Synthesis of glucose-templated lysine analogs and incorporation into the antimicrobial dipeptide sequence kW-OBn

Article abstract of DOI:10.1016/j.carres.2008.04.018

The synthesis of two glucose-templated (GlcT) lysine analogs GlcTK and GlcTk in which the side chain of d- and l-lysine (k and K) is conformationally constrained via incorporation into a d-glucose scaffold is described. A key-step in the synthesis is a high yielding, reductive ring opening of an exocyclic glucose-derived epoxide to form a α-hydroxy ester that can be converted into GlcTK and GlcTk. To demonstrate the use of these building blocks in peptide synthesis, we replaced d-lysine in the antimicrobial dipeptide sequence kW-OBn (W = l-tryptophan) and determined the antibacterial activity against various gram-positive and gram-negative organisms. Our results show that the replacement of d-lysine by unprotected GlcTk in dipeptide kW-OBn results in reduced antibacterial activity.

Full text of DOI:10.1016/j.carres.2008.04.018

Available online at www.sciencedirect.com
Carbohydrate Research 343 (2008) 1644–1652
Synthesis of glucose-templated lysine analogs and incorporation
into the antimicrobial dipeptide sequence kW-OBn
Kaidong Zhang,a Dhananjoy Mondal,a George G. Zhanelb and Frank Schweizera,b,
*
aDepartment of Chemistry, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2
bDepartment of Medical Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada R3E 3P4
Received 8 February 2008; received in revised form 9 April 2008; accepted 13 April 2008
Available online 22 April 2008
Abstract—The synthesis of two glucose-templated (GlcT) lysine analogs GlcTK and GlcTk in which the side chain of D- and L-lysine
(k and K) is conformationally constrained via incorporation into a D-glucose scaffold is described. A key-step in the synthesis is a
high yielding, reductive ring opening of an exocyclic glucose-derived epoxide to form a a-hydroxy ester that can be converted into
GlcTK and GlcTk. To demonstrate the use of these building blocks in peptide synthesis, we replaced D-lysine in the antimicrobial
dipeptide sequence kW-OBn (W = L-tryptophan) and determined the antibacterial activity against various gram-positive and gram-
negative organisms. Our results show that the replacement of D-lysine by unprotected GlcTk in dipeptide kW-OBn results in reduced
antibacterial activity.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Carbohydrates; Lysine; Neoglycoconjugates; Unnatural amino acids; Glycoconjugates; Glycopeptides
1. Introduction
tial electrostatic interactions between the positively
charged basic side chains of lysine, arginine, and orni-
thine to the negatively charged lipid membrane of
pathogens, followed by adoption of an amphipathic a-
helical or b-sheet structure.6,7 The ability to kill target
bacteria rapidly, the unusually broad spectrum of activ-
ity against some of the more serious antibiotic resistant
pathogens, and the relative difficulty with which mutants
develop resistance in vitro make AMPs attractive targets
for drug development.6,7
However, in vivo efficacy studies of several cationic
peptide antibiotics have been disappointing most likely
due to the fact that many AMPs exhibit poor bioavail-
ability, susceptibility to proteolytic cleavage, and low
in vivo antimicrobial activity.8 Moreover, the size of
most AMPs is so large that production costs represent
additional concern. To overcome some of these
drawbacks, ultrashort cationic antimicrobial peptides
(UAMPs) and lipopeptides in the form of di-, tri-, and
tetrapeptides have recently emerged as a novel class of
potential antimicrobial drug candidates.9–11 In particu-
lar, the small size and facile preparation reduce produc-
tion costs while the presence of only a few amide bonds
The explosive growth of multi-drug resistant (MDR)
bacteria in hospitals and the community have led to
an emerging crisis where an increasing number of
antimicrobial classes cease to be of clinical usefulness.1
Despite this growing concern, only one new class of
antibiotics, the oxazolidinones, has entered the clinic
during the past two decades.2 Cationic antimicrobial
peptides (AMPs) with their ability to combat MDR
bacteria have been proposed as new antibacterial
agents.3–5 AMPs are defined as peptides containing a
net excess of positively charged residues, approximately
50% hydrophobic residues and a size ranging from 12 to
50 residues.6
Although the mode of action of AMPs is not fully
understood, most AMPs appear to manifest their bio-
logical action by enhancing the permeability of lipid
membranes of bacterial cells. This typically involves ini-
*
Corresponding author. Tel.: +1 2044747012; e-mail: schweize@cc.
umanitoba.ca
0008-6215/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2008.04.018

K. Zhang et al. / Carbohydrate Research 343 (2008) 1644–1652
1645
and the low molecular weight may improve the
2. Results and discussion
pharmacokinetic and pharmacodynamic properties of
UAMPs.
The synthesis started with the readily available D-gluco-
configured lactone 119 (Scheme 1), which reacts with the
enolate of methyl bromoacetate generated from lithium
bis-(trimethylsilyl)amide (LiN(SiMe3)2) in tetrahydrofu-
ran (THF) at ꢀ78 °C, to produce the exocyclic epoxide 2
in 80% yield as a single stereoisomer.17,20 Trimethylsilyl
trifluoromethanesulfonate (TMSOTf)-promoted reduc-
tive ring opening of epoxide 2 with tributyltin hydride
in dichloromethane at 0 °C afforded silylether 3 and
alcohol 5 (ratio: 3:5 = 3.5:1) in a combined yield of
92%. Compounds 3 and 5 were obtained as a single dia-
stereoisomer. Silyl ether 3 was hydrolyzed quantitatively
into alcohol 5 by exposure to trifluoroacetic acid con-
taining wet THF.
The stereochemistry at C-2 of alcohol 5 was deter-
mined by conversion of 5 into the known benzyl ester
4.21 This was achieved in a two-step procedure. First,
methyl ester 5 was hydrolyzed to the corresponding acid
with lithium hydroxide in wet THF. Subsequently, ester-
fication of the acid using cesium carbonate and benzyl
bromide in DMF afforded benzyl ester 4 as a single
product in 89% yield. Hydroxy ester 5 served as the
starting material to install an amino function at C-2.
In situ activation of the hydroxyl group as a trifluoro-
methanesulfonate ester using trifluoromethanesulfonic
anhydride in pyridine followed by nucleophilic displace-
ment with sodium azide–15-crown-5 provided azide 6 in
82% isolated yield. Catalytic hydrogenation of 6 using
Pearlman’s catalyst followed by protection of the amino
function as t-butoxycarbamate provided the protected
amino ester 7 in 90% yield.
In this respect, the potent antimicrobial activity of the
dipeptide D-lysine-L-tryptophan-OBn (kW-OBn) against
Staphylococcus aureus (S. aureus), methicillin-resistant
S. aureus (MRSA), and methicillin-resistant Staphylo-
coccus epidermidis (MRSE) strains attracted our atten-
tion.9 In particular, we were interested in exploring
structure activity relationships of this dipeptide via
replacement of D-lysine (k) by carbohydrate-templated
D-lysine analogs CTLys (Fig. 1).18 CTLys are a novel
class of polyfunctional lysine analogs in which the car-
bohydrate scaffold constrains the side chain while at
the same time introducing molecular diversity such as
poly(hydroxylation) and artificial glycosylation. It is
noteworthy that post-translational hydroxylation of ly-
sine12 and arginine12 and other amino acids13–15 has
been shown to enhance the biological activity of
AMPs.12 Furthermore, CTLys contain the 1,3-hydroxy-
amine binding motifs16 of aminoglycoside antibiotics
that have been proposed to interact as bidentate RNA
hydrogen bond acceptor to the phosphodiester back-
bone or Hoogsteen face of guanosine (Fig. 1).16 More-
over, additional modification and derivatization of the
carbohydrate scaffold may be used to tailor the physical,
chemical, biological, and pharmacodynamic properties
of CTLys-containing peptides.
In this paper, we report the synthesis of two glucose-
templated lysines, GlcTks and GlcTKs (Fig. 1), and de-
scribe the use of these building blocks in peptide synthe-
sis. Moreover, we replaced D-lysine (k) by GlcTk in the
antimicrobial dipeptide kW-OBn and determined the
antibacterial activities against various gram-positive
and gram-negative bacteria. A preliminary report on
the synthesis of glucose-templated lysine analogs with-
out experimental details and biological evaluation ap-
peared recently.17
Compound 7 served as precursor for introducing the
second amino functionality into the carbohydrate scaf-
fold. This was achieved by selective tosylation16 of the
primary hydroxy group using p-toluenesulfonyl chloride
in pyridine followed by nucleophilic displacement of the
NH
2
NH
NH
2
2
NH
2
NH
NH
2
2
O
O
HO
OH
OH
OH
HO
OH
O
O
O
CTLys with 1,3 hydroxyamine
binding motif
carbohydrate-templated
lysine (CTLys)
lysine (K,k)
NH
2
NH
2
NH
NH
2
2
O
O
HO
HO
HO
HO
OH
OH
OH
OH
O
O
D-Glucose-templated
L-lysine (GlcTK)
D-Glucose-templated
D-lysine (GlcTk)
Figure 1. Examples of carbohydrate-templated lysine analogs (CTLys) and glucose-templated lysine analogs GlcTk and GlcTK.

1646
K. Zhang et al. / Carbohydrate Research 343 (2008) 1644–1652
OBn
OBn
OBn
R
2
R
O
1
a
b
O
BnO
BnO
BnO
O
BnO
BnO
R
3
BnO
O
O
OBn
BnO
O
OBn
CO Me
2
2
1
3: R = OTMS; R = H; R = OCH
1
2
3
3
4: R = OH; R = H; R = OBn
c
1
2
3
d
5: R = OH; R = H; R = OCH3
1
2
3
e
OH
OBn
N
3
NHBoc
O
N
g
f
3
R
O
1
O
R
HO
HO
O
2
BnO
BnO
HO
HO
OCH
3
OCH
OCH
3
3
OH
OBn
OH
O
O
7
(1.2:1)
8: R = NHBoc, R = H
6
1
2
9: R = H, R = NHBoc
1
2
NHFmoc
R
1
R
O
2
HO
OH
HO
h
OH
O
10: R = NHBoc, R = H
1
1
2
11: R = H, R = NHBoc
2
Scheme 1. Reagents and conditions: (a) LiHMDS, BrCH2COOMe, THF, ꢀ78 °C, 80%; (b) TMSOTf, Bu3SnH, CH2Cl2, 0 °C, 72%; (c) CF3COOH,
THF–H2O (5:1), rt, quant.; (d) (i) LiOH, THF–H2O (1:1); (ii) Cs2CO3, BnBr, DMF, rt, 89% (over 2 steps); (e) (i) Tf2O, dry pyridine, CH2Cl2, rt; (ii)
NaN3, 15-crown-5, CH2Cl2, rt, 81% (over 2 steps); (f) (i) Pd(OH)2–C, H2 (10 psi), MeOH, rt; (ii) (Boc)2O, TEA, MeOH, 90% (over 2 steps); (g) (i)
TsCl, pyridine, 0 °C to rt, 64%; (ii) NaN3, DMF, 100 °C, 52% (8) and 43% (9); (h) (i) LiOH, THF–H2O (1:1); (ii) Pd(OH)2–C, H2 (10 psi), MeOH, rt;
(iii) Fmoc-OPfp, NaHCO3, acetone–H2O (1:1), rt, 63% (10, over 3 steps) or 65% (11, over 3 steps).
tosylate with sodium azide at elevated temperature in
DMF. It is noteworthy that the displacement of the pri-
mary tosylate by sodium azide does not result in epimer-
ization at C-2 and provides only azide 8, if the reaction
is performed below 80 °C. However, we observed sub-
stantial epimerization at 100 °C affording diastereomeric
azides 8 and 9 in 53% and 42% yields, respectively. Both
diastereomers can be separated by flash chromatogra-
phy. Exposure of esters 8 and 9 to basic conditions
(LiOH, THF–H2O) followed by catalytic hydrogenation
using Pearlman’s catalyst (Pd(OH)2–C, H2, MeOH) and
protection of the primary amino function as 9H-fluore-
nylmethoxycarbamate using 9-fluorenylmethyl pentaflu-
orophenyl carbonate provided the glucose-templated D-
lysine analogs GlucTk 10 and glucose-templated L-ly-
sine analogs GlucTK 11 in 63% and 65% isolated yields,
respectively. Building blocks 10 and 11 are suitably pro-
tected to be used as novel lysine mimetics in solution
phase peptide synthesis.
To demonstrate the use of chimeric glucose-lysine
analogs in peptide synthesis, we decided to convert azide
8 into the di-N-Boc-protected acid 13 (Scheme 2). This
was achieved via a three-step procedure. Reduction of
azide 8 by catalytic hydrogenation followed by protec-
N
3
NHBoc
O
NHBoc
NHBoc
OCH
NHBoc
NHBoc
O
a
HO
HO
b
HO
HO
O
HO
HO
3
OCH
3
OH
OH
OH
O
OH
13
O
O
8
12
c
H N
2
NH
2
O
O
H
N
HO
HO
NHBoc
NHBoc
O
N
R
O
H
N
d
HO
HO
OH
O
R
(CF COOH)
OH
3
3
O
N
H
16: R = NHBn
17: R = OBn
14: R = NHBn
15: R = OBn
Boc
Scheme 2. Reagents and conditions: (a) (i) Pd(OH)2–C, H2 (10 psi), MeOH, rt; (ii) Boc2O, Et3N, MeOH, rt (90%, over 2 steps); (b) LiOH, THF–H2O
(1:1), 0 °C, 90%; (c) TBTU, H-Trp(Boc)-NHBn, DIPEA, DMF, rt, 77% (14) and TBTU, H-Trp(Boc)-OBn, DIPEA, DMF, rt, 83% (15); (d) TFA,
CH2Cl2, rt, quant.

K. Zhang et al. / Carbohydrate Research 343 (2008) 1644–1652
1647
tion using di-t-butyl dicarbonate and triethylamine in
methanol afforded the di-t-butylcarbamate-protected es-
ter 12 in 90% yield. Saponification of ester 12 using lith-
ium hydroxide in aqueous THF provided acid 13 in 90%
yield. To study the influence of the constrained sugar
moiety and the presence of the 1,3-hydroxyamine motifs
on the bioactivity of small antibacterial peptides, we
decided to incorporate acid 13 into the amphiphilic anti-
microbial dipeptide sequences kW-OBn14 and kW-
NHBn. The latter was selected to study how the nature
of the C-terminus influences the antimicrobial activity.
Amidation of 13 was achieved by the coupling of 13
to H-Trp(Boc)-NHBn and H-Trp(Boc)-OBn using 2-
(1H-benzotriazole-1yl)-1,1,3,3-tetra-methyluronium tet-
rafluoroborate (TBTU) as coupling reagent in DMF
to produce protected dipeptides 14 and 15 in 77% and
83% isolated yields, respectively. We did not observe es-
ter formation involving the unprotected hydroxyl
groups during peptide coupling demonstrating no need
for protection of the secondary hydroxyl groups of 13
in peptide synthesis. Exposure of dipeptides 14 and 15
to a 50% mixture of trifluoroacetic acid in dichlorometh-
ane afforded unprotected dipeptides 16 and 17 in quan-
titative yields (Scheme 2).
activity of kW-OBn. For instance, dipeptide kW-OBn
exhibits strong S. aureus activity while this activity is
abolished in peptide kW-NHBn. The reasons for this pe-
culiar behavior are currently not understood.
In summary, we have developed a synthetic pathway
into suitably protected glucose-templated C-glycosidic
lysine analogs with natural and unnatural C(a) configu-
ration. The carbohydrate scaffold induces a conforma-
tional constraint into the side chain of lysine while, at
the same time, introducing artificial post-translational
modifications such as hydroxylation and glycosylation.
To demonstrate the use of carbohydrate-templated ly-
sine analogs in peptide synthesis and to explore the bio-
logical properties, we replaced lysine in the antibacterial
dipeptide kW-OBn by GlcTk. Peptide synthesis was
achieved without the need for hydroxyl group protec-
tion. Determination of the biological activity against
various bacterial strains demonstrated that the replace-
ment of lysine by GlcTk in the dipeptide kw-OBn re-
duces strongly the antibacterial activity. We are
currently studying the lysinemimetic and glycomimetic
properties of GlcTk and GlcTK in other peptides.
With peptides 16 and 17 in hand, we investigated the
antibacterial activity of these peptides against various
bacterial strains and compared them to the known anti-
microbial dipeptide kW-OBn 189 as well as kW-NHBn
19 (Table 1). Our results show that replacement of lysine
by GlcTk reduces the antibacterial activity of kW-OBn
by 4–32-fold against gram-positive cocci and 2–16-fold
versus gram-negative bacilli. It is possible that the low
antibacterial activity of dipeptide 17 is the result of de-
creased amphiphilicity and hydrophobicity. For in-
stance, substitution of D-lysine by GlcTk introduces
three hydroxyl groups. This results in significantly in-
creased hydrophilicity of GlcTk-W-OBn when com-
pared to kW-OBn. Amphiphilicity and hydrophobicity
are crucial for the antibacterial activity of AMPs.5–7
However, other factors such as the bulkiness of the su-
gar fragment or the restricted rotation of the lysine mo-
tif may also contribute to the reduced activity.
Interestingly, we observed that the nature of the C-ter-
minus contributes substantially to the antimicrobial
3. Experimental
3.1. General methods
CH2Cl2 was distilled from calcium hydride. Organic
solutions were concentrated under diminished pressure
at <40 °C (bath temperature). NMR spectra were
recorded at 300 or 500 MHz for 1H and at 75 MHz
for 13C. Chemical shifts are reported relative to CHCl3
(dH 7.26, dc (center of triplet) 77.0 ppm) or to CH3OH
(dH 3.35, dC (center of septet) 49.0 ppm) or to acetone
as internal standard (D2O). TLC was performed on E.
Merck Silica Gel 60 F254 with detection by charring
with 8% H2SO4 acid. Silica gel (0.040–0.063 mm) was
used for column chromatography. Lactone 1 was pur-
chased from Toronto Research Chemicals. Reference
peptides 18 and 19 were prepared according to the liter-
ature procedure9 and obtained as TFA salts.
3.2. (2S)-Methyl 2-trimethylsilyloxy-2-(2,3,4,6-tetra-O-
benzyl-b-D-glucopyranosyl)-ethanoate (3) and (2S)-methyl
2-hydroxy-2-(2,3,4,6-tetra-O-benzyl-b-D-glucopyranos-
yl)-ethanoate (5)
Table 1. Representative minimal inhibitory concentrations (MIC) in
lg/mL for various bacterial strains
Peptide S. aureusa MRSAb MRSEc E. colid P. aeruiginosae
To a mixture of epoxide 221 (480 mg, 0.79 mmol) and
tributyltin hydride (0.84 mL, 3.15 mmol) in CH2Cl2
(15 mL) was added dropwise trimethylsilyl trifluoro-
methanesulfonate (TMSOTf, 0.42 mL, 2.36 mmol) at
0 °C. The mixture was stirred for 30 min at this temper-
ature and quenched with satd aq NaHCO3 (15 mL). The
organic layer was separated and the aqueous phase was
extracted with CH2Cl2 (3 ꢁ 10 mL). The combined
16
17
18
19
256
256
16
>512
256
64
128
128
4
512
512
32
>512
>512
512
512
512
256
512
<512
a ATCC 29213.
b Methicillin-resistant S. aureus (ATCC 33592).
c Methicillin-resistant S. epidermidis (ATCC 14990).
d ATCC 25922.
e ATCC 27853.

1648
K. Zhang et al. / Carbohydrate Research 343 (2008) 1644–1652
organic layers were dried (Na2SO4), concentrated, and
purified by flash column chromatography (hexanes–
EtOAc, 2:1) to afford 3 (388 mg, 72%) and 5 (96 mg,
20%) as a colorless syrup. The trimethylsilyl ether 3
was converted to 5 (337 mg, quant.) by treatment with
trifluoroacetic acid (0.19 mL, 5 equiv) in aq THF
(THF–H2O, 5:1) for overnight. Compound 3: 1H
NMR (300 MHz, CDCl3): d 0.20 (s, 9H), 3.46–3.54
(m, 1H), 3.64 (dd, 1H, J = 9.2, 9.6 Hz), 3.68–3.85 (m,
8H), 4.55 (d, 1H, J = 12.2 Hz), 4.62 (d, 1H, J =
12.2 Hz), 4.63–4.74 (m, 3H), 4.86 (d, 1H, J = 10.9 Hz),
4.88 (d, 1H, J = 10.9 Hz), 4.96 (d, 1H, J = 11.4 Hz),
5.00 (d, 1H, J = 11.4 Hz), 7.22–7.41 (m, 20H); 13C
NMR (75 MHz, CDCl3): d 0.9, 52.0, 68.8, 71.7, 73.2,
74.6, 75.0, 75.6, 77.7, 78.7, 79.9, 80.2, 87.4, 127.3–
128.5 (aromatic carbons), 138.2, 138.5, 138.5, 138.6,
172.4; MS (ES+): m/z 707.42 [M+Na]+; Anal. Calcd
for C40H48O8Si: C, 70.15; H, 7.06. Found: C, 70.25;
H, 7.14; compound 5: ½aꢂD +27.0 (c 1.2, CHCl3); H
NMR (300 MHz, CDCl3): d 2.85 (br s, 1H), 3.46–3.54
(m, 1H), 3.59 (dd, 1H, J = 8.4, 8.6 Hz), 3.63–3.72 (m,
3H), 3.73–3.87 (m, 5H), 4.47–4.59 (m, 3H), 4.61 (d,
1H, J = 11.0 Hz), 4.77 (d, 1H, J = 11.0 Hz), 4.85 (d,
1H, J = 10.8 Hz), 4.89—4.98 (m, 3H), 7.19–7.39 (m,
20H); 13C NMR (75 MHz, CDCl3): d 52.7, 69.0, 69.6,
73.3, 75.1, 75.2, 75.6, 77.8, 78.3, 79.5, 79.5, 86.9,
127.5–128.6 (aromatic carbons), 138.1, 138.2, 138.3,
138.6, 173.3; MS (ES+): m/z 635.13 [M+Na]+; Anal.
Calcd for C37H40O8: C, 72.53; H, 6.58. Found: C,
72.61; H, 6.46.
CDCl3): d 67.3, 68.6, 69.6, 73.3, 75.1, 75.2, 75.6, 77.7,
78.1, 79.5, 79.6, 86.8, 127.6–128.6 (aromatic carbons),
135.3, 138.1, 138.2, 138.3, 138.6, 172.7; MS (ES+): m/z
689.44 [M+H]+; Anal. Calcd for C43H44O8: C, 74.98;
H, 6.44. Found: C, 74.93; H, 6.52.
3.4. (2R)-Methyl 2-azido-2-(2,3,4,6-tetra-O-benzyl-b-D-
glucopyranosyl)-ethanoate (6)
Compound 5 (50 mg, 0.08 mmol) was dissolved in dry
CH2Cl2 (2.0 mL) and cooled to 0 °C. Pyridine (66 lL,
0.8 mmol) was added. The reaction mixture was stirred
for 5 min, and then trifluoromethanesulfonic anhydride
(55 lL, 0.32 mmol) was slowly added. The reaction mix-
ture was stirred under 0 °C for 1 h. Water (5.0 mL) was
added followed by extraction with CH2Cl2 (3 ꢁ 5 mL).
The organic layer was dried (over Na2SO4), concen-
trated, and re-dissolved in anhyd CH2Cl2 (2.0 mL).
NaN3 (16 mg, 0.24 mmol) and 15-crown-5 (4 lL,
0.2 mmol) were added. The reaction mixture was stirred
at room temperature for 24 h and then water was added.
The aqueous phase was extracted with CH2Cl2
(3 ꢁ 20 mL) and the combined organic layers were dried
(Na2SO4) and concentrated. Flash column chromato-
graphy (hexanes–EtOAc, 4:1) yielded azide 6 (42 mg,
25
1
25
1
81%) as a pale-yellow oil. ½aꢂD +6.0 (c 0.5, CHCl3); H
NMR (300 MHz, CDCl3) d 3.52–3.56 (m, 4H), 3.64
(dd, 1H, J = 9.6, 8.6 Hz), 3.71–3.77 (m, 3H), 3.80 (dd,
1H, J = 9.4, 8.6 Hz), 3.92 (dd, 1H, J = 1.9, 9.4 Hz),
4.37 (d, 1H, J = 1.9 Hz), 4.55 (d, 1H, J = 12.1 Hz),
4.57–4.65 (m, 2H), 4.69 (d, 1H, J = 10.6 Hz), 4.83 (d,
1H, J = 10.9 Hz), 4.88 (d, 1H, J = 10.9 Hz), 4.93–4.97
(m, 2H), 7.23–7.36 (m, 20H); 13C NMR (75 MHz,
CDCl3): d 52.6, 62.8, 68.9, 73.5, 74.7, 75.1, 75.6, 77.3,
78.2, 79.6, 79.7, 87.1, 127.6–128.5 (aromatic carbons),
137.9, 138.0, 138.2, 138.3, 167.8; MS (ES+): m/z
660.03 [M+Na]+; Anal. Calcd for C37H39N3O7: C,
69.68; H, 6.16; N, 6.59. Found: C, 69.72; H, 6.21; N,
6.63.
3.3. (2S)-Benzyl 2-hydroxy-2-(2,3,4,6-tetra-O-benzyl-b-
D-glucopyranosyl)-ethanoate (4)
LiOH (24 mg, 1.02 mmol) was added to a mixture of
compound
5 (104 mg, 0.17 mmol) in THF–H2O
(3.0 mL, 1:1). The mixture was stirred at room temper-
ature for 12 h and formic acid in water was added to
quench the reaction until acidic. After extraction with
EtOAc (5 ꢁ 20 mL), the organic layer was evaporated
and the crude residue was dissolved in DMF (3.0 mL).
Cs2CO3 (72 mg, 0.22 mmol) was added 30 min prior
to the addition of BnBr (40 lL, 0.34 mmol). The mixture
was stirred at room temperature for 2 h and water was
added. The aqueous layer was extracted with EtOAc
(3 ꢁ 20 mL) and the combined organic layers were dried
(Na2SO4) and concentrated. Flash column chromato-
graphy (hexanes–EtOAc, 4:1) yielded 4 (104 mg, 89%)
as a clear oil. 1H NMR (300 MHz, CDCl3): d 3.30–
3.36 (m, 2H), 3.48 (dd, 1H, J = 1.7, 11.1 Hz), 3.59–
3.64 (m, 2H), 3.67 (dd, 1H, J = 1.9, 8.6 Hz), 3.74 (dd,
1H, J = 8.8, 9.1 Hz), 3.83 (dd, 1H, J = 9.1, 8.6 Hz),
4.52–4.58 (m, 3H), 4.61 (d, 1H, J = 10.7 Hz), 4.78 (d,
1H, J = 10.9 Hz), 4.84 (d, 1H, J = 10.9 Hz), 4.90–4.97
(m, 3H), 5.10 (d, 1H, J = 12.0 Hz), 5.33 (d, 1H,
J = 12.0 Hz), 7.19–7.38 (m, 25H); 13C NMR (75 MHz,
3.5. (2R)-Methyl 2-(t-butoxycarbonylamino)-2-(b-D-
glucopyranosyl)-ethanoate (7)
Compound 6 (223 mg, 0.32 mmol) was dissolved in
MeOH (6.0 mL) and aq hydrochloric acid (0.5 mmol).
Pearlman’s catalyst (160 mg) was added and the mixture
was hydrogenated for 6 h at atmospheric pressure. The
mixture was filtered, concentrated, and re-dissolved in
MeOH (2.0 mL). Et3N (0.5 mL) and Boc2O (550 mg,
2.5 mmol) were added and the mixture was stirred at
room temperature for 12 h. The crude residue was con-
centrated and purified with silica gel column chromato-
graphy (MeOH–EtOAc, 1:6) to afford 10 (100 mg, 90%)
25
as a syrup. ½aꢂD ꢀ29.0 (c 1.45, CH3OH); 1H NMR
(300 MHz, CD3OD): d 1.46 (s, 9H), 3.07 (dd, 1H,
J = 9.3, 9.2 Hz), 3.23–3.36 (m, 2H), 3.43–3.59 (m, 3H),

K. Zhang et al. / Carbohydrate Research 343 (2008) 1644–1652
1649
3.74 (s, 3H), 3.89 (dd, 1H, J = 1.9, 11.8 Hz), 4.66 (d, 1H,
J = <1 Hz); 13C NMR (75 MHz, CD3OD): d 28.7, 52.6,
55.4, 63.6, 71.6, 72.0, 79.7, 80.8, 82.2, 82.6, 158.2, 173.0;
MS (ES+): m/z 352.29 [M+H]+; HRMS (ES+) calcd for
C14H26NO9 [M+H]+: 352.1608; found: 352.1607.
quant.), which was dissolved in MeOH (4.0 mL) and
hydrogenated for 20 min using 20 wt % Pd(OH)2–C.
The solution was filtered and the solvent was evaporated
in vacuo. The solid residue was dissolved in aqueous
acetone (3.0 mL, acetone–H2O, 1:1) and treated with
9-fluorenylmethyl pentafluorophenyl carbonate (91 mg,
0.24 mmol) and NaHCO3 (31 mg, 0.37 mmol) for 4 h
at room temperature. Water (10.0 mL) was added
and the aqueous layer was extracted with EtOAc
(6 ꢁ 10 mL). Finally, the solvent was dried (Na2SO4)
and concentrated. The crude product was purified by
flash column chromatography (MeOH–EtOAc, 1:1) to
afford compound 10 (45 mg, 63%) as a colorless thick
3.6. (2R)-Methyl 2-(t-butoxycarbonylamino)-2-(6-azido-
6-deoxy-b-D-glucopyranosyl)-ethanoate (8) and (2S)-
methyl 2-(t-butoxycarbonylamino)-2-(6-azido-b-D-gluco-
pyranosyl)-ethanoate (9)
To a solution of compound 7 (100 mg, 0.29 mmol) in
dry pyridine (8.0 mL) was added toluenesulfonyl chlo-
ride (128 mg, 0.68 mmol) and the reaction was stirred
for 2 h at 0 °C, then raised to room temperature for
6 h. The solvent was removed in vacuo and then purified
by gradient flash column chromatography (EtOAc to
EtOAc–MeOH, 20:1) to afford a crude mixture contain-
ing the 6-O-tosyl-b-D-glucopyranoside derivative
(92 mg, 64%). Sodium azide (118 mg, 1.82 mmol) was
added to the solution of tosylate in dry DMF (3.0 mL)
and the mixture was heated to 100 °C for overnight.
The solvent was removed in vacuo and the residue was
purified by gradient flash column chromatography
(EtOAc to EtOAc–MeOH, 20:1) to afford compounds
8 (36 mg, 52%) and 9 (30 mg, 43%) as a pale-yellow oil
1
liquid. H NMR (300 MHz, CD3OD): d 1.43 (s, 9H),
2.98–3.13 (m, 2H), 3.18 (m, 1H), 3.22–3.44 (m, 2H),
3.62–3.80 (m, 2H), 4.23 (t, 1H, J = 6.8 Hz), 4.32–4.48
(m, 3H), 7.28–7.87 (m, 8H); 13C NMR (75 MHz,
CDCl3): d 28.9, 43.4, 48.5, 56.4, 67.8, 72.0, 73.1, 79.0,
80.4, 80.8, 83.0, 120.9–128.8 (aromatic carbons),
142.6–145.4 (aromatic carbons), 157.7, 159.1, 169.5;
MS (ESꢀ): m/z 557.09 [MꢀH]ꢀ.
3.8. (2S)-2-(t-Butoxycarbonylamino)-2-[6-deoxy-6-(9H-
fluoren-9-ylmethoxycarbonylamino)-b-D-glucopyranosyl]-
ethanoic acid (11)
25
in a ratio 1.2:1, respectively. Compound 8: ½aꢂD ꢀ15.0
1
(c 0.25, CH3OH); H NMR (300 MHz, CDCl3): d 1.44
Ester 9 (40 mg, 0.11 mmol) was treated with lithium
hydroxide (15 mg, 0.66 mmol) for 1 h at 0 °C in aq
THF (4.0 mL, 1:1), and then acidified with 99% formic
acid (5 drops). The solution was extracted with EtOAc
(6 ꢁ 10 mL) and the combined organic layers were dried
(Na2SO4) and concentrated to afford crude acid (35 mg),
which was dissolved in MeOH (4.0 mL) and hydroge-
nated for 20 min using 20 wt % Pd(OH)2–C. The solu-
tion was filtered and the solvent was evaporated in
vacuo. The solid residue was dissolved in aqueous ace-
tone (3.0 mL, 2:1) and treated with 9-fluorenylmethyl
pentafluorophenyl carbonate (75 mg, 0.18 mmol) and
NaHCO3 (30 mg, 0.36 mmol) for 4 h at room tempera-
ture. Water (10.0 mL) was added and the aqueous layer
was extracted with EtOAc (6 ꢁ 10 mL). Finally, the sol-
vent was dried (Na2SO4) and concentrated. The crude
product was purified by flash column chromatography
(MeOH–EtOAc, 1:1) to afford compound 11 (39 mg,
(s, 9H), 3.32 (dd, 1H, J = 5.9, 13.4 Hz), 3.45 (dd, 1H,
J = 3.2, 9.2 Hz), 3.49–3.58 (m, 4H), 3.70 (dd, 1H, J =
3.2, 9.2 Hz), 3.75 (s, 3H), 4.00–4.16 (br s, 1H), 4.72–
4.78 (m, 2H), 4.88 (br s, 1H), 5.63 (d, 1H, J = 8.4 Hz);
13C NMR (75 MHz, CDCl3): d 28.3, 51.3, 52.6, 53.9,
70.4, 70.6, 77.9, 79.5, 80.6, 80.8, 155.7, 169.6; HRMS
(ES+) calcd for C14H24N4NaO8 [M+Na]+: 399.1492;
25
found: 399.1489; compound 9: ½aꢂD +74.0 (c 0.1,
1
CH3OH); H NMR (300 MHz, CDCl3): d 1.46 (s, 9H),
3.25 (t, 1H, J = 9.3 Hz), 3.33 (dd, 2H, J = 6.2,
13.2 Hz), 3.42 (m, 2H), 3.47 (br s, 1H), 3.50 (m, 1H),
3.60 (t, 1H, J = 8.9 Hz), 3.76 (dd, 1H, J = 2.3, 9.5 Hz),
3.80 (s, 3H), 4.67 (dd, 1H, J = 2.3, 8.0 Hz), 4.70 (br s,
1H), 5.57 (d, 1H, J = 8.0 Hz); 13C NMR (75 MHz,
CDCl3): d 28.2, 51.4, 53.0, 53.8, 69.9, 70.8, 77.2, 79.4,
80.0, 81.4, 157.4, 169.9; HRMS (ES+) calcd for
C14H24N4NaO8 [M+Na]+: 399.1492; found: 399.1490.
25
65%) as a thick colorless liquid. ½aꢂD +5.0 (c 0.7,
3.7. (2R)-2-(t-Butoxycarbonylamino)-2-[6-deoxy-6-(9H-
fluoren-9-ylmethoxycarbonylamino)-b-D-glucopyranosyl]-
ethanoic acid (10)
CH3OH); 1H NMR (300 MHz, CD3OD): d 1.45 (s,
9H), 3.13 (ABq, 1H, J = 9.3 Hz), 3.17–3.29 (m, 2H),
3.35 (m, 2H), 3.38–3.45 (m, 2H), 3.55–3.70 (m, 1H),
3.75 (d, 1H, J = 9.4 Hz), 4.20 (t, 1H, J = 6.9 Hz),
4.28–4.44 (m, 3H), 7.28–7.42 (m, 4H), 7.65 (dd, 2H,
J = 1.6, 6.8 Hz), 7.78 (d, 2H, J = 7.4 Hz); 13C NMR
(75 MHz, CD3OD): d 28.8, 42.7, 48.6, 56.2, 67.9, 71.2,
71.7, 78.7, 80.2, 80.8, 81.5, 120.9–128.8 (aromatic car-
bons) 142.6–145.4 (aromatic carbons), 158.9, 159.3,
169.2; MS (ESꢀ): m/z 556.90 [MꢀH]ꢀ; Anal. Calcd
Ester 8 (60 mg, 0.16 mmol) was treated with lithium
hydroxide (21 mg, 0.96 mmol) for 1 h at 0 °C in aqueous
THF (4.0 mL, 1:1), and then acidified with formic acid
(99%, 100 lL). The solution was extracted with EtOAc
(6 ꢁ 10 mL) and the combined organic layers were dried
(Na2SO4) and concentrated to afford crude acid (58 mg,

1650
K. Zhang et al. / Carbohydrate Research 343 (2008) 1644–1652
for C28H34N2O10: C, 60.21; H, 6.14; N, 5.02. Found: C,
60.32; H, 6.19; N, 4.98.
for C18H32N2O10: C, 49.53; H, 7.39; N, 6.42. Found:
C, 49.49; H, 7.27; N, 6.58.
3.11. N-[(2R)-2-(t-Butoxycarbonylamino)-2-[6-deoxy-6-
(t-butoxycarbonylamino)-b-D-glucopyranosyl]-acetyl]-
Trp(Boc)-NHBn (14)
3.9. (2R)-Methyl 2-(t-butoxycarbonylamino)-2-[6-deoxy-
6-(t-butoxycarbonylamino)-b-D-glucopyranosyl]-ethano-
ate (12)
To the mixture of Fmoc-Trp(Boc)-OH (205 mg,
0.39 mmol) and TBTU (451 mg, 1.4 mmol) in DMF
(5.0 mL) were added benzylamine (165 mL, 1.51 mmol)
and N,N-diisopropylethylamine (334 lL, 1.87 mmol)
and stirred for 2 h at room temperature. The solvent
was removed in vacuo and the residue was purified by
flash column chromatography (hexanes–EtOAc, 2:1) to
yield the Fmoc-Trp(Boc)-NHBn (211 mg, 88%). The
solution of Fmoc-Trp(Boc)-NHBn (151 mg, 0.25 mmol)
and piperidine (0.5 mL) in DMF (2.0 mL) was stirred
for 1 h at room temperature. The solvent was removed
in vacuo and the crude product was purified by flash col-
umn chromatography (MeOH–EtOAc, 1:20) to afford
the H-Trp(Boc)-NHBn (129 mg, 96%). To the solution
of 13 (23 mg, 0.05 mmol) in DMF (2.0 mL) were added
H-Trp(Boc)-NHBn (26 mg, 0.09 mmol), TBTU (58 mg,
0.18 mmol), and N,N-diisopropylethylamine (43 mL,
0.24 mmol). The mixture was stirred for 4 h at room
temperature before removing the solvent under reduced
pressure. The crude product was purified by flash col-
umn chromatography using EtOAc as eluent to afford
A solution of azide 8 (170 mg, 0.50 mmol) in MeOH
(20.0 mL) was treated with Pd(OH)2–C under an atmo-
sphere of hydrogen for 1 h at room temperature. The
reaction mixture was filtered through Celite, and rinsed
with MeOH. The filtrate was evaporated under reduced
pressure to give a colorless foam (155 mg, 0.44 mmol),
which was treated with di-t-butyl dicarbonate (193 mg,
0.88 mmol) and Et3N (0.3 mL, 2.2 mmol) in MeOH
for 4 h at room temperature. Finally, the solvent was
evaporated under reduced pressure to give a colorless
liquid. The crude product was then purified by flash col-
umn chromatography (MeOH–EtOAc, 1:1) to afford
25
compound 12 (183 mg, 90%) as a colorless liquid. ½aꢂD
1
+4.0 (c 0.8, CH3OH); H NMR (300 MHz, CD3OD):
d 1.50 (br s, 18H), 3.02 (br t, 1H, J = 7.2 Hz), 3.08 (br
t, 1H, J = 9.3 Hz), 3.24 (m, 1H), 3.36 (m, 1H), 3.50
(dd, 1H, J = 1.6, 10.0 Hz), 3.56 (br d, 1H, J = 9.0 Hz),
3.64 (m, 1H), 3.78 (s, 3H), 4.69 (br s, 1H); 13C NMR
(75 MHz, CD3OD): d 28.8, 28.9, 43.0, 52.8, 55.5, 71.7,
72.9, 79.3, 80.3, 80.9, 81.0, 82.1, 158.1, 158.7, 172.0;
MS (ES+): m/z 473.24 [M+Na]+; Anal. Calcd for
C19H34N2O10: C, 50.66; H, 7.61; N, 6.22. Found: C,
50.54; H, 7.59; N, 6.28.
25
14 (29 mg, 77%) as a thick colorless liquid. ½aꢂD ꢀ5.0
1
(c 1.0, CHCl3); H NMR (300 MHz, CD3OD): d 1.38
(s, 9H), 1.42 (s, 9H), 1.69 (s, 9H), 2.88–3.09 (m, 2H),
3.18 (dd, 2H, J = 7.6, 14.2 Hz), 3.25–3.33 (m, 2H),
3.33–3.35 (m, 4H), 3.36–3.48 (m, 3H), 4.29–4.40 (m,
2H), 4.44 (br s, 1H), 4.84 (m, 1H), 7.08 (br d, 2H,
J = 7.0 Hz), 7.20–7.37 (m, 5H), 7.52 (s, 1H), 7.66 (br
d, 1H, J = 7.6 Hz), 8.13 (br d, 1H, J = 8.1 Hz); 13C
NMR (75 MHz, CD3OD): d 28.5, 28.6, 28.8, 43.0,
44.1, 54.8, 72.0, 72.5, 79.0, 80.3, 81.0, 81.1, 84.9, 116.2,
117.2, 120.2, 123.7, 125.6, 128.2, 128.3, 129.5, 131.9,
136.9, 139.3, 151.0, 158.9, 160.6, 172.2, 173.3; MS
(ES+): m/z 834.17 [M+Na]+; Anal. Calcd for
C41H57N5O12: C, 60.65; H, 7.08; N, 8.63. Found: C,
60.59; H, 7.07; N, 8.77.
3.10. (2R)-2-(t-Butoxycarbonylamino)-2-[6-deoxy-6-(t-
butoxycarbonylamino)-b-D-glucopyranosyl]-ethanoic acid
(13)
Ester 12 (80 mg, 0.18 mmol) was dissolved in aq THF
(4.0 mL, THF–H2O, 1:1) and treated with LiOH
(26 mg, 1.07 mmol) at 0 °C. The reaction mixture was
stirred at this temperature for 1 h after which the mix-
ture was acidified to pH 3 with 99% formic acid, and ex-
tracted with EtOAc (5 ꢁ 10 mL). The combined organic
layers were dried (over anhyd Na2SO4) and concen-
trated under reduced pressure to give a colorless foam,
which was dissolved in EtOAc (3.0 mL). Crystallization
commenced within 1 h. After completion of crystalliza-
tion, the solid was collected by filtration and rinsed with
EtOAc (2 ꢁ 2 mL) to afford 13 (70 mg, 90%) as white
3.12. N-[(2R)-2-(t-Butoxycarbonylamino)-2-[6-deoxy-6-
(t-butoxycarbonylamino)-b-D-glucopyranosyl]-acetyl]-
Trp(Boc)-OBn (15)
25
crystals. Mp 205–207 °C; ½aꢂD ꢀ7.0 (c 0.9, CH3OH);
To
a
solution of Fmoc-Trp(Boc)-OH (100 mg,
1H NMR (300 MHz, CD3OD): d 1.45 (s, 9H), 1.46 (s,
9H), 2.98 (m, 1H), 3.04 (t, 1H, J = 9.2 Hz), 3.15–3.22
(m, 1H), 3.33 (m, 1H), 3.44 (dd, 1H, J = 1.6, 10.0 Hz),
3.63 (br ABq, 2H, J = 9.6 Hz), 4.61 (br s, 1H); 13C
NMR (75 MHz, CD3OD): d 28.7, 28.8, 43.0, 55.2,
71.8, 72.9, 79.3, 80.3, 80.8, 80.9, 82.3, 158.1, 158.7,
173.2; MS (ES+): m/z 459.23 [M+Na]+; Anal. Calcd
0.19 mmol) in DMF (5.0 mL) was added Cs2CO3
(249 mg, 0.77 mmol) followed by the addition of benzyl
bromide (90 lL, 0.76 mmol) and then stirred for 4 h at
room temperature. The solvent was removed in vacuo
and the residue was purified by flash column chromato-
graphy (hexane–EtOAc, 2:1) to yield the Fmoc-
Trp(Boc)-OBn (107 mg, 92%). The solution of Fmoc-

K. Zhang et al. / Carbohydrate Research 343 (2008) 1644–1652
1651
Trp(Boc)-OBn (107 mg, 0.17 mmol) and piperidine
(0.5 mL) in DMF (2.0 mL) was stirred for 1 h at room
temperature. The solvent was removed in vacuo and
the crude product was purified by flash column chroma-
tography (MeOH–EtOAc, 1:20) to afford the H-
Trp(Boc)-OBn (65 mg, 95%). Compound 13 (30 mg,
0.07 mmol) was dissolved in DMF (2.0 mL) and H-
Trp(Boc)-OBn (36 mg, 0.09 mmol), TBTU (81 mg,
0.25 mmol), and N,N-diisopropylethylamine (60 lL,
0.34 mmol) were added and then stirred for 4 h at room
temperature. The solvent was removed under reduced
pressure and the crude product was purified by flash col-
umn chromatography using EtOAc as eluent to afford
3H), 3.48–3.60 (m, 2H), 4.30 (br s, 1H), 5.14 (dd, 1H,
J = 5.7, 10.3 Hz), 5.24 (br s, 2H), 7.17 (t, 1H, J =
7.6 Hz), 7.22 (br s, 1H), 7.28 (t, 1H, J = 7.6 Hz), 7.36
(dd, 2H, J = 3.8, 7.6 Hz), 7.42–7.49 (m, 3H), 7.53 (d, 1
H J = 8.2 Hz), 7.70 (d, 1H, J = 8.2 Hz); 13C NMR
(75 MHz, D2O): d 27.2, 40.9, 53.3, 53.6, 68.4, 69.3,
70.7, 76.0, 76.5, 77.5, 109.1, 112.6, 118.9, 119.7, 122.4,
125.0, 126.7, 128.7, 129.1, 129.2, 135.4, 136.8, 166.2,
173.3; MS (ES, [M+Na]+); MS (ES+): m/z 513.23
[M+H]+.
3.15. Determination of the MIC values for peptides 16–19
25
15 (46 mg, 83%) as a thick liquid. ½aꢂD ꢀ32.0 (c 1.5,
Bacterial isolates were obtained from the American
Type Culture Collection (ATCC). Isolates were kept
frozen in skim milk at ꢀ80 °C until minimum inhibitory
concentration (MIC) testing was carried out. Following
two subcultures from frozen stock, the in vitro activities
of peptides were determined by macrobroth dilution in
accordance with the Clinical and Laboratory Standards
Institute (CLSI) 2006 guidelines.22 Stock solutions of
peptides were prepared and dilutions made as described
by CLSI. Test tubes contained doubling antimicrobial
dilutions of cation adjusted Mueller–Hinton broth and
inoculated to achieve a final concentration of approxi-
mately 5 ꢁ 105 CFU/mL, then incubated in ambient
air for 24 h prior to reading. Colony counts were per-
formed periodically to confirm inocula. Quality control
was performed using ATCC QC organisms.
1
CH3OH); H NMR (300 MHz, CD3OD): d 1.38 (br s,
18H), 1.63 (s, 9H), 3.00–3.15 (m, 2H), 3.18 (d, 2H,
J = 7.0 Hz), 3.24 (br s, 1H), 3.33–3.40 (m, 5H), 4.89
(m, 1H), 5.03 (s, 2H), 7.06–7.12 (m, 2H), 7.15–7.31 (m,
5H), 7.40 (br s, 1H), 7.50 (m, 1H), 8.05 (d, 1H,
J = 8.2 Hz); 13C NMR (75 MHz, CD3OD): d 26.9,
27.3, 27.4, 27.6, 40.8, 54.6, 54.9, 66.7, 69.8, 70.1, 76.8,
77.3, 78.8, 79.0, 79.7, 83.4, 114.7, 114.8, 118.2, 122.2,
123.4, 124.0, 127.4, 127.8, 127.9, 134.5, 134.9, 149.3,
155.4, 157.1, 170.0, 171.3; MS (ES+): m/z 835.33
[M+Na]+; Anal. Calcd for C41H56N4O13: C, 60.58; H,
6.94; N, 6.89. Found: C, 60.57; H, 6.88; N, 6.93.
3.13. N-[(2R)-2-(Amino)-2-[6-deoxy-6-(amino)-b-D-
glucopyranosyl]-acetyl]-Trp-NHBnꢃ(CF3CO2H)3 (16)
A solution of TFA–CH2Cl2 (1:1, 4.0 mL) was added to
compound 14 (80 mg, 0.10 mmol) at room temperature.
After 3 h, the solution was diluted with toluene
(2 ꢁ 5 mL), concentrated in vacuo, provided 16
1H NMR (300 MHz, D2O): d 2.52–2.74 (m, 2H), 2.88–
3.04 (m, 2H), 3.28–3.40 (m, 2H), 3.42–3.56 (m, 2H),
3.70–3.77 (m, 1H), 4.32–4.41 (m, 1H), 4.45–4.54 (m,
2H), 5.00–5.11 (m, 1H), 7.16–7.49 (m, 8H), 7.60–7.68
(m, 1H), 7.78 (dd, 1H, J = 7.8, 17.8 Hz); 13C NMR
(75 MHz, D2O): d 27.9, 40.9, 43.4, 53.4, 54.8, 69.4,
70.8, 76.0, 76.6, 77.4, 109.0, 112.6, 118.9, 119.7, 122.3,
125.1, 126.8, 127.4, 127.8, 129.1, 136.8, 137.8, 166.1,
173.3; MS (ES+): m/z 511.99 [M+H]+.
Acknowledgment
25
(73 mg, quant.) as a salt. ½aꢂD ꢀ8.0 (c 0.45, CH3OH);
The authors thank the Natural Sciences and Engineer-
ing Research Council of Canada (NSERC) for financial
support in the form of a Discovery Grant.
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solution of TFA–CH2Cl2 (1:1, 4.0 mL) at room temper-
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salt. ½aꢂD ꢀ16.0 (c 0.3, CH3OH); H NMR (300 MHz,
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9.6 Hz), 3.18 (br d, 1H, J = 14.0 Hz), 3.22–3.42 (m,
25
1

1652
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