Synthesis of new polymerizable metal-chelating lipids

Full text of DOI:10.1021/jo982397j

J . Org. Chem. 1999, 64, 2969-2974
2969
In this paper, we report the synthesis of several metal-
chelating polymerizable lipids. The procedures are gen-
eral enough to allow for the variation of the metal-
chelating headgroup as well as the polymerizable fatty
acid moiety. We routinely prepare these lipids in our
laboratory in moderately good quantity (>250 mg) in high
purity.
Syn th esis of New P olym er iza ble
Meta l-Ch ela tin g Lip id s
Bidhan C. Roy and Sanku Mallik*
Department of Chemistry, North Dakota State University,
Fargo, North Dakota 58105
Received December 8, 1998
Resu lts a n d Discu ssion
The attractive properties of functionalized lipids (bio-
adaptability, two-dimensional fluidity, inherent potential
of self-organization, and pattern formation) make them
aptly suited for numerous biological applications, e.g., for
functionalization of membranes,1 for drug delivery sys-
tems,2 for immobilizing receptors on lipid surfaces,3 for
liposome targeting with antibodies,4 and for biosensor
applications.5
Interest in fabricating stable lipid-based systems (mono-
layers, supported bilayers, and liposomes) has led to the
synthesis of polymerizable lipids. A variety of polymer-
izable moieties, e.g., conjugated diene, conjugated diyne,
styrene, and acrylate moieties, have been incorporated.
The polymerizable fragment can be positioned near the
headgroup, near the hydrophobic tail, or at the middle
of the fatty acids.6 The nature as well as position of the
polymerizable moiety determines the supramolecular
structure formed and polymerization properties of the
lipids.
Recent interest in protein targeting to lipid monolay-
ers7 and liposomes8 requires the synthesis of metal-
chelating lipids. Though some saturated metal-chelating
lipids are reported in the literature,9 reports for the
synthesis of polymerizable counterparts are relatively
few.10 Usually, these synthetic routes provide substan-
tially low yields and the structural diversity of the metal-
chelating lipids are often limited.
We have successfully prepared the polymerizable lipids
1-5 (Figure 1). These lipids have the conjugated diyne
moiety as the polymerizable functionality. Conjugated
dialkynes polymerize efficiently under ultraviolet radia-
tion to generate crystalline cross-linked polymers.11 Lipid
1 is chiral with nitrilotriacetate as the headgroup. The
compound 2 is a chiral lipid with iminodiacetate as the
metal-chelating headgroup. Lipids 3 and 4 have one fatty
acid chain; lipids 4 and 5 have the capability to position
two metal ions 8-12 Å apart in the headgroup. For this
study, the lipid 1 has been prepared as a mixture of
diastereomers and lipids 2 and 5 have been synthesized
in racemic forms using the commercially available race-
mic 2,3-diaminopropanoic acid.
Syntheses of the lipids 1 and 2 are depicted in Scheme
1. The nonpolymerizable counterpart of the lipid 1 has
been reported in the literature.12 Recently, the synthesis
of a polymerizable lipid similar to 1 has been reported.10
A modified procedure was followed to accomplish com-
patibility amongst the protecting groups. The Cbz pro-
tected L-lysine (6) was alkylated with bromoacetic acid
to produce the nitrilotriacetate group. The three carboxy-
lic acid groups were protected as methyl esters to improve
solubility of the product in organic solvents. The hydro-
genation reaction was carried out in the presence of a
dilute formic acid solution (4% in methanol) to prevent
oligomerization of the amino ester 7.
The amino ester 7 was reacted with Boc-protected
racemic 2,3-diaminopropanoic acid (9)13 using BOP
(Castro’s reagent)14 as the coupling reagent. After depro-
tection, the diamine 8 was combined with the polymer-
izable acid (commercially available from GFS Chemicals,
Inc., Gainesville, FL) to afford the ester protected lipid.
Ester groups were hydrolyzed under mild conditions
(LiOH, MeOH-THF)15 to yield the polymerizable lipid 1
as a waxy white solid. The synthesis of the racemic lipid
2 started from the selectively functionalized diamine 10.16
A strategy similar to that of 1 afforded the lipid 2 as a
white waxy solid.
* To whom correspondence should be addressed. Tel: 701-231-8829.
Fax: 701-231-8831. E-mail: mallik@badlands.nodak.edu.
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Kienanen, K.; Aizawa, M. Anal. Chem. 1998, 70, 260-264. Yu, Y.-Y.;
Wie, B. J . V.; Koch, A. R.; Moffett, D. F.; Davies, W. C. Biosens.
Bioelectr. 1997, 12, 135-144.
(6) Cevc, G., Ed. Phospholipids Handbook; Marcel Dekker, Inc.:
New York, 1993.
Scheme 2 shows the synthesis of the polymerizable
lipid 3. This lipid has only one polymerizable fatty acid
moiety. For 3, the polymerizable acid was linked to the
metal-chelating headgroup through the amide linkage.
(7) Pack, D. W.; Chen, G.; Malony, K. M.; Chen, C.-T.; Arnold, F.
H. J . Am. Chem. Soc. 1997, 119, 2479-2487. Dietrich, C.; Schmitt, L.;
Tempe, R. Biochemistry 1996, 35, 1100-1105.
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1998, 14, 3889-3894. Esch, J . H.; Hoffmann, M. A. M.; Notle, R. J . M.
J . Org. Chem. 1995, 60, 1599-1610. Schmitt, L.; Dietrich, C.; Tampe,
R. J . Am. Chem. Soc. 1994, 116, 8485-8491. Singh, A.; Tsao, L.-I.;
Markowitz, M.; Gaber, B. P. Langmuir 1992, 8, 1570-1577.
(10) Dorn, I. T.; Hofmann, U. G.; Peltonen, J . P.; Tampe, R.
Langmuir 1998, 14, 4836-4842. Marlowitz, M.; Puranik, D. B.; Singh,
A. Chem. Phys. Lipids 1995, 76, 63-71. Singh, A.; Tsao, L.-I.; Puranik,
D. B. Synth. Commun. 1995, 25, 573-586. Storrs, R. W.; Tropper, F.
D.; Li, H. Y.; Song, C. K.; Kuniyoshi, J . K.; Sipkins, D. A.; Li, K. C. P.;
Bednarski, M. K. J . Am. Chem. Soc. 1995, 117, 7301-7306.
(11) Kobayashi, S.; Uyama. H. Polish J . Chem. 1994, 68, 417-444.
(12) Gritsch, S.; Neumaler, K.; Schmitt, L.; Tampe, R. Biosens.
Bioelectr. 1995, 10, 805-812.
(13) Serghaert, C.; Maes, P.; Tartar, A. J . Chem. Soc., Perkin Trans.
1 1986, 1061-1064.
(14) Castro, B.; Dormoy, J .-R.; Dourtoglou, B.; Evin, G.; Selve, C.;
Ziegler, J .-C. Synthesis 1976, 751-757.
(15) Mallik, S.; Mallik, I. Synlett 1996, 734-737.
(16) Zuckermann, R. N.; Ronald, N.; Martin, E. J .; Spellmeyer, D.
C.; Stauber, G. B.; Gregory, B.; Shoemaker, K. R. J . Med. Chem. 1994,
17, 2678-2685.
10.1021/jo982397j CCC: $18.00 © 1999 American Chemical Society
Published on Web 03/31/1999

2970 J . Org. Chem., Vol. 64, No. 8, 1999
Notes
F igu r e 1. Structures of the metal-chelating polymerizable lipids synthesized.
bromoacetic acid was carried out according to literature proce-
dure.9 The resultant triacid (3.0 g, 7.57 mmol) was dissolved in
80 mL of dry methanol, p-toluenesulfonic acid (8.69 g, 45.65
mmol) was added, and the solution was heated under reflux for
36 h. Methanol was removed in vacuo. The viscous colorless
liquid was extracted with chloroform, and the combined organic
phase was successively washed with water, 5% aqueous NaH-
CO3, and water and then dried (Na2SO4). The crude product was
purified by silica gel column chromatography with 2% methanol/
chloroform as the solvent (Rf ) 0.3) to afford the pure product
as a viscous liquid. Yield (Cbz-7): 2.80 g (84%). 1H NMR (400
MHz, CDCl3/D2O): δ 1.21-1.37 (m, 4H), 1.51-1.54 (m, 2H),
2.97-3.02 (m, 2H), 3.24 (t, 1H, J ) 7.1 Hz), 3.35-3.38 (m, 4H),
3.47 (s, 3H), 3.51 (s, 6H), 4.96 (s, 2H), 7.15-7.20 (m, 5H). 13C
NMR (75 MHz, CDCl3): δ 23.1, 29.5, 30.1, 41.0, 51.6, 51.9, 52.7,
64.8, 66.7, 128.2, 128.7, 136.9, 156.6, 171.9, 173.1.
The lipids discussed thus far have one metal-chelating
moiety in the headgroup. We are interested in positioning
more than one transition metal ion in a defined pattern
using organic molecules.17 Lipids 4 and 5 were designed
(using the molecular modeling software Spartan 5.0.3,
Wavefunction, Inc., Irvine, CA) to place two metal ions
8-12 Å apart. The synthesis started by reacting tert-butyl
3,5-bis(bromomethyl)benzoate (14)18 with diethyl imino-
diacetate (Scheme 3). It should be noted that the bromide
can be displaced by a variety of other nucleophilic ligands.
After removal of the tert-butyl groups, the resultant
carboxylic acid 15 was combined with the monoprotected
diamine 1016 using BOP reagent. Further elaboration of
the intermediate 16 afforded the lipids 4 and 5 as waxy
white solids.
The triester from the above reaction (Cbz-7, 2.25 g, 5.15 mmol)
was dissolved in 4.4% formic acid solution in MeOH. A small
portion of Pd-black was added, and hydrogen was bubbled
through the solution. Reaction was carried out at room temper-
ature for 6 h. Pd-black was filtered off, and solvent was removed
in vacuo to give the product 7 as a viscous oil. This was taken
Exp er im en ta l Section
Gen er a l Meth od s. Commercial reagents were purchased
from either Aldrich or Acros Chemical Co. and were used without
further purification unless specified otherwise. Experiments
were conducted under an atmosphere of dry nitrogen. Melting
points were determined on a micromelting point apparatus, and
all melting points are uncorrected. 1H NMR and 13C NMR
spectra are recorded using 270, 300, or 400 MHz spectrometers
in one of the following solvents: D2O, CDCl3, CD3OD, and
DMSO-d6 with TMS internal standard. Many of the reported
lipids here contained exchangeable protons (from amine and
amide groups). To remove these signals from the 1H NMR
spectra, one drop of D2O was added to the solvents CDCl3 and
DMSO-d6. MS analyses were obtained from the Mass Spectros-
copy facilities at University of California, Riverside or at the
University of Minnesota, Minneapolis. Elemental analyses were
carried out by in-house materials characterization laboratory.
TLC was performed with Adsorbosil Plus 1P, 20 × 20 cm plate,
0.25 mm (Alltech Associates, Inc.). Chromatography plates were
visualized by either UV light or iodine chamber. For workup,
the organic layer was dried over anhydrous Na2SO4 and con-
centrated in vacuo. The syntheses of polymerizable lipids were
carried out at low temperature in the dark. The compounds were
stored at -20 °C in solution under nitrogen to avoid the
initiation of polymerization.
1
directly to the next step. Yield (7): 1.75 g (88%). H NMR (400
MHz, CDCl3/D2O): δ 1.35-1.60 (m, 4H), 1.65-1.72 (m, 2H),
2.95-3.00 (m, 2H), 3.42 (t, 1H, J ) 6.7 Hz), 3.58 (s, 4H), 3.67 (s,
6H), 3.70 (s, 3H).
Meth yl 6-(2,3-Dia m in op r op a n oyla m in o)-2-bis((m eth oxy-
ca r bon yl)m eth yl) a m in oh exa n oa te (8). To a solution of acid
913 (1.48 g, 4.89 mmol) and 7‚(HCOOH)2 (1.7 g, 4.89 mmol) was
added BOP reagent (2.16 g, 4.89 mmol) followed by the addition
of Et3N (2.27 mL, 19.55 mmol). The reaction was continued for
12 h at room temperature and then quenched with a saturated
solution of NaCl. The product was extracted with ethyl acetate.
The combined organic layer was washed successively with 5%
citric acid, water, 5% NaHCO3, and water. The organic layer
was dried (Na2SO4) and evaporated to provide the crude product.
Purification was achieved by silica gel column chromatography
with 2% MeOH/CHCl3 as the eluant (Rf ) 0.4) to give the pure
product as a viscous liquid. Yield (Boc-8): 2.22 g (77%). 1H NMR
(400 MHz, CDCl3/D2O): δ 1.41-1.57 (m, 22H), 1.67-1.72 (m,
2H), 3.22-3.30 (m, 3H), 3.42-3.48 (m, 2H), 3.67 (m, 4H), 3.66-
3.82 (m, 9H), 4.17-4.20 (m, 1H). 13C NMR (100 MHz, CDCl3):
δ 22.87, 28.37, 28.39, 29.71, 39.19, 39.24, 51.59, 51.86, 52.49,
64.74, 170.57, 171.65, 171.93, 173.12, 173.15.
The di-Boc protected compound (Boc-8, 2 g, 3.40 mmol) was
dissolved in cold trifluoroacetic acid (0-5 °C, 10 mL) and stirred
at room temperature for 2.5 h. TFA was evaporated to dryness
in high vacuo. To remove the residual TFA, the viscous oil was
repeatedly treated with dry CH2Cl2 (10 × 2 mL per batch) and
the dichloromethane was removed under high vacuo. The
resultant oily liquid was quite pure and was taken to the next
Meth yl 6-Am in o-2-(bis((m eth oxyca r bon yl)m eth yl)a m i-
n o)h exa n oa te (7). The reaction of ꢀ-N-Cbz-lysine (6) with
(17) Sun, S.; Saltmarsh, J .; Mallik, S.; Thomasson, K. J . Chem. Soc.,
Chem. Commun. 1998, 519-520.
(18) Guether, R.; Nieger, M.; Rissanen, K.; Voegtle, F. Chem. Ber.
1994, 127, 743-758.

Notes
Sch em e 1. Syn th esis of th e P olym er iza ble
J . Org. Chem., Vol. 64, No. 8, 1999 2971
Sch em e 2. Syn th esis of th e Lip id 3
Meta l-Ch ela tin g Lip id s 1 a n d 2
0.55 g, 1.73 mmol) were dissolved in MeCN (30 mL) in the
presence of Et3N (1.2 mL). BOP reagent (0.76 g, 1.73 mmol) was
added, and the reaction was stirred for 24 h at room tempera-
ture. The workup procedure was same as described for 8. The
crude product was purified by column chromatography (silica
gel, 3% MeOH/CHCl3, Rf ) 0.4). Yield (ester-1): 0.70 g (92%).
1H NMR (400 MHz, CDCl3/D2O): δ 0.85 (t, 6H, J ) 6.8 Hz),
1.22-1.39 (m, 36H), 1.48-1.52 (m, 10H), 1.53-1.65 (m, 6H),
2.15-2.25 (m, 12H), 3.19-3.26 (m, 2H), 3.37-3.41 (m, 2H),
3.59-3.63 (m, 5H), 3.66-3.69 (m, 10H), 3.71-3.74 (m, 1H),
4.38-4.42 (m, 1H). 13C NMR (100 MHz, CDCl3): δ 14.20, 19.22,
19.28, 22.75, 24.73, 25.40, 25.66, 28.12, 28.20, 28.27, 28.42, 28.54,
28.56, 28.65, 28.91, 28.94, 29.17, 29.38, 29.56, 29.66, 31.91, 33.48,
36.48, 51.61, 51.87, 52.53, 64.24, 65.22, 65.48, 77.24, 77.76, 77.79,
170.16, 172.00, 174.59, 176.94.
A solution of lithium hydroxide (0.32 g, 7.7 mmol) in 1:1
MeOH/THF (30 mL) was added to a solution of the trimethyl
ester (ester-1, 0.85 g, 0.86 mmol) at 5 °C under nitrogen. The
reaction was stirred at room temperature for 24 h. The solvents
were evaporated in vacuo, and the white solid was dissolved in
water and acidified to pH ) 3.0 with 1 N HCl. The white
precipitate was filtered and washed with water (250 mL) to
afford the product 1 as a waxy white solid. Yield (lipid-1): 710
1
mg (84%). H NMR (400 MHz, DMSO-d6/D2O): δ 0.85 (t, 6H, J
) 6.6 Hz), 1.24-1.31 (m, 42H), 1.42-1.46 (m, 14H), 2.03 (t, 2H,
J ) 7.1 Hz), 2.10 (t, 2H, J ) 7.1 Hz), 2.25-2.29 (m, 8H), 2.95-
3.05 (m, 1H), 3.25-3.33 (m, 2H), 3.40-3.59 (m, 4H), 4.25-4.31
(m, 1H). 13C NMR (100 MHz, DMSO-d6): δ 14.51, 18.86, 22.67,
23.65, 24.92, 25.51, 25.68, 28.24, 28.27, 28.30, 28.60, 28.64, 28.66,
28.76, 28.97, 29.00, 29.25, 29.47, 29.51, 29.93, 31.87, 35.88, 54.53,
55.12, 65.91, 65.93, 78.41, 78.46, 170.17, 172.68, 173.27, 173.92,
174.45. FAB m/z (M + H) calcd for C55H88N4O9 949.5, found
949.5. Anal. Calcd for C55H88N4O9‚HCl: C, 67.01; H, 9.10; N,
5.68. Found: C, 66.79; H, 8.75; N, 5.46.
E t h yl 2-(2-(2-(2-(2,3-b is((ter t-b u t oxy)ca r b on yla m in o)-
p r op a n oyla m in o)eth oxy)eth oxy)eth yl)((eth oxyca r bon yl)-
m eth yl)am in o)acetic Acid (11). The mono-Boc-protected amine
1016 (2.38 g, 10.44 mmol), ethylbromoacetate (3.48 g, 20.88
mmol), and anhydrous K2CO3 were mixed in MeCN, and the
resulting mixture was sonicated (125 W bath sonicator) at room
temperature for 12 h. Suspended solids were filtered off, and
solvent was removed under vacuo. Crude viscous oil was purified
by silica gel column chromatography (2% MeOH/CHCl3, Rf )
0.4) to afford the pure product. Yield (Boc-11): 3.60 g (82%). 1H
NMR (400 MHz, CDCl3/D2O): δ 1.18 (t, 6H, J ) 7.0 Hz), 1.35
(s, 9H), 2.89 (t, 2H, J ) 5.5 Hz), 3.20-3.24 (m, 2H), 3.42-3.46
step. Yield (8): 2.30 g (93%). 1H NMR (400 MHz, DMSO-d6/D2O)
δ1.23-1.47 (m, 4H), 1.58-1.62 (m, 2H), 3.00-3.09 (m, 1H),
3.13-3.32 (m, 3H), 3.39 (t, 1H, J ) 7.2 Hz), 3.55-3.61 (m, 13H),
4.05-4.08 (m, 1H). 13C NMR (100 MHz, CD3OD): δ 22.72, 23.00,
29.41, 39.55, 50.63, 50.76, 50.91, 52.18, 52.23, 172.03, 173.29,
173.31.
2-B is ((c a r b o x y m e t h y l)a m in o )-6-(2,3-b is (8′10′-h e n e -
icosa d iyn oyla m in o)p r op a n oyla m in o)h exa n oic Acid (Lip id
1). Under nitrogen, the above-mentioned amine salt (8, 0.62 g,
0.86 mmol) and polymerizable acid (8,10-heneicosadiynoic acid,

2972 J . Org. Chem., Vol. 64, No. 8, 1999
Sch em e 3. Syn th esis of th e Lip id s 4 a n d 5
Notes
(m, 2H), 3.50 (s, 4H), 3.52-3.58 (m, 6H), 4.15 (q, 4H, J ) 7.0
Hz). 13C NMR (100 MHz, CDCl3): δ 14.26, 28.42, 40.36, 53.55,
55.84, 60.47, 70.17, 70.21, 70.24, 70.33, 79.05, 156.07, 171.35.
The Boc group of the amine ester (Boc-11, 2.22 g, 5.28 mmol)
was cleaved with TFA (10 mL). The workup procedure was the
same as described for 8. The free amine was generated from the
TFA salt by passing through a weakly basic anion-exchange
column (Dowex 66) and eluting with methanol. Eluant was
collected until pH ) 7.0, and methanol was evaporated in vacuo
to afford the free amine. Yield (t-Bu-11): 2.88 g (100%). 1H NMR
(400 MHz, CDCl3/D2O): δ 1.27 (t, 6H, J ) 7.2 Hz), 3.21-3.26
(m, 2H), 3.61-3.68 (m, 6H), 3.75 (t, 2H, J ) 4.8 Hz), 3.81-3.86
(m, 2H), 4.14 (q, 4H, J ) 7.2 Hz), 4.30 (s, 4H). 13C NMR (100
MHz, CDCl3): δ 13.7, 40.2, 54.0, 55.82, 63.1, 65.3, 66.5, 70.3,
70.4, 166.0.
The coupling of amine (t-Bu-11, 2.25 g, 4.12 mmol) and acid
9 (1.25 g, 4.12 mmol) was carried out with BOP reagent (1.82 g,
4.12 mmol) in the presence of excess Et3N (5.81 g, 57.5 mmol)
in MeCN. The workup procedure was the same as 8. The crude
oily liquid was purified by silica gel column chromatography (5%
MeOH/CHCl3, Rf ) 0.4) to give compound 11 as viscous oily
liquid. Yield (11): 2.46 g, (98%). 1H NMR (400 MHz, CDCl3/
D2O): δ 1.21 (t, 6H, J ) 7.2 Hz), 1.37 (s, 18H), 3.00 (t, 2H, J )
5.2 Hz), 3.35-3.41 (m, 3H), 3.42-3.49 (m, 3H), 3.51-3.54 (m,
4H), 3.59 (t, 2H, J ) 5.2 Hz), 3.64 (s, 4H), 4.09-4.19 (m, 5H).
13C NMR (100 MHz, CDCl3): δ 14.21, 23.03, 23.75, 28.34, 28.39,
28.91, 30.45, 38.72, 39.18, 53.75, 55.37, 60.92, 69.67, 69.75, 70.34,
170.57, 170.61, 170.93.
2-((2-(2-(2-(2,3-Bis(10′,12′-p en t a cosa d iyn oyla m in o)p r o-
pan oylam in o)eth oxy)eth oxy)eth yl)(car boxym eth yl)am in o)-
a cetic Acid (Lip id 2). The di-Boc-protected compound 11 (2.00
g, 3.30 mmol) was cleaved with TFA (8 mL). The workup
procedure was same as described for 8. The crude product was
used in the next step without any further purification. Yield
(TFA-3): 2.40 g (97%). 1H NMR (400 MHz, DMSO-d6/D2O): δ
1.23 (t, 6H, J ) 7.0 Hz), 3.19 (t, 2H, J ) 5.0 Hz), 3.24-3.30 (m,
3H), 3.32-3.39 (m, 1H), 3.46-3.50 (m, 6H), 3.63 (t, 2H, J ) 5
Hz), 3.94 (s, 4H), 4.10-4.20 (m, 5H).
The coupling of amine (TFA-3) salt (0.7 g, 0.94 mmol) and
10,12-pentacosadiynoic acid (0.7 g, 1.88 mmol) was achieved with
BOP reagent (0.83 g, 1.88 mmol) in the presence of excess Et3N
(1.5 mL, 9.42 mmol) in MeCN/CHCl3 (1:1). The workup proce-
dure is the same as described for 8. The crude product was
purified by silica gel column chromatography with 4% MeOH
in CHCl3 (Rf ) 0.4) to give a colorless waxy semisolid. Yield
(ester-2): 0.95 g (90%). 1H NMR (400 MHz, CDCl3/D2O): δ 0.82
(t, 6H, J ) 7.0 Hz), 1.05-1.25 (m, 50H), 1.27-1.35 (m, 8H),
1.40-1.48 (m, 8H), 1.51-1.56 (m, 4H), 2.10-2.19 (m, 12H), 2.93
(t, 2H, J ) 5.4 Hz), 3.32-3.39 (m, 2H), 3.42-3.48 (m, 4H), 3.52
(s, 4H), 3.54-3.58 (m, 6H), 4.08-4.13 (m, 4H), 4.39-4.43 (m,

Notes
J . Org. Chem., Vol. 64, No. 8, 1999 2973
1H). 13C NMR (100 MHz, CDCl3): δ 14.17, 14.30, 19.22, 19.23,
22.73, 25.55, 25.69, 28.36, 28.38, 28.40, 28.84, 28.90, 29.00, 29.14,
29.24, 29.26, 29.30, 29.38, 29.52, 29.65, 29.66, 29.69, 31.95, 36.49,
36.52, 39.31, 42.25, 53.67, 54.83, 55.84, 60.62, 65.28, 65.30, 65.36,
65.38, 69.59, 70.20, 70.25, 70.29, 77.37, 77.40, 77.57, 77.59,
170.22, 171.40, 174.31, 175.23.
28.82, 28.93, 29.17, 29.39, 29.56, 29.64, 31.97, 35.23, 36.58, 39.25,
65.20, 65.28, 65.53, 70.17, 70.21, 70.39, 77.17, 77.79, 159.50,
173.80
The ester (ester-3, 0.25 g, 0.37 mmol) was dissolved in 88%
cold formic acid (5 mL) and stirred for 48 h under nitrogen at
room temperature. Formic was evaporated in high vacuo, the
resultant waxy solid was repeatedly treated with dry CH2Cl2
(10 × 2 mL each time), and the dichloromethane was removed
under vacuo. The product was obtained as a yellowish semisolid.
Yield (lipid-3‚formate): 0.18 g (88%). 1H NMR (400 MHz, CDCl3/
D2O): δ 0.86 (t, 3H, 7.0 Hz), 1.17-1.42 (m, 20H), 1.47-1.65 (m,
6H), 2.15-2.33 (m, 10H), 3.42-3.46 (m, 2H), 3.53-3.64 (m, 8H),
8.05 (s, 1H). 13C NMR (100 MHz, CDCl3): δ 14.22, 19.20, 19.26,
22.76, 24.68, 25.75, 28.19, 28.26, 28.42, 28.45, 28.53, 28.64, 28.71,
28.93, 28.97, 29.19, 29.39, 29.58, 29.65, 31.95, 34.05, 65.23, 65.29,
65.48, 69.82, 69.94, 70.02, 77.37, 77.77, 168.64, 174.93, 178.39.
FAB m/z (M + H) calcd for C31H52N2O7 581.4, found 581.4. Anal.
Calcd for C31H52N2O7: C, 65.93; H, 9.28; N, 4.96. Found: C,
65.69; H, 9.18; N, 4.70.
Selective hydrolysis of the ester (0.4 g, 0.38 mmol) was carried
out with lithium hydroxide (0.08 g, 1.90 mmol) in THF-MeOH
mixture. After standard workup (same as lipid 1), a white waxy
solid of lipid 2 was obtained. Yield 0.34 g (89%). 1H NMR (400
MHz, CDCl3/D2O): δ 0.85 (t, 6H, J ) 7.0 Hz), 1.18-1.29 (m,
44H), 1.32-1.38 (m, 8H), 1.44-1.52 (m, 8H), 1.54-1.59 (m, 4H),
2.14-2.23 (m, 12H), 3.33-3.38 (m, 2H), 3.42-3.45 (m, 2H),
3.50-3.55 (m, 4H), 3.57-3.61 (m, 5H), 3.80-3.85 (m, 2H), 4.15
(s, 4H). 13C NMR (100 MHz, CDCl3): δ 14.23, 19.29, 22.78, 25.62,
25.74, 28.44, 28.45, 28.94, 28.95, 29.08, 29.20, 29.30, 29.35, 29.44,
29.58, 29.71, 29.73, 29.74, 36.47, 36.53, 54.53, 65.29, 65.37, 69.65,
70.07, 77.32, 77.51, 77.68, 77.70, 170.29, 170.76, 174.92, 175.56.
FAB m/z (M + H): calcd for C63H106N4O9 1063.7, found 1063.7.
Anal. Calcd for C63H106N4O9: C, 71.15; H, 10.05; N, 5.27.
Found: C, 70.79; H, 9.99; N, 5.12.
Sod iu m
3,5-Bis(b is((et h oxyca r b on yl)m et h yl)a m in o-
m eth yl)ben zoa te (15). tert-Butyl 3,5-bis(bromomethyl)ben-
zoate18 (1.21 g, 3.28 mmol) and diethyl iminodiacetate (1.25 g,
6.61 mmol) were dissolved in MeCN, and excess K2CO3 (4.6 g,
32.87 mmol) was added. The resulting reaction mixture was
sonicated (125 W bath sonicator) overnight at room temperature.
Solids were filtered, and solvent was removed in vacuo. The
crude oily product was purified via silica gel column chroma-
tography (2% MeOH/CHCl3, Rf ) 0.3). Yield (t-Bu-ester-15): 1.89
g (98%). 1H NMR (400 MHz, CDCl3): δ 1.25 (t, 12H, J ) 10.4
Hz) 1.57 (s, 9H), 3.54 (s, 8H), 3.95 (s, 4H), 4.15 (q, 8H, J ) 10.4
Hz), 7.62 (s, 1H), 7.86 (s, 2H). 13C NMR (100 MHz, CDCl3): δ
14.3, 28.2, 54.3, 57.5, 60.5, 76.9, 77.2, 81.0, 129.2, 132.4, 133.8,
138.8, 165.8, 171.0.
The ester (t-Bu-ester-15, 1.26 g, 2.17 mmol) was dissolved in
10 mL of TFA and stirred at room temperature for 2.5 h. TFA
was evaporated in vacuo. It was then dissolved in CHCl3 (30
mL) and stirred with an aqueous 5% solution of NaHCO3 for
0.5 h. The organic layer was washed with water, dried (Na2-
SO4), and evaporated to give compound 15, which was used in
the next step without any further purification. Yield (15): 1.08
g (91%). 1H NMR (400 MHz, CDCl3): δ 1.26 (t, 12H, J ) 7.1
Hz), 3.56 (s, 8H), 3.98 (s, 4H), 4.07 (q, 8H, J ) 7.1 Hz) 7.65 (s,
1H), 7.80 (s, 2H). 13C NMR (100 MHz, CDCl3): δ 14.2, 54.2, 57.6,
60.5, 129.7, 130.9, 134.3, 138.8, 169.7, 171.0.
ter t-Bu t yl 2-(2-(2-(2-Am in oet h oxy)et h oxy)et h yl)((ter t-
bu tyloxyca r bon yl)m eth yla m in o) a ceta te (13). Excess di-
amine 12 (40 g, 270 mmol) and Et3N (11 mL, 77 mmol) were
dissolved in chloroform (100 mL) and cooled to -45 °C. Cbz
chloride (13.13 g, 77 mmol in 70 mL of CHCl3) was added by
syringe pump over 3 h. The resulting solution was stirred at
room temperature for 12 h, and the organic layer was washed
with water, dried (Na2SO4), and then evaporated. The crude
product was purified by silica gel column chromatography (20%
MeOH/CHCl3, Rf ) 0.2) to afford the pure compound as a
colorless viscous oil. Yield (Cbz-13): 12.61 g (58%). 1H NMR (400
MHz, CDCl3/D2O): δ 2.82 (t, 2H, J ) 5.1 Hz), 3.35-3.39 (m,
2H,), 3.47 (t, 2H, J ) 5.1 Hz), 3.53-3.66 (m, 6H), 5.07 (s, 2H),
7.31-7.34 (m, 5H).
To a solution of mono-Cbz-diamine (4.00 g, 15.02 mmol) and
Proton Sponge (6.42 g, 30.00 mmol) in CHCl3 (30 mL) at 5 °C
was added dropwise a solution of tert-butyl bromoacetate in
CHCl3. After 12 h, the precipitate was filtered, and the solvent
was removed in vacuo. The solid was suspended in AcOEt,
cooled, and filtered. The AcOEt layer was washed with water,
and the crude product was purified by silica gel column chro-
matography (with 17% MeOH/chloroform, Rf ) 0.3) to afford the
pure compound as a viscous oil. Yield (t-Bu-ester-13): 6.41 g
(87%). 1H NMR (270 MHz, CDCl3/D2O): δ 1.42 (s, 18H), 2.92 (t,
2H, J ) 5 Hz), 3.34-3.40 (m, 2H), 3.46 (s, 4H), 3.51-3.62 (m,
8H), 5.08 (s, 2H), 7.34 (m, 5H). 13C NMR (75 MHz, CDCl3): δ
28.3, 41.1, 53.4, 56.8, 66.7, 70.2, 70.3, 70.5, 70.6, 81.0, 128.2,
128.6, 128.6, 136.8, 156.8, 171.0.
Eth yl 2-(((5-(N-(2-(2-(2-a m in oeth oxy)eth oxy)eth yl)ca r -
b a m oyl)-3-((b is(et h oxyca r b on yl) m et h yla m in o)m et h yl)-
ph en yl)m eth yl)(eth oxycar bon yl)m eth ylam in o)acetate (16).
The coupling reaction between 15 (1.35 g, 1.82 mmol) and amine
10 (0.45 g, 1.82 mmol) was achieved with BOP reagent (1.09 g,
2.47 mmol) using Et3N (0.7 mL) as a base and MeCN (30 mL)
as the solvent. The workup procedure was the same as 8. The
crude product was purified with column chromatography (silica
gel, 5% MeOH/CHCl3, Rf ) 0.5) to afford the pure compound as
This compound from the previous reaction (t-Bu-ester-13) was
dissolved in 60 mL of MeOH/H2O (20:1), a spatula tip of 5% wet
Pd/C was added, and H2 was bubbled. Total removal of the Cbz
group was achieved after 6 h as monitored by TLC (same solvent
as the reaction). The catalyst was filtered, and the solvent was
removed in a rotary evaporator. The solid was recrystallized from
hexane-chloroform mixture to provide compound 13 as a white
solid. Yield (13): 2.78 g (98%); mp: 60-61 °C. 1H NMR (400
MHz, CDCl3/D2O): δ 1.42 (s, 18H), 2.89 (t, 2H, J ) 4.6 Hz), 3.15
(t, 2H, J ) 4.8 Hz), 3.51-3.55 (m, 6H), 3.65 (t, 2H, J ) 4.8 Hz),
3.74 (t, 2H, J ) 4.8 Hz), 3.94 (t, 2H, J ) 4.8 Hz). 13C NMR (100
MHz, CDCl3): δ 28.2, 40.1, 53.4, 56.0, 67.6, 68.0, 70.4, 70.6, 81.8,
171.0.
1
a viscous oil. Yield (Boc-16): 1.74 g (90%). H NMR (400 MHz,
CDCl3/D2O): δ 1.24 (t, 12H, J ) 7.3 Hz), 1.41 (s, 9H), 3.28-
3.30 (m, 2H), 3.50-3.54 (m, 10H), 3.63 (s, 4H), 3.65 (s, 4H), 3.90
(s, 4H), 4.13 (q, 8H, J ) 7.3 Hz), 7.47 (s, 1H), 7.82 (s, 2H). 13C
NMR (100 MHz, CDCl3): δ 14.3, 28.4, 28.5, 40.3, 54.3, 57.7, 60.6,
70.2, 70.3, 127.2, 132.6, 135.1, 138.8, 156.0, 171.1.
The Boc group of Boc-protected amine-ester (Boc-16, 1.60 g,
2.12 mmol) was cleaved with TFA (5 mL) using the same
procedure as 15. The resultant TFA salt was treated with 4%
aqueous NaHCO3 and extracted with CHCl3. The organic layer
was dried and evaporated to afford the pure 16 as a viscous pale
yellow liquid. Yield (16): 1.35 g (97%). 1H NMR (400 MHz,
CDCl3/D2O): δ 1.26 (t, 12H, J ) 7.1 Hz), 3.17 (t, 2H, J ) 4.7
Hz), 3.63-3.71 (m, 10H), 3.88 (s, 8H), 4.19 (q, 8H, J ) 7.1 Hz),
4.31 (s, 4H), 7.67 (s, 1H), 8.01 (s, 2H). 13C NMR (100 MHz, CD3-
OD): δ 13.01, 53.69, 58.27, 58.30, 61.82, 66.53, 69.98, 129.20,
132.05, 136.02, 138.11, 159.77, 168.26.
2-((3-((Bis(ca r boxym eth yl)a m in o)m eth yl)-5-(N-(2-(2-(2-
(8′,10′-h en eicosa d iyn oyla m in o)eth oxyeth oxy)eth yl)ca r ba -
m oyl)p h en yl)m eth yl)(ca r boxym eth yl)a m in o)a cetic Acid
(Lip id 4). Polymerizable acid (8,10-heneicosadiynoic acid, 0.226
g, 0.71 mmol) and free amine 16 (0.46 g, 0.71 mmol) were coupled
with BOP reagent (0.315 g, 0.712 mmol) in the presence of Et3N
(7.12 mmol). After standard workup (same as lipid 3), the crude
2-((Ca r boxym eth yl)(2-(2-(2-(8′,10′-h en eicosa d iyn oyla m i-
n o)eth oxy)eth oxy)eth yl)a m in o)a cetic Acid (Lip id 3). The
coupling of amine 13 (0.93 g, 2.51 mmol) and polymerizable acid
(8,10-heneicosadiynoic acid, 0.80 g, 2.51 mmol) was achieved
with BOP reagent (1.11 g, 2.51 mmol) as described for 8. The
crude product was purified by silica gel column chromatography
(with 2% MeOH/chloroform, Rf ) 0.4) to give a waxy solid. Yield
(ester-3): 1.40 g (86%). 1H NMR (400 MHz, CDCl3): δ 0.85 (t,
3H, J ) 6.8 Hz), 1.21-1.34 (broad s, 18H), 1.44-1.51 (m, 22H),
1.53-1.62 (m, 2H), 2.14-2.24 (m, 6H), 2.94 (t, 2H, J ) 5.9 Hz),
3.43 (t, 2H, J ) 5.0 Hz), 3.46 (s, 4H), 3.51 (t, 2H, J ) 4.8 Hz),
3.58-3.60 (m, 6H). 13C NMR (100 MHz, CDCl3): δ 14.21, 19.18,
19.27, 22.76, 24.10, 25.65, 28.10, 28.22, 28.39, 28.40, 28.41, 28.64,

2974 J . Org. Chem., Vol. 64, No. 8, 1999
Notes
product was purified by column chromatography (silica gel, 2%
2-(((3-(Bis(carboxymethylamino)methyl)-5-(N-(2-(2-(2-(2,3-bis-
(10′,12′-p en ta cosa d iyn oyla m in o)p r op a n oyla m in o)eth oxy)-
eth oxy)eth yl)ca r ba m oyl)p h en yl)m eth yl)(ca r boxym eth yl)-
a m in o)a cetic Acid (Lip id 5). The polymerizable acid (10,12-
pentacosadiynoic acid, 0.35 g, 0.47 mmol) and the amine 17 (0.35
g, 0.47 mmol) were coupled with BOP reagent (0.47 g, 47 mmol)
in the presence of Et3N (0.3 mL, 14.10 mmol), as described for
lipid 3. The crude product was purified by column chromatog-
raphy (silica gel, 5% MeOH/CHCl3, Rf ) 0.6), and the pure
product was obtained as a waxy white solid. Yield (ester-5): 0.67
g (97%). 1H NMR (400 MHz, CDCl3/D2O): δ 0.84 (t, 6H, J ) 7.0
Hz), 1.15-1.24 (m, 56H), 1.30-1.35 (m, 8H), 1.40-1.48 (m, 8H),
1.53-1.56 (m, 4H), 2.11-2.21 (m, 12H), 3.34-3.39 (m, 2H),
3.44-3.51 (m, 11H), 3.55-3.65 (m, 9H), 3.92 (s, 4H), 4.11 (q,
MeOH/CHCl3, Rf ) 0.3) to give the pure amide as a waxy white
1
solid. Yield (ester-4): 0.55 g (89%). H NMR (400 MHz, CDCl3/
D2O): δ 0.86 (t, 3H, J ) 6.8 Hz), 1.22-1.36 (m, 34H), 2.02-2.29
(m, 8H), 3.41-3.43 (m, 2H), 3.53-3.57 (m, 8H), 3.64-3.67 (m,
10H), 4.00 (s, 4H), 4.15 (q, 8H, J ) 6.8 Hz), 7.54 (s, 1H), 7.85 (s,
2H). 13C NMR (100 MHz, CDCl3): δ 14.21, 19.25, 19.39, 22.76,
24.68, 25.05, 28.21, 28.41, 28.43, 28.50, 28.56, 28.61, 28.63, 28.65,
28.94, 28.96, 29.18, 29.39, 29.56, 29.66, 30.64, 31.22, 31.97, 33.32,
65.22, 65.25, 65.42, 77.31, 129.64, 133.48, 134.73, 141.78, 152.66,
158.09, 173.43.
Selective hydrolysis of the ester groups (ester-4, 0.52 g, 0.58
mmol) was carried out with LiOH (0.29 g, 7.0 mmol) in THF/
H2O as described for lipid 1. Lipid 4 was obtained as a waxy
white solid. Yield (4): 0.35 g (79%). 1H NMR (400 MHz, DMSO-
d6/D2O): δ 0.84 (t, 3H, J ) 4.0 Hz), 1.20-1.29 (m, 16H), 1.39-
1.45 (m, 6H), 2.02 (t, 2H, J ) 7.2 Hz), 2.17-2.25 (m, 6H), 3.16
(t, 2H, J ) 5.6 Hz), 3.34-3.40 (m, 12H), 3.47-3.51 (m, 6H), 3.87
(s, 4H), 7.46 (s, 1H), 7.67 (s, 2H). 13C NMR (100 MHz, DMSO-
d6): δ 14.52, 18.80, 18.83, 22.66, 25.64, 28.19, 28.25, 28.53, 28.60,
28.74, 28.96, 29.22, 29.44, 29.48, 31.86, 35.78, 38.99, 54.02, 57.50,
65.89, 69.48, 69.72, 70.10, 78.49, 78.57, 127.08, 132.31, 135.11,
139.42, 167.01, 172.75, 172.78. FAB m/z (M + H) calcd for
8H, J ) 7.0 Hz), 4.34-4.38 (m, 1H),7.49 (s, 1H), 7.75 (s,2H). 13
C
NMR (100 MHz, CDCl3): δ 14.18, 14.28, 19.23, 19.25, 22.74,
25.55, 25.69, 28.37, 28.41, 28.84, 28.91, 29.00, 29.15, 29.23, 29.26,
29.29, 29.39, 29.53, 29.66, 29.67, 29.69, 31.97, 36.50, 36.56, 39.22,
39.92, 42.15, 43.35, 54.30, 55.18, 57.70, 60.62, 65.28, 65.36, 65.38,
69.56, 70.04, 70.32, 70.35, 77.35, 77.63, 126.95, 132.55, 134.94,
138.84, 167.67, 170.22, 171.08, 174.48, 175.52.
Selective hydrolysis of the ester groups (ester-5, 0.16 g, 0.117
mmol) was carried out with LiOH (0.04 g, 0.95 mmol) in 4 mL
of THF-MeOH at room temperature. The workup procedure was
same as that described for lipid 1. The lipid 5 was a waxy white
solid. Yield (5): 0.128 g (92%). 1H NMR (400 MHz, DMSO-d6/
D2O): δ 0.85 (t, 6H, J ) 6.8 Hz), 1.20-126 (m, 44H), 1.28-1.31
(m, 8H) 1.38-1.46 (m, 12H), 2.02 (t, 2H, J ) 7.4 Hz), 2.09 (t,
2H, J ) 7.4 Hz), 2.25 (t, 8H, J ) 6.9 Hz), 3.16-3.21 (m, 2H),
3.22-3.29 (m, 3H), 3.34-3.44 (m, 9H), 3.48-3.53 (m, 8H), 3.87
(s, 4H), 4.27-4.31 (m, 1H), 7.48 (s, 1H), 7.69 (s, 2H). 13C NMR
(100 MHz, DMSO-d6): δ 14.54, 18.79, 22.70, 25.65, 25.83, 28.28,
28.36, 28.75, 28.81, 28.84, 28.95, 28.97, 28.99, 29.22, 29.24, 29.31,
29.32, 29.33, 29.47, 29.56, 29.61, 31.89, 54.05, 57.54, 65.91, 69.42,
69.45, 69.51, 70.10, 78.49, 127.13, 127.66, 135.15, 139.44, 172.75,
172.79, 172.81. FAB m/z (M + H) calcd for C76H120N6O14 1341.9,
found 1341.9. Anal. Calcd for C76H120N6O14‚HCl: C, 66.23; H,
8.84; N, 6.09. Found: C, 66.56; H, 8.64; N, 6.20.
C
44H66N4O12 843.4, found 843.4. Anal. Calcd for C44H66N4O12
:
C, 62.69; H, 7.89; N, 6.65. Found: C, 62.40; H, 7.87; N, 6.50.
E t h yl 2-(5-(N-(2-(2-(2-(2,3-Dia m in op r op a n oyla m in o)-
eth oxy)eth yl)ca r ba m oyl)-3-bis (eth oxyca r bon yl)m eth yl)-
a m in o)a ceta te (17). Coupling of amine 16 (1.30 g, 1.98 mmol)
and acid 9 (0.61 g, 1.98 mmol) was carried out with BOP reagent
(0.88 g, 1.98 mmol). The crude product was purified by silica
gel column chromatography (2% MeOH/CHCl3, Rf ) 0.4) to afford
the amide as a colorless viscous liquid. Yield (Boc-17): 1.72 g
1
(92%). H NMR (400 MHz, CDCl3/D2O): δ 1.22 (t, 12H, J ) 7.1
Hz), 1.37 (s, 18H), 3.31-3.39 (m, 3H), 3.41-3.51 (m, 11H), 3.56-
3.63 (m, 8H), 3.87 (s, 4H), 4.08-4.16 (m, 9H), 7.45 (s, 1H), 7.78
(s, 2H). 13C NMR (100 MHz, CDCl3): δ 14.28, 28.34, 28.36, 39.28,
39.89, 42.67, 54.31, 57.70, 60.60, 69.64, 70.02, 70.30, 70.34, 76.32,
126.91, 132.51, 134.99, 138.93, 167.68, 170.71, 171.11.
The Boc groups (Boc-17, 1.65 g, 1.76 mmol) were cleaved with
TFA (8 mL). After evaporation of TFA, the resultant viscous oil
was dissolved in excess CHCl3, washed with 4% NaHCO3
solution, dried, and evaporated to afford compound 17 as a
viscous yellow liquid. Yield (17): 1.2 g (92%). 1H NMR (400 MHz,
CDCl3/D2O): δ 1.24 (t, 12H, J ) 7.2 Hz), 3.42-3.46 (m, 3H),
3.49 (s, 9H), 3.56 (t, 2H, J ) 5.1 Hz), 3.61-3.67 (m, 8H), 3.89 (s,
4H), 4.00-4.16 (m, 9H), 7.46 (s, 1H), 7.81 (s, 2H). 13C NMR (100
MHz, CDCl3): δ 14.25, 38.64, 38.76, 39.82, 39.94, 54.35, 57.74,
60.64, 69.68, 70.15, 70.17, 70.24, 77.38, 127.07, 132.43, 134.99,
138.86, 167.67, 167.75, 171.24.
Ack n ow led gm en t. This work was supported by an
IDeA grant from NIH to North Dakota and an AREA
grant (NIH R01-54321-01) and a CAREER award to
S.M.
J O982397J