A highly convergent synthesis of myristoyl-carba(dethia)-coenzyme A

Article abstract of DOI:10.1002/ejoc.200901410

Co-translational myristoylation of the N-terminal glycine residues of diverse signalling proteins is required for membrane attachment and proper function of these molecules. The transfer of myristate from myristoyl-coenzyme A (myr-CoA) is catalysed by the enzyme N-myristoyltransferase (Nmt). Nmt has been implicated in a number of human diseases, including cancer and epilepsy, as well as in pathogenic mechanisms such as fungal and virus infections, including HIV and hepatitis B. Rational design has led to the development of potent competitive inhibitors, including several nonhydrolysable acyl-CoA substrate analogues. Linear synthetic strategies, following the route of the original CoA synthesis, however, generate such analogues in very low overall yields that typically are not sufficient for in vivo studies. Here we present a new, highly convergent synthesis of myristoylcarba(dethia)-coenzyme A (1) that allows this substrate analogue to be obtained in a yield 11 times higher than that of the reported linear synthesis. In addition, enzymatic cleavage of the adenosine-2′,3′-cyclophosphate in the last step of the synthesis proved to be an efficient way to obtain the isomerically pure 3′-phosphate 1 free of the 2′-phosphate 13.

Full text of DOI:10.1002/ejoc.200901410

FULL PAPER
DOI: 10.1002/ejoc.200901410
A Highly Convergent Synthesis of Myristoyl-carba(dethia)-coenzyme A
Lutz Tautz[a,b] and Janos Rétey*[b]
Keywords: N-Myristoylation / Coenzyme A / Antifungal agents / Antiviral agents / Inhibitors
Co-translational myristoylation of the N-terminal glycine res-
idues of diverse signalling proteins is required for membrane
attachment and proper function of these molecules. The
transfer of myristate from myristoyl-coenzyme A (myr-CoA)
is catalysed by the enzyme N-myristoyltransferase (Nmt).
Nmt has been implicated in a number of human diseases,
including cancer and epilepsy, as well as in pathogenic
mechanisms such as fungal and virus infections, including
HIV and hepatitis B. Rational design has led to the develop-
ment of potent competitive inhibitors, including several non-
hydrolysable acyl-CoA substrate analogues. Linear synthetic
strategies, following the route of the original CoA synthesis,
however, generate such analogues in very low overall yields
that typically are not sufficient for in vivo studies. Here
we present a new, highly convergent synthesis of myristoyl-
carba(dethia)-coenzyme A (1) that allows this substrate ana-
logue to be obtained in a yield 11 times higher than that of
the reported linear synthesis. In addition, enzymatic cleav-
age of the adenosine-2Ј,3Ј-cyclophosphate in the last step of
the synthesis proved to be an efficient way to obtain the iso-
merically pure 3Ј-phosphate 1 free of the 2Ј-phosphate 13.
tococcus neoformans, the cause of a fungal infection,[6] or
in viruses such as HIV-I,[7,8] polio virus[9] and hepatitis B
virus.[10] The assembly of HIV infectious virions, for in-
stance, is dependent on the myristoylation of gag, gag-pol
and nef viral polyprotein precursors by cellular Nmt.[7] Nmt
is thus regarded as a potential drug target for anticancer,
antiepilepsy, antifungal and antiviral agents.
Introduction
Post- or co-translational lipidation is required for the
proper functioning of many proteins involved in signal
transduction and cellular growth control.[1] The co-transla-
tional transfer of a myristoyl group from myristoyl-coen-
zyme A (myr-CoA) to an N-terminal glycine residue of a
substrate protein is catalysed by the enzyme N-myristoyl-
transferase (Nmt; EC 2.1.3.97).[2] Nmt recognises the
amino-terminal motif G(X)3(S/T/A/C/N) of the nascent
peptide, after the initiating methionine has been removed
by methionine aminopeptidase during translation. Known
human N-myristoylated proteins include kinases such as the
Src family kinases and the catalytic subunit of cAMP-de-
pendent protein kinase, as well as phosphatases such as cal-
cineurin.[3] Nmt has been implicated in human diseases
such as colon cancer, in which N-myristoylation of the
oncogene product p60c-src is required for its malign ef-
fect,[4] or epilepsy, in which myristoylation of calcineurin is
involved in neuronal apoptosis.[5] Myristoylated proteins
also play important roles in several pathogens such as Cryp-
Inhibitors of Nmt have been reported (reviewed in
ref.[11–13]) and include substrate analogues such as peptides,
peptidomimetics and myr-CoA analogues, as well as several
reported small molecules, including benzofurans and
benzothiazole derivatives, showing promising antifungal ac-
tivity in vivo.[14,15] Rational design has led to the develop-
ment of several non-hydrolysable acyl-coenzyme A ana-
logues as potent competitive inhibitors of Nmt, including
S-(2-oxopentadecyl)-CoA,[16] S-(3-oxohexadecyl)-CoA and
myristoyl-carba(dethia)-CoA.[17] One major drawback,
however, is the limited amounts of such compounds access-
ible through existing synthetic strategies. Myristoyl-carba-
(dethia)-CoA (1, Figure 1), in which the sulfur atom of the
myr-CoA thioester bond is replaced by a methylene group,
for example, was generated by a linear synthesis[17] that ap-
plies the route of Moffatt and Khorana’s original coen-
zyme A synthesis.[18] The low overall yield of 1.7% (with
regard to myristic acid) does not offer any easy means to
obtain 1 or similar molecules in quantities necessary for in
vivo studies. Here we present a novel and highly convergent
strategy for easy synthesis of myristoyl-carba(dethia)-CoA
(1) and other carba(dethia)-CoA derivatives as single iso-
mers in high overall yields.
[a] Infectious and Inflammatory Disease Center, Burnham Insti-
tute for Medical Research,
La Jolla, CA 92037, USA
[b] Institute of Organic Chemistry, Department of Biochemistry,
University of Karlsruhe,
76131 Karlsruhe, Germany
E-mail: Janos.Retey@ioc.uka.de
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.200901410.
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A Highly Convergent Synthesis of Myristoyl-carba(dethia)-coenzyme A
Figure 1. Myristoyl-carba(dethia)-CoA (1). The methylene group that replaces the sulfur atom of the thioester bond in myr-CoA is
highlighted in bold.
eine in the penultimate step (ca. 30% reported,[17] 10% in
Results and Discussion
our hands), we sought a strategy to introduce the phosphate
Our goal of an efficient synthesis of myristoyl-carba- group earlier in the synthesis. In nature, the biosynthesis of
(dethia)-CoA (1) prompted us to explore more convergent coenzyme A starts with the phosphorylation of pantothenic
synthetic opportunities to access this non-hydrolysable sub- acid (vitamin B5).[19] Accordingly, Martin et al.[20] reported
strate analogue and potent inhibitor of Nmt. Because the a method for the generation of acetyl-carba(dethia)-CoA,
modest overall yield of the linear synthesis[17] is primarily in which the pantothenic acid moiety is first phosphoryl-
due to the very low levels of conversion during the phos- ated, then coupled to adenosinephosphate, and in the last
phorylation reaction of myristoyl-carba(dethia)-panteth- step coupled with the acetate moiety through aminolysis
Scheme 1. Convergent strategy applied from Martin et al.’s acetyl-carba(dethia)-CoA synthesis,[20] involving an aminolysis reaction of
amine 8 and thioester 6 in the penultimate step.
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L. Tautz, J. Rétey
FULL PAPER
of the thioester-activated pantothenic acid. Inspired by the mixture for 6 and 8 turned out to be problematic. We used
convergent strategy, we tested whether this route would also different mixtures of water/buffer with acetonitrile, DMF,
be suitable for effectively generating myristoyl-carba(de- DMSO or pyridine, at room temperature or 50 °C, with use
thia)-CoA (1).
of NaOH or DMAP to adjust the pH between 10.5 and 11.
The S-propyl thiopantothenate (2) (Scheme 1) was syn- Although product 9 was always formed, yields were found
thesised as reported by Martin et al.[20] Unlike in that syn- to be extremely low (between 5 and 22%).
thesis, however, phosphorylation of
2 was then ac-
complished by application of a phosphoramidite method
based on PIII chemistry.[21] Deprotection of the allyl phos-
phate 3 afforded the phosphate 4 with an overall yield of
74% in the phosphorylation step, representing a significant
improvement over the reported 17% obtained with dimethyl
phosphorochloridate.[20] Compound 4 was then treated with
the morpholidate 5[22] in dry pyridine, with formation of a
pyrophosphate bond to yield the thioester 6.
Scheme 2. Synthesis of amine 8, starting from myristic acid (7).
The amine 8 was generated from myristic acid (7) as de-
scribed,[22,23] with slight variations and yield improvements
To overcome the solubility problems, we changed the
(Scheme 2 and Supporting Information). The subsequent synthetic strategy to that effect that the aminolysis reaction
aminolysis of 6 with 8 (Scheme 1) constituted the key reac- was carried out before formation of the pyrophosphate
tion of this convergent strategy. Martin et al.[20] reported a (Scheme 3). The thioester 10 was therefore generated and
90% conversion to the amide in phosphate buffer at pH = then used in the aminolysis reaction. Both 8 and 10 could
10.5 within 20 h to generate the acetyl-CoA analogue. In readily be dissolved in acetonitrile/water at pH = 10.5. The
our case, however, finding a common solvent or solvent reaction was monitored by HPLC, during which the pH
Scheme 3. Improved strategy with application of the aminolysis reaction earlier in the synthesis; acidic hydrolysis of the adenosine-2Ј,3Ј-
cyclophosphate results in a mixture of the phosphate isomers 1 and 13.
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A Highly Convergent Synthesis of Myristoyl-carba(dethia)-coenzyme A
was kept at 10.5. After 4 d, the dibenzyl phosphate 11 was
In addition to the failure to separate the two phosphate
isolated in 72% yield. After cleavage of the benzyl groups isomers, we also saw scope for improvement and simplifi-
and acidic hydrolysis of the ketal, 12 could be coupled with cation of the synthesis. The aminolysis conditions favoured
the morpholidate 5. Subsequent acidic hydrolysis of the cy- by Martin et al. allowed the formation of the amide bond
clophosphate yielded myristoyl-carba(dethia)-CoA (1) and in the presence of the unprotected phosphate group. Be-
its 2Ј-phosphate isomer myristoyl-carba(dethia)-isoCoA cause we had already modified the synthetic route such that
1
(13) in a ratio of 2:1, according to H and 31P NMR spec- a side reaction with the free phosphate group of the adeno-
troscopy (Figures S1 and S2 in the Supporting Infor- sine moiety was no longer a concern, we were now also
mation). The overall yield of the final product 1, with re- able to use peptide chemistry to couple amine 8 with the
gard to myristic acid, was therefore 13%. However, pantothenic acid phosphate entity more efficiently
attempts to separate the two isomers by ion-exchange (Scheme 4). In addition to the expected higher yields, rela-
chromatography with a DEAE-cellulose column as de- tive to the aminolysis reaction, there was also no need for
scribed for the coenzyme A synthesis,[18] or by use of the two extra steps to generate the thioester. We first gener-
Dowex® 1ϫ8 (Cl– form), were not successful, most proba- ated -pantothenic acid 4Ј-dibenzyl phosphate (15) by ap-
bly because of the high surface activities of these com- plying the phosphoramidite method developed by
pounds. Use of HPLC on a C18 reversed-phase column as Bannwarth and Trzeciak,[21] which had been shown to be
described[17] was also not capable of separating the isomers, superior to other phosphorylation strategies. Free -panto-
prompting us to search for an alternative strategy.
thenic acid was obtained from its sodium salt 14 upon elu-
Scheme 4. Final route to generate isomerically pure myristoyl-carba(dethia)-CoA (1). The key intermediate 12 was obtained through the
use of peptide chemistry to coupling of amine 8 with pantothenic acid phosphate 15. The final step of the synthesis involves an enzymatic
cleavage of the adenosine-2Ј,3Ј-cyclophosphate with RNase T2, which exclusively generated the desired product 1.
Eur. J. Org. Chem. 2010, 1728–1735
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L. Tautz, J. Rétey
FULL PAPER
tion from a Dowex® 50ϫ8 (H+ form) column and removal phosphate 15, together with the specific enzymatic cleavage
of water by lyophilisation, followed by drying in high of the adenosine-2Ј,3Ј-cyclophosphate with RNase T2, re-
vacuum. Under water-free conditions, in the presence of the sulted in a significant improvement over and simplification
weak base tetrazole, the primary hydroxy group of -panto- of existing methods. The use of peptide chemistry saved two
thenic acid was selectively phosphitylated with dibenzyl- synthetic steps relative to the modified aminolysis-based
N,N-diisopropylphosphoramidite. Subsequent oxidation route[20] and should be easily applicable to the synthesis of
with m-chloroperbenzoic acid (mCPBA) led to the benzyl- any acyl-carba(dethia)-CoA analogue, regardless of the na-
ester-protected phosphate 15. In the key reaction of this ture of the acyl group. Syntheses of analogues containing
new synthetic strategy, the carboxy group of 15 was first hydrophobic acyl groups should thus in particular benefit
activated with 1,1Ј-carbonyldiimidazole (CDI) to form the from our new strategy. Conveniently, the key reactant pan-
corresponding N,N-dimethylformamide. The amine 8 was tothenic acid phosphate can be easily obtained in good
then added, and the ketal-protected dibenzyl phosphate 11 yield by application of the phosphoramidite method, which
was obtained in 86% yield (in comparison with 72% by proved to be far superior to the reported phosphorylation
aminolysis). Cleavage of the benzyl groups with hydrogen of pantothenic acid with methyl phosphorochloridate.[20]
in the presence of palladium on charcoal, and acidic hydrol- Most importantly, the use of RNase T2 in the last step of
ysis of the ketal were achieved quantitatively to yield the the synthesis exclusively generates the desired 3Ј-phosphate
phosphate 12. Adenosine-2Ј,3Ј-cyclophosphate 5Ј-phospho- isomer, eliminating the need (and challenge) to separate the
morpholidate (5) was generated as reported[22] and was cou- final product from its 2Ј-phosphate isomer.
pled with 12 in dry pyridine as described above.
Because attempts to separate the two phosphate isomers
Experimental Section
myristoyl-carba(dethia)-CoA (1) and myristoyl-carba(de-
thia)-isoCoA (13) had not been successful before, we sought
a way to generate only the desired product 1. Utilisation of
the enzyme ribonuclease T2 (RNase T2) turned out to be
an elegant solution, providing the pure isomer 1 through
regioselective opening of the cyclophosphate. First used by
Keep et al.,[24] RNase T2 cleaves nucleoside-2Ј,3Ј-cyclo-
phosphates specifically to afford the 3Ј-phosphates. The en-
zymatic reaction was performed in potassium phosphate
buffer at 30 °C, and the pH was optimised to 6.3. Use of
lower pH values as described in the original report[24] led
to increased acidic hydrolysis, and consequently to forma-
tion of the 2Ј-phosphate isomer. After 24 h, over 90% (ac-
cording to analytical HPLC) of the adenosine-2Ј,3Ј-cyclo-
phosphate had been converted. The product was purified
by preparative HPLC with use of a C18 reversed-phase col-
umn and an acetonitrile/potassium phosphate gradient. The
desired myristoyl-carba(dethia)-CoA (1) was isolated in
51% yield with regard to phosphate 12, and 19% with re-
gard to myristic acid (7). More importantly, 1 was gener-
ated isomerically pure without any further need for separa-
General: All reactions sensitive to air or moisture were carried out
under argon in oven-dried glassware unless otherwise noted. Anhy-
drous solvents were purchased from Fluka and used without fur-
ther drying. All other reagents were purchased from Acros Or-
ganics, Fluka, or Sigma-Aldrich, and were used as supplied unless
otherwise stated. Analytical thin-layer chromatography (TLC) was
performed by using Polygram Sil G/UV254 silica gel plates (Mach-
erey-Nagel). For column chromatography, Merck silica gel 60 (40–
63 µm) was used. Reversed-phase (RP) column chromatography
was performed with an HP 1050 HPLC system with automated
liquid sampler and diodearray detector (Hewlett-Packard). For an-
alytical HPLC, a Grom column (250ϫ4 mm) with Grom-Sil 100
ODS-0 AB (5 µm) was used. For preparative HPLC, a Macherey-
Nagel column (250ϫ20 mm) with Nucleosil 100 C18 (7 µm) was
used. 1H and 13C NMR spectra were recorded with Bruker AM 400
or Bruker DRX 500 spectrometers. Chemical shifts are reported
in ppm from tetramethylsilane with reference to internal residual
solvent. The following abbreviations are used to designate the sig-
nal multiplicities: s = singlet, d = doublet, dd = doublet of doublet,
t = triplet, and m = multiplet. Coupling constants are reported
in Hertz (Hz). High-resolution mass spectrometry (HRMS-EI and
HRMS-FAB) and MS-FAB spectra were were carried ot with a
Finnigan MAT 90 spectrometer. The relative intensities (in %) are
reported in reference to the base peak. Melting points were ob-
tained with a Büchi B-535 apparatus.
1
tion from its 2Ј-phosphate isomer. H and 31P NMR spec-
troscopy (Figures S3 and S4 in the Supporting Information)
clearly demonstrated the presence of only one isomer.
S-Propyl 3-{(2R)-4-[Bis(allyloxy)phosphoryloxy]-2-hydroxy-3,3-di-
methylbutyrylamino}thiopropionate (3): Compound
1.95 mmol) and diallyl-N,N-diisopropylphosphoramidite (514 µL,
2 (540 mg,
Conclusions
We have developed a new convergent synthesis that al- 1.95 mmol) were dissolved in dry acetonitrile (5 mL) under an inert
gas in a round flask (100 mL) fitted with a septum. A tetrazole
solution in acetonitrile (0.45 , 4.33 mL, 1.95 mmol) was added,
and the mixture was stirred at room temperature for 5 min.
mCPBA (722 mg of a mixture of 70% mCPBA, 10% 3-chloroben-
zoic acid and 20% water, 505 mg, 2.92 mmol) was then added, and
the solution was stirred at room temperature for 2 h. After evapora-
tion of the solvent and drying in high vacuum, the product was
purified by silica gel flash chromatography [cyclohexane/ethyl ace-
tate, 10:1, 2:1, 1:1, 1:5, 0:1; Rf(cyclohexane/ethyl acetate 1:1) =
lows the potent Nmt inhibitor myristoyl-carba(dethia)-CoA
(1) to be generated in a total of 14 steps and a very good
overall yield of 19% (with regard to myristic acid). In rela-
tion to the linear synthesis, based on Moffatt’s and Khor-
ana’s original method for generating coenzyme A,[18] this
constitutes an 11-fold increase in yield, while the number of
synthetic steps has been reduced by three. An effective new
route to obtain the key intermediate myristoyl-carba(de-
thia)-pantethein-4Ј-phosphate (12), using peptide chemistry
0.06]. Compound 3 was obtained as a colourless oil. Yield: 629 mg
1
to link the readily accessible amine 8 and pantothenic acid (74%). H NMR (CD3OD, 500 MHz): δ = 0.95 (s, 3 H, -C-CH3),
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A Highly Convergent Synthesis of Myristoyl-carba(dethia)-coenzyme A
0.97 (t, J = 7.4 Hz, 3 H, CH3-CH2-), 1.01 (s, 3 H, -C-CH3), 1.59
(m, 2 H, CH3-CH2-), 2.82 (t, J = 6.7 Hz, 2 H, -SCO-CH2-), 2.87
tion (100 m), and the mixture was stirred at room temperature for
4 d. During this time, the pH value was checked several times and
(m, 2 H, -CH2-SCO-), 3.50 (m, 2 H, CH2-NHCO-), 3.86 (s, 1 H, adjusted to 11.0. The solution was then neutralised with HCl (1 ),
-CHOH-), 3.96 (ABX, J1 = 9.3, J2 = 4.5 Hz, 2 H, -CH2-OH), 4.58
(m, 4 H, 2 -CH2-CH=CH2), 5.34 (ABX, 4 H, 2 -CH2-CH=CH2),
and the solvent was removed by lyophilisation. The product was
purified by preparative HPLC on a C18 reversed-phase column
5.99 (m, 2 H, 2 -CH2-CH=CH2) ppm. 31P NMR (CD3OD, with a potassium phosphate buffer/acetonitrile gradient [A: 10 m
500 MHz): δ = 0.05 (1 P) ppm. HRMS (70 eV): calcd. 437.1637; KPH (pH = 6.0); B: acetonitrile/10 m KPH (pH = 6.0), 80:20).
found 437.1632.
Compound 9 eluted between 60 and 70% B. After concentration
of the eluate by lyophilisation, buffer salts were removed by HPLC
on a C18 reversed-phase column with a water/acetonitrile gradient,
S-Propyl 3-[(2R)-2-Hydroxy-3,3-dimethyl-4-phosphonooxybutyryl-
amino]thiopropionate (4): Compound
3 (630 mg, 1.44 mmol),
1
and 9 was obtained as its potassium salt. Yield: 18 mg (22%). H
Pd[P(C6H5)3]4 (167 mg, 5 mol-% per allyl group) and dimethylbar-
bituric acid (225 mg, 1.46 mmol) were dissolved in dry THF (7 mL)
under an inert gas in a round flask (50 mL). After the mixture
had been stirred at room temperature overnight, the solvent was
evaporated, and the residue was dried in high vacuum, after which
the product was purified by preparative HPLC on a C18 reversed-
phase column with use of a water/acetonitrile gradient (A: H2O +
0.1% TFA; B: acetonitrile + 0.1% TFA). Compound 4 eluted be-
NMR (D2O, 500 MHz): δ = 0.74 (s, 3 H, -C-CH3), 0.83 (t, J =
6.9 Hz, 3 H, CH3-CH2-), 0.88 (s, 3 H, -C-CH3), 1.19 [s, 22 H,
-(CH2)11-], 1.48 [m, 2 H, -C(OCH2)2-CH2-CH2-], 1.59 [m, 4 H,
-CH2-C(OCH2)2-CH2-], 2.44 (m, 2 H, -NHCO-CH2-), 3.12 (m, 2
H, CH2-NHCO-), 3.45 (m, 2 H, -NHCO-CH2-CH2-), 3.67 (ABX,
J1 = 9.4, J2 = 4.9 Hz, 2 H, -CH2-OH), 3.96 [s, 4 H, -C(OCH2)2-],
4.00 (s, 1 H, -CHOH-), 4.25 (m, 2 H, 5Ј-position ribose), 4.62 (m,
1 H, 4Ј-position ribose), 5.23 (m, 1 H, 3Ј-position ribose), 5.40 (m,
1 H, 2Ј-position ribose), 6.33 (d, J = 4.1 Hz, 1 H, 1Ј-position
ribose), 8.25 (s, 1 H, adenine ring C-2 position), 8.45 (s, 1 H,
adenine ring C-8 position) ppm. 31P NMR (D2O, 500 MHz): δ =
–11.18 to –10.41 (2 P, pyrophosphate), 20.13 (1 P, cyclophosphate)
ppm. MS-FAB (glycerol): m/z (%) = 1068.9 (91), 1051.1 (20) [M +
3 Na – 3 H]+, 1025.3 (100) [M + K + H]+, 1007.4 (26) [M + Na –
H]+, 986.4 (1.5) [M + H]+, 985.4 (4) [M]+, 981.4 (94), 937.4 (77).
1
tween 25% and 35% B. Yield: 505 mg (98%). H NMR (CD3OD,
500 MHz): δ = 0.94 (s, 3 H, -C-CH3), 0.96 (t, J = 7.4 Hz, 3 H,
CH3-CH2-), 1.00 (s, 3 H, -C-CH3), 1.59 (m, 2 H, CH3-CH2-), 2.82
(t, J = 6.7 Hz, 2 H, -SCO-CH2-), 2.87 (m, 2 H, -CH2-SCO-), 3.50
(m, 2 H, CH2-NHCO-), 3.84 (ABX, J1 = 9.5, J2 = 5.1 Hz, 2 H,
-CH2-OH), 3.91 (s, 1 H, -CHOH-) ppm. 31P NMR (CD3OD,
500 MHz): δ = 1.58 (1 P) ppm. MS-FAB (glycerol): m/z (%) = 358.2
(92) [M + H]+, 282.1 (2.5) [M – C3H7S]+, 260.2 (100) [M –
H2PO4]+.
S-Propyl 3-{(2R)-4-[Bis(benzyloxy)phosphoryloxy]-2-hydroxy-3,3-
dimethylbutyrylamino}thiopropionate (10): Compound 2 (540 mg,
S-Propyl 4Ј-(Adenosine-2Ј,3Ј-cyclophosphate-5Ј-pyrophosphate)thio-
pantothenate (6): The bis(N,NЈ-dicyclohexyl-4-morpholinecarbox-
amidium) salt of adenosine-2Ј,3Ј-cyclophosphate-5Ј-phosphomor-
pholidate (5, 462 mg, 0.373 mmol) and compound 4 (134 mg,
0.373 mmol) were each repeatedly dissolved in dry pyridine (5 mL),
concentrated through evaporation and dried in high vacuum. The
reactants were then transferred to a round flask (100 mL) and dis-
solved in dry pyridine (5 mL), before being stirred under an inert
gas at room temperature for 2 d. After evaporation of the solvent,
the residue was washed with diethyl ether and dried in high vac-
uum. The product was then purified by preparative HPLC on a
C18 reversed-phase column with a potassium phosphate buffer/ace-
tonitrile gradient [A: 10 m KPH (pH = 6.0); B: acetonitrile/10 m
KPH (pH = 6.0), 80:20]. Compound 6 eluted between 20 and 40%
B. After concentration of the eluate by lyophilisation, buffer salts
were removed by HPLC on a C18 reversed-phase column with use
of a water/acetonitrile gradient. Compound 6 was obtained as its
tripotassium salt. Yield: 213 mg (66%). 1H NMR (D2O, 500 MHz):
δ = 0.63 (s, 3 H, -C-CH3), 0.74 (s, 3 H, -C-CH3), 0.77 (t, J = 7.4 Hz,
3 H, CH3-CH2-), 1.39 (m, 2 H, CH3-CH2-), 2.69 (m, 4 H, -CH2-
SCO-CH2-), 3.38 (m, 2 H, CH2-NHCO-), 3.56 (ABX, J1 = 9.8, J2
= 4.8 Hz, 2 H, -CH2-OH), 3.88 (s, 1 H, -CHOH-), 4.16 (m, 2 H,
5Ј-position ribose), 4.53 (m, 1 H, 4Ј-position ribose), 5.14 (m, 1 H,
3Ј-position ribose), 5.33 (m, 1 H, 2Ј-position ribose), 6.24 (d, J =
4.1 Hz, 1 H, 1Ј-position ribose), 8.16 (s, 1 H, adenine ring C-2
position), 8.34 (s, 1 H, adenine ring C-8 position) ppm. 31P NMR
(D2O, 500 MHz): δ = –10.67 to –9.90 (2 P, pyrophosphate), 20.41
(1 P, cyclophosphate) ppm. MS-FAB (glycerol): m/z (%) = 862.9
(44) [M + 3 K – 2 H]+, 825.0 (100), [M + 2 K – H]+, 787.0 (33)
[M + K]+, 749.3 (0.2) [M + H]+, 748.0 (0.08) [M]+.
1.95 mmol)
and
dibenzyl-N,N-diisopropylphosphoramidite
(641 µL, 1.95 mmol) were dissolved in dry acetonitrile (5 mL) un-
der an inert gas in a round flask (100 mL) with a septum. A tetra-
zole solution in acetonitrile (0.45 , 4.33 mL, 1.95 mmol) was
added, and the mixture was stirred at room temperature for 5 min.
mCPBA (722 mg of a mixture of 70% mCPBA, 10% 3-chloroben-
zoic acid and 20% water, 505 mg, 2.92 mmol) was then added, and
the solution was stirred at room temperature for 2 h. After evapora-
tion of the solvent and drying in vacuo, the product was purified
by silica gel flash chromatography [cyclohexane/ethyl acetate, 10:1,
2:1, 1:1, 1:5, 0:1; Rf(cyclohexane/ethyl acetate, 1:1) = 0.015]. Com-
pound 10 was obtained as a colourless oil. Yield: 748 mg (71%).
1H NMR (CDCl3, 250 MHz): δ = 0.77 (s, 3 H, -C-CH3), 0.95 (t, J
= 7.4 Hz, 3 H, CH3-CH2-), 1.06 (s, 3 H, -C-CH3), 1.58 (m, 2 H,
CH3-CH2-), 2.78 (t, J = 6.3 Hz, 2 H, -SCO-CH2-), 2.84 (m, 2 H,
-CH2-SCO-), 3.54 (m, 2 H, -CH2-NHCO-), 3.80 (ABX, J1 = 8.1,
J2 = 10.1 Hz, 2 H, -CH2-O-), 3.91 (s, 1 H, -CHOH-), 5.03 (m, 4 H,
2 -CH2-C6H5), 7.33 (m, 10 H, 2 -C6H5) ppm. 31P NMR (CDCl3,
500 MHz): δ = 1.04 (1 P) ppm. MS-FAB (3-NBA): m/z (%) = 560.2
(6) [M + Na]+, 538.2 (28) [M + H]+, 260.2 (56) [M – dibenzyl
phosphate], 180.3 (100) [M – dibenzyl phosphate – {C(O)-
SCH2CH2CH3} + Na]+.
Dibenzyl (3R)-3-Hydroxy-2,2-dimethyl-4-oxo-4-{3-oxo-3-[3-(2-tri-
decyl-1,3-dioxolan-2-yl)propylamino]propylamino}butyl Phosphate
(11) by Aminolysis: Compounds 10 (100 mg, 0.186 mmol) and 8
(58 mg, 0.186 mmol) were dissolved in acetonitrile/water (1:1,
6 mL) in a round flask (25 mL). The pH was adjusted to 11.0 with
an NaOH solution (100 m), and the mixture was stirred at room
temperature for 4 d. During this time, the pH value was checked
several times and adjusted to 11.0. The solution was then neutral-
ised with HCl (1 ), and the solvent was removed by lyophilisation.
The product was purified by preparative HPLC on a C18 reversed-
phase column with a water/acetonitrile gradient (A: H2O; B: aceto-
nitrile). Compound 11 eluted between 94 and 100% B. After con-
centration of the eluate by lyophilisation, compound 11 was ob-
Adenosine-2Ј,3Ј-cyclophosphate of 2-Methyl-2-tridecyl-1,3-dioxolan-
dethio-CoA (9): Compounds 6 (66 mg, 0.076 mmol) and 8 (24 mg,
0.076 mmol) were heated to 50 °C in pyridine (2 mL) in a round
flask (25 mL) and dissolved completely by brief sonication with a
sonicator bath. The pH was then adjusted to 11.0 with NaOH solu-
Eur. J. Org. Chem. 2010, 1728–1735
© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
1733

L. Tautz, J. Rétey
FULL PAPER
tained as a white solid. Yield: 103 mg (72%). 1H NMR (CDCl3,
500 MHz): δ = 0.80 (s, 3 H, CH3- pantetheine), 0.87 (t, J = 7.0 Hz,
3 H, CH3-CH2-), 1.05 (s, 3 H, CH3- pantetheine), 1.24 [s, 20 H,
-(CH2)10-], 1.29 [m, 2 H, -CH2-CH2-C(OCH2)2-], 1.55 [m, 4 H,
-CH2-C(OCH2)2-CH2-CH2-], 1.62 [m, 2 H, -C(OCH2)2-CH2-], 2.41
and 13 as a white solid. Yield: 38 mg (61%). H NMR (CD3OD,
500 MHz): δ = 0.83 (s, 3 H, CH3- pantetheine), 0.89 (t, J = 7.1 Hz,
3 H, CH3-CH2-), 1.06 (s, 3 H, CH3- pantetheine), 1.27 [s, 20 H,
-(CH2)10-], 1.52 (m, 2 H, -CH2-CH2-CO-), 1.70 (m, 2 H, -CO-CH2-
CH2-), 2.44 (m, 6 H, -NHCO-CH2-, -CH2-CO-CH2-), 3.13 (m, 2
1
(m, 2 H, -NHCO-CH2-), 3.21 (m, 2 H, -NHCO-CH2-CH2-), 3.55 H, -CO-CH2-CH2-CH2-), 3.45 (m, 2 H, -NHCO-CH2-CH2-), 3.78
[m, 2 H, -C(OCH2)2-CH2-CH2-CH2-], 3.78 (ABX, J1 = 10.1, J2 = (ABX, J1 = 9.7, J2 = 4.8 Hz, 2 H, -CH2-O-), 4.07 (s, 1 H,
7.4 Hz, 2 H, -CH2-O-), 3.88 (s, 1 H, -CHOH-), 3.90 [s, 4 H, -CHOH-), 4.21 (m, 2 H, ribose 5Ј-position, 2Ј-isomer), 4.27 (m, 2
-C(OCH2)2-], 5.03 (m, 4 H, 2 -CH2-C6H5), 6.21 (m, 1 H, -NH-), H, ribose 5Ј-position, 3Ј-isomer), 4.49 (m, 1 H, ribose 4Ј-position,
7.28 (m, 1 H, -NH-), 7.34 (m, 10 H, 2 -C6H5) ppm. 31P NMR 2Ј-isomer), 4.62 (m, 1 H, ribose 4Ј-position, 3Ј-isomer), 4.72 (m, 1
(CDCl3, 500 MHz): δ = 0.72 (1 P) ppm. MS-FAB (3-NBA): m/z H, ribose 3Ј-position, 2Ј-isomer), 5.15 (m, 1 H, ribose 2Ј-position,
(%) = 813.4 (2.5) [M + K]+, 799.4 (11), 798.4 (45), 797.4 (100) [M
+ Na]+, 776.4 (25), 775.4 (55) [M + H]+, 774.4 (3) [M]+.
2Ј-isomer), 6.13 (d, J = 5.6 Hz, 1 H, ribose 1Ј-position, 3Ј-isomer),
6.28 (d, J = 5.6 Hz, 1 H, ribose 1Ј-position, 2Ј-isomer), 8.19 (s, 1
H, adenine C-2 position), 8.57 (s, 1 H, adenine C-8 position) ppm.
31P NMR (CD3OD, 500 MHz): δ = –9.26 to –8.67 (m, 2 P, pyro-
phosphate), 1.91/2.18 (s, 1 P, 2Ј-/3Ј-position) ppm.
(3R)-3-Hydroxy-2,2-dimethyl-4-oxo-4-[3-oxo-3-(4-oxoheptadecyl-
amino)propylamino]butyl Dihydrogen Phosphate (12): Compound
11 (100 mg, 0.129 mmol) and palladium black (10%, ca. 10 mg)
were added to dry methanol (5 mL) in a two-necked flask (50 mL)
fitted with septum and stopcock. After purging of the flask several
times with argon and then with hydrogen, the reaction mixture was
stirred under hydrogen at room temperature overnight. The catalyst
was then removed by filtration, and the solvent was evaporated.
Remaining benzyl alcohol was removed in high vacuum. For hy-
drolysis of the ketal protecting group, the colourless oil was dis-
solved in ethanol (80%) and applied to a column (25ϫ300 mm)
with Dowex® 50ϫ8 (H+ form). The product was eluted with eth-
anol (80%). The eluate was then concentrated, and the residue was
dissolved in ethanol (10 mL) and, after addition of toluene (2 mL),
concentrated. After drying in high vacuum, compound 12 was ob-
tained as a colourless oil. Yield: 76 mg (99%). 1H NMR ([D6]-
DMSO, 500 MHz): δ = 0.78 (s, 3 H, CH3- pantetheine), 0.83 (t, J
= 6.9 Hz, 3 H, CH3-), 0.86 (s, 3 H, CH3- pantetheine), 1.21 [s, 20
3-[4-(Bis-benzyloxy-phosphoryloxy)-2(R)-hydroxy-3,3-dimethyl-but-
yrylamino]propionate (15): -Pantothenic acid sodium salt (14, 1 g,
4.15 mmol) was dissolved in water and applied to a column
(25ϫ300 mm) with Dowex® 50X8 (H+ form). The free acid was
eluted with water (250 mL), and the water was removed by lyo-
philisation. The residue was dried in high vacuum. The free acid
(900 mg, 4.11 mmol) and dibenzyl-N,N-diisopropylphosphoramid-
ite (1.352 mL, 4.11 mmol) were dissolved in dry acetonitrile (5 mL)
under inert gas in a round flask (100 mL) with septum. A tetrazole
solution in acetonitrile (0.45 , 9.133 mL, 4.11 mmol) was added,
and the solution was stirred for 15 min at room temperature.
mCPBA (1.52 g of a mixture of 70 % mCPBA, 10 % 3-chloro-
benzoic acid and 20% water, 1.064 g, 6.16 mmol) was then added,
and the mixture was stirred for another 2 h at room temperature.
The solvent was evaporated, and the residue was purified by silica
H, -(CH2)10-], 1.41 (m, 2 H, -CH2-CH2-CO-), 1.54 (m, 2 H, -CO- gel flash chromatography (ethyl acetate, methanol). Compound 15
CH2-CH2-), 2.22 (m, 2 H, -NHCO-CH2-), 2.36 (m, 4 H, -CH2-CO- was obtained as a colourless oil. Yield: 836 mg (42%). 1H NMR
CH2-), 2.95 (m, 2 H, -CO-CH2-CH2-CH2-), 3.25 (m, 2 H, -NHCO- (CD3OD, 500 MHz): δ = 0.89 (s, 3 H, -C-CH3), 0.95 (s, 3 H,
CH2-CH2-), 3.63 (ABX, J1 = 9.7, J2 = 5.8 Hz, 2 H, -CH2-O-), 3.67 -C-CH3), 2.51 (t, J = 6.6 Hz, 2 H, HOOC-CH2-), 3.46 (m, 2 H,
(s, 1 H, -CHOH-), 7.70 (t, J = 5.8 Hz, 1 H, -NH-), 7.83 (t, J = HOOC-CH2-CH2-), 3.84 (s, 1 H, -CHOH-), 3.92 (ABX, J1 = 9.4,
6.8 Hz, 1 H, -NH-) ppm. 13C NMR ([D6]DMSO, 500 MHz): δ =
J2 = 4.5 Hz, 2 H, -CH2-O-), 5.04 (s, 2 H, -CH2-C6H5), 5.06 (s, 2
14.38 (CH3-CH2), 19.91 (CH3-), 21.23 (CH3-), 22.54 (CH2), 23.66 H, -CH2-C6H5), 7.36 (m, 10 H, 2 -C6H5) ppm. 13C NMR (CD3OD,
(CH2), 23.76 (CH2), 29.06 (CH2), 29.15 (CH2), 29.33 (CH2), 29.36 500 MHz): δ = 18.64 (CH3-), 19.91 (CH3-), 33.31 (HOOC-CH2-),
(CH2), 29.45 (4 CH2), 31.74 (CH2), 35.29 (CH2), 35.56 (CH2), 38.29 34.38 (HOOC-CH2-CH2-), 38.65 [-C(CH3)2-], 69.34 (-CH2-C6H5),
(CH2), 39.03 [-C(CH3)2-], 39.55 (CH2), 42.32 (CH2), 71.65 (CH2), 69.38 (-CH2-C6H5), 73.41 (-CH2-O-), 73.95 (-CHOH-), 127.75 (4
74.32 [-CH(OH)-], 170.84 (-HN-CO-), 172.53 (-HN-CO-), 210.56 aromat. tert.), 128.27 (6 aromat. tert.), 135.81 (2 aromat. quart.),
(-CO-) ppm. 31P NMR ([D6]DMSO, 500 MHz): δ = 0.29 (1 P) ppm. 173.80 (-NHCO-), 174.06 (HOOC-) ppm. 31P NMR (CD3OD,
MS-FAB (3-NBA): m/z (%) = 596.4 (8) [M + 2 Na]+, 595.4 (22),
589.4 (5) [M + K]+, 575.4 (9), 574.4 (34), 573.4 (100) [M + Na]+,
571.4 (9), 551.4 (13) [M + H]+, 550.4 (1) [M]+.
500 MHz): δ = 0.16 (1 P) ppm. MS-FAB (3-NBA): m/z (%) = 504.2
(3), 503.2 (20) [M + H + Na]+, 502.2 (59) [M + Na]+, 482.2 (8),
481.2 (28), 480.2 (100) [M + H]+, 479.3 (3) [M]+.
Myristoyl-carba(dethia)-CoA (1) and Myristoyl-carba(dethia)-iso-
Dibenzyl (3R)-3-Hydroxy-2,2-dimethyl-4-oxo-4-{3-oxo-3-[3-(2-tri-
CoA (13), 2Ј-Phosphate Isomer: To remove traces of water, com-
decyl-1,3-dioxolan-2-yl)propylamino]propylamino}butyl Phosphate
pounds 5 (130 mg, 0.105 mmol) and 12 (36 mg, 0.065 mmol) were (11) by Peptide Chemistry: Compound 15 (150 mg, 0.312 mmol)
dissolved in dry pyridine (5 mL) and the mixtures concentrated
three times. After drying in high vacuum, the reactants were dis-
solved in dry pyridine (5 mL) and stirred at room temperature un-
der argon for 2 d. After evaporation of the solvent, the residue was
washed with diethyl ether and dried in high vacuum. The residue
was purified by preparative HPLC on a C18 reversed-phase column
with a potassium phosphate buffer/acetonitrile gradient [A: 10 m
potassium phosphate (pH = 6.0); B: acetonitrile/10 m potassium
phosphate (pH = 6.0), 80:20]. The adenosine-2Ј,3Ј-cyclophosphate
eluted between 55 and 70% buffer B. After concentration of the
eluate by lyophilisation, the residue was stirred in methanol with
TFA (2%) at room temperature overnight to open the cyclophos-
phate. Preparative HPLC on a C18 reversed-phase column with a
water/acetonitrile gradient yielded a mixture of the two isomers 1
was dissolved in dry DMF (6 mL) under an inert gas in a round
flask (50 mL) fitted with septum and bubble counter and heated to
40 °C. CDI (100 mg, 0.624 mmol) in dry DMF (4 mL) was added,
and the mixture was stirred for 90 min, until gas formation had
stopped. The amine 8 (98 mg, 0.312 mmol), dissolved in dry DMF
(3 mL), was then added, and the reaction mixture was stirred at
40 °C overnight. The solvent was evaporated, and the residue was
purified by preparative HPLC on a C18 reversed-phase column
with a water/acetonitrile gradient (A: H2O; B: acetonitrile). Com-
pound 11 eluted between 94 and 100% B. After concentration of
the eluate by lyophilisation, compound 11 was obtained as a white
solid. Yield: 208 mg (86%). 1H NMR (CDCl3, 500 MHz): δ = 0.80
(s, 3 H, CH3- pantetheine), 0.87 (t, J = 7.0 Hz, 3 H, CH3-CH2-),
1.05 (s, 3 H, CH3- pantetheine), 1.24 [s, 20 H, -(CH2)10-], 1.29 [m,
1734
www.eurjoc.org
© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2010, 1728–1735

A Highly Convergent Synthesis of Myristoyl-carba(dethia)-coenzyme A
2 H, -CH2-CH2-C(OCH2)2-], 1.55 [m, 4 H, -CH2-C(OCH2)2-CH2- Supporting Information (see footnote on the first page of this arti-
CH2-], 1.62 [m, 2 H, -C(OCH2)2-CH2-], 2.41 (m, 2 H, -NHCO- cle): Figures S1–S4, showing 1H NMR and 31P NMR spectra of
CH2-), 3.21 (m,
-C(OCH2)2-CH2-CH2-CH2-], 3.78 (ABX, J1 = 10.1, J2 = 7.4 Hz, 2
H, -CH2-O-), 3.88 (s, 1 H, -CHOH-), 3.90 [s, 4 H, -C(OCH2)2-],
2
H, -NHCO-CH2-CH2-), 3.55 [m,
2
H,
the isomeric mixture of myristoyl-carba(dethia)-CoA (1) and myr-
istoyl-carba(dethia)-isoCoA (13) and of the pure isomer myristoyl-
carba(dethia)-CoA (1); details for the synthesis of 3-(2-tridecyl-1,3-
5.03 (m, 4 H, 2 -CH2-C6H5) 6.21 (m, 1 H, -NH-), 7.28 (m, 1 H, dioxolan-2-yl)propan-1-amine (8).
-NH-), 7.34 (m, 10 H, 2 -C6H5) ppm. 31P NMR (CDCl3, 500 MHz):
δ = 0.72 (1 P) ppm. MS-FAB (3-NBA): m/z (%) = 813.4 (2.5) [M
+ K]+, 799.4 (11), 798.4 (45), 797.4 (100) [M + Na]+, 776.4 (25),
Acknowledgments
775.4 (55) [M++H]+, 774.4 (3) [M]+.
Myristoyl-carba(dethia)-CoA (1) by Enzymatic Cleavage of Cyclo-
phosphate: To remove traces of water, compounds 5 (130 mg,
0.105 mmol) and 12 (36 mg, 0.065 mmol) were dissolved in dry pyr-
idine (5 mL) and the mixtures concentrated three times. After dry-
ing in high vacuum, the reactants were dissolved in dry pyridine
(5 mL) under argon and stirred at room temperature for 2 d. After
evaporation of the solvent, the residue was washed with diethyl
ether and dried in high vacuum. The residue was purified by pre-
parative HPLC on a C18 reversed-phase column with a potassium
phosphate buffer/acetonitrile gradient [A: 10 m potassium phos-
phate (pH = 6.0); B: acetonitrile/10 m potassium phosphate (pH
= 6.0), 80:20]. The adenosine-2Ј,3Ј-cyclophosphate eluted between
55 and 70% buffer B. After concentration of the eluate by lyophili-
sation, the residue was dissolved in EDTA/water (5 m, 1 mL), the
pH was adjusted to 6.3, and ribonuclease T2 (150 U) was added.
The solution was incubated at 30 °C for 24 h, and the reaction was
monitored by analytical HPLC. The enzyme was then denatured
by a short incubation at 80 °C and could be removed by centrifuga-
tion. After sterile filtration, the mixture was separated by prepara-
tive HPLC on a C18 reversed-phase column with a potassium
phosphate/acetonitrile gradient [A: 10 m potassium phosphate
(pH = 6.0); B: acetonitrile/potassium phosphate, 80:20]. Product 1
eluted between 44 and 49% B within 24 min. The combined prod-
uct fractions were concentrated by lyophilisation, and buffer salts
were removed by preparative HPLC on a C18 reversed-phase col-
umn with a water/acetonitrile gradient (A: H2O; B: acetonitrile).
After lyophilisation and drying in high vacuum, the potassium salt
This work was supported by the U. S. National Institutes of Health
(grant CA132121, to L. T.).
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1
of 1 was obtained as a white solid. Yield: 33 mg (51%). H NMR
(CD3OD, 500 MHz): δ = 0.86 (s, 3 H, CH3- pantetheine), 0.89 (t,
J = 7.0 Hz, 3 H, CH3-CH2-), 1.06 (s, 3 H, CH3- pantetheine), 1.27
[s, 20 H, -(CH2)10-], 1.52 (m, 2 H, -CH2-CH2-CO-), 1.70 (m, 2 H,
-CO-CH2-CH2-), 2.44 (m, 6 H, -NHCO-CH2-, -CH2-CO-CH2-),
3.13 (m, 2 H, -CO-CH2-CH2-CH2-), 3.45 (m, 2 H, -NHCO-CH2- [15] K. Kawasaki, M. Masubuchi, K. Morikami, S. Sogabe, T. Aoy-
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CH2-), 3.81 (ABX, J1 = 9.4, J2 = 4.5 Hz, 2 H, -CH2-O-), 4.06 (s, 1
H, -CHOH-), 4.35 (m, 2 H, ribose 5Ј-position), 4.50 (m, 1 H, ribose
4Ј-position), 4.75 (m, 1 H, ribose 2Ј-position), 6.09 (d, J = 5.2 Hz,
1 H, ribose 1Ј-position), 8.26 (s, 1 H, adenine C-2 position), 8.68
(s, 1 H, adenine C-8 position) ppm. 13C NMR (CD3OD, 500 MHz):
δ = 13.45 (CH3-CH2), 21.15 (CH3-), 22.74 (CH2), 23.20 (CH3-),
23.50 (CH2), 23.85 (CH2), 23.86 (CH2), 29.33 (CH2), 29.49 (CH2),
29.60 (CH2), 29.65 (CH2), 29.67 (CH), 29.75 (CH2), 29.76 (CH2),
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38.73 (CH2), 39.12 [-C(CH3)2-], 39.54 (CH2), 42.53 (CH2), 72.36
(CH2), 74.09 [-CH(OH)-], 74.96 (CH), 83.28 (CH), 88.41 (CH),
88.43 (C), 110.00 (C), 172.82 (-HN-CO-), 174.61 (-HN-CO-),
212.31 (-CO-) ppm. 31P NMR (CD3OD, 500 MHz): δ = –9.57 (m,
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3Ј-position ribose) ppm. MS-FAB (glycerol): m/z (%) = 1035.9
(100) [M – 2 H + 2 K]+, 998.3 (88) [M + K]+, 959.3 (8) [M]+.
HRMS-FAB (glycerol): calcd. for C36H64N7O17P3K 998.3209;
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Received: December 4, 2009
Published Online: February 16, 2010
Eur. J. Org. Chem. 2010, 1728–1735
© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
1735