyllactose with a final concentration greater than 30 g L−1 of culture medium, by continuously feeding the culture with an excess of lactose. A detailed report of the production conditions is provided in Drouillard et al. (2010).

#### **4.1.3 Synthesis of various sialyloligosaccharides by using purified recombinant 2,3 sialyltransferase from** *Photobacterium* **sp. JT-ISH-224**

Using the procedure described in section 4.1.1, we could enzymatically produce 3′ sialyllactose (sialoside 6) by using 2,3-sialyltransferase instead of 2,6-sialyltransferase. While using a recombinant 2,3-sialyltransferase derived from *Photobacterium* sp. JT-ISH-224 to produce 3′-sialyllactose, we detected a by-product in the enzymatic reaction mixture and determined its structure to be 2,3′-disialyllactose (sialoside 7; Mine et al., 2010a). This recombinant 2,3-sialyltransferase can also transfer Neu5Ac from CMP-Neu5Ac to the anomeric hydroxyl groups of mannose and 6-mannobiose to produce sialosides 8 & 9, respectively (Mine et al., 2010b), and transfer Neu5Ac to inositols to produce sialosides 10 & 11 (Mine et al., 2010c). The structures of sialosides 6–11 are shown in Figure 5.

(A) 3′-sialyllactose (sialoside 6), (B) 2, 3′-disialyllactose (sialoside 7), (C) sialyl-6-mannobiose (sialoside 8), (D) sialyl-mannose (sialoside 9), (E) sialyl-1D-*chiro*-inositol (sialoside 10), (F) sialyl-*epi*-inositol (sialoside 11).

Fig. 5. Structures of sialosides 6–11.

Purification of Marine Bacterial Sialyltransferases and Sialyloligosaccharides 81

The reaction solution after enzymatic reaction of substrates with recombinant 2,6 sialyltransferase from *P. damselae* JT0160 strain contained unreacted lactose and CMP-Neu5Ac, free Neu5Ac as result of hydrolysis of CMP-Neu5Ac, and the product. The contents of the fractions eluted with 5, 10, 50, 100, 500, and 1000 mM potassium phosphate buffer (pH 6.8) are shown. A, lactose; N, Neu5Ac; D, CMP-Neu5Ac; R, reaction solution

Many mono-sialyloligosaccharides composed of di-, tri- or tetra-saccharide eluted with 5 to 10 mM potassium phosphate buffer. We also demonstrated that disialyloligosaccharides, such as sialosides 3 (Fig. 4) and 7 (Fig. 5), eluted with 100 mM potassium phosphate buffer. In contrast, many of the unreacted acceptor substrates passed through the column because of their electrically neutral property. Unreacted CMP-Neu5Ac and free Neu5Ac, resulting from the hydrolysis of CMP-Neu5Ac during the reaction, were eluted with 500 and 50 mM potassium phosphate buffer, respectively (Fig. 7). Therefore, it is easy to separate these compounds in the enzymatic reaction mixture with this column chromatography process.

**A N D R FT Wash 5mM 10mM 50mM 100mM 500mM 1M**

The reaction solution after enzymatic reaction with recombinant 2,3-sialyltransferase from *Photobacterium.*sp. JT-ISH-224 strain contained unreacted lactose, CMP-Neu5Ac, free Neu5Ac, and both the mono-sialyloligosaccharide (sialoside 6) as main product (black arrow) and the di-sialyloligosaccharide (sialoside 7) as by-product (red arrow). The contents of the fractions eluted with 5, 10, 50, 100, 500, and 1000 mM potassium phosphate buffer (pH 6.8) are shown. A, lactose; N, Neu5Ac; D, CMP-Neu5Ac; R; reaction solution after enzymatic

During the stepwise elution described above, we sometimes observed that both the reaction product and free Neu5Ac were present in the same fraction. In this case, the separation of these compounds can be improved by increasing the volume of 10 mM potassium

This basic procedure for the separation of sialyloligosaccharide in the enzymatic reaction mixture is more effective when the enzyme reaction produces a single monosialyloligosaccharide. If the reaction mixture contains a variety of mono-sialyloligosaccharides, it is preferable to perform the preparative chromatography using a different column, such as

Fig. 7. Separation of mono-sialyloligosaccharide and di-sialyloligosaccharide from the

reaction solution by using anion–exchange column chromatography.

phosphate buffer (e.g., using 3–5 column volumes of the buffer).

TSKgel Amide-80 (Tosoh Bioscience, Tokyo, Japan) (Endo et al., 2009).

reaction, FT, flow-through.

after enzymatic reaction; FT, flow-through.

#### **4.2 Purification of sialyloligosaccharides by use of column chromatography**

In general, for the separation of oligosaccharides, it is convenient to utilize highperformance liquid chromatography (HPLC), and various types of columns, such as reversephase columns, ion-exchange columns, and gel-filtration columns, that are commercially available. Because Neu5Ac is negatively charged, it is comparatively easy to separate sialyloligosachharide(s) from other neutral oligosaccharides by using anion–exchange column chromatography (Sabesan & Paulson, 1986). For further purification of the compound, gel–filtration column chromatography is effective.

The conditions and method used for each column chromatography step are described below.

#### **4.2.1 Separation of the sialyloligosaccharide and unreacted substrates from the enzymatic reaction mixture**

The basic procedure for anion–exchange column chromatography is as follows:


The column volume required for separation is dictated by the scale of the synthetic reaction. For 10 mg or less of acceptor substrate, all of the reaction product will bind to the resin described above. For more than 100 mg of acceptor substrate, it is desirable to either perform the chromatography process at least twice, or to increase the amount of resin by using a larger column (e.g., ø 2.5 cm × 10 cm).

An example of results obtained for the separation of sialyloligosaccharide by using the above procedure is shown in Figure 6.

**A N D R FT Wash 5mM 10mM 50mM 100mM 500mM 1M**

Fig. 6. TLC analysis of fractions separated by using anion–exchange column chromatography.

In general, for the separation of oligosaccharides, it is convenient to utilize highperformance liquid chromatography (HPLC), and various types of columns, such as reversephase columns, ion-exchange columns, and gel-filtration columns, that are commercially available. Because Neu5Ac is negatively charged, it is comparatively easy to separate sialyloligosachharide(s) from other neutral oligosaccharides by using anion–exchange column chromatography (Sabesan & Paulson, 1986). For further purification of the

The conditions and method used for each column chromatography step are described below.

1. The reaction mixture was diluted with 10 mL of deionized water and introduced onto an Econo column (ø1.0 cm × 10 cm; Bio-Rad Laboratories) containing AG1-X2 ion–

3. Elution of the sialyloligosaccharide was performed twice with 10 mL each of 5, 10, 50,

4. An aliquot of each eluted fraction was analyzed by using TLC, as described in section

The column volume required for separation is dictated by the scale of the synthetic reaction. For 10 mg or less of acceptor substrate, all of the reaction product will bind to the resin described above. For more than 100 mg of acceptor substrate, it is desirable to either perform the chromatography process at least twice, or to increase the amount of resin by

An example of results obtained for the separation of sialyloligosaccharide by using the

**A N D R FT Wash 5mM 10mM 50mM 100mM 500mM 1M**

Fig. 6. TLC analysis of fractions separated by using anion–exchange column

**4.2.1 Separation of the sialyloligosaccharide and unreacted substrates from the** 

The basic procedure for anion–exchange column chromatography is as follows:

2. The column was washed with 3 column volumes (~ 30 mL) of deionized water.

**4.2 Purification of sialyloligosaccharides by use of column chromatography** 

compound, gel–filtration column chromatography is effective.

exchange resin (phosphate form; 200–400 mesh).

using a larger column (e.g., ø 2.5 cm × 10 cm).

above procedure is shown in Figure 6.

100, 500, or 1000 mM potassium phosphate buffer (pH 6.8).

**enzymatic reaction mixture** 

4.1.1.

chromatography.

The reaction solution after enzymatic reaction of substrates with recombinant 2,6 sialyltransferase from *P. damselae* JT0160 strain contained unreacted lactose and CMP-Neu5Ac, free Neu5Ac as result of hydrolysis of CMP-Neu5Ac, and the product. The contents of the fractions eluted with 5, 10, 50, 100, 500, and 1000 mM potassium phosphate buffer (pH 6.8) are shown. A, lactose; N, Neu5Ac; D, CMP-Neu5Ac; R, reaction solution after enzymatic reaction; FT, flow-through.

Many mono-sialyloligosaccharides composed of di-, tri- or tetra-saccharide eluted with 5 to 10 mM potassium phosphate buffer. We also demonstrated that disialyloligosaccharides, such as sialosides 3 (Fig. 4) and 7 (Fig. 5), eluted with 100 mM potassium phosphate buffer. In contrast, many of the unreacted acceptor substrates passed through the column because of their electrically neutral property. Unreacted CMP-Neu5Ac and free Neu5Ac, resulting from the hydrolysis of CMP-Neu5Ac during the reaction, were eluted with 500 and 50 mM potassium phosphate buffer, respectively (Fig. 7). Therefore, it is easy to separate these compounds in the enzymatic reaction mixture with this column chromatography process.

**A N D R FT Wash 5mM 10mM 50mM 100mM 500mM 1M**

Fig. 7. Separation of mono-sialyloligosaccharide and di-sialyloligosaccharide from the reaction solution by using anion–exchange column chromatography.

The reaction solution after enzymatic reaction with recombinant 2,3-sialyltransferase from *Photobacterium.*sp. JT-ISH-224 strain contained unreacted lactose, CMP-Neu5Ac, free Neu5Ac, and both the mono-sialyloligosaccharide (sialoside 6) as main product (black arrow) and the di-sialyloligosaccharide (sialoside 7) as by-product (red arrow). The contents of the fractions eluted with 5, 10, 50, 100, 500, and 1000 mM potassium phosphate buffer (pH 6.8) are shown. A, lactose; N, Neu5Ac; D, CMP-Neu5Ac; R; reaction solution after enzymatic reaction, FT, flow-through.

During the stepwise elution described above, we sometimes observed that both the reaction product and free Neu5Ac were present in the same fraction. In this case, the separation of these compounds can be improved by increasing the volume of 10 mM potassium phosphate buffer (e.g., using 3–5 column volumes of the buffer).

This basic procedure for the separation of sialyloligosaccharide in the enzymatic reaction mixture is more effective when the enzyme reaction produces a single monosialyloligosaccharide. If the reaction mixture contains a variety of mono-sialyloligosaccharides, it is preferable to perform the preparative chromatography using a different column, such as TSKgel Amide-80 (Tosoh Bioscience, Tokyo, Japan) (Endo et al., 2009).

Purification of Marine Bacterial Sialyltransferases and Sialyloligosaccharides 83

the supernatant was loaded onto a Dowex 1 (HCO3 form, Sigma-Aldrich Japan) column (ø 5 x 20 cm). After the column was washed with distilled water, the acidic oligosaccharides retained on the Dowex 1 resin were eluted with 100 mM NaHCO3. The eluted fractions containing acidic oligosaccharides were pooled and the NaHCO3 was removed by treatment with Amberlite IR120 (H+ form) until the pH reached 3. The pH was then adjusted to 6.0

Anion–exchange column chromatography is a powerful method for separating a single mono-sialyloligosaccharide from the other components in the sample solution; however, it is impossible to separate one mono-sialyloligosaccharide from another monosialyloligosaccharide, such as 6′-sialyllactose or 3′-sialyllactose, by using this method. In such cases, HPLC can be used successfully for the separation, as described by Endo et al. (Endo et al., 2009). In this protocol, the HPLC system is equipped with a TSKgel Amide-80 column (particle size 5 mm, ø 4.6 × 250 mm; Tosoh Bioscience), and is run at 40°C with a flow rate of 1mLl/min, and the eluates are monitored with a UV (195 nm) detector. The elution conditions are as follows: 0 to 15 min isocratic elution with 75% acetonitrile in 15 mM potassium phosphate buffer (pH 5.2); and 15 to 45 min linear gradient elution with a

gradient of 75% to 50% acetonitrile in 15 mM potassium phosphate buffer (pH 5.2).

Some examples of HPLC chromatograms produced for sialyl-compounds synthesized as

with NaOH and the acidic oligosaccharide fraction was freeze-dried.

**4.2.3 Separation of various sialyl-compounds by using HPLC** 

described in section 4.1 are shown in Figure 9.

Fig. 9. Chromatograms of some sialyl-compounds.

For further purification of the sialyloligosaccharide, we performed gel–filtration column chromatography. The procedure is as follows:


The purpose of this process is to remove salt carried from the former chromatography process. The product was eluted in the 30th to 50th fractions (Fig. 8). When Neu5Ac was mixed with the product, it could be separated from the product under a lower flow rate (e.g., 1.0 mL/min). The purity of sialyloligosaccharides obtained by using a combination of the two chromatography processes described above is usually more than 95% (data not shown).

Fig. 8. TLC analysis of the fractions separated by use of gel–filtration column chromatography.

The product is usually contained in the 30th to 50th fraction eluted during the gel–filtration column chromatography; a typical example is shown. A; lactose, D; CMP-Neu5Ac, N; Neu5Ac, C; 6′-sialyllactose standard.

#### **4.2.2 Alternative anion-exchange column chromatography method for large-scale purification of 6′-sialyllactose**

As mentioned in section 4.1.2, a large volume of solution containing 6′-sialyllactose could be prepared by using high cell-density cultures of a genetically engineered *E. coli* strain. In such cases, we performed an alternative anion–exchange column chromatography process. At the end of the fermentation, the whole culture was permeabilized by autoclaving at 100°C for 50 min. The mixture was centrifuged at 7,000*g* for 30 min and the supernatant containing the oligosaccharides was removed. The pH of the extracellular fraction was lowered to 3.0 by the addition of a strong cation-exchange resin (Amberlite IR120 H+ form, Sigma-Aldrich Japan, Tokyo), and the proteins that were precipitated by this process were removed by centrifugation. The pH of the clear supernatant was then adjusted to 6.0 by the addition of a weak anion exchanger (Dowex 66 free base form; Sigma-Aldrich Japan) and, after decanting,

For further purification of the sialyloligosaccharide, we performed gel–filtration column

2. The dried residue was dissolved in 2.5 mL of deionized water and then loaded onto a Sephadex G-15 column (ø 1.6 × 70 cm) and eluted with deionized water under a 2.5

3. The fractions containing glycosidic Neu5Ac were pooled and evaporated to dryness. The purpose of this process is to remove salt carried from the former chromatography process. The product was eluted in the 30th to 50th fractions (Fig. 8). When Neu5Ac was mixed with the product, it could be separated from the product under a lower flow rate (e.g., 1.0 mL/min). The purity of sialyloligosaccharides obtained by using a combination of the two chromatography processes described above is usually more than 95% (data not shown).

**A D N C 30 31 32 35 40 44 45 46 48**

Fig. 8. TLC analysis of the fractions separated by use of gel–filtration column chromatography.

The product is usually contained in the 30th to 50th fraction eluted during the gel–filtration column chromatography; a typical example is shown. A; lactose, D; CMP-Neu5Ac, N;

As mentioned in section 4.1.2, a large volume of solution containing 6′-sialyllactose could be prepared by using high cell-density cultures of a genetically engineered *E. coli* strain. In such cases, we performed an alternative anion–exchange column chromatography process. At the end of the fermentation, the whole culture was permeabilized by autoclaving at 100°C for 50 min. The mixture was centrifuged at 7,000*g* for 30 min and the supernatant containing the oligosaccharides was removed. The pH of the extracellular fraction was lowered to 3.0 by the addition of a strong cation-exchange resin (Amberlite IR120 H+ form, Sigma-Aldrich Japan, Tokyo), and the proteins that were precipitated by this process were removed by centrifugation. The pH of the clear supernatant was then adjusted to 6.0 by the addition of a weak anion exchanger (Dowex 66 free base form; Sigma-Aldrich Japan) and, after decanting,

**4.2.2 Alternative anion-exchange column chromatography method for large-scale** 

**Fraction No.**

1. The fractions containing glycosidic Neu5Ac were evaporated to dryness.

mL/min flow rate and collected in increments of 1 mL.

chromatography. The procedure is as follows:

Neu5Ac, C; 6′-sialyllactose standard.

**purification of 6′-sialyllactose** 

the supernatant was loaded onto a Dowex 1 (HCO3 form, Sigma-Aldrich Japan) column (ø 5 x 20 cm). After the column was washed with distilled water, the acidic oligosaccharides retained on the Dowex 1 resin were eluted with 100 mM NaHCO3. The eluted fractions containing acidic oligosaccharides were pooled and the NaHCO3 was removed by treatment with Amberlite IR120 (H+ form) until the pH reached 3. The pH was then adjusted to 6.0 with NaOH and the acidic oligosaccharide fraction was freeze-dried.

#### **4.2.3 Separation of various sialyl-compounds by using HPLC**

Anion–exchange column chromatography is a powerful method for separating a single mono-sialyloligosaccharide from the other components in the sample solution; however, it is impossible to separate one mono-sialyloligosaccharide from another monosialyloligosaccharide, such as 6′-sialyllactose or 3′-sialyllactose, by using this method. In such cases, HPLC can be used successfully for the separation, as described by Endo et al. (Endo et al., 2009). In this protocol, the HPLC system is equipped with a TSKgel Amide-80 column (particle size 5 mm, ø 4.6 × 250 mm; Tosoh Bioscience), and is run at 40°C with a flow rate of 1mLl/min, and the eluates are monitored with a UV (195 nm) detector. The elution conditions are as follows: 0 to 15 min isocratic elution with 75% acetonitrile in 15 mM potassium phosphate buffer (pH 5.2); and 15 to 45 min linear gradient elution with a gradient of 75% to 50% acetonitrile in 15 mM potassium phosphate buffer (pH 5.2).

Some examples of HPLC chromatograms produced for sialyl-compounds synthesized as described in section 4.1 are shown in Figure 9.

Fig. 9. Chromatograms of some sialyl-compounds.

Purification of Marine Bacterial Sialyltransferases and Sialyloligosaccharides 85

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The sample solution (150 mL) containing sialyl-compound(s) was separated as described in section 4.2.3. A; an example of the separation of two mono-sialyloligosaccharides, B; examples of the chromatogram of various mono-sialyloligosaccharides.

### **5. Conclusion**

It is now possible to produce large amounts of sialyloligosaccharides by using newly developed methods, including chemoenzymatic methods and fermentation methods. It is also possible to produce huge quantities of sialyltransferase enzymes. However, large-scale production of other glycosyltransferases, such as *N*-acetylglucosaminyltransferase or fucosyltransferase, is still difficult. For this reason, it is of great importance to identify enzymes that could be used in the production of other glycosyltransferases and to establish mass-production methods for these enzymes.

#### **6. Acknowledgment**

The authors would like to thank Ms. Hitomi Kajiwara for her valuable comments and all of their collaborators.

#### **7. References**


The sample solution (150 mL) containing sialyl-compound(s) was separated as described in section 4.2.3. A; an example of the separation of two mono-sialyloligosaccharides,

It is now possible to produce large amounts of sialyloligosaccharides by using newly developed methods, including chemoenzymatic methods and fermentation methods. It is also possible to produce huge quantities of sialyltransferase enzymes. However, large-scale production of other glycosyltransferases, such as *N*-acetylglucosaminyltransferase or fucosyltransferase, is still difficult. For this reason, it is of great importance to identify enzymes that could be used in the production of other glycosyltransferases and to establish

The authors would like to thank Ms. Hitomi Kajiwara for her valuable comments and all of

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B; examples of the chromatogram of various mono-sialyloligosaccharides.

**5. Conclusion** 

**6. Acknowledgment** 

their collaborators.

**7. References** 

mass-production methods for these enzymes.

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**5** 

*Colombia* 

**Simple Preparation of New** 

Vladimir V. Kouznetsov, Carlos E. Puerto Galvis,

**Potential Bioactive Nitrogen-Containing** 

 Leonor Y. Vargas Méndez and Carlos M. Meléndez Gómez  *School of Chemistry, Industrial University of Santander, Bucaramanga,* 

**Molecules and Their Spectroscopy Analysis** 

The impact of research on the small molecules chemistry is difficult to quantify and currently, it is still one of the most active areas of organic chemistry, medicinal chemistry and lately chemical biology. In recent years, a lot of interest has been shown in the preparation of nitrogen-containing compounds due to their numerous biologically significant activities. But it is the separation and purification process of the new synthetized

Many texts about the simple and optimal preparation of bioactive compounds have been published, and in this chapter the multicomponent reactions and efficient linear process, which allow the synthesis of this kind of structures, will be discussed. However, the purpose of this chapter is to reveal those important aspects that finally determined why a molecule can be used and distributed as a drug: their preparation, purification, and

In almost all organic synthetic methodologies the purification process use simple column chromatography techniques (gravity or external pressure) using different support materials (solid adsorbents) as the stationary phase. Column chromatography is advantageous over most of the other chromatographic techniques because it can be used in both analytical and preparative applications. After the preparation and purification of a new compound has been realized, it becomes the characterization step. New purified molecules must be strongly characterized to determine its structural configuration. Among different analysis techniques, NMR experiments and X-Ray crystallography are the most efficient ways to determine the relative stereochemistry and, in suitable cases, also the absolute configuration

In the development of our medicinal program directed to small molecules for drug delivery, the strategies for the preparation of nitrogen-containing molecules such as substituted indoles, tetrahydroquinolines, and N-substituted amides of carboxylic acids are illustrated in this chapter as well as their synthetic applications and analytic characterization. The discussion is complemented with a deep explanation of the analytical techniques employed

organic molecules, the ones that take a key role in drug design and development.

**1. Introduction** 

characterization.

of the obtained products.

