Chemical synthesis of UDP-4-O-methyl-GlcNAc, a potential chain terminator of chitin synthesis

Article abstract of DOI:10.1016/j.carres.2004.03.014

Chitin synthase converts uridine diphosphoryl-N-acetylglucosamine (UDP-GlcNAc) to chitin (poly-β-(1→4)-GlcNAc). During polymerization, elongation occurs at the 4-OH (nonreducing) terminus of the growing chitin chain. Blockage of the 4-OH via incorporation of UDP-N-acetyl-4-O- methylglucosamine (UDP-4-OMe-GlcNAc, 3) can potentially terminate chitin polymerization, and represents a novel strategy for chitin synthase inhibition. The chemical synthesis of 3 and preliminary evaluation of its possible incorporation by chitin synthase are reported herein.

Full text of DOI:10.1016/j.carres.2004.03.014

Carbohydrate
RESEARCH
Carbohydrate Research 339 (2004) 1531–1536
Chemical synthesis of UDP-4-O-methyl-GlcNAc, a potential
chain terminator of chitin synthesis
*
Robert Chang, Philip Moquist andNathaniel S. Finney
Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093-0358, USA
Received23 September 2003; accepted5 March 2004
Abstract—Chitin synthase converts uridine diphosphoryl-N-acetylglucosamine (UDP-GlcNAc) to chitin (poly-b-(1 fi 4)-GlcNAc).
During polymerization, elongation occurs at the 4-OH (nonreducing) terminus of the growing chitin chain. Blockage of the 4-OH
via incorporation of UDP-N-acetyl-4-O-methylglucosamine (UDP-4-OMe-GlcNAc, 3) can potentially terminate chitin polymeri-
zation, andrepresents a novel strategy for chitin synthase inhibition. The chemical synthesis of 3 andpreliminary evaluation of its
possible incorporation by chitin synthase are reportedherein.
Ó 2004 Elsevier Ltd. All rights reserved.
Keywords: Chitin synthase; Glucosamine; 4-O-Methyl-GlcNAc; UDP-sugar analog; Synthesis
1. Introduction
involvedthe synthesis of substrate analogs or structure-
7–12
baseddesign.
As part of our ongoing research pro-
Chitin synthase (CS) is the enzyme that converts uridine
diphosphoryl-N-acetylglucosamine (UDP-GlcNAc) 1 to
chitin,1–5 the b-(1 fi 4)-linkedpolymer of N-acetylglu-
cosamine. It is a member of a class of enzymes known as
polymerizing, or processive, glycosyltransferases, which
are also responsible for the synthesis of other important
gram on chitin synthase,13–15 we present a novel
approach to the manipulation of CS basedon termina-
tion of the polymerization reaction.
It is generally acceptedthat chitin is elongatedat the
nonreducing terminus16––the sugar donor UDP-Glc-
NAc 1 delivers a sugar residue to the 4-hydroxyl group
of the growing chitin chain (Scheme 1). We envisioned
that if the 4-OMe UDP-GlcNAc analog 3 couldserve as
a substrate for CS, then the resulting product 2 could
not undergo subsequent elongation (Scheme 2), thus
terminating chitin synthesis.17 This blocking strat-
egy couldalso be applicable to other polymerizing
6
biopolymers such as cellulose andhyaluronan. Absent
in mammals, chitin is an essential component of the
fungal cell wall. Consequently, the inhibition of CS has
been recognizedas a promising strategy for the devel-
opment of antifungal therapeutics. To date, the devel-
opment of chitin synthase inhibitors has primarily
O
OH
OH
OH
NH
AcHN
AcHN
O
HO
HO
O
HO
O
O
O
O
R
HO
HO
O
HO
O
O
N
O
O
O
O
HO
AcHN
O P O P
O
AcHN
AcHN
OH
OH
OH OH
HO OH
n
1
chitin
* Corresponding author. Tel.: +1-858-822-1293; fax: +1-858-822-0386; e-mail: nfinney@chem.ucsd.edu
0008-6215/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2004.03.014

1532
R. Chang et al. / Carbohydrate Research 339 (2004) 1531–1536
OH
O
HO
HO
OH
AcHN
O UDP
AcHN
AcHN
1
HO
HO
HO
O
O
HO
HO
O
O
chitin synthase
AcHN
OH
OH
= GlcNAc
growing chitin chain
Scheme 1.
OH
O
blocked at C4: chitin elongation terminated
H3CO
HO
OH
AcHN
3
O
UDP
AcHN
HO
AcHN
HO
O
O
H3CO
HO
HO
O
O
chitin synthase
AcHN
OH
OH
= GlcNAc
growing chitin chain
2
Scheme 2.
glycosyltransferases, such as cellulose synthase andhy-
aluronan synthase. In addition, this compound could be
valuable for studying other UDP-GlcNAc transferase,
such as those responsible for the ubiquitous mono-
GlcNac glycosylation of proteins.6 We report here the
chemical synthesis of 3 (Scheme 3), as well as the results
of preliminary evaluation of 3 as a substrate for chitin
synthase.
tion of precipitatedradioactive chitin at fixedtime (1 h,
which is below the cutoff for linear time dependence)
with fixedconcentration of 3 (0 or 3 mM) in the presence
of variedconcentration of UDP-GlcNAc (Fig. 1). Our
initial studies have led to three interesting observations.
First, preliminary measurements suggest that 3 does not
function as a competitive inhibitor. While this could
mean it is a noncompetitive inhibitor, it is also consis-
tent with 3 serving as a noninhibitory terminating sub-
strate. (The kinetic profile for such a scenario is complex
andwill require much further stuyd.) Secon,d we
observe a linear decrease in precipitated chitin as a
function of the mole fraction of 3 incorporatedin the
assay solution (data not shown), which is consistent with
3 terminating growing chitin chains in a statistical
manner. Finally, enzyme activity is constant over time––
chitin production remains linear out to P2 h in the
presence of inhibitor (as it does in the absence of
inhibitor).
2. Results and discussion
The synthesis of 3 began with commercially available
glucosamine 4, which is easily convertedto the known
triol 817 in four steps (35% overall yieldfrom 4). Reg-
ioselective O-benzylation of 8 by treatment with Bu2SnO
and benzyl bromide in the presence of TBAI afforded 9
in 71% yield.18–21 Subsequent O-methylation of the
4-hydroxyl group of 9 with NaH andCH I proceeded
3
slowly, providing 10 in modest yield after 3 days.
Removal of the N-phthalimido protecting group by
hydrazinolysis, acylation of the resulting amine with
acetic anhydride, and TBS removal with TBAF pro-
ceeded smoothly, furnishing 12 in three steps (78%
overall yieldfrom 10). Phosphorylation with tetrabenzyl
pyrophosphate gave the benzyl-protectedanomeric
phosphate 13 as the desired a anomer in high yield.22
Global debenzylation was achieved by hydrogenation
over palladium catalyst to provide 14 as the ammonium
salt. Finally, coupling of the phosphate 14 with UMP-
morpholidate provided 3 in 37% yield,23;24 representing a
4% overall yieldfrom glucosamine 4.
Taken together, these data are consistent with a sce-
nario in which incorporation of 3 leads to termination of
the growing chain, after which the 4-OMe-GlcNAc
terminatedchitooligomers are release,d regenerating
active enzyme. It is important to emphasize that these
are only preliminary results andthat two significant
challenges must be overcome to clarify the behavior of
3. Chitin synthase is an integral membrane protein
assayedas a part of a particulate membrane prepara-
tion. This makes obtaining accurate kinetic data (as
opposedto the single time point/variable substrate
concentration data described here) very difficult. Given
that the kinetics of chain termination are complex––we
are not aware of an appropriate establishedkinetic
model––this is a significant hurdle. Similarly, isolation
Chitin synthase assays were carriedout by an estab-
25
lishedmethod basedon precipitation andquantifica-

R. Chang et al. / Carbohydrate Research 339 (2004) 1531–1536
1533
1. NaOMe, MeOH
2. phthalic anhydride
OAc
OAc
O
OH
NEt3, MeOH, ∆
NH2NH2•HOAc
3. Ac2O, pyr
O
O
AcO
AcO
AcO
HO
HO
DMF
OAc
OH
OH
AcO
PhthN
PhthN
6
NH2
44%
97%
4
5
TBSCl
OH
OAc
imidazole
CH2Cl2
NaOMe
MeOH
99%
O
O
HO
HO
AcO
AcO
OTBS
OTBS
OTBS
82%
PhthN
8
PhthN
7
OBn
O
OBn
Bu2SnO
TBAI, BnBr
toluene
CH3I, NaH
DMF
O
HO
H3CO
BnO
OTBS
BnO
PhthN
9
PhthN
10
71%
59%
OBn
OBn
1. NH2NH2, MeOH, ∆
TBAF
2. Ac2O, pyridine
THF 0 °C
O
O
H3CO
BnO
H3CO
BnO
OH
OTBS
79%
99%
AcHN
AcHN
11
12
OBn
O
OH
LDA, [(BnO)2P]2O
THF, –78 - 0 °C
H2, Pd/C
O
H3CO
BnO
H3CO
HO
MeOH, NEt3
O
P
OBn
O
P
AcHN
AcHN
14
91%
99%
O
OBn
O
OH
O–Et3NH+
13
O
OH
UMP-morpholidate
1-H-tetrazole
pyridine
NH
O
H3CO
HO
O
P
O
P
N
O
O
37%
AcHN
O
O
O
OH OH
HO OH
3
Scheme 3.
andidentification of the putative 4-OMe-GlcNAc ter-
minatedoligomers is not a trivial objective, since it
requires separation of small quantities of short chitin
chains from a complex reaction mixture––something
that has not previously been accomplished––followed by
appropriate characterization. Again, while not insur-
mountable, this is a significant obstacle. Once these is-
sues have been addressed, we anticipate that application
of this chain-termination strategy shouldalso have
application to other important polymerizing glyco-
syltransferases.
3. Experimental
3.1. General methods
All reactions were carriedout in oven or flame-dried
glassware, under an atmosphere of N2, unless otherwise
noted. THF and CH2Cl2 were dried by passage through
an activated column of alumina, and pyridine was dis-
tilledfrom CaH 2. All other reagents were usedas
receivedunless otherwise state.d Thin-layer chroma-
tography (TLC) was performedon Silica Gel 60 plates

1534
R. Chang et al. / Carbohydrate Research 339 (2004) 1531–1536
)4.1, 17.6, 25.3, 57.5, 70.7, 73.6, 73.7, 74.1, 74.2, 78.3,
93.2, 122.8, 123.1, 127.2, 127.5, 127.6, 127.7, 127.9,
128.3, 133.6, 137.5, 138.0, 167.4; MALDI-HRMS:
Calcdfor C 34H41NO7Si (M+Naþ) m=z 626.2544; found
m=z 626.2552.
3.3. 3,6-Di-O-benzyl-1-O-tert-butyldimethylsilyl-2-
deoxy-4-O-methyl-2-phthalimido-b-D-glucopyranose (10)
NaH (0.23 g, 5.70 mmol, 5.0 equiv) was added to a
solution of 9 (0.69 g, 1.14 mmol, 1 equiv), iodomethane
(7.1 mL, 114.00 mmol, 100.0 equiv), andDMF (11.4 mL)
at 0 °C. The reaction was allowedto warm to room
temperature andwas stirredfor 3 days. The solution was
then pouredinto ice-coldbrine (50 mL), extractedwith
EtOAc (50 mL), washedwith brine (3 · 50 mL), dried
over Na2SO4, andconcentratedunder reducedpressure
to affordan oily residue. Purification by silica chroma-
tography (5:95 EtOAc–hexanes) afforded 10 as a yellow
solid(0.37 g, 59%). 1H NMR (CDCl3): d )0.11 (s, 3H),
0.04 (s, 3H), 0.65 (s, 9H), 3.45 (dd, 1H, J 8.4 Hz, 9.6 Hz),
3.54 (m, 1H), 3.57 (s, 3H), 3.77 (m, 2H), 4.08 (dd, 1H,
J 8.4 Hz, 11.2 Hz), 4.23 (dd, 1H, J 8.4 Hz, 11.2 Hz),
4.45–4.81 (m, 4H), 4.32 (d, 1H, J 8.0 Hz), 6.90 (m, 4H),
7.37 (m, 10H); 13C NMR (CDCl3): d )5.5, )4.1, 17.6,
25.4, 57.8, 60.5, 68.8, 73.3, 74.2, 75.0, 78.4, 81.6, 93.1,
122.8, 123.0, 127.1, 127.3, 127.4, 127.7, 127.8, 128.1,
133.5, 137.9, 138.1, 167.4; MALDI-HRMS: Calcdfor
C35H43NO7Si (M+Naþ) m=z 640.2701; found m=z
640.2718.
Figure 1. Preliminary evaluation of 3 as a substrate for chitin synthase.
(F254, EM Science) that were visualizedwith UV light or
stainedwith KMnO , ninhydrin, or phosphomolybdic
4
acid. Flash column chromatography was performed
using silica gel (Selecto Scientific, 32–63 lm) or reversed-
phase silica gel (EM Science, Silica Gel 60, RP-18) as
indicated. IR spectra were recorded using a Nicolet 550
spectrometer. 1H NMR data were acquired on a Varian
Mercury-400 (400 MHz) spectrometer andare reported
in ppm downfield from Me4Si relative to residual
monoprotium solvent (CHCl3 at 7.26 ppm, CHD2OD at
3.30 ppm, HOD at 4.67 ppm). Proton-decoupled 13C
NMR spectra were obtainedon a Varian Mercury-400
(100 MHz) spectrometer andare reportedin ppm rela-
tive to solvent as internal standard (CDCl3 at 77.0 ppm,
CD3OD at 49.0 ppm, added CH3OH at 49.5 ppm for
D2O). High resolution mass spectra were obtainedon an
Ionspec Ultima FTMS operating in the MALDI mode
(MALDI-FTMS) at the Scripps Research Institute, La
Jolla, CA.
3.4. 2-Acetamido-3,6-di-O-benzyl-1-O-tert-butyldimeth-
ylsilyl-2-deoxy-4-O-methyl-b-D-glucopyranose (11)
A suspension of 10 (0.23 g, 0.36 mmol, 1 equiv) in
hydrazine (1.13 mL, 36.00 mmol, 100.0 equiv) and
MeOH (3.6 mL) was heatedto 55 °C for 24 h andcon-
centrated under reduced pressure. The residue was
diluted with EtOAc (50 mL), washed with brine (50 mL),
dried over Na2SO4, andconcentratedto obtain an oily
residue. Purification by silica chromatography (1:1
EtOAc–hexanes) afforded the free amine, which was
subsequently dissolved in 1:1 pyridine–Ac2O (2 mL) and
stirredovernight. The solution was concentratedand
chromatographedon silica gel (3:7 EtOAc–hexanes) to
afford 11 (0.15 g, 79%) as a clear oil. 1H NMR (CDCl3):
d 0.08 (s, 3H), 0.11 (s, 3H), 0.87 (s, 9H), 1.84 (s, 3H),
3.33 (m, 1H), 3.46 (m, 1H), 3.51 (s, 3H), 3.69 (m, 2H),
3.97 (dd, 1H, J 8.4 Hz, 10.0 Hz), 4.56–4.84 (m, 4H), 4.97
(d, 1H, J 7.2 Hz), 5.37 (d, 1H, J 8.0 Hz), 7.34 (m, 10H);
13C NMR (CDCl3): d )5.2, )4.0, 18.0, 23.6, 25.7, 58.8,
60.3, 69.1, 73.3, 74.2, 74.8, 80.1, 80.7, 94.9, 127.3, 127.4,
127.5, 127.9, 128.1, 128.2, 138.2, 138.4, 169.7; MALDI-
3.2. 3,6-Di-O-benzyl-1-O-tert-butyldimethylsilyl-2-
deoxy-2-phthalimido-b-D-glucopyranose (9)
In a vessel equippedwith a Dean–Stark trap, a sus-
pension of dibutyltin oxide (1.10 g, 4.40 mmol, 2.2 equiv)
and 8 (0.85 g, 2.00 mmol, 1 equiv) was refluxedin tolu-
ene (40 mL) for 12 h. TBAI (1.63 g, 4.40 mmol, 2.2 equiv)
and BnBr (0.52 mL, 4.40 mmol, 2.2 equiv) were added,
and the reaction was stirred for an additional 4 h. The
reaction mixture was then concentrated under reduced
pressure, andthe resiude was suspeneddin EtOAc.
Insoluble material was removedby filtration, andthe
filtrate was concentrated under reduced pressure.
Purification by silica gel chromatography (1:9 EtOAc–
1
hexanes) afforded 9 as a yellow oil (0.86 g, 71%). H
NMR (CDCl3): d )0.08 (s, 3H), 0.06 (s, 3H), 0.68 (s,
9H), 3.11 (br s, 1H), 3.68 (m, 1H), 3.82 (m, 3H), 4.14
(dd, 1H, J 8.4 Hz, 11.2 Hz), 4.29 (dd, 1H, J 8.4 Hz,
10.8 Hz), 4.53–4.78 (m, 4H), 5.38 (d, 1H, J 8.0 Hz), 6.97
(m, 5H), 7.37 (m, 10H); 13C NMR (CDCl3): d )5.5,
HRMS: Calcdfor
C
29H43NO6Si (M+Naþ) m=z
552.2752; found m=z 552.2736.

R. Chang et al. / Carbohydrate Research 339 (2004) 1531–1536
1535
3.5. 2-Acetamido-3,6-di-O-benzyl-2-deoxy-4-O-methyl-
glucopyranose (12)
D
-
6.8 Hz); 13C NMR (CDCl3): d 9.3, 22.9, 47.4, 55.6, 60.9,
62.2, 73.3, 73.5, 81.3, 95.0, 173.6; MALDI-HRMS:
Calcdfor C H18NO9P (M+Naþ) m=z 338.0611; found
9
TBAF (0.30 mL, 1.0 M solution in THF, 0.30 mmol,
1.2 equiv) was added to a solution of 11 (0.13 g,
0.25 mmol, 1 equiv) in THF (2.5 mL) at 0 °C. After 1 h,
the solution was concentratedandchromatographedon
silica (2:98 MeOH–CH2Cl2) to afford 12 (0.10 g, 99%) as
a white solid. 1H NMR (CDCl3): d 1.83 (s, 3H), 3.36 (dd,
1H, J 8.5 Hz, 10.0 Hz), 3.51 (s, 3H), 3.69 (m, 3H), 3.95
(m, 1H), 4.09 (m, 1H), 4.55–4.88 (m, 4H), 5.22 (app t, 1H,
J 3.2 Hz), 5.31 (d, 1H, J 8.8 Hz), 7.35 (m, 10H); 13C NMR
(CDCl3): d 23.5, 52.9, 60.6, 69.0, 70.8, 73.7, 74.8, 79.1,
80.7, 92.0, 127.5, 127.6, 127.7, 128.1, 128.2, 128.4, 138.0,
138.3, 170.0; MALDI-HRMS: Calcdfor C 23H29NO6
(M+Naþ) m=z 438.1887; found m=z 438.1888.
m=z 338.0612.
3.8. Uridinediphosphoryl 4-O-methyl-N-acetyl-glucos-
amine, ammonium salt (3)
A solution of 14 (0.044 g, 0.11 mmol, 1 equiv) in dry
pyridine (0.550 mL) was charged to a solution of
4-morpholine-N,N0-dicyclohexylcarboxamidinium uri-
dine 50-monophosphomorpholidate (0.151 g, 0.22 mmol,
2.0 equiv) and1- H-tetrazole (0.023 g, 0.33 mmol,
3.0 equiv) in dry pyridine (0.550 mL) and stirred for
5 days at room temperature. The solution was then
concentrated under reduced pressure and applied to a
size-exclusion gel (Bio-Rad, Bio-Gel, P-2 Fine,
2.5 · 100 cm), elutedwith 0.25 M NH 4HCO3 and
lyophilizedto obtain 3 (0.027 g, 37%) as a fluffy white
3.6. 2-Acetamido-3,6-di-O-benzyl-2-deoxy-4-O-methyl-a-
D
-glucopyranose 1-dibenzylphosphate (13)
1
solid. H NMR (D2O): d 1.93 (s, 3H), 3.21 (dd, 1H, J
A solution of 12 (0.053 g, 0.13 mmol, 1 equiv) in THF
(2.6 mL) was cooledto )78 °C under N2 andLDA
(0.070 mL, 2.0 M in 4:2:1.5 THF–heptane–ethylbenzene,
0.14 mmol, 1.1 equiv) was added dropwise. After 15 min,
9.2 Hz, 10 Hz), 3.43 (s, 3H), 3.63 (dd, 1H, J 4.0 Hz,
12.8 Hz), 3.73 (m, 3H), 3.82 (dt, 1H, J 3.6 Hz, 10.4 Hz),
4.12 (m, 3H), 4.22 (m, 2H), 5.35 (dd, 1H, J 3.2 Hz,
7.2 Hz), 5.83 (m, 2H), 7.82 (d, 1H, J 8.0 Hz); 13C NMR
(D2O): d 22.8, 54.4, 60.8, 65.5, 70.2, 71.3, 72.6, 74.4,
79.6, 83.7, 88.8, 89.1, 95.0, 103.1, 142.0, 152.2, 166.6,
a
solution of tetrabenzyl pyrophosphate (0.086 g,
0.16 mmol, 1.3 equiv) in THF (0.800 mL) was added.
The reaction was warmedto 0 °C andstirredfor 3 h. The
solution was subsequently diluted with CH2Cl2 (20 mL),
175.1; MALDI-HRMS: Calcdfor
C 18H29N3O17P2
(M+Hꢀ) m=z 620.0899; found m=z 620.0910.
washedwith satdaq NaHCO
(1 · 20 mL), brine
3
(1 · 20 mL), dried over Na2SO4, andconcentratedto
obtain a yellow oil. Chromatography on silica gel (5:95
MeOH–CH2Cl2) afforded 13 (0.080 g, 91%) as a yellow
3.9. Chitin synthase assay
The assay protocol usedis basedon the procedure of
Orlean,25 modified after helpful discussions with Prof.
Peter Orlean (University of IL) andDr. Enrico Cabib
(NIH). S. cerevisiae strains (PP-1D, wildtype) were
kindly provided by Prof. Orlean and were stored at
)70 °C on freezer stabs. Active yeast cultures were
temporarily maintainedon agar plates andstoredat
4 °C. Cells were culturedin 200 mL YEPG (1% yeast
extract, 2% bactopeptone, 2% glucose) medium at 30 °C
andallowedto grow to saturation. An aliquot (10–
12 mL) of the satdmedium was transferredto 400 mL of
YEPG medium to give an optical density of 0.15–0.20
andallowedto grow to an optical density of 0.65–0.70.
The cells were washedwith coldwater andTM buffer
(50 mM Tris Æ HCl, 2.5 mM MgCl2, pH 7.5) by suspen-
sion andcentrifugation (15 min, 2000 g). The wet weight
of the cells at this point was typically ꢁ1 g; this weight
was usedto determine the volume of buffer in which the
final pellet was suspended (see below).
1
solid. H NMR (CDCl3): d 1.63 (s, 3H), 3.50 (s, 4H),
3.65 (dd, 1H, J 4.0 Hz, 11.6 Hz), 3.81 (m, 1H), 4.22 (m,
2H), 4.45–4.84 (m, 5 H), 4.97–5.09 (m, 4H), 5.19 (d, 1H,
J 8.8 Hz), 5.64 (dd, 1H, J 3.3 Hz, 5.4 Hz), 7.31 (m, 20H);
13C NMR (CDCl3): d 23.1, 52.3, 60.7, 67.9, 69.6, 73.1,
73.4, 74.6, 78.9, 79.4, 97.5, 127.4, 127.5, 127.7, 127.9,
128.2, 128.3, 128.4, 128.5, 128.6, 135.2, 137.7, 138.0,
169.7. MALDI-HRMS: Calcdfor
C
37H42NO9P
(M+Naþ) m=z 698.2489; found m=z 698.2495.
3.7. 2-Acetamido-2-deoxy-4-O-methyl-a-D-glucopyra-
nose-1-phosphate, monotriethylammonium salt (14)
A solution of 13 (0.074 g, 0.11 mmol, 1 equiv) in MeOH
was placedunedr an H
atmosphere H2 (1 atm) in
2
presence of Pd–C (0.015 g, 5% w/w). After 6 h, the
reaction was filteredthrough a padof Celite. Et 3N
(0.017 mL, 0.12 mmol, 1.1 equiv) was added to the fil-
trate, andthe mixture was concentratedto afford 14
The cells were suspended in 2 mL of TM buffer in a
50-mL plastic centrifuge tube, andglass beads (0.45 mm)
were added until the volume of beads reaches about
3 mm below the liquid’s surface. The tube was then
vortexed20 · 30 s, with 30 s of cooling on ice between
each vortex; vortexing was performedin a 4 °C cold
1
(0.046 g, quant) as a white solid. H NMR (CD3OD): d
1.30 (t, 9H, J 7.6 Hz), 2.21 (s, 3H), 3.16 (q, 6H, J
7.6 Hz), 3.45 (m, 1H), 3.56 (s, 3H), 3.66 (dd, 1H, J
4.4 Hz, 11.6 Hz), 3.80 (m, 3H), 3.95 (dt, 1H, J 2.8 Hz,
10.4 Hz), 4.27 (d, 1H, J 8.4 Hz), 5.42 (dd, 1H, J 3.2 Hz,

1536
R. Chang et al. / Carbohydrate Research 339 (2004) 1531–1536
room. The broken cells were removedfrom the bottom
of the tube with a glass Pasteur pipet, andthe glass beads
were rinsed5–7 · 1.5 mL with TM buffer. The pooled
rinsings were centrifugedat 2000 g for 4 min, the super-
natant was removed, and the remaining cell-wall pre-
cipitate was washedonce more with TM buffer. The cell-
wall free supernatants were combinedandcentrifugedat
60,000g for 1 h.
structure support (GM62116, GM61894, CHE-
9709183). We thank Prof. Peter Orlean (U of IL) and
Dr. Enrico Cabib (NIH) for assay advice, Prof. Orlean
for donation of yeast strains, Prof. Scott Singleton
(UNC Chapel Hill) for helpful discussion and Prof. Jack
Kyte andMr. Steven Aadms (UCSD) for technical
assistance.
The enzyme pellet was suspended in 1.6 mL · (gram
wet weight of cells) TM buffer andhomogenizedthor-
oughly with a glass Dounce homogenizer. The mem-
branes are pretreatedwith trypsin (quantifiedby weight,
10 min, 30 °C) andthen treatedwith 3.0 · (mass of
trypsin) trypsin inhibitor. Typically, four different con-
centrations of trypsin/trypsin inhibitor were testedfor
each new membrane preparation, andthe combination
with the highest activity at 30 min was usedin the assay.
Concentrations of trypsin typically testedwere 0.5, 1.0,
2.0, and4.0 mg/mL, andtrypsin was aeddto the
membrane preparation at a concentration of 1 mL of
trypsin solution for every 5 mL of membrane prepara-
tion. Individual assays were performed in 1.5-mL Ep-
pendorf centrifuge tubes.
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We thank the NIH (GM60875), the Hellman Founda-
tion andthe UC Systemwide Biotechnology Program
for direct support andthe NIH andNSF for infra-