Synthesis of NTA3-DTDA - A chelator-lipid that promotes stable binding of his-tagged proteins to membranes

Article abstract of DOI:10.1071/CH06112

A six-step reaction sequence is described for the preparation of compound 1 (NTA₃-DTDA), a membrane-penetrating and potent chelator that can be incorporated into liposomes and plasma membrane vesicles containing antigens and thus allowing targe

Full text of DOI:10.1071/CH06112

Full Paper
CSIRO PUBLISHING
Aust. J. Chem. 2006, 59, 302–306
www.publish.csiro.au/journals/ajc
Synthesis of NTA3-DTDA — A Chelator-Lipid that Promotes Stable
Binding of His-Tagged Proteins to Membranes
Joseph G. Altin,A Martin G. Banwell,B,C Phillip A. Coghlan,B Christopher J. Easton,B
Michael R. Nairn,B and Daniel A. OffermannB
A School of Biochemistry and Molecular Biology, Faculty of Science, Australian National University,
Canberra ACT 0200, Australia.
B Research School of Chemistry, Institute of Advanced Studies, Australian National University,
Canberra ACT 0200, Australia.
C Corresponding author. Email: mgb@rsc.anu.edu.au
A six-step reaction sequence is described for the preparation of compound 1 (NTA3-DTDA), a membrane-
penetrating and potent chelator that can be incorporated into liposomes and plasma membrane vesicles containing
antigens and thus allowing targeted delivery of such assemblies to a variety of cells for the purposes of eliciting
anti-tumour responses. Full spectroscopic characterization of this dendritic-type compound as well as certain of its
precursors is reported.
Manuscript received: 3 April 2006.
Final version: 26 April 2006.
Introduction
acid)-ditetradecylamine (NTA3-DTDA or 1; Fig. 1) is a very
useful material in this regard. It can, for example, be used to
anchor, via metal ion bridges, oligo-histidine-tagged forms
of antibody fragments that target dendritic cells onto either
tumour cell-derived PMVs or onto antigen-containing SLs.
Such targeting elicits strong anti-tumour responses. How-
ever, further developments in this promising area have been
restricted by the lack of access to gram quantities of pure
samples of compound 1. Indeed, until now only milligram
samples of this material have been available for research
purposes and these were contaminated with mono-, di-, and
The delivery of antigen-containing stealth liposomes (SLs)
and tumour-derived plasma membrane vesicles (PMVs) to
various immune cells can enhance anti-tumour immunity.
The approach has potential applications in cancer vaccine
development and in establishing tumour immunotherapy
regimes.[1–3] Clearly, the cytoselective targeting of such SLs
and PMVs would significantly enhance the therapeutic value
of the encapsulated or membrane-associated antigens.[1–3]
Recent work by one of us (J.G.A.)[2,3] has revealed that the
membrane-penetrating and potent chelator 3-(nitrilotriacetic
CO2H
HO2C
N
CO2H
O
N
H
O
H
N
N
O
CO2H
CO2H
N
H
N
O
H
N
O
N
HO2C
CO2H
CO2H
N
HO2C
1
Fig. 1.
© CSIRO 2006
10.1071/CH06112
0004-9425/06/050302

Synthesis of NTA3-DTDA
303
CO2H
CO2H
the same conditions as employed in the analogous conversion
5 → 6 and with methanol as reaction solvent. The product
primary amine 8 was then subjected to treatment with triflu-
oroacetic acid (TFA) in order to effect removal of the TMSE
groups and thus generating the target nona-acid 2. However,
ESI mass spectral analysis of this product inevitably revealed
that it was contaminated with significant quantities of the cor-
responding mono-, di-, and tri-methyl ester derivatives.These
by-products presumably arise through carry over of methanol
from the preceding hydrogenolysis step and the participation
of this alcohol in acid-catalyzed trans-esterification and/or
esterificationreactionswhencompound 8istreatedwithTFA.
Various attempts, including those involving the application
of freeze drying techniques, were made to remove residual
methanol from product 8 but methyl ester by-products con-
tinued to contaminate the free acid 2 derived from it. These
difficulties were ultimately addressed by using the much
more weakly nucleophilic solvent 2,2,2-trifluoroethanol in
the hydrogenolysis step. This led to amine 8, the pivotal pre-
cursor to the final target 1, in 98% yield. Cleavage of the
ester residues within compound 8 using TFA then proceeded
to give the polyacid 2 in quantitative yield and, therefore,
uncontaminated with ester by-products.The compound could
be purified by simple washing with toluene, acetonitrile, then
diethyl ether. This process avoided the need to use HPLC
techniques for purification which, as established during ear-
lier studies,[4] necessarily involved methanol as an eluting
solvent and so resulting in further contamination of the tar-
get material by mono-, di-, and tri-methyl ester derivatives.
The ESI mass spectrum of compound 2 revealed a protonated
molecular ion at m/z 995.5 and an accurate mass measure-
ment on this species established that it was of the expected
composition. The 13C NMR spectrum displayed only five
of the expected twelve carbonyl carbon resonances but this
is undoubtedly due to the high degree of similarity of the
relevant atoms within this pseudo-symmetrical system and
the resulting overlap of signals. Indeed, this sort of situation
was encountered throughout the entire series of compounds
described herein.
The completion of the synthesis of compound 1 is shown
in Scheme 2 and involved, in the initial stages, the prepa-
ration of a suitably functionalized synthon corresponding to
the lipophilic tail of this target. Thus, commercially available
1-bromotetradecane was treated with ammonia according
to the procedure of Altin, White, and Easton[5] and in this
way the secondary amine 10 was obtained. Reaction of this
last compound with succinic anhydride in the presence of
triethylamine then afforded succinate 11 (42%). This then
engaged in an EDAC·HCl-promoted coupling reaction with
compound 8 so as to generate, in 72% yield, the TMSE-
protected form, 12, of the final target. Exposure of compound
12 to neat TFA at 0–18◦C for 4 h then gave compound 1 in
92% yield and as a white solid with a broad melting range.
The negative ion ESI mass spectrum of this material revealed
an (M − H)− species at m/z 1485 while a doubly charged
ion was observed at m/z 742 and represented the base peak.
The 13C NMR spectrum of this C72-containing and pseudo-
symmetrical compound displayed thirty-two resonances as
HO2C
N
O
N
H
H
N
CO2H
CO2H
H2N
N
O
O
N
N
HO2C
H
CO2H
CO2H
N
HO2C
2
Fig. 2.
tri-methyl ester derivatives arising from the use of methanol
as a solvent at a pivotal point in the synthesis.[3,4] Accord-
ingly, we detail below a synthesis of NTA3-DTDA that allows
access to pure material in gram quantities. By related means
we have also been able to obtain congener 2 (Fig. 2), incorpo-
rating the polar head group of compound 1 and required for
undertaking control experiments related to the establishment
of the in vivo mode of action of the latter material.
Results and Discussion
The reaction sequence leading to the 2-(trimethysilyl)ethyl
(TMSE) ester protected form, 8, of the head group compound
2 is shown in Scheme 1.Thus, following previously described
protocols[5] that were inspired by the work of Tampé and
co-workers,[6] the commercially available and carbobenzy-
loxy (Cbz) protected form, 3, of lysine was treated with
an excess of α-bromoacetic acid in aqueous alkali. As a
result, and after work-up with mineral acid, the previously
reported[7] triacid 4 was obtained in 93% yield. Esterification
of compound 4 with 2-trimethylsilylethanol using N-(3-
dimethylaminopropyl)-Nꢀ-ethylcarbodiimide hydrochloride
(EDAC·HCl)[8] as coupling agent then afforded triester 5 in
90% yield. This product was characterized by 1H NMR, 13
C
NMR, and IR spectroscopy, as well as by low- and high-
resolution ESI mass spectrometry (MS). All such data were
in full accord with the assigned structure. Hydrogenolytic
cleavage of the Cbz-group within compound 5 was accom-
plished under standard conditions using an atmosphere of
dihydrogen in the presence of 10% palladium on carbon, and
so giving the primary amine 6 (98%) which was coupled,
using EDAC·HCl, with one-third of a molar equivalent of the
precursor triacid 4. In this manner the dendritic-type com-
pound 7 was obtained in 81% yield. Removal of Cbz-group
within dendrimer 7 was achieved hydrogenolytically under

304
J. G. Altin et al.
CO2TMSE
CO2H
NH2
CO2R
N
(a)
(c)
Cbz
Cbz
N
H
N
H
CO2R
H2N
N
CO2TMSE
RO2C
TMSEO2C
4 R ϭ H
5 R ϭ TMSE
3
6
(b)
Compound
(d)
N
4
CO2TMSE
CO2R
CO2R
TMSEO2C
CO2TMSE
RO2C
N
O
N
N
O
N
H
H
(e)
H
N
H
N
CO2TMSE
CO2TMSE
Cbz
CO2R
H2N
N
H
N
O
H
O
N
O
H
O
N
N
N
TMSEO2C
N
RO2C
CO2R
CO2TMSE
CO2R
CO2R
N
TMSEO2C
CO2TMSE
RO2C
8 R ϭ TMSE
2 R ϭ H
7
(f)
Scheme 1. Reagents and conditions: (a) α-Bromoacetic acid (4.3 mol equiv.), NaOH, water, 18–50◦C, 18 h. (b) EDAC·HCl (4.6 mol equiv.),
DMAP (3.3 mol equiv.), 2-(trimethylsilyl)ethanol (4.4 mol equiv.), CH2Cl2, 0–18◦C, 72 h. (c) H2 (1 atm), 10% Pd on C, MeOH, 18◦C, 16 h.
(d) Compound 4 (0.27 mol equiv.), EDAC·HCl (1.3 mol equiv.), DMAP (1.0 mol equiv.), CH2Cl2, 0–18◦C, 72 h. (e) H2 (1 atm), 10% Pd on
C, TFE, 18◦C, 16 h. (f) TFA, 0–18◦C, 4 h.
(a)
1-Bromotetradecane
N
H
9
10
(b)
O
N
OH
O
11
CO2R
Compound
(c)
8
RO2C
N
CO2R
O
N
H
O
H
N
N
CO2R
CO2R
N
H
N
O
H
O
N
O
N
N
RO2C
CO2R
CO2R
12 R ϭ TMSE
1 R ϭ H
(d)
RO2C
Scheme 2. Reagents and conditions: (a) See ref. [5]. (b) See ref. [5]. (c) Compound 8 (0.5 mol equiv.), EDAC·HCl
(1.5 mol equiv.), DMAP (1.5 mol equiv.), CH2Cl2, 0–18◦C, 16 h. (d) TFA, 0–18◦C, 4 h.

Synthesis of NTA3-DTDA
305
a result of the overlap of various signals arising from the
presence of many near equivalent carbons within the struc-
ture. Reversed-phase HPLC analysis of this material revealed
it was an essentially homogeneous material while biologi-
cal evaluations established that the product had the expected
capacity to anchor histidine-tagged proteins onto SLs and
PMVs.
dichloromethane (500 mL) maintained under argon was cooled to 0◦C
then N-(3-dimethylaminopropyl)-Nꢀ-ethylcarbodiimide hydrochloride
(EDAC·HCl) (18.00 g, 93.9 mmol) was added and the resulting mix-
ture was left to stir at 0◦C for 0.5 h. 2-(Trimethylsilyl)ethanol (10.50 g,
88.8 mmol) was then added to the reaction mixture and the resulting
solution was stirred at 0◦C for a further 1 h then warmed to 18◦C and
allowed to stir at this temperature for 3 days. The by now light-yellow
reaction mixture was treated with distilled water (500 mL) and the sepa-
rated aqueous layer was extracted with dichloromethane (3 × 100 mL).
The combined organic fractions were dried (Na2SO4), filtered and
concentrated under reduced pressure to afford a clear, colourless oil.
Subjection of this material to flash chromatography (silica, 1:9 →
1:1 v/v ethyl acetate/hexane gradient elution) and concentration of the
appropriate fractions (Rf 0.4 in 1:3 v/v ethyl acetate/hexane) afforded
compound 5 (12.60 g, 90%) as a clear, colourless oil. (HRMS (+ve ESI):
Found [M + H]+ 697.3707, [M + Na]+ 719.3545. C33H60N2O8Si3
requires [M + H]+ 697.3736, [M + Na]+ 719.3555.) δH (300 MHz,
CDCl3) 7.38–7.29 (5H, complex m, –C6H5), 5.09 (2H, s, –CH2Ph),
4.91 (1H, m, –NH), 4.21–4.10 (6H, complex m, 3 × –OCH2–), 3.61
(4H, s, 2 × –NCH2CO2–), 3.40 (1H, t, J 7.7, –NCH–), 3.18 (2H, m,
–NHCH2–), 1.76–1.32 (6H, complex m, –NCH2(CH2)3–), 1.04–0.93
(6H, complex m, 3 × –CH2Si–), 0.04 (9H, s, 3 × –CH3), 0.02 (18H,
s, 6 × –CH3). δC (75 MHz, CDCl3) 172.9, 171.6, 156.5, 136.8, 128.5,
128.1, 128.0, 66.5, 64.7, 62.9, 62.8, 52.8, 40.8, 30.1, 29.4, 23.1, 17.6,
17.4, –1.5. νmax (neat)/cm−1 3381, 2953, 2899, 1728, 1526, 1455, 1413,
1250, 1217, 1159, 1061, 1042, 988, 937, 860, 838, 756, 696, 664, 608.
m/z (+ve ESI) 719 ([M + Na]+, 1%), 698 ([M + H]+, 100).
Conclusions
A practical method for the preparation of the important SL-
and PMV-targeting compound NTA3-DTDA (1) that deliv-
ers gram quantities of pure forms of this material has been
established. Such methodology should be capable of ready
adaptation to the construction of related assemblies and so
allowing the fine-tuning of the biological properties of this
fascinating class of compound.
Experimental
General Procedures
Proton (1H) and carbon (13C) NMR spectra were recorded on either
a Varian Inova 500 spectrometer operating at 500 MHz for proton and
126 MHz for carbon or a Gemini 300 NMR spectrometer, operating at
300 MHz (for proton) and 75 MHz (for carbon). Unless otherwise spec-
ified, spectra were acquired at 20◦C in deuterochloroform (CDCl3) that
had been filtered through basic alumina immediately before use. Chem-
ical shifts are recorded as δ values in parts per million (ppm). Infrared
spectra (νmax) were recorded on a Perkin–Elmer 1800 Series FTIR
Spectrometer and samples were analyzed as thin films on KBr plates.
Low resolution mass spectra were recorded on a Micromass–Waters
LC-ZMD single quadrupole liquid chromatograph-MS or VG Quat-
tro II triple quadrupole MS instrument using electrospray techniques.
High resolution mass spectra were recorded on anAUTOSPEC spectro-
meter. Dichloromethane was distilled from calcium hydride and THF
was distilled, under nitrogen, from sodium benzophenone ketyl. Where
necessary, reactions were performed under a nitrogen atmosphere.
Compound 6
A magnetically stirred solution of compound 5 (11.75 g, 16.9 mmol)
in methanol (1000 mL) was treated with 10% palladium on charcoal
(700 mg). The ensuing mixture was degassed, flushed with dihydrogen,
stirred for 16 h at 18◦C under an atmosphere of dihydrogen then filtered
andthefiltrateconcentratedunderreducedpressure. Inthismanner com-
pound 6 (9.28 g, 98%) was obtained as a clear, colourless oil. (HRMS
(+ve ESI): Found [M + H]+ 563.3382. C25H54N2O6Si3 requires
[M + H]+ 563.3368.)δH (300 MHz, CDCl3)4.22–4.12(6H, complexm,
3 × –OCH2–), 3.59 (4H, s, 2 × –NCH2CO2–), 3.41 (1H, t, J 7.7,
–NCH–), 2.86–2.71 (2H, complex m, NH2CH2–), 1.78–1.34 (6H, com-
plex m, –NCH2(CH2)3–), 1.04–0.93 (6H, complex m, 3 × –CH2Si–),
0.04 (9H, s, 3 × –CH3), 0.03 (18H, s, 6 × –CH3) (signal due to amine
protons not observed). δC (75 MHz, CDCl3) 173.0, 171.6, 64.9, 62.8,
62.7, 52.8, 41.8, 33.1, 30.4, 23.2, 17.6, 17.4, −1.5. νmax (neat)/cm−1
2953, 2899, 1745, 1592, 1454, 1416, 1388, 1346, 1250, 1159, 1061,
1042, 974, 937, 860, 837, 764, 695, 664, 608. m/z (+ve ESI) 564 (47%),
563 ([M + H]+, 100).
Compound 4
A
magnetically stirred solution of α-bromoacetic acid (25.0 g,
180 mmol) in NaOH (90 mL of a 1.5 M aqueous solution) was cooled to
0◦C then a solution of compound 3 (12.0 g, 42 mmol) in NaOH (150 mL
of a 1.5 M aqueous solution) was added dropwise over 2 h. The ensuing
mixture was allowed to warm to 18◦C, stirred at this temperature for
16 h, and then at 50◦C for 2 h. The cooled reaction mixture was treated,
dropwise, with HCl (240 mL of a 1 M aqueous solution), and the ensu-
ing white precipitate filtered off and washed with HCl (120 mL of a
0.1 M, aqueous solution) then distilled water (2 × 120 mL). The result-
ing solid was dried under high vacuum to afford the title compound
4[7] (15.75 g, 93%) as a white solid, mp 162.4–167.4◦C (lit.[7] 170◦C).
(HRMS (+ve ESI): Found [M + H]+ 397.1610, [M + Na]+ 419.1428.
C18H24N2O8 requires [M + H]+ 397.1611, [M + Na]+ 419.1430.) δH
(300 MHz, CD3OD) 7.38–7.28 (5H, complex m, –C6H5), 5.06 (2H,
s, –CH2Ph), 3.62 (4H, ABq, J 18, 2 × –NCH2CO2H), 3.46 (1H, dd,
J 8.1 and 6.3, –NCH–), 3.12 (2H, t, J 6.6, –NHCH2–), 1.87–1.37
(6H, complex m, –NCH2(CH2)3–) (signals due to exchangeable protons
not observed). δC (75 MHz, CD3OD) 175.8(1), 175.7(8), 158.9, 138.4,
129.4, 128.9, 128.8, 67.3, 66.7, 55.3, 41.4, 30.5(4), 30.4(8), 24.6. νmax
(nujol mull)/cm−1 3374, 2924, 2855, 1698, 1535, 1456, 1377, 1265,
1144, 1022, 975, 892, 776, 739, 698. m/z (+ve ESI) 793 ([2M + H]+,
6%), 419 (9), 397 ([M + Na]+, 63), 353 (100).
Compound 7
A magnetically stirred solution of compound 4 (300 mg, 0.76 mmol) and
DMAP (350 mg, 2.86 mmol) in dichloromethane (150 mL) maintained
under argon was cooled to 0◦C then EDAC·HCl (710 mg, 3.70 mmol)
was added and the ensuing mixture left to stir at 0◦C for 0.5 h.
A solution of compound 6 (1.60 g, 2.84 mmol) in dichloromethane
(100 mL) was then added and the reaction mixture stirred at 0◦C
for 1 h, allowed to warm to 18◦C, left at this temperature for 3
days, then quenched with water (250 mL). The separated aqueous
layer was extracted with dichloromethane (3 × 50 mL) and the com-
bined organic fractions then dried (Na2SO4), filtered, and concentrated
under reduced pressure to afford a light-yellow oil. Subjection of
this material to flash chromatography (silica, 1:3 → 1:1 v/v ethyl
acetate/hexane gradient elution) and concentration of the appropri-
ate fractions (Rf 0.7 in 3:1 v/v ethyl acetate/hexane) afforded com-
pound 7 (1.24 g, 81%) as a clear, colourless oil. (HRMS (+ve ESI):
Found [M + H]+ 2030.1138, [M + Na]+ 2052.1040. C93H180N8O23Si9
requires [M + H]+ 203.1163, [M + Na]+ 2052.0983.) δH (300 MHz,
CDCl3) 7.42 (2H, broad s, –NH), 7.36–7.27 (6H, complex m, –C6H5
and –NH), 5.15 (1H, t, J 5.7, –NH), 5.07 (2H, s, –CH2Ph), 4.20–4.10
(18H, complex m, 9 × –OCH2–), 3.58 (12H, s, 6 × –NCH2CO2–), 3.36
(5H, t, J 7.5, Cbz–NHCH2– and 3 × –NCHCO2–), 3.28–3.12 (10H,
Compound 5
A magnetically stirred solution of compound 4 (8.00 g, 20.2 mmol)
and 4-(N,N-dimethylamino)pyridine (DMAP) (8.10 g, 66.3 mmol) in

306
J. G. Altin et al.
complex m, 2 × –NCH2C(O)NH– and 3 × –C(O)NHCH2–), 3.12–
3.02 (1H, broad s, –NCHC(O)N–), 1.74–1.28 (24H, complex m, 4 ×
–NCH2(CH2)3–), 1.04–0.92 (18H, complex m, 9 × –CH2Si–), 0.04
(27H, s, 9 × –CH3), 0.02 (54H, s, 18 × –CH3). δC (75 MHz, CDCl3)
172.9(2), 172.8(8), 172.4, 171.6, 156.5, 136.8, 128.5, 128.1, 128.0, 66.5,
65.7, 64.8, 64.7, 62.9, 62.8, 56.2, 50.7, 40.6, 39.2, 30.1, 29.9, 29.5, 29.3,
28.8, 28.2, 24.0, 23.1, 23.2, 17.6, 17.3, −1.4. νmax (neat)/cm−1 2953,
2899, 1729, 1667, 1535, 1453, 1345, 1250, 1217, 1157, 1061, 1042,
976, 936, 860, 837, 760, 695. m/z (+ve ESI) 2032 ([M + H]+, 5%),
1016 ([M + 2H]2+, 100).
(HRMS (+ve ESI): Found [M + H]+ 2387.5506. C117H235N9O23Si9
requires [M + H]+ 2387.5498.) δH (300 MHz, CDCl3) 7.60 (4H, broad
s, 3 × NH), 4.21–4.10 (18H, complex m, 9 × –OCH2), 3.60 (12H,
s, 6 × –NCH2CO2–), 3.37 (4H, t, J 7.5, 4 × –NCHCO2–), 3.31–
3.14 (16H, complex m, 2 × –NCH2C(O)NH–, 4 × –C(O)NHCH2–, and
–C(O)N(CH2–)2), 2.68–2.60 (2H, complex m, –C(O)CH2CH2C(O)–),
2.58–2.48 (2H, complex m, –C(O)CH2CH2C(O)–), 1.90–1.18 (72H,
complex m, 4 × –NCH2(CH2)3– and 2 × –CH2(CH2)12CH3), 1.04–
0.93(18H, complexm, 9 × –CH2Si–), 0.90(6H, t, J6.2, 2 × –CH3), 0.04
(27H, s, 9 × –CH3), 0.03 (54H, s, 18 × –CH3). δC (75 MHz, CDCl3)
172.8(4), 172.8(2), 172.7, 171.5, 171.4, 171.1, 65.5, 64.8, 64.7, 62.8(0),
62.7(9), 62.7(0), 62.6(6), 60.4, 56.1, 52.7, 48.1, 46.3, 39.3, 38.9, 31.9,
29.7, 29.7, 29.6(4), 29.6(3), 29.6(1), 29.5(8), 29.4(6), 29.4(0), 29.0,
27.8, 27.1, 27.0, 23.4, 23.2, 22.7, 21.0, 17.6, 17.3, 14.1, −1.5. νmax
(neat)/cm−1 3301, 3073, 2953, 2926, 2855, 1746, 1651, 1546, 1461,
1427, 1379, 1250, 1158, 1061, 1042, 976, 937, 860, 837, 762, 695, 663,
608. m/z (+ve ESI) 2389 ([M + H]+, << 1%), 1195 (28%), 537 (100).
Compound 8
A magnetically stirred solution of compound 7 (224 mg, 0.11 mmol) in
2,2,2-trifluoroethanol (20 mL) was treated with 10% palladium on char-
coal (15 mg), the ensuing mixture degassed, flushed with dihydrogen,
and then stirred under an atmosphere of dihydrogen at 18◦C for 16 h.The
reaction mixture was then filtered through a pad of Celite and the filtrate
concentrated under reduced pressure to afford compound 8 (204 mg,
Compound 1
97%) as a clear, colourless oil. (HRMS (+ve ESI): Found [M + H]+
12
13C1H174N8O21Si9 requires [M + H]+ 1897.0829.)
A chilled sample of compound 12 (395 mg, 0.165 mmol) was cooled
to 0◦C, treated with trifluoroacetic acid (5 mL) and the ensuing mix-
ture allowed to stand for 2 h at 0◦C and then for an additional 2 h
at 18◦C. All volatiles were then removed under reduced pressure to
afford a pale-brown resin that was washed with toluene (2 × 10 mL)
and acetonitrile (2 × 10 mL) to afford compound 1 (225 mg, 92%) as
a white solid, mp 169.0–193.9◦C (dec.). (HRMS (–ve ESI): Found
[M − H]− 1484.9028. C72H127N9O23 requires [M − H]− 1484.8967.)
δH (300 MHz, [D6]DMSO) 8.27 (2H, m, 2 × –NH), 8.01 (1H, m, –NH),
7.74 (1H, m, –NH), 3.47 (12H, ABq, J 17.9, –NCH2CO2–), 3.38–
2.90 (14H, complex m, 2 × –NCH2C(O)NH–, 3 × –C(O)NHCH2–, and
–C(O)N(CH2–)2), 2.48–2.40 (2H, complex m, –C(O)CH2CH2C(O)–),
2.27 (2H, m, –C(O)CH2CH2C(O)–), 1.68–1.10 (78H, complex m, 2 ×
–CH2(CH2)12CH3), 0.84 (6H, t, J 6.8, 2 × –CH3) (signals due to nine
carboxylic acid protons not observed). δC (75 MHz, [D6]DMSO) 174.0,
173.3, 171.7, 171.4, 171.1, 170.7, 64.8, 64.4, 55.8, 53.4, 47.2, 45.3,
31.4, 30.7, 29.4, 29.1(8), 29.1(5), 29.0(7), 28.9, 28.6, 27.8, 27.4, 26.5,
26.3, 23.5, 23.2, 22.2, 14.0. νmax (nujol mull)/cm−1 3305, 2924, 2854,
1922, 1730, 1631, 1460, 1377, 1249, 977, 896, 722. m/z (–ve ESI) 1485
([M − H]−, 5%), 769 (6), 742 ([M − 2H]2−, 100), 495 (60), 371 (16).
HPLC Rt 12.7 min (Alltima (Alltech) C-18, 5 µm, 240 × 4.6 mm col-
umn, 959:40:1 v/v/v methanol/water/TFA (as isocratic solvent), flow
rate 1 mL min−1).
1897.0812.
C
84
δH (300 MHz, CDCl3) 7.97 (2H, m, –NH2), 7.74 (1H, m, –NH), 7.51
(1H, m, –NH), 6.53 (1H, m, –NH), 4.20–4.04 (18H, complex m,
9 × –OCH2–), 3.55 (12H, s, 6 × –NCH2CO2–), 3.48–2.88 (16H, com-
plex m, H2NCH2–, 4 × –NCHC(O)–, 2 × –NCH2C(O)NH–, and 3 ×
–C(O)NHCH2–), 1.82–1.22 (24H, complex m, 4 × –NCH2(CH2)3–),
1.02–0.88 (18H, complex m, 9 × –CH2Si–), 0.01 (27H, s, 9 × –CH3),
−0.01 (54H, s, 18 × –CH3). δC (75 MHz, CDCl3) 173.0, 171.8, 171.7,
171.6, 65.0, 64.7, 63.0(4), 62.9(7), 62.9(2), 62.8(6), 52.8, 39.3, 30.1,
29.4, 29.0, 23.8, 23.3, 17.6, 17.4, −1.4. νmax (neat)/cm−1 3281, 3063,
2953, 2899, 1745, 1668, 1542, 1421, 1384, 1345, 1250, 1158, 1061,
1042, 976, 937, 860, 837, 763, 694, 663, 608. m/z (+ve ESI) 1899
(64%), 1898 (96), 1897 ([M + H]+, 100), 961 (65), 950 (27), 614 (36),
564 (40).
Compound 2
A sample of compound 8 (200 mg, 0.10 mmol) was cooled to 0◦C,
treated with trifluoroacetic acid (5 mL), and the resulting mixture
stirred at 0◦C for 2 h then at 18◦C for a further 2 h. The reaction
mixture was concentrated at reduced pressure to a pale-brown resin
that was washed successively with toluene (2 × 10 mL), acetonitrile
(2 × 10 mL), and diethyl ether (10 mL), and so affording compound 2
(98 mg, 100%) as a white solid, mp 79.0–98.5◦C (dec.). (HRMS (+ve
ESI): Found [M + H]+ 995.4449. C40H66N8O21 requires [M + H]+
995.4421.) δH (300 MHz, D2O) 3.97 (15H, m, 6 × –CH2CO2– and 3 ×
–NCHCO2–), 3.65 (4H, ABq, J 17.0, 2 × –NCH2C(O)NH–), 3.53 (1H,
m, –NCHC(O)NH–), 3.20 (6H, broad s, 3 × –NHCH2–), 2.92 (2H, t,
J 7.4, H2NCH2–), 2.00–1.44 (24H, complex m, 4 × –NCH2(CH2)3–)
(signals due to exchangeable protons not observed). δC (75 MHz,
[D6]DMSO) 174.4, 174.2, 174.1, 171.7, 171.2, 64.9, 64.6, 55.8, 54.6,
54.4, 38.4, 29.5, 28.9, 27.0, 23.3, 22.9. νmax (nujol mull)/cm−1 3267,
2924, 2855, 1923, 1724, 1644, 1458, 1377, 1240, 1024, 981, 897. m/z
(+ve ESI) 996 (49%), 995 ([M + H]+, 100), 498 (11).
Acknowledgments
We thank Lipotek Pty Ltd for financial support including the
provision of a post-doctoral stipend to D.A.O.
References
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Compound 12
A magnetically stirred solution of acid 11 (242 mg, 0.48 mmol) and 4-
(N,N-dimethyl)aminopyridine (87 mg, 0.71 mmol) in dichloromethane
(25 mL) maintained under argon was cooled to 0◦C then EDAC·HCl
(136 mg, 0.71 mmol) was added and the ensuing mixture left to stir
at 0◦C for 0.5 h. A solution of amine 8 (440 mg, 0.23 mmol) in
dichloromethane (25 mL) was added and the ensuing mixture stirred at
0◦C for 1 h, allowed to warm to 18◦C, stirred at this temperature for 16 h,
then quenched with water (50 mL). The separated aqueous layer was
extracted with dichloromethane (3 × 25 mL) and the combined organic
fractions then dried (Na2SO4), filtered, and concentrated under reduced
pressure to afford a clear, colourless oil. Subjection of this material to
flash chromatography (silica, 1:1 → 3:1 v/v ethyl acetate/hexane gradi-
entelution)andconcentrationoftheappropriatefractions(Rf 0.8inethyl
acetate) afforded compound 12 (399 mg, 72%) as a clear, colourless oil.