Sialic Acid Synthases from C. jejuni for the Enzymatic Synthesis of … · 2020. 10. 3. · Sialic...
1
D O NHAc OH O CO 2 - HO OH HO O HO OH N N NH 2 O O NHAc OH O HO OH HO CO 2 - O HO OH O O HO HO OH OH O O N N N () 5 N O 50mM Tris pH 8.5 30°C α 2,6SiaT CMP Mg 2+ Acceptor E (2,6) Sialic Acid Synthases from C. jejuni for the Enzymatic Synthesis of Sialoconjugates Mark Schnellbächer , Wolf-Dieter Fessner* Institut für Organische Chemie und Biochemie, Technische Universität Darmstadt, Petersenstraße 22, 64287 Darmstadt Introduction Sialic acids are a family of 9-carbon monosaccharides that represent one of the most important constituents of cell-surface glycoconjugates in vertebrates, where they mediate recognition phenomena in physiological events, e.g. cell cell adhesion and intercellular ‑ ‑ communication processes. However, they are also of importance in the pathogenesis of a variety of diseases, including inflammatory disease, cancer metastasis and bacterial or viral infection. Sialylated glycoconjugates are commerically significant for the synthesis of novel pharmaceuticals. The pathogenic microorganism Campylobacter jejuni has three genes encoding for PEP dependent sialic acid synthases (SiaS). ‑ [1] We have been applied these synthetases to the stereoselective preparation of novel sialic acid analogs, which can be used for the synthesis of novel sialoconjugates in an enzymatic cascade reaction. Synthetic Concept For the synthesis of novel sialylated conjugates by enzymatic cacscade reaction (Figure 1) three different enzymes are needed, for which we used a sialic acid synthetase (SiaS), a CMP sialic acid synthetase (CSS) from ‑ Neisseria meningitidis group B [2,3] and an α (2,6) Sialotransferase ( ‑ ‑ α2,6SiaT) from Photobacterium leiognathi. [4] For the first reaction step we employed the three SiaS enzymes (NeuB1-3) from C. jejuni, which were subcloned and heterologously expressed in E.coli on large scale. Biotrans 2011, Italy, Giardini Naxos Figure 1. General scheme for the enzymatic cascade reation (PEP= phosphoenolpyruvate; CTP= cytidine 5`-triphosphate) Results and Discussion A variety of monosaccharides and their derivatives (Table 1) were used to investigate the substrate specificity of the SiaS enzymes (NeuB1-3). Experiments were conducted with different ratios of sugar electrophile and PEP nucleophile. Activity of SiaS enzymes was tested at temperatures from r.t. up to 60°C. The scale up reactions with entries 10 11 (Figure 2a) were made under identical conditions, ‑ ‑ and products (A) were purified by BioGel and ion exchange chromatography. Interestingly, ‑ a by-product (B) accumulated during the reaction, which proved difficult to be separated from products. Independent experiments demonstrated that its origin arose from an unprecedented enzymatic reaction between the Tris buffer and PEP (Figure 2b). Thus, for preparative synthesis of (A) the buffer system had to be changed to 50mM triethanolamine to avoid by-product forming. Figure 2. [a] Scale-up with entry 9 (R 1 =OH;R 2 =H) and 10 (R 1 =H;R 2 =OH) [b] unprecedented formation of by-product from Tris and PEP For the subsequent cascade step, the CSS-catalyzed nucleotide activation of the sialic acid (Figure 3), we first tested the product of entry 10 (5‑epi Neu5Ac). The reaction was ‑ performed at 37°C with TLC monitoring against Neu5Ac as a positive control. In comparision, the CSS-catalyzed activation of 5‑epi Neu5Ac ( ‑ C) proved rather slow, and complete conversion was not attained. Figure 3. Activation of 5-epi-Neu5Ac with CMP-sialic acid synthetase (CSS) However, when the CMP-activation of 5‑epi Neu5Ac ( ‑ D) was coupled to sialotransfer catalyzed by α(2,6)SiaT in the presence of a fluorescence-labeled lactosyl-acceptor successful formation of an expected sialotrisaccharide was observed (Figure 4). Purification of the fluorescent sialoconjugate (E) was achieved by simple chromatography over RP 18 ‑ silica using a methanol gradient. The structure of isolated product was fully corroborated by NMR spectroscopic analysis. ‑ Figure 4. [a] Transfer of CMP-activated 5- epi-Neu5Ac using α-(2,6)-Sialyltransferase ( α2,6SiaT) [b] 2D-NMR spectra from purified product E (left: COSY, right: HSQC) Table 1. Substrate specificity of recombinant sialic acid synthetases (NeuB1-3) from C. jejuni. Reference [1] Linton, D.; Karlyshev, A.V.; Hitchen, P.G.; Morris, H.R.; Dell, A.; Gregson, N.A.; Wren, B.W. Mol. Microbiol. 2000, 35, 1120-1134 [2] M. Knorst, W.-D. Fessner, Adv. Synth. Catal. 2001, 343, 698. [3] N. He; D. Yi; W.-D. Fessner, Adv. Synth. Catal. 2011, 353, 2384 [4] D. Yi; W.-D. Fessner, see Poster # [5] Sundaram, A.K.; Pitts, L.; Muhammad, K.; Wu, J.; Betenbaugh, M.; Woodard, R.W.; Biochem. J., 2004, 383, 83-89 These experiments document that the three SiaS enzymes possess only narrow tolerance with regard to the constitutional and configurational structure of the sugar component. Entries 1 8 were unacceptable as substrates for all of the enzymes. NeuB1 accepts only ‑ ManNAc, its natural substrate (entry 9), [5] while NeuB2 3 both used GlcNAc and GalNAc. ‑ Conversion of the latter to the corresponding sialic acid analogs was rather slow and did not reach quantitative conversion. Verification of product structure required a scale-up of sialic acids synthesis for NMR and MS analysis. 2 2 6 5,6 2 5,6 3,4 7 1 Acridone peaks [a] [b] [a] [b] D-Hexose PEP P i CTP PP i SiaS Sialic acid analog CSS CMP- sialate Acceptor CMP SiaT Sialo- conjugate O NHAc OH R 1 HO OH PEP P i O NHAc OH OH CO 2 - NeuB3 50mM Tris pH 8.0 37°C R 2 HO OH OH NH 2 + HO H N OH O O HO OPO 3 2- O 2 NeuB3 A B HO OH HO Mg 2+ 37°C PP i 50mM Tris pH 8.5 37°C CSS CTP inorganic pyrophosphatase PO 4 2- Mg 2+ C O NHAc OH OH CO 2 - HO OH HO D O NHAc OH O CO 2 - HO OH HO O HO OH N N NH 2 O C T P C M P Product E Lactosyl- acceptor 1 3 4 5 7 Product D Educt C Reference Neu5Ac Entry Substrate Product 1 2 3 4 5 6 7 8 9 10 11 D-Glucose 5-epi-KDN X X X D-Mannose KDN X X X D-Galactose 5,7-bisepi-KDN X X X 2-Desoxy-glucose 5-dexoxy-KDN X X X 2-Desoxy-galactose 5-dexoxy-7-epi-KDN X X X Glucosamine 5-Amino-5-epi-KDN X X X Mannosamine 5-Amino-KDN X X X Galactosamine 5-Amino-7-epi -KDN X X X N-Acetylmannosamine Neu5Ac quant. X X N-Acetylglucosamine 5-epi -Neu5Ac X yes yes N-Acetylgalactosamine 5,7-bisepi-Neu5Ac X yes yes NeuB1 [a] NeuB2 [b] NeuB3 [b] X: no reaction [a] : 50 mM Bicine pH 8.0 [b] : 50 mM Tris pH 7.0-8.5
Transcript of Sialic Acid Synthases from C. jejuni for the Enzymatic Synthesis of … · 2020. 10. 3. · Sialic...
-
D
O
NHAcOH
O
CO2-
HO
OH
HO
O
HO OH
N
N
NH2
O
O
NHAc
OH
O
HO
OH
HO
CO2-
O
HO
OH
OO
HO
HO
OH
OH
O O N
NN
()5
N
O
50mM Tris pH 8.530°C
α 2,6SiaT
CMP
Mg2+
Acceptor
E
(2,6)
Sialic Acid Synthases from C. jejuni for the Enzymatic Synthesis of Sialoconjugates
Mark Schnellbächer, Wolf-Dieter Fessner* Institut für Organische Chemie und Biochemie, Technische Universität Darmstadt, Petersenstraße 22, 64287 Darmstadt
IntroductionSialic acids are a family of 9-carbon monosaccharides that represent one of the most important constituents of cell-surface glycoconjugates in vertebrates, where they mediate recognition phenomena in physiological events, e.g. cell cell adhesion and intercellular ‑ ‑communication processes. However, they are also of importance in the pathogenesis of a variety of diseases, including inflammatory disease, cancer metastasis and bacterial or viral infection. Sialylated glycoconjugates are commerically significant for the synthesis of novel pharmaceuticals.
The pathogenic microorganism Campylobacter jejuni has three genes encoding for PEP dependent sialic acid synthases (SiaS).‑ [1] We have been applied these synthetases to the stereoselective preparation of novel sialic acid analogs, which can be used for the synthesis of novel sialoconjugates in an enzymatic cascade reaction.
Synthetic ConceptFor the synthesis of novel sialylated conjugates by enzymatic cacscade reaction (Figure 1) three different enzymes are needed, for which we used a sialic acid synthetase (SiaS), a CMP sialic acid synthetase (CSS) from ‑ Neisseria meningitidis group B[2,3] and an α (2,6) Sialotransferase (‑ ‑ α2,6SiaT) from Photobacterium leiognathi.[4] For the first reaction step we employed the three SiaS enzymes (NeuB1-3) from C. jejuni, which were subcloned and heterologously expressed in E.coli on large scale.
Biotrans 2011, Italy, Giardini Naxos
Figure 1. General scheme for the enzymatic cascade reation (PEP= phosphoenolpyruvate;
CTP= cytidine 5`-triphosphate)
Results and DiscussionA variety of monosaccharides and their derivatives (Table 1) were used to investigate the substrate specificity of the SiaS enzymes (NeuB1-3). Experiments were conducted with different ratios of sugar electrophile and PEP nucleophile. Activity of SiaS enzymes was tested at temperatures from r.t. up to 60°C.
The scale up reactions with entries 10 11 (Figure 2a) were made under identical conditions, ‑ ‑and products (A) were purified by BioGel and ion exchange chromatography. Interestingly, ‑a by-product (B) accumulated during the reaction, which proved difficult to be separated from products. Independent experiments demonstrated that its origin arose from an unprecedented enzymatic reaction between the Tris buffer and PEP (Figure 2b). Thus, for preparative synthesis of (A) the buffer system had to be changed to 50mM triethanolamine to avoid by-product forming.
Figure 2. [a] Scale-up with entry 9 (R1=OH;R2=H) and 10 (R1=H;R2=OH)
[b] unprecedented formation of by-product from Tris and PEP
For the subsequent cascade step, the CSS-catalyzed nucleotide activation of the sialic acid (Figure 3), we first tested the product of entry 10 (5‑epi Neu5Ac). The reaction was ‑performed at 37°C with TLC monitoring against Neu5Ac as a positive control. In comparision, the CSS-catalyzed activation of 5‑epi Neu5Ac (‑ C) proved rather slow, and complete conversion was not attained.
Figure 3. Activation of 5-epi-Neu5Ac with CMP-sialic acid synthetase (CSS)
However, when the CMP-activation of 5‑epi Neu5Ac (‑ D) was coupled to sialotransfer catalyzed by α(2,6)SiaT in the presence of a fluorescence-labeled lactosyl-acceptor successful formation of an expected sialotrisaccharide was observed (Figure 4). Purification of the fluorescent sialoconjugate (E) was achieved by simple chromatography over RP 18 ‑silica using a methanol gradient. The structure of isolated product was fully corroborated by NMR spectroscopic analysis.‑
Figure 4. [a] Transfer of CMP-activated 5-epi-Neu5Ac using α-(2,6)-Sialyltransferase (α2,6SiaT) [b] 2D-NMR spectra from purified product E (left: COSY, right: HSQC)
Table 1. Substrate specificity of recombinant sialic acid synthetases (NeuB1-3) from C. jejuni.
Reference[1] Linton, D.; Karlyshev, A.V.; Hitchen, P.G.; Morris, H.R.; Dell, A.; Gregson, N.A.;
Wren, B.W. Mol. Microbiol. 2000, 35, 1120-1134[2] M. Knorst, W.-D. Fessner, Adv. Synth. Catal. 2001, 343, 698.[3] N. He; D. Yi; W.-D. Fessner, Adv. Synth. Catal. 2011, 353, 2384[4] D. Yi; W.-D. Fessner, see Poster #[5] Sundaram, A.K.; Pitts, L.; Muhammad, K.; Wu, J.; Betenbaugh, M.; Woodard, R.W.;
Biochem. J., 2004, 383, 83-89
These experiments document that the three SiaS enzymes possess only narrow tolerance with regard to the constitutional and configurational structure of the sugar component. Entries 1 8 were unacceptable as substrates for all of the enzymes. NeuB1 accepts only ‑ManNAc, its natural substrate (entry 9),[5] while NeuB2 3 both used GlcNAc and GalNAc. ‑Conversion of the latter to the corresponding sialic acid analogs was rather slow and did not reach quantitative conversion. Verification of product structure required a scale-up of sialic acids synthesis for NMR and MS analysis.
2
2
6
5,6
2
5,63,47
1
Acridone peaks
[a]
[b]
[a]
[b]
D-Hexose
PEP PiCTP
PPi
SiaS Sialic acidanalog
CSS
CMP-sialate
Acceptor CMP
SiaT Sialo-conjugate
O
NHAc
OH
R1HO
OH
PEP Pi
O
NHAc
OH
OH
CO2-NeuB350mM Tris pH 8.0
37°C
R2
HO OH
OH
NH2
+ HO
HN
OH
O O
HOOPO32-
O
2
NeuB3
A
B
HO
OH
HO
Mg2+
37°C
PPi
50mM Tris pH 8.537°C
CSS
CTP
inorganicpyrophosphatase
PO42-
Mg2+
C
O
NHAc
OH
OH
CO2-
HO
OH
HO
D
O
NHAcOH
O
CO2-
HO
OH
HO
O
HO OH
N
N
NH2
O
CTP
CMP
Product E
Lactosyl-acceptor
13
4
5
7
Product D
Educt C
Reference
Neu5Ac
Entry Substrate Product
1234567891011
D-Glucose 5-epi-KDN X X XD-Mannose KDN X X XD-Galactose 5,7-bisepi-KDN X X X2-Desoxy-glucose 5-dexoxy-KDN X X X2-Desoxy-galactose 5-dexoxy-7-epi-KDN X X XGlucosamine 5-Amino-5-epi-KDN X X XMannosamine 5-Amino-KDN X X XGalactosamine 5-Amino-7-epi-KDN X X XN-Acetylmannosamine Neu5Ac quant. X XN-Acetylglucosamine 5-epi-Neu5Ac X yes yesN-Acetylgalactosamine 5,7-bisepi-Neu5Ac X yes yes
NeuB1[a] NeuB2[b] NeuB3[b]
X: no reaction [a]: 50 mM Bicine pH 8.0 [b]: 50 mM Tris pH 7.0-8.5
Folie 1
![IOS Press Fucose and sialic acid expressions in human ...U/20 μg protein) [8] to remove the rest of α2,6- and α2,3-sialic acids accessible for lectins. 2.5. The lectin-ELISA procedure](https://static.fdocument.org/doc/165x107/60fbc4a99cf27c5a96062a1c/ios-press-fucose-and-sialic-acid-expressions-in-human-u20-g-protein-8.jpg)
![The host immune response in respiratory virus infection ... · (IFN-α/β receptor), activating the JAK/STAT pathway [3]. ... croup, tonsilitis Seasonal influenza Sialic acids Fever,](https://static.fdocument.org/doc/165x107/5c7b1b0809d3f277748b45cf/the-host-immune-response-in-respiratory-virus-infection-ifn-receptor.jpg)
