Bifunctional bisamphiphilic transmembrane building blocks for artificial signal transduction

Article abstract of DOI:10.1055/s-2008-1067155

A convergent modular total synthesis is described which affords bisamphiphilic bifunctional transmembrane building blocks useful for artificial signal transduction across membranes. The concept relies on direct Glaser-Hay coupling of lithocholic or cholenic acid propargyl esters, carrying recognition or signaling units on their opposite hydroxy functionalities. For catechol recognition, a 2-(aminomethyl)phenylboronic acid was introduced, while the ammonium alcohol was targeted by a bisphosphonate dianion. Signaling units comprised an N-protected cysteine (thiol nucleophile) and an N-protected cysteine/pyridine disulfide, respectively (disulfide substrate). AU these polar headgroups were connected to the hydroxy of the lipid core by ester formation. Georg Thieme Verlag Stuttgart.

Full text of DOI:10.1055/s-2008-1067155

PAPER
2247
Bifunctional Bisamphiphilic Transmembrane Building Blocks for Artificial
Signal Transduction
Transmembrane
Buildiccks hael Maue, Kai Bernitzki, Manuel Ellermann, Thomas Schrader*
Fachbereich Chemie, Universität Duisburg-Essen, Universitätsstr. 5, 45117 Essen, Germany
Fax +49(201)1834252; E-mail: Thomas.Schrader@uni-due.de
Received 24 May 2008
Dedicated to Prof. R. W. Hoffmann on the occasion of his 75th birthday
lar’ disulfides, detectable by a specific absorption intensi-
Abstract: A convergent modular total synthesis is described which
affords bisamphiphilic bifunctional transmembrane building blocks
useful for artificial signal transduction across membranes. The con-
cept relies on direct Glaser–Hay coupling of lithocholic or cholenic
ty rise at 432 nm.3 This overall strategy requires the total
synthesis of large bisamphiphilic building blocks, which
exactly span a lipid bilayer (typically DMPC or DPPC).
acid propargyl esters, carrying recognition or signaling units on These must carry on one end a recognition head group and
their opposite hydroxy functionalities. For catechol recognition, a
on the other end a reactive group for the SN2 signaling re-
2-(aminomethyl)phenylboronic acid was introduced, while the am-
action.
monium alcohol was targeted by a bisphosphonate dianion. Signal-
The synthetic concept should involve a modular approach,
allowing exchange of recognition and signaling tips at
will and at the same time be convergent, i.e., minimize the
number of consecutive steps and avoid excessive protect-
ing group manipulations.
ing units comprised an N-protected cysteine (thiol nucleophile) and
an N-protected cysteine/pyridine disulfide, respectively (disulfide
substrate). All these polar headgroups were connected to the hy-
droxy of the lipid core by ester formation.
Key words: signal transduction, phosphonate, Glaser–Hay cou-
pling, disulfide coupling
Lithocholic acid has already been successfully used for
extracellular membrane signaling, but introduces a kink
and this brings potential destabilization just outside the
phosphatidylcholine headgroups. Replacement of this
commercially available steroid by a cholenic acid tem-
plate requires three additional steps but, nevertheless, was
realized, because of its perfectly flat overall topology with
an equatorial placement of its secondary hydroxy group
(Figure 1).4
All living organisms rely on the fundamental process of
signal transduction across their cell membranes.1 Al-
though ~60% of all manufactured drugs target one of the
large family of G-protein-coupled receptors (GPCRs), a
functional synthetic biomimetic version has proven elu-
sive to date. Building on the creative approach delineated
by the Hunter group, the authors of this manuscript have
designed unsymmetrical bisamphiphilic transmembrane
building blocks that can be embedded in lipid bilayers.2
Two recognition head groups for catechol and ammonium
alcohol recognition are tailored for ditopic adrenaline
binding bringing both transmembrane units into close
proximity in the fluidic membrane. At the opposite end, a
thiol nucleophile is attached to one unit, which is designed
to nucleophilically displace thiopyridine from an adjacent
The overall strategy is as follows: each steroid is first
elongated at its carboxy terminus (C1) to a propargyl ester
or less reactive amide derivative, then adorned at its O-ter-
minus (C3¢) with an appropriate functional head group, ei-
ther for primary messenger recognition, or for second
messenger generation. In the key step, two of these lipids
are connected by alkyne coupling, before in the final
steps, all protecting groups are cleaved off.
disulfide substrate at the other unit. Adrenaline injection The propargyl ester or amide formation from both steroids
into a suspension of unilamellar liposomes with embed- proceeds smoothly with N,N¢-dicyclohexylcarbodiimide/
ded transmembrane building blocks, should exclusively 4-(dimethylamino)pyridine and affords spacer elements
result in thiopyridine displacement from the ‘intracellu- 2a–c in high yields (70–81%). In order to avoid intermo-
O
O
4
4
1
1
17'
17'
OH
OH
3'
HO
HO 3'
3β-hydroxy-5-cholenic acid
lithocholic acid
H
Figure 1 Rigid lipid templates for all transmembrane building blocks with two orthogonal attachment points
SYNTHESIS 2008, No. 14, pp 2247–2256
x
x.
x
x
.
2
0
0
8
Advanced online publication: 02.07.2008
DOI: 10.1055/s-2008-1067155; Art ID: C03208SS
© Georg Thieme Verlag Stuttgart · New York

2248
M. Maue et al.
PAPER
O
O
HX
OH
HO
X
HO
DCC, DMAP
CH2Cl2, r.t.
(70–81%)
1a,b
2a–c
= lithocholic acid, X = O
= lithocholic acid, X = NMe
= cholenic acid, X = O
1a:
1b:
= lithocholic acid
= cholenic acid
2a:
2b:
2c:
Scheme 1 Steroid elongation to propargyl esters or N-methylamides
O
H
N
PG
OH
STr
O
O
H
N
X
PG
O
2a–c
HCTU, Cl-HOBt, DIPEA,
DMF–CH2Cl2, r.t.
(58–92%)
STr
3a–c
3a:
3b:
3c:
= lithocholic acid, X = O, PG = Boc
= lithocholic acid, X = NMe, PG = Bocm
= cholenic acid, X = O, PG = Ac
Scheme 2 Cysteine attachment by esterification
COOH
O
O
O
(MeO)2OP
PO(OMe)2
X
(MeO)2P
O
2a–c
HCTU, Cl-HOBt, DIPEA,
4a–c
DMF–CH2Cl2, r.t.
(42–79%)
(MeO)2P
4a:
4b:
4c:
= lithocholic acid, X = O
= lithocholic acid, X = NMe
= cholenic acid, X = O
O
Scheme 3 Bisphosphonate attachment by esterification
O
O
O
H
N
i
X
2a,b
3a,b
PG
O
+
X
OH
STr
5a,b
5a:
5b:
= lithocholic acid, X = O, PG = Boc; 58%
= lithocholic acid, X = NMe, PG = Boc; 65%
Scheme 4 Directed Glaser–Hay coupling towards true membrane-spanning building blocks with boronic acid head groups. Reagents and con-
ditions: (i) 2a,b (2 equiv), 3a,b (1 equiv), CuCl (5 equiv), TMEDA (5 equiv), O2, CH2Cl2, r.t.
O
O
O
O
H
O
N
3a,c + 4a,c
X
X
O
P(OMe)2
PG
O
STr
6a,b
P(OMe)2
O
6a:
6b:
= lithocholic acid, X = O, PG = Boc; 39%
= cholenic acid, X = O, PG = Ac; 65%
Scheme 5 Directed Glaser–Hay coupling towards true membrane-spanning building blocks with bisphosphonate head groups. Reagents and
conditions: 3a,c (2 equiv), 4a,c (1 equiv), CuCl (5 equiv), TMEDA (5 equiv), O2, CH2Cl2, r.t.
Synthesis 2008, No. 14, 2247–2256 © Thieme Stuttgart · New York

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Transmembrane Building Blocks
2249
lecular hydrogen bonding between adjacent lipid moi- Fmoc-protected glycine is followed by Fmoc removal
eties, N-methylpropargylamine was employed in this with piperidine and reductive amination with 2-
reaction, so that the resulting amides retain their mobility formylphenylboronic acid in the presence of molecular
within the fluidic lipid phase, a prerequisite for effective sieves.7 Finally, trifluoroacetic acid/triisopropylsilane
signaling (Scheme 1).
completely deprotects the cysteine and releases the cate-
chol-selective functional transmembrane unit 10b (10%
from 5b, Scheme 6).
For the signaling units the steroid’s O-terminus was sub-
sequently esterified with N/S-diprotected cysteine
(Scheme 2), assisted by the powerful combination of O- The bisphosphonate targets 12 and 13 emerge from het-
(6-chlorobenzotriazol-1-yl)-N,N,N¢,N¢-tetramethyluroni-
erodimers 6 in short sequences: it is critical to begin with
um hexafluorophosphate/6-chloro-1-hydroxybenzotriaz- phosphonate dealkylation (Scheme 7), most conveniently
ole (HCTU) with 6-chloro-1-hydroxybenzotriazole (Cl- with SN2 attack by lithium bromide in dipolar aprotic
HOBt), furnishing 3a–c in high yields (58–92%).
acetonitrile to give 11a,b (99%).8
Ditopic adrenaline recognition requires the attachment of Subsequent acid cleavage of all cysteine protecting
an amino alcohol and a catechol host unit to 2a–c. To this groups leads to bisphosphonate target 12a,b with a nu-
end, the steroid’s secondary hydroxy was esterified with a cleophilic thiol on one end (99%). From 11 the corre-
3,5-bisphosphonate-functionalized benzoic acid (Scheme sponding disulfide target 13a,b can also be synthesized by
3), following almost the same HCTU/Cl-HOBt protocol, an elegant short procedure (Scheme 8). Although it re-
described above, to give 4a–c (42–79%).
quires deprotection of the thiol and subsequent acid-cata-
lyzed displacement of thiopyridine from the dimeric
aldrithiol, both steps can be carried out in one pot, starting
with detritylation by trifluoroacetic acid/triisopropylsi-
lane, followed by addition of the disulfide reagent after
two hours at 0 °C (99% over both steps).9
Alternatively, a phenylboronic acid was intended to be at-
tached to the elongated steroid by way of a compact gly-
cine spacer. However, although this by itself proved
feasible, the subsequent key alkyne coupling could not be
effected in the presence of the free boronic acid and the or-
der of coupling steps had to be inverted, placing the intro- For all important combinations, the synthetic concept,
duction of the sensitive boronic acid at the end of the total outlined in the beginning, could be realized: Starting from
synthesis.
well-accessible steroidal carboxylic acids, propargyl ester
or N-methylamide formation and subsequent esterifica-
tion at the opposite hydroxy end generates lipids with at-
tached recognition and signaling moieties in protected
form. The key process is a directed Glaser–Hay coupling
to furnish the desired heterodimeric products in good
yields. Further elaboration to the dianionic
bisphosphonate10 or free 2-(aminomethyl)phenylboronic
acid head groups proceeds in a straightforward manner. A
final cysteine deprotection and/or disulfide coupling gen-
erates the reactive nucleophile and its substrate in excel-
lent yields.
Thus, the next step for all target molecules is the alkyne
coupling, which transforms the monosteroidal lipids into
disteroidal, true transmembrane units. Since they are all
unsymmetric, a directed coupling procedure would be the
most elegant solution. A prominent example in the litera-
ture is the palladium-catalyzed cross-coupling of alkynes
with 1-iodoalkynes.5 However, all attempts to perform the
coupling step under Cadiot–Chodciewiczk conditions
produced complex mixtures, which contained the desired
unsymmetrical dialkynes in unacceptable low amounts
(<30%). In sharp contrast, symmetrical Glaser–Hay cou-
plings proceeded smoothly with several terminal alkynes Preliminary experiments with extruded unilamellar DPPC
3 and 4.6 It was therefore attempted to shift the 1:2 ratio liposomes of ~200 nm diameter, carrying these transmem-
between hetero- and homodimers by employing a twofold brane units, indeed show a reproducible increase in UV/
excess of the least valuable species, whose excess could vis absorption at 342 nm, followed by a second thiopyri-
be recovered during the chromatographic purification dine displacement after injection of a water-soluble reduc-
step. This procedure was, indeed, successful and fur- ing agent (sulfonated phosphine) for extracellular
nished the desired heterocoupled dialkynes in up to 65% disulfides.
isolated yields.
Scheme 4 shows the preparation of dialkynes 5a,b with
only one protected cysteine headgroup, which served for
the consecutive construction of the boronic acid recogni-
tion unit from lithocholic acid’s secondary free hydroxy
group (58–65%).
Melting points were determined on a Reichert Kofler Thermoplan
melting point apparatus and are uncorrected. 1H NMR spectra were
recorded on Bruker Avance ARX-200, AMX-300, DRX-400, and
DRX-500; the residual hydrogen signal of the deuterated solvent
was used as reference. 13C NMR spectra were obtained on the same
spectrometers, 31P NMR spectra on a Bruker Advance ARX-200
and 11B NMR spectra on a Bruker Avance DRX-400 and DRX-500.
MS (including HRMS) were obtained on a Finnegan MAT 711 (EI)
and Finnegan MAT95 S (ESI). Reaction progress was monitored by
Scheme 5 similarly depicts the heterodimer formation be-
tween cysteine- and bisphosphonate-carrying steroid pro-
pargyl esters 6a,b (39–65%).
analytical TLC on precoated aluminum plates (silica gel 60 F254
,
From 5, the synthetic route towards the boronic acid target
10 runs quite straightforward: N-ethyl-N¢-[3-(dimethyl-
amino)propyl]carbodiimide (EDCI) assisted coupling of
Merck) and products were visualized by CAM reagent [Ce(SO4)2
(2 g), (NH4)2MoO4 (50 g), concd H2SO4 (50 mL), H2O (400 mL)].
The solvent systems are stated for each procedure. Column chroma-
tography was carried out on silica gel 60 (Merck). All compounds
Synthesis 2008, No. 14, 2247–2256 © Thieme Stuttgart · New York

2250
M. Maue et al.
PAPER
5a,b
O
H
EDCl, CH2Cl2,
r.t.
N
Fmoc
OH
O
O
O
O
H
H
N
N
X
PG
O
O
Fmoc
X
STr
7a,b
7a:
7b:
= lithocholic acid, X = O, PG = Boc
= lithocholic acid, X = NMe, PG = Boc
7a,b
piperidine (5% in DMF)
r.t.
Boc
HN
O
O
O
O
NH2
X
O
X
O
STr
8a,b
8a:
8b:
= lithocholic acid, X = O
= lithocholic acid, X = NMe
8b
O
Et3N (2 equiv), MS (3Å),
MeOH–CH2Cl2 (2:1), 0 °C
H
OH
B
(1.1 equiv)
O
OH
OH
HO
B
Boc
HN
O
O
O
H
N
N
O
N
O
Me
Me
STr
9b
TIPS (4 equiv),
TFA (6 equiv),
0 °C
(10% from 5b)
OH
HO
B
O
O
O
O
H
H2N
N
N
O
O
N
Me
Me
SH
10b
9b, 10b:
= lithocholic acid
Scheme 6 Elaboration of the core transmembrane unit 5a,b with free steroidal hydroxy group into the boronic acid building block 10b by
glycine coupling, deprotection, and reductive amination
were obtained as colorless crystals with the exception of a few light-
ly yellow crystals.
stirred at r.t. for 16 h. The precipitate was collected by filtration and
washed thoroughly with CH2Cl2. The solvent was evaporated and
the resulting highly viscous oil was dried in vacuo. The crude prod-
uct was then purified by column chromatography.
Esterification/Amidation with Propargylic Alcohol/N-Methyl-
propargylamine; General Procedure 1
To a stirred mixture of carboxylic acid (13.28 mmol), propargylic
alcohol/N-methylpropargylic amine (19.92 mmol), and DMAP
(2.66 mmol) in CH2Cl2 (30 mL), was added dropwise a soln of DCC
(14.61 mmol) in CH2Cl2 (15 mL) over 2 h and the mixture was
Prop-2-ynyl 4-(3-Hydroxy-10,13-dimethylhexadecahydro-1H-
cyclopenta[a]phenanthren-17-yl)pentanoate (2a)
Yield: 3.84 g (70%); mp 93 °C; Rf = 0.40 (hexane–EtOAc, 1:1).
Synthesis 2008, No. 14, 2247–2256 © Thieme Stuttgart · New York

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Transmembrane Building Blocks
2251
6a,b
LiBr (2.5 equiv), MeCN
reflux
(99%)
OLi
MeO
PG
HN
O
O
O
O
P
O
X
X
O
O
STr
MeO
11a,b
P
LiO
O
11a:
11b:
= lithocholic acid, X = O, PG = Boc
= cholenic acid, X = O, PG = Ac
Scheme 7 Phosphonate methyl ester cleavage in bisphosphonates 6
OLi
MeO
R
O
O
O
P
O
O
HN
X
X
O
O
SH
12a,b
MeO
P
LiO
TIPS (4 equiv),
TFA (6 equiv),
0 °C
O
(99%)
11a,b
S
S
N
TIPS (4 equiv), TFA (6 equiv), 0 °C
(99% over both steps)
(5 equiv)
N
OLi
MeO
R
O
O
O
O
P
O
HN
X
X
O
O
13a,b
S
S
MeO
N
P
LiO
O
12a, 13a:
12b, 13b:
= lithocholic acid, X = O, R = H
= cholenic acid, X = O, R = Ac
Scheme 8 Final acidic trityl cleavage towards the free thiol nucleophiles 12, optionally followed by disulfide formation to yield the SN2 sub-
strates 13
1H NMR (CDCl3): d = 0.64 (s, 3 H), 0.88–2.42 (m, 35 H), 2.46 (t,
1H NMR (CDCl3): d = 0.58 (s, 3 H), 0.86 (m, 6 H), 0.87–2.37 (m,
29 H), 2.13 (t, 4J = 2.5 Hz, 1 H), 2.92 + 3.00 (s, 3 H), 3.50 (m, 1 H),
3.98 + 4.16 (s, 2 H).
4JH,H = 2.3 Hz, 1 H), 3.57–3.67 (m, 1 H), 4.67 (d, 4JH,H = 2.3 Hz, 2
H).
13C NMR (CDCl3): d = 12.2, 18.4, 21.0, 23.5, 24.4, 26.6, 27.3, 28.3,
29.8, 30.7, 31.0, 31.1, 34.7, 35.5, 35.5, 36.0, 36.6, 40.3, 40.6, 42.3,
42.9, 51.9, 53.6, 56.1, 56.6, 72.0, 74.8, 78.0, 173.6.
13C NMR (CDCl3): d = 12.1, 18.6, 20.9, 23.5, 24.3, 25.1, 25.7, 26.5,
27.3, 28.3, 30.2, 30.4, 30.6, 31.0, 31.3, 33.4, 34.0, 34.5, 34.6, 35.5,
35.6, 35.9, 36.2, 36.5, 39.6, 40.3, 40.5, 42.2, 42.8, 49.0, 56.1, 56.6,
71.8, 72.8, 78.3, 79.1, 173.4.
MS (EI): m/z = 396 [M+ – H2O].
HRMS (ESI+, MeOH): m/z [M + Na]+ calcd for C28H45NNaO2:
4-(3-Hydroxy-10,13-dimethylhexadecahydro-1H-cyclopen-
ta[a]phenanthren-17-yl)-N-methyl-N-(prop-2-ynyl)pentan-
amide (2b)
450.3343; found: 450.3351.
Prop-2-ynyl 4-(3-Hydroxy-10,13-dimethyl-
2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclo-
Yield: 4.60 g (81%); Rf = 0.17 (hexane–EtOAc, 2:1).
penta[a]phenanthren-17-yl)pentanoate (2c)
Yield: 4.44 g (82%); mp 81 °C; Rf = 0.21 (hexane–EtOAc, 2:1).
Synthesis 2008, No. 14, 2247–2256 © Thieme Stuttgart · New York

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M. Maue et al.
PAPER
1H NMR (CDCl3): d = 0.66 (s, 3 H), 0.91 (d, 3JH,H = 6.2 Hz, 3 H),
2.70 (dd, 2JH,H = 12.3 Hz, 3JH,H = 2.1 Hz, 1 H), 4.55–4.67 (m, 1 H),
4
4
3
0.99 (s, 3 H), 1.00–2.43 (m, 26 H), 2.46 (t, JH,H = 2.5 Hz, 1 H),
4.69 (d, J = 2.5 Hz, 2 H), 5.40 (t, JH,H = 6.8 Hz, 1 H), 6.01 (d,
3.42–3.58 (m, 1 H), 4.64 (d, 4JH,H = 2.5 Hz, 2 H), 5.32 (d, 3JH,H = 5.3
3JH,H = 6.6 Hz, 1 H), 7.20–7.35 (m, 9 H), 7.42 (d, 3JH,H = 8.1 Hz, 6
Hz, 1 H).
H).
13C NMR (CDCl3): d = 11.9, 18.3, 19.4, 21.1, 24.2, 28.1, 30.8, 31.0,
31.6, 31.9, 31.9, 35.3, 36.5, 37.3, 39.8, 42.3, 42.4, 50.1, 51.7, 55.8,
56.7, 71.7, 74.7, 77.8, 121.6, 140.8, 173.4.
13C NMR (CDCl3): d = 12.1, 18.5, 19.5, 21.2, 23.3, 24.4, 27.8, 27.8,
28.3, 31.1, 31.2, 32.1, 34.3, 35.5, 36.8, 37.1, 38.0, 38.1, 39.9, 42.6,
50.2, 51.4, 51.9, 56.0, 56.9, 67.0, 74.9, 75.9, 78.1, 123.1, 127.1,
128.2, 129.7, 139.5, 144.6, 169.7, 170.1, 173.5.
Esterification with Acyl-Cys(Trt)-OH/5-Carboxy-m-xylylene-
bisphosphonic Acid Tetramethyl Ester; General Procedure 2
Ac-Cys(Trt)-OH, Boc-Cys(Trt)-OH, or 5-carboxy-m-xylylenebis-
phosphonic acid tetramethyl ester (1.67 mmol) was dissolved in
CH2Cl2–DMF (2:1, 25 mL) and DIPEA (4.16 mmol) was added.
The soln was cooled to 0 °C in ice water. HCTU (1.67 mmol) and
Cl-HOBt (3.47 mmol) were added. The mixture was stirred at 0 °C
for 10 min. The activated carboxylic acid was treated with 2a–c
(1.39 mmol). Stirring was continued for 24 h and the mixture was
allowed to warm up to r.t. It was diluted with CH2Cl2 (10 mL) and
washed with sat. aq NaHCO3, 1 M AcOH, and brine. The separated
organic extract was dried (MgSO4). The filtrate was evaporated to
dryness and the resulting crude product was purified by column
chromatography.
HRMS (ESI+, MeOH): m/z [M + Na]+ calcd for C51H61NNaO5S:
822.4163; found: 822.4180.
10,13-Dimethyl-17-[1-methyl-4-oxo-4-(prop-2-ynyloxy)butyl]-
hexadecahydro-1H-cyclopenta[a]phenanthren-3-yl 3,5-
Bis[(dimethoxyphosphoryl)methyl]benzoate (4a)
Yield: 0.72 g (68%); mp 87 °C; Rf = 0.24 (CH2Cl2–MeOH, 19:1).
1H NMR (CDCl3): d = 0.64 (s, 3 H), 0.90 (d, 3JH,H = 6.4 Hz, 3 H),
0.93 (s, 3 H), 1.00–2.41 (m, 28 H), 2.45 (t, 4JH,H = 2.1 Hz, 1 H), 3.18
(d, 2JH,P = 21.8 Hz, 4 H), 3.67 (d, 3JH,P = 10.8 Hz, 12 H), 4.64 (s, 2
H), 4.88–4.97 (m, 1 H), 7.43 (s, 1 H), 7.82 (s, 2 H).
13C NMR (CDCl3): d = 12.2, 18.4, 21.0, 23.4, 24.3, 26.4, 26.8, 27.2,
1
28.3, 31.0, 31.1, 32.7 (d, JC,P = 138.4 Hz), 32.4, 34.7, 35.2, 35.4,
35.9, 40.2, 40.6, 42.1, 42.9, 51.8, 53.1 (d, 2JC,P = 7.1 Hz), 56.1, 56.5,
74.8, 75.4, 78.0, 129.6–129.7 (m, 1 C), 131.9, 132.2–132.3 (m, 1 C),
135.2–135.3 (m, 1 C), 165.6, 173.4.
Prop-2-ynyl 4-{3-[2-(tert-Butoxycarbonylamino)-3-(trityl-
thio)propanoyloxy]-10,13-dimethylhexadecahydro-1H-cyclo-
penta[a]phenanthren-17-yl}pentanoate (3a)
31P NMR (CDCl3): d = 28.6.
HRMS (ESI+, MeOH): m/z [M + Na]+ calcd for C40H60NaO10P2:
Yield: 1.32 g (92%); mp 75 °C; Rf = 0.51 (hexane–EtOAc, 3:1).
1H NMR (CDCl3): d = 0.65 (s, 3 H), 0.92–2.43 (m, 43 H), 2.45 (t,
4JH,H = 2.4 Hz, 1 H), 2.50 (dd, 2JH,H = 11.7 Hz, 3JH,H = 3.9 Hz, 1 H),
2.58 (dd, 2JH,H = 11.8 Hz, 3JH,H = 5.5 Hz, 1 H), 4.24 (br s, 1 H), 4.66
(d, 4JH,H = 2.2 Hz, 2 H), 4.66–4.75 (m, 1 H), 5.09 (d, 3JH,H = 7.9 Hz,
1 H), 7.20 (t, 3JH,H = 7.3 Hz, 3 H), 7.27 (t, 3JH,H = 7.5 Hz, 6 H), 7.39
(d, 3JH,H = 7.7 Hz, 6 H).
13C NMR (CDCl3): d = 12.2, 18.4, 21.0, 23.4, 24.3, 26.4, 26.5, 27.1,
28.3, 28.4, 31.0, 31.1, 32.2, 34.5, 34.7, 35.0, 35.4, 35.9, 40.3, 40.6,
42.0, 42.9, 51.9, 52.8, 56.2, 56.7, 66.7, 74.8, 76.0, 77.9, 79.9, 126.9,
128.1, 129.6, 144.5, 155.1, 170.3, 173.4.
785.3554; found: 785.3583.
10,13-Dimethyl-17-{1-methyl-4-[methy](prop-2-ynyl)amino]-4-
oxobutyl}-hexadecahydro-1H-cyclopenta[a]phenanthren-3-yl
3,5-Bis[(dimethoxyphosphoryl)methyl]benzoate (4b)
Yield: 0.85 g (79%); mp 92 °C; Rf = 0.16 (CH2Cl2–MeOH, 19:1).
1H NMR (CDCl3): d = 0.57 (s, 3 H,), 0.84 (m, 6 H), 0.90–0.98 (m,
1 H), 1.00–1.21 (m, 8 H), 1.23–1.42 (m, 9 H), 1.44–1.47 (m, 1 H),
1.60–1.63 (m, 1 H), 1.66–1.88 (m, 5 H), 1.90–1.93 (m, 1 H), 2.14–
4
2.21 (m, 1 H), 2.14 (t, JHH = 2.47 Hz, 1 H), 2.23–2.39 (m, 1 H),
HRMS (ESI+, MeOH): m/z [M + Na]+ calcd for C54H69NNaO6S:
882.4738; found: 882.4730.
2.89 (s, 1 H), 2.98 (s, 2 H), 3.12 (d, 2JPH = 21.8 Hz, 4 H), 3.59 (d,
2JPH = 10.8 Hz, 12 H), 3.96 (m, 2 H,), 4.80–4.89 (m, 1 H), 7.41 (s,
1 H), 7.73 (s, 2 H).
4-{3-[2-(tert-Butoxycarbonylamino)-3-(tritylthio)propanoyl-
oxy]-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenan-
thren-17-yl}-N-methyl-N-(prop-2-ynyl)pentanamide (3b)
Yield: 0.88 g (60%); Rf = 0.1 (hexane–EtOAc, 3:1).
1H NMR (CDCl3): d = 0.59 (s, 3 H), 0.85 (m, 6 H), 0.84–2.55 (m,
27 H), 1.37 (s, 12 H), 2.13 (t, 4J = 2.5 Hz, 1 H), 2.92 + 3.00 (s, 3 H),
3.97 + 4.15 (s, 2 H), 4.21 (m, 1 H), 4.67 (m, 1 H), 5.03 (d, 3J = 6.3
Hz, 1 H), 7.08–7.36 (m, 15 H).
13C NMR (CDCl3): d = 11.9, 18.4, 20.8, 23.2, 24.1, 26.2, 26.9, 28.1,
1
29.9, 30.1, 30.8, 31.3, 32.1, 33.2 (d, JPC = 115.3 Hz), 33.3, 34.3,
34.5, 35.0, 35.3, 35.7, 36.0, 38.5, 39.3, 39.9, 40.3, 41.8, 42.6, 52.9
2
(d, JPC = 6.1 Hz), 55.9, 56.3, 71.6, 72.7, 75.1, 78.1, 78.8, 129.4,
131.6, 131.9, 135.1, 165.6, 173.2.
31P NMR (CDCl3): d = 28.6.
HRMS (ESI+, MeOH): m/z [M + Na]+ calcd for C41H63NNaO9P2:
798.3876; found: 798.3881.
13C NMR (CDCl3): d = 12.2, 14.3, 18.6, 21.0, 23.4, 24.3, 26.4, 26.5,
27.1, 28.4, 30.3, 30.5, 31.1, 31.4, 32.2, 33.4, 34.5, 34.7, 35.0, 35.7,
35.9, 36.3, 39.6, 40.3, 40.6, 42.0, 42.9, 52.7, 56.3, 56.7, 71.7, 72.8,
76.0, 79.1, 79.9, 126.9, 128.1, 129.6, 144.5, 155.1, 170.3, 173.4.
HRMS (ESI+, MeOH): m/z [M + Na]+ calcd for C55H72N2NaO5S:
895.5054; found: 895.5039.
10,13-Dimethyl-17-[1-methyl-4-oxo-4-(prop-2-ynyloxy)butyl]-
2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclo-
penta[a]phenanthren-3-yl 3,5-Bis[(dimethoxyphosphoryl)-
methyl]benzoate (4c)
Yield: 0.45 g (42%); mp 136 °C; Rf = 0.24 (CH2Cl2–MeOH, 19:1).
1H NMR (CDCl3): d = 0.66 (s, 3 H), 0.91 (d, 3JH,H = 6.6 Hz, 3 H),
Prop-2-ynyl 4-{3-[2-Acetamido-3-(tritylthio)propanoyloxy]-
10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetrahydro-
1H-cyclopenta[a]phenanthren-17-yl}pentanoate (3c)
Following general procedure 2 using Ac-Cys(Trt)-OH (1.22 mmol)
and the respective relative amounts of DIPEA (4.58 mmol), HCTU
(1.68 mmol) and Cl-HOBt (3.36 mmol) as well as 2c (1.53 mmol);
yield: 0.56 g (58%); mp 74 °C; Rf = 0.58 (hexane–EtOAc, 3:1).
2
0.93–2.47 (m, 29 H), 3.16 (d, JH,P = 21.8 Hz, 4 H), 3.66 (d,
3JH,P = 10.8 Hz, 12 H), 4.64 (d, 4JH,H = 2.2 Hz, 2 H), 4.76–4.86 (m,
1 H), 5.38 (d, 3JH,H = 4.4 Hz, 1 H), 7.43 (s, 1 H), 7.81 (s, 2 H).
13C NMR (CDCl3): d = 11.9, 18.4, 19.4, 21.1, 24.3, 27.9, 28.2, 30.9,
1
31.0, 31.9, 32.0, 32.7 (d, JC,P = 138.3 Hz), 35.4, 36.7, 37.1, 38.3,
39.8, 42.5, 50.1, 51.8, 53.0 (d, 2JC,P = 7.4 Hz), 55.8, 56.7, 74.8, 74.9,
77.9, 122.8, 129.6–129.6 (m, 1 C), 131.7–131.8 (m, 1 C), 132.2–
132.3 (m, 1 C), 135.3, 139.7, 165.4, 173.4.
1H NMR (CDCl3): d = 0.71 (s, 3 H), 0.80–2.47 (m, 35 H), 2.49 (t,
4JH,H = 2.5 Hz, 1 H), 2.55 (dd, 2JH,H = 12.3 Hz, 3JH,H = 4.5 Hz, 1 H),
Synthesis 2008, No. 14, 2247–2256 © Thieme Stuttgart · New York

PAPER
Transmembrane Building Blocks
2253
31P NMR (CDCl3): d = 28.6.
HRMS (ESI+, MeOH): m/z [M + Na]+ calcd for C40H58NaO10P2:
3.71 (d, 3JH,P = 10.8 Hz, 12 H), 4.26 (br s, 1 H), 4.71–4.78 (m, 5 H),
4.97 (m, 1 H), 5.11 (d, 3JH,H = 7.6 Hz, 1 H), 7.22 (t, 3JH,H = 7.1 Hz,
3 H), 7.29 (t, 3JH,H = 7.2 Hz, 6 H), 7.41 (d, 3JH,H = 7.6 Hz, 6 H), 7.47
(s, 1 H), 7.85 (s, 2 H).
783.33974; found: 783.33953.
Glaser–Hay Coupling; General Procedure 3
13C NMR (CDCl3): d = 12.2, 18.5, 21.1, 21.1, 23.5, 23.5, 24.4, 26.5,
26.6, 26.9, 27.2, 27.2, 28.4, 28.5, 31.1, 31.1, 31.1, 32.8 (d,
1JC,P = 138.4 Hz), 32.5, 34.6, 34.8, 34.8, 35.1, 35.3, 35.5, 36.0, 36.0,
40.3, 40.4, 40.7, 42.1, 42.2, 43.0, 52.2, 52.9, 53.1 (d, 2JC,P = 6.1 Hz),
56.2, 56.3, 56.6, 56.7, 66.8, 70.4, 70.4, 73.9, 74.0, 75.5, 76.1, 80.0,
127.0, 128.1, 129.7, 132.0, 132.3–132.4 (m, 1C), 135.3–135.4 (m,
1C), 144.6, 155.2, 165.7, 170.4, 173.3, 173.3.
The acetylene compounds and CuCl (exact amounts see below)
were dissolved/suspended in CH2Cl2 (25 mL). TMEDA was added
to the stirred mixture, resulting in slow dissolution of all CuCl.
Upon completion, the atmosphere above the green soln was saturat-
ed with O2 (balloon). Stirring was continued at r.t. for 6 h, until a
deep blue color was reached. The reaction was quenched with sat.
aq NH4Cl. The organic soln was subsequently washed with sat. aq
NH4Cl (3 ×), the separated organic extract was dried (MgSO4), and
the solvent was evaporated in vacuo. The slightly green crude prod-
uct was purified by column chromatography.
31P NMR (CDCl3): d = 28.6.
HRMS (ESI+, MeOH): m/z [M
+
H]+ calcd for
C94H128NO16P2S:1621.84571; found: 1621.85069.
17-{4-[6-(4-{3-[2-Acetamido-3-(tritylthio)propanoyloxy]-10,13-
dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-
1H-cyclopenta[a]phenanthren-17-yl}pentanoyloxy)hexa-2,4-
diynyloxy]-1-methyl-4-oxobutyl}-10,13-dimethyl-
2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclo-
penta[a]phenanthren-3-yl 3,5-Bis[(dimethoxyphosphoryl)-
methyl]benzoate (6b)
6-[4-(3-Hydroxy-10,13-dimethylhexadecahydro-1H-cyclopen-
ta[a]phenanthren-17-yl)pentanoyloxy]hexa-2,4-diynyl 4-{3-[2-
(tert-Butoxycarbonylamino)-3-(tritylthio)propanoyloxy]-10,13-
dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-
yl}pentanoate (5a)
Following general procedure
3 using 2a (0.21 mmol), 3a
(0.11 mmol), CuCl (0.80 mmol), and TMEDA (0.8 mmol);
yield: 0.09 g (65%); Rf = 0.14 (hexane–EtOAc, 3:1).
Following general procedure
3 using 3c (0.08 mmol), 4c
(0.08 mmol), CuCl (0.61 mmol), and TMEDA (0.61 mmol);
yield: 0.05 g (39%); Rf = 0.08 (CH2Cl2–MeOH, 19:1).
1H NMR (CDCl3): d = 0.63 (s, 3 H), 0.65 (s, 3 H), 0.91 (m, 12 H),
0.84–2.00 (m, 53 H), 1.43 (d, 3J = 8.02 Hz, 9 H), 2.25–2.41 (m, 4
H), 2.51–2.56 (m, 2 H), 3.62 (m, 1 H), 4.25 (m, 1 H), 4.73 (m, 1 H),
4.74 (s, 4 H), 5.09 (d, 3J = 6.87 Hz, 1 H), 7.21 (t, 3J = 7.22 Hz, 3 H),
7.28 (t, 3J = 8.36 Hz, 6 H), 7.39 (d, 3J = 7.79 Hz, 6 H).
1H NMR (CDCl3): d = 0.70 (s, 6 H), 0.71 (s, 6 H), 0.88–2.51 (m, 59
2
3
H), 2.55 (dd, JH,H = 12.3 Hz, JH,H = 4.9 Hz, 1 H), 2.60 (ddd,
2JH,H = 12.3 Hz, JH,H = 5.6 Hz, JH,H = 2.5 Hz, 1 H), 3.22 (d,
2JH,P = 21.8 Hz, 4 H), 3.71 (d, 3JH,P = 10.9 Hz, 12 H), 4.55–4.69 (m,
2 H), 4.74 (s, 4 H), 4.80–4.92 (m, 1 H), 5.36–5.46 (m, 2 H), 6.06 (d,
3JH,H = 7.7 Hz, 1 H), 7.23 (t, 3JH,H = 7.4 Hz, 3 H), 7.29 (t, 3JH,H = 7.4
Hz, 6 H), 7.40 (d, 3JH,H = 8.4 Hz, 6 H), 7.47 (s, 1 H), 7.86 (s, 2 H).
3
3
17-{4-[(6-{[4-(3-Hydroxy-10,13-dimethylhexadecahydro-1H-
cyclopenta[a]phenanthren-17-yl)pentanoyl](methyl)ami-
no}hexa-2,4-diynyl)(methyl)amino]-1-methyl-4-oxobutyl}-
10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-
3-yl 2-(tert-Butoxycarbonylamino)-3-(tritylthio)propanoate
(5b)
13C NMR (CDCl3): d = 11.9, 18.4, 19.4, 21.1, 24.3, 27.9, 28.2, 30.9,
1
31.0, 31.9, 32.0, 32.7 (d, JC,P = 138.3 Hz), 35.4, 36.7, 37.1, 38.3,
39.8, 42.5, 50.1, 51.8, 53.0 (d, 2JC,P = 7.4 Hz), 55.8, 56.7, 74.8, 74.9,
77.9, 122.8, 129.6–129.6 (m, 1 C), 131.7–131.8 (m, 1 C), 132.2–
132.3 (m, 1 C), 135.3, 139.7, 165.4, 173.4.
Following general procedure
3 using 2b (0.39 mmol), 3b
(0.39 mmol), CuCl (1.94 mmol), and TMEDA (1.94 mmol);
yield: 0.29 g (58%); Rf = 0.12 (hexane–EtOAc, 1:1).
31P NMR (CDCl3): d = 28.9.
1H NMR (CDCl3): d = 0.57 (s, 6 H), 0.84 (m, 12 H), 0.83–2.55 (m,
HRMS (ESI+, MeOH): m/z [M
+
Na]+ calcd for
68 H), 2.89 + 2.98 (s, 6 H), 3.53 (m, 1 H), 4.04 + 4.21 (s, 4 H), 4.17
C91H117NO15P2SNa: 1580.7511; found: 1580.7467.
3
(m, 1 H), 4.66 (m, 1 H), 5.08 (d, J = 8.3 Hz, 1 H), 7.08–7.36 (d,
3J = 6.8 Hz, 15 H).
Esterification with Fmoc-Gly-OH; General Procedure 4
Fmoc-Gly-OH (0.15 mmol) and the hydroxy compounds
(0.1 mmol) were dissolved in CH2Cl2 (25 mL). A soln of EDCl
(0.125 mmol) in CH2Cl2 (15 mL) was added dropwise over a peri-
od of 6 h. The mixture was stirred at r.t. for 16 h. The clear soln was
then washed H2O (2 ×) and the aqueous phase was extracted with
CH2Cl2. The combined organic extracts were dried (MgSO4) and
the solvent evaporated in vacuo. The crude product was purified by
column chromatography.
13C NMR (CDCl3): d = 12.1, 14.3, 18.6, 19.8, 20.9, 21.0, 21.1, 21.4,
23.4, 23.5, 24.3, 26.4, 26.5, 27.1, 27.3, 28.3, 28.4 30.2, 30.4, 30.6,
31.0, 31.3, 32.2, 33.6, 34.4, 34.7, 35.1, 35.0, 35.5, 35.9, 36.5,36.9,
40.3, 40.5, 41.9, 42.1, 42.8, 52.7, 56.1, 56.3, 56.4, 56.6, 60.5, 66.7,
67.4, 67.9, 69.0, 71.8, 72.5, 73.7, 74.8, 76.0, 77.5, 78.0, 125.6,
125.8, 126.9, 127.9, 128.2, 128.4, 129.0, 129.6, 130.0, 137.8, 144.5,
155.1, 170.3, 173.4.
HRMS (ESI+, MeOH): m/z [M + Na]+ calcd for C83H115N3NaO7S:
1320.8348; found: 1320.8342.
6-(4-{3-[2-(tert-Butoxycarbonylamino)-3-(tritylthio)propanoyl-
oxy]-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phen-
anthren-17-yl}pentanoyloxy)hexa-2,4-diynyl 4-(3-{2-[(9H-
Fluoren-9-ylmethoxy)carbonylamino]acetoxy}-10,13-dimethyl-
hexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pen-
tanoate (7a)
17-{4-[6-(4-{3-[2-(tert-Butoxycarbonylamino)-3-(tritylthio)pro-
panoyloxy]-10,13-dimethylhexadecahydro-1H-cyclopen-
ta[a]phenanthren-17-yl}pentanoyloxy)hexa-2,4-diynyloxy]-1-
methyl-4-oxobutyl}-10,13-dimethylhexadecahydro-1H-cyclo-
penta[a]phenanthren-3-yl 3,5-Bis[(dimethoxyphosphoryl)-
methyl]benzoate (6a)
Yield: 0.09 g (55%); Rf = 0.1 (hexane–EtOAc, 3:1).
1H NMR (300 MHz, CDCl3): d = 0.63 + 0.66 (s, 6 H), 0.92 (m, 12
H), 0.93–2.58 (m, 68 H), 3.96 (d, 3J = 5.7 Hz, 2 H), 4.25 (t, 3J = 7.1
Hz, 1 H), 4.38 (d, 3J = 7.6 Hz, 2 H), 4.74 (s, 4 H) 4.82 (m, 2 H), 5.10
Following general procedure
(0.05 mmol), CuCl (0.25 mmol), and TMEDA (0.25 mmol);
yield: 0.05 g (65%); Rf = 0.05 (CH2Cl2–MeOH, 19:1).
3 using 3a (0.05 mmol), 4a
3
3
1H NMR (CDCl3): d = 0.67 (s, 3 H), 0.67 (s, 3 H), 0.94–2.45 (m, 76
(d, J = 7.9 Hz, 1 H), 5.33 (t, J = 5.4 Hz, 1 H), 7.27–7.76 (m,
3J = 7.6 Hz, 23 H).
2
3
H), 2.53 (dd, JH,H = 21.8 Hz, JH,H = 4.1 Hz, 1 H), 2.60 (dd,
2JH,H = 21.9 Hz, 3JH,H = 5.3 Hz, 1 H), 3.22 (d, 2JH,P = 21.9 Hz, 4 H),
Synthesis 2008, No. 14, 2247–2256 © Thieme Stuttgart · New York

2254
M. Maue et al.
PAPER
13C NMR (100 MHz, CDCl3): d = 12.3, 18.5, 21.1, 21.1, 23.5, 24.4,
26.5, 26.6, 26.7, 27.2, 28.6, 29.9, 31.1, 31.2, 32.4, 34.6, 34.8, 35.2,
35.5, 36.0, 36.1, 40.3, 40.4, 40.7, 42.2, 43.0, 43.3, 47.2, 52.2, 52.9,
56.2, 56.3, 56.6, 56.8, 66.9, 67.5, 70.5, 74.0, 76.2, 77.5, 120.3,
125.4, 127.0, 127.3, 128.0, 128.2, 129.8, 169.7, 170.4, 173.4.
35.2, 35.7, 35.9, 37.0, 40.3, 40.3, 40.6, 42.1, 42.9, 44.4, 52.8, 53.5,
53.5, 55.1, 56.3, 56.4, 56.6, 56.7, 66.7, 73.8, 75.2, 76.1, 126.9,
128.1, 129.7, 170.3, 173.4.
2-[(2-{17-[4-({6-[(4-{3-[(2-(tert-Butoxycarbonylamino)-3-(tri-
tylthio)propanoyloxy]-10,13-dimethylhexadecahydro-1H-cy-
clopenta[a]phenanthren-17-yl}pentanoyl)(methyl)amino]hexa-
2,4-diynyl}(methyl)amino)-1-methyl-4-oxobutyl]-10,13-di-
methylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yloxy}-
2-oxoethylamino)methyl]phenylboronic Acid (9b)
A mixture of the amine 8b (0.04 mmol), 2-formylphenylboronic
acid (0.04 mmol), Et3N (0.08 mmol), and molecular sieves (3 Å)
were dissolved in dry MeOH. The mixture was stirred at r.t. for 3 h,
NaB(OAc)3H (0.24 mmol) was added to the soln. Stirring was con-
tinued for 1 h. Subsequently, the mixture was filtrated through
Celite. The filtrate was evaporated under reduced pressure and dried
in vacuo. The residue was washed several times with hot MeCN in
order to remove traces of less polar byproducts; yield: 0.02 g
(34%); Rf = 0.28 (CH2Cl2–MeOH, 9:1).
17-(4-[(6-{[4-(3-{2-[(9H-Fluoren-9-ylmethoxy)carbonylami-
no]acetoxy}-10,13-dimethylhexadecahydro-1H-cyclopen-
ta[a]phenanthren-17-yl)pentanoyl](methyl)amino}hexa-2,4-
diynyl)(methyl)amino]-1-methyl-4-oxobutyl)-10,13-dimethyl-
hexadecahydro-1H-cyclopenta[a]phenanthren-3-yl 2-(tert-
Butoxycarbonylamino)-3-(tritylthio)propanoate (7b)
Yield: 0.13 g (85%); Rf = 0.3 (hexane–EtOAc, 1:1).
1H NMR (CDCl3): d = 0.56 (s, 6 H), 0.83–2.55 (m, 82 H, steroid),
2.89 + 2.97 (s, 6 H), 3.30–4.00 (m, 4 H), 4.04 + 4.22 (s, 4 H), 4.30
(m, 2 H), 4.71 (m, 2 H), 5.06 (d, 3J = 8.3 Hz, 1 H), 5.39 (t, 3J = 5.0
Hz, 1 H), 7.08–7.72 (m, 19 H).
13C NMR (CDCl3): d = 12.1, 14.3, 18.6, 20.9, 21.1, 23.4, 24.2, 26.3,
26.6, 27.0, 28.4, 30.4, 31.0, 32.2, 34.4, 34.6, 34.7, 34.9, 35.6, 35.8,
36.9, 40.2, 40.4, 41.9, 42.7, 43.1, 47.2, 52.7, 56.1, 56.3, 56.6, 60.4,
66.6, 67.2, 67.9, 73.7, 75.9, 77.2, 79.9, 120.1, 125.2, 126.8, 127.1,
127.8, 128.1, 129.5, 141.3, 143.9, 144.4, 155.0, 156.4, 169.5, 170.2,
171.2, 173.3.
1H NMR (300 MHz, CDCl3): d = 0.66 (s, 6 H), 0.93 (m, 12 H),
0.80–2.59 (m, 58 H), 1.44 (s, 12 H), 2.97 + 3.09 (s, 6 H), 3.87–4.29
(m, 8 H), 4.73 + 4.83 (s, 2 H), 5.11 (d, 3J = 8.1 Hz, 1 H), 7.10–7.79
(m, 3J = 7.8 Hz, 15 H).
13C NMR (100 MHz, CDCl3): d = 1.1, 11.1, 11.4, 12.2, 14.2, 16.2,
18.7, 21.0, 21.6, 22.6, 22.8, 23.1, 23.4, 23.5, 23.9, 24.3, 25.9, 26.4,
26.6, 26.7, 27.1, 27.3, 28.0, 28.4, 29.0, 29.5, 29.8, 30.4, 30.5, 30.7,
31.1, 31.4, 32.2, 32.6, 33.7, 34.5, 34.7, 35.1, 35.5, 35.7, 35.9, 36.6,
37.0, 38.9, 40.3, 40.5, 40.6, 42.0, 42.2, 42.9, 48.5, 52.8, 54.1, 56.2,
56.4, 56.5, 56.6, 56.7, 63.1, 66.7, 67.2, 67.5, 73.8, 76.0, 76.5, 80.0,
126.9, 128.1, 129.6, 144.5, 155.1, 170.3, 173.4.
HRMS (ESI+, MeOH): m/z [M
C100H128N4NaO10S:1600.9277; found: 1600.9275.
+
Na]+ calcd for
Fmoc Deprotection; General Procedure 5
The Fmoc-protected amine (0.09 mmol) was dissolved in DMF
(5 mL). Piperidine (0.2 mL) was added at r.t. Stirring was continued
for 20 min. The mixture was diluted with the same amount of
CH2Cl2 and piperidine was extracted with 0.001 M aq HCl. The or-
ganic phase was dried (MgSO4), the solvent was evaporated in vac-
uo and the crude product was purified by column chromatography.
HRMS (ESI+, MeOH–CH2Cl2): m/z (M+ – H2O) calcd for
C98H128N4BO10S: 1563.9439; found: 1563.9401.
Cleavage of Phosphonic Acid Methyl Esters; General Proce-
dure 6
6-(4-{3-[2-(tert-Butoxycarbonylamino)-3-(tritylthio)propanoyl-
oxy]-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phen-
anthren-17-yl}pentanoyloxy)hexa-2,4-diynyl 4-[3-(2-Amino-
acetoxy)-10,13-dimethylhexadecahydro-1H-cyclopen-
ta[a]phenanthren-17-yl]pentanoate (8a)
The respective bisphosphonic acid tetramethyl ester (0.12 mmol)
was dissolved in MeCN (25 mL) under argon. Dry LiBr
(0.27 mmol) was added and the mixture was refluxed for 2–4 h. The
product appeared as a colorless precipitate. The solvent was decant-
ed carefully and the product was washed MeCN (2 ×) and Et2O
(3 ×). The colorless pure products were dried in vacuo.
Yield: 0.11 g (92%); Rf = 0.1 (hexane–EtOAc, 1:1).
1H NMR (300 MHz, CDCl3): d = 0.62 (s, 6 H), 0.90 (m, 12 H),
0.93–2.56 (m, 57 H), 1.42 (s, 12 H), 3.37 (s, 2 H), 4.24 (m, 1 H),
17-(4-[6-(4-{3-[2-(tert-Butoxycarbonylamino)-3-(tritylthio)pro-
panoyloxy]-10,13-dimethylhexadecahydro-1H-cyclopen-
ta[a]phenanthren-17-yl}pentanoyloxy)hexa-2,4-diynyloxy]-1-
methyl-4-oxobutyl)-10,13-dimethylhexadecahydro-1H-cyclo-
penta[a]phenanthren-3-yl 3,5-Bis{[hydroxy(methoxy)phospho-
ryl]methyl}benzoate Dilithium Salt (11a)
3
4.71 (s, 4 H), 4.72 (m, 2 H), 5.12 (d, J = 8.0 Hz, 1 H), 7.10–7.38
(m, 3J = 7.0 Hz, 15 H).
13C NMR (100 MHz, CDCl3): d = 12.1, 18.4, 21.0, 23.4, 24.3, 26.4,
27.1, 28.3, 28.4, 31.0, 32.2, 32.4, 34.5, 34.7, 35.1, 35.4, 35.9, 40.3,
40.6, 42.0, 42.9, 52.1, 52.7, 56.1, 56.2, 56.7, 58.3, 66.7, 70.4, 73.9,
75.3, 76.0, 77.4, 80.0, 126.9, 128.1, 129.6, 144.5, 155.1, 170.3,
173.3.
Yield: 0.19 g (99%); mp >300 °C.
1H NMR (MeOH-d4–CDCl3): d = 0.58 (s, 6 H), 0.71–2.58 (m, 80
2
3
H), 2.93 (d, JH,P = 20.8 Hz, 4 H), 3.45 (d, JH,P = 10.5 Hz, 6 H),
4.05–4.18 (m, 1 H), 4.50–4.65 (m, 6 H), 7.03–7.38 (m, 15 H), 7.59
(s, 3 H).
17-[4-([6-({4-[3-(2-Aminoacetoxy)-10,13-dimethylhexadecahy-
dro-1H-cyclopenta[a]phenanthren-17-yl]pentanoyl}(meth-
yl)amino)hexa-2,4-diynyl](methyl)amino)-1-methyl-4-oxo-
butyl]-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phen-
anthren-3-yl 2-(tert-Butoxycarbonylamino)-3-(tritylthio)pro-
panoate (8b)
13C NMR (MeOH-d4–CDCl3): d = 11.3, 17.4, 17.5, 20.3, 22.4, 22.6,
23.6, 25.8, 26.1, 26.4, 26.5, 27.4, 27.6, 30.3, 30.4, 30.4, 31.5, 32.4
(d, 1JC,P = 115.3 Hz), 33.4, 34.0, 34.1, 34.3, 34.3, 34.5, 34.8, 35.4,
35.4, 39.7, 39.8, 40.0, 41.4, 41.6, 42.3, 50.8 (d, 2JC,P = 6.1 Hz), 51.4,
51.4, 52.7, 55.6, 55.6, 56.0, 56.2, 66.4, 69.3, 69.4, 73.2, 73.2, 74.6,
75.5, 77.3, 79.4, 126.3, 127.1, 127.4, 127.6, 129.0, 129.7, 135.4,
144.0, 155.4, 166.5, 170.2, 173.1.
Yield: 0.12 g (98%); Rf = 0.08 (CH2Cl2–MeOH, 30:1).
1H NMR (CDCl3): d = 0.69 (s, 6 H), 0.85–2.65 (m, 80 H), 3.02 +
3.11 (s, 6 H), 3.62 (s, 2 H), 4.22 + 4.34 (s, 4 H), 4.30 (m, 1 H), 4.67
(m, 2 H), 4.78 (m, 1 H), 5.16 (d, 3J = 5.1 Hz, 1 H), 7.17–7.76 (m, 15
H).
31P NMR (MeOH-d4–CDCl3): d = 25.0.
13C NMR (CDCl3): d = 12.2, 18.6, 21.0, 23.4, 24.4, 25.1, 26.5, 26.6,
26.8, 27.2, 28.5, 29.9, 30.5, 31.1, 32.2, 32.4, 34.1, 34.5, 34.7, 35.1,
HRMS (ESI–, MeOH): m/z [M]– calcd for C92H121NO16P2S:
795.3952; found: 795.3945.
Synthesis 2008, No. 14, 2247–2256 © Thieme Stuttgart · New York

PAPER
Transmembrane Building Blocks
2255
17-{4-[6-(4-{3-[2-Acetamido-3-(tritylthio)propanoyloxy]-10,13-
dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-
1H-cyclopenta[a]phenanthren-17-yl}pentanoyloxy)hexa-2,4-
diynyloxy]-1-methyl-4-oxobutyl}-10,13-dimethyl-
2,3,4,7,8,9,10,11, 12,13,14,15,16,17-tetradecahydro-1H-cyclo-
penta[a]phenanthren-3-yl 3,5-Bis{[hydroxy(methoxy)phospho-
ryl]methyl}benzoate Dilithium Salt (11b)
HRMS (ESI–, MeOH): m/z [M]– calcd for C68H100NO14P2S:
1248.6334; found: 1248.6325.
17-[4-(6-{4-[3-(2-Acetamido-3-mercaptopropanoyloxy)-10,13-
dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-
1H-cyclopenta[a]phenanthren-17-yl]pentanoyloxy}hexa-2,4-
diynyloxy)-1-methyl-4-oxobutyl]-10,13-dimethyl-
2,3,4,7,8,9,10,11,1213,14,15,16,17-tetradecahydro-1H-cyclo-
penta[a]phenanthren-3-yl 3,5-Bis{[hydroxy(methoxy)phospho-
ryl]methyl}benzoate Dilithium Salt (12b)
Following general procedure 7 using 11b (0.1 mmol); yield: 0.12 g
(99%); mp >150 °C.
1H NMR (MeOH-d4–CDCl3): d = 0.63 (s, 6 H), 0.64 (s, 6 H), 0.85–
2.45 (m, 69 H), 2.85–2.97 (m, 6 H), 3.53 (d, 3JH,P = 10.5 Hz, 6 H),
5.29–5.38 (m, 2 H), 7.53 (s, 1 H), 7.62 (s, 2 H).
31P NMR (MeOH-d4–CDCl3): d = 25.6.
Yield: 0.19 g (99%); mp >300 °C.
1H NMR (MeOH-d4–CDCl3): d = 0.51 (s, 6 H), 0.62–2.54 (m, 69
H), 2.80 (d, 2JH,P = 20.3 Hz, 4 H), 3.36 (d, 3JH,P = 10.2 Hz, 6 H), 4.37
(br s, 1 H), 4.46–4.67 (m, 5 H), 5.16–5.26 (m, 2 H), 7.03–7.24 (m,
15 H), 7.41 (s, 1 H), 7.51 (s, 2 H).
13C NMR (MeOH-d4–CDCl3): d = 12.1, 15.2, 18.5, 19.6, 21.4, 22.6,
24.6, 27.9, 28.2, 28.4, 31.2, 31.3, 32.2, 34.0, 34.2 (d, 1JC,P = 130.2
Hz), 35.7, 35.7, 36.9, 37.0, 37.2, 37.4, 38.2, 38.5, 40.1, 40.1, 42.8,
50.4, 50.5, 51.7 (d, 2JC,P = 5.5 Hz), 52.2, 52.4, 56.1, 57.0, 57.1, 66.3,
67.3, 70.5, 74.1, 75.1, 76.1, 123.1, 123.3, 127.2, 128.3, 129.9,
130.6, 136.0–136.1 (m, 1C), 136.3, 139.7–139.7 (m, 1C), 140.0,
144.7, 167.2, 170.5, 171.3, 171.5, 174.0.
HRMS (ESI–, MeOH): m/z [M]– calcd for C70H98NO15P2S:
1286.6127; found: 1286.6124.
31P NMR (MeOH-d4–CDCl3): d = 25.4.
HRMS (ESI–, MeOH): m/z [M]– calcd for C89H112NO15P2S:
Disulfide Coupling; General Procedure 8
S-Trityl cleavage was effected with TFA–TIPS in CH2Cl2 as de-
scribed in general procedure 7. After stirring for 2 h, aldrithiole
(0.1 mmol) was added to the mixture. Stirring was continued at r.t.
for a further 16 h. The purification step again depends on the nature
of the bisphosphonate ester group, as outlined in general procedure
7.
1528.7222; found: 1528.7200.
Boc/Trityl Deprotection; General Procedure 7
The Boc- or trityl-protected starting material (0.1 mmol) and i-
Pr3SiH (0.2 mmol) were dissolved in CH2Cl2–TFA (1:1, 10 mL).
The color of the soln immediately changed to a dark yellow. The
mixture was stirred at r.t. for 10 min, at which point the yellow color
had disappeared and stirring was continued for a further 4 h. The
mixture was diluted with the same amount of toluene and the sol-
vents were evaporated in vacuo; this procedure was repeated three
times. Final purification was achieved by two different procedures,
depending on the nature of the bisphosphonate ester groups: The
dried lithium salts were extracted several times with Et2O in order
to remove Ph3CH. After this operation the product was dried in vac-
uo. The neutral esters were purified by column chromatography.
1-[17-(4-(6-{4-[3-(3,5-Bis{[hydroxy(methoxy)phosphoryl]meth-
yl}benzoyloxy)-10,13-dimethylhexadecahydro-1H-cyclopen-
ta[a]phenanthren-17-yl]pentanoyloxy}hexa-2,4-diynyloxy)-1-
methyl-4-oxobutyl)-10,13-dimethylhexadecahydro-1H-cyclo-
penta[a]phenanthren-3-yloxy]-1-oxo-3-(pyridin-2-yldisulfa-
nyl)propan-2-aminium Trifluoroacetate Dilithium Salt (13a)
Yield: 0.13 g (99%); mp >300 °C.
1H NMR (MeOH-d4–CDCl3): d = 0.37 (s, 6 H), 0.43 (s, 6 H), 0.62–
2.17 (m, 68 H), 2.76 (d, 2JH,P = 20.2 Hz, 4 H), 3.33 (d, 3JH,P = 10.6
Hz, 6 H), 4.39–4.63 (m, 6 H), 6.96 (t, 3JH,H = 5.3 Hz, 1 H), 7.16 (d,
3JH,H = 7.6 Hz, 1 H), 7.30 (s, 1 H), 7.40 (t, 3JH,H = 7.6 Hz, 1 H), 7.46
(s, 2 H), 8.25 (d, 3JH,H = 5.3 Hz, 1 H).
1-(17-{4-[(6-{[4-(3-{2-[2-(Dihydroxyboryl)benzylamino]acet-
oxy}-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenan-
thren-17-yl)pentyl](methyl)amino}hexa-2,4-diynyl)(meth-
yl)amino]-1-methyl-4-oxobutyl}-10,13-dimethylhexadeca-
hydro-1H-cyclopenta[a]phenanthren-3-yloxy)-3-mercapto-1-
oxopropan-2-aminium Trifluoroacetate (10b)
31P NMR (MeOH-d4–CDCl3): d = 24.7.
HRMS (ESI–, MeOH): m/z [M]– calcd for C73H103N2O14P2S2:
1357.6320; found: 1357.6324.
Following general procedure 7 using 9b (0.1 mmol); yield: 0.033 g
(33%).
1H NMR (300 MHz, CDCl3): d = 0.64 (s, 6 H), 0.91 (m, 12 H),
0.85–2.80 (m, 58 H), 2.97 + 3.06 (s, 6 H), 3.63 (m, 1 H), 4.10–4.45
(m, 8 H), 4.85 + 4.95 (m, 2 H), 7.10–7.64 (m, 4 H).
HRMS (ESI+, MeOH–CH2Cl2): m/z (M+ + Na + 2 MeOH – H2O)
calcd for C70H107N4NaBO8S: 1197.7800; found: 1197.7807.
17-{4-[6-(4-{3-[2-Acetamido-3-(pyridin-2-yldisulfanyl)pro-
panoyloxy]-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-
tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl}pen-
tanoyloxy)hexa-2,4-diynyloxy]-1-methyl-4-oxobutyl}-10,13-
dimethyl-2,3-4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-
1H-cyclopenta[a]phenanthren-3-yl 3,5-Bis{[hydroxy(meth-
oxy)phosphoryl]methyl}benzoate Dilithium Salt (13b)
Yield: 0.13 g (99%); mp 163 °C.
1-{17-[4-(6-{4-[3-(3,5-Bis{[hydroxy(methoxy)phosphoryl]-
methyl}benzoyloxy)-10,13-dimethylhexadecahydro-1H-cyclo-
penta[a]phenanthren-17-yl]pentanoyloxy}hexa-2,4-diynyloxy)-
1-methyl-4-oxobutyl]-10,13-dimethylhexadecahydro-1H-cyclo-
penta[a]phenanthren-3-yloxy}-3-mercapto-1-oxopropan-2-
aminium Trifluoroacetate Dilithium Salt (12a)
Following general procedure 7 using 11a (0.1 mmol); yield: 0.13 g
(99%); mp >300 °C.
1H NMR (MeOH-d4–CDCl3): d = 0.57 (s, 6 H), 0.61 (s, 6 H), 0.74–
2.39 (m, 66 H), 2.74–2.84 (m, 2 H), 2.93 (d, 2JH,P = 20.5 Hz, 4 H),
3.55 (d, 3JH,P = 10.5 Hz, 6 H), 4.53–4.96 (m, 6 H), 7.54 (s, 1 H), 7.61
(s, 2 H).
1H NMR (MeOH-d4–CDCl3): d = 0.62 (s, 6 H), 0.64 (s, 6 H), 0.84–
2.42 (m, 38 H), 2.93 (d, 2JH,P = 20.4 Hz, 4 H), 3.53 (d, 3JH,P = 10.4
Hz, 6 H), 4.53–4.80 (m, 7 H), 5.27–5.34 (m, 2 H), 7.11 (t, 3JH,H = 5.0
Hz, 4JH,H = 0.8 Hz, 1 H), 7.52–7.67 (m, 5 H), 8.42 (d, 3JH,H = 4.7 Hz,
1 H).
13C NMR (MeOH-d4–CDCl3): d = 11.5, 16.9, 17.9, 19.0, 19.0, 20.8,
22.1, 24.0, 26.1, 27.4, 27.6, 27.9, 30.7, 30.7, 31.6, 31.7, 32.8, 34.1,
35.1, 36.4, 36.4, 36.7, 36.8, 37.7, 37.9, 39.5, 42.2, 49.3, 49.8, 49.9,
51.2, 51.8, 54.1, 55.6, 56.5, 56.5, 69.9, 73.5, 74.6, 75.7, 77.4, 117.7,
122.5, 122.8, 128.2, 130.3, 134.8, 135.5, 139.0, 139.4, 164.0, 166.4,
169.5, 171.3, 173.5.
31P NMR (MeOH-d4–CDCl3): d = 24.6.
31P NMR (MeOH-d4–CDCl3): d = 24.5.
Synthesis 2008, No. 14, 2247–2256 © Thieme Stuttgart · New York

2256
M. Maue et al.
PAPER
HRMS (ESI–, MeOH): m/z [M]– calcd for C76H102NO15P2S2:
1394.6161; found: 1394.6131.
(4) Bharucha, K. R.; Buckley, G. C.; Cross, C. K.; Rubin, L. J.;
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Acknowledgment
Financial support by the DFG (Deutsche Forschungsgemeinschaft)
is gratefully acknowledged.
(6) Hay, A. S. J. Org. Chem. 1962, 27, 3320.
(7) Gray, C. W.; Houston, T. A. J. Org. Chem. 2002, 67, 5426.
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Synthesis 2008, No. 14, 2247–2256 © Thieme Stuttgart · New York