Rapid combinatorial synthesis of aminoglycoside antibiotic mimetics: Use of a polyethylene glycol-linked amine and a neamine-derived aldehyde in multiple component condensation as a strategy for the discovery of new inhibitors of the HIV RNA Rev responsive element

Article abstract of DOI:10.1021/ja9612817

A library of neomycin B mimetics has been prepared rapidly without chromatography using a neamine-derived aldehyde, tert-butyl isocyanide or isocyanoacetic acid methyl ester, a glycine-conjugated polyethylene glycol (PEG) methyl ether, and various Cbz-N-protected amino acids as substrates in a Ugi-type one-pot reaction. The product linked to PEG was isolated by precipitation in ether. A simultaneous base-catalyzed hydrolysis and de-O-acetylation followed by hydrogenation provided an easy access to a library of neomycin B mimetics, which were screened for binding to the Rev responsive element of HIV mRNA (RRE). Several products were found to be more active than neamine with the IC₅₀ values in the micromolar range.

Full text of DOI:10.1021/ja9612817

10150
J. Am. Chem. Soc. 1996, 118, 10150-10155
Rapid Combinatorial Synthesis of Aminoglycoside Antibiotic
Mimetics: Use of a Polyethylene Glycol-Linked Amine and a
Neamine-Derived Aldehyde in Multiple Component
Condensation as a Strategy for the Discovery of New Inhibitors
of the HIV RNA Rev Responsive Element
William K. C. Park,† Manfred Auer,‡ Herbert Jaksche,‡ and Chi-Huey Wong*,†
Contribution from the Department of Chemistry, The Scripps Research Institute,
10666 North Torrey Pines Road, La Jolla, California 92037, and Sandoz Research Institute,
Brunner Str. 59, A-1235 Wien, Austria
ReceiVed April 18, 1996X
Abstract: A library of neomycin B mimetics has been prepared rapidly without chromatography using a neamine-
derived aldehyde, tert-butyl isocyanide or isocyanoacetic acid methyl ester, a glycine-conjugated polyethylene glycol
(PEG) methyl ether, and various Cbz-N-protected amino acids as substrates in a Ugi-type one-pot reaction. The
product linked to PEG was isolated by precipitation in ether. A simultaneous base-catalyzed hydrolysis and de-O-
acetylation followed by hydrogenation provided an easy access to a library of neomycin B mimetics, which were
screened for binding to the Rev responsive element of HIV mRNA (RRE). Several products were found to be more
active than neamine with the IC50 values in the micromolar range.
The replication of human immunodeficiency virus type 1
(HIV-1) is dependent on the function of the viral transactivator
protein Rev.1,2 Rev acts in the nucleus through the recognition
of a highly structured target RNA sequence (Rev response
element, RRE) by an arginine-rich sequence in the N-terminus
of the protein (Kd ≈ 1 nM for the protein-RNA interaction).3
Thus blocking this highly specific interaction by a small
molecule such as an aminoglycoside is an attractive strategy
for the inhibition of HIV.4 Among many aminoglycosides that
have been examined, neomycin B (1) is by far the most effective
inhibitor (IC50 ) 0.1 to 1 µM), and this aminoglycoside
antibiotic has been shown to compete with Rev for binding to
RRE (Figure 1).1
Direct use of neomycin B as an inhibitory drug, however,
has been discouraged due in large part to its toxicity.5 In
addition, neomycin B is relatively unstable (the ribosyl glyco-
sidic bond is sensitive to acids) and, like many other aminogly-
coside antibiotics, is prone to enzymatic modifications in ViVo
(e.g. phosphorylation and acetylation) that leads to the problem
of drug resistance.6a Therefore, it is highly desirable to find
compounds which are less toxic, more stable, and more active
than neomycin B.6b
Figure 1. The RRE region of the HIV mRNA interacts competitively
with Rev and neomycin B.
biosamine (3) has no inhibitory activity (Figure 2). Furthermore,
2 contains the trans-1,3-hydroxy amine and 1,3-diamine com-
ponents that are common structures of many aminoglycosides
that exhibit inhibitory activity against other RNAs andribozymes.6c
For these reasons we intend to develop effective and rapid
synthetic methodologies for the preparation of a library of
compounds containing neamine as a common core and screen
this library of aminoglycoside mimetics for binding to RRE and
other specific RNA sequences as an approach to the develop-
ment of novel antiviral agents and ribozyme inhibitors.
Although 1 is a fairly good inhibitor of the Rev-RRE
interaction by binding to the RRE, neamine (2) alone interacts
only weakly with the mRNA (IC50 ≈ 100 µM) and neo-
† The Scripps Research Institute.
‡ Sandoz Research Institute.
In a representative synthesis of a neomycin B mimetic library,
refluxing neomycin B in acidic methanol (1 N HCl in methanol)
gave neamine 2 (81%) and neobiosamine 3 (72%) in the
X Abstract published in AdVance ACS Abstracts, October 1, 1996.
(1) (a) Zapp, M. L.; Stern, S.; Green, M. R. Cell 1993, 74, 969. (b)
Werstuck, G.; Zapp, M. L.; Green, M. R. Chem. Biol. 1996, 3, 129.
(2) (a) Sharp, P. A.; Marcinak, R. A. Cell 1989, 59, 229. (b) Rosen, C.
A.; Pavlakis, G. N. AIDS 1990, 4, 499. (c) Vashnav, Y. N.; Wong-Staal, F.
Annu. ReV. Biochem. 1991, 60, 577. (d) Cullen, B. R. Cell 1993, 73, 417.
(3) Olsen, H. S.; Cocharane, A. W.; Dillon, P. J.; Nalin, C. M.; Rosen,
C. A. Genes DeV. 1990, 4, 1357.
(6) (a) Mollering, R. C. ReV. Infec. Dis. 1983, 5, S212. Roestamadji, J.;
Graspas, I.; Mobashery, S. J. Am. Chem. Soc. 1995, 117, 11060. (b) For
short oligonucleotides as inhibitors, see: Cload, S. T.; Schepartz, A. J. Am.
Chem. Soc. 1994, 116, 437. For peptides as inhibitors, see: Harada, K.;
Martin, S. S.; Frankel, A. D. Nature 1996, 380, 175. (c) Moazed, D.; Noller,
H. F. Nature 1987, 327, 389. von Ahsen, U.; Davies, J.; Schroeder, R.
Nature 1991, 353, 368. von Ahsen, U.; Noller, H. F. Science 1993, 260,
1500.
(4) Green, M. R. AIDS Res. ReV. 1993, 3, 4.
(5) Koeda, T.; Umemura, K.; Yokoda, M. In Aminoglycoside Antibiotics;
Umezawa, H., Hooper, I. R., Eds.; Springer-Verlag: Berlin, Heidelberg,
New York, 1982; Vol. 62, p 293.
S0002-7863(96)01281-4 CCC: $12.00 © 1996 American Chemical Society

Combinatorial Synthesis of Aminoglycoside Mimetics
J. Am. Chem. Soc., Vol. 118, No. 42, 1996 10151
Scheme 1. Synthesis of Neomycin B Mimetics Using
Four-Component Condensationa
Figure 2. Structure of neomycin B (1) and its substructures neamine
2 and neobiosamine 3.
Figure 3. Conditions: (i) CbzCl, acetone, saturated Na2CO3(aq),
toluene, 0 °C, 86%; (ii) Ac2O, 10% (v/v) DMF/py, room temperature,
overnight, 78%; (iii) allyl bromide, n-Bu4NI, (Me3Si)2NLi, DMSO,
room temperature, 76%; (iv) O3, DCM, -76 °C, 95%.
hydrochloride forms.7 The acid lability of the R-glycosidic
linkage between 2 and 3 comes from the lack of the stabilization
effect assisted by hydrogen bonding from the neighboring amino
group as seen in the other R-glycosidic linkages of neomycin
B.5 Upon evaporation, neamine 2 crystallized out while
neobiosamine 3 remained in solution. The amino groups of
neamine were then protected with the benzyloxycarbonyl (Cbz)
group to give 4. Acetylation of 4 was performed in a 1:9
mixture of DMF and pyridine to give a single product with only
the 5-OH group free (5) in 78% yield. This regioselective
acetylation is believed to originate from both steric hindrance
of the OH at C-5 and its hydrogen bonding with the NH at
C-2′ (Figure 3). Allylation of 5 in the presence of lithium bis-
(trimethylsilyl)amide as base in DMSO gave the desired
allylated product 6.8 This alkylation step facilitates not only
further functionalization of the allyl group of the neamine
derivative but also provides a new opportunity for the synthesis
of neamine derivatives which may be screened for stable
neomycin B mimetics.
Further transformation of 6 to aldehyde 7 was achieved by
ozonolysis in 95% yield. An Ugi-type of multiple component
condensation (MCC)9,10 was then used to construct the library.
Boc-N-protected glycine was coupled to the PEG methyl ether
(MW ca. 5000) with DCC in the presence of DMAP to give
the product in 88% yield. Treatment of this product with TFA
followed by a brief exposure to the basic resin Amberlite 400
(OH- form) in methanol gave the free amine-containing
compound 8.
a 11a Gly, 11b Ala, 11c Val, 11d Phe, 11e Trp, 11f His, 11g Tyr,
11h Thr, 11i Ser, 11j Asp, 11k Gln, 11l Lys, and 11m Arg (side chains
of some amino acids were also protected with Cbz, e.g. Lys or Arg).
12: R ) tert-butyl. 13: R ) CH2C(O)OCH3 and R′ ) side chain of
the corresponding amino acid (a-m).
Figure 4. Structures of the peptido aminoglycosides 15a-m, 17a-
m, 19a-m, and 21a-m as neomycin B mimetics. The removal of Cbz
(i) group was carried out by hydrogenation (10% Pd-C in AcOH) as
the final step.
tert-butyl isocyanide 9, and the glycine-PEG derivative 8 were
reacted with various N-protected amino acids 11a-m (Scheme
1). Similarly, a second library was prepared using isocyanoace-
tic acid methyl ester 10 instead of 9.
The individual condensation products 12a-m and 13a-m
in the reaction mixture were easily isolated by precipitation with
ether and then treated with either LiOH in 10% (v/v) H2O/
MeOH or NaOMe solution in methanol (1 M) to give 14a-m
and 18a-m or 16a-m and 20a-m, respectively. This treat-
ment conveniently released the peptido aminoglycosides from
the PEG as well as the acetyl groups. The PEG units were
removed from the desired products by precipitation, and the
filtrates were then concentrated by evaporation. These products
were hydrogenated with 10% Pd-C in AcOH to give the
corresponding peptido aminoglycosides 15a-m, 17a-m, 19a-
m, and 21a-m, respectively in the AcOH salt forms as shown
in Figure 4.
Two isocyanides were used in two sets of the MCC: tert-
butyl isocyanide 9 and isocyanoacetic acid methyl ester 10. The
latter was prepared by N-formylation of glycine followed by
dehydration.11 In the first MCC set, the neamine derivative 7,
(7) Rinehart, K. L., Jr. The Neomycins and Related Antibiotics; John
Wiley and Sons, Inc.: New York, 1964; p 93.
(8) Allylation of 5 with H2CdCHCH2Br in the presence of NaOH and
DMF at refluxing temperature for 4 h or in the presence of NaH and DMF
for 1 h failed. Allylation of 5 with H2CdCHCH2Br in the presence of Bu4-
NI, LiN(SiMe3)2, and DMF at rt for 1 h gave predominantly the de-O-
acetylated products.
(9) Keating, T. A.; Armstrong, R. W. J. Am. Chem. Soc. 1995, 117, 7842.
(10) Ugi, I.; Lohberger, S.; Karl, R. In ComprehensiVe Organic
Chemistry; Trost, B. M., Ed.; Pergamon Press: New York, 1991; Vol. 2,
p 1083.
(11) (a) Schollkopf, U.; Hoppe, D. Personal communication. (b) Scholl-
kopf, U.; Hoppe, D. Liebigs Ann. Chem. 1972, 763, 1.
Chromatography was not used in the isolation of products
from these reactions. Each of the final products 15a-m, 17a-
m, 19a-m, and 21a-m was relatively pure (>90% purity)
based on the mass analysis. Without further purification, each

10152 J. Am. Chem. Soc., Vol. 118, No. 42, 1996
Park et al.
Table 1. Percent Inhibition of the Rev-RRE Interaction with 200 µM Concentration of Peptido Aminoglycosides 15a-m, 17a-m, 19a-m,
and 21a-m,13 70.5% inhibition was observed with 200 µM 1
a
b
c
d
e
f
g
h
i
j
k
l
m
15
17
19
21
<5
14.5
17.5
24.5
<5
12
<5
72
55
36
36.5
<5
8
<5
78
37
<5
71
32
66.5
54
39
39
87
65
58
91
<5
81.5
51
41
<5
81
52
<5
24
47.5
<5
19
<5
48.5
60.5
41.5
74.5
70.5
53
<5
41.5
55
56
16.5
<5
aldehyde and a PEG-linked amine as key substances in the
multiple component Ugi condensation. No chromatography is
necessary, and each final product is pure enough for charac-
terization and evaluation of biological activity. The new
stereogenic center formed in the reaction is greater than 90%
diasteromerically pure. The procedure reported here is superior
to solution- or solid-phase methods.13 The use of polyethylene
glycol holds the advantages of both solution- and solid-phase
chemistry, and the desired product can be easily isolated by
precipitation with ether.14 Monitoring the progress of the
reaction is also easy because the NMR spectral data can be
obtained without cleaving the product from the polymer.15 In
addition several different solvents such as trimethyl orthofor-
mate,16 2,2,2-trifluoroethanol, or methanol10 can be used as
reaction media. This is particularly beneficial for the Ugi-type
reactions since the solvents are known to promote the Schiff
base formation.
Figure 5. Structures of the peptido aminoglycosides 15j and 19h,j,k
which are active to inhibit the interaction between Rev and mRNA of
HIV.
Work is in progress to screen other members of the library
and study the detailed course of actions of the active peptido
aminoglycosides.
Table 2. Percent Inhibition of the Resynthesized and Purified
Active Peptido Aminoglycosides 15j, 19h, 19j, and 19k at 200 µM
General Methods
A Bruker AMX-400 spectrometer was used for 400 MHz 1H NMR
and 100 MHz 13C NMR analyses. High-resolution mass spectra
(HRMS) were obtained on a VG ZAB-ZSE mass spectrometer. For
the MS of the compounds that are obtained from the MCC, normal
molecular ion peaks (M + H+, M - H+, M + Na+, or M + Cs+) were
recorded.
A representative procedure of the filterbinding assay used in the
study is described as follows: Rev-RRE complexes were detected on
nitrocellulose filters and were subsequently quatitated by measuring
the retained radioactivity on the filter. The peptido aminoglycosides
were dissolved in a 1:1 mixture of DMSO and H2O at a concentration
of 10 mM. The inhibitory potency of the neomycin B mimetic on the
Rev-RRE interaction was analyzed by preincubation of the RRE (8
pM) with 200 µM testing compounds. After 10 min Rev protein was
added to a final concentration of 3.6 nM in a total volume of 250 µL
(2% DMSO). The incubation proceeded for additional 15 min.
Subsequently an aliquot of 200 µL was filtered through a prewetted
nitrocellulose filter using a S&S MINIFOLD filtration unit without
compd
% inhibn
compd
% inhibn
15j
19h
91
87
19j
19k
85
81
product was initially tested for its potential to inhibit the
interaction of the full-length RRE (252 nucleotides) with HIV-1
Rev in a standard “filterbinding assay”, in which recombinant
Rev protein (13 kDa) was bound to the radio labeled RRE RNA
transcripts.12 The results are shown in Table 1.
These results suggest that the most potent compounds of the
series require a free carboxylic acid moiety instead of a methyl
ester at position R. A smaller functionality at R′ is preferred
to the tert-butyl group. This result also indicates that the side
chain R′′ may interact more efficiently with the RRE if R′ is
less hindered: Thr, Asp, and Gln in series 19 (Figure 5) were
found to be active while only Asp in series 15 showed more
than 80% inhibition. In comparison, of the 52 compounds
tested, nine of them showed activity as good as or better than
neomycin B (70.5%) and were much better than neamine and
4′-deoxyneamine (10.5% and <5%, respectively, at 200 µM in
the filterbinding assay).
In order to confirm these initial screening results compounds
15j, 19h, 19j, and 19k were resynthesized, purified, and retested
for their binding affinities. The results are tabulated in Table
2.
The percent inhibitions of the resynthesized compounds are
consistent with those of the earlier results as shown in Table 1.
All of theses compounds showed slightly better activity than
that of neomycin B.
(13) (a) For comparison, the four-component condensation reaction was
carried out using a â-alanine-linked Wang resin as the amine component,
7 as an aldehyde, and tert-butyl isocyanide and Cbz-N-protected lysine as
an acid in MeOH, trimethyl orthoformate, or 2,2,2-trifluoroethanol. None
of the solvents provided an adequate solvation for the reaction. The same
reaction was then carried out in 30% (v/v) of each of the above solvents in
DMF and a significantly lowered reactivity was observed. Because DMF
is not a good solvent for the reaction as a Schiff base formation is required.
After 7 days ca. 30% of the unreacted â-alanine was recovered from the
cleaved products. In addition, it is difficult to monitor the reaction progress
using the solid-phase method unless the intermediate is cleaved from the
support. The solution-phase MCC requires chromatography to isolate the
desired product. (b) For recent developments on solid-phase combinatorial
chemistry, see: Thompson, L. A.; Ellman, J. A. Chem. ReV. 1996, 96, 555.
Armstrong, R. W.; Combs, A. P.; Tempest, P. A.; Brown, S. D.; Keating,
T. A. Acc. Chem. Res. 1996, 29, 123. Keating, T. A.; Armstrong, R. W. J.
Am. Chem. Soc. 1996, 118, 2574.
In summary we have presented a new method for the rapid
synthesis of aminoglycoside mimetics using a neamine-derived
(14) Han, H.; Wolfe, M. M.; Brenner, S.; Janda, K. D. Proc. Natl. Acad.
Sci. U.S.A. 1995, 92, 6419.
(12) (a) Daly, T. J.; Cook, K. S.; Gray, G. S.; Maione, T. E.; Rusche, J.
R. Nature 1989, 342, 816. (b) Daly, T. J.; Doten, R. C.; Rennert, P.; Auer,
M.; Jaksche, H.; Donner, A.; Fisk, G.; Rusche, J. R. Biochemistry 1993,
32, 10479.
(15) Douglas, S. P.; Whitfield, D. M.; Krepinsky, J. J. J. Am. Chem.
Soc. 1991, 113, 5095.
(16) Look, G. C.; Murphy, M. M.; Campbell, D. A.; Gallop, M. A.
Tetrahedron Lett. 1995, 36, 2937.

Combinatorial Synthesis of Aminoglycoside Mimetics
J. Am. Chem. Soc., Vol. 118, No. 42, 1996 10153
washing. After filtration the membrane was removed, dried at 45 °C
for 20 min, and counted using a Packard Matrix 96 direct beta counter.
All measurements were performed in 96-well microtiter plates and
duplicated minimum values.
133.4, 128.6, 128.5, 128.4, 128.1, 128.0, 117.7, 98.3, 81.6, 79.4, 77.6,
75.7, 74.1, 70.9, 69.8, 68.8, 66.8, 66.7, 54.0, 50.2, 50.0, 41.6, 34.1,
20.8, 20.5, 20.4; HRMS for C53H60N4O17 (M + Cs) calcd 1157.3008,
found 1157.3050.
Compound 7. A solution of 6 (1.00 g, 0.977 mmol) in dichlo-
romethane (20 mL) was cooled to -76 °C. Ozone gas was passed
through the solution at that temperature until it became a light-blue
color. The color was diminished as oxygen gas was bubbled through
the solution. The reaction was quenched with triphenyl phosphine
(0.384 g, 1.47 mmol, 1.5 equiv), and the resutant reaction solution was
evaporated under reduced pressure to give a white solid. The desired
product was obtained by column chromatography using a 7:3 mixture
of toluene and acetone as eluent: yield 0.952 g, 0.928 mmol, 95%; 1H
NMR (CDCl3) δ 9.24 (s, 1H, HC(O)), 7.24-7.30 (m, 20 H, aromatic),
5.42 (m, 1H, NH), 4.70-5.15 (m, 11H), 2.98-4.40 (m, 11H), 2.10
(m, 1H, H-2eq), 1.74, 1.81, 1.84 (s, each 3H, CH3), 1.28 (m, 1H, H-2ax);
HRMS for C52H58N4O18 (M + Cs) calcd 1159.2800, found 1159.2844.
Compound 8. To a solution of polyethylene glycol methyl ether
(MW ca. 5000, 1.12 g) in DCM was added t-BocGlyOH (38.5 mg,
0.22 mmol), DCC (45.4 mg, 0.22 mmol), and DMAP (26.9 mg, 0.22
mmol). The reaction mixture was stirred at room temperature overnight.
A white solid that formed during the reaction was filtered, and the
filtrate was evaporated under reduced pressure to give a fluffy white
solid. This solid (1.15 g) was then redissolved in DCM and treated
with 95% TFA (2 mL) at ambient temperature for 1 h. The reaction
mixture was evaporated and further dried in Vacuo overnight to give 8
Neamine 2. A solution of 1 (20.0 g, 20.8 mmol) in methanol (500
mL) was brought to boil, to which a concentrated HCl (18 g, 12.1 N)
was added dropwise. The solution was refluxed for 6 h before all
reactants were completely consumed. The reaction solution was cooled
to room temperature to give a light-yellow solution. This solution was
then evaporated to approximately half of its volume and cooled to 0
°C. The solid precipitate was filtered to give the desired product 2:
yield 7.8 g, 17.1 mmol, 82%; H NMR (D2O) δ 5.94 (d, J1′,2′ ) 3.8
Hz, 1H, H-1′), 4.01-4.06 (m, 3H, H-5′,5,3′), 3.47-3.53 (m, 1H, H-4),
3.36 (m, 1H, H-1), 3.28 (ddd, J3,4 ) 4.5 Hz, 1H, H-3), 2.52 (ddd, J2eq,1
1
) J2eq,3 ) 2.6 Hz, 1H, H-2eq), 1.94 (ddd, J2ax,1 ) J2ax,3 ) 7.9, J2ax,2eq
)
15.7 Hz, 1H, H-2ax); 13C NMR (D2O) δ 102.0, 83.53, 81.36, 78.65,
76.98, 75.38, 59.74, 55.94, 54.66, 46.45, 34.43; ESI for C12H26N4O6
calcd 323 (M + H+), found 323.
Compound 4. To an aqueous saturated Na2CO3 solution (20 mL)
of 2 (3.9 g, 8.36 mmole) was dropwise added at 0 °C over a half hour
period a solution of CbzCl (6.40 g, 37.6 mmol) in acetone (15 mL)
and toluene (2 mL). This reaction mixture was vigorously stirred
overnight in a cold room (4 °C) to give a white precipitate. The
precipitate was then filtered and pulverized with an HCl solution (1
N) until the filtrate became neutral. The white solid was dried for 2
days in Vacuo: yield 6.40 g, 7.44 mmol, 86%; 1H NMR (DMSO-d6) δ
7.33-7.39 (broad m, 20 H, aromatic), 6.86-7.19 (broad s, 4H, NH),
4.89-5.20 (broad m, 9H, benzyl 4 × CH2, H-1′), 3.11-3.73 (broad
1
as a white solid in a TFA salt form:17 yield 1.15 g; H NMR (CDCl3)
δ 3.68 (2H, glycyl CH2), 3.66 (broad s, ca. 450H, PEG-CH2), 3.33 (s,
3H, OCH3).
m, 16H), 1.76 (broad m, 1H, H-2eq), 1.34 (broad dd, J2ax,1 ) J2ax,3
)
8.25, J2ax,2eq ) 15.9 Hz, 1H, H-2ax); 13C NMR (DMSO-d6) δ 156.6,
156.3, 155.8, 142.6, 137.4, 137.3, 137.1, 128.7, 128.6, 128.4, 128.1,
127.8, 127.5, 126.7, 126.5, 98.7, 76.6, 74.2, 71.5, 70.7, 65.4, 65.3, 65.2,
62.9, 56.2, 51.3, 50.4, 42.1, 36.1; HRMS for C44H50N4O14 (M + Cs)
calcd 991.2378, found 991.2348.
Compound 10. To an ethyl formate solution (120 mL) containing
glycine methyl ester monohydrochloride (20.0 g, 0.159 mol) was added
p-TsOH (20 mg), and the solution was brought to boil. Boiling
triethylamine (1.1 equiv) was added dropwise, and the reaction mixture
was refluxed overnight. The reaction mixture was then cooled to room
temperature. A white triethylamine hydrochloride salt was filtered,
and the filtrate was concentrated to ca. 30 mL. The resultant solution
was further cooled to -5 °C and filtered. The filtrate was concentrated
to afford a light brown liquid. Vacuum distillation of this liquid gave
formylacetic acid methyl ester as a colorless oil at 105 °C, 0.15
mmHg: yield 17.7 g, 0.151 mol, 95%. To the solution of formyl acetic
acid methyl ester (10.0 g, 85.4 mmol) and triethylamine (2.5 equiv) in
DCM (200 mL) was added POCl3 (1.0 equiv) dropwise at 0 °C. The
solution turned red immediately. After all of the POCl3 was added,
the reaction mixture was stirred for an additional 1 h at room
temperature. To the reaction mixture was slowly added a Na2CO3
solution (10 g, 200 mL of H2O). The reaction mixture was stirred for
1/2 h. The organic phase was separated from aqueous phase, washed
with a saturated NaCl solution, and dried over K2CO3. The resultant
solution was then filtered, and the filtrate was evaporated under reduced
pressure to give a dark brown oil. The final product (liquid) was
obtained from a fractional distillation (54 °C, 0.15 mmHg): yield 6.22
Compound 5. A solution of acetic anhydride (0.808 mL, 8.57
mmol, 3.6 equiv) in DMF (3 mL) was added through a pressure-
equalized dropping funnel to a solution of 4 (2.04 g, 2.38 mmol) in
pyridine (4 mL, 20 equiv) at ambient temperature. The solution was
stirred overnight to result in a light-yellow solution. This solution
mixture was acidified with an aqueous HCl solution (1 N), diluted with
EtOAc (30 mL), neutralized with a saturated Na2CO3 solution (2 × 10
mL), and washed with water (2 × 10 mL) and a saturated NaCl solution
(10 mL). The organic layer was then dried over MgSO4 and filtered.
The filtrate was evaporated under reduced pressure to give a white
precipitate before all the sovent was removed. This white solid was
isolated by filtration: yield 1.82 g, 1.85 mmol, 78%; 1H NMR (CDCl3)
δ 7.20-7.31 (m, 20 H, aromatic), 5.48, 5.65, 5.83 (broad s, each 1H,
NH), 4.94-5.34 (m, 10 H), 4.84 (m, 3H), 4.91 (m, 1H), 4.70 (m, 1H),
3.65 (m, 3H), 3.94 (m, 2H), 3.48 (m, 1H), 3.34 (m, 1H), 3.24 (m, 1H),
2.96 (m, 1H), 2.12 (broad d, 1H, H-2eq), 1.74, 1.82, 1.93 (s, each 3H,
CH3), 1.48 (broad m, 1H, H-2ax); 13C NMR (CDCl3) δ 169.9, 169.8,
169.2, 156.4, 156.0, 155.7, 137.4, 137.3, 137.2, 137.0, 128.4, 127.8,
127.7, 127.5, 127.4, 98.2, 81.4, 76.4, 73.6, 71.7, 68.7, 68.0, 65.4, 65.3,
65.1, 53.7, 50.0, 48.8, 34.1, 20.9, 20.5, 20.4; HRMS for C50H56N4O17
(M + Cs) calcd 1117.2695, found 1117.2664.
1
g, 61.5 mmol, 72%; H NMR (CDCl3) δ 4.21 (s, 3H, CH3), 3.77 (d,
2H, glycyl CH2); 13C NMR (CDCl3) δ 161.1, 164.4, 53.3, 43.3; FABMS
for C4H5NO2 (M + Cs+) calcd 231.95, found 231.
A representative MCC: 7 (70 mg, 0.0682 mmol) and 8 (300 mg,
∼0.060 mmol) were dissolved in degassed 2,2,2-trifluoroethanol (500
mL), and the mixture was stirred at room temperature for 2 h. To this
solution were added 9 (5.7 mg, 0.0682 mmol) and 11a (14.3 mg, 0.0682
mmol). The reaction mixture was stirred at ambient temperature for 5
days. The isolation of the desired product was accomplished by
precipitation and filtration with cold ether. The precipitated product
was dried in Vacuo to give 12a as a white solid (290 mg). At this
stage the product was divided into two portions to carry out further
modifications.
(i) 12a (140 mg) was dissolved in THF (1 mL) at 0 °C, and LiOH
solution (0.2 mL, 1 N, 10% (v/v) H2O/MeOH) was added to the
solution. The mixture was then stirred for 4 h while the temperature
was slowly elevated to room temperature. After the solution was
neutralized with acidic resin (Amberlite IR 120) and filtered, the filtrate
Compound 6. To a solution of 5 (1.0 g, 1.02 mmol) and tetrabutyl
ammonium iodide (420 mg, 1.13 mmol) in DMSO (3 mL) was added
allyl bromide (112 µL, 1.20 equiv). The reaction mixture was stirred
for 5 min at room temperature. While the solution was vigorously
stirred, lithium bis(trimethylsilyl)amide (1.3 mL, an 1 M solution in
THF) was added at once. The reaction was complete within half an
hour. The reaction mixture was then diluted with EtOAc (30 mL) and
washed with HCl (1 N, 2 × 20 mL), saturated NaHCO3 (2 × 20 mL),
water (2 × 20 mL), and saturated NaCl (20 mL). The organic phase
was then dried over MgSO4 and filtered. The filtrate was evaporated
under reduced pressure to give a crude product. The desired product
was isolated by column chromatography using a 1:9 mixture of actone
1
and toluene: yield 0.790 g, 0.771 mmol, 76%; H NMR (CDCl3) δ
7.26-7.33 (m, 20H, aromatic), 5.48, 5.73 (s, each 1H, NH), 4.70-
5.15 (m, 16 H), 3.85-4.10 (m, 7H), 2.90-3.48 (m, 4H), 2.06 (m, 1H,
H-2eq), 1.81, 1.87, 1.93 (s, each 3H, CH3), 1.28 (m, 1H, H-2ax); 13C
NMR (CDCl3) δ 171.1, 170.6, 169.8, 156.6, 155.8, 155.6, 136.3, 136.2,
(17) Trifluoroacetic acid as a salt was removed by treating the PEG
derivative with a basic ion-exchange resin (Amberlite IRA-400(OH)) in
methanol for less than 5 min.

10154 J. Am. Chem. Soc., Vol. 118, No. 42, 1996
Park et al.
was evaporated under reduced pressure to afford a white solid. The
solid was then washed with cold ether (2 × 10 mL), and insoluble
PEG material was removed by filtration. The filtrate was then
concentrated and further dried in Vacuo to give 14a as a white solid
(17 mg). This product was then directly used for the next step without
further purification. ESI for C64H77N7O20 (M - H+), calcd 1234, found
1234.
To a solution of 14a (15 mg, 0.0259 mmol) in acetic acid (glacial,
2 mL) was added 10% Pd/C (10 mg). The solution was stirred under
hydrogen at ambient temperature for 3 h. The reaction mixture was
then filtered through a Celite bed, and the filtrate was concentrated
under reduced pressure to give 15a as light-yellow syrup: yield 20
mg, 0.0228 mmol, 88% (based on the assumtion that five acetic acid
molecules are complexed with a molecule of 15a); ESI for C23H45-
N7O10 (M - H+) calcd 578.3, found 578.
(ii) To the PEG-linked compound 12a (140 mg) in MeOH (1 mL)
was added NaOMe (100 mL, 1 M, MeOH) at 0 °C for 3 h. After the
solution was neutralized with an acidic resin (Amberlite IR 120) and
filtered, the filtrate was evaporated under reduced pressure to afford a
white solid. The solid was then washed with cold ether (2 × 10 mL),
and insoluble PEG material was removed by filtration. The filtrate
was then concentrated and further dried in Vacuo overnight to give
16a as a white solid (13 mg, 0.0103 mmol). FABMS for C64H78N7O20
(M + Cs+) calcd 1396.9, found 1396. The solution of 16a was stirred
under hydrogen at ambient temperature for 3 h. The reaction mixture
was filtered through a Celite bed, and the filtrate was concentrated under
reduced pressure to give 17a as light-yellow syrup: yield 9 mg, 0.0101
mmol, 98% (based on the assumption that five acetic acid molecules
are complexed with a molecule of 17a); FABMS for C24H47N7O10 (M
+ Cs+) calcd 725, found 725.
1.73 (m, 1H, H-2ax), 1.67-1.70 (m, 2H, guanidyl CH2), 1.25-1.34
(m, 11H, t-Bu, guanidyl CH2).
Preparation of Active Compounds 15j, 19h, 19j, and 19k.18
Compound 16j. To a solution of 7 (200 mg, 0.195 mmol) in 2,2,2-
trifluoroethanol (1.0 mL) was added glycine methyl ester (14 mg, 0.156
mmol) at room temperature. The solution was stirred for 2 h before
tert-butyl isocyanide (13 mg, 18 µL, 0.156 mmol) and CbzAsp(OBn)-
OH (11j, 55.7 mg, 0.156 mmol) were added. The reaction mixture
was stirred for additional 5 days at room temperature to complete. The
mixture was then poured into a 1 N HCl and extracted from ethyl
acetate. The organic layer was washed with 1 N NaHCO3 solution
and water followed by saturated NaCl. The organic layer was dried
over MgSO4 and filtered. The filtrate was then concentrated under
reduced pressure to give a light-yellow crude product. The desired
product 16j was isolated by silica gel column chromatography using
9:1 and 8:2 gradient solvent mixtures of toluene and acetone: yield
170.6 mg, 0.114 mmol, 73%; 1H NMR (CD3OD) δ 7.20-7.64 (m, 30
H, Ph), 4.69-5.42 (m, 15H), 3.55-4.26 (m, 8 H), 3.77 (broad s, 3H,
OCH3), 3.29-3.30 (broad s, 3H, OCH3), 1.88 (s, 3H, C(O)CH3), 1.80
(s, 3H, C(O)CH3), 1.28-1.33 (s, 9H, t-Bu); 13C NMR (CD3OD) δ 173.1,
172.8, 171.9, 171.7, 171.5, 168.2, 163.1, 159.2, 158.9, 158.4, 158.0,
157.8 (CdO), 138.4, 138.1, 138.0, 137.2, 133.8, 133.1, 133.0, 132.3,
130.1, 130.0, 129.9, 129.8, 129.6, 129.5, 129.3, 129.2, 129.1, 128.9,
128.7, 98.92, 84.9, 78.4, 78.2, 73.1, 72.9, 72.0, 71.1, 69.7, 68.7, 38.5,
68.1, 67.9, 67.8, 67.7, 67.3, 66.7, 62.7, 54.7, 53.6, 52.0, 50.9, 50.8,
49.6, 49.4, 49.2, 41.9, 37.7, 34.5, 30.8, 29.9, 29.0, 28.91, 28.9, 28.8,
28.7, 28.7, 28.6, 21.0, 20.8, 20.7; HRMS for C79H91N7O25 (M + Cs)
calcd 1670.5119, found 1670.5139.
Compound 15j. After the above methyl ester 16j (100 mg, 0.0650
mmol) was dissolved in THF (1 mL), a LiOH solution (0.2 mL, 1 N,
10% (v/v) H2O/MeOH) was added to the solution at 0 °C. The mixture
was then stirred for 12 h while the temperature was slowly elevated to
room temperature. After the solution was neutralized with acidic resin
(H+, Amberlite IR 120) and filtered, the filtrate was evaporated under
reduced pressure to afford 14j as a white solid. The product was then
isolated by a column chromatography using a 9:1 mixture of CHCl3
and CH3OH as eluent: yield 70 mg, 0.053 mmol, 82%; ESI for
C65H77N7O22 (M - H+) calcd 1306, found 1306. To a solution of 14j
(45 mg, 0.0344 mmol) in acetic acid (glacial, 2 mL) was added 10%
Pd/C (10 mg). The solution was stirred under hydrogen at ambient
temperature for 3 h. The reaction mixture was then filtered through a
Celite bed, and the filtrate was concentrated under reduced pressure to
give 15j as a white solid: yield 31 mg, 0.0331 mmol, 96%; 1H NMR
(D2O) δ 5.84 (d, J1′,2′ ) 4 Hz, 1H, H-1′), 3.64-4.36 (m, 10 H), 2.83
(m, 1H), 2.61, (m, 1H), 2.42 (m, 1H, H-2eq), 1.76 (ddd, J2ax,3 ) J2ax,3
) 7.9 Hz, J2ax,2eq ) 15.6 Hz, 1H, H-2ax), 1.27 (s, 9H, t-Bu); 13C NMR
(D2O) δ 171.6, 170.9, 170.4, 170.3 (CdO), 135.6, 134.4, 131.4, 98.5,
97.2, 94.9, 80.5, 77.6, 76.6, 74.9, 74.8, 73.1, 71.9, 71.6, 70.7, 61.7,
55.9, 54.9, 52.1, 50.7, 24.8; ESI for C25H47N7O12 calcd 638 (M + H+),
found 638.
Compound 20h. To a solution of 7 (250 mg, 0.244 mmol) in 2,2,2-
trifluoroethanol (1.0 mL) was added glycine methyl ester (22 mg, 0.244
mmol) at room temperature. The solution was stirred for 2 h before
10 (50 mg, 0.448 mmol) and 11h CbzThr(OBn)OH (123.6 mg, 0.488
mmol) were added. After the reaction, the desired product 20h was
isolated similarly by silica gel column chromatography using 9:1 and
8:2 gradient solvent mixtures of toluene and acetone: yield 210 mg,
0.136 mmol, 56%; 1H NMR (CD3OD) δ 7.67-7.64 (m, 30H, Ph),
4.69-5.42 (m, 15H), 3.55-4.26 (m, 8 H), 3.77 (broad s, 3H, OCH3),
1.89 (s, 3H, C(O)CH3), 1.77 (s, 3H, C(O)CH3), 1.59-1.61 (m, 1H),
1.27 (1H, H-2a); 13C NMR (CD3OD) δ 174.2, 172.6, 172.0, 171.5,
171.3, 171.1, 164.2, 159.1, 158.9, 158.5, 158.1, 157.8 (CdO), 139.6,
138.4, 138.3, 138.1, 133.8, 133.1, 132.2, 130.0, 129.9, 129.8, 129.5,
129.4, 129.3, 129.1, 128.9, 128.7, 99.4, 98.3, 84.7, 84.3, 83.8, 81.4,
78.6, 78.4, 78.0, 77.2, 76.5, 76.0, 75.7, 73.0, 72.9, 72.8, 72.3, 72.3,
72.2, 70.9, 70.7, 70.4, 69.9, 69.7, 69.3, 68.4, 68.1, 68.0, 67.9, 67.8,
67.7, 67.6, 67.4, 62.7, 60.7, 52.6, 50.9, 50.8, 41.9, 34.9, 34.5, 33.1,
30.4, 21.4, 21.3, 21.0, 20.8, 20.7, 17.2, 16.9, 16.7; HRMS for
C78H89N7O26 (M + Cs) calcd 1672.4912, found 1672.4840.
The physical data of some other products obtained from the multiple-
component condensation are as follows.
15k: FABMS for C26H50N8O11 (M + Cs) calcd 782, found 782; (M
+ H+) 650.
16a: FABMS for C64H78N7O20 (M + Cs) calcd 1396.9, found 1396.
16b: FABMS for C65H79N7O20 (M + Cs) calcd 1410.4, found 1410.
16c: FABMS for C67H83N7O20 (M + Cs) calcd 1438.5, found 1438.
16d: FABMS for C71H83N7O20 (M + Cs) calcd 1486.5, found 1486.
16e: FABMS for C73H83N8O20 (M + Cs) calcd 1524.5, found 1525.
16f: FABMS for C68H81N9O20 (M + Cs) calcd 1476.5, found 1476.
16j: FABMS for C67H81N7O22 (M + Cs) calcd 1468.0, found 1468.
16k: FABMS for C67H82N8O21 (M + Cs) calcd 1467.5, found 1467.
16l: FABMS for C76H92N8O22 (M + Cs) calcd 1601.5, found 1601.
16m: FABMS for C84H98N10O24 (M + Cs) calcd 1630.6, found 1630.
17a: FABMS for C24H47N7O10 (M + Cs) calcd 725, found 725.
17c: FABMS for C26H50N8O11 (M + Cs) calcd 767, found 736
(M + Cs+ - OCH3).
17g: FABMS for C31H52N7O11 (M + Cs) calcd 831, found 831.
21b: FABMS for C64H75N7O22 (M + Cs) calcd 1426, found 1426.
21c: FABMS for C66H79N7O22 (M + Cs) calcd 1454, found 1454.
21d: FABMS for C70H79N7O22 (M + Cs) calcd 1502, found 1502.
21e: FABMS for C72H79N8O22 (M + Cs) calcd 1541, found 1409
(M + H+).
21f: FABMS for C66H75N9O22 (M + Cs) calcd 1478, found 1478.
21i: FABMS for C64H75N7O23 (M + Cs) calcd 1310.5, found 1310.
21k: ESI for C66H78N8O23 (M + H+) calcd 1352, found 1353 (M +
H+); 1375 (M + Na+).
21l: FABMS for C74H85N8O24 (M + Cs) calcd 1603.5, found 1603.
15e: 1H NMR (D2O) δ 7.12-7.51 (m, 4H, aromatic), 5.61 (d, J1′,2′
) 2.5 Hz, 1H, H-1′), 2.36-2.40 (m, 1H, H-2eq), 1.73 (m, 1H, H-2ax),
1.28-1.32 (m, 2H, indolyl CH2), 1.21-1.27 (m, 9H, t-Bu).
15l: 1H NMR (D2O) δ 5.75 (d, J1′,2′ ) 2.5 Hz, 1H, H-1′), 2.95-
3.00 (m, 2H, lysyl CH2), 2.40-2.43 (m, 1H, H-2eq), 1.79-1.86 (m,
2H, lysyl CH2), 1.66-1.72 (m, 1H, H-2ax), 1.45 (m, 2H, lysyl CH2),
1.27-1.31 (m, 11H, t-Bu, lysyl CH2).
17b: 1H NMR (D2O) δ 5.77 (d, J1′,2′ ) 2.5 Hz, 1H, H-1′), 3.28 (s,
3H, OCH3), 2.36-2.40 (ddd, J2eq,2ax ) 14.4 Hz, 1H, H-2eq), 1.71-
1.78 (m, 1H, H-2ax), 1.42-1.46 (m, 3H, alanyl CH3), 1.29-1.31 (m,
9H, t-Bu).
17m: 1H NMR (D2O) δ 5.74 (d, J1′,2′ ) 2.5 Hz, 1H, H-1′), 3.28 (s,
3H, OCH3), 2.36-2.40 (m, 1H, H-2eq), 2.03 (m, 2H, guanidyl CH2),
(18) For the purpose of characterizations of the MCC products before
deprotection, glycine methyl ester was used as the amine component instead
of the PEG attached glycine.

Combinatorial Synthesis of Aminoglycoside Mimetics
J. Am. Chem. Soc., Vol. 118, No. 42, 1996 10155
Compound 19h. The methyl ester of 20h was deprotected as usual
to give 18h as a white solid. The product was then isolated by a column
chromatography using a 9:1 mixture of CHCl3 and CH3OH as eluent.
Yield: 82 mg, 0.0592 mmol, 91%; ESI for C70H80N7O23 (M - H+),
calcd 1385, found 1385. To a solution of 18h (50 mg, 0.0361 mmol)
in acetic acid (glacial, 2 mL) was added 10% Pd/C (10 mg). The
solution was stirred under hydrogen at ambient temperature overnight.
The reaction mixture was then filtered through a Celite bed, and the
filtrate was concentrated under reduced pressure to give 19h as a white
(D2O) δ 5.79 (d, J1′,2′ ) 4 Hz, 1H, H-1′), 3.99-4.59 (m, 2H), 3.00-
4.20 (m, 21H), 2.64-2.72 (m, 3H), 2.35 (m, 1H, H-2eq), 1.70 (m, 1H,
H-2ax); 13C NMR (D2O) δ 179.1, 178.4, 176.9 (CdO), 159.9, 97.6,
85.6, 79.7, 78.8, 75.7, 75.1, 73.2, 73.0, 71.6, 70.7, 56.0, 54.6, 45.4,
42.4, 38.9, 31.8; ESI for C23H41N7O14 calcd 640 (M + H+), found 640.
Compound 20k. To a solution of 7 (150 mg, 0.146 mmol) in 2,2,2-
trifluoroethanol (1.0 mL) was added glycine methyl ester (22 mg, 0.244
mmol) at room temperature. The solution was stirred for 2 h before
10 (50 mg, 0.448 mmol) and 11k CbzGlnOH (137 mg, 0.488 mmol)
were added. The reaction mixture was stirred for 7 days at room
temperature to complete. After workup and isolation, the desired
product 20k was obtained: yield 123 mg, 0.0833 mmol, 57%; 1H NMR
(CD3OD) δ 6.93-7.48 (m, 30H, Ph), 4.81-5.23 (m, 16H), 3.29-4.06
(m, 28H), 3.68, 3.66 (s, 3H ea, C(O)OCH3), 3.14-3.18 (m, 1H), 1.54-
1.95 (m, 6H), 1.96 (s, 3H, C(O)CH3), 1.77 (s, 3H, C(O)CH3), 0.78-
1.30 (m, 9H); 13C NMR (CD3OD) δ 174.8, 172.6, 172.1, 171.5, 171.3,
171.1, 164.2, 159.1, 158.9, 158.5, 158.1, 157.8 (CdO), 139.6, 138.4,
138.3, 138.1, 133.8, 133.1, 132.2, 130.0, 129.9, 129.8, 129.5, 129.4,
129.3, 129.1, 128.9, 128.7, 99.4, 98.3, 84.7, 84.3, 83.8, 81.4, 78.6, 78.4,
78.0, 77.2, 76.5, 76.0, 75.7, 73.0, 72.9, 72.8, 72.3, 72.3, 72.2, 70.9,
70.7, 70.4, 69.9, 69.7, 69.3, 68.4, 68.1, 68.0, 67.9, 67.8, 67.7, 67.6,
67.4, 62.7, 60.7, 52.6, 50.9, 50.8, 41.9, 34.9, 34.5, 33.1, 30.4, 21.4,
21.3, 21.0, 20.8, 20.7, 17.2, 16.9, 16.7; ESI for C72H84N8O26 (M +
Cl-), calcd 1514, found 1514, 1327, 1249.
1
solid: yield 28 mg, 0.0303 mmol, 84%; H NMR (D2O) δ 5.80 (d,
J1′,2′ ) 4 Hz, 1H, H-1′), 3.27-4.59 (m, 24 H), 2.64-2.72 (m, 3H),
2.35 (m, 1H, H-2eq), 1.76 (m, 1H, H-2ax); 13C NMR (D2O) δ 179.1,
178.4, 172.9, 172.0 (CdO), 98.5, 96.5, 85.2, 84.9, 80.8, 77.6, 75.9,
75.5, 74.9, 73.2, 71.7, 70.9, 70.8, 55.9, 52.4, 51.1, 50.9, 45.9, 42.5,
25.4; ESI for C23H49N7O13 calcd 626 (M + H+), found 626.
Compound 20j. To a solution of 7 (165 mg, 0.161 mmol) in 2,2,2-
trifluoroethanol (1.0 mL) was added glycine methyl ester (22 mg, 0.244
mmol) at room temperature. The solution was stirred for 2 h before
10 (50 mg, 0.448 mmol) and 11j CbzAsp(OBn)OH (174.4 mg, 0.488
mmol) were added. The reaction mixture was stirred for 7 days at
room temperature to complete. Following the same workup, 20j was
isolated by using 9:1 and 8:2 gradient solvent mixtures of toluene and
acetone: yield 185 mg, 0.119 mmol, 71%; 1H NMR (CD3OD) δ 7.24-
7.64 (m, 30H, Ph), 4.92-5.10 (m, 15H), 3.55-4.26 (m, 8H), 3.64
(broad s, 3H, OCH3), 3.59 (broad s, 3H, OCH3), 2.92-3.02 (m, 2H),
2.78-2.81 (m, 1H), 1.97 (s, 3H, C(O)CH3), 1.89 (s, 3H, C(O)CH3),
1.77 (s, 3H, C(O)CH3), 1.21-1.26 (m, 3H), 0.78-0.88 (m, 4H); 13C
NMR (CD3OD) 173.2, 172.4, 172.3, 172.2, 171.9, 171.6, 171.5, 171.0,
169.2, 164.1, 158.9, 158.6, 158.5, 158.4, 158.1, 138.6, 138.4, 138.1,
138.0, 137.9, 137.3, 137.2, 133.8, 133.2, 133.1, 130.2, 130.1, 130.0,
129.9, 129.7, 129.6, 129.3, 129.1, 129.0, 126.4, 97.74, 84.2, 77.7, 77.5,
77.0, 76.1, 75.3, 72.7, 72.4, 71.9, 70.8, 70.6, 69.8, 68.2, 68.1, 68.0,
68.0, 65.3, 62.4, 53.2, 52.8, 51.1, 51.0, 43.1, 42.5, 40.7, 37.5, 37.1,
36.8, 34.7, 33.1, 30.8, 30.5, 30.4, 28.2, 27.9, 24.7, 21.6, 21.5, 20.9,
20.8, 20.4; HRMS for C78H87N7O27 (M + Cs) calcd 1686.4704, found
1686.4755.
Compound 19k. After 20k (65 mg, 0.0462 mmol) was hydrolyzed
and neutralized as usual, 18k was obtained as a white solid, which (50
mg, 0.0336 mmol) was hydrogenated for 3h to give 19k as a white
solid: yield 30 mg, 0.0336 mmol, quantitative; 1H NMR (D2O) δ 5.67
(d, J1′,2′ ) 4 Hz, 1H, H-1′), 4.30-4.65 (m, 1H), 3.14-4.22 (m, 18H),
2.38 (m, 1H, H-2eq), 1.70-1.76 (m, 1H, H-2ax); 13C NMR (D2O) δ
179.1, 178.5, 172.9, 172.1 (CdO), 98.5, 84.8, 80.8, 77.6, 75.9, 75.5,
74.9, 73.2, 72.9, 71.7, 71.5, 70.9, 70.8, 55.9, 52.4, 51.1, 50.9, 50.8,
45.9, 42.4, 20.2; ESI for C24H44N8O13 calcd 653 (M + H+), found 651.
Acknowledgment. This paper is dedicated to Prof. Nelson
Leoner on the occasion of his 80th birthday and for his
pioneering contribution in the field of nucleic acid recognition.
We thank Sandoz for financial support of this research. W. K.
C. Park also thanks Dr. A. Do¨mling for valuable discussions.
Compound 19j. After 20j (60 mg, 0.0386 mmol) was treated with
base as usual, compound 18j was obtained (50 mg, 0.0337 mmol) and
hydrogenated for 3 h. The reaction mixture was then filtered through
a Celite bed, and the filtrate was concentrated under reduced pressure
to give 19j as a white solid: yield 22 mg, 0.0234 mmol, 70%; 1H NMR
JA9612817