Synthesis of 1-C-linked diphosphate analogues of UDP-N-Ac-glucosamine and UDP-N-Ac-muramic acid Andrej Babic ˇ a, * , Stanislav Gobec a , Christine Gravier-Pelletier b , Yves Le Merrer b , Slavko Pec ˇar a, c a Faculty of Pharmacy, University of Ljubljana, As ˇkerc ˇeva 7, 1000 Ljubljana, Slovenia b Universite´ Paris Descartes, UMR 8601 CNRS, Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, 45 rue des Saints-Pe `res, 75006 Paris, France c Jozef Stefan Institute, Jamova cesta 39, 1000 Ljubljana, Slovenia article info Article history: Received 22 March 2008 Received in revised form 12 June 2008 Accepted 3 July 2008 Available online 8 July 2008 Keywords: C-Linked glycosides substrate analogues Diphosphate isosteres Microwave-assisted synthesis abstract UDP-N-acetyl-glucosamine and UDP-N-acetyl-muramic acid are two important cytoplasmic precursors of bacterial peptidoglycan. The convergent synthesis of their analogues is reported. The a-1-C-linked-N- acetyl-glucosamine was synthesized using microwave-assisted Keck radical allylation. Oxidation of al- kene derivatives to the corresponding carboxylic acids allowed attachment to O- and N-sulfamoyluridine giving stable diphosphate mimetics. Ó 2008 Elsevier Ltd. All rights reserved. 1. Introduction N-Acetyl-glucosamine (GlcNAc) and N-acetyl-muramic acid (MurNAc) are present in almost all eubacteria as constituent units of the bacterial cell wall peptidoglycan. 1 In the biosynthesis of polysaccharides, glycolipids, glycoproteins and the bacterial cell wall, GlcNAc and MurNAc are usually utilized, in the form of precursors containing nucleoside diphosphates, by a variety of enzymes, in particular glycosyltransferases. 2–5 These enzymes are important targets for possible modulation of certain biochemical processes. We have focused on the enzymes involved in the cyto- plasmic steps of peptidoglycan biosynthesis. 1,6 Since MurA, MurC and MurG enzymes transfer or modify UDP-GlcNAc or UDP-Mur- NAc, it was reasoned that analogues of these compounds could mimic their substrates and serve as useful starting points for designing new enzyme inhibitors. The design and synthesis of nucleotide diphosphate sugar ana- logues are imposing challenges. Replacement of the diphosphate moiety is not straightforward. Ionic, steric and H-bond forming characteristics, as well as the distance between nucleoside and sugar moieties, all play an important role and affect the binding to the target enzymes. We have introduced the O- and N- sulfamoyl amide group in lieu of the diphosphate part of UDP as depicted in Figure 1 . This was based on similar approaches that showed inhibition of protein glycosylation. 7 To render the target compounds chemically and enzymatically stable, C-linked glycosides were chosen as a synthetically feasible alternative to natural a-O-glycosides. 5,8–10 However, to achieve this, maintaining the a-configuration at the anomeric position of the N- Ac-glucosamine would be crucial. Fortunately, synthetic strategies for making C-linked derivatives of N-Ac-glucosamine have been thoroughly investigated. 11–18 Keck radical allylation 19 was chosen as the key reaction to introduce the a-carbon–carbon bond at the anomeric position. O HO RO NH HO O N NH OH HO O X O O S H N O O O OH O HO RO NH HO O P O N NH OH HO O O O O P O OH OH O Ac Ac X = NH, O R = H, Figure 1. Design of O- and N-sulfamoyl amide UDP-GlcNAc and UDP-MurNAc analogues. * Corresponding author. Tel.: þ386 1 4769 601; fax: þ386 1 4258 031. E-mail address: [email protected](A. Babic ˇ). Contents lists available at ScienceDirect Tetrahedron journal homepage: www.elsevier.com/locate/tet 0040-4020/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.tet.2008.07.009 Tetrahedron 64 (2008) 9093–9100
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lable at ScienceDirect
Tetrahedron 64 (2008) 9093–9100
Contents lists avai
Tetrahedron
journal homepage: www.elsevier .com/locate/ tet
Synthesis of 1-C-linked diphosphate analogues of UDP-N-Ac-glucosamineand UDP-N-Ac-muramic acid
Andrej Babic a,*, Stanislav Gobec a, Christine Gravier-Pelletier b, Yves Le Merrer b, Slavko Pecar a,c
a Faculty of Pharmacy, University of Ljubljana, Askerceva 7, 1000 Ljubljana, Sloveniab Universite Paris Descartes, UMR 8601 CNRS, Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, 45 rue des Saints-Peres, 75006 Paris, Francec Jo�zef Stefan Institute, Jamova cesta 39, 1000 Ljubljana, Slovenia
a r t i c l e i n f o
Article history:Received 22 March 2008Received in revised form 12 June 2008Accepted 3 July 2008Available online 8 July 2008
0040-4020/$ – see front matter � 2008 Elsevier Ltd.doi:10.1016/j.tet.2008.07.009
a b s t r a c t
UDP-N-acetyl-glucosamine and UDP-N-acetyl-muramic acid are two important cytoplasmic precursorsof bacterial peptidoglycan. The convergent synthesis of their analogues is reported. The a-1-C-linked-N-acetyl-glucosamine was synthesized using microwave-assisted Keck radical allylation. Oxidation of al-kene derivatives to the corresponding carboxylic acids allowed attachment to O- and N-sulfamoyluridinegiving stable diphosphate mimetics.
� 2008 Elsevier Ltd. All rights reserved.
OHORO
NH
HON
NH
OO
O
H
OHORO NH
HO
O P
O
N
NH
OHHO
OO
O
O
PO
OHOH
O
Ac
Ac
X = NH, O
1. Introduction
N-Acetyl-glucosamine (GlcNAc) and N-acetyl-muramic acid(MurNAc) are present in almost all eubacteria as constituent unitsof the bacterial cell wall peptidoglycan.1 In the biosynthesis ofpolysaccharides, glycolipids, glycoproteins and the bacterial cellwall, GlcNAc and MurNAc are usually utilized, in the form ofprecursors containing nucleoside diphosphates, by a variety ofenzymes, in particular glycosyltransferases.2–5 These enzymes areimportant targets for possible modulation of certain biochemicalprocesses. We have focused on the enzymes involved in the cyto-plasmic steps of peptidoglycan biosynthesis.1,6 Since MurA, MurCand MurG enzymes transfer or modify UDP-GlcNAc or UDP-Mur-NAc, it was reasoned that analogues of these compounds couldmimic their substrates and serve as useful starting points fordesigning new enzyme inhibitors.
The design and synthesis of nucleotide diphosphate sugar ana-logues are imposing challenges. Replacement of the diphosphatemoiety is not straightforward. Ionic, steric and H-bond formingcharacteristics, as well as the distance between nucleoside andsugar moieties, all play an important role and affect the binding tothe target enzymes. We have introduced the O- and N- sulfamoylamide group in lieu of the diphosphate part of UDP as depicted in
: þ386 1 4258 031.).
All rights reserved.
Figure 1. This was based on similar approaches that showedinhibition of protein glycosylation.7
To render the target compounds chemically and enzymaticallystable, C-linked glycosides were chosen as a synthetically feasiblealternative to natural a-O-glycosides.5,8–10 However, to achieve this,maintaining the a-configuration at the anomeric position of the N-Ac-glucosamine would be crucial. Fortunately, synthetic strategiesfor making C-linked derivatives of N-Ac-glucosamine have beenthoroughly investigated.11–18 Keck radical allylation19 was chosenas the key reaction to introduce the a-carbon–carbon bond at theanomeric position.
O OHHO
XS
N
O O O OHR = H,
Figure 1. Design of O- and N-sulfamoyl amide UDP-GlcNAc and UDP-MurNAcanalogues.
A. Babic et al. / Tetrahedron 64 (2008) 9093–91009094
Here we describe the synthesis of the UDP-GlcNAc and UDP-MurNAc analogues where the uridine and C-1-linked sugar moie-ties are connected by an O- or N-sulfamoylamide bridge, which actsas a diphosphate isostere.
2. Results and discussion
The target compounds were synthesized using a convergentstrategy. The O- and N-sulfamoyl uridine moieties were synthe-sized separately from 1-C-linked N-acetyl-a-D-glucosamine de-rivatives. Both moieties were then joined by a simple couplingreaction followed by deprotection to afford the end compounds.
Scheme 1 presents the synthesis of the desired a-1-C-linked-GlcNAc. According to known procedures, N-acetyl-glucosamine 1was first converted to chloride derivative 2 in acetyl chloride sat-urated with hydrogen chloride gas.14 The reaction proceeds aspreviously described albeit the instability of 2 prevents normalchromatographic purification. In the next reaction step we formedthe desired carbon–carbon bond using Keck radical allylation. De-spite the fact that this reaction proceeds via a free radical mecha-nism, it is not devoid of stereoselectivity and a number of authorshave reported this reaction using chloride 2 with different resultinga/b anomer ratios.13–15,17 To achieve as high an a/b anomer ratio aspossible, only 3 equiv of allyltributyltin was used, and only0.15 equiv of azobisisobutyronitrile (AIBN) initiator, as reported byCui and Horton.17 This lowered the yield slightly compared to thatreported by Bouvet and Ben but the 1H NMR spectrum indicatedpure a-anomer of 3. As previously observed very little side productswere formed when THF was used as a solvent in the reaction.14
In addition to conventional oil-bath heating, the reaction wasalso attempted under microwave-assisted conditions. Using es-sentially the same amount of reagents and solvent, a significantreduction in reaction time was achieved, the reaction being com-plete in only 10 min at 100 �C compared to 7 h at 70 �C under theconventional procedure. A slight improvement in reaction yieldwas also achieved, but more importantly, and to our surprise, onlythe a-anomer was detected. Given the very short reaction time andrelatively low temperature needed to achieve total conversion ofthe starting material, it appears that this novel use of microwaveirradiation for Keck radical allylation is preferable to conventionalheating.
In the next step, 1-C-allyl-N-acetyl-glucosamine 3 was oxidizedto the corresponding carboxylic acid 4 using the sodium periodateand ruthenium trichloride oxidation system. Diluted dioxane/watercould be used instead of the tricomponent tetrachlorocarbon,acetonitrile and water mixture to perform this reaction in verygood yield.21,22
Alkene 3 was used as the starting material for the synthesis of1-C-linked N-acetyl-muramic acid 7. The standard two-stepdeprotection and reprotection protocol for peracetylated alkenederivative 3 was used (Scheme 2).
Acetyl groups were removed with sodium methoxide in abso-lute methanol at ambient temperature in quantitative yield. Afterthe usual work up with Amberlyst� 15 ion exchange resin, thecrude product was reprotected at the 40- and 60-hydroxy positionwith benzaldehyde and trifluoroacetic acid as catalyst.14 This gavebenzylidene derivative 5 with the 30-hydroxy group convenientlyunprotected and available for further synthesis.
The muramic acid moiety was introduced to the free 30-hydroxygroup through the Williamson ether synthesis. Racemic (R,S)-2-chloropropionic acid was used as a reagent, since it was known thatstereoselectivity could be achieved for this reaction.23–25 As in thecase of 2-acetamido-1-O-benzyl-4,6-O-benzylidene-2-deoxy-a-D-glucosamine,25 the (R)-muramic acid derivative 6 was obtainedfrom 5 in good yield with a diastereoisomeric ratio of 4.3:1. Theratio was determined from the benzylidene proton signal of themuramic and isomuramic derivatives mixture at 5.59 and5.42 ppm, respectively. To ensure that the desired diastereoisomerwith R-lactoyl configuration was the major reaction product,6 equiv of sodium hydride was used as a base and 5 equiv of race-mic (R,S)-2-chloropropionic acid, which was added quickly to thesuspension. The obtained crude sodium salt was treated withmethyl iodide in DMF at ambient temperature to give 6 as a di-astereoisomeric mixture. The diastereoisomeric mixture wasseparated using flash chromatography under essentially the samechromatographic conditions as for the methyl ester of (R,S)-1-O-benzyl-4,6-O-benzylidene-N-acetyl-muramic acid described pre-viously.25 Carboxylic acid 7 was obtained in the same way ascompound 4 in dioxane/water with the sodium periodate and ru-thenium trichloride oxidation system, which left all the protectinggroups intact.
The N- and O-sulfamoyluridine derivatives 12 and 13 weresynthesized from commercially available 20,30-isopropylidene-uri-dine 8. The 50-N-sulfamoyl derivative 12 was obtained in four re-action steps (Scheme 3). The 50-hydroxy group was first convertedinto a p-toluenesulfonyl ester 9 with p-toluenesulfonylchloride andpyridine as the catalyst/base. The following nucleophilic sub-stitution with sodium azide gave azido derivative 10 in high yield.The ensuing catalytic hydrogenation proceeded to completionwithin 3 h, in complete contrast to the report that the azido de-rivative 10 was entirely resistant to reduction.26 It was establishedthat a dilute solution of the azido derivative 10 in methanol is re-quired for fast catalytic hydrogenation. Compound 11 was found tobe pure by 1H NMR spectroscopy and was used without purificationin the next reaction step due to its inherent instability. Treatment of11 with sulfamoyl chloride27 at 0 �C in dry dichloromethane, withtriethylamine as the base, gave the desired 50-N-sulfamoyl-20,30-O-isopropylidene-uridine 12 in relatively low yield. On the otherhand, 50-O-sulfamoyl-20,30-O-isopropylidene-uridine 13 was syn-thesized in a straightforward direct sulfamoylation of 20,30-O-iso-propylidene-uridine under reaction conditions similar to thoseused for 12.
A. Babic et al. / Tetrahedron 64 (2008) 9093–9100 9095
The 20,30-O-isopropylidene uridine derivatives 12 and 13 werethen coupled with the protected C-linked MurNAc acid 7 andGlcNAc 4 to give N-sulfamoyl compound 14 and O-sulfamoyl de-rivatives 15 and 18 (Schemes 4 and 5). The coupling reaction was
tested with several coupling reagents, however, using equimolaramounts of carboxylic acid and N- or O-sulfamoyl-20,30-O-iso-propylidene-uridine and small excesses of DCC and DMAP(1.2 equiv) gave the best yields in short reaction times at room
, rt, 24 h; (b) 70% TFA, rt, 1 h; (c) 1 M LiOH, rt, 2 h.
A. Babic et al. / Tetrahedron 64 (2008) 9093–91009096
temperature. However, the use of DCC as the coupling agent madethe isolation and purification difficult. Dicyclohexylurea had tobe removed completely by precipitation and filtration prior tochromatographic purification. Only then could the products 14, 15and 18, be isolated in a pure form without the co-elution ofdicyclohexylurea.
The deprotection procedure for C-linked N-acetyl-glucosaminederivatives 14 and 15 (Scheme 4) proceeded smoothly. Acetylgroups were removed in the same manner as for compound 4 withsodium methoxide in dry methanol at room temperature. Thenacidic hydrolysis of the isopropylidene protecting group was per-formed in 70% trifluoroacetic acid. The crude products were puri-fied by gel filtration using Sephadex LH-20, which afforded purecompounds 16 and 17.
In a similar manner the coupling reaction between the C-linkedN-acetyl-muramic acid derivatives 7 and O-sulfamoyl-20,30-O-iso-propylidene-uridine 13, using DCC and DMAP, gave 18 (Scheme 5).The benzylidene and isopropylidene protecting groups were re-moved in 70% trifluoroacetic acid and then the ester group washydrolyzed under basic conditions (1 M LiOH). The product 19 wasisolated as the dilithium salt after pH adjustment with Amberlyst15 and gel filtration on LH-20.
3. Conclusions
The synthesis of new 1-C-linked O- and N-sulfamoylamido an-alogues of UDP-GlcNAc and UDP-MurNAc is presented. The keycompound 3 has been synthesized using the Keck radical allylationunder microwave-accelerated conditions and converted into 1-C-linked-MurNAc and GlcNAc carboxylic acid derivatives. Couplingwith protected N- and O-sulfamoyl uridine derivatives and theensuing deprotection reactions gave new diphosphate analogues ofUDP-GlcNAc and UDP-MurNAc that could provide starting pointsfor further development of new inhibitors of the bacterial cell wallbiosynthesis.
4. Experimental
4.1. General
Chemicals from Sigma–Aldrich and Fluka were used withoutfurther purification. Analytical TLC was performed on Merck silicagel (60 F254) plates (0.25 mm) and components visualized withultraviolet light and dyed with 20% sulfuric acid in ethanol, rho-damine G6, 2,4-dinitrophenylhydrazine and ninhydrin. Flashchromatography was performed using Merck silica gel (0.040–0.063 mm). Gel filtration was performed using LH-20 stationaryphase and methanol as eluent. 1H, 13C, DEPT-135, gradient COSY andgradient HSQC NMR spectra were recorded on a Bruker AM250 andAVANCE DPX300 spectrometers in CDCl3, DMSO-d6, D2O, MeOH-d4
and acetone-d6 solution with TMS as the internal standard. Mi-croanalyses were performed on a Perkin–Elmer C, H, N analyzer240C. Mass spectra were obtained using a VG-Analytical AutospecQ and Q-TOF Premier mass spectrometers.
4.3.1. Method A (classical heating)2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-a-D-glucopyranosyl chlor-
ide (1.2 g, 3.3 mmol) and AIBN (170 mg, 1.1 mmol) were suspendedin allyltributyltin (3.86 mL, 12.6 mmol) and distilled THF (7.0 mL).The reaction mixture was flushed with argon for 10 min and thenstirred at 70 �C for 7 h after which TLC analysis indicated the ab-sence of starting material. The solvent was evaporated under re-duced pressure and the oily residue dissolved in acetonitrile(50 mL) and extracted with pentane (5�30 mL). Flash chromato-graphy (dichloromethane/acetone 10:1) afforded a colourless waxysolid, which crystallized overnight giving a colourless solid (0.60 g,1.5 mmol, 49% yield).
4.3.2. Method B (microwave-accelerated)2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-a-D-glucopyranosyl chlor-
ide (365 mg, 1.0 mmol) and AIBN (25 mg, 0.15 mmol) were sus-pended in allyltributyltin (1.0 mL, 3.0 mmol) and distilled THF(2.0 mL). The reaction mixture was flushed with argon for 10 minand then stirred in a microwave reactor at 100 �C for 10 min. Thepower was set at 10 W. The reactor was cooled with compressed airduring the reaction to reduce temperature fluctuations. The solventwas evaporated under reduced pressure and the oily residue dis-solved in acetonitrile (20 mL) and extracted with pentane(5�15 mL). Flash chromatography (dichloromethane/acetone 10:1)afforded a colourless waxy solid, which crystallized overnight giv-ing colourless solid (185 mg, 0.51 mmol, 51% yield). The 1H NMRspectra are identical using method A or B. Mp 104–105 �C. IR (KBr,cm�1): 3420, 2944, 1740, 1667, 1515, 1431, 1386, 1244, 1152, 1093,1035, 988, 936, 924, 814, 668, 608. [a]D
3-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-a-D-glucopyranosyl)propene (210 mg, 0.54 mmol) was dissolved in water (20 mL) anddioxane (20 mL). While being stirred vigorously, sodium periodate(460 mg, 2.15 mmol) and ruthenium(III) chloride (6 mg, 0.03 mmol)were added. The same amount of sodium periodate was addedagain after 1 h. The reaction was complete in 4 h and the solventswere evaporated in vacuo. The dark green solid was resuspended inethyl acetate and filtered. The crude product obtained after evapo-ration of ethyl acetate was purified with flash chromatography(dichloromethane/methanol 15:1 to 8:1). Colourless oil (157 mg,0.40 mmol, 74% yield), which slowly solidified was obtained. Mp148–150 �C. IR (KBr, cm�1): 3547, 2939, 2363, 1748, 1558, 1374, 1239,1139, 1088, 1043, 913, 602. [a]D
Compound 5 was synthesized according to Ref. 14.3-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-a-D-glucopyranosyl)
propene (200 mg, 0.54 mmol) was dissolved in dry methanol(2.0 mL). Under an argon atmosphere 30% sodium methoxide(100 mL) was added and the reaction mixture stirred at ambienttemperature. After 2 h, the reaction was judged complete by TLCand the solution was neutralized using Amberlyst IR-15 ion ex-change resin and concentrated to dryness under reduced pressureto obtain colourless oil. 3-(2-Acetamido-2-deoxy-a-D-glucopyr-anosyl) propene was judged pure by 1H and 13C NMR spectroscopyand was dissolved in benzaldehyde (1.8 mL) and cooled to 0 �Cunder argon. Anhydrous trifluoroacetic acid (100 mL) was thenadded dropwise and the temperature was raised to ambient. After3 h, the reaction mixture was evaporated to dryness under reducedpressure, which gave a colourless solid (168 mg, 0.50 mmol, 92%yield). Mp >240 �C. IR (KBr, cm�1): 3286, 1996, 1630, 1542, 1375,1136, 1094, 1039, 993, 757, 697. [a]D
3-(2-Acetamido-4,6-benzylidene-2-deoxy-a-D-glucopyranosyl)propene (420 mg, 1.26 mmol) was suspended in dioxane (40 mL)stored over molecular sieves. While being stirred under an argonatmosphere, sodium hydride (544 mg, 23 mmol) was added care-fully to the suspension and the temperature raised to 50 �C. After0.5 h, the reaction mixture was cooled to ambient temperature and2-chloropropionic acid (580 mL, 6.3 mmol) was added dropwise tothe suspension. The reaction mixture was heated at 50 �C for 4 h atwhich time TLC analysis indicated total absence of starting mate-rial. Water (1.0 mL) was added carefully at ambient temperature toquench the excess sodium hydride and the reaction mixture wasevaporated to dryness under reduced pressure. The crude productwas suspended in brine (30 mL), cooled on ice for 1 h and theprecipitated solids filtered off. After extensive drying in vacuo for24 h the sodium salt was dissolved in DMF (15 mL) and methyliodide (400 mL, 6.4 mmol) was added in one portion at room tem-perature. The reaction mixture was stirred for 24 h. The solventswere evaporated and the crude product purified by flash chroma-tography (dichloromethane/hexane/ethyl acetate 1:1:1) yieldinga colourless powder (253 mg, 0.60 mmol, 48% yield). Mp 209–210 �C. IR (KBr, cm�1): 3311, 3083, 2948, 2873,1758,1736,1651,1553,1450,1372,1341, 1316,1211,1174,1132,1094, 996, 922, 752, 697, 648.[a]D
3-(2-Acetamido-4,6-benzylidene-2-deoxy-3-((R)-1-(methoxy-carbonyl)ethoxy)-a-D-glucopyranosyl) propene (100 mg, 0.24 mmol)was dissolved in water (20 mL) and dioxane (20 mL). Sodium peri-odate (440 mg, 2.06 mmol) and ruthenium(III) chloride (5 mg,0.02 mmol) were then added consecutively and the dark solutionwasstirred overnight. The solution was concentrated to dryness underreduced pressure and the dark green solid resuspended in ethyl ac-etate and filtered off. Ethyl acetate was evaporated and the crudeproduct applied to a flash chromatography column. The product waseluted by gradient elution (dichloromethane/methanol 15:1 to 5:1)yielding colourless solid (98 mg, 0.22 mmol, 93% yield). Mp 158–160 �C. IR (KBr, cm�1): 3339, 2938, 1734, 1655,1546,1450,1372,1314,1278, 1212, 1130, 1093, 1010, 914, 877, 749, 697. [a]D
50-Azido-50-deoxy-20,30-O-isopropylidene-uridine (650 mg,2.1 mmol) was dissolved in methanol (150 mL) and argon was
A. Babic et al. / Tetrahedron 64 (2008) 9093–91009098
bubbled through the solution for 10 min. Pd/C (50 mg, 10%) wasadded and the suspension stirred under hydrogen. After 3 h, TLCindicated the disappearance of the starting azide and the Pd/C wasfiltered off. The solvent was evaporated under reduced pressure atnot more than 40 �C and the colourless solid (585 mg, 2.07 mmol,98%) dried in vacuo. 1H NMR spectroscopy indicated a high degreeof purity so the product was used without purification for furthersynthesis. Mp 85–87 �C. [a]D
50-Amino-50-deoxy-20,30-O-isopropylidene-uridine (100 mg,0.35 mmol) was dissolved in dry dichloromethane (8 mL) and tri-ethylamine (180 mL). Under an argon atmosphere, the solution wascooled to 0 �C in an ice bath. Sulfamoyl chloride (65 mg, 0.56 mmol)was added to the solution. The temperature was then raised to am-bient temperature. After being stirred for 5 h, MeOH was used toquench the reaction mixture, which was concentrated to drynessunder reduced pressure. Flash chromatography afforded a colourlessoil (36 mg, 0.10 mmol, 29%). Mp 110–112 �C. IR (KBr, cm�1): 1690,1460, 1386, 1334, 1273, 1215, 1159, 1089, 860, 816, 550. [a]D
20,30-O-Isopropylidene-uridine (300 mg, 1.06 mmol) was sus-pended in dichloromethane (20 mL). After the addition of tri-ethylamine (1.0 mL), the solution was cooled to 0 �C. Sulfamoylchloride (150 mg, 1.3 mmol) was added to the stirred reactionmixture. After 1 h another portion of sulfamoyl chloride (600 mg,1.3 mmol) was added and the reaction mixture allowed to warm toambient temperature. After stirring overnight, methanol (0.5 mL)was added and the reaction mixture filtered through a pad of Celite545 and evaporated to dryness. The crude product was purified byflash chromatography (ethyl acetate/methanol 20:1), which affor-ded a colourless solid (220 mg, 0.61 mmol, 57% yield). Mp 90–92 �C.IR (KBr, cm�1): 1686, 1560, 1465, 1380, 1274, 1216, 1183, 1070, 992,932, 814, 668, 553. [a]D
0.15 mmol) and 4-dimethylaminopyridine (20 mg, 0.15 mmol) weresuspended in dry dichloromethane (15 mL). The suspension dis-solved in 15 min and precipitate started forming after 1 h. The re-action mixture was left stirring overnight and then concentrated to3 mL and the precipitate filtered off and washed with the minimalvolume of dichloromethane. The solution was concentrated to dry-ness. Flash chromatography (dichloromethane/methanol 10:1–7:1)afforded a colourless solid (49 mg, 0.07 mmol, 51% yield). Mp 148–150 �C. IR (KBr, cm�1): 1746, 1692, 1647, 1561, 1458, 1378, 1236, 1150,1043, 824, 728, 562. [a]D
20,30-O-Isopropylidene-50-O-(2-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-a-D-glucopyranosyl)acetamidosulfamoyl) uridine (37 mg,0.050 mmol) was dissolved in dry methanol (2.0 mL) and flushedwith argon. Sodium methoxide solution in methanol (150 mL, 30%)was added dropwise at ambient temperature. After being stirred for4 h, the reaction mixture was diluted with methanol (5 mL) andAmberlyst 15 ion exchange resin was added to adjust the pH of thesolution to 7. The resin was filtered off and methanol evaporatedunder reduced pressure. The obtained white solid was dissolved inbidistilled water (800 mL) and trifluoroacetic acid (1850 mL) andstirred at ambient temperature. After 1 h, the solvents were evap-orated under reduced pressure and at temperature not exceeding30 �C. Traces of water were removed by toluene co-evaporationunder reduced pressure, which gave a brownish solid. The crudeproduct was purified on a Sephadex LH-20 gel filtration column withmethanol as mobile phase. The fractions containing the productwere pooled and evaporated under reduced pressure to afforda colourless solid (17 mg, 0.030 mmol, 60% yield). Mp 158–159 �C. IR(KBr, cm�1): 2930, 2345, 1686, 1467, 1441, 1273, 1160. [a]D
4.15. 4-Dimethylaminopyridine salt of 20,30-O-isopropylidene-50-O-(2-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-a-D-glucopyranosyl)acetamidosulfamoyl)uridine (15)
A. Babic et al. / Tetrahedron 64 (2008) 9093–9100 9099
acetic acid (90 mg, 0.23 mmol), dicyclohexylcarbodiimide (74 mg,0.36 mmol) and 4-dimethylaminopyridine (44 mg, 0.36 mmol)were suspended in dry dichloromethane (20 mL). The suspensiondissolved in 15 min and a precipitate started forming after 0.5 h.The reaction mixture was left stirring overnight. The solvent wasevaporated to concentrate the mixture to 3 mL. Then the pre-cipitate was filtered off and washed with a minimal volume ofdichloromethane. The solution was concentrated to dryness. Gra-dient flash chromatography (dichloromethane/methanol 10:1 to7:1) afforded colourless solid (98 mg, 0.13 mmol, 57% yield). Mp124–126 �C. IR (KBr, cm�1): 1747, 1692, 1648, 1561, 1458, 1378,1236, 1151, 1043, 824, 728, 562. [a]D
20,30-O-Isopropylidene-50-O-(2-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-a-D-glucopyranosyl)acetamidosulfamoyl) uridine (65 mg,0.088 mmol) was dissolved in dry methanol (4.0 mL) and flushedwith argon. Sodium methoxide solution in methanol (300 mL, 30%)was added dropwise at ambient temperature. After being stirred for4 h, the reaction mixture was diluted with methanol (10 mL) andAmberlyst 15 ion exchange resin was added to adjust the pH of thesolution to 7. The resin was filtered off and methanol evaporatedunder reduced pressure. The obtained white solid was dissolved inbidistilled water (800 mL) and trifluoroacetic acid (1.85 mL) andstirred at ambient temperature. After 1 h, the solvents were evap-orated under reduced pressure and at temperature not exceeding30 �C. Traces of water were removed by toluene co-evaporationunder reduced pressure, which gave a gummy white solid. The crudeproduct was purified using Sephadex LH-20 gel filtration columnand methanol as mobile phase. The fractions containing the productwere pooled and evaporated under reduced pressure to afforda colourless solid (35 mg, 0.062 mmol, 70% yield). Mp 193–196 �C. IR(KBr, cm�1): 2929, 2345,1686,1547,1467,1390,1273,1220, 814, 766.[a]D
2-(2-Acetamido-4,6-benzylidene-2-deoxy-3-((R)-1-(methoxy-carbonyl)ethoxy)-a-D-glucopyranosyl)acetic acid (50 mg, 0.11 mmol),20,30-O-isopropylidene-50-O-sulfamoyluridine (42 mg, 0.11 mmol),dicyclohexylcarbodiimide (26 mg, 0.12 mmol) and 4-dimethyl-aminopyridine (15 mg, 0.12 mmol) were suspended in drydichloromethane (10 mL). The suspension dissolved in 15 minand a precipitate started forming after 1 h. The reaction mixturewas left stirring overnight and concentrated to 2 mL and theprecipitate filtered off and washed with a minimal volume ofdichloromethane. The solution was concentrated to dryness.Gradient flash chromatography (dichloromethane/methanol 10:1to 7:1) afforded colourless solid (60 mg, 0.077 mmol, 70% yield).Mp 193–194 �C. IR (KBr, cm�1): 1691, 1571, 1460, 1380, 1277,1145, 1092, 1001, 832, 760, 698. [a]D
20,30-O-Isopropylidene-50-O-(2-(2-acetamido-4,6-benzylidene-2-deoxy-3-((R)-1-(methoxycarbonyl)ethoxy)-a-D-glucopyranosyl)-acetamidosulfamoyl)uridine (20 mg, 0.026 mmol) was suspendedin water (0.5 mL) and trifluoroacetic acid (1.5 mL) at 0 �C. Thesuspension dissolved immediately upon the addition of the tri-fluoroacetic acid and stirred for 1 h. The solvents were evaporatedunder reduced pressure using a rotary evaporator at temperaturenot exceeding 30 �C. Water and benzaldehyde traces were removedwith toluene co-evaporation (2�5 mL), affording a colourless oil,which was dried for several hours in vacuo. Lithium hydroxide(1 M, 1.0 mL) and methanol (1.0 mL) were added at room temper-ature and the solution was stirred for 2 h. The solution was dilutedwith methanol (2 mL) and the pH adjusted to 7 with Amberlyst� 15ion exchange resin. After filtration, the solvent was evaporatedunder reduced pressure. The crude product was purified usingSephadex LH-20 gel filtration column (flow rate 5 mL/h) withmethanol as mobile phase. The fractions containing the productwere pooled and evaporated under reduced pressure to afforda colourless solid (16 mg, 0.024 mmol, 94% yield). Mp 211–213 �C.IR(KBr, cm�1): 3422, 2390, 1687, 1459, 1209, 1145, 849. [a]D
A. Babic et al. / Tetrahedron 64 (2008) 9093–91009100
71.50, 68.92, 62.47, 54.60, 36.58, 22.60, 19.79. LRMS (ESI), m/z 641.2(MþH)þ, 647.2 (MþLi)þ, 653.2 (Mþ2Li)þ. HRMS (ESI), m/z calcd forC22H33N4O16S1 641.1612 (MþH)þ, found 641.1617, m/z calcd forC22H32N4O16SLi 647.1694 (MþLi)þ, found 647.1697. HPLC: ColumnC18 Phenomex Luna 10 m; mobile phase: 10% acetonitrile, 90%trifluoroacetic acid (0.1%), flow rate 1.0 mL/min; injection volume:10 mL; retention time: 7.61 min (96.26% at 254 nm, 98.97 at 210 nm).
Acknowledgements
This work was supported by the European Union FP6 IntegratedProject EUR-INTAFAR (Project no. LSHM-CT-2004-512138) underthe thematic priority Life Sciences, Genomics and Biotechnologyfor Health, Institute Charles Nodier and the Ministry of HigherEducation, Science and Technology of the Republic of Slovenia. Theauthors thank Dr. Roger H. Pain for critical reading of themanuscript.
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