Second-generation dimeric inhibitors of chitin synthase

Article abstract of DOI:10.1016/j.bmc.2004.09.027

Chitin synthase (CS) is essential for fungal cell wall biosynthesis and is an attractive medicinal target. Expanded results from our efforts to develop mechanism based inhibitors of CS are presented here. Specifically, we describe uridine dimers linked by tartrate amides as potential pyrophosphate mimics.

Full text of DOI:10.1016/j.bmc.2004.09.027

Bioorganic & Medicinal Chemistry 12 (2004) 6451–6460
Second-generation dimeric inhibitors of chitin synthase
a,b,
*
a
Adam R. Yeager and Nathaniel S. Finney
a
University of California, San Diego, Department of Chemistry and Biochemistry, 9500 Gilman Dr. La Jolla, CA 92093-0358, USA
b
Organisch-chemisches Institut, Universit a¨ t Z u¨ rich, Winterthurerstrasse 190, 8057 Z u¨ rich, Switzerland
Received 8 June 2004; revised 2 September 2004; accepted 16 September 2004
Abstract—Chitin synthase (CS) is essential for fungal cell wall biosynthesis and is an attractive medicinal target. Expanded results
from our efforts to develop mechanism based inhibitors of CS are presented here. Specifically, we describe uridine dimers linked by
tartrate amides as potential pyrophosphate mimics.
ꢀ
2004 Elsevier Ltd. All rights reserved.
1
. Introduction and background
OH
HO
HO
AcHN
O
O
N
Chitin synthase (CS) is the enzyme responsible for the
conversion of uridine diphosphoryl-N-acetylglucos-
amine (UDP-GlcNAc) into chitin—polymeric chains of
b-1,4-linked N-acetylglucosamine (GlcNAc) (Fig. 1).
The biosynthesis of chitin is essential for fungal growth
and reproduction, and because it is absent in humans CS
O
O—
P
NH
O
O
O
O
chitin synthase
—O P
O
O
UDP-GlcNAc
UDP
HO OH
1
represents an important antifungal target. In broad
HO
HO
terms, three approaches to the development of CS inhib-
itors warrant consideration: inhibition based on enzyme
structure, inhibition based on substrate analogs, and
inhibition based on mechanism.
NHAc
O
NHAc
HO
HO
O
HO
O
O
HO
O
OH
O
HO
O
AcHN
AcHN
OH
OH
n
chitin
The first of these is not yet a viable option. While the
crystal structures of several glycosyltransferases have
recently been solved, polymerizing transferases such as
CS are large integral membrane proteins and are likely
to elude crystallographic characterization for some time
Figure 1. Biosynthesis of chitin.
therefore been focused on a mechanism based approach
5
to the development of CS inhibitors.
2
to come. The second option, inhibition by substrate
analogs, appears more feasible, but is complicated by
the fact that CS (like most transferases) has low affinity
for its substrate: KM values for CS are ꢀ1mM, consist-
ent with very weak substrate binding. While the best
inhibitors of CS are the naturally occurring polyoxin
and nikkomycins (Chart 1), which are generally re-
Developing mechanism based inhibitors necessarily
requires an understanding the mechanism of the en-
zyme. It was, in fact, the conflicting mechanistic hypoth-
6
eses in the literature, combined with the complete
absence of experimental mechanistic investigation that
provided the initial incentive for our studies of CS.
Mechanistic proposals for CS must address the extended
structure of the polysaccharide chain, in which adjacent
glycosyl units have opposed orientations (Fig. 1). As has
3
garded as UDP-GlcNAc analogs, the rational design
of CS inhibitors along these lines has not produced com-
4
pounds with comparable potency. Our research has
6
a,b,c
been noted,
multiple active sites with appropriate
Keywords: Glycosyltransferase, Mechanism; Inhibitor; Phosphate
mimic.
proximity and spatial orientation could allow for the
sequential transfer of two sugar residues without the
need for rotation of the growing chain or the enzyme
*
Corresponding author. Tel.: +41 44 635 4283; fax: +41 44 635
888; e-mail addresses: finney@unizh.edu; nfinney@chem.ucsd.edu
6
0
968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.09.027

6452
A. R. Yeager, N. S. Finney / Bioorg. Med. Chem. 12 (2004) 6451–6460
O
inhibition, providing the first evidence of a two active-site
5
c
HO2C
CO2H
mechanism for CS (Chart 2). These data are consistent
with the hypothesis that CS operates via a two site mech-
anism, distinct from that of the more well understood sin-
gle sugar glycosyltransferases. The best dimeric inhibitor
of the first series, compound 2 (IC50 = 1.1mM), was an
order of magnitude more potent than the corresponding
monomeric control (6, IC50 = 11.8mM). While the differ-
ence in inhibitory activity is informative, 2 is still a much
NH
O
OH
O
N
O
O
H2N
O
N
H
OH NH2
HO
OH
polyoxin D
K ~ 1µM
i
weaker inhibitor than any the polyoxins or nikkomycins
3
OHC
(
K ꢀ 0.001ꢁ0.1mM). It is anticipated that dimerization
i
HO
NH
O
N
CH3
O
CO2H
of more potent monomeric fragments will lead to signif-
icantly enhanced inhibition of CS. This letter describes
our most recent efforts in that direction.
N
O
N
H
OH NH2
HO
OH
nikkomycin X
K ~ 0.14 µM
i
2. Design, synthesis, and evaluation of uridine–tartrate
monomers of UDP-analogs
Chart 1. Naturally occurring CS inhibitors.
The structural resemblance to UDP is regarded as the
determining factor of the activities of the polyoxins and
(
Fig. 2). Consequently, even without specific structural
7
information, if CS possesses two active sites in close
proximity it should be possible to enhance binding affin-
ity through multivalent interactions between multiple
UDP-binding motifs.
nikkomycins. While these represent ideal monomeric
precursors to dimeric inhibitors of CS, dimerization of
these compounds is precluded by their lack of availability
3
,8
(due to price and/or synthetic complexity). As an alter-
native, we have constructed dimeric UDP analogs via the
union of uridine and tartaric acid fragments (Chart 3). It
was anticipated that the tartrate moiety would provide
additional polar interactions similar to the UDP-Glc-
NAc pyrophosphate (as well as the polyoxamic acid side
To this end, we have previously disclosed a series of di-
meric uridine compounds with tether-length-dependant
active site
HO
2
HO
9
UDP
chain). Uridine fragments were therefore dimerized
through tartrate amide linkages (Chart 4) and evaluated
for enhanced chitin synthase inhibition.
O
HO
O
HO
HO
O
AcNH
UDP
active site
1
Initially, two uridine–tartrate amides (7, 8) were chosen
as monomeric substrate analogs (Schemes 1 and 2). The
monomers were prepared by the condensation of a pro-
2
UDP
= GlcNAc
0
HO
HO
tected 5 -aminouridine fragment (18) with a protected
NHAc
tartrate monoester. Amine 18 was prepared from the
corresponding azido diol (15) by sequential TES protec-
tion, Boc protection of the uracil imide and hydrogeno-
HO
HO
O
HO
O
O
O
O
HO
AcNH
AcNH
OH
10,11
lysis of the azide.
prepared by the mono-saponification of the acetonide
Tartrate acids 19 and 20 were
Figure 2. Proposed two site mechanism for CS.
1
2
protected (R,R)-and (S,S)-diethyl tartrates. Condensa-
tion of amine 18 with acid 19 or 20 provided esters 21
1
3
and 22 (Scheme 2). Treatment of 21 and 22 with
O
N
NH
HO
N
OH
O
H
N
O
O
O
O
O
O
N
H
NH
O
O
n
HO
O
HO
OH
O
N
O
HN
O
O
1
2
3
4
5
n=1 32%
n=2 45%
n=3 5%
n=4 4%
n=5 1%
2
H N
N
H
O
O
OH
HO
OH
O
7
NH
O
N
O
NH
O
O
H3CO
O
N
H
HO
O
N
O
H2N
N
H
6
6%
HO
OH
O
OH
HO
OH
Chart 2. First-generation dimeric inhibitors, monomeric control, and
inhibition of CS activity. Percentile values are % inhibition of CS
activity at 1mM inhibitor.
8
Chart 3. Uridine–tartrate UDP analogs.

A. R. Yeager, N. S. Finney / Bioorg. Med. Chem. 12 (2004) 6451–6460
6453
O
O
HN
NH
9
1
1
L= (CH2)2
0 L= (CH2)3
1 L= (CH2)4
O
OH
OH
O
H
N
H
N
O
N
N
O
O
O
N
H
L
N
H
12 L= (CH2)2O(CH2)2
OH
O
O
OH
HO
OH
HO
OH
O
O
NH
HO
N
OH
NH
OH
O
OH
O
H
H
N
N
O
N
O
N
O
O
H3CO
N
H
O
N
H
O
O
OH
3
O
OH
14
HO
OH
HN
HO
OH
1
O
Chart 4. Uridine–tartrate dimers, linked by diamines.
O
N
O
N
O
N
NH
NH
NBoc
O
O
O
N3
O
X
O
O
b
O
a
O
1
8
EtO
N
H
TBSO
1
9 or 20
O
O
RO OR
TESO OTES
OTBS
Me Me
1
1
5 R = H
6 R = TES
17 X = N3
18 X = NH2
21 R,R
a
c
22 S,S (not shown)
O
O
O
O
OH
NBoc
d, e
OEt
EtO
EtO
OH
OH
O
O
EtO
EtO
N
O
O
O
b
O
c,d
OH
O
2
H N
N
H
TBSO
7, 8
Me Me
O
O
1
9
OTBS
Me Me
2
3 R,R
O
O
O
OH
24 S,S (not shown)
d, e
OEt
Scheme 2. Synthesis of uridine–tartrate monomers. Reagents: (a)
HBTU, NEt , CH CN, 90% (21), 82% (22); (b) NH OH, EtOH, 58%
O
O
OH
O
3
3
4
Me Me
(
23), 14% (24); (c) TBAF/THF, 79%; (d) TFA, 64% over two steps (7),
0% over two steps (8).
2
0
7
Scheme 1. Synthesis of uridine and tartrate intermediates. Reagents:
a) TBSCI, imidazole, pyridine, 95%; (b) Boc O, DMAP, pyridine,
9%; (c) H , Pd/C, MeOH, 94%; (d) dimethoxy-propane, p-TsOH,
(
2
9
2
Acid 25 was obtained by saponification of ester 21.
Amide bond formations of 25 with commercially avail-
able diamines were achieved using HBTU-mediated
peptide coupling. The monomeric control compound
14 was prepared by the same method. The shortest
dimer (13) was created through the coupling of amine
acetone, 99%; (e) KOH, ETOH, 30–50%.
1
5
ammonia resulted in the free amide compounds 23 and
24. Subsequent removal of TBS, Boc, and acetonide
groups provided amides 7 and 8.
18 with acid 25. Deprotections of the dimers (and mono-
mer) were performed under the same conditions
Uridine–tartrate amide compounds 7 and 8 exhibited
greater CS inhibition (12% and 20% at 1mM, respec-
tively), than did uridine glycol monomer 6 (6% at
employed for 7 and 8, and the final products were puri-
6
fied by both silica and reverse-phase chromatography.
1
1
mM). The enhanced inhibition upon addition of the
tartrate groups provided incentive for the synthesis of
related dimers, as dimerization is expected to amplify
the increases inhibition exponentially. (In an ideal case,
4. Evaluation of uridine–tartrate dimers
2
K (dimer) = [K (monomer)] .)
i
The uridine–tartrate dimeric compounds were evaluated
as inhibitors of chitin synthase (Fig. 2). Monomeric
control 14 had reduced activity (8%) relative to the uri-
dine–tartaric amide monomer 8 (12%), indicating that
addition of the methyl ether served to diminish inhibi-
tion. However, dimeric compound 9 is more active than
monomer 14, indicating that there is still a benefit from
dimerization. As the length of the diamine linkers
i
3
. Design, and synthesis of uridine–tartrate dimers
Dimeric uridine–tartrate conjugates 9–12 were prepared
by coupling 25 with a series of diamine linkers of vary-
1
4
ing lengths (two to eight atoms; Chart 3; Scheme 3).

6454
A. R. Yeager, N. S. Finney / Bioorg. Med. Chem. 12 (2004) 6451–6460
O
NBoc
O
O
N
O
a
O
b
21
HO
N
H
TBSO
O
O
OTBS
Me Me
25
O
NBoc
O
Me Me
TBSO
N
OTBS
O
O
O
O
N
O
H
H
c, d
N
N
L
N
N
H
TBSO
9 - 12
H
O
O
O
O
O
O
OTBS
Me Me
BocN
2
2
2
2
6 L = (CH2)2
7 L = (CH2)3
8 L = (CH2)4
O
9 L = (CH2)2O(CH2)2
O
NBoc
O
TBSO
N
OTBS
NH
O
O
N
b
O
c, d
18 + 25
N
H
TBSO
13
14
O
O
O
O
OTBS
O
Me Me
BocN
30
O
NBoc
c, d
O
O
N
O
b
O
H3CO
21
N
N
H
TBSO
H3CO
H
NH2
O
O
OTBS
Me Me
31
Scheme 3. Synthesis of uridine–tartrate dimers. Reagents: (a) KOH, EtOH, 99%; (b) HBTU, NEt
3
, CH CN, 60–80%; (c) TBAF, THF, 90%; (d)
3
TFA, 70–80%.
˚
17
increases (from 9 to 12), the inhibitory activities of the
compounds decline, becoming essentially non-existent
for 11 and 12. Only the shortest dimer of the series, 13
uridine carbamate compound (1, ꢀ14A), and they
exhibit similar inhibitory activity despite significant dif-
ferences in the structure of the linker connecting the uri-
dine fragments. While this strengthens the conclusions
drawn from previous results, it also indicates that in-
ter-uridine distance is still the primary determinant of
inhibitory activity and that the use of tartrate linkers
as pyrophosphate analogs provides no benefit.
(
uridine–tartrate–uridine), had significant activity to-
ward CS (35%). Not only did dimer 13 have substan-
tially more activity than the monomeric control, it
surpassed the uridine–tartrate amides and the longer
dimers 9–12 (Fig. 3).
The extended distance between uridine fragments for
˚
dimer 13 (ꢀ12A) is very similar to that of the shortest
5. Conclusions
These data reinforce our earlier conclusions regarding
the mechanism by which chitin synthase produces chitin:
they are consistent with a two site mechanism for CS,
and in addition appear to further delineate the distance
between two uridine binding sites. While it is unfortu-
nate that the incorporation of tartrate fragments does
not significantly enhance the affinity of uridine dimers
for CS, this is in keeping with previous findings that
the success of tartrate as a pyrophosphate mimic is very
case dependent. With regard to the design of future
inhibitors, it appears that there is no need to further
explore ÔlongÕ dimers. Instead, work will focus on more
4
4
3
3
2
2
1
1
5
0
5
0
5
0
5
0
5
0
8
9
10
11
12
13
14
1
8
rigid dimers, and on identifying linkers that make a
Figure 3. Inhibition of CS by 8–14.
positive contribution to the binding affinity.

A. R. Yeager, N. S. Finney / Bioorg. Med. Chem. 12 (2004) 6451–6460
6455
6. Experimental
d 40.4, 70.4, 72.3, 72.4, 73.1, 81.9, 90.1, 102.4, 142.0,
1
ꢁ
52.0, 166.8, 174.1, 176.8. FTIR (KBr), cm : 3375
(br), 1688 (s), 1548 (s), 1474 (s).
1
6
.1. General
All reactions were carried out in oven or flame dried
glassware, under an atmosphere of nitrogen, except
where noted. THF and CH Cl were dried by passage
6.2.2. Fully deprotected uridine tartrate amide 8. Fully
protected amide 24 (0.08g, 0.10mmol) was dissolved in
THF (1mL) TBAF (0.22mL of 1M solution in THF,
0.22mmol) was added. After 20min, the reaction was
concentrated and purified by silica gel chromatography
(5–10% MeOH/CH Cl ) to provide the intermediate diol
2
2
through an activated column of alumina, and pyridine
and acetonitrile were distilled from CaH . All amines
2
used in amide bond forming reactions were dried over
P O in vacuo. All other reagents were used as obtained,
2
2
1
(0.04g, 76%) as a white solid. H NMR (400MHz,
2
5
unless otherwise stated. Thin layer chromatography was
performed on silica gel 60 (F254, 250nm, EM Science)
plates and visualized with UV light or stained with
CD OD): d 1.48 (s, 6H), 1.57 (s, 9H), 3.56 (dd, 1H,
3
J = 2.5, 10Hz), 3.64 (dd, 1H, J = 3.3, 7.5Hz), 4.04 (m,
2H), 4.25 (t, 1H, J = 3.1Hz), 4.59 (m, 2H), 5.77 (d,
1H, J = 3Hz), 5.82 (d, 1H, J = 5Hz), 7.75 (d, 1H,
KMnO , ninhydrin, or PMA. Flash column chromato-
4
1
3
graphy was performed using silica gel (Selecto Scientific,
32–63nm) or reverse phase (EM Science, silica gel 60,
RP-18) as indicated. IR spectra were recorded using a
J = 5Hz). C NMR (100MHz, CD OD): d 26.6, 27.7,
3
41.7, 72.3, 74.4, 78.9, 79.1, 83.7, 87.8, 92.2, 102.4,
113.9, 142.9, 148.8, 149.8, 162.1, 172.0, 174.6. FTIR
1
Nicolet 550 spectrometer. H NMR data were acquired
ꢁ1
(KBr), cm : 3356 (br), 2995 (s), 2944 (s), 1789 (s),
1676 (s), 1557 (s), 1458 (s), 1384 (s). HRMS (MALDI-
on a Varian Mercury-400 (400MHz) spectrometer and
are reported in parts per million relative to the solvent
+
FTMS), m/z: calcd for C H N O Na (MNa ):
2
1
30
4
11
(
4
CHCl at 7.26ppm, CHD OH at 3.30ppm, D O at
2
537.1818, found 537.1808. TLC (15% CH OH/CH Cl ),
3 2 2
R : 0.51. The resulting diol (0.04g, 0.05mmol) was dis-
f
3
2
1
.67ppm). Proton decoupled C NMR spectra were ob-
3
tained on a Varian Mercury-400 (100MHz) spectrome-
ter and are reported in parts per million relative to
solvent as internal standard (CDCl3 at 77.0ppm,
CD OH at 49.0ppm, added CH OH at 49.5ppm for
solved in TFA (0.5mL), stirred for 8h, and concen-
trated. The resulting oil was purified by silica gel
chromatography (8% H O/CH CN) to provide 8
2
3
1
(0.02g, 96%) as a white solid. H NMR (400MHz,
D O): d 3.49 (s, 2H), 3.98 (s, 2H), 4.20 (s, 1H), 4.40 (s,
3
3
D O). High resolution mass spectra were obtained on
2
2
an Ionspec Ultima FTMS (MALDI-FTMS) at The Scri-
pps Research Institute, La Jolla, CA, or the Pasarow
Mass Spectrometry Laboratory at the University of Cal-
ifornia, Los Angeles. Chitin synthase activity assays
1H), 4.42 (s, 1H), 5.65 (d, 1H, J = 4.4Hz), 5.72 (d, 1H,
J = 8.0Hz), 7.55 (d, 1H, J = 6.8Hz).
1
3
C NMR
(100MHz, D O): d 41.2, 71.1, 72.9, 72.9, 73.7, 82.6,
2
90.8, 102.8, 142.6, 151.9, 166.5, 174.5, 177.3. FTIR
(KBr), cm : 3388 (br), 1684 (s), 1553 (s), 1492 (s),
5
,19
ꢁ1
were performed as previously described.
1
422 (s), 1387 (s), 1265 (s). HRMS (MALDI-FTMS),
+
6
6
.2. Experimental procedures
.2.1. Fully deprotected uridine tartrate amide 7. The
m/z: calcd for C H N O (MH ): 375.1146, found
14 18 4 9
375.1148. TLC (20% H O/CH CN), R : 0.35.
2 3 f
fully protected amide 23 (0.08g, 0.06mmol) was dis-
solved in THF (1mL) and TBAF (0.13mL of 1M solu-
tion in THF, 0.13mmol) was added. After 40min, the
reaction was concentrated and purified by silica gel
chromatography (5–10% MeOH/CH Cl ) to provide
6.2.3. General procedure for deprotection of compounds
26–31 to provide 9–14. The dimers were dissolved in
THF and TBAF (1M in THF, 1.1equiv per TBS group)
was added. After approximately 90min, the reactions
were concentrated and purified by silica gel chromato-
graphy (8% H O/CH CN) to provide an intermediate
2
2
1
the diol intermediate (0.02g, 79%) as a white solid. H
2
3
NMR (400MHz, CD OD): d 1.46 (s, 3H), 1.47 (s,
diol, which was then stirred in neat TFA for 24h. The
sample was then concentrated and purified by silica gel
chromatography (12% H O/CH CN) followed by re-
verse-phase chromatography (to provide white solids)
prior to use in CS assays.
3
3
2
7
H), 3.59 (m, 2H), 4.03 (m, 2H), 4.23 (m, 1H), 4.59 (s,
H), 5.76 (d, 1H, J = 2.8Hz), 5.81 (d, 1H, J = 5.3Hz),
.75 (d, 1H, J = 5.3Hz). C NMR (100MHz, CD OD):
2
3
1
3
3
d 26.5, 27.7, 41.9, 72.3, 74.5, 78.7, 79.1, 83.8, 87.8, 92.2,
9
1
1
5.6, 102.3, 113.9, 142.8, 148.8, 149.8, 162.1, 172.1,
ꢁ
1
74.6. FTIR (KBr), cm : 3362 (br), 2995 (s), 2934 (s),
6.2.4. Dimer 9. White solid, 0.05g, 43% over two steps.
H NMR (400MHz, D O): d 3.30 (s, 4H), 3.52 (q, 4H,
1
789 (s), 1676 (s), 1536 (s), 1457 (s). HRMS (MALDI-
2
+
FTMS), m/z: calcd for C H N O Na (MNa) :
11
537.1828, found 537.1809. TLC (10% MeOH/CH Cl ),
2 2
R : 0.56. The resulting diol (0.03g, 0.05mmol) was
J = 3, 12.6Hz), 3.99 (m, 4H), 4.20 (m, 2H), 4.42 (m,
4H,), 5.66 (d, 2H, J = 2.8Hz), 5.74 (d, 2H, J = 5.0Hz),
2
1
30
4
1
3
7.54 (d, 2H, J = 5Hz). C NMR (100MHz, D O): d
f
2
dissolved in TFA (0.5mL), stirred for 8h, and concen-
trated. The resulting oil was purified by silica gel chro-
matography (15% H O/CH CN) to provide 7 (0.01g,
39.2, 40.9, 70.9, 72.9, 73.0, 73.6, 82.5, 90.7, 102.9,
ꢁ
1
142.6, 151.8, 166.4, 174.4, 174.5. FTIR (KBr), cm
3449 (br), 1649 (s), 1544 (s). HRMS (MALDI-FTMS),
:
2
3
1
+
m/z: calcd for C H N O Na (MNa) 797.2196, found
7
9%) as a white solid. H NMR (400MHz, D O):
2
28 38
8
18
d 3.47 (dd, 1H, J = 2.0, 9.3Hz), 3.54 (dd, 1H,
J = 2.8, 8.0Hz), 3.99 (d, 2H, J = 1.5Hz), 4.21 (t,
797.2213. TLC (20% H O/CH CN), R : 0.34.
2 3 f
1
J = 1.3Hz), 5.68 (d, 1H, J = 2.8Hz), 5.74 (d, 1H,
H, J = 3.0Hz), 4.41 (d, 1H, J = 0.8Hz), 4.43 (d, 1H,
6.2.5. Dimer 10. White solid, 0.06g, 37% over two steps.
H NMR (400MHz, D O): d 1.63 (s, 2H), 3.19 (m, 4H),
3.52 (m, 4H), 4.01 (m, 4H), 4.23 (m, 2H), 4.43 (s, 2H),
1
2
1
3
Hz), 7.54 (d, 1H, 5Hz). C NMR (100MHz, D O):
5
2

6456
A. R. Yeager, N. S. Finney / Bioorg. Med. Chem. 12 (2004) 6451–6460
4
(
.45 (s, 2H), 4.50 (s, 4H), 5.69 (d, 2H, J = 2.8Hz), 5.77
d, 2H, J = 5.0Hz), 7.57 (d, 2H, J = 5.0Hz). C NMR
reaction allowed to stir for 16h, concentrated, and puri-
fied by silica gel chromatography (40% EtOAc/hexanes)
to afforded 16 (1.97g, 95%), as a white foam. H NMR
1
3
1
(
100MHz, D O): d 28.9, 36.9, 39.2, 40.9, 70.9, 73.0,
2
7
1
1
3.1, 73.6, 82.6, 90.6, 102.9, 142.7, 152.2, 166.8, 174.2,
(400MHz, CDCl ): d 0.10 (m, 12H), 0.89 (s, 9H), 0.90 (s,
3
ꢁ
1
74.7. FTIR (KBr), cm : 3445 (br), 1676 (s), 1562 (s),
9H), 3.61 (dd, 1H, J = 2, 8.3Hz), 3.82 (dd, 1H, J = 2, 8.5
Hz), 3.96 (m, 1H), 4.13 (m, 1H), 4.21 (t, 1H, J = 2Hz),
5.65 (d, 1H, J = 1.8Hz), 5.77 (d, 1H, 5Hz), 7.70 (d,
475 (s). HRMS (MALDI-FTMS), m/z: calcd for
+
C H N O Na (MNa) 811.2353, found 811.2387.
18
TLC (20% H O/CH CN), R : 0.40.
2
2
9
40
8
1
3
1H, J = 5Hz), 9.70 (s, 1H). C NMR (100MHz,
CDCl ): d ꢁ4.8, ꢁ4.7, ꢁ4.3, ꢁ4.1, 18.1, 18.1, 25.8,
3
f
3
6
.2.6. Dimer 11. White solid, 0.06g, 30% over two steps.
51.0, 71.0, 75.0, 81.2, 91.1, 102.1, 140.1, 150.0, 163.3.
FTIR (film) cm : 2855–3187 (br), 2121 (s), 1693 (br),
1
ꢁ1
H NMR (400MHz, D O): d 1.40 (s, 4H), 3.12 (m, 4H),
2
3
.48 (m, 4H), 3.97 (m, 4H), 4.19 (t, 2H, J = 2.8Hz), 4.38
s, 2H), 4.40 (s, 2H), 5.65 (d, 2H, J = 2.8Hz), 5.73 (d,
1474 (s). HRMS (MALDI-FTMS), m/z: calcd for
C H N O Si Na
520.2409. TLC (40% EtOAc/hexanes), R : 0.40.
f
+
(M+Na )
(
520.2388,
found
2
1
39
5
5
2
1
3
H, J = 5.0Hz), 7.54 (d, 2H, J = 5.0Hz). C NMR
2
(
100MHz, D O): d 26.6, 39.4, 40.9, 70.9, 73.0, 73.1,
2
7
1
1
3.6, 82.6, 90.6, 102.9, 142.7, 151.9, 166.4, 174.0,
6.2.11. Fully protected azidouridine 17. The TBS-pro-
tected azide 16 (8.05g, 16.2mmol) was dried by co-evap-
oration with tetrahydrofuran and dissolved in dry
ꢁ
1
74.7. FTIR (KBr), cm : 3371 (br), 1667 (s), 1553 (s),
475 (s). HRMS (MALDI-FTMS), m/z: calcd for
+
C H N O (MH) 803.2690, found 803.2693. TLC
3
pyridine (20mL) under N . DMAP (0.40g, 3.24mmol,
2
0
43
8
18
(
18% H O/CH CN), R : 0.13.
2
the mixture was cooled to 0ꢁC, and Boc O (10.6g,
3
f
2
4
8.6mmol) was added. The reaction was allowed to stir
6
.2.7. Dimer 12. White solid, 0.08g, 38% over two steps.
Dimer 12 was purified by reverse-phase silica gel chro-
for 16h at room temperature, concentrated, and purified
by silica gel chromatography (35–40% EtOAc/hexanes)
to provide 17 (9.60g, 99%) as a white foam. H NMR
1
matography (0–10% H O/CH CN) before use in chitin
2
synthase assays. H NMR (400MHz, D O): d 3.30 (s,
3
1
(400MHz, CDCl ): d 0.08 (m, 12H), 0.88 (s, 9H), 0.90
2
3
4
4
7
3
1
3
H), 3.48 (m, 8H), 3.97 (m, 4H), 4.19 (m, 2H), 4.41 (s,
H), 5.66 (d, 2H, J = 2.8Hz), 5.73 (d, 2H, J = 5.0Hz),
(s, 9H), 1.59 (s, 9H), 3.58 (dd, 1H, J = 2.2, 8.3Hz),
3.75 (dd, 1H, J = 2.3, 7.5Hz), 3.95 (m, 1H), 4.11 (m,
1H), 4.17 (m, 1H), 5.72 (d, 1H, J = 2.3Hz), 5.78 (d,
1
3
.54 (d, 2H, J = 5.0Hz). C NMR (100MHz, D O): d
2
1
3
9.6, 41.1, 69.4, 71.1, 73.1, 73.7, 82.7, 90.7, 102.9,
1H, 5Hz), 7.62 (d, 1H, 5.2Hz). C NMR (100MHz,
CDCl ): d ꢁ4.8, ꢁ4.7, ꢁ4.6, ꢁ4.2, 18.0, 18.0, 25.8,
ꢁ
1
42.7, 151.9, 166.4, 174.3, 174.7. FTIR (film), cm
:
365 (br), 1681 (s), 1541 (s), 1475 (s). HRMS (MAL-
3
27.4, 51.3, 71.4, 74.8, 82.0, 86.5, 90.2, 101.8, 139.1,
147.3, 147.9, 160. FTIR (film), cm : 3091 (s), 2942
+
ꢁ1
DI-FTMS), m/z: calcd for C H N O Na (MNa)
3
0
42
8
19
8
R : 0.38.
41.2458, found 841.2466. TLC (20% H O/CH CN),
2
(s), 2855 (s), 2121 (s), 1789 (s), 1728 (s), 1693 (s). HRMS
(MALDI-FTMS), m/z: calcd for C H N O Si Na
3
f
26 47
5
7
2
+
MNa ) 620.2883, found 620.2912. TLC (40% EtOAc/
(
hexanes), R : 0.67.
6
.2.8. Dimer 13. White solid, 0.17g, 61% over two steps.
f
1
H
NMR (400MHz, D O):
d
3.39 (dd, 2H,
J = 2.3,8.3Hz), 3.52 (dd, 2H, J = 3.0, 6.5Hz), 3.93 (m,
H), 4.13 (t, 2H, J = 3.0Hz), 4.41 (s, 2H), 5.62 (d, 1H,
J = 2.3Hz), 5.66 (d, 1H, J = 5.0Hz), 7.47 (d, 1H,
2
6.2.12. Fully protected aminouridine 18. The fully pro-
tected azide 17 (1.99g, 3.33mmol) was dissolved in
MeOH (10mL) and 10% Pd/C (0.08g) was added. The
4
1
3
J = 5.0Hz). C NMR (100MHz, D O): d 40.7, 70.9,
solution was flushed with H (3·) and allowed to stir
2
2
7
3.1, 73.6, 82.5, 90.5, 102.8, 142.6, 151.7, 166.7, 174.5.
under H for 4h. The reaction was then concentrated
and purified by silica gel chromatography (8–15%
CH OH/CH Cl ) to afford 18 (1.70g, 94%) as a white
2
ꢁ
1
FTIR (KBr), cm : 3441 (br), 1702 (s), 1553 (s), 1466
s). HRMS (MALDI-FTMS), m/z: calcd for
C H N O Na (MNa) 623.1566, found 623.1542.
(
3
2
2
+
solid. Extended reaction times provided unwanted by-
products resulting from Boc migration from the uracil
ring to the primary amine. H NMR (400MHz,
2
2
28
6
14
1
6
.2.9. Monomer 14. White solid, 0.23g, 61% over two
1
steps. H NMR (400MHz, D O): d 3.24 (s, 3H), 3.42
CD OD): d 0.07 (m, 12H), 0.88 (s, 9H), 0.90 (s, 9H),
2
3
(
m, 6H), 3.99 (m, 2H), 4.20 (m, 1H), 4.42 (s, 1H), 4.44
s, 1H), 5.67 (d, 1H, J = 1.5Hz), 5.74 (d, 1H,
C NMR
1.60 (s, 9H), 2.89 (dd, 1H, J = 3.3, 7.5Hz), 3.05 (dd,
1H, J = 2.0, 7.5Hz), 3.93 (m, 1H), 4.02 (m, 1H), 4.23
(t, 1H, J = 2.7Hz), 5.75 (m, 2H), 7.811 (d, 1H, J =
(
J = 5.0Hz), 7.55 (d, 1H, J = 5.0 Hz).
1
3
1
3
(
100MHz, D O): d 39.3, 40.9, 58.6, 70.9, 71.0, 73.0,
5.3Hz). C NMR (100MHz, CD OD): d ꢁ4.7, ꢁ4.5,
2
3
7
1
1
3.1, 73.6, 82.6, 90.6, 102.9, 142.7, 151.9, 166.4, 174.3,
ꢁ4.5, ꢁ4.3, 18.0, 18.1, 25.8, 26.0, 28.0, 42.1, 72.2, 73.8,
ꢁ
1
74.6. FTIR (KBr), cm : 3353 (br), 1676 (s), 1545 (s),
84.2, 86.8, 92.0, 101.8, 141.2, 147.3, 148.2, 160.0. FTIR
(KBr), cm : 2935 (s), 2861 (s), 1797 (s), 1731 (s), 1681
ꢁ
1
475 (s). HRMS (MALDI-FTMS), m/z: calcd for
+
C H N O (MNa) 433.1565, found 433.1576. TLC
1
(s), 1458 (s). HRMS (MALDI-FTMS), m/z: calcd for
C H N O Si Na (MH ) 572.3182, found 572.3174.
6
25
4
10
+
(
20% H O/CH CN), R : 0.38.
3
2
f
26 50
3
7
2
TLC (15% CH OH/CH Cl ), R : 0.49.
3
2
2
f
6
.2.10. TBS protected azidouridine 16. Compound 15
5
(1.13g, 4.2mmol) was dried by co-evaporation with
THF, dissolved in dry pyridine (5mL) under N , and
6.2.13. L-(ꢁ)-Diethyltartrate acetonide. L-(+)-Diethyltar-
trate (30.40g, 147.60mmol) was dissolved in dry benz-
ene (80mL). Dimethoxypropane (145.00mL, 1.18mol)
and p-TsOH (2.79g, 14.80mmol) were added and the
2
imidazole (1.70g, 25.2mmol) was added. After cooling
to 0ꢁC, TBSCl (3.80g, 25.2mmol) was added, and the

A. R. Yeager, N. S. Finney / Bioorg. Med. Chem. 12 (2004) 6451–6460
6457
reaction was heated to reflux (90ꢁC) with a Dean–Stark
(10.98g, 44.6mmol) in EtOH (70mL). After 2h, it was
diluted with Et O (800mL), and extracted with H O
apparatus. After 1.5h removal of CH OH/C H (21mL)
was complete and the reaction was concentrated. The
resulting oily solid was dissolved in Et O (500mL) and
3
6
6
2
2
(2 · 400mL). The aqueous layer was washed with
Et O (3 · 500mL) to provide unreacted starting mate-
2
2
washed with H O (1 · 400mL), NaHCO (1 · 400mL),
rial. The aqueous layer was acidified with 2M HCl, ex-
tracted with Et O (3 · 400mL), dried (Na SO ), and
2
3
and brine (1 · 400mL), then dried (Na SO ), and con-
2
4
2
2
4
centrated to yield the acetonide of L-(ꢁ)-diethyltartrate.
concentrated to yield monoester 20 (5.10g, 50%) as a
thick black liquid, pure by H NMR. H NMR
1
1
1
(
36.00g, 99%) as a thick brown liquid, pure by
H
1
NMR. H NMR (400MHz, CDCl ): d 1.30 (t, 3H,
(400MHz, CDCl ): d 1.33 (t, 3H, J = 7.2Hz), 1.50 (s,
3
3
J = 6.8Hz), 1.47 (s, 6H), 4.27 (m, 2H), 4.77 (m, 2H).
C NMR (100MHz, CDCl ): d 14.1, 26.3, 61.7, 113.5,
3H), 1.52 (s, 3H), 4.29 (q, 2H, J = 7.2, 14.4Hz), 4.79
(d, 1H, J = 5.6Hz), 4.87 (d, 1H, J = 5.2Hz). C NMR
1
3
13
3
ꢁ1
1
69.3. FTIR (film), cm : 2991 (s), 2951 (s), 1765 (s),
(100MHz, CDCl ): d 14.2, 26.4, 26.4, 62.2, 114.1,
3
ꢁ
1
169.3, 174.2. FTIR (film) cm : 3431 (br), 1741 (s),
1
HRMS (MALDI-FTMS), m/z: calcd for C H O Na
469 (s), 1381 (s), 1221 (br), 1108 (s), 1036 (s), 852 (s).
1645 (s), 1389 (s), 1213 (s), 1108 (s). HRMS (MALDI-
FTMS), m/z: calcd for C H O Na (MNa) 241.0688,
1
1
18
6
+
MNa) 269.0996, found 269.0992. TLC (10% MeOH/
+
(
CH Cl ), R : 0.9.
9
14
6
found 241.0690. TLC (10% MeOH/CH Cl ), R : 0.10.
2
2
2
f
2
f
6.2.14. Tartrate monoester 19. KOH (2.50g, 44.6mmol)
was dissolved in EtOH (70mL) and added dropwise
6.2.17. Fully protected uridine tartrate monoester 21. The
acid 19 and amine 18 were each dried by co-evaporation
with THF. The acid (0.78g, 3.38mmol) was dissolved in
dry CH CN (12mL) under N . HBTU (1.28g,
(30min) to a solution of diester intermediate 19
(10.98g, 44.6mmol) in EtOH (70mL). After 2h, it was
3
2
diluted with Et O (800mL), and extracted with H O
2
3.38mmol), the amine (1.61g, 2.80mmol), and NEt3
(0.48mL, 3.38mmol) were added. The reaction was stir-
red for 16h, concentrated, and then partitioned between
EtOAc (400mL) and brine (400mL). The organic layer
2
(
2 · 400mL). The aqueous layer was washed with
Et O (3 · 500mL) to provide unreacted starting mate-
rial. The aqueous layer was acidified with 2M HCl, ex-
tracted with Et O (3 · 400mL), dried (Na SO ), and
concentrated to yield crude monoester 19 (5.10g, 50%)
2
was washed with NaHCO3 (1 · 400mL) and H O
2
2
4
2
(1 · 400mL), then dried (Na SO ), and concentrated.
2
4
1
1
as a thick black liquid, pure by H NMR. H NMR
400MHz, CDCl ): d 1.32 (t, 3H, J = 6.8Hz), 1.49 (s,
The resulting solid was purified by silica gel chromato-
graphy (3% MeOH/CH Cl ) to yield 21 (1.83g, 90%)
(
3
2
2
1
3
(
H), 1.51 (s, 3H), 4.29 (q, 2H, J = 7.2, 14.4Hz), 4.79
d, 1H, J = 5.6Hz), 4.86 (d, 1H, J = 5.2Hz). C NMR
as a white foam. H NMR (400MHz, CDCl ): d 0.04
3
1
3
(m, 12H), 0.85 (s, 9H), 0.89 (s, 9H), 1.3 (t, 3H, J = 7.7,
7.7Hz), 1.46 (s, 3H), 1.47 (s, 3H), 1.57 (s, 9H), 3.53
(m, 2H), 3.87 (t, 1H, J = 3.3, 4.3Hz), 4.06 (m, 1H),
4.27 (q, 2H, J = 8, 17Hz), 4.49 (t, 1H, J = 5.7Hz), 4.70
(q, 2H, J = 6.2, 17Hz), 5.49 (d, 1H, J = 6.7Hz), 5.76
(d, 1H, J = 9Hz), 7.01 (t, 1H, J = 6.7Hz), 7.29 (d, 1H,
(
1
100MHz, CDCl ): d 14.2, 26.4, 26.4, 62.2, 76.5, 77.1,
3
ꢁ
1
14.0, 169.4, 173.7. FTIR (film), cm : 35 19 (br),
3
004 (s), 2951 (s), 1746 (s), 1641 (s), 1396 (s), 1238
br), 1125 (s). HRMS (MALDI-FTMS), m/z:
(
C H N O (MNH ) 236.1136, found 236.1134. TLC
+
9
18
1
6
4
1
3
(
10% MeOH/CH Cl ), R : 0.10.
2
J = 9Hz). C NMR (100MHz, CDCl ): d ꢁ4.6, ꢁ4.3,
2
f
3
ꢁ
4.1, 18.9, 19.0, 26.4, 26.5, 26.5, 26.5, 26.8, 27.7, 42.3,
53.2, 68.8, 74.4, 75.2, 78.5, 79.3, 86.0, 87.7, 90.6, 102.7,
114.5, 142.9, 148.6, 149.8, 171.6, 171.8. FTIR (film)
cm : 3380 (br), 2943 (s), 2847 (s), 1798 (s), 1728 (s),
1693 (s), 1536 (s), 1449 (S), 1379 (s) 1256 (s). HRMS
(MALDI-FTMS), m/z: calcd for C H N O Si Na
6
.2.15. D-(ꢁ)-Diethyltartrate acetonide. D-(ꢁ)-Diethyl-
tartrate (30.40g, 147.60mmol) was dissolved in dry benz-
ene (80mL). Dimethoxypropane (145.00mL, 1.18mol)
and p-TsOH (2.79g, 14.80mmol) were added and the
reaction was heated to reflux (90ꢁC) in a flask equipped
with a Dean–Stark apparatus. After 1.5h removal of
CH OH/C H (21mL) was complete and the reaction
ꢁ
1
3
0
53
3
10
2
+
(MNa ꢁBoc) 694.3167, found 694.3143. TLC (10%
MeOH/CH Cl ), R : 0.87.
2
3
6
6
2
f
was concentrated. The resulting oily solid was dissolved
in Et O (1 · 500mL) and washed with H O
6.2.18. Fully protected uridine tartrate monoester 22.
Acid 20 and amine 18 were each dried by co-evaporation
with THF. The acid (1.20g, 5.40mmol) was dissolved in
dry CH CN (15mL) under N . HBTU (2.20g,
5.88mmol), the amine (2.82g, 4.90mmol), and NEt3
(0.82mL, 5.88mmol) were added. The reaction was stir-
red for 15h, concentrated, and partitioned between
EtOAc (400mL) and brine (400mL). The organic layer
2
2
(
(
1 · 400mL), NaHCO
(1 · 400mL), and brine
3
1 · 400mL), dried (Na SO ), and concentrated to yield
2
4
the acetonide of D-diethyl tartrate (36.00g, 99%) as a
thick brown liquid, pure by H NMR. [ H NMR
3
2
1
1
(
6
400MHz, CDCl ): d 1.33 (t, 6H, J = 7.2Hz), 1.51 (s,
3
1
3
H), 4.29 (q, 4H, J = 7.6, 14.2Hz), 4.78 (s, 2H).
C
NMR (100MHz, CDCl ): d 14.1, 26.4, 61.8, 113.5,
3
ꢁ
1
69.3. FTIR (film), cm : 2959 (br), 2871 (s), 1757 (s),
1
was washed with NaHCO3 (1 · 400mL) and H O
2
1
(
669 (s), 1461 (s), 1381 (s), 1213 (s), 1117 (s). HRMS
MALDI-FTMS), m/z: calcd for C H O Na (MNa) ,
(1 · 400mL), then dried (Na SO ), and concentrated.
2
4
+
The resulting solid was purified by silica gel chromato-
graphy (35–40% EtOAc/hexanes) to yield 22 (3.05g,
1
1
18
6
269.0996, found 269.0998. TLC (40% EtOAc/hexanes),
R : 0.80.]
1
82%) as a white foam. H NMR (400MHz, CDCl ): d
f
3
ꢁ
0.03 (s, 3H), 0.00 (s, 3H) 0.05 (s, 6H), 0.82 (s, 6H),
6
.2.16. Tartrate monoester 20. KOH (2.50g, 44.6mmol)
was dissolved in EtOH (70mL) and added dropwise
30min) to a solution of diester intermediate of 20
0.86 (s, 6H), 1.27 (t, 3H, J = 4.5Hz), 1.43 (s, 3H), 1.44
(s, 3H), 1.54 (s, 9H), 3.88 (q, 1H, J = 1.5, 2.5Hz), 4.04
(t, 1H, J = 2Hz), 4.23 (q, 2H, J = 4.4, 8.9Hz), 4.4 (t, 1H,
(

6458
A. R. Yeager, N. S. Finney / Bioorg. Med. Chem. 12 (2004) 6451–6460
J = 3.4Hz), 4.64 (d, 1H, J = 3.8Hz), 4.70 (d, 1H,
J = 3.5Hz), 5.48 (d, 1H, J = 3.8Hz), 5.72 (d,
3.54 (m, 2H), 4.09 (t, 1H, J = 4.0, 4.3Hz), 4.13 (d, 1H,
J = 2.8Hz), 4.34 (m, 1H), 4.53 (d, 1H, J = 3.8Hz), 4.66
(d, 1H, J = 3.8Hz), 5.87 (m, 2H), 7.78 (d, 1H,
1
H, J = 5Hz), 7.08 (t, 1H, J = 3.8Hz), 7.28 (d, 1H,
1
3
13
J = 5Hz). C NMR (100MHz, CDCl ): d ꢁ4.9, ꢁ4.7,
J = 5Hz), 8.48 (m, 1 H). C NMR (100MHz, CD OD):
3
3
ꢁ
4.6, ꢁ4.4, 14.1, 17.9, 18.0, 25.7, 25.8, 26.2, 26.6, 27.4,
d ꢁ0.46, ꢁ4.2, ꢁ4.1, 18.9, 19.0, 42.3, 68.8, 74.4, 75.2,
4
1
0.9, 61.8, 73.0, 77.5, 77.7, 84.0, 86.6, 92.9, 101.9,
13.2, 141.2, 147.1, 147.9, 159.9, 169.6, 169.6. FTIR
79.1, 85.8, 87.7, 90.6, 102.8, 113.6, 142.8, 148.6, 149.7,
ꢁ
1
161.8, 172.8, 174.7. FTIR (KBr), cm : 3398 (Br),
2944 (s), 2861 (s), 1805 (s), 1739 (s), 1681 (s), 1458 (s),
1367 (s), 1268 (s). HRMS (MALDI-FTMS), m/z: Calcd
ꢁ
1
(
film) cm : 3406 (br), 2493 (s), 2873 (s), 1728 (s),
693 (s), 1632 (s), 1527 (s), 1387 (s). HRMS (MALDI-
FTMS), m/z: calcd for C H N O Si Na
MNa ꢁBoc) 694.3162, found 694.3155. TLC (15%
CH OH/CH Cl ), R : 0.91.
1
+
for C H N O Si Na (MNaꢁBoc) , 666.2854, found
3
0
53
3
10
2
28 49
3
10
2
+
(
666.2826. TLC (15% MeOH/CH Cl ), R : 0.30.
2 2 f
3
2
2
f
6.2.22. General procedure for dimerization of 26–29. Acid
21 was azeotropically dried with THF and dissolved in a
minimum amount of dry CH CN. HBTU (1.50equiv)
3
6
0
.2.19. Uridine tartrate amide 23. Compound 21 (0.20g,
.26mmol) was dissolved in EtOH (1mL). Ammonium
hydroxide (0.13mL of a 37% solution, 1.32mmol) was
added, and the reaction was allowed to stir 30h then
concentrated. Purification by silica gel chromatography
was added and the reactions were allowed to stir for
10min. The appropriate diamine (0.50equiv) and NEt3
(1.00equiv) were added and the reactions were allowed
to stir for 15–24h. The mixtures was partitioned be-
tween EtOAc (400mL) and brine (400mL), the organic
layers were washed with NaHCO3 (1 · 400mL) and
H O (1 · 400mL), dried (Na SO ), and concentrated.
(
3% MeOH/CH Cl ) provided amide 23 (0.11g, 58%) as
2 2
1
a white solid. H NMR (400MHz, CD OD): d ꢁ0.01 (s,
3
3
0
4
2
H), 0.08 (s, 3H), 0.12 (s, 3H). 0.13 (s, 3H), 0.88 (s, 9H),
.94 (s, 9H), 1.47 (s, 6H), 1.56 (s, 9H), 3.53 (m, 2H),
.086 (m, 1H), 4.13 (m, 1H), 4.34 (m, 1H), 4.58 (q,
H, J = 4.0, 3.8Hz), 5.86 (m, 2H), 7.80 (d, 1H,
2
2
4
The resulting solids were purified by silica gel chroma-
tography (2–5% MeOH/CH Cl ) to yield white solids.
2
2
1
3
J = 5.3Hz). C NMR (100MHz, CD OD): d ꢁ4.6,
3
1
6.2.23. Dimer 26. White solid, 0.19g, 63%. H NMR
ꢁ
4.3, ꢁ4.1, 18.9, 19.0, 26.4, 26.5, 26.5, 26.6, 27.8, 42.3,
7
1
4.4, 75.2, 78.8, 79.1, 86.1, 87.7, 90.5, 102.7, 113.9,
42.8, 148.6, 149.8, 161.8, 172.1, 174.5. FTIR (film),
(400MHz, CDCl ): d ꢁ0.02 (s, 6H), 0.02 (s, 6H), 0.07
3
(s, 12H), 0.84 (s, 12H), 0.88 (s, 12H), 1.46 (s, 3H), 1.48
(s, 3H), 1.56 (s, 18H), 3.33 (q, 4H, J = 3.9, 7.8Hz),
3.52 (m, 2H), 3.59 (m, 2H), 3.91 (t, 2H, J = 2.3Hz),
4.06 (t, 2H, J = 2.5Hz), 4.29 (t, 2H, J = 3.0Hz), 5.62
(d, 2H, J = 3.8Hz), 5.77 (d, 2H, J = 5.0Hz), 7.33 (t,
ꢁ
1
cm : 3353 (br), 2934 (s), 2864 (s), 1789 (s), 1693 (s),
527 (s), 1457 (s). HRMS (MALDI-FTMS), m/z: calcd
765.3543, found
1
for C H N O Si Na (MNa)
+
3
3
58
4
11
2
7
65.3538. TLC (10% MeOH/CH Cl ), R : 0.56.
2
2
f
2
H, J = 3.8Hz), 7.45 (d, 2H, J = 5Hz), 7.56 (t, 2H,
1
3
6
0
.2.20. Uridine tartrate amide 24. Compound 22 (0.62g,
.80mmol) was dissolved in EtOH (1mL). Ammonium
J = 3.5Hz). C NMR (100MHz, CDCl ): d ꢁ4.8,
3
ꢁ4.6, ꢁ4.5, ꢁ4.3, 18.0, 18.1, 25.8, 25.8, 26.1, 26.1,
27.5, 38.6, 41.0, 72.9, 73.8, 83.8, 86.6, 91.5, 102.0,
112.4, 140.8, 147.3, 148.1, 160.1, 169.8, 170.2. FTIR
hydroxide (0.77mL of a 37% solution, 8.0mmol) was
added, and the reaction was allowed to stir 15h then
concentrated. Purification by silica gel chromatography
ꢁ
1
(film), cm : 3345 (br), 3109 (w), 2934 (s), 2847 (s),
1789 (s), 1728 (s), 1684 (s), 1545 (s), 1379 (s), 1274 (s).
HRMS (MALDI-FTMS), m/z: calcd for C H N O
10-
(
3% MeOH/CH Cl ) provided amide 24 (0.08g, 14%) as
2 2
1
a white solid. H NMR (400 MHz, CD OD): d ꢁ0.01 (s,
3
30 53
3
+
+
3
0
H), 0.07 (s, 3H), 0.12 (s, 3H). 0.14 (s, 3H), 0.87 (s, 9H),
.94 (s, 9H), 1.47 (s, 3H), 1.48 (s, 3H), 1.56 (s, 9H), 3.50
Si Na (MNa ꢁ2Boc) 1333.66814, found 1333.6315.
2
TLC (15% CH OH/CH Cl ) R : 0.91.
f
3
2
2
(
4
(
7
dd, 1H, J = 6.2, 13Hz), 3.58 (dd, 1H, J = 7.2, 16Hz),
.08 (t, 1H, J = 6.2Hz), 4.14 (d, 1H, J = 3.3Hz), 4.35
m, 1H), 4.56 (q, 2H, J = 6.8, 14Hz), 5.85 (m, 2H),
1
6.2.24. Dimer 27. White solid, 0.30g, 46%. H NMR
(400MHz, CDCl ): d ꢁ0.04 (s, 6H), 0.00 (s, 6H). 0.05
3
1
3
.80 (d, 1H, J = 9.3Hz). C NMR (100MHz, CD OD):
3
(s, 12H), 0.82 (s, 12H), 0.87 (s, 2H), 1.44 (s, 6H), 1.46
(s, 6H), 1.54 (s, 18H), 3.32 (m, 4H), 3.48 (m, 2H),
3.58 (m, 2H), 3.90 (s, 2H), 4.04 (m, 2H), 4.26 (t,
2H, J = 2.8Hz), 5.62 (d, 2H, J = 3.8Hz), 5.75 (d, 2H,
J = 5.1Hz), 7.34 (t, 2H, J = 3.8Hz), 7.45 (d, 2H,
d ꢁ4.6, ꢁ4.2, ꢁ4.1, 18.8, 18.9, 26.5, 26.7, 27.8, 28.7,
4
1
(
2.2, 74.1, 75.1, 78.9, 79.1, 86.0, 87.6, 90.4, 102.7,
13.9, 142.9, 148.6, 149.7, 161.8, 172.0, 174.3. FTIR
ꢁ
1
KBr), cm : 3371 (br), 2943 (s), 2864 (s), 1789 (s),
684 (s), 1545 (s), 1466 (s). HRMS (MALDI-FTMS),
1
3
1
J = 5Hz), 7.58 (t, 2H, J = 3.5Hz).
C
NMR
+
+
m/z: C H N O Si Na (MNa ꢁBoc) 665.3009, found
(100MHz, CDCl ): d ꢁ4.8, ꢁ4.6, ꢁ4.5, ꢁ4.4, 17.9,
2
8
50
4
9
2
3
6
65.2983. TLC (10% CH OH/CH Cl ), R : 0.60.
f
18.0, 25.8, 26.1, 27.4, 29.6, 35.8, 38.6, 41.0, 72.8, 73.8,
7
3
2
2
7.1, 77.5, 83.8, 86.5, 91.3, 102.0, 112.3, 140.8, 147.2,
ꢁ
1
6
(
.2.21. Fully protected uridine tartrate 25. Compound 21
0.50g, 0.65mmol) was dissolved in EtOH (5mL) and
148.0, 160.0, 169.5, 170.1. FTIR (film) cm : 3353
(br), 2855 (s), 1807 (s), 1728 (s), 1693 (s), 1553 (s),
1457 (s), 1274 (s). HRMS (MALDI-FTMS), m/z: calcd
potassium hydroxide (0.04g, 0.69mmol) was added.
After 110min, the reaction was concentrated and puri-
fied by silica gel chromatography (8–15% MeOH/
CH Cl ) to provide acid 25 (0.41g, 85%) as a white
+
+
for C H N O Si Na (MNa ꢁ2Boc) 1347.6438,
3
0
53
3
10
2
found 1347.6403. TLC (15% CH OH/CH Cl ), R : 0.92.
f
3
2
2
2
2
1
1
6.2.25. Dimer 28. White solid, 0.22g, 29%. H NMR
foam. H NMR (400MHz, CD OD): d 0.00 (s, 3H),
3
0
0
.09 (s, 3H), 0.13 (s, 3H), 0.13 (s, 3H), 0.88 (s, 9H),
.94 (s, 9H), 1.44 (s, 3H), 1.47 (s, 3H), 1.57 (s, 9H),
(400MHz, CDCl ): d 0.00 (s, 6H), 0.04 (s, 6H), 0.09 (s,
12H), 0.86 (s, 12H), 0.90 (s, 12H), 1.49 (s, 12H), 1.58
3

A. R. Yeager, N. S. Finney / Bioorg. Med. Chem. 12 (2004) 6451–6460
6459
(
(
s, 18H), 3.28 (m, 4H), 3.37 (m, 4H), 3.53 (m, 4H), 3.63
m, 4H), 3.91 (s, 2H), 4.08 (m, 2H), 4.28 (m, 2H), 4.52 (s,
16h the mixture was partitioned between EtOAc
(400mL) and brine (400mL), the organic layer was
4
6
H), 5.66 (d, 2H, J = 3.8Hz), 5.78 (d, 2H, J = 5.3Hz),
.97 (t, 2H, J = 3.8Hz), 7.48 (d, 2H, J = 5.3Hz), 7.64
washed with NaHCO3 (1 · 400mL) and H O
2
(1400mL), dried (Na SO ), and concentrated. The
resulting solid was purified by silica gel chromatography
(5% MeOH/CH Cl ) to yield 31 (0.82g, 77%) as a white
2
4
1
3
(
t, 2H, J = 3.5Hz). C NMR (100MHz, CDCl ): d
3
ꢁ
4.8, ꢁ4.7, ꢁ4.5, ꢁ4.4, 17.9, 18.0, 25.7, 26.1, 26.9,
2
2
1
2
1
7.4, 38.7, 40.9, 72.8, 73.9, 77.0, 77.4, 83.8, 86.5, 91.0,
01.9, 112.2, 140.7, 147.2, 148.0, 160.0, 169.4, 169.7.
solid. H NMR (400MHz, CDCl ) d: ꢁ0.04 (s, 3H), 0.00
3
(s, 3H), 0.05 (s, 6H), 0.82 (s, 6H), 0.86 (s, 6H), 1.44 (s,
3H), 1.45 (s, 3H), 1.54 (s, 9H), 3.32 (s, 3H), 3.44 (m,
5H), 3.64 (m, 1H), 3.91 (m, 1H), 4.04 (m, 1H), 4.22 (t,
1H, J = 3.0Hz), 4.51 (s, 2H), 5.65 (d, 1H, J = 3.8Hz),
5.75 (d, 1H, J = 5.0Hz), 7.10 (m, 1H), 7.46 (d, 1H,
ꢁ
1
FTIR (KBr), cm : 3362 (br), 2834 (s), 1807 (s), 1728
s), 1702 (s), 1536 (s), 1492 (s). HRMS (MALDI-
FTMS), m/z: calcd for C H N O Si Na
MNa ꢁ2Boc) 1361.6594, found 1361.6630. TLC
10% CH OH/CH Cl ), R : 0.69.
(
3
0
53
3
10
2
+
+
(
(
1
3
J = 5.0Hz), 7.63 (m, 1H). C NMR (100MHz, CDCl3):
d ꢁ4.9, ꢁ4.7, ꢁ4.5, ꢁ4.4, 17.9, 18.0, 25.7, 25.8, 25.9,
26.0, 26.1, 27.4, 39.0, 40.9, 58.7, 70.6, 72.7, 73.9, 77.0,
77.5, 83.9, 86.4, 90.8, 102.0, 112.3, 140.5, 147.2, 148.1,
3
2
2
f
1
.2.26. Dimer 29. White solid, 0.53g, 62%. H NMR
6
(
400MHz, CDCl ): d 0.00 (s, 6H), 0.04 (s, 6H), 0.09 (s,
3
ꢁ1
1
2H), 0.86 (s, 12H), 0.90 (s, 12H), 1.48 (s, 12H), 1.58
160.0, 169.2, 169.9. FTIR (film), cm : 3353 (br), 2951
(s), 2855 (s), 1798 (s), 1728 (s), 1693 (s), 1545 (s), 1466
(s), 1387 (s), 1265 (s). HRMS (MALDI-FTMS), m/z:
(
(
s, 18H), 3.56 (m, 12H), 3.91 (t, 2H, J = 5.2Hz), 4.08
m, 2H), 4.30 (t, 2H, J = 6.3Hz), 4.55 (s, 4H), 5.64 (d,
+
+
2H, J = 6.3Hz), 5.79 (d, 2H, J = 9.3Hz), 7.20 (t, 2H,
J = 6.0Hz), 7.47 (d, 2H, J = 9.3Hz), 7.59 (m, 2H). C
calcd for C H N O Si Na (MNa ꢁBoc) 723.3427,
3
0
53
3
10
2
1
3
found 723.3435. TLC (10% CH OH/CH Cl ), R : 0.81.
2
3
2
f
NMR (100MHz, CDCl ): d ꢁ4.9, ꢁ4.7, ꢁ4.5, ꢁ4.4,
3
1
7
1
3
7.9, 18.0, 25.8, 25.9, 26.1, 27.4, 39.0, 40.9, 69.2, 72.8,
3.8, 77.1, 77.5, 83.8, 86.5, 91.2, 101.9, 112.4, 140.7,
47.2, 148.0, 160.0, 169.5, 169.8. FTIR (KBr), cm :
362 (br), 2934 (s), 1798 (s), 1719 (s), 1693 (s), 1536
Acknowledgements
ꢁ
1
The authors 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, 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 for
helpful discussion, and Prof. Jack Kyte and Mr. Steven
Adams (UCSD) for technical assistance.
(
s), 1449 (s), 1397 (s), 1265 (s). HRMS (MALDI-
+
FTMS), m/z: calcd for C H N O Si Na (MNa ꢁ
3
0
53
3
10
2
+
2
Boc)
CH OH/CH Cl ), R : 0.86.
1377.6544, found 1377.6492. TLC (15%
3
2
2
f
6
.2.27. Dimer 30. Acid 21 (0.37g, 0.50mmol) and amine
0.34g, 0.60mmol) 18 were azeotropically dried with
THF and then combined and dissolved in dry CH CN
(
3
(
3mL). HATU (0.28g, 0.75mmol) and NEt (0.10mL,
3
0
.75mmol) were added and the reaction was allowed
to stir. After 19h the mixture was partitioned between
EtOAc (400mL) and brine (400mL), the organic layer
was washed with NaHCO (1 · 400mL) and H O
References and notes
3
2
1
. For representative overviews of fungal chitin synthase (EC
2.4.1.16): (a) Munro, C. A.; Gow, N. A. R. Med. Mycol.
(
1 · 400mL), then dried (Na SO ), and concentrated.
2
4
The resulting solid was purified by silica gel chromato-
graphy (5% MeOH/CH Cl ) to yield 30 (0.82g, 77%)
2
001, 39, 41–53; (b) Valdivieso, M. H.; Duran, A.;
2
2
Roncero, C. EXS 1999, 87, 55–69; (c) Merz, R. A.;
Horsch, M.; Nyhlen, L. E.; Rast, D. M. EXS 1999, 87, 9–
37; (d) Cabib, E. Adv. Enzymol. 1987, 59, 59–101.
. Glycosyltransferase structural determination is a growing
field, as demonstrated by the following reports: (a)
Strynadka, N. C.; Withers Stephen, G.; Wakarchuk, W.
W. Nat. Struct. Biol. 2004, 11, 163–170; (b) Davies, G. J.
Nat. Struct. Biol. 2001, 8, 98–100; (c) Charnock, S. J.;
Henrissat, B.; Davies, G. J. Plant Phys. 2001, 125, 527–
531. However, to date no structures of polymerizing
transferases have been determined.
3. Zhang, D.; Miller, M. J. Curr. Pharm. Des. 1999, 5, 73–
99.
4. (a) Grugier, J.; Xie, J.; Duarte, I.; Valery, J.-M. J. Org.
Chem. 2000, 65, 979–984; (b) Schafer, A.; Thiem, J. J. Org.
Chem. 2000, 65, 24–29; (c) Xie, J.; Thellend, A.; Becker,
H.; Vidal-Cros, A. Carbohydr. Res. 2001, 334, 177–182; (d)
Obi, K.; Uda, J.-I.; Iwase, K.; Sugimoto, O.; Ebisu, H.;
Matsuda, A. Bioorg. Med. Chem. Lett. 2000, 10, 1451–
1
as a white solid. H NMR (400MHz, CDCl ): d ꢁ0.03
3
(
(
(
s, 6H), 0.02 (s, 6H). 0.06 (s, 12H), 0.83 (s, 12H), 0.87
s, 12H), 1.44 (s, 6H), 1.54 (s, 18H), 3.51 (m, 4H), 3.91
t, 2H, J = 2.3Hz), 4.04 (t, 2H, J = 2.5Hz), 4.37 (t, 2H,
2
J = 3.3Hz), 4.50 (s, 2H), 5.54 (d, 2H, J = 3.3Hz), 5.74
1
d, 2H, J = 5.3Hz), 7.34 (d, 2H, J = 3.5Hz). C NMR
3
(
(
100MHz, CDCl ): d ꢁ4.8, ꢁ4.7, ꢁ4.5, ꢁ4.4, 17.9,
3
1
7
1
2
1
8.0, 25.7, 25.8, 26.0, 27.4, 27.5, 38.6, 41.0, 73.0, 73.4,
7.3, 83.5, 86.5, 92.6, 102.0, 112.4, 141.0, 147.2, 148.0,
ꢁ
1
59.9, 169.6. FTIR (film) cm : 3380 (br), 3091 (w),
951 (s), 2864 (s), 1798 (s), 1728 (s), 1684 (s), 1536 (s),
387 (s), 1274 (s). HRMS (MALDI-FTMS), m/z: calcd
+
+
for C H N O Si Na (MNa ꢁ2Boc) 1119.5328,
3
0
53
3
10
2
found 1119.5328. TLC (15% CH OH/CH Cl ), R : 0.94.
2
3
2
f
6.2.28. Monomer 31. Acid 21 (0.99g, 1.34mmol) was
azeotropically dried with THF and dissolved in dry
CH CN (3mL). HATU (0.76g, 2.01mmol) was added,
3
and the reaction was stirred for 10min. The amine
(0.13mL, 1.47mmol) and NEt3 (0.28mL, 2.01mmol)
were added and the reaction was allowed to stir. After
1
454; (e) Behr, J.-B.; Gautier-Lefebvre, I.; Mvondo-Evina,
C.; Guillerm, G.; Ryder, N. S. J. Enzym. Inhib. 2001, 16,
07–112; (f) Wang, R.; Steensma, D. H.; Takaoka, Y.;
Yun, J. W.; Kajimoto, T.; Wong, C.-H. Bioorg. Med.
Chem. 1997, 5, 661–672.
1

6460
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5
. (a) Yeager, A. R.; Finney, N. S. In Abstract of Papers,
21st ORGN-102, American Chemical Society, 2001, San
Behr, J.-B.; Gourlain, T.; Helimi, A.; Guillerm, G. Bioorg.
Med. Chem. Lett. 2003, 13, 1713–1716.
2
Diego, CA, 2002; (b) Chang, R.; Yeager, A. R.; Finney, N.
S. Org. Biomol. Chem. 2003, 1, 39–41; (c) Yeager, A. R.;
Finney, N. S. J. Org. Chem. 2004, 69, 613–618.
10. For a convenient preparation of 15, see Ref. 5b.
11. Imide carbonate protection was necessary to prevent
unwanted acylation of the uracil ring during amide bond
formation.
12. Musich, J. A.; Rapoport, H. J. Am. Chem. Soc. 1978, 100,
4865–4872.
13. Lundt, B. F.; Johansen, N. L.; Volund, A.; Markussen, J.
Int. J. Pept. Protein Res. 1978, 12, 258–268.
14. The less expensive R,R-tartrate was chosen for dimeriza-
tion, as the R,R- and S,S-amides provided inhibition
within experimental error of one another.
6
. For previous discussion of possible mechanisms for CS
and/or other polymerizing transferases, see: (a) Saxena, I.
M., Jr.; Brown, R. M., Jr.; Fevre, M.; Geremia, R. A.;
Henrissat, B. J. Bacteriol. 1995, 177, 1419–1424; (b)
Saxena, I. M.; Brown, R. M., Jr.; Dandekar, T. Phyto-
chemistry 2001, 57, 1135–1148; (c) Saxena, I. M.; Brown,
R. M., Jr. Curr. Opin. Plant Biol. 2000, 3, 523–531; (d)
Kamst, E.; Spaink, P. H. Trends Glycosci. Glycotechnol.
1
999, 11, 187–199, See also Refs. 2b and 2c.
15. (a) Dourtoglou, V.; Ziegler, J. C.; Gross, B. Tetrahedron
Lett. 1978, 19, 1269–1272; (b) Knorr, R.; Trzeciak, A.;
Bannwarth, W.; Gillessen, D. Tetrahedron Lett. 1989, 30,
7
. The peptidyl nucleoside compounds have multiple key
functional groups that increase binding ability, overcom-
ing the generally weak interaction of the native substrate.
See Ref. 3 and the following representative examples: (a)
Isono, K.; Azuma, T.; Suzuki, S. Chem. Pharm. Bull. 1971,
1927–1930.
16. Despite the fact that dimers were typically pure by
1
H
NMR after chromatography on silica, further purification
on reverse-phase silica was essential for obtaining repro-
ducible assay results.
19, 505–512; (b) Krainer, E.; Becker, J. M.; Naider, F. J.
Med. Chem. 1991, 34, 174–180; (c) Naider, F.; Shenbaga-
murthi, P.; Steinfeld, A. S.; Smith, H. A.; Boney, C.;
Becker, J. M. Antimicrob. Agents Chemother. 1983, 24,
787–796; (d) Khare, R. K.; Becker, J. M.; Naider, F. J.
Med. Chem. 1988, 31, 650–656.
. For recent examples of synthetic studies on the polyoxins
and related compounds, see: (a) Dondoni, A.; Franco, S.;
Junquera, F.; Merchan, F. L.; Merino, P.; Tejero, T. J.
Org. Chem. 1998, 62, 5497–5507; (b) More, J. D.; Finney,
N. S. Synlett 2003, 9, 1307–1310.
0
0
17. Maximum 5 -5 distances were calculated using Chem3D
3.0 (Cambridgesoft, Cambridge, MA). The maximum
distance was determined after manually adjusting dihe-
dral angles to attain the most extended conformation
possible.
8
9
18. (a) Finkelstein, A. V.; Janin, J. Protein Eng. 1989, 3, 1–3;
(b) Searle, M. S.; Williams, D. H. J. Am. Chem. Soc. 1992,
114, 10690–10697; (c) Searle, M. S.; Williams, D. H.;
Gerhard, U. J. Am. Chem. Soc. 1992, 114, 10697–
10704.
. Previous studies of the use of the tartrate moiety as a
diphosphate have provided mixed results. See Ref. 4 and
19. Orlean, P. J. Biol. Chem. 1987, 262, 5732–5739.