Synthesis and activity of 5′-uridinyl dipeptide analogues mimicking the amino terminal peptide chain of nucleoside antibiotic mureidomycin A

Article abstract of DOI:10.1016/S0968-0896(03)00270-0

A series of 5′-uridinyl dipeptides were synthesised which mimic the amino terminal chain of nucleoside antibiotic mureido omycin A. Aminoacyl-β-alanyl- and aminoacyl-N-methyl-β-alanyl- dipeptides were attached either via an ester linkage to the 5′-hydroxyl of uridine, or via an amide linkage to 5′-amino-5′-deoxyuridine. The most active inhibitor of Escherichia coli phospho-MurNAc-pentapeptide translocase (MraY) was 5′-O-(L-Ala-N-methyl-β-alanyl)-uridine (13l), which also showed 97% enzyme inhibition at 2.35 mM concentration, and showed antibacterial activity at 100 μg/mL concentration against Pseudomonas putida. Both the central N-methyl amide linkage and a 5′ uridine ester linkage were required for highest biological activity. Enzyme inhibition was shown to be competitive with Mg²⁺. It is proposed that the primary amino terminus of the inhibitor binds in place of the Mg²⁺ cofactor at the MraY active site, positioned via a cis-N-methyl amide linkage.

Full text of DOI:10.1016/S0968-0896(03)00270-0

Bioorganic & Medicinal Chemistry 11 (2003) 3083–3099
Synthesis and Activity of 50-Uridinyl Dipeptide Analogues
Mimicking the Amino Terminal Peptide Chain of Nucleoside
Antibiotic Mureidomycin A
Nigel I. Howard and Timothy D. H. Bugg*
Department of Chemistry, University of Warwick, Coventry CV4 7AL, UK
Received 31 January 2003;accepted 8 April 2003
Abstract—A series of 50-uridinyl dipeptides were synthesised which mimic the amino terminal chain of nucleoside antibiotic mur-
eido omycin A. Aminoacyl-b-alanyl- and aminoacyl-N-methyl-b-alanyl- dipeptides were attached either via an ester linkage to the
50-hydroxyl of uridine, or via an amide linkage to 50-amino-50-deoxyuridine. The most active inhibitor of Escherichia coli phospho-
MurNAc-pentapeptide translocase (MraY) was 50-O-(l-Ala-N-methyl-b-alanyl)-uridine (13l), which also showed 97% enzyme
inhibition at 2.35 mM concentration, and showed antibacterial activity at 100 mg/mL concentration against Pseudomonas putida.
Both the central N-methyl amide linkage and a 50 uridine ester linkage were required for highest biological activity. Enzyme inhi-
bition was shown to be competitive with Mg2+. It is proposed that the primary amino terminus of the inhibitor binds in place of the
Mg2+ cofactor at the MraY active site, positioned via a cis-N-methyl amide linkage.
# 2003 Elsevier Science Ltd. All rights reserved.
Introduction
which catalyses the first step of the lipid cycle of bac-
terial peptidoglycan biosynthesis, namely the reaction of
UDPMurNAc-l-Ala-g-d-Glu-m-DAP-d-Ala-d-Ala with
lipid carrier undecaprenyl phosphate, to give lipid
intermediate I (see Fig. 1B).8 They show no inhibition of
bacterial teichoic acid or mammalian N-linked glyco-
protein biosynthesis,9 unlike MraY inhibitor tunicamy-
cin, which is a potent inhibitor of the mammalian
phospho-GlcNAc transferase enzyme.10 Using a con-
tinuous fluorescent assay, Brandish et al. have reported
previously that mureidomycin A is a slow-binding inhi-
bitor (Ki 35 nM, Ki* 2 nM) of solubilised Escherichia coli
MraY.11,12 The molecular basis for the EI to EI* tran-
sition during slow-binding inhibition is unknown.
Mureidomycins A–D (1–4) are peptidyl nucleoside
antibiotics isolated from Streptomyces flavidovirens
SANK 60486 (see Fig. 1A).1À3 Their structure consists
of a 30-deoxyuridine nucleoside linked via a 40,50-
enamide linkage to a peptide chain containing two
meta-tyrosine residues, one methionine residue, and
one N-methyl-diaminobutyric acid residue.2 They show
antibacterial activity against strains of Pseudomonas
(MIC 0.1–3 mg/mL), and have been shown to protect
mice against infection by Pseudomonas aeruginosa
(ED50 50–100 mg/kg).3 Two closely related families of
natural products, the pacidamycins 1–7,4À6 and the
napsamycins A–D,7 also with selective anti-pseudo-
monal activity, have been isolated from Streptomyces
coeruleorubidus AB1183F-64 and Streptomyces sp. HIL
Y-82, respectively.
We have previously studied the role of the unusual
enamide functional group in the biological activity of
mureidomycin A. Although an enamide-containing
model compound based on a tetrahydrofuran ring
showed high chemical reactivity, a uridine-based enam-
ide analogue showed low chemical reactivity, and
showed no inhibition of E. coli MraY.13 In the pacida-
mycin family, chemical reduction of the enamide has
been reported, to give a dihydropacidamycin-D which
showed only slightly reduced activity towards MraY.14
The mureidomycins selectively inhibit phospho-Mur-
NAc-pentapeptide translocase (MraY), an enzyme
*Corresponding author. Tel.: +44-2476-573018;fax: +44-2476-
524112;e-mail: t.d.bugg@warwick.ac.uk
0968-0896/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00270-0

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N. I. Howard, T. D. H. Bugg / Bioorg. Med. Chem. 11 (2003) 3083–3099
Figure 1. (A) Structures of mureidomycins A–D. Absolute stereochemistry is based on stereochemical elucidation of pacidamycins.14 (B) Reaction
catalysed by phosphoMurNAc-pentapeptide translocase (MraY, translocase I). (C) Structures of target 50-uridinyl dipeptides.
Thus, the enamide functional group does not appear to
be primarily responsible for the biological activity of
these compounds.
idomycin18 analogues lacking the N-methyl and C-
methyl groups of the N-methyl-diaminobutryic acid
moiety show no biological activity at 100 mg/mL con-
centration, suggesting an important role for these
methyl groups.
We have also reported previously that 3-aminopropio-
nyl (5) and 7-aminoheptanoyl (6) 50-uridine esters
showed some inhibitory activity (IC50 0.26 and 1.5 mM,
respectively) towards E. coli MraY, suggesting that the
amino terminus of the peptide chain may be
important for enzyme inhibition.15 A number of
synthetic dihydropacidamycins containing various
amino acid sidechains have been reported to show
comparable anti-pseudomonal activity and MraY
inhibition,14,16 indicating that the nature of the amino
acid sidechains is not critical for activity in this series.
However, dihydropacidamycin17 and dihydromure-
In order to investigate the role of the amino-terminal
peptide chain of mureidomycin A for MraY inhibition
and antibacterial activity, we decided to synthesise a
series of 50-uridinyl dipeptide analogues in which the
diaminobutyric acid unit is simplified to b-alanine or
N-methyl b-alanine (see Fig. 1C). We report the synth-
esis and biological evaluation of a series of 50-O-(amino-
acyl-b-alanyl)-uridine derivatives in which the dipeptide
is attached via either an ester or an amide linkage to the
50-position of uridine.

N. I. Howard, T. D. H. Bugg / Bioorg. Med. Chem. 11 (2003) 3083–3099
3085
Results
76% overall yield. The t-butyl ester was found to couple
satisfactorily to carboxylic acids, whereas the corre-
sponding methyl ester suffered from intramolecular
reaction to form b-lactam by-products.
Synthesis of 50-uridinyl dipeptides
Attachment of the dipeptide via an amide linkage
required the preparation of 50-amino-50-deoxyuridine.
Attempts to couple the known 20-O,30-O-isopropylidene
derivative18 to a carboxylic acid using DCC or mixed
anhydride couplings gave in our hands only 10–20%
yields of coupled product. The major side product was
found to be a secondary amine arising from intramole-
cular Michael addition of the free amine to C-6 of the
uracil base, observed previously in the literature.19
Therefore, 50-azido-50-deoxyuridine was prepared by
treatment of uridine with di-isopropyl-azodicarboxy-
late, triphenylphosphine and HN3, in 64% yield.20 Pro-
tection of the 20- and 30-hydroxyl groups with
t-butyldimethylsilyl chloride,21 followed by reduction of
the azide using H2S via the method of Dempcy et al.,22
gave the known TBDMS-protected amine21 (7) in 80%
yield. The TBDMS-protected amine (7) was found not
to suffer from intramolecular reaction during amide
bond couplings.
As illustrated in Scheme 1, six N-Boc-amino acids (l-
Phe, d-Phe, l-Tyr-OtBu, Gly, l-Ala, b-Ala) were cou-
pled with b-alanine methyl ester and N-methyl b-alanine
t-butyl ester to give 12 protected dipeptides (8a–l).
Three coupling methods were used: mixed anhydride
coupling using isobutyl chloroformate24 (method A) or
isopropenyl chloroformate25 (method B);formation of
the N-hydroxysuccinimide active ester by isobutyl
chloroformate mixed anhydride coupling (method C) or
using isopropenyl succinimido carbonate26 (method D),
followed by coupling;or PyBOP coupling 27 (method E).
Coupling of the N-methyl amine was problematic using
method A, but gave yields of 70–95% using methods C
or E. Each of the protected dipeptides was treated with
1 M sodium hydroxide in 1:1 H2O/dioxane to give the
free acids (9a–l) in 70–99% yield.
Amide coupling of the 12 free acids to protected
50-amino-50-deoxyuridine (8) proceeded slowly, pre-
sumably due to steric hindrance. Mixed anhydride
coupling with isopropenyl chloroformate (method B)
N-Methyl b-alanine t-butyl ester was prepared by addi-
tion of N-benzylmethylamine to t-butyl acrylate,23 fol-
lowed by debenzylation via catalytic hydrogenation, in
Scheme 1. Synthesis of amide-linked 50-uridinyl dipeptides. Method A: isobutyl chloroformate, Et3N;method B: isopentenyl chloroformate, Et N;
3
method C: coupling of N-hydroxysuccinimide ester to amine;method D: isopropenyl succinimidocarbonate;method E: PyBOP coupling. AA,
amino acid (a,b, l-Phe; c,d, d-Phe; e,f, l-Tyr; g,h, Gly; i,j, b-Ala; k,l, l-Ala;see Table 1);R, H ( a,c,e,g,i,k) or CH3 (b,d,f,h,j,l);P, CH 3 or tBu.

3086
N. I. Howard, T. D. H. Bugg / Bioorg. Med. Chem. 11 (2003) 3083–3099
gave 33–66% yields of 10a–l;also PyBOP coupling
(method E) gave the desired coupled products 10f and
10k in 60 and 48%, respectively. Deprotection was
achieved by treatment with tetrabutylammonium fluor-
ide (31–71% yield), followed by acidic deprotection in
50% aqueous formic acid (92–99% yield), followed by
purification via cation exchange chromatography on
Dowex 50W resin, to give the desired products 11a–l
(see Scheme 1).
apparent in the 50-esters, where there is duplication of
signals for ribose carbons (by 0.4–1.0 ppm for 12a–l,
and by 0.2–0.6 ppm for 13a–l) as well as for b-alanine
and amino acid carbon signals. The two observed sig-
nals for the N-methyl carbon are typically separated by
2.0–3.0 ppm, for example 36.3 and 34.1 ppm in 12l, and
35.3 and 33.3 ppm in 13l. No apparent changes were
observed when NMR spectra were recorded at 37 ꢀC.
Biological evaluation
Ester coupling of the 12 free acids to 20-O,30-O-iso-
propylidene uridine also proceeded slowly. Best yields
were obtained via a mixed anhydride coupling method
using isopropenyl chloroformate (method B), which
gave 20–74% yields of the 12 protected products 12a–l,
as shown in Scheme 2. Coupling to the 50-O position,
rather than the uracil imide N, was confirmed in each
case by a shift of the H-50 1H NMR signal from 3.6 to
4.2 ppm, and corresponding shifts in the 13C NMR
spectra of the coupled products. Deprotection was
achieved using 50% aqueous formic acid, in 83–99%
yield, to give the desired ester linked products 13a–l.
The compounds were assayed as inhibitors of solubi-
lised E. coli phospho-MurNAc-pentapeptide translo-
case, using a radiochemical assay, monitoring transfer
of 14C from 14C-UDPMurNAc-pentapeptide to lipid-
linked product.11,15 Inhibitors were added at 2.35 mM
and 235 mM concentrations. The majority of the amide-
linked analogues (see Table 1) showed only 15–30%
inhibition at 2.35 mM concentration, with 13j (b-Ala-N-
Me-b-Ala-) and 13e (l-Tyr-b-Ala-) showing 43 and
48% inhibition, respectively. The majority of the ester-
linked analogues (see Table 1) also showed 15–
30% inhibition at 2.35 mM concentration, however
15l (l-Ala-N-Me-b-Ala-) showed 97% inhibition at
2.35 mM.
From inspection of the 13C NMR spectra for the uridine
50-dipeptides (see Tables 3–6 in the Experimental), it is
apparent that the compounds containing N-methyl-b-
alanine show duplication of signals, due to cis and trans
rotamers of the N-methyl amide linkage. This is especially
The latter compound 15l, which was consistently the
most potent enzyme inhibitor, was tested at variable
concentrations of MgCl2. As shown in Table 2, at
235 mM inhibitor and 25 mM Mg2+, 8% inhibition was
observed, but 33% inhibition was observed at 5 mM
Mg2+, consistent with the hypothesis that the amino
terminus of MrdA binds in place of the Mg2+ cofactor
Table 1. Inhibition (%) of solubilised E. coli MraY by 50-uridinyl
dipeptide analogues 11a–l and 13a–la
AA
R
MraY inhibition of
amide 11 (%)
@ 2.35 mM
MraY inhibition of
ester 13 (%)
@ 2.35 mM
a
b
c
d
e
f
g
h
i
l-Phe
l-Phe
d-Phe
d-Phe
l-Tyr
l-Tyr
Gly
H
Me
H
Me
H
Me
H
Me
H
27
NT
22
NT
48
NT
NT
NT
16
43
21
13
15
27
8
24
18
40
23
34
33
24
97
Gly
b-Ala
b-Ala
l-Ala
l-Ala
j
k
l
Me
H
Me
14
aAssays as described in the Experimental. AA, amino-terminal amino
acid;R, beta-alanine N-substituent;NT, not tested.
Table 2. Inhibition (%) of solubilised E. coli MraY by analogues 13l
a
at 5–25 mM concentrations of MgCl2
Concentration of
MgCl2 (mM)
Inhibition
@ 2.35 mM 13l (%)
Inhibition
@ 235 mM 13l (%)
5
12.5
25
97
96
97
33
28
8
Scheme 2. Synthesis of ester-linked 50-uridinyl dipeptides. Method B:
isopentenyl chloroformate, Et3N. AA, amino acid (a,b, l-Phe; c,d, d-
Phe; e,f, l-Tyr; g,h, Gly; i,j, b-Ala; k,l, l-Ala;see Table 1);R, H
(a,c,e,g,i,k) or CH3 (b,d,f,h,j,l).
aAssays as described in the Experimental. Data corrected for variation
in enzyme activity at 5–25 mM MgCl2.

N. I. Howard, T. D. H. Bugg / Bioorg. Med. Chem. 11 (2003) 3083–3099
3087
at the MraY active site.15 Under the same assay condi-
tions, 235 mM mureidomycin A showed 97% inhibition
of MraY at 25 mM MgCl2.
to 20-O,30-O-isopropylidene-uridine using the isopro-
penyl chloroformate mixed anhydride method, followed
by acidic deprotection.
Compounds 11a–l and 13a–l were tested for anti-
bacterial activity at 100 mg/mL against E. coli K12,
Pseudomonas putida, Bacillus subtilis, and Streptococcus
pneumoniae, using a filter disk assay on Luria Broth
agar plates. The only compound to show antibacterial
activity was the l-Ala-N-Me-b-Ala-containing analogue
13l, which showed strong activity (27 mm zone of
inhibition) against P. putida at 100 mg/mL. The anti-
pseudomonal selectivity of this compound matches that
of the parent compound mureidomycin A.3
Biological evaluation
Compounds 14–16 were assayed as inhibitors of solubi-
lised E. coli phospho-MurNAc-pentapeptide translo-
case, using the radiochemical assay. They showed 14, 27
and 21% enzyme inhibition respectively at 2.35 mM
concentration, thus the conformational restraint has
not apparently assisted the biological activity of these
compounds. No antibacterial activity was observed for
14–16 at 100 mg/mL against E. coli K12, P. putida, B.
subtilis, and S. pneumoniae, using a filter disk assay.
Synthesis of conformationally restrained analogues
As described above, highest biological activity was
shown by a compound containing the N-methyl-amide
functional group, also found in mureidomycin A. A
plausible hypothesis is that the N-methyl amide forms a
cis–amide bond which positions the amino terminus
correctly for enzyme inhibition. As mentioned above,
NMR spectra of N-methyl-b-alanine-containing analo-
gues show duplication of peaks, corresponding to cis
and trans amide rotamers which interconvert slowly.
Discussion
In order to examine the role of the amino terminal
peptide chain of mureidomycin A, we have synthesised
a series of 50-uridinyl dipeptide analogues. Most potent
inhibition of MraY is observed with analogue 11l
containing N-methyl-b-alanine and an ester linkage to
the 50-position of uridine. Significantly lower activity is
observed if either the N-methyl amide or the ester link-
age is not present, implying that both functional groups
are important for biological activity. The same analogue
shows anti-pseudomonal activity, with a similar selec-
tivity to the natural product. Enzyme assays at variable
Mg2+ concentrations indicate that increasing con-
centrations of Mg2+ reduce the potency of enzyme
inhibition, consistent with the hypothesis that the amino
terminus binds in place of the Mg2+ cofactor.15
In order to examine this hypothesis, conformationally
restrained analogues 14–16 were designed, as shown in
Scheme 3, in which the amino functional group is held
in the desired conformation. Analogue 14, containing a
piperidine-3-carboxylic acid unit attached to the
50-position of uridine, was synthesised by coupling of N-
Boc-nipecotic acid to 20-O,30-O-isopropylidene-uridine,
followed by acidic deprotection. Analogues 15 and 16
contain a d-lactam ring formed from l- or d-ornithine,
attached via a two-carbon linkage to the 50-position of
uridine. The d-lactam ring was formed by cyclisation of
l- or d-ornithine, using the method of Pellegata et al.28
and alkylated using ethyl bromoacetate, after Semple et
al.29 After alkaline deprotection, the acids were coupled
On the basis of these and other literature data, a
hypothesis can be made for the molecular mechanism of
action of mureidomycin A, illustrated in Figure 2. It has
been demonstrated previously that the unusual enamide
functional group is not primarily responsible for biolo-
gical activity.13,14 It appears that the a-amino terminus
Scheme 3. Synthesis of conformationally restrained analogues 14–16. Conditions: (a) 20,30-isopropylidene uridine, coupling method B;(b) 50%
HCO2H/H2O.

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N. I. Howard, T. D. H. Bugg / Bioorg. Med. Chem. 11 (2003) 3083–3099
aminoheptanoyl-uridine (6) shown previously to inhibit
MraY,15 were also less active than 13l.
We have found that the chemical nature of the linkage
of the dipeptide to the 50-position of uridine is impor-
tant for biological activity. Thus, replacement of the 50-
ester linkage of 13l with a 50-amide linkage (13l) leads to
a loss of biological activity. Since the amide linkage is
held in a trans conformation, but the ester linkage is
conformationally flexible, it appears that the ester link-
age permits an active conformation which is not possi-
ble for the amide. This in turn implies that a (secondary)
role of the 50-enamide linkage in the natural product is
to permit the adoption of an active conformation,
rather than to promote reactivity.
The molecular basis for the slow-binding inhibition
kinetics shown by mureidomycin A has yet to be estab-
lished, a plausible explanation being a conformational
change of the enzyme–inhibitor complex to form the
EI* complex. Although these synthetic analogues of
MrdA are not potent enzyme inhibitors, they are the
first simplified synthetic analogues to show antibacterial
activity, thus inhibition of MraY is a realistic target for
antibacterial chemotherapy.
Experimental
General
Figure 2. (A) Proposed Mg2+ binding site at the active site of MraY.
(B) Proposed binding of analogue 13l to Mg2+ binding site of MraY,
via cis-amide linkage. (C) Binding of mureidomycin A to Mg2+ bind-
ing site of MraY, via cis-amide linkage.
Chemicals were purchased from Acros, Aldrich, Avo-
cado, BDH, Lancaster, Novobiochem, or Sigma Che-
mical Companies, unless otherwise stated. Flash
chromatography was performed using Merck Kieselgel
60 (230–400 mesh). 300 MHz NMR spectra were recor-
ded on either a Bruker AC300 or Bruker DPX 300
Fourier Transform Spectrometer. 20,30-Di-tertbutyldi-
methylsilyl-50-amino-50-deoxyuridine (7) was prepared
by the route of Peterson et al.,21 but using hydrogen
sulfide gas to reduce 50-azide substituent as described by
Dempcy et al.22 b-Alanine methyl ester was prepared by
treatment of b-alanine with thionyl chloride and
methanol, in 89% yield. Isopropenyl succinimido car-
bonate was prepared by the method of Takeda et al.26
is important for biological activity;thus we propose that
the protonated ammonium ion binds in place of the
Mg2+ cofactor at the MraY active site. The MraY amino
acid sequence contains a DDxxD motif at residues 115–
119 which, as proposed previously,15 may form a Mg2+
binding site at Asp-115 and Asp-116, as illustrated in
Figure 2A. The N-methyl amide functional group is also
important for biological activity, consistent with obser-
vations by other researchers that analogues lacking this
group are biologically inactive.17,18 Since two rotamers
are observed in solution by 13C NMR spectroscopy for
compounds containing the N-methyl amide, we propose
that this compound exists partly in a cis-amide con-
former, whose structure positions the amino terminus in
the Mg2+ binding site (see Fig. 2B). Our initial attempts
to synthesise conformationally restrained analogues
have not resulted in increased biological activity;how-
ever, in principle this strategy could lead to the design of
more potent inhibitors for MraY.
Preparation of N-methyl ꢀ-alanine tert-butyl ester. A
modification of the method of Leonard and Durand
was used.23 A solution of tert-butyl acrylate (14.65 mL,
0.1 mol) in anhydrous methanol (20 mL) was added to a
solution of N-benzylmethylamine (14.2 mL, 0.11 mol) in
anhydrous methanol (20 mL) at 0 ꢀC. After 15 min, the
reaction was allowed to warm to room temperature and
the solution stirred for 20 h. The solvent was carefully
removed under reduced pressure and the resulting oil
purified by distillation to give N-methyl, N-benzyl-b-
alanine tert-butyl ester as a colourless oil, 22.66 g, 91%.
dH (300 MHz, CDCl3) 7.25–7.33 (5H, m), 3.51 (2H, s,
PhCH2), 2.72 (2H, t, J=6.9 Hz, CH2N), 2.46 (2H, t,
J=6.9 Hz, CH2CO), 2.42 (3H, s, NCH3), 1.46 (9H, s,
tBu) ppm dC (75 MHz, CDCl3) 172.1, 139.2, 129.1,
128.5, 128.4, 127.1, 80.4, 62.2, 53.2, 42.0, 34.2, 28.3
ppm; m/z (ES+) 250.1 (MH+). 10% Palladium on car-
Mureidomycin A could also adopt a cis-amide con-
former, as shown in Figure 2C. The terminal amino acid
in MrdA is meta-tyrosine, whereas in this study analo-
gues containing l-Phe (13b) or l-Tyr (13f) at this posi-
tion were less active than 13l containing l-Ala, found in
pacidamycin D.14 Analogues 13i and 13j containing b-
alanine, matching the length of the acyl chain of 50-O-7-

N. I. Howard, T. D. H. Bugg / Bioorg. Med. Chem. 11 (2003) 3083–3099
3089
bon (1.50 g) was suspended in a solution of N-phenyl-N-
methyl-b-alanine-tert-butyl ester (15.00 g, 60.16 mmol)
and anhydrous methanol (100 mL). The solution was
degassed, then saturated with hydrogen gas, and stirred
for 2 days. The reaction mixture was filtered through a
Celite pad, and solvents carefully removed under
reduced pressure. The resulting oil was purified by
Kugelrohr distillation to give N-methyl-b-alanine-tert-
butyl ester as a colourless oil, 9.01 g, 84%. dH
(300 MHz, CDCl3) 2.75 (2H, t, J=6.9 Hz, CH2N), 2.37
(5H, CH2CO and NCH3), 1.91 (1H, br, NH), 1.40 (9H,
s, tBu) ppm; dC (75 MHz, CDCl3) 172.3, 80.6, 47.3, 36.3,
35.6, 28.2 (tBu) ppm; m/z (ES+) 160.0 (MH+).
(1H, t, J=3.6 Hz, NH), 5.12 (1H, d, J=4.0 Hz, NH),
4.17 (1H, br, Tyr a-CH), 3.56 (3H, s, OMe), 3.22–3.47
(2H, m, NCH2), 2.91 (2H, d, J=6.2 Hz, Tyr b-CH2),
t
2.28–2.42 (2H, m, CH2CO), 1.32 (9H, s, Bu), 1.24 (9H,
t
s, Bu) ppm; dC (75 MHz, CDCl3) 172.9, 171.7, 154.6,
131.9, 130.0, 124.6, 80.3, 78.7, 56.3, 52.1, 38.5, 35.1,
34.0, 29.2, 28.6 ppm; m/z (FAB) 423 (MH+).
N-Boc-b-Ala-b-Ala-OMe (8i) was prepared by method
A, in 60% yield. dH (300 MHz, CDCl3) 6.81 (1H, br,
NH), 5.40 (1H, br, NH), 3.54 (3H, s, OCH3), 3.37 (2H,
q, J=6.9, NCH2), 3.23 (2H, q, J=6.9 Hz, NCH2), 2.42
(2H, t, J=6.9 Hz, CH2CO), 2.24 (2H, t, J=6.9 Hz,
t
CH2CO), 1.30 (9H, s, Bu) ppm; dC (75 MHz, CDCl3)
173.0, 171.9, 156.4, 79.3, 52.0, 36.4, 35.9, 34.6, 33.6,
Preparation of N-Boc-aminoacyl-ꢀ-alanyl esters (8a–l).
N-Boc-amino acids (l-phenylalanine, d-phenylalanine,
l-tyrosine(O-tBu), glycine, b-alanine, or l-alanine) were
coupled with either b-alanine methyl ester or N-methyl
b-alanine t-butyl ester, using coupling method A, C, D
or E, as described below.
28.6 ppm; m/z (CI+, NH3) 275 (MH+).
Method C (N-hydroxysuccinimide ester coupling). Pre-
t
paration of N-Boc-L-Phe-N-Me-ꢀ-Ala-OtBu (8b). Boc-
l-phenylalanine (265 mg, 1.0 mmol), isopropenyl
succinimido carbonate (199 mg, 1.0 mmol) and 4-dime-
thylaminopyridine (12 mg, 0.1 mmol) were dissolved in
1,4-dioxane (6.0 mL) and stirred at room temperature
for 19 h. The solvent was removed in vacuo, and the
residue taken up in ethyl acetate (100 mL), and washed
with 1 M hydrochloric acid (50 mL), water (50 mL), 5%
sodium bicarbonate solution (50 mL), brine (50 mL).
The organic phase was dried (Na2SO4), and evaporated
in vacuo to give Boc-l-Phe N-hydroxysuccinimide ester
as a white solid (351 mg, 97%). dH (300 MHz, CDCl3)
7.15–7.32 (5H, m, Ar), 4.98 (1H, d, J=4.0 Hz, NH),
4.83 (1H, q, J=4.0 Hz, Phe a-CH), 3.22 and 3.09 (2 Â
1H, dd, J=4.1, 11.8 Hz, Phe b-CH2), 2.76 (4H, s, succ-
Method A (isobutyl chloroformate mixed anhydride cou-
pling).24 General method. N-tBoc-amino acid (2 mmol)
and N-methyl morpholine (219 L, 2 mmol) were dis-
solved in anhydrous THF (10 mL), and the solution
cooled to 0 ꢀC. Isobutylchloroformate (286 mL,
2.2 mmol) was added, the resulting suspension was stir-
red for 20 min, and a solution of b-alanine methyl ester
hydrochloride (279 mg, 2 mmol) and N-methyl morpho-
line (219 mL, 2 mmol) in anhydrous DMF (5 mL) was
added, and the solution stirred for 30 min at 0 ꢀC, then
allowed to warm to room temperature, and stirred for
20–24 h. The solvents were removed in vacuo, and the
residue taken up in ethyl acetate (50 mL) and washed
with water (25 mL), 5% potassium hydrogen sulphate
solution (25 mL), 5% sodium bicarbonate solution
(25 mL), water (25 mL), brine (25 mL). The organic
phase was dried (Na2SO4), concentrated in vacuo, and
residue purified by flash column chromatography (3%
MeOH/DCM) to give the requisite protected dipeptides.
t
CH2), 1.31 (9H, s, Bu) ppm; dC (75 MHz, CDCl3)
169.3, 168.2, 155.1, 135.2, 130.1, 129.0, 127.7, 80.8, 53.1,
38.4, 28.6, 25.9 ppm. N-tBoc-l-phenylalanine-N-hydroxy-
succinimide ester (362 mg, 1.0 mmol) and N-methyl-b-
alanine-tert-butyl ester (159 mg, 1.0 mmol) were dis-
solved in acetonitrile (6 mL) and stirred at room tem-
perature for 24 h. The solvent was removed in vacuo,
and the residue taken up in ethyl acetate (100 mL) and
washed with 1 M hydrochloric acid (50 mL), water
(50 mL), 5% sodium bicarbonate solution (50 mL),
brine (50 mL). The organic phase was dried (Na2SO4),
evaporated in vacuo, and the residue taken up in ethyl
acetate and purified by flash column chromatography
(1:1 ethyl acetate/hexane) to give a white solid, 306 mg,
75%. dH (300 MHz, CDCl3) 7.08–7.26 (5H, m, Ar), 5.56
(1H, d, J=6.2 Hz, NH), 4.70 (1H, m, Phe a-CH), 3.30–
3.60 (2H, m, NCH2), 2.89 (2H, m, Phe b-CH2), 2.79,
2.66 (2 Â s, rotamers, N–CH3), 2.15–2.40 (2H, m,
N-Boc-l-Phe-b-Ala-OMe (8a) was prepared by method
A, in 91% yield. dH (300 MHz, CD3OD) 7.11–7.22 (5H,
m, Ar), 4.21 (1H, t, J=4.1 Hz, Phe a-CH), 3.61 (3H, s,
OMe), 3.28–3.49 (2H, m, NCH2), 3.05 and 2.79 (2 Â
1H, d, J=4.1, 10.2 Hz, Phe b-CH2), 2.34–2.48 (2H, m,
t
CH2CO), 1.32 (9H, s, Bu) ppm; dC (75 MHz, CDCl3)
174.3, 173.5, 157.5, 138.6, 130.4, 129.4, 127.7, 80.6, 57.4,
52.2, 39.4, 36.2, 34.5, 28.6 ppm; m/z (ES+) 351 (MH+).
N-Boc-d-Phe-b-Ala-OMe (8c) was prepared by method
A, in 71% yield. dH (300 MHz, CDCl3) 7.08–7.22 (5H,
m, Ar), 6.68 (1H, br, NH), 5.41 (1H, d, J=6.23 Hz,
NH), 4.26 (1H, m, Phe a-CH), 3.51 (3H, s, OMe) 3.18–
3.48 (2H, m, NCH2), 2.90 (2H, d, J=4.2 Hz, Phe b-
t
t
CH2CO), 1.26 (9H, s, Bu), 1.21 (9H, s, Bu) ppm; dC
(75 MHz, CDCl3) 171.8, 171.2, 155.3, 136.8, 129.7,
128.6, 127.1, 80.9, 79.6, 51.7, 44.8, 40.1, 35.9, 33.7, 33.6,
28.5, 28.3 ppm; m/z (CI+, NH3) 407 (MH+).
t
CH2), 2.22–2.45 (2H, m, CH2CO), 1.31 (9H, s, Bu)
ppm; dC (75 MHz, CDCl3) 172.8, 171.8, 155.7, 137.2,
129.6, 128.8, 127.1, 80.1, 56.2, 52.0, 39.2, 35.1, 34.0,
28.6 ppm; m/z (ES+) 351 (MH+).
Method D (isopropenyl succinimido carbonate coupling).
Preparation of N-Boc-L-Ala-N-Me-ꢀ-Ala-OtBu (8i).
tBoc-l-alanine (189 mg, 1.0 mmol), isopropenyl succini-
mido carbonate26 (199 mg, 1.0 mmol) and 4-dimethyla-
minopyridine (12 mg, 0.1 mmol) were dissolved in dry
acetonitrile (6 mL) and stirred at room temperature for
5 h. A solution of N-methyl-b-alanine-tert-butyl ester
N-Boc-l-Tyr(O-tBu)-b-Ala-OMe (8e) was prepared by
method A, in 81% yield. dH (300 MHz, CDCl3) 7.00
(2H, d, J=8.3 Hz, Ar), 6.81 (2H, d, J=8.2 Hz, Ar), 6.37

3090
N. I. Howard, T. D. H. Bugg / Bioorg. Med. Chem. 11 (2003) 3083–3099
(159 mg, 1.0 mmol) in acetonitrile/1,4-dioxane (3 mL)
(2:1 v/v) was added and the solution stirred for 20 h. The
solvent was evaporated in vacuo, and the residue taken
up in ethyl acetate (100 mL), and washed with 1 M
hydrochloric acid (50 mL), water (50 mL), 5% sodium
bicarbonate solution (50 mL), brine (50 mL). The
organic phase was dried (Na2SO4), evaporated to give a
yellow oil, which was taken up in ethyl acetate and
purified by flash column chromatography (hexane/ethyl
acetate 1:1) to give a colourless oil (126 mg, 38%). dH
(300 MHz, CDCl3) 5.60 (1H, br, NH), 4.50 (1H, m, Ala
a-CH), 3.41–3.75 (2H, m, NCH2), 3.02 and 2.86 (2 Â s,
rotamers, N–CH3), 2.35–2.52 (2H, m, CH2CO), 1.36
155.3, 154.4, 131.9, 131.7, 130.2, 125.2, 81.2, 80.8, 79.5,
51.8, 45.3, 44.8, 39.9, 39.6, 35.8, 33.6, 34.6, 33.7, 29.1,
28.3, 28.2 ppm; m/z (FAB) 479 (MH+).
N-Boc-Gly-b-Ala-OMe (8g) was prepared by method E,
in 49% yield. dH (300 MHz, CDCl3) 7.03 (1H, br, NH),
5.70 (1H, br, NH), 3.71 (2H, d, J=6.9 Hz, Gly a-CH2),
3.59 (3H, s, OCH3), 3.43 (2H, q, J=6.9 Hz, NCH2),
t
2.49 (2H, t, J=6.9 Hz, CH2CO), 1.33 (9H, s, Bu) ppm;
dC (75 MHz, CDCl3) 173.0, 170.2, 156.5, 80.2, 52.0,
44.4, 35.2, 34.1, 28.6 ppm; m/z (CI+, NH3) 261 (MH+).
N-Boc-Gly-N-Me-b-Ala-OtBu (8h) was prepared by
method E, in 71% yield. dH (300 MHz, CDCl3) 5.48
(1H, br, NH), 3.79, 3.68 (2 Â 1H, d, J=6.9 Hz, Gly a-
CH2), 3.40, 3.29 (2H, t, J=6.9 Hz, NCH2), 2.79, 2.69 (2
 s, rotamers, NCH3), 2.27 (2H, m, CH2CO), 1.22
t
and 1.39 (2 Â 9H, s, Bu), 1.21 (3H, d, J=6.8 Hz, Ala
CH3) ppm; dC (75 MHz, CDCl3) 173.0, 171.4, 155.5,
81.1, 79.8, 46.7, 45.0, 36.1, 34.9, 33.8, 28.7, 28.4,
19.1 ppm; m/z (CI+, NH3) 331 (MH+).
t
(18H, s, 2 Â Bu) ppm; dC (75 MHz, CDCl3) 171.1,
Method
E
(PyBOP coupling).27 General method.
170.2, 168.5, 156.0, 81.4, 80.8, 79.4, 44.7, 44.5, 42.6,
42.2, 34.9, 33.2, 34.2, 33.8, 28.4, 28.1 ppm; m/z (FAB)
317 (MH+).
N-tBoc-amino acid (2 mmol), b-alanine methyl ester
hydrochloride (278 mg, 2 mmol) or N-methyl-b-alanine-
tert-butyl ester (318.5 mg, 2 mmol), PyBOP (1.040 g,
2 mmol) were dissolved in anhydrous dichloromethane
(10 mL). Diisopropylethylamine (1.045 mL, 6 mmol) in
anhydrous dichloromethane (2 mL) was added drop-
wise. The solution was stirred for 2 days. The solution
was diluted with ethyl acetate (30 mL), and washed with
water (20 mL), 5% potassium hydrogen sulphate solu-
tion (20 mL), 5% sodium bicarbonate solution (20 mL),
brine (20 mL). The organic phase was dried (Na2SO4),
and concentrated in vacuo. The residue was taken up in
ethyl acetate and purified by flash column chromato-
graphy (ethyl acetate) to give the requisite protected
dipeptide.
N-Boc-b-Ala-N-Me-b-Ala-OtBu (8j) was prepared by
method E, in 97% yield. dH (300 MHz, CDCl3) 5.46 (1H,
br, NH), 3.52 (2H, m, NCH2), 3.30 (2H, m, NCH2), 2.96
and 2.88 (2 Â s, rotamers, NCH3), 2.52 and 2.27 (4H, m,
2 Â CH2CO), 1.34 (9H, s, tBu), 1.32 (9H, s, tBu) ppm; dC
(75 MHz, CDCl3) 171.8, 171.3, 170.5, 156.2, 81.5, 80.8,
79.0, 45.6, 44.4, 36.5, 36.0, 36.0, 33.3, 34.6, 34.0, 33.2,
28.6, 28.2 ppm; m/z (FAB+) 331 (MH+).
Preparation of N-Boc-aminoacyl-ꢀ-alanines (9a–l). Gen-
eral procedure. The fully protected dipeptide (8a–l) was
dissolved in 1 M sodium hydroxide/1,4-dioxane (1:1 v/v)
(8 mL/mmol) and stirred at room temperature for 15–
16 h. The solution was acidified to pH 2 with 1 M
hydrochloric acid and extracted with ethyl acetate
(20 mL/mmol). The organic phase was dried (Na2SO4)
and concentrated in vacuo to give the free acid.
N-Boc-l-Ala-b-Ala-OMe (8c) was prepared by method
E, in 75% yield. dH (300 MHz, CDCl3) 7.01 (1H, br,
NH), 5.51 (1H, br, NH), 4.04 (1H, m, Ala a-CH), 3.59
(3H, s, OMe), 3.42 (2H, m, NCH2), 2.47 (2H, t,
t
J=6.9 Hz, CH2CO), 1.33 (9H, s, Bu), 1.24 (3H, d,
J=6.91 Hz, Ala CH3) ppm; dC (75 MHz, CDCl3) 173.8,
173.2, 155.9, 80.2, 52.1, 50.6, 35.2, 34.1, 28.6, 18.7 ppm;
m/z (FAB) 275 (MH+).
N-tBoc-l-Phe-b-Ala (9a). 90% yield. dH (300 MHz,
CDCl3) 7.73 (1H, br, NH), 7.09 (5H, br, Ar), 5.94 (1H,
br, NH), 4.38 (1H, br, Phe a-CH), 3.29 (2H, br, NCH2),
2.96 and 2.77 (2 Â 1H, dd, J=4.1, 10.2 Hz, Phe b-CH2),
2.20 (2H, br, CH2CO), 1.20 (9H, s, tBu) ppm; dC
(75 MHz, CDCl3) 178.5, 172.9, 156.4, 137.4, 129.8,
128.7, 127.0, 80.2, 56.2, 39.5, 37.0, 28.7 ppm; m/z (ES+)
337.2 (MH+).
N-Boc-d-Phe-N-Me-b-Ala-OtBu (8d) was prepared by
method E, in 98% yield. dH (300 MHz, CDCl3) 7.08–
7.26 (5H, m, Ar), 6.04 (1H, d, J=6.2 Hz, NH), 4.71
(1H, m, Tyr a-CH), 3.32–3.74 (2H, m, NCH2), 2.80–
3.08 (5H, m, Tyr b-CH2 and NCH3), 2.25–2.50 (2H, m,
t
t
CH2CO), 1.41 (9H, s, Bu), 1.32 (9H, s, Bu) ppm; dC
(75 MHz, CDCl3) 172.0, 171.8, 171.3, 155.3, 137.0,
136.8, 129.8, 128.6, 127.1, 80.9, 79.6, 51.8, 45.3, 44.9,
40.5, 40.1, 36.0, 33.7, 34.6, 33.6, 28.6, 28.3 ppm; m/z
(FAB) 407 (MH+).
N-tBoc-l-Phe-N-Me-b-Ala (9b) 98% yield. dH
(300 MHz, acetone-d6) 7.11–7.32 (5H, m, Ar), 6.34 (1H,
br, NH), 6.25 (1H, d, J=6.9 Hz, NH), 4.73–4.89 (1H,
m, Phe a-CH), 3.39–3.72 (2H, m, NCH2), 2.83–3.03
(5H, m, Phe b-CH2 and N–CH3), 2.42–2.69 (2H, m,
t
CH2CO), 1.32 (9H, s, Bu) ppm; dC (75 MHz, acetone-
N-Boc-l-Tyr(O-tBu)-N-Me-b-Ala-OtBu (8f) was pre-
pared by method E, in 91% yield. dH (300 MHz, CDCl3)
6.98 (2H, d, J=8.4 Hz), 6.71 (2H, d, J=8.4 Hz), 5.52
(1H, d, J=6.9 Hz, NH), 4.59 (1H, m, Tyr a-CH), 3.18–
3.49 (2H, m, NCH2), 2.79 (2H, m, Tyr b-CH2), 2.70,
2.53 (2 Â s, rotamers, NCH3), 2.10–2.35 (2H, m,
d6) 174.2, 173.2, 156.6, 138.7, 138.5, 130.8, 129.5, 127.8,
79.8, 52.9, 46.2, 45.7, 39.8, 39.6, 36.5, 34.2, 33.7, 32.7,
28.9 ppm; m/z (FAB) 351 (MH+).
N-tBoc-d-Phe-b-Ala (9c) 76% yield. dH (300 MHz, ace-
tone-d6) 7.45 (1H, br, NH), 7.11–7.28 (5H, m, Ar), 6.11
(1H, br, NH), 4.29 (1H, br, Phe a-CH), 3.41 (2H, m,
NCH2), 3.12 and 2.89 (2 Â 1H, dd, J=4.1, 10.2 Hz, Phe
t
t
CH2CO), 1.25 (9H, s, Bu), 1.23 (9H, s, Bu), 1.13 (9H,
t
s, Bu) ppm; dC (75 MHz, CDCl3) 171.9, 171.1, 170.3,

N. I. Howard, T. D. H. Bugg / Bioorg. Med. Chem. 11 (2003) 3083–3099
3091
b-CH2), 2.45 (2H, t, J=6.9 Hz, CH2CO), 1.30 (9H, s,
tBu) ppm; dC (75 MHz, acetone-d6) 173.9, 173.0, 156.6,
139.1, 130.6, 129.4, 127.6, 79.7, 57.1, 39.4, 36.2, 34.6,
28.9 ppm; m/z (FAB) 337 (MH+).
tBu) ppm; dC (75 MHz, CDCl3) 175.1, 173.9, 172.6,
156.6, 79.6, 45.7, 44.7, 36.7, 36.6, 36.5, 33.5, 34.0, 33.3,
33.1, 32.6, 28.4 ppm; m/z (ES+) 275 (MH+).
N-tBoc-l-Ala-b-Ala (9k) 75% yield. dH (300 MHz,
CD3OD) 3.98 (1H, m, Ala a-CH), 3.36 (2H, m, NCH2),
2.33 (2H, t, J=6.9 Hz, CH2CO), 1.43 (9H, s, Bu), 1.26
N-tBoc-d-Phe-N-methyl-b-Ala (9d). 99% yield. dH
(300 MHz, CDCl3) 7.12–7.29 (5H, m, Ar), 5.96 (1H, d,
J=6.9 Hz, NH), 4.75–4.96 (1H, m, Phe a-CH), 3.39–
3.73 (2H, m, NCH2), 2.82–3.02 (2H, m, Phe b-CH2),
2.83, 2.74 (2 Â s, rotamers, N–CH3), 2.36–2.59 (2H, m,
t
(3H, d, J=6.8 Hz, Ala CH3) ppm; dC (75 MHz,
CD3OD) 179.1, 176.2, 158.0, 81.1, 50.4, 37.7, 37.8, 29.2,
19.1 ppm; m/z (FAB) 261 (MH+).
t
CH2CO), 1.35 (9H, s, Bu) ppm; dC (75 MHz, CDCl3)
174.7, 173.6, 172.3, 156.6, 136.1, 136.0, 129.2, 128.2,
126.7, 79.8, 51.4, 44.9, 44.5, 39.5, 39.3, 35.5, 33.6, 32.4,
31.6, 28.1 ppm; m/z (FAB) 351 (MH+).
N-tBoc-l-Ala-N-methyl-b-Ala (9l) 85% yield. dH
(300 MHz, CDCl3) 5.81 (1H, m, NH), 4.47–4.68 (1H, m,
Ala a-CH), 3.46–3.74 (2H, m, NCH2), 3.08 and 2.89 (2
 s, rotamers, N–CH3), 2.49–2.70 (2H, m, CH2CO),
N-tBoc-l-Tyr(O-tBu)-b-Ala (9e) 99% yield. dH
(300 MHz, CDCl3) 6.37 (1H, br, NH), 7.01 (2H, d,
J=8.3 Hz), 6.83 (2H, d, J=8.3 Hz), 5.94 (1H, br, NH),
4.39 (1H, br, Tyr a-CH), 3.30 (2H, br, NCH2), 2.96 and
2.73 (2 Â 1H, m, Tyr b-CH2), 2.22 (2H, m, CH2CO),
1.35 (9H, 2 Â s, Bu), 1.25 and 1.21 (2 Â d, J=6.8 Hz,
t
Ala CH3) ppm; dC (75 MHz, CDCl3) 175.0, 173.8, 155.8,
79.9, 47.8, 46.7, 45.6, 45.2, 36.3, 34.1, 33.3, 32.3, 28.6,
18.6, 18.1 ppm; m/z (FAB) 275 (MH+).
1.24 (9H, s, Bu), 1.21 (9H, s, Bu) ppm; dC (75 MHz,
CDCl3) 178.9, 171.7, 156.3, 154.6, 132.5, 130.2, 124.6,
80.0, 78.7, 56.1, 38.9, 36.6, 29.1, 28.7 ppm; m/z (FAB)
409 (MH+).
Preparation of 50 - N - (N - Boc - aminoacyl - ꢀ - alanyl)-50-
amino-20-O,30-O-di(tert-butyldimethylsilyl) 50-deoxyuri-
dines (10a–l). N-Boc-aminoacyl-b-alanine (9a–l) was
coupled with 50-amino-20-O-,30-O-di(tert-butyldimethylsi-
lyl) 50-deoxyuridine (7), using coupling method B or E.
See Table 3 for 13C NMR data.
t
t
N-tBoc-l-Tyr(O-tBu)-N-methyl-b-Ala (9f) 99% yield.
d
H (300 MHz, CDCl3) 7.03 (2H, d, J=8.3 Hz), 6.79 (2H,
d, J=8.3 Hz), 5.84 (1H, d, J=6.9 Hz, NH), 4.70 (1H, m,
Tyr a-CH), 3.25–3.60 (2H, m, NCH2), 2.76–2.92 (2H,
m, Tyr b-CH2), 2.65 (3H, s, NCH3), 2.35–2.51 (2H, m,
CH2CO), 1.32 (9H, s, Bu), 1.21 (9H, s, Bu) ppm; dC
(75 MHz, CDCl3) 174.8, 173.9, 172.8, 155.7, 154.5,
131.6, 130.2, 124.6, 80.1, 78.8, 52.0, 50.4, 45.6, 45.1,
39.6, 39.4, 36.3, 33.5, 33.2, 32.2, 29.1, 28.6 ppm; m/z
(FAB) 423 (MH+).
Method B (isopropenyl chloroformate mixed anhydride
coupling). Preparation of 50-N-(N-Boc-L-Phe-ꢀ-Ala-)-50-
amino-20-O-,30-O-di(tert-butyldimethylsilyl) 50-deoxyuri-
dine (10a). N-tBoc-l-Phe-b-Ala (9a) (183 mg, 561 mmol)
was dissolved in anhydrous THF (2.0 mL) and neu-
tralised with N-methyl morpholine (62 mL, 561 mmol).
The solution was cooled to 0 ꢀC and isopropenyl-
chloroformate (62 mL, 561 mmol) was added, and the
resulting suspension stirred for 4 min. A solution of 50-
amino-20,30-O-bis(tert-butyldimethyl silyl)-50-deoxyuri-
dine (8) (265 mg, 561 mmol) and triethylamine (78 mL,
561 mmol) in anhydrous THF (2.0 mL) was added, and
the suspension stirred at 0 ꢀC for 15 min, then room
temperature for 2 days. The solvent was removed in
vacuo, and the residue taken up in ethyl acetate
(50 mL), and washed with water (20 mL), 5% potassium
hydrogen sulphate solution (20 mL), 5% sodium bicar-
bonate solution (20 mL), water (20 mL), and brine
(20 mL). The organic phase was dried (Na2SO4), and
concentrated in vacuo. The residue was taken up in 3%
MeOH/DCM and purified by flash column chromato-
graphy (3% MeOH/DCM) to give 10a as a white foam,
214 mg, 48%. dH (300 MHz, CDCl3) 7.29 (1H, d,
J=8.2 Hz, uracil H-6), 7.08–7.22 (5H, m, Ar), 6.89 (1H,
br, NH), 5.69 (1H, d, J=8.2 Hz, uracil H-5), 5.42 (1H,
br, NH), 5.37 (1H, br, H-10), 4.42 (1H, br, H-20), 4.20
(1H, br, Phe a-CH), 4.05 (1H, br, H-40), 3.89 (1H, br,
H-30), 3.58 (1H, m, H-50), 3.31 (3H, br, H-50 and b-Ala
NCH2), 2.96 (2H, br, Phe b-CH2), 2.26 (2H, br, b-Ala
CH2CO), 1.29 (9H, s), 0.83 (9H, s), 0.78 (9H, s), 0.04
(3H, s), À0.01 (3H, s), À0.02 (3H, s), À0.04 (3H, s)
ppm; m/z (FAB) 790 (MH+);HRMS 790.4243, calcd
790.4243.
t
t
N-tBoc-Gly-b-Ala (9g) 72% yield. dH (300 MHz,
CD3OD) 3.71 (2H, s, Gly a-CH2), 3.45 (2H, t,
J=6.9 Hz, NCH2), 2.43 (2H, t, J=6.9 Hz, CH2CO),
t
1.47 (9H, s, Bu) ppm; dC (75 MHz, CD3OD) 178.5,
172.8, 158.8, 81.2, 45.1, 37.5, 29.1 ppm; m/z (FAB) 247
(MH+).
N-tBoc-Gly-N-methyl-b-Ala (9h) 87% yield. dH
(300 MHz, CDCl3) 5.69 (1H, br, NH), 3.89 and 3.76 (2
 d, rotamers, J=6.8 Hz, Gly a-CH2), 3.35–3.51 (2H, m,
NCH2), 2.87, 2.75 (2 Â s, rotamers, NCH3), 2.40 (2H, m,
t
CH2CO), 1.24 (9H, s, Bu) ppm; dC (75 MHz, CDCl3)
174.5, 173.4, 169.5, 156.6, 79.9, 46.4, 45.0, 42.6, 42.2,
35.3, 33.5, 32.7, 32.3, 28.5 ppm; m/z (FAB) 261 (MH+).
N-tBoc-b-Ala-b-Ala (9i) 97% yield. dH (300 MHz,
CDCl3) 7.05 (1H, br, NH), 5.48 (1H, br, NH), 3.43 (2H,
q, J=6.9 Hz, NCH2), 3.29 (2H, q, J=6.9 Hz, NCH2),
2.52 (2H, t, J=6.9 Hz, CH2CO), 2.32 (2H, t, J=6.9 Hz,
t
CH2CO), 1.34 (9H, s, Bu) ppm; dC (75 MHz, CDCl3)
175.6, 172.7, 156.9, 80.0, 38.8, 37.3, 35.4, 34.1,
28.7 ppm; m/z (ES+) 261 (MH+).
N-tBoc-b-Ala-N-methyl-b-Ala (9j) 99% yield. dH
(300 MHz, CDCl3) 5.57 (1H, br, NH), 3.56 (2H, m,
NCH2), 3.32 (2H, m, NCH2), 2.99, 2.88 (2 Â s, rota-
mers, NCH3), 2.45–2.62 (4H, m, CH2CO), 1.36 (9H, s,
50-N-(N-Boc-d-Phe-b-Ala-)-50-amino-20-O-,30-O-di(tert-
butyldimethylsilyl) 50-deoxyuridine (10c) was prepared

3092
N. I. Howard, T. D. H. Bugg / Bioorg. Med. Chem. 11 (2003) 3083–3099
by method B from acid 9c in 56% yield. dH (300 MHz,
CDCl3) 7.08–7.26 (6H, m, Ar and uracil H-6), 6.69 (1H,
br, NH), 5.67 (1H, d, J=8.2 Hz, uracil H-5), 5.24 (1H,
br, H-10), 5.17 (1H, br, NH), 4.50 (1H, br, H-20), 4.19
(1H, br, Phe a-CH), 3.99 (1H, m, H-40), 3.83 (1H, m, H-
30), 3.55 (1H, m, H-50), 3.20–3.48 (3H, br, H-50 and b-
Ala NCH2), 2.93 (2H, br, Phe b-CH2), 2.28 (2H, br, b-
Ala CH2CO), 1.30 (9H, s), 0.82 (9H, s), 0.77 (9H, s),
0.02 (3H, s), À0.02 (3H, s), À0.03 (3H, s), À0.06 (3H, s)
ppm; m/z (FAB) 790 (MH+);HRMS 790.4242, calcd
790.4243.
NH), 5.30 and 5.05 (2 Â d, J=2.9 Hz, H-10), 4.72 and
4.40 (2 Â dd, J=2.9, 3.5 Hz, H-20), 3.98–4.10 (1H, m,
H-40), 3.89 (1H, m, H-30), 3.79 (1H, m, H-50), 3.45–3.65
(2H, m, NCH2), 3.15–3.43 (3H, m, H-50 and NCH2),
2.91 and 2.88 (2 Â s, N–CH3), 2.42–2.72 (4H, m,
CH2CO), 1.33 and 1.32 (9H, 2 Â s), 0.82 (9H, s), 0.78
and 0.76 (9H, 2 Â s), À0.01 (3H, s), À0.03 (3H, s),
À0.03 (3H, s), À0.06 (3H, s) ppm; m/z (FAB) 728
(MH+);HRMS 728.4086, calcd 728.4086.
50-N-(N-Boc-l-Ala-b-Ala-)-50-amino-20-O-,30-O-di(tert-
butyldimethylsilyl) 50-deoxyuridine (10k) was prepared
by method E from acid 9k in 48% yield. dH (300 MHz,
CDCl3) 9.92 (1H, br, NH), 7.37 (1H, d, J=8.2 Hz, ura-
cil H-6), 7.14 (1H, br, NH), 6.96 (1H, br, NH), 5.72
(1H, d, J=8.2 Hz, uracil H-5), 5.39 (1H, d, J=2.9 Hz,
H-10), 5.30 (1H, br, NH), 4.42 (1H, br, H-20), 3.96–4.09
(2H, m, H-40 and Ala a-CH), 3.88 (1H, m, H-30), 3.43–
3.72 (2H, m, H-50), 3.20–3.39 (2H, m, b-Ala NCH2),
2.36 (2H, t, J=6.9 Hz, CH2CO), 1.33 (9H, s), 1.26 (3H,
d, J=6.9 Hz, Ala CH3), 0.83 (9H, s), 0.79 (9H, s), À0.01
(3H, s), À0.03 (3H, s), À0.03 (3H, s), À0.06 (3H, s)
ppm; m/z (FAB) 714 (MH+);HRMS 714.3929, calcd
714.3930.
50-N-(N-Boc-l-Tyr(O-tBu)-b-Ala-)-50-amino-20-O-,30-O-
di(tert-butyldimethylsilyl) 50-deoxyuridine (10e) was
prepared by method B from acid 9e in 66% yield. dH
(300 MHz, CDCl3) 7.31 (1H, d, J=8.2 Hz, uracil H-6),
7.11 (1H, br, NH), 6.99 (2H, d, J=8.3 Hz, Ar), 6.85
(1H, br, NH), 6.80 (2H, d, J=8.3 Hz, Ar), 5.69 (1H, d,
J=8.2 Hz, uracil H-5), 5.43 (1H, br, NH), 5.38 (1H, br,
H-10), 4.42 (1H, br, H-20), 4.18 (1H, br, Tyr a-CH), 4.07
(1H, br, H-40), 3.87 (1H, br, H-30), 3.59 (1H, m, H-50),
3.33 (3H, br, H-50 and b-Ala NCH2), 2.86–2.99 (2H, m,
Tyr b-CH2), 2.26 (2H, br, b-Ala CH2CO), 1.30 (9H, s),
1.22 (9H, s), 0.81 (9H, s), 0.76 (9H, s), 0.01 (3H, s),
À0.02 (3H, s), À0.04 (3H, s), À0.06 (3H, s) ppm; m/z
(FAB) 862 (MH+).
50-N-(N-Boc-l-Ala-N-methyl-b-Ala-)-50-amino-20-O-,30-
O-di(tert-butyldimethylsilyl) 50-deoxyuridine (10l) was
prepared by method B from acid 9l in 46% yield. dH
(300 MHz, CDCl3) 9.69 (1H, br, NH), 7.29 and 7.11 (2
 d, J=8.2 Hz, rotamers, uracil H-6), 7.17 (1H, br,
NH), 5.69 (2H, m, uracil H-5, and NH), 5.38 and 5.07
(2 Â d, J=4.9 Hz, H-10), 4.78, and 4.47 (2 Â t,
J=6.1 Hz, Ala a-CH), 4.67 and 4.34 (2 Â t, J=5.0 Hz,
H-20), 3.80–4.13 (3H, m, H-30, H-40, H-50), 3.30–3.72
(3H, m, H-50 and NCH2), 3.03 and 2.90 (2 Â s, N–CH3),
2.34–2.58 (2H, m, CH2CO), 1.42 and 1.37 (9H, 2 Â s),
1.25 and 1.19 (3H, 2 Â d, J=6.8 Hz, Ala CH3), 0.83
(9H, s), 0.78 (9H, s), À0.01 (3H, s), À0.03 (3H, s), À0.06
(3H, s) ppm; m/z (FAB) 728 (MH+);HRMS 728.4086,
calcd 728.4086.
50-N-(N-Boc-l-Tyr(O-tBu)-N-methyl-b-Ala-)-50-amino-
20-O-,30 -O-di(tert-butyldimethylsilyl) 50 -deoxyuridine
(10f) was prepared by method E from acid 9f in 60%
yield. dH (300 MHz, CDCl3) 10.42 (NH), 7.33 (2H, m,
uracil H-6, NH), 7.01 (2H, d, J=8.2 Hz, Ar), 6.79 (2H,
d, J=8.2 Hz, Ar), 5.79 (1H, d, J=6.1 Hz, NH), 5.69
(1H, d, J=8.1 Hz, uracil H-5), 5.48 (1H, d, J=2.7 Hz,
H-10), 4.62 (1H, m, H-20), 4.22 (1H, t, J=4.1 Hz, Tyr a-
CH), 4.02 (1H, br, H-40), 3.89 (1H, br, H-30), 3.29–3.47
(4H, br, H-50, b-Ala NCH2), 2.82 (2H, m, Tyr b-CH2),
2.79 and 2.62 (2 Â s, rotamers, N–CH3), 2.38 (2H, br, b-
Ala CH2CO), 1.32 (9H, s), 1.22 (9H, s), 0.84 (9H, s),
0.79 (9H, s), À0.01 (3H, s), À0.03 (3H, s), À0.04 (3H, s),
À0.07 (3H, s) ppm; m/z (FAB) 786 (MH+).
Preparation of 50-N-(aminoacyl-ꢀ-alanyl-)-50-amino-50-
deoxyuridines (11a–l). General procedure. The fully pro-
tected 50-uridinyl-dipeptide (10a–l) was dissolved in either
(a) THF (3mL/100 mmol), and tetrabutylammonium
fluoride (1 M in THF, 1.5 equivalents) added, or (b)
acetonitrile (3 mL/100 mmol), and tetraethylammonium
fluoride (0.5 M in MeCN, 1.5 equivalents) added, and
the solution stirred for 16–20 h. The solvents were
removed in vacuo, and the residue taken up in 10%
MeOH/DCM, and purified by flash column chromato-
graphy (10% MeOH/DCM) to give the desilylated pro-
duct. The N-tBoc protected 50-uridinyl-dipeptides were
then dissolved in 50% aqueous formic acid (ꢁ10 mL/
mmol) and stirred at room temperature for 16 h. The
resulting solutions were applied to an ion-exchange
chromatography column (Dowex1 50WX8-100, 3 Â
1 cm) and eluted with 50% aqueous formic acid (3 Â
5 mL), 3% ammonia solution (3 Â 5 mL), and 5%
ammonia solution (4 Â 5 mL). The fractions that con-
tained compounds that were both UV active and nin-
hydrin positive were combined and freeze-dried to give
the free amine product. See Table 4 for 13C NMR data.
50-N-(N-Boc-b-Ala-b-Ala-)-50-amino-20-O-,30-O-di(tert-
butyldimethylsilyl) 50-deoxyuridine (10i) was prepared
by method B from acid 9i in 35% yield. dH (300 MHz,
CDCl3) 9.39 (1H, br, NH), 7.20 (1H, d, J=8.2 Hz, ura-
cil H-6), 7.09 (1H, br, NH), 6.55 (1H, br, NH), 5.69
(1H, d, J=8.2 Hz, uracil H-5), 5.21 (1H, br, NH), 5.18
(1H, d, J=4.8 Hz, H-10), 4.53 (1H, t, J=4.7 Hz, H-20),
4.02 (1H, m, H-40), 3.89 (1H, m, H-30), 3.61 (1H, m, H-
50), 3.41 (2H, q, J=6.7 Hz, NCH2), 3.31 (3H, m, H-50
and NCH2), 2.39 (2H, m, CH2CO), 2.30 (2H, t,
J=6.6 Hz, CH2CO), 1.32 (9H, s), 0.83 (9H, s), 0.78 (9H,
s), À0.01 (3H, s), À0.03 (3H, s), À0.03 (3H, s), À0.06
(3H, s) ppm; m/z (FAB) 714 (MH+);HRMS 714.3929,
calcd 714.3930.
50-N-(N-Boc-b-Ala-N-methyl-b-Ala-)-50-amino-20-O-,30-
O-di(tert-butyldimethylsilyl) 50-deoxyuridine (10j) was
prepared by method B from acid 9j in 33% yield. dH
(300 MHz, CDCl3) 9.62 (1H, br, NH), 7.20 (1H, br,
NH), 7.29 and 7.09 (2 Â d, J=8.2 Hz, rotamers, uracil
H-6), 5.69 (1H, d, J=8.2 Hz, uracil H-5), 5.39 (1H, br,

N. I. Howard, T. D. H. Bugg / Bioorg. Med. Chem. 11 (2003) 3083–3099
Table 3. 13C NMR data for 10a–l (75 MHz, CDCl3)
3093
10a
10c
10e
10f
10i
10j
10k
10l
Uracil
C-2
C-4
C-5
151.1
164.0
103.0
143.3
94.4
73.4
73.4
84.4
41.5
18.4
18.3
26.1
À4.0
À4.2
À4.4
ND
172.4
39.1
36.1
151.0
163.5
103.1
143.9
95.8
73.4
72.9
84.9
41.4
18.4
18.3
26.1
À4.0
À4.2
À4.3
À4.4
172.1
39.0
36.1
—
155.8
80.3
28.7
171.8
56.3
36.1
137.2
129.7
124.6
127.2
151.0
164.0
103.0
143.2
94.4
73.4
73.4
84.4
41.5
18.4
18.3
26.1
À4.0
À4.3
À4.4
ND
151.0
164.0
102.9
142.5
93.1
74.0
73.4
84.2
41.6
150.9
163.4
103.1
144.3
96.3
73.4
72.7
85.0
41.3
18.4
18.3
26.1
À4.1
À4.2
À4.3
À4.5
172.5
37.2
36.0
—
150.9
163.7
103.0
143.2
94.9
73.3
71.4
84.5
41.5
18.4
18.3
26.1
À4.2
À4.4
À4.6
151.0
163.8
103.1
143.3
94.7
73.6
73.3
84.4
41.4
18.4
18.3
26.1
À4.0
À4.3
À4.4
151.9
163.4
103.0
142.9
94.1
73.4
72.0
84.4
41.3
18.4
18.4
26.1
À4.0
À4.1
À4.4
C-6
Ribose
C-10
C-20
C-30
C-40
C-50
Si-tBu
18.3
18.2
26.1
À4.0
Si-Me
À4.1
À4.4
ND
172.3
34.7
b-Ala
C¼O
a-C
b-C
N-Me
C¼O
t-Bu
172.4
38.4
36.1
—
171.9
36.7
47.4/45.3
36.4/33.8
156.6
79.9
28.8
171.7
36.1/35.1
34.2/33.2
172.7
36.4
36.4
—
155.9
80.3
28.8
173.6
50.8
18.9
173.2
37.0/34.9
47.1/46.1
36.6/34.4
155.8
79.9
28.7
170.5
46.9/46.6
20.9/18.7
46.7/46.3
36.4/34.8
155.8
80.4
—
Boc
AA
155.8
80.3
28.7
172.2
56.5
36.0
137.1
129.6
124.6
128.9
155.8
80.2
28.7
172.2
56.6
36.1
132.0
130.1
124.6
154.4
78.7
29.2
156.5
80.3
28.8
172.0
36.7
35.9
28.7
C¼O
a-C
b-C
171.8
52.2/51.9
39.6/39.3
131.7
130.2
124.6
154.6
78.8
29.1
50-N-(l-Phe-b-Ala-)-50-amino-50-deoxyuridine (11a) was
obtained as a white foam (48 mg, 46%). dH (300 MHz,
D2O) 7.42 (1H, d, J=8.2 Hz, uracil H-6), 6.96–7.23 (5H,
m, Ar), 5.63 (1H, d, J=8.2 Hz, uracil H-5), 5.54 (1H, d,
J=3.9 Hz, H-10), 4.12 (1H, t, J=4.1 Hz, H-20), 3.99 (1H,
m, H-40), 3.94 (1H, t, J=4.1 Hz, H-30), 3.68 (1H, t,
J=6.6 Hz, Phe a-CH), 3.30–3.48 (3H, m, H-50, NCH2),
3.17 (1H, td, J=6.5, 13.1 Hz, H-50), 2.89 and 2.77 (2 Â
1H, dd, J=6.5, 13.1 Hz, Phe b-CH2), 2.22 (2H, t,
J=6.6 Hz, CH2CO) ppm; m/z (FAB) 462 (MH+);
HRMS 462.1988, calcd 462.1989.
51%). dH (300 MHz, D2O) 7.42 (1H, d, J=8.2 Hz, uracil
H-6), 6.91 (2H, d, J=8.2 Hz, Ar), 6.69 (2H, d,
J=8.2 Hz, Ar), 5.62 (1H, d, J=8.1 Hz, uracil H-5), 5.53
(1H, d, J=1.1 Hz, H-10), 4.10 (1H, dd, J=1.1, 4.2 Hz,
H-20), 4.00 (2H, m, H-40 and Tyr a-CH), 3.89 (1H, t,
J=4.1 Hz, H-30), 3.30–3.48 (3H, m, H-50 and NCH2),
3.22 (1H, dt, J=6.5, 13.1 Hz, H-50), 2.96 and 2.82 (2 Â
1H, dd, J=4.0, 10.3 Hz, Tyr b-CH2), 2.33 (2H, t,
J=6.6 Hz, CH2CO) ppm; m/z (FAB) 478 (MH+);
HRMS 478.1938, calcd 478.1938.
50-N-(b-Ala-b-Ala-)-50-amino-50-deoxyuridine (11i) was
obtained as a white foam (6 mg, 43%). dH (300 MHz,
D2O) 7.54 (1H, d, J=8.1 Hz, uracil H-6), 5.74 (1H, d,
J=8.2 Hz, uracil H-5), 5.63 (1H, d, J=2.7 Hz, H-10),
4.23 (1H, t, J=2.9 Hz, H-20), 3.97 (2H, m, H-30 and H-
40), 3.42 (2H, m, H-50), 3.33 (2H, t, J=6.8 Hz, NCH2),
3.11 (2H, m, NCH2), 2.54 (2H, t, J=6.8 Hz, CH2CO),
2.38 (2H, t, J=6.7 Hz, CH2CO) ppm; m/z (FAB) 386
(MH+).
50-N-(d-Phe-b-Ala-)-50-amino-50-deoxyuridine (11c) was
obtained as a white foam (18 mg, 67%). dH (300 MHz,
D2O) 7.47 (1H, d, J=8.2 Hz, uracil H-6), 7.02–7.28 (5H,
m, Ar), 5.68 (1H, d, J=8.1 Hz, uracil H-5), 5.58 (1H, d,
J=4.0 Hz, H-10), 4.13 (1H, t, J=4.1 Hz, H-20), 3.96 (2H,
m, H-30 and H-40), 3.88 (1H, t, J=6.5 Hz, Phe a-CH),
3.39 (3H, m, H-50 and NCH2), 3.19 (1H, dt, J=6.5,
11.8 Hz, H-50), 2.92 (2H, m, Phe b-CH2), 2.31 (2H, m,
CH2CO) ppm; m/z (FAB) 462 (MH+).
50-N-(b-Ala-N-methyl-b-Ala-)-50-amino-50-deoxyuridine
(11j) was obtained as a white solid (12 mg, 39%). dH
(300 MHz, D2O) 7.54 (1H, d, J=8.1 Hz, uracil H-6), 5.74
(1H, d, J=8.1 Hz, uracil H-5), 5.64 (1H, d, J=1.9 Hz, H-
10), 4.26 (1H, dd, J=2.0, 5.9 Hz, H-20), 3.98 (2H, m, H-30
and H-40), 3.35–3.60 (4H, m, H-50 and NCH2), 3.10 (2H,
m, NCH2), 2.89 and 2.78 (2 Â s, N–CH3), 2.68 and 2.49
(2 Â t, J=6.8 Hz, CH2CO), 2.39 (3H, t, J=6.6 Hz,
CH2CO) ppm; m/z (FAB) 400 (MH+).
50-N-(l-Tyr-b-Ala-)-50-amino-50-deoxyuridine (11e). For
this compound, the desilylated N-tBoc-O-tert-butyl
ether protected compound was dissolved in 95% tri-
fluoroacetic acid (10 mL/mmol) and stirred at 0 ꢀC for
30 min. The solution was diluted with water (30 mL/
mmol) and freeze-dried. The residue was dissolved in
50% formic acid and purified by ion-exchange chroma-
tography as above, to give 11e as a white foam (7 mg,

3094
N. I. Howard, T. D. H. Bugg / Bioorg. Med. Chem. 11 (2003) 3083–3099
50-N-(l-Ala-b-Ala-)-50-amino-50-deoxyuridine (11k) was
obtained as a white foam (18 mg, 30%). dH (300 MHz,
D2O) 7.58 (1H, d, J=8.2 Hz, uracil H-6), 5.79 (1H, d,
J=8.1 Hz, uracil H-5), 5.68 (1H, d, J=2.9 Hz, H-10),
4.23 (1H, t, J=3.0 Hz, H-20), 4.01 (2H, m, H30 and H40),
3.87 (1H, q, J=6.8 Hz, Ala a-CH), 3.24–3.54 (4H, m,
H-50 and NCH2), 2.39 (2H, t, J=6.6 Hz, CH2CO), 1.32
(3H, d, J=6.8 Hz, Ala CH3) ppm; m/z (FAB) 386
(MH+);HRMS 386.1676, calcd 386.1676.
dried (Na2SO4), and concentrated in vacuo. The residue
was purified by flash column chromatography (3%
MeOH/DCM) to give the coupled product. See Table 4
for 13C NMR data.
50-O-(N-Boc-l-Phe-b-Ala)-20 -O-,30 -O-isopropylidine
uridine (12a) was obtained as a colourless oil (495 mg,
72%). dH (300 MHz, CDCl3) 10.32 (1H, br, NH), 7.09–
7.26 (6H, m, uracil H-6, Ar), 6.77 (1H, br, NH), 5.68
(1H, d, J=8.2 Hz, uracil H-5), 5.50 (1H, s, H-10), 5.44
(1H, d, J=8.0 Hz, NH), 5.04 (1H, d, J=4.9 Hz, H-20),
4.78 (1H, t, J=4.9 Hz, H-30), 4.11–4.40 (4H, m), 3.40
(2H, m, NCH2), 2.94 (2H, d, J=6.2 Hz, Phe b-CH2),
2.39 (2H, m, CH2CO), 1.49 (3H, s), 1.32 (9H, s), 1.29
(3H, s) ppm; m/z (FAB) 603 (MH+);HRMS 603.2665,
calcd 603.2666.
50-N-(l-Ala-N-methyl-b-Ala-)-50-deoxy-uridine
(11l)
was obtained as a white foam (43 mg, 68%). dH
(300 MHz, D2O) 7.53 (1H, d, J=8.1 Hz, uracil H-6),
5.78 (1H, d, J=8.2 Hz, uracil H-5), 5.65 (1H, d,
J=1.7 Hz, H-10), 4.21 (1H, m, H-20), 4.18 (1H, m, Ala
a-CH), 3.92 (2H, m, H-30 and H-40), 3.64 (1H, q,
J=6.9 Hz, H-50), 3.24–3.50 (3H, m, H-50 and NCH2),
2.91 and 2.79 (2 Â s, rotamers, N–CH3), 2.52 and 2.39
(2 Â t, J=6.8 Hz, CH2CO), 1.23 and 1.22 (2 Â d,
J=6.8 Hz, Ala CH3) ppm; m/z (FAB) 400 (MH+);
HRMS 400.1832, calcd 400.1852.
50-O-(N-Boc - l - Phe - N - methyl - b - Ala)-20-O-,30-O-iso-
propylidine uridine (12b) was obtained as a colourless
oil (458 mg, 74%). dH (300 MHz, CDCl3) 10.42 (1H, br,
NH), 7.28 (1H, d, J=8.2 Hz, uracil H-6), 7.05–7.25 (5H,
m, Ar), 5.58–5.79 (3H, m), 5.09 and 4.99 (1H, d,
J=5.3 Hz, rotamers, H-20), 4.82 (1H, H-30), 4.72 (1H, q,
J=6.1 Hz, Phe a-CH), 4.01–4.32 (3H, m, H-40 and H-
50), 3.51 and 3.40 (2H, m, NCH2), 2.89 (2H, m, Phe b-
CH2), 2.79 and 2.66 (2 Â s, N–CH3), 2.45 (2H, m,
CH2CO), 1.49 (3H, s), 1.31 and 1.28 (9H, 2 Â s), 1.21
(3H, s) ppm; m/z (FAB) 617 (MH+);HRMS 617.2822,
calcd 617.2823.
Preparation of 50-O-(N-Boc-aminoacyl-ꢀ-alanyl)-20-O-,
30-O-isopropylidine uridines (12a–l). General procedure.
The N-tBoc-protected dipeptide (9a–l) was dissolved in
anhydrous DCM (20 mL/g), neutralised with N-methyl
morpholine (1 equiv), and the resulting solution cooled
to 0 ꢀC. Isopropenylchloroformate (1 equiv) was added,
and the solution stirred for 4 min. A solution of 20, 30-O-
isopropylideneuridine (1 equiv) and 4-dimethyl-amino-
pyridine (5 mg/mmol dipeptide) in anhydrous THF
(10 mL/mmol) was added, and the resulting suspension
stirred for 20 min at 0 ꢀC, then 2 days at room tem-
perature. The solvents were removed in vacuo, and the
residue taken up in ethyl acetate (25 mL/mmol) and
washed with water (20 mL/mmol), 5% potassium
hydrogen sulphate solution (20 mL/mmol), 5% sodium
bicarbonate solution (20 mL/mmol), water (20 mL/
mmol), and brine (20 mL/mmol). The organic phase was
50-O-(N-Boc-d-Phe-b-Ala)-20 -O-,30 -O-isopropylidene
uridine (12c) was obtained as a white foam (19 mg,
49%). dH (300 MHz, CDCl3) 9.64 (1H, br, NH), 7.04–
7.24 (6H, m, uracil H-6, Ar), 6.49 (1H, br, NH), 5.68
(1H, d, J=8.2 Hz, uriacil H-5), 5.42 (1H, d, J=0.8 Hz,
H-10), 5.20 (1H, d, J=7.1 Hz, NH), 5.07 (1H, dd,
J=0.8, 5.8 Hz, H-20), 4.79 (1H, t, J=5.9 Hz, H-30),
4.11–4.31 (4H, m), 3.49 (1H, br), 3.29 (1H, br), 2.92
(2H, m, Phe b-CH2), 2.33 (2H, m, CH2CO), 1.50 (3H,
s), 1.33 (9H, s), 1.27 (3H, s) ppm; m/z (FAB) 603
(MH+);HRMS 603.2665, calcd 603.2666.
50-O-(N-Boc - d - Phe - N - methyl-b-Ala)-20-O-,30-O-iso-
propylidene uridine (12d) was obtained as a white foam
(49 mg, 72%). dH (300 MHz, CDCl3) 10.42 (1H, br, NH),
7.05–7.30 (6H, m, uracil H-6, Ar), 5.70 (2H, m, uracil H-
5, NH), 5.54 (1H, s, H-10), 5.02 (1H, d, J=4.9 Hz, H-20),
4.80 (1H, m, H-30), 4.73 (1H, q, J=5.9 Hz, Phe a-CH),
4.12–4.36 (3H, m), 3.59–3.34 (2H, m, NCH2), 2.88 (2H,
d, J=6.0 Hz, Phe b-CH2), 2.80 and 2.69 (3H, 2 Â s,
rotamers, N–CH3), 2.48 (2H, m, CH2CO), 1.51 (3H, s),
1.34 (9H, s), 1.31 (3H, s) ppm; m/z (FAB) 617 (MH+);
HRMS 617.2824, calcd 617.2822.
Table 4. 13C NMR data for 11a–l (75 MHz, D2O)
11a
11c
11e
11j
11k
11l
Uracil
C-2
C-4
C-5
C-6
151.5 ND
166.5 ND
102.5 102.4 102.4
142.0 142.0 141.8
ND
ND
151.9
166.4
102.6
142.6
91.0
73.7
70.8
82.1
41.1
ND
164.5
102.6
142.6
91.1
73.3
70.9
82.1
41.1
ND
35.3 34.4/33.9
36.2 45.9/45.7
—
ND
49.3 47.1/46.9
16.9 17.5/16.5
152.4
167.3
102.6
142.5
91.0
73.3
70.9
82.0
41.0
Ribose C-10
90.7
73.7
71.0
81.9
41.6
91.0
73.6
71.0
81.6
41.6
90.6
73.8
71.1
82.1
41.9
ND
36.7
39.4 46.3/45.1
—
ND
54.7
36.1
C-20
C-30
C-40
C-50
b-Ala
C¼O 174.2 ND
174.7
30.2
174.5
50-O-(N-Boc - l - Tyr(O - tBu) - b - Ala) - 20 - O - ,30 - O - iso-
propylidene uridine (12e) was obtained as a colourless
oil (70 mg, 21%). dH (300 MHz, CDCl3) 7.21 (1H, d,
J=8.2 Hz, uracil H-6), 7.03 (2H, d, J=8.2 Hz, Ar), 6.83
(2H, d, J=8.2 Hz, Ar), 6.66 (1H, br, NH), 5.67 (1H, d,
J=8.2 Hz, uracil H-5), 5.45 (1H, br, NH), 5.27 (1H, s,
H-10), 5.05 (1H, dd, J=0.8, 6.1 Hz, H-20), 4.79 (1H, t,
J=6.0 Hz, H-30), 4.14–4.34 (4H, m, H40), 3.37 (2H, m,
NCH2), 2.87 (2H, d, J=6.2 Hz, Tyr b-CH2), 2.20–2.47
a-C
b-C
36.0
39.4
—
37.5
38.0
—
N-Me
35.9/33.2
171.3
35.9
35.6/33.6
171.3
AA
C¼O 173.4 ND
a-C
b-C
55.5
35.3
54.7
36.0
34.4
135.6 135.6 131.0
129.5 129.4 116.1
129.2 129.0
127.8 127.4

N. I. Howard, T. D. H. Bugg / Bioorg. Med. Chem. 11 (2003) 3083–3099
3095
(2H, br, CH2CO), 1.50 (3H, s), 1.36 (9H, s), 1.30 (3H, s),
1.23 (9H, s) ppm; m/z (FAB) 675 (MH+).
20), 4.84 (1H, m, H-30), 4.15–4.53 (3H, m), 4.04 and 3.91 (2
 d, J=6.1 Hz, Gly a-CH2), 3.68 and 3.57 (2  m, NCH2),
2.96 and 2.93 (2 Â s, N–CH3), 2.62 (2H, t, J=6.0 Hz,
CH2CO), 1.55 (3H, s), 1.42 (9H, s), 1.33 (3H, s) ppm; m/z
(FAB) 527 (MH+);HRMS 527.2354, calcd 527.2353.
50-O-(N-Boc-l-Tyr(O-tBu)-N-methyl-b-Ala)-20-O-,30-O-
isopropylidene uridine (12f) was obtained as a colour-
less oil (336 mg, 55%). dH (300 MHz, CDCl3) 10.32 (1H,
br, NH), 7.24, 7.20 (1H, d, J=8.2 Hz, uracil H-6), 7.06
(2H, d, J=8.2 Hz, Ar), 6.81 (2H, d, J=8.2 Hz, Ar),
5.59–5.75 (3H, m), 5.02 (1H, d, J=6.2 Hz, H-20), 4.82
(1H, m, H-30), 4.68 (1H, q, J=6.1 Hz, Tyr a-CH), 4.01–
4.35 (3H, m), 3.45 (2H, m, NCH2), 2.84 (2H, m, Tyr b-
CH2), 2.79, 2.60 (3H, 2 Â s, rotamers, N–CH3), 2.48
(2H, m, CH2CO), 1.52 (3H, s), 1.36 (9H, s), 1.29 (3H, s),
1.25 (9H, s) ppm; m/z (FAB) 689 (MH+);HRMS
689.3398, calcd 689.3398.
50-O-(N-Boc-b-Ala-b-Ala)-20 -O-,30 -O-isopropylidene
uridine (12i) was obtained as a white foam (74 mg,
49%). dH (300 MHz, CDCl3) 9.72 (1H, br, NH), 7.21
(1H, d, J=8.1 Hz, uracil H-6), 6.49 (1H, br, NH), 5.69
(1H, d, J=8.1 Hz, uracil H-5), 5.49 (1H, d, J=0.78 Hz,
H-10), 5.22 (1H, br, NH), 5.05 (1H, dd, J=0.8, 5.9 Hz,
H-20), 4.83 (1H, dd, J=4.2, 5.9 Hz, H-30), 4.18–4.34
(3H, m), 3.47 (2H, q, J=6.0 Hz, NCH2), 3.32 (2H, q,
J=6.0 Hz, NCH2), 2.54 (2H, t, J=6.0 Hz, CH2CO),
2.30 (2H, t, J=6.1 Hz, CH2CO), 1.50 (3H, s), 1.37 (9H,
s), 1.31 (3H, s) ppm; m/z (FAB) 527 (MH+);HRMS
527.2352, calcd 527.2353.
50-O-(N-Boc-Gly-b-Ala)-20-O-,30-O-isopropylidene uri-
dine (12g) was obtained as a colourless oil (56 mg,
34%). dH (300 MHz, CDCl3) 9.89 (1H, br, NH), 7.22
(1H, d, J=8.1 Hz, uracil H-6), 6.82 (1H, br, NH), 5.67
(1H, d, J=8.2 Hz, uracil H-5), 5.49 (1H, d, J=0.8 Hz,
H-10), 5.37 (1H, br, NH), 5.08 (1H, dd, J=0.8, 5.0 Hz,
H-20), 4.83 (1H, t, J=4.9 Hz, H-30), 4.18–4.35 (3H, m),
3.70 (2H, d, J=6.1 Hz, Gly a-CH2), 3.47 (2H, q,
J=6.1 Hz, NCH2), 2.53 (2H, t, J=6.1 Hz, CH2CO),
1.49 (3H, s), 1.39 (9H, s), 1.31 (3H, s) ppm; m/z (FAB)
513 (MH+).
50-O-(N-Boc-b-Ala-N-methyl-b-Ala)-20 -O-,30 -O-iso-
propylidene uridine (12j) was obtained as a colourless
oil (73 mg, 20%). dH (300 MHz, CDCl3) 10.05 (1H, br,
NH), 7.23 (1H, d, J=8.1 Hz, uracil H-6), 5.73 (1H, d,
J=8.1 Hz, uracil H-5), 5.60 (1H, d, J=0.8 Hz, H-10),
5.49 (1H, br, NH), 5.12 and 5.09 (1H, dd, J=0.9,
6.2 Hz, H-20), 4.90 (1H, m, H-30), 4.21–4.41 (3H, m),
3.60 (2H, q, J=6.1 Hz, NCH2), 3.39 (2H, q, J=5.9 Hz,
NCH2), 2.95 and 2.89 (2 Â s, rotamers, N–CH3), 2.59
(3H, m), 2.49 (1H, t, J=5.9 Hz, CH2CO), 1.56 (3H, s),
1.43 (9H, s), 1.36 (3H, s) ppm; m/z (FAB) 541 (MH+);
HRMS 541.2510, calcd 541.2509.
50-O-(N-Boc-Gly-N-methyl-b-Ala)-20 -O-,30 -O-iso-
propylidene uridine (12h) was obtained as a white foam
(190 mg, 56%). dH (300 MHz, CDCl3) 10.05 (1H, br,
NH), 7.20 (1H, d, J=8.1 Hz, uracil H-6), 5.72 (1H, d,
J=8.2 Hz, uracil H-5), 5.64 (1H, br, NH), 5.75 (1H, s,
H-10), 5.13 and 5.04 (2 Â dd, rotamers, J=0.7, 6.1 Hz, H-
50-O-(N-Boc-l-Ala-b-Ala)-20 -O-,30 -O-isopropylidene
uridine (12k) was obtained as a colourless oil (85 mg,
Table 5. 13C NMR data for 12a–l (75 MHz, CDCl3)
12a
12b12c
12d
150.5
163.5
103.2
143.6
96.4
84.6
80.8
85.5
64.0
115.1
27.6
25.7
171.7
35.0
39.5
12e
150.6
164.2
102.9
143.5
95.8/95.5
84.8/84.6
81.5/81.3
85.8/85.6
64.6/64.5
114.8
27.5
25.6
172.3
33.1/32.2
45.2/44.9
36.3/34.0
155.5
80.1
28.7
171.6
51.9/51.8
40.4/40.1
136.9
129.8
128.8
127.2
12f
150.7
12g
150.6
164.1
102.9
143.7
96.1/95.5
84.8/84.3
81.4/80.6
85.8/85.3
64.5/63.5
114.7
27.5
25.6
172.5
33.3/32.2
45.2/45.0
36.2/34.0
155.5
80.3
28.7
171.6
52.0/51.6
40.1/39.8
131.7
130.3
124.6
12h
150.6
12i
12j
12k
12l
Uracil
C-2
C-4
150.8
164.1
103.1
143.6
95.9
84.6
80.9
85.5
64.0
114.9
27.5
25.8
171.9
34.9
39.3
150.6
164.2
102.9
149.9
163.5
102.3
150.5
163.9
103.1
143.5
96.3
84.8
81.5
85.9
64.3
115.0
27.5
25.7
172.3
35.3
37.1
—
150.5
164.0
103.0
150.7
164.1
103.1
143.5
96.0
84.8
81.3
85.8
64.3
114.9
27.5
25.7
173.5
34.4
35.2
—
150.4
163.9
103.0
163.9
103.1
143.4
95.9
84.6
80.9
85.5
64.0
114.9
27.6
25.7
172.0
35.0
38.7
—
155.8
80.9
28.7
171.9
56.1
34.5
132.0
130.1
124.6
154.6
78.8
29.1
164.0
103.1
143.5
96.2
84.8
81.4
85.9
64.3
115.0
27.5
25.7
172.3
35.2
34.4
—
C-5
C-6
143.7
143.3
143.7
143.7
Ribose
C-10
96.0/95.4
84.8/84.4
81.5/80.9
85.9/85.4
64.6/63.8
114.8
27.5
25.6
172.4
33.2/32.2
45.2/45.0
36.2/32.2
155.5
80.2
28.7
171.6
95.9/95.1
84.2/83.7
80.7/80.1
85.2/84.7
63.8/63.2
114.2
26.9
25.0
171.1
32.4/32.0
42.2/41.9
34.7/33.1
155.8
79.7
28.1
96.7/95.7
84.9/84.6
81.4
86.0/85.9
64.6/64.3
114.8
27.5
25.7
172.3
34.1/33.3
45.4/44.6
36.3/33.5
156.5
79.5
28.8
171.8
32.7
36.7/36.5
96.4/95.7
84.8/84.3
81.3/80.5
85.9/85.4
64.3/63.6
115.0
27.6
25.7
173.4
33.6/32.4
45.4/45.1
36.3/34.1
155.8
80.4
28.8
171.7
46.8/46.5
20.1/19.2
C-20
C-30
C-40
C-50
Isoprop
b-Ala
C¼O
a-C
b-C
N-Me
C¼O
t-Bu
—
—
Boc
AA
155.8
80.6
28.7
ND
56.1
155.8
80.8
28.7
171.8
56.2
34.4
137.1
129.6
129.0
127.3
156.5
80.6
28.7
170.2
44.5
156.5
80.6
28.8
172.3
34.6
36.6
155.8
80.4
28.7
172.2
50.3
19.1
C¼O
a-C
170.0
44.4/43.9
51.9/51.7
40.5/40.1
136.9
129.8
128.7
127.2
b-C
34.4
137.1
129.7
128.9
127.2
154.5
78.8
29.1

3096
N. I. Howard, T. D. H. Bugg / Bioorg. Med. Chem. 11 (2003) 3083–3099
25%). dH (300MHz, CDCl3) 10.17 (1H, br, NH), 7.29
(1H, d, J=8.1 Hz, uracil H-6), 6.95 (1H, br, NH), 5.68
(1H, d, J=8.1 Hz, uracil H-5), 5.54 (1H, d, J=0.8Hz, H-
10), 5.34 (1H, br, NH), 5.04 (1H, dd, J=0.9, 5.9 Hz, H-20),
4.81 (1H, dd, J=6.0 Hz, H-30), 4.19–4.30 (3H, m), 4.09
(1H, br, Ala a-CH), 3.46 (2H, q, J=5.9 Hz, NCH2), 2.53
(2H, t, J=6.0 Hz, CH2CO), 1.51 (3H, s), 1.37 (9H, s),
1.32 (3H, s), 1.28 (3H, d, J=5.9Hz, Ala CH3) ppm; m/z
(FAB) 527 (MH+);HRMS 527.2353, calcd 527.2354.
7.52 (1H, d, J=8.2 Hz, uracil H-6), 7.04–7.26 (5H, m,
Ar), 5.68 (2H, m, H-10 and uracil H-5), 4.55 (1H, t,
J=6.1 Hz, Phe a-CH2), 4.21–4.32 (3H, m), 4.02–4.20
(2H, m), 3.79 and 3.23 (2 Â 1H, td, J=5.9, 11.9 Hz,
NCH2), 3.02 and 2.96 (2H, d, J=6.20 Hz, Phe b-CH2),
2.79 and 2.73 (3H, 2 Â s, rotamers, N–CH3), 2.52 (2H,
t, J=5.9 Hz, CH2CO) ppm; m/z (FAB) 477 (MH+);
HRMS 477.1986, calcd 477.1985.
50-O-(Gly-b-Ala) uridine (13f) was obtained as a pale
yellow solid (29 mg, 99%). dH (300 MHz, D2O) 7.60
(1H, d, J=8.1 Hz, uracil H-6), 5.76 (1H, d, J=8.1 Hz,
uracil H-5), 5.71 (1H, d, J=3.8 Hz, H-10), 4.23–4.32
(3H, m, H-20 and H-50), 4.12–4.19 (2H, m, H-30 and H-
40), 3.63 (2H, s, Gly a-CH2), 3.40 (2H, t, J=6.5 Hz,
NCH2), 2.56 (2H, t, J=6.5 Hz, CH2CO) ppm; m/z
(FAB) 373 (MH+);HRMS 373.1360, calcd 373.1359.
50-O-(N-Boc-l-Ala-N-methyl-b-Ala)-20 -O-,30 -O-iso-
propylidene uridine (12l) was obtained as a colourless
oil (66 mg, 38%). dH (300 MHz, CDCl3) 9.72 (1H, br,
NH), 7.25 and 7.13 (2 Â d, J=8.1 Hz, rotamers, uracil
H-6), 5.69 and 5.66 (2 Â d, J=8.2 Hz, uracil H-5), 5.54
and 5.51 (2 Â s, H-10), 5.08 and 4.99 (2 Â d, J=6.1 Hz,
H-20), 4.85 (1H, m, H-30), 4.12–4.72 (4H, m), 3.74 and
3.55 (2H, q, J=5.0 Hz, NCH2), 3.02 and 2.90 (2 Â s, N–
CH3), 2.64 and 2.58 (2 Â t, J=6.1 Hz, CH2CO), 1.50
(3H, s), 1.39 and 1.37 (9H, 2 Â s), 1.29 (3H, s), 1.26 and
1.20 (2 Â d, J=6.1 Hz, Ala CH3) ppm; m/z (FAB) 541
(MH+);HRMS 541.2510, calcd 541.2509.
50-O-(Gly-N-methyl-b-Ala) uridine (13g) was obtained
as a white foam (140 mg, 94%). dH (300 MHz, D2O)
7.59 and 7.53 (2 Â d, J=8.1 Hz, rotamers, uracil H-6),
5.69 (2H, m, H-10 and uracil H-5), 4.23 (3H, m, H-20
and H-50), 4.10 (2H, m, H-30 and H-40), 3.98 and 3.82 (2
 s, Gly a-CH2), 3.45–3.58 (2H, m, NCH2), 2.87 and
2.79 (2 Â s, rotamers, N–CH3), 2.67 and 2.58 (2 Â t,
J=6.0 Hz, CH2CO) ppm; m/z (FAB) 387 (MH+);
HRMS 387.1515, calcd 387.1516.
Preparation of 50-O-(aminoacyl-ꢀ-alanyl)-uridines (13a–
l). General procedure. The protected 50-O-uridinyl-
dipeptide (12a–l) was dissolved in 50% aqueous formic
acid (20 mL/mmol) and stirred at room temperature for
20–24 h. The resulting solutions were diluted with water
(100 mL/mmol), and freeze-dried. The residues were
dissolved in water, filtered through a cotton wool plug,
and the filtrate freeze-dried to give the requisite product.
See Table 6 for 13C NMR data.
50-O-(b-Ala-b-Ala) uridine (13h) was obtained as a
white foam (20 mg, 94%). dH (300 MHz, D2O) 7.57 (1H,
d, J=8.1 Hz, uracil H-6), 5.75 (2H, m, H-10 and
uracil H-5), 4.27 (3H, m, H-20 and H-50), 4.11 (2H,
br, H-30 and H-40), 3.33 (2H, t, J=5.9 Hz, NCH2),
3.10 (2H, t, J=5.9 Hz, NCH2), 2.52 (4H, m, CH2CO)
ppm; m/z (FAB) 387 (MH+);HRMS 387.1516, calcd
387.1516.
50-O-(l-Phe-b-Ala) uridine (13a) was obtained as a
white foam (88 mg, 97%). dH (300 MHz, D2O) 7.44 (1H,
d, J=8.1 Hz, uracil H-6), 6.95–7.22 (5H, m, Ar), 5.67
(2H, m, H-10, uracil H-5), 3.92–4.27 (6H, m), 3.40–3.09
(1H, m, NCH2), 2.94 (2H, m, Phe b-CH2), 2.32 (2H, m,
CH2CO) ppm; m/z (FAB) 463 (MH+);HRMS
463.1828, calcd 483.1829.
50-O-(b-Ala-N-methyl-b-Ala) uridine (13i) was obtained
as a white foam (52 mg, 89%). dH (300 MHz, D2O) 7.59
and 7.56 (2 Â d, J=8.1 Hz, rotamers, uracil H-6), 5.82
(2H, m, H-10 and uracil H-5), 4.23 (3H, m, H-20 and H-
50), 4.11 (2H, m, H-30 and H-40), 3.52 (2H, m, NCH2),
3.11 (2H, t, J=5.9 Hz, NCH2), 2.94 and 2.74 (2 Â s,
rotamers, N–CH3), 2.77 and 2.66 (2H, t, J=6.0 Hz,
CH2CO), 2.53 (2H, t, J=6.0 Hz, CH2CO) ppm; m/z
(FAB) 401 (MH+);HRMS 401.1673, calcd 401.1672.
50-O-(l-Phe-N-methyl-b-Ala) uridine (13b) was obtained
as a white foam (322 mg, 84%). dH (300 MHz, D2O)
7.50 (1H, d, J=8.1 Hz, uracil H-6), 7.02–7.22 (5H, m,
Ar), 5.64 (2H, m, H-10, uracil H-5), 4.49 (1H, t,
J=6.1 Hz, Ala a-CH2), 4.11–4.23 (4H, m), 4.06 (1H, t,
J=6.1 Hz, H-40), 3.73 and 3.22 (2 Â 1H, td, J=5.9,
11.9 Hz, NCH2), 2.99 and 2.94 (2H, d, J=6.15 Hz, Phe
b-CH2), 2.74 and 2.69 (2 Â s, rotamers, N–CH3), 2.50
(2H, t, J=6.0 Hz, CH2CO) ppm; m/z (FAB) 477
(MH+);HRMS 477.1985, calcd 477.1985.
50-O-(l-Ala-b-Ala) uridine (13k) was obtained as a
white foam (59 mg, 93%). dH (300 MHz, D2O) 7.55 (1H,
d, J=8.1 Hz, uracil H-6), 5.76 (1H, d, J=8.1 Hz, uracil
H-5), 5.70 (1H, d, J=1.9 Hz, H-10), 4.25 (3H, m, H-20
and H-50), 4.12 (2H, m, H-30 and H-40), 3.87 (1H, q,
J=6.1 Hz, Ala a-CH), 3.39 (2H, m, NCH2), 2.52 (2H, t,
J=6.0 Hz, CH2CO), 1.31 (3H, d, J=6.1 Hz, Ala CH3)
ppm; m/z (FAB) 387 (MH+);HRMS 387.1516, calcd
387.1516.
50-O-(d-Phe-b-Ala) uridine (13c) was obtained as a
white foam (13 mg, 94%). dH (300 MHz, D2O) 7.52 (1H,
d, J=8.2 Hz, uracil H-6), 7.03–7.28 (5H, m, Ar), 5.68
(2H, m, H-10 and uracil H-5), 3.96–4.29 (6H, m), 3.45
and 3.18 (2 Â 1H, td, J=6.1, 12.3 Hz, NCH2), 2.97 (2H,
d, J=6.1 Hz, Phe b-CH2), 2.43 (2H, t, J=6.0 Hz,
CH2CO) ppm; m/z (FAB) 463 (MH+);HRMS
463.1830, calcd 463.1829.
50-O-(l-Ala-N-methyl-b-Ala) uridine (13l) was obtained
as a white foam (79 mg, 84%). dH (300 MHz, D2O) 7.61
and 7.57 (2 Â d, J=8.1 Hz, uracil H-6), 5.79 (1H, d,
J=8.1 Hz, uracil H-5), 5.73 (1H, d, J=1.5 Hz, H-10),
4.30 (4H, m), 4.12 (2H, m, H-30 and H-40), 3.65 and 3.47
(2 Â 1H, td, J=6.0, 12.0 Hz, NCH2), 2.97 and 2.82 (2 Â
50-O-(d-Phe-N-methyl-b-Ala) uridine (13d) was obtained
as a white foam (372 mg, 91%). dH (300 MHz, D2O)

N. I. Howard, T. D. H. Bugg / Bioorg. Med. Chem. 11 (2003) 3083–3099
3097
s, rotamers, N–CH3), 2.70 and 2.58 (2H, t, J=5.9 Hz,
CH2CO), 1.38 and 1.36 (2 Â d, J=6.2 Hz, Ala CH3)
ppm; m/z (FAB) 401 (MH+);HRMS 401.1674, calcd
401.1672.
methyl morpholine (774 mL, 7.04 mmol), and isopro-
penyl chloroformate (385 mL, 3.52 mmol) added. The
resulting suspension was stirred for 2 min, and a solution
of 20, 30-O-isopropylideneuridine (1.00 g, 3.52 mmol) in
anhydrous THF (10 mL) was added. The suspension
was stirred for 2 days. Solvents were removed in vacuo,
and the residue taken up in ethyl acetate (50 mL) and
washed with water (25 mL), 5% potassium hydrogen
sulphate solution (25 mL), 5% sodium bicarbonate
solution (35 mL), and brine (25 mL). The organic phase
was dried (Na2SO4) and concentrated in vacuo. The
residue was taken up in 3% MeOH/DCM and purified
by flash column chromatography (3% MeOH/DCM) to
give 50-O-(N-tBoc-3-piperidinecarboxyl)- 20,30-O-iso-
propylideneuridine as a white solid, 625 mg, 36%. dH
(300 MHz, CDCl3) 10.29 (1H, br, NH), 7.27 (1H, d,
J=8.1 Hz, uracil H-6), 5.68 (1H, d, J=8.1 Hz, uracil H-
5), 5.60 (1H, s, H-10), 4.99 (1H, d, J=6.8 Hz, H-20), 4.78
(1H, br, H-30), 4.23 (3H, m, H-40 and H-50), 4.02 (1H,
m, NCH2), 3.84 (1H, d, J=10.6 Hz, NCHequiv), 2.91
(1H, dd, J=8.7, 10.6 Hz, NCHax), 2.72 (1H, td, J=9.1,
0.8 Hz, NCHax), 2.38 (1H, m, CHCO), 1.93 (1H, m),
1.61 (2H, m), 1.48 (3H, s), 1.34 (10H, s), 1.24 (3H, s)
ppm; m/z (FAB) 496 (MH+);HRMS 496.2296, calcd
496.2295.
In the cases of 50-O-(l-Tyr-b-Ala) uridine (13e) and 50-O-
(l-Tyr-b-Ala) uridine (13f), the N-tBoc/O-tert-butyl
ether/isopropylidene protected compound 12e/f was dis-
solved in 95% trifluoroacetic acid (10 mL/mmol) and
stirred at 0 ꢀC for 30min. The solutions were diluted with
water (50 mL/mmol) and freeze-dried. The residues were
dissolved in water, filtered through a cotton wool plug,
and the filtrate freeze-dried to give the requisite product.
50-O-(l-Tyr-b-Ala) uridine (13e) was isolated as a white
foam (30 mg, 92%). dH (300 MHz, D2O) 7.46 (1H, d,
J=8.1 Hz, uracil H-6), 6.88 (2H, d, J=8.2 Hz, Ar), 6.63
(2H, d, J=8.2 Hz, Ar), 5.64 (2H, m, H-10 and uracil H-
5), 4.23 (2H, m, H-50), 4.15 (2H, m, H-20 and H-40), 4.07
(1H, t, J=4.9 Hz, H-30), 3.94 (1H, t, J=6.0 Hz, Tyr a-
CH), 3.48 and 3.10 (2 Â 1H, m, NCH2), 2.89 (2H, m,
Tyr b-CH2), 2.27–2.53 (2H, m, CH2CO) ppm; m/z
(ES+) 479 (MH+);HRMS (FAB) 479.1777, calcd
479.1778.
50-O-(l-Tyr-N-methyl-b-Ala) uridine (15f) was isolated
as a white foam (98 mg, 83%). dH (300 MHz, D2O) 7.40
(1H, d, J=8.1 Hz, uracil H-6), 6.84 (2H, d, J=8.3 Hz,
Ar), 6.56 (2H, d, J=8.3 Hz, Ar), 5.58 (2H, m, H-10 and
uracil H-5), 4.37 (1H, t, J=6.1 Hz, Tyr a-CH), 4.18
(2H, m, H-50), 4.11 (2H, m, H-20 and H-40), 3.96 (1H, m,
H-30), 3.65 and 3.18 (2 Â 1H, td, J=6.0, 12.0 Hz,
NCH2), 2.89 and 2.83 (2H, d, J=6.1 Hz, Tyr b-CH2),
2.65 and 2.60 (3H, 2 Â s, rotamers, N–CH3), 2.43 (2H,
t, J=5.9 Hz, CH2CO) ppm; m/z (FAB) 493 (MH+);
HRMS 493.1935, calcd 493.1934.
The protected 50-ester (100 mg, 202 mmol), was dissolved
in aqueous formic acid (6 mL, 50% v/v) and stirred at
room temperature for 16 h. The solution was diluted
with water (5 mL) and freeze-dried to give 50-O-(3-
piperidinecarboxyl) uridine (14) as a white foam, 72 mg,
99%. dH (300 MHz, D2O) 7.54 (1H, d, J=8.2 Hz, uracil
H-6), 5.75 (1H, d, J=8.2 Hz, uracil H-5), 5.68 (1H, d,
J=0.7 Hz, H-10), 4.29 (3H, m, H-20 and H-50), 4.12 (2H,
br, H-30 and H-40), 3.38 (1H, dd, J=1.3, 13.4 Hz,
NCHequiv), 3.17 (2H, m), 2.89 (2H, m), 1.99 (1H, br),
1.79 (1H, m), 1.63 (2H, m) ppm; dC (75 MHz, D2O)
173.6, 166.4, 151.6, 142.4, 102.5, 91.1, 81.1, 73.5, 69.6,
64.5, 44.3, 44.1, 38.3, 24.7, 20.9 ppm; m/z (FAB) 356
(MH+);HRMS 356.1457, calcd 356.1458.
Preparation of 50-O-(3-piperidinecarboxyl) uridine (14).
1-(tBoc)-3-piperidinecarboxylic acid (807mg, 3.52mmol)
in anhydrous THF (15 mL) was neutralised with N-
Table 6. 13C NMR data for 13a–l (75 MHz, D2O)
13a
13b13c
13d
151.4
166.3
102.5
142.0
90.8
73.6
69.7
81.2
64.4
13e
151.0
166.3
102.5
142.0
91.1/90.7
73.6
69.6
13f
151.5
166.2
102.4
141.8
90.7
73.7
69.7
81.3
64.5
13g
151.4
166.1
102.4
141.7
91.0/90.6
73.7/73.5
69.6/69.4
81.1/80.8
64.4
173.4
31.8
44.9
36.0/33.2
169.3
51.8
35.7
125.1
115.9
131.1
155.6
13h
151.8
13i
151.7
166.4
102.5
142.1
90.7
13j
150.5
13k
151.7
166.6
102.5
142.2
90.9/90.7
73.7/73.5
69.6
81.4/81.3
64.2/64.0
173.9
32.2/30.2
45.3/44.2
35.7/33.0
172.2
29.7
36.0/35.8
13l
151.7
Uracil
C-2
C-4
C-5
151.4
166.2
103.1
141.8
90.6
73.7
69.7
81.3
64.3
151.4
166.3
102.5
151.7
166.5
102.5
166.5
102.5
142.2
90.8
73.6
69.6
81.4
64.0
173.7
33.6
35.4
—
164.6
102.5
142.1
90.7
73.6
69.6
81.4
64.0
173.8
33.7
36.0
—
166.4
102.5
142.1
90.7
73.6
69.6
81.3
64.0
173.6
33.6
35.4
—
C-6
141.9
142.2
Ribose
C-10
C-20
C-30
C-40
C-50
C¼O
a-C
91.1/90.8
73.7/73.5
69.7/69.5
81.3/81.1
64.6/64.0
173.5
31.9/31.7
45.0/44.7
36.1/33.2
169.3
51.9
36.6
133.6
129.7
129.4
128.4
91.1/90.8
73.6/73.5
69.6
81.3
64.1
73.6
69.7/69.6
81.3/81.1
64.0
173.6
31.8
40.3
34.6/32.9
167.0
44.4
81.2
64.3/64.0
173.8
31.9
45.1/44.7
36.0/33.3
169.2
51.9/51.8
36.6
133.6
129.8
129.4
128.4
b-Ala
173.3
35.1
37.2
173.5
35.3
37.3
173.4
35.2
36.4
173.6
32.0/31.8
44.8/44.6
35.3/33.3
170.7
47.4
16.3/15.5
b-C
N-Me
C¼O
a-C
—
—
—
AA
169.2
54.6
33.3
134.0
129.6
129.3
128.2
169.2
54.6
33.5
133.9
129.6
129.4
128.2
169.3
54.5
33.4
125.5
116.0
130.0
155.4
167.2
40.6
166.5
32.2
35.2
171.0
49.3
16.8
b-C

3098
N. I. Howard, T. D. H. Bugg / Bioorg. Med. Chem. 11 (2003) 3083–3099
Preparation of 50-O-((S)-3-amino-2-piperidin-1-yl)acetyl)
uridine (15). 20-[N-7-tBoc-(S)-3-amino-2-oxo-piperidin-
1-yl] acetic acid (87 mg, 320 mmol), prepared from
l-ornithine by the literature methods,28,29 was dissolved
in anhydrous dichloromethane (3 mL), neutralised with
N-methyl morpholine (35 mL, 320 mmol) and cooled to
0 ꢀC. Isopropenyl chloroformate (35 mL, 320 mmol) was
added and the solution stirred for 4 min. A solution of
20,30-O-isopropylideneuridine (91 mg, 320 mmol) and
4-dimethylaminopyridine (3 mg) in anhydrous tetra-
hydrofuran (4 mL) was added. The solution was stirred
for 20 min at 0 ꢀC, than allowed to warm to room tem-
perature and stirred for 2 days. Solvents were removed
in vacuo, and the residue taken up in ethyl acetate
(20 mL), and washed with water (15 mL), 5% potassium
hydrogen sulphate (15 mL), 5% sodium bicarbonate
(15 mL), water (15 mL), and brine (15 mL). The organic
phase was dried (Na2SO4) and concentrated in vacuo.
The residue was purified by flash column chromato-
graphy (3% MeOH/DCM) to give the protected 50-ester
as a colourless oil, 26 mg, 15%.
phosphate, 150 mg phosphatidylglycerol and 25 mM
MgCl2 in 100 mM Tris buffer pH 7.5, and were incu-
bated at 37 ꢀC for 30 min. Assays were stopped by
addition of 50 mL 6 M pyridinium acetate pH 4.5. Lipid-
linked product was extracted into 100 mL n-butanol, and
14C determined by scintillation counting. All assays
were performed in duplicate.% Inhibition was calcu-
lated as {(cpm in the absence of inhibitor À cpm in the
presence of inhibitor)/cpm in the absence of inhibitor}
 100.
Antibacterial testing
Stock 100 mg/mL aqueous solutions of the inhibitor
samples were prepared. 50 mL of sample was placed on a
15 mm round filter paper and the filter paper placed on
a ‘lawn’ of bacterial culture (100 mL of culture at A600
0.1) on a Luria Broth agar plate and incubated at 37 ꢀC
for 16 h. Ampicillin (100 mg/mL) and mureidomycin A
(100, 10, 1 mg/mL) controls were used. Antibacterial
activity was measured by the size of the zone of clear-
ance around the filter paper. The strains used for testing
were E. coli K12, P. putida ATCC 4359 (Gram-negative);
B. subtilis W23 and S. pneumoniae (Gram-positive).
dH (300 MHz, CDCl3) 9.40 (1H, br, NH), 7.21 (1H, d,
J=8.4 Hz, uracil H-6), 5.71 (1H, d, J=8.3 Hz, uracil H-
5), 5.63 (1H, d, J=6.8 Hz, H-10), 5.44 (1H, d, J=5.0 Hz,
NH), 5.00 (1H, m, H-20), 4.79 (1H, m, H-30), 4.33 (1H,
m, H-50), 4.14–4.30 (3H, m), 4.04 (1H, br, pip a-CH),
3.87 and 3.81 (2 Â d, J=13.3 Hz, NCH2CO), 3.30 (2H,
m, NCH2), 2.38 (1H, m), 1.90 (2H, m), 1.71 (1H, m),
1.47 (3H, s), 1.36 (9H, s), 1.28 (3H, s) ppm; m/z (FAB)
539 (MH+);HRMS 539.2352, calcd 539.2353.
Acknowledgements
We would like to thank EPSRC, the Wellcome Trust
(Grant 054393), Dr. Laura Zawadzke and Bristol-
Myers Squibb Pharmaceuticals for financial support,
Dr. Adrian Lloyd (University of Warwick) for assis-
tance with enzyme assays, and Prof. C. Dowson
(Department of Biological Sciences, University of War-
wick) for assistance with antibacterial testing.
The protected 50 ester (20 mg, 37 mmol) was dissolved in
aqueous formic acid (4 mL, 50% v/v) and stirred at
room temperature for 36 h. The solution was diluted
with water (6 mL) and freeze-dried to give 50-O-((S)-3-
amino-2-piperidin-1-yl)acetyl) uridine (15) as a white
foam (16 mg, 98%). dH (300 MHz, D2O) 7.54 (1H, d,
J=8.3 Hz, uracil H-6), 5.79 (1H, d, J=8.3 Hz, uracil H-
5), 5.71 (1H, s, H-10), 3.88–4.45 (8H, m), 3.32 (2H, m,
pip NCH2), 2.21 (1H, m), 1.91 (3H, m) ppm; dC
(75 MHz, D2O) 170.4, 168.2, 166.5, 152.0, 142.3, 102.6,
90.8, 81.3, 73.5, 69.6, 64.6, 50.2, 49.6, 49.4, 25.0,
20.2 ppm; m/z (FAB) 399 (MH+);HRMS 399.1516,
calcd 399.1516.
References and Notes
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Y.;Haneishi, T. J. Antibiot. 1989, 42, 662.
2. Isono, F.;Inukai, M.;Takahashi, S.;Haneishi, T.;
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50-O-((R)-3-Amino-2-piperidin-1-yl)acetyl) uridine (16)
was prepared as above from N-7-tBoc-(R)-3-amino-2-
piperidinone, and was obtained as a white foam (1.6 mg,
3%). dH (300 MHz, D2O) 7.54 (1H, d, J=8.3 Hz, uracil
H-6), 5.79 (1H, d, J=8.3 Hz, uracil H-5), 5.71 (1H, s, H-
10), 3.88–4.45 (8H, m), 3.32 (2H, m, pip NCH2), 2.21
(1H, m), 1.91 (3H, m) ppm; m/z (FAB) 399 (MH+);
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14C-UDPMurNAc-pentapeptide, 10 mM undecaprenyl

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