A novel methodology for the synthesis of fumarates and maleates

Article abstract of DOI:10.1055/s-2006-948166

The stereoselectivity of both the Wittig and the Horner-Wadsworth-Emmons reactions allows for the synthesis of orthogonally protected fumarates and maleates, respectively, from α-keto esters. This methodology has been shown to be useful in the synthesis of a derivative of the pH sensitive cis-aconitic linker, important for prodrug approaches in drug delivery. We have incorporated this linker into a cholesterol-based PEGylated lipid, for its potential use in liposomal drug and gene delivery. The synthesized lipids have also been shown to be pH degradable using HPLC analyses. Georg Thieme Verlag Stuttgart.

Full text of DOI:10.1055/s-2006-948166

LETTER
1933
A Novel Methodology for the Synthesis of Fumarates and Maleates
S
ynthesis of Fumarate
s
a
M
aleat
y
a
Imperial College Genetic Therapies Centre, Department of Chemistry, Flowers Building, Armstrong Road, Imperial College London,
London, SW7 2AZ, UK
Fax +44(20)75945803; E-mail: a.miller@imperial.ac.uk
b
IC-VEC Ltd., Flowers Building, Armstrong Road, London, SW7 2AZ, UK
E-mail: m.jorgensen@icvec.com
Received 24 February 2006
LD particles usually enter cells by a mechanism known as
endocytosis, a process that leads to their encapsulation
within intracellular compartments known as endosomes.9
These endosomes ‘mature’ with time and eventually fuse
Abstract: The stereoselectivity of both the Wittig and the Horner–
Wadsworth–Emmons reactions allows for the synthesis of ortho-
gonally protected fumarates and maleates, respectively, from a-keto
esters. This methodology has been shown to be useful in the synthe-
sis of a derivative of the pH sensitive cis-aconitic linker, important with lysosomes; a process whereby the pH of the compart-
ment changes from pH 7.4 to slightly acidic pH 5.10 Many
for prodrug approaches in drug delivery. We have incorporated this
linker into a cholesterol-based PEGylated lipid, for its potential use
of the triggerable systems currently being studied focus on
in liposomal drug and gene delivery. The synthesized lipids have
this subtle pH change to facilitate the shedding of a
also been shown to be pH degradable using HPLC analyses.
‘stealth’ layer. The main classes of bioconjugate linkers
Key words: Wittig, Horner–Wadsworth–Emmons (HWE), ozonol-
ysis, cis-aconitic, PEGylated lipids
(usually incorporated between the lipid and PEG
moieties) that have been shown to undergo acid-mediated
hydrolysis include di-ortho esters,11 hydrazones,12 vinyl
ethers13 and the cis-aconitic acid linker.14,15
The use of cationic liposomes for the delivery of nucleic
The cis-aconityl moiety has been shown to hydrolyse
acids has shown great promise in recent years,1 but has yet
under mildly acidic conditions through the intramolecular
to provide a therapeutic solution. For efficient in vivo ap-
formation of an unsaturated, cyclic anhydride15 and
plications, which often require extended blood-circulation
release the potent anti-cancer drugs doxorubicin and
times,2 liposome-nucleic acid (LD) systems require the
daunomycin from HMPA polymers.16
attachment of a hydrophilic polymeric ‘stealth’ layer
In this communication, we present a methodology that al-
lows for the facile synthesis of orthogonally protected
maleate and fumarate functional groups. This methodolo-
gy evolved from the earlier synthesis of a pH-sensitive
sugar–lipid conjugate within our laboratories,17 and from
a study whereby we demonstrated the controlled and se-
lective incorporation of the pH sensitive cis-aconitic link-
er into a PEGylated cholesterol-based lipid (1a–c,
Figure 1).
[commonly, polyethylene glycol (PEG)] to the liposome
surface; usually through a PEG-functionalized lipid.3 The
down-side to these ‘PEGylated’ systems is that PEG
inhibits the release of encapsulated nucleic acids at the
site of therapeutic action.4
One solution to this problem has been to develop triggered
release systems, wherein the PEG layer is naturally
detached from the surface of the LD particles at the site of
therapeutic action.5 Researchers have made use of in-
trinsic cellular properties for this trigger effect, such as,
the change in redox potentials,6 varying enzyme
concentrations7 and changes in pH.8
Previous methods for introducing the cis-aconitic linker
required simple nucleophilic reactions of appropriate
amines with the commercially available cis-aconitic
O
O
H
O
O
N
OH
O
H
H
H
H
n
N
N
O
H
O
1a, n = ~16 (PEG MW 750 g/mol)
1b, n = ~44 (PEG MW 2000 g/mol)
1c, n = ~112 (PEG MW 5000 g/mol)
Figure 1 The pH-sensitive cholesterol based PEGylated lipid (1)
SYNLETT 2006, No. 12, pp 1933–1937
0
1
.0
8
.2
0
0
6
Advanced online publication: 24.07.2006
DOI: 10.1055/s-2006-948166; Art ID: D05906ST
© Georg Thieme Verlag Stuttgart · New York

1934
W. S. Price et al.
LETTER
anhydride.18 However, these conjugates are poorly char-
acterized and cis-aconitic anhydride is known to undergo
spontaneous cis–trans isomerization, leading to the un-
desired, non-pH-sensitive trans product.19 Our previous
synthesis of a glycolipid relied upon incorporating a de-
rivative of the cis-aconityl linker through a de novo syn-
thesis. One of the two key steps involved the treatment of
an a-keto ester with a Horner–Wadsworth–Emmons
(HWE) reagent, which generated an orthogonally protect-
ed maleate. However, this specific approach relied on a
protecting group strategy not viable for general utility.17
Table 1 a-Keto Ester Formation
Entry
R
R¢
Compound Yield (%)
1
2
3
4
Boc
Fmoc
Boc
Fmoc
Me
Me
Bn
Bn
5a
5b
5c
5d
67
69
58
55
We reasoned that treatment of a-keto esters with Wittig or
HWE reagents would result in the selective formation of
the respective fumarates and maleates. Furthermore, the
incorporation of orthogonally protected acids would also
allow for selective deprotections and subsequent regio-
selectivity in further synthetic manipulations.
Second, the a-keto phosphoranes (3a and 3b, Boc- and
Fmoc-protected amines, respectively) were subjected to
ozonolysis, forming the very electrophilic diketo nitrile
(4a,b, not isolated). The nitrile intermediates were treated
in situ with the required alcohol forming the a-keto esters
5a–d (Scheme 2 and Table 1).21 To introduce an ortho-
gonal protection, two different esters were synthesized, a
methyl ester and a benzyl ester, allowing for either acidic,
basic or, in the case of R¢ = Bn, hydrogenolysis deprotec-
tion protocols. Lower yields were obtained for the forma-
tion of the benzyl esters owing to the lower
nucleophilicity of benzyl alcohol.
Using the simple, 2-step route to a-keto esters developed
by Wasserman,20 we first coupled the commercially
available N-protected 6-amino hexanoic acid (2) to tri-
phenyl(phosphoranylidene)acetonitrile to form a-keto
phosphorane 3. The crude compounds were purified by
silica gel flash chromatography to yield the desired prod-
ucts in 90% yields (Scheme 1).
By treatment with classical Wittig and HWE reagents, the
a-keto esters were selectively transformed into the respec-
tive fumarates and maleates. For the synthesis of the
fumarates, the Wittig reagent tert-butoxycarbonyl-
methylene was added slowly to a solution of the a-keto
ester 5a–d in THF at 0 °C (Scheme 3).22 1H NMR was
used to determine the ratios of the Z- and E-products in the
crude mixture, with the distinctive alkene protons show-
ing at dH = 5.6 and 6.5 ppm, respectively. Simple NOE
measurements confirmed the stereochemistry. The two
isomers were easily separated using flash chromatogra-
phy on silica gel, giving excellent yields and good selec-
tivities for the E-products (6a–d, Table 2).
PPh3
i
OH
RHN
RHN
CN
O
O
3a, R = Boc
2a, R = Boc
3b, R = Fmoc
2b, R = Fmoc
Scheme 1 Reagents and conditions: i) N-Boc- or N-Fmoc-protected
amino hexanoic acid 2 (1 equiv), triphenyl(phosphoranylidene)aceto-
nitrile (1 equiv), EDCI (1.3 equiv), DMAP (0.1 equiv), CH2Cl2, 0 °C
to r.t., 12 h.
PPh3
O
O
i
ii
Table 2 Selective Synthesis of Orthogonally Protected Fumarates
5
RHN
CN
RHN
5
CN
RHN
5
OR'
Entry
R
R¢
Compound Yield (%) E:Z
O
O
O
5a, R = Boc, R' = Me
5b, R = Fmoc, R' = Me
5c, R = Boc, R' = Bn
5d, R = Fmoc, R' = Bn
3a, R = Boc
3b, R = Fmoc
4a, R = Boc
4b, R = Fmoc
1
2
3
4
Boc
Fmoc
Boc
Fmoc
Me
Me
Bn
Bn
6a
6b
6c
6d
72
71
76
79
10:1
10:1
8:1
Scheme 2 Reagents and conditions: i) a-keto phosphorane 3,
CH2Cl2, O3, –78 °C, 20 min; ii) flushed mixture with dry N2 before
adding the relevant alcohol (1.2 equiv).
8:1
O
O
OR'
RHN
RHN
OR'
i
O
O
O
5a, R = Boc, R' = Me
5b, R = Fmoc, R' = Me
5c, R = Boc, R' = Bn
5d, R = Fmoc, R' = Bn
6a, R = Boc, R' = Me
6b, R = Fmoc, R' = Me
6c, R = Boc, R' = Bn
6d, R = Fmoc, R' = Bn
Scheme 3 Reagents and conditions: i) a-keto ester 5 (1 equiv), tert-butylcarbonylmethylene triphenylphosphorane (2 equiv), THF, 0 °C, 15 h.
Synlett 2006, No. 12, 1933–1937 © Thieme Stuttgart · New York

LETTER
Synthesis of Fumarates and Maleates
1935
O
O
OR'
O
i
RHN
RHN
OR'
O
O
5a, R = Boc, R' = Me
5b, R = Fmoc, R' = Me
5c, R = Boc, R' = Bn
5d, R = Fmoc, R' = Bn
7a, R = Boc, R' = Me
7b, R = Fmoc, R' = Me
7c, R = Boc, R' = Bn
7d, R = Fmoc, R' = Bn
Scheme 4 Reagents and conditions: i) a-keto ester 5 (1 equiv), tert-butyl-P,P¢-dimethyl phosphonacetate (1 equiv), NaH (1 equiv), THF,
0 °C, 3 h.
The maleates were formed using the HWE reagent, tert- maleate 9, was proved by ozonolysis followed by an oxi-
butyl-P,P¢-dimethylphosphonoacetate. This reagent was dative work-up,24 to afford a-keto methyl ester 5a in 96%
first added dropwise to a solution of NaH in dry THF gen- yield (Scheme 6).
erating the anion, which was then added dropwise to the
The resultant acid 9 was activated with isobutyl chloro-
formate and then the in situ generated mixed anhydride
was treated with cholesteryloxy-3-carbonyl-1,2-diamino-
ethane, affording cholesterol-derivative 10 in good yield
a-keto ester.23 Again, 1H NMR (and NOE measurements)
was used to determine the ratios of the E- and Z-products
from the crude mixture, with the two isomers easily sepa-
rated by flash chromatography on silica gel. The reaction
(79%). The removal of the Boc group was achieved with
gave excellent selectivity for the maleates 7a–d, in good
a 1 M solution of hydrogen chloride in dry EtOAc.25 The
resultant amine hydrochloride 11 was reacted with the
yields (Table 3). The reduced yields for 7c and 7d can be
reasonably explained by the steric interactions between
respective commercially available (Nektar Corporation,
the benzyl and tert-butyl groups. It is important to note
USA) NHS-activated PEG esters giving the PEGylated
lipids 12a–c (PEG 750, 2000 and 5000, respectively).
that such steric interactions did not influence the E/Z-
selectivites. Interestingly for these maleates the alkene
Analytical HPLC was used to monitor the PEG coupling
proton signal could be observed as a triplet owing to small
reactions. The final step required base-mediated hydroly-
sis of the methyl esters of 12a–c to give the acid-labile
4J coupling (J = 1.4 Hz.).
PEGylated lipids 1a–c as the regio- and stereopure
maleates.
Table 3 Selective Synthesis of Orthogonally Protected Maleates
Entry
R
R¢
Compound Yield (%)
E:Z
The rate of hydrolysis of the acid sensitive PEG lipids
1a–c was evaluated at pH 5 and pH 7.4 in 0.1 M phosphate
buffered saline (PBS) incubated at 37 °C. The percentage
of degradation was determined by HPLC (Figure 2).
1
2
3
4
Boc
Fmoc
Boc
Fmoc
Me
Me
Bn
Bn
7a
7b
7c
7d
78
68
50
51
4:96
5:95
4:96
5:96
The methodology developed to provide the selective in-
corporation of the maleic moiety required for the forma-
tion of the cis-aconitic linker was next applied to the
synthesis of three novel pH-triggerable, cholesterol-based
PEG lipids (1a–c). In addition, the methodology devel-
oped to generate the fumaric moiety should find applica-
tions in the synthesis of non-pH-sensitive controls for our
biological studies. To determine the effect of the PEG
length on the hydrolysis rates of the linker, three PEG
polymers were used: PEG 750 (n = 16), PEG 2000
(n = 44) and PEG 5000 (n = 112), to give their respective
lipids 1a, 1b and 1c.
Figure 2 The degradation of the acid labile lipids 1a–c
At physiological pH and temperature the lipids appear to
be stable, with £1% hydrolysis over five hours. In a more
acidic environment of pH 5, however, up to 25% of the
lipids are hydrolyzed within the same time frame, thus
demonstrating the pH sensitivity of the lipids. One obser-
vation that is clear from the data is that there was a signif-
icant difference in the hydrolysis rates of the linker
depending on the length of the PEG polymer used. One
possible explanation is that in solutions the lipids organize
into entropically stabilized structures called micelles
where the longer PEG lipids, arranged on the outside of
the micelle, have sufficiently large ‘hydrated’ spheres that
Thus, the a-keto methyl ester 5a, was treated with tri-
methyl phosphonoacetate under the HWE conditions to
afford the corresponding dimethyl maleate 8 (confirmed
1
by H NMR and NOE measurements) in excellent yield
(79%, Scheme 5). Maleate 8 was then subjected to regio-
selective hydrolysis of the less substituted methyl ester by
using 1.1 equivalents of the LiOH. The regioselectivity of
Synlett 2006, No. 12, 1933–1937 © Thieme Stuttgart · New York

1936
W. S. Price et al.
LETTER
O
O
O
O
O
i
5
5
ii
BocHN
O
O
BocHN
O
5
BocHN
O
OH
O
9
5a
8
O
O
O
5
5
H2N
O
BocHN
O
O
iii
iv
O
H
H
N
N
N
Chol
N
H
Chol
H
O
11
10
O
O
O
O
5
v
O
N
H
O
O
n
H
N
N
Chol
H
12a, n = ~16 (PEG MW 750 g/mol)
12b, n = ~44 (PEG MW 2000 g/mol)
12c, n = ~112 (PEG MW 5000 g/mol)
O
O
O
5
O
N
OH
vi
O
n
H
H
N
N
H
Chol
O
1a, n = ~16 (PEG MW 750 g/mol)
1b, n = ~44 (PEG MW 2000 g/mol)
1c, n = ~112 (PEG MW 5000 g/mol)
Scheme 5 Reagents and conditions: i) trimethyl phosphonoacetate (1 equiv), a-keto ester 5a (1.1 equiv), NaH (1 equiv), THF, 0 °C, 2 h
(79%); ii) LiOH (1.1 equiv), THF–H2O 3:1, 0 °C, 1 h (78%); iii) isobutyl chloroformate (1.1 equiv), NMM (2 equiv), after 10 min N¢-chole-
steryloxy-3-carbonyl-1,2-diaminoethane (1.1 equiv) was added, CH2Cl2, 0 °C to r.t., 12 h (79%); iv) 1 M HCl in EtOAc, 1 h (95%); v) PEG-
NHS (0.9 equiv), NMM (1 equiv), CH2Cl2, r.t., 12 h, 12a (69%), 12b (67%), 12c (58%); vi) 10% LiOH in THF–H2O 3:1, 0 °C, 20 min, 1a
(79%), 1b (72%), 1c (71%); NOTE: Chol = cholesteryloxy.
limit the free exchange of protons to the acid-sensitive
linkers. Although the lipids were shown to be acid labile
the true test is to see how such molecules will behave in
LD particles for nucleic acid delivery in in vitro cellular
models.
References and Notes
(1) (a) Kosterelos, K.; Miller, A. D. Chem. Soc. Rev. 2005, 970.
(b) Fenske, D. B.; Cullis, P. R. Methods Enzymol. 2005, 7.
(2) Gabizon, A.; Papahadjopoulos, D. Proc. Natl. Acad. Sci.
U.S.A. 1998, 6949.
(3) Woodle, M. C.; Matthey, K. K.; Newman, M. S.; Hidayat, J.
E.; Collins, L. R.; Redemann, C.; Martin, F. J.;
Papahadjopolous, D. Biochim. Biophys. Acta 1992, 1105,
193.
O
O
5
BocHN
O
i
BocHN
5
O
(4) (a) Holland, J. W.; Hui, C.; Cullis, P. R.; Madden, T. D.
Biochemistry 1996, 35, 2618. (b) Keller, M.; Harbottle, R.
P.; Perouzel, E.; Colin, M.; Shah, L.; Rahim, A.; Vaysse, L.;
Bergau, A.; Moritz, S.; Brahimi-Horn, C.; Coutelle, C.;
Miller, A. D. ChemBioChem 2003, 4, 286.
(5) Guo, X.; Szoka, F. C. Jr. Acc. Chem. Res. 2003, 36, 335.
(6) Kirpotin, D.; Hong, K.; Mullah, N.; Papahadjopoulos, D.;
Zalipsky, S. FEBS Lett. 1996, 388, 115.
OH
O
9
5a
O
Scheme 6 Reagents and conditions: i) maleate 9, CH2Cl2, O3,
–78 °C, 20 min; ii) flushed mixture with dry N2 before adding PPh3
(4 equiv, 96%).
In conclusion, we have developed a facile, stereoselective
synthesis for the formation of fumarates and maleates
using Wittig and HWE reactions, respectively, in combi-
nation with Wasserman’s a-keto ester procedure. We
have presented the utility of this methodology for the
development of steroidal lipids that are temporarily
PEGylated through an engineered, pH-sensitive linker
based on the cis-aconityl group.
(7) Pak, C. C.; Ali, S.; Janoff, A. S.; Meers, P. Biochim. Biophys.
Acta 1998, 1372, 13.
(8) Kratz, F.; Beyer, U.; Schutte, M. T. Crit. Rev. Ther. Drug
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(11) (a) Guo, X.; Mackay, J. A.; Szoka, F. C. Biophys. J. 2003,
84, 1784. (b) Guo, X.; Szoka, F. C. Bioconj. Chem. 2001, 12,
291.
Synlett 2006, No. 12, 1933–1937 © Thieme Stuttgart · New York

LETTER
Synthesis of Fumarates and Maleates
1937
(12) (a) Hamann, P. R.; Hinman, L. M.; Hollander, I.; Beyer, C.
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Zhang, Z. Y.; Berstrand, N.; Edwards, K.; Kim, J. M.;
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(14) (a) Alshamkhani, A.; Duncan, R. Int. J. Pharm. 1995, 122,
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(15) Bender, M. L. J. Am. Chem. Soc. 1957, 79, 1528.
(16) (a) Ulbrich, K.; Etrych, T.; Chytil, P.; Jelinkova, M.; Rihova,
B. J. Controlled Release 2003, 33. (b) Yoo, H. S.; Lee, E.
A.; Park, T. G. J. Controlled Release 2002, 17.
(c) Drummond, D. C.; Daleke, D. L. Chem. Phys. Lipids
1995, 27.
atmosphere. After 5 h the reaction was allowed to stir at r.t.
for a further 10 h. The crude mixture was then concentrated
in vacuo and purified using flash chromatography on silica
gel to afford the desired fumarate.
IR (CH2Cl2 film): 3394, 2977, 2935, 1715, 1647, 1518,
1455, 1366, 1273, 1152, 974, 869 cm–1. 1H NMR (400 MHz,
CDCl3): d = 1.32–1.38 [2 H, m, CH2 (CH2)2C=C], 1.40–1.52
(4 H, m, 2 × CH2), 1.43 (9 H, s, 3 × CH3), 1.48 (9 H, s,
3 × CH3), 2.72 (2 H, t, J = 7.6 Hz, CH2C=C), 3.07–3.10 (2
H, m, CH2NH), 3.77 (3 H, s, CH3), 4.52 (1 H, br, NH), 6.65
(1 H, s, trans-alkene). 13C NMR (100 MHz, CDCl3):
d = 26.73, 27.49 (2 × CH2), 28.08, 28.48 [2 × C(CH3)3],
28.78 (CH2), 29.72 (CH2), 40.49 (CH2NH), 52.35 (CH3),
78.94, 81.34 [2 × C(CH3)3], 128.80 (CH=C), 145.80
(CH=C), 155.93 (CO2NH), 164.95, 167.65 (2 × CO). MS
(CI): m/z calcd for C19H34N1O6: 372.2386; found: 372.2382
[M+ + H]; m/z (%) 389 (15) [M+ + NH4], 372 (100) [M+ + H],
333 (76) [M+ – (CH3)3 + NH4], 272 (52) [M+ – (CH3)3CO2].
(23) Typical Procedure for the Formation of Maleates.
To a stirred suspension of NaH [0.01 g, 0.25 mmol (60% in
mineral oil, w/w)] in anhyd THF (1 mL) at 0 °C and under a
nitrogen atmosphere, was added tert-butyl-P,P-
(17) Fletcher, S.; Jorgensen, M. R.; Miller, A. D. Org Lett. 2004,
4245.
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Commun. 1981, 102, 1048.
(19) Remenyi, J.; Balazs, B.; Toth, S.; Falus, A.; Toth, G.;
Hudecz, F. Biochem. Biophys. Res. Commun. 2003, 303,
556.
(20) Wasserman, H. H.; Ho, W.-B. J. Org. Chem. 1994, 4364.
(21) General Procedure for the Formation of the a-Keto
Esters.
dimethylphosphonoacetate (0.05 mL, 0.25 mmol) in anhyd
THF (1 mL) dropwise. After 30 min, the a-keto phosphorane
(0.25 mmol) in THF (2 mL) was added dropwise over 1 h.
After 3 h the excess NaH was neutralized with H2O (0.1 mL)
and the crude mixture concentrated in vacuo, azetroped with
MeOH. The crude mixture was purified using flash
chromatography on silica gel (hexane–EtOAc, 7:1) to afford
the desired maleate. IR (neat): 3400, 2977, 2934, 2863,
1715, 1519, 1367, 1248, 1159, 1066, 1003, 781 cm–1. 1H
NMR (400 MHz, CDCl3): d = 1.32–1.38 [2 H, m, CH2
(CH2)C=C], 1.40–1.52 (4 H, m, 2 × CH2), 1.43 (9 H, s,
3 × CH3), 1.48 (9 H, s, 3 × CH3), 2.31 (2 H, t, J = 7.2 Hz,
CH2C=C), 3.07–3.10 (2 H, m, CH2NH), 3.77 (3 H, s, CH3),
O2 gas was bubbled through a solution of cyano keto
phosphorane (2 mmol) in CH2Cl2 (25 mL) at r.t. for 10 min,
cooled to –78 °C for 5 min. The solution then had O3
bubbled through for 40 min at –78 °C until the color of the
solution had changed to a murky green, via a bright yellow.
Bubbling N2 gas through the solution still at –78 °C for 10
min quenched the excess O3, once the solution returned to
bright yellow in color the alcohol (2.4 mmol) was added.
The solution was then purged with N2 and stirred at –78 °C
for 2 h until the solution turned colorless. The crude product
was purified using flash chromatography on silica gel to
afford the a-keto ester as a clear oil.
4.52 (1 H, br, NH), 5.69 (1 H, t, J = 1.4 Hz, cis-alkene). 13
NMR (100 MHz, CDCl3): d = 25.98 (CH2), 26.64 (CH2),
C
27.91 [t-Bu C(CH3)3], 28.32 [Boc C(CH3)3], 29.68 (CH2),
34.08 (CH2C=C), 40.30 (CH2NH), 52.00 (CH3), 78.96 [Boc
C(CH3)3], 81.33 [t-Bu C(CH3)3], 121.90 (CH=C), 147.83
(CH=C), 155.90 (CONH), 164.07 (CO), 169.25 (CO). MS
(CI): m/z calcd for C19H37N2O6: 389.2651; found: 389.2645
+
+
[M+ + NH4 ]; m/z (%) = 389 (60) [M+ + NH4 ], 372 (80) [M+
+ H], 333 (65), 316 (35), 277 (100) and 216 (33).
(24) Diaz, M.; Branchadell, V.; Oliva, A.; Ortuno, R. M.
Tetrahedron 1995, 51, 11841.
(22) Typical Procedure for the Formation of a Fumarate (6a).
To a solution of the a-keto ester (0.25 mmol) in anhyd THF
(6 mL) was added tert-butoxycarbonylmethylene triphenyl
phosphorane (0.188 g, 0.5 mmol) dropwise (via syringe
pump 2 mL/h) in anhyd THF (4 mL) at 0 °C under a nitrogen
(25) Gibson, F. S.; Bergmeier, S. C.; Rapoport, H. J. Org. Chem.
1994, 59, 3216.
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