Current Protocols in Nucleic Acid Chemistry || A Simple Chemical Synthesis of Sugar Nucleoside Diphosphates in Water

UNIT 13.12A Simple Chemical Synthesis of SugarNucleoside Diphosphates in Water

Hidenori Tanaka,1 Yayoi Yoshimura,1 and Ole Hindsgaul1

1Carlsberg Laboratory, Copenhagen, Denmark

ABSTRACT

Chemoenzymatic oligosaccharide synthesis is attractive since it eliminates the tediousmultistep protection-deprotection requirements of pure chemical synthesis. Chemoenzy-matic synthesis using glycosyltransferases, however, requires not only the correct enzymeto control both regio- and stereospecificity, but also the glycosyl donor to provide thesugar that is added. This unit describes a simple synthesis of sugar-nucleoside diphos-phates (sugar-NDPs), the type of glycosyl donor (e.g., UDP-Glc, UDP-Gal, ADP-Glc)required by most glycosyltransferases, by using a chemical coupling reaction in water.The preparation of sugar-NDPs by this method therefore does not require any skillsin synthetic organic chemistry. Curr. Protoc. Nucleic Acid Chem. 54:13.12.1-13.12.10.C© 2013 by John Wiley & Sons, Inc.

Keywords: chemical synthesis � chemoenzymatic oligosaccharide synthesis � glycosyl-transferase � pyrophosphate formation � sugar-nucleoside diphosphate � sugarnucleotide

INTRODUCTION

Sugar-nucleoside diphosphates (sugar-NDPs) are important intermediates in carbohy-drate metabolism and act as activated glycosyl donors in oligosaccharide biosynthesis(Palcic, 2011; Schmaltz et al., 2011). They can be prepared either chemically or thoughenzymatic methods, some of which include recycling systems that enable the regenera-tion of sugar-NDPs from the released NMPs. The enzymatic preparation of sugar-NDPsrequires access to the necessary enzymes, if these are available, but has the advantages ofbeing simple and can be performed in situ in the glycosyltransferase reaction. Chemicalsynthesis is more complex but it is also more powerful for the preparation of oligosac-charide analogs in cases where the enzymes do not tolerate a modification in the sugarpart of the sugar-NDP substrate.

Methods for the chemical synthesis of sugar-NDPs have been comprehensively reviewed(Wagner et al., 2009). The most common procedures involve the formation of thepyrophosphate linkage by the coupling of an activated nucleoside 5′-monophosphate(NMP) with a sugar 1-phosphate (Fig. 13.12.1). Phosphomorpholidates and phosphoim-idazolides are the most frequently used activated phosphates. N,N′-Carbonyldiimidazole(CDI) is a common activating reagent for the generation of the phosphoimidazolidefrom the phosphate group of NMPs (Cramer et al., 1961; Simon et al., 1990). The CDIactivation proceeds under mild conditions and usually does not require protection ofthe hydroxyl groups. However, both activation and coupling reaction are performed inanhydrous organic solvents, requiring the preparation of tri- or tetra-alkylammoniumsalts of the NMP and the sugar 1-phosphate to achieve solubility. This unit describes asimple synthesis of sugar-NDPs in water using a new activating reagent, 2-imidazoyl-1,3-dimethylimidazolinium intermediate (ImIm), generated in situ from 2-chloro-1,3-dichloromethylimidazolinium chloride (DMC; Isobe and Ishikawa, 1999) and imidazole.This reagent directly couples sodium or potassium salts of an NMP and sugar-1-phosphate

Current Protocols in Nucleic Acid Chemistry 13.12.1-13.12.10, October 2013Published online October 2013 in Wiley Online Library (wileyonlinelibrary.com).DOI: 10.1002/0471142700.nc1312s54Copyright C© 2013 John Wiley & Sons, Inc.

NucleosidePhosphorylationand RelatedModifications

13.12.1

Supplement 54

13.12.2

Supplement 54 Current Protocols in Nucleic Acid Chemistry

organic solvent(anhydrous)

organicsolvent

(anhydrous)

isolated

CDI

NMP activated NMP

sugar-NDPsugar 1-P

Figure 13.12.1 Classical approach to sugar-NDP synthesis by coupling an activated NMP witha sugar 1-phosphate in organic solvents.

generatedin situ

NMP activated NMP

water or D2O1-2 hr

addsugar 1-P

sugar-NDP(sugar donor)

oligosaccharide

sugar acceptor

glycosyltransferase

ImIm

Advantages: one pot reaction, no organic solvents, commercial reagents, simple

Figure 13.12.2 One-pot synthesis of a sugar-NDP using ImIm as the activating reagent in water.

in a one-pot reaction (see Fig. 13.12.2). The yields are substantially higher when the re-action is performed in D2O rather than in H2O, as slower hydrolysis of intermediatesoccurs in the less nucleophilic D2O. The solution of the crude product (without any work-up) can be used directly as a source of glycosyl donor in glycosyltransferase-mediatedoligosaccharide synthesis (Tanaka et al., 2012).

BASICPROTOCOL 1

SYNTHESIS OF SUGAR-NDPs IN WATER (D2O) USING ImIm REAGENT

Nucleoside 5′-monophosphate disodium salts and sugar 1-phosphate dipotassium saltsare commercially available from several chemical companies and can be used directlyfor the coupling reaction without any pre-treatment such as conversion into their tetra-or tri-alkylammonium salts.


13.12.3

Current Protocols in Nucleic Acid Chemistry Supplement 54

Materials

2-Chloro-1,3-dimethylimidazolinium chloride (DMC, Sigma-Aldrich)Imidazole (Sigma-Aldrich)Deuterium oxide (D2O, D: 99.9%, Cambridge Isotope Laboratories)NMP disodium salt:

Uridine 5′-monophosphate disodium salt (UMP, 22% water content, Carbosyth)Adenosine 5′-monophosphate disodium salt (AMP, 21% loss on drying,

Carbosyth)Guanosine 5′-monophosphate disodium salt hydrate (GMP, 22% loss on drying,

Sigma-Aldrich)Sugar 1-phosphate:

α-D-Glucose 1-phosphate dipotassium salt (Glc 1-P, 12% water content,Carbosyth)

α-D-Galactose 1-phosphate dipotassium salt pentahydrate (Gal 1-P,Sigma-Aldrich)

α-D-Mannose 1-phosphate dipotassium salt (Man 1-P, 0.02% loss on drying,Carbosyth)

50 mM Tris(hydroxylmethyl)aminomethane hydrogen chloride (Tris·Cl) buffer, pH8.0

20 U/μL calf intestinal alkaline phosphatase (AP, Life Technologies)MilliQ waterDEAE Sephacel ion-exchange column (2.6 cm × 16 cm; GE Healthcare)30 and 400 mM ammonium acetate aqueous solution

1.5-mL microcentrifuge tubesMagnetic stir barMagnetic stirrer15-mL centrifuge tubes30◦C incubatorESI-MS500-mL round-bottom flasksRotary evaporatorLyophilizer

Couple NMP with sugar 1-phosphate using ImIm activation1. Place 33.8 mg DMC (200 μmol), 27.2 mg imidazole (400 μmol), and NMP dis-

odium salt (47.2 mg, 100 μmol UMP; 49.5 mg, 100 μmol AMP) into a 1.5-mLmicrocentrifuge tube with a magnetic stir bar.

For 52.9 mg GMP (100 μmol), use 67.7 mg DMC (400 μmol) and 54.4 mg imidazole(800 μmol).

DMC is very hygroscopic so it should be weighed quickly. It should be stored under argon.

2. Add 50 μL D2O to the tube.

In the case of GMP, which has a lower solubility, add 100 μL D2O.

3. Stir 1 hr with a magnetic stirrer at 37◦C.

Stir for 2 hr when GMP is used.

4. Add sugar 1-phosphate (9.6 mg, 25 μmol Glc 1-P; 10.7 mg, 25 μmol Gal 1-P; 8.4 mg,25 μmol Man 1-P) and keep stirring for an additional 18 hr at 37◦C.

Isolate sugar-NDP (optional)5. Transfer the reaction mixture into a 15-mL centrifuge tube and dilute with 5.0 mL

of 50 mM Tris·Cl buffer (pH 8.0).

A SimpleChemical

Synthesis of SugarNucleoside

Diphosphates inWater

13.12.4


6. Add 2.5 μL AP (50 U) to the mixture.

Add 5 μL AP (100 U) to the mixture when GMP is used.

AP is used for cleaving the phosphate group in unreacted NMP to facilitate isolation ofcoupling product by ion exchange chromatography.

7. Incubate 24 hr at 30◦C.

8. Dilute reaction mixture with 5.0 mL MilliQ water.

9. Purify solution by ion-exchange chromatography on a DEAE Sephacel ion-exchangecolumn (flow rate: 2.0 mL/min, linear gradient 30 mM to 400 mM aq. ammoniumacetate), collecting 20-mL fractions.

10. Check fractions by ESI-MS to identify product-containing fractions.

Using UV detection at 254 nm is an alternative way to identify the product.

11. Collect the identified fractions in a 500-mL round-bottom flask.

12. Concentrate on a rotary evaporator and transfer to a 15-mL centrifuge tube.

13. Remove water and residual ammonium acetate by lyophilization.

14. Dissolve the residue in 2.0 mL MilliQ water and lyophilize.

15. Repeat step 14 to complete removal of ammonium acetate.

16. Dissolve the residue in 5.0 mL MilliQ water

17. Estimate the sugar NDP concentration and yield by UV absorbance at λmax 260 nm(uracil and adenine) or λmax 252 nm (guanine) using 0.5 mM aqueous solution ofthe appropriate NMP as reference standard.

18. Remove water by lyophilization.

19. Characterize the product by NMR and ESI-MS.

UDP-Glc di-ammonium salt (43% isolated yield); 1H-NMR (400 MHz, D2O): δ = 8.04(d, 1H, J = 8.2 Hz, H6), 6.08 (m, 2H, H5, H1′), 5.71 (dd, 1H, J1,2 = 3.6 Hz, J1,P = 7.2 Hz,H1′′), 4.48 (m, 2H, H2′, H3′), 4.39 (m, 1H, H4′), 4.32 (m, 2H, H5a′, H5b′), 4.01 (m, 1H,H5′′), 3.97 (m, 1H, H6a′′), 3.91-3.85 (m, 2H, H3′′, H6b′′), 3.64 (m, 1H, H2′′), 3.57 (m,1H, H4′′); 31P{1H}-NMR (162 MHz, D2O): δ = −11.1 (JP,P = 20.7 Hz), −12.8; m/z(ESI): found [M-2NH4+H]– 565.1, C15H30N4O17P2 calcd. for [M-2NH4+H]– 565.1.

UDP-Gal di-ammonium salt (35% isolated yield); 1H-NMR (400 MHz, D2O): δ = 8.05(d, 1H, J = 8.1 Hz, H6), 6.08 (m, 2H, H1′, H5), 5.75 (dd, J1,2 = 3.6 Hz, J1,P = 7.2 Hz, 1H,H1′′), 4.48 (m, 2H, H2′, H3′), 4.40 (m, 1H, H4′), 4.33 (m, 2H, H5a′, H5b′), 4.28 (m, 1H,H5′′), 4.14 (m, 1H, H4′′), 4.02 (dd, 1H, J2,3 = 10.4 Hz, J3,4 = 3.2 Hz, H3′′), 3.94-3.81 (m,3H, H2′′, H6a′′, H6b′′); 31P{1H}-NMR (162 MHz, D2O): δ = −11.1 (JP,P = 20.7 Hz),−12.6; m/z (ESI): found [M-2NH4+H]– 565.2, C15H30N4O17P2 calcd. for [M-2NH4+H]–

565.1.

ADP-Glc di-ammonium salt (32% isolated yield); 1H-NMR (400 MHz, D2O): δ = 8.62(s, 1H, H8), 8.39 (s, 1H, H2), 6.25 (d, J1,2 = 6.0 Hz, 1H, H1′), 5.71 (dd, 1H, J1,2 = 3.4 Hz,J1,P = 6.8 Hz, H1′′), 4.87 (m, 1H, H2′), 4.65 (m, 1H, H3′), 4.51 (m, 1H, H4′), 4.34 (m, 2H,H5a′, H5b′), 4.00-3.82 (m, 4H, H4′′, H5′′, H6a′′, H6b′′), 3.62 (m, 1H, H2′′), 3.55 (m, 1H,H3′′); 31P{1H}-NMR (162 MHz, D2O): δ = −11.1 (JP,P = 20.7 Hz), −12.8; m/z (ESI):found [M-2NH4+H]– 588.2, C16H31N7O15P2 calcd. for [M-2NH4+H]– 588.1.

GDP-Man di-ammonium salt (25% isolated yield); 1H-NMR (400 MHz, D2O): δ = 8.27(s, 1H, H8), 6.05 (d, 1H, J1,2 = 6.0 Hz, H1′), 5.63 (m, 1H, H1′′), 4.90 (m, 1H, H2′),4.63 (m, 1H, H3′), 4.46 (m, 1H, H4′), 4.32 (m, 2H, H5a′, H5b′), 4.16 (m, 1H, H2′′), 4.03(m, 1H, H3′′), 4.00-3.94 (m, 2H, H5′′, H6a′′), 3.87 (m, 1H, H6b′′), 3.80 (m, 1H, H4′′);31P{1H}-NMR (162 MHz, D2O): δ = −11.4 (JP,P = 20.8 Hz), −13.8; m/z (ESI): found[M-2NH4+H]– 604.1, C16H31N7O16P2 calcd. for [M-2NH4+H]– 604.1.


13.12.5


BASICPROTOCOL 2

USE OF SYNTHETIC CRUDE UDP-GAL AS A DONOR-SUBSTRATE IN AGALACTOSYLTRANSFERASE REACTION

An important feature of the present synthetic protocol is that the crude reaction mixtureof the sugar-NDP can be used directly as the source of glycosyl-donor in enzymaticoligosaccharide synthesis. This is because all chemically reactive species have beenhydrolyzed by the end of the coupling reaction. This protocol describes transfer ofgalactose from crude UDP-Gal to GlcNAc using a β(1,4)-galactosyltransferase on amilligram scale (Fig. 13.12.3).

Materials

2-Chloro-1,3-dimethylimidazolinium chloride (DMC, Sigma-Aldrich)Imidazole (Sigma-Aldrich)Uridine 5′-monophosphate disodium salt (UMP, 22% water content, Carbosyth)Deuterium oxide (D2O, D: 99.9%, Cambridge Isotope Laboratories)α-D-Galactose 1-phosphate dipotassium salt pentahydrate (Gal 1-P, Sigma-Aldrich)MilliQ waterGlcNAc-TMR (Zhang et al., 1995)4-Morpholinepropanesulfonic acid (MOPS, Sigma)Manganese (II) chloride tetrahydrate (MnCl2, Sigma)Bovine serum albumin (BSA, Sigma)0.65 μg/μL bovine β-1,4-galactosyltransferase (β-1,4-GalT, Sigma-Aldrich)Chloroform (CHCl3, LAB-SCAN)Methanol (MeOH, LAB-SCAN)2,5-Dihydroxybenzoic acid (DHB, Sigma-Aldrich)Acetonitrile (ACN, LAB-SCAN)Trifluoroacetic acid (TFA, Sigma-Aldrich)

1.5-mL microcentrifuge tubesMagnetic stir bar and stirrer37◦C incubatorSilica TLC plate (Merck)Sep-Pak C18 Plus Light Cartridge (Waters)20-mL plastic syringe (Beckton Dickinson) connected to the Sep-pak cartridgeRotary evaporator (BUCHI) connected to a vacuum pump (KNF Lab)Lyophilizer

Prepare crude synthetic UDP-Gal for the enzymatic reaction1. Place 9.0 mg DMC (53 μmol), 7.3 mg imidazole (107 μmol), and 13.3 mg UMP

disodium salt (28 μmol) in a 1.5-mL microcentrifuge tube equipped with a magneticstir bar.

2. Add 13 μL D2O to the tube.

3. Stir 1 hr with magnetic stirrer at 37◦C.

4. Add 3 mg Gal-1-P (7 μmol) and keep stirring for an additional 18 hr at 37◦C.

5. Add 585 μL D2O and record 1H and 31P -NMR spectra to identify and quantifycomponents (Fig. 13.12.4).

6. Dilute the NMR sample to a final volume of 1.3 mL with MilliQ water.

The final crude synthetic UDP-Gal mixture is used as a source of glycosyl donor. Ac-cording to NMR analysis (Fig. 13.12.4), the final crude mixture (1.3 mL) consists ofUDP-Gal (3.2 mM), GalP (1.6 mM), galactose 1,2-cyclic- phosphate (0.6 mM), UMP(5.5 mM), UMP dimer (6.5 mM), 1,3-dimethyl-2-imidazolidinone (41 mM), and imidazole-HCl (82 mM).

A SimpleChemical



13.12.6


�-1,4-GalT

12 hr, 37 °C

crude UDP-Gal

Figure 13.12.3 Enzymatic synthesis of LacNAc-TMR using crude UDP-Gal and β(1,4)-galactosyltransferase.

imidazole-HCl

1,3-dimethyl-2-imidazolidinone

Gal 1,2-cyclic-phosphate

UMP

Gal 1-P

UMP dimer

UDP-Gal

A B

0

8 6 4 10 5 0 �5 �10 �15 [ppm][ppm]2

2

0

5

10

15

4

6

8

10

12

[p] [p]

Figure 13.12.4 (A) Analysis of crude synthetic UDP-Gal by 1H NMR spectroscopy and (B) by 31P NMR spectroscopy.

Perform enzyme reaction with crude UDP-Gal and β-1,4-GalT7. Dissolve 1.7 mg GlcNAc-TMR in 57 μL MilliQ water in a 1.5-mL microcentrifuge

tube.

8. Add 100 μL of the following buffer mixture: MOPS (500 mM), MnCl2 (200 mM),and BSA (10 mg/mL), pH 7.0.

9. Add 813 μL of crude reaction mixture of UDP-Gal (2.6 μmol) from step 6.

10. Add 30 μL β-1,4-GalT (19.5 μg).

The final reaction mixture (1 mL) consists of GlcNAc-TMR (1.7 mg, 2.0 μmol), UDP-Gal (2.6 μmol), MOPS (50 mM), MnCl2 (20 mM), BSA (1.0 mg/mL), and β-1,4-GalT(19.5 μg).

β-1,4-GalT was expressed, re-folded, and purified as described previously (Ramakrishnaet al., 2001).

11. Incubate reaction mixture at 37◦C.


13.12.7


12. Monitor reaction by TLC in 65:35:5 (v/v/v) CHCl3/MeOH/H2O.

Rf values are 0.5 for GlcNAc-TMR and 0.3 for LacNAc-TMR, respectively.

13. Add 15 μL β-1,4-GalT (9.8 μg) and 390 μL UDP-Gal (1.5 μmol) from step 6 after6-hr incubation.

14. Incubate reaction mixture for an additional 6 hr (total 12 hr) at 37◦C.

UMP and UMP dimer in the reaction mixture inhibits β-1,4 GalT. The inhibition constants(Ki) are 149 μM (UMP) and 36 μM (UMP dimer), compared with Km value of UDP-Gal(22 μM). However, the enzymatic reaction proceeds completely with a longer incubation.

Purify LacNAc-TMR15. Purify LacNAc-TMR, the product of the β -1,4 GalT reaction, by washing the

Sep-Pak C18 cartridge with 20 mL MeOH followed by 20 mL MilliQ water.

16. Transfer the enzymatic reaction mixture in 10 mL MilliQ water onto Sep-Pak car-tridge via a 20-mL plastic syringe.

17. Wash the Sep-Pak cartridge with 100 mL MilliQ water.

18. Elute LacNAc-TMR with 30 mL MeOH.

19. Concentrate on a rotary evaporator.

20. Dissolve the residue with MilliQ water and lyophilize.

21. Characterize the product by 1H-NMR and MALDI-TOF MS using DHB as thematrix.

As matrix for MALDI-TOF MS, DHB solution was prepared by dissolving DHB in aACN/MilliQ (50:50 v/v) solution containing 0.1% TFA at a concentration of 10 mg/mL.

LacNAc-TMR (1.8 μmol, yield 90%); 1H-NMR (400 MHz, CD3OD): δ 8.52, 8.05, 7.36,7.26, 7.02, and 7.94 (9H, TMR), 4.39 (d, 1H, J1,2 = 8.4 Hz, H-1GlcNAc), 4.38 (d, 1H,J1,2 = 7.5 Hz, H-1Gal), 3.91-3.29 (m, 26H), 2.86-2.83 (m, 1H), 2.52-2.46 (m, 2H), 2.25-2.20(m, 2H), 2.03-1.95 (m, 1H), 1.96 (s, 3H, –C(=O)CH3

GlcNAc), 1.61-1.57 (m, 2H), 1.57-1.48(m, 2H), 1.40-1.31 (br, 8H); m/z (MALDI-TOF): found [M+H]+ 994.77, C50H68N5O16

calcd. for [M+H]+ 994.47.

COMMENTARY

Background InformationThere are several synthetic strategies for

the preparation of sugar-NDPs (reviewed byWagner et al., 2009). Most approaches in-volve the formation of the pyrophosphatelinkage between the sugar 1-phosphate andthe NMP. One of these phosphates has tobe activated prior to coupling. Nucleoside5′-phospho-morpholidates and -imidazolidesare the most common activated species sincethey can be easily prepared from the NMPwith or without hydroxyl protecting groups.Both activated forms of NMPs have been ex-tensively used for sugar-NDP synthesis butfrequently require long reaction times (∼1week) and give only modest yields (∼30% to50%). The addition of catalysts like nitrogen-containing heterocycles to the coupling re-actions can effect substantial improvementsin both rate and yield (Wittmann and Wong,

1997; Tsukamoto and Kahne, 2011). Recently,sulfonyl imidazolium salts were developed asefficient coupling reagents (Mohamady andTaylor, 2012).

The method described here using ImImas the activating agent enables sugar-NDPsynthesis using commercial sodium or potas-sium salts of the NMP and sugar 1-phosphateto carry out both phosphate activation andsubsequent coupling in a one-pot fashion.The formation of this reagent was discov-ered serendipitously when the authors werefollowing the work by Noguchi et al. (2009)and Tanaka et al. (2009a,b) that applied DMCin some novel transformations of unprotectedcarbohydrates in water. The presence of imi-dazole in these reactions led to the formationof ImIm as evidenced by NMR and MS.

The greatest advantage of this methodlies in its simplicity and the completely

A SimpleChemical



13.12.8


Imim (3)exact mass: 165.11

(1.0 L/mol of DMC)room temperature � 5 min

165.1

Imlm(3)

imidazole (2)

DMC(1)

Im · HCl

A

B

0

75 100 125 150 175 200 225 250 275 m/z

1

2

3

Intens.�106

Figure 13.12.5 (A) Reaction of DMC (1) with imidazole (2). (B) Detection of ImIm intermediate(3) by ESI-MS.

direct manner in which it is carried out. Asa bonus, the crude coupling product can beused directly as a source of glycosyl donor inglycosyltransferase-mediated oligosaccharidesynthesis because all of the chemically reac-tive species have been neutralized by the end ofthe coupling reaction. The crude reaction prod-ucts contain the NMP and the NMP-dimer,both of which can inhibit glycosyltransferases.In practice, this means that the enzymatic syn-thesis reaction must be carried out for a longertime to reach completion.

Critical Parameters andTroubleshooting

The formation of the ImIm intermediate 3,generated from DMC 1 and imidazole 2, can beconfirmed by 1H NMR spectroscopy and ESI-MS (Fig. 13.12.5). NMP activation using thein situ–formed ImIm 3 can be monitored byboth 31P and 1H NMR spectroscopy. Samplespectra taken during the activations of UMPare shown in Figure 13.12.6. 31P NMR clearlyshows that the signal corresponding to UMP 4

(δ = 2.3 ppm) decreased and simultaneously apeak assigned to the UMP-imidazolide (UMP-Im 5: δ = −8.2 ppm) increased for up to 1 hrwhere the maximum conversion was achieved.Although complete consumption of intermedi-ate 3 required ∼2 hr, confirmed by 1H NMR,1 hr was found to be optimal for the activa-tion step to produce 5 before addition of sugar1-phosphate. The result is that remainingImIm causes dimerization of some of the ex-cess 4 to give UMP-dimer 6. Reaction times>1 hr resulted in increased formation of 6 re-sulting from competing hydrolysis of imida-zolide 5.

Substrate solubility is critical to both theactivation step using ImIm and subsequentcoupling step. In dissolving GMP disodiumsalt, two times the volume of water was re-quired since its solubility is relatively lowcompared to UMP and AMP. This led to lowerconversion into the reactive GMP-Im. There-fore, a larger excess of reagents (GMP, DMC,and imidazole) and a longer activation time(2 hr) was required. Additionally, it was found


13.12.9


(1.0 L/mol of UMP)37 °C

15 min

30 min

1 hr

2 hr

4 hr

8 hr

16 hr

15 min

UMP (4) � Im-HCl

30 min

1 hr

2 hr

4 hr

8 hr

16 hr

A

B C

0 �5 �10 [ppm]

8.0 7.5 7.0 6.5 6.0[ppm]

Figure 13.12.6 (A) Activation of UMP (4) by using ImIm (3). (B) Monitoring of UMP-Im (5) formation by 31P NMRspectroscopy and (C) by 1H NMR spectroscopy.

that the counter-cation of the NMPs and sugar1-Ps was also very important in this method.Sodium, potassium, or tetra- or trialkylammo-nium salt can be used, but ammonium (NH4

+)salts resulted in undesired side reactions. Theammonium ion reacts with ImIm and NMP-Imresulting in substantially reduced yields.

Caution should be exercised during the iso-lation of sugar-NDPs after AP treatment. Thecomplete separation of the sugar-NDP andNMP-dimer by ion-exchange chromatographyis difficult. The use of ammonium bicarbon-ate instead of ammonium acetate gives muchbetter separation on the DEAE Sephacel, butintroduces a severe risk—the resultant solu-tion containing the sugar-NDP becomes ba-sic (pH 8 to 8.5) and must be neutralized bycareful addition of Dowex (H+) resin followedby passage through a Dowex (Na+) column

before concentration or lyophilization. Other-wise, intramolecular cyclization occurs easilyunder the basic conditions by the attack of thehydroxyl group at the 2 position of the sugar(in the case of glucose and galactose) at thephosphorus atom that is linked to the sugaranomeric position giving the sugar 1,2-cyclic-phosphate and NMP.

Anticipated ResultsModerate (∼40%) isolated yields of the fi-

nal sugar-NDP can be expected using the pro-cedure described in this unit, which requiresvery concentrated solutions of the reagents andreactants. Lower yields will be obtained wheneither of the phosphates have poor solubilityin water, such as is the case with GMP. Bet-ter yields can be expected if the phosphatesare used as their alkylammonium salts in

A SimpleChemical



13.12.10


organic solvents, since hydrolysis of interme-diates would be less and therefore less reagentand reactants would be required. However, thiswould introduce additional complexity to theprocedure.

Time ConsiderationsThe entire procedure for the synthesis of

crude sugar-NDP is performed in 1 day: if it isinitiated at noon, the sugar-NDP is ready foruse the next morning. The purification of thesugar-NDP including AP treatment can takeup to 4 days as it requires the lyophilizationstep to be performed three times. The glyco-syltransferase reaction can take from 1 day to1 week, depending on the specific activity ofthe enzymes used.

AcknowledgmentsThe authors would like to thank Prof.

Monica M. Palcic for her guidance in all ofthe enzymology aspects of this work.

Literature CitedCramer, F., Schaller, H., and Staab, H.A.

1961. Darstellung von Imidazoliden der Phos-phorsaure. Chem. Ber. 94:1612-1621.

Isobe, T. and Ishikawa, T. 1999. 2-Chloro-1,3-dimethylimidazolinium chloride. 1. A powerfuldehydrating equivalent to DCC. J. Org. Chem.64:6984-6988.

Mohamady, S. and Taylor, S.D. 2012. Rapidand efficient synthesis of nucleoside polyphos-phates and their conjugates using sulfonyl imi-dazolium salts. Curr. Protoc. Nucl. Acid Chem.51:13.11.1-13.11.24.

Noguchi, M., Tanaka, T., Gyakushi, H., Kobayashi,A., and Shoda, S. 2009. Efficient synthesis ofsugar oxazolines from unprotected N-acetyl-2-amino sugars by using chloroformamidiniumreagent in water. J. Org. Chem. 74:2210-2212.

Palcic, M.M. 2011. Glycosyltransferases as biocat-alysts. Curr. Opin. Chem. Biol. 15:226-233.

Ramakrishna, B., Shah, P.S., and Qasba, P.K.2001. α-Lactalbumin (LA) stimulates milkβ-1,4-galactosyltransferase I (β4Gal-T1) totransfer glucose from UDP-glucose to N-

acetylglucosamine: Crystal structure of β4Gal-T1·LA complex with UDP-Glc. J. Biol. Chem.276:37665-37671.

Schmaltz, R.M., Hanson, S.R., and Wong, C.-H.2011. Enzymes in the synthesis of glycoconju-gates. Chem. Rev. 111:4259-4307.

Simon, E.S., Grabowski, S., and Whitesides, G.M.1990. Convenient syntheses of cytidine 5′-triphosphate, guanosine 5′-triphosphate, anduridine 5′-triphosphate and their use in thepreparation of UDP-glucose, UDP-glucuronicacid, and GDP-mannose. J. Org. Chem. 55:1834-1841.

Tanaka, H., Yoshimura, Y., Jørgensen, M.R.,Cuesta-Seijo, J.A., and Hindsgaul, O. 2012. Asimple synthesis of sugar nucleoside diphos-phates by chemical coupling in water. Angew.Chem. Int. Ed. 51:11531-11534.

Tanaka, T., Huang, W.C., Noguchi, M., Kobayashi,A., and Shoda, S. 2009a. Direct syn-thesis of 1,6-anhydro sugars from unpro-tected glycopyranoses by using 2-chlror-1,3-dimethylimidazolium chloride. TetrahedronLett. 50:2154-2157.

Tanaka, T., Nagai, H., Noguchi, M., Kobayashi, A.,and Shoda, S. 2009b. One-step conversion ofunprotected sugars to β-glycosyl azides using2-chloroimidazolinium salt in aqueous solution.Chem. Commun. 23:3378-3379.

Tsukamoto, H. and Kahne, D. 2011. N-Methyl-imidazolium chloride-catalyzed pyrophosphateformation: Application to the synthesis of lipidI and NDP-sugar donors. Bioorg. Med. Chem.Lett. 21:5050-5053.

Wagner, G.K., Pesnot, T., and Field, R.A. 2009.A survey of chemical methods for sugar-nucleotide synthesis. Nat. Prod. Rep. 26:1172-1194.

Wittmann, V. and Wong, C.-H. 1997. 1H-Tetrazoleas catalyst in phosphomorpholidate couplingreactions: Efficient synthesis of GDP-fucose,GDP-mannose, and UDP-galactose. J. Org.Chem. 62:2144-2147.

Zhang, Y., Le, X., Dovichi, N.J., Compston, C.A.,Palcic, M.M., Diedrich, P., and Hindsgaul,O. 1995. Monitoring biosynthetic transforma-tions of N-acetyllactosamine using fluorescentlylabeled oligosaccharides and capillary elec-trophoretic separation. Anal. Biochem. 227:368-376.

Documents

Current Protocols in Nucleic Acid Chemistry || A Simple Chemical Synthesis of Sugar Nucleoside Diphosphates in Water