Synthesis and biological activities of flavolipids

Article abstract of DOI:10.1016/j.tet.2010.09.088

Syntheses of the bacterial surfactants 6S,6S-, 9S,9S-, and 9U,9U-flavolipids confirmed the structures proposed for them from spectroscopic analysis of a flavolipid mixture and made pure flavolipids available for the first time. All three synthetic flavolipids and a straight chain analogue were found to be weakly cytotoxic and to inhibit metastatic cancer cell migration, with 9U,9U-flavolipid (the most abundant natural flavolipid) having the most activity. Biosynthetic routes to the branched side-chains of the flavolipids are suggested, and it is proposed that branched chains are employed to hinder biodegradation.

Full text of DOI:10.1016/j.tet.2010.09.088

Tetrahedron 66 (2010) 9107e9112
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis and biological activities of flavolipids
,
a
b
Samiul M. Ahad a, Alison L. Ange a, Robert B. Bates a , Bonnie L. Bell , Adria A. Bodour ,
Bryan R. Bourne a, Cristina G. Contreras a, Emily L. Goldberg a, A.A. Leslie Gunatilaka c, Sheryl King a,
Albert K. Lee a, Rebecca L. Lowa, Raina M. Maier b, Kathryn M. Marlor a, Marilyn T. Marron c,
Ryan C. Scolnik a, Matthew J. Streeter a, Malgorzata Strelczuk a, Long N. Trinh a, Vu K. Truong a,
Sage P. Vissering a, Megan C. Weick a, Maria T. Williams a
*
a Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ 85721, United States
b Department of Soil, Water and Environmental Science, University of Arizona, Tucson, AZ 85721, United States
c Southwest Center for Natural Products Research, University of Arizona, Tucson, AZ 85721, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 April 2010
Received in revised form 24 September 2010
Accepted 27 September 2010
Available online 1 October 2010
Syntheses of the bacterial surfactants 6S,6S-, 9S,9S-, and 9U,9U-flavolipids confirmed the structures
proposed for them from spectroscopic analysis of a flavolipid mixture and made pure flavolipids available
for the first time. All three synthetic flavolipids and a straight chain analogue were found to be weakly
cytotoxic and to inhibit metastatic cancer cell migration, with 9U,9U-flavolipid (the most abundant
natural flavolipid) having the most activity. Biosynthetic routes to the branched side-chains of the fla-
volipids are suggested, and it is proposed that branched chains are employed to hinder biodegradation.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Biosurfactant
Flavobacterium
Flavolipid
Siderophore
1. Introduction
Flavolipid names include the number of carbons in each
branched fatty acid and whether it is saturated (S) or has E-a,b-
From the surface exudate of soil bacteria of the genus Fla-
vobacterium from Mt. Lemmon, Arizona, 37 closely related new
carboxylic acids termed ‘flavolipids’ (1) were recently character-
ized.1 (The name ‘flavolipid’ has also been used for other com-
pounds.2) The Mt. Lemmon flavolipids are of interest for possible
use in bioremediation as reported by Bodour and co-workers1 and
because we have found them to be weakly cytotoxic and to inhibit
metastatic cancer cell migration.
unsaturation (U).1 The number of carbon atoms in the branched
chain ‘secondary fatty acids’ of the flavolipids varies from six to ten,
consistent with fatty acid biosynthesis from valine via isobutyryl
CoA (/6, 8, and 10) and isoleucine via isovaleryl CoA (/7 and
9).3,4 Decarboxylation of another amino acid, lysine, is very likely
the source of the cadaverine (1,5-diaminopentane) grouping in
flavolipids.
2. Results
OH
H
R
N
N
CO2H
OH
We now report the synthesis of two minor flavolipids (6S,6S-
flavolipid, 2; 9S,9S-flavolipid, 3) and the major one (9U,9U-fla-
volipid, 4, 23% of the flavolipid mixture1). The syntheses confirm
the structures determined by NMR and mass spectral methods for
these flavolipids and make available pure flavolipids, which differ
in chain length (2 vs 3) and unsaturation (3 vs 4) for testing pur-
poses. The two synthetic methods we employed were based on
methods used by Miller and Lee5 and Phanstiel and co-workers6e8
to make related compounds with unbranched side-chains; we also
synthesized one of these unbranched siderophores (acinetoferrin
C5 homologue, ACH, 56) to be able to compare the properties of
straight chain (5) and branched chain (4) siderophores.
1
flavolipids
10
6
O
O
2
R
U-flavolipid chain (CH3)2CH(CH2)nCH=CH- n=0-4
S-flavolipid chain (CH3)2CH(CH2)nCH2CH2- n=0-4
13
* Corresponding author. Tel.: þ1 520 621 6317; fax: þ1 520 621 6407; e-mail
address: batesr@u.arizona.edu (R.B. Bates).
0040-4020/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2010.09.088

9108
S.M. Ahad et al. / Tetrahedron 66 (2010) 9107e9112
For the cadaverine unit, the first synthesis used bromide 75 and
OH
N
H
N
the second used cadaverine derivative 8.6 In the preparation of 8
from cadaverine, it was important to remove all of the di-Boc-ca-
daverine from the mono-Boc-cadaverine chromatographically to
avoid later byproducts.
CO2H
OH
2
6S,6S-flavolipid
9S,9S-flavolipid
O
O
2
OH
N
H
N
CO2H
3
OBz
OH
O
O
2
Br
NHBoc
HN
NHBoc
CO2H
8
OH
N
7
9
H
N
CO2H
4
9U,9U-flavolipid
The branched acid (4-methylpentanoic acid, 9) needed for
OH
O
O
O
O
2
2
6S,6S-flavolipid (2) synthesis was commercially available. The
branched acid 12, synthesized as shown in Scheme 1 from 5-
methylhexanol (10) by Swern oxidation10 followed by reaction of
aldehyde 11 with the dianion of diethylphosphonoacetic acid,11 was
used for 9S,9S- and 9U,9U-flavolipids (3 and 4).
OH
N
H
N
CO2H
5
acinetoferrin C5
homologue (ACH)
OH
6S,6S-Flavolipid (2) was assembled as shown in Scheme 2 using
benzyl (Bn) protection of the N-hydroxyl groups. 1-(3-Dimethyla-
minopropyl)-3-ethylcarbodiimide hydrochloride (EDC) was used in
the coupling to give hydroxamide derivative 13 to facilitate the
removal of the urea byproduct; this intermediate was made by Sibi
and co-workers by a different method.12 When intermediate 14 was
purified to 99þ% by chromatography (the main impurity was the
O-alkylation product, formed in about 20% yield5), the remaining
reactions went in good yields. The NMR and mass spectra
(including the MS/MS fragmentation peaks) of synthetic 6S,6S-
flavolipid (2) were as expected, confirming the structure proposed
earlier (the chemical shifts of the synthetic flavolipids were slightly
different than reported for the flavolipid mixture1 since the NMRs
of the synthetics were measured in CD3OD and the natural fla-
volipid mixture was measured in CD3OD plus a few drops of D2O to
dissolve the sample more completely).1 In both synthetic routes,
after the protecting groups on the hydroxamic acids were removed,
it was important to measure the NMR spectra in deuteromethanol,
since in deuterochloroform the absorptions of the protons near the
hydroxamic acid group were greatly exchange-broadened.
For the citric acid unit, both our synthetic routes used citric acid
derivative 67 with tert-butyl (t-Bu) ester protection of the central
carboxyl group to avoid the imide formation,8 which occurs when
the central carboxyl is benzyl-protected. During our early attempts
to use benzyl ester protection of the central carboxyl group of citric
acid (abandoned due to severe problems with imide formation), we
increased the yield of anhydromethylenecitric acid, the first in-
termediate in the preparation of the reagent used for benzyl pro-
tection, from 50%9 to 70% by heating citric acid with a 2-fold excess
of paraformaldehyde at 190 ꢀC with shaking for 2 h on a Parr ap-
paratus, cooling, adding water (100 mL for each gram of crude
product), decanting from undissolved paraformaldehyde, and col-
lecting the crystalline product in two crops.
O
O
O
CO2But
OH
N
O
2
6
(EtO)2P(O)CHLiCO2Li
CO2H
Swern
OH
CHO
10
11
12
Scheme 1. Synthesis of E-7-methyl-2-octenoic acid (12).
R = Me2CH(CH2)2
BnONH2
1"
OBn
NH
OBn
7
, K2CO3
RCO2H
R
1
R
R
N
NHBoc
1
1'
5
EDC
9
O
13
O
14
TFA
OR"
N
6
OBn
N
, Et3N
H
N
NH3+ TFA-
R
CO2R'
OH
O
O
O
15
2
R'=But, R"=Bn
R'=But, R"=H
R'=R"=H
16
H2/Pd
TFA
17
2
Scheme 2. Synthesis of 6S,6S-flavolipid (2).

S.M. Ahad et al. / Tetrahedron 66 (2010) 9107e9112
9109
The method of Scheme 2 was unsuitable for 9U,9U-flavolipid (4)
since the carbonecarbon double bonds would be reduced to single
bonds during the removal of the benzyl groups. Instead, the
Phanstiel method6e8 shown in Scheme 3, using benzoyl (Bz) in-
stead of benzyl (Bn) protection of the N-hydroxyl groups, was used
to synthesize 9U,9U-flavolipid (4), which was then hydrogenated to
9S,9S-flavolipid (3). The NMR, mass spectra, and MS/MS spectra of
synthetic 9U,9U- and 9S,9S-flavolipids (4 and 3) were as expected,
confirming the structures proposed earlier.1
cancer cell lines: NCI-H460 (non-small cell lung cancer) and MDA-
MB-231 (metastatic breast carcinoma). Cells were exposed to serial
dilutions of test compounds for 72 h and then cell viability was
determined as follows. The cells were plated at low density in RPMI
1640 media supplemented with 10% fetal bovine serum. The fol-
lowing day compounds 2e5 were added and after 72 h, the viability
was evaluated by the MTT assay.13 All compounds were tested at 10
and 20 mM and found to be weakly cytotoxic to these cell lines with
inhibition ranging from 20% to 60% at 20 mM (data not shown).
Scheme 3. Synthesis of 6U,6U-siderolipid (4) and 9S,9S-siderolipid (3).
All four synthetic lipids 2e5 were purified by repeated chro-
matography on Sephadex LH-20, eluting with 8% ethanol in tolu-
ene, and eventually crystallized as waxy solids. They were stored
at ꢁ80 ꢀC to minimize cyclization to imides.8
9U,9U-Flavolipid (4), the most abundant natural flavolipid, was the
most active.
Compounds 2e5 were also tested for their cell migration in-
hibitory activity in the metastatic prostate cancer cell line, PC-3M.14
All showed weak to moderate activity compared to the DMSO
control, with 9U,9U-flavolipid 4 again being the most active (Fig. 1).
The three synthetic flavolipids 2e4 and straight chain analogue
ACH 5 were evaluated for in vitro cytotoxic activity against two
Fig. 1. Effect of compounds 2e5 on migratory activity of the metastatic prostate cancer cell line, PC-3M. (A) DMSO control (negative); (B) LY294002 control (positive) at 7.5
(C) 6S,6S-flavolipid (2) at 20 M; (D) 9S,9S-flavolipid (3) at 20 M; (E) 9U,9U-flavolipid (4) at 20 M; (F) ACH (5) at 20 M.
mM;
m
m
m
m

9110
S.M. Ahad et al. / Tetrahedron 66 (2010) 9107e9112
3. Discussion
readily with straight chains.21 This allows them longer lifetimes in
the environment to carry out their roles as iron chelators. It may
also account for the greater activity of 9U,9U-flavolipid (4) in
comparison to the straight chain analogue ACH (5) in both the
cytotoxicity and cancer cell migration tests performed in this study.
The syntheses of pure 6S,6S- (2), 9S,9S- (3), and 9U,9U-fla-
volipids (4) verify the structures proposed for them from NMR and
mass spectral analysis of the natural mixture of 37 flavolipids.1
The findings that these three pure flavolipids are cytotoxic and
inhibit cell migration prompted us to synthesize a straight chain
analogue (ACH, 5) and test it also, to learn the effects of branched
chains versus straight chains in these biological tests. All four
compounds had some activity in both tests, with 9U,9U-flavolipid
(4) being more active than the other compounds. In view of these
results, we next consider possible reasons why flavobacteria might
4. Experimental
4.1. General procedures
NMR spectra were obtained at 500 or 600 MHz in CDCl3 except
for hydroxamic acids, for which CD3OD was used to avoid line-
broadening caused by rotation in the hydroxamic acid unit. ESI mass
spectra were measured on a Bruker 9.4 Tesla Apex Qh spectrometer.
Flavolipids and other compounds without tert-butyl ester protection
of the central citric acid carboxyl group were stored in a ꢁ80 ꢀC
freezer since they slowly lose water to give imides at room tem-
perature.8 Sephadex LH-20 was purchased from SigmaeAldrich;
other chemicals were purchased from Aldrich.
favor the chain lengths they do, why most have a,b-unsaturation,
and why the branched chains of the flavolipids might be preferable
to the straight chains of the related siderophores.
3.1. Chain length
Many bacteria produce siderophores to scavenge iron since iron
availability in soils is limited.15,16 Much of the iron in the environ-
ment is in tightly bound mineral forms and it is possible that the
hydrophobic nature of the flavolipids with their 16e20 chain
lengths1 provides an advantage in associating with and extracting
iron from hydrophobic surfaces. Rhamnolipid biosurfactants, which
also have metal-chelating ability, have been shown to associate
strongly with mineral surfaces and to extract metals from these
surfaces.17,18 The flavolipids may similarly enhance extraction from
mineral surfaces. Following extraction, the cell-membrane-com-
ponent-like nature of flavolipids could facilitate delivery back to the
producing cell. The mechanism of delivery of iron to the cell has
been shown for acinetoferrin (23, produced by the pathogenic
bacterium Acinetobacter haemolyticus), whose high membrane af-
finity (partition coefficient Kx¼6.8ꢂ105) allows it to deliver scav-
enged iron back to the cell via the membrane.19
4.2. 5-Methylhexanal (11)10
[Note: this aldehyde evaporates rapidly at room temperature
and should be kept in a tightly closed container.] Oxalyl chloride
(5.6 mL, 64.6 mmol) in CH2Cl2 (100 mL) was cooled in dry ice and
dimethylsulfoxide (DMSO, 4.8 mL, 67.8 mmol) was added drop-
wise. After 10 min, triethylamine (45 mL, 322 mmol) was added
and the cold bath was removed. After standing overnight, the so-
lution was washed 2ꢂ with saturated NH4Cl, 2ꢂ with saturated
NaCl, and dried over Na2CO3. CH2Cl2 and volatile byproducts were
slowly distilled off through a Vigreux column. Ether (3ꢂ50 mL) was
added and distilled off. To remove further Et3N, ether (50 mL) was
added and the solution was rewashed with saturated NH4Cl
(2ꢂ10 mL), brine (2ꢂ10 mL), dried, and most of the ether was dis-
OH
N
H
N
CO2H
23
acinetoferrin
OH
O
O
2
OH
N
H
N
CO2H
24
nannochelins
A-C: R = H or Me
OH
O
O
2
RO
O
3.2.
a
,b-Unsaturation
tilled off, leaving what NMR showed to be a quantitative yield of 4:1
aldehyde 11/ether, used in the next reaction. 1H NMR (CDCl3)
0.89
d
The natural flavolipid mixture contains 11 U,U-flavolipids, which
comprise 65% of the natural flavolipid mixture.1 We conclude that
(d, J¼6.8 Hz, H6), 1.21 (m, H4), 1.55 (nonet, J¼6.5 Hz, H5), 1.64 (m,
H3), 2.42 (m, H2), 9.77 (t, J¼2.0 Hz, H1).
a,b-unsaturation is important for making Flavoacterium competitive
4.3. (E)-7-Methyl-2-octenoic acid (12)11
in the environment. It is noteworthy that there are other bacterial
siderophores with a,b-unsaturated carbonyls in two chains: acine-
toferrin (23),6 which has three methylenes instead of five in the
diamine, and nannochelins AeC (24), which are weak growth
To tetrahydrofuran (THF, 250 mL) chilled in dry ice/acetone,
butyllithium solution (2.5 M in hexane, 95 mL, 238 mmol) was
added. Diethylphosphonoacetic acid (7.2 mL, 107 mmol) was added
dropwise with stirring. After 0.5 h, 5-methylhexanal (11, 12.2 g,
107 mmol) was added dropwise. After 0.5 h, the solution was stir-
red overnight at 25 ꢀC. Excess NaHCO3 was added and most of the
THF was evaporated in a hood. The aqueous solution was washed
with ether (3ꢂ100 mL), acidified with concd HCl to pH 4, and
extracted with ether (3ꢂ100 mL). Distillation of the ether through
a Vigreux column left acid 12 (7.0 g, 42%). It could be used directly
or purified by silica gel chromatography, eluting with 15% ethyl
inhibitors of bacteria and fungi.20 It is difficult to see how trans-
a,b-
unsaturation, which changes the molecular geometry of these side-
rophores verylittle, could enhance iron scavenging; perhaps it serves
a second purpose such as inhibiting cell division in other bacteria
through Michael-type additions tothe carbonecarbon double bonds.
3.3. Branched chains
We propose that flavolipids have rare branched-chain fatty acids
to avoid the oxygenase-catalyzed biodegradation, which occurs
acetate in hexanes. 1H NMR (CDCl3)
d
0.88 (d, J¼7.7 Hz, H8), 1.20 (q,

S.M. Ahad et al. / Tetrahedron 66 (2010) 9107e9112
9111
J¼7.4 Hz, H6), 1.47 (p, J¼7.5 Hz, H5), 1.55 (n, J¼6.7 Hz, H7), 2.22 (q,
J¼7.2 Hz, H4), 5.83 (d, J¼15.5 Hz, H2), 7.08 (dt, J¼15.5, 6.7 Hz, H3),
oil (2.2 g) to be a mixture of the desired diamide 16 with DCU,
which was used in the next reaction. 1H NMR (CD3OD)
0.88 (d,
d
10.10 (s, CO2H); 13C NMR (CDCl3)
d
22.5 (C8), 25.7 (C5), 27.8 (C7),
J¼6.0 Hz, H16), 1.45 (s, t-Bu), 2.37 (m, H13), 2.56 and 2.66 (d,
32.5 (C4), 38.4 (C6),120.7 (C2),152.2 (C3),171.6 (C1); MS (ESIꢁ): m/z
155.1079 (calcd for C9H15O2, 155.1078) [MꢁH]ꢁ.
J¼14.5 Hz, H3), 3.20 (q, J¼6.4 Hz, H6), 3.62 (t, J¼7.0 Hz, H10), 4.80 (s,
CH2O), 6.65 (m, NH), 7.3e7.4 (m, ArH); 13C NMR (CDCl3)
d 22.9
(C16), 25.2 (C8), 27.7 (C15), 28.3 (C17), 29.2 (C7), 30.1 (C9), 30.9 (C3),
31.6 (C13), 35.0 (C14), 40.3 (C6), 45.2 (C10), 75.4 (C2), 77.3 (C21),
83.5 (C18), 129.9 (C23), 130.2 (C25), 130.8 (C24), 136.3 (C22), 172.1
(C4), 174.3 (C1), 177.2 (C12); MS (ESIþ): m/z 825.5363 (calcd for
C46H73N4O9, 825.5372) [MþH]þ.
4.4. N-Benzyloxy-4-methylpentanamide (13)12
A solution of 4-methylpentanoic acid (9, 3.53 mL, 28.1 mmol),
O-benzylhydroxylamine hydrochloride (4.50 g, 28.1 mmol), EDC
(5.4 g, 28.1 mmol), triethylamine (3.9 mL, 28.1 mmol), and 4-
(dimethylamino)pyridine (DMAP, 343 mg, 2.8 mmol) in dichloro-
methane (DCM, 65 mL) was stirred overnight at 25 ꢀC. After solvent
evaporation, the residue was taken up in EtOAc and washed with
1 M HCl, 3ꢂNaHCO3, water, and brine. Evaporation gave 13 (5.7 g,
4.8. 6S,6S-Flavolipid tert-butyl ester (17)
Benzyl-protected ester 16 (1.90 g, 2.30 mmol) in EtOH (50 mL)
was stirred with 10% Pd/C catalyst under H2 for 2 h. Filtering off
solids, washing them with EtOH, and evaporating left a quantitative
92%). 1H NMR (CDCl3)
d
0.88 (d, J¼7.1 Hz, H5), 1.52 (q, J¼6.2 Hz, H3),
1.55 (n, J¼7.0 Hz, H4), 2.04 (m, H2), 4.92 (s, OCH2), 7.38 (br s, ArH);
yield of ester 17 as a colorless oil. 1H NMR (CD3OD)
d 0.93 (d,
13C NMR (CDCl3)
d
22.1 (C5), 27.5 (C4), 31.0 (C2), 34.1 (C3), 77.8 (C20),
J¼6.5 Hz, H16), 1.48 (s, t-Bu), 1.63 (p, J¼7.4 Hz, H9), 2.47 (t, J¼7.7 Hz,
128.3 (C40), 128.3 (C60), 129.0 (C50), 135.4 (C30), 171.3 (C1); MS
H13), 2.58 and 2.67 (d, J¼14.5 Hz, H3), 3.16 (t, J¼7.0 Hz, H6), 3.59 (t,
(ESIþ): m/z 222 [MþH]þ.
J¼7.0 Hz, H10); 13C NMR (CD3OD)
d 22.9 (C16), 25.1 (C8), 28.3 (C15),
28.3 (C17), 29.3 (C7), 30.1 (C9), 31.6 (C13), 40.4 (C6), 45.2 (C10), 75.4
(C2), 83.5 (C18), 172.2 (C4), 180.5 (C12) MS (ESIþ): m/z 645.4426
(calcd for C32H61N4O9, 645.4433) [MþH]þ.
4.5. N-Boc-N0-benzyloxy-N0-(40-methylpentanoyl)-cadaverine
(14)
A mixture of 13 (3.2 g, 14.5 mmol), 5-(Boc-amino)-1-pentyl
bromide (2.14 g, 8.1 mmol), K2CO3 (8.0 g, 58 mmol), and KI
(240 mg, 1.5 mmol) in acetone (50 mL) was refluxed overnight.
After evaporation of the acetone, ether (75 mL) was added and the
solution was washed with 0.5 M NaOH (3ꢂ10 mL). Evaporation of
the ether left 3.2 g (97%) of a 4:1 mixture of 14 with the O-alkyl-
ation byproduct. Repeated silica gel chromatography, eluting with
20% ethyl acetate/hexanes, gave (after the O-alkylation product)
4.9. 6S,6S-Flavolipid (2)
tert-Butyl ester 17 (1.50 g, 2.33 mmol) was stirred with tri-
fluoroacetic acid (TFA, 5 mL) for 30 min. TFA was evaporated and
chloroform (2ꢂ5 mL) was added and evaporated, leaving 2 (1.37 g,
100%) as an oil, which was chromatographed on Lipophilic
Sephadex to give waxy crystals, mp 91e92 ꢀC. 1H NMR (CD3OD)
d
0.92 (d, J¼6.0 Hz, H16), 1.32 (p, J¼7.0 Hz, H8), 1.48 (q, J¼6.8 Hz,
99þ% pure 14. 1H NMR (CDCl3)
d
0.88 (d, J¼5.8 Hz, H50), 1.30 (p,
H14), 1.52 (p, J¼7.3 Hz, H7), 1.57 (n, J¼6.4 Hz, H15), 1.62 (p, J¼6.9 Hz,
H9), 2.46 (t, J¼7.8 Hz, H13), 2.61 and 2.71 (d, J¼14.5 Hz, H3), 3.16 (t,
J¼7.3 Hz, H3), 1.44 (s, Boc), 1.50 (q, J¼6.6 Hz, H30), 1.55 (n, J¼7.4 Hz,
H40), 1.64 (p, J¼7.5 Hz, H4), 2.38 (t, J¼7.8 Hz, H20), 3.10 (q, J¼6.6 Hz,
H1), 3.62 (t, J¼7.3 Hz, H5), 4.63 (br s, NH), 4.80 (s, ArCH2), 7.38e7.39
J¼7.0 Hz, H6), 3.58 (t, J¼7.0 Hz, H10); 13C NMR (CD3OD)
d 22.9 (C16),
25.1 (C8), 28.3 (C15), 29.3 (C7), 30.1 (C9), 31.6 (C13), 39.7 (C14), 40.3
(C6). 43.3 (C3), 45.5 (C10), 73.9 (C2), 172.7 (C4), 176.5 (C12), 177.0
(C1); MS (ESIꢁ): m/z 587.3665 (calcd for C28H51N4O9, 587.3662)
[MꢁH]ꢁ; MS/MS on 587: 569(7) [MꢁH2O]ꢁ; 551(100) [Mꢁ2H2O]ꢁ;
471(4) [MꢁH2Oꢁketene]ꢁ; 435(9) [Mꢁ3H2Oꢁketene]ꢁ; 309(18)
[C16H22O4N2]ꢁ.
(m, ArH); 13C NMR (CDCl3)
d
22.3 (C50), 26.5 (C3), 27.9 (C40), 28.4
(C5000), 29.3 (C4), 29.5 (C2), 30.4 (C20), 33.4 (C30), 40.4 (C1), 45.0 (C5),
76.3 (C200), 79.1 (C4000), 128.7 (C400), 128.9 (C600), 129.1 (C500), 134.6
(C300), 156.0 (C2000), 175.3 (C10); MS (ESIþ): m/z 407.2901 (calcd for
C23H39N2O4, 407.2904) [MþH]þ.
4.6. N0-Benzyloxy-N0-(40-methylpentanoyl)-cadaverinium
triflate (15)
4.10. 7-Methyl-2-octenoyl chloride (18)
To (E)-7-methyl-2-octenoic acid (12, 4.5 g, 28.8 mmol) in dry
benzene (60 mL) was added DMF (10 drops) followed by oxalyl
chloride (14.6 g, 57.6 mmol) dropwise. After 30 min, the volatiles
were blown off with argon, leaving acid chloride 18 containing
DMF, which was used directly in the next reaction. 1H NMR (CDCl3)
Boc derivative 14 (1.48 g, 3.65 mmol) was stirred with tri-
fluoroacetic acid (TFA, 2ꢂ10 mL) for 1 h and the TFA was evaporated
in a hood. Ether was added and evaporated three times to remove
excess TFA, leaving amine salt 15 (1.53 g, 100%). 1H NMR (CDCl3)
d
0.85 (d, J¼6.9 Hz, H50), 2.32 (t, J¼7.3 Hz, H20), 2.90 (br s, H1), 3.61
d
0.88 (d, J¼6.3 Hz, H8), 1.20 (m, H6), 1.5 (m, H5, H7), 2.28 (q,
J¼6.1 Hz, H4), 6.08 (d, J¼14.2 Hz, H2), 7.24 (dt, J¼14.2, 6.6 Hz, H3);
13C NMR (CDCl3)
22.3 (C8), 25.3 (C5), 27.6 (C7), 32.5 (C4), 38.1 (C6),
(m, H5), 4.77 (s, ArCH2), 7.35 (br s, ArH), 8.00 (br s, NH); 13C NMR
d
22.0 (C50), 26.0 (C3), 27.6 (C40), 27.9 (C2), 29.7 (C4), 30.2 (C20), 33.7
d
(C30), 44.5 (C5), 76.5 (C200), 128.3 (C400), 129.2 (C500), 129.3 (C600),
134.6 (C300), 177.1 (C10); MS (ESIþ): m/z 307.2380 (calcd for
C18H31N2O2, 307.2380) [RNH3]þ.
1256.0 (C2), 157.5 (C3), 165.1 (C1).
4.11. (E)-N-Boc-N0-benzoyloxy-N0-(70-methyl-20-octenoyl)-
cadaverine (19)
4.7. O,O-Dibenzyl-6S,6S-flavolipid tert-butyl ester (16)
A solution of benzoyl peroxide (4.6 g, 19 mmol) in DCM (70 mL)
was added dropwise to a vigorously stirred mixture of 8 bi-
carbonate in a carbonate buffer solution (pH 10.5, 100 mL).8 After
16 h, a solution of freshly prepared acid chloride 18 (2.25 g,
12.9 mmol) in DCM (10 mL) was added dropwise. After 3 h, the
organic layer was separated and the aqueous layer was washed
2ꢂDCM. The organic layer was dried (Na2SO4), the solution was
decanted, and the solvent was evaporated. The residue was chro-
matographed on silica gel, eluting with 50% EtOAc/hexanes, giving
To 15 (1.85 g, 4.4 mmol) in CH2Cl2 (20 mL) at 0 ꢀC was added
Et3N (4 mL). This solution was added to a freshly prepared solution
of citric acid derivative 6 (2 mmol) in dioxane (50 mL).7 After stir-
ring overnight, dicyclohexylurea (DCU) was filtered off and the
solvents were evaporated. The residual oil was dissolved in CHCl3
and washed quickly with 0.5 M HCl. The aqueous layer was
extracted with CHCl3, the combined organic layers were dried over
Na2SO4, and the solvent was evaporated. NMR showed the residual

9112
S.M. Ahad et al. / Tetrahedron 66 (2010) 9107e9112
19 as an oil (3.5 g, 59%). 1H NMR (CDCl3)
d
0.81 (d, J¼6.9 Hz, H80),
J¼14.0 Hz, H3), 3.16 (t, J¼6.5 Hz, H6), 3.65 (t, J¼6.6 Hz, H10), 6.61 (br
d, J¼15.0 Hz, H13), 6.84 (dt, J¼15.0, 7.3 Hz, H14); 13C NMR (CD3OD)
1.13 (q, J¼7.8 Hz, H60), 1.43 (s, t-Bu), 1.69 (p, J¼7.2 Hz, H4), 2.13 (q,
J¼7.1 Hz, H40), 3.11 (m, H1), 3.87 (m, H5), 4.61 (br s, NH), 6.05 (d,
J¼15.0 Hz, H20), 7.02 (dt, J¼15.0, 7.0 Hz, H30), 7.53 (t, J¼7.5 Hz, 500),
d
23.1 (C19), 25.1 (C8), 27.5 (C16), 28.3 (C18), 29.2 (C7), 30.0 (C9),
33.8 (C15), 39.8 (C17), 40.3 (C6), 43.3 (C3), 45.2 (C10), 75.5 (C2),
120.7 (C13), 148.2 (C14), 168.5 (C12), 177.4 (C4), 177.0 (C1); MS
(ESIꢁ): m/z 667.4276 (calcd for C34H59N4O9, 667.4288) [MꢁH]ꢁ; MS/
MS on 667: 631, 529, 511, 475, 349.1
7.68 (t, J¼7,5 Hz, 600), 8.11 (d, J¼7.5 Hz, 400); 13C NMR (CDCl3)
d 22.4
(C80), 22.5 (C4), 23.8 (C3), 27.8 (C50), 27.8 (C70), 28.4 (C5000), 29.6 (C2),
32.7 (C40), 38.3 (C60), 40.3 (C1), 48.3 (C5), 79.0 (C4000), 118.0 (C20),
128.9 (C500), 129.6 (C300), 130.0 (C400), 133.4 (C600), 149.4 (C30), 156.0
(C4), 164.5 (C10), 170.3 (C200); MS (ESIþ): m/z 461.3002 (calcd for
C26H41N2O5, 461.3010) [MþH]ꢁ; [MþH]þ.
4.16. 9S,9S-Flavolipid (3)
9U,9U-Flavolipid (4, 76 mg) in EtOH (5 mL) was stirred with 10%
Pd/C under H2 for 2 h. Filtering off the solids, washing with EtOH,
and evaporating the solvent gave 3 (72 mg, 94%) as waxy crystals,
4.12. (E)-N-Boc-N0-hydroxy-N0-(70-methyl-20-octenoyl)-
cadaverine (20)
mp 96e98 ꢀC. 1H NMR (CD3OD)
d
0.87 (d, J¼6.6 Hz, H19), 1.18 (q,
Using ‘iron-free’ glassware and silica gel,6 a 10% solution of
NH4OH/MeOH (60 mL) was added to benzoyl-protected hydroxa-
mic acid 19 (3.45 g, 7.5 mmol) at 0 ꢀC. After stirring for 2 h, the
volatiles were evaporated. Benzene and then chloroform were
added and evaporated and the residue was chromatographed on
silica gel, eluting with 25% EtOAc/hexanes, to give 20 as an oil (2.7 g,
J¼6.9 Hz, H17), 1.32 (br s, H8, H15, H16), 1.51 (p, J¼6.6 Hz, H7), 1.53
(n, J¼7.0 Hz, H18), 1.59 (p, J¼7.0 Hz, H14), 1.62 (p, J¼7.3 Hz, H9), 2.45
(t, J¼7.2 Hz, H13), 2.60 and 2.71 (d, J¼15.1 Hz, H3), 3.17 (t, J¼6.6 Hz,
H6), 3.59 (t, J¼6.9 Hz, H10); 13C NMR (CD3OD)
d 23.2 (C19), 25.1
(C8), 26.2 (C16), 27.5 (C14), 28.4 (C18), 29.3 (C7), 30.0 (C9), 30.9
(C15), 33.5 (C13), 40.2 (C17), 40.3 (C6), 43.3 (C3), 45.2 (C10), 75.5
(C2), 172.5 (C4), 176.2 (C12), 177.0 (C1); MS (ESIꢁ): m/z 671.4593
(calcd for C34H63N4O9, 671.4601) [MꢁH]ꢁ; MS/MS on 671: 635, 513,
477, 351.1
100%). 1H NMR (CD3OD)
d
0.89 (d, J¼6.5 Hz, H80), 1.42 (s, t-Bu), 2.23
(q, J¼6.9 Hz, H40), 3.02 (t, J¼6.7 Hz, H 1), 3.65 (t, J¼6.6 Hz, H5), 6.61
(br d, J¼15.5 Hz, H20), 76.84 (dt, J¼15.5, 6.8 Hz, H30); 13C NMR
(CD3OD) d
23.1 (C80), 25.0 (C3), 27.5 (C50), 28.9 (C500), 28.8 (C70), 29.1
(C2), 30.7 (C4), 33.8 (C40), 39.7 (C60), 41.3 (C1), 79.9 (C400), 120.7
(C20), 148.1 (C30), 158.7 (C200), 168.5 (C10); MS (ESIþ): m/z 357.2748
(calcd for C19H37N2O4, 357.2748) [MþH]þ.
Acknowledgements
This work was supported by grant number 0714245 from the
National Science Foundation and by a Camille and Henry Dreyfus
Foundation Senior Scientist Mentor Initiative Award to R.B.B.
4.13. (E)-N0-Hydroxy-N0-(70-methyl-20-octenoyl)-
cadaverinium triflate (21)
Supplementary data
Boc derivative 20 was treated analogously to 14, producing
a quantitative yield of triflate 21. 1H NMR (CD3OD)
d 0.89 (d,
Supplementary data associated with this article can be found in
the online version at doi:10.1016/j.tet.2010.09.088.
J¼6.6 Hz, H80), 1.56 (n, J¼6.7 Hz, H70), 2.23 (q, J¼7.3 Hz, H40), 2.92 (t,
J¼7.3 Hz, H5), 3.68 (t, J¼6.8 Hz, H1), 6.63 (d, J¼15.5 Hz, H20), 6.84
(dt, J¼15.5, 7.0 Hz, H30); 13C NMR (CD3OD)
d
23.1 (C80), 24.6, 27.2,
References and notes
27.5 (C50), 28.2 (C70), 30.9, 33.8 (C40), 39.8 (C60), 40.8 (C1), 118.1 (q,
J¼87.0 Hz, CF3), 119.6 (C20), 148.4 (C30), 163.2 (q, J¼13.5 Hz, CCF3),
172.6 (C10); MS (ESIþ): m/z 257.2222 (calcd for C14H29N2O2,
257.2224) [RNH3]þ.
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4.14. 9U,9U-Flavolipid tert-butyl ester (22)
This was prepared in 60% yield analogously to 16 but starting
with 20. 1H NMR (CD3OD)
d
0.89 (d, J¼6.6 Hz, H8), 1.45 (s, Boc), 1.67
(p, J¼6.7 Hz, H4), 2.23 (q, J¼7.0 Hz, H4), 2.58 and 2.67 (d, J¼14.7 Hz,
H200), 3.16 (t, J¼6.3 Hz, H10), 3.65 (t, J¼6.6 Hz, H50), 6.63 (br d,
J¼15.5 Hz, H2), 6.83 (dt, J¼15.5, 7.0 Hz, H3); 13C NMR (CD3OD)
d
23.1 (C19), 25.1 (C8), 27.5 (C16), 28.3 (C18), 28.3 (C20), 29.2 (C7),
30.1 (C9), 30.9 (C3), 33.8 (C15), 39.8 (C17), 40.3 (C6), 45.2 (C10), 75.4
(C2), 83.5 (C21), 120.7 (C13), 148.2 (C14), 168.5 (C12), 172.1 (C4),
174.3 (C1); MS (ESIþ): m/z 725.5043 (calcd for C38H69N4O9,
725.5059) [MþH]þ.
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Boc derivative 22 (976 mg, 1.35 mmol) was stirred with TFA
(5 mL) for 15 min and the TFA was evaporated. Benzene and then
2ꢂ chloroform was added and evaporated and the residue was
chromatographed on Lipophilic Sephadex, eluting with 8% EtOH/
toluene to give 4 (585 mg, 65%), mp 98e100 ꢀC. 1H NMR (CD3OD)
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0.89 (d, J¼6.1 Hz, H19), 1.22 (q, J¼7.4 Hz, H17), 1.34 (p, J¼7.6 Hz,
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H8), 1.48 (p, J¼7.9 Hz, H16), 1.53 (p, J¼7.5 Hz, H7), 1.55 (n, J¼7.0 Hz,
H17), 1.66 (p, J¼7.4 Hz, H9), 2.23 (q, J¼6.8 Hz, H15), 2.61 and 2.71 (d,