Selective protection strategies in the synthesis of TRIS-fatty ester derivatives

Article abstract of DOI:10.1071/CH02124

A methodology for the selective synthesis of lipophilic acyl derivatives of the glycinamido triol (1) with either one, two, or three fatty ester groups has been established. Peracylation of (1), with palmitoyl chloride gave the triacylated derivative. Conversion of (1) into the acetonide, followed by acylation with either palmitoyl chloride or lauroyl chloride, and acetal hydrolysis provided the monoacylated derivatives. Treatment of (1) with trimethyl orthoacetate gave the orthoacetate derivative. Mild hydrolysis provided the monoacetate/diol. Acylation of the two hydroxyl groups with palmitoyl chloride gave the dipalmitate/acetate. Selective cleavage of the acetate group afforded the dipalmitate of (1). Analogous chemistry with trimethyl orthoformate provided the same dipalmitate via the orthoformate, monoformate/diol, and dipalmitate/formate. A more robust synthesis of the dipalmitate was achieved by converting the hydroxyl group of the acetonide of (1) into a tert-butyldiphenylsilyl ether, followed by acetal hydrolysis, palmitoylation of the liberated hydroxyl groups, and desilylation.

Full text of DOI:10.1071/CH02124

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Full Papers
Aust. J. Chem. 2002, 55, 629–634
Selective Protection Strategies in the Synthesis of
TRIS-Fatty Ester Derivatives
Raju Adhikari,A Craig L. Francis,A,B Gregory W. SimpsonA and Qi YangA
A CSIRO Molecular Science, Ian Wark Laboratory, Bag 10, Clayton South, Clayton, Vic. 3169, Australia.
B Author to whom correspondence should be addressed (e-mail: craig.francis@csiro.au).
A methodology for the selective synthesis of lipophilic acyl derivatives of the glycinamido triol (1) with either one,
two, or three fatty ester groups has been established. Peracylation of (1), with palmitoyl chloride gave the triacylated
derivative. Conversion of (1) into the acetonide, followed by acylation with either palmitoyl chloride or lauroyl
chloride, and acetal hydrolysis provided the monoacylated derivatives. Treatment of (1) with trimethyl orthoacetate
gave the orthoacetate derivative. Mild hydrolysis provided the monoacetate/diol. Acylation of the two hydroxyl
groups with palmitoyl chloride gave the dipalmitate/acetate. Selective cleavage of the acetate group afforded the
dipalmitate of (1). Analogous chemistry with trimethyl orthoformate provided the same dipalmitate via the
orthoformate, monoformate/diol, and dipalmitate/formate. A more robust synthesis of the dipalmitate was achieved
by converting the hydroxyl group of the acetonide of (1) into a tert-butyldiphenylsilyl ether, followed by acetal
hydrolysis, palmitoylation of the liberated hydroxyl groups, and desilylation.
Manuscript received: 19 June 2002.
Final version: 6 August 2002.
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Introduction
The equivalence of the three TRIS hydroxyl groups
avoids the problem of structural isomers thereby enabling
easier production of mono-, di-, and tri-fatty acyl forms
without the separation difficulties generally associated with
glycerol derivatives. In addition, tri-fatty acyl derivatives can
be formed using TRIS, which is not possible with glycerol.
The amine group of TRIS provides an ideal attachment
site for peptides and drugs with or without the use of spacer
molecules (such as succinate or simple amino acids) to give
conjugates of the general structure in Figure 1. TRIS is
readily available, inexpensive, non-toxic, and it has the added
advantage of being generally acceptable to the
pharmaceutical industry.
Increased lipophilicity would improve the therapeutic
benefit of many drugs.[1] Lipophilicity can be increased by a
variety of strategies including chemical modification by the
attachment of fatty acyl groups. The bond between the fatty
acid(s)-containing moiety and the drug may be chemically or
biologically labile resulting in a pro-drug, or it may be
biologically stable and a new drug entity is formed.
Fatty acyl groups can be attached to drugs through mono-
and di-glycerides. This method has been used with a variety
of drugs including acyclovir, AZT, chlorambucil, L-Dopa,
and methotrexate.[1] Whilst these derivatives have enhanced
biological properties, their preparation is often difficult and
unsuitable for commercial synthesis. The hydroxyl groups of
glycerol are non-equivalent and the preparation and isolation
of mono- and di-esters is complicated by the presence of
structural isomers.
OR1
R = H or fatty acyl group
X = drug
Y = optional linker
H
X
Y
N
OR2
OR3
Fig. 1. General conjugate structure.
Studies in our laboratories have developed a method of
conjugating fatty acids to compounds using 2-amino-
2-hydroxymethyl-1,3-propanediol (TRIS).[1–7] TRIS is a
common buffer molecule which has similarities to glycerol
(Diagram 1) allowing esterification by one to three fatty
acids to mimic mono-, di-, and triglycerides.
Using this approach, we have prepared fatty acyl
conjugates of a variety of drugs including morphine,
indomethacin, chlorambucil, AZT, and methotrexate, and
assessed their biological activities.[1,3,4,6,7]
To synthesize these conjugates, we required methods for
preparation of the TRIS-fatty acid moieties. This paper
describes the synthetic methodology we established to
produce N-protected precursors of the type N-(benzyloxy-
carbonyl)-glycyl-TRIS-fatty ester(s) where derivatives
bearing one, two, or three fatty ester groups were selectively
synthesized.
OH
OH
OH
OH
OH
H2N
OH
Glycerol
Tris
Diagram 1
© CSIRO 2002
10.1071/CH02124
0004-9425/02/100629

630
R. Adhikari et al.
some simple protection/deprotection chemistry, was re-
quired. The approach we used for the synthesis of mono-
acylated derivatives involved the acetonide protecting group
(Scheme 2).
OR1
OR2
OR3
O
H
N
O
N
H
O
(1) R1 = R2 = R3 = H
O
O
(2) R1 = R2 = R3 = CO(CH2)14CH3 (palmitoyl)
(3) R1 = R2 = CO(CH2)14CH3, R3 = H
(4) R1 = CO(CH2)14CH3, R2 = R3 = H
H
CH3COCH3
O
N
O
(1)
N
H
CuSO4
OH
O
p-TsOH
(5)
Diagram 2
acyl chloride
Et3N
cat. DMAP
Results and Discussion
The key, N-protected, starting triol (1) (Diagram 2) was
prepared using a different method from those reported,[8–10]
by treating N-(benzyloxycarbonyl)-glycine-N-carboxyan-
hydride with TRIS in aqueous dioxan (Scheme 1).
O
O
H
O
N
O
N
H
OR
O
(6) R = palmitoyl
(7) R = lauroyl
O
O
OH
OH
OH
O
N
O
H2N
p-TsOH
H2O
O
O
TRIS
OH
−CO2
H
N
O
OH
OR
N
OH
OH
OH
H
O
O
H
N
O
(4) R = palmitoyl
(8) R = lauroyl
N
H
O
(1)
Scheme 2
Scheme 1
Two hydroxyl groups of triol (1) were blocked as the
acetonide (5).[9] The remaining hydroxyl group of (5) was
acylated with palmitoyl chloride to give (6). Removal of the
acetonide group by hydrolysis afforded the monopalmitate
(4). The monolaurate (8) was prepared in an analogous
manner with lauroyl chloride via (7).
For a specific synthesis of diacylated derivatives of (1),
the preferred method would be selective reaction of one
hydroxyl group and acylation of the remaining hydroxyls
followed by deprotection of the first hydroxyl group.
Previous work in our laboratories has successfully achieved
monoprotection of triols of similar structure to (1) through
the formation of caged orthoacetates followed by controlled
hydrolysis to afford monoacetate diols.[11–13] After some
experimentation we were able to develop conditions to
produce orthoacetate (9) and convert it into the desired
dipalmitate (3) (Scheme 3).
Treatment of (1) with trimethyl orthoacetate in DMF with
formic acid catalyst gave the orthoacetate (9) in a mixture
containing some of the desired monoacetate (10) due to
hydrolysis under the reaction conditions. Treatment of this
mixture with aqueous acid fully converted (9) into (10).
Monoacetate (10) was readily converted into the dipalmitate
(11) by treatment with palmitoyl chloride/triethylamine/
catalytic 4-dimethylaminopyridine (DMAP) in DCM. We
experienced some difficulty in selectively removing the
acetate group to give the dipalmitate (3). Attempted selective
reactions using sodium or potassium hydroxide in a range of
The simplest derivative to prepare is the one in which
fatty acyl groups are attached to all three TRIS hydroxyl
groups. Three palmitoyl groups were attached to (1) by
treatment with 3.4 equivalents of palmitoyl chloride and
triethylamine in dichloromethane (DCM)/N,N-dimethyl-
formamide (DMF), producing tripalmitate (2).
Synthesis of mono- and di-acylated derivatives was more
complex. Initially, direct acylation of (1) was investigated.
This was expected to produce a mixture of the mono-, di-,
and tri-acylated derivatives (4), (3), and (2) respectively,
which could in principle be separated by chromatography.
Treatment of triol (1) with one equivalent of palmitoyl
chloride, with the aim of isolating the monopalmitate (4)
from the crude product mixture, was briefly investigated and
found to be unsatisfactory. Crude mixtures contained (4) and
(mainly) dipalmitate (3) with traces of tripalmitate (2) and
starting material (1). Attempts at selective crystallization
returned a product mainly consisting of (3). Chromatography
of the mother liquor returned only 15–20% yield of (4).
Repeating the above reaction with 2.2 equivalents of
palmitoyl chloride provided a crude product mixture
containing, by high-pressure liquid chromatography (HPLC)
analysis, a mixture of (2), (3), and (4) in a ratio of 29, 47, and
24%, respectively, which were then separated by column
chromatography on silica gel.
Ultimately, for larger scale work, a specific synthesis of
either monoacylated or diacylated derivatives, involving

Selective Protection Strategies in the Synthesis of TRIS-Fatty Ester Derivatives
631
(1)
O
O
H
R =
O
N
R
O
N
H
O
R’C(OMe)3
cat. HCO2H
OH
O
H
R =
O
N
*
(5)
O
Pm = palmitoyl
OR’’
OR’’
O
O
H2O
R’
TBDPSCl imidazole
R
N
H
R
N
O
cat.HCO2H
H
O
O
OH
(9) R’ = Me
O
R’
p-TsOH
(12) R’ = H
R
O
R
N
H
OH
N
H2O
(10) R' = Me, R'' = H
H
OTBDPS
OTBDPS
NH3/MeOH
a
(11) R' = Me, R'' = palmitoyl
(17)
(18)
(3)
(13) R' = H, R'' = H
a
Et3N
NaHCO3/H2O/MeOH
palmitoyl
chloride
(14) R' = H, R'' = palmitoyl
cat.
DMAP
a = palmitoyl chloride/Et3N/cat. DMAP
OPm
OPm
Scheme 3
TBAF
R
OPm
R
N
H
OPm
N
H
solvents resulted in some hydrolysis of the palmitate ester
groups. The use of methanol/ammonia gave the desired
product but in a disappointing 64% yield.
OH
OTBDPS
(3)
(19)
Scheme 4
We considered that the use of formate in place of acetate
as a protecting group would result in a higher yield in the
important deprotection step. We were unaware of any reports
of the use of orthoformates in a protection strategy for triols
of this type, however, we were pleased to find that the
approach was successful.
We repeated the chemistry described in Scheme 3
substituting orthoformate for orthoacetate to obtain the
dipalmitate/formate (14), via orthoformate (12) and
formate/diol (13). The formate (14) was readily de-
formylated by the action of sodium bicarbonate in aqueous
methanol to afford (3).
The success of these orthoester routes raised the question
of the generality of this approach and we briefly attempted to
prepare trimethyl orthopalmitate to investigate the direct
preparation of the monopalmitate (4), via the orthopalmitate
(15) (Diagram 3). Trimethyl orthopalmitate has been
reported[14] but in our hands only hydrolysed material,
methyl palmitate, was isolated.
The free hydroxyl group of (5) was protected as the
tert-butyldiphenylsilyl (TBDPS) ether to give (17), followed
by removal of the acetonide protecting group to afford the
monoprotected ether (18). Palmitoylation to give (19)
followed by removal of the silyl group using
tetrabutylammonium fluoride gave the desired dipalmitate
(3) in good yield (55% in five steps from (1)).
In summary, selective routes have been developed for the
synthesis of tripalmitate (2), dipalmitate (3), monopalmitate
(4), and monolaurate (8). All of these products could be
easily deprotected, when required, by standard hydro-
genolysis over Pd/C catalyst to provide the free amino
compounds for attachment to drugs.[15]
The synthetic methods described in this paper should be
generally applicable to a variety of fatty acids, and other
amino acids (in place of glycine), enabling preparation of
further analogues.
O
Experimental
H
N
OMe
O
O
O
N
H
General
NH2+Cl−
O
O
14
14
Melting points (m.p.) were determined on
a
Gallenkamp
(15)
(16)
MPD350.BM2.5 apparatus and are uncorrected. Petroleum spirit refers
to a fraction boiling between 40–60°C. All organic extracts were dried
over anhydrous magnesium sulfate unless otherwise stated. Developed,
analytical thin-layer chromatography (TLC) plates (Macherey–Nagel
Polygram pre-coated plastic sheets, 0.2 mm layer of silica gel with
fluorescent indicator UV254) were visualized with either ultraviolet
(UV) light (254 nm) or a solution of 10% (w/v) phosphomolybdic acid
in ethanol and heating. Flash chromatography[16] was performed using
Merck Silica gel 60, particle size 0.040–0.063 mm (230–400 mesh
ASTM). Radial chromatography was performed on a Harrison
Research Chromatotron (7924T) using 1, 2, or 4 mm thick silica plates
(silica gel 60 PF254, Merck No. 7749). Analytical HPLC was
performed with a Waters 700 Satellite WISP autosampler, a Waters
Diagram 3
Attempts at the direct conversion of the intermediate
iminoester hydrochloride (16) into orthopalmitate (15) also
failed.
Whilst the orthoester strategy described in Scheme 3
above provided the desired compound, the yields were not
always reproducible. Hence, we developed a longer but more
reliable approach using the acetonide (5), which we had in
hand from the monoacylation chemistry (Scheme 4).

632
R. Adhikari et al.
600E Multisolvent delivery system, a Waters 2487 dual absorbance
detector at 254 nm and a 150 × 4.6 mm Alltima C18 column. A flow
rate of 1 mL/min was used. A gradient system was used commencing
with 50% acetonitrile/25% tetrahydrofuran/25% water (containing
0.05% trifluoroacetic acid (TFA)) for 2 min followed by a 5 min
gradient to 50% acetonitrile/50% tetrahydrofuran (containing 0.05%
TFA). Data was processed using a Waters Maxima 820 or a Waters
Millenium 32 chromatography workstation. Proton nuclear magnetic
resonance (1H NMR) and carbon nuclear magnetic resonance (13C
NMR) spectra were recorded at room temperature on a Bruker AC-200
or Varian Gemini-200 spectrometer operating at 200 and 50.3 MHz,
respectively, using CDCl3 as solvent and internal reference unless
otherwise specified. Solute concentrations were approximately
10 mg/mL in 5 mm tubes. Mass spectra were recorded on a Fisons
Instruments VG Platform quadrupole using either atmospheric pressure
chemical ionization (APCI) or electrospray ionization (ESI) in the
positive (+) ion and/or negative (-) ion mode with a cone voltage of
30 eV for both APCI and ESI, using either 1:1 acetonitrile/water or
methanol as solvent. Samples were usually introduced dissolved in
methanol/DCM. Only the major fragments are given with their relative
abundances shown in parentheses. The high resolution accurate mass
work was carried out on a Bruker Bio Apex FTICR-MS at Monash
University. Microanalyses were performed at the University of Otago,
New Zealand.
t, J 7 Hz, CH2CO; 1.50–1.70, m, CH2CH2CO; 1.26, m, 12 × CH2; 0.88,
t, J 6 Hz, Me. Mass spectrum (APCI+) m/z 1028 (M+H, 100%);
(APCI–) m/z 1062 (M+Cl–, 100%).
N-[2-[(1-oxohexadecyl)oxy]-1,1-bis[[(1-oxohexadecyl)oxy]methyl]-
ethyl]-Nα-(benzyloxycarbonyl)glycinamide (2),
N-[1-hydroxymethyl-[2-[(1-oxohexadecyl)oxy]-1-[(1-oxohexadecyl)-
oxymethyl]ethyl]-Nα-(benzyloxycarbonyl)glycinamide (3), and
N-[1,1-bis(hydroxymethyl)-2-[(1-oxohexadecyl)oxy]ethyl]-Nα-(benzyl
oxycarbonyl)glycinamide (4) mixture
The titled mixture was prepared from the triol (1) using a similar
procedure to that for tripalmitate (2) above, except with 2.2 molar
equivalents of palmitoyl chloride. The crude product (HPLC analysis
indicated
a mixture of tripalmitate (2), dipalmitate (3), and
monopalmitate (4) in a ratio of 29, 47, and 24%, respectively) was
dissolved in DCM and filtered through a silica pad, eluting with 3%
methanol in DCM to remove the polar impurities. This product was
further purified by flash chromatography on silica gel, eluting with
0–5% methanol/DCM and monitoring by UV absorption at 254 nm.
Fractions containing (2) were combined and evaporated and the residue
was recrystallized from ethanol to give the tripalmitate (2) (30.8 g,
19%) as a colourless solid. Later fractions containing (3) were
combined and evaporated and the residue recrystallized from ethanol to
give dipalmitate (3) (44.1 g, 35%) as a colourless solid. M.p. 76–77°C
(Found: C, 70.1; H, 10.4; N, 3.6%. C46H80N2O8 requires C, 70.0; H,
10.2; N, 3.6%) (Found: m/z 789.599. C46H81N2O8 requires m/z
789.599). 1H NMR (CDCl3) δ 7.35, m, Ph; 6.62, s, TRIS-NH; 5.35, m,
gly-NH; 5.14, s, CH2Ph; 4.39 and 4.19, AB, J 11.5 Hz, 2 ×
CH2O(palmitate); 3.83, d, J 5.8 Hz, CH2N; 3.77, s, CH2OH; 2.31, t, J
7.5 Hz, 2 × CH2CO; 1.50–1.71, m, 2 × CH2CH2CO; 1.25, m, 24 × CH2;
0.88, t, J 6.5 Hz, 2 × Me. Mass spectrum (APCI+) m/z 811 (M+Na,
100%); (APCI–) m/z 823 (M+Cl–, 58%), 255 (100). Elution with higher
proportions of methanol stripped the diffuse band of (4) (not
quantified) from the column.
N-[2-Hydroxy-1,1-bis(hydroxymethyl)ethyl]-Nα-
(benzyloxycarbonyl)glycinamide (1)
2-Amino-2-(hydroxymethyl)-1,3-propanediol (TRIS) (21.07 g,
0.18 mmol) was dissolved in water (100 mL). Dioxan (300 mL) was
added and the stirred solution cooled with an ice bath. Benzyl
2,5-dioxo-oxazolidine-3-carboxylate (N-benzyloxycarbonyl-glycine-
N-carboxyanhydride) (40.9 g, 0.18 mol) was added portionwise over 30
min and rinsed in with dioxan (10–15 mL). The reaction was stirred and
allowed to warm to room temperature, and then stirred for a further 4 h.
The reaction mixture was concentrated under vacuum. The residue was
taken up in water (120 mL) and extracted with ethyl acetate (1 ×
500 mL, 4 × 400 mL, 2 × 300 mL). The combined organic extracts were
evaporated under vacuum to give a colourless solid that was triturated
with methanol/DCM/petroleum spirit (60–80) (approx. ratio, 1:2:2;
approx. volume, 150 mL). The colourless solid was filtered off. The
filtrate was evaporated and the residue triturated as above to give
another crop of colourless solid. 1H NMR analysis indicated both crops
were identical; therefore, they were combined to afford the title
compound (1) (27.8 g, 51%) as a colourless solid. m.p. 130–131°C
(lit.[8–10] 130–131°C). 1H NMR ((CD3)2SO) δ 7.47, t, J 5.8 Hz, gly-NH;
7.35, m, ArH; 7.16, s, TRIS-NH; 5.04, s, CH2Ph; 4.72, t, J 5.8 Hz, 3 ×
OH; 3.65, d, J 5.8 Hz, gly-CH2; 3.54, d, J 5.8 Hz, 3 × CH2O.
N-(2,2-Dimethyl-5-hydroxymethyl-[1,3]dioxan-5-yl)-Nα-
(benzyloxycarbonyl)glycinamide (5)
Anhydrous cupric sulfate (10.8 g) and p-toluenesulfonic acid
monohydrate (p-TsOH) (0.440 g, 2.32 mmol) were added to a stirred
solution of (1) (8.5 g, 27.24 mmol) in acetone (850 mL) at room
temperature. The solution was stirred for 36 h before an excess of solid
potassium carbonate was added. After a further 2 h of stirring, the
cupric sulfate and excess base were filtered off and the filtrate
concentrated. The residual oil was dissolved in chloroform (300 mL)
and washed with 5% sodium bicarbonate (1 × 50 mL), water, and then
dried over potassium carbonate. The solvent was removed under
vacuum to give a viscous residue, which was triturated with petroleum
spirit (2 × 50 mL) to give the title compound (5) (8.82 g, 92%) as a
colourless crystalline solid. M.p. 92–94°C (Found: C, 58.1; H, 7.2; N,
7.9%. C17H24N2O6 requires C, 57.9; H, 6.9; N, 7.9%). 1H NMR δ 7.35,
m, Ph; 6.80, s, TRIS-NH; 5.40, m, gly-NH; 5.18, s, CH2Ph; 4.54, t, J
6.25 Hz, OH; 3.93, d, J 6.25 Hz, CH2O; 3.82, s, 2 × CH2O(acetonide);
3.70, d, J 6.25 Hz, CH2N; 1.42, s, CH3; 1.38, s, CH3. Mass spectrum
(CI+) m/z 353 (M+1, 7%), 335 (9), 295 (100).
N-[2-[(1-oxohexadecyl)oxy]-1,1-bis[[(1-oxohexadecyl)oxy]methyl]-
ethyl]-Nα-(benzyloxycarbonyl)glycinamide (2)
The triol (1) (50.00 g, 0.16 mol) was dissolved in DMF (350 mL) with
stirring. DCM (1.60 L) then triethylamine (100.5 mL, 0.721 mol) was
added and the stirred solution cooled with an ice–salt bath. Palmitoyl
chloride (165 mL, 0.544 mol) was added dropwise over 1.5 h. The
dropping funnel was rinsed with DCM (100 mL). The reaction was
stirred at –3°C for a further hour and then at 4°C for 48 h. Triethylamine
hydrochloride was filtered off and rinsed with ethyl acetate. The filtrate
was evaporated under vacuum and the residue dissolved in ethyl acetate
(600 mL) with warming. The ethyl acetate extract was washed with
water (600 mL) then with saturated sodium chloride solution (600 mL).
DCM (500 mL) was added to the organic layer and the combined
organic phase was washed with saturated sodium chloride solution,
dried and evaporated to give a crude product, which was flash
chromatographed on silica gel. Elution with 0.1–0.4% methanol in
DCM, followed by recrystallization from ethanol afforded the title
compound (2) (151.0 g, 92%) as a colourless solid. M.p. 60–61°C
(Found: C, 72.4; H, 11.0; N, 2.8%. C62H110N2O9 requires C, 72.5; H,
10.8; N, 2.7%). 1H NMR δ 7.35, m, ArH; 6.50, s, TRIS-NH; 5.28, m,
gly-NH; 5.14, s, CH2Ph; 4.41, s, 3 × CH2O; 3.81, d, J 6 Hz, CH2N; 2.31,
N-(2,2-Dimethyl-5-[(1-oxohexadecyl)oxymethyl]-[1,3]dioxan-5-yl)-
Nα-(benzyloxycarbonyl)glycinamide (6)
DMAP (0.425 g, 3.48 mmol) was added to a solution of (5) (12.25 g,
34.76 mmol), triethylamine (13.6 mL, 97.8 mmol) and palmitoyl
chloride (10.6 mL, 35 mmol) in DCM (185 mL) at 0°C. The resulting
solution was stirred at 0°C for 10 min and then for a further 2 h at room
temperature. The reaction mixture was diluted with DCM (100 mL) and
washed with saturated ammonium chloride solution (500 mL). The
aqueous layer was extracted with DCM (100 mL) and the combined
extracts were washed with water, dried over potassium carbonate/
magnesium sulfate, filtered and the solvent was removed under vacuum
to yield a light yellow solid. Recrystallization from ether/petroleum
spirit afforded the title compound (6) (17.2 g, 84%) as a colourless,
crystalline solid. M.p. 68–70°C (Found: C, 67.0; H, 9.3; N, 4.6%.

Selective Protection Strategies in the Synthesis of TRIS-Fatty Ester Derivatives
633
C33H54N2O7 requires C, 67.1; H, 9.2; N, 4.7%). 1H NMR δ 7.30, m, Ph;
6.44, s, TRIS-NH; 5.24, m, gly-NH; 5.08, s, CH2Ph; 4.40, s,
CH2O(palmitate); 4.21 and 3.65, AB, J 12.5 Hz, 2 × CH2O; 3.74, d, J
6.25 Hz, CH2N; 2.25, t, J 6.3 Hz, OCOCH2; 1.50–1.70, m, CH2CH2CO;
1.32, s, CH3; 1.38, s, CH3; 1.10, m, 12 × CH2; 0.88, t, J 6.2 Hz, CH3.
Mass spectrum (APCI+) m/z 613 (M+Na, 3%), 591 (M+H, 1), 533
(100); (APCI–) m/z 625 (M+Cl–, 100%). The mother liquor was
evaporated and the yellow, oily residue was recrystallized as above to
give a second batch of (6) (2.47 g, 12%) as a slightly yellow-coloured
solid.
337 for M+H) and the hydrolysis product (10) (acetyl methyl peak at δ
2.02, m/z 355 for M+H). The crude material was immediately mixed
with water (15 mL) and formic acid (12 drops) added. The resulting
mixture was stirred at room temperature for 4–5 h until a clear solution
was obtained. The water was removed under vacuum and the residue
extracted with ethyl acetate (200 mL). The ethyl acetate extract was
washed with water (2 × 50 mL) and dried. The filtrate was concentrated
under vacuum to yield the title compound (10) (0.68 g, 64%) as an oil
which was used without further purification. 1H NMR δ 7.25, m, ArH;
6.80, s, TRIS-NH; 5.61, m, gly-NH; 5.03, s, CH2Ph; 4.16, s, CH2OAc;
3.75, d, J 6 Hz, CH2N; 3.68 and 3.51, AB, J 12 Hz, 2 × CH2O; 2.02, s,
COCH3. Mass spectrum (CI+) m/z 355 (M+H, 12%), 337 (M–H2O,
100).
The above procedure is hereinafter referred to as ‘standard
acylation’.
N-(2,2-Dimethyl-5-[(1-oxododecyl)oxymethyl]-[1,3]dioxan-5-yl)-Nα-
(benzyloxycarbonyl)glycinamide (7)
N-[1,1-bis(hydroxymethyl)-2-(formyloxy)ethyl]-Nα-
(benzyloxycarbonyl)glycinamide (13)
Treatment of (5) with lauroyl chloride by the standard acylation
procedure afforded the title compound (7) (15.27 g, 84%) as a
colourless solid. M.p. 56.5–57.5°C (Found: C, 65.4; H, 8.8; N, 5.2%.
C29H46N2O7 requires C, 65.1; H, 8.7; N, 5.2%). 1H NMR δ 7.35, m, Ph;
6.36, s, TRIS-NH; 5.31, m, gly-NH; 5.14, s, CH2Ph; 4.46, s,
CH2O(laurate); 4.28 and 3.73, AB, J 12.5 Hz, 2 × CH2O; 3.83, d, J
6.25 Hz, CH2N; 2.32, t, J 6.5 Hz, OCOCH2; 1.60, m, CH2CH2CO;
1.49, s, CH3; 1.41, s, CH3; 1.28, m, 8 × CH2; 0.88, t, J 6.2 Hz, CH3. Mass
spectrum (APCI–) m/z 569 (M+Cl–, 100%).
Use of trimethyl orthoformate in the above procedure gave the title
compound (13) (0.59 g, 56%) as an oil which was used without further
purification. 1H NMR δ 8.00, s, formate; 7.25, m, ArH; 6.76, s,
TRIS-NH; 5.49, m, gly-NH; 5.04, s, CH2Ph; 4.22, s, CH2O(formate);
3.78, d, J 6 Hz, CH2N; 3.70 and 3.53, AB, J 12 Hz, 2 × CH2O. Mass
spectrum (CI+) m/z 341 (M+H, 12%), 323 (M–H2O, 100). H NMR
1
spectroscopic and mass spectrometric (CI+) analysis of the intermediate
indicated a mixture of predominantly the cyclic orthoformate (12) (CH
peak at δ 5.18, m/z 323 for M+H) and the hydrolysis product (13)
(formate resonance at δ 8.00, m/z 341 for M+H).
N-[1,1-bis(hydroxymethyl)-2-[(1-oxohexadecyl)oxy]ethyl]-Nα-
(benzyloxycarbonyl)glycinamide (4)
N-[1-acetoxymethyl-2-[(1-oxohexadecyl)oxy]-1-[(1-oxohexadecyl)-
oxymethyl]ethyl]-Nα-(benzyloxycarbonyl)glycinamide (11)
A solution of the monopalmitate acetonide (6) (10.0 g, 16.95 mmol)
and p-TsOH (0.67 g, 3.52 mmol) in acetone (170 mL)/water (30 mL)
was refluxed overnight. The acetone was removed under vacuum and
the concentrate diluted with water (50 mL). The solution was extracted
with ether (2 × 200 mL), and the extracts were washed with water, dried
over potassium carbonate and concentrated under vacuum. The solid
residue was radially chromatographed on silica gel, eluting with 1-3%
methanol in DCM to give the title compound (4) (7.75 g, 83%) as a
colourless solid. A small portion was further purified by crystallization
from chloroform/petroleum spirit to afford a crystalline solid. M.p.
64–66°C (Found: C, 65.2; H, 8.9; N, 5.0%. C30H50N2O7 requires C,
65.4; H, 9.1; N, 5.1%). 1H NMR δ 7.36, s, Ph; 6.78, s, TRIS-NH; 5.34,
m, gly-NH; 5.14, s, CH2Ph; 4.26, s, CH2O(palmitate); 3.86, d, J 6 Hz,
CH2N; 3.71 and 3.56, AB, J 12.4 Hz, 2 × CH2O; 2.34, t, J 7.3Hz,
OCOCH2 ; 1.50–1.70, m, CH2CH2CO; 1.25, m, 12 × CH2; 0.88, t, J 6.5
Hz, CH3. Mass spectrum (APCI+) m/z 551 (M+H, 30%), 533 (100);
(APCI–) m/z 585 (M+Cl–, 100%).
Standard acylation of (10) afforded the title compound (11) (1.56 g,
97%) as a pale yellow-coloured solid. A portion of the product was
crystallized from chloroform/petroleum spirit to give a colourless solid.
M.p. 62–63°C (Found: C, 69.7; H, 9.9; N, 3.4%. C48H82N2O9 requires
C, 69.4; H, 9.9; N, 3.4%) (Found: m/z 853.593. C48H82N2O9Na requires
1
m/z 853.592). H NMR δ 7.36, m, ArH; 6.50, s, TRIS-NH; 5.29, m,
gly-NH; 5.14, s, CH2Ph; 4.42, s, 6H, 2 × CH2O(palmitate) +
CH2O(acetate); 3.80, d, J 5.8 Hz, CH2N; 2.31, t, J 7.5 Hz, 2 × CH2CO;
2.07, s, CH3CO; 1.50–1.70, m, 2 × CH2CH2CO; 1.25, m, 24 × CH2;
0.88, t, J 6.5 Hz, 2 × Me. Mass spectrum (APCI+) m/z 853 (M+Na,
62%), 831 (M+H, 38), 575 (100); (APCI–) m/z 866 (M+Cl–, 100%).
N-[1-hydroxymethyl-2-[(1-oxohexadecyl)oxy]-1-[(1-oxohexadecyl)-
oxymethyl]ethyl]-Nα-(benzyloxycarbonyl)glycinamide (3)
Method A. Pre-cooled (–10°C) saturated methanolic ammonia
solution (35 mL) was added to a solution of (11) (0.7 g, 0.84 mmol) in
DCM (10 mL) at –10°C. The resulting mixture was left to stand at
–10°C for 50 h. The solvent was removed under vacuum to give a
colourless solid, which was purified by column chromatography on
silica gel, providing the title compound (3) (0.3 g, 64%) as a colourless
solid.
N-[1,1-bis(hydroxymethyl)-2-[(1-oxododecyl)oxy]ethyl]-Nα-
(benzyloxycarbonyl)glycinamide (8)
Use of the monolaurate acetonide (7) in the above procedure gave the
title compound (8) (4.11 g, 88%) as a clear, colourless gum (Found: m/z
1
517.289. C26H42N2O7+Na requires m/z 517.289). H NMR δ 7.35, s,
Method B. A solution of (14) (0.2 g, 0.244 mmol) in methanol
(28 mL) was mixed with a solution of sodium hydrogen carbonate (1 g)
in water (15 mL) and stirred overnight at room temperature. The solvent
was evaporated under vacuum and the solid residue was extracted with
chloroform (1 × 50 mL) and the organic extract washed with water and
dried. Solvent removal under vacuum gave the title compound (3)
(0.17 g, 88%) as a colourless solid.
Ph; 6.80, s, TRIS-NH; 5.46, m, gly-NH; 5.14, s, CH2Ph; 4.27, s,
CH2O(laurate); 3.86, d, J 6 Hz, CH2N; 3.72 and 3.56, AB, J 12.5 Hz,
2 × CH2O; 2.34, t, J 7.3 Hz, OCOCH2; 1.50–1.70, m, CH2CH2CO;
1.25, m, 8 × CH2; 0.90, t, J 6.5 Hz, CH3. Mass spectrum (APCI+) m/z
517 (M+Na, 39%), 495 (M+H, 77), 477 (M–OH, 100); (APCI–) m/z
529 (M+Cl–, 100%).
Method C. Tetrabutylammonium fluoride (17.9 mL, 2 equiv./
mol.) was added to a solution of (19) (9.2 g, 8.95 mmol) in
tetrahydrofuran (95 mL) at 0°C and the resulting mixture was stirred at
room temperature for 3 h. Removal of solvent under vacuum gave a
residue, which was extracted with ether (400 mL). The combined ether
extracts were washed with saturated sodium chloride solution (2 ×
100 mL), water (2 × 100 mL) and dried. The solvent was removed under
reduced pressure to give an oil, which was further purified by column
chromatography on silica gel. Elution with 2% methanol in DCM gave
the title compound (3) (5.0 g, 71%) as a colourless solid.
N-[1,1-bis(hydroxymethyl)-2-acetoxyethyl]-Nα-(benzyloxycarbonyl)-
glycinamide (10)
Trimethyl orthoacetate (1 mL) and formic acid (2 drops) was added to
a stirred solution of (1) (1.0 g, 3.2 mmol) in dry DMF (10 mL) under a
nitrogen atmosphere and the mixture stirred at 50°C for 14 h. The
solvent and excess reagent were removed under vacuum to yield a
1
colourless semi-solid (1.0 g). Crude H NMR spectroscopic and mass
spectrometric (CI+) analysis indicated the sample to be a mixture of
predominantly the cyclic orthoacetate (9) (methyl peak at δ 1.43, m/z

634
R. Adhikari et al.
N-[1-formyloxymethyl-2-[(1-oxohexadecyl)oxy]-1-[(1-oxohexadecyl)-
oxymethyl]ethyl]-Nα-(benzyloxycarbonyl)glycinamide (14)
gly-NH; 5.10, s, CH2Ph; 4.47, s, 2 × CH2O(palmitate); 3.88, s, CH2OSi;
3.76, d, J 5.1 Hz, CH2N; 2.23, t, J 7.5 Hz, 2 × CH2CO; 1.44–1.65, m, 2
× CH2CH2CO; 1.25, m, 24 × CH2; 1.06, s, C(CH3)3; 0.88, t, J 6.2 Hz, 2
× Me. Mass spectrum (APCI+) m/z 1050 (M+H, 22%), 772 (100);
(APCI–) m/z 1062 (M+Cl–, 47%), 824 (100).
Standard acylation of (13) afforded the title compound (14) (0.77 g,
87%) as a colourless solid. M.p. 47–49°C (Found: C, 68.6; H, 10.1; N,
3.2%. C47H80N2O9 requires C, 69.1; H, 9.9; N, 3.4%). 1H NMR δ 8.01,
s, formate; 7.36, m, ArH; 6.51, s, TRIS-NH; 5.28, m, gly-NH; 5.14, s,
CH2Ph; 4.55, s, CH2O(formate); 4.41, s, 2 × CH2O(palmitate); 3.81, d,
J 5.8 Hz, CH2N; 2.32, t, J 7.5 Hz, 2 × CH2CO; 1.50-1.70, m, 2 ×
CH2CH2CO; 1.25, m, 24 × CH2; 0.88, t, J 6 Hz, 2 × Me. Mass spectrum
(ESI+) m/z 818 (M+H, 100%).
Acknowledgments
The authors acknowledge the financial support of F. H.
Faulding and Co. Limited, 1538 Main North Road, Salisbury
South, S.A. 5106, and the excellent technical assistance of
Noel Hart and Marianne Bliese.
N-(2,2-Dimethyl-5-(t-butyldiphenylsilyloxymethyl)-[1,3]dioxan-5-yl)-
Nα-(benzyloxycarbonyl)glycinamide (17)
Imidazole (3.4 g, 50 mmol) was added to a solution of (5) (8.0 g,
22.7 mmol) in DMF (50 mL). t-Butyldiphenylchlorosilane (6.5 mL,
25 mmol) was added and the reaction mixture was stirred for 10 min at
0°C and thereafter at room temperature for 2 h. Solvent was removed
under vacuum and the residue was extracted with DCM (400 mL). The
extract was washed with water, dried over potassium carbonate and the
solvent was removed under vacuum to yield a colourless semi solid
(approx. 14 g) which was crystallized from chloroform/petroleum spirit
afford the title compound (17) (13.5 g, 92%) as a colourless crystalline
solid. M.p. 104–105°C (Found: m/z 591.291. C33H43N2O6Si requires
m/z 591.289). 1H NMR δ 7.30–7.70, m, 3 × Ph; 6.06, s, TRIS-NH; 5.27,
m, gly-NH; 5.10, s, CH2Ph ; 4.30 and 3.83, AB, J 11.8 Hz, 2 × CH2O;
3.97, s, CH2OSi; 3.78, J 7 Hz, CH2N; 1.49, s, CH3; 1.37, s, CH3; 1.07,
s, C(CH3)3. Mass spectrum (APCI+) m/z 613 (M+Na, 45%), 551 (100).
References
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N-[1,1-bis(hydroxymethyl)-2-(t-butyldiphenylsilyloxymethyl)ethyl]-
Nα-(benzyloxycarbonyl)glycinamide (18)
A solution of (17) (9.0 g, 15.25 mmol) and p-TsOH (0.365 g,
1.92 mmol) in acetone/water (85:15, 300 mL) was refluxed for 12 h.
The acetone was removed under vacuum and the concentrate was
diluted with water (50 mL) and extracted with ether (3 × 100 mL). The
combined extracts were dried over potassium carbonate and
concentrated to yield the title compound (18) (7.0 g, 83%) as a
colourless, viscous oil which was used without further purification.
(Found: m/z 559.259. C30H39N2O6Si requires m/z 551.258.) 1H NMR δ
7.28–7.78, m, 3 × Ph; 6.91, s, TRIS-NH; 5.38, m, gly-NH; 5.06, s,
CH2Ph; 3.84, d, J 5.8 Hz, CH2N; 3.79 and 3.56, AB, J 11.7 Hz, 2 ×
CH2O; 3.69, s, CH2OSi; 1.07, s, C(CH3)3. Mass spectrum (APCI+) m/z
551 (M+H, 100%).
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N-[1-t-butyldiphenylsilyloxymethyl-[2-[(1-oxohexadecyl)oxy]-1-[(1-
oxohexadecyl)oxymethyl]ethyl]-Nα-(benzyloxycarbonyl)-
glycinamide (19)
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Standard acylation of (18) afforded the title compound (19) (11.4 g,
93%) as a light yellow coloured solid. A portion of the product was
crystallized from chloroform/petroleum spirit to give a colourless solid.
M.p. 40–42°C (Found: m/z 1027.720. C62H99N2O8Si requires m/z
1027.717). 1H NMR δ 7.31–7.66, m, 3 × Ph; 6.30, s, TRIS-NH; 5.24, m,