Fully orthogonally protected 2-deoxystreptamine from kanamycin

Article abstract of DOI:10.1021/jo8004414

(Chemical Equation Presented) A fully orthogonally protected and enantiopure 2-deoxy-streptamine derivative is prepared in a few straightforward steps from commercially available kanamycin. Resolution of a sterically hindered diacetate was effected by a Verenium esterase and was followed by a chemoselective Staudinger reduction-acylation protocol.

Full text of DOI:10.1021/jo8004414

SCHEME 1. Synthesis of meso 2-DOS Derivatives from
Kanamycin
Fully Orthogonally Protected 2-Deoxystreptamine
from Kanamycin
M. Waqar Aslam,† Guuske F. Busscher,† David P. Weiner,‡
Rene´ de Gelder,† Floris P. J. T. Rutjes,† and
Floris L. van Delft*,†
Institute for Molecules and Materials, Radboud UniVersity
Nijmegen, ToernooiVeld 1, 6525 ED Nijmegen, The
Netherlands, and Verenium Corporation, 4955 Directors
Place, San Diego, California 92121
F.VanDelft@science.ru.nl
ReceiVed February 25, 2008
A fully orthogonally protected and enantiopure 2-deoxy-
streptamine derivative is prepared in a few straightforward
steps from commercially available kanamycin. Resolution
of a sterically hindered diacetate was effected by a Verenium
esterase and was followed by a chemoselective Staudinger
reduction-acylation protocol.
Consequently, a more practical strategy involves the preparation
of analogues based solely on the central aminohexitol known
as 2-deoxystreptamine (2-DOS). The fundamental role of 2-DOS
in binding to RNA6 makes it a privileged scaffold for develop-
ment of active structural analogues of the natural aminoglycosides.
Despite appreciable synthetic effort,7 the most efficient route
toward 2-DOS remains acidic degradation of neomycin.8
However, meso 2-DOS thus obtained requires multiple protect-
ing group manipulations before incorporation in novel RNA
ligands is ensured. Recently we reported the synthesis of a
versatile enantiopure 2-DOS building block by stepwise deg-
radation of neomycin.9 Herein, we describe a route toward fully
orthogonally protected and enantiopure 2-DOS from kanamycin
A, a member of the 4,6-substituted aminoglycosides.
Because kanamycin is a 2-DOS 4,6-linked aminoglycoside,
it can be conveniently converted into selectively 5-O-protected
2-DOS via global protection followed by hydrolysis of the
glycosidic bond (Scheme 1). Thus, Cu-catalyzed diazotransfer10
on kanamycin A was followed by global O-allylation and
benzylation, to give the fully protected derivatives 1a and 1b,
respectively, in good yields. Next, acidic hydrolysis of glycosidic
bonds of 1a (1 N HCl, MeOH, reflux) gave an anomeric mixture
Since the discovery of streptomycin in 1944,1 aminoglyco-
sides find clinical use due to the bactericidal activity and low
cost. The mode of action involves binding to the A-site of
prokaryotic rRNA, thereby interfering with the fidelity of
translation and ultimately causing bacterial cell death.2 In
addition, a number of aminoglycosides have been found to
display specific interactions with viral RNA.3 More recently,
emergence of resistance and high human toxicity4 has stimulated
the development of new analogues of aminoglycosides devoid
of such limitations.5 The multiple hydroxy and amino functions,
however, thwart a straightforward synthesis of structural
analogues due to inevitable protecting group transformations.
† Radboud University Nijmegen.
‡ Verenium Corporation.
(1) Schatz, A.; Waksman, S. A. Proc. Soc. Exp. Biol. Med. 1944, 55, 244.
(2) (a) Beaucaire, G. J. Chemother. 1995, 7 (suppl 2), 111. (b) Zhao, F.;
Zhao, Q.; Blount, K. F.; Han, Q.; Tor, Y.; Hermann, T. Angew. Chem., Int. Ed.
2005, 44, 447. (c) Magnet, S.; Blanchard, J. S. Chem. ReV. 2005, 105, 477.
(3) (a) Zapp, M. L.; Stern, S.; Green, M. R. Cell 1993, 74, 969. (b) Mei,
H. Y.; Cui, M.; Heldsinger, A.; Lemrow, S. M.; Loo, J. A.; Sannes-Lowery,
K. A.; Sharmeen, L.; Czarnik, A. W. Biochemistry 1998, 37, 1420.
(4) (a) Zembower, T. R.; Noskin, G. A.; Postelnick, M. J.; Nguyen, C.;
Peterson, L. R. Int. J. Antimicrob. Agents 1998, 10, 95. (b) Nagai, J.; Takano,
M. Drug Metab. Pharmacokinet. 2004, 19, 159.
(6) (a) Yoshizawa, S.; Fourmy, D.; Eason, R. G.; Puglisi, J. D. Biochemistry
2002, 41, 6263. (b) Liu, X.; Thomas, J. R.; Hergenrother, P. J. J. Am. Chem.
Soc. 2005, 127, 12434.
(7) Busscher, G. F.; Rutjes, F. P. J. T.; van Delft, F. L. Chem. ReV. 2005,
10, 775.
(8) Georgiadis, M. P.; Constantiou-Kokotou, V.; Kokotos, G. J. Carbohydr.
Chem. 1991, 10, 739.
(5) (a) Rai, R.; Chen, H. N.; Czyryca, P. G.; Li, J.; Chang, C. W. T. Org.
Lett. 2006, 8, 887. (b) Wang, J.; Li, J.; Chen, H. N.; Chang, H.; Tanifum, C. T.;
Liu, H. H.; Czyryca, P. G.; Chang, C. W. T. J. Med. Chem. 2005, 48, 6271. (c)
Ding, Y.; Hofstadler, S. A.; Swayze, E. E.; Risen, L.; Griffey, R. H. Angew.
Chem., Int. Ed. 2003, 42, 3409.
(9) van den Broek, S. A. M. W.; Gruyters, B. W. T.; Rutjes, F. P. J. T.; van
Delft, F. L.; Blaauw, R. H. J. Org. Chem. 2007, 72, 3577.
(10) Nyffeler, P. T.; Liang, C. H.; Koeller, K. M.; Wong, C. H. J. Am. Chem.
Soc. 2002, 124, 10773.
10.1021/jo8004414 CCC: $40.75
Published on Web 06/04/2008
2008 American Chemical Society
J. Org. Chem. 2008, 73, 5131–5134 5131

TABLE 1. Enzymatic Desymmetrization of meso Diacetates 5a,b
entry substrate
enzymea
time conversion yield (%) ee (%)b
1
2
3
4
5
6
7
8
5a
5a
5a
5a
5a
5a
5b
6
Verenium 1
Verenium 2
Verenium 3
Verenium 4
Verenium 5
Verenium 6
Verenium 5
6
6
6
6
2
6
7
73
36
ndc
19
FIGURE 1. Representation of the Crystal Structure of 9.17
95
100
40
92
22
>99
rapidly. We next screened a set of esterases containing the
GGG(A)X-motif,13 specifically selected for the capacity to
hydrolyze sterically hindered substrates, as provided by Vere-
nium Corporation. Out of 17 enzymes, six were able to convert
5a and 5b, although four esterases (entries 1, 2, 4, 6) hydrolyzed
both acetate groups and one esterase (entry 3) gave a mixture
of monoacetate, diacetate, and diol.
Verenium 5 10
a Enzyme, phosphate buffer pH 7.5, MeCN, 37 °C. b Determined by
chiral HPLC. c Mixture of monoacetate, diacetate, and diol.
SCHEME 2. Synthesis of meso Compounds 4a and 4b
To our delight, in a mixture of acetonitrile and phosphate
buffer (pH 7.5, 37 °C) Verenium esterase 5 was able to
hydrolyze a single acetate group of substrate 5a with more than
95% conversion and 92% isolated yield. The resulting monoac-
etate 7a was formed in excellent enantiomeric excess as judged
by HPLC. Reaction of the same enzyme with more sterically
hindered benzylated substrate 5b (entry 7) gave only 22%
isolated yield. In addition, with substrate 6 less than 10%
conversion was detected, further supporting the role of steric
factors. Further attempts to optimize the reaction conditions,
i.e., variation in temperature, pH, or cosolvents, were not
successful. Finally, attempts to perform asymmetric acylation
of 4a by subjection to Verenium esterase 5 in neat vinyl acetate
were fruitless; no conversion was detected. At this point, the
absolute configuration of 7a was established as depicted in Table
1 by comparison of optical rotation with a sample prepared by
a degradative route from D-neamine (see Supporting Informa-
tion).
To arrive at fully orthogonally protected 2-DOS, differentia-
tion between the two amines is required. A chemoselective azide
reduction10,14 was attempted. Thus, subjection of 7a to a one-
pot Staudinger reduction-Boc protection15 afforded compound
8 in 40% yield (Scheme 3).
By the same sequence of events, additional proof of the
absolute configuration of 8 was obtained by X-ray crystal-
lography. To this end, the aza-ylide intermediate resulting from
selective azide reduction was treated with p-BrCbzCl, prepared
by treating p-bromobenzyl alcohol with diphosgene,16 to give
p-BrCbz-protected amine 9 in 27% yield (Scheme 3). Crystal-
lization from MeCN followed by X-ray diffraction gave the
structure depicted in Figure 1.
of methyl glycosides 2a and 3a along with the desired 5-O-
protected 2-DOS 4a. However, separation of 4a from the
unwanted methyl glycosides 2a and 3a was thwarted as a result
of near identical retention times on silica. Fortunately, time-
consuming chromatographic separation could be avoided by
transient trimethylsilylation of the free hydroxyls of 5-O-
protected 4a using hexamethyldisilazane (HMDS) and TMSCl.
After separation, rapid and quantitative acidic hydrolysis of silyl
ethers gave 4a in pure form. Thus, 5-O-allyl-protected 2-DOS
4a was obtained from kanamycin A in five chemical steps in
36% overall yield. A similar sequence of events gave benzylated
compound 4b. Since compounds 4a and 4b are meso, enzymatic
pathways were explored to convert 4a and 4b into building
blocks suitable for the assembly of enantiopure aminoglycoside
analogues. For reasons of comparison, the 5-deoxy derivative
5c was also prepared by a known route.11
A well-established technology for the desymmetrization of
diols involves enzymatic hydrolysis.12 To this end, 4a and 4b
were acetylated to 5a and 5b and an alternative substrate for
enzymatic desymmetrization, compound 6, was obtained by
Staudinger reduction, followed by Cbz-protection and acetyla-
tion. Initially, a number of commercially available esterases and
lipases were investigated, but none of the enzymes demonstrated
hydrolytic activity on 5a or 5b. The lack of reactivity is likely
due to steric hindrance of the 5-O-allyl group, since enzymatic
conversion of the analogous 2,5-dideoxy derivative 5c took place
In conclusion, a fully orthogonally protected and enantiopure
2-DOS derivative was prepared in eight steps and 12% overall
yield from kanamycin. Desymmetrization of the sterically
hindered meso diol by enzymatic resolution was achieved by a
(13) (a) Kourist, R.; Krishna, S. H.; Patel, J. S.; Bartnek, F.; Hitchman, T. S.;
Weiner, D. P.; Bornscheuer, U. T. Org. Biomol. Chem. 2007, 5, 3310.
(14) Li, J.; Chen, H. N.; Chang, H.; Wang, J.; Chang, C. W. T. Org. Lett.
2005, 7, 3061.
(11) Seeberger, P. H.; Baumann, M.; Zhang, G. T.; Kanemitsu, T.; Swayze,
E. E.; Hofstadler, S. A.; Griffey, R. H. Synlett 2003, 9, 1323.
(15) Ariza, X.; Urp´ı, F.; Viladomat, C.; Vilarrasa, J. Tetrahedron Lett. 1998,
39, 9101.
(12) (a) Drauz, K.; Waldmann, H. Enzyme Catalysis in Organic Synthesis:
A ComprehensiVe Handbook; Wiley-VCH: Weinheim, Germany, 2002. (b)
Chenevert, R.; Jacques, F Tetrahedron: Asymmetry 2006, 17, 1017.
(16) Waterfield, W. R. J. Am. Chem. Soc. 1963, 85, 2731.
(17) Spek, A. L. J. Appl. Crystallogr. 2003, 36, 7.
5132 J. Org. Chem. Vol. 73, No. 13, 2008

SCHEME 3. Chemoselective Azide Reduction
1,3-Diazido-1,3-dideamino-5-O-benzyl-2-deoxystreptamine (4b).
Prepared by an analogous procedure with benzyl bromide instead
of allyl bromide to afford pure 4b as a white solid (32 mg, 65%).
Rf 0.11 (EtOAc/heptane, 1:3). Mp 135-136 °C. 1H NMR (CDCl3,
400 MHz) δ 7.49-7.28 (m, 5H), 4.83 (s, 2H), 3.51-3.31 (m, 4H),
3.24 (t, J ) 7.8 Hz, 1H), 2.19 (dt, J ) 13.2, 3.9 Hz, 1H), 1.27 (dt,
J ) 13.2, 12.9 Hz, 1H). 13C NMR (CDCl3, 300 MHz) δ 128.3,
˜
127.8, 127.5, 82.7, 75.0, 59.8, 31.3. HRMS (ESI) m/z calcd for
C13H16N6O3 (M + Na)+: 327.1182, found: 327.1177.
1,3-Diazido-1,3-dideamino-5-O-allyl-4,6-di-O-acetyl-2-deox-
ystreptamine (5a). To a solution of 4a (205 mg) in pyridine (10
mL) was added DMAP (catalytic) and acetic anhydride (1 mL) at
0 °C. The reaction mixture was allowed to warm to room
temperature and stirred for two hours. Solvents were evaporated
under vacuum and residue was purified by column chromatography
(EtOAc/heptane, 1:4) to afford 5a (235 mg, 86%) as a white solid.
Rf 0.28 (EtOAc/heptane, 1:3). 1H NMR (CDCl3, 400 MHz) δ 5.75
(ddt, J ) 17.3, 10.4, 5.7 Hz, 1H), 5.17 (qdd, J ) 1.2, 1.4, 10.3,
13.2, 2H), 5.01 (t, J ) 9.9 Hz, 2H), 4.07 (dt, J ) 1.4, 5.6 Hz, 2H),
3.45 (m, 3H), 2.28 (dt, J ) 13.3, 4.6 Hz, 1H), 2.13 (s, 6H), 1.54
(dt, J ) 13.2, 12.6 Hz, 1H). 13C NMR (CDCl3, 300 MHz) δ 168.9,
133.4, 116.7, 78.5, 74.2, 73.1, 57.8, 31.4, 20.3. HRMS (ESI) m/z
calcd for C13H18N6O5 (M + Na)+ 361.1236, found 361.1250.
1,3-Diazido-1,3-dideamino-5-O-benzyl-4,6-di-O-acetyl-2-deox-
ystreptamine (5b). Prepared by an analogous procedure from 4b
to afford 5b (935 mg, 81%) as a white solid. Rf 0.25 (EtOAc/
heptane, 1:3). 1H NMR (CDCl3, 400 MHz) δ 7.38-7.18 (m, 5H),
5.06 (t, J ) 9.9 Hz, 2H), 4.60 (s, 2H), 3.52-3.39 (m, 3H) 2.29 (dt,
J ) 13.5, 4.1 Hz, 1H), 2.02 (s, 6H), 1.57 (dt, J ) 13.0, 12.1 Hz,
1H). 13C NMR (CDCl3, 300 MHz) δ 168.9, 128.0, 127.4, 127.2,
79.0, 74.3, 57.8, 31.3, 20.2. HRMS (ESI) m/z calcd for C17H20N6O5
(M + Na)+ 411.151, found 411.136.
General Procedure for Enzymatic Resolution. To a solution
of a diacetate 5 or 6 (0.088 mmol) in CH3CN (200 µL) was added
phosphate buffer (pH 7.5, 2 mL) and a Verenium enzyme (20 mg).
The reaction mixture was shaken for 48 h at 37 °C and then filtered
over Celite (flushed with excess of EtOAc). Solvents were
evaporated under vacuum, and the residue was purified by column
chromatography (EtOAc/heptane, 1:8 to 1:5). HPLC-analysis was
performed on a CHIRALCEL OD-H column using racemic
compound as reference (column CHIRALCEL OD-H, hexane/i-
PrOH (9:1), flow rate of 1 mL/min).
Verenium esterase. Finally, chemoselective azide reduction/Boc-
protection led to the first fully orthogonally protected 2-DOS
based building block, suitable for the synthesis of aminogly-
coside type RNA-binding ligands.
Experimental Section
1, 3,6′,3′′-Tetraazido-1,3,6′,3′′-tetradeaminokanamycin. To a
solution of NaN3 (53.5 g, 0.82 mol) in a mixture of H2O/CH2Cl2
(270 mL, 1:1 v/v) at 0 °C was added Tf2O (68.3 mL, 0.41 mol, 1.5
equiv/NH2 based on 50% conversion). The reaction mixture was
stirred at room temperature for 2 h. After quenching with aqueous
NaHCO3, the layers were separated and the aqueous layer was
extracted with CH2Cl2 (200 mL). The organic layers were combined
to afford 335 mL of TfN3 solution. Then, to a solution of kanamycin
(20 g, 34.3 mmol) and CuSO4 (219 mg) in H2O (335 mL) were
added the TfN3 solution, MeOH (1.1 L), and Et3N (43 mL). The
reaction mixture was stirred overnight at room temperature. Then
solid NaHCO3 (35 g) was added carefully, and the organic solvents
were evaporated. The aqueous residue was extracted with EtOAc
(5 × 300 mL), and the organic layers were combined, dried
(Na2SO4), and concentrated in vacuo to give yellow oil. Purification
by column chromatography (pure EtOAc to 10% MeOH/EtOAc)
afforded the tetraazidoderivative of kanamycin (17 g, 84%) as a
(1R,3S,4R,5S,6S)-1-N-Boc-3-azido-3-deamino-5-O-allyl-6-O-
acetyl-2-deoxystreptamine (8). Compound 7a (20 mg, 0.067
mmol) was dissolved in THF (1 mL) and cooled to -78 °C. To
this cooled solution was added 1 M THF solution of PMe3 (67 µL,
0.067 mmol), and the reaction mixture was stirred at -78 °C for 5
min. The reaction was warmed to room temperature and stirred
for 2 h. The reaction was then quenched with Boc-ON (41 mg,
0.167 mmol) and stirred for 4 h. Solvents were then evaporated
under vacuum, and the residue was purified with column chroma-
tography (EtOAc/heptane, 1/3) to afford 8 (10 mg, 40%) as a white
1
colorless oil. Rf 0.44 (MeOH/EtOAc, 1/9). H NMR (MeOD, 400
MHz) δ 5.24(d, J ) 3.8 Hz, 1H), 5.18 (d, J ) 3.8 Hz, 1H),
4.08-3.98 (m, 2H), 3.78-3.28 (m, 15H), 2.33 (dt, J ) 4.2, 12.6
Hz, 1H), 1.56 (q, J ) 12.4 Hz, 1H). 13C NMR (MeOD, 300 MHz)
δ 100.4, 98.0, 83.0, 80.2, 73.8, 72.9, 71.9, 71.5, 70.4, 70.1, 67.9,
66.4, 60.0, 58.8, 50.8, 31.5. HRMS (ESI) m/z calcd for
C18H28N12O11 (M + Na)+ 611.201, found 611.193.
1,3-Diazido-1,3-dideamino-5-O-allyl-2-deoxystreptamine (4a).
Acetyl chloride (1.5 mL) was added to MeOH (15 mL) at 0 °C.
The obtained 1 N HCl/MeOH was added to 1a (200 mg, 0.23
mmol), and the reaction mixture was refluxed overnight. The
reaction was then quenched with NaHCO3, concentrated, and
extracted with EtOAc. The organic layer was washed with water
and brine, then dried (Na2SO4), and concentrated in vacuo. Crude
product was treated with 5 equiv of HMDS and catalytic trimeth-
ylsilyl chloride in acetonitrile. After purification by column
chromatography (EtOAc/heptane, 1:15) the resulting bis-TMS-ether
was treated with 1 N HCl in MeOH to afford pure 4a as a white
solid (36 mg, 61%). Rf 0.14 (EtOAc/heptane, 1/3). Mp 85-87 °C.
1H NMR (CDCl3, 400 MHz) δ 6.01-5.86 (m, 1H), 5.28 (qdd, J )
1.2, 1.4, 10.36, 21.2 Hz, 2H), 4.35 (m, 2H), 4.18 (dt, J ) 1.4, 5.6
Hz, 2H), 3.50-3.34 (m, 4H), 3.15 (t, J ) 8.9 Hz, 1H), 2.20 (dt, J
) 4.6, 12.9 Hz, 1H), 1.38 (dt, J ) 12.6, 13.2 Hz, 1H). 13C NMR
(CDCl3, 300 MHz) δ 134.1, 117.3, 82.5, 75.5, 73.9, 59.8.5, 31.3.
HRMS (ESI) m/z calcd for C9H14N6O3 (M - H)- 253.1049, found
253.1059.
1
solid. Rf 0.17 (EtOAc/heptane, 1:3). Mp 138-139 °C. H NMR
(CDCl3, 300 MHz) δ 5.85 (m, 1H), 5.22 (qdd, J ) 1.25, 1.43, 10.37,
16.44 Hz, 2H), 4.73 (dd, J ) 9.5, 10.6 Hz, 1H), 4.20 (ddd, J )
1.55, 2.97, 5.80 Hz, 2H), 3.41 (m, 3H), 2.7 (d, J ) 2.36 Hz, 1H),
2.26 (dt, J ) 4.20, 12.83 Hz, 1H) 2.09 (s, 3H), 1.41 (s, 9H). 1.28
(m, 1H). 13C NMR (CDCl3, 300 MHz) δ 170.6, 164.7, 133.8. 117.0,
80.8, 79.5, 76.7, 74.8, 73.7, 69.6, 48.8, 33.0, 27.7, 20.0. HRMS
(ESI) m/z calcd for C16H26N4O6 (M + Na)+ 393.191, found 393.177.
[R]20 +1.6 (EtOAc). HRMS (ESI) m/z calcd for C16H26N4O6 (M
D
+ Na)+ 393.1750, found 393.1770.
(1R,3S,4R,5S,6S)-1-N-(4-BrCbz)-3-Azido-3-deamino-5-O-al-
lyl-6-O-acetyl-2-deoxystreptamine (9). Prepared by an analogous
manner from 7a by quenching with p-bromobenzyloxycarbonyl
chloride (17 mg, 0.072 mmol) instead of BOC-ON to afford 9a (9
mg, 27%) as a white crystalline solid. Rf 0.17 (EtOAc/heptane, 1:3).
Mp 127-128 °C. 1H NMR (CDCl3, 300 MHz) δ 7.47 (d, J ) 8.2
Hz, 2H), 7.19 (d, J ) 8.2 Hz, 2H), 5.86 (tdd, J ) 5.6, 10.3, 17.1
J. Org. Chem. Vol. 73, No. 13, 2008 5133

Hz, 1H), 5.21 (qdd, J ) 1.2, 1.4, 10.3, 15.1 Hz, 2H), 5.01 (m, 2H),
4.74 (dd, J ) 9.4, 10.7 Hz, 1H), 4.20 (ddd, J ) 1.4, 2.7, 5.8 Hz,
2H), 3.28-3.57 (m, 3H), 2.65 (d, J ) 1.8 Hz, 1H), 2.30 (dt, J )
3.9, 12.8 Hz, 1H), 1.9 (s, 3H), 1.31 (dd, J ) 12.4, 25.1 Hz, 1H).
13C NMR (CDCl3, 300 MHz) δ 133.7, 131.2, 129.2, 117.0, 80.7,
75.7, 74.5, 73.8, 65.6, 59.4, 49.7, 32.9, 20.3. HRMS (ESI) m/z calcd
for C19H23N4O6Br (M + Na)+ 505.0699, found 505.0691.
The authors thank Flash Bartnek and Jesal Patel (Verenium
Corporation) for preparation and production of enzymes.
Supporting Information Available: General procedures,
synthesis, and characterizations of all new compounds. This
material is available free of charge via the Internet at
http://pubs.acs.org.
Acknowledgment. The Higher Education Commission of
Pakistan (HEC) is acknowledged for financial support (M.W.A.).
JO8004414
5134 J. Org. Chem. Vol. 73, No. 13, 2008