- Email: [email protected]
Efficient chemoenzymatic synthesis of UDP-α-6-N3-glucose
Accepted Manuscript Efficient chemoenzymatic synthesis of UDP-α-6-N3-glucose Jiajia Wang, Dongzhe Zhang, Yinhang Wen, Xuefeng Cao, Jing Ma, Peng George Wang PII: DOI: Reference:
S0960-894X(19)30066-6 https://doi.org/10.1016/j.bmcl.2019.02.002 BMCL 26282
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date: Revised Date: Accepted Date:
14 November 2018 24 December 2018 1 February 2019
Please cite this article as: Wang, J., Zhang, D., Wen, Y., Cao, X., Ma, J., George Wang, P., Efficient chemoenzymatic synthesis of UDP-α-6-N3-glucose, Bioorganic & Medicinal Chemistry Letters (2019), doi: https://doi.org/10.1016/ j.bmcl.2019.02.002
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Efficient chemoenzymatic synthesis of UDP-α-6-N3-glucose Jiajia Wang Wang c,*
#,a,c
, Dongzhe Zhang #,a, Yinhang Wen a, Xuefeng Cao c, Jing Ma b,* , Peng George
a. School of Basic Medical Sciences, Henan University Joint National Laboratory for Antibody Drug Engineering, Kaifeng, Henan, 475004, People’s Republic of China b. Institute of Chemical Biology, College of Pharmacy, Henan University, Kaifeng, People’s Republic of China c. Department of Chemistry and Center of Diagnostics & Therapeutics, Georgia State University, 50 Decatur St SE, Atlanta, Georgia, 30303, United States To whom correspondence should be addressed: Jing Ma, [email protected]; Peng George Wang, Email: [email protected];
Abstract A novel chemo-enzymatic synthetic method for UDP-α-6-N3-glucose was developed by combining the versatility of chemical synthesis and natural enzyme stereoselectivity of Bifidobacterium longum (BLUSP). This flexible and efficient platform expanded the substrate scope for UDP-sugars on an improved scale, particularly for UDP-sugar substrates containing bioorthogonal functional groups. Metabolic labeling strategy, where unnatural sugar nucleotides containing bioorthogonal reactive groups are introduced to glycoconjugates, is popular for the identification and visualization of glycoconjugates in vivo [1]. Alternatively, chemoenzymatic labeling transfers sugar nucleotides with bioorthogonal functional groups, such as azide- or alkyne-groups, onto a target acceptor, providing a robust strategy to facilitate proteomic and DNA analysis in vitro [2-4]. Bioorthogonal functional groups in the above mentioned strategies can be conjugated with either fluorescent tags for direct visualization or affinity probes for enrichment by click chemistry, both of which precisely indicate the location and/or characterization of the modified proteins. The sixth DNA base, 5-hydroxymethylcytosine (5hmC) after the fifth base 5methylcytosine (5mC), is commonly found in all cytosine variants of the DNA sequence [5]. This base has attracted significant attention because it can be used as an epigenetic marker in embryonic stem cells and mammalian brains [6-8]. Recent studies have demonstrated that 5hmC act as signal sites of active 5mC demethylation [9] and may directly modulate gene expression [10]. Therefore, a rapid and effective method to quantify and map the distribution of 5hmC at the DNA level is necessary for understanding its role in gene expression and regulation. However, a major challenge with respect to distinguishing 5hmC from 5mC remains. Traditional approaches, such as anti-5hmC antibodies and bisulfite sequencing, suffer from low specificity toward 5mC in immunoprecipitation assays [8, 11]. Although many methods have been developed to detect 5hmC, many well-documented drawbacks remains [12-15]. 1
Recently, Song et al. showed that wild type β-glucosyltransferase (β-GT) can transfer the engineered glucose moiety, UDP-α-6-N3-Glc which contains a biorthogonal group, onto the hydroxyl group of 5hmC. The azido handle can be selectively tagged with biotin using copper-free click chemistry for further detection, enrichment, and sequence analysis of 5hmC-containing DNA fragments in mammalian genomes [16] (Figure 1). Moreover, it is highly sensitive for clinical analysis if 5hmC is attached to a fluorescent reporter, for instance, in solid tumor samples and white blood cells where 5hmC modification is extremely low [17-19]. Moreover, Narek et al. recently demonstrated that O-GlcNAc transferase (OGT) can catalyze intracellular protein modification with UDPα-6-N3-Glc using the substrate promiscuity of OGT under certain conditions, which provided a fresh consideration of the O-GlcNAcylation [20]. Therefore, the development of an efficient procedure for the preparation of UDP-α-6-N3-Glc would be important for meeting the needs of the growing glycoscience community.
Detection UDP-α-6N3 -Glucose
Affinity purification Biotin-alkyne
Sequencing
lysate or 5-hmc
UDP-α-6N3 -Gl ucose:
Figure 1. Selective labeling of 5-hmC in genomic DNA using UDP-α-6N3-glucose.
To date, various methodologies have been developed to generate UDP-sugars. Chemical synthesis allows for the preparation of a diverse range of natural and unnatural structures but is challenging due to poor anomeric stereo selectivity, the need for multistep procedures with advanced purification requirements, and low isolated yields of the last two key synthetic steps. For example, Narek et al. only isolated the final desired products with a yield of 7% after stirring for 8 days, and Song et al. purified UDP-sugar with C18 reverse-phase HPLC with an unreported yield [16, 20, 21]. Meanwhile, enzymatic methods can directly convert the free sugar into desired sugar nucleotides without the need for protecting groups and afford the product with perfect regio- and stereo-selectivity. However, enzyme utilization is much less flexible because of the limited substrate specificity [22-24]. Recently, a hybrid chemoenzymatic strategy, combining chemical and enzymatic methods, has been successfully applied for the efficient synthesis of different sugar libraries. Using this strategy, glycan core precursors were first prepared via chemical synthesis and the precursors were subsequently extended to diverse and complex glycans via catalysis with robust glycosyltransferases [25-27]. Herein, a chemoenzymatic strategy for the synthesis of UDP-α-6N3-glucose is reported, showing efficient preparation of the desired product in a 190 mg scale with relatively simple operation that will facilitate chemoenzymatic labeling of glycoproteins or DNA. 2
Scheme 1. Chemoenzymatic synthesis of UDP-α-6-N3-Glucose.
Starting from the commercially available of 1,2:5,6-di-O-isopropylidene-α-Dglucofuranose 1, compound 3 was afforded by selective removal of the isopropylidene protecting groups at the 5,6-sites in the presence of acetic acid aqueous solution and following tosylation at the 6-hydroxy position. Subsequently, the treatment of 3 with NaN3 afforded the azido substituted furanose 4, which was then converted to the pyranose form of 6-N3-glucose 5 by heating to reflux in an 80% acetic acid aqueous solution. From intermediate 5, 6-N3-glucose-1-phospahte 7 was chemically prepared according to the procedure reported by Edgar et al. [28]. Briefly, the unprotected sugar 5 was reacted with p-toluenesulfonyl hydrazine to furnish a glycosylsulfonylhydrazide adduct in 92% yield, and further oxidized using anhydrous copper chloride in the presence 2-methyl-2-oxazoline and excess crystalline phosphoric acid to afford the corresponding α/β mixtures of 6-N3-glucose-1-phospahte 7. After complete reaction, the reaction mixture was poured into dichloromethane to precipitate the crude 7, which was then treated with water and saturated barium hydroxide to precipitate the excess phosphoric acid. The insoluble barium salt was removed by centrifugation and the entire process was repeated until no more precipitate was generated. The supernatant liquid was collected and concentrated before precipitation from ethanol. Due to their possible toxicity to the enzyme, the barium salts of 7 were re-dissolved in water and treated with 1M sodium carbonate to produce sodium salts of 6-N3-glucose-1-phospahte as a candidate for conversion of UDP-α-6-N3-glucose with an α/β ratio 3:1.
3
After obtaining 6-N3-glucose-1-phospahte, we expressed the relevant Bifidobacterium longum (BLUSP) enzyme, which has been reported to yield UDP-Glc, UDP-Gal, UDPMan, and their derivatives from the corresponding 1-phosphate substrates in a one-pot multienzyme manner [29]. The enzymatic reaction mixture containing substrate 7, BLUSP, MgCl2, uridine triphosphate (UTP), and Tris-HCl (pH 7.5) was incubated overnight at 37 ℃. When the majority of 7 was converted to the desired UDP-α-6-N3glucose 8 according to TLC analysis, the reaction was quenched by adding an equivalent amount of cold ethanol. After purification with a Bio-gel P-2 column, 190 mg of the target compound 8 was obtained representing a yield of 46%. Purity analysis using capillary electrophoresis (CE) confirmed the satisfactory purity of the product with a retention time of 11.5 min. In the associated HRMS spectrum, two peaks at m/z 590.0531 [M-H] and m/z 612.0349 [M-2H+Na] were detected. Moreover, the structure of UDP-α6-N3-glucose was confirmed by 1H NMR, 13C NMR, and 31P NMR (see Supporting Information). In summary, these results indicated that UDP-α-6-N3-glucose can be
YPLIU #115-126 RT: 2.49-2.73 AV: 12 NL: 2.91E4 T: FTMS - p ESI Full ms [200.00-2000.00] 590.0531 z=1
100 95 90 85 80 75 70
Relative Abundance
65 612.0349 z=1
60 55 50 45 471.0873 z=1
40 35 30 25 20 15
669.9936 z=1 460.9269 z=1 445.0500 z=1
472.0907 z=1 490.1547 z=2
504.9422 z=1
10
584.8830 591.0564 z=1 z=1 526.9242 z=1
5
529.0459 z=?
555.0248 z=1
540
560
574.1506 z=?
592.0572 z=1
669.0454 z=? 727.9521 613.0383 671.9907 z=1 z=1 691.9753 634.0169 z=1 z=1 z=1 710.0117 648.0117 z=1 z=?
0 460
480
500
520
580
600
620
640
660
680
m/z
Figure 2. CE and HRMS analysis data of UDP-α-6-N3-glucose.
4
700
720
efficiently prepared using the chemoenzymatic approach developed herein. In conclusion, we developed a chemoenzymatic strategy for the efficient preparation of UDP-α-6-N3-glucose. As no known kinase can specifically produce α-6-N3-glucose-1phosphate, we used a versatile chemical phosphorylation strategy to produce 6-N3glucose-1-phosphate, 7. The described protocol circumvents the traditional challenges of chemical synthesis by eliminating the need for protecting groups and advanced purification equipment. Although the obtained product is a mixture of α and β isomers, the stereo-pure product was obtained in good yield from α/β mixtures of the monosaccharide-1-P precursors from combination with the natural stereo-selective BLUSP enzyme. Thus, a facile and applicable approach for the production of the biologically useful nucleotide sugar UDP-α-6-N3-glucose on a preparative scale was demonstrated. This approach can be implemented for the biosynthesis of any nucleotide sugar and its derivatives, particularly for molecules containing biorthogonal function groups used to identify target proteins by in vitro incorporation [4, 20, 30]. Author Contributions: # These authors contributed equally.
Acknowledgments: This work was supported by the Natural Science Foundation of China (31000371, 21807025) and Key Scientific Research Projects of Universities in Henan (19B15003). The authors have declared no conflict of interest. Reference 1.
Palaniappan, K.K. and C.R. Bertozzi, Chemical Glycoproteomics. Chemical Reviews, 2016. 116(23): p. 14277-14306.
2.
Clark, P.M., et al., Direct in-gel fluorescence detection and cellular imaging of OGlcNAc-modified proteins. J Am Chem Soc, 2008. 130(35): p. 11576-7.
3.
Wen, L., et al., Two-Step Chemoenzymatic Detection of N-Acetylneuraminic Acidalpha(2-3)-Galactose Glycans. J Am Chem Soc, 2016. 138(36): p. 11473-6.
4.
Wen, L., et al., A One-Step Chemoenzymatic Labeling Strategy for Probing Sialylated Thomsen-Friedenreich Antigen. ACS Cent Sci, 2018. 4(4): p. 451-457.
5.
Kothari, R.M. and V. Shankar, 5-Methylcytosine content in the vertebrate deoxyribonucleic acids: species specificity. J Mol Evol, 1976. 7(4): p. 325-9.
6.
Kriaucionis, S. and N. Heintz, The Nuclear DNA Base 5-Hydroxymethylcytosine Is Present in Purkinje Neurons and the Brain. Science, 2009. 324(5929): p. 929-930. 5
7.
Tahiliani, M., et al., Conversion of 5-Methylcytosine to 5-Hydroxymethylcytosine in Mammalian DNA by MLL Partner TET1. Science, 2009. 324(5929): p. 930-935.
8.
Ito, S., et al., Role of Tet proteins in 5mC to 5hmC conversion, ES-cell self-renewal and inner cell mass specification. Nature, 2010. 466(7310): p. 1129-U151.
9.
Hashimoto, H., et al., Recognition and potential mechanisms for replication and erasure of cytosine hydroxymethylation. Nucleic Acids Res, 2012. 40(11): p. 4841-9.
10.
Branco, M.R., G. Ficz, and W. Reik, Uncovering the role of 5-hydroxymethylcytosine in the epigenome. Nature Reviews Genetics, 2012. 13(1): p. 7-13.
11.
Huang, Y., et al., The behaviour of 5-hydroxymethylcytosine in bisulfite sequencing. PLoS One, 2010. 5(1): p. e8888.
12.
Jin, S.G., S. Kadam, and G.P. Pfeifer, Examination of the specificity of DNA methylation profiling techniques towards 5-methylcytosine and 5-hydroxymethylcytosine. Nucleic Acids Res, 2010. 38(11): p. e125.
13.
Wang, H., et al., Comparative characterization of the PvuRts1I family of restriction enzymes and their application in mapping genomic 5-hydroxymethylcytosine. Nucleic Acids Res, 2011. 39(21): p. 9294-305.
14.
Leitch, H.G., et al., Naive pluripotency is associated with global DNA hypomethylation. Nat Struct Mol Biol, 2013. 20(3): p. 311-6.
15.
Spruijt, C.G., et al., Dynamic readers for 5-(hydroxy)methylcytosine and its oxidized derivatives. Cell, 2013. 152(5): p. 1146-59.
16.
Song, C.X., et al., Selective chemical labeling reveals the genome-wide distribution of 5hydroxymethylcytosine. Nature Biotechnology, 2011. 29(1): p. 68-72.
17.
Nestor, C.E., et al., Tissue type is a major modifier of the 5-hydroxymethylcytosine content of human genes. Genome Res, 2012. 22(3): p. 467-77.
18.
Shahal, T., et al., Spectroscopic quantification of 5-hydroxymethylcytosine in genomic DNA. Anal Chem, 2014. 86(16): p. 8231-7.
19.
Chen, H.Y., et al., Spectroscopic quantification of 5-hydroxymethylcytosine in genomic DNA using boric acid-functionalized nano-microsphere fluorescent probes. Biosens Bioelectron, 2017. 91: p. 328-333.
20.
Darabedian, N., et al., The Metabolic Chemical Reporter 6-Azido-6-deoxy-glucose Further Reveals the Substrate Promiscuity of O-GlcNAc Transferase and Catalyzes the Discovery of Intracellular Protein Modification by O-Glucose. J Am Chem Soc, 2018. 140(23): p. 7092-7100.
6
21.
Nifker, G., et al., One-Pot Chemoenzymatic Cascade for Labeling of the Epigenetic Marker 5-Hydroxymethylcytosine. Chembiochem, 2015. 16(13): p. 1857-1860.
22.
Li, L., et al., Efficient enzymatic synthesis of guanosine 5'-diphosphate-sugars and derivatives. Org Lett, 2013. 15(21): p. 5528-30.
23.
Muthana, M.M., et al., Efficient one-pot multienzyme synthesis of UDP-sugars using a promiscuous UDP-sugar pyrophosphorylase from Bifidobacterium longum (BLUSP). Chem Commun (Camb), 2012. 48(21): p. 2728-30.
24.
Muthana, M.M., et al., Improved one-pot multienzyme (OPME) systems for synthesizing UDP-uronic acids and glucuronides. Chem Commun (Camb), 2015. 51(22): p. 4595-8.
25.
Wang, S., et al., Facile Chemoenzymatic Synthesis of O-Mannosyl Glycans. Angew Chem Int Ed Engl, 2018. 57(30): p. 9268-9273.
26.
Yang, J., et al., Structure-based engineering of E. coli galactokinase as a first step toward in vivo glycorandomization. Chem Biol, 2005. 12(6): p. 657-64.
27.
Wen, L., et al., Toward Automated Enzymatic Synthesis of Oligosaccharides. Chem Rev, 2018.
28.
Edgar, L.J., S. Dasgupta, and M. Nitz, Protecting-group-free synthesis of glycosyl 1phosphates. Org Lett, 2012. 14(16): p. 4226-9.
29.
Muthana, M.M., et al., Efficient one-pot multienzyme synthesis of UDP-sugars using a promiscuous UDP-sugar pyrophosphorylase from Bifidobacterium longum (BLUSP). Chemical Communications, 2012. 48(21): p. 2728-2730.
30.
Li, J., et al., An OGA-Resistant Probe Allows Specific Visualization and Accurate Identification of O-GlcNAc-Modified Proteins in Cells. ACS Chem Biol, 2016. 11(11): p. 3002-3006.
7
8
S0960-894X(19)30066-6 https://doi.org/10.1016/j.bmcl.2019.02.002 BMCL 26282
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date: Revised Date: Accepted Date:
14 November 2018 24 December 2018 1 February 2019
Please cite this article as: Wang, J., Zhang, D., Wen, Y., Cao, X., Ma, J., George Wang, P., Efficient chemoenzymatic synthesis of UDP-α-6-N3-glucose, Bioorganic & Medicinal Chemistry Letters (2019), doi: https://doi.org/10.1016/ j.bmcl.2019.02.002
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Efficient chemoenzymatic synthesis of UDP-α-6-N3-glucose Jiajia Wang Wang c,*
#,a,c
, Dongzhe Zhang #,a, Yinhang Wen a, Xuefeng Cao c, Jing Ma b,* , Peng George
a. School of Basic Medical Sciences, Henan University Joint National Laboratory for Antibody Drug Engineering, Kaifeng, Henan, 475004, People’s Republic of China b. Institute of Chemical Biology, College of Pharmacy, Henan University, Kaifeng, People’s Republic of China c. Department of Chemistry and Center of Diagnostics & Therapeutics, Georgia State University, 50 Decatur St SE, Atlanta, Georgia, 30303, United States To whom correspondence should be addressed: Jing Ma, [email protected]; Peng George Wang, Email: [email protected];
Abstract A novel chemo-enzymatic synthetic method for UDP-α-6-N3-glucose was developed by combining the versatility of chemical synthesis and natural enzyme stereoselectivity of Bifidobacterium longum (BLUSP). This flexible and efficient platform expanded the substrate scope for UDP-sugars on an improved scale, particularly for UDP-sugar substrates containing bioorthogonal functional groups. Metabolic labeling strategy, where unnatural sugar nucleotides containing bioorthogonal reactive groups are introduced to glycoconjugates, is popular for the identification and visualization of glycoconjugates in vivo [1]. Alternatively, chemoenzymatic labeling transfers sugar nucleotides with bioorthogonal functional groups, such as azide- or alkyne-groups, onto a target acceptor, providing a robust strategy to facilitate proteomic and DNA analysis in vitro [2-4]. Bioorthogonal functional groups in the above mentioned strategies can be conjugated with either fluorescent tags for direct visualization or affinity probes for enrichment by click chemistry, both of which precisely indicate the location and/or characterization of the modified proteins. The sixth DNA base, 5-hydroxymethylcytosine (5hmC) after the fifth base 5methylcytosine (5mC), is commonly found in all cytosine variants of the DNA sequence [5]. This base has attracted significant attention because it can be used as an epigenetic marker in embryonic stem cells and mammalian brains [6-8]. Recent studies have demonstrated that 5hmC act as signal sites of active 5mC demethylation [9] and may directly modulate gene expression [10]. Therefore, a rapid and effective method to quantify and map the distribution of 5hmC at the DNA level is necessary for understanding its role in gene expression and regulation. However, a major challenge with respect to distinguishing 5hmC from 5mC remains. Traditional approaches, such as anti-5hmC antibodies and bisulfite sequencing, suffer from low specificity toward 5mC in immunoprecipitation assays [8, 11]. Although many methods have been developed to detect 5hmC, many well-documented drawbacks remains [12-15]. 1
Recently, Song et al. showed that wild type β-glucosyltransferase (β-GT) can transfer the engineered glucose moiety, UDP-α-6-N3-Glc which contains a biorthogonal group, onto the hydroxyl group of 5hmC. The azido handle can be selectively tagged with biotin using copper-free click chemistry for further detection, enrichment, and sequence analysis of 5hmC-containing DNA fragments in mammalian genomes [16] (Figure 1). Moreover, it is highly sensitive for clinical analysis if 5hmC is attached to a fluorescent reporter, for instance, in solid tumor samples and white blood cells where 5hmC modification is extremely low [17-19]. Moreover, Narek et al. recently demonstrated that O-GlcNAc transferase (OGT) can catalyze intracellular protein modification with UDPα-6-N3-Glc using the substrate promiscuity of OGT under certain conditions, which provided a fresh consideration of the O-GlcNAcylation [20]. Therefore, the development of an efficient procedure for the preparation of UDP-α-6-N3-Glc would be important for meeting the needs of the growing glycoscience community.
Detection UDP-α-6N3 -Glucose
Affinity purification Biotin-alkyne
Sequencing
lysate or 5-hmc
UDP-α-6N3 -Gl ucose:
Figure 1. Selective labeling of 5-hmC in genomic DNA using UDP-α-6N3-glucose.
To date, various methodologies have been developed to generate UDP-sugars. Chemical synthesis allows for the preparation of a diverse range of natural and unnatural structures but is challenging due to poor anomeric stereo selectivity, the need for multistep procedures with advanced purification requirements, and low isolated yields of the last two key synthetic steps. For example, Narek et al. only isolated the final desired products with a yield of 7% after stirring for 8 days, and Song et al. purified UDP-sugar with C18 reverse-phase HPLC with an unreported yield [16, 20, 21]. Meanwhile, enzymatic methods can directly convert the free sugar into desired sugar nucleotides without the need for protecting groups and afford the product with perfect regio- and stereo-selectivity. However, enzyme utilization is much less flexible because of the limited substrate specificity [22-24]. Recently, a hybrid chemoenzymatic strategy, combining chemical and enzymatic methods, has been successfully applied for the efficient synthesis of different sugar libraries. Using this strategy, glycan core precursors were first prepared via chemical synthesis and the precursors were subsequently extended to diverse and complex glycans via catalysis with robust glycosyltransferases [25-27]. Herein, a chemoenzymatic strategy for the synthesis of UDP-α-6N3-glucose is reported, showing efficient preparation of the desired product in a 190 mg scale with relatively simple operation that will facilitate chemoenzymatic labeling of glycoproteins or DNA. 2
Scheme 1. Chemoenzymatic synthesis of UDP-α-6-N3-Glucose.
Starting from the commercially available of 1,2:5,6-di-O-isopropylidene-α-Dglucofuranose 1, compound 3 was afforded by selective removal of the isopropylidene protecting groups at the 5,6-sites in the presence of acetic acid aqueous solution and following tosylation at the 6-hydroxy position. Subsequently, the treatment of 3 with NaN3 afforded the azido substituted furanose 4, which was then converted to the pyranose form of 6-N3-glucose 5 by heating to reflux in an 80% acetic acid aqueous solution. From intermediate 5, 6-N3-glucose-1-phospahte 7 was chemically prepared according to the procedure reported by Edgar et al. [28]. Briefly, the unprotected sugar 5 was reacted with p-toluenesulfonyl hydrazine to furnish a glycosylsulfonylhydrazide adduct in 92% yield, and further oxidized using anhydrous copper chloride in the presence 2-methyl-2-oxazoline and excess crystalline phosphoric acid to afford the corresponding α/β mixtures of 6-N3-glucose-1-phospahte 7. After complete reaction, the reaction mixture was poured into dichloromethane to precipitate the crude 7, which was then treated with water and saturated barium hydroxide to precipitate the excess phosphoric acid. The insoluble barium salt was removed by centrifugation and the entire process was repeated until no more precipitate was generated. The supernatant liquid was collected and concentrated before precipitation from ethanol. Due to their possible toxicity to the enzyme, the barium salts of 7 were re-dissolved in water and treated with 1M sodium carbonate to produce sodium salts of 6-N3-glucose-1-phospahte as a candidate for conversion of UDP-α-6-N3-glucose with an α/β ratio 3:1.
3
After obtaining 6-N3-glucose-1-phospahte, we expressed the relevant Bifidobacterium longum (BLUSP) enzyme, which has been reported to yield UDP-Glc, UDP-Gal, UDPMan, and their derivatives from the corresponding 1-phosphate substrates in a one-pot multienzyme manner [29]. The enzymatic reaction mixture containing substrate 7, BLUSP, MgCl2, uridine triphosphate (UTP), and Tris-HCl (pH 7.5) was incubated overnight at 37 ℃. When the majority of 7 was converted to the desired UDP-α-6-N3glucose 8 according to TLC analysis, the reaction was quenched by adding an equivalent amount of cold ethanol. After purification with a Bio-gel P-2 column, 190 mg of the target compound 8 was obtained representing a yield of 46%. Purity analysis using capillary electrophoresis (CE) confirmed the satisfactory purity of the product with a retention time of 11.5 min. In the associated HRMS spectrum, two peaks at m/z 590.0531 [M-H] and m/z 612.0349 [M-2H+Na] were detected. Moreover, the structure of UDP-α6-N3-glucose was confirmed by 1H NMR, 13C NMR, and 31P NMR (see Supporting Information). In summary, these results indicated that UDP-α-6-N3-glucose can be
YPLIU #115-126 RT: 2.49-2.73 AV: 12 NL: 2.91E4 T: FTMS - p ESI Full ms [200.00-2000.00] 590.0531 z=1
100 95 90 85 80 75 70
Relative Abundance
65 612.0349 z=1
60 55 50 45 471.0873 z=1
40 35 30 25 20 15
669.9936 z=1 460.9269 z=1 445.0500 z=1
472.0907 z=1 490.1547 z=2
504.9422 z=1
10
584.8830 591.0564 z=1 z=1 526.9242 z=1
5
529.0459 z=?
555.0248 z=1
540
560
574.1506 z=?
592.0572 z=1
669.0454 z=? 727.9521 613.0383 671.9907 z=1 z=1 691.9753 634.0169 z=1 z=1 z=1 710.0117 648.0117 z=1 z=?
0 460
480
500
520
580
600
620
640
660
680
m/z
Figure 2. CE and HRMS analysis data of UDP-α-6-N3-glucose.
4
700
720
efficiently prepared using the chemoenzymatic approach developed herein. In conclusion, we developed a chemoenzymatic strategy for the efficient preparation of UDP-α-6-N3-glucose. As no known kinase can specifically produce α-6-N3-glucose-1phosphate, we used a versatile chemical phosphorylation strategy to produce 6-N3glucose-1-phosphate, 7. The described protocol circumvents the traditional challenges of chemical synthesis by eliminating the need for protecting groups and advanced purification equipment. Although the obtained product is a mixture of α and β isomers, the stereo-pure product was obtained in good yield from α/β mixtures of the monosaccharide-1-P precursors from combination with the natural stereo-selective BLUSP enzyme. Thus, a facile and applicable approach for the production of the biologically useful nucleotide sugar UDP-α-6-N3-glucose on a preparative scale was demonstrated. This approach can be implemented for the biosynthesis of any nucleotide sugar and its derivatives, particularly for molecules containing biorthogonal function groups used to identify target proteins by in vitro incorporation [4, 20, 30]. Author Contributions: # These authors contributed equally.
Acknowledgments: This work was supported by the Natural Science Foundation of China (31000371, 21807025) and Key Scientific Research Projects of Universities in Henan (19B15003). The authors have declared no conflict of interest. Reference 1.
Palaniappan, K.K. and C.R. Bertozzi, Chemical Glycoproteomics. Chemical Reviews, 2016. 116(23): p. 14277-14306.
2.
Clark, P.M., et al., Direct in-gel fluorescence detection and cellular imaging of OGlcNAc-modified proteins. J Am Chem Soc, 2008. 130(35): p. 11576-7.
3.
Wen, L., et al., Two-Step Chemoenzymatic Detection of N-Acetylneuraminic Acidalpha(2-3)-Galactose Glycans. J Am Chem Soc, 2016. 138(36): p. 11473-6.
4.
Wen, L., et al., A One-Step Chemoenzymatic Labeling Strategy for Probing Sialylated Thomsen-Friedenreich Antigen. ACS Cent Sci, 2018. 4(4): p. 451-457.
5.
Kothari, R.M. and V. Shankar, 5-Methylcytosine content in the vertebrate deoxyribonucleic acids: species specificity. J Mol Evol, 1976. 7(4): p. 325-9.
6.
Kriaucionis, S. and N. Heintz, The Nuclear DNA Base 5-Hydroxymethylcytosine Is Present in Purkinje Neurons and the Brain. Science, 2009. 324(5929): p. 929-930. 5
7.
Tahiliani, M., et al., Conversion of 5-Methylcytosine to 5-Hydroxymethylcytosine in Mammalian DNA by MLL Partner TET1. Science, 2009. 324(5929): p. 930-935.
8.
Ito, S., et al., Role of Tet proteins in 5mC to 5hmC conversion, ES-cell self-renewal and inner cell mass specification. Nature, 2010. 466(7310): p. 1129-U151.
9.
Hashimoto, H., et al., Recognition and potential mechanisms for replication and erasure of cytosine hydroxymethylation. Nucleic Acids Res, 2012. 40(11): p. 4841-9.
10.
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