A new synthesis of peptidyl epoxysuccinates for probing cysteine protease-inhibitor P3/S3 binding interactions

Article abstract of DOI:10.1055/s-1999-3648

A new synthesis of peptidyl epoxysuccinates has been developed involving the N-acylation of amino acid benzyl esters (12) with the tartrate ester derived epoxy acid 13 followed by deprotection of the benzyl esters and acylation with amines 21-28. This syn

Full text of DOI:10.1055/s-1999-3648

1500
PAPER
A New Synthesis of Peptidyl Epoxysuccinates for Probing Cysteine Protease-
Inhibitor P3/S3 Binding Interactions
William R. Roush,* Alejandro Alvarez Hernandez, Gerardo Zepeda
Department of Chemistry, University of Michigan, Ann Arbor, MI 48109–1055, USA
Fax +1(734)6479279; E-mail: roush@umich.edu
Received 16 March 1999; revised 30 April 1999
The regiochemistry of the inhibition reaction of papain
Abstract: A new synthesis of peptidyl epoxysuccinates has been
with E-64 was determined by Rich,31 who established that
developed involving the N-acylation of amino acid benzyl esters
substitution of the epoxide by the active site cysteine res-
idue occurs at C(2), adjacent to the negatively charged
(12) with the tartrate ester derived epoxy acid 13 followed by depro-
tection of the benzyl esters and acylation with amines 21–28. This
synthesis is ideally suited for the rapid synthesis of peptidyl ep- carboxylate. This conclusion has been verified by three
oxysuccinate inhibitors of cysteine proteases, in order to probe se-
lectivity issues concerning inhibitor/enzyme P3/S3 binding
interactions.
different crystal structures of the papain∑E64 or pa-
pain∑E64c complexes.32-34 Examination of these X-ray
structures reveals that the peptide backbone of E-64 or E-
Key words: peptidyl epoxysuccinates, E-64 analogs, cysteine pro-
64c (5) orients in a direction opposite to that of chloro- or
fluoromethyl ketone inhibitors, 7,35,36 which are presumed
tease inhibitors, peptide synthesis
to bind in a manner analogous to the natural peptide sub-
strates. Consequently, the L-leucyl side chain of 1 does not
optimally occupy the enzyme S2 binding pocket.
Cysteine proteases are an important class of enzymes in-
volved in the degradative processing of peptides and pro-
teins. 1,2 They are ubiquitous in nature and play vital roles
in numerous pathological processes including arthritis,
osteoporosis, Alzheimer’s disease, cancer cell invasion,
and apoptosis. 1-3 Cysteine proteases are also essential to
the life cycles of many pathogenic protozoa, 4,5 including
Trypanosoma cruzi, the etiologic agent of Chagas’ dis-
ease, and Plasmodium falciparum, the most dangerous of
the malarial parasites. 6 Cruzain7 and falcipain, 8 the major
cysteine proteases of T. cruzi and P. falciparum, have
been identified as potential therapeutic targets for treat-
ment of Chagas’disease and malaria, respectively.6,7,9-12
O
H
O
N
CO2H
N
HS-Cys25
alkylation
site
H
O
H
S
3 pocket
1, E-64c
S2 pocket
Armed with these insights, we embarked on a program de-
signed to optimize the interactions of the epoxysuccinyl
inhibitors with the S2 and S3 binding pockets of the target-
ed cysteine proteases. Initially, we utilized Yokoo’s proce-
In connection with ongoing efforts to develop potent and
selective inhibitors13 of cruzain and falcipain, 14-16 we be-
came interested in a class of naturally occurring peptidyl
epoxysuccinate derivatives, of which E-64 (1),17-19 cathe-
statin C (2),20,21 and circinamide (3)22 are representative
examples. These compounds are potent, highly selective
irreversible inhibitor of cysteine proteases. Considerable
effort has been devoted to the development of E-64 ana-
logs as therapeutic agents. 23-27 Loxistatin (4),28 a prodrug
analog of E-64, was developed for treatment of muscular
dystrophy and was taken into human clinical trials in Ja-
pan. 29 Potent and selective inhibitors of cathepsin B also
37
dure for synthesis of E-64c analogs.
As illustrated
below by the synthesis of 11 containing a L-homopheny-
lalanine residue at P2, this route involves the coupling of
the BOC protected amino acid (6) with an amine (in this
case, isopentyl amine, 7) envisaged to serve as the P3 sub-
stituent. Deprotection of the resulting peptide 8 followed
by N-acylation with the p-nitrophenyl ester 938 and ester
hydrolysis then completed the synthesis of 11.
H
N
O
H
N
7
1) TFA, CH2Cl2
NH2
HO2C
BOC
N
H
BOC
have been developed based on the epoxysuccinyl motif.
EDC, HOBT
DMF, 23 °C
95%
2) 9, EtOAc
3) KOH, EtOH
90%
26,30
Ph
6
8
Ph
O
O
O
O
O
O
O
NH2
O
O
H
H
N
H
H
O
2
O
N
O
H2N
N
N
N
OR
OEt
N
O
N
H
OH
H
H
O
O
NH2
O
O
O2N
10, R = Et
11, R = H
9
Ph
1, E-64
2, cathestatin C
HO
O
O
O
O
O
H
O
H
N
H2N
H2N
N
N
OH
N
OR
H
O
O
While this sequence proved extremely useful for the syn-
thesis of a series of peptidyl epoxysuccinates by varying
4, loxistatin, R = Et
5, E64c, R = H
3, circinamide
Synthesis 1999, No. SI, 1500–1504 ISSN 0039-7881 © Thieme Stuttgart · New York

PAPER
A New Synthesis of Peptidyl Epoxysuccinates
1501
Table 1 Synthesis of Amides 14 and Carboxylic Acids 15
the amino acid (P2 residue) at the beginning of the synthe-
sis, this route proved less convenient for synthesis of ana-
logs designed to probe the structural requirements of the
P3/S3 interactions, since the P3 substituent (e.g., 7 in the
above scheme) is introduced in the very first step. This
prompted us to develop a new synthesis of peptidyl succi-
nate derivatives that would permit the P3 substituent to be
introduced at the end of the synthesis. Thus, acylation of
Amidesa Solvent Yieldb
% d.e.
Carboxylic Yieldb
Acidsc
14a
14a
14b
14c
14c
14d
14d
14e
14e
CHCl3 80–83%
DMF 77–80%
CHCl3 74–78%
CHCl3 76–83%
96%
–
98%
>99%
>98%
94%
92%
96%
86–94%
15a
94–99%
15b
15c
94–99%
95%
DMF
CHCl3 72%
DMF 56%
CHCl3 77–80%
DMF 54–68%
68%
L-amino acid benzyl esters 1239,40 (typically as the p-tolu-
15d
15e
96–98%
40
enesulfonate salts)
with mono acid 1341 provided
96%
amides 14 in good to excellent yield (Table 1). The opti-
mized coupling conditions called for use of EDC, HOBT
and N-methylmorpholine (NMM) in CHCl3 at 0–23 °C.
The reaction was very sluggish when performed using
a All acylation reactions of 12a–12e and 13 were performed using
EDC and HOBT in the indicated solvent. N-Methylmorpholine
Et3N as the base, as the Et3N salt of 13 is insoluble in (NMM) was used as the base for the reactions performed in CHCl3,
whereas Et3N was used for the couplings performed in DMF.
CHCl3. The amount of racemization observed was also
somewhat enhanced when Et3N was used. Chloroform
b Yields of product isolated by chromatography.
c Deprotection of the benzyl esters of amides 14a–14e were performed
was used as solvent, rather than DMF which is more com-
by hydrogenation over 10% Pd/C using 40 psi H2 in a 1 : 1 mixture of
monly employed for peptide couplings, 42,43 because prod-
uct isolation was easier owing to the presence of fewer
EtOAc and MeOH.
side products, and consequently the isolated yields were
generally better for the reactions performed in CHCl3. The
epoxide unit of 14 is susceptible to ring opening by
HOBT, and this side reaction was much more serious performed by activation of the carboxyl end of a growing
when the reactions were performed in DMF than in peptide chain). 42
CHCl3. However, it proved necessary to use HOBT in
these couplings in order to minimize racemization of the
TBSO
NH2
N3
NH2
NH2
a-amino acid ester. Nevertheless, as shown in the Table,
2–3% racemization was observed during these couplings,
as evidenced by the presence of a minor set of epoxide C–
H resonances in the 1H NMR spectrum of the purified re-
action product. 44
n
n
X
21, n = 1
22, n = 2
23, n = 3
24, n = 4
25, n = 2
26, n = 3
27, X = CH
28, X = N
Deprotection of any functional groups introduced in the P3
amine unit (see 20d below, for example) and then hydrol-
ysis of the ethyl ester provides the targeted epoxysucci-
nate derivatives. All of the ethyl esters 16–20 so prepared
(Table 2) have been converted into the corresponding car-
boxylic acids and tested as inhibitors of cysteine proteas-
es. Of the E-64 analogs described herein, 30 is the most
potent inhibitor of cruzain, with 11 being the second most
potent. 45 Details of the enzyme inhibition studies of these
and related inhibitors will be reported in due course. 46
O
(13)
OEt
O
O
H
N
O
H
2 (40 psi)
HO2C
BnO2C
NH2
BnO2C
OEt
EDC, HOBT, NMM
CHCl3, 0 ° to 23 °C
O
R
R
10% Pd/C
EtOAc, MeOH
14
12
O
O
O
O
H
H
O
R'-NH2
HO2C
N
R'
N
OEt
OEt
N
H
O
O
R
R
EDC, HOBT
15
23 °C
12, 14 and 15:
16, R = CH2CH(CH3)2 (Leu)
17, R = CH(CH3)C2H5 (ILeu)
18, R = CH2C6H5 (Phe)
19, R = CH2C6H4OH (Tyr)
20, R = CH2CH2C6H5 (HPhe)
(a) R = CH2CH(CH3)2 (Leu)
(b) R = CH(CH3)C2H5 (ILeu)
(c) R = CH2C6H5 (Phe)
(d) R = CH2C6H4OH (Tyr)
(e) R = CH2CH2C6H5 (HPhe)
O
O
H
O
1) aq. HF, CH3CN
N
OEt
TBSO
N
H
O
2) KOH, EtOH
90%
O
O
20d
H
Hydrogenolysis of amides 14 provided carboxylic acids
15 in excellent yield (Table 1), which were used immedi-
ately in coupling reactions with amines 7 and 21–28. Rep-
resentative results are summarized in Table 2. Many of the
acylations gave much better yields in CHCl3 (using EDC
and NMM) than in DMF (using EDC and Et3N). This ef-
fect was especially pronounced in the acylations of the
leucine derivative 15a, for which comparative data are
shown in the Table. Importantly, no evidence of addition-
al racemization of the a-N-acylamino acid was observed
(which typically compromises peptide synthesis when
O
Ph
N
OR
HO
N
H
O
29, R = Et
30, R = H
Ph
In summary, we have developed a new synthesis of pepti-
dyl epoxysuccinate derivatives. Although the overall effi-
ciency of this synthesis of peptidyl epoxysuccinate
derivatives is somewhat lower than Yokoo’s method for
synthesis of E-64c analogs,37 it is a highly attractive alter-
Synthesis 1999, No. SI, 1500–1504 ISSN 0039-7881 © Thieme Stuttgart · New York

1502
W. R. Roush et al.
PAPER
13C NMR (125 MHz, CDCl3): d = 14.02, 31.51, 33.51, 51.78, 52.84,
53.77, 62.29, 64.49, 126.38, 128.27, 128.45, 128.65, 128.69,
134.97, 140.28, 165.74, 166.39, 171.13.
Table 2 Synthesis of Peptidyl Amides by the Coupling of Carboxy-
lic Acids 15 and Primary Aminesa
Carboxylic R’NH2 Conditionsa
Prod- Yieldb
uct
IR (CHCl3) 3684, 3400, 3025, 3016, 1745, 1689, 1523, 1426, 1227,
1205, 1028 cm-1.
Acids
15a
15a
15a
15a
15a
15a
15a
15a
15a
7
21
21
22
22
23
23
24
24
CHCl3, NMM, 0° to 23°C
CHCl3, NMM, 0° to 23°C
DMF, Et3N, 23°C
CHCl3, NMM, 0° to 23°C
DMF, Et3N, 23°C
CHCl3, NMM, 0° to 23°C
DMF, Et3N, 23°C
CHCl3, NMM, 0° to 23°C
DMF, Et3N, 23°C
4
82%
76%
33%
65%
28%
72%
32%
65%
24%
HRMS (CI, CH4) calcd. for C23H25NO6 411.1675, found 411.1672.
16b
16b
16c
16c
16d
16d
16e
16e
Anal. C23H25NO6 (411.1) calcd C 67.14, H 6.12, N 3.42; found C
67.17, H 6.49, N 3.40.
[2S-[2a,3b(S)]}-3({[3-Phenyl-1-(carboxyl)propyl]amino}carbo-
nyl)oxirane Carboxylic Acid Ethyl Ester (15e)
A mixture of benzyl ester 14e (0.30 g, 0.7 mmol) and 10% Pd/C
(150 mg) in 40 mL of EtOAc and MeOH (40 mL) was shaken in a
Parr–Knorr apparatus under hydrogen (40 psi) for 10 min. The cat-
alyst was removed by filtration through a pad of Celite and the so-
lution concentrated in vacuo to afford a solid (0.22 g, 96%), mp
110–112 °C. [a]25D = +74.7 (c = 3.0, CH3OH).
15b
15b
15b
7
25
26
CHCl3, NMM, 0° to 23°C
CHCl3, NMM, 0° to 23°C
CHCl3, NMM, 0° to 23°C
17a
17f
17g
70%
47%
43%
1H NMR (500 MHz, CDCl3): d = 1.33 (t, J = 7 Hz, 3 H), 2.11 (m, 1
H), 2.29 (m, 1 H), 2.68 (t, J = 7 Hz, 2 H), 3.23 (s, 1 H), 3.69 (s, 1
H), 4.29 (m, 2H), 4.66 (m, 1 H), 6.53 (d, J = 8 Hz, 1H), 7.24 (m,
5H), 10.10 (br, 1 H).
13C NMR (CDCl3, 100 MHz): d = 14.02, 31.70, 33.14, 51.62, 53.69,
62.40, 126.52, 128.30, 128.70, 140.08, 166.29, 166.35, 175.52.
15d
15d
22
23
DMF, Et3N, 0° to 23°C
DMF, Et3N, 0° to 23°C
19c
19d
63%
81%
15e
15e
15e
15e
15e
15e
15e
7
CHCl3, NMM, 0° to 23°C
CHCl3, NMM, 0° to 23°C
CHCl3, NMM, 0° to 23°C
CHCl3, NMM, 0° to 23°C
CHCl3, NMM, 0° to 23°C
DMF, Et3N, 23°C
10
76%
70%
64%
66%
52%
80%
55%
21
22
23
24
27
28
20b
20c
20d
20e
20h
20i
IR (KBr) 3403, 3057, 30,30, 2982, 2932, 1751, 1742, 1648, 1551,
1498, 1442, 1395, 1370, 1346, 1239, 1214, 1169, 1028, 898 cm-1.
HRMS (CI, CH4), calcd for C16H19NO6 321.1212, found 321.1203.
DMF, Et3N, 23°C
a All acylation reactions summarized in Table 2 were performed using
EDC and HOBT in the indicated solvent. N-Methylmorpholine
(NMM) was used as the base for the reactions performed in CHCl3,
whereas Et3N was used for the couplings performed in DMF.
b Yields of product isolated by chromatography.
{2S-[2a,3b(S)]}-3-{[(3-Phenyl-1-{[(5-t-butyldimethylsilanoxyl-
pentyl)amino]carbonyl} propyl)amino]carbonyl}oxirane Car-
boxylic Acid Ethyl Ester (20d)
To a solution of amine 2347 (0.44 g, 2.0 mmol) acid 15e (0.64 g, 2
mmol), HOBT (0.26 g, 2 mmol) and N-methylmorpholine (0.22
mL, 2 mmol) in CHCl3 (10 mL) under N2 at 0 °C was added EDC
(0.42 g, 2.2 mmol). The resulting mixture was stirred for 1 h at this
temperature then at 23 °C for 9h. The mixture was then partitioned
between EtOAc (30 mL) and H2O (30 mL). The organic portion was
washed with brine, dried (Na2SO4) and concentrated in vacuo. The
residue was purified by flash chromatography on silica gel (elution
with hexane–EtOAc 7–3) to afford 20d (0.42 g, 66%) which was
used immediately in the next step.
native particularly for the rapid synthesis of analogs de-
signed to probe selectivity issues concerning inhibitor/
enzyme P3/S3 binding interactions.
1H NMR (400 MHz, CDCl3): d = 0.04 (s, 6 H), 0.88 (s, 9 H), 1.31
(t, J = 7.6 Hz, 3 H), 1.36 (m, 2 H), 1.51 (m, 4 H), 1.97 (m, 1 H), 2.16
(m, 2 H), 2.62 (m, 2 H), 3.25 (m, 2 H), 3.27 (d, J = 1.6 Hz, 1 H),
3.60 (t, J = 6 Hz, 2 H), 3.63 (d, J = 1.6 Hz, 1 H), 3.63 (d, Jab = 1.6
Hz, 1 H), 4.26 (m, 2 H), 4.34 (m, 1 H), 5.78 (t, J = 5.6 Hz, 1 H), 6.20
(d, J = 8 Hz, 1 H), 7.23 (m, 5H).
Representative Experimental Procedures
{2S-[2a,3b(S)]}-Oxirane Carboxylic Acid, 3[({3-Phenyl-1-[(ben-
zyloxy)carbonyl]propyl}amino)carbonyl] Ethyl Ester (14e)
To a solution of the p-toluenesulfonic acid salt of L-homophenyla-
lanine benzyl ester40 (0.44 g, 1.0 mmol), monoethyl epoxysuccinate
1341 (0.16 g, 1 mmol), HOBT (0.13 g, 1 mmol) and N-methylmor-
pholine (0.11 mL, 1 mmol) in CHCl3 (5 mL) under nitrogen at 0 °C
was added EDC (0.21 g, 1.1 mmol). The resulting mixture was
stirred for 1 h at this temperature then at 23 °C for 9 h. The mixture
was partitioned between EtOAc (30 mL) and H2O (30 mL). The or-
ganic portion was washed with brine, dried (Na2SO4) and concen-
trated in vacuo. The residue was purified by flash chromatography
on silica gel (elution with hexane–EtOAc 7–3 then 5:5) to afford a
white solid (0.32 g, 77%). mp 102 °C.
{2S-[2a,3b(S)]}-3-{[(3-Phenyl-1-{[(5-hydroxylpentyl)ami-
no]carbonyl}propyl)amino] carbonyl}oxirane Carboxylic Acid
Ethyl Ester (29)
A 48% solution of HF (0.31 mL, 0.84 mmol) was added to a suspen-
sion of 20d (0.29g, 0.56 mmol) in MeCN (5 mL) and CH2Cl2 (5 mL)
at 0 °C and the mixture was then allowed to warm to 23 °C and
stirred for 15 min. The mixture was diluted with CH2Cl2 (30 mL)
and washed with concentrated NaHCO3 (2 x 20 mL), brine (20 mL),
dried (Na2SO4) and concentrated in vacuo. The residue was redis-
solved in CH2Cl2 and MeOH and passed through a short pad of sil-
ica gel to give, after concentration in vacuo, a solid (0.21 g, 95%)
mp 157 °C; [a]25D = +31.6 (c = 1.2, CHCl3).
[a]25 D = +50.8 (c = 2.5, CHCl3).
1H NMR (500 MHz, CDCl3): d = 1.33 (t, J = 7.0 Hz, 3 H), 2.06 (m,
1 H), 2.22 (m, 1 H), 2.56 (m, 2 H), 3.21 (d, J = 2.0 Hz, 1 H), 3.65
(d, J = 2.0 Hz, 1 H), 4.27 (m, 2 H), 4.69 (m, 1 H), 5.17 (d, J = 12.0
Hz, 1 H), 5.22 (d, J = 12.0 Hz, 1 H), 6.53 (d, J = 80 Hz, 1 H), 7.08–
7.41 (m, 10 H).
1H NMR (CDCl3, 400 MHz): d = 1.32 (t, J = 7.2 Hz, 3 H), 1.40 (m,
2 H), 1.54 (m, 4 H), 1.84 (br s, 1H), 1.97 (m, 1 H), 2.15 (m, 1 H),
2.62 (m, 2 H), 3.26 (m, 2 H), 3,32 (d, J = 2 Hz, 1 H), 3.63 (m, 2 H),
Synthesis 1999, No. SI, 1500–1504 ISSN 0039-7881 © Thieme Stuttgart · New York

PAPER
A New Synthesis of Peptidyl Epoxysuccinates
1503
3.65 (d, J = 2 Hz, 1 H), 4.26 (m, 2 H), 4.38 (m, 1 H), 6.28 (br s, 1
H), 6.85 (br s, 1 H), 7.23 (m, 5 H).
(10) Engel, J. C.; Doyle, P. S.; Palmer, J.; Hsieh, I.; Bainton, D. F.;
McKerrow, J. H. J. Cell Sci. 1998, 111, 597.
(11) Engel, J. C.; Doyle, P. S.; Hsieh, I.; McKerrow, J. H. J. Exp.
Med. 1998, 188, 725.
(12) Rosenthal, P. J.; Wollish, W. S.; Palmer, J. T.; Rasnick, D. J.
Clin. Invest. 1991, 88, 1467.
(13) Reviews of cysteine protease inhibitors: Otto, H.-H.;
Schirmeister, T. Chem. Rev. 1997, 97, 133.
13C NMR (CDCl3, 100 MHz): d = 14.02, 22.95, 28.99, 31.75, 31.98,
33.75, 39.50, 52.62, 52.80, 53.72, 62.34, 62.43, 126.37, 128.28,
128.63, 140.46, 166.10, 166.49, 170.33.
IR (CHCl3) 3440, 3390, 1755, 1680, 1525, 1460, 1375, 1310, 1080,
1030, 935, 905 cm-1.
Rasnick, D. In Cysteine Proteases: Evolution, Function and
Inhibitor Design; J. H. McKerrow and M. N. G. James, Eds.;
ESCOM Science Publishers: Leiden, 1996; Vol. 6; pp 47.
Rich, D. H. In Comprehensive Medicinal Chemistry; P. G.
Sammes, Ed.; Pergamon: Oxford, 1990; Vol. 2; pp 391.
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10994.
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Bioorg. Med. Chem. Lett. 1998, 8, 2809.
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Hansell, E.; Rosenthal, P. J. Bioorg. Med. Chem. 1998, 6,
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(17) Hanada, K.; Tamai, M.; Yamagishi, M.; Ohmura, S.; Sawada,
J.; Tanaka, I. Agric. Biol. Chem. 1978, 42, 523.
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Tanaka, I. Agric. Biol. Chem. 1978, 42, 529.
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HRMS (CI, NH3), calcd for C21H31N2O6 (M+1)+407.2182, found
407.2162.
{2S-[2a,3b(S)]}-3-{[(3-Phenyl-1-{[(5-hydroxylpentyl)ami-
no]carbonyl}propyl)amino]carbonyl}oxirane Carboxylic Acid
(30)
A 1N solution of KOH in EtOH (0.5 mL, 0.50 mmol) was added to
a solution of 29 (0.20g, 0.50 mmol) in THF (5 mL) and EtOH (5
mL) at 0 °C and stirred for 30 min. The solvent was then evaporated
in vacuo and the residue dissolved in cold water (5 mL), acidified to
pH 2 with 10% KHSO4 and extracted with EtOAc (5 × 10 mL). The
combined organic portions were washed with brine (20 mL), dried
(Na2SO4) and concentrated in vacuo to obtain 30 as a syrup which
was crystallized with EtOAc and hexane to give a white solid (0.18
25
g, 95%). mp 133 °C. [a] D = +56.0 (c = 2.5, CH3OH).
1H NMR (CDCl3 and CD3OD 400 MHz): d = 1.40 (m, 2 H), 1.54
(m, 4 H), 1.84 (br s, 1H), 1.97 (m, 1 H), 2.15 (m, 1 H), 2.62 (m, 2
H), 3.26 (m, 2 H), 3,32 (d, J = 2 Hz, 1 H), 3.63 (m, 2 H), 3.65 (d,
J = 2 Hz, 1 H), 4.38 (m, 1 H), 6.29 (br s, 1 H), 6.87 (br d, J = 8 Hz,
1 H), 7.23 (m, 5 H).
13C NMR (100 MHz, DMSO-d6): d = 23.07, 29.08, 31.66, 32.31,
34.22, 51.61, 52.92, 53.04, 60.90, 79.32, 126.10, 128.45, 128.56,
141.40. 165.52, 169.07, 170.79
(22) Shin, H. J.; Matsuda, H.; Murakami, M.; Yamaguchi, K.
Tetrahedron 1997, 53, 5747.
IR (KBr) n = 3300, 3088, 2941, 2861, 1742, 1666, 1644, 1556,
1497, 1455, 1250 cm-1.
(23) Tamai, M.; Hanada, K.; Adachi, T.; Oguma, K.; Kashiwagi,
K.; Omura, S.; Ohzeki, M. J. Biochem. 1981, 90, 255.
(24) Barrett, A. J.; Kembhavi, A. A.; Brown, M. A.; Krischke, H.;
Knight, C.; Tamai, M.; Hanada, K. Biochem. J. 1982, 201,
189.
(25) Sumiya, S.; Yoneda, T.; Kitamura, K.; Murata, M.; Yokoo, C.;
Tamai, M.; Yamamoto, A.; Inoue, M.; Ishida, T. Chem.
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(26) Turk, D.; Podobnik, M.; Popovic, T.; Katunuma, N.; Bode,
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HRMS (CI, CH4), calcd for C19H27N2O6 (M+H)+379.1869, found
379.1868.
Anal. C19H26N2O6 (378.43) calcd C 60.31, H 6.93, N 7.40; found C
60.43, H 7.32, N 7.09.
Acknowledgement
This work was supported by NIH Program Project Grant AI35707.
G.Z. Thanks the United States-Mexico Foundation for Science for
financial support.
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Synthesis 1999, No. SI, 1500–1504 ISSN 0039-7881 © Thieme Stuttgart · New York

1504
W. R. Roush et al.
PAPER
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diastereomers of 14 was confirmed in several cases by
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Synthesis 1999, No. SI, 1500–1504 ISSN 0039-7881 © Thieme Stuttgart · New York