Synthesis of fluorescently labeled UDP-GlcNAc analogues and their evaluation as chitin synthase substrates

Article abstract of DOI:10.1021/jo0483670

(Chemical Equation Presented) Chitin synthase (CS) polymerizes UDP-GlcNAc to form chitin (poly-β(1,4)-GlcNAc), a key component of fungal cell wall biosynthesis. Little is known about the substrate specificity of chitin synthase or the scope of substrate m

Full text of DOI:10.1021/jo0483670

Synthesis of Fluorescently Labeled UDP-GlcNAc Analogues and
Their Evaluation as Chitin Synthase Substrates
Adam R. Yeager and Nathaniel S. Finney*,†
Department of Chemistry and Biochemistry, University of California, San Diego,
9500 Gilman Dr., La Jolla, California 92093
finney@unizh.ch
Received September 15, 2004
Chitin synthase (CS) polymerizes UDP-GlcNAc to form chitin (poly-â(1,4)-GlcNAc), a key component
of fungal cell wall biosynthesis. Little is known about the substrate specificity of chitin synthase
or the scope of substrate modification the enzyme will tolerate. Following a previous report
suggesting that 6-O-dansyl GlcNAc is biosynthetically incorporated into chitin, we became interested
in developing an assay for CS activity based on incorporation of a fluorescent substrate. We describe
the synthesis of two fluorescent UDP-GlcNAc analogues and their evaluation as chitin synthase
substrates.
Introduction
Chitin synthase (CS) converts cytosolic uridine diphos-
phoryl-N-acetylglucosamine (UDP-GlcNAc) to chitin, poly-
â(1,4)-GlcNAc (Figure 1). CS is an attractive therapeutic
target, as chitin is an integral structural component of
the fungal cell wall but is not found in mammals. Current
methods for CS activity determination are based on the
incorporation of radiolabeled substrate into the growing
chitin chains.1,2 While these methods provide reasonably
reliable and reproducible results, there is a clear need
for an assay that is both faster and avoids the hazards
associated with radioactivity.3 We present here the
design, synthesis, and evaluation of the first fluorescently
labeled UDP-GlcNAc analogues (3 and 4) as part of our
ongoing efforts to develop inhibitors of CS.
FIGURE 1. Synthesis of chitin.
† Present address: Organisch-Chemisches Institut, Universita¨t
Zu¨rich, Winterthurerstrasse 190, 8057 Zu¨rich.
Background. Most studies of chitin synthase rely on
a similar assay protocol: incubation of CS (obtained as
a crude membrane preparation) with a radiolabeled
substrate (14C-UDP-GlcNAc) results in the synthesis of
radiolabeled chitin.1,2 The chitin is isolated by precipita-
tion and filtration of the reaction mixture, facilitated by
the insolubility of chitin chains in almost all solvents.
(1) Orlean, P. J. Biol. Chem. 1987, 262, 5732-5739.
(2) Cabib, E. Antimicrob. Agents Chemother. 1991, 35, 170-173.
(3) To date, only one alternative to the radiolabeled substrate has
been reported. For an indirect assay that measures the amount of
chitin bound to a wheat germ agglutinin-covered surface by monitoring
the formation of a horseradish peroxidase-conjugate, see: Lucero, H.
A.; Kuranda, M. J.; Bulik, D. A. Anal. Biochem. 2002, 305, 97-105.
10.1021/jo0483670 CCC: $30.25 © 2005 American Chemical Society
Published on Web 01/27/2005
J. Org. Chem. 2005, 70, 1269-1275
1269

Yeager and Finney
CHART 1. Fluorescently Labeled GlcNAc (1) and
UDP-GlcNAc (2)
CHART 2. Fluorescently Labeled UDP-GlcNAc
Analogues
Measurement of the radioactivity content of the filter
pads provides a means to quantify CS activity. Scintil-
lation counting is the bottleneck in the assay process,
limiting the total number of data points that can be
collected in a day.
A fluorescence-based assay could dramatically increase
the amount of data acquired per unit of time, in part
through the use of fluorescent plate readers.4-6 Increas-
ing the rate of data acquisition would facilitate routine
kinetic characterization of inhibitors (for which only IC50
values are typically reported) and would permit large
libraries of compounds to be screened for activity against
the enzyme, accelerating the discovery of novel inhibitors.
Fluorescence-based assays have been used in this capac-
ity for nonpolymerizing (single sugar) glycosyltransferase
systems.7
viding a more direct measurement of CS activity, and
(2) it provides compatibility with the most common in
vitro CS assay protocol. This naturally led us to consider
compounds such as 2 and 3 (Chart 2) as potential
substrates. While there was no prior indication that
substitution of the 2-NHAc group would be tolerated by
CS,11 4 was prepared and evaluated on the basis of its
synthetic accessibility.
Synthesis of Dansyl UDP-GlcNAc Analogue 3. The
susceptibility of the dansylate in 1 to nucleophilic dis-
placement precluded the direct synthesis of the analogue
2. As an alternative, the 6-OH group was converted to
an amine and subsequently dansylated, forming a much
more robust sulfonamide bond.
In addition, a fluorescence-based assay would avoid the
well documented hazards associated with the use of
radioactive materials.8 While it has been demonstrated
that less hazardous 3H-labeled UDP-GlcNAc can be used
in place of 14C-labeled substrate,9,10 the use of any
radioactive material introduces other obvious problems
involving procurement, transportation, and secure stor-
age.
Analogue Design. Developing a fluorescence assay for
CS activity based on a fluorescent substrate analogue
requires that analogues be processed by the enzyme with
efficiency similar to that of UDP-GlcNAc. Carrano et al.
have reported that fluorescently labeled GlcNAc analogue
(1, Chart 1) is biosynthetically incorporated into chitin
by Candida albicans protoplasts, with the implication
that 1 is transiently converted in vivo to 2.10
Treatment of GlcNAc with concentrated HCl in BnOH
provided the anomeric benzyl compound 5 in 71% yield
(Scheme 1).12,13 Selective tosylation of the primary alcohol
was effected with TsCl in cold (0 °C) pyridine, and
subsequent displacement of the primary tosyl group with
azide anion was achieved using NaN3 in warm (70 °C)
DMF to provide 7. The resulting azide was reduced under
Staudinger conditions (PPh3, THF, H2O).14 Selective
dansylation of 8 was carried out by treatment with
dansyl-Cl in cold (0 °C) pyridine. Acylation of the diol
(as well as the sulfonamide) was performed under
standard conditions to provide 10 (95% yield).
Standard catalytic or transfer hydrogenation methods
failed to remove the anomeric benzyl group.15-22 After
(11) For an example of a lectin-aminoglycoside interaction that is
enhanced by the addition of a large amine substituent, see: Liang,
R.; Loebach, J.; Horan, N.; Ge, M.; Thompson, C.; Yan, L.; Kahne, D.
Proc. Natl. Acad. Sci. 1997, 94, 10554-10559.
The indication that CS can tolerate such substitution
at the 6-OH position of UDP-GlcNAc provides a basis for
the design of new potential substrates. The use of
analogues of UDP-GlcNAc rather than GlcNAc is appeal-
ing in that (1) it obviates the need for additional
metabolic transformations prior to polymerization, pro-
(12) Kuhn, R.; Baer, H. H.; Seeliger, A. ANN 1958, 611, 236-241.
(13) A variety of anomeric protecting groups were examined (TBS,
allyl, boc, etc.), and the benzyl group proved to be superior with regard
to subsequent reaction selectivity and other deprotection steps.
(14) Staudinger, H.; Meyer, J. Helv. Chim. Acta 1919, 2, 635.
(15) Kiso, M.; Fujita, M.; Tanahashi, M.; Fujishima, Y.; Oqawa, Y.;
Hasegawa, A. Carbohydr. Res. 1988, 177, 51-67.
(16) Sharma, M.; Bernacki, R. J.; Paul, B.; Korytnyk, W. Carbohydr.
Res. 1990, 198, 205-221.
(4) Washiya, K.; Furuike, T.; Nakajima, F.; Lee, Y. C.; Nishimura,
S.-I. Anal. Biochem. 2000, 283, 39-48.
(5) Shedletzky, E.; Unger, C.; Delmer, D. P. Anal. Biochem. 1997,
249, 88-93.
(17) Thomas, R. L.; Rutan, J. F.; Abbas, S. A.; Matta, K. L.
Carbohydr. Res. 1989, 189, 21-30.
(6) Borg-von Zepelin, M.; Wagner, T. Mycoses 1995, 38, 339-347.
(7) Hector, R.; Braun, P. C. J. Clin. Microb. 1986, 24, 620-624.
(8) Regulatory Guide 8.29. Instructions Concerning Risks from
Occupational Radiation Exposure; U.S. Nuclear Regulatory Commis-
sion, Office of Public Affairs (OPA), Washington, D.C., 20555, 1996.
(9) (a) Chang, R.; Yeager, A. R.; Finney, N. S. Org. Biomol. Chem.
2003, 1, 39-41. (b) Yeager, A. R.; Finney, N. S. J. Org. Chem. 2004,
69, 613-618.
(18) Nicolaou, K. C.; Hummel, C. W.; Bockovich, N. J.; Wong, C.-H.
Chem. Commun. 1991, 870.
(19) Hecker, S. J.; Minich, M. L. J. Org. Chem. 1990, 55, 6051-
6054.
(20) Anantharamaiah, G. M.; Sivanandaiah, K. M. Ind. J. Chem. B
1976, 14B, 343-345.
(21) Alais, J.; Veyrieres, A. Tetrahedron Lett. 1987, 28, 3345-3348.
(22) Park, M. H.; Takeda, R.; Nakanishi, K. Tetrahedron Lett. 1987,
28, 3823-3824.
(10) Carrano, L.; Tavecchia, P.; Sponga, F.; Spreafico, F. J. Antibiot-
ics 1997, 50, 177-179.
1270 J. Org. Chem., Vol. 70, No. 4, 2005

Fluorescently Labeled UDP-GlcNAc Analogues
SCHEME 1. Synthesis of 3a
a Reagents and conditions: (a) BnOH, HCl (aq), 71%; (b) TsCl, Py, 0 °C, 81%; (c) NaN3, DMF, 70 °C, 82%; (d) PPh3, H2O, THF, 90%;
(e) dansylCl, py, 0 °C, 93%; (f) Ac2O, NEt3, DMAP, CH2Cl2, 95%; (g) FeCl3, CH2Cl2, 45-79%; (h) [(BnO)2PO]2O, LDA, THF, 78 °C, 53%;
(i) Pd/C, H2, MeOH, 74%; (j) 7:3:1 MeOH/H2O/NEt3, 99%; (k) UMP-morpholidate, tetrazole, py, 3-5 days, 8%.
SCHEME 2. Synthesis of Compound 4a
extensive experimentation, it was determined that only
iterative treatment with excess FeCl3 in CH2Cl2 effected
cleavage to a significant extent.22,23 Treatment of the
lithium salt of 11 with tetrabenzylpyrophosphate (ob-
tained from the DCC coupling of dibenzyl pyrophos-
phate)24,25 provided the R-dibenzyl phosphate 12. The
benzyl phosphate protecting groups were easily removed
by hydrogenolysis (1 atm H2, cat. Pd/C), and global
deacylation was achieved by stirring in 7:3:1 MeOH/H2O/
NEt3.26 The deprotected phosphate, 14, was coupled with
uridine monophosphate by stirring in minimal pyridine
for several days with the morpholidate of uridine mono-
phosphate to obtain dansylated UDP-GlcNAc (3).27-29
Synthesis of Dansyl UDP-GlcNAc Analogue 4. The
synthesis of the second fluorescently labeled UDP-
GlcNAc analogue, 4, was much less complicated than that
of 3 (Scheme 2). Reaction of amine 15 (obtained in three
steps from glucosamine)30,31 with dansyl-Cl provided 16
(68%). Removal of the anomeric acetate with hydrazine
acetate in DMF gave 17 in near quantitative yield.32-34
Phosphorylation, deprotection, and coupling of 17 were
performed in analogy to the synthesis of 3.
a Reagents and conditions: (a) DansylCl, py, 68%; (b) H2NNH2-
HOAc, DMF, 99%; (c) [(BnO)2PO]2O, LDA, THF, -78 °C, 90%; (d)
H2, Pd/C, MeOH, 99%; (e) 7:3:1 MeOH/H2O/NEt3, 99%; (f) UMP-
morpholidate, tetrazole, py, 16%
GlcNAc (initially 1 mM) was replaced with 3 or 4. Control
experiments were performed in which the omitted UDP-
GlcNAc was not replaced with 3 or 4.
Evaluation of 3 and 4 as CS Substrates. Com-
pounds 3 and 4 were evaluated as substrates for CS (see
Experimental Section for details). To test the ability of
the fluorescent analogues to replace the native substrate,
assays were conducted in which a fraction of the UDP-
Five different proportions (100, 90, 75, 60, and 40%)
of the normal 0.66 mM per assay of UDP-GlcNAc were
examined for chitin synthesis (Figure 2). Only a small
decrease in activity is observed when the amount of UDP-
GlcNAc is reduced (Figure 2, circles), consistent with the
fact that the substrate is initially present in saturating
concentrations. Experiments in which the omitted UDP-
GlcNAc is replaced with fluorescently labeled substrate
(Figure 2: 3, squares; 4, triangles) show a more pro-
nounced decrease in CS activity. As the proportion of the
fluorescent compounds is increased, the activity of the
enzyme decreases: at 40% UDP-GlcNAc/60% dansyl-
labeled substrate, the activity decreases 44% with 3 and
37% with 4.
(23) Rodebaugh, R.; Debenham, J. S.; Fraser-Reid, B. Tetrahedron
Lett. 1996, 37, 5477-5478.
(24) Elhalabi, J.; Rice, K. G. Carbohydr. Res. 2002, 337, 1935-1940.
(25) Khorana, H. G.; Todd, A. R. J. J. Chem. Soc. 1953, 2257-2260.
(26) Zamyatina, A.; Gronow, S.; Oertelt, C.; Puchberger, M.; Brade,
H.; Kosma, P. Angew. Chem., Int. Ed. 2000, 39, 4150-4153.
(27) Heidlas, J. E.; Lees, W. J.; Pale, P.; Whitesides, G. M. J. Org.
Chem. 1992, 57, 146-151.
(28) Wong, C.-H.; Wittman, V. J. Org. Chem. 1997, 57, 2144-2147.
(29) Moffat, J. G. Methods Enzymol. 1966, 8, 136-142.
(30) Medgyes, A.; Farkas, E.; Liptak, A.; Pozsgay, V. Tetrahedron
1997, 53, 4159-4178.
(31) Bergmann, M.; Zervas, L. Chem. Ber. 1931, 64, 973-980.
(32) Coward, J. K.; Lee, J. J. Org. Chem. 1992, 57, 4126-4135.
(33) Excoffier, G.; Gagnaire, D.; Utille, J.-P. Carbohydr. Res. 1975,
39, 368-373.
(34) Khan, R.; Konowicz, P. A.; Gardossi, L.; Matulova, M.; de
Gennaro, S. Austral. J. Chem. 1996, 49, 293-298.
The decrease in 3H incorporation is the result expected
if 3 and 4 are incorporated into the growing chitin chains.
However, no fluorescence is observed upon examination
J. Org. Chem, Vol. 70, No. 4, 2005 1271

Yeager and Finney
SCHEME 3. Biosynthesis of UDP-GlcNAc in
Fungia
FIGURE 2. Evaluation of 3 and 4 as substrates for CS.
a Enzymes and cosubstrates: (a) glutamine-fructose 6-phos-
phate amido transferase, glutamine; (b) glucosamine phosphate
N-acetyltransferase; (c) 3-phospho-N-acetylglucosamine mutase;
(d) UDP-N-acetylglucosamine pyrophosphorylase, uridine tri-
phosphate.
of the chitin isolated by filtration. We estimate that 1%
incorporation of 3 or 4 should be visible,35 and the absence
of emission suggests that if 3 or 4 are incorporated, they
are incorporated with <1% of the efficiency of UDP-
GlcNAc. Assuming this estimate to be correct, the
decrease in CS activity in the presence of 3 and 4 can
have one of two explanations. The first is that 3 and 4
bind more tightly than UDP-GlcNAc but turn over only
very slowly; this would account for a decrease in activity
without commensurate appearance of fluorescent prod-
uct. An alternative interpretation is that 3 and 4 bind to
CS with moderate affinity but do not turn over at all,
that is, they are inhibitors.
synthase, although it is possible that they are actually
substrates, albeit very inefficient ones. In either case,
they are not useful for the development of a fluorescently
based assay for chitin synthase activity. However, given
the precedent for other glycosyltransferases accepting
unnatural substrates, it remains possible that 3 and 4
will prove to be useful for developing other fluorescence-
based enzyme assays.37
Our current data do not allow us to distinguish
between these two possibilities. However, given that our
objective was to develop a high-throughput assay for CS
activity, the difference between an inhibitor and a
substrate that binds but does not turn over within the
limits of detection is essentially a semantic one.
In retrospect, the possibility that 3 and 4 are inhibitors
is not entirely surprising, as the biosynthesis of UDP-
GlcNAc in fungi proceeds through GlcNAc-6-phosphate
rather than free GlcNAc (Scheme 3).36 As such, if dansyl
GlcNAc (1) really is converted in vivo to the correspond-
ing nucleotide sugar (2) and then to chitin, this must
proceed through adventitious exploitation of an alterna-
tive biosynthetic route. This is possible but rather
improbable, and we believe it is more likely that the
previous report of efficient direct fungal incorporation of
1 into chitin is in error.10
Experimental Section
UDP-GlcNAc Analogue 3. Monophosphate 14 (0.08 g,
0.132 mmol) was azeotropically dried with THF and dissolved
in dry pyridine (0.53 mL) under N2. Tetrazole (0.03 g, 0.40
mmol) and uridine 5′-monophosphate morpholidate (4-mor-
pholine-N,N′-dicyclohexyl-carboxamidine salt) (0.18 g, 0.26
mmol) were added, and the reaction was concentrated after
120 h. Purification by silica gel chromatography (10-15% H2O/
CH3CN with 1% NEt3) yielded 0.03 g, contaminated with
UMP-morpholidate and starting material. This mixture was
purified by size exclusion chromatography (250 mM NH4OAc)
to yield 0.02 g, still contaminated with a small amount of
dansyl starting material, which was removed after purification
by reverse-phase silica gel chromatography (10% CH3CN/H2O)
1
to yield 3 (0.01 g, 8.2%) as a yellow film. H NMR (400 MHz,
CD3OD), δ: 2.05 (s, 3H), 2.87 (s, 6H), 3.03 (m, 2H), 3.64 (m,
2H), 3.90 (m, 2H), 4.10 (s, 1H), 4.22 (m, 3H), 4.34 (s, 1H), 5.44
(s, 1H), 5.81 (d, 1H, J ) 7.2 Hz), 5.93 (d, 1H, J ) 4.4 Hz), 7.25
(d, 1H, J ) 7.2 Hz), 7.55 (m, 2H), 7.99 (d, 1H, J ) 8.8 Hz),
8.18 (d, 1H, J ) 6.8 Hz), 8.39 (d, 1H, J ) 8.4 Hz), 8.52 (d, 1H,
J ) 8.4 Hz). 13C NMR (100 MHz, CD3OD), δ: 14.5, 23.1, 30.8,
45.2, 45.8, 55.3, 66.1, 71.1, 72.6, 72.7, 73.1, 75.5, 84.9, 89.7,
96.1, 103.1, 116.3, 120.6, 124.2, 128.9, 129.8, 130.8, 130.8,
131.0, 136.7, 142.4, 152.4, 152.8, 166.0, 174.1. FTIR (KBr),
cm-1: 3423 (br), 1684 (s), 1457 (s), 1256 (s), 1117 (s), 933 (s).
HRMS(MALDI-FTMS), m/z: calcd for C29H38N5O18P2S (M -
H)- 838.1413, found 838.1296. TLC (20% H2O/CH3CN), Rf:
0.55.
Conclusion
We have described the synthesis and evaluation of the
first fluorescent analogues of UDP-GlcNAc. These com-
pounds appear to act as weak inhibitors toward chitin
(35) Under our standard assay conditions (see Experimental Sec-
tion), a single assay vial produces ∼5 × 10-8 mol of CS. 100%
incorporation of 3 or 4 would thus provide ∼5 × 10-8 mol of dansyl
fluorophore distributed on a ∼1 cm diameter filter pad. This in turn
would be roughly equivalent to viewing 1 mL of a 10-5 M solution of
dansyl fluorophore. Emission from such a solution, even in water, can
easily be seen. Our estimate that 1% of this emission (i.e., viewing 1
mL of a 10-7 solution) would also be visible is based on the assumption
that the final environment of the dansyl group is more like dioxane (φ
∼ 0.66) than pure H2O (φ ∼ 0.07). See Supporting Information for an
expanded discussion of these calculations and assumptions.
(36) Biochemical Pathways: An Atlas of Biochemistry and Molecular
Biology; Michal, G., Ed.; John Wiley and Sons: New York, 1999; pp
27-44.
UDP-GlcNAc Analogue 4. Phosphate 20 (0.04 g, 0.06
mmol) was dried overnight in vacuo and dissolved in dry
pyridine (500 mL) under N2. Uridine 5′-monophosphate mor-
(37) For examples of assays based on unnatural substrate incorpo-
ration followed by postglycosylation modification with a visualizing
agent, see: (a) Khidekel, N.; Arndt, S.; Lamarre-Vincent, N.; Lippert,
A.; Poulin-Kerstien, K. G.; Ramakrishnan, B.; Qasba, P. K.; Hsieh-
Wilson, L. C. J. Am. Chem. Soc. 2003, 125, 16162-16163. (b) Hang,
H. C.; Yu, C.; Pratt, M. R.; Bertozzi, C. R. J. Am. Chem. Soc. 2004,
126, 6-7.
1272 J. Org. Chem., Vol. 70, No. 4, 2005

Fluorescently Labeled UDP-GlcNAc Analogues
pholidate (4-morpholine-N,N′-dicyclohexylcarboxamidine salt)
(0.09 g, 0.13 mmol) and tetrazole (0.01 g, 0.19 mmol) were
added and allowed to stir for 153 h. The mixture was
concentrated and purified by reverse-phase silica gel chroma-
tography (2-100% CH3CN/H2O) to obtain 0.04 g of product,
contaminated with the 4-morpholine-N,N′-dicyclohexylcar-
boxamidine salt. This mixture was subjected to size exclusion
chromatography (250 mM NH4HCO3) and again to reverse-
phase silica gel chromatography to obtain 4 (0.01 g, 16%) as a
pale yellow film. 1H NMR (400 MHz, CD3OD), δ: 2.86 (s, 6H),
3.20 (m, 2H), 3.60 (m, 1H), 3.65 (m, 1H), 3.81 (m, 1H), 3.92
(m, 1H), 4.12 (s, 1H), 4.25 (s, 2H), 4.35 (s, 1H), 5.60 (s, 1H),
5.80 (s, 1H), 5.94 (s, 1H), 7.23 (d, 1H, J ) 7.2 Hz), 7.55 (m,
2H), 8.00 (m, 1H), 8.33 (d, 1H, J ) 6.4 Hz), 8.47 (m, 2H). 13C
NMR (100 MHz, CD3OD), δ: 46.3, 60.2, 63.2, 71.6, 72.7, 73.2,
75.1, 76.1, 90.3, 103.6, 116.7, 121.7, 124.6, 129.4, 130.1, 131.1,
Amine 8. Triphenylphosphine (4.58 g, 17 mmol) and H2O
(5 mL) were added to a solution of azide 7 (2.86 g, 8.51 mmol)
in THF (15 mL). The reaction was concentrated after 20 h.
The resulting residue was dissolved in H2O (500 mL) and
washed with CH2Cl2 (5 × 250 mL). The aqueous layer was
concentrated to yield 8 (2.37 g, 90%) as a white solid. 1H NMR
(400 MHz, CD3OD), δ: 1.95 (s, 3H), 2.74 (dd, 1H, J ) 7.4, 13.6
Hz), 2.99 (dd, 1H, J ) 2.4, 13.6 Hz), 3.23 (t, 1H, J ) 9.2 Hz),
3.61 (m, 1H), 3.70 (dd, 1H, J ) 8.8, 10.4 Hz), 3.88 (dd, 1H, J
) 3.6, 10.8 Hz), 4.61 (dd, 2H, J ) 12.2, 88.4 Hz), 4.85 (d, 1H,
J ) 3.2 Hz), 7.24-7.38 (m, 5H). 13C NMR (100 MHz, CD3OD),
δ: 23.1, 44.3, 55.9, 70.9, 72.9, 74.1, 74.5, 98.1, 129.1, 129.5,
129.7, 139.3, 173.8. FTIR (film), cm-1: 3301 (br), 3091 (s), 2899
(s), 1641 (s), 1571 (s), 1457 (s), 1396 (s), 1108 (s), 1055 (s).
HRMS(MALDI-FTMS), m/z: calcd for C15H22N2O5 (M + H)+
311.1607, found 311.1594. TLC (10% MeOH/CH2Cl2), Rf: 0.01.
Sulfonamide 9. Amine 8 (2.37 g, 7.65 mmol) was dissolved
in dry pyridine (100 mL) under N2 and cooled to 0 °C. Dansyl-
Cl (3.10 g, 11.48 mmol) was dissolved in dry pyridine (100 mL)
and added to the amine solution dropwise over 0.75 h. After
20 h at 4 °C, the reaction was concentrated and purified by
silica gel chromatography (5-15% MeOH/CH2Cl2) to yield 9
(3.88 g, 93%) as a yellow foam. 1H NMR (400 MHz, CDCl3), δ:
1.96 (s, 3H), 2.88 (s, 6H), 3.20 (dd, 2H, J ) 12.2, 98.4 Hz),
3.65 (m, 1H), 3.70 (m, 1H), 4.05 (m, 1H), 4.44 (dd, 2H, J )
11.6, 86.4 Hz), 4.77 (d, 1H, J ) 3.2 Hz), 6.11 (d, 1H, J ) 8.8
Hz), 7.14 (d, 1H, J ) 7.2 Hz), 7.26 (m, 5H), 7.49 (m, 2H), 8.23
(d, 1H, J ) 7.2 Hz), 8.35 (d, 1H, J ) 8.4 Hz), 8.51 (d, 1H, J )
8.8 Hz). 13C NMR (100 MHz, CDCl3), δ: 23.3, 43.5, 69.6, 70.4,
71.2, 72.5, 96.9, 115.1, 118.9, 123.1, 127.8, 128.2, 128.3, 129.3,
129.5, 129.7, 130.1, 134.8, 137.0, 151.6, 171.4. FTIR (film),
cm-1: 3345 (br), 2951 (br), 1667 (s), 1571 (s), 1466 (s), 1326
(s), 1151 (s), 1055 (s). HRMS(MALDI-FTMS), m/z: calcd for
C27H33N3O7S (M)+ 543.2039, found 543.2025. TLC (10% MeOH/
CH2Cl2), Rf: 0.46.
Sulfonamide 10. DMAP (0.02 g, 0.15 mmol), NEt3 (0.82
mL, 5.88 mmol), and acetic anhydride (0.56 mL, 5.88 mmol)
were added to a solution of 9 (0.80 g, 1.47 mmol) in dry CH2-
Cl2 (2 mL), and the reaction was allowed to stir for 0.5 h. The
reaction was diluted with CH2Cl2 (200 mL), washed with
NaHCO3 (1 × 200 mL) and brine (1 × 200 mL), dried (Na2-
SO4), and concentrated to yield 10 (0.93 g, 95%) as a yellow
solid. 1H NMR (400 MHz, CDCl3), δ: 1.91 (s, 3H), 2.04 (s, 3H),
2.14 (s, 3H), 2.19 (s, 3H), 2.89 (s, 6H), 4.13 (dd, 1H, J ) 9.6,
15.2 Hz), 4.24 (d, 1H, J ) 13.2 Hz), 4.35 (m, 1H), 4.47 (d, 1H,
J ) 11.6 Hz), 4.90 (m, 2H), 5.02 (t, 1H, J ) 9.6 Hz), 5.30 (t,
1H, J ) 9.6 Hz), 5.70 (d, 1H, J ) 9.6 Hz), 7.18 (d, 1H, J ) 8.0
Hz), 7.37 (m, 5H), 7.56 (m, 2H), 7.82 (d, 1H, J ) 8.4 Hz), 8.35
(d, 1H, J ) 7.2 Hz), 8.59 (d, 1H, J ) 8.8 Hz). 13C NMR (100
MHz, CDCl3), δ: 20.8, 20.9, 23.2, 24.7, 30.4, 45.4, 47.5, 51.9,
69.1, 69.7, 70.7, 71.1, 95.5, 115.3, 117.5, 122.9, 128.1, 128.4,
128.5, 128.8, 129.4, 129.7, 131.5, 131.6, 134.1, 136.2, 152.1,
169.6, 170.1, 170.3, 170.9. FTIR (film), cm-1: 3345 (br), 2951
(s), 1754 (s), 1693 (s), 1545 (s), 1370 (s), 1239 (s), 1178 (s), 1055
(s). HRMS(MALDI-FTMS), m/z: calcd for C33H39N3O10NaS (M
+ Na)+ 692.2248, found 692.2272. TLC (10% MeOH/CH2Cl2),
Rf: 0.72.
131.4, 131.4, 138.6, 143.0, 153.0, 166.5. FTIR (KBr), cm-1
:
3467 (br), 1684 (s), 1466 (s), 1309 (s), 1256 (s), 1125 (s). HRMS-
(MALDI-FTMS), m/z: calcd for C28H37N3O18P2S (M)- 797.1268,
found 797.1150. TLC (20% H2O/CH3CN), Rf: 0.52.
Benzyl-Protected GlcNAc 5. N-Acetyl glucosamine (6.00
g, 27.12 mmol) was dissolved in benzyl alcohol (50 mL), and
concentrated HCl (2.9 mL) was added. The mixture was heated
to 90 °C for 3 h, cooled to room temperature, and then poured
onto 500 mL Et2O and stored overnight at -20 °C. The
resulting precipitate was recovered by filtration and rinsing
with Et2O and hexanes to yield 17.64 g of crude material,
which was purified by silica gel chromatography (8-15%
MeOH/CH2Cl2) to yield 5 (5.98 g, 71%) as a white foam. 1H
NMR (400 MHz, CD3OD), δ: 1.95 (s, 3H), 3.37 (t, 1H, J ) 9.2
Hz), 3.68 (m, 3H), 3.82 (d, 1H, J ) 10.8 Hz), 3.89 (dd, 1H, J )
3.4, 10.8 Hz), 4.61 (dd, 2H, J ) 11.8, 98.4 Hz), 4.85 (s, 1H),
7.24-7.37 (m, 5H). 13C NMR (100 MHz, CD3OD), δ: 23.1, 31.4,
55.9, 63.2, 70.6, 72.9, 73.1, 74.5, 97.9, 126.4, 129.4, 129.6, 129.7,
139.3, 173.8. FTIR (film), cm-1: 3301 (br), 2917 (s), 1650 (s),
1562 (s), 1387 (s), 1239 (s), 1143 (s), 1045 (s). HRMS(MALDI-
FTMS), m/z: calcd for C15H22N1O6 (M + H)+ 312.1447, found
312.1437. TLC (10% MeOH/CH2Cl2), Rf: 0.24.
Tosylate 6. Benzylated GlcNAc 5 (0.59 g, 1.90 mmol) was
cooled to 0 °C in dry pyridine (6.0 mL). TsCl (0.54 g, 2.84 mmol)
was dissolved in dry pyridine (5 mL) and added to the solution
of 5 over 0.5 h. The mixture was allowed to stir for 14 h at 4
°C, concentrated, and purified by silica gel chromatography
(10% MeOH/CH2Cl2) to obtain 6 (0.72 g, 81%) as a white foam.
1H NMR (400 MHz, CD3OD), δ: 1.93 (s, 3H), 2.42 (s, 3H), 3.27
(t, 1H, J ) 9.2 Hz), 3.62 (t, 1H, J ) 8.8 Hz), 3.71-3.76 (m,
1H), 3.78-3.83 (m, 1H), 4.19 (dd, 1H, J ) 5.8, 10.6 Hz), 4.29
(dd, 1H, J ) 2.0, 10.4 Hz), 4.48 (dd, 2H, J ) 12.4, 79.2 Hz),
4.70 (d, 1H, J ) 3.2 Hz), 7.27 (m, 5H), 7.39 (d, 2H, J ) 8.0
Hz), 7.78 (d, 2H, J ) 8.0 Hz). 13C NMR (100 MHz, CD3OD), δ:
21.8, 22.7, 55.2, 70.5, 71.0, 71.6, 72.0, 72.6, 97.5, 128.9, 129.0,
129.3, 129.4, 130.9, 134.4, 128.6, 146.4, 173.4. FTIR (film),
cm-1: 3388 (br), 2934 (br), 1676 (s), 1553 (s), 1370 (s), 1186
(s), 1125 (s), 1082 (s). HRMS(MALDI-FTMS), m/z: calcd for
C22H27N1O8S (M)+ 465.1457, found 465.1458. TLC (10% MeOH/
CH2Cl2), Rf: 0.3.
Azide 7. NaN3 (6.82 g, 104.9 mmol) was added to a solution
of tosylate 6 (4.88 g, 10.49 mmol) in DMF (15 mL), and the
mixture was heated to 70 °C for 20 h. Concentration and silica
gel chromatography (10% MeOH/CH2Cl2) afforded 7 (2.86 g,
Alcohol 11. FeCl3 (0.94 g, 5.74 mmol) was added to a
solution of 10 (0.39 g, 0.57 mmol) in dry CH2Cl2 (2 mL) and
allowed to stir for 18.5 h, at which time an additional FeCl3
(0.50 g, 3.03 mmol) was added. After 24 h, the mixture was
diluted with EtOAc (600 mL) and washed with brine (2 × 400
mL). The brine layers were combined and back-extracted with
EtOAc (2 × 300 mL). The organic layers were combined and
washed with saturated EDTA (3 × 400 mL) and brine (2 ×
200 mL), dried (Na2SO4), and concentrated. Purification by
silica gel chromatography (4% MeOH/CH2Cl2) provided 11
(0.26 g, 45-79%) as a yellow solid. 1H NMR (400 MHz, CDCl3),
δ: 1.98 (s, 3H), 2.05 (s, 3H), 2.14 (s, 3H), 2.20 (s, 3H), 2.88 (s,
6H), 4.01 (dd, 1H, J ) 9.4, 15.2 Hz), 4.21 (d, 1H, J ) 15 Hz),
4.31 (m, 1H), 4.46 (t, 1H, J ) 9.8 Hz), 4.98 (t, 1H, J ) 9.8 Hz),
5.20 (d, 1H, J ) 3.2 Hz), 5.35 (t, 1H, J ) 10.2 Hz), 5.91 (d, 1H,
1
82%) as a white foam. H NMR (400 MHz, CD3OD), δ: 1.94
(s, 3H), 3.34 (t, 1H, J ) 8.8 Hz), 3.41 (dd, 1H, J ) 6.2, 13.0
Hz), 3.49 (dd, 1H, J ) 2.4, 13.8 Hz), 3.67 (dd, 1H, J ) 8.8,
10.4 Hz), 3.79 (m, 1H), 3.91 (dd, 1H, J ) 3.6, 10.8 Hz), 4.62
(dd, 2H, J ) 12.4, 88.4 Hz), 7.25-7.37 (m, 5H). 13C NMR (100
MHz, CD3OD), δ: 23.1, 53.2, 55.7, 70.9, 72.9, 73.6, 97.9, 129.3,
129.6, 129.7, 139.0, 173.8. FTIR (film), cm-1: 3432 (br), 3310
(s), 2943 (s), 2095 (s), 1667 (s), 1623 (s), 1562 (s), 1457 (s), 1387
(s), 1291 (s), 1047 (s). HRMS(MALDI-FTMS), m/z: calcd for
C15H22N2O5 (M + H)+ 337.1512, found 337.1508. TLC (15%
MeOH /CH2Cl2), Rf: 0.65.
J. Org. Chem, Vol. 70, No. 4, 2005 1273

Yeager and Finney
J ) 9.2 Hz), 7.17 (d, 1H, J ) 7.2 Hz), 7.56 (m, 2H), 7.81 (d,
1H, J ) 8.4 Hz), 8.32 (d, 1H, J ) 7.2 Hz), 8.57 (d, 1H, J ) 8.4
Hz). 13C NMR (100 MHz, CDCl3), δ: 20.9, 21.0, 23.3, 24.8, 45.4,
47.7, 52.3, 68.8, 70.7, 70.8, 91.3, 115.2, 117.5, 123.0, 128.7,
129.5, 129.7, 131.5, 131.6, 132.2, 134.0, 152.1, 169.9, 170.2,
170.7, 171.0. FTIR (film), cm-1: 3388 (br), 2951 (s), 2812 (s),
1737 (s), 1676 (s), 1545 (s), 1370 (s), 1239 (s), 1186 (s), 1047
(s). HRMS(MALDI-FTMS), m/z: calcd for C26H33N3O10S (M)+
579.1887, found 579.1871. TLC (10% MeOH/CH2Cl2), Rf: 0.73.
pyridine (1 mL) and allowed to stir for 11 h. Concentration
followed by silica gel chromatography (1-5% MeOH/CH2Cl2)
provided 16 (0.39 g, 68%) as a pale yellow foam. 1H NMR (400
MHz, CDCl3), δ: 1.31 (s, 3H), 1.74 (s, 3H), 2.01 (s, 3H), 2.05
(s, 3H), 2.86 (s, 6H), 3.69 (t, 1H, J ) 9.2 Hz), 3.80 (m, 1H),
4.03 (dd, 1H, J ) 2.0, 12.6 Hz), 4.23 (dd, 1H, J ) 2.0, 12.6
Hz), 5.07 (t, 1H, 9.4 Hz), 5.17 (t, 1H, J ) 10.0 Hz), 5.59 (d,
1H, J ) 8.8 Hz), 7.16 (d, 1H, J ) 7.2 Hz), 7.50 (m, 2H), 8.08
(d, 1H, J ) 8.8 Hz), 8.27 (d, 1H, J ) 7.2 Hz), 8.52 (d, 1H, J )
8.4 Hz). 13C NMR (100 MHz, CDCl3), δ: 19.9, 20.4, 20.7, 20.8,
45.4, 57.0, 61.5, 67.7, 72.5, 72.7, 92.1, 115.2, 119.0, 123.3, 128.3,
129.2, 129.3, 129.8, 130.3, 135.9, 151.7, 169.0, 170.3, 171.3.
Dibenzyl Phosphate 12. Lithium diispropylamide (0.72,
2.00 M in THF, 1.44 mmol) was added to a solution of 11 (0.42
g, 0.72 mmol) in THF (4 mL) at -78 °C, and the reaction
mixture was stirred for 0.5 h. Tetrabenzyl pyrophosphate (0.70
g, 1.29 mmol) was added and stirring continued at -78 °C for
1 h. The reaction was allowed to stir at 4 °C for 10 h and then
was diluted with EtOAc (300 mL) and washed with NaHCO3
(2 × 200 mL) and brine (1 × 200 mL). The organic fraction
was dried (Na2SO4) and concentrated to 0.81 g, which was
purified by silica gel chromatography (0-1% MeOH/CHCl3)
FTIR (film), cm-1
: 3315 (br), 2952 (s), 2853 (s), 2787 (s),
1755(s), 1590 (s), 1450 (s), 1384 (s), 1235 (s), 1079 (s), 1054
(s). HRMS(MALDI-FTMS), m/z: calcd for C26H32N2O11S (M+)
580.1721, found 580.1727. TLC (10% MeOH/CH2Cl2), Rf: 0.60.
Alcohol 17. Tetraacetate 16 (0.39 g, 0.63 mmol) was
azeotropically dried with THF and dissolved in dry DMF (1
mL). Hydrazine acetate (0.07 g, 0.75 mmol, azeotropically dried
with THF) was added and the reaction allowed to stir for 16
h. The mixture was diluted with EtOAc (300 mL) and washed
with brine (2 × 300 mL). The aqueous fractions were pooled
and back-extracted with EtOAc (1 × 300 mL). Organic frac-
tions were combined, dried (Na2SO4), and concentrated to yield
1
to yield 12 (0.31 g, 53%) as a pale yellow foam. H NMR (400
MHz, CDCl3), δ: 1.72 (s, 3H), 2.05 (s, 3H), 2.14 (s, 6H), 2.88
(s, 6H), 4.16 (m, 2H), 4.39 (m, 1H), 4.47 (m, 1H), 5.16 (m, 5H),
5.71 (m, 2H), 7.18 (d, 1H, J ) 7.6 Hz), 7.35 (m, 5H), 7.55 (m,
2H), 7.86 (d, 1H, J ) 8.4 Hz), 8.20 (d, 1H, J ) 7.2 Hz), 8.57 (d,
1H, J ) 9.4 Hz). 13C NMR (100 MHz, CDCl3), δ: 20.8, 20.9,
22.8, 24.8, 45.4, 47.3, 51.9, 52.0, 69.9, 70.0, 70.1, 70.2, 71.0,
95.5, 95.5, 115.3, 117.6, 122.9, 128.0, 128.1, 128.5, 128.5, 128.6,
128.8, 129.3, 129.8, 130.5, 131.5, 134.3, 135.2, 135.3, 135.4,
135.4, 152.0, 169.8, 170.0, 170.2, 170.7. FTIR (film), cm-1: 3301
(br), 2943 (s), 1763 (s), 1684 (s), 1562 (s), 1457 (s), 1361 (s),
1239 (s), 1151 (s). HRMS(MALDI-FTMS), m/z: calcd for
C40H46N3O13PSNa (MNa+) 862.2381, found 862.2372. TLC
(10% MeOH /CH2Cl2), Rf: 0.77.
1
17 (0.36 g, 99%) as a pale yellow foam. H NMR (400 MHz,
CDCl3), δ: 1.09 (s, 3H), 1.93 (s, 3H), 2.06 (s, 3H), 2.86 (s, 6H),
3.42 (m, 1H), 4.02 (d, 1H, J ) 10.4 Hz), 4.18 (m, 2H), 4.88, (t,
1H, J ) 9.6 Hz), 5.19 (t, 1H, J ) 9.8 Hz), 5.28 (m, 1H), 5.56
(d, 1H, J ) 10.4 Hz), 7.17 (d, 1H, J ) 7.6 Hz), 7.54 (m, 2H),
8.18 (d, 1H, J ) 8.4 Hz), 8,27 (d, 1H, J ) 7.4 Hz), 8.54 (d, 1H,
J ) 8.4 Hz). 13C NMR (100 MHz, CDCl3), δ: 19.7, 20.6, 20.8,
45.4, 56.2, 61.9, 67.1, 68.4, 69.7, 92.2, 115.1, 119.1, 123.2, 128.3,
129.2, 129.7, 129.8, 130.7, 134.5, 151.7, 169.3, 170.3, 170.6.
FTIR (film), cm-1: 3439 (br), 3266 (br), 2952 (s), 1747 (s),
1665(s), 1582 (s), 1458 (s), 1376 (s), 1334 (s), 1244 (s), 1153
(s), 1046 (s). HRMS(MALDI-FTMS), m/z: calcd for C24H30-
N2O10S (MNa+) 561.1513, found 561.1522. TLC (10% MeOH/
CH2Cl2), Rf: 0.51.
Phosphate 13. To a solution of phosphate 12 (0.12 g, 0.14
mmol) in MeOH (1 mL) was added Pd/C (0.02 g, 10% w/w Pd/
C). The reaction was flushed with H2 (three times) and stirred
under H2 (1 atm) for 4 h. NEt3 (0.02 mL, 0.14 mmol) was
added, and the mixture was filtered through Celite, rinsing
with MeOH. Concentration of the filtrate yielded 13 (0.08 g,
Dibenzyl Phosphate 18. Compound 17 (0.30 g, 0.55 mmol)
was azeotropically dried with THF and cooled to -78 °C in
dry THF (4 mL), and LDA (0.41 mL of 2 M THF solution) was
added. After 10 min, tetrabenzyl pyrophosphate (0.42 g, 0.77
mmol) was added. The reaction was allowed to stir for 40 min
and then warmed to 4 °C and allowed to stir 12 h. The mixture
was then concentrated, diluted with EtOAc (500 mL), washed
with NaHCO3 (1 × 300 mL) and brine (1 × 300 mL), and then
dried (Na2SO3) and concentrated to 0.55 g of crude material.
Purification by silica gel chromatography (1-2% MeOH/CH2-
Cl2) provided 18 (0.40 g, 90%) as a pale yellow foam. 1H NMR
(400 MHz, CDCl3), δ: 0.97 (s, 3H), 1.92 (s, 3H), 1.99 (s, 3H),
2.84 (s, 6H), 3.5 (s, 1H), 3.85 (d, 1H, J ) 11.8 Hz), 4.07 (d, J
) 10.4 Hz), 4.14 (dd, 1H, J ) 3.6, 12.4 Hz), 4.90 (t, 1H, J )
9.8 Hz), 5.11 (m, 5H), 5.87 (dd, 1 H, J ) 3.2, 6.0 Hz), 6.05 (s,
1H), 7.12 (d, 1H, J ) 7.6 Hz), 7.35 (m, 10H), 7.47 (t, 1H, J )
7.2 Hz), 7.53 (t, 1H, J ) 8.0 Hz), 8.16 (d, 1H, J ) 8.8 Hz), 8.26
(d, 1H, J ) 7.2 Hz), 8.53 (d, 1H, J ) 8.4 Hz). 13C NMR (100
MHz, CDCl3), δ: 19.4, 20.5, 20.7, 45.4, 55.8, 61.0, 67.7, 69.0,
69.1, 69.8, 70.1, 97.1, 115.1, 119.1, 123.2, 129.7, 128.1, 128.2,
128.3, 128.4, 128.5, 128.5, 129.2, 129.6, 129.7, 130.5, 134.7,
135.2, 135.2, 135.3, 135.3, 151.7, 169.0, 170.0, 170.2. FTIR
(film), cm-1: 3135 (br), 2947 (s), 2834 (s), 2781 (s), 1750 (s),
1592 (s), 1457 (s), 1374 (s), 1336 (s), 1246 (s). HRMS(MALDI-
FTMS), m/z: calcd for C38H43N2O13PSNa (MNa+) 821.2116,
found 821.2119. TLC (10% MeOH/CH2Cl2), Rf: 0.83.
1
74%) as a pale yellow foam. H NMR (400 MHz, CD3OD), δ:
1.30 (t, 9H, J ) 7.2 Hz), 1.95 (s, 3H), 1.98 (s, 3H), 2.06 (s, 3H),
2.19 (t, 3H), 2.87 (s, 6H), 4.28 (m, 3H), 4.84 (m, 1H), 5.04 (t,
1H, J ) 10.0 Hz), 5.33 (t, 1H, J ) 10.0 Hz), 5.52 (dd, 1H, J )
3.4, 7 Hz), 7.25 (d, 1H, J ) 7.2 Hz), 7.58 (t, 1H, J ) 8.2 Hz),
7.65 (t, 1H, J ) 8.0 Hz), 7.86 (d, 1H, J ) 8.4 Hz), 8.31 (d, 1H,
J ) 7.2 Hz), 8.59 (d, 1H, J ) 8.4 Hz). 13C NMR (100 MHz,
CD3OD), δ: 9.3, 20.8, 21.0, 22.7, 24.9, 45.7, 47.5, 53.2, 70.9,
72.2, 72.6, 94.7, 116.4, 118.7, 124.3, 129.7, 130.3, 130.8, 132.2,
135.7, 153.3, 171.4, 171.7, 172.8, 173.3. FTIR (KBr), cm-1
:
3398 (br), 2985 (s), 2944 (s), 2803 (s), 1747 (s), 1665 (s), 1557
(s), 1458 (s), 1343 (s), 1235 (s), 1169 (s), 1046 (s). HRMS-
(MALDI-FTMS), m/z: calcd for C26H34N3O13PSNa (MNa+)
682.1442, found 682.1447. TLC (15% H2O/CH3CN), Rf: 0.24.
Phosphate 14. Tetraacetate 13 (0.02 g, 0.02 mmol) was
dissolved in 7:3:1 MeOH/H2O/NEt3 and stirred for 36 h. The
mixture was concentrated, redissolved in MeOH, and filtered
through Celite. Concentration of the filtrate yielded 14 (0.01
g, 99%) of the diol as a pale yellow foam. 1H NMR (400 MHz,
CD3OD), δ: 1.98 (s, 3H), 2.85 (s, 6H), 2.96 (dd, 1H, J ) 6.8,
12.6 Hz), 3.13 (d, 1H, J ) 7.2 Hz), 3.23 (t, 1H, J ) 9.2 Hz),
3.65 (t, 1H, J ) 9.2 Hz), 3.89 (m, 2H), 5.37 (s, 1H), 7.23 (d,
1H, J ) 7.6 Hz), 7.55 (m, 2H), 8.18 (d, 1H, J ) 7.2 Hz), 8.37
(d, 1H, J ) 8.4 Hz), 8.52 (d, 1H, J ) 8.4 Hz). 13C NMR (100
MHz, CD3OD), δ: 9.7, 23.4, 45.7, 46.2, 46.3, 48.0, 55.8, 55.9,
73.1, 73.2, 73.5, 95.4, 116.8, 121.0, 124.6, 129.4, 130.3, 131.3,
131.3, 131.5, 137.2, 153.3, 174.1. FTIR (KBr), cm-1: 3432 (br),
1658 (s), 1562 (s), 1475 (s), 1326 (s), 1151 (s), 942 (s), 793 (s).
HRMS(MALDI-FTMS), m/z: calcd for C20H28N3O10PSNa
(MNa+) 556.1125, found 556.1129. TLC (20% H2O/CH3CN), Rf:
0.22.
Phosphate 19. Dibenzyl phosphate 18 (0.39 g, 0.49 mmol)
was dissolved in MeOH (2 mL); Pd/C (0.10 g, 10% w/w Pd/C)
was added, and the mixture was flushed with H2 (three times)
and allowed to stir under H2 (1 atm) for 8 h. NEt3 (0.0 mL,
0.49 mmol) was added, and the mixture was filtered through
a pad of Celite and concentrated to yield 19 (0.29 g, 99%) as a
pale yellow solid. 1H NMR (400 MHz, CD3OD), δ: 0.69 (s, 3H),
1.31 (t, 9H, J ) 7.2 Hz), 1.85 (s, 3H), 2.00 (s, 3H), 3.17 (q, 6H,
Sulfonamide 16. Amine 14 (0.38 g, 0.99 mmol)30,31 was
added to a solution of dansyl-Cl (0.40 g, 1.48 mmol) in dry
1274 J. Org. Chem., Vol. 70, No. 4, 2005

Fluorescently Labeled UDP-GlcNAc Analogues
J ) 7.6, 14.2 Hz), 3.45 (m, 1H), 3.57 (d, 1H, J ) 10.8 Hz), 4.05
(m, 1H), 4.22 (m, 1H), 4.8 (t, 1H, J ) 10.0 Hz), 5.16 (t, 1H, J
) 10.0 Hz), 5.68 (m, 1H), 7.26 (d, 1H, J ) 7.2 Hz), 7.57 (m,
2H), 8.24 (m, 2H), 8.21 (d, 1H, J ) 8.8 Hz). 13C NMR (100
MHz, CD3OD), δ: 9.7, 20.0, 21.1, 21.2, 46.3, 47.8, 57.8, 57.9,
63.2, 69.7, 70.6, 71.7, 96.9, 97.1, 116.8, 121.2, 124.8, 129.5,
130.4, 131.1, 131.4, 137.9, 153.3, 171.3, 171.3, 172.4. FTIR
(film), cm-1: 3441 (br), 2995 (s), 2943 (s), 1754 (s), 1580 (s),
1457 (s), 1379 (s), 1335 (s), 1248 (s) cm-1. HRMS(MALDI-
FTMS), m/z: calcd for C24H31N2O13PSNa (MNa+) 641.1177,
found 641.1173. TLC (10% MeOH/CH2Cl2), Rf: 0.11.
The enzyme pellet was suspended in 1.6 mL × {gram wet
weight of cells} of TM buffer and homogenized throughly with
a glass Dounce homogenizer. The membranes were pretreated
with trypsin (quantified by weight, 10 min, 30 °C) and then
treated with 3.0 × {mass of trypsin} of trypsin inhibitor.
Typically, four different concentrations of trypsin/trypsin
inhibitor were tested for each new membrane preparation, and
the combination with the highest activity at 30 min was in
used in the assay. Concentrations of trypsin typically tested
were 0.5, 1.0, 2.0, and 4.0 mg/mL, and trypsin was added to
the membrane preparation at a concentration of 1 mL of
trypsin solution for every 5 mL of membrane preparation.
Individual assays were performed in 1.5 mL eppendorf cen-
trifuge tubes.
Phosphate 20. Phosphate 19 (0.05 g, 0.06 mmol) was
dissolved in 7:3:1 MeOH/H2O/NEt3 and allowed to stir for 72
h. After concentration, compound 20 (0.04 g, 99%) was
1
obtained as a pale yellow solid. H NMR (400 MHz, CD3OD),
The assay solution used contained UDP-GlcNAc (1.0 mM),
GlcNAc (40mM), and digitonin (0.2% w/v) dissolved in pH 7.5
tris buffer (50 mM) containing MgCl2 (5.0 mM). Radioactive
substrate (typically 0.125 µCi, transferred to the eppendorf as
a solution that was then evaporated to dryness under vacuum)
in 40 mL of assay solution (containing necessary inhibitor) was
transferred to each tube. Trypsin-treated membrane (20 mL)
was then added, and the mixture was incubated for 1 h at 30
°C. The reaction was stopped by the addition of 1 mL of cold
(0 °C) aqueous trichloroacetic acid (10% v/v), filtered onto glass
fiber filter disks (Whatman GF/C, 25 mm), and rinsed with
7:3 EtOH/1 M acetic acid (4 × 1 mL), and the remaining
radioactivity on the filter paper was measured by scintillation
counting.
Enzyme activity (in the absence of inhibitor) was typically
25 000-30 000 cpm/h, with a time-independent background
of 500-600 cpm. Under these conditions, enzyme activity was
linear (on the basis of plots of incorporated radioactivity vs
time) to at least 3 h. Control reactions run in in the presence
of 0.1 mM polyoxin D or nikkomycin Z, both of which were
known competitive inhibitors (Ki ≈ 10 µM) of chitin synthase,
always showed g99% inhibition.
δ: 1.27 (t, 9H, J ) 7.2 Hz), 1.90 (s, 3H), 2.86 (s, 6H), 3.11 (q,
6H, J ) 7.2, 14.4 Hz), 3.60 (m, 3H), 3.75 (d, 1H, J ) 11.6 Hz),
3.85 (m, 1H), 5.50 (dd, 1H, J ) 3.6, 6.6 Hz), 7.24 (d, 1H, J )
7.6 Hz), 7.57 (m, 2H), 8.32 (d, 1H, J ) 7.2 Hz), 8.42 (d, 1H, J
) 8.4 Hz), 8.51 (d, 1H, J ) 8.4 Hz). 13C NMR (100 MHz, CD3-
OD), δ: 9.2, 24.2, 45.8, 46.9, 60.0, 60.1, 62.6, 72.2, 73.3, 74.0,
95.7, 95.8, 116.0, 121.0, 124.2, 128.7, 129.5, 130.5, 130.9, 130.9,
138.2, 152.7, 179.7. FTIR (KBr), cm-1: 3458 (br), 2943 (s), 2750
(s), 2681 (s), 2497 (s), 1580 (s), 1483 (s), 1317 (s), 1151 (s) cm-1
.
HRMS(MALDI-FTMS), m/z: calcd for C18H25N2O10PSNa
(MNa+): 515.0860, found 515.0882. TLC (20% H2O/CH3CN),
Rf: 0.40.
Chitin Synthase Assay. The assay protocol used is based
on the procedure of Orlean,1,9 modified after helpful discussions
with Prof. Peter Orlean (University of Illinois) and Dr. Enrico
Cabib (NIH).1 Saccharomyces cerevisiae strains (PP-1D, wild
type) were kindly provided by Prof. Orlean and were stored
at -70 °C on freezer stabs. Active yeast cultures were
temporarily maintained on agar plates, stored at 4 °C. Cells
were cultured in 200 mL of YEPG (1% yeast extract, 2%
bactopeptone, 2% glucose) medium at 30 °C and allowed to
grow to saturation. An aliquot (10-12 mL) of the saturated
medium was transferred to 400 mL of YEPG medium to give
an optical density of 0.15-0.20 and allowed to grow to an
optical density of 0.65-0.70. The cells were washed with cold
water and TM buffer (50 mM tris‚HCl, 2.5 mM MgCl2, pH 7.5)
by suspension and centrifugation (15 min, 2000×g). The wet
weight of the cells at this point was typically around 1 g; this
weight was used to determine the volume of buffer in which
the final pellet was suspended (vide infra).
The cells were suspended in 2 mL of TM buffer in a 50 mL
plastic centrifuge tube, and glass beads (0.45 mm) were added
until the volume of beads reached about 3 mm below the
liquid’s surface. The tube was then vortexed 20 times for 30
s, with 30 s of cooling on ice between each vortex; vortexing
was performed in a 4 °C cold room. The broken cells were
removed from the bottom of the tube with a glass Pasteur
pipet, and the glass beads were rinsed 5-7 times with 1.5 mL
of TM buffer. The pooled rinsings were centrifuged at 2000g
for 4 min; the supernatant was removed, and the remaining
cell wall precipitate was washed once more with TM buffer.
The cell-wall-free supernatants were combined and centrifuged
at 60 000g for 1 h.
Acknowledgment. We thank the NIH (GM60875),
the Hellman Foundation, and the UC Systemwide
Biotechnology Program for direct support and the NIH
and NSF for infrastructure support (GM62116, GM61894,
CHE9709183). We thank Prof. Peter Orlean (University
of Illinois) and Dr. Enrico Cabib (NIH) for assay advice,
Prof. Orlean for donation of yeast strains, Prof. Scott
Singleton (University of North Carolina, Chapel Hill)
for helpful discussion, and Prof. Jack Kyte and Mr.
Steven Adams (University of California, San Diego) for
technical assistance.
Supporting Information Available: General experimen-
1
tal details and copies of H NMR spectra for all compounds.
This material is available free of charge via the Internet at
http://pubs.acs.org.
JO0483670
J. Org. Chem, Vol. 70, No. 4, 2005 1275