Short and efficient synthesis of alkyne-modified amino glycoside building blocks

Article abstract of DOI:10.1002/ejoc.200900076

In the light of recent progress in RNA biology, the need for molecules that bind to RNA and thus may be suited to manipulating RNA-mediated processes is steadily increasing. We present a very short and efficient synthetic route to alkynemodified neamine a

Full text of DOI:10.1002/ejoc.200900076

FULL PAPER
DOI: 10.1002/ejoc.200900076
Short and Efficient Synthesis of Alkyne-Modified Amino Glycoside Building
Blocks
Claudine M. Klemm,[a] Arne Berthelmann,[a] Saskia Neubacher,[a] and Christoph Arenz*[a]
Keywords: RNA / RNA recognition / Glycosides / Amino glycosides / Click chemistry / Alkynes
In the light of recent progress in RNA biology, the need for
per-catalysed 1,3-dipolar cycloaddition to diazides (“click
molecules that bind to RNA and thus may be suited to manip- chemistry”). The conjugate dimers thus formed inhibited
ulating RNA-mediated processes is steadily increasing. We
present a very short and efficient synthetic route to alkyne-
modified neamine and 2-deoxystreptamine derivatives on a
half-gram scale. These derivatives are suitable for con-
structing a library of potential divalent RNA binders by cop-
Dicer-mediated micro-RNA maturation with IC50 values be-
tween 0.6 and 15 µ
M.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2009)
macular degeneration act by a completely unspecific acti-
vation of the extracellular toll-like receptor TLR3.[16]
Thus, the manipulation of RNA-mediated processes by
small molecules may offer alternative approaches towards
pharmacological applications. In contrast to nucleic acids
and nucleic acid mimics, amino glycosides that target pro-
karyotic ribosomal RNA have been widely used in the treat-
ment of human disease for more than half a century.[17] In
spite of their significant toxicity, amino glycosides play a
leading role in the treatment of enterococcal, mycobacterial
and severe Gram-negative bacterial infections and are the
most commonly used antibiotics worldwide.[18]
Since the discovery of streptomycin by Schatz and Waks-
man,[17] a reasonable effort has been made to expand the
class of naturally occurring amino glycosides by synthetic
derivatives and analogues. Owing to their complex struc-
tures, only a few attempts to synthesize de novo amino
sugars or amino cyclitols have been reported.[19–21] More
recent strategies involve the chemical modification of ex-
isting amino glycoside building blocks.[22] Examples include
conjugation with intercalating residues or the dimerization
of whole amino glycosides[23] or substructures like 2-deoxy-
streptamine (2-DOS)[24,25] and neamine.[26–28] However, ow-
ing to the laborious chemo- and regioselective protection
and deprotection procedures involved in this synthetic strat-
egy, most libraries based on the combinatorial conjugation
of amino glycoside building blocks reported so far barely
exceed 20 members. Such libraries should be far too small
for the identification of selective RNA binders. Moreover,
because RNA structures are very dynamic and difficult to
predict, a rational design of selective small-molecule RNA
binders is not in sight. Hence, the synthesis of libraries of
small molecules targeting RNA is gaining ever increasing
attention. In the course of our efforts to identify RNA
binders that inhibit miRNA maturation,[29,30] we decided to
Introduction
During the last decade, the scientific view of RNA
underwent a dramatic change. Although the discovery of
self-splicing catalytic RNA[1] and the existence of ribo-
switch-mediated regulation of gene expression[2] had already
been widely accepted several years ago, these observations
seemed to be restricted to a few species and thus were
mainly of academic interest. Since the discovery of RNA
interference,[3] and more recently the discovery of micro-
RNAs (miRNAs) in mammals,[4] a whole plethora of RNA-
related phenomena has (re)gained increased scientific atten-
tion. Today RNA is not only a state-of-the-art tool and a
potential drug candidate, for example, siRNAs, antisense
molecules, aptamers[5] and ribozymes,[6] RNA itself is a po-
tential drug target, like ribosomal RNA, mRNA,[7] viral
RNA,[8–10] miRNAs[11–13] and riboswitches.[14] To study the
function of potential RNA drug targets by creating loss-
of-function phenotypes, the use of antisense molecules or
siRNAs that either block or degrade the target RNA is the
most straightforward approach. However, in spite of the
tremendous effort put into the development of therapeutic
oligonucleotides in recent years, there are only a few prod-
ucts on the market to date and no systemically acting oligo-
nucleotide has gained FDA approval so far.[15] Moreover,
very recent results have added new complexity to the use of
RNA as a drug because it has been shown that a number
of the siRNAs that are in clinical trials against age-related
[a] Institute for Chemistry, Humboldt-University of Berlin,
Brook-Taylor-Str. 2, 12489 Berlin, Germany
Fax: +49-30-20936947
E-mail: christoph.arenz@chemie.hu-berlin.de
Supporting information for this article is available on the
WWW under http://www.eurjoc.org/ or from the author.
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Synthesis of Alkyne-Modified Amino glycoside Building Blocks
synthesize a library of neamine and 2-deoxystreptamine (2-
DOS) dimers by the copper-catalyzed 1,3-dipolar cycload-
dition of alkynes to bifunctional azides (Scheme 1).
Scheme 2. Silyl shift as observed for some of the 2-DOS derivatives.
neamine 6, which was subsequently converted into either
the corresponding azide 7a or the Boc-protected amine 7b,
as described previously (Scheme 3).[37] The protected
neamine was treated with an excess of cyclohexanone di-
methyl ketal to give the 3Ј,4Ј:5,6-diketal. The diketal was
then allowed to equilibrate to the more stable 5,6-mono-
ketal 8a or 8b under acidic conditions. For 8a, this detour
resulted in a doubling of the reaction yield compared with
Scheme 1. Alkyne-modified neamine and 2-deoxystreptamine for
dimerization by “click chemistry”.
Results and Discussion
An elegant method to overcome the synthetic limitations the literature.[38]
of extensive protecting group chemistry is the use of chemo-
In the case of the Boc-protected derivative 8b, however,
selective conjugation procedures like the Sharpless–Meldal the yields were considerably lower. Notably, the uncon-
variant of the Huisgen reaction,[31,32] which makes the post- verted 7b was nearly quantitatively recovered upon LC pu-
conjugation steps obsolete. Recently, a library of 105 2- rification. To form the alkyne-modified 2-DOS, in the next
DOS conjugates constructed by this so-called “click chemis- step, the vicinal diol was cleaved by using sodium periodate
try” was reported by Hergenrother and co-workers.[25] and 2-DOS was liberated from the intermediate amino alde-
Interestingly, several members of this library showed a hyde under alkaline conditions to form the protected 2-
highly size-selective binding affinity towards artificial ter- DOS building blocks 9a and 9b. With these compounds in
minal RNA hairpin loops, underscoring the benefits of a hand, we introduced the alkyne moiety by using sodium
powerful synthetic strategy towards amino glycoside mim- hydride and propargyl bromide. To our surprise, we were
ics. Inspired by these results, we wanted to expand this ap- unable to isolate the alkyne 10a after column chromatog-
proach to an analogous conjugation of the more complex raphy. A more detailed investigation revealed that 10a com-
neamine building block. This endeavour was mainly driven pletely underwent an intermolecular cycloaddition reaction
by the idea that the larger interaction surface of the within 12 h at room temperature to form the triazole 11 (see
neamine scaffold relative to that of 2-DOS might lead to the Supporting Information for more details). This cycload-
even more potent and more selective RNA-binding conju- dition occurred in methanolic solution, even when the reac-
gates.
tion vessel was protected from light and after several rounds
Although the neamine and the 2-DOS moiety can be of extraction with EDTA to ensure complete removal of
readily obtained by acidolysis of neomycin B on a copper and other divalent cations. A similar reaction at ele-
multigram scale,[33,34] derivatization essentially includes re- vated temperature has been described previously.[39] How-
gio- and enantio-controlled arrangement of protecting ever, this phenomenon was restricted to the propargyl-sub-
groups. As an example, the esterase-mediated racemic reso- stituted 2-DOS derivative. Longer residues (pentynyl or
lution of peracetylated 2-DOS diazide is a common route hexynyl residues) did not react in this way (data not shown).
to the enantiopure protected 2-DOS building block.[35] Finally, deprotection of the Boc-protected derivative 10b af-
However, this procedure includes the use of expensive en- forded the desired optically active building block 2 in good
zymes and laborious protection and deprotection pro- yield.
cedures. Moreover, in our hands, the optically pure and par-
To synthesize an alkyne-linked enamine, we first envi-
tially deprotected compound 5 underwent racemization sioned a synthesis analogous to the method published by
through the migration of the silyl group under basic depro- Riguet et al.[40] In this approach, the amino groups of
tection conditions (Scheme 2).[36]
neamine were protected with trityl groups, which are re-
As an alternative to the racemic resolution of acetylated movable under very mild conditions. In fact, owing to their
2-DOS we decided to make use of the 2,6-dideoxydiamino- intrinsic lability we had problems with spontaneous cleav-
glucose residue of neamine as a protecting group.[37] Neo- age of the trityl protecting groups during liquid chromatog-
mycin B sulfate was subjected to acid hydrolysis to yield raphy, even when neutral or alkaline aluminium oxide was
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C. M. Klemm, A. Berthelmann, S. Neubacher, C. Arenz
FULL PAPER
Scheme 3. Synthesis of neamine 1 and the 2-DOS derivative 2.
used as the stationary phase. The main difficulties with this of this side-product were observed when 2 equiv. of propar-
methodology, however, arose with the partial protection of gyl bromide were added. This, however, led to only moder-
the hydroxy functions as PMB ethers, which resulted in a ate reaction yields of the desired product 13. After cleavage
mixture of the two- and the desired three-fold protected ne- of the silyl ether and acidic treatment of the intermediate
amine. Instead of the reported equimolar product forma- 14, the neamine 1 was isolated in good yield.
To test whether the newly developed neamine building
block 1 and the 2-DOS derivative 2 are amenable to the
copper-catalyzed 1,3-cycloaddition to diazides, conjugation
was attempted with three different linkers (Scheme 5). In
contrast to the method described by Hergenrother and co-
workers,[25] we obtained the best conjugation yields in a
mixture of DMF and water using a mixture of copper(II)
sulfate and sodium ascorbate in the presence of the
tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA)
ligand.[42]
tion, in the best case we obtained a molar ratio of about
4:1, which makes this approach very ineffective (Scheme 4).
The conjugates were purified in parallel and the pro-
cedure included liquid chromatography on silica gel fol-
lowed by chromatography on RP-18, mainly to remove co-
eluted silica gel. After purification we noticed a massive loss
of substance with the isolated yields varying between 15
and 35%. In contrast to the 2-DOS derivative 2, which was
Scheme 4.
Owing to these difficulties, we decided to rely on the completely converted into the divalent conjugates in all
chemistry established for the synthesis of the 2-DOS deriva- cases according to TLC, the conjugation yields for the
tive 2. We treated the intermediate ketal 8b with 1.2 equiv. neamine building block 1 varied and in some cases the
of TBDMS chloride, which gave the regioisomer 12 in 84% mono-conjugation products were also observed. Possibly,
isolated yield.[41] When 12 was treated with sodium hydride the unprotected amino groups of 1 are able to complex
and 10 equiv. of propargyl bromide, a disubstituted product copper ions, thus poisoning the catalyst. However, because
was formed, which was not subjected to detailed structural we need only milligram amounts of each substance for bio-
characterization (data not shown). Only minimal amounts logical studies, the product loss was favoured over the de-
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Synthesis of Alkyne-Modified Amino glycoside Building Blocks
A. We will report on the biological investigation of such
molecules, including the issue of selectivity between dif-
ferent miRNAs, in due course.
Experimental Section
General: All reagents were purchased in the highest quality avail-
able and were used without further purification. Thin-layer
chromatography was carried out on aluminium plates from Merck
(Kieselgel 60 F254). Flash chromatography was performed on silica
gel 60 from Merck and octadecyl-modified silica gel (end-capped)
1
from Machery & Nagel. H and 13C NMR spectra were recorded
Scheme 5. Synthesis of the neamine and 2-DOS conjugates.
with a Bruker DPX 300 spectrometer. Chemical shifts are reported
in parts per million relative to a residual solvent signal: 1H (CDCl3)
= 7.26 ppm, 13C (CDCl3) = 77.0 ppm; 1H (CD3OD) = 3.31 ppm,
13C (CD3OD) = 49.05 ppm; 1H (D2O) = 4.79 ppm. High-resolution
mass spectra (HRMS) were obtained by electrospray ionization
(ESI) with a Hewlett–Packard GCMS 5995-A spectrometer. Op-
tical rotations were measured with a Perkin–Elmer Polarimeter 241
in a 10 cm cell.
protection of each compound after the coupling step. When
the coupling step was performed on a larger scale, however,
the isolated yields were approximately 75% (see the Exp.
Sect.).
Neamine (6)[34] and compounds 7a,[34] 7b,[37] 8b,[37] and 12[41] were
synthesized as described previously. The conjugates 2A2 and 2B2
have been described before.[25]
Biological Studies
With the idea in mind that a small molecule binding to
a hairpin-shaped precursor miRNA (pre-miRNA) may lead
to inhibition of the Dicer-mediated cleavage and thus the
formation of a mature miRNA, we tested the six conjugates
in a fluorescence-based assay developed in our group.[29] In
a preliminary study, we showed that the promiscuous RNA
binder kanamycin A is able to inhibit the maturation of
the let-7 RNA with an IC50 between 50 and 100 µ.[30] In
contrast, all six 2-DOS and neamine conjugates showed
much better inhibition of let-7 than kanamycin A. In fact,
the IC50 values for the 2-DOS and the neamine conjugates
were in the ranges of 10–20 and 0.5–1.5 µ, respectively
(Table 1).
1,3,2Ј,6Ј-Tetraazido-5,6-O-cyclohexylideneneamine (8a): 1,1-Di-
methoxycyclohexane (2.98 g, 3.15 mL, 20.7 mmol) and p-tolu-
enesulfonic acid monohydrate (cat. amount) were added to a solu-
tion of 1,3,2Ј,6Ј-tetraazidoneamine (7a; 1.47 g, 3.45 mmol) in dry
dimethylformamide (2.5 mL). The reaction was heated at 50 °C and
25 mbar for 5 h in a rotary evaporator and then quenched by the
addition of triethylamine (1 mL). After evaporation the yellow oil
was dissolved in chloroform (30 mL) and washed consecutively
with water (10 mL) and sat. NaHCO3 (10 mL). The organic layer
was dried with magnesium sulfate and the solvent was removed in
vacuo. Finally, the 1,3,2Ј,6Ј-tetraazido-3Ј,4Ј,5,6-di-O-cyclohexylid-
eneneamine [Rf = 0.79 (50% ethyl acetate in cyclohexane)] was fully
converted into the desired 1,3,2Ј,6Ј-tetraazido-5,6-O-cyclohexylid-
eneneamine by the following procedure: dry dimethylformamide
(25 mL), p-toluenesulfonic acid monohydrate (cat. amount) and
dry methanol (0.7 mL) were added to 1,3,2Ј,6Ј-tetraazido-3Ј,4Ј,5,6-
di-O-cyclohexylideneneamine. This solution was heated for 8 h in
a rotary evaporator (50 °C, 25 mbar) and quenched with triethyl-
amine (1 mL). After evaporation, the crude product was purified
by chromatography on silica gel (25Ǟ33% ethyl acetate in cyclo-
hexane) to yield 8a (1.49 g, 2.94 mmol, 85%) as a colourless oil. Rf
= 0.37 (50% ethyl acetate in cyclohexane). [α]2D2 = +93.7 (c = 1.0,
chloroform). 1H NMR (300 MHz, CDCl3): δ = 1.36–1.55 (m, 3 H,
2eq-CH2, CH2), 1.57–1.70 (m, 8 H, CH2), 2.32 (td, J = 5.0, 13.3 Hz,
1 H, 2ax-CH2), 3.25 (dd, J = 3.6, 10.5 Hz, 1 H, 2Ј-CH2), 3.42 (m,
1 H, 6-CH), 3.47–3.61 (m, 5 H, 3-, 4-, 5Ј-CH, 6Ј-CH2), 3.62–3.69
(m, 1 H, 1-CH), 3.79–3.85 (m, 1 H, 5-CH), 3.90–4.02 (m, 2 H, 3Ј-,
4Ј-CH), 5.48 (d, J = 3.6 Hz, 1 H, 1Ј-CH) ppm. 13C NMR (75 MHz,
CD3CN): δ = 2ϫ23.6 and 24.7 (CH2), 33.6 (2-CH2), 35.9 and 36.1
(CH2), 51.0 (6Ј-CH2), 57.0 (1-CH), 60.7 (3-CH), 62.4 (2Ј-CH), 70.9,
71.0 and 71.2 (3Ј-, 4Ј-, 5Ј-CH), 76.9 (5-CH), 79.1 and 79.2 (4-, 6-
CH), 96.1 (1Ј-CH), 113.5 (Cq) ppm. HRMS: calcd. for
C18H30N13O6 [M + NH4]+ 524.2442; found 524.2454.
Table 1. Inhibition of miRNA maturation by the click conjugates.
Conjugate
Inhibition of miRNA maturation: IC50 [µ]
1A1
1B1
1C1
2A2
2B2
2C2
1.31Ϯ0.02
0.63Ϯ0.04
1.10Ϯ0.06
15.1Ϯ0.37
11.1Ϯ0.11
14.7Ϯ3.09
Conclusion
In this study we have developed a short, straightforward
and cost-effective synthesis of alkyne-conjugated neamine
and 2-DOS. These compounds were easily synthesized in
half-gram amounts. These compounds were subjected to
copper-catalyzed 1,3-dipolar cycloaddition with aromatic
and aliphatic dazides to yield conjugate dimers. Thus, we
have developed a method for the synthesis of a small library
of 2-DOS and neamine dimers. The binding affinity of these
dimers to RNA was then tested and the inhibition values
for Dicer-mediated let-7 maturation were in most cases one-
1,3-Diazido-5,6-O-cyclohexylidene-2-deoxystreptamine (9a): A solu-
tion of 1,3,2Ј,6Ј-tetraazido-5,6-O-cyclohexylideneneamine (2.67 g,
5.28 mmol) in methanol (190 mL) was cooled to 0 °C. After ad-
dition of NaIO4 (8.47 g, 39.6 mmol), the reaction mixture was
to-two orders of magnitude better than that of kanamycin stirred for 15 h at room temperature. The formation of the desired
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C. M. Klemm, A. Berthelmann, S. Neubacher, C. Arenz
FULL PAPER
dialdehyde was monitored by TLC [Rf = 0.74 (50% ethyl acetate
in cyclohexane)]. The precipitate was filtered through Celite and
the solution was concentrated in vacuo. The residue was dissolved
in ethyl acetate (260 mL) and washed with water (260 mL) and sat.
NaHCO3 (290 mL). The organic layer was dried with Na2SO4 and
the solvents were evaporated. The solid was then dissolved in meth-
anol (260 mL) and n-butylamine (1.15 g, 1.56 mL, 15.8 mmol) was
added dropwise. After 2 h stirring at room temperature n-bu-
tylamine (0.74 g, 1 mL, 10.1 mmol) was added again and the mix-
ture was stirred for an additional 24 h. The crude product was puri-
fied by chromatography on silica gel (0Ǟ5% ethyl acetate in cyclo-
hexane) to yield 9a (1.30 g, 4.40 mmol, 83%) as yellow crystals. Rf
= 0.43 (30% ethyl acetate in cyclohexane). [α]2D2 = +13.3 (c = 1.0,
chloroform). 1H NMR (300 MHz, CDCl3): δ = 1.38–1.47 (m, 3 H,
2eq-CH2, CH2), 1.61–1.69 (m, 8 H, CH2), 2.33 (td, J = 4.9, 13.6 Hz,
1 H, 2ax-CH2), 3.05 (br., 1 H, OH), 3.36–3.47 (m, 3 H, 4-, 5-, 6-
CH), 3.61–3.80 (m, 2 H, 1-, 3-CH) ppm. 13C NMR (75 MHz,
CDCl3): δ = 23.6, 23.7 and 24.9 (CH2), 33.7 (2-CH2), 36.2 and 36.2
(CH2), 57.4 (1-CH), 62.4 (3-CH), 74.2 (4-CH), 79.1 and 79.2 (5-,
6-CH), 113.6 (Cq) ppm. HRMS: calcd. for C12H18N6O3 [M + H]+
295.1513; found 295.1518.
ganic layers were dried with Na2SO4 and the solvents evaporated.
Purification by chromatography on silica gel (0Ǟ2% methanol in
chloroform) provided 10b as a colourless solid (1.11 g, 2.31 mmol,
72 %). Rf = 0.34 (30 % ethyl acetate in cyclohexane). 1H NMR
(300 MHz, CD3OD): δ = 1.44 (m, 21 H, CH2, CH3, 2eq-CH2), 1.65
(m, 8 H, CH2), 2.40–2.58 (td, J = 4.6, 13.2 Hz, 1 H, 2ax-CH2), 2.81
(t, J = 2.4 Hz, 1 H, ϵCH), 3.38–3.71 (m, 5 H, 1-, 3-, 4-, 5-, 6-CH),
4.37 (dq, J = 2.4, 15.6 Hz, 2 H, O-CH2) ppm. 13C NMR (75 MHz,
CD3OD): δ = 2ϫ24.9 and 26.2 (CH2), 3ϫ28.8 and 3ϫ28.8 (CH3),
2ϫ37.3 and 37.5 (2-CH2, CH2), 50.1 and 52.8 (1-, 3-CH), 59.1 (O-
CH2), 2ϫ75.7 [Cq(CH3)3], 79.8, 80.3, 80.6, 81.0 and 82.3 (4-, 5-, 6-
CH and CϵCH), 112.3 (Cq), 157.7 and 158.0 [Cq(O)] ppm. HRMS:
calcd. for C25H41N2O7 [M + H]+ 481.2908; found 481.2914.
Pentacyclic Internal Cycloaddition Product 11: 1,3-Diazido-5,6-O-
cyclohexylidene-2-deoxystreptamine (0.20 g, 0.68 mmol) and a
small portion of tetrabutylammonium iodide were dissolved in dry
toluene (1 mL) in a dry Schlenk flask. Then NaH (0.14 g,
3.40 mmol, 60% in mineral oil) and after 15 min propargyl bromide
(0.11 g, 0.08 mL, 0.75 mmol, 80% in toluene) were added over a
period of 5 min. After 24 h the reaction was quenched by the ad-
dition of water (1 mL). Subsequently the solution was poured into
a mixture of diethyl ether (10 mL) and water (10 mL) and extracted
three times with diethyl ether (3ϫ5 mL). The collected organic
phases were washed with brine (5 mL) and dried with magnesium
sulfate. After removal of the solvent, the residue was purified by
chromatography on silica gel (0Ǟ5% ethyl acetate in cyclohexane)
to give 11 (0.22 g, 0.67 mmol, 99%) as a colourless solid. Rf = 0.44
(30% ethyl acetate in cyclohexane) and 0.02 after 16 h in CD3OD.
1H NMR (300 MHz, CD3OD): δ = 1.40–1.50 (m, 2 H, CH2), 1.60–
1.95 (m, 9 H, CH2, 2eq-CH2), 3.19 (td, J = 4.6, 13.1 Hz, 1 H, 2ax-
CH2), 3.69 (dd, J = 8.8, 10.1 Hz, 1 H, 6-CH), 3.82 (t, J = 9.2 Hz,
1 H, 5-CH), 3.94 (t, J = 9.3 Hz, 1 H, 4-CH), 4.03–4.12 (m, 1 H, 5-
CH), 4.22–4.34 (m, 1 H, 1-CH), 4.98 (td, J = 0.9, 15.5 Hz, 1 H, O-
CHH), 5.21 (d, J = 15.5 Hz, 1 H, O-CHH), 7.54 (s 1 H,
Cq=CH) ppm. 13C NMR (75 MHz, CD3OD): δ = 2ϫ24.7 and 26.0
(CH2), 31.6 (2-CH2), 37.1 and 37.2 (CH2), 58.2 and 58.9 (1-, 3-
CH), 63.4 (O-CH2), 77.5 (4-CH), 78.7 (5-CH), 81.6 (6-CH), 114.3
(Cq), 129.5 and 132.7 (Cq=CH) ppm. HRMS (ESI): calcd. for
1,3-Bis-N-(tert-butyloxycarbonyl)-5,6-O-cyclohexylidene-2-deoxy-
streptamine (9b): NaIO4 (18.8 g, 88.5 mmol) was added to an ice-
cold solution of 8b (9.44 g, 11.8 mmol) in tetrahydrofuran/water
(1:1, 200 mL). The solution was stirred overnight at room tempera-
ture. The white precipitate was filtered through Celite and the fil-
trate was concentrated to dryness in vacuo. The white residue was
dissolved in ethyl acetate (100 mL) and the resulting solution was
washed with water (200 mL) and brine (200 mL). The organic layer
was dried (Na2SO4) and the solvent was removed under reduced
pressure. The residue was dissolved in methanol (275 mL) and n-
butylamine (2.59 g, 35.4 mmol) was added slowly. After stirring
overnight at room temperature the solvent was removed under re-
duced pressure. The residue was purified by flash chromatography
(30 Ǟ 50 % ethyl acetate in cyclohexane) to yield 9b (2.49 g,
5.54 mmol, 47%) as a white solid. Rf = 0.18 (40% ethyl acetate in
cyclohexane). [α]2D2 = –23.0 (c = 1.0, chloroform). 1H NMR
(300 MHz, CDCl3): δ = 1.37 (m, 21 H, CH2, CH3, 2eq-CH2), 1.52–
1.64 (m, 8 H, CH2), 1.92 (br., 1 H, OH), 2.40–2.58 (m, 1 H, 2ax-
CH2), 3.31–3.37, 4.41–3.48, 3.51–3.57 and 3.63–3.72 (m, 5 H, 1-,
3-, 4-, 5-, 6-CH), 4.74 (d, J = 5.9 Hz, 2 H, NH) ppm. 13C NMR
(75 MHz, CDCl3): δ = 23.6, 23.6 and 2 ϫ 24.9 (CH2), 6 ϫ 28.3
(CH3), 36.1 and 36.3 (2-CH2, CH2), 49.2 (1-CH), 53.4 (3-CH), 74.1
(4-CH), 77.9 (5-CH), 79.8 and 80.4 [Cq(CH3)3], 80.7 (6-CH), 112.6
(Cq), 155.3 and 156.5 [Cq(O)] ppm. HRMS: calcd. for C22H39N2O7
[M + H]+ 443.2752; found 443.2754; calcd. for C22H42N3O7 [M +
NH4]+ 460.3017; found 460.3021; calcd. for C22H38N2NaO7 [M +
Na]+ 465.2571; found 465.2578.
C
15H21N6O3 [M + H]+ 333.1670; found 333.1669; calcd. for
C15H20N6NaO3 [M + Na]+ 355.1489; found 355.1489.
4-O-Propargyl-2-deoxystreptamine (2): The fully protected alkyne
10b (0.72 g, 1.52 mmol) was dissolved in dichloromethane (8 mL)
and trifluoroacetic acid (4 mL) was added. After 30 min the mix-
ture was concentrated in vacuo and purified by chromatography on
silica gel [0Ǟ10% NH4OH (28–30%) in methanol] followed by a
purification with reversed-phase chromatography to remove eluted
silica gel. Yield: 0.55 g, 1.29 mmol, 85%, colourless solid. Rf = 0.38
[10% NH4OH (28–30%) in methanol]. [α]2D2 = +31.5 (c = 1.0,
1,3-Bis-N-(tert-butyloxycarbonyl)-5,6-O-cyclohexylidene-4-O-pro-
pargyl-2-deoxystreptamine (10b): Streptamine derivative 9b (1.43 g,
3.23 mmol) was dissolved in dry tetrahydrofuran (45 mL) and co-
oled to 0 °C and then NaH (0.26 g, 6.47 mmol, 60% in mineral
oil) was added. After stirring for 1 h at 0 °C a catalytic amount of
tetrabutylammonium iodide (TBAI) and a solution of propargyl
bromide (0.96 g, 6.47 mmol, 80 % in toluene) in dry tetra-
hydrofuran (5 mL) were added. The ice-bath was removed and the
reaction was stirred for 3 d at room temperature. The addition of
TBAI was repeated every 24 h. Water (5 mL) was used to quench
the reaction, which was then poured into a mixture of water
(30 mL) and diethyl ether (60 mL). The aqueous layer was ex-
tracted with diethyl ether (2ϫ50 mL) and the organic layer was
washed with sat. NaHCO3 (30 mL). All the aqueous layers were
re-extracted with diethyl ether (2ϫ50 mL) and the combined or-
1
water). H NMR (300 MHz, CD3OD): δ = 1.84 (q, J = 12.3 Hz, 1
H, 2eq-CH2), 2.43 (m, 1 H, 2ax-CH2), 2.99 (t, J = 2.3 Hz, 1 H,
ϵCH), 3.16–3.28 (m, 2 H, 1-, 3-CH), 3.39–3.57 (m, 3 H, 4-, 5-, 6-
CH), 4.60 (m, 2 H, O-CH2) ppm. 13C NMR (75 MHz, CD3OD): δ
= 30.0 (2-CH2), 50.7 and 51.6 (1-, 3-CH), 60.6 (O-CH2), 74.4 (5-
CH), 77.1 (ϵCH), 77.7 and 80.1 (4-, 6-CH), 80.7 (Cqϵ) ppm.
HRMS: calcd. for C9H17N2O3 [M + H]+ 201.1239; found 201.1234.
1,3,2Ј,6Ј-Tetrakis-N-tert-(butyloxycarbonyl)-5,6-O-cyclohexylidene-
3Ј-O-(tert-butyldimethylsilyl)-4Ј-O-propargylneamine (13): NaH
(0.09 g, 2.18 mmol, 60% suspension in mineral oil) was added to
an ice-cold solution of 12 (1.00 g, 1.09 mmol) in anhydrous tetra-
hydrofuran (15 mL) under argon. After stirring for 1 h at room
temperature, a small amount of tetrabutylammonium iodide and,
within 5 min, propargyl bromide (0.32 g, 2.18 mmol) were added.
2792
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© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2009, 2788–2794

Synthesis of Alkyne-Modified Amino glycoside Building Blocks
After stirring the solution for 2 d at room temperature, the reaction H]+ 361.2082; found 361.2080; calcd. for C15H30N4O6 [M + 2H]2+
was quenched by the addition of methanol (1 mL). The solution 181.1077; found 181.1076.
was poured into a mixture of water (30 mL) and diethyl ether
General Procedure for the Synthesis of the Conjugates: TBTA
(30 mL). The aqueous layer was separated and extracted with di-
(15 mol-%, 0.09  solution in DMF) was added to a solution of the
ethyl ether (2ϫ30 mL). The organic layer was back-extracted with
alkyne-substituted 2-deoxystreptamine (2) or neamine (1) (33 µmol,
brine (30 mL), dried (Na2SO4) and concentrated to dryness under
0.05  in DMF) and the corresponding diazide (15 µmol). After
reduced pressure. The yellow residue was purified by flash
degassing, a freshly prepared sodium ascorbate solution (30 mol-
chromatography (20Ǟ40% ethyl acetate in cyclohexane) to yield
%, 1  in water) was added followed by 15 mol-% of a CuII sulfate
13 (0.53 g, 0.56 mmol, 51%) as a white foam. Rf = 0.50 (33% ethyl
solution (0.35  in water) and the mixture was shaken for 10 d at
1
acetate in cyclohexane). H NMR (300 MHz, CDCl3): δ = 0.03 [s,
50 °C. Then the solution was evaporated to a minimum and the
6 H, Si(CH3)2], 0.81 [s, 9 H, SiCq(CH3)3], 1.38–1.59 (m, 47 H, CH2,
residue was purified by column chromatography on silica gel using
CH3, 2eq-CH2), 2.18 (m, 1 H, CϵCH), 2.48–2.52 (m, 1 H, 2ax-
a gradient from 0 to 15% (for 2-DOS derivatives) or 0 to 30% (for
CH2), 3.44–4.19 (m, 13 H, 1-, 3-, 4-, 5-, 6-, 2Ј-, 3Ј-, 4Ј-, 5Ј-CH, 6Ј-
neamine derivatives) aqueous ammonium hydroxide solution (28–
CH2, O-CH2), 4.65–4.98 (m, 5 H, NH, 1Ј-CH) ppm. 13C NMR
30%) in methanol. To separate any silica gel eluted, the residue
(75 MHz, CDCl3): δ = –5.1 and –4.1 [Si(CH3)2], 18.2 [SiCq-
obtained was dissolved in water (2 mL) and purified again through
(CH3)3], 23.7, 24.9 and 26.8 (CH2), 3ϫ25.8 [SiCq(CH3)3], 28.1–
a reversed-phase column. The isolated substances were pure by
28.4 (12ϫCH3), 2ϫ36.0 and 36.2 (2-CH2, CH2), 38.5 (6Ј-CH2),
NMR and HPLC–MS or UPLC–MS.
46.7 (O-CH2), 49.0, 53.9 and 55.0 (1-, 3-, 2Ј-CH), 71.0, 71.4, 71.5,
When the reactions were carried out on a larger scale (165 µ of
alkyne in a 0.15  DMF stock solution), the isolated yields were
all around 75%, respectively.
72.1 and 72.9 (4-, 5-, 3Ј-, 4Ј-, 5Ј-CH), 78.4, 79.0, 80.0 and 80.8
(CqCH3), 79.8 (CϵCH), 80.3 (6-CH), 81.4 (CϵCH), 99.2 (1Ј-CH),
112.4 (Cq), 2ϫ154.9, 155.1 and 155.4 [Cq(O)] ppm. HRMS: calcd.
for C47H83N4O14Si [M + H]+ 955.5670; found 955.5670; calcd. for
C47H86N5O14Si [M + NH4]+ 972.5935; found 972.5932; calcd. for
C47H82N4NaO14Si [M + Na]+ 977.5489; found 977.5484.
Conjugate 1A1: 1H NMR (300 MHz, D2O): δ = 1.86 (q, J =
12.6 Hz, 2 H, 2eq-CH2), 2.41 (dt, J = 12.4, 4.1 Hz, 2 H, 2ax-CH2),
3.24–3.68 (m, 16 H, 1-, 3-, 4-, 5-, 6-, 4Ј-CH, 6Ј-CH2), 3.91–4.09 (m,
4Ј-O-Propargylneamine (1): Tetrabutylammonium fluoride trihydr- 6 H, 2Ј-, 3Ј-, 5Ј-CH), 4.54 (s, 4 H, O-CH2), 5.91 (d, J = 3.8 Hz, 2
ate (TBAF; 0.97 g, 3.08 mmol) was added to a solution of 13 H, 1Ј-CH), 7.98–8.01 and 8.13–8.16 (m, 8 H, CHar), 8.71 (s, 2 H,
(0.67 g, 0.7 mmol) in dry tetrahydrofuran (15 mL) and the solution Cq=CH) ppm. 13C NMR (75 MHz, D2O): δ = 28.1 (2-CH2), 41.9
was stirred for 20 h at room temperature. The ice-cold solution was (O-CH2), 47.7 (6Ј-CH2), 48.3 and 49.6 (1-, 3-CH), 53.4, 68.0, 68.9,
quenched with water (10 mL), extracted with DCM (3ϫ40 mL) 70.9, 72.4, 75.1 and 77.3 (4-, 5-, 6-, 2Ј-, 3Ј-, 4Ј-, 5Ј-CH), 95.7 (1Ј-
and dried (Na2SO4). The solvent was removed under reduced pres- CH2), 2ϫ121.7 (CHar), 125.0 (Cq=CH), 2ϫ129.6 (CHar), 138.7,
sure and the resulting residue was purified by flash chromatography 139.7 and 140.0 (2ϫCq-ar, Cq=CH) ppm. HRMS: calcd. for
(33Ǟ50% ethyl acetate in cyclohexane) to yield the desilylated in- C42H65N14O14S [M + H]+ 1021.4520; found 1021.4542.
termediate (0.47 g, 0.56 mmol, 81%) as a white solid. Rf = 0.29
Conjugate 1B1: 1H NMR (300 MHz, D2O): δ = 1.86 (q, J =
1
(50% ethyl acetate in cyclohexane). H NMR (300 MHz, CDCl3):
12.6 Hz, 2 H, 2eq-CH2), 2.46 (dt, J = 12.4, 4.1 Hz, 2 H, 2ax-CH2),
δ = 1.38–1.57 (m, 47 H, CH2, CH3, 2eq-CH2), 2.18 (t, J = 2.21 Hz,
3.25–3.68 (m, 16 H, 1-, 3-, 4-, 5-, 6-, 4Ј-CH, 6Ј-CH2), 3.93–4.10 (m,
1 H, CϵCH), 2.38–2.44 (m, 1 H, 2ax-CH2), 3.22–4.35 (m, 13 H, 1-,
6 H, 2Ј-, 3Ј-, 5Ј-CH), 4.55 (s, 4 H, O-CH2), 5.93 (br., 2 H, 1Ј-
3-, 4-, 5-, 6-, 2Ј-, 3Ј-, 4Ј-, 5Ј-CH, 6Ј-CH2, O-CH2), 4.84–5.17 (m,
CH), 7.78–7.83, 8.06–8.12 and 8.42 (m, 8 H, CHar), 8.71 (s, 2 H,
5 H, NH, 1Ј-CH) ppm. 13C NMR (75 MHz, CDCl3): δ = 23.5, 23.6
Cq=CH) ppm. 13C NMR (75 MHz, D2O): δ = 28.1 (2-CH2), 42.0
and 24.8 (CH2), 28.1–28.3 (12ϫCH3), 2ϫ35.9 and 36.2 (2-CH2,
(O-CH2), 47.8 (6Ј-CH2), 48.4 and 49.6 (1-, 3-CH), 53.4, 68.0, 68.1,
CH2), 38.7 (6Ј-CH2), 47.3 (O-CH2), 48.9, 50.9 and 54.6 (1-, 3-, 2Ј-
70.9, 72.4, 75.1 and 77.3 (4-, 5-, 6-, 2Ј-, 3Ј-, 4Ј-, 5Ј-CH), 95.8 (1Ј-
CH), 71.0, 71.8, 71.9, 72.2 and 72.3 (4-, 5-, 3Ј-, 4Ј-, 5Ј-CH), 79.4,
CH2), 119.9 (CHar), 125.0 (Cq=CH), 126.6, 128.5 and 131.8 (CHar),
79.6, 79.8 and 79.9 (CqCH3), 80.2 (CϵCH), 80.4 (6-CH), 81.4
136.9, 138.6 and 140.9 (2ϫCq-ar, Cq=CH) ppm. HRMS: calcd. for
(CϵCH), 98.5 (1Ј-CH), 112.4 (Cq), 155.0, 155.1, 156.5 and 156.8
C42H65N14O14S [M + H]+ 1021.4520; found 1021.4528.
[Cq(O)] ppm. HRMS: calcd. for C41H69N4O14 [M
+
Conjugate 1C1: 1H NMR (300 MHz, D2O): δ = 1.90 (q, J =
12.6 Hz, 2 H, 2eq-CH2), 2.47 (dt, J = 12.4, 4.1 Hz, 2 H, 2ax-CH2),
2.84 (br., 4 H, CH2), 3.26–3.70 (m, 16 H, 1-, 3-, 4-, 5-, 6-, 4Ј-CH,
6Ј-CH2), 3.94–4.09 (m, 6 H, 2Ј-, 3Ј-, 5Ј-CH), 4.54 (s, 4 H, O-CH2),
5.94 (d, J = 3.8 Hz, 2 H, 1Ј-CH), 7.13–7.16 and 7.28–7.32 (m, 8 H,
CHar), 8.24 (s, 2 H, Cq=CH) ppm. 13C NMR (75 MHz, D2O): δ =
28.1 (2-CH2), 36.1 (CH2), 42.0 (O-CH2), 47.7 (6Ј-CH2), 48.4 and
49.6 (1-, 3-CH), 52.4 [CH2Cq(O)], 53.4, 68.0, 68.9, 71.0, 72.4, 75.1
and 77.2 (4-, 5-, 6-, 2Ј-, 3Ј-, 4Ј-, 5Ј-CH), 95.7 (1Ј-CH2), 2ϫ118.2
(CHar), 127.8 (Cq=CH), 2ϫ129.2 (CHar), 134.0, 137.7 and 139.5
H]+ 841.4805; found 841.4805; calcd. for C41H72N5O14 [M +
NH4]+ 858.5070; found 858.5076; calcd. for C41H68N4NaO14 [M +
Na]+ 863.4624; found 863.4629.
This white solid (0.22 g, 0.26 mmol) was stirred in DCM (1.5 mL),
water (0.7 mL) and trifluoroacetic acid (1.0 mL) for 2 h at room
temperature. The solution was concentrated to dryness and the resi-
due was purified by flash chromatography [0Ǟ20% NH4OH (28–
30%) in methanol]. To separate any silica gel eluted, the residue
obtained was purified again by flash chromatography on a reversed
phase to yield 1 (0.19 g, 0.23 mmol, 88%) as a white solid. Rf =
(2ϫCq-ar, Cq=CH), 165.8 [Cq(O)] ppm. HRMS: calcd. for
1
0.17 [10% NH4OH (28–30%) in methanol]. H NMR (300 MHz,
C48H75N16O14 [M + H]+ 1099.5643; found 1099.5676.
D2O): δ = 1.82–1.95 (q, J = 12.5 Hz, 1 H, 2eq-CH2), 2.46–2.50 (td,
J = 3.8, 12.4 Hz, 1 H, 2ax-CH2), 3.00 (t, J = 2.5 Hz, 1 H, CϵCH), Conjugate 2A2: 1H NMR (300 MHz, D2O): δ = 1.81 (q, J =
3.17–3.66 and 3.87–4.10 (m, 13 H, 1-, 3-, 4-, 5-, 6-, 2Ј-, 3Ј-, 4Ј-, 5Ј- 12.5 Hz, 2 H, 2eq-CH2), 2.43 (dt, J = 4.2, 12.5 Hz, 2 H, 2ax-CH2),
CH, 6Ј-CH2, O-CH2), 5.94 (d, J = 3.7 Hz, 1 H, 1Ј-CH) ppm. 13C 3.24–3.43 (m, 4 H, 1-, 3-CH), 3.51–3.66 (m, 6 H, 4-, 5-, 6-CH),
NMR (75 MHz, D2O): δ = 29.2 (2-CH2), 37.0 (6Ј-CH2), 47.4 (O- 4.95 (d, J = 12.3 Hz, 2 H, O-CHH), 5.14 (d, J = 12.3 Hz, 2 H, O-
CH2), 48.4, 48.8 and 53.6 (1-, 3-, 2Ј-CH), 68.8, 69.5, 71.0, 72.6, CHH), 7.91 (d, J = 8.8 Hz, 4 H, CHar), 8.06 (d, J = 8.8 Hz, 4 H,
75.0, 75.2 and 78.9 (4-, 5-, 6-, 3Ј-, 4Ј-, 5Ј-CH, CϵCH), 76.6 CHar), 8.53 (s, 2 H, Cq=CH) ppm. 13C NMR (75 MHz, D2O): δ =
(CϵCH), 97.0 (1Ј-CH) ppm. HRMS: calcd. for C15H29N4O6 [M + 28.1 (2-CH2), 49.0 and 49.8 (1-, 3-CH), 64.6 (O-CH2), 72.4, 75.4
Eur. J. Org. Chem. 2009, 2788–2794
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
2793

C. M. Klemm, A. Berthelmann, S. Neubacher, C. Arenz
FULL PAPER
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calcd. for C30H41N10O8S [M + H]+ 701.2824; found 701.2820.
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Conjugate 2B2: 1H NMR (300 MHz, D2O): δ = 1.82 (q, J =
12.5 Hz, 2 H, 2eq-CH2), 2.43 (dt, J = 4.1, 12.2 Hz, 2 H, 2ax-CH2),
3.25–3.43 (m, 4 H, 1-, 3-CH), 3.52–3.69 (m, 6 H, 4-, 5-, 6-CH),
4.92–4.97 (m, 2 H, O-CHH), 5.12–5.16 (m, 2 H, O-CHH), 7.65–
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calcd. for C30H41N10O8S [M + H]+ 701.2824; found 701.2823.
Conjugate 2C2: 1H NMR (300 MHz, D2O): δ = 1.79 (q, J =
12.5 Hz, 2 H, 2eq-CH2), 2.41 (dt, J = 4.3, 12.5 Hz, 2 H, 2ax-CH2),
2.78 (br., 4 H, CH2), 3.22–3.34 (m, 4 H, 1-, 3-CH), 3.50–3.64 (m,
6 H, 4-, 5-, 6-CH), 4.88–4.92 (m, 2 H, O-CHH), 5.07–5.11 (m, 2
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8 H, CHar), 8.06 (s, 2 H, Cq=CH) ppm. 13C NMR (75 MHz, D2O):
δ = 28.1 (2-CH2), 36.1 (CH2), 49.0 and 49.8 (1-, 3-CH), 52.4
[CH2Cq(O)], 64.6 (O-CH2), 72.4, 75.4 and 79.7 (4-, 5-, 6-CH),
2ϫ121.4 (CHar), 126.6 (Cq=CH), 2ϫ129.1 (CHar), 134.0, 139.4
and 143.8 (2ϫCq-ar, Cq=CH), 165.9 [Cq(O)] ppm. HRMS: calcd.
for C36H51N12O8 [M + H]+ 779.3947; found 779.3964.
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Supporting Information (see also the footnote on the first page of
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The authors gratefully acknowledge funding by the Deutsche For-
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