The facile preparation of primary and secondary amines via an improved Fukuyama-Mitsunobu procedure. Application to the synthesis of a lung-targeted gene delivery agent

Article abstract of DOI:10.1039/b418168a

An efficient modification of the Fukuyama-Mitsunobu procedure has been developed whereby primary or secondary amines can be synthesized from alkyl alcohols and the corresponding nosyl-protected/activated amine. Most importantly, the use of the DTBAD and diphenylpyridinylphosphine, as Mitsunobu reagents, generates reaction by-products that can be easily removed, providing a remarkably clean product mixture. This improved technique was implemented in the synthesis of a complex lipopeptide designed to target α ₉β₁-integrin proteins predominant on upper airway epithelial cells. The Royal Society of Chemistry 2005.

Full text of DOI:10.1039/b418168a

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A R T I C L E
The facile preparation of primary and secondary amines via an
improved Fukuyama–Mitsunobu procedure. Application to the
synthesis of a lung-targeted gene delivery agent
Cristina Guisado,a Jodie E. Waterhouse,a Wayne S. Price,a Michael R. Jorgensen*b and
Andrew D. Miller*a
a Imperial College Genetic Therapies Centre, Department of Chemistry, Flowers Building,
Armstrong Road, Imperial College London, London, UK SW7 2AZ.
E-mail: a.miller@imperial.ac.uk
b IC-Vec Ltd., Flowers Building, Armstrong Road, London, UK SW7 2AZ.
E-mail: m.jorgensen@imperial.ac.uk
Received 2nd December 2004, Accepted 25th January 2005
First published as an Advance Article on the web 15th February 2005
An efficient modification of the Fukuyama–Mitsunobu procedure has been developed whereby primary or secondary
amines can be synthesized from alkyl alcohols and the corresponding nosyl-protected/activated amine. Most
importantly, the use of the DTBAD and diphenylpyridinylphosphine, as Mitsunobu reagents, generates reaction
by-products that can be easily removed, providing a remarkably clean product mixture. This improved technique was
implemented in the synthesis of a complex lipopeptide designed to target a9b1-integrin proteins predominant on
upper airway epithelial cells.
the acidic component (pKa < 11) of the reaction in the
Introduction
form of N-alkylsulfonamides,8 amides,10 phthalimides11 or N-
trifluoroacetamides.12 Perhaps the most useful activating group
is the 2-nitrobenzenesulfonamide (Ns- or nosyl group), de-
veloped by Fukuyama and Kan,13 which also functions as
a protecting group that can be efficiently removed with soft
nucleophiles via Meisenheimer complexes.13,14 Both primary and
secondary amines can be synthesized under standard Mitsunobu
conditions15 from alkyl alcohols using the corresponding nosy-
lamide or N-alkylnosylamide, respectively.
Numerous reagents have been developed to improve on the
versatility of the original DEAD and TPP combination e.g.
1,1ꢀ-(azodicarbonyl)dipiperidine (ADDP),16 N,N,Nꢀ,Nꢀ-tetra-
methylazodicarboxamide (TMAD)12 and cyanomethylenetri-
butyl-phosphorane (CMBP),17 with modified phosphines such
as tributylphosphines (TBP),18 trimethylphosphine (TMP),18
4-(dipenylphosphinyl)benzoic acid 2-(trimethylsilyl)ethyl ester
(DPPBE)19 and 1,2-bis[diphenylphosphino] ethane.20 None of
these aforementioned reagents has appeared to have gained
widely accepted use in the literature.
However, as previously noted, one major problem with the
Mitsunobu reaction is the isolation and purification of the de-
sired product from the complex reaction mixtures; in particular,
from reagent by-products such as triphenylphosphine oxide
and dicarboethoxyhydrazine derived from TPP and DEAD,
respectively. This problematic aspect has led to the development
of some Mitsunobu reagents that can be removed from the
reaction mixtures by simple post-reaction procedures.21 For
example, fluorous analogues of DEAD can be removed by flu-
orous extraction techniques21b,c and chemically-tagged reagents
(and resultant by-products) can be selectively removed by post-
reaction sequestations.21a,f Polymer-bound PPh3 and the resul-
tant post-reaction polymer-bound triphenylphosphine oxide are
eliminated from the reaction mixture by simple filtration.21e,f
During the synthesis of lung-targeting gene delivery agent 1
(Fig. 1) significant synthetic problems were experienced when
we attempted to install the secondary amine;22,23 the methods
attempted often leading to the elimination of cholesterol or
production of other by-products. Furthermore, due to their
inherent ambiphilic nature the purification of lipids can be an
extremely difficult task, especially when dealing with complex
mixtures of product, by-products and reactants. As a result an
With the ever increasing amount of biologically relevant nitroge-
nous molecules, the preparation of the amine functional group
is probably one of the most significant aspects in organic chem-
istry today. Furthermore, polymethylene polyamines have been
shown to be involved in many important biological functions
and are promising antiparasitic agents, antimalarials, antidiar-
rhoeals, anti-HIV agents and metal chelators.1 Another impor-
tant application of the polyamines is their recent exploitation as
gene delivery agents, especially when they are conjugated to lipid
moieties such as cholesterol. In this case the polyamine has the
dual purpose of binding and condensing nucleic acids, as well
as structurally providing the hydrophilic end of the ambiphilic
lipid important in the formation of liposomes and micelles.2
The efficiency of polyamine synthesis generally relies on the
effectiveness of the transformation steps of primary alcohols to
primary amines and, subsequently, primary amines to secondary
amines. Although the investigation into new methods for the
synthesis of amines is recognised as an important area of
organic chemistry, there are few efficient installations of nitro-
gen onto functionalised organic molecules. Furthermore, the
manipulation, handling and purification difficulties relating to
nitrogenous containing compounds are well accepted by organic
chemists.
The installation of primary amines3 can be achieved via
Gabriel synthesis4 or by transformation of an alkyl halide to an
azide5 followed by reduction to the primary amine.6 The alky-
lation of p-toluensulfonamide7 or N-Boc-p-toluensulfonamide8
under Mitsunobu conditions are also useful methods, although
they require relatively harsh deprotection conditions.
The synthesis of secondary amines from the corresponding
primary amines can also be achieved by several methods:9 N-
Alkylation with alkyl halides or sulfonates, reductive alkylations
with aldehydes or ketones and reduction of N-alkylamides.
The method of Weinreb and coworkers for the alkylation
of N-methyl-p-toluenesulfonamide8 with various primary and
secondary alcohols under Mitsunobu conditions has led to the
development of many different Mitsunobu modifications over
the past 15 years.
For the synthesis of amines from alcohols using the Mit-
sunobu reaction, the amines must be activated/protected as
T h i s j o u r n a l i s
T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 0 4 9 – 1 0 5 7
1 0 4 9
©

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Fig. 1 Lung-targeting gene delivery agent JW001 1.
efficient modification of the Fukuyama–Mitsunobu reaction for
generation of primary and secondary amines from primary or
secondary alcohols was established (Scheme 1).22
The modified lipid 1 incorporates an integrin-mediated target-
ing ligand, based upon the protein tenascin C, that can target the
a9b1-integrin commonly found in lung epithelial cells (Fig. 1).22
Such an agent could be important for the delivery of suitable
therapeutic DNA to the lung for treatment of the devastating
respiratory disease cystic fibrosis.
for the conversion of 2 to the desired amine 4 was achieved
by a combination of several cumbersome, often low yielding,
functional group interconversion steps.23 These proceeded via
the conversion of the alcohol to the corresponding mesylate,
nucleophilic substitution by an azide anion and subsequent
reduction of the azide (adaptation of the Staudinger reaction)
to the primary amine giving the desired product in 45% overall
yield.23 The seemingly simple N-alkylation of the amine (to
give the desired secondary amine) frequently results in the
production of undesired by-products, including polyalkylated
amine products. Despite the ease with which this reaction
proceeds, the inherent lack of control results in complex
mixtures and low isolated yields. In an effort to improve on this
strategy we employed an adapted and optimised version of the
Fukuyama–Mitsunobu procedure.24 Using this method it is
Results and discussion
A large laboratory stock of cholesterol-derived alcohol 222,23
was deemed to be a good starting point for the synthesis of
tenascin lipopeptide 1 (Scheme 1). Initially our synthetic route
Scheme 1 The synthesis of lung-targeting gene delivery agent JW001 1. Reaction conditions: (a) DTBAD, Ph2PyP, CH2Cl2, rt, 90%; (b) 3,6,9-
trioxa-11-benzyloxyundecan1-ol, DTBAD, Ph2PyP, CH2Cl2, rt, 71%; (c) sodium naphthalenide, THF, −30 ◦C, 74%; (d) (Boc)2O, Et3N, DCM,
84%; (e) p-nitrophenyl chloroformate, DMAP, DCM, 92%; (f) i. 9, piperidine, DMF; ii. 8, Et3N, DMF, 18 h; iii. Resin cleavage: 5% H2O in TFA,
1.5 h, 10%.
1 0 5 0
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 0 4 9 – 1 0 5 7

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Table 1 Conversion of primary and secondary alcohols to primary
amines
possible to convert a primary alcohol to a protected primary
amine in one step. This primary amine can also be easily con-
verted to a secondary amine under analogous conditions. This
procedure bypasses some of the caveats of previous protocols
for synthesis of linear polyamines; it affords better yields of the
primary amines and also asserts more chemoselective control
over their subsequent alkylation.
Initially we utilised the standard Fukuyama–Mitsunobu
procedure24 whereby cholesteryl-alcohol 2 was reacted with 2-
nitrobenzenesulfonamide 3 under standard DEAD–PPh3 con-
ditions (Scheme 1). However, poor conversion of 2 to 2-nosyl
amine occurred and 4 was obtained in only 43% yield. Further-
more, purification of 4 from the reaction by-products was prob-
lematic. In an effort to simply the purification of the mixtures
resulting from standard Mitsunobu conversions15, Kiankarimi
et al.25 introduced a procedure whereby they substituted
DEAD for di-tert-butylazodicaboxylate (DTBAD). By adding
an additional post-reaction acid quench step, the unreacted
DTBAD and dicarbo-tert-butyloxyhydrazine would decompose
to isobutene, nitrogen (or hydrazine) and carbon dioxide, thereby
considerably cleaning-up the reaction mixture. Furthermore, the
substitution of PPh3 for diphenyl-2-pyridylphosphine allowed
removal of all unreacted phosphine and its oxide by-product
by means of an acidic extractive work up. Employing these
conditions, together with slow addition (ca. 40 min) of the
DTBAD reagent in anhydrous dichloromethane, we generated 4
cleanly in much improved yield (90%). It was later noted that, by
using 2 eq. each of DTBAD and diphenyl-2-pyridylphosphine,
respectively, the slow addition step could be omitted. Similarly,
nosyl-protected secondary amine 5 was synthesized in 71% yield
from 4 and monobenzyl-protected tetraethylene glycol.26
Deprotection of both the nosyl and benzyl protecting groups
in 5 was achieved in one step with sodium naphthalenide
affording 6 in 74% yield. Boc-protection of the secondary amine
and subsequent activation of the primary alcohol as the p-
nitrophenol carbonate gave 8 in 77% yield for the two steps. The
lipidic carbonate 8 allows for facile conjugation of an alcohol
to a primary amine through formation of a carbamate linkage.
Thus 8 was now activated for solid phase synthetic coupling to
the fully protected, resin bound tenascin peptide 9.
Entry
R1
R2
Sulfonamide
Amine
1
PhCH2
H
H
H
H
H
H
H
H
H
H
H
CH3
CH3
10a (97%)
10b (90%)
10c (89%)
10d (96%)
10e (95%)
10f (30%)
10g (90%)
10h (96%)
10i (75%)
10j (92%)
10k (84%)
10l (70%)
10m (85%)
11a (89%)
11b (95%)
11c (91%)
11d (92%)
11e (93%)
11f (80%)
11g (86%)
11h (90%)
11i (100%)
11j (91%)
11k (80%)
11l (80%)
11m (84%)
2
p-MeOC6H4CH2
p-NO2C6H4CH2
Ph
3
4
5
p-MeOC6H4
6
p-NO2C6H4
=
7
PhCH CH
8
CH3CH2CH2CH2
CH3(CH2)2(CH3)CH
CH3CH2C≡C
9
10
11
12
13
=
CH3CH2CH CH
CH3CH2CH2CH2
PhCH2
extensively studied in the literature.8,13 Under our conditions,
treatment of nosyl-protected phenylethylamine 10a with
primary alcohols n-pentanol (entry 14) and phenylethylamine
(entry 15) gave sulfonamides 12a and 12b respectively in good
yields (Table 2). However, reaction of 10a with secondary
alcohols hexan-2-ol (entry 16) and 1-phenylpropan-2-ol (entry
17) gave only average yields of the corresponding sulfonamides
12c and 12d. It should be noted however, that no further
attempts were made to optimise this procedure for the synthesis
of secondary amines. Nonetheless, the ease of purification
would still make this modification an attractive alternative to
standard Mitsunobu procedures.
Table 2 Conversion of primary and secondary alcohols to secondary
amines
The tenascin peptide22a (amino acid sequence: NH2–
GGGPLAEIDGIELA–CO2H) was synthesized on acid labile
Wang resin (Novabiochem, C Biosciences, UK) according
to a standard Fmoc peptide synthesis protocol22b affording
the corresponding fully protected and resin-bound tenascin 9
(Scheme 1). After removal of the N-terminal Fmoc-protecting
group, the liberated amine was reacted with carbonate 8 giving
the fully-protected tenascin lipopeptide on resin. Finally, the
resin cleavage (and concomitant removal of all protecting
groups) with trifluoroacetic acid (TFA) gave the desired tenascin
product 1 in 10% yield after purification by semi-preparative
reverse phase HPLC.
Entry R1
R2
Sulfonamide Amine
14
15
16
17
CH3CH2CH2CH2
PhCH2
CH3CH2CH2CH2
Ph CH2
H
H
12a (74%)
12b (64%)
13a (90%)
13b (96%)
13c (96%)
13d (94%)
CH3 12c (50%)
CH3 12d (40%)
Thus, having developed an extremely facile procedure for
producing primary and secondary amines on cholesterol-
based lipids, previously a problematic synthetic step in our
laboratories,23 we were interested to investigate its versatility
further. 2-Nitrobenzenesulfonamide 3 was treated with a variety
of primary (entries 1–11) and secondary alcohols (entries 12–13)
under the new DTBAD (2 eq.)–diphenyl-2-pyridylphosphine
(2 eq.) conditions, thereby obtaining the respective nosyl-
protected amines 10 (Table 1). Deprotection of the nosyl group
was achieved by treating 10 with thiophenol and potassium
carbonate in acetonitrile at 50 ◦C overnight, giving the amines 11
(Table 1). Both nosylamine 10 and deprotected 11 were obtained
in good to excellent yields, from both primary and secondary
alcohols. An exception to this trend was the reaction with
p-nitrobenzylalcohol (entry 6) which gave the corresponding
sulfonamide 10f in a poor 30% yield.
In conclusion, we have developed an improved Fukuyama–
Mitsunobu procedure for the high yielding synthesis of primary
and secondary amines. The combination of nosyl-chemistry and
“cleaner” Mitsunobu reagents described here is applicable to
the sysnthesis of amines in both natural product and medicinal
chemistry. Lastly, this method has permitted the synthesis of
the lung-targeted gene delivery agent 1 which is currently
undergoing biological evaluation.
Experimental section
General
The Fukuyama–Mitsunobu reaction of N-nosyl-Nꢀ-
alkylamines with primary and secondary alcohols has been
Dried dichloromethane was distilled with phosphorus pentox-
ide, other solvents were purchased predried as required. All the
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 0 4 9 – 1 0 5 7
1 0 5 1

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reactions were performed under nitrogen using dry solvents
unless otherwise stated. Thin layer chromatography (tlc) was
performed on pre-coated Merck-Kieselgel 60 F254 aluminium
backed plated and revealed with ultraviolet light, iodine, acidic
ammonium molybdate(IV), acidic ethanolic vanillin, iodine,
bromocresol green, Dragendorff’s reagent, ninhydrin, chloranil
or other agents. Flash column chromatography was accom-
removed under a reduced pressure to give 2 (44 g, 87%) as a
colourless oil; tmax(film)/cm−1 3339, 2922, 1671, 1472 and 1376;
dH(300 MHz, CDCl3) 0.67 (3H, s), 0.87 (6H, d, J 6.5), 0.91 (3H,
d, J 6.5), 1.00 (3H, s), 1.62 − 1.03 (21H, m), 1.70–2.06 (5H,
m), 2.23–2.36 (2H, m), 3.33 (2H, t, J 4.5), 3.72 (2H, t, J 5.0),
4.50 (1H, m), 5.00 (1H, br), 5.37 (1H, m); m/z (FAB) 496 (M +
Na)+, 474 (M + H)+, 369 (chol)+. Found: (M + H)+ 474.3654,
C30H52NO3 requires (M + H)+ 474.3947.
1
plished on Merck-Kieselgel 60 (230–400 mesh). H- and 13C-
NMR were recorded on either Bruker DRX300, Jeol GX-270Q,
or Bruker Advance400 Ultrashield TM machine using residual
isotopic solvent as an internal reference (s = singlet, d = doublet,
t = triplet, q = quartet, quin = quintet, br = broad singlet).
FAB mass spectra were recorded using VG-7070B, Jeol SX-102
instruments and ESI mass spectrometry was carried out using a
Bruker Daltronics ESI 6000 spectrometer. Infrared spectra were
recorded using Jasco 620 FTIR spectrometer. Where appropriate
a Pharmacia LKB–Ultrospec III (deuterium lamp at 300 nm)
was used to read the UV absorbance. All chemicals were pur-
chased from Sigma Aldrich or Lancaster if not otherwise stated.
N1-Cholesteryloxycarbonyl-1-amino-2ꢀꢀ-
nitrobenzenesulfonamidoethane 4
A mixture of 2-nitrobenzylsulfonamide 3 (0.202 g, 1 mmol),
diphenyl-2-pyridylphosphine (0.263 g, 1 mmol) and 2 (0.237 g,
0.5 mmol) was dissolved in anhydrous dichloromethane (10 mL)
under a nitrogen atmosphere. To this stirred mixture di-tert-
butylazadicarboxylate (0.230 g, 1 mmol) was added in one
portion. After stirring for 2 h, HCl in dioxane (4 M, 5 mL) was
added and the mixture left to stir for 1 h. The mixture was then
concentrated in vacuo, dissolved in dichloromethane (20 mL)
and washed with aq. HCl (4 M, 3 × 15 mL). The organic phase
was collected, dried with magnesium sulfate and concentrated in
vacuo. The crude residue was purified by flash chromatography
on silica gel (hexane–EtOAc 2 : 1), to yield 4 (0.304 g, 90%)
as an off white solid, mp 185 ◦C; tmax(film)/cm−1 3347, 2946,
2868, 2253, 1770, 1696, 1540, 1440, 1364, 1254, 1167, 908 and
733; dH(400 MHz, CDCl3) 0.67 (3H, s), 0.85 (3H, d, J 6.4),
0.86 (3H, d, J 6.4), 0.90 (3H, d, J 6.8), 0.99 (3H, s), 1.02–1.63
(21H, m), 1.77–2.06 (5H, m), 2.22–2.32 (2H, m), 4.42–4.46 (1H,
m,), 4.98 (1H, br), 5.33 (1H, m), 5.77 (1H, br), 7.71–7.76 (2H,
m), 7.84–7.88 (1H, m), 8.11–8.15 (1H, m); dC(100 MHz, CDCl3)
11.8, 18.7, 19.3, 21.0, 22.5, 22.8, 23.8, 24.3, 28.0, 28.1, 28.2, 31.8,
31.9, 35.8, 36.2, 36.5, 36.9, 38.4, 39.5, 39.7, 40.7, 42.3, 43.7, 50.0,
56.1, 56.7, 74.8, 122.6, 125.4, 131.0, 132.8, 133.5, 133.6, 139.7,
148.0, 156.4; m/z (ESI) 655.9 (M − H)−, C36H54N3O6S requires
(M − H)− 656.4.
Peptide synthesis: materials and methods
The FastMocTM reagent HBTU was obtained from Advanced
Chemtech Europe (Cambridge, UK) and CN Bioscience (Not-
tingham, UK). The solvents, DMF and acetonitrile, were
purchased from Rathburn (Walker-Burn, Scotland). All the
reagents used in the syntheses were of the highest purity. The
following amino acids and resins were obtained from Nova
Biochem: Fmoc–Leu–OH, Fmoc–Glu(OtBu)–OH, Fmoc–Ile–
OH, Fmoc–Gly–OH, Fmoc–Asp(OtBu)–OH, Fmoc–Ala–OH,
Fmoc–Pro–OH, Fmoc–Gly3–OH, Gly–(OtBu)·HCl and Fmoc–
Ala-Wang (100–200) preloaded resin.
Peptides and lipopeptides were synthesised using two different
reaction vessels. The first used the Isolute SPE Filtration
Columns and their appropriate accessories (caps and stoppers)
combined with a VacMaster-10 sample processing station for
removing solvents, all purchased from Jones Chromatography
(Mid Glamorgan, UK). The second was using a glass reaction
vessel treated overnight with a solution of 50 mL toluene–
dichlorodimethylsilane 20%. The vessel was then washed with
methanol (2 × 30 mL), DMF (2 × 30 mL) and dichloromethane
(1 × 30 mL) before drying.
N1-Cholesteryloxycarbonyl-3-aza-N3-2ꢀꢀ-nitrobenzenesulfonyl-
6,9,12-trioxa-15-benzyloxy-1-amino-tetradecane 5
A solution of diphenyl 2-pyridylphosphine (0.028 g, 0.11 mmol)
and DTBAD (0.024 g, 0.11 mmol) in dry dichloromethane
(5 mL) was transferred under nitrogen into a solution of 3,6,9-
trioxa-11-benzyloxyundecan-1-ol (0.020 g, 0.07 mmol) in dry
dichloromethane (2.5 mL). A solution of sulfonamide 4 (0.050 g,
0.07 mmol) in dry dichloromethane (2.5 mL) was also added to
the reaction mixture and left stirring overnight. The solvent
was removed under a reduced pressure and the resultant residue
was purified by column chromatography (hexane–EtOAc 2 :
3) to produce 5 (0.048 g, 71%) as a colourless viscous oil;
mmax(film)/cm−1 3345, 3045, 2945, 2873, 1711, 1455, 1123 and
1010; dH(400 MHz, CDCl3) 0.68 (3H, s), 0.85 (6H, d, J 6.5), 0.90
(3H, d, J 6.5), 0.99 (3H, s), 1.00–1.62 (21H, m), 1.78–2.09 (5H,
m), 2.15–2.38 (2H, m), 3.38 (2H, m), 3.45 (m, 2H), 3.52 (2H,
t, J 4.8), 3.58 (2H, m), 3.59 (12H, m), 4.47 (1H, m), 4.58 (2H,
s), 5.38 (1H, m), 5.55 (1H, br), 7.27–7.37 (5H, m), 7.71–7.76
(2H, m), 7.84–7.88 (1H, m), 8.11–8.15 (1H, m); dC(100 MHz,
CDCl3) 11.8, 18.7, 19.3, 21.0, 22.5, 22.7, 23.8, 24.3, 28.0, 28.2,
29.7, 31.9, 35.8, 36.2, 36.6, 37.0, 39.5, 39.8, 42.3, 48.3, 49.6,
50.1, 56.2, 56.7, 69.5, 69.8, 70.3, 70.4, 70.5, 70.6, 70.7, 73.2,
76.6, 119.5, 122.5, 126.3, 127.6, 128.3, 130.6, 132.7, 135.3, 138.0,
139.6, 148.0, 148.1, 155.9; m/z (FAB) 969 (M + H)+, 369 (chol)+.
The preloaded resin derivatised with the first Fmoc-protected
amino acid was added to either reaction vessel. It was washed
with MeOH (4 × 2 min with 30 mL) and DMF (4 × 2 min
with 30 mL). To allow the resin to swell, DMF (10–20 mL) was
added to the resin for 30 min to 1 h. After the first deprotection
of Fmoc, the loading of the resin was calculated. For it, the
collected solutions from Fmoc-deprotection were filled up to
250 mL with MeOH in a volumetric flask, diluted 1 : 25 and the
absorbance at 300 nm was measured by a UV spectrometer. The
loading on the resin was calculated by the following equation:
A · d · v · 1 × 10−3
n =
,
e · 1
where
e300 = 7800 mol−1 cm−1 d = 25 (dilution) v = 250 mL
A(k300 nm) = absorbance n = mol × 10−3
1 = 1 cm
N1-Cholesteryloxycarbonyl-1-amino-2-hydroxyethane 2
Ethanolamine (15 mL, 246.0 mmol) was dissolved in dichloro-
methane (35 mL) and was cooled to 0 ◦C using an ice bath.
A solution of cholesteryl chloroformate (50 g, 112.0 mmol) in
dichloromethane (300 mL) was added dropwise over 1 h; during
that time a white precipitate formed. The reaction warmed to rt
and continued stirring for 18 h. The precipitate was removed by
filtration and the solution was washed with saturated NaHCO3
(2 × 75 mL), water (2 × 75 mL), dried (MgSO4) and the solvent
N1-Cholesteryloxycarbonyl-3-aza-6,9,12-trioxa-1-amino-15-
hydroxytetradecane 6
A dry round bottomed flask was charged with naphthalene
(4.5 g, 34.7 mmol) and sodium metal (0.8 g, 34.7 mmol) under
nitrogen. Dry THF (100 mL) was added to the flask a◦nd
vigorously stirred for 1 h. The solution was cooled to −30 C
using dry ice and acetone. A solution of 5 (1.6 g, 1.7 mmol) in
1 0 5 2
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 0 4 9 – 1 0 5 7

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dry THF (10 mL) was added to the flask over 5 min, and stirred
for 45 min maintaining −30 ◦C. Once the reaction had gone
to completion, a solution of 2,6-di-tert-butyl-4-methyl phenol
(7.48 g, 34.7 mmol) in THF (2 mL) was slowly added and
allowed to warm to rt. The solvent was removed under a reduced
pressure and the resultant residue was dry loaded onto a silica
flash column (DCM–MeOH–NH3 97 : 2.5 : 0.5) giving 6 (0.83 g,
74%) as a viscous white solid; mmax(film)/cm−1 3610, 3345, 2936,
2863, 1698, 1456 and 1260; dH(400 MHz, CDCl3) 0.67 (3H, s),
0.85 (6H, d, J 6.5), 0.90 (3H, d, J 6.5), 0.99 (3H, s), 1.01–1.65
(21H, m), 1.76–2.08 (5H, m), 2.17–2.41 (2H, m), 2.74 (4H, m),
2.98 (1H, br), 3.30 (2H, m), 3.58–3.79 (14H, m), 4.50 (1H, m),
5.36 (1H, m), 5.72 (1H, br); dC(100 MHz, CDCl3) 11.8, 18.7,
19.3, 21.0, 22.5, 22.8, 23.8, 24.2, 28.0, 28.2, 29.6, 31.8, 35.7,
36.0, 36.5, 36.9, 38.6, 39.5, 39.7, 40.3, 42.2, 48.4, 48.7, 49.9,
56.1, 56.6, 61.3, 69.7, 70.1, 70.3, 70.5, 73.0, 74.1, 122.4, 139.9,
156.3; m/z (FAB) 649 (M + H)+, 369 (chol)+. Found: (M + H)+
649.5144, C38H68N2O6 requires (M + H)+ 649.5156.
followed by washing with DMF (5 × 2 min). After each Fmoc
deprotection step, the collected cleavage and washing solutions
were diluted and the absorbance was determined as for the initial
loading calculations in order to check the completeness of the
Fmoc cleavage. A Kaiser test was performed; if it was negative
the deprotection step was repeated. Once the Fmoc groups were
removed a solution of Fmoc protected amino acid (3 eq.), HBTU
(3 eq.) and DIPEA (5 eq.) in DMF (5 to 10 mL) was added to
the resin and shaken for 45 min. The resin was washed with
DMF (5 × 2 min) and another Kaiser test performed, again if
a negative result was not obtained the coupling was repeated.
This process was repeated until the correct amino acid sequence
was obtained, where it was then washed with dichloromethane
(2 × 5 mL, 2 min) and MeOH (2 × 5 mL, 2 min). Batches of
50 mg were deprotected and cleaved for characterisation. The
crude peptide was purified by reversed-phase HPLC (Vydac C4
column at a flow rate of 1 mL min−1, 214 nm), using a gradient
30–100% acetonitrile over 40 min (water as the second solvent).
G3PLAEIDGIELA (0.010 g, 27%) eluting at Rt = 6.24 min, 99%
purity; m/z (ESI) 1311 (M + H)+, C57H95N14O21 requires (M +
H)+ 1311.
N1-Cholesteryloxycarbonyl-3-aza-N3-tbutoxycarbonyl-6,9,12-
trioxa-1-amino-15-hydroxytetradecane 7
To a solution of 6 (0.030 g, 0.046 mmol) in dry dichloromethane
(1 mL), di-tert-butyldicarbonate (0.010 g, 0.046 mmol) and
triethylamine (4.1 mL, 0.05 mmol) were added under nitrogen.
The reaction was stirred for 10 h until the reaction had gone
to completion. The solvent was removed in vacuo and the
residue was dry loaded onto a silica flash column (hexane–
EtOAc 1 : 1) affording 7 (0.028 g, 84%) as a white viscous solid;
mmax(film)/cm−1 3611, 2947, 2871, 1698, 1652, 1456 and 1265;
dH(400 MHz, CDCl3) 0.69 (3H, s), 0.88 (6H, d, J 6.5), 0.93 (3H,
d, J 6.5), 1.02 (3H, s), 1.03–1.72 (21H, m), 1.47 (9H, s), 1.76–
2.10 (5H, m), 2.22–2.48 (2H, m), 3.14 (1H, br), 3.26–3.48 (6H,
m), 3.52–3.85 (14H, m), 4.49 (1H, m), 5.35 (1H, m), 5.65 (1H,
br); dC(100 MHz, CDCl3) 11.5, 18.4, 19.0, 20.7, 22.3, 22.5, 23.5,
24.0, 27.7, 27.9, 28.1, 31.5, 35.5, 36.2, 36.7, 38.3, 39.3, 39.4, 39.5,
39.7, 42.0, 47.9, 48.2, 49.7, 55.9, 56.3, 61.2, 69.5, 70.0, 70.1, 72.3,
73.7, 79.5, 122.1, 139.5, 155.9; m/z (FAB) 771 (M + Na)+, 749
(M + H)+, 649 (M-Boc + H)+, 369 (chol)+. Found: (M + H)+
749.5708, C43H76N2O8 requires (M + H)+ 749.5680.
Lipopeptide 1
The tenascin peptidoresin 9 (0.050 g, 0.03 mmol) was swelled
with DMF (7 mL, 30 min), the terminal Fmoc was deprotected
using 25% piperidine in DMF (2 × 5 min, 5 mL) and washed
with DMF (5 × 2 min, 5 mL). A Kaiser test was performed and
produced a positive result. Dry DMF (1.5 mL) and triethylamine
(11 mL, 0.075 mmol) were transferred to the resin and shaken
for 5 min. A premixed solution containing carbonate 8 (0.054 g,
0.06 mmol) and triethylamine (11 mL, 0.075 mmol) in dry DMF
(1 mL) was also transferred into the reaction vessel under argon
(including 2 mL of washings) and shaken for 18 h. The resin
was washed with DMF (5 × 2 min, 10 mL) and a Kaiser test
performed. The colourless beads produced a negative result
indicating that the lipid had been successfully coupled. The
resin was washed further with MeOH (3 × 2 min, 5 mL)
and dichloromethane (3 × 5 mL, 2 min). The resin was air-
dried then cleaved for 1.5 h using 95% TFA–water (2 mL). The
crude lipopeptide was precipitated in ice cold MTBE (10 mL),
centrifuged (3600 rpm, 4 ◦C, 2 × 5 min) and freeze dried to
produce a white powder. The crude lipopeptide was purified
by reversed-phase HPLC (Vydac C4 colum at a flow rate of
1 mL min−1, 214 nm), using a gradient 30–100% acetonitrile over
40 min (water as the second solvent). Lipopeptide 1 (0.0058 g,
9.7%) was eluted at Rt = 32.6 min, 99% purity; m/z (ESI-MS)
1985.8 (M + H)+, 1574.8 (M–CholOCO)+, 1311.7 (tenascin +
H)+. C96H162N16O28 requires (M + H)+ 1986.
N1-Cholesteryloxycarbonyl-3-aza-N3-tbutoxycarbonyl-6,9,12-
trioxa-1-amino-15-O15-4ꢀꢀ-nitrophenyloxycarbonyltetradecane 8
To a solution of 7 (0.050 g, 0.067 mmol) in dry dichloromethane
(3.5 mL) was added DMAP (0.016 g, 0.13 mmol), triethylamine
(19 mL, 0.13 mmol) and p-nitrophenyl chloroformate (0.041 g,
0.2 mmol) under nitrogen. The reaction was stirred for 10 h,
the solvent removed under a reduced pressure and purified by
column chromatography (hexane–EtOAc 2 : 3) giving 8 (0.056 g,
92%) as a viscous white solid; mmax(film)/cm−1 3348, 2942, 1770,
1696, 1616 and 1593; dH(400 MHz, CDCl3) 0.69 (3H, s), 0.87
(6H, d, J 6.5), 0.91 (3H, d, J 6.5), 1.00 (3H, s), 1.01–1.72 (21H,
m), 1.46 (9H, s), 1.78–2.14 (5H, m), 2.21–2.44 (2H, m), 3.26–
3.52 (6H, m), 3.56–3.78 (10H, m), 3.80 (2H, m), 4.42–4.54 (3H,
m), 5.35 (1H, m), 5.63 (1H, br), 7.38 (2H, m), 8.27 (2H, m);
dC(100 MHz, CDCl3) 11.5, 18.4, 19.0, 20.7, 22.3, 22.5, 23.5, 24.0,
27.7, 27.9, 28.1, 31.5, 35.5, 36.2, 36.7, 38.3, 39.2, 39.4, 39.5, 39.7,
42.1, 48.0, 48.2, 49.7, 55.8, 56.3, 69.3, 69.9, 70.2, 70.4, 73.9, 79.3,
122.2, 124.9, 125.7, 139.6, 145.0, 152.1, 156.5, 163.0; m/z (FAB)
936 (M + Na)+, 914 (M + H)+, 814 (M–Boc + H)+, 369 (chol)+.
Found: (M + H)+ 914.5772, C50H79N3O12 requires (M + H)+
914.5742.
General procedure for Mitsunobu reaction of primary and
secondary alcohols with 2-nitrobenzenesulfonamide
A mixture of 2-nitrobenzenesulfonamide 3 (0.202 g, 1.0 mmol),
diphenyl-2-pyridylphosphine (0.263 g, 1.0 mmol) and the al-
cohol (0.5 mmol) was dissolved in anhydrous dichloromethane
(10 mL) under an atmosphere of nitrogen. To this solution di-
tert-butylazodicarboxylate (0.230 g, 1.0 mmol) was added in one
portion and the resulting mixture was stirred at rt for 2 h. HCl in
dioxane (5 mL, 4 M) was added to the mixture and, after stirring
for 1 h, the excess solvent was evaporated. The residue was
dissolved in dichloromethane and washed with 4 M HCl twice.
The organic layer was dried with anhydrous magnesium sulfate
and the solvent evaporated. Flash column chromatography gave
the desired sulfonamide.
Tenascin peptide 9
2-Nitro-N-phenethyl-benzenesulfonamide 10a14,18,27
This peptide was synthesised using preloaded Fmoc–Ala-Wang
resin (0.4 g, 0.2 mmol, 0.5 mmol g−1). Once the resin was swollen
the peptide sequence was built up by alternating coupling and
Fmoc deprotection steps. Fmoc deprotection was achieved by
cleaving with a solution of 25% piperidine in DMF (2 × 5 min)
Flash column chromatography (hexane–EtOAc 4 : 1) gave 10a
(97%) as a white waxy oil; dH(400 MHz, CDCl3) 2.76 (2H, t,
J 6.8), 3.32 (2H, q, J 6.8), 5.24 (1H, t, J 5.6), 7.00–7.02 (2H,
m), 7.10–7.18 (3H, m), 7.63–7.65 (2H, m), 7.73–7.76 (1H, m),
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 0 4 9 – 1 0 5 7
1 0 5 3

View Article Online
8.01–8.03 (1H, m); dC(100 MHz, CDCl3) 36.0, 45.0, 125.4, 126.9,
128.7, 128.8, 131.0, 132.8, 133.5, 133.9, 137.4, 147.9; m/z (ESI)
328.9 (M + Na)+, C14H14N2O4SNa requires (M + Na)+ 329.06.
131.1, 132.8, 133.5, 148.1; m/z (FAB) 273 (M + H)+, 186 (Ns)+.
Found: (M + H)+ 273.0901, C11H17N2O4S requires (M + H)+
273.0909.
N-(2-Methyl-pentyl)-2-nitro-benzenesulfonamide 10i
N-[2-(4-Methoxy-phenyl)-ethyl]-2-nitro-benzenesulfonamide 10b
Flash column chromatography (hexane–EtOAc 4 : 1) gave 10b
Flash column chromatography (hexane–EtOAc 7 : 1) gave 10i
(75%) as a colourless oil; dH(400 MHz, CDCl3) 0.81 (3H, t,
J 7.2), 0.86 (3H, d, J 6.4), 1.03–1.30 (4H, m), 1.58–1.66 (1H,
m), 2.86 (1H, dt, J 12.8, J 6.4), 2.98 (1H, dt, J 12.4, J 6.4), 5.30
(1H, t, J 6.4), 7.72–7.74 (2H, m), 7.81–7.84 (1H, m), 8.08–8.10
(1H, m); dC(100 MHz, CDCl3) 14.0, 17.4, 19.8, 32.9, 36.1, 49.7,
125.3, 131.0, 132.8, 133.6, 148.1; m/z (ESI) 308.9 (M + Na)+,
C12H18N2O4SNa requires (M + Na)+ 309.1.
◦
(90%) as a white solid, mp 151 C; dH(400 MHz, CDCl3) 2.77
(2H, t, J 6.8), 3.35 (2H, q, J 6.8), 3.77 (3H, s), 5.29 (1H, t, J 4.8),
6.76 (2H, d, J 8.4), 6.99 (2H, d, J 8.4), 7.71 (2H, dd, J 5.6,
J 3.2), 7.83 (1H, dd, J 6.0, J 3.2), 8.08 (1H, dd, J 6.0, J 3.2);
dC(100 MHz, CDCl3) 35.1, 45.3, 55.3, 114.2, 125.4, 129.3, 129.7,
131.0, 132.8, 133.4, 134.0, 158.5; m/z (ESI) 358.9 (M + Na)+,
C15H16N2O5SNa requires (M + Na)+ 359.07.
2-Nitro-N-pent-2-ynyl-benzenesulfonamide 10j
2-Nitro-N-[2-(4-nitro-phenyl)-ethyl]-benzenesulfonamide 10c
Flash column chromatography (hexane–EtOAc 6 : 1) gave 10j
(92%) as a colourless oil; dH(400 MHz, CDCl3) 0.81 (3H, t, J 7.6),
1.82 (2H, qt, J 7.2, J 2.4), 3.95 (2H, dt, J 6.0, J 2.4), 5.60 (1H, t,
J 6.0), 7.73–7.77 (2H, m), 7.89–7.92 (1H, m), 8.18–8.20 (1H, m);
dC(100 MHz, CDCl3) 12.0, 13.4, 34.0, 73.0, 87.3, 125.4, 131.7,
132.9, 133.6, 134.4, 148.0; m/z (FAB) 269 (M + H)+. Found:
(M + H)+ 269.0597, C11H13N2O4S requires (M + H)+ 269.0596.
Flash column chromatography (hexane–EtOAc 4 : 1) gave 10c
(89%) as a yellow solid, mp 115 ◦C; dH(400 MHz, CDCl3) 2.98
(2H, t, J 6.8), 3.45 (2H, q, J 6.8), 5.36 (1H, t, J 6.0), 7.31 (2H,
d, J 8.4), 7.70–7.73 (2H, m), 7.83–7.85 (1H, m), 8.05–8.07 (1H,
m), 8.09 (2H, d, J 8.8); dC(100 MHz, CDCl3) 36.2, 44.5, 123.9,
125.5, 129.7, 130.8, 132.9, 133.7, 133.8, 145.3, 147.1; m/z (ESI)
349.9 (M − H)−, C14H12N3O6S requires (M − H)− 350.1.
N-Benzyl-2-nitro-benzenesulfonamide 10d27,28
2-Nitro-N-pent-2-enyl-benzenesulfonamide 10k
Flash column chromatography (hexane–EtOAc 7 : 1) gave 10k
(84%) as a colourless oil; dH(400 MHz, CDCl3) 0.92 (3H, t, J
7.6), 1.95–2.03 (2H, m), 3.77 (2H, t, J 6.8), 5.21–5.29 (2H, m),
5.45–5.52 (1H, m), 7.72–7.76 (2H, m), 7.85–7.87 (1H, m), 8.12–
8.14 (1H, m); dC(100 MHz, CDCl3) 13.9, 20.7, 40.7, 122.7, 125.4,
131.2, 132.8, 133.6, 134.0, 136.7, 148.1; m/z (FAB) 271 (M +
H)+, 186 (M − Ns)+. Found: (M + H)+ 271.0766, C11H15N2O4S
requires (M + H)+ 271.0753.
Flash column chromatography (hexane–EtOAc 4 : 1) gave 10d
(96%) as a pale oil; dH(400 MHz, CDCl3) 4.32 (2H, d, J 6.0),
5.72 (1H, t, J 6.0), 7.22 (5H, br), 7.63 (1H, td, J 7.6, J 1.6), 7.67
(1H, td, J 7.6, J 1.6), 7.81 (1H, dd, J 7.6, J 1.6), 8.00 (1H, dd,
J 7.6, J 1.6); dC(100 MHz, CDCl3) 47.9, 125.3, 127.9, 128.1,
128.7, 131.0, 132.8, 133.4, 134.0, 135.7, 147.9; m/z (FAB)
293 (M + H)+, 91 (PhCH2)+. Found: (M + H)+ 293.0600,
C13H13N2O4S requires (M + H)+ 293.0596.
N-(4-Methoxy-benzyl)-2-nitro-benzenesulfonamide 10e29
N-(1-Methyl-pentyl)-2-nitro-benzenesulfonamide 10l
Flash column chromatography (hexane–EtOAc 6 : 1) gave 10l
(70%) as a colourless oil; dH(400 MHz, CDCl3) 0.77 (3H, t, J 7.2),
1.08 (3H, d, J 6.8), 1.14–1.26 (4H, m), 1.40–1.45 (2H, m), 3.45–
3.55 (1H, m), 5.11 (1H, d, J 8.0), 7.71–7.74 (2H, m), 7.82–7.85
(1H, m), 8.13–8.15 (1H, m); dC(100 MHz, CDCl3) 13.9, 21.8,
22.2, 27.6, 27.8, 37.0, 51.2, 125.3, 130.6, 132.9, 133.4, 135.1,
147.8; m/z (ESI) 308.9 (M + Na)+, C12H18N2O4SNa requires
(M + Na)+ 309.1.
Flash column chromatography (hexane–EtOAc 4 : 1) gave 10e
(95%) as a pale solid; dH(400 MHz, CDCl3) 3.75 (3H, s), 4.25
(2H, d, J 6.0), 5.62 (1H, t, J 6.0), 6.75 (2H, d, J 8.8), 7.12 (2H, d,
J 8.8), 7.64 (1H, td, J 7.6, J 1.6), 7.68 (1H, td, J 7.6, J 1.6), 7.82
(1H, dd, J 8.0, J 1.6), 8.01 (1H, dd, J 7.6, J 1.6); dC(100 MHz,
CDCl3) 47.5, 55.3, 114.1, 125.3, 127.7, 129.3, 131.1, 132.7, 133.3,
134.1, 147.9, 159.4; m/z (FAB) 323 (M + H)+, 290 (M − OMe)+.
2-Nitro-N-(4-nitro-benzyl)-benzenesulfonamide 10f
N-(1-Methyl-2-phenyl-ethyl)-2-nitro-benzenesulfonamide 10m
Flash column chromatography (hexane–EtOAc 4 : 1) gave 10f
(30%) as a yellow solid, mp 132 ◦C; dH(400 MHz, CDCl3) 4.44
(2H, d, J 6.4), 5.89 (1H, t, J 6.4), 7.46 (2H, d, J 8.8), 7.68 (1H,
td, J 7.6, J 1.6), 7.74 (1H, td, J 7.6, J 1.6), 7.87 (1H, dd, J 7.6,
J 1.2), 8.03 (1H, dd, J 7.6, J 1.6), 8.13 (2H, d, J 8.8); m/z (ESI)
335.9 (M − H)−, C13H10N3O6S requires (M − H)− 336.0.
Flash column chromatography (hexane–EtOAc 4 : 1) gave 10m
(85%) as a pale solid, mp 138 ◦C; dH(400 MHz, CDCl3) 1.22
(3H, d, J 6.4), 2.68–2.81 (2H, m), 3.74–3.81 (1H, m), 5.30 (1H,
d, J 7.2), 7.00–7.03 (2H, m), 7.07–7.10 (3H, m), 7.64–7.66 (2H,
m), 7.74–7.77 (1H, m), 8.00–8.02 (1H, m); dC(100 MHz, CDCl3)
22.2, 43.5, 52.8, 125.5, 126.8, 128.4, 129.2, 130.5, 132.9, 133.2,
134.6, 137.0, 147.4; m/z (ESI) 318.9 (M − H)−, C15H15N2O4S
requires (M − H)− 319.1.
2-Nitro-N-(3-phenyl-allyl)-benzenesulfonamide 10g
Flash column chromatography (hexane–EtOAc 4 : 1) gave 10g
(90%) as a yellow solid; dH(400 MHz, CDCl3) 3.85 (2H, td, J 6.4,
J 1.2), 5.43 (1H, t, J 6.0), 5.94 (2H, dt, J 15.6, J 6.4), 6.42 (1H,
d, J 15.6), 7.12–7.21 (5H, m), 7.58–7.60 (2H, m), 7.74–7.77 (1H,
m), 8.04–8.06 (1H, m); dC(100 MHz, CDCl3) 46.1, 123.5, 125.4,
126.5, 128.1, 128.6, 131.2, 132.8, 133.6, 133.7, 134.2, 135.9,
148.0; m/z (ESI) 340.9 (M + Na)+, C15H14N2O4SNa requires
(M + Na)+ 341.06.
General procedure for Mitsunobu reaction for primary and
secondary alcohols and Nosyl-protected primary amines
A mixture of 2-nitro-N-phenethyl-benzenesulfonamide (0.306 g,
1.0 mmol), diphenyl-2-pyridylphosphine (0.263 g, 1.0 mmol)
and the primary alcohol (0.5 mmol) was dissolved in anhydrous
dichloromethane (10 mL) under an atmosphere of nitrogen. To
this solution di-tert-butylazodicarboxylate (0.230 g, 1.0 mmol)
was added slowly (syringe pump, 30–45 min) and the resulting
mixture was stirred at rt for 2 h. HCl in dioxane (5 mL, 4 M) was
added to the mixture and, after stirring for 1 h, the excess solvent
was evaporated. The residue was dissolved in dichloromethane
and washed twice with 4 M HCl. The organic layer was dried
with anhydrous magnesium sulfate and the solvent evaporated.
Flash column chromatography gave the desired sulfonamide.
2-Nitro-N-pentyl-benzenesulfonamide 10h30
Flash column chromatography (hexane–EtOAc 4 : 1) gave 10h
(96%) as a colourless oil; dH(400 MHz, CDCl3) 0.81 (3H, t, J 7.2),
1.21–1.26 (4H, m), 1.46–1.53 (2H, m), 3.07 (2H, q, J 6.8), 5.28
(1H, t, J 6.0), 7.72–7.74 (2H, m), 7.81–7.84 (1H, m), 8.09–8.11
(1H, m); dC(100 MHz, CDCl3) 13.8, 22.1, 28.6, 29.1, 43.9, 125.4,
1 0 5 4
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 0 4 9 – 1 0 5 7

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2-Nitro-N-pentyl-N-phenethyl-benzenesulfonamide 12a
113.9, 129.8, 131.9, 158.1; m/z (ESI) 151.9 (M + H)+, C9H14NO
requires (M + H)+ 152.1.
2-(4-Nitro-phenyl)-ethylamine 11c33
Flash column chromatography (hexane–EtOAc 4 : 1) gave 12a
(74%) as a pale solid; dH(400 MHz, CDCl3) 0.77 (3H, t, J 6.8),
1.10–1.25 (4H, m), 1.43–1.50 (2H, m), 2.77 (2H, t, J 8.4), 3.24
(2H, t, J 8.0), 3.42 (2H, dd, J 10.0, J 8.0), 7.06–7.19 (5H, m),
7.49 (1H, dd, J 7.2, J 2.0), 7.54 (1H, td, J 7.2, J 2.0), 7.57 (1H,
td, J 7.6, J 1.6), 7.85 (1H, dd, J 7.6, J 1.6); dC(100 MHz, CDCl3)
14.0, 22.3, 27.8, 28.7, 35.2, 47.8, 48.8, 124.2, 126.7, 128.6, 128.8,
130.6, 131.7, 133.5, 133.7, 138.2, 148.1; m/z (FAB) 377 (M +
H)+. Found: (M + H)+ 377.1543, C19H25N2O4S requires (M +
H)+ 377.1535.
Flash column chromatography (CH2Cl2–MeOH–NH3 92 : 7 :
1) gave 11c (91%) as a yellow solid; dH(400 MHz, CDCl3) 2.86
(2H, t, J 6.8), 3.02 (2H, t, J 6.8), 7.36 (2H, d, J 8.8), 8.16 (2H,
d, J 8.8); dC(100 MHz, CDCl3) 39.9, 43.1, 123.7, 129.6, 146.7,
147.9; m/z (ESI) 166.9 (M + H)+, C8H18N2O2 requires (M +
H)+ 167.1.
Benzylamine 11d34
2-Nitro-N,N-diphenethyl-benzenesulfonamide 12b
Flash column chromatography (CH2Cl2–MeOH–NH3 92 : 7 : 1)
gave 11d (92%) as a yellow liquid; dH(400 MHz, CDCl3) 3.81 (2H,
s), 7.16–7.20 (2H, m), 7.24–7.30 (3H, m); dC(100 MHz, CDCl3)
46.6, 126.8, 127.1, 128.6, 143.4; m/z (EI) 107 (M)+. Found: (M)+
107.0727, C7H9N requires (M)+ 107.0735.
Flash column chromatography (hexane–EtOAc 6 : 1) gave 12b
(64%) as a pale oil; dH(400 MHz, CDCl3) 2.76 (4H, t, J 8.4),
3.47 (4H, dd, J 9.2, J 7.6), 7.05–7.19 (10H, m), 7.46–7.57 (3H,
m),7.85 (1H, dd, J 7.6, J 1.6); dC(100 MHz, CDCl3) 35.2, 49.3,
124.3, 126.8, 128.7, 128.8, 130.6, 131.7, 133.5, 138.1, 148.1; m/z
(ESI) 432.9 (M + Na)+, C22H22N2O4SNa requires (M + Na)+
433.1.
4-Methoxy-benzylamine 11e35
Flash column chromatography (CH2Cl2–MeOH–NH3 92 : 7 :
1) gave 11e (93%) as a yellow oil; dH(400 MHz, CDCl3) 3.79–
3.81 (2H, m), 3.80 (3H, s), 6.87 (2H, d, J 8.8), 7.23 (2H, d, J
8.8); dC(100 MHz, CDCl3) 46.0, 55.3, 114.0, 128.3, 135.6, 158.6;
m/z (EI) 255 (dimer–NH3)+, 136 (M)+, 77 (Ph)+. Found: (M)+
137.0810, C8H11NO requires (M)+ 137.0841.
N-(1-Methyl-pentyl)-2-nitro-N-phenethyl-benzenesulfonamide
12c
Flash column chromatography (hexane–EtOAc 6 : 1) gave 12c
(50%) as a pale oil; dH(400 MHz, CDCl3) 0.77 (3H, t, J 7.2), 1.08
(3H, d, J 6.8), 1.14–1.26 (4H, m), 1.40–1.45 (2H, m), 2.66 (2H, t,
J 8.4), 3.43 (2H, dd, J 9.2, J 7.6), 3.45–3.55 (1H, m), 7.71–7.74
(2H, m), 7.82–7.85 (1H, m), 8.13–8.15 (1H, m); dC(100 MHz,
CDCl3) 13.9, 21.8, 22.2, 27.6, 27.8, 37.0, 45.0, 51.2, 125.4, 126.9,
128.7, 128.8, 131.0, 132.8, 133.5, 133.9, 137.4, 147.9; m/z (ESI)
412.9 (M + Na)+, C20H26N2O4SNa requires (M + Na)+ 413.2.
4-Nitro-benzylamine 11f36
Flash column chromatography (CH2Cl2–MeOH–NH3 92 : 7 : 1)
gave 11f (80%) as a yellow oil; dH(400 MHz, CDCl3) 4.03 (2H,
s), 7.52 (2H, d, J 8.8), 8.21 (2H, d, J 8.4); m/z (ESI) 176.6 (M +
Na)+, C8H18N2O2Na requires (M + Na)+ 175.1.
3-Phenyl-allylamine 11g37
N-(1-Methyl-2-phenyl-ethyl)-2-nitro-N-phenethyl-
benzenesulfonamide 12d
Flash column chromatography (CH2Cl2–MeOH–NH3 92 : 7 : 1)
gave 11g (86%) as a slightly yellow liquid; dH(400 MHz, CDCl3)
3.48 (2H, d, J 4.8), 6.32 (2H, dt, J 15.6, J 5.6), 6.50 (1H, d,
J 16.0), 7.20–7.38 (5H, m); dC(100 MHz, CDCl3) 44.3, 126.2,
127.3, 128.5, 128.8, 129.6, 131.0, 131.4, 137.1; m/z (EI) 133
(M)+, 77 (Ph)+. Found: (M)+ 133.0885, C9H11N requires (M)+
133.0891.
Flash column chromatography (hexane–EtOAc 7 : 1) gave 12d
(40%) as a pale oil; dH(400 MHz, CDCl3) 1.18 (3H, d, J 6.4),
2.68 (1H, dd, J 12.8, J 9.6), 2.98–3.03 (3H, m), 3.47–3.58 (2H,
m), 4.20–4.25 (1H, m), 7.12–7.14 (2H, m), 7.15–7.27 (6H, m),
7.32–7.35 (2H, m), 7.57–7.67 (3H, m), 7.96 (1H, dd, J 7.6,
J 1.6); dC(100 MHz, CDCl3) 18.7, 28.2, 38.4, 42.5, 45.7,
56.2, 124.2, 126.7, 128.6, 128.7, 128.8, 129.2, 130.9, 131.6,
133.4, 134.0, 138.0, 138.6, 148.1; m/z (ESI) 446.9 (M + Na)+,
C23H24N2O4SNa requires (M + Na)+ 447.1.
Pentylamine 11h38
Due to the low boiling point of the amine 11h, the reaction
was carried out in CD3CN (0.6 mL) in a NMR tube and the
yield (90%) was determinated by 1H-NMR spectrum, using
benzylamine as internal standard; dH(400 MHz, CD3CN) 0.89
(3H, t, J 4.8), 1.26–1.39 (6H, m), 2.57 (2H, t, J 6.8); dC(100 MHz,
CD3CN) 13.1, 22.0, 28.6, 33.1, 41.6; m/z (ESI) 88.2 (M + H)+,
C5H14N requires (M + H)+ 88.2.
General procedure for deprotection reaction
A mixture of the sulfonamide 10 or 12 (0.6 mmol), thiophenol
(0.195 g, 1.8 mmol) and potassium carbonate (0.326 g, 2.4 mmol)
in acetonitrile (10 mL) was stirred at 50 ◦C for 5–12 h. Once tlc
indicated that the reaction had gone to completion, the reaction
mixture was directly purified by flash chromatography on silica
gel to give the correspondent amine 11 or 13.
2-Methyl-pentylamine 11i39
Due to the low boiling point of the amine 11i, the reac-
tion was carried out in CD3CN (0.6 mL) in a NMR tube
and the yield (100%) was determinated by 1H-NMR spec-
trum, using benzylamine as internal standard; dH(400 MHz,
CD3CN) 0.85 (3H, d, J 6.4), 0.89 (3H, t, J 7.2), 1.01–1.08
(1H, m), 1.24–1.42 (4H, m), 2.37 (1H, dd, J 12.4, J 6.8),
2.51 (1H, dd, J 12.4, J 5.6); dC(100 MHz, CD3CN) 13.4, 16.5,
19.6, 35.7, 36.1, 47.9; m/z (ESI) 102.2 (M + H)+, C5H16N
requires (M + H)+ 102.1.
Phenethylamine 11a31
Flash column chromatography (CH2Cl2–MeOH–NH3 92 : 7 : 1)
gave 11a (89%) as a yellow oil; dH(400 MHz, CDCl3) 2.76 (2H, t,
J 6.8), 2.98 (2H, t, J 6.8), 7.22–7.25 (3H, m), 7.31–7.34 (2H, m);
dC(100 MHz, CDCl3) 40.2, 43.7, 126.1, 128.5, 128.7, 139.9; m/z
(EI) 121 (M)+, 92 (PhCH2)+, 77 (Ph). Found: (M)+ 121.0897,
C8H11N requires (M)+ 121.0891.
2-(4-Methoxy-phenyl)-ethylamine 11b32
Pent-2-ynylamine 11j40
Flash column chromatography (CH2Cl2–MeOH–NH3 92 : 7 : 1)
gave 11b (95%) as a yellow solid; dH(400 MHz, CDCl3) 2.68 (2H,
t, J 6.8), 2.92 (2H, t, J 6.8), 3.78 (3H, s), 6.84 (2H, d, J 8.8),
7.11 (2H, d, J 8.8); dC(100 MHz, CDCl3) 39.2, 43.7, 55.3,
Due to the low boiling point of the amine 11j, the reaction
was carried out in CD3CN (0.6 mL) in a NMR tube and the
yield (91%) was determinated by 1H-NMR spectrum, using
benzylamine as internal standard; dH(400 MHz, CD3CN) 1.08
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 0 4 9 – 1 0 5 7
1 0 5 5

View Article Online
(3H, t, J 7.6), 2.13–2.16 (2H, m), 3.28 (2H, t, J 2.0); dC(100 MHz,
CD3CN) 11.5, 13.2, 30.7, 80.4, 82.6; m/z (ESI) 84.2 (M + H)+,
C5H10N requires (M + H)+ 84.7.
Acknowledgements
We would like to thank IC-Vec Ltd. for funding this research and
Mitsubishi Chemical Corporation for supporting the Imperial
College Genetic Therapies Centre. Mass spectral analysis was
kindly performed by Mr John Barton (Imperial College) and
additional experimentation by Dr Arnaud Cheguillaume (IC-
Vec Ltd.).
Pent-2-enylamine 11k41
Due to the low boiling point of the amine 11k, the reaction
was carried out in CD3CN (0.6 mL) in a NMR tube and the
yield (80%) was determinated by 1H-NMR spectrum, using
benzylamine as internal standard; dH(400 MHz, CD3CN) 0.95
(3H, t, J 7.6), 2.01–2.06 (2H, m), 3.20–3.22 (2H, m), 5.33–5.44
(2H, m); dC(100 MHz, CD3CN) 13.4, 19.9, 38.1, 125.3, 126.0;
m/z (ESI) 86.2 (M + H)+, C5H11N requires (M + H)+ 86.1.
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