Synthesis of paromomycin derivatives modified at C(5″) to selectively target bacterial rRNA

Article abstract of DOI:10.1016/j.carres.2006.09.014

The furanosyl moiety (ring III) of C(6′)-deoxyparomomycin and paromomycin was modified in search of aminoglycoside antibiotics with altered selectivity. The key intermediates were the N-Boc-protected derivative of C(6′)-deoxyparomomycin and the benzyliden

Full text of DOI:10.1016/j.carres.2006.09.014

Carbohydrate Research 342 (2007) 499–519
Synthesis of paromomycin derivatives modified at C(500) to
selectively target bacterial rRNA
a
a
b
a,
*
´
Iwona Kudyba, Deborah Perez Fernandez, Erik C. Bo¨ttger and Andrea Vasella
aLaboratorium fu¨r Organische Chemie, ETH Zu¨rich, HCI, CH-8093 Zu¨rich, Switzerland
bInstitut fu¨r Medizinische Mikrobiologie, Universita¨t Zu¨rich, Gloriastrasse 30/32, CH-8006 Zu¨rich, Switzerland
Received 31 August 2006; accepted 18 September 2006
Available online 17 October 2006
Dedicated to the memory of Professor Nikolay K. Kochetkov
Abstract—The furanosyl moiety (ring III) of C(60)-deoxyparomomycin and paromomycin was modified in search of aminoglycoside
antibiotics with altered selectivity. The key intermediates were the N-Boc-protected derivative of C(60)-deoxyparomomycin and the
benzylidene-protected paromomycin. Their H2C(500)–OH group was oxidised with trichlorocyanuric acid or [bis(acetoxy)iodo]-
benzene in the presence of catalytic amounts of TEMPO to yield the corresponding aldehydes and acids, which were transformed
into the protected alkoxy imines, amides and the amine. Standard deprotection gave the title compounds derived from C(60)-deoxy-
paromomycin and derived from paromomycin that proved less active than paromomycin and its C(60)-deoxy analogue.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Keywords: Aminoglycoside antibiotics; Paromomycin; Selectivity; Oxidation; Oxime; Carboxamide; Mycobacterium smegmatis
1. Introduction
structure based design of new drugs have thus been
pursued to obtain antibiotics that discriminate between
prokaryotic and eukaryotic rRNA,18–24 and recent key
insights into the crystal structure of the complex formed
by paromomycin and the 16S rRNA A-site25–29 allow
for a rational approach to such modifications. Thus,
bacterial ribosomes possess a guanine base (G) at posi-
tion 1491 (numbered according to the E. coli sequence),
while eukaryotic cytoplasmic ribosomes are character-
ised by 1491A and mitochondrial ones by
1491C.11,12,28,30–32 The above mentioned crystal struc-
ture shows a distance between C(500)–O and N(7) of
2.41 A, indicating that C(5 )–OH of ring III of paromo-
mycin forms a hydrogen bond to N(7) of G1491 in the
bacterial ribosome. In eukaryotic cytoplasmic ribo-
somes, there may be a corresponding hydrogen bond
between C(500)–OH and N(7) of A1491. We planned to
impair the (speculative) interaction of paromomycin
with N(7) of A1491 by replacing C(500)–OH by a substi-
tuent that cannot act as hydrogen bond donor, and to
differentiate between G, A and C on the basis of their
different charge density distribution.33 Aromatic
Paromomycin, a broad-spectrum aminoglycoside anti-
biotic,1,2 binds to the 16S rRNA at the tRNA acceptor
A site and impairs the translation process by causing
misreading and by hindering translocation.3–6 The selec-
tivity of paromomycin, that is the discrimination
between prokaryotic and eukaryotic ribosomes, and of
aminoglycoside antibiotics in general, is significantly
limited by the cross-species conservation of the structure
of the decoding site of prokaryotic and eukaryotic ribo-
somes7,8 with the consequence of significant drug-associ-
ated toxicity.9,10 Experimental results of the interactions
between aminoglycosides and mutated A-sites indicate
that rRNA sequence differences largely account for the
selectivity of aminoglycosides with respect to bacterial
and human ribosomes.11–17 Modifications of strategic
structural elements of aminoglycoside antibiotics, or
00
˚
*
Corresponding author. Tel.: +41 446325130; e-mail: vasella@
org.chem.ethz.ch
0008-6215/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2006.09.014

500
I. Kudyba et al. / Carbohydrate Research 342 (2007) 499–519
intermolecular interactions (p–p or CH–p interactions34)
appeared promising, and we speculated that they may
also compensate for the partial loss of activity resulting
from deoxygenation of C(60) of paromomycin.35 Based
on similar assumptions, Foloppe et al. screened a library
of small molecules to find compounds with good stack-
ing interactions with G1491.21 We report on the prepa-
ration of paromomycin and C(60)-deoxyparomomycin
derivatives bearing either a C(500)-hydroximo-, O-substi-
tuted C(500)-hydroximo-, anilino or a corresponding car-
boxyl, or aminocarbonyl function and on the influence
of these modifications at position C(500) on the antibiotic
activity. This position was already modified by a few
research groups. Thus, Baasov et al. prepared a series
of branched neomycin (C(60)@NH2) derivatives pos-
sessing an additional glycosyl moiety at O–C(500)36,37
to obtain antibiotics resistant towards aminoglycoside
phosphotransferase APH(30) that phosphorylates the
C(500)–OH group. Hanessian et al. prepared C(500)–
NH2 derivatives of paromomycin and neomycin for
the same purpose.38 The same hydroxymethyl group
was also modified to obtain conformationally biased
Me
Me
O
O
HO
HO
HO
HO
NHBoc
NH2
H2N
BocHN
N
RO
RO
O
O
O
O
d)
N
NHBoc
NH2
O
O
BocNH
OH
OH
HO
HO
H2N
O
O
OH
3a−f
O
O
OH
4a−g
BocHN
H2N
OH
OH
c)
3b
3g
b)
Me
OH
O
O
HO
HO
HO
HO
NH2
NHBoc
H2N
BocHN
HO
O
O
O
a)
HO
NH2
O
NHBoc
O
O
OH
OH
HO
HO
HO
H2N
HO BocHN
H2SO4 H2O
O
O
OH
O
O
OH
H2N
H2N
H2N
BocHN
1
2
OH
OH
e)
Me
Me
O
O
HO
HO
HO
HO
H2N
NHBoc
H2N
BocHN
O
O
O
O
d)
NH2
HO2C
NH2
NHBoc
HO2C
O
O
OH
OH
HO BocHN
O
O
OH
O
O
OH
BocHN
6
5
OH
OH
f)
Me
Me
O
O
HO
HO
HO
HO
NHBoc
H2N
d)
H2N
BocHN
O
O
O
O
NHBoc
NH2
RHNOC
RHNOC
NH2
O
O
OH
OH
HO BocHN
OH
O
O
OH
O
O
BocHN
7a−c
c)
OH
8a−c, g
OH
7b
7g
a: R = Bn, b: R = CH2C6H4(4-NO2), c: R = Ph, d: R = Me,
e: R = CH2C6F4, f: R = Tr, g: R= CH2C6H4(4-NH2),
Scheme 1. Reagents and conditions: (a) six steps, 18% overall yield; (b) TCCA, TEMPO, EtOAc, 25 ꢁC; then RONH2ÆHCl, 1:1 pyridine–MeOH,
25 ꢁC; 44–72% from 2; (c) H2, Pd/C, MeOH or 80% aq AcOH, 25 ꢁC, 72–88%; (d) 97:3, CF3 COOH/anisole, 25 ꢁC, 56–91%; (e) BAIB, TEMPO, 1:1
MeCN–water, 23 ꢁC; 54%; (f) EDAC, HOBt, DMF, 0 ꢁC; then DIPEA, RNH2, 25 ꢁC, 40–45%.

I. Kudyba et al. / Carbohydrate Research 342 (2007) 499–519
501
neomycin and paromomycin derivatives. Asensio et al.
designed and synthesised neomycin derivatives with a
covalent bond between C(500)–OH and C(20)–NH2 to
mimic the corresponding hydrogen bond that appears
to be formed while interacting with the ribosome, with
the intention of overcoming bacterial resistance based
OH
R1O
O
N3
O
R2O
HO
HO
HO
N3
NH2
H2N
O
RO
RO
O
O
d)
N
N
O
O
N3
NH2
O
OH
OH
HO
N3
N3
HO
H2N
O
O
OH
O
OH
O
H2N
12a−b
OH
OH
10a−c R1, R2 = PhCH
11a−c R1 = R2 = H
c)
b)
OH
O
O
Ph
O
HO
HO
O
HO
N3
H2N
O
NH2
N3
O
a)
HO
N3
O
O
N3
NH2
HO2C
1
O
O
OH
OH
HO
N3
HO
H2N
O
O
OH
O
O
OH
H2N
9
14
OH
OH
e)
f)
R1O
O
O
Ph
O
O
R2O
HO
RO
N3
N3
N3
N3
O
O
O
h)
N3
HO2C
R3OC
O
N3
O
OH
O
OR
HO
N3
N3
OR
N3
O
O
OH
O
O
OR
N3
13
OH
OR
18 R1, R2 = PhCH, R = Ac, R3 = OH
19 R1, R2 = PhCH, R = Ac, R3 = NH2
20 R1, R2 = PhCH, R = H, R3 = NH2
21 R1 = R2 = R = H, R3 = NH2
i)
j)
g)
c)
d)
OH
O
R1O
O
R2O
HO
HO
HO
N3
N3
NH2
H2N
O
d)
O
O
N3
O
RHNOC
N3
NH2
RHNOC
O
OH
O
OH
HO
N3
HO
H2N
O
O
OH
O
O
OH
17a−c
H2N
OH
OH
15b−15c R1, R2= PhCH
c)
16b−16c R1 = R2= H
a: R = H, b: R = CH2C6H4(4-NO2), c: R = Ph
Scheme 2. Reagents and conditions: (a) two steps, 37% overall yield; (b) TCCA, TEMPO, EtOAc, 25 ꢁC, then RONH2ÆHCl, 1:1 pyridine–MeOH,
25 ꢁC, 46–69% from 9; (c) TsOHÆH2O, MeOH, 25 ꢁC, 62–89%; (d) HS(CH2)3SH, MeOH, Et3N, 25 ꢁC, 71–86%; (e) BAIB, TEMPO, 1:1 MeCN–
water, 23 ꢁC, 54%; (f) PMe3, THF, 1 N NaOH, 50 ꢁC, 84%, then H2, 10% Pd/C, 80% aq AcOH, 23 ꢁC, 64%; (g) EDAC, HOAt, DIPEA, RNH2,
DMF, 25 ꢁC, 34–64%; (h) Ac2O, pyridine, 25 ꢁC, 18 h, 76%; (i) N,N0-carbonyldiimidazole, THF, 25 ꢁC, 2 h, then NH4OAc, 16 h, 76%; (j) NaOMe,
MeOH, CH2Cl2, 25 ꢁC, 16 h, 92%.

502
I. Kudyba et al. / Carbohydrate Research 342 (2007) 499–519
on the deactivation of the antibiotics by O-adenyltrans-
ferase ANT(4).39,40 For the same reason, C(500)-methyl,
propyl or carboxylate derivatives were synthesised.40
Tor, Hermann et al. studied the selective binding of con-
formationally constrained paromomycin and neomycin
derivatives to A-site and HIV-1 TAR RNA targets.41,42
deprotection with TFA gave the acid 6 from 5, and
the amides 8a–c,g from 7a–c,g.
The analogous, paromomycin-derived alkoxy imines
12a–b and the amides 17a–c were obtained by first trans-
forming paromomycin sulfate into the corresponding
known pentaazide by diazo transfer from TfN3 in the
presence of CuSO4Æ5H2O and t-BuOK, followed by
benzylidenation according to a known procedure35 to
obtain 9 (Scheme 2). The protected alkoxy imines
10a–c were synthesised similarly as described above for
the 60-deoxy analogues. Selective oxidation of 9 with tri-
chlorocyanuric acid (TCCA) in the presence of a cata-
lytic amount of TEMPO44 was directly followed by
treatment of the resulting crude aldehyde with substi-
tuted hydroxylamines in a mixture of pyridine and
methanol, to yield 46–69% of the protected N-alkoxy
imines 10a–c. Debenzylidenation gave the N-alkoxy imi-
nes 11a (89%) and 11b (83%). The N-phenoxy imine 11c
was not stable in solution and decomposed within hours
at ambient temperature. The pentaazides 11a and 11b
were reduced with propane-1,3-dithiol47 to the N-alkoxy
imines 12a (76%) and 12b (71%).
The benzylidene derivative 9 was also transformed
into the acid 14, the amides 17a–c, and the aniline
24 (Schemes 2 and 3). Regioselective oxidation of 9
with [bis(acetoxy)iodo]benzene (BAIB) and catalytic
amounts of TEMPO45 followed by standard Staudinger
reaction and debenzylidenation yielded 29% of the acid
14. The amides 17b,c were prepared by oxidation of 9
to the benzylidene protected acid 13 followed by trans-
formation into the amides 15b,c, using N-ethyl-N0-
(3-dimethylaminopropyl)-carbodiimide (EDAC) in the
presence of 1-hydroxy-7-azabenzotriazole (HOAt) as
coupling agent. Hydrolytic cleavage of the phenyl di-
oxane ring of 15b and 15c to provide 16b and 16c, and
reduction of the azido groups led in yields of 59 and
56% to 17b and 17c, respectively.
2. Results and discussion
The synthesis of the desired derivatives from the N-Boc
protected C(60)-deoxyparomomycin 235 possessing only
one primary hydroxyl group appeared straightforward,
and was undertaken first (Scheme 1). The alkoxy imines
4a–g were obtained by selective oxidation of the
H2C(500)–OH group of 2 with trichlorocyanuric acid
(TCCA) in the presence of catalytic amounts of TEM-
PO,43,44 followed by treatment of the crude product (a
mixture of aldehyde, hydrate and methyl hemiacetals)
with the O-substituted hydroxylamines in a mixture of
pyridine and methanol, and deprotection of the resulting
protected oxime ethers 3a–f that were isolated in yields
of 44–72%. The aniline 3g was obtained in 88% yield
by reduction of the nitrophenyl ether 3c with H2 in the
presence of Pd/C. Minor amounts (< 5%) of the (Z) iso-
mers of the alkoxy imines 3a–g were isolated by FC. Re-
moval of the N-Boc group of 3a–g with TFA led to the
pentakis-trifluoroacetate of the C(60)-deoxy alkoxy imi-
nes 4a–g. The O-phenyl oxime 4c and the O-trityl ana-
logue 4f decomposed in aqueous solution at 25 ꢁC
within minutes (4c), or days (4f).
The N-Boc protected C(60)-deoxy acid 5 (Scheme 1)
was prepared in a yield of 54% by regioselective oxida-
tion of the deoxyparomomycin derivative 2 with [bis-
(acetoxy)iodo]benzene (BAIB) and catalytic amounts
of TEMPO.45 The carboxamides 7a–c were obtained
by coupling of the acid 5 using N-ethyl-N0-(3-dimethyl-
aminopropyl)-carbodiimide (EDAC) in the presence of
hydroxybenzotriazole (HOBt). The aniline 7g was
obtained in 72% yield by reduction of the nitrophenyl
ether 3c with H2 in the presence of Pd/C. Standard
The primary amide 17a was prepared in a slightly
modified way. Oxidation of 9 followed by acetylation
gave the protected acid 18 that was transformed into
the amide 19 (76% yield) by treatment with N,N0-carb-
onyldiimidazole and ammonium acetate. Standard
OH
R2O
O
O
R1O
HO
HO
HO
NH2
H2N
N3
N3
O
O
PhHN
PhHN
N3
a)
c)
O
O
NH2
N3
9
O
O
OH
OH
HO
H2N
HO
N3
O
O
OH
O
O
OH
H2N
OH
OH
22 R1, R2 = PhCH
23 R1 = R2 = H
24
b)
Scheme 3. Reagents and conditions: (a) TCCA, TEMPO, EtOAc, 25 ꢁC; then PhNH2, NaBH3CN, 55%; (b) TsOHÆH2O, MeOH, 25 ꢁC, 91%;
(c) PMe3, THF, 1 N NaOH, 50 ꢁC, 84%.

I. Kudyba et al. / Carbohydrate Research 342 (2007) 499–519
503
deacetylation of 19 followed by debenzylidenation gave
the pentaazide 21 that was reduced to yield 57% (from
19) of the amide 17a (Scheme 2).
The protected aniline 22 was obtained in a yield of
30% by reductive amination with aniline and sodium
cyanoborohydride of the aldehyde obtained from 9,48
and transformed by standard debenzylidenation and
Staudinger reaction/hydrolysis into the desired the ani-
line 24 (67%) (Scheme 3).
play a role in stabilising the active conformation of par-
omomycin. This interpretation is in agreement with
observations of Asensio et al.40 and Tor et al.41,42 about
paromomycin and neomycin derivatives deoxygenated
at C(500). So far, only derivatives of paromomycin
and neomycin in which the C(500) hydroxy group was
replaced by an unsubstituted amino group38 displayed
similar antibacterial activities as the parent compounds.
4. Experimental
3. Biological studies
4.1. General methods
See Ref. 35. 1H and 13C NMR spectra for Boc-protected
compounds (3a–g, 5, 7a–c,g) are not described; signals
were broad, even when the spectra were recorded at
100 ꢁC in Me2SO-d6. Minor (Z) isomers of alkoxyimines
were formed in max. 5% yield. They were separated
from the (E) isomers but not characterised.
The deoxyparomomycin 2 was prepared from com-
mercially available 150–54 according to a known proce-
dure35 (Note that the structure of 1 and its derivatives
in this paper has to be corrected; the absolute configura-
tion of ring IV should be L).
The experimental model used to investigate ribosomal
drug susceptibility is a single rRNA allelic derivative
of the Gram positive eubacterium Mycobacterium
smegmatis.46 Genetic manipulations of its single rRNA
operon by site directed mutagenesis and recA-mediated
gene conversion resulted in homogeneous populations
of mutant ribosomes.14,15,49 Ribosomal drug susceptibil-
ity was studied by determining the minimal inhibitory
concentrations, as described in detail in Refs. 14 and 15.
All derivatives of paromomycin and (60)-deoxypa-
romomycin modified at position C(50) proved less active
against M. smegmatis wild type, as compared to the par-
ent compounds (see Table 1). The derivatives 4a,b,d–g,
6, 8a–c,g were 8–16-fold less active than (60)-deoxypa-
romomycin. The relatively most active compound
among the paromomycin derivatives is 12a. It showed
a 16 times lower activity than paromomycin. No activity
towards ribosomes with a single mutated base at posi-
tion 1491 (G1491A, G1491C) was found, suggesting
that the hydrogen bond between C(500)–OH and N(7)
of G1491 is crucial for binding and activity of paromo-
mycin and its derivatives. Deoxygenation of the
H2C(500)–OH group may also lead to elimination of
the intramolecular H-bond to C(20)–NH2, which may
4.2. General procedure for the transformation of primary
alcohols into N-alkoxyimino derivatives (GP 1)
Under N2, a cooled (6 0 ꢁC) 0.05 M soln of the alcohol
(1 equiv) in EtOAc was treated with trichloroisocyanu-
ric acid (TCCA; 1.1 equiv) and a catalytic amount of
TEMPO (0.05 equiv), allowed to warm to 25 ꢁC, stirred
for 25–40 min and basified with 1 N NaOH. After sepa-
ration of the phases, the aq layer was extracted with
EtOAc (3·). The combined org. layers were dried
(MgSO4) and evaporated. A 0.05 M soln of the residue
(crude aldehyde, its hydrate and methyl hemiacetal) in
1:1 MeOH–pyridine was treated with the appropriate
N-alkoxyamine, and stirred at 25 ꢁC for 24 h. MeOH
was evaporated, the residue was taken up in brine and
EtOAc, the phases were separated and the aq layer
was extracted with EtOAc (3·). The combined org. lay-
ers were dried (MgSO4) and evaporated to give the
crude N-alkoxyimines.
Table 1. Ribosomal drug susceptibilitya
Wild type
1491G
Mutant
G1491A
Mutant
G1491C
Paromomycin
1
64
>512
512
>512
>512
>512
>512
>512
>512
>512
>512
>512
>512
>512
>512
>512
60-Deoxyparomomycin
32–64
256
512
256–512
>512
16–32
64
32–64
128
4a,c,d
4b
4g
>512
>512
>512
>512
>512
>512
256–512
>512
>512
>512
4.2.1. 500-(N-Benzyloxyimino)-1,3,20,2000,6000-pentakis-[N-
(tert-butoxycarbonyl)]-60,500-dideoxyparomomycin (3a).
GP 1 and FC (2:2:0.1 ! 2:2:0.3 CHCl3–EtOAc–MeOH)
gave 3a (465 mg, 72%). White solid: mp 173–176 ꢁC;
6, 8a–c,g
12a
12b
14
25
½aꢀD +47.6 (c 0.145, MeOH); Rf = 0.62 (2:2:0.5,
17a
17b
17c
CHCl3–EtOAc–MeOH); IR (ATR); m 3374m, 2979w,
512
128–56
256–512
2931w, 2071w, 1682s, 1523w, 1426s, 1393m, 1367s,
1307w, 1254w, 1165s, 1118m, 1041s, 1024s, 975s cmꢁ1
;
;
24
HRESIMS: 1204.6092 (22 [M+H]+, C55H91N6NaO
þ
a Ribosomal drug susceptibility is given as determination of minimal
inhibitory concentrations (MIC [lg/mL]).
23
Calcd 1204.6136), 1226.5926 (100, [M+Na]+,

504
I. Kudyba et al. / Carbohydrate Research 342 (2007) 499–519
C55H90N6NaO23þ; Calcd 1226.3193). Anal. Calcd for
C55H90N6O23 (1204.32): C, 54.90; H, 7.54; N, 6.98.
Found: C, 54.71; H, 7.58; N, 6.72.
1194.5140). Anal. Calcd for C55H85F5N6O23 (1294.28):
C 51.08, H 6.62, N 6.50. Found: C 51.27, H 6.60, N 6.44.
4.2.6. 1,3,20,2000,6000-Pentakis-[N-(tert-butoxycarbonyl)]-
60,500-dideoxy-500-[N-(trityloxy)imino]paromomycin (3f).
4.2.2.
1,3,20,2000,60-Pentakis-[N-(tert-butoxycarbonyl)]-
60,500-dideoxy-500-[N-(4-nitrobenzyloxy)imino]paromomy-
cin (3b). GP 1 and FC (2:2:0.2 ! 3:4:0.5, CHCl3–
EtOAc–MeOH) gave 3b (1406 mg, 62%). White solid:
GP
1
and FC (2:2:0.1 ! 2:2:0.3, CHCl3–EtOAc–
MeOH) gave 3f (162 mg, 44%). White solid: mp 140–
25
142 ꢁC; ½aꢀD +35.2 (c 0.44, MeOH); Rf = 0.72 (2:2:0.7,
25
CHCl3–EtOAc–MeOH); IR (ATR): m 3397w, 2977w,
mp 150–153 ꢁC; ½aꢀD +41.8 (c 0.43, MeOH); Rf = 0.79
2932w, 1687s, 1506m, 1449w, 1392m, 1366m, 1280m,
(2:2:0.75, CHCl3–EtOAc–MeOH); IR (ATR): m 3359w,
2977w, 2933w, 1689s, 1603w, 1519s, 1454w, 1392m,
1365m, 1345m, 1283m, 1248m, 1162s, 1041s, 1020s,
993s, 950w, 919w cmꢁ1; HRESIMS: 1270.5785 (100,
1247m, 1159s, 1039s, 1022s, 987s, 961m, 920m cmꢁ1
;
;
HRESIMS: 1355.6913 (38, [M+H]+, C67H99N6O
þ
23
Calcd 1355.6762), 1377.6558 (100, [M+Na]+,
C67H98N6NaO23þ; Calcd 1377.6581). Anal. Calcd for
C67H98N6O23 (1355.52): C, 59.37; H, 7.29; N, 6.20.
Found: C, 59.21; H, 7.13; N, 6.06.
[M+Na]+, C55H89N7NaO þ; Calcd 1270.5806). Anal.
25
Calcd for C55H89N7O25 (1248.32): C, 52.92; H, 7.19;
N, 7.85. Found: C, 54.00; H, 7.29; N, 7.66.
4.2.7. 1,3,20,2000,6000-Pentakis-[N-(tert-butoxycarbonyl)]-
60,500-dideoxy-500-[N-(4-aminobenzyloxy)imino]paromo-
mycin (3g). Under H2, a suspension of 3c (0.136 mmol,
170 mg) and 10% Pd/C (17 mg) in MeOH (6 mL) was
stirred at 25 ꢁC for 12 h, filtered and evaporated. FC
(2:2:0.2 ! 2:2:0.5, CHCl3–EtOAc–MeOH) gave 3g
4.2.3. 1,3,20,2000,6000-Pentakis-[N-(tert-butoxycarbonyl)]-
60,500-dideoxy-500-[(N-phenoxy)imino]paromomycin (3c).
GP 1 and FC (3:4:0.1 ! 3:4:0.5 CHCl3–Et2O–MeOH)
gave 3c (226 mg, 70%). White solid: mp 171–173 ꢁC;
25
½aꢀD +11.7 (c 0.21, MeOH); Rf = 0.54 (3:4:0.75,
CHCl3–Et2O–MeOH); IR (ATR): m 3363w, 2979w,
25
(153 mg, 88%). White solid: mp 160–162 ꢁC; ½aꢀD
2923w, 1682s, 1593w, 1512m, 1454w, 1392m, 1366m,
+38.3 (c 0.36, MeOH); Rf = 0.46. (2:2:0.75, CHCl3–
EtOAc–MeOH); IR (ATR): m 3363m, 2979w, 2932w,
1686s, 1614w, 1520m, 1480w, 1427m, 1393m, 1367s,
1283m, 1248m, 1160s, 1044s, 1033s, 1018s, 922w cmꢁ1
;
;
HRESIMS: 1211.5807 (100, [M+Na]+, C54H88N6NaO
þ
23
Calcd 1211.5799). Anal. Calcd for C54H88N6O23
(1189.3029): C, 54.54; H, 7.46; N, 7.07. Found: C,
54.37; H, 7.50; N, 6.95.
1308w, 1281w, 1252m, 1164s, 1117s, 1042s, 974s cmꢁ1
;
;
HRESIMS: 1218.6255 (100, [M+H]+, C55H92N7O
þ
23
Calcd
1218.6245),
1240.6205
(69,
[M+Na]+,
C55H91N7NaO23þ; Calcd 1240.6064). Anal. Calcd for
C55H91N7O23 (1218.34): C, 54.22; H, 7.53; N, 8.05.
Found: C, 54.15; H, 7.52; N, 7.89.
4.2.4. 1,3,20,2000,6000-Pentakis-[N-(tert-butoxycarbonyl)]-
60,500-dideoxy-500-[N-(methoxy)imino]paromomycin (3d).
GP 1 and FC (3:4:0.1 ! 3:4:0.5, CHCl3–Et2O–MeOH)
25
4.2.8. 1,3,20,2000,6000-Pentaazido-40,60-O-benzylidene-1,3,20,
2000,6000-pentadeamino-500-deoxy-500-[N-(hydroxy)imino]paro-
momycin (10a). GP 1 and FC (amino phase gel, 9:1,
CH2Cl2–MeOH) gave 10a (260 mg, 46%). White solid:
gave 3d (60 mg, 62%). Oil. ½aꢀD +34.0 (c 1.17, MeOH);
Rf = 0.50 (3:4:0.75, CHCl3–Et2O–MeOH); IR (ATR);
m 3364m, 2978w, 2935w, 2070m, 1681s, 1521m, 1426s,
1393m, 1366s, 1309w, 1254m, 1164s, 1118s, 1040s,
976s cmꢁ1; HRESIMS: 1149.5652 (100, [M+Na]+,
C49H86N6NaO23þ; Calcd 1149.5642). Anal. Calcd for
C49H86N6O23 (1127.23): C, 52.21; H, 7.69; N, 7.46.
Found: C, 52.28; H, 7.64; N, 7.24.
25
mp 130–135 ꢁC; ½aꢀD +130.7 (c 0.14, MeOH);
Rf = 0.18 (amino phase gel, 9:1, CH2Cl2–MeOH); IR
(ATR): m 3406w, 2924w, 2099s, 1455w, 1373w, 1333w,
1255m, 1155w, 1089m, 1018s cmꢁ1
;
1H NMR
(500 MHz, CD3OD) see Table 6, additionally, d 7.50–
7.47 (m, 2 arom. H), 7.36–7.32 (m, 3 arom. H), 5.58
(s, CHPh); 13C NMR (125 MHz, CD3OD) see Table 7,
additionally, d 139.15 (s), 130.02 (d), 129.10 (2d),
4.2.5. 1,3,20,2000,6000-Pentakis-[N-(tert-butoxycarbonyl)]-
60,500-dideoxy-500-[N-(pentafluorobenzyloxy)imino]paromo-
mycin (3e). GP
CHCl3–EtOAc–MeOH) gave 3e (284 mg, 49%). White
1
and FC (2:2:0.1 ! 2:2:0.3,
127.60 (2d), 104.13 (d, CHPh); HRMALDIMS: 429.1026
25
þ
solid: mp 180–184 ꢁC; ½aꢀD +134.95 (c 0.24, MeOH);
(100), 869.2661 (79, [M+Na]+, C30H39N16NaO
;
14
Rf = 0.85 (2:2:0.5, CHCl3–EtOAc–MeOH); IR (ATR):
Calcd 869.2651). Anal. Calcd for C30H38N16O14ÆMeOH
(878.76): C, 42.37; H, 4.82, N, 25.50. Found: C, 42.03;
H, 4.77; N, 25.60.
m
3362w, 2978w, 2936w, 1688s, 1505s, 1454w,
1392m, 1366s, 1305m, 1278m, 1248m, 1161s, 1126s,
1037s, 1022s, 959m, 939m cmꢁ1; HRMALDIMS:
1315.5502 (100, [M+Na]+, C55H85F5N6NaO23þ; Calcd
4.2.9. 1,3,20,2000,6000-Pentaazido-40,60-O-benzylidene-1,3,20,
2000,6000-pentadeamino-500-deoxy-500-[N-(4-nitrobenzyloxy)-
imino]paromomycin (10b). GP 1 and FC (4:1 ! 4:2,
CHCl3–MeCN) gave 10b (145 mg, 69%). White solid:
1315.5484), 1215.4987 (23, [MꢁCO2CMe3+H+Na]+,
þ
C50H77F5N6NaO21
;
Calcd 1215.4960), 1194.5194
(45, [MꢁCO2CMe3+2H]+, C45H69F5N6O
; Calcd
þ
21

I. Kudyba et al. / Carbohydrate Research 342 (2007) 499–519
505
28
mp 131–134 ꢁC; ½aꢀD +114.9 (c 0.125, MeOH);
Rf = 0.60 (4:3, CHCl3–MeCN); IR (ATR): m 3418w,
2924w, 2099s, 1692w, 1606w, 1520m, 1454w, 1372w,
4.4. General procedure for the formation of the amides
7a–c (GP 2)
1346m, 1254m, 1155m, 1097m, 1044m, 1012s cmꢁ1; H
Under N2, a 0.05 M soln of 5 in DMF was treated at
0 ꢁC with HOBt (2 equiv) and EDAC (2 equiv), and stir-
red at 0 ꢁC for 30 min. DIPEA (2 equiv) and the corre-
sponding amine (2 equiv) were added, the mixture was
stirred at 25 ꢁC for 18 h, cooled to 0 ꢁC, treated with
1 N HCl and diluted with AcOEt and H2O. The layers
were separated and the aq layer was extracted with
AcOEt (2·). The combined org. layers were washed with
brine, dried (MgSO4), filtered and evaporated.
1
NMR (500 MHz, CD3OD) see Table 6, additionally,
d 8.25–8.23 (m, 2 arom. H), 7.62–7.60 (m, 2 arom. H),
7.49–7.47 (m, 2 arom. H), 7.35–7.33 (m, 3 arom. H),
5.55 (s, CHPh), 5.22 (s, CH2Ph); 13C NMR (125 MHz,
CD3OD) see Table 7, additionally, d 148.98 (s),
147.06 (s), 139.14 (s), 130.04 (d), 129.89, 129.12,
127.62, 124.63 (4 · 2d), 104.20 (d, CHPh), 75.68
(t, CH2Ph); HRESIMS: 1004.2979 (100, [M+Na]+,
þ
C37H43N17NaO16
; Calcd 1004.2971). Anal. Calcd
for C37H43N17O16Æ0.5MeOH (997.86): C, 45.14; H,
4.55; N, 24.86. Found: C, 45.51; H, 4.58; N,
24.32.
4.4.1. 1,3,20,2000,6000-Pentakis-[N-(tert-butoxycarbonyl)]-
60-deoxy-500-(N-benzyl)paromomycincarboxamide (7a).
GP 2 on 49 mg of 5 and FC (1:1:0 ! 6:6:0.5, CHCl3–
AcOEt–MeOH) gave 7a (24 mg, 45%). Yellowish solid;
26
4.2.10. 1,3,20,2000,6000-Pentaazido-40,60-O-benzylidene-1,3,
20,2000,6000-pentadeamino-500-deoxy-500-[N-(phenoxy)imino]-
paromomycin (10c). GP 1 and FC (2:2:0.1 ! 2:2:0.3,
CHCl3–EtOAc–MeOH) gave 10c (378 mg, 69%). White
½aꢀD +12.8 (c 0.23, MeOH); Rf = 0.36 (3:3:0.5, CHCl3–
AcOEt–MeOH); IR (ATR): m 3379w, 2970w, 2933w,
1681s, 1512m, 1452m, 1410m, 1391m, 1366s, 1283w, 1246m,
1162s, 1027s, 989s cmꢁ1; HRMALDIMS: 858.4365
25
(40, [Mꢁring IꢁCO2CMe3+3H]+, C39H64- N5O
;
þ
solid: mp 115–118 ꢁC; ½aꢀD +85.7 (c 0.3, MeOH);
16
Rf = 0.67 (2:2:0.5, CHCl3–EtOAc–MeOH); IR (ATR):
m 3422w, 2105s, 1587w, 1487w, 1474w, 1371w, 1331w,
Calcd 858.4348), 1103.5588 (49, [MꢁCO2- CMe3+2H]+,
þ
C50H83N6O21
; Calcd 1103.5616), 1225.5927 (100,
1255w, 1158w, 1020m cmꢁ1
;
1H NMR (400 MHz,
[M+Na]+, C55H90N6NaO
;
Calcd 1225.5955),
þ
23
1226.5961 (62, [M+H+Na]+, C55H91N6- NaO23
;
þ
CD3OD) see Table 6, additionally, d 7.50–7.46 (m, 2
arom. H), 7.35–7.31 (m, 5 arom. H), 7.27–7.15 (m, 2
arom. H), 7.04–7.00 (m, 1 arom. H), 5.52 (s, CHPh);
13C NMR (100 MHz, CD3OD) see Table 7, addition-
ally, d 160.63 (s), 139.12 (s), 130.02 (d), 130.43, 129.10,
127.59, 124.50 (4 · 2d), 104.13 (d, CHPh). Anal. Calcd
for C36H42N16O14 (922.82): C, 46.85; H, 4.59; N,
24.29. Found: C, 46.74; H, 4.70; N, 24.28.
Calcd 1226.6033). Anal. Calcd for C55H90N6- O23ÆH2O
(1203.34): C, 54.09; H, 7.59; N, 6.88. Found: C, 54.20;
H, 7.23; N, 6.75.
4.4.2. 1,3,20,2000,6000-Pentakis-[N-(tert-butoxycarbonyl)]-
60-deoxy-500-[N-(4-nitrobenzyl)]paromomycincarboxamide
(7b). GP 2 on 81 mg of 5 and FC (1:1:0 ! 9:9:1.2,
CHCl3–AcOEt–MeOH) gave 7b (36 mg, 40%). Yellow-
27
4.3. 1,3,20,2000,6000-Pentakis-[N-(tert-butoxycarbonyl)]-60-
deoxy-500-carboxyparomomycin (5)
ish solid; ½aꢀD +27.8 (c 0.265, MeOH); Rf = 0.28
(3:3:0.5, CHCl3–AcOEt–MeOH); IR (ATR): m 3356w,
2977w, 2933w, 1682s, 1609w, 1515s, 1455w, 1392m,
1366m, 1345m, 1279m, 1248m, 1160s, 1111m, 1034s,
996m cmꢁ1; HRMALDIMS: 803.3705 (36, [Mꢁring
A soln of 2 (1.2 mmol, 1.32 g) in 1:1 MeCN–water
(14 mL) was treated with bisacetoxyiodobenzene
(4.8 mmol, 1.55 g) and TEMPO (0.54 mmol, 84 mg),
stirred at 23 ꢁC for 48 h, cooled to 0 ꢁC, treated with
1 N HCl (5 mL) and diluted with AcOEt (50 mL) and
H2O (30 mL). The layers were separated, and the aq
layer was extracted with AcOEt (2 · 50 mL). The com-
bined org. layers were washed with brine (80 mL), dried
(MgSO4), filtered and evaporated. FC (1:1:0 ! 8:8:5,
CHCl3–AcOEt–MeOH) gave 5 (722 mg, 54%). White
Iꢁ2CO2CMe3+4H]+, C34H55N6O þ; Calcd 803.3675),
16
903.4260 (33, [Mꢁring I–CO2CMe3+3H]+, C39H63-
N6O18þ; Calcd 903.4199), 970.4210 (49, [Mꢁ3CO2-
CMe3+3H+Na]+, C40H65N7NaO þ; Calcd 970.4233),
19
1070.4805 (60, [Mꢁ2CO2CMe3+2H+Na]+, C45H73N7-
NaO21þ; Calcd 1070.4757), 1170.5469 (73, [MꢁCO2-
CMe3+H+Na]+, C50H81N7NaO þ; Calcd 1170.5282),
23
1270.5927 (100, [M+Na]+, C55H89N7NaO þ; Calcd
25
26
solid: mp 195 ꢁC (dec); ½aꢀD +4.0 (c 0.285, MeOH);
1270.5806), 1271.5850 (63, [M+H+Na]+, C55H9N7-
þ
Rf = 0.41 (1:1:1, CHCl3–AcOEt–MeOH); IR (ATR): m
3359w, 2977w, 2933w, 1682s, 1629w, 1511m, 1455w,
1391m, 1283m, 1367s, 1283w, 1249m, 1161s, 1041s
cmꢁ1; HRMALDIMS: 454.0200 (100), 1112.5374 (57,
NaO25
;
Calcd 1271.5884). Anal. Calcd for
C55H89N7O25ÆH2O (1266.36): C, 52.17; H, 7.24; N,
7.74. Found: C, 52.07; H, 7.29; N, 7.45.
[MꢁH]ꢁ, C48H82N5O ꢁ; Calcd 1112.5350), 1113.5395
4.4.3. 1,3,20,2000,6000-Pentakis-[N-(tert-butoxycarbonyl)]-
60-deoxy-500-(N-phenyl)paromomycincarboxamide (7c).
GP 2 with HOAt instead of HOBt on 46 mg of 5 and
FC (1:1:0 ! 6:6:0.1, CHCl3–AcOEt–MeOH) gave 7c
24
(30, [M]ꢁ, C48H83N5O24; Calcd 1113.5428). Anal. Calcd
for C48H83N5O24 (1114.20): C, 51.74; H, 7.51; N, 6.29.
Found: C, 51.68; H, 7.27; N, 6.18.

506
I. Kudyba et al. / Carbohydrate Research 342 (2007) 499–519
28
(21 mg, 43%). Yellowish solid: mp 195–200 ꢁC; ½aꢀD
+5.3 (c 0.515, MeOH); Rf = 0.28 (3:3:0.5, CHCl3–AcOEt–
MeOH); IR (ATR): m 3364w, 2978w, 2933w, 1682s,
1601w, 1511s, 1446m, 1392m, 1366s, 1345m, 1281m,
Rf = 0.36 (1:4:3, CHCl3–MeOH–25% aq NH3); IR
(ATR): m 3500–2800w, 1667s, 1527w, 1403w, 1335m,
1273w, 1210s, 1158s, 1115s, 1080m, 1038m, 965m,
932m cmꢁ1 1H NMR (500 MHz, D2O) see Table 2,
;
1248m, 1161s, 1032s cmꢁ1; HRMALDIMS: 711.3158
additionally, d 7.51–7.43 (m, 5 arom. H), 5.22 (s,
CH2Ph); 13C NMR (126 MHz, D2O) see Table 3, addi-
(42, [Mꢁ5CO2CMe3+5H+Na]+, C30H50N6NaO
;
þ
13
2
Calcd 711.3303), 811.3654 (46, [Mꢁ4 CO2CMe3+4H+
tionally, d 165.82 (q, JC,F 27.5, 5C@O), 139.45 (s),
Na]+, C34H56N6O þ; Calcd 811.3701), 911.4227 (65,
131.64 (2d), 131.33 (d), 131.04 (2d), 120.16, (dt, JC,F
2
15
[Mꢁ3CO2CMe3+3H+Na]+, C39H64N6NaO þ; Calcd
34.8, JC,F 285.6, 5CF2), 111.18, (td, JC,F 39.1, JC,F
262.5, 5CF3), 78.64 (t, CH2Ph); HRMALDIMS:
580.2828 (100), 688.3378 (49), 725.3315 (29, [M+Na]+,
C30H50N6NaO13þ; Calcd 725.3334). Anal. Calcd for
C49H53N6F35O23 (1758.26): C, 34.46; H, 4.04; N, 4.78.
Found: C, 34.49; H, 4.05; N, 4.66.
1
2
1
17
911.4226), 1011.4785 (81, [Mꢁ2CO2CMe3+2H+Na]+,
C44H72N6NaO19þ; Calcd 1011.4750), 1012.4813 (42,
[Mꢁ2 CO2CMe3+3H+Na]+, C44H73N6NaO þ; Calcd
19
1012.4828), 1111.5192 (74, [MꢁCO2CMe3+H+Na]+,
C49H80N6NaO21þ; Calcd 1111.5274), 1211.5815 (100,
[M+Na]+, C54H88N6-NaO
;
Calcd 1211.5799),
þ
23
1212.5840 (60, [M+H+Na]+, C54H89N6NaO þ; Calcd
4.6.2. 1,3,20,2000,6000-Pentaammonium-60,500-dideoxy-500-[N-
(4-nitrobenzyloxy)imino]paromomycin pentakis(trifluoro-
acetate) (4bÆ5CF3CO2H). GP 3, FC (RP C-18,
9:1 ! 8:2, H2O–MeOH) and lyophilisation gave
4bÆ5CF3CO2H (48 mg, 91%). White solid: mp 130 ꢁC
23
1212.5877). Anal. Calcd for C54H88N6O23ÆH2O
(1207.32): C, 53.72; H, 7.51; N, 6.96. Found: C, 53.30;
H, 7.56; N, 6.69.
25
4.5. 1,3,20,2000,6000-Pentakis-[N-(tert-butoxycarbonyl)]-60-
deoxy-500-[N-(4-aminobenzyl)]paromomycincarboxamide
(7g)
(dec); ½aꢀD +57.5 (c 0.135, MeOH); Rf = 0.30 (1:4:3,
CHCl3–MeOH–25% aq NH4); IR (ATR): m 3372m
(br), 1670s, 1519m, 1433w, 1348m, 1188s, 1131s,
1033m, 1013m cmꢁ1 1H NMR (600 MHz, D2O) see
;
Under H2, a suspension of 7b (0.030 mmol, 44 mg) in
80% aq AcOH (2 mL) and 10% Pd/C (8 mg) was stirred
at 23 ꢁC for 18 h, filtered through a pad of Celite and
evaporated. FC (1:0:0 ! 6:6:1, CHCl3–EtOAc–MeOH)
Table 2, additionally, d 8.28 (d, J 8.7, 2 arom. H), 7.62
(d, J 8.7, 2 arom. H), 5.29, 5.28 (2d, J 19.3, CH2Ph);
13C NMR (150 MHz, D2O) see Table 3, additionally,
2
d 165.64 (q, JC,F 292.8, 6C@O), 150.15 (s), 147.66 (s),
25
1
gave 7g (31 mg, 72%). White solid; ½aꢀD +23.3 (c 0.30,
131.41 (2d), 126.70 (2d), 119.0 (q, JC,F 34.7, 6CF3),
MeOH); Rf = 0.34. (3:3:1, CHCl3–EtOAc–MeOH);
HRMALDIMS: 1040.5061 (33, [Mꢁ2CO2CMe3+2H+
77.25 (t, CH2Ph); HRMALDIMS: 580.28þ28 (100),
748.3370 (16, [M+Na]+, C30H49N7NaO
; Calcd
15
Na]+, C45H75N7NaO þ; Calcd 1040.5015), þ1118.5717
770.3184). Anal. Calcd for C30H49N7O15Æ6CF3CO2H
(1335.88): C, 35.23; H, 4.87; N, 6.85; Found: C, 35.20;
H, 4.05; N, 6.96.
19
(46, [MꢁCO2CMe3+2H]+, C50H84N7O
;
Calcd
21
1118.5720), 1140.5595 (41, [MꢁCO2CMe3+H+Na]+,
C50H83N7NaO21þ; Calcd 114þ0.5540), 1240.6040 (100,
[M+Na]+, C55H91N7NaO
;
Calcd 1240.6064),
4.6.3. 1,3,20,2000,6000-Pentaammonium-60,500-dideoxy-500-[N-
(phenoxy)imino]paromomycin pentakis(trifluoroacetate)
23
1241.6067 (67, [M+H+Na]+, C55H92N7NaO þ; Calcd
23
1241.6142). Anal. Calcd for C55H91N7O23ÆH2O
(1236.36): C, 53.43; H, 7.58; N, 7.93. Found: C, 53.48;
H, 7.78; N, 7.49.
(4cÆ5CF3CO2H). Decomposed at
minutes.
25 ꢁC within
4.6.4. 1,3,20,2000,6000-Pentaammonium-60,500-dideoxy-500-[N-
(methoxy)imino]paromomycin pentakis(trifluoroacetate)
(4dÆ5CF3CO2H). GP 3, FC (RP C-18, 9:1 ! 8:2,
H2O–MeOH) and lyophilisation gave (4dÆ5CF3CO2H)
4.6. General procedure for the de-N-bocylation (GP 3)
A soln of the paromomycin derivatives 3a–g, 6, 8a–c,g
(1 equiv) in TFA was treated with anisole (6 equiv)
and stirred for 1–10 min. After evaporation of the sol-
vent (water bath max. 30 ꢁC), a soln of the residue in
H2O was washed with EtOAc (1·), and taken to dryness
to give the crude pentaammonium salts.
25
(76 mg, 83%). White solid: mp 140 ꢁC (dec); ½aꢀD
+38.4 (c 0.34, MeOH); Rf = 0.36 (1:4:3, CHCl3–
MeOH–25% aq NH3); IR (ATR): m 3400–2700w,
1668s, 1623m, 1520w, 1432w, 1164s, 1188s, 1128s,
1
1033s cmꢁ1; H NMR (500 MHz, D2O) see Table 2,
additionally, d 4.83 (s, OMe); 13C NMR (126 MHz,
2
4.6.1.
1,3,20,2000,6000-Pentaammonium-60,500-dideoxy-500-
D2O) see Table 3, additionally, d 165.67 (q, JC,F 35.5,
1
(benzyloxyimino)paromomycin pentakis(pentafluoroprop-
ionate) (4aÆ5C2F5CO2H). GP 3, FC (RP C-18,
9:1:0.1 ! 8:2:0.1, H2O–MeOH–C3F7CO2H) and lyo-
philisation gave (4aÆ5C2F5CO2H) (42 mg, 79%). White
5C@O), 119.0 (q, JC,F 291.8, 5CF3), 64.54 (s, OMe);
HRMALDIMS: 580.2822 (100), 649.3004 (62,
[M+Na]+, C24H46N6NaO þ; Calcd 649.3021). Anal.
13
Calcd for C34H51F15N6O23ÆH2O (1214.78): C, 34.91; H,
4.38; N, 6.85. Found: C, 34.62; H, 4.40; N, 6.92.
25
solid: mp 140 ꢁC (dec); ½aꢀD +38.9 (c 0.17, MeOH);

I. Kudyba et al. / Carbohydrate Research 342 (2007) 499–519
507
Table 2. 1H NMR chemical shifts [ppm] and coupling constants [Hz] for 4a–b, 4d–g in D2O
4a
5C2F5CO2H
4b
5CF3CO2Ha
4d
5CF3CO2Ha
4e
5CF3CO2Ha
4f
4ga
5CF3CO2Ha
H-10
H-20
H-30
H-40
H-50
Me-60
H-1
5.88
5.82
3.08
3.83
3.05
3.68
1.26
3.32
1.81
2.45
3.47
3.92
3.87
3.63
5.42
4.36
4.62
4.82
7.86
5.18
3.55
4.16
3.74
3.94–3.91
3.32
3.24
4.1
10.4
9.0
9.0
6.2
12.7
4.2
10.5
12.7
12.7
4.2
9.2
8.9
5.84
5.93
3.26
3.93
3.27
3.76
1.34
3.55–3.40
1.88
2.50
3.54
3.94–3.90
3.92
3.69
5.45
4.47
4.71
4.80
7.79
5.18
3.59
4.21
3.82
4.04–4.00
3.40
3.33
4.1
10.0
8.8
8.9
6.3
12.6
4.2
10.6
12.6
12.6
4.2
10.2
8.8
5.51
2.91
3.80
3.03
3.66
1.23
3.78–3.76
1.79
5.38
2.72
3.53
3.13–3.06
3.84
1.27
2.94–2.82
1.30–1.28
2.03–2.00
2.94–2.82
3.43
3.73–3.66
3.30
5.35
3.23–3.19
3.94–3.86
3.16
3.75
1.32
3.31–3.24
3.99–3.82
3.22
3.69
1.26
3.31–3.24
1.80
2.41
3.45
3.82
3.99–3.82
3.62
5.38
4.40
4.60
4.70–4.66
7.63
5.25
3.54
4.16
3.23–3.19
1.82
Hax-2
Heq-2
H-3
H-4
2.48–2.45
3.46–3.35
3.94–3.86
3.94–3.86
3.68
2.43
3.33–3.27
3.40–3.33
3.78–3.76
3.60
5.36
4.00
4.49
4.86
7.92
5.20
3.55
4.20
3.78–3.76
4.05
3.32
3.22
4.0
10.4
9.0
H-5
H-600
H-100
H-200
H-300
H-4
H-500
H-1000
H-2000
H-3000
H-4000
H-5000
Ha-6000
Hb-6000
5.45
3.94–3.86
4.82–4.80
4.82
4.41
4.66–4.63
4.66–4.63
7.64
4.98
3.13–3.06
4.02
3.73–3.66
4.15–4.05
3.02
3.13–3.06
3.8
10.2
9.3
9.5
7.82
5.23–5.21
3.60–3.58
4.21
3.79–3.78
3.94–3.86
3.30
3.77
4.22
3.24
3.24
3.23–3.19
4.1
b
0
J1 ,2
0
0
0
0
0
4.0
b
0
J2 ,3
0
J3 ,4
9.0
9.0
6.2
2.9
b
8.9
8.9
6.3
12.6
4.2
10.5
12.6
12.6
4.1
10.4
9.1
9.0
2.6
4.6
5.7
5.8
1.7
2.9
1.2
3.1
1.5
5.7
0
J4 ,5
9.1
6.2
12.5
4.2
10.5
12.5
12.5
0
J5 ,6
6.3
b
J1,2ax
J1,2eq
J1,6
b
10.3
12.9
12.9
b
9.9
b
J2ax,2eq
J2ax,3
J2eq,3
J3,4
b
b
4.1
b
b
b
9.2
9.4
9.9
1.9
b
J4,5
J5,6
00 00
J1 ,2
8.8
2.4
4.4
5.5
8.9
2.9
4.5
4.9
4.7
1.7
3.1
1.4
3.1
1.6
5.4
4.2
9.0
2.5
4.5
5.6
5.5
1.7
2.9
1.2
3.1
1.2
5.3
4.3
8.9
4.2
4.3
3.4
4.2
1.7
2.9
1.1
3.1
1.4
5.9
4.2
13.6
00 00
J2 ,3
4.2
b
00 00
J3 ,4
00 00
J4 ,5
5.4
b
6.3
1.8
3.3
b
000,2000
000,3000
000,4000
0004000
J1
J2
J2
J3
J4
J5
J5
J6
2.9
b
2.9
b
3.3
b
000,5000
000,6000
000,6000
b
5.2
b
a
4.2
13.7
4.3
13.5
b
000a,6000
b
13.7
13.8
13.7
a Assignment based on DQFCOSY and HSQC spectrum.
b Not determined.
4.6.5. 1,3,20,2000,6000-Pentaammonium-60500-dideoxy-500-[N-
(pentafluorobenzyloxy)imino]paromomycin pentakis(tri-
fluoroacetate) (4eÆ5CF3CO2H). GP 3, FC (RP C-18,
9:1 ! 8:2, H2O–MeOH) and lyophilisation gave
4eÆ5CF3CO2H (68 mg, 88%). White solid: mp 180 ꢁC
2700w, 1667s, 1524m, 1507m, 1434w, 1375w, 1186s,
1126s, 1033s, 1015s, 960m, 939m cmꢁ1 1H NMR
;
(500 MHz, D2O) see Table 2, additionally, d 5.31 (s,
PhCH2); 13C NMR (126 MHz, D2O): see Table 3, addi-
2
tionally, d 165.65 (q, JC,F 35.5, 5C@O), 149.7–148.7,
25
(dec); ½aꢀD +32.7 (c 0.425, MeOH); Rf = 0.50 (1:4:3,
147.7–146.7, 141.8–138.9 (3m, 4 arom. H), 130.07 (s, 1
1
CHCl3–MeOH–25% aq NH3); IR (ATR): m 3500–
arom. H), 119.01, (q, JC,F 291.7, 5CF3), 65.67 (s,

508
I. Kudyba et al. / Carbohydrate Research 342 (2007) 499–519
Table 3. 13C NMR chemical shifts (ppm) for 4a,b,d–g in D2O
4a
5C2F5CO2H
4b
5CF3CO2Ha
4d
5CF3CO2Ha
4e
5CF3CO2Ha
4f
4ga
5CF3CO2Ha
C-10
C-20
C-30
C-40
C-50
C-60
C-1
C-2
C-3
C-4
C-5
C-600
C-100
C-200
C-300
C-400
C-5000
C-1000
C-2
C-3000
C-4000
C-5000
C-6000
96.90
56.28
71.35
76.83
72.58
19.16
52.43
31.24
51.32
78.56
87.48
75.28
112.93
76.61
81.71
80.74
152.95
98.86
53.41
70.27
70.30
72.31
43.14
96.90
56.28
71.21
76.92
72.58
19.21
52.34
30.89
51.28
77.79
88.00
75.21
112.99
76.54
81.64
80.92
153.20
98.93
53.49
70.21
70.31
72.47
43.19
96.83
56.25
71.33
76.73
72.83
19.16
52.31
30.74
51.32
77.76
87.73
75.02
113.03
76.28
81.71
80.71
151.90
98.73
53.42
70.29
70.08
72.72
43.11
96.59
56.22
71.25
76.75
72.83
19.11
52.31
30.59
51.27
77.49
87.80
75.05
112.94
76.63
81.56
80.62
153.24
98.91
53.43
70.17
70.36
72.41
43.07
96.84
56.18
71.35
76.63
72.56
19.11
52.27
31.45
51.27
78.56
88.07
75.49
112.67
76.39
81.42
81.85
146.30
98.83
53.44
70.07
70.16
72.41
43.07
100.76
57.87
74.57
79.39
77.54
19.44
52.94
29.25
52.46
80.43
92.42
77.92
112.17
77.95
87.86
81.47
155.62
101.91
54.84
71.45
72.82
76.51
43.49
a Assignment based on DQFCOSY and HSQC spectrum.
CH2C6F5); HRMALDIMS: 580þ.2835 (100), 794.3057
Calcd 794.3043),
(500 MHz, D2O) see Table 2, additionally, d 7.28 (d,
J 8.5, 2 arom. H), 6.86 (d, J 8.5, 2 arom. H), 5.05
(s, CH2Ph); 13C NMR (125 MHz, D2O) see Table 3,
additionally, d 134.26 (s), 134.02 (2d), 129.76 (s),
118.97 (2d), 78.71 (t, CH2Ph); HRMALDIMS: 718.3624
(21, [M+H]+, C30H46F5N6O13
;
815.2845 (56, [M+Na]+, C30H45F5N6NaO þ; Calcd
13
815.2862). Anal. Calcd for C40H50F20N6O23ÆH2O
(1380.83): C, 34.79; H, 4.80; N, 6.09. Found: C, 34.66;
H, 4.55; N, 6.27.
(66, [M+Na]+, C49H86N6NaO þ; Calcd 1149.5642).
23
4.6.6. 1,3,20,2000,6000-Pentaammonium-60,500-dideoxy-500-[N-
(trityloxy)imino]paromomycin pentakis(trifluoroacetate)
(4fÆ5CF3CO2H). GP 3, FC (RP C-18, 9:1 ! 8:2,
H2O–MeOH) and lyophilisation gave 4fÆ5CF3CO2H
4.6.8. 1,3,20,2000,6000-Pentaammonium-60-deoxy-500-carboxy-
paromomycin pentakis(trifluoroacetate) (6Æ5CF3CO2H).
GP 3 on 40 mg of 5, FC (RP C-18, 1:17, H2O–MeOH)
and lyophilisation gave 6Æ5CF3CO2H (22 mg, 52%).
25
23
(70 mg, 84%). White solid: mp 150 ꢁC (dec); ½aꢀD
White solid: mp 145 ꢁC (dec); ½aꢀD +28.6 (c 0.044,
+44.9 (c 0.17, MeOH); Rf = 0.70 (1:4:3, CHCl3–
MeOH–25% aq NH3); IR (ATR): 3500–2700w, 1671s,
1524w, 1592w, 1444w, 1332w, 1198s, 1132s, 1022m,
H2O); Rf = 0.20 (7:3, MeOH–25% aq NH3); IR (ATR):
m 2917w (br), 1668s, 1627w, 1520m, 1434w, 1334w,
1
1188s, 1126s, 1035s cmꢁ1; H NMR (500 MHz, D2O)
1018m, 918w cmꢁ1
;
1H NMR (500 MHz, D2O) see
see Table 4. 13C NMR (125 MHz, D2O) see Table 5,
Table 2, additionally, d 7.48-7.38 (m, 15 arom. H); 13C
NMR (126 MHz, D2O) see Table 3, additionally, d
additionally d 163.00 (q, JC,F 37.6, 5C@O), 116.37,
2
1
(q, JC,F 291.9, 5CF3). HRMALDIMS: 614.2869 (100,
2
165.60 (q, JC,F 35.6, 5C@O), 152.34 (3s), 131.38
[M+H]+, C23H45N5O þ; Calcd 614.2885); 615.2903
14
1
(6d), 130.95 (6d), 130.53 (3d), 116.69, (q, JC,F 292.5,
(28, [M+2H]+, C23H45N5O þ; Calcd 615.2963). Anal.
14
5CF3), 94.01 (s, CPh3); HRMALDIMS: 580.2843
Calcd for C23H43N5O14Æ6CF3CO2HÆH2O (1315.77): C,
31.95; H, 3.91; N, 5.32. Found: C, 31.79; H, 3.90; N 5.32.
(100), 877.3939 (54, [M+Na]+, C42H58N6NaO þ; Calcd
13
877.3960).
4.6.9. 1,3,20,2000,6000-Pentaammonium-60-deoxy-500-[N-benz-
yl]paromomycincarboxamide pentakis(trifluoroacetate)
(8aÆ5CF3CO2H). GP 3 on 9 mg of 7a and lyophilisa-
tion gave 8aÆ5CF3CO2H (8 mg, 84%). White solid;
Rf = 0.30 (4:1, MeOH–25% aq NH3); 1H NMR
(500 MHz, D2O) see Table 4, additionally d 7.48–7.41
(m, 2 arom. H), 7.40–7.37 (m, 3 arom. H), 4.70 (d,
J 15.1 Hz, PhCHaCHbNHCO), 4.38 (d, J 15.1 Hz,
4.6.7.
60,500-Dideoxy-500-[N-(4-aminobenzyloxy)imino]-
paromomycin (4g). GP 3, FC (1:4:2, CHCl3–MeOH–
25% aq NH3) and lyophilisation gave 4g (19 mg, 85%).
White solid: mp 160 ꢁC (dec); Rf = 0.54 (1:4:3, CHCl3–
MeOH–25% aq NH3); IR (ATR): m 3500–2700m,
1682m, 1612m, 1544s, 1519s, 1406m, 1335m, 1129m,
1116m, 1044s, 1033s, 1013s, 923m cmꢁ1 1H NMR
;

I. Kudyba et al. / Carbohydrate Research 342 (2007) 499–519
509
PhCHaHbNHCO); 13C NMR (125 MHz, D2O) see
Table 5, additionally d 140.18 (s), 131.75 (2d), 130.63
(d), 130.13 (2d), 45.79 (t, PhCH2NHCO); 19F NMR
4.6.10. 1,3,20,2000,6000-Pentaammonium-60-deoxy-500-[N-(4-
nitrobenzyl)]paromomycincarboxamide pentakis(hepta-
fluorobutanoate) (8bÆ5C3F7CO2H). GP 3 on 31 mg of
7b, FC (RP C-18, 2:3:0.013, H2O–MeOH–C3F7CO2H)
and lyophilisation gave 8bÆ5C3F7CO2H (25 mg, 55%).
(282 MHz, D2O) ꢁ73.98 (s, CF3CO2H); HRMALDI-
MS: 558.2714 (100, [Mꢁring I+2H]+, C24H40N5O
;
;
þ
þ
10
23
Calcd 558.2775), 703.3497 (32, [M+H]+, C30H51N6O
White solid: mp 151 ꢁC (dec); ½aꢀD +21.1 (c 0.04,
13
Calcd 703.3514). Anal. Calcd for C30H50N6O13Æ6CF3-
CO2H (1386.89): C, 36.37; H, 4.07; N 6.06. Found: C,
36.23; H 3.95; N, 6.18.
H2O); Rf = 0.28 (1:4:1, CHCl3–MeOH–25% aq NH3);
IR (ATR): m 2934w (br), 1667s, 1521w, 1403w, 1335m,
1271w, 1211s, 1158s, 1115s, 1080m, 1046m, 1018m,
Table 4. 1H NMR chemical shifts (ppm) and coupling constants (Hz) for 6, 8a–c,g in D2O
6
8a
5CF3CO2Ha
8b
5C3F7CO2Ha
8c
8g
5CF3CO2Ha
H-10
H-20
H-30
H-40
H-50
Me-60
H-1
Hax-2
Heq-2
H-3
H-4
H-5
5.73
3.33–3.18
3.74
3.33–3.18
3.65–3.60
1.21
3.33–3.18
1.76
2.90
3.44
3.89
3.83
3.65–3.60
5.39
4.20
4.46
4.37
5.20
3.50
4.13
3.70
4.18
5.89
3.35
3.88
3.24
3.75–3.71
1.32
3.40–3.34
1.88
2.51
3.56
4.03
3.97
3.75–3.71
5.53
4.48
4.70
4.60
5.28
3.61
4.23
5.86
3.29–3.17
3.84
3.29–3.17
3.77–3.67
1.30
3.36
1.84
2.69
3.52
4.00–3.94
4.00–3.94
3.77–3.67
5.54
4.51
4.73
4.65
5.30
3.63
4.25
5.67
3.20
3.76
3.17
3.69–3.52
1.27
3.11–3.01
1.45
5.51
3.22–3.00
3.71–3.64
3.22–3.00
3.57–3.48
1.27
3.22–3.00
1.44
2.13
3.22–3.00
3.85–3.76
3.85–3.76
3.57–3.48
5.41
4.43–4.41
4.65
4.55
5.09
3.22–3.00
4.09
3.71–3.64
4.16–4.14
3.22–3.00
3.22–3.00
2.16
3.11–3.01
3.88–3.73
3.88–3.75
3.54
5.50
4.48
4.72
4.66
5.08
3.26
H-600
H-100
H-2
H-300
H-400
H-1000
H-2000
H-3000
H-4000
H-5000
Ha-6000
Hb-6000
4.09
3.79
3.80
4.27
3.29–3.17
3.29–3.17
4.0
10.6
8.9
b
3.69–3.52
4.08–4.05
2.95
2.62
4.1
10.6
9.3
9.3
6.3
12.6
4.0
4.24–4.21
3.21–3.20
3.21–3.20
4.0
10.6
8.9
9.1
6.3
12.7
3.33–3.18
3.33–3.18
4.1
11.0
9.2
b
0
0
0
0
0
0
J1 ;2
3.7
b
0
J2 ;3
b
b
0
J3 ;4
0
J4 ;5
0
J5 ;6
6.2
12.6
4.3
b
6.3
12.4
4.3
10.7
12.6
ꢂ13.0
4.2
6.1
12.5
J1,2ax
J1,2eq
J1,6
4.1
b
ꢂ4.9
b
9.9
12.7
12.6
4.0
b
J2ax,2eq
J2ax,3
J2eq,3
J3,4
12.6
12.6
4.3
10.1
9.0
9.0
3.4
5.0
5.2
12.6
12.7
4.1
10.1
8.9
8.9
1.7
4.4
7.3
12.5
12.5
ꢂ4.9
b
ꢂ13.0
b
b
b
b
J4,5
J5,6
00 00
J1 ,2
ꢂ13.0
b
9.9
b
1.6
4.4
7.3
1.7
3.1
00 00
J2 ;3
00 00
J3 ;4
4.2
7.8
1.6
3.1
b
5.9
7.1
b
000;2000
000;3000
000;4000
0004000
J1
J2
J2
J3
J4
J5
J5
J6
1.6
3.2
1.6
3.1
3.1
b
ꢂ1.2
ꢂ1.2
ꢂ1.4
3.2
3.1
3.1
3.1
b
3.1
b
000;5000
000;6000
000;6000
1.6
3.7
7.4
b
1.2
b
1.3
3.6
7.8
b
b
b
b
3.2
8.8
13.4
a
b
b
b
000a;6000
b
a Assignment based on DQFCOSY and HSQC spectrum.
b Not determined.

510
I. Kudyba et al. / Carbohydrate Research 342 (2007) 499–519
Table 5. 13C NMR chemical shifts (ppm) for 6 and 8a–c in D2O
124.08 (2d); HRMALDIMS: 689.3341 (100, [M+H]+,
þ
C29H49N6O13
;
Calcd 689.3358), 690.3370 (34,
6
8a
5CF3CO2Ha
8b
5C3F7CO2Ha
8c
[M+2H]+, C29H50N6O þ; Calcd 690.3436), 711.3196
5CF3CO2H
13
(42, [M+Na]+, C29H48N6NaO þ; Calcd 711.3177).
C-10
C-20
C-30
C-40
C-50
C-60
C-1
C-2
C-3
C-4
C-5
C-600
C-100
C-200
C-300
C-400
C-5000
C-1000
C-2
C-3000
C-4000
C-5000
C-6000
96.17
54.02
68.49
74.49
72.67
16.67
49.77
28.08
48.65
75.85
83.74
69.95
109.23
73.99
80.00
81.95
176.95
95.87
50.72
67.71
67.23
70.29
40.41
97.81
56.57
71.16
76.85
75.04
19.20
52.43
30.62
51.27
77.79
87.15
72.76
113.30
75.64
80.52
82.11
173.68
97.44
53.47
69.97
69.54
73.39
42.96
97.80
56.56
71.13
76.91
75.12
19.16
52.50
30.86
51.27
78.18
87.28
72.66
113.39
75.67
80.65
82.05
174.14
97.50
53.51
70.01
69.48
73.49
42.95
100.21
57.40
71.90
77.52
76.33
19.22
53.26
35.15
51.75
81.31
87.80
72.88
112.74
77.06
82.41
83.22
173.29
99.32
54.27
71.80
70.48
73.66
42.89
13
4.6.12. 60-Deoxy-500-[N-(4-aminobenzyl)]paromomycin-
carboxamide (8g). GP 3 on 12 mg of 7g, FC (THF;
1:1, THF–MeOH; 1:0 ! 22:3, MeOH–25% aq NH3)
and lyophilisation gave 8g (7 mg, 99%). Yellowish solid;
Rf = 0.29 (4:1, MeOH–25% aq NH3); IR (ATR): m
3227w (br), 2920w, 1660w, 1617w, 1519w, 1451w,
1404w, 1338w, 1103s, 1041s cmꢁ1
;
1H NMR
(300 MHz, D2O) see Table 4, additionally d 7.18 (d,
J 8.3 Hz, 2 arom. H), 6.83 (d, J 8.4 Hz, 2 arom. H),
4.46 (d, J 14.9 Hz, ArCHaHbNHCO), 4.24 (d, J
14.7 Hz, ArCHaHbNHCO); HRMALDIMS: 263.0540
(100), 718.3616 (95, [M+H]+, C30H52N7O þ; Calcd
13
718.3623), 719.3649 (31, [M+2H]+, C30H53N7O
;
þ
13
Calcd 719.3701), 740.3432 (76, [M+Na]+, C30H51N7-
NaO13þ; Calcd 740.3443).
4.7. 500-Anilino-1,3,20,2000,6000-pentaazido-40,60-O-benzyl-
idene-1,3,20,2000,6000-pentadeamino-500-deoxyparomomycin
(22)
a Assignment based on DQFCOSY and HSQC spectrum.
965m, 931m cmꢁ1; 1H NMR (500 MHz, D2O) see Table
4, additionally d 8.29 (d, J 8.8 Hz, 2 arom. H), 7.57 (d,
J 8.9 Hz, 2 arom. H), 4.70 (d, J 16.0 Hz, ArCHaCHb-
NHCO), 4.54 (d, J 15.9 Hz, ArCHaHbNHCO); 13C
NMR (125 MHz, D2O) see Table 5, additionally d 165.89
A cooled (60 ꢁC) soln of 9 (1.041 g, 1.25 mmol) in
EtOAc (10 mL) was treated with trichlorocyanuric acid
(TCCA, 304 mg, 1.375 mmol) and TEMPO (25 mg,
0.125 mmol), allowed to warm to 25 ꢁC, stirred for
20 min and basified with 1 N NaOH (5 mL). After sep-
aration of the phases, the aq layer was extracted with
EtOAc (3 · 5 mL). The combined org. layers were dried
(MgSO4) and evaporated. A soln of the residue (crude
aldehyde, its hydrate and methyl hemiacetal) in 1:1
THF–ClCH2CH2Cl (6 mL) was treated with aniline
2
(t, JC,F 24.7, 5C@O), 149.74 (s), 148.07 (s), 130.78
2
1
(2d), 126.77 (2d), 120.17 (qt, JC,F 33.9, JC,F 286.8,
5CF3CF2CF2CO2H), 113.5–108.6 (m, CF3CF2CF2-
CO2H), 45.31 (t, PhCH2NHCO); 19F NMR (282 MHz,
D2O) ꢁ80.76 (t, J 8.3, CF3CF2CF2CO2H); ꢁ118.08
(q, J 8.2, CF3CF2CF2CO2H); ꢁ127.01 (br s, CF3CF2-
CF2CO2H); HRMALDIMS: 603.2598 (66, [Mꢁring
(109 lL,
1.62 mmol)
and
NaBH3CN
(80 mg,
I+H]+, C24H39N6O þ; Calcd 603.2626), 748.3345 (100,
12
1.62 mmol), stirred at 25 ꢁC for 24 h and neutralised
with 1 N NaOH. After evaporation of THF, the aq layer
was extracted with EtOAc (3 · 5 mL). The combined
org. layers were dried (MgSO4) and evaporated. FC
(2:2:0.1 ! 2:2:0.3, CHCl3–EtOAc–MeOH) gave 22
[M+H]+, C30H50N7O þ; Calcd 748.3365), 749.3373
15
(36, [M+2H]+, C30H51N7O
;
Calcd 749.3443),
þ
15
770.3134 (37, [M+Na]+, C30H49N7NaO15
; Calcd
þ
12
770.3184). Anal. Calcd for C30H49N7O15Æ7C3F7CO2H
(2246.02): C, 31.02; H, 2.51; N 4.37. Found: C, 31.07;
H 2.26; N, 4.73.
25
(343 mg, 30%). White solid: mp 130–140 ꢁC; ½aꢀD
+131.7 (c 0.28, MeOH); Rf = 0.62 (2:2:0.5, CHCl3–
EtOAc–MeOH); IR (ATR): m 3395w, 2927w, 2104s,
1738w, 1603w, 1508w, 1452w, 1366w, 1260w, 1230w,
4.6.11. 60-Deoxy-500-(N-phenyl)paromomycincarboxam-
ide (8c). GP 3 on 19 mg of 7c, FC (THF; 1:1, THF–
MeOH; 1:0 ! 22:3, MeOH–25% aq NH3) and lyophili-
sation gave 8c (11 mg, quant.). White solid; Rf = 0.41
(4:1, MeOH–25% aq NH3); IR (ATR): m 3187w (br),
2904w, 1670w, 1599w, 1542w, 1496w, 1447w, 1384w,
1125w, 1089m, 1030m cmꢁ1 1H NMR (400 MHz,
;
CD3OD) see Table 7, additionally, d 7.48–7.46 (m, 2
arom. H); 7.35–7.31 (m, 3 arom. H), 7.15–7.11 (m, 2
arom. H), 6.71–6.64 (m, 3 arom. H), 5.51 (s, CHPh);
13C NMR (100 MHz, CD3OD) see Table 8, addition-
ally, d 149.88 (s, C@O), 139.15 (s), 130.21 (2d), 130.00
(s), 129.09 (2d), 127.59 (2d), 118.58 (2d), 114.60 (2d),
104.11 (d, CHPh); HRMALDIMS: 420.1þ610 (100),
1322w, 1257w, 1102s, 1038s cmꢁ1
;
1H NMR
(500 MHz, D2O) see Table 4, additionally d 7.57–7.54
(m, 2 arom. H), 7.48–7.45 (m, 2 arom. H), 7.32–7.28
(m, arom. H); 13C NMR (125 MHz, D2O) see Table 5,
additionally d 138.82 (s), 132.14 (2d), 128.86 (d),
909.3363 (81, [M+H]+, C36H45N16O
;
Calcd
13
909.3352). Anal. Calcd for C36H44N16O13ÆCH3OH

I. Kudyba et al. / Carbohydrate Research 342 (2007) 499–519
511
(940.88): C, 47.23; H, 5.14; N, 24.82. Found: C, 47.08;
H, 5.19; N, 24.66.
(2d); 101.58 (d, PhCH), 75.16, 74.47 and below solvent
peak (C-4, C-6, C-200, C-300), 21.10, 20.92, 20.74, 20.50
(5q, 5CH3CO); HRMALDIMS: 596.1840 (10þ0,
;
9
4.8. 1,3,20,2000,6000-Pentadeamino-1,3,20,2000,6000-pentaazido-
40,60-O-benzylidene-500-carboxyparomomycin (13)
[Mꢁring IIIꢁring IV+Na+H]+, C23H27N9NaO
Calcd 596.1829), 612.1571 (52, [Mꢁring IIIꢁring IV+
K+H]+, C23H27KN9O þ; Calcd 612.1569), 1080.2995
9
A soln of 9 (1.061 g, 1.27 mmol) in 1:1 MeCN–water
(24 mL) was treated with bisacetoxyiodobenzene
(1.189 g, 3.69 mmol) and TEMPO (91 mg, 0.58 mmol),
stirred at 23 ꢁC for 48 h, cooled to 0 ꢁC, treated with
1 N HCl (30 mL) and diluted with AcOEt (50 mL).
The layers were separated and the aq layer was extracted
with AcOEt (2 · 50 mL). The combined org. layers were
washed with brine (100 mL), dried (MgSO4), filtered and
evaporated. FC (1:1:0 ! 2:2:1, CHCl3–AcOEt–MeOH)
gave 13 (585 mg, 54%). White solid: mp 175 ꢁC (dec);
(94, [M+Na]+, C40H47N15NaO þ; Calcd 1080.3019),
20
1081.3050 (47, [M+Na+H]+, C40H48N15NaO þ; Calcd
20
1081.3098), 1096.2719 (77, [M+K+H]+, C40H47KN15O
Calcd 1096.2759).
;
þ
20
4.10. 6,30,200,3000,4000-Penta-O-acetyl-1,3,20,2000,6000-penta-
deamino-1,3,20,2000,6000-pentaazido-40,60-O-benzylidene-500-
paromomycincarboxamide (19)
Under Ar, a soln of 18 (98 mg, 0.093 mmol) in THF
(5 mL) was treated with N,N0-carbonyldimidazole
(36 mg, 0.22 mmol), stirred at 25 ꢁC for 2 h. The reac-
tion mixture was treated with AcONH4 (32 mg,
0.42 mmol), stirred at 25 ꢁC for 16 h, diluted with
AcOEt (12 mL) and washed with H2O (10 mL). The
two layers were separated and the aq layer was extracted
with AcOEt (2 · 12 mL). The combined org. layers were
washed with brine (20 mL), dried (MgSO4), filtered and
evaporated. FC (1:1, cyclohexane–AcOEt) gave 19
(74 mg, 76%). White solid: mp 128–133 ꢁC; Rf = 0.36
(4:6, CHCl3–AcOEt); IR (ATR): m 2101s, 1745s,
1698m, 1453w, 1431w, 1370m, 1332 w, 1218s, 1168m,
25
½aꢀD +76.0 (c 0.265, MeOH); Rf = 0.20 (2:2:1, CHCl3–
AcOEt–MeOH); IR (ATR): m 3360w, 2934w, 2855w,
2099s, 1602m, 1453w, 1413w, 1375w, 1328w, 1257m,
1142m, 1121m, 1088s, 1026s, 992s, 921m cmꢁ1 1H
;
NMR (500 MHz, CD3OD) see Table 10, additionally d
7.45–7.51 (m, 2 arom. H), 7.32–7.37 (m, 3 arom. H);
5.59 (s, PhCH), 13C NMR (125 MHz, CD3OD) see
Table 11, additionally d 139.16 (s), 130.02, 129.11
(3d), 127.61 (2d), 103.17 (d, PhCH); HRMꢁALDIMS:
846.2534 (100, [MꢁH]ꢁ, C30H36N15O
;
Calcd
15
846.2521). Anal. Calcd for C30H37N15O15Æ2MeOH
(911.79): C, 42.15; H, 4.97; N, 23.04; found: C, 42.21;
H, 4.64; N, 2.54.
1130m, 1093m, 1029s, 998s, 968m, 931m cmꢁ1 1H
;
NMR (300 MHz, CDCl3) d 7.46–7.43 (m, 2 arom. H),
7.37–7.34 (m, 3 arom. H), 6.98 (br s, CONH2), 5.62
4.9. 6,30,200,3000,4000-Penta-O-acetyl-1,3,20,2000,6000-pentade-
amino-1,3,20,2000,6000-pentaazido-40,60-O-benzylidene-500-
carboxyparomomycin (18)
ðdd; J2 ;3 10:4 Hz; J3 ;4 9:6 Hz; H-30Þ, 5.60–5.56 (m, 1H),
0
0
0
0
5.50 (s, PhCH), 5.35 (br s, H-100), 5.08 ðt; J2
¼
000;3000
2:9 Hz; H-3000Þ, 5.05 ðd; J1
1:9 Hz; H-1000Þ,
000;4000
J3
000;2000
Under Ar, a soln of 13 (137 mg, 0.161 mmol) in pyridine
(6 mL) was treated with Ac2O (1.7 mL, 17.9 mmol), stir-
red at 25 ꢁC for 18 h, diluted with AcOEt (30 mL) and
washed with cold 1 N HCl (20 mL). The layers were
separated and the aq layer was extracted with AcOEt
(2 · 30 mL). The combined org. layers were washed with
brine (50 mL), dried (MgSO4), filtered and evaporated.
FC (7:3, cyclohexane–AcOEt then 3:3:0.05 ! 3:3:1,
CHCl3–AcOEt–MeOH) gave 18 (131 mg, 76%). Yellow-
ish solid; Rf = 0.47 (2:2:1, CHCl3–AcOEt–MeOH); IR
(ATR): m 2941w, 2101s, 1744s, 1609m, 1429w, 1370m,
4.99 (dd, J5,6 10.2 Hz, J1,6 9.1 Hz, H-6), 4.78–4.67
(m, 4H), 4.34–4.13 (m, 3H), 4.13–4.03 (m, H-5000),
4.01 (J4,5 = J5,6 9.0 Hz, H-5), 3.80–3.35 (m, 8H), 3.15
ðdd; J1 ;2 3:9 Hz; J2 ;3 10:4 Hz; H-20Þ,
2.39
(dt,
0
0
0
0
J1,2eq = J2eq3 4.4 Hz, J2ax2eq 13.2 Hz, Heq-2); 2.21, 2.18,
2.16, 2.14, 2.13 (5s, 5CH3CO), 1.63 (q, J2ax,2eq
=
J1,2ax = J3,2ax 12.9 Hz, Hax-2); 13C NMR (75 MHz,
CDCl3) see Table 11, additionally d 170.08, 169.87,
169.60, 168.49 (5s, 5CH3CO), 136.78 (s), 129.06 (d),
128.15 (2d), 126.14 (2d), 101.58 (d, PhCH), 20.91,
20.77, 20.59, 20.25 (5q, 5CH3CO); HRMALDIMS:
1216s, 1125m, 1093m, 1028s, 970m cmꢁ1
;
1H NMR
1079.3177 (100, [M+Na]+, C40H48N16NaO þ; Calcd
19
(300 MHz, CDCl3) d 7.47–7.43 (m, 2 arom. H), 7.40–
7.30 (m, 3 arom. H), 5.63–5.48 (m, H-30, H-100), 5.50
(s, PhCH), 5.10–5.06 (m, 1H), 5.00–4.90 (m, 2H),
4.75–4.72 (m, 2H), 4.34–4.25 (m, 2H), 4.11–4.08 (m,
1H), 4.03–3.97 (m, 1H), 3.80–3.30 (m, 9H), 3.20–3.13
(m, H-20), 2.45–2.35 (m, Heq-2), 2.20, 2.18, 2.16, 2.15,
2.13 (5s, 5CH3CO), 1.65 (q, J2ax,2eq = J1,2ax = J3,2ax
12.1 Hz, Hax-2); 13C NMR (75 MHz, CDCl3) see
Table 11; additionally d 169.87, 169.52, 168.40 (5s,
5CH3CO), 136.77 (s); 129.02 (d), 128.11 (2d); 126.13
1079.3179), 1080.3236 (50, [M+Na+H]+, C40H49N16-
NaO19þ; Calcd 1080.3258).
4.11. 1,3,20,2000,6000-Pentadeamino-1,3,20,2000,6000-pentaazido-
40,60-O-benzylidene-500-paromomycincarboxamide (20)
A soln of 19 (53 mg, 0.05 mmol) in 4:1 MeOH–
CH2Cl2 was treated with NaOMe (32 mg, 0.60 mmol),
stirred at 25 ꢁC for 16 h, neutralised with Amberlite
IR-120 (H+ form), filtered and evaporated. FC

512
I. Kudyba et al. / Carbohydrate Research 342 (2007) 499–519
(1:1:0 ! 12:12:0.5, CHCl3–AcOEt–MeOH) gave 20
(39 mg, 92%). White solid; Rf = 0.18 (3:3:0.5, CHCl3–
AcOEt–MeOH); 1H NMR (300 MHz, CD3OD) see
Table 10, additionally d 7.51–7.48 (m, 2 arom. H),
7.35–7.33 (m, 3 arom. H), 5.59 (s, PhCH).
(s), 139.17 (s), 130.04 (d), 129.89 (2d), 129.89 (2d),
129.13 (2d), 127.62 (2d), 125.67 (d), 121.73 (d), 103.16
(d, PhCH); HRMALDIMS: 945.2941 (100, [M+Na]+,
þ
C36H42N16NaO14
; Calcd 945.2964), 946.2972 (45,
[M+H+Na]+, C36H43N16NaO
;
Calcd 946.3046).
þ
14
Anal. Calcd for C36H42N16O14ÆH2O (940.83): C,
45.96; H, 4.71; N 23.82. Found: C, 46.27; H 4.92; N,
23.32.
4.12. General procedure for the formation of the amides
15b–15c (GP 4)
Under N2, a 0.1 M soln of 13 in DMF was treated
with HOAt (1.1 equiv), EDAC (1.1 equiv), DIPEA
(2 equiv) and the corresponding amine (1.1 equiv), and
the mixture was stirred at 25 ꢁC for 25 h, cooled to
0 ꢁC, treated with 1 N HCl and diluted with AcOEt
and H2O. The two layers were separated and the aq
layer was extracted with AcOEt (2·). The combined
org. layers were washed with brine, dried (MgSO4),
filtered and evaporated.
4.13. General procedure for the cleavage of 40,60-O-
benzylidene acetals (GP 5)
A 0.05 M soln of 40,60-O-benzylidene acetals (1 equiv) in
MeOH was treated with TsOHÆH2O (1–2 equiv), stirred
at 25 ꢁC for 1–6 h, basified with 1 N NaOH or satd
NaHCO3 and extracted with EtOAc (3·). The combined
org. layers were dried (MgSO4) and evaporated to give
the crude product.
4.12.1.
1,3,20,2000,6000-Pentadeamino-1,3,20,2000,6000-penta-
4.13.1. 1,3,20,2000,6000-Pentaazido-1,3,20,2000,6000-pentadeam-
ino-500-deoxy-500-[N-(hydroxy)imino]paromomycin (11a).
GP 5 and FC (amino phase gel, 7:3, CH2Cl2–MeOH)
gave 11a (79 mg, 89%). White solid: mp 123–125 ꢁC.
Rf = 0.24 (amino phase gel, 7:3, CH2Cl2–MeOH); IR
(ATR): m 3351w, 2925w, 2099s, 1633w, 1368w, 1331w,
azido-40,60-O-benzylidene-500-[N-(4-nitrobenzyl)]paromo-
mycincarboxamide (15b). GP 4 on 200 mg of 13 and
FC (1:1:0 ! 6:6:0.05, CHCl3–AcOEt–MeOH) gave 15b
26
(148 mg, 64%). Yellowish solid: mp 128–132 ꢁC; ½aꢀD
+85.7 ꢁC (c 0.21, MeOH); Rf = 0.48 (3:3:0.5, CHCl3–
AcOEt–MeOH); IR (ATR): m 3403w, 2931w, 2099s,
1666w, 1605w, 1519m, 1455w, 1372w, 1344m, 1256m,
1259m, 1138m, 1125m, 1024s cmꢁ1
;
1H NMR
(500 MHz, CD3OD) see Table 6; 13C NMR (125 MHz,
CD3OD) see Table 7; HRMALDIMS: 757.2381 (30,
1140m, 1123m, 1087m, 1026s, 992s, 911w cmꢁ1 1H
;
NMR (500 MHz, CD3OD) see Table 10, additionally d
8.22 (d, J 8.8 Hz, 2 arom. H), 7.57 (d, J 8.9 Hz, 2 arom.
H), 7.50–7.48 (m, 2 arom. H), 7.36–7.32 (m, 3 arom. H),
5.57 (s, PhCH), 4.63 (d, J 15.8, ArCHaHbNHCO), 4.47
(d, J 15.8, ArCHaHbNHCO); 13C NMR (125 MHz,
CD3OD) see Table 11, additionally d 148.70 (s), 147.58
(s), 139.13 (s), 130.03 (d), 129.57 (2d), 129.11 (2d),
127.61 (2d), 124.84 (2d), 103.17 (d, PhCH), 43.34
(t, ArCH2NHCO); HRMALþDIMS: 1004.2966 (100,
[MꢁH]ꢁ, C23H33N16O ꢁ; Calcd 757.2362). Anal. Calcd
14
for C23H34N16O14 2.5MeOH (838.72): C, 36.52; H, 5.29;
N, 26.72. Found: C, 36.97; H, 5.24; N, 26.87.
4.13.2.
1,3,20,2000,6000-Pentaazido-1,3,20,2000,6000-pentade-
amino-500-deoxy-500-[N-(4-nitrobenzyloxy)imino]paromo-
mycin (11b). GP 5 and FC (2:2:0.1 ! 2:2:0.4, CHCl3–
EtOAc–MeOH) gave 11b (231 mg, 83%). White
solid: mp 128–130 ꢁC; Rf = 0.26 (2:2:0.5, CHCl3–
EtOAc–MeOH); IR (ATR): m 3407w, 2933w, 2104s,
[M+Na]+, C37H43N17NaO
;
Calcd 1004.2971),
16
1005.3012 (50, [M+H+Na]+, C37H44N17NaO þ; Calcd
1606w, 1520w, 1346m, 1260w, 1143w, 1030m cmꢁ1; H
1
16
1005.3050). Anal. Calcd for C37H43N17O16ÆCH3OH
(1013.88): C, 45.02; H, 4.67; N 23.49. Found: C, 45.06;
H 4.61; N, 23.16.
NMR (300 MHz, (CD3)2CO): d 8.30–8.20 (m, 2 arom.
00 00
H), 7.78–7.64 (m, 2 arom. H), 7.75 ðd; J4 ;5 4:7 Hz;
H-500Þ, 5.79 ðd; J1 ;2 4:4 Hz; H-10Þ, 5.55 (br s, H-100),
0
0
000;2000
5.26 (s, CH2Ph), 5.22 ðd; J1
1:9 Hz; H-1000Þ, 5.18
4.12.2.
1,3,20,2000,6000-Pentadeamino-1,3,20,2000,6000-penta-
(d, J 4.35 Hz, 1H), 4.96 (d, J 4.05 Hz, 1H), 4.76 (d,
J 5.0 Hz, 1H), 4.73 (d, J 4.0 Hz, 1H), 4.67 (dd, J
4.7, 7.2 Hz, 1H), 4.62 (dd, J 4.7, 6.8 Hz, 1H), 4.51
(d, J 4.7 Hz, 1H), 4.46 (dt, J 1.24, 4.8 Hz, 1H),
4.04–4.95 (m, H-5000), 4.93–4.84 (m, H-50), 4.84
azido-40,60-O-benzylidene-500-(N-phenyl)paromomycincarb-
oxamide (15c). GP 4 on 95 mg of 13 and FC
(1:1:0 ! 6:6:0.05, CHCl3–AcOEt–MeOH) gave 15c
(35 mg, 34%). Yellowish solid; Rf = 0.57 (3:3:0.5,
CHCl3–AcOEt–MeOH); IR (ATR): m 3387w, 2933w,
2877w, 2099s, 1732w, 1659w, 1597w, 1537w, 1495w,
1441w, 1373w, 1315w, 1254m, 1137m, 1122m, 1085m,
ðt; J5 ;6 a ¼ J6 a;6 b 9:3 Hz; H-60aÞ, 4.74 (dd,
J
8.4,
0
0
0
0
0
0
8.7 Hz, 1H), 4.76–4.71 (m, 1H), 4.68 ðdd; J5 ;6 b 5:0
Hz; J6 a;6 b 9:3 Hz; H-60bÞ, 4.66–4.57 (m, H-40,H-3);
0
0
1
1026s, 994s, 918m cmꢁ1; H NMR (300 MHz, CD3OD)
4.62 ðdd; J5
12:8 Hz; H-6000aÞ, 4.55–
000;6000
a
;6000b
8:4 Hz; J6a000
000;6000
b
;6000b
12:8
see Table 10, additionally d 7.65–7.62 (m, 2 arom. H),
7.50–7.47 (m, 2 arom. H), 7.34–7.30 (m, 5 arom. H),
7.14–7.09 (m, arom. H), 5.57 (s, PhCH); 13C NMR
(75 MHz, CD3OD) see Table 11, additionally d 139.33
4.48 (m, H-1), 4.44 ðdd; J5
4:7 Hz; J6a000
Hz; H-6000bÞ, 4.08 ðdd; J3 ;2 10:4 Hz; J1 ;2 4:5 Hz; H-20Þ,
2.21 (dt, J1,2eq = J3,2eq 4.3 Hz, J2ax,2eq 12.4 Hz,
Heq-2); 1.45 (q, J2ax,2eq = J1,2ax = J3,2ax 12.4 Hz,
0
0
0
0

I. Kudyba et al. / Carbohydrate Research 342 (2007) 499–519
513
Table 6. 1H NMR chemical shifts (ppm) and coupling constants (Hz) for 10a–c, 11a, 22 and 23 in CD3OD
10aa
10ba
10ca
22a
11a
5.85
3.10
4.10
3.46–3.37
3.95
3.83
23a
5.85
2.90
3.93
3.30
3.99
3.80
H-10
H-20
H-30
H-40
H-50
Ha-60
Hb-60
H-1
5.85
3.26
4.10
3.55
4.13–4.08
4.22
5.79
3.16
4.10
3.48
4.13
5.85
3.20
4.11
3.41
4.15–4.05
4.18
5.88
3.00
4.09
3.32
4.12–4.08
4.18
4.22
3.77
3.54–3.50
1.37
3.73
3.46–3.38
1.38
3.65
3.41–3.34
1.37
3.71–3.65
3.46–3.39
1.38
3.73
3.50
1.36
3.72–3.67
3.43–3.38
1.38
Hax-2
Heq-2
H-3
H-4
2.21
3.45–3.40
3.61
3.67
3.38
5.44
4.41
4.61–4.79
4.61–4.79
7.41
2.21
3.53
3.59
3.69
3.38
5.44
4.38
4.61–4.58
4.61–4.58
7.61
—
5.11
2.21
3.53
3.61
3.72
3.41
5.52
4.46
4.76
2.43
3.55–3.50
3.60
3.71–3.65
3.44
5.36
4.00
4.45
4.29
3.54
2.18
2.19
3.46–3.37
3.46–3.37
3.68–3.65
3.46–3.37
5.43
3.52–3.46
3.72–3.67
3.72–3.67
3.43–3.38
5.37
4.36
4.41
4.28
3.52
H-5
H-600
H-100
H-200
H-300
H-400
H-500a
H-5000b
H-1
4.39
4.61–4.56
4.61–4.56
7.42
—
5.12
4.81–4.78
7.88
—
—
5.13
3.22
5.14
3.24
5.13
5.18
H-2000
H-3000
H-4000
H-5000
Ha-6000
Hb-6000
3.69–3.65
4.94
3.46–3.44
4.02
3.66
3.36
3.64–3.61
4.92
3.46–3.38
3.99
3.64–3.61
3.32
3.72–3.69
4.95
3.46–3.41
4.02
3.65
3.20
3.71–3.65
4.96
3.46–3.39
4.04
3.67
3.33
3.68–3.65
3.93
3.46–3.45
4.00
3.65
3.37
3.72–3.67
3.46–3.44
3.96–3.94
4.03
3.64
3.35
0
0
0
0
0
0
J1 ;2
3.9
10.1
9.5
9.4
4.9
10.2
10.1
12.6
4.2
9.5
12.6
12.6
4.2
9.7
8.9
8.1
3.9
10.1
8.8
9.7
5.0
10.2
10.2
12.4
4.2
9.8
12.4
12.2
4.4
9.8
8.8
9.8
3.9
10.1
9.5
9.5
4.9
10.0
10.0
12.2
4.3
3.8
10.2
9.3
9.5
3.8
10.5
8.9
10.0
2.3
3.7
10.5
8.8
9.9
0
J2 ;3
0
J3 ;4
0
J4 ;5
0
J5 ;6 a
4.9
b
2.4
b
0
0
J5 ;6 b
4.9
0
0
J6 a;6 b
J1,2ax
J1,2eq
J1,6
10.0
b
11.9
12.4
4.5
9.8
12.4
12.4
11.9
12.6
4.2
b
4.2
8.6
12.5
12.5
4.2
8.5
9.8
9.8
1.3
4.5
7.1
3.3
8.1
12.9
1.8
3.3
3.3
2.0
8.7
4.1
12.9
9.5
J2ax,2eq
J2ax,3
J2eq,3
J3,4
12.2
12.2
4.3
9.8
8.9
8.9
1.4
3.5
1.5/3.9
6.2
—
—
1.9
3.7
3.7
2.2
8.6
4.5
12.9
12.6
12.6
4.2
b
4.2
b
b
b
b
b
J4,5
J5,6
00 00
J1 ;2
00 00
J2 ;3
00 00
J3 ;4
1.4
3.5
b
1.4
3.7
b
1.5
3.8
b
1.6
4.6
6.9
3.6
7.7
13.0
1.8
b
00 00
J4 ;5 a
00 00
J4 ;5 b
6.8
—
—
1.9
3.8
3.8
2.3
8.3
4.6
12.9
6.3
—
—
1.9
3.8
3.8
2.4
8.7
4.2
13.1
6.3
—
—
1.9
3.8
3.8
2.3
10.5
4.9
12.9
00
00
J5 a;5 b
000;2000
000;3000
0004000
J1
J2
J3
J4
J5
J5
J6
b
000;5000
000;6000
000;6000
1.9
8.5
4.5
a
b
000a,6000
b
12.9
a Assignment based on DQFCOSY and HSQC spectrum.
b Not determined.
Hax-2); 13C NMR (75 MHz, (CD3)2CO) see Table 7,
additionally, d 145.95 (2s), 129.04 (2d), 124.58 (2d),
74.64 (t, CH2Ph); HRESIMS: 916.2664 (100, [M+
4.13.3. 500-Anilino-1,3,20,2000,6000-pentaazido-1,3,20,2000,6000-
pentadeamino-500-deoxyparomomycin (23). GP 5 and
FC (2:2:0.1 ! 2:2:0.4, CHCl3–EtOAc–MeOH) gave 23
(126 mg, 92%). White solid: mp 125–127 ꢁC;
Na]+, C30H39N17NaO þ; Calcd 916.2658).
16

514
Table 7. 13C NMR chemical shifts (ppm) for 10a–c, 11a,b, 22 and 23 in CD3OD
10aa 10ba 10ca 22a
98.66 98.86 98.76 98.81
I. Kudyba et al. / Carbohydrate Research 342 (2007) 499–519
11a
97.77
11bb
96.40
23a
98.26
C-10
C-20
C-30
C-40
C-50
C-60
C-1
65.15
69.96
82.88
64.54
69.83
61.95
33.09
61.38
77.72
85.51
77.65
110.98
74.80
79.53c
79.78c
149.80
99.68
62.14
70.97
69.83
75.84
52.35
65.19
69.80
82.99
69.86
69.86
62.00
33.08
61.35
77.79c
85.60
77.76c
111.01
74.88
79.38d
79.47d
151.34
99.78
61.94
71.03
69.86
75.92
52.54
64.51
69.79
82.89
64.51
69.79
62.00
33.10
61.37
77.92
85.56
77.74
111.34
74.89
79.31c
79.43c
153.51
99.85
62.00
71.04
69.76
75.99
52.44
64.89
69.38
82.85
64.58
69.82
62.05
33.05
61.42
77.98
86.15
77.90
111.86
74.86
79.06
80.70
47.40
99.62
61.79
71.24
69.69
75.93
52.60
64.69
71.82c
72.62c
74.05
62.54
62.16
33.15
61.51
77.59d
85.59
76.56d
110.63
74.89
79.75e
79.93e
149.86
99.79
61.98
69.77
70.99
75.72
52.29
63.46
71.19c
71.55c
73.14
61.91
60.63
32.09
60.37
75.54
84.34
74.50
107.26
74.02
78.39
79.24
150.54
98.74
60.63
68.85
70.01
74.39
51.33
64.36
71.92c
71.98c
74.07
62.59
62.03
33.02
61.38
77.70
85.86
77.01
111.22
74.92
78.93
80.65
48.36
99.66
61.75
69.57
71.16
75.73
52.45
C-2
C-3
C-4
C-5
C-600
C-100
C-200
C-300
C-400
C-5
C-1000
C-2000
C-3000
C-4000
C-5000
C-6000
a Assignment based on DQFCOSY and HSQC spectrum.
b Solvent (CD3)2CO.
c –e May be interchanged.
25
½aꢀD +156.8 (c 0.65, MeOH); Rf = 0.13 (2:2:0.5, CHCl3–
EtOAc–EtOAc); IR (ATR): m 3384w, 2936w, 2098s,
1602w, 1506w, 1373w, 1326w, 1255m, 1123m, 1017s
4.13.5.
1,3,20,2000,6000-Pentadeamino-1,3,20,2000,6000-pen-
taazido-500-(N-phenyl)paromomycincarboxamide (16c).
GP 5 on 86 mg of 15c and FC (1:1:0 ! 6:6:0.4, CHCl3–
EtOAc–MeOH) gave 16c (29 mg, 63%). White solid;
Rf = 0.46 (2:2:1, CHCl3–EtOAc–MeOH); 1H NMR
(300 MHz, CD3OD) see Table 10, additionally d 7.65–
7.61 (m, 2 arom. H), 7.34–7.28 (m, 2 arom. H), 7.13–
7.08 (m, arom. H).
cmꢁ1; H NMR (400 MHz, CD3OD) see Table 6, addi-
1
tionally, d 7.13–7.07 (m, 2 arom. H), 6.71–6.61 (m, 3
arom. H); 13C NMR (100 MHz, CD3OD) see Table 7,
additionally, d 149.72 (s), 130.09 (2d), 114.60 (2d),
111.22 (d); HRMALDIMS: 844.2868 (100, [M+Na]+,
C29H40N16NaO13þ; Calcd 844.2858). Anal. Calcd for
C29H40N16O13 (820.73): C, 42.44; H, 4.91; N, 27.31.
Found: C, 42.27; H, 4.98; N, 26.76.
4.13.6.
azido-500-paromomycincarboxamide (21). GP
1,3,20,2000,6000-Pentadeamino-1,3,20,2000,6000-penta-
on
5
35 mg of 20 and FC (1:1:0 ! 12:12:1.5, CHCl3–
EtOAc–MeOH) gave 21 (24 mg, 72%). White solid;
Rf = 0.41 (2:2:1, CHCl3–EtOAc–MeOH); 1H NMR
(300 MHz, CD3OD) see Table 10.
4.13.4.
1,3,20,2000,6000-Pentadeamino-1,3,20,2000,6000-pen-
taazido-500-[N-(4-nitrobenzyl)]paromomycincarboxamide
(16b). GP 5 on 86 mg of 15b and FC (1:1:0 !
6:6:0.5, CHCl3–EtOAc–MeOH) gave 16b (62 mg,
26
79%). White solid; ½aꢀD +97.9 ꢁC (c 0.235, MeOH);
4.14. General procedure for the reduction of azides with
propane-1,3-dithiol (GP 6)
Rf = 0.45 (2:2:1, CHCl3–EtOAc–MeOH); 1H NMR
(500 MHz, CD3OD) see Table 10, additionally d 8.21
(d, J 8.8 Hz, 2 arom. H), 7.56 (d, J 8.9 Hz, 2 arom.
H), 4.58 (d, J 15.9 Hz, ArCHaHbNHCO), 4.51 (d,
J 15.9 Hz, ArCHaHbNHCO); 13C NMR (125 MHz,
CD3OD) see Table 11, additionally d 148.67 (s), 147.55
(s), 129.51 (2d), 124.76 (2d), 43.38 (t, ArCH2NHCO);
A 0.1 M soln of the protected N-alkoxyimino or amido
azide (1 equiv) in MeOH was treated with Et3N
(25 equiv) and propane-1,3-dithiol (25 equiv), stirred at
25 ꢁC for 24–48 h and evaporated. A soln of residue in
H2O was washed with EtOAc (1·). The aq layer was
evaporated to give the crude product.
HRMALDIMS: 916.2636 (100, [M+Na]+, C30H39-
þ
N17NaO16
;
Calcd
916.2658),
917.2650
Calcd 917.2737).
(39,
[M+H+Na]+, C30H40N17NaO
;
þ
4.14.1. 1,3,20,2000,6000-Pentaammonium-500-deoxy-500-(hydr-
oxyimino)paromomycin pentaacatate (12aÆ5HOAc). GP
6 FC (9:1 ! 7:3, MeOH–25% aq NH3) and dissolution
in 10% aq HOAc, partial evaporation and lyophilisation
16
Anal. Calcd for C30H39N17O16ÆCH3OH (927.78): C,
40.22; H, 4.68; N 25.72. Found: C, 40.21; H 4.67; N,
25.44.

I. Kudyba et al. / Carbohydrate Research 342 (2007) 499–519
515
gave 12aÆ5HOAc (53 mg, 76%). White solid: mp 112 ꢁC
25 aq NH3); IR (ATR): m 3500–2700m, 1538s, 1399s,
1336m, 1140m, 1118m, 1011s cmꢁ1 1H NMR
25
(dec); ½aꢀD +42.7 (c 0.19, H2O); Rf = 0.20 (7:3, MeOH–
;
(500 MHz, D2O) see Table 8; additionally, d 1.92 (s,
5OAc); 13C NMR (125 MHz, D2O) see Table 9, addi-
tionally, d 184.91 (s, 5C@O), 25.78 (q, 5 Me); HRM-
ALDIMS: 596.2136 (100), 651.2797 (14, [M+Na]+,
C23H44N6NaO14þ; Calcd 651.2813). Anal. Calcd for
C33H64N6O19 (928.89): C, 42.67; H, 6.94; N, 9.05.
Found: C, 42.40; H, 6.83; N, 9.34.
Table 8. 1H NMR chemical shifts (ppm) and coupling constants (Hz)
for 12a,b and 24 in D2O
12aÆ5HOAca
12bÆ5HOAca
24a
H-10
H-20
H-30
H-40
H-50
Ha-60
Hb-60
H-1
5.75
3.34
3.89
3.52
3.84–3.80
3.89
3.82–3.78
3.31–3.23
1.67
2.33
3.31–3.23
3.81
3.89
3.65
5.44
4.46
4.67
4.80–4.74
7.72
—
5.32
3.60
4.23
3.84–3.80
4.30
3.43
3.38
4.0
10.6
10.7
5.79
3.11
3.87
3.31
3.73–3.70
3.88
3.71
3.34–3.30
1.80
2.44
3.46
3.98
3.87
3.65
5.42
4.37
4.68
4.81
7.86
—
5.20
3.55
4.17
3.75
3.99–3.97
3.32
3.24
4.0
10.3
8.9
8.9
3.3
6.4
5.34
2.52
3.51
3.19
3.76
3.81
3.67
2.73
1.19
1.94
2.87
3.24
3.68–3.61
3.42
5.31
4.38
4.49
4.29
3.44
3.35
4.94
3.02–3.00
4.00
3.68–3.61
3.90
2.98
2.88
3.7
10.3
9.2
9.8
6.2
4.14.2. 1,3,20,2000,6000-Pentaammonium-500-deoxy-500-[N-(4-
nitrobenzyloxy)imino]paromomycin pentaacetate (12bÆ
5HOAc). GP 6, FC (9:1 ! 7:3, MeOH–25% aq NH3)
and dissolution in 10% aq HOAc, partial evaporation
and lyophilisation gave 12bÆ5HOAc (76 mg, 71%).
H
H
ax-2
eq-2
H-3
H-4
H-5
25
White solid: mp 113 ꢁC (dec); ½aꢀD +52.1 (c 0.165,
H2O); Rf = 0.26 (7:3, MeOH–25% aq NH3); IR
(ATR): m 3500–2700m, 2920m, 1699w, 1544s, 1518s,
H-600
H-100
H-200
H-300
H-4
H-500a
H-500b
H-1000
H-2000
H-3000
H-4000
H-5000
Ha-6000
Hb-6000
1
1399s, 1344s, 1103w, 1010s cmꢁ1; H NMR (600 MHz,
D2O) see Table 8, additionally, d 8.28–8.26 (m, 2 arom.
H), 7.63–7.61 (m, 2 arom. H), 5.29 (s, CH2Ph), 1.94
(s, 5OAc); 13C NMR (150 MHz, D2O) see Table 9,
additionally, d 182.62 (s, 5C@O), 150.17 (s), 147.60
(s), 131.43 (2d), 126.70 (2d), 77.30 (s,CH2Ph); 25.42
(q, 5 Me); HRMALDIMS: 596.2751 (100), 764.3265
(12, [M+H]+, C30H50N7O16þ; Calcd 764.3314), 786.3115
(23, [M+Na]+, C30H49N7NaO þ; Calcd 786.3133).
16
Anal. Calcd for C40H69N7O21ÆH2O (1082.02): C,
44.40; H, 6.61; N, 9.06. Found: C, 44.54; H, 6.26; N,
9.04.
0
0
0
0
0
0
J1 ;2
0
J2 ;3
0
J3 ;4
0
J4 ;5
9.7
6.9
b
Table 9. 13C NMR chemical shifts (ppm) for 12a,b and 24 in D2O
0
J5 ;6 a
0
0
J5 ;6 b
2.1
12aÆ5HOAca
12bÆ5HOAca
24a
0
0
J6 a;6 b
J1,2ax
J1,2eq
J1,6
10.0
12.7
4.2
9.9
12.7
12.7
4.2
8.9
8.8
8.9
2.5
4.8
6.2
5.8
—
10.6
12.6
4.1
12.1
12.1
4.1
C-10
C-20
C-30
C-40
C-50
C-60
C-1
C-2
C-3
C-4
C-5
C-600
C-100
C-200
C-300
C-400
C-5000
C-1
C-2000
C-3000
C-4000
C-5000
C-6000
98.28
56.40
72.18
71.85
76.24
62.89
51.68b
32.78
52.72b
81.54
87.72
75.58
112.83
76.24
81.76
80.77
152.37
98.87
53.46
70.41
71.36
72.76
43.15
97.57
56.17
71.54
71.93
76.73
63.10
52.38
32.78
51.47
79.35
87.73
75.15
112.89
76.42
81.60
80.93
153.29
98.88
51.47
70.27b
70.24b
72.61
43.16
101.57
57.94
75.97
72.65
75.80
63.53
53.01
37.72
52.81
85.81
86.90
79.70
111.93
75.66
80.69
82.03
49.26
101.69
53.44
73.13
71.20
76.69
43.62
9.1
9.2
J2ax,2eq
J2ax,3
J2eq,3
J3,4
12.6
12.6
4.1
10.1
8.9
9.1
3.0
4.6
5.0
12.1
12.1
4.3
9.4
9.6
9.2
2.2
4.8
6.3
J4,5
J5,6
00 00
J1 ;2
00 00
J2 ;3
00 00
J3 ;4
00 00
J4 ;5 a
5.0
—
4.7
6.8
00 00
J4 ;5 b
00
00
J5 a;5 b
—
—
1.7
2.9
1.2
3.1
1.7
5.9
4.1
13.2
1.8
3.2
b
000;2000
000;3000
000;4000
0004000
J1
J2
J2
J3
J4
J5
J5
J6
1.7
3.0
1.1
3.0
1.6
5.9
4.1
13.8
3.2
1.6
7.8
4.4
13.5
000;5000
000;6000
000;6000
a
b
000a;6000
b
13.7
a Assignment based on DQFCOSY and HSQC spectrum.
b Assignments can be interchanged.
a Assignment based on DQFCOSY and HSQC spectrum.
b Not determined.

516
I. Kudyba et al. / Carbohydrate Research 342 (2007) 499–519
4.14.3. 500-Paromomycincarboxamide (17a). GP 6 on
24 mg of 21, FC (1:0 ! 1:1, THF–MeOH, then
9:1 ! 8:2, MeOH–25% aq NH3), evaporation and lyo-
1487w, 1399s, 1336w, 1135s, 1016s cmꢁ1
;
1H
NMR (500 MHz, D2O) see Table 12; 13C NMR
(125 MHz, D2O) see Table 13; HRMþALDIMS:
Calcd
25
philisation gave 17a (17 mg, 86%). White solid; ½aꢀD
629.2978 (100, [M+H]+, C23H45N6O
;
14
629.2994), 651.2797 (32, [M+Na]+, C23H44N6NaO
Calcd 651.2813).
;
þ
+33.0 (c 0.205, H2O); Rf = 0.22 (7:3, MeOH–25% aq
NH3); IR (ATR): m 3500–2700m, 2920m, 1681m, 1579m,
14
Table 10. 1H NMR chemical shifts (ppm) and coupling constants (Hz) for 13, 15b–c, 16b–c, 20, 21 in CD3OD
13a
15ba
15c
20
16ba
16c
5.75
3.01
3.92
3.56–3.32
3.92–3.82
3.90–3.60
3.90–3.60
3.56–3.32
1.43
21
5.68
3.04
3.92
3.79–3.35
3.96–3.89
3.83
3.79–3.35
3.79–3.35
1.41
H-10
H-20
H-30
H-40
H-50
Ha-60
Hb-60
H-1
5.65
3.23
4.09
3.59–3.53
4.13
5.58
2.97
4.06
3.45
4.15–4.12
4.19
3.76–3.70
3.44–3.42
1.46
5.78
3.14
4.09
3.83–3.36
4.18–4.08
4.21
3.83–3.36
3.83–3.36
1.47
5.68
3.21
5.65
2.94
3.91
3.36
3.94–3.90
3.83
3.77–3.71
3.44–3.39
1.42
4.22–4.00
3.58–3.42
4.22–4.00
4.23
3.80–3.63
3.58–3.42
1.46
4.23
3.82–3.73
3.52–3.43
1.49
Hax-2
H
eq-2
2.21
2.23
2.22
2.22
2.19
2.19
2.18
H-3
H-4
H-5
H-600
H-100
H-200
H-3
3.59–3.53
3.86
3.82–3.73
3.82–3.73
5.47
3.54–3.49
3.67
3.74
3.55
5.54
3.83–3.36
3.83–3.36
3.83–3.36
3.83–3.36
5.52
3.58–3.42
3.80–3.63
3.80–3.63
3.55
5.52
4.32
3.58–3.47
3.77–3.71
3.77–3.71
3.60–3.58
5.55
3.56–3.32
3.90–3.60
3.90–3.60
3.62
5.54
4.40
3.79–3.35
3.79–3.35
3.79–3.35
3.79–3.35
5.51
4.32
4.66
4.38
4.68
4.42
4.73
4.35
4.67
4.31
4.62
4.63
4.72
H-400
H-1000
H-2000
H-3000
H-4000
H-5000
Ha-6000
Hb-6000
4.56
5.21
3.86
3.99
3.52–3.43
4.02
3.63
3.47
3.8
4.63
5.20
3.70–3.67
3.98
3.48–3.47
4.03
3.62
3.39
3.8
10.1
4.68
5.18
4.55
5.19
3.80–3.63
3.97
3.58–3.42
4.02
3.80–3.63
3.42
3.7
10.0
b
4.61
5.18
3.67
3.98
3.58–3.47
4.03
3.61
3.38
3.7
10.6
8.8
10.0
2.5
b
4.66
5.17
4.52
5.17
3.79–3.35
3.96
3.79–3.35
4.06–3.99
3.79–3.35
3.79–3.35
3.4
3.83–3.36
3.97
3.83–3.36
4.01–3.96
3.83–3.36
3.39
4.0
10.0
9.7
b
3.70–3.68
3.98
3.50–3.42
4.01–3.90
3.51
3.56–3.32
3.9
10.4
0
0
0
0
0
0
J1 ;2
0
J2 ;3
10.1
2.8
9.8
10.6
9.0
b
0
J3 ;4
9.3
9.4
5.0
b
9.1
b
b
0
J4 ;5
b
b
b
0
J5 ;6 a
4.9
4.7
b
5.0
b
2.2
b
0
0
J5 ;6 b
12.5
10.1
12.8
4.4
b
0
0
J6 a;6 b
J1,2ax
J1,2eq
J1,6
9.7
12.6
4.3
9.7
12.5
4.3
b
9.3
12.8
4.4
11.9
12.6
4.4
b
12.1
12.5
4.4
b
12.6
4.1
9.1
9.6
9.3
J2ax,2eq
J2ax,3
J2eq,3
J3,4
12.8
12.8
4.4
9.4
9.4
b
12.8
12.6
4.3
9.4
9.3
9.3
2.7
4.2
5.3
12.5
12.5
4.3
b
12.8
12.8
4.4
b
12.6
12.6
4.4
b
12.6
12.6
4.1
b
12.5
12.5
4.4
b
b
b
b
b
b
b
b
b
J4,5
J5,6
9.3
2.8
4.0
5.6
1.9
9.1
1.9
4.1
6.1
1.7
00 00
J1 ;2
00 00
J2 ;3
00 00
J3 ,4
2.9
4.3
4.3
2.0
3.9
3.9
3.9
2.4
7.8
5.6
12.8
2.2
3.7
5.7
1.6
2.2
4.3
5.9
2.0
3.8
2.2
4.0
5.9
1.9
000;2000
000;3000
000;4000
0004000
J1
J2
J2
J3
J4
J5
J5
J6
2.0
3.8
b
3.7
b
3.7
b
4.1
b
3.7
b
ꢂ1.4
3.8
2.3
8.2
4.9
12.9
3.7
b
3.7
2.2
b
3.8
4.1
b
3.7
b
000;5000
000;6000
000;6000
2.2
8.2
4.8
12.9
b
b
b
b
7.8
b
a
5.6
b
5.0
12.8
b
000a;6000
b
12.6
a Assignment based on DQFCOSY and HSQC spectrum.
b Not determined.

I. Kudyba et al. / Carbohydrate Research 342 (2007) 499–519
517
Table 11. 13C NMR chemical shifts (ppm) for 13, 15b–c, 16b, 18 in
CD3OD
Table 12. 1H NMR chemical shifts (ppm) and coupling constants (Hz)
for 14, 17a–c in D2O
13a
99.50
15ba
99.92
15c
99.52
16ba
98.44
18b
98.17
19b
97.97
14a
17aa
17ba
17c
C-10
C-20
C-30
C-40
C-50
C-60
C-1
C-2
C-3
C-4
C-5
C-600
C-100
C-200
C-300
C-400
C-5000
C-1
C-2000
C-3000
C-4000
C-5000
C-6000
H-10
H-20
H-30
H-40
H-50
Ha-60
Hb-60
H-1
5.78
5.48
3.02
3.55
3.71
3.87–3.84
3.89
3.79
3.08–3.01
1.44
2.14
3.08–3.01
3.66
3.84
3.47
5.51
4.43
4.70
4.62
5.13
3.25
4.12
3.76–3.75
4.23
3.37
3.30
3.8
5.67
3.12
3.79
3.42
3.80–3.68
3.85
3.80–3.68
3.24–3.15
1.59
2.24
3.24–3.15
3.65
3.87
3.80–3.68
5.50
4.49
4.69
4.62
5.17
3.40
4.15
5.92
3.43
3.96
3.42
3.82–373
3.91
3.82–373
3.39
1.94
2.52
3.62
4.04
4.14
3.82–373
5.56
4.58
4.80
4.73
5.32
3.65
4.25
3.74
4.21
3.04
2.80
4.0
10.7
65.25
69.40
83.05
64.67
69.87
61.28
32.96
61.34
75.35
85.35
76.25
105.79
76.33
81.43
83.23
178.90
100.06
62.56
71.20
70.09
75.35
52.29
65.06
69.27
83.01
64.65
69.81
61.94
32.80
61.03
78.19
85.56
76.48
108.90
75.40
80.34
82.21
172.94
100.02
62.16
71.09
69.81
75.71
52.40
65.30
69.58
82.98
64.67
69.87
62.02
32.89
61.23
77.84
85.80
76.57
109.05
75.48
80.69
82.72
170.74
100.29
62.23
71.10
69.87
75.77
52.09
64.61
72.02
72.02
74.22
62.47
61.84
32.91
61.41
76.40
85.66
76.19
108.15
75.25
80.23
82.04
173.18
99.76
62.15
71.13
69.83
75.72
52.40
61.42
68.62
78.78
63.41
68.62
58.03
31.23
61.30
68.68
78.46
63.22
68.52
57.90
31.28
58.93
75.96
81.83
74.99
105.24
74.00
78.79
80.62
170.80
100.13
57.07
68.52
65.50
72.99
50.44
3.45–3.30
3.99–3.90
3.50
3.80–3.72
3.99–3.90
3.80–3.72
3.45–3.30
1.84
2.46
3.55–3.48
3.99
3.99–3.90
3.80–3.72
5.48
4.36
4.58
4.45
5.30
3.61
4.24
3.80
4.30
58.95
c
Hax-2
H
eq-2
81.65
c
H-3
H-4
H-5
106.05
c
H-600
H-100
H-200
H-300
H-4000
H-1000
H-2000
H-3
H-4000
H-5000
Ha-6000
Hb-6000
c
78.78
181.47
100.04
57.29
68.62
65.80
73.05
50.54
3.80–3.68
4.24–4.21
3.24–3.15
3.24–3.15
3.9
10.7
9.3
9.3
3.44–3.40
3.45–3.30
4.0
b
a Assignment based on DQFCOSY and HSQC spectrum.
b Solvent CDCl3.
c Not assigned.
0
0
0
0
0
0
J1 ;2
0
J2 ;3
9.9
9.9
9.1
2.1
0
J3 ;4
9.4
9.4
b
9.1
10.7
b
0
J4 ;5
4.14.4. 500-N-(4-Nitrobenzyl)paromomycincarboxamide
(17b). GP 6 on 33 mg of 16b, FC (1:0 ! 1:1, THF–
MeOH, then 85:15 ! 8:2, MeOH–25% aq NH3),
evaporation and lyophilisation gave 17b (21 mg, 75%).
J5 ;6 a
2.1
b
0
b
b
b
b
0
0
J5 ;6 b
5.4
0
0
J6 a;6 b
J1,2ax
J1,2eq
J1,6
12.0
12.7
4.1
12.1
12.6
3.9
b
12.5
b
12.7
4.3
10.7
12.7
12.7
4.3
9.1
9.1
10.1
b
25
b
White solid; ½aꢀD +22.8 (c 0.21, H2O); Rf = 0.17
9.4
J2ax,2eq
J2ax,3
J2eq,3
J3,4
12.5
12.5
b
12.7
12.7
4.1
9.3
9.2
9.2
2.5
4.6
6.0
1.8
3.3
3.3
1.7
7.8
3.6
13.6
12.6
12.6
3.9
10.1
9.2
9.2
1.6
4.5
7.2
(8:2, MeOH–25% aq NH3); IR (ATR): m 3500–
2700w, 2903w, 1666w, 1602w, 1516m, 1345m, 1108s,
1
1028s cmꢁ1; H NMR (500 MHz, D2O) see Table 12,
9.2
9.2
b
additionally, d 8.25 (d, J 8.8 Hz, 2 arom. H), 7.56
(d, J 8.8 Hz, 2 arom. H), 4.71 (d, J ꢂ 16.7 Hz, ArCHa-
HbNHCO), 4.47 (d, J 15.7 Hz, ArCHaHbNHCO); 13C
NMR (125 MHz, D2O) see Table 13, additionally, d
149.71 (s), 148.12 (s), 130.95 (2d), 126.73 (2d), 45.22
(t, ArCH2NHCO); HRMALDIMS: 764.3294 (100,
J4,5
J5,6
00 00
J1 ;2
00 00
J2 ;3
00 00
J3 ;4
2.9
4.5
5.7
1.5
3.1
4.3
7.9
1.7
3.1
3.1
2.0
3.3
8.2
13.6
000;2000
000;3000
0004000
J1
J2
J3
J4
J5
J5
J6
1.6
3.1
3.1
b
[M+H]+, C30H50N7O þ; Calcd 764.3314), 765.3321
3.1
16
(36, [M+2H]+, C30H51N7O16
;
Calcd 765.3392),
þ
000;5000
000;6000
000;6000
ꢂ1.2
b
b
b
786.3138 (40, [M+Na]+, C30H49N7NaO
; Calcd
þ
3.3
a
16
7.5
b
b
786.3133).
000a;6000
b
a Assignment based on DQFCOSY and HSQC spectrum.
b Not determined.
4.14.5. 500-N-Phenylparomomycincarboxamide (17c).
GP 6 on 20 mg of 16c, FC (1:3:0 ! 1:3:1, CHCl3–
MeOH–25% aq NH3), evaporation and lyophilisation
25
gave 17c (15 mg, 89%). Yellowish solid; ½aꢀD +23.6 (c
(125 MHz, D2O) see Table 13, additionally, d 138.79
0.265, H2O); Rf = 0.21 (8:2, MeOH–25% aq NH3); IR
(ATR): m 3500–2700w, 1670w, 1599w, 1492w, 1415m,
(s), 132.22 (2d), 128.95 (d), 124.10 (2d); HRMþALDIMS:
705.3288 (100, [M+H]+, C29H49N6O14
;
Calcd
1047s cmꢁ1; H NMR (500 MHz, D2O) see Table 12,
705.3307), 706.3320 (34, [M+2H]+, C29H50N6O
;
1
þ
14
additionally, d 7.59–7.56 (m, 2 arom. H), 7.49–7.45 (m,
Calcd
706.3385),
727.3083
(38,
[M+Na]+,
2 arom. H), 7.32–7.29 (m, arom. H); 13C NMR
C29H48N6NaO14þ; Calcd 727.3126).

518
I. Kudyba et al. / Carbohydrate Research 342 (2007) 499–519
Table 13. 13C NMR chemical shifts (ppm) for 14 and 17a–c in D2O
at 23 ꢁC for 20 h, filtered through a pad of Celite and
evaporated. FC (RP C-18, 1:8:0.05, MeOH–H2O–
AcOH) gave 14 (21 mg, 64%). White solid: mp 227 ꢁC
(dec); Rf = 0.1. (75:25, MeOH–25% aq NH3); IR
(ATR): m 3041m, 1663s, 1516w, 1433M, 1409m, 1183s,
14a
99.31
17aa
17ba
99.28
17c
98.72
C-10
C-20
C-30
C-40
C-50
C-60
C-1
C-2
C-3
C-4
C-5
C-600
C-100
C-200
C-300
C-400
C-5000
C-1
C-2000
C-3000
C-4000
C-5000
C-6000
100.77
57.51
72.27
74.54
75.81
63.14
53.12
35.67
52.26
84.33
86.68
77.05
111.06
75.92
81.54
82.66
177.41
100.89
54.47
72.27
70.86
73.79
35.67
56.54
71.47
71.99
75.37
63.04
52.58
31.31
51.52
80.60
86.44
76.34
112.02
76.34
82.33
84.15
180.00
98.29
53.43
70.39
69.82
73.03
43.00
56.90
72.01
72.60
75.97
62.93
51.74
33.78
53.11
82.57
87.37
75.85
112.71
75.41
80.30
82.13
174.53
98.43
53.94
71.13
69.94
73.62
43.06
56.57
71.36
76.54
74.87
62.99
52.60
30.64
51.40
79.38
86.88
71.97
113.13
76.18
81.38
82.26
172.96
97.93
53.44
70.04
69.47
73.39
42.81
1
1124s, 1047s, 1026s cmꢁ1; H NMR (500 MHz, D2O)
see Table 12, additionally, d 1.90 (s, CH3CO2H); 13C
NMR (125 MHz, D2O) see Table 13, d 180.14 (s,
CH3CO2H), 25.78 (q, CH3CO2H); HRMALDIMS:
620.2816 (100, C23H44N5O15þ; Calcd 630.2834).
References
1. Mingeot-Leclercq, M. P.; Glupczynski, Y.; Tulkens, P. M.
Antimicrob. Agents Chemother. 1999, 43, 727–737.
2. Kotra, L. P.; Haddad, J.; Mobashery, S. Antimicrob.
Agents Chemother. 2000, 44, 3249–3256.
3. Magnet, S.; Blanchard, J. S. Chem. Rev. 2005, 105, 477–
497.
4. Jana, S.; Deb, J. K. Appl. Microbiol. Biotechnol. 2006, 70,
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5. Vicens, Q.; Westhof, E. Chembiochem 2003, 4, 1018–1023.
6. Ogle, J. M.; Carter, A. P.; Ramakrishnan, V. Trends
Biochem. Sci. 2003, 28, 259–266.
a Assignment based on DQFCOSY and HSQC spectrum.
7. Recht, M. I.; Douthwaite, S.; Puglisi, J. D. EMBO J. 1999,
18, 3133–3138.
8. Bo¨ttger, E. C.; Springer, B.; Prammananan, T.; Kidan, Y.;
Sander, P. EMBO Rep. 2001, 2, 318–323.
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567, 359–360.
10. Mingeot-Leclercq, M. P.; Tulkens, P. M. Antimicrob.
Agents Chemother. 1999, 43, 1003–1012.
11. Hobbie, S. N.; Pfister, P.; Brull, C.; Westhof, E.; Bo¨ttger,
¨
E. C. Antimicrob. Agents Chemother. 2005, 49, 5112–5118.
12. Hobbie, S. N.; Pfister, P.; Brull, C.; Sander, P.; Francois,
¨
4.15. General procedure for the Staudinger reaction
(GP 7)
A 0.02 M soln of the azide (1 equiv) in THF was treated
with 1 N NaOH (0.5 mL) and 1 M PMe3 in THF
(6 equiv), stirred for 2–4 h at 50 ꢁC and evaporated. A
soln of the residue in H2O was washed with EtOAc
(1·) and evaporated to give the crude amines.
B.; Westhof, E.; Bo¨ttger, E. C. Antimicrob. Agents
Chemother. 2006, 50, 1489–1496.
13. Recht, M. I.; Douthwaite, S.; Dahlquist, K. D.; Puglisi, J.
D. J. Mol. Biol. 1999, 286, 33–43.
14. Pfister, P.; Hobbie, S.; Vicens, Q.; Bo¨ttger, E. C.; Westhof,
E. Chembiochem 2003, 4, 1078–1088.
15. Pfister, P.; Hobbie, S.; Bru¨ll, C.; Corti, N.; Vasella, A.;
Westhof, E.; Bo¨ttger, E. C. J. Mol. Biol. 2005, 346, 467–
475.
16. Ryu, D. H.; Rando, R. R. Bioorg. Med. Chem. 2001, 9,
2601–2608.
4.15.1. 500-Anilino-500-deoxyparomomycin (24). GP 7
and FC (1:4:1 ! 1:4:2, CHCl3–MeOH–25% aq NH3)
gave 23 (25 mg, 73%). White solid: mp 153 ꢁC (dec);
25
½aꢀD +58.7 (c 0.35, H2O); Rf = 0.37 (1:4:2.5, CHCl3–
MeOH–25% aq NH3); IR (ATR): m 3292m, 2937m,
1647w, 1602m, 1497w, 1456w, 1381w, 1330w, 1257w,
1092w, 1013s cmꢁ1 1H NMR (400 MHz, D2O) see
;
Table 6, additionally, d 7.29–7.25 (m, 2 arom. H),
6.85–6.81 (m, 3 arom. H); 13C NMR (100 MHz, D2O)
see Table 7, additionally, d 150.55 (s), 132.36 (2d),
111.53 (d), 116.71 (2d); HRMALDIMS: 714.3340 (100,
17. Destasio, E. A.; Dahlberg, A. E. J. Mol. Biol. 1990, 212,
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18. Vourloumis, D.; Takahashi, M.; Winters, G. C.; Simon-
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19. Knowles, D. J. C.; Foloppe, N.; Matassova, N. B.;
Murchie, A. I. H. Curr. Opin. Pharmacol. 2002, 2, 501–
506.
[M+Na]+, C29H50N6NaO þ; Calcd 714.3334). Anal.
13
Calcd for C39H70N6O18Æ3H2O (1045.04): C, 44.82; H,
7.33; N, 8.04. Found: C, 44.86; H, 6.95; N, 8.24.
20. Haddad, J.; Kotra, L. P.; Llano-Sotelo, B.; Kim, C.;
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