Synthesis of a phosphonate-linked aminoglycoside-coenzyme a bisubstrate and use in mechanistic studies of an enzyme involved in aminoglycoside resistance

Article abstract of DOI:10.1002/chem.200802172

Aminoglycoside N-6′-acetyltransferases (AAC(6′)s) are important determinants of antibiotic resistance. A good mechanistic understanding of these enzymes is essential to overcome aminoglycoside resistance. We have previously reported the synthesis of amide- and sulfonamide-linked aminoglycoside-coenzyme A conjugates, which were useful mechanistic and structural probes of AAC(6′)s. We report here the synthesis of a phosphonate-linked aminoglycoside-coenzyme A variant, which is expected to be a superior mimic of the tetrahedral intermediate proposed for catalysis by AAC(6′)s. This synthetic target is especially challenging for a number of reasons, including the presence of multiple functional groups, the water solubility of both starting materials, and incompatibility of P^III chemistry with water. We have overcome these challenges by adding the expensive coenzyme A in the last step by means of an elegant Michael-type addition onto a vinylphosphonate in water. Overall, a single protection step was needed. The decreased inhibitory potency of this bisubstrate compared to that of the amide-linked analogue suggests that Enterococcus faecium AAC(6′)-Ii may not stabilize the proposed tetrahedral intermediate, and may act mainly through proximity catalysis.

Full text of DOI:10.1002/chem.200802172

DOI: 10.1002/chem.200802172
Synthesis of a Phosphonate-Linked Aminoglycoside–Coenzyme A
Bisubstrate and Use in Mechanistic Studies of an Enzyme Involved in
Aminoglycoside Resistance
Feng Gao,[a, b] Xuxu Yan,[a] and Karine Auclair*[a]
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Abstract: Aminoglycoside N-6’-acetyl-
transferases (AAC(6’)s) are important
determinants of antibiotic resistance. A
good mechanistic understanding of
these enzymes is essential to overcome
aminoglycoside resistance. We have
previously reported the synthesis of
amide- and sulfonamide-linked amino-
be a superior mimic of the tetrahedral
intermediate proposed for catalysis by
AAC(6’)s. This synthetic target is espe-
cially challenging for a number of rea-
sons, including the presence of multiple
functional groups, the water solubility
of both starting materials, and incom-
patibility of PIII chemistry with water.
We have overcome these challenges by
adding the expensive coenzyme A in
the last step by means of an elegant
Michael-type addition onto a vinyl-
phosphonate in water. Overall, a single
protection step was needed. The de-
creased inhibitory potency of this bi-
substrate compared to that of the
amide-linked analogue suggests that
Enterococcus faecium AAC(6’)-Ii may
not stabilize the proposed tetrahedral
intermediate, and may act mainly
through proximity catalysis.
glycoside–coenzyme A
conjugates,
which were useful mechanistic and
structural probes of AAC(6’)s. We
report here the synthesis of a phos-
Keywords: coenzyme A · enzyme
inhibitors
·
Michael addition ·
ACHTUNGTRENNUNG
neamine · medicinal chemistry
ACHTUNGTRENNUNG
Introduction
Aminoglycosides are useful antibiotics that target the 16S ri-
bosomal RNA A site of bacteria. Aminoglycoside resistance
is however spreading rapidly. It is mainly caused by the ex-
pression of drug-modifying enzymes, such as nucleotidyl-
transferases (ANTs), phosphoryltransferases (APHs) and
acetyltransferases (AACs). Among them, aminoglycoside N-
6’-acetyltransferases (AAC(6’)s) are some of the most clini-
cally relevant. AAC(6’)s selectively transfer an acetyl group
from acetyl coenzyme A (AcCoA) to the 6’-NH2 of most
aminoglycosides. It is proposed
that a tetrahedral intermediate
is formed during the acetyl
transfer by these enzymes
(shown here). The acetylated
They were also useful mechanistic probes of Salmonella en-
terica AAC(6’)-Iy.[3] To our surprise, the bisubstrate with a
sulfonamide linker (2) was 40-fold less potent against
AAC(6’)-Ii than the amide-linked analogue (1b). These re-
sults support the earlier suggestion by Wright and co-work-
ers that AAC(6’)-Ii may catalyze the acetyl transfer reaction
mainly through proximity effects.[4,5] Alternatively, sulfona-
mides may not be good mimics of the enzymatic intermedi-
ate.
aminoglycosides
display
a
greatly reduced antibacterial
activity. We have reported the
use of amide-[1] and sulfon-
AHCTUNGTRENNUNG
amide-linked[2] aminoglycoside–
CoA bisubstrates as structural
and mechanistic probes for these enzymes (e.g. see 1 and 2).
Most of these conjugates were nanomolar inhibitors of
AAC(6’)-Ii and successfully crystallized with the enzyme.[1]
Indeed, very few examples of the use of sulfonamides as
tetrahedral intermediate mimetics have been reported.[6–9]
Phosphoryl groups, on the other hand, have been extensive-
ly used for this purpose.[10–21]
Aminoglycoside–CoA
bisub-
strates that mimic the tetrahe-
[a] Dr. F. Gao, X. Yan, Prof. K. Auclair
Department of Chemistry, McGill University
801 Sherbrooke Street West, Montrꢁal, Quꢁbec
H3A 2K6 (Canada)
dral intermediate better than
amide-linked bisubstrates rep-
resent useful probes to clarify
the mechanism of AAC(6’)s.
Fax : (+1)514-398-3797
We therefore embarked on the
E-mail: karine.auclair@mcgill.ca
syntheses of bisubstrates 3a–c,
which pose a number of chal-
lenges: 1) both starting materi-
als, aminoglycosides and CoA,
have multiple reactive functional groups; 2) the starting ma-
terials are only soluble in aqueous solutions, while the more
efficient PIII chemistry is not compatible with such condi-
[b] Dr. F. Gao
Currently at Albany Molecular Research Inc.
26 Corporate Circle, P.O. Box 15098, Albany
New York 12212 (USA)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200802172. It contains general
experimental methods as well as 1D and 2D NMR spectra of the
final product and selected intermediates.
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K. Auclair et al.
tions; 3) the functional groups of CoA cannot withstand the
typical conditions employed for protection/deprotection of
aminoglycosides; and finally 4) CoA is expensive and only
available in small amounts. We report here a five-step syn-
thesis of the phosphonate-linked neamine–CoA conjugate
3c.
Results and Discussion
Targets 3a–c were designed based on our previously report-
ed structure–activity relationships for amide-linked bisub-
strates.[1,22] Compared to the proposed enzymatic tetrahedral
intermediate (shown above), 3a–c have extra carbon atoms
between N6’ of the aminoglycoside and S of CoA. As sug-
gested by our previous results with the bisubstrate inhibitors
1a–d, these extra atoms are not expected to compromise in-
hibition, and n=2 may even strengthen it as was the case
for 1b.[1]
Scheme 1. First synthetic plan for the preparation of bisubstrate 3a. Re-
agents and conditions: i) NBS, PPh3; ii) PBr3, CCl4; iii) I2, PPh3; iv) tri-
chlorocyanuric acid, DMF.
The key intermediate in the
synthesis of bisubstrate 3a was
methyl iodomethylphosphonyl
chloride (4; Scheme 1A), which
may be derived from dimethyl
iodomethylphosphonate 8c. a-
Hydroxymethylphosphonate
7
was used in numerous attempts
to prepare the corresponding a-
halomethylphosphonates 8a–c
without success (Scheme 1B).
Conventional halogenation pro-
cedures led to complex mix-
tures, even with very mild
chlorination reagents such as
trichlorocyanuric acid.[23] Alter-
natively, attempts to convert
chloromethyl dimethylphospho-
nate (9) into iodomethyl di-
Scheme 2. Second synthetic plan for the preparation of bisubstrate 3a. Compound 5 is 1,3,2’-tri-N-(tert-butoxy-
carbonyl)neamine[2] (see Scheme 3). Conditions tested separately: i) TEA/H2CO3 buffer, pH~8.4; ii) H2O/ace-
tone, DIPEA; iii) H2O/acetone, TEA; iv) H2O, 0.1n NaOH.
ACHTUNGTRENNUNG
methylation product 10 (Scheme 1C).
As an alternative route, the Michaelis–Arbuzov reaction
of trimethylphosphite and diiodomethane was attempted,
but proceeded with an extremely low yield due to the de-
composition of diiodomethane and the rearrangement of tri-
methylphosphite to dimethyl methylphosphonate.[24–26] To
optimize this reaction, various conditions and catalysts were
tested. The addition of copper powder to slow down the de-
composition of diiodomethane, reaction at a lower tempera-
ture (1308C instead of the literature reported 1608C)[24] and
a shorter reaction time (3 h instead of 12 h) afforded
iodomethyl dimethylphosphonate (8c) in about 30% yield
(Scheme 2). A typical two-step chlorination procedure[15,27]
was adopted to prepare phosphonyl chloride 4. A number of
nucleophilic reagents such as NaOH/MeOH,[15,16] LiN3/
DMF,[28] TMSBr/Py,[29] tert-butylamine,[27] thiophenol/
TEA,[30] and NaI/acetone[31,32] were reported to cleave di-
methyl phosphonates to the monophosphonic acids. We
found that tert-butylamine worked the best to convert 8c to
11. The phosphonic acid 11 was next converted to phos-
phonyl chloride 4 in good yield by using oxalyl choride.
Treatment of 1,3,2’-tri-N-(tert-butoxycarbonyl)neamine (5)[2]
with phosphonyl chloride 4 in the presence of base afforded
the N-6’-phosphonamidate intermediate 12, which yielded
13 after removal of the Boc protecting groups. The iodo-
AHCTUNGTREGmNNUN ethylphosphonamidate 13 was treated with CoA (shown
À
as CoA SH in Scheme 2) under various conditions, none of
which yielded the desired product; instead the demethylated
product 14 was observed. It was reasoned that steric hin-
drance may prevent approach by the thiol. To avoid this
problem, we embarked on the synthesis of 3b. It was envis-
aged that Michael addition of CoA onto vinylphosphonate
18 (Scheme 3A) would likely be a reasonable route.
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Medicinal Chemistry
FULL PAPER
tion. We reasoned that the
more electronegative O (vs. N)
would make vinyl phosphonate
23 (Scheme 5) a better Michael
acceptor than 17 (Scheme 4).
Moreover, this modification
was not expected to decrease
inhibition because both N-6’-
OH- and N-6’-NH2-containing
aminoglycosides bind AAC(6’)
tightly, with the former being
inhibitors and the latter being
substrates. Thus, after methan-
AHCTUNGTERGoNNUN lysis of paromomycin to pa-
Scheme 3. Synthetic plan for the preparation of bisubstrate 3b.
romamine,[34] reaction with Boc
anhydride provided 1,3,2’-tri-N-
To investigate the feasibility of this route, model reactions
were carried out with commercial dimethyl vinylphos-
ACHTUNGTRENNUNGphonate and N-acetylcysteCAHTUNGTRNEaNUGN mine (as a surrogate for CoA;
Scheme 3B). Surprisingly, the addition was complete within
one hour at RT in both organic
and aqueous solutions.
(tert-butoxycarbonyl)paromamine (21), which was reacted
with 16 to yield 22. Removal of the Boc protecting groups
generated 23, which was treated with CoA under basic aque-
ous solution to produce the desired bisubstrate 3c as two
Thus, the synthesis of bisub-
strate 3b was attempted as
shown in Scheme 4. Mono-de-
methylation of commercially
available dimethyl vinylphosph-
onate yielded phosphonic acid
19,[33] which was converted to
phosphonyl chloride 16. Treat-
ment of compound 5 with phos-
phonyl chloride 16 in the pres-
ence of base afforded the N-6’-
phosphonamidate intermediate
20, which yielded phosphonami-
date 17 after deprotection. The
Michael addition of CoA onto
vinylphosphonamidate 17 was
not successful under the condi- Scheme 4. Attempted synthesis of bisubstrate 3b. Conditions tested separately: i) TEA H2CO3 buffer, pH~
8.4; ii) H2O/acetone, TEA; iii) H2O/acetone, DIPEA; iv) H2O, 0.1m NaOH; v) H2O/CAN, AIBN, 508C.
tions tested (Scheme 4), and
mostly demethylated product
was observed. Alternatively,
radical reactions were also
tested, but did not produce the
desired product either.
This was unexpected consid-
ering the high efficiency of the
model reaction (Scheme 3B),
and made us realize that the
model reaction may not have
been representative, because
À
the P N bond was replaced
À
with a P O bond. This prompt-
ed us to attempt the synthesis
of 3c, in which neamine is re-
placed with paromamine to
better mimic the model reac- Scheme 5. Synthesis of bisubstrate 3c. The yield for the last step is non-optimized.
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K. Auclair et al.
Table 1. Gradient profile for HPLC purification.
diastereoisomers (~1:1) in 31% total yield (non-optimized)
after HPLC purification. The regioselectivity of the phos-
phorylation is demonstrated by a (31P, 1H) HMBC correlation
spectrum, in which the 6’-CH2 is correlated to the linker
phosphorus (see Supporting Information). The high cost of
CoA (and the low activity, see below) prevented us from
making enough material to allow separate studies of the dia-
stereoisomers.
Time
AHCTUNGTERG[NNUN min]
A [%] (0.05% TFA[a]
in H2O)
B [%] (0.04% TFA
in ACN)
0
20
30
99
60
99
1
40
1
[a] TFA=trifluroacetic acid.
The affinity of bisubstrates 3c for AAC(6’)-Ii was mea-
sured both using the standard enzyme assay[22] (Ki =2.0Æ
0.9 mM) and isothermal calorimetry (Kd =0.81Æ0.04 mM).
To our surprise, the expected better mimetic of the tetrahe-
dral intermediate, 3c, was a much weaker inhibitor than the
corresponding amide-linked bisubstrate (1b, Ki =0.04Æ
0.02 mM). Even if one diastereoisomer was more potent
than the other, based on the Ki of the diastereomeric mix-
ture, it is unlikely to compete with 1b. The extra atoms be-
tween the aminoglycoside and CoA compared to the tetra-
hedral intermediate cannot explain the reduced activity of
inhibitor 3c, because 3c has a linker (phosphonate) of the
same length as that of 1b (amide-linked), yet 1b is a low
nanomolar inhibitor, the most potent AAC(6’)-Ii inhibitor
reported to date.[1] Moreover, the crystal structure of
AAC(6’)-Ii in complex with 1b clearly shows that the extra
methylene groups reach out into a grove without affecting
the relative positions of the aminoglycoside and CoA.[1]
Based on enzyme kinetic studies, Wright and co-workers[5]
have suggested that this enzyme may not stabilize the tetra-
hedral intermediate and only provide catalysis through a
proximity effect. Our results are in good agreement with
this suggestion. Indeed, the reduction in affinity observed
when the amide linker (as in 1b) is replaced with a phos-
phonate such as 3c, may result from repulsion of the phos-
phonate negative charge within the enzyme catalytic site.
Examination of the various crystal structures reported for
this enzyme are also in agreement with this proposition, as
no residues able to stabilize the intermediate have been
identified.[1,35,36] In summary, we have reported the first syn-
thesis of a phosphonate-linked aminoglycoside–CoA deriva-
tive (3c) by means of an elegant Michael-type addition of
the CoA thiol onto a vinylphosphonate in water. Our route
includes only one protection step and the expensive CoA is
added in the last step.
two diastereoisomers have retention time at 13.30 and 13.75 min, respec-
tively. The purity of the bisubstrate was evaluated by HPLC with the
same equipment and column, but using isocratic eluent with a ratio of
phase A/phase B of either 85:15 (method A) or 64:40 (method B). The
1
purity of 3c used in the enzymatic inhibition assays was >95%. H NMR
(D2O, 500 MHz): d=8.55 (s, 1H), 8.31 (s, 1H), 6.08 (d, J=4.0 Hz, 1H),
5.64 (brs, 1H), 4.49 (brs, 1H), 4.24 (brs, 2H), 4.16 (brs, 2H), 3.90–3.20
(m, 21H), 2.66 (m, 2H), 2.54 (t, J=6.5 Hz, 2H), 2.34 (m, 3H), 2.17 (m,
2H), 1.79 (q, J=12.5 Hz, 1H), 0.78 (brs, 3H), 0.70 ppm (brs, 3H);
13C NMR (D2O, 400 MHz by HMQC and HMBC): d=174.7, 174.4 (C=O
of CoA), 150.2, 148.7, 144.9, 142.7, 118.5 (aromatic carbons of adenine),
96.7, 87.5, 83.3, 79.3, 74.9, 74.3, 73.8, 72.2, 71.8, 71.6, 68.6, 68.5, 65.1, 64.7,
53.8, 53.7, 53.2, 51.3, 39.9, 38.2, 35.4, 35.3, 30.2, 27.6, 24.8, 23.4, 23.3, 20.8,
18.4 ppm; 31P NMR (D2O,81 MHz): d=35.7, 35.6 (diastereoisomers);
ESI-MS: m/z calcd for C36H66N10O25P4S [M+H]+: 1195.3; found: 1195.1;
HR-MS: m/z calcd for C36H66N10O25P4S [M+H]+: 1195.2896; found:
1195.2945.
Paromamine:[34] Paromomycin sulfate (5 g, 7.0 mmol) was dissolved in
MeOH (100 mL), and conc. HCl (12m, 4 mL) was added. The mixture
was heated to reflux for 6 h, and concentrated to a volume of 20 mL. Di-
ethyl ether (50 mL) was added and the product was allowed to crystallize
at about 88C for 2 h. Filtration was used to collect the paromamine hy-
drogen chloride salt as white crystals (3.65 g, yield: 84%). 1H NMR
(D2O, 400 MHz): d=5.52 (d, J=4.0, 1H), 3.82–3.69 (m,4H), 3.62 (dd, J=
6.8, 5.2 Hz, 1H), 3.55 (t, J=8.8 Hz, 1H), 3.46 (m, 2H), 3.40–3.32 (m,
2H), 3.21 (m, 1H), 2.38 (td, J=12.0, 4.0 Hz, 1H); 1.75 ppm (q, J=
12.0 Hz, 1H); 13C NMR (D2O, 75 MHz): d=97.6 (C1’), 80.6 (C5’), 74.7
(C4’), 74.3 (C3’), 73.6 (C5), 69.6 (C6), 68.8 (C4), 60.3 (C6’), 54.1 (C2’),
49.4 (C1), 48.9 (C3), 28.2 (C2) ppm; ESI-MS: m/z calcd for C12H25N3O7
[M+H]+: 323.2; found: 323.2.
Methyl chloro iodomethylphosphonate (4): Compound 11 (0.33 g,
1.4 mmol) was dissolved in DMF (2.5 mL, 0.03 mmol) and dry DCM
(10 mL). Oxalyl chloride (0.15 mL, 2 mmol) was added dropwise. The so-
lution was stirred at 08C for 20 min, warmed to RT, and stirred for 1 h.
The reaction mixture was concentrated to give phosphonochloridate 4 as
a yellowish oil, which was used immediately in a reaction with 1,3,2’-tri-
N-(tert-butoxycarbonyl)neamine. 1H NMR (CDCl3, 300 MHz): d=3.96
(d, J=10.8 Hz, 3H), 3.46 ppm (d, J=8.8 Hz, 2H); 13C NMR (CDCl3,
75 MHz): d=54.7 (d, J=5.8 Hz), À9.3 ppm (d, J=155 Hz); 31P NMR
(CDCl3, 81 MHz): d=34.0 ppm; ESI-MS: m/z calcd for C2H5ClIO2P
[M+H]+: 254.9; found: 254.9.
Dimethyl hydroxymethylphosphonate (7): This compound was prepared
by using
a
previously reported procedure.[15] Yield: 76%. 1H NMR
(CDCl3, 200 MHz): d=4.89 (brs, 1H), 3.96 (d, J=5.8 Hz, 2H), 3.82 ppm
(d, J=10.6 Hz, 6H); 13C NMR (CDCl3, 75 MHz): d=54.3 (d, J=5.6 Hz),
62.0 ppm (d, J=155 Hz); 31P NMR (CDCl3, 81 MHz): d=27.9 ppm; ESI-
MS: m/z calcd for C3H9O4P [M+H]+: 141.1; found: 141.1.
Experimental Section
Preparation of bisubstrate 3c: Crude product 23 (55 mg, ca. 0.06 mmol)
was dissolved in a mixture of water (5 mL) and acetone (1 mL), CoA
(sodium salt, 15 mg, 0.02 mmol) was directly added to this solution, fol-
lowed by the addition of diisopropylethylamine (DIPEA, 0.25 mL). The
mixture was sonicated for 10 min and stirred for 16 h at 48C. The solu-
tion was then poured into water (30 mL) and washed with EtOAc
(10 mLꢂ2). The aqueous layer was separated and freeze-dried to pro-
duce a yellowish powder, which was further purified by reverse phase
HPLC to yield the bisubstrate 3c as a pair of diastereoisomers (6 mg,
31% yield). HPLC separation was achieved on an Agilent Zorbax SB-C8
Dimethyl iodomethylphosphonate (8c): Diiodomethane (16 g, 60 mmol)
and copper beads (1 g, diameter <0.5 mm) were mixed and heated to
1308C. Trimethyl phosphite (7.7 g, 60 mmol) was added through an auto-
matic syringe pump at speed of 5 mLh.À1 The mixture was refluxed for
3 h. After distillation of the by-product dimethyl methylphosphonate
(house vacuum at about 708C), the remaining yellowish liquid afforded
the desired compound in low yield (32%, purity >95% by NMR).
1H NMR (CDCl3, 300 MHz): d=3.70 (d, J=11.0 Hz, 6H), 2.95 ppm (d,
J=10.2 Hz, 2H); 13C NMR (CDCl3, 75 MHz): d=54.1 (d, J=5.8 Hz),
À15.8 ppm (d, J=155 Hz); 31P NMR (CDCl3, 81 MHz): d=23.9 ppm;
HRMS: m/z calcd for C3H8IO3P [M+H]+: 250.9334; found: 250.9329.
column (4.6ꢂ250 mm, 5 m). Samples were eluted at
a flow rate of
3 mLminÀ1, by using the linear gradients shown in Table 1 below. The
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Medicinal Chemistry
FULL PAPER
Dimethyl chloromethylphosphonate (9): This compound was prepared as
(1 mL, 1:1). The reaction was monitored by NMR spectroscopy. There
was no reaction before addition of TEA. The reaction was, however,
complete within 2 h after addition of TEA (100 mL, 0.7 mmol). The mix-
ture was purified on silica gel (Rf: 0.43, CHCl3/MeOH, 10/1) to yield the
desired product (89 mg, 70%). 1H NMR (CDCl3, 200 MHz): d=6.28
(brs, 1H), 3.60 (d, J=10.6 Hz, 6H), 3.31 (q, J=6.4 Hz, 2H), 2.90 (t, J=
6.8 Hz, 2H), 2.85 (d, J=6.4 Hz, 2H), 1.95 (s, 3H), 1.80 ppm (m, 2H);
13C NMR (CDCl3, 75 MHz): d=176.0, 54.2 (d, J=5.2 Hz), 40.5, 33.2,
30.6, 29.2 (d, J=10.2 Hz), 22.5 ppm; 31P NMR (CDCl3, 81 MHz): d=
34.1 ppm; ESI-MS: m/z calcd for C8H18NO4PS [M+H]+: 256.1; found:
256.1.
1
previously reported.[16] Yield: 62%. H NMR (CDCl3, 200 MHz): d=3.71
(d, J=10.8 Hz, 6H), 3.46 ppm (d, J=10.6 Hz, 2H); 13C NMR (CDCl3,
75 MHz): d=53.9 (d, J=5.6 Hz), 32.7 ppm (d, J=156 Hz); 31P NMR
(CDCl3, 81 MHz): d=22.2 ppm; ESI-MS: m/z calcd for C3H835ClO4P:
158.0; found: 158.0.
Methyl iodomethylphosphonate (11): Compound 8c (2.50 g, 1 mmol) was
dissolved in tert-butylamine (20 mL) and the resulting solution was re-
fluxed for 16 h. The reaction mixture was concentrated (10 mL) and fil-
tered to collect the crystalline methyl iodomethylphosphonate tert-butyl-
ACHTUNGTRENNUNGamine salt (3.13 g, quantitative yield). This salt (2.0 g, 6.5 mmol) was dis-
Methyl vinylphosphonate (19):[33] Dimethyl vinylphosphonate (4.0 g,
29.4 mmol) was dissolved in tert-butylamine (20 mL) and was heated to
reflux for 16 h at 508C. The reaction mixture was concentrated (ca.
10 mL) and filtered to collect the methyl vinylphosphonate tert-butylami-
solved in chloroform (60 mL) and Dowex 8–100 resin (10 g, 200–400
mesh, wet) was added. After 30 min of shaking, the resin was removed
by filtration, and the filtrate was dried over Na2SO4. After evaporation of
the solvent, the desired compound was obtained as a yellowish oil (1.1 g,
72%). 1H NMR (CDCl3, 200 MHz): d=11.60 (brs, 1H), 3.81 (d, J=
10.8 Hz, 3H), 3.00 ppm (d, J=8.8 Hz, 2H); 31P NMR (CDCl3, 81 MHz):
d=24.1 ppm; ESI-MS: m/z calcd for C2H6IO3P [M+H]+: 236.9; found:
236.9.
nium salt as
a
white crystal (3.2 g, yield: 56%). 1HNMR (CDCl3,
200 MHz): d=8.59 (brs, 3H), 6.20–5.60 (m, 3H), 3.52 ppm (d, J=
10.8 Hz, 3H); 31PNMR (CDCl3, 81 MHz): d=13.8 ppm. This salt (1.95 g,
10 mmol) was dissolved in chloroform (60 mL) and Dowex 8–100 resin
(8 g, 200–400 mesh, wet) was added. The heterogeneous mixture was
shaken for 30 min. The resin was removed by filtration, the filtrate was
dried over Na2SO4 and solvents were evaporated under vacuum to give
1,3,2’-Tri-N-(tert-butoxycarbonyl)-6’-N-(methyl iodomethyl phosphonyl)
neamine (12): Compound 5 (125 mg, 0.2 mmol) and diisopropylethyl-
ACHTUNGTRENNUNGamine (50 mL, 0.25 mmol) were dissolved in anhydrous DMF (20 mL).
1
the desired compound as a yellowish oil (0.81 g, 66%). H NMR (CDCl3,
The mixture was cooled to 08C, and methyl iodomethylphosphonate
chloride (4, 50 mg, 0.2 mmol) was added dropwise. The reaction mixture
was stirred for 1 h at 08C and for another 2 h at RT. Reaction completion
was monitored by TLC, Rf =0.40 (tailing, CHCl3/MeOH/NH4OH, 4/1/
0.1); ESI-MS: m/z calcd for C29H54IN4O14P [M+Na]+: 863.2; found
863.2. The reaction mixture was evaporated to dryness to give a white
powder. This solid was triturated with water (20 mL) to remove the am-
monium salt. The remaining solid material was dried to afford the crude
200 MHz): d=11.99 (brs, 1H), 6.30–5.98 (m, 3H), 3.71 ppm (d, J=
10.8 Hz, 3H); 13C NMR (CDCl3, 75 MHz): d=127.0, 124.5, 52.3 ppm (d,
J=5.8); 31P NMR (CDCl3, 81 MHz): d=21.1 ppm; ESI-MS: m/z calcd
for C2H6IO3P [M+H]+: 236.9; found: 236.9.
1,3,2’-Tri-N-(tert-butoxycarbonyl)-6’-N-(methyl vinylphosphonyl) ne-
AHCTUNGERTGaNNUN mine (20): Compound 5 (125 mg, 0.2 mmol) and diisopropylethylamine
(50 mL, 0.25 mmol) were dissolved in anhydrous DMF (20 mL). The mix-
ture was cooled to 08C, and methyl vinylphosphonyl chloride (16, 28 mg,
0.2 mmol) was added. The reaction mixture was stirred for 1 h at 08C
and for another 2 h at RT. Reaction completion was monitored by TLC:
Rf =0.38 (CHCl3/MeOH/NH4OH, 4/1/0.1); ESI-MS: m/z calcd for
C30H55N4O14P [M+Na]+: 749.3; found: 749.2; 31P NMR ([D6]DMSO,
81 MHz): d=25.1, 25.7 ppm.
product as
a
white powder (138 mg, 82%). 31P NMR ([D6]DMSO,
81 MHz): d=28.3 and 28.9 ppm (diastereoisomers). Attempted purifica-
tion on silica gel led to decomposition. Compound 12 was therefore used
as a crude sample in the next step.
6’-N-(Methyl iodomethyl phosphonyl) neamine (13): Crude 12 (80 mg,
ca. 0.06 mmol) was mixed with DCM (10 mL) containing anisole (1
drop). To the mixture, TFA (2 mL) was added and the mixture became
homogeneous immediately. The reaction was complete after 1 h at RT as
monitored by TLC and ESI-MS. Evaporation of the volatiles afforded a
white salt, which was dissolved in water (20 mL) and washed with chloro-
form (3ꢂ5 mL). The aqueous phase was lyophilized to afford the product
as a white powder (48 mg, ca, 60%). 31P NMR (D2O, 81 MHz): d=26.4,
27.2 ppm; ESI-MS: m/z calcd for C14H30IN4O8P [M+H]+: 541.1; found:
541.0. This compound is also prone to decomposition on silica gel; it was
therefore directly used in the next step.
1,3,2’-Tri-N-(tert-butoxycarbonyl) paromamine (21): Paromomycin hydro-
gen chloride salt (0.86 g, 2 mmol) and NaOH (0.60 g, 15 mmol) were dis-
solved in MeOH (80 mL). The mixture was stirred for a few minutes to
yield an homogeneous solution. Boc anhydride (2.0 g, 9.2 mmol) was
added in three batches. The mixture was stirred for 16 h and then evapo-
rated. The residue was triturated with petroleum ether (60 mLꢂ3) and
washed with water (50 mLꢂ2), to yield the desired product (0.93 g, yield:
1
75%). H NMR ([D6]DMSO, 400 MHz): d=6.63 (d, J=7.5 Hz, 1H), 6.46
(brs, 1H), 6.30 (d, J=7.5 Hz, 1H), 4.97 (brs, 1H), 4.82 (brs, 1H), 4.73
(brs, 1H), 4.65 (brs, 1H), 4.58 (brs, 1H), 4.18 (brs, 1H), 3.63–3.52 (m,
3H), 3.35–3.04 (m, 8H), 1.75 (brs, 1H), 1.37 (s, 27H), 1.18 ppm (q, J=
10.5 Hz, 1H); 13C NMR ([D6]DMSO, 75 MHz): d=156.5, 155.9, 155.6,
99.8, 82.6, 78.4, 78.2, 77.7, 74.4, 73.2, 71.8, 70.6, 61.0, 56.3, 51.4, 50.3, 36.0,
29.0, 28.8 ppm; ESI-MS: m/z calcd for C27H49N3O13 [M+H]+: 624.3;
found: 624.3.
Methyl chloro vinylphosphonate (16): Compound 19 (250 mg, 2 mmol)
and dry DMF (25 mL) were mixed in dry DCM (15 mL). The mixture
was cooled to 08C, to which oxalyl chloride (0.26 mL, 3 mmol) was
added dropwise. The reaction mixture was stirred for 30 min and then for
another hour at RT. All the volatiles were evaporated under vacuum
(458C, 15 mmHg) to yield the desired product as yellowish oil (0.32 g).
This crude product was directly used in the next reaction. 1HNMR
(CDCl3, 200 MHz): d=6.80–6.22 (m, 3H), 3.95 ppm (d, J=10.8 Hz, 3H);
31P NMR (CDCl3, 81 MHz): d=29.9 ppm.
1,3,2’-Tri-N-(tert-butoxycarbonyl)-6’-O-(methyl vinyl phosphonyl) parom-
amine (22): Into a flame-dried flask, compound 21 (0.32 g, 0.5 mmol) was
dissolved in anhydrous DMF (20 mL), and activated 4 ꢃ molecular
sieves (3 g) were added. The mixture was allowed to stir for 2 h and then
transferred by cannula into another dry flask. A freshly prepared solution
of compound 19 (67 mg, 0.48 mmol) in DCM (10 mL) was added drop-
wise to the above solution at 08C. The reaction was complete after 3 h as
revealed by TLC (Rf: 0.42, CHCl3/MeOH/NH4OH, 5:1:0.1). Evaporation
of the solvent under vacuum produced the crude product as a white solid
powder. ESI-MS: m/z calcd for C30H54N3O15P [M+Na]+: 750.3; found:
750.2. Purification using silica column was not possible due to decomposi-
tion, thus the crude product was used directly in the next step.
6’-N-(Methyl vinylphosphonyl) neamine (17). Crude 20 (75 mg) was
mixed with DCM (10 mL) containing anisole (1 drop). TFA (2 mL) was
added to the mixture and it became homogeneous immediately. The reac-
tion was completed after 1 h at RT as monitored by TLC and ESI-MS.
Evaporation of the volatiles afforded a white salt, which was dissolved in
water (20 mL) and washed with chloroform (3ꢂ5 mL). The aqueous
phase was lyophilized to afford the crude product as a white powder
(36 mg, ca. 34%). 31P NMR (D2O, 81 MHz): d=24.2, 24.8 ppm; ESI-MS:
m/z calcd for C15H31N4O8P [M+H]+: 427.2; found: 427.2. Attempts to
purify this compound on silica gel led to decomposition, and therefore
the crude product was used in the reaction with CoA.
6’-O-(Methyl vinyl phosphonyl) paromamine (23): Crude compound 22
(0.16 g) was dissolved in DCM (5 mL) along with anisole (3 drops). TFA
(2 mL) was added to the mixture which became homogeneous immedi-
ately. The mixture was stirred for 1 h at RT and evaporation of the sol-
vent yielded the crude product 23 (200 mg, yield: 100%). 31P NMR (D2O,
Dimethyl 2-((2-acetamidoethyl)sulfanyl)ethylphosphonate (18): Di-
ACHTUNGTRENNUNGmethylvinylphosphonate (68 mg, 0.5 mmol) and N-acetylcysteamine
(60 mg, 0.5 mmol) were dissolved in
a mixture of [D6]acetone/D2O
Chem. Eur. J. 2009, 15, 2064 – 2070
ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
2069

K. Auclair et al.
81 MHz): d=23.9, 23.8 ppm; ESI-MS: m/z calcd for C15H30N3O9P
[M+H]+: 428.3; found: 428.1.
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Received: October 20, 2008
Published online: January 16, 2009
2070
www.chemeurj.org
ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2009, 15, 2064 – 2070