Structural requirements for vanA activity of vancomycin analogues

Article abstract of DOI:10.1016/S0040-4020(02)00743-3

We have prepared several sets of glycopeptide analogues in order to probe the molecular basis for the activity of derivatives that overcome vanA resistance. The results described in this paper provide compelling evidence that good vanA activity is due to

Full text of DOI:10.1016/S0040-4020(02)00743-3

Tetrahedron 58 (2002) 6585–6594
Structural requirements for VanA activity
of vancomycin analogues
Zhong Chen, Ulrike S. Eggert, Steven D. Dong, Simon J. Shaw, Binyuan Sun,
*
John V. LaTour and Daniel Kahne
Department of Chemistry, Princeton University, Princeton, NJ 08544, USA
Received 18 June 2002; accepted 25 June 2002
Dedicated with affection to Professor Yoshito Kishi
Abstract—We have prepared several sets of glycopeptide analogues in order to probe the molecular basis for the activity of derivatives that
overcome vanA resistance. The results described in this paper provide compelling evidence that good vanA activity is due to a mechanism of
action that does not involve peptide binding. Hypothesizing that this mechanism of action involves an interaction of the disaccharide portion
of vancomycin analogues with bacterial transglycosylases, we have prepared a compound in which the vancomycin aglycone is coupled to a
known transglycosylase inhibitor that is structurally unrelated to the disaccharides that have been previously investigated. The activity of this
compound is excellent. This work provides a clear prescription for the design of better glycopeptide analogues. q 2002 Published by Elsevier
Science Ltd.
1. Introduction
of them may promote dimerization, we questioned whether
the entropic advantages of membrane anchoring/dimeriza-
tion would be sufficient to overcome the extremely weak
binding of vancomycin to D-Ala–D-Lac.† Further investi-
gation revealed that biological activity against resistant
microorganisms was maintained when the vancomycin
binding pocket was damaged so that it could not bind
even D-Ala–D-Ala, let alone D-Ala–D-Lac. This finding led
us to propose that 2 is active against resistant strains because
it has a second mechanism of action, one that involves a
direct interaction between the substituted disaccharide and
some other target important in peptidoglycan synthesis.8 To
test this hypothesis, we have prepared and evaluated the sets
of vancomycin analogues described below.
Vancomycin is a glycopeptide antibiotic that inhibits
peptidoglycan biosynthesis by binding to the D-Ala–D-Ala
dipeptide termini of peptidoglycan precursors.1 It is widely
used to treat Gram-positive infections, and has become
increasingly important in recent years because it is the only
antibiotic capable of curing many multi-drug resistant
infections. The emergence of resistance to vancomycin (1)
in enterococcal strains has aroused considerable concern.2
The predominant form of vancomycin resistance in
enterococcal strains occurs when these bacteria incorporate
genes encoding proteins that produce peptidoglycan pre-
cursors terminating in D-Ala–D-Lac instead of D-Ala–D-
Ala.3 Efforts to overcome resistance have led to a class
of vancomycin derivatives containing hydrophobic sub-
stituents such as chlorobiphenyl on the vancosamine
nitrogen (e.g. 2).4 The activity of these derivatives was
initially attributed to a combination of two factors: their
ability to anchor to the bacterial membrane near the
peptidoglycan precursor; and their ability to dimerize,
which was proposed to increase their avidity for peptido-
glycan precursors presented in multiple copies on the
membrane surface.5,6
2. Results and discussion
Vancomycin analogues 2–8 (Figs. 1 and 2) were syn-
thesized following routes described previously.9–12 Com-
pound 2 is the vanA-active chlorobiphenyl vancomycin
analogue discovered by researchers at Lilly;4 2a is the
corresponding desleucyl derivative, which is incapable of
peptide binding.13 Compound 3 resembles compound 2
except that the chlorobiphenyl substituent has been moved
from the vancosamine to the C6 position of the penultimate
sugar, glucose; 3a is the corresponding desleucyl derivative.
While hydrophobic substituents do facilitate anchoring of
vancomycin derivatives to membranes, and although some
†
Keywords: vancomycin; glycopeptide analogues; vanA activity.
*
It has recently been shown that LY329332, which is similar in structure to
chlorobiphenyl vancomycin, does not dimerize or insert into
membranes.7
Corresponding author. Tel.: þ1-609-258-6368; fax: þ1-609-258-2617;
e-mail: dkahne@princeton.edu
0040–4020/02/$ - see front matter q 2002 Published by Elsevier Science Ltd.
PII: S0040-4020(02)00743-3

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Z. Chen et al. / Tetrahedron 58 (2002) 6585–6594
Figure 1. Lipid derivatives of vancomycin and their desleucyl analogues.
Compound 4 also resembles 2 except that the glucose has
been replaced by ethylene glycol; 4a is the corresponding
desleucyl derivative. This set of compounds was designed to
test whether a particular carbohydrate structure is required
for biological activity against resistant bacterial strains. We
reasoned that a mechanism involving non-specific mem-
brane interactions would not be sensitive to the position of
the lipid substituent on the molecule, but a mechanism
involving specific interactions with some other target would
be. Minimum inhibitory concentrations for compounds 1–4
were measured using a standard microdilution assay and the
results are summarized in Table 1.
The activity of these compounds indicates that the structure
of the substituted disaccharide is important for biological
activity. Moving the hydrophobic substituent from the
terminal vancosamine sugar to the glucose abolishes
peptide-binding-independent activity (compare 2a and 3a),
consistent with the hypothesis of a second mechanism. The
monosaccharide derivatives 4/4a provide even more
compelling evidence that vanA activity involves a specific
interaction with some as yet unidentified target rather than a
non-specific membrane interaction. In compounds 4/4a the
substituted vancosamino sugar is attached to the aglycone
via a flexible linker containing four atoms, thus mimicking
the number of atoms between C1 of the vancosamino sugar
and the aglycone in compounds 2/2a. However, even though
the sugar in 4/4a is identical to the terminal sugar in 2/2a
and can be displayed at the same distance from the
aglycone, the biological activity of these pairs of com-
pounds is very different.
Table 1. Minimum inhibitory concentrations (MICs, mg/mL) of com-
pounds 1–4 against sensitive and VanA vancomycin-resistant strains of
E. faecium
Compound
E. faecium
Resistant (VanA)
Sensitive
We then reasoned that if the substituted disaccharide did, in
fact, have some biological activity that was distinct from
peptide binding, it should be possible to alter the way
in which it is attached to the aglycone. We prepared
compounds 5 and 6 (Schemes 1 and 2) to evaluate this
hypothesis. In compound 5, the substituted disaccharide is
attached via an ethylene glycol linker to the fourth amino
Vancomycin (1)
2
3
2
,0.025
,0.03
0.8
10
64
2048
12.5
16
63
40
4
2a
3a
4a
1024
.200
50

Z. Chen et al. / Tetrahedron 58 (2002) 6585–6594
6587
Figure 2. Linked vancomycin derivatives which incorporate both natural and unnatural disaccharides.
acid of the crosslinked heptapeptide aglycone, whereas in
compound 6 the same disaccharide is linked via an aryl
spacer to the carboxy terminus. As shown in Table 2, both
compounds have reasonable activity against vanA-resistant
E. faecium, with the activity of compound 6 being
comparable to that of 2. Therefore, the orientation of the
substituted disaccharide with respect to the aglycone can be
altered without destroying activity against resistant strains.
This result supports the hypothesis that these vancomycin
derivatives are comprised of two separate determinants of
biological activity.
suggested that it inhibits the transglycosylation step of
peptidoglycan synthesis even though it cannot bind the
peptide portion of the peptidoglycan precursor substrate.8,14
This result suggested to us that the target of the substituted
disaccharide was a component of the transglycosylation
complex—perhaps even a bacterial transglycosylase itself.
Since the substituted disaccharide on compound 5 was not
optimized to block transglycosylation, but merely happened
to do so (possibly because it bears a chance resemblance to
the carbohydrate substrates of bacterial transglycosylases),
we surmised that it should be possible to make a compound
with better activity against resistant bacterial strains by
attaching a carbohydrate that was designed to block
transglycosylation. We prepared compound 7 (Scheme 3)
in which a functionalized disaccharide designed to inhibit
bacterial transglycosylases (at least when coupled to a
phospholipid)15 was attached to the aglycone at A4 via an
ethylene glycol linker, exactly as in the linked chlorobi-
phenyl derivative 5.9 We chose compound 5 as the standard
to which to compare 7 because having an ethylene glycol
linker rather than a glycosidic linkage to the aglycone
facilitates the synthesis. We also prepared compound 8
(Scheme 4), which bears a much closer resemblance to
compound 5 than 7 does. The MICs of 7 and 8 were
Mechanistic investigations of 2a in our laboratory have
Table 2. Minimum inhibitory concentrations (MICs, mg/mL) of com-
pounds 5–8
Compound
E. faecium
Sensitive
Resistant (VanA)
5
6
7
8
,0.01
0.16
0.1
63
16
16
.500
1

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Z. Chen et al. / Tetrahedron 58 (2002) 6585–6594
Scheme 1. Synthesis of linked vancomycin derivative 5. (a) (i) Tf2O, DTBMP, 278 to 2208C, Et2O/CH2Cl2, 84%; (ii) NaI, acetone, 99%; (iii) NaOMe,
MeOH, 82%. (b) (i) Cs2CO3, DMF, 83%; (ii) PdCl2(PPh3)2, Bu3SnH, DMF/AcOH, 79%; (iii) 40-chlorobiphenyl-4-carboxaldehyde, DIPEA, NaBH3CN, DMF,
46%.
Scheme 2. Synthesis of carboxy-linked derivative 6. (a) (i) 40-Chlorobiphenyl-4-carboxaldehyde, DIPEA, NaBH3CN, DMF; (ii) TBAF, 58% over two steps.
(b) TBTU, HOBt, DMF, 23%.
evaluated and compared to that of 5. Compound 8 has poor
activity against vanA strains even though it is structurally
related to 5. Compound 7, in contrast, shows a four-fold
improvement in activity against vanA-resistant bacterial
strains compared with compound 5.
3. Conclusion
While these results do not prove that the vanA activity of the
chlorobiphenyl vancomycin disaccharide is due to its ability
to inhibit bacterial transglycosylases, they do show that
Scheme 3. Synthesis of linked hybrid 7. (a) (i) NH4HCO3, (Boc)2O, pyridine, CH3CN, 76%; (ii) NH2NH2, THF/CH3OH, 74%; (iii) 3-(trifluoromethyl)benzoic
acid, HATU, DIPEA, DMF, 93%; (iv) Ac2O, DMAP, pyridine, 92%; (v) Hg(CF3CO2)2, (C2H5)2O·BF3, 2-chloroethanol, 83%. (b) (i) Me3P, H2O/THF/EtOH;
(ii) 4-chloro-3-(trifluoromethyl)phenyl isocyanate, CH2Cl2/DMF, 72% over two steps; (iii) NaI, acetone; (iv) NaOMe, MeOH, 86% over two steps. (c) (i)
Cs2CO3, DMF, 84%; (ii) PdCl2(PPh3)2, Bu3SnH, DMF/AcOH, 66%.
Scheme 4. Synthesis of linked vancomycin derivative 8. (a) (i) Tf2O, DTBMP, 278 to 2208C, Et2O/CH2Cl2, 91%, 2:1 a/b; (ii) NaI, acetone, 99%;
(iii) NaOMe, MeOH, 94%. (b) (i) Cs2CO3, DMF, 82%; (ii) PPh3, THF/H2O, 81%; (iii) PdCl2(PPh3)2, Bu3SnH, DMF/AcOH, 78%; (iv) 40-chlorobiphenyl-4-
carboxaldehyde, DIPEA, NaBH3CN, DMF, 62%.

Z. Chen et al. / Tetrahedron 58 (2002) 6585–6594
6589
the hypothesis has prescriptive value for how to design
glycopeptide antibiotics. It should be possible to make still
better vancomycin derivatives by attaching even better
inhibitors of transglycosylation (and possibly transpeptida-
tion) to the vancomycin aglycone. Whether it will also be
possible to make vanA-active peptide-binding antibiotics
without necessarily starting from the natural vancomycin
aglycone is of considerable interest to us at present and
would permit access to an even wider range of structural
variants with different properties.
5.22 (m, 2H), 5.08 (d, J¼4.5 Hz, 1H), 4.98 (apt, J¼9.5 Hz,
1H), 4.94 (s, 1H), 4.78 (br s, 1H), 4.54–4.43 (m, 4H), 4.27
(dd, J¼12.5, 5.0 Hz, 1H), 4.17–4.10 (m, 2H), 3.87 (ddd,
J¼11.0, 8.0, 6.5 Hz, 1H), 3.80 (dd, J¼9.5, 8.0 Hz, 1H), 3.70
(ddd, J¼9.5, 5.0, 2.3 Hz, 1H), 3.31 (ddd, J¼10.0, 8.0,
5.5 Hz, 1H), 3.24 (ddd, J¼10.0, 8.0, 7.0 Hz, 1H), 2.17 (s,
3H), 2.10 (s, 3H), 2.04 (s, 3H), 2.02 (s, 3H), 2.17–1.95 (m,
2H), 1.68 (s, 3H), 1.13 (d, J¼6.5 Hz, 3H); 13C NMR
(CDCl3, 500 MHz) d 171.9, 171.3, 170.6, 170.4, 133.6,
118.4, 102.2, 97.9, 76.1, 75.0, 74.6, 72.5, 71.6, 69.4, 65.9,
64.2, 62.7, 53.6, 36.0, 24.8, 21.5, 21.5, 21.4, 21.3, 17.9,
1.9; HRMS (FAB) calcd for C27H40INNaO14 [MþNa]þ
752.1391, found 752.1384.
4. Experimental
4.1. General
2-Iodoethyl 2-O-(3-allyloxycarbonylamino-4-O-acetyl-
2,3,6-trideoxy-3-C-methyl-a-L-lyxo-hexopyranosyl)-3,4,6-
tri-O-acetyl-b-D-glucopyranoside in 1 mg/mL NaOMe/
MeOH (2 mL) was stirred at room temperature for
60 min, quenched with NH4OAc (20 mg), concentrated
and purified by flash chromatography (10% MeOH/CH2Cl2)
to give 19 mg (82%) of 11: Rf¼0.32 (12% MeOH/CH2Cl2);
1H NMR (CD3OD, 500 MHz) d 5.95 (m, 1H), 5.41 (d,
J¼4.5 Hz, 1H), 5.31 (dq, J¼17.5, 1.5 Hz, 1H), 5.19 (dq,
J¼10.5, 1.5 Hz, 1H), 4.56–4.50 (br m, 3H), 4.43 (d,
J¼7.5 Hz, 1H), 4.19 (ddd, J¼11.5, 7.0, 6.0 Hz, 1H), 3.90–
3.84 (m, 2H), 3.67 (m, 1H), 3.52–3.44 (m, 3H), 3.39–3.33
(m, 3H), 3.28 (m, 2H), 2.09 (br d, J¼13.8 Hz, 1H), 1.87 (dd,
J¼13.8, 4.5 Hz, 1H), 1.66 (s, 3H), 1.23 (d, J¼6.5 Hz, 3H);
13C NMR (CD3OD, 500 MHz) d 134.7, 117.5, 103.0,
98.5, 79.8, 78.1, 77.5, 73.5, 71.9, 71.8, 66.0, 65.2, 62.9,
54.8, 36.0, 24.3, 18.0, 2.7; HRMS (FAB) calcd for
C19H32INNaO10 [MþNa]þ 584.0969, found 584.0961.
4.1.1. 2-Iodoethyl 2-O-(3-allyloxycarbonylamino-2,3,6-
trideoxy-3-C-methyl-a-L-lyxo-hexopyranosyl)-b-D-gluco-
pyranoside (11). Sulfoxide 1016 (156 mg, 0.394 mmol), was
azeotroped three times with toluene and dissolved in Et2O
(3 mL). 2-Chloroethyl 3,4,6-tri-O-acetyl-b-D-glucopyrano-
side12 (9, 104 mg, 0.282 mmol) and 2,6-di-t-butyl-4-methyl
pyridine (174 mg, 0.845 mmol) were azeotroped three times
with toluene, dissolved in 1:2 CH2Cl2/Et2O (7.5 mL), and
cooled to 2788C. Triflic anhydride (33 mL, 0.197 mmol) was
added, then 10 was added over 15 min, and the reaction was
stirred at 2788C for 20 min, warmed to 2208C over 1.5 h,
stirred for another 30 min, and then quenched with saturated
NaHCO3 (10 mL). The aqueous layer was extracted with
CH2Cl2 (3£10 mL) and the combined organic layers were
dried over Na2SO4, concentrated and purified by flash
chromatography (27% EtOAc/CH2Cl2) to give 151 mg
(84%) of 2-chloroethyl 2-O-(3-allyloxycarbonylamino-4-O-
acetyl-2,3,6-trideoxy-3-C-methyl-a-L-lyxo-hexopyranosyl)-
3,4,6-tri-O-acetyl-b-D-glucopyranoside as a colorless solid:
Rf¼0.39 (30% EtOAc/CH2Cl2); 1H NMR (CDCl3,
500 MHz) d 5.89 (m, 1H), 5.28 (br dd, J¼17.5, 1.5 Hz,
1H), 5.23 (apt, J¼9.5 Hz, 1H), 5.20 (br d, J¼9.7 Hz, 1H),
5.08 (d, J¼4.5 Hz, 1H), 4.99 (apt, J¼9.5 Hz, 1H), 4.91 (s,
1H), 4.79 (br s, 1H), 4.54–4.43 (m, 4H), 4.27 (dd, J¼12.5,
5.0 Hz, 1H), 4.17 (dt, J¼11.0, 5.0 Hz, 1H), 4.13 (dd, J¼12.5,
2.4 Hz, 1H), 3.82 (dd, J¼9.5, 8.0 Hz, 1H), 3.78 (dt, J¼11.0,
4.5 Hz, 1H), 3.72–3.62 (m. 3H), 2.17 (s, 3H), 2.09 (s, 3H),
2.03 (s, 3H), 2.02 (s, 3H), 2.17–1.95 (m, 2H), 1.67 (s, 3H),
1.12 (d, J¼6.5 Hz, 3H); 13C NMR (CDCl3, 500 MHz) d
172.0, 171.3, 170.5, 170.4, 155.2, 133.6, 118.4, 102.3, 97.9,
76.2, 74.8, 74.8, 72.5, 70.5, 69.4, 65.9, 64.1, 62.7, 53.7, 43.3,
36.0, 24.7, 21.5, 21.4, 21.4, 21.3, 17.8; HRMS (FAB) calcd
for C27H40ClNNaO14 [MþNa]þ 660.2035, found 660.2028.
4.1.2. Compound 5. Disaccharide 11 (13 mg, 0.023 mmol)
and the protected vancomycin aglycone 12 (45 mg,
0.032 mmol), synthesized as described,16 were combined
and azeotroped with toluene three times. Cs2CO3 (9.1 mg,
0.028 mmol) was added, the mixture was azeotroped twice
with toluene, DMF (0.2 mL) was added, and the reaction
was stirred at room temperature for 12 h and then quenched
with AcOH (one drop). The reaction mixture was pre-
cipitated with water (10 mL), centrifuged, and the precipi-
tate purified by flash chromatography (8–14% MeOH/
CH2Cl2) to give 35 mg (83%) of a white solid: Rf¼0.29
(15% MeOH/CH2Cl2); 1H NMR (CD3OD, 500 MHz) d 7.61
(s, 1H), 7.58 (d, J¼8.2 Hz, 1H), 7.30 (d, J¼8.2 Hz, 1H),
7.09 (s, 1H), 6.97 (d, J¼8.9 Hz, 1H), 6.89 (d, J¼2.1 Hz,
1H), 6.42 (d, J¼2.1 Hz, 1H), 5.80–6.14 (m, 6H), 5.72 (s,
1H), 5.06–5.46 (m, 15H), 4.40–4.80 (m, 17H), 4.34–4.42
(m, 1H), 4.17 (s, 1H), 4.08–4.18 (m, 1H), 3.88 (d, J¼
11.3 Hz, 1H), 3.64–3.70 (m, 1H), 3.48–3.54 (m, 2H), 3.40
(s, 1H), 2.93 (s, 3H), 2.36–2.46 (m, 1H), 2.12 (d, J¼
13.7 Hz, 1H), 1.80–1.90 (m, 2H), 1.60 (s, 3H), 1.50–1.60
(m, 1H), 1.20 (d, J¼6.1 Hz, 3H), 0.97 (d, J¼4.8 Hz,
3H), 0.93 (d, J¼5.2 Hz, 3H); HRMS (FAB) calcd for
A solution of 2-chloroethyl 2-O-(3-allyloxycarbonylamino-
4-O-acetyl-2,3,6-trideoxy-3-C-methyl-a-L-lyxo-hexo-
pyranosyl)-3,4,6-tri-O-acetyl-b-D-glucopyranoside (60 mg,
0.094 mmol) and NaI (300 mg, 2 mmol) in acetone
(0.8 mL) was refluxed for 60 h, cooled to room temperature,
concentrated, and then the residue was dissolved in CH2Cl2
and filtered. The filtrate was concentrated and purified by
flash chromatography (25% EtOAc/CH2Cl2) to yield 68 mg
(99%) of 2-iodoethyl 2-O-(3-allyloxycarbonylamino-4-O-
acetyl-2,3,6-trideoxy-3-C-methyl-a-L-lyxo-hexopyrano-
syl)-3,4,6-tri-O-acetyl-b-D-glucopyranoside as a colorless
C88H103Cl2N9NaO29
1842.6154.
[MþNa]þ
1842.6136,
found
To this material (30 mg, 0.016 mmol) in 1:1 DMF/AcOH
(1.5 mL) was added PdCl2(PPh3)2 (8 mg, 0.011 mmol).
After the solution was degassed, Bu3SnH in 50 mL portions
was added every 5 min until the reaction was complete. The
reaction mixture was precipitated with acetone (40 mL) and
1
solid: Rf¼0.36 (25% EtOAc/CH2Cl2); H NMR (CDCl3,
500 MHz) d 5.89 (m, 1H), 5.28 (dq, J¼17.5, 1.5 Hz, 1H),

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Z. Chen et al. / Tetrahedron 58 (2002) 6585–6594
the precipitate was suspended in water (5 mL) and kept at
48C overnight, after which the suspension was filtered. The
filtrate was concentrated and purified by reverse phase
HPLC (0–30% CH3CN/H2O with 0.1% TFA) to give 21 mg
(79%) of the deprotected product as its TFA salt: Rf¼0.41
3.47 (t, J¼9.1 Hz, 1H), 3.36 (s, 1H), 3.31–3.27 (m, 1H),
3.03 (t, J¼7.7 Hz, 2H), 2.75 (t, J¼7.5 Hz, 2H), 2.08–2.00
(m, 4H), 1.77 (s, 3H), 0.93 (d, J¼6.6 Hz, 3H); 13C NMR
(125.8 MHz, CD3OD) d 153.9, 141.3, 139.5, 137.9, 134.2,
1328, 131.1, 129.4, 128.8, 127.8, 106.6, 104.6, 101.2, 97.8,
78.7, 78.4, 77.4, 70.9, 69.7, 64.2, 61.8, 60.2, 58.8, 56.2,
43.6, 39.6, 34.2, 33.1, 29.8, 26.2, 24.1, 22.9, 19.6, 16.4,
13.3; HRMS (FAB) calcd for C37H50ClN2O10 717.3154
[MþH]þ: 717.3151.
1
(6:6:1:2 CHCl3/MeOH/H2O/saturated NH4OH); H NMR
(DMSO-d6, 500 MHz) d 9.04 (s, 1H), 8.77 (s, 1H), 8.55 (d,
J¼5.2 Hz, 1H), 7.84 (s, 1H), 7.46–7.71 (m, 5H), 7.28–7.34
(m, 2H), 7.14 (s, 1H), 6.70–6.79 (m, 3H), 6.39 (s, 1H), 6.26
(s, 1H), 5.94–6.00 (m, 1H), 5.77 (d, J¼7.9 Hz, 1H), 5.62 (s,
1H), 5.31 (d, J¼4.0 Hz, 1H), 5.10–5.23 (m, 3H), 4.89 (s,
1H), 4.40–4.49 (m, 5H), 4.17–4.24 (m, 4H), 4.01 (s, 1H),
3.86–3.94 (m, 1H), 3.65 (d, J¼10.4 Hz, 1H), 3.43–3.46 (m,
1H), 3.07–3.16 (m, 3H), 2.60 (s, 3H), 2.11–2.17 (m, 1H),
1.84–1.87 (m, 1H), 1.53–1.67 (m, 4H), 1.44 (s, 3H), 1.06
(d, J¼6.2 Hz, 3H), 0.93 (d, J¼6.1 Hz, 3H), 0.88 (d,
J¼6.1 Hz, 3H); HRMS (FAB) calcd for C68H80Cl2N9O25
[MþH]þ 1492.4642, found 1492.4648.
4.1.4. Compound 6. To a solution of the vancomycin
aglycone (15)17 (16 mg, 0.014 mmol) and disaccharide 14
(5 mg, 0.007 mmol) in DMF (1 mL) was added HOBt
(3 mg, 0.018 mmol) and TBTU (6 mg, 0.018 mmol)
followed by N-methyl morpholine (4 mL, 0.035 mmol).
The reaction was stirred overnight at room temperature. The
solution was purified by reverse-phase HPLC, eluting with a
linear gradient (10–60% CH3CN/H2O with 0.1%TFA) to
give 3 mg (23%) of the desired product 6: 1H NMR
(CD3OD, 500 MHz) d 8.96 (br d, J¼5.5 Hz, 1H), 8.65 (br s,
1H), 8.22 (br t, 5.5 Hz, 1H), 7.74 (d, J¼8.3 Hz, 2H), 7.70 (s,
2H), 7.66 (d, J¼8.6 Hz, 2H), 7.61 (d, J¼8.6 Hz, 3H), 7.49
(d, J¼8.3 Hz, 2H), 7.24 (d, J¼8.9 Hz, 1H), 7.10 (s, 1H),
6.83, (br s, 1H), 6.80–6.77 (m, 1H), 6.61 (s, 2H), 6.45 (d,
J¼5.7 Hz, 2H), 5.46 (d, J¼4.4 Hz, 1H), 5.42 (s, 1H), 5.35
(s, 1H), 5.31 (s, 1H), 5.17 (d, J¼8.0 Hz, 1H), 4.73 (br s, 1H),
4.67 (d, J¼5.5 Hz, 1H), 4.58 (q, J¼6.5 Hz, 1H), 4.33 (d,
J¼9.6 Hz, 1H), 4.23 (s, 1H), 4.21 (s, 2H), 4.04 (t, 6.5 Hz,
1H), 3.86 (s, 6H, –OCH3), 3.75 (br d, J¼13.0 Hz, 2H), 3.71
(d, J¼8.4 Hz, 1H), 3.66 (dd, J¼12.3, 5.4 Hz, 1H), 3.60 (s,
1H), 3.55 (t, J¼8.8 Hz, 2H), 3.45 (t, J¼8.8 Hz, 2H), 3.20–
3.14, (m, 1H), 2.95 (d, J¼15.6 Hz, 1H), 2.78 (s, 3H), 2.66 (t,
J¼8.2 Hz, 2H), 2.19 (dd, J¼13.8, 4.4 Hz, 1H), 2.04 (d,
J¼13.1 Hz, 1H), 1.96 (s, 1H), 1.95–1.89 (m, 3H), 1.87 (s,
3H), 1.71–1.63 (m, 2H), 1.16 (d, J¼6.4 Hz, 3H), 0.97 (d,
J¼4.3 Hz, 3H), 0.93 (d, J¼4.3 Hz, 3H); HRMS (MALDI)
calcd for C90H99Cl3N10NaO26 1863.5690 [MþNa]þ:
1863.5744.
To the aforementioned TFA salt (10 mg, 0.006 mmol) in
DMF (0.35 mL) was added DIPEA (5.4 mL, 0.031 mmol)
followed by 40-chlorobiphenyl-4-carboxaldehyde (62 mL of
a 0.1 M solution in DMF). The reaction was stirred at 608C
for 30 min, NaBH3CN (19 mL of a 1.0 M solution in THF)
was added, the reaction was stirred at 658C for 4.5 h, cooled
to room temperature, and poured into Et2O (12 mL). The
resulting precipitate was purified by reverse phase HPLC
(10–60% CH3CN/H2O with 0.1% AcOH) to give 5 mg
(46%) of the AcOH salt of 5 as a white solid: Rf¼0.66
(6:6:1:2 CHCl3/MeOH/H2O/(saturated NH4OH)); 1H NMR
(DMSO-d6, 500 MHz) d 8.69 (s, 1H), 8.50 (s, 1H), 7.86 (s,
1H), 7.70 (s, 1H), 7.68 (s, 1H), 7.62 (m, 2H), 7.30–7.53 (m,
10H), 7.16 (s, 1H), 6.70–6.80 (m, 2H), 6.39 (s, 1H), 6.26 (s,
1H), 5.77 (d, J¼7.9 Hz, 1H), 5.58 (s, 1H), 5.30 (s, 1H), 5.24
(s, 1H), 5.09–5.13 (m, 2H), 4.82 (s, 1H), 4.17–4.42 (m,
10H), 3.89–3.93 (m, 1H), 3.67 (d, J¼10.7 Hz, 1H), 3.45 (m,
1H), 3.02–3.13 (m, 4H), 2.29 (s, 3H), 3.12–3.18 (m, 1H),
1.91 (s, 3H), 1.62–1.78 (m, 3H), 1.37–1.51 (m, 4H), 1.09
(d, J¼6.4 Hz, 3H), 0.89 (d, J¼6.4 Hz, 3H), 0.85 (d, J¼
6.4 Hz, 3H); HRMS (FAB) calcd for C81H89Cl3N9O25
[MþH]þ 1692.5035, found 1692.4990.
4.1.5. 2-Chloroethyl 2-O-(2-deoxy-3,4,6-tri-O-acetyl-2-
[3-trifluoromethyl-benzamido]-b-D-glucopyranosyl)-3-
azido-3-deoxy-4-O-methyl-b-D-glucopyranosiduronamide
(17). To a solution of phenyl 2-O-(2-deoxy-2-phthalimido-
3,4,6-tri-O-acetyl-b-D-glucopyranosyl)-3-azido-3-deoxy-4-
O-methyl-1-thio-b-D-glucopyranosiduronic acid (16, 2.50 g,
3.37 mmol) in 20 mL CH3CN were added pyridine
(0.175 mL), di-tert-butyl-dicarbonate (0.96 g, 4.38 mmol)
and NH4HCO3 (0.35 g, 4.38 mmol), the reaction was stirred
at room temperature for 10 h, quenched with methanol
(1 mL), diluted with CH2Cl2 (30 mL), washed with
saturated NaHCO3 (50 mL£2), brine (50 mL), dried over
Na2SO4, filtered, and concentrated. The crude product was
purified by flash chromatography (90% EtOAc/petroleum
ether) to afford 1.90 g (76%) of phenyl 2-O-(2-deoxy-2-
phthalimido-3,4,6-tri-O-acetyl-b-D-glucopyranosyl)-3-azido-
3-deoxy-4-O-methyl-1-thio-b-D-glucopyranosiduronamide:
1H NMR (CDCl3, 500 MHz) d 7.7–7.9 (m, 4H), 7.4–7.5
(m, 2H), 7.25–7.35 (m, 3H), 6.13 (brd, J¼2.6 Hz, 1H), 5.85
(dd, J¼9.2, 10.6 Hz, 1H), 5.73 (d, J¼8.4 Hz, 1H), 5.60 (brd,
J¼2.9 Hz, 1H), 5.24 (dd, J¼9.2, 9.9 Hz, 1H), 4.65 (d, J¼
9.2 Hz, 1H), 4.43 (dd, J¼8.4, 10.6 Hz, 1H), 4.30 (dd, J¼4.8,
12.5 Hz, 1H), 4.21 (dd, J¼2.6, 12.5 Hz, 1H), 3.92 (m,
1H), 3.63 (d, J¼9.5 Hz, 1H), 3.43 (s, 3H), 3.35–3.45
4.1.3. 4-(3-Aminopropyl)-2,6-dimethoxyphenyl 2-(3-N-
[4-(4-chlorophenyl)-benzylamino]-2,3,6,trideoxy-3-C-
methyl-a-L-lyxo-hexopyranosyl)-b-D-glucopyranoside
(14). To a solution of disaccharide 13 (90 mg, 0.14 mmol)11
in DMF (5 mL) was added 40-chlorobiphenyl-4-carboxalde-
hyde (38 mg, 0.18 mmol) and DIPEA (122 mL, 0.7 mmol).
The mixture was stirred at 558C for 30 min, NaBH3CN
(0.7 mL of a 1 M solution in THF) was added and the
mixture was stirred at 558C for 3 h. AcOH (0.4 mL) was
then added and the product was partially purified by reverse-
phase HPLC, eluting with a linear gradient (15–80%
CH3CN/H2O with 0.1% AcOH). The lyophilized powder
was dissolved in DMF (1 mL) and TBAF (1 mL of a 1 M
solution in THF) was added. The mixture was heated at
558C overnight. The product was purified by reverse-phase
HPLC, eluting with a linear gradient (10–60% CH3CN/H2O
1
with 0.1% AcOH) to yield 58 mg (58%) of 14: H NMR
(500 MHz, D2O) d 7.72 (d, J¼8.4 Hz, 2H), 7.67 (d, J¼
8.4 Hz, 2H), 7.44 (d, J¼8.1 Hz, 2H), 7.38 (d, J¼8.4 Hz,
2H), 6.76 (s, 2H), 5.32 (s, 1H), 5.23 (d, J¼7.3 Hz, 1H),
4.45–4.44 (m, 1H), 3.93–3.88 (m, 7H), 3.79–3.61 (m, 5H),

Z. Chen et al. / Tetrahedron 58 (2002) 6585–6594
6591
(m, 2H), 3.16 (t, J¼9.5 Hz, 1H), 2.08 (s, 3H), 2.04 (s,
3H), 1.88 (s, 3H); 13C NMR (CDCl3, 125 MHz) d
170.9, 170.3, 170.2, 169.7, 168.3, 134.6, 133.0, 132.2,
131.6, 129.3, 128.5, 123.8, 97.8, 85.7, 81.7, 72.1, 70.8,
69.1, 68.4, 62.2, 60.6, 54.9, 21.0, 20.8, 20.6; HRMS (FAB)
calcd for C33H35N5NaO13S [MþNa]þ 764.1850, found
764.1872.
(s, 1H), 8.24 (d, J¼7.7 Hz, 1H), 7.95 (d, J¼7.7 Hz, 1H),
7.7–7.8 (m, 2H), 7.4–7.5 (m, 3H), 7.25–7.35 (m, 3H), 5.57
(dd, J¼9.5, 10.3 Hz, 1H), 5.45 (d, J¼8.4 Hz, 1H), 5.17 (d,
J¼9.9 Hz, 1H), 5.08 (apt, J¼10.3 Hz, 1H), 4.2–4.4 (m, 3H),
4.0–4.1 (m, 2H), 3.92 (apt, J¼9.9 Hz, 1H), 3.5–3.6 (m,
4H), 3.44 (apt, J¼9.5 Hz, 1H), 2.09 (s, 3H), 2.06 (s, 3H),
1.93 (s, 3H); 13C NMR (DMF-d7, 125 MHz) d 171.3, 171.0,
170.7, 170.2, 166.5, 136.9, 134.9, 132.4, 131.6, 131.0,
130.8, 130.0, 129.1, 128.0, 126.5, 125.1, 125.1, 124.3,
100.6, 85.2, 81.9, 79.5, 77.0, 74.0, 72.4, 70.3, 69.5, 63.3,
60.3, 56.1, 21.2, 21.1, 20.9; HRMS (FAB) calcd for
C33H36F3N5NaO12S [MþNa]þ 806.1931, found 806.1968.
To a solution of this disaccharide (1.90 g, 2.56 mmol) in a
5:1 mixture of CH3OH/CH2Cl2 (30 mL) was added
hydrazine (3.9 mL, 125 mmol). The reaction mixture was
stirred 20 h at room temperature, concentrated, and purified
by reverse-phase HPLC, eluting with a linear gradient
(0–80% CH3CN/H2O). The fractions containing the
product were combined and concentrated to give 0.90 g
(74%) of phenyl 2-O-(2-amino-2-deoxy-b-D-glucopyrano-
syl)-3-azido-3-deoxy-4-O-methyl-1-thio-b-D-glucopyrano-
To a solution of phenyl 2-O-(2-deoxy-3,4,6-tri-O-acetyl-2-
[3-trifluoromethyl-benzamido]-b-D-glucopyranosyl)-3-
azido-3-deoxy-4-O-methyl-1-thio-b-D-glucopyranosiduro-
namide (100 mg, 0.128 mmol) in 2-chloroethanol (5 mL),
BF3·Et2O (0.16 mL, 1.28 mmol) and Hg(TFA)2 (61 mg,
0.192 mmol) were added. After stirring for 4.5 h, the
reaction mixture was concentrated and purified by flash
chromatography (5–10% CH3OH/CH2Cl2) to afford 80 mg
1
siduronamide: H NMR (CD3OD, 500 MHz) d 7.50–7.55
(m, 2H), 7.25–7.35 (m, 3H), 4.86 (d, J¼9.9 Hz, 1H), 4.72
(d, J¼8.1 Hz, 1H), 3.90 (dd, J¼1.8, 12.1 Hz, 1H), 3.75–
3.85 (m, 2H), 3.73 (dd, J¼5.1, 11.7 Hz, 1H), 3.69 (t,
J¼9.5 Hz, 1H), 3.52 (s, 3H), 3.40 (t, J¼9.5 Hz, 1H), 3.25–
3.35 (m, 3H), 2.64 (t, J¼8.1 Hz, 1H); 13C NMR (CD3OD,
125 MHz) d 173.1, 134.9, 133.3, 130.1, 128.9, 103.7, 87.6,
82.8, 79.7, 78.4, 78.0, 76.4, 72.0, 70.4, 63.1, 60.7, 58.9;
HRMS (FAB) calcd for C19H27N5NaO8S [MþNa]þ
508.1478, found 508.1481.
1
(83%) of compound 17: H NMR (CD3OD, 500 MHz) d
8.13 (s, 1H), 8.06 (d, J¼8.1 Hz, 1H), 7.83 (d, J¼7.7 Hz,
1H), 7.66 (t, J¼7.7 Hz, 1H), 5.42 (dd, J¼9.5, 10.6 Hz, 1H),
5.12 (d, J¼3.3 Hz, 1H), 5.06 (apt, J¼9.5 Hz, 1H), 4.97 (d,
J¼8.4 Hz, 1H), 4.26 (d, J¼3.7 Hz, 2H), 4.15 (dd, J¼8.4,
10.6 Hz, 1H), 4.12 (d, J¼9.9 Hz, 1H), 3.8–4.0 (m, 3H),
3.7–3.8 (m, 2H), 3.64 (apt, J¼9.5 Hz, 1H), 3.46 (dd, J¼3.7,
10.6 Hz, 1H), 3.44 (s, 3H), 3.19 (apt, J¼9.9 Hz, 1H), 2.08
(s, 3H), 2.03 (s, 3H), 1.94 (s, 3H); 13C NMR (CD3OD,
125 MHz) d 173.9, 172.4, 172.1, 171.4, 168.9, 136.7, 132.2,
132.0, 130.7, 129.4, 125.5, 124.4, 103.8, 99.6, 82.4, 81.0,
74.1, 73.3, 71.8, 70.7, 70.1, 66.0, 63.2, 60.9, 56.4, 43.4,
20.9, 20.7, 20.7; HRMS (FAB) calcd for C29H35ClF3N5-
NaO13 [MþNa]þ 776.1770, found 776.1809.
To a solution of phenyl 2-O-(2-amino-2-deoxy-b-D-gluco-
pyranosyl)-3-azido-3-deoxy-4-O-methyl-1-thio-b-D-gluco-
pyranosiduronamide (0.90 g, 1.85 mmol) and a,a,a-tri-
fluoro-m-toluic acid (0.42 g, 2.22 mmol) in DMF (60 mL)
were added HATU (0.84 g, 2.22 mmol) and DIPEA
(0.39 mL, 2.22 mmol). The reaction was stirred 12 h at
room temperature, quenched with CH3OH (10 mL), and
purified by flash chromatography (10% CH3OH/CH2Cl2) to
give 1.13 g (93%) of phenyl 2-O-(2-deoxy-2-[3-trifluoro-
methyl-benzamido]-b-D-glucopyranosyl)-3-azido-3-deoxy-
4.1.6. 2-Iodoethyl 2-O-(2-deoxy-2-[3-trifluoromethyl-
benzamido]-b-D-glucopyranosyl)-3-(4-chloro-3-trifluoro-
methyl-phenyl)-ureido-3-deoxy-4-O-methyl-b-D-gluco-
pyranosiduronamide (18). To a solution of 17 (80 mg,
0.106 mmol) in a mixture of 1:1 EtOH/THF (6 mL),
P(CH3)3 (0.212 mL of a 1.0 M solution in THF) was
added. The reaction was stirred for 1.5 h, H2O (0.212 mL)
was added, and stirring continued for another 12 h. The
crude amine was concentrated, azeotroped with toluene
three times and dissolved in a mixture of 7:1.5 CH2Cl2/DMF
(8.5 mL). 4-Chloro-3-(trifluoromethyl)-phenyl isocyanate
(28 mg, 0.127 mmol) was added, the solution was stirred
for 3.5 h, quenched with CH3OH (2 mL), concentrated, and
purified by flash chromatography (10% CH3OH/CH2Cl2) to
afford 72 mg of 2-chloroethyl 2-O-(2-deoxy-2-[3-trifluoro-
methyl-benzamido]-3,4,6-tri-O-acetyl-b-D-glucopyranosyl)-
3-(4-chloro-3-trifluoromethyl-phenyl)-ureido-3-deoxy-4-O-
methyl-b-D-glucopyranosiduronamide (72% over two
1
4-O-methyl-1-thio-b-D-glucopyranosiduronamide: H NMR
(CD3OD, 500 MHz) d 8.26 (s, 1H), 8.18 (d, J¼8.1 Hz, 1H),
7.82 (d, J¼7.7 Hz, 1H), 7.66 (t, J¼7.7 Hz, 1H), 7.4–7.5 (m,
2H), 7.22–7.32 (m, 3H), 5.00 (d, J¼8.4 Hz, 1H), 4.87 (d,
J¼8.8 Hz, 1H), 3.98 (dd, J¼8.4, 10.3 Hz, 1H), 3.94 (dd,
J¼2.6, 11.7 Hz, 1H), 3.7–3.8 (m, 3H), 3.54–3.64 (m, 2H),
3.4–3.5 (m, 4H), 3.3–3.4 (m, 2H); 13C NMR (CD3OD,
125 MHz) d 173.0, 169.1, 137.1, 135.0, 133.2, 132.3, 131.9,
130.6, 130.1, 129.2, 128.7, 126.6, 125.7, 125.6, 124.5, 102.2,
87.4, 82.6, 79.5, 78.1, 77.1, 75.5, 72.5, 70.6, 63.2, 60.6, 58.6;
HRMS (FAB) calcd for C27H30F3N5NaO9S [MþNa]þ
680.1614, found 680.1590.
To a solution of phenyl 2-O-(2-deoxy-2-[3-trifluoromethyl-
benzamido]-b-D-glucopyranosyl)-3-azido-3-deoxy-4-O-
methyl-1-thio-b-D-glucopyranosiduronamide (1.13 g,
1.72 mmol) and DMAP (0.42 g, 3.44 mmol) in pyridine
(40 mL) was added Ac2O (8.11 mL, 86.0 mmol). After 2 h
at room temperature, the reaction was quenched with
CH3OH (10 mL) and concentrated. The crude solid was
washed with H2O and CH2Cl2 to give 1.23 g (92%) of
phenyl 2-O-(2-deoxy-3,4,6-tri-O-acetyl-2-[3-trifluoro-
methyl-benzamido]-b-D-glucopyranosyl)-3-azido-3-deoxy-
4-O-methyl-1-thio-b-D-glucopyranosiduronamide: 1H
NMR (DMF-d7, 500 MHz) d 9.04 (d, J¼8.8 Hz, 1H), 8.27
1
steps): H NMR (CD3OD, 500 MHz) d 7.91 (s, 1H), 7.80
(d, J¼8.1 Hz, 1H), 7.35–7.45 (m, 2H), 7.1–7.2 (m, 3H),
5.63 (br s, 1H), 5.25 (br s, 1H), 5.20 (d, J¼2.9 Hz, 1H), 5.00
(apt, J¼9.9, 9.9 Hz, 1H), 4.2–4.3 (m, 2H), 4.16 (d, J¼
9.5 Hz, 1H), 3.7–4.0 (m, 8H), 3.3–3.5 (m, 4H), 2.08
(s, 3H), 2.01 (s, 3H), 1.85 (s, 3H); 13C NMR (CD3OD,
125 MHz) d 174.4, 172.4, 171.9, 171.5, 168.4, 157.0,
140.2, 135.9, 132.7, 131.8, 131.6, 130.3, 129.0, 128.9,
126.3, 125.3, 124.7, 124.1, 123.4, 123.3, 117.9, 103.0,

6592
Z. Chen et al. / Tetrahedron 58 (2002) 6585–6594
100.2, 81.4, 79.9, 73.2, 72.4, 70.5, 70.4, 63.3, 60.8, 57.6,
54.1, 43.5, 20.9, 20.7, 20.6; HRMS (MALDI) calcd for
C37H40Cl2F6N4NaO14 [MþNa]þ 971.1715, found 971.1758.
5.40 (s, 1H), 5.31 (d, J¼11.4 Hz, 2H), 5.2–5.3 (m, 2H), 4.96
(d, J¼8.4 Hz, 1H), 4.87 (m, 1H), 4.7–4.8 (m, 1H), 4.77 (d,
J¼5.1 Hz, 1H), 4.68 (d, J¼5.1 Hz, 1H), 4.46–4.64 (m, 3H),
4.0–4.3 (m, 4H), 3.32 (s, 3H), 3.0–3.1 (m, 1H), 1.8–1.9 (m,
2H), 1.4–1.6 (m, 2H), 0.93 (d, J¼6.6 Hz, 3H), 0.89 (d,
J¼6.6 Hz, 3H); HRMS (MALDI) calcd for C84H85Cl3F6-
N12Na O28 [MþNa]þ 1951.4458, found 1951.4357.
To 2-chloroethyl 2-O-(2-deoxy-2-[3-trifluoromethyl-benza-
mido]-3,4,6-tri-O-acetyl-b-D-glucopyranosyl)-3-(4-chloro-
3-trifluoromethyl-phenyl)-ureido-3-deoxy-4-O-methyl-b-
D-glucopyranosiduronamide (72 mg, 0.076 mmol) in
acetone (0.7 mL) was added NaI (171 mg, 1.14 mmol).
The reaction was refluxed for 24 h, concentrated, and the
crude iodide was dissolved in 1 mg/mL NaOMe/MeOH and
stirred for 2 h before quenching with NH4OAc (40 mg). The
solution was concentrated and purified by flash chroma-
tography (15% CH3OH/CH2Cl2) to give 66 mg of com-
pound 18 (86% over two steps): 1H NMR (CD3OD,
500 MHz) d 8.03 (s, 1H), 7.90 (d, J¼7.9 Hz, 1H), 7.48 (s,
1H), 7.41 (d, J¼7.9 Hz, 1H), 7.1–7.3 (m, 3H), 5.29 (d,
J¼3.4 Hz, 1H), 4.20 (d, J¼9.8 Hz, 1H), 4.01 (apt, J¼
10.5 Hz, 1H), 3.85–3.97 (m, 4H), 3.80 (d, J¼9.5 Hz, 1H),
3.68 (dd, J¼6.6, 11.7 Hz, 1H), 3.25–3.45 (m, 10H); 13C
NMR (CD3OD, 125 MHz) d 174.6, 168.5, 157.2, 140.3,
136.5, 132.7, 132.0, 131.7, 131.5, 130.1, 128.9, 128.6,
126.4, 125.5, 124.6, 124.3, 123.4, 118.0, 103.2, 100.0, 81.9,
78.8, 78.3, 74.6, 72.6, 71.3, 63.2, 60.8, 59.5, 54.2, 2.7;
HRMS (MALDI) calcd for C31H34ClF6IN4NaO11
[MþNa]þ 937.0754, found 937.0709.
4.1.8. 2-Iodoethyl 2-O-(3-azido-2,3,6-trideoxy-a-L-ribo-
hexopyranosyl)-b-D-glucopyranoside (20). Sulfoxide 19
(72 mg, 0.221 mmol) was azeotroped with toluene three
times and dissolved in Et2O (3 mL). Nucleophile 9 (58 mg,
0.158 mmol) and 2,6-di-t-butyl-4-methyl pyridine (97 mg,
0.474 mmol) were azeotroped with toluene three times,
dissolved in 1:2 CH2Cl2/Et2O (6 mL), and the solution was
cooled to 2788C. Triflic anhydride (18.6 mL, 0.111 mmol)
was added followed by the sulfoxide solution (added
dropwise over 20 min), and the reaction was stirred at
2788C for 20 min and then slowly warmed to 2208C over
1.5 h. After stirring for another 30 min, the reaction was
quenched with saturated NaHCO3 (10 mL) and the aqueous
layer was extracted with CH2Cl2 (3£8 mL). The combined
organic layers were dried over Na2SO4, concentrated and
purified by flash chromatography (15% EtOAc/CH2Cl2) to
give 81 mg (91%) of a 2:1 mixture of a and b 2-chloroethyl
2-O-(3-azido-4-O-acetyl-2,3,6-trideoxy-L-ribo-hexopyrano-
syl)-3,4,6-tri-O-acetyl-b-D-glucopyranoside as a colorless
solid: Rf¼0.45–0.39 (20% EtOAc/CH2Cl2); LRMS (ESI)
calcd for C22H32ClN3NaO12 [MþNa]þ 588, found 588.
4.1.7. Compound 7. Disaccharide 18 (22 mg, 0.024 mmol)
and protected vancomycin aglycone 1216 (48 mg, 0.035
mmol) were combined and azeotroped with toluene three
times. Cs2CO3 (10 mg, 0.031 mmol) was added followed
by DMF (1 mL) and the reaction was stirred at room
temperature for 12 h, quenched with a drop of AcOH,
concentrated, and purified by flash chromatography (15%
CH3OH/CH2Cl2) to afford 44 mg (84%) of the coupled
product: 1H NMR (CD3OD, 500 MHz) d 7.99 (s, 1H), 7.91
(d, J¼7.7 Hz, 1H), 7.62 (d, J¼3.7 Hz, 1H), 7.42 (s, 2H),
7.37 (d, J¼7.7 Hz, 1H), 7.28 (d, J¼8.4 Hz, 1H), 7.1–7.2 (m,
3H), 7.07 (s, 1H), 7.04 (d, J¼8.4 Hz, 1H), 6.92 (d, J¼
8.8 Hz, 1H), 6.66 (d, J¼2.2 Hz, 1H), 6.40 (d, J¼1.8 Hz,
1H), 5.7–6.2 (m, 6H), 5.66 (s, 1H), 5.1–5.5 (m, 11H),
5.0–5.1 (m, 2H), 4.27 (m, 1H), 4.16 (s, 1H), 3.94
(d, J¼11.0 Hz, 1H), 2.90 (s, 3H), 1.81 (t, J¼11.0 Hz, 1H),
0.95 (d, J¼6.2 Hz, 3H), 0.90 (d, J¼7.3 Hz, 3H); HRMS
(MALDI) calcd for C100H105Cl3F6N12NaO30 [MþNa]þ
2195.5922, found 2195.5891.
The a/b mixture of 2-chloroethyl 2-O-(3-azido-4-O-acetyl-
2,3,6-trideoxy-L-ribo-hexopyranosyl)-3,4,6-tri-O-acetyl-b-
D-glucopyranoside (81 mg, 0.143 mmol) and NaI (300 mg,
2 mmol) was dissolved in acetone (0.8 mL) and heated at
reflux for 72 h after which the reaction mixture was cooled
to room temperature and the solvent was evaporated.
CH2Cl2 was added to the residue and the mixture was
filtered. The filtrate was concentrated and purified by flash
chromatography (12% EtOAc/CH2Cl2) to give 61 mg of
2-chloroethyl 2-O-(3-azido-4-O-acetyl-2,3,6-trideoxy-a-L-
ribo-hexopyranosyl)-3,4,6-tri-O-acetyl-b-D-glucopyrano-
side as a colorless solid: Rf¼0.34 (15% EtOAc/CH2Cl2); 1H
NMR (CDCl3, 500 MHz) d 5.25 (apt, J¼9.5 Hz, 1H), 4.99
(apt, J¼9.5 Hz, 1H), 4.93 (br d, J¼3.7 Hz, 1H), 4.61 (dd,
J¼9.8, 3.5 Hz, 1H), 4.55 (d, J¼8.0 Hz, 1H), 4.46 (dq, J¼
9.8, 6.2 Hz, 1H), 4.27 (dd, J¼12.5, 5.0 Hz, 1H), 4.14–4.08
(m, 3H), 3.95 (ddd, J¼11.0, 9.0, 6.0 Hz, 1H), 3.76 (dd,
J¼9.5, 8.0 Hz, 1H), 3.71 (ddd, J¼9.5, 5.0, 2.3 Hz, 1H), 3.32
(m, 2H), 2.14 (s, 3H), 2.10 (s, 3H), 2.04 (s, 3H), 2.02 (s, 3H),
2.16–1.97 (m, 2H), 1.16 (d, J¼6.2 Hz, 3H); 13C NMR
(CDCl3, 500 MHz) d 171.3, 170.9, 170.5, 170.4, 102.3,
96.3, 76.1, 75.8, 74.5, 72.4, 72.0, 69.3, 62.8, 62.7, 55.9,
33.4, 21.5, 21.5, 21.4, 21.3, 17.9, 1.8; LRMS (ESI) calcd for
C22H32IN3NaO12 [MþNa]þ 680, found 680.
To a solution of this compound (22 mg, 0.010 mmol) in 1:1
DMF/AcOH (2 mL) was added PdCl2(PPh3)2 (7 mg,
0.010 mmol) followed by Bu3SnH in 50 mL portions
every 5 min until the reaction was complete. The mixture
was pipetted into acetone (40 mL), the resulting precipitate
was dissolved in 1:1 H2O/CH3OH (1.5 mL) and purified
by reverse-phase HPLC, eluting with a linear gradient
(10–70% CH3CN/H2O with 0.1% AcOH). The fractions
containing the product were combined and concentrated to
give 12 mg of compound 7 (66%): 1H NMR (DMF-d7,
500 MHz) d 8.74 (s, 1H), 8.67 (m, 1H), 8.30 (m, 1H), 7.71
(s, 1H), 7.67 (d, J¼6.6 Hz, 2H), 7.49 (d, J¼8.1 Hz, 2H),
7.45 (d, J¼8.8 Hz, 1H), 7.3–7.4 (m, 3H), 7.28 (t, J¼7.3 Hz,
1H), 7.21 (s, 2H), 6.88 (dd, J¼2.2, 8.4 Hz, 1H), 6.81 (d,
J¼8.1 Hz, 1H), 6.71 (d, J¼11.7 Hz, 1H), 6.59 (s, 1H), 6.51
(m, 3H), 5.96 (d, J¼7.7 Hz, 1H), 5.86 (s, 1H), 5.46 (s, 1H),
A solution of 2-chloroethyl 2-O-(3-azido-4-O-acetyl-2,3,6-
trideoxy-a-L-ribo-hexopyranosyl)-3,4,6-tri-O-acetyl-b-D-
glucopyranoside (40 mg, 0.061 mmol) in 1 mg/mL NaOMe/
MeOH (3 mL) was stirred at room temperature for 3.5 h,
quenched with NH4OAc (20 mg), concentrated, and purified
by flash chromatography (gradient 7–10% MeOH/CH2Cl2)
to give 28 mg (94%) of 20 as a colorless solid: Rf¼0.35
(12% MeOH/CH2Cl2); 1H NMR (CD3OD, 500 MHz) d 5.25

Z. Chen et al. / Tetrahedron 58 (2002) 6585–6594
6593
(d, J¼4.0 Hz, 1H), 4.46 (d, J¼8.0 Hz, 1H), 4.32 (dq, J¼9.5,
6.2 Hz, 1H), 4.10 (m, 1H), 3.98–3.86 (m, 3H), 3.68 (m, 1H),
3.52 (m, 1H), 3.42–3.26 (m, 6H), 2.24 (br dd, J¼15.1,
2.5 Hz, 1H), 2.01 (ddd, J¼15.1, 4.0, 4.0 Hz, 1H), 1.21 (d,
J¼6.2 Hz, 3H); 13C NMR (CD3OD, 500 MHz) d 103.2,
97.2, 79.4, 78.6, 78.0, 74.0, 72.3, 71.8, 65.4, 62.9, 60.2,
33.7, 18.2, 2.3; HRMS (FAB) calcd for C14H24IN3O8Na
[MþNa]þ 512.0506, found 512.0498.
NH4OH); 1H NMR (DMSO 500 MHz) d 8.77 (s, 1H),
8.57 (d, J¼4.9 Hz, 1H), 7.86 (s, 1H), 7.50–7.70 (m, 4H),
7.47 (d, J¼9.8 Hz, 1H), 7.20–7.40 (m, 2H), 7.15 (s, 2H),
6.70–6.80 (m, 3H), 6.40 (s, 1H), 6.26 (s, 1H), 5.99 (t, J¼
5.5 Hz, 1H), 5.79 (d, J¼7.9 Hz, 1H), 5.00–5.30 (m, 5H),
4.88 (s, 1H), 4.58 (t, J¼6.1 Hz, 1H), 4.53 (d, J¼7.6 Hz, 1H),
4.30–4.50 (m, 4H), 3.90–4.10 (m, 3H), 3.67 (d, J¼4.6 Hz,
1H), 3.40–3.50 (m, 2H), 3.00–3.20 (m, 2H), 2.10–2.20
(1H, m), 1.90–2.10 (m, 2H), 1.60–1.70 (m, 2H), 1.50–1.60
(m, 2H), 1.13 (d, J¼6.1 Hz, 3H), 0.92 (d, J¼6.1 Hz,
3H), 0.88 (d, J¼6.1 Hz, 3H); HRMS (ESI) calcd for
C67H78Cl2N9NaO25 [MþNa]þ 1500.4291, found
1500.4250.
4.1.9. Compound 8. Disaccharide 20 (12 mg, 0.024 mmol)
and the protected aglycone 12 (48 mg, 0.034 mmol) were
combined and azeotroped with toluene three times, where-
upon Cs2CO3 (9.6 mg, 0.029 mmol) was added and the
mixture was azeotroped with toluene twice. DMF (0.2 mL)
was added, the reaction was stirred at room temperature for
12 h, quenched with a drop of AcOH, and purified by flash
chromatography (gradient 7–14% MeOH/CH2Cl2) to give
35 mg (82%) of the linked product as a white solid: Rf¼0.28
(12% MeOH/CH2Cl2); 1H NMR (CD3OD, 500 MHz) d 7.66
(d, J¼8.2 Hz, 1H), 7.59 (s, 1H), 7.56 (d, J¼8.5 Hz, 1H),
7.42 (s, 1H), 7.33 (d, J¼8.5 Hz, 1H), 7.07 (s, 1H), 6.94 (d,
J¼8.5 Hz, 1H), 6.66 (d, J¼2.1 Hz, 1H), 6.39 (d, J¼2.1 Hz,
1H), 5.76–6.12 (m, 5H), 5.72 (s, 1H), 5.00–5.44 (m, 14H),
4.40–4.74 (m, 14H), 4.28–4.40 (m, 2H), 4.14 (s, 1H), 3.81
(d, J¼10.1 Hz, 1H), 3.64 (m, 1H), 3.49 (t, J¼4.6 Hz, 1H),
3.39 (t, J¼8.9 Hz, 1H), 2.90 (s, 3H), 2.20 (dd, J¼14.7,
2.2 Hz, 1H), 1.95 (s, 1H), 1.80–1.88 (m, 1H), 1.44–1.56
(m, 2H), 1.15 (d, J¼6.1 Hz, 3H), 0.95 (d, J¼6.1 Hz,
3H), 0.90 (d, J¼5.5 Hz, 3H); HRMS (FAB) calcd for
To the axial amine (TFA salt, 5 mg, 0.003 mmol) in DMF
(0.3 mL) was added DIPEA (2.7 mL, 0.016 mmol) followed
by 40-chlorobiphenyl-4-carboxaldehyde (31 mL of a 0.1 M
solution in DMF). The reaction mixture was stirred at 608C
for 30 min, NaBH3CN (9.4 mL of a 1 M solution in THF)
was added, stirring continued for another 70 min, and then
the solution was cooled to room temperature and poured
into Et2O (12 mL). The precipitate was purified by reverse
phase HPLC (gradient 10–60% CH3CN/H2O with 0.1%
AcOH) to give 3.4 mg (62%) of 8 as its AcOH salt: Rf¼0.65
1
(6:6:1:2 CHCl3/MeOH/H2O/saturated NH4OH); H NMR
(DMSO-d6, 500 MHz) d 8.68 (s, 1H), 8.51 (s, 1H), 7.83
(s, 1H), 7.40–7.60 (m, 13H), 7.16 (s, 1H), 6.94 (s, 1H),
6.70–6.80 (m, 2H), 6.39 (s, 1H), 6.26 (s, 1H), 5.76
(t, J¼7.9 Hz, 1H), 5.56 (s, 1H), 5.37 (s, 1H), 5.23 (s, 1H),
5.15–5.25 (m, 2H), 4.82 (s, 1H), 3.90–4.50 (m, 9H), 2.29
(s, 3H), 1.70–1.80 (m, 1H), 1.40–1.50 (m, 2H), 1.06 (d, J¼
6.1 Hz, 3H), 0.90 (d, J¼6.7 Hz, 3H), 0.86 (d, J¼6.7 Hz,
3H); LRMS (ESI) calcd for C80H87Cl3N9O25 [MþH]þ
1677, found 1677.
C83H95Cl2N11NaO27
1770.5713.
[MþNa]þ
1770.5674,
found
The coupled product from above (20 mg, 0.011 mmol) and
PPh3 (15 mg, 0.057 mmol) were heated in 6:1 THF/H2O
(0.35 mL) at 558C for 10 h, and cooled to room temperature.
Following precipitation with Et2O (10 mL), the reaction
mixture was purified by reverse phase HPLC (gradient
10–100% CH3CN/H2O with 0.1% AcOH) to give 16 mg
(81%) of the amine as its AcOH salt: Rf¼0.28 (16:4:1:1
4.1.10. Determination of minimum inhibitory concen-
trations.18 The strains used for all compounds except 3, 3a
were Enterococcus faecium 49624, E. faecium CL 4931
(VanA). The strains used for 3, 3a were E. faecium RLA1,
E. faecium CL 5242 (VanA). Test compounds were
dissolved in DMSO. Minimum inhibitory concentration
was determined by a standard broth microdilution assay
using Brain Heart Infusion medium. Viable cells were
stained blue by adding 50 mL 1 mg/mL 3-[4,5-dimethyl-
thiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to
each well. The minimum inhibitory concentration is defined
as the lowest concentration of compound that prevented
visible growth.
1
CHCl3/MeOH/H2O/saturated NH4OH); H NMR (CD3OD,
500 MHz) d 7.68 (d, J¼7.9 Hz, 1H), 7.60 (s, 1H), 7.57
(d, J¼8.2 Hz, 1H), 7.43 (s, 1H), 7.32 (d, J¼8.2 Hz, 1H),
7.05 (s, 1H), 6.94 (d, J¼8.9 Hz, 1H), 6.66 (d, J¼2.1 Hz,
1H), 6.38 (d, J¼2.1 Hz, 1H), 5.76–6.10 (m, 6H), 5.71 (s,
1H), 5.55 (s, 1H), 5.02–5.44 (m, 16H), 4.30–4.76 (m, 19H),
4.10–4.18 (m, 3H), 3.84 (d, J¼10.7 Hz, 1H), 3.65 (m, 1H),
3.40–3.60 (m, 3H), 3.40 (dd, J¼10.1, 4.3 Hz, 1H), 2.89 (s,
3H), 2.30–2.40 (m, 1H), 1.95–2.05 (m, 1H), 1.45–1.55 (m,
2H), 1.23 (d, J¼6.1 Hz, 3H), 0.94 (d, J¼5.5 Hz, 3H), 0.89
(d, J¼5.5 Hz, 3H); HRMS (FAB) calcd for C83H98Cl2N9O27
[MþH]þ 1722.5949, found 1722.5934.
Acknowledgments
To a solution of the protected heptapeptide linked to the
amino disaccharide (14 mg, 0.008 mmol) in 1:1 DMF/
AcOH (1.5 mL) was added PdCl2(PPh3)2 (5.7 mg,
0.08 mmol). The solution was degassed, Bu3SnH in 50 mL
portions was added every 5 min until the reaction was
complete, the product was precipitated with acetone
(30 mL), redissolved in water (5 mL) and filtered. The
filtrate was concentrated and purified by reverse-phase
HPLC (gradient 0–30% CH3CN/H2O with 0.1% TFA) to
give 10 mg (78%) of the deprotected product as the TFA
salt: Rf¼0.41 (6:6:1:2 CHCl3/MeOH/H2O/saturated
This work was supported by NIH grant R01GM66174.
References
1. Barna, J. C. J.; Williams, D. H. Ann. Rev. Microbiol. 1984, 38,
339–357.
2. Murray, B. E. N. Engl. J. Med. 2000, 342, 710–721.
3. Bugg, T. D.; Dutka-Malen, S.; Arthur, M.; Courvalin, P.;
Walsh, C. T. Biochemistry 1991, 30, 2017–2021.

6594
Z. Chen et al. / Tetrahedron 58 (2002) 6585–6594
4. Nagarajan, R. J. Antibiot. 1993, 46, 118–195.
Goldman, R. C.; Silhavy, T. J.; Kahne, D. Science 2001, 294,
361–364.
5. Cooper, M. A.; Williams, D. H. Chem. Biol. 1999, 6, 891–898.
6. Allen, N. E.; LeTourneau, D. L.; Hobbs, J. N. J. Antimicrob.
Agents Chemother. 1997, 41, 66–71.
15. Sofia, M. J.; Allanson, N.; Hatzenbuhler, N. T.; Jain, R.;
Kakarla, R.; Kogan, N.; Liang, R.; Liu, D. S.; Silva, D. J.;
Wang, H. M.; Gange, D.; Anderson, J.; Chen, A.; Chi, F.;
Dulina, R.; Huang, B. W.; Kamau, M.; Wang, C. W.; Baizman,
E.; Branstrom, A.; Bristol, N.; Goldman, R.; Han, K. H.;
Longley, C.; Midha, S.; Axelrod, H. R. J. Med. Chem. 1999,
42, 3193–3198.
7. Kim, S. J.; Cegelski, L.; Studelska, D. R.; O’Connor, R. D.;
Mehta, A. K.; Schaefer, J. Biochemistry 2002, 41, 6967–6977.
8. Ge, M.; Chen, Z.; Onishi, H. R.; Kohler, J.; Silver, L. L.;
Kerns, R.; Fukuzawa, S.; Thompson, C.; Kahne, D. Science
1999, 284, 507–511.
9. Sun, B.; Chen, Z.; Eggert, U. S.; Shaw, S. J.; LaTour, J. V.;
Kahne, D. J. Am. Chem. Soc. 2001, 123, 12722–12723.
10. Kerns, R.; Dong, S. D.; Fukuzawa, S.; Carbeck, J.; Kohler, J.;
Silver, L.; Kahne, D. J. Am. Chem. Soc. 2000, 122,
12608–12609.
16. Thompson, C.; Ge, M.; Kahne, D. J. Am. Chem. Soc. 1999,
121, 1237–1244.
17. Nagarajan, R.; Schabel, A. A. J. Chem. Soc. Chem. Comm.
1988, 19, 1306–1307.
18. NCCLS, Methods for Dilution Antimicrobial Susceptibility
Tests for Bacteria That Grow Aerobically (Approved
Standard, NCCLS Document M7-A4). 4th ed. National
Committee for Clinical Laboratory Standards: Wayne, PA,
1997.
11. Eggert, U.S. PhD Thesis, Princeton University 2001.
12. Chen, Z. PhD Thesis, Princeton University 2001.
13. Allen, N. E.; LeTourneau, D. L.; Hobbs, J. N. J. Antibiot.
1997, 50, 677–684.
14. Eggert, U. S.; Ruiz, N.; Falcone, B. V.; Branstrom, A. A.;