Synthesis of C-11 linked active ester derivatives of vitamin D3 and their conjugations to 42-residue helix-loop-helix peptides

Article abstract of DOI:10.1016/j.tet.2010.04.051

Derivatives of vitamin D₃ carrying an 8-carbon linker at C-11 terminating in an active ester were synthesized from commercial vitamin D₃ using a disassembly-reassembly strategy. Vitamin D₃ was cleaved at the C6-C7 double b

Full text of DOI:10.1016/j.tet.2010.04.051

Tetrahedron 66 (2010) 4577e4586
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Tetrahedron
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Synthesis of C-11 linked active ester derivatives of vitamin D3 and their
conjugations to 42-residue helixeloopehelix peptides
,
,
b
a,
Qi Zhang a y, Thomas Norberg a , Jonas Bergquist , Lars Baltzer
*
*
a Department of Biochemistry and Organic Chemistry, Uppsala University, Box 576, SE-751 23 Uppsala, Sweden
b Department of Physical and Analytical Chemistry, Uppsala University, Box 579, 75123 Uppsala, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history:
Derivatives of vitamin D3 carrying an 8-carbon linker at C-11 terminating in an active ester were syn-
thesized from commercial vitamin D3 using a disassemblyereassembly strategy. Vitamin D3 was cleaved
at the C6eC7 double bond and the ‘upper’ fragment was converted, via a series of reactions, to de-
rivatives substituted at C-11 with an 8-carbon linker terminating in an ethyl ester. Reassembly with
modified ‘lower’ fragments using HornereWittig olefination followed by linker ester hydrolysis and re-
esterification with p-nitrophenol gave C-11 substituted p-nitrophenyl esters. These vitamin D derivatives
were conjugated to 42-amino acid helixeloopehelix peptides by reaction of their p-nitrophenyl esters
with lysyl side-chain amino groups on the peptides. The vitamin Ddpeptide conjugates, being potential
specific binder candidates for vitamin D-binding protein, were characterized by mass spectroscopy and
CD measurements.
Received 8 October 2009
Received in revised form 23 March 2010
Accepted 12 April 2010
Available online 11 May 2010
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
Alzheimer’s disease, amyotrophic lateral sclerosis (ALS), and Par-
kinson’s disease. Accurate and fast measurement of DBP is then an
The vitamin D family of molecules is associated with the regu-
lation of calcium and phosphorus metabolism in higher organisms,
and various vitamin D derivatives have been shown to affect dis-
ease conditions, such as rickets, osteoporosis, breast cancer, pros-
tate cancer, psoriasis, and Alzheimer’s disease. The influences of
vitamin D derivatives on these and other diseases have spurred
a wide range of research efforts where a large number of vitamin D
derivatives have been synthesized and biologically evaluated (for
a recent comprehensive review of the synthetic work, see Okamura
and Zhue1).
Biological evaluations of vitamin D derivatives have involved
both in vivo and in vitro studies, the latter often investigating the
ability of various vitamin D derivatives to bind to receptor pro-
teins. Two extensively studied such receptor proteins are the vi-
tamin D receptor (VDR) and the vitamin D-binding protein (DBP).
The latter is the major carrier of vitamin D and its metabolites in
serum, and as such it is an important regulator of vitamin D levels
in the body. Considering the importance of vitamin D in health
and disease, it is not surprising that abnormal serum DBP levels
have been correlated to disease conditions.2e4 We are interested
in the possibility to use DBP levels in body fluids as biomarker for
essential requirement, and we have therefore set out to design
peptide conjugates5 of vitamin D derivatives that could poten-
tially, by way of their increased binding strength and multi-
functionality, be important tools in such measurements. We have
shown previously6 that the conjugation of benzenesulfonamide
with an affinity, KD, of 1.5 mM for human Carbonic anhydrase II
(HCAII) to a 42-residue polypeptide via an aliphatic spacer, gave
rise to a polypeptide conjugate binder for HCAII with a dissocia-
tion constant of 20 nM. The small molecule ligand and the poly-
peptide both contributed to binding in a cooperative manner, the
polypeptide mainly through hydrophobic interactions between
HCAII and the hydrophobic faces of the amphiphilic helices of the
helixeloopehelix motif. We hypothesized that amphiphilic heli-
ces bind well to proteins through hydrophobic interactions,
whereas the polar sides of the helices make them soluble in aq
solution. The versatility of polypeptides makes them highly suit-
able for further functionalization, e.g., for the incorporation of
fluorophores.7,8 They are therefore very well suited for bio-
analytical applications, such as the identification and quantifica-
tion of proteins related to disease.9
We here report synthesis of the vitamin D derivatives 1, 2, and 3,
carrying an active ester-containing linker attached to the C-11 of
the vitamin D skeleton. To our knowledge, this is the first example
of vitamin D derivatives with a C11-attached linker. We also report
conjugation of the synthesized vitamin D derivatives to 42-residue
polypeptides, and characterization of the conjugates by MALDI-TOF
* Corresponding authors. E-mail addresses: thomas.norberg@biorg.uu.se
(T. Norberg), lars.baltzer@biorg.uu.se (L. Baltzer).
y
Present address: School of Chemistry and Materials Science, Shaanxi Normal
University, Shaanxi Xi’an, 710062, People’s Republic of China
0040-4020/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2010.04.051

4578
Q. Zhang et al. / Tetrahedron 66 (2010) 4577e4586
mass spectroscopy and CD spectroscopy. The evaluation of the
peptide conjugates as specific binders of DBP and other proteins
will be reported in future communications.
the literature,1 and the following account of synthetic details
therefore applies only to the functionalizationereassembly parts of
our syntheses of 1, 2, and 3 (see Schemes 1e6).
O
O
O
OH
O
O
O
H
H
H
NO2
NO2
NO2
HO
HO
1
2
3
2. Results and discussion
The bromide 1015 was considered a suitable first intermediate
for C-11 chain extension. It was synthesized starting from the
known compound 616, oxidation of which (with PDC) gave the al-
dehyde 717, which in turn was converted to an acetal 8 by refluxing
with 1,2-ethanediol, toluene, and a catalytic amount of p-TSA.
Cleavage of the benzyl protecting group in 8 to give 9, followed by
treatment with PPh3 and CBr4 finally gave bromide 10.15
2.1. Design and synthesis of C-11 linked active ester
derivatives of vitamin D3
The design of a useful vitamin Ddpeptide conjugate requires
a derivative of vitamin D that binds strongly and specifically to
DBP. Also, this derivative should carry a linking moiety pro-
truding from a region of the molecule not involved in the binding
to DBP. The linking moiety should terminate in a functionality,
which is reactive toward a peptide attachment point, such as
a lysyl side-chain amino group. Inspection of the crystal structure
of the DBPeligand complex10 reveals that C-11 of the vitamin D3
molecule is not strongly involved in DBP binding, and attachment
here of a linking moiety should therefore not affect vitamin D3
binding to any greater extent. Indeed, vitamin D derivatives with
simple C-11 substituents have been synthesized11e14 and
shown11,12 to bind DBP similarly to unsubstituted controls. Cal-
culations using PyMOL software gave at hand, that an 8-carbon
chain extending from C-11 extrudes far enough from the
DBPdvitamin D3 surface that attachment of a peptide ligand at the
end of the chain should not interfere with the binding, but rather
strengthen it by providing additional peptideeprotein interactions
with the DBP surface. We therefore decided to synthesize the C-11
substituted active ester vitamin D3 derivatives 1, 2, and 3. To ac-
complish this, we used vitamin D3 as starting material and followed
a strategy of disassemblyereassembly previously used in vitamin D
syntheses.1 The strategy is sketched below (Fig.1) for compound 2.
Briefly, commercial vitamin D3 was cleaved at the C6eC7 double
bond and the ‘upper’ fragment was converted to the C-11
substituted ethyl ester 13 by a series of reactions, the crucial one
(attachment of the C-11 substituent), being a conjugate cuprate
addition. Reassembly of the ‘upper’ and ‘lower’ fragments using
HornereWittig olefination followed by ester hydrolysis and re-
esterification gave the C-11 substituted p-nitrophenyl ester 2.
The initial vitamin D3 cleavage steps (giving, after modification,
compounds 4 and 16) have been previously described in detail in
The Grundmann ketone 418 afforded the
a,b-unsaturated ketone
519 under Saegusa conditions.13,14 Reaction of 5 with the cuprate
prepared from bromide 10 by treatment with t-BuLi and the com-
plex CuI/n-Bu3P, gave a single C-11 substituted stereoisomer 11 in
moderate yield. The alpha-C-11 structure of compound 11 was
evident from its 1H NMR spectrum, which showed one large
(13.3 Hz) and one small (4.6 Hz) vicinal coupling constant for
H11eH9a/H9b, and also a strong NOE interaction between H11
(multiplet at 2.03 ppm) and the H18 methyl group (singlet at
0.63 ppm). It’s not surprising that only the
obtained since the copper reagent attacks molecule 5 from the less
hindered -face.
a-stereoisomer was
a
Several different conditions were tried to release the aldehyde
from compound 11 (Scheme 2). Strongly acidic deprotection con-
ditions (1 N HCl aq/THF/40 ꢀC), gave, after subsequent Wittig 2-
carbon extension (using (ethoxycarbonylmethylidene)-triphenyl-
phosphorane20,21), not the desired product 12 but instead the 14-
position isomer 12a in 88% yield. However, milder deprotection
conditions (DDQ/MeCN/H2O/rt/24 h or PPTs/acetone/H2O/reflux/
15 h) gave the desired product 12 in good (76e82%) yield.
To avoid possible side-reactions involving the double bond in 12,
hydrogenation (Pd/C) of 12 was performed to give the expected
compound 13 (Scheme 3). A trial HornereWittig reaction of 13 with
the simple lithium phosphinoxy carboanion prepared from 1422
afforded compound 15. Hydrolysis followed by re-esterification
with p-nitrophenol gave the active ester 1, a truncated analog of
VD3 suitable for protein conjugation.
A similar strategy was used to synthesize the vitamin D3 de-
rivative 2. Coupling of the phosphine oxide 1623 with ketone 13
using n-BuLi at ꢁ78 ꢀC gave the ethyl ester 17 (Scheme 4).
O
EtO
6
H
O
O2N
O
H
13
OH
O
6
+
+
H
H
P(O)Ph2
OH
HO
HO
HO
HO
Vitamin D3
2
9
Figure 1. Synthetic strategy for preparation of compound 2.

Q. Zhang et al. / Tetrahedron 66 (2010) 4577e4586
4579
3
3
O
O
a, b
c
3
+
Br
O
O
H
H
H
O
O
O
4
5
10
11
g
O
O
O
d
f
e
OBn
3
OH
OBn
HO
OBn
4
3
O
O
9
6
7
8
Scheme 1. (a) (i) LDA/THF/ꢁ78 ꢀC/2.5 h; (ii) TMSCl/ꢁ78 ꢀC to 0 ꢀC; (b) Pd(OAc)2/THF/CH3CN/rt/overnight, 79% from 4; (c) (i) 10/t-BuLi/ꢁ85 ꢀC/Et2O/1.5 h; (ii) CuI n-Bu3P/ꢁ85 ꢀC to
ꢁ50 ꢀC/1 h; (iii) 5/-78 ꢀC/30 min, 69%; (d) PDC/CH2Cl2/rt/overnight, 85%; (e) 1,2-ethanediol/p-TSA/toluene/reflux/16 h, 98%; (f) Pd/C/H2/MeOH/rt/9 h, 91%; (g) PPh3/CBr4/CH2Cl2/
0
ꢀC/1 h, 82%.
R
EtO2C
O
4
R
R
OHC
O
4
H
12
4
Different Conditions
Ph3P=CHCO2Et
O
H
CH2Cl2
r.t., overnignt
H
O
O
R
11
EtO2C
4
R=
H
12a
O
Scheme 2. Chain extension of 11 and attempted acetal removal under different conditions.
R
R
EtO2C
EtO2C
a
4
b
4
+
H
Ph2P
H
O
O
O
12
13
14
R
R
O
EtO2C
c, d
4
O
H
H
NO2
R=
1
15
Scheme 3. (a) Pd/C/H2/MeOH/2 h, 88%; (b) (i) 14/n-BuLi/ꢁ78 ꢀC/40 min; (ii) 13/ꢁ78 ꢀC to 0 ꢀC/5 h, 50%; (c) LiOH$H2O/THF/H2O/rt/36 h; (d) p-nitrophenol/DCC/DMAP/CH2Cl2/rt/
24 h, 78%.
EtO2C
P(O)Ph2
6
EtO2C
6
a
+
H
H
13
O
17
16
TBSO
TBSO
O2N
b, c
O2N
O
O
O
O
6
6
H
H
18
2
TBSO
HO
Scheme 4. (a) (i) 16/n-BuLi/ꢁ78 ꢀC/40 min; (ii) 13/ꢁ78 ꢀC/2 h, 82%; (b) LiOH$H2O/THF/H2O/rt/72 h; (c) p-nitrophenol/DCC/DMAP/CH2Cl2/rt/overnight, 78%.

4580
Q. Zhang et al. / Tetrahedron 66 (2010) 4577e4586
EtO2C
P(O)Ph2
P(O)Ph2
6
b
a
c, d
H
2
TBSO
HO
16
19
20
HO
Scheme 5. (a) TBAF/THF/5 h, 97%; (b) (i) n-BuLi/ꢁ78 ꢀC/1 h; (ii) 13/ꢁ78 ꢀC/2 h, 75%; (c) LiOH$H2O/THF/H2O/rt/36 h; (d) p-nitrophenol/DCC/CH2Cl2/rt/overnight, 71%.
O
O
a
c, d
OR
b
e
OTES
OTES
4
H
H
H
O
O
O
21 R = H
23
24
22 R = TES
EtO2C
EtO2C
EtO2C
6
6
5
j
f, g
h
OTES
OH
OH
H
H
H
O
O
O
25
26
27
O
EtO2C
6
O
6
O
PPh2
OR'
OH
H
k
l
H
n, o
m
19
NO2
TESO
28
RO
HO
29 R = R' = TES
30 R = R' = H
3
Scheme 6. (a) NaIO4/RuCl3/CCl4/CH3CN/phosphate buffer/45 ꢀC/2e4 d, 43%e55%; (b) TESCl/imidazole/DMF/rt/overnight, 97%; (c) (i) LDA/THF/ꢁ78 ꢀC/1 h; (ii) TMSCl/ꢁ78 ꢀC to 0 ꢀC; (d)
Pd(OAc)2/THF/CH3CN/rt/overnight, 75% from 22; (e) (i) 10/t-BuLi/ꢁ85 ꢀC/Et2O/1.5 h; (ii) CuI$n-Bu3P/ꢁ85 ꢀC to ꢁ50 ꢀC/1 h; (iii) 23/ꢁ78 ꢀC/2 h, 73%; (f) PPTs/acetone/H2O/reflux/17 h; (g)
Ph3P]HCO2Et/CH2Cl2/rt/overnight, 83% (from 24); (h) Pd/C/H2/MeOH/2 h, 100%; (j) TESCl/imidazole/DMF/rt/6 h, 97%; (k) TESCl/imidazole/DMF/rt/overnight, 69%; (l) (i) 28/n-BuLi/
ꢁ78 ꢀC/40 min; (ii) 27/ꢁ78 ꢀC/2 h, 84%; (m) PPTs/acetone/H2O/reflux/3 h, 100%; (n) LiOH$H2O/THF/H2O/rt/44 h; (o) p-nitrophenol/DCC/DMAP/CH2Cl2/rt/2.5 h., 76% (from 30).
Hydrolysis followed by re-esterification with p-nitrophenol gave
the active ester 18. However, attempted removal of the TBS group
from 18 gave unexpectedly low yields (w25%) despite several at-
tempts using different conditions.
Therefore, cleavage of the TBS group was instead effected earlier,
at the stage of the phosphine oxide 16, which gave 19 in good yield
(97%) on treatment with TBAF in THF (Scheme 5). Conversion of 19
into the dianion by treatment with 2 equiv of n-BuLi ꢁ78 ꢀC and
subsequent WittigeHorner reaction with the ketone 13 gave the
desired triene 20 in 75% yield. Hydrolysis followed by re-esterifi-
cation with p-nitrophenol gave the final active ester 2, a VD3 de-
rivative suitable for protein conjugation.
Considering the low yield in the final TBS deprotection step
during the synthesis of 2, we now introduced the more acid-sen-
sitive TES group to protect the hydroxy group in phosphine oxide
19, giving phosphine oxide 28. The compounds 27 and 28 un-
derwent HornereWittig reaction yielding triene 29. To our satis-
faction, the TES groups in 29 were smoothly cleaved by acid to give
30 in high yield. The ethyl ester in 30 was then finally saponified
and converted to the target VD3 active ester 3, suitable for protein
conjugation.
2.2. Peptide design, synthesis, and conjugation
In consideration of the fact that the 25-hydroxy derivative of
vitamin D3 binds more strongly to DBP than vitamin D3 itself24e27
we decided to also prepare the 25-hydroxylated vitamin D3 de-
rivative 3. The synthesis (Scheme 6) started again from Grundmann
ketone 4,18 which was selectively hydroxylated at C-25 by ruthe-
nium-catalyzed oxidation28,29 to give 21. After TES ether protection
of the C-25 hydroxy group, the resulting ketone 2230 was converted
to corresponding enone 23.31,13,14 Michael addition with the curp-
rate generated from bromide 10 using t-BuLi/CuI$n-Bu3P31 gave
compound 24 (again only the alpha C-11 isomer). Hydrolysis of the
acetal and TES ether groups of 24 in acetone/H2O catalyzed by PPTs
and treatment as before of the intermediate aldehyde with
(ethoxycarbonylmethylidene)-triphenylphosphorane20,21 gave es-
ter 25. Subsequent hydrogenation (Pd/C) followed by TES-imidaz-
ole/DMF re-protection of the C-25 hydroxyl group afforded the
expected ‘upper’ fragment 27.
The design and synthesis of 42-residue helixeloopehelix pep-
tides and their conjugations to small molecules containing active
esters has been described in detail previously.32e34 In short, the
peptide primary sequences were designed to fold into two am-
phiphilic helices connected by a short loop. The amino acids were
selected based on their propensity for helix or loop formation.
Residues capable of salt bridge formation, N- and C-terminal cap-
ping groups, a conjugation point (a lysyl 3-amino group), and
a fluorophore group (for protein binding monitoring) were in-
troduced in selected positions. In this paper, an example conjuga-
tion of 2 to the peptide 4-D15L8 (Fig. 2a, this peptide has a dansyl
fluorophore in position 15 and a free lysyl 3-amino group in position
8) is described in detail. Several other analogous peptides were
conjugated in a similar way to either 1, 2, and 3 to form a library of
peptide conjugates. The evaluation of the binding properties of

Q. Zhang et al. / Tetrahedron 66 (2010) 4577e4586
4581
solution of a-cyano-4-hydroxycinnamic acid in 1:1 acetonitril/wa-
ter, the mixture was spotted onto a source plate, dried, the plate
was inserted into the ion source of a Bruker Daltonics Ultraflex II
TOF/TOF instrument, and spectra were recorded in the positive ion
mode. High resolution MS spectra were run on an Agilent LC/MSD
TOF instrument (Agilent Technologies, Santa Clara, CA, USA), with
detection in the positive ion mode. Agilent TOF software and Agi-
lent QS software were used to record and analyze mass spectra,
respectively. Standard autotune of masses was performed in the
TOF-MS instrument before the experimental runs, and typical mass
errors of 1e3 ppm were achieved in the calibration.
Thin layer chromatography (TLC) was performed on Silica Gel
F254 aluminum plates (Merck, Darmstadt, Germany) with detection
by UV-light and/or by staining with 8% phosphomolybdic acid in
ethanol. Column chromatography was performed in the flash mode
(unless otherwise stated) on Matrex silica gel 60 (35e70 mm).
Figure 2. a) The amino acid sequence of peptide 4-D15L8, where lysine residues in
bold represent addressable sites for modification. The one letter code for amino acids is
used: A¼Ala, C¼Cys, D¼Asp, E¼Glu, F¼Phe, G¼Gly, H¼His, I¼Ile, K¼Lys, L¼Leu,
N¼Asn, P¼Pro, Q¼Gln, R¼Arg, V¼Val, X¼Nle. (b) Cartoon of peptide 4-D15L8 conju-
gated with 2. The tertiary structure shown is based on the crystal structure of the four-
helix-bundle rop protein (PDB entry code: 1ROP) 36. The vitamin D residue from 2 and
the dansyl residue were attached at Lys-8 (purple) and Lys-15 (red), respectively. The
peptide is folded only when dimerized, but for reasons of clarity the monomeric form
is shown.
Dry THF and dry Et2O were distilled from Na/Ph2CO under N2.
Dry CH2Cl2 was distilled over CaH2 under N2. All other solvents and
reagents were commercially available and used as received without
any further purification. Abbreviations: THF¼tetrahydrofuran,
LDA¼lithium diethylamide, TMSCl¼trimethylsilyl chloride, t-
Buli¼tert-butylithium, PDC¼pyridinium dichromate, p-TSA¼p-tol-
uenesulfonic acid, DDQ¼dichlorodibensoquinone, PPTs¼pyr-
idinium p-toluenesulfonate, DCC¼dicyclohexyl carbodiimide,
DMAP¼4-(N,N-dimethylamino)pyridine,
TBS¼tert-Butyldime-
these conjugates toward VDB and other proteins will be reported in
a coming publication.
The peptide 4-D15L8 was synthesized on solid phase using
Fmoc chemistry and a dansyl fluorophore group was introduced
tylsilyl, TES¼triethylsilyl, TBAF¼tetrabutylammonium fluoride,
TBTU¼O-(7-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetra-
fluoroborate, DIPEA¼diisopropylethylamine, DMSO¼dimethyl
sulfoxide.
after selective deprotection of the 3-amino group of Lys15. The
peptide was then cleaved from the resin by 95% trifluoroacetic acid,
purified by reversed-phase HPLC and identified by MALDI-TOF
mass spectrometry. After attempting several different conjugation
conditions, the derivative 2 was reacted (twofold excess) with 4-
D15L8 in DMSO containing 5% DIPEA, giving the desired conjugate
(Fig. 2b) in 38% yield after HPLC purification.
The secondary structures of unconjugated 4-D15L8 and 4-D15L8
conjugated with 2 were investigated by CD spectroscopy. Both
peptides gave rise to minima at 208 and 222 nm and a maximum at
4.1.1. De-A, B-cholest-9(11)-en-8-one (5). This compound was pre-
pared from compound 418 essentially as described by Okamura
et al.19
4.1.2. 6-Benzyloxyhexanal (7). 6-Benzyloxyhexanol (6)16 (3 g,
14.42 mol) was dissolved in dry CH2Cl2 (50 mL) and PDC (10.8 g,
28.72 mmol) was added. The reaction mixture was stirred at room
temperature overnight, then CH2Cl2 (200 mL) was added and the
reaction solution was filtered through a Celite pad. The filtrate was
concentrated and the residue was purified by column chromatog-
raphy (10:1 n-pentane/EtOAc) to give 717 as colorless oil (2.52 g,
190 nm characteristic of a-helices. Their mean residue ellipticities at
222 nmwereꢁ12,519 andꢁ17,520 deg cm2 dmolꢁ1 (10
mM inH2O at
298 K), respectively, which are in the same range as those previously
85%). 1H NMR (400 MHz, CDCl3)
d
9.75 (t, J¼2.2 Hz, 1H), 7.34e7.20
reported35, indicating that both peptides were helical dimers.
(m, 5H), 4.49 (s, 2H), 3.47 (t, J¼6.4 Hz, 2H), 2.40e2.48 (m, 2H),
1.72e1.61 (m, 4H), 1.46e1.38 (m, 2H).
3. Conclusion
4.1.3. 1,1-Ethylenedioxy-6-benzyloxyhexane (8). A catalytic amount
of p-TSA (380 mg) was added to a stirred solution of aldehyde 7
(2.038 g, 9.89 mmol) and 1,2-ethanediol (2.76 mL, 49.5 mmol) in
toluene (20 mL) and the reaction mixture was refluxed for 16 h
with a DeaneStark water trap. The reaction mixture was then
cooled, washed with saturated aq NaHCO3 solution (15 mL), and
brine, then dried (MgSO4) and concentrated. The residue was pu-
rified by column chromatography (10:1 n-pentane/EtOAc) to give 8
as colorless oil (2.423 g, 98%). 1H NMR (400 MHz, CDCl3)
Three C-11 linked active ester derivatives of vitamin D3 were
designed and synthesized, utilizing
a
strategy of dis-
assemblyereassembly. Conjugations of these vitamin D3 de-
rivatives to 42-residue peptides gave a library of conjugates
suitable for future binding screening.
4. Experimental section
4.1. General materials and methods
d
7.37e7.28 (m, 5H), 4.86 (t, J¼4.8 Hz, 1H), 4.52 (s, 2H), 4.00e3.94
(m, 2H), 3.90e3.84 (m, 2H), 3.49 (t, J¼6.4 Hz, 2H),1.71e1.63 (m, 4H),
1.46e1.45 (m, 4H); 13C NMR (100 MHz, CDCl3)
138.90, 128.60,
Concentrations were performed with a rotatory evaporator at
reduced pressure (bath temperature <40 ꢀC). The 1H NMR and 13C
NMR spectra were recorded for CDCl3 solutions (internal CHCl3, dH
7.26, internal CDCl3 dC 77.66, 77.34, 77.02 ppm) at 25 ꢀC using
Varian Unity 400 or 500 MHz instrument, unless otherwise stated.
Assignments were corroborated by appropriate 2-D experiments.
For ES-MS spectra, dilute methanol solutions of the analyte were
infused, using a syringe pump, directly into the ion source of
a SCIEX API 150-EX mass spectrometer. For MALDI-TOF spectra,
a dilute aq solution of the sample was mixed (1:1) with a saturated
d
127.88, 127.74, 104.82, 73.11, 70.56, 65.09, 34.11, 29.95, 26.41, 24.20;
ESIMS m/z 273.2 ([MþNa]þ); ESIHRMS calcd for C15H23O3 ([MþH]þ)
251.1648, found 251.1842.
4.1.4. 1,1-Ethylenedioxy-6-hydroxyhexane (9). A suspension of
compound 8 (3.5 g, 14 mmol) and Pd/C (350 mg, 10 wt %) in MeOH
(28 mL) was stirred for 9 h under H2 atmosphere at room temper-
ature, then the mixture was filtered through a Celite pad and the
filtrate was concentrated. The residue was purified by column

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Q. Zhang et al. / Tetrahedron 66 (2010) 4577e4586
chromatography (1.5:1 n-pentane/EtOAc) to give 937 as a colorless
oil (2.04 g, 91%). 1H NMR (400 MHz, CDCl3)
3.96e3.90 (m, 2H), 3.87e3.82 (m, 2H), 3.61 (t, J¼6.6 Hz, 2H), 1.75
(br s, 1H), 1.67e1.62 (m, 2H), 1.59e1.52 (m, 2H), 1.47e1.37 (m, 4H).
(100 MHz, CDCl3) d 211.8, 167.0, 149.4, 121.6, 62.2, 60.4, 56.8, 49.4,
d
4.82 (t, J¼5.0 Hz, 1H),
48.0, 46.7, 39.7, 37.6, 36.9, 36.2, 35.7, 32.3, 29.5, 28.2, 28.2, 28.0, 27.1,
24.0, 23.0, 22.8, 19.2, 19.1, 14.5, 13.5; ESIMS m/z 455.0 ([MþNa]þ);
ESIHRMS calcd for C28H49O3 ([MþH]þ) 433.3683, found 433.3847.
4.1.5. 1,1-Ethylenedioxy-6-bromohexane (10). A mixture of
9
4.1.8. 11a-(7-Ethoxycarbonylhept-5-en-1-yl)-de-A,B-14-epi-8-cho-
(995 mg, 6.22 mmol) and PPh3 (2.45 g, 9.33 mmol) in CH2Cl2
(31 mL) was stirred in an ice-water bath while CBr4 (3.1 g,
9.33 mmol) was added in portions. After completed addition, stir-
ring was continued at 0 ꢀC for 1 h. The mixture was diluted with n-
pentane (200 mL), filtered through a Celite pad and the filtrate was
concentrated. The residue was purified by column chromatography
(10:1 n-pentane/EtOAc) to give 1015 as a colorless oil (1.14 g, 82%).
lestanone (12a). Compound 11 (63 mg, 0.16 mmol) was dissolved in
THF (0.8 mL) and 1 N HCl aq (0.8 mL) was added. The reaction
mixture was stirred vigorously at 40 ꢀC for 3 h, then diluted with
diethyl ether and washed with, successively, saturated aq NaHCO3,
water, and brine, then dried (MgSO4), filtered, and the filtrate was
concentrated. The residual oil was then converted into 12a (54 mg,
26
80%) by a similar Wittig-reaction as described above (12). [a]
D
1H NMR (400 MHz, CDCl3)
d
4.85 (t, J¼4.6 Hz, 1H), 3.98e3.92 (m,
þ62.7 (c 2.52, CHCl3); 1H NMR (400 MHz, CDCl3)
d
6.97 (dt, J¼15.6,
2H), 3.89e3.83 (m, 2H), 3.41 (t, J¼6.8 Hz, 2H), 1.91e1.84 (m. 2H),
1.70e1.65 (m, 2H), 1.49e1.44 (m, 4H).
6.8 Hz, 1H), 5.82 (d, J¼16.0 Hz, 1H), 4.19 (q, J¼7.2 Hz, 2H), 2.43e2.35
(m, 2H), 2.20 (ddd, J¼1.6, 6.8, 14.4 Hz, 2H), 2.10e2.01 (m, 2H),
1.93e1.82 (m, 2H), 1.73 (dt, J¼13.6, 2.4 Hz, 1H), 1.68e1.59 (m, 1H),
1.54e0.94 (cluster of signals,19H),1.28 (t, J¼7.2 Hz, 3H), 1.06 (s, 3H),
0.88 (d, J¼6.4 Hz, 3H), 0.86 (d, J¼6.4 Hz, 3H), 0.85 (d, J¼6.4 Hz, 3H);
4.1.6. 11a-[(6,6-Ethylenedioxy)hex-1-yl]-de-A, B-cholest-8-one (11).
A solution of bromide 10 (1.7 g, 7.62 mmol) in dry diethyl ether
(16 mL) was added dropwise to a cooled (ꢁ85 ꢀC) t-BuLi solution
(9 mL, 15.24 mmol, 1.7 M in heptane) during 1 h under N2. The
mixture was stirred for 30 min. Separately and simultaneously, n-
Bu3P (0.95 mL, 3.81 mmol) was added to a suspension of CuI
(725 mg, 3.81 mmol) in dry diethyl ether (21 mL). At ꢁ85 ꢀC, the
solution of the complex CuI/n-Bu3P complex was transferred by
cannula to the initial reaction mixture. The resulting solution was
warmed to ꢁ50 ꢀC during 1 h, then again cooled to ꢁ78 ꢀC and
a solution of 5 (500 mg,1.91 mmol) in dry diethyl ether (10 mL) was
added dropwise. After stirred for 30 min at ꢁ78 ꢀC, the reaction was
quenched by addition of saturated aq NH4Cl (5 mL), and was then
diluted with diethyl ether (50 mL), washed with, successively,
saturated aq NH4Cl, water and brine, and dried (MgSO4) and
evaporated. The residue was purified by column chromatography
13C NMR (100 MHz, CDCl3)
d 215.4, 167.0, 149.5, 121.6, 60.4, 60.2,
56.7, 47.7, 46.1, 45.9, 39.7, 36.9, 35.7, 34.3, 33.3, 32.4, 29.5, 28.7, 28.2,
28.2, 26.8, 24.4, 23.0, 22.8, 22.5, 22.2, 19.4, 14.5; ESIMS m/z 455.0
([MþNa]þ); ESIHRMS calcd for C28H49O3 ([MþH]þ) 433.3683, found
433.3610.
4.1.9. 11a-(7-Ethoxycarbonylhept-1-yl)-de-A,B-cholest-8-one (13). A
solution of compound 12 (41 mg, 0.095 mmol) in MeOH (1 mL) was
mixed with Pd/C (6 mg, 10 wt %). The suspension was stirred for 2 h
under H2 atmosphere at room temperature, then the suspension
was filtered through a Celite pad and the filtrate was concentrated.
The residue was purified by column chromatography (17:1 n-
pentane/EtOAc) to give 13 as colorless oil (36 mg, 88%). [
a
]
25 þ11.1
D
(c 0.75, CHCl3); 1H NMR (400 MHz, CDCl3)
d
4.12 (q, J¼7.2 Hz, 2H),
(8:1 n-pentane/EtOAc) to give 11 as colorless oil (531 mg, 69%).
2.42 (dd, J¼11.6, 7.6 Hz, 1H), 2.34 (dd, J¼5.0, 13.4 Hz, 1H), 2.27
(t, J¼7.2 Hz, 2H), 2.16 (dd, J¼4.2, 13.0 Hz, 1H), 2.07e2.00 (m, 1H),
1.95e1.83 (m, 2H, including 1.86 (t, J¼12.6 Hz, 1H)), 1.76e1.00
(cluster of signals, 28H, including 1.24 (t, J¼7.2 Hz, 3H)), 0.96 (d,
J¼5.6 Hz, 3H), 0.87 (d, J¼6.4 Hz, 6H), 0.63 (s, 3H); 13C NMR
26
[
a]
þ9.1 (c 0.78, CHCl3); 1H NMR (500 MHz, CDCl3)
d 4.83 (t,
D
J¼4.8 Hz,1H), 3.96 (m, 2H), 3.84 (m, 2H), 2.42 (dd, J¼7.4,11.7 Hz,1H,
H-14), 2.35 (dd, J¼4.8, 13.4 Hz, 1H, H-9a), 2.16 (dd, J¼4.0, 12.9 Hz,
1H, H-12a), 2.03 (m, 1H, H-11), 1.89 (m, 1H), 1.87 (ddd, J¼1.0, 12.3,
13.4 Hz, 1H, 9b), 1.70 (m, 1H), 1.67e1.62 (signal cluster, 2H),
1.56e1.45 (signal cluster, 2H), 1.44e1.22 (signal cluster, 14H),
1.20e1.08 (signal cluster, 3H), 1.02 (m, 1H), 0.95 (d, J¼6.0 Hz, 3H),
0.87 (d, J¼6.6 Hz, 3H), 0.86 (d, J¼6.6 Hz, 3H), 0.63 (s, 3H, H-18); 13C
(100 MHz, CDCl3)
d 215.5, 174.1, 60.4, 60.2, 56.7, 47.7, 46.1, 45.9,
39.7, 37.0, 35.7, 34.6, 34.3, 33.3, 29.8, 29.4, 29.3, 28.7, 28.2, 27.0,
25.2, 24.4, 23.0, 22.8, 22.5, 22.2, 19.4, 14.5; ESIMS m/z 457.0
([MþNa]þ); ESIHRMS calcd for C28H51O3 ([MþH]þ) 435.3839,
found 435.3892.
NMR (100 MHz, CDCl3)
d 211.8, 104.8, 65.1, 62.1, 56.7, 49.4, 48.0,
46.6, 39.6, 37.6, 36.9, 36.2, 35.7, 34.0, 29.8, 28.2, 28.0, 27.2, 24.2,
24.0, 23.0, 22.8, 19.1, 19.1, 13.5; ESIMS m/z 429.4 ([MþNa]þ);
ESIHRMS calcd for C26H47O3 ([MþH]þ) 407.3526, found 407.4081.
4.1.10. 11a
-[7-(4-Nitrophenyloxy)carbonylhept-1-yl]-8-(300-methyl-
but-200-enylidene)-de-A,B-cholestane (1). A solution of 1422 (107 mg,
0.40 mmol) in dry THF (1 mL) was added dropwise to a solution of n-
BuLi (0.26 mL, 0.42 mmol, 1.6 M) in dry THF (1 mL) at ꢁ78 ꢀC under
N2. After stirring at ꢁ78 ꢀC for 40 min, a solution of compound 10
(115 mg, 0.26 mmol) in dry THF (1 mL) was added by syringe. The
reaction mixture was allowed to warm to 0 ꢀC during 5 h, then
saturated aq NH4Cl (3 mL) was added to quench the reaction. The
mixture was diluted by diethyl ether (20 mL), washed with saturated
aq NH4Cl and brine, and dried (MgSO4) and concentrated. The resi-
due was purified by column chromatography (100:1 n-pentane/
EtOAc) to give 15 as colorless oil (64 mg, 50%). 1H NMR (400 MHz,
4.1.7. 11a-(7-Ethoxycarbonylhept-5-en-1-yl)-de-A,B-cholest-8-one
(12). DDQ (8.3 mg, 0.036 mmol) was added to a solution of 3
(73.5 mg, 0.18 mmol) in CH3CN/H2O (v/v¼9/1, 1 mL). The reaction
mixture was stirred at room temperature for 24 h, after which the
reaction was quenched by saturated aq Na2SO3 (2 mL). The mixture
was diluted with diethyl ether (20 mL), washed with saturated aq
Na2SO3, H2O and brine, and dried (MgSO4), filtered, and the filtrate
was concentrated. The residual oil was dissolved in dry CH2Cl2
(2 mL) and (ethoxycarbonylmethylidene)-triphenylphosphor-
ane20,21 (125 mg, 0.36 mmol) was added. The mixture was stirred at
room temperature for 24 h, and then concentrated and the residue
was purified by column chromatography (20:1 n-pentane/EtOAc) to
CDCl3)
d
6.15 (d, J¼10.8 Hz, 1H), 5.79 (d, J¼11.6 Hz, 1H), 4.14 (q,
J¼7.2 Hz, 2H), 2.85 (dd, J¼3.4, 13.4 Hz, 1H), 2.31 (t, J¼7.4 Hz, 2H),
2.05e1.89 (m, 3H), 1.83 (s, 3H), 1.77 (s, 3H), 1.66e1.13 (cluster of
signals, 30H), 0.95 (d, J¼6.0 Hz, 3H), 0.89 (d, J¼6.8 Hz, 3H), 0.88 (d,
give 12 as colorless oil (56.1 mg, 72%). [
a
]
26 þ10.4 (c 1.43, CHCl3); 1H
D
NMR (400 MHz, CDCl3)
d
6.94 (dt, J¼15.6, 7.0 Hz, 1H), 5.80 (d,
J¼6.4 Hz, 3H), 0.56 (s, 3H); 13C NMR (100 MHz, CDCl3)
d 174.2, 139.8,
J¼15.6 Hz, 1H), 4.18 (q, J¼7.2 Hz, 2H), 2.42 (dd, J¼11.8, 7.4 Hz, 1H),
2.35 (dd, J¼13.6, 4.8 Hz, 1H), 2.22e2.14 (m, 3H), 2.05e2.01 (m, 1H),
1.90e1.83 (m, 2H, including 1.88 (t, J¼12.6 Hz, 1H)), 1.77e1.66 (m,
1H), 1.59e1.02 (cluster of signals, 23H), 0.96 (d, J¼5.6 Hz, 3H), 0.87
(d, J¼6.8 Hz, 3H), 0.86 (d, J¼6.8 Hz, 3H), 0.64 (s, 3H); 13C NMR
132.8, 120.7, 116.7, 60.4, 56.8, 56.6, 48.0, 45.8, 39.8, 38.1, 36.4, 36.0,
35.0, 34.7, 30.1, 29.5, 29.4, 28.3, 28.3, 27.4, 26.6, 25.3, 24.1, 23.1, 22.8,
22.3, 19.2, 18.4, 14.5, 13.1; ESIMS m/z 509.0 ([MþNa]þ).
Solid LiOH$H2O (18 mg, 0.43 mmol) was added to a solution of
15 (38 mg, 0.078 mmol) in THF/H2O (v/v¼1/1, 2 mL). The reaction

Q. Zhang et al. / Tetrahedron 66 (2010) 4577e4586
4583
mixture was stirred at room temperature for 36 h, then poured into
H2O (20 mL) and washed with diethyl ether (5 mL). The aqueous
layer was concentrated to about 5 mL, the pH value was adjusted to
about four and the mixture was extracted with ethyl acetate
(30 mLꢂ3). The combined organic extracts were washed with brine
(1ꢂ10 mL), dried (MgSO4), filtered, and concentrated. The residue
(a colorless oil), p-nitrophenol (12.6 mg, 0.0906 mmol), DCC
(18.7 mg, 0.0906 mmol), and DMAP (1.8 mg, 0.0151 mmol) were
dissolved in dry CH2Cl2 (1 mL). The reaction mixture was stirred at
room temperature for 24 h, then diluted with diethyl ether (20 mL)
and filtered. The filtrate was washed by H2O until the washings
were no longer yellow in color, then with brine, dried (MgSO4), and
The residue was purified by column chromatography (50:1 n-
25
pentane/EtOAc) to give 18 as a colorless oil (52 mg, 78%). [a]
D
þ25.2 (c 0.83, CHCl3); 1H NMR (400 MHz, CDCl3)
8.29 (d, J¼9.2 Hz,
d
2H), 7.30 (d, J¼9.2 Hz, 2H), 6.18 (d, J¼10.8 Hz, 1H), 6.03 (d,
J¼11.2 Hz, 1H), 5.03 (s, 1H), 4.80 (s, 1H), 3.87e3.82 (m, 1H), 2.87 (dd,
J¼13.4, 3.8 Hz, 1H), 2.62 (t, J¼7.6 Hz, 2H), 2.48 (dd, J¼13.2, 3.6 Hz,
1H), 2.40 (dt, J¼13.6, 5.0 Hz, 1H), 2.26 (dd, J¼9.0, 12.6 Hz, 1H),
2.13e2.03 (m, 2H), 2.00e1.89 (m, 3H), 1.83e1.76 (m, 2H), 1.63e1.13
(cluster of signals, 25H), 1.06e1.02 (m, 1H), 0.95 (d, J¼6.0 Hz, 3H),
0.90 (s, 9H), 0.89 (d, J¼6.8 Hz, 6H), 0.56 (s, 3H), 0.093 (s, 3H), 0.086
(s, 3H); 13C NMR (100 MHz, CDCl3)
d 171.5, 155.8, 145.6, 145.5, 141.0,
136.7, 125.4, 122.7, 121.6, 118.1, 112.5, 70.8, 56.8, 56.6, 47.9, 47.1, 46.0,
39.8, 38.0, 36.6, 36.4, 36.2, 35.0, 34.6, 33.0, 30.6, 29.4, 29.3, 28.3,
27.4, 26.1, 25.0, 24.1, 23.1, 22.8, 22.3, 19.2, 18.4, 13.1, ꢁ4.3, ꢁ4.4;
ESIMS m/z 762.5 ([MþH]þ); ESIHRMS calcd for C47H76NO5Si
([MþH]þ) 762.5494, found 762.5255.
concentrated. The residue was purified by column chromatography
25
(50:1 n-pentane/EtOAc) to give 1 as colorless oil (35 mg, 78%). [a]
D
þ13.8 (c 1.05, CHCl3); 1H NMR (400 MHz, CDCl3)
8.29 (d, J¼8.8 Hz,
d
2H), 7.29 (d, J¼9.6 Hz, 2H), 6.14 (d, J¼10.8 Hz, 1H), 5.79 (d,
J¼11.2 Hz, 1H), 2.85 (dd, J¼3.8, 13.4 Hz, 1H), 2.62 (t, J¼7.6 Hz, 2H),
2.05e1.90 (m, 3H), 1.81 (s, 3H), 1.77 (s, 3H), 1.61e1.01 (cluster of
signals, 27H), 0.95 (d, J¼6.0 Hz, 3H), 0.88 (d, J¼6.8 Hz, 3H), 0.88 (d,
4.1.13. (S)-(Z)-[2-(5-Hydroxy-2-methylene-cyclohexylidene)ethyl]di-
phenylphosphine oxide (19). Bu4NF in THF (0.33 mL, 0.33 mmol,
1 M) was added to a stirred solution of 1623 (100 mg, 0.22 mmol) in
THF (1.1 mL). After being stirred at room temperature for 5 h, the
reaction mixture was diluted with CH2Cl2, washed with H2O and
brine, dried (MgSO4), and concentrated. The residue was purified
J¼6.4 Hz, 3H), 0.56 (s, 3H); 13C NMR (100 MHz, CDCl3)
d 171.6, 155.7,
145.5, 139.7, 132.8, 125.5, 122.7, 120.7, 116.8, 56.8, 56.6, 48.0, 45.8,
39.8, 38.1, 36.4, 36.4, 36.0, 35.0, 34.6, 30.1, 29.5, 29.3, 28.3, 28.3, 27.4,
26.7, 25.0, 24.1, 23.1, 22.9, 22.3, 19.2, 18.4, 13.1; ESIMS m/z 601.8
([MþNa]þ); ESIHRMS calcd for C37H56NO4 ([MꢁH2þH]þ) 578.4211,
found 578.4209.
by column chromatography (20:1 CH2Cl2/MeOH) to give 19 as
23
a white wax (72 mg, 97%). [
a
]
þ9.1 (c 1.61, CHCl3); 1H NMR
D
(400 MHz, CDCl3)
d
7.72e7.67 (m, 4H), 7.52e7.42 (m, 6H), 5.35 (q,
4.1.11. 3
b
-(tert-Butyldimethylsilyloxy)-11
a
-(7-ethoxycarbonylhept-
J¼7.2 Hz, 1H), 4.93 (s, 1H), 4.70 (s, 1H), 3.72e3.66 (m, 1H), 2.73 (br s,
1H), 3.29 (dd, J¼7.6, 14.0 Hz, 2H), 2.46 (br d, J¼12.8 Hz, 1H),
2.23e2.15 (m, 2H), 1.82e1.75 (m, 2H), 1.61e1.54 (m, 1H); 13C NMR
1-yl)-vitamin D3 (17). A solution of 1623 (455 mg, 1.01 mmol) in dry
THF (1 mL) was added slowly to a solution of n-BuLi (0.63 mL,
1.01 mmol, 1.6 M) in dry THF (1 mL) at ꢁ78 ꢀC under N2. After
stirring at ꢁ78 ꢀC for 40 min, a solution of compound 13 (110 mg,
0.25 mmol) in dry THF (1 mL) was added dropwise. The reaction
mixture was stirred at ꢁ78 ꢀC for another 2 h, and then warmed to
ꢁ50 ꢀC during 3 h. Saturated aq NH4Cl (3 mL) was added to quench
the reaction. The mixture was diluted with diethyl ether (25 mL),
washed with saturated aq NH4Cl and brine, dried (MgSO4), and
concentrated. The residue was purified by column chromatography
(100 MHz, CDCl3)
d
145.5 (d, J¼3.0 Hz, P split), 142.7 (d, J¼12.1 Hz, P
split), 133.3 (d, J¼40.2 Hz, P split), 132.3 (d, J¼40.9 Hz, P split), 132.1
(d, J¼2.3 Hz, P split), 132.1 (d, J¼3.0 Hz, P split), 131.3 (d, J¼9.1 Hz, P
split), 131.1 (d, J¼9.1 Hz, P split), 128.9 (d, J¼6.8 Hz), 128.8 (d,
J¼6.1 Hz, P split), 114.0 (d, J¼9.1 Hz, P split), 111.8, 69.1 (d, J¼2.2 Hz,
P split), 46.2 (d, J¼2.3 Hz, P split), 35.3, 32.2, 31.2 (d, J¼69.1 Hz, P
split); ESIMS m/z 339.1 ([MþH]þ); ESIHRMS calcd for C21H24O2P
([MþH]þ) 339.1515, found 339.1417.
(50:1 n-pentane/EtOAc) to give 17 as colorless oil (138 mg, 82%).
25
[
a]
þ31.2 (c 0.64, CHCl3); 1H NMR (400 MHz, CDCl3)
d
6.18 (d,
4.1.14. 11a-(7-Ethoxycarbonylhept-1-yl)-vitamin D3 (20). A solution
D
J¼10.8 Hz, 1H), 6.02 (d, J¼11.2 Hz, 1H), 5.03 (s, 1H), 4.80 (s, 1H), 4.15
(q, J¼7.2 Hz, 2H), 3.87e3.82 (m, 1H), 2.87 (dd, J¼13.4, 3.8 Hz, 1H),
2.48 (dd, J¼13.0, 3.8 Hz, 1H), 2.39 (dt, J¼13.2, 4.8 Hz, 1H), 2.34e2.26
(m, 3H), 2.14e1.91 (m, 5H), 1.67e1.01 (cluster of signals, 31H, in-
cluding 1.28 (t, J¼7.2 Hz, 3H)), 0.95 (d, J¼6.4 Hz, 3H), 0.91 (s, 9H),
0.89 (d, J¼6.4 Hz, 6H), 0.56 (s, 3H), 0.10 (s, 3H), 0.09 (s, 3H); 13C NMR
of 19 (60 mg, 0.177 mmol) in dry THF (1.2 mL) was added slowly to
a solution of n-BuLi (0.23 mL, 0.363 mmol, 1.6 M) in dry THF (1 mL)
at ꢁ78 ꢀC under N2. After stirring at ꢁ78 ꢀC for 1 h, a solution of
compound 13 (40 mg, 0.092 mmol) in dry THF (1 mL) was added
dropwise. The reaction mixture was stirred at ꢁ78 ꢀC for another
2 h, then saturated aq NH4Cl (2 mL) was added to quench the re-
action. The mixture was diluted with diethyl ether (25 mL), washed
with saturated aq NH4Cl and brine, dried (MgSO4), and concen-
trated. The residue was purified by column chromatography (5:1 n-
(100 MHz, CDCl3) d 174.2, 145.7, 141.1, 136.6, 121.6, 118.1, 112.4, 70.8,
60.4, 56.8, 56.6, 47.9, 47.1, 46.0, 39.8, 38.1, 36.6, 36.4, 36.2, 35.0, 34.6,
33.0, 30.1, 29.5, 29.42 28.3, 27.4, 26.1, 25.2, 24.1, 23.1, 22.8, 22.3, 19.2,
18.4, 14.5, 13.1, ꢁ4.3, ꢁ4.4; ESIMS m/z 669.5 ([MþH]þ); ESIHRMS
calcd for C43H77O3Si ([MþH]þ) 669.5643, found 669.5412.
pentane/EtOAc) to give 20 as colorless oil (38 mg, 75%). [
a
]
26 þ9.7 (c
D
0.62, CH2Cl2); 1H NMR (400 MHz, CDCl3)
d
6.27 (d, J¼10.8 Hz, 1H),
6.04 (d, J¼11.6 Hz, 1H), 5.07 (s, 1H), 4.84 (s, 1H), 4.15 (q, J¼7.1 Hz,
2H), 3.98e3.95 (m, 1H), 2.88 (dd, J¼13.4, 4.2 Hz, 1H), 2.61 (dd,
J¼13.2, 4.0 Hz, 1H), 2.44e2.39 (m, 1H), 2.34e2.30 (m, 3H, including
2.32 (t, J¼7.4 Hz, 2H)), 2.23e2.18 (m, 1H), 2.04 (dd, J¼3.8, 13.0 Hz,
1H), 2.00e1.89 (m, 3H), 1.73e1.13 (cluster of signals, 31H, including
1.28 (t, J¼7.2 Hz, 3H)),1.04e0.99 (m,1H), 0.95 (d, J¼6.4 Hz, 3H), 0.89
(d, J¼6.8 Hz, 3H), 0.88 (d, J¼6.4 Hz, 3H), 0.56 (s, 3H); 13C NMR
4.1.12. 3b-(tert-Butyldimethylsilyloxy)-11a-[7-(4-nitrophenyloxy)
carbonylhept-1-yl]-vitamin D3 (18). Solid LiOH$H2O (37 mg,
0.90 mmol) was added to a solution of 17 (60 mg, 0.090 mmol) in
THF/H2O (v/v¼1/1, 2 mL). The reaction mixture was stirred at room
temperature and monitored by TLC. When the reaction was com-
plete (72 h), the pH value was adjusted to about five with diluted aq
HCl (0.1 M). The mixture was extracted with diethyl ether
(3ꢂ40 mL). The combined organic extracts were washed with brine
(1ꢂ10 mL), dried (MgSO4), filtered, and concentrated. The residue
(a colorless oil), p-nitrophenol (15.3 mg, 0.11 mmol), DCC (22.7 mg,
0.11 mmol), and DMAP (2.2 mg, 0.018 mmol) were dissolved in dry
CH2Cl2 (1 mL). The reaction mixture was stirred at room tempera-
ture overnight, then diluted with diethyl ether (20 mL) and filtered.
The filtrate was washed by H2O until the washings were no longer
yellow in color, then with brine, dried (MgSO4), and concentrated.
(100 MHz, CDCl3) d 174.2, 145.4, 141.9, 135.3, 122.6, 117.7, 112.6, 69.4,
60.4, 56.9, 56.6, 48.0, 46.2, 46.1, 39.8, 38.0, 36.4, 36.2, 35.4, 35.1,
34.6, 32.2, 30.0, 29.5, 29.4, 28.3, 28.2, 27.4, 25.2, 24.1, 23.1, 22.8, 22.3,
19.2, 14.5, 13.0; ESIMS m/z 555.5 ([MþH]þ); ESIHRMS calcd for
C37H63O3 ([MþH]þ) 555.4778, found 555.4787.
4.1.15. 11
a-[7-(4-Nitrophenyloxy)carbonylhept-1-yl]-vitamin
D3
(2). Solid LiOH$H2O (16 mg, 0.38 mmol) was added to a solution of
20 (20 mg, 0.036 mmol) in THF/H2O (v/v¼1/1, 1 mL). The reaction

4584
Q. Zhang et al. / Tetrahedron 66 (2010) 4577e4586
mixture was stirred at room temperature and monitored by TLC.
When the reaction was complete (36 h), the pH value was adjusted
to about five with diluted aq HCl (0.1 M). The mixture was extracted
with ethyl acetate (3ꢂ10 mL), and the combined organic extracts
were washed with brine (1ꢂ5 mL), dried (MgSO4), filtered, and
concentrated. The residue (a colorless oil), p-nitrophenol (10 mg,
0.072 mmol), and DCC (15 mg, 0.072 mmol) were dissolved in dry
CH2Cl2 (1 mL). The reaction mixture was stirred at room tempera-
ture overnight, then diluted with diethyl ether (20 mL) and filtered.
The filtrate was washed with H2O until the washings were no
longer yellow in color, then with brine, dried (MgSO4), and con-
concentrated. The residual oil was dissolved in dry CH2Cl2 (2 mL)
and (ethoxycarbonylmethylidene)-triphenylphosphorane (160 mg,
0.46 mmol) was added. The resulting mixture was stirred at room
temperature overnight, and then concentrated and the residue was
purified by column chromatography (1:1 n-pentane/EtOAc) to give
25 as colorless oil (86 mg, 83%). [
(400 MHz, CDCl3)
a
]
24 þ14.0 (c 1.96, CH2Cl2); 1H NMR
D
d
6.94 (dt, J¼15.6, 7.0 Hz, 1H), 5.80 (d, J¼15.6 Hz,
1H), 4.17 (q, J¼7.2 Hz, 2H), 2.42 (dd, J¼11.6, 7.2 Hz, 1H), 2.34 (dd,
J¼13.4, 5.0 Hz, 1H), 2.21e2.13 (m, 3H), 2.08e2.00 (m, 1H), 1.94e1.83
(m, 2H, including 1.86 (t, J¼12.8 Hz, 1H)), 1.75e1.65 (m, 1H),
1.45e1.23 (cluster of signals, 22H, including 1.27 (t, J¼7.2 Hz, 3H)),
1.21 (s, 6H), 1.10e1.04 (m, 1H), 0.97 (d, J¼5.6 Hz, 3H), 0.63 (s, 3H);
centrated. The residue was purified by column chromatography
25
(5:1 n-pentane/EtOAc) to give 2 as colorless oil (16.5 mg, 71%). [
a
]
13C NMR (100 MHz, CDCl3)
d 211.6, 166.9, 149.4, 121.6, 71.2, 62.2,
D
ꢁ2.9 (c 1.09, CHCl3); 1H NMR (400 MHz, CDCl3)
d
8.29 (d, J¼9.2 Hz,
60.4, 56.8, 49.5, 48.0, 46.7, 44.6, 37.6, 36.9, 36.5, 35.7, 32.3, 29.7,
29.5, 28.2, 28.0, 27.1, 21.0, 19.2, 19.1, 14.5, 13.5; ESIMS m/z 471.2
([MþNa]þ); ESIHRMS calcd for C28H49O4 ([MþH]þ) 449.3632,
found 449.3576.
2H), 7.30 (d, J¼9.2 Hz, 2H), 6.26 (d, J¼11.2 Hz, 1H), 6.03 (d,
J¼11.2 Hz, 1H), 5.07 (s, 1H), 4.84 (s, 1H), 3.98e3.96 (m, 1H), 2.89 (dd,
J¼13.2, 4.0 Hz, 1H), 2.65e2.58 (m, 3H, including 2.63 (t, J¼7.4 Hz,
2H)), 2.46e2.39 (m, 1H), 2.31 (dd, J¼7.4, 13.0 Hz, 1H), 2.20 (ddd,
J¼5.0, 8.2, 13.4 Hz, 1H), 2.04 (dd, J¼3.4, 12.6 Hz, 1H), 2.00e1.89 (m,
3H), 1.83e1.13 (cluster of signals, 25H), 1.06e0.98 (m, 1H), 0.95 (d,
J¼6.4 Hz, 3H), 0.89 (d, J¼6.4 Hz, 3H), 0.88 (d, J¼6.8 Hz, 6H), 0.56 (s,
4.1.19. 11a-(7-Ethoxycarbonylhept-1-yl)-25-hydroxy-de-A,B-cholest-
8-one (26). To a solution of compound 25 (50 mg, 0.11 mmol) in
MeOH (1 mL) was added Pd/C (5 mg, 10 wt %). The resulting sus-
pension was stirred for 2 h under H2 atmosphere at room temper-
ature, then the suspension was filtered through a Celite pad and the
filtrate was concentrated. The residue was purified by column
3H); 13C NMR (100 MHz, CDCl3)
d 171.6, 155.8, 145.5, 145.3, 141.8,
135.4,125.4, 122.6, 122.6, 117.7,112.7, 69.4, 56.9, 56.7, 47.9, 46.2, 46.1,
39.8, 38.0, 36.4, 36.2, 35.4, 35.1, 34.6, 32.2, 30.0, 29.5, 29.3, 28.3,
28.2, 27.4, 25.0, 24.1, 23.1, 22.8, 22.3, 19.2, 13.1; ESIMS m/z 648.5
([MþH]þ); ESIHRMS calcd for C41H62NO5 ([MþH]þ) 648.4629,
found 648.4643.
chromatography (1.5:1 n-pentane/EtOAc) to give 26 as colorless oil
25
(50 mg, 100%). [
a
]
þ12.3 (c 1.34, CH2Cl2); 1H NMR (400 MHz,
D
CDCl3)
d
4.11 (q, J¼7.1 Hz, 2H), 2.42 (dd, J¼11.8, 7.4 Hz, 1H), 2.34 (dd,
J¼4.8,13.6 Hz,1H), 2.27 (t, J¼7.6 Hz, 2H), 2.15 (dd, J¼4.0,12.8 Hz,1H),
2.08e1.98 (m, 1H), 1.94e1.83 (m, 2H, including 1.86 (t, J¼12.8 Hz,
1H)), 1.76e1.66 (m, 1H), 1.64e1.57 (m, 2H), 1.45e1.22 (cluster of
signals, 24H, including 1.24 (t, J¼7.2 Hz, 3H)), 1.20 (s, 6H), 1.09e1.02
(m, 1H), 0.96 (d, J¼5.2 Hz, 3H), 0.62 (s, 3H); 13C NMR (100 MHz,
4.1.16. 25-Triethylsilyloxy-de-A, B-cholest-9(11)-en-8-one (23). This
compound was prepared from compound 418 essentially as pre-
viously described.13,14,28e31
4.1.17. 11
a-[(6,6-Ethylenedioxy)hex-1-yl]-25-triethylsilyloxy-de-A, B-
CDCl3) d 211.7, 174.0, 71.2, 62.2, 60.4, 56.8, 49.5, 48.0, 46.7, 44.6, 37.8,
cholest-8-one (24). A solution of bromide 10 (350 mg, 1.50 mmol) in
dry diethyl ether (3.2 mL) was added slowly to a cooled (-85 ꢀC) t-
BuLi solution (1.8 mL, 3.0 mmol, 1.7 M in heptane) under N2 during
1 h. The mixture was stirred for 30 min. Separately and simulta-
neously, n-Bu3P (0.19 mL, 0.75 mmol) was added to a suspension of
CuI (143 mg, 0.75 mmol) in dry diethyl ether (4.3 mL). At ꢁ85 ꢀC, the
solution of CuI/n-Bu3P complex was transferred by cannula to the
initial reaction mixture. The resulting solution was warmed to
ꢁ50 ꢀC during 1 h, then again cooled to ꢁ78 ꢀC and a solution of 23
(147 mg, 0.38 mmol) in dry diethyl ether (5.3 mL) was added drop-
wise. After stirred for 2 h at ꢁ78 ꢀC, the reaction was quenched by
addition of methanol (0.5 mL), and was then diluted with diethyl
ether (20 mL), washed with, successively, saturated aq NH4Cl, water
and brine, and dried (MgSO4), and evaporated. The residue was
purified by column chromatography (3:1 n-pentane/diethyl ether) to
36.9, 36.5, 35.7, 34.6, 29.8, 29.7, 29.5, 29.4, 29.3, 28.0, 27.3, 25.2, 21.0,
19.2, 19.1, 14.5, 13.5; ESIMS m/z 473.4 ([MþNa]þ); ESIHRMS calcd for
C28H51O4 ([MþH]þ) 451.3789, found 451.3740.
4.1.20. 11a-(7-Ethoxycarbonylhept-1-yl)-25- triethylsilyloxy-de-A,B-
cholest-8-one (27). TESCl (0.03 mL, 0.18 mmol) was added to a so-
lution of compound 26 (53 mg, 0.12 mmol) and imidazole (24.5 mg,
0.36 mmol) in dry DMF (1 mL) under N2 atmosphere. The reaction
solution was stirred at room temperature for 6 h. The resulting
mixture was quenched by 1 mL H2O, diluted by 50 mL diethyl ether
and washed by water (1 mLꢂ4), brine (1 mLꢂ1) and dried over
MgSO4. After concentrated in vacuum, the residue was purified by
column chromatography (10:1 n-pentane/EtOAc) to give 27 as
25
colorless oil (64 mg, 97%). [
a
]
D
þ8.6 (c 0.95, CH2Cl2); 1H NMR
(400 MHz, CDCl3)
d
4.12 (q, J¼7.2 Hz, 2H), 2.41 (dd, J¼11.6, 7.2 Hz,
give 24 as colorless oil (146 mg, 73%). [
a
]
24 þ9.5 (c 2.08, CH2Cl2); 1H
1H), 2.33 (dd, J¼5.0, 13.4 Hz, 1H), 2.26 (t, J¼7.4 Hz, 2H), 2.14 (dd,
J¼3.8, 13.0 Hz, 1H), 2.04e1.98 (m, 1H), 1.94e1.81 (m, 2H, including
1.84 (t, J¼12.8 Hz, 1H)), 1.74e1.20 (cluster of signals, 26H, including
1.22 (t, J¼7.0 Hz, 3H)), 1.18 (s, 6H), 1.06e1.99 (m, 1H), 0.97e0.93 (m,
12H, including 0.95 (t, J¼8.0 Hz, 9H)), 0.61 (s, 3H), 0.57 (q, J¼8.0 Hz,
D
NMR (500 MHz, CDCl3)
d
4.83 (t, J¼4.8 Hz, 1H), 3.96 (m, 2H), 3.84 (m,
2H), 2.43 (dd, J¼7.4,11.6 Hz,1H, H-14), 2.36 (dd, J¼4.6,13.4 Hz,1H, H-
9a), 2.15 (dd, J¼3.8,12.9 Hz, 1H), 2.03 (m,1H, H-11), 1.90 (m, 1H),1.89
(ddd, 0.9, 12.3, 13.3 Hz, 1H, H-9b), 1.70 (m, 1H), 1.64 (m, 2H),
1.53e1.21 (cluster of signals, 19H), 1.18 (s, 6H), 0.94 (m, 12H), 0.63 (s,
6H); 13C NMR (100 MHz, CDCl3)
d 211.8, 174.1, 73.6, 62.2, 60.4, 56.9,
3H, H-18), 0.56 (q, J¼7.8 Hz, 6H); 13C NMR (100 MHz, CDCl3)
d
211.7,
49.5, 48.0, 46.7, 45.7, 37.8, 37.0, 36.6, 35.7, 34.6, 30.3, 30.0, 29.8, 29.4,
29.3, 28.0, 27.3, 25.2, 21.0, 19.2, 19.1, 14.5, 13.5, 7.4, 7.1; ESIMS m/z
587.4 ([MþNa]þ); ESIHRMS calcd for C34H65O4Si ([MþH]þ)
565.4653, found 565.4581.
104.8, 73.6, 65.1, 62.2, 56.9, 49.5, 48.0, 46.7, 45.7, 37.7, 37.0, 36.6, 35.7,
34.1, 30.3, 30.1, 29.9, 28.0, 27.2, 24.2, 21.0, 19.2, 19.1, 13.5, 7.4, 7.1;
ESIMS m/z 559.4 ([MþNa]þ); ESIHRMS calcd for C32H61O4Si
([MþH]þ) 537.4340, found 537.4277.
4.1.21. (S)-(Z)-[2-(5-Triethylsilyloxy-2-methylene-cyclohexylidene)
ethyl]diphenylphosphine oxide (28). TESCl (0.05 mL, 0.30 mmol)
was added to a stirred solution of 19 (67 mg, 0.20 mmol) and im-
idazole (41 mg, 0.60 mmol) in dry DMF (1 mL). After being stirred at
room temperature overnight, the reaction mixture was concen-
trated in vacuum and the residue was purified by column chro-
matography (1:2 n-pentane/EtOAc) to give 28 as colorless oil
4.1.18. 11a-(7-Ethoxycarbonylhept-5-en-1-yl)-25-hydroxy-de-A,B-
cholest-8-one (25). Compound 24 (125 mg, 0.23 mmol) was dis-
solved in acetone/H2O (v/v 9:1, 3 mL). After added PPTs (12 mg,
0.047 mmol), the reaction mixture was heated to reflux for 17 h,
then cooled down, diluted with diethyl ether, washed in turn with
saturated aq NaHCO3, water and brine, dried (MgSO4), and

Q. Zhang et al. / Tetrahedron 66 (2010) 4577e4586
4585
24
(62 mg, 69%). [
CDCl3)
a]
þ44.9 (c 2.47, CH2Cl2); 1H NMR (400 MHz,
The reaction mixture was stirred at room temperature and moni-
tored by TLC. When the reaction was complete (44 h), the pH value
was adjusted to about two with diluted aq HCl (0.5 M). The mixture
was extracted with ethyl acetate (4ꢂ20 mL), and the combined
organic extracts were washed with brine (1ꢂ5 mL), dried (MgSO4),
filtered, and concentrated. The residue (a colorless oil), p-nitro-
phenol (9 mg, 0.065 mmol), DCC (8 mg, 0.039 mmol), and DMAP
(0.16 mg, 0.0013 mmol) were dissolved in dry CH2Cl2 (1 mL). The
reaction mixture was stirred at room temperature for 2.5 h, then
diluted with diethyl ether (2 mL) and filtered. The filtrate was
washed by H2O until the washings were no longer yellow in color,
then with brine, dried (MgSO4), and concentrated. The residue was
purified by column chromatography (2:1 n-pentane/EtOAc) to give
D
d
7.76e7.70 (m, 4H), 7.55e7.45 (m, 6H), 5.38 (q, J¼7.3 Hz,
1H), 4.94 (s, 1H), 4.71 (s, 1H), 3.72e3.49 (m, 1H), 3.43e3.34 (m, 1H),
3.22 (ddt, J¼6.4, 2.0, 16.8 Hz,1H), 2.39 (br d, J¼12.4 Hz,1H), 2.24 (dt,
J¼4.4, 13.2 Hz, 1H), 2.16e2.10 (m, 1H), 1.82e1.78 (m, 1H), 1.70e1.63
(m, 1H), 1.54e1.44 (m, 1H), 0.93 (t, J¼8.0 Hz, 9H), 0.56 (q, J¼8.0 Hz,
6H); 13C NMR (100 MHz, CDCl3)
d
145.2 (d, J¼2.3 Hz, P split), 142.9
(d, J¼12.1 Hz, P split), 133.5 (d, J¼62.2 Hz, P split), 132.6 (d,
J¼62.9 Hz, P split), 132.0 (d, J¼3.0 Hz, P split), 131.9 (d, J¼2.3 Hz, P
split), 131.5 (d, J¼9.1 Hz, P split), 131.2 (d, J¼9.1 Hz, P split), 128.8 (d,
J¼11.4 Hz, P split), 128.6 (d, J¼11.3 Hz, P split), 113.5 (d, J¼8.4 Hz, P
split), 111.9, 70.6 (d, J¼2.3 Hz, P split), 47.1 (d, J¼2.2 Hz, P split), 36.6,
32.8, 31.4 (d, J¼69.8 Hz,
P split), 7.1, 5.1; ESIMS m/z 475.2
([MþNa]þ); ESIHRMS calcd for C27H38O2PSi ([MþH]þ) 453.2380,
3 as colorless oil (12.6 mg, 76%). [
a
]
23 ꢁ5.0 (c 0.42, CH2Cl2); 1H NMR
D
found 453.2308.
(400 MHz, CDCl3)
d
8.29 (d, J¼8.8 Hz, 2H), 7.30 (d, J¼9.6 Hz, 2H),
6.26 (d, J¼10.8 Hz, 1H), 6.05 (d, J¼11.6 Hz, 1H), 5.07 (s, 1H), 4.84 (s,
1H), 3.99e3.94 (m, 1H), 2.89 (dd, J¼13.4, 3.4 Hz, 1H), 2.65e2.58 (m,
3H, including 2.63 (t, J¼7.4 Hz, 2H)), 2.44e2.39 (m, 1H), 2.31 (dd,
J¼7.4, 12.6 Hz, 1H), 2.23e2.16 (m, 1H), 2.06e1.06 (cluster of signals,
39H, including 1.24 (s, 6H)), 0.97 (d, J¼6.4 Hz, 3H), 0.56 (s, 3H); 13C
4.1.22. 3b-Triethylsilyloxy-11a-(7-ethoxycarbonylhept-1-yl)-25-trie-
thylsilyloxy-vitamin D3 (29). A solution of 28 (61.8 mg, 0.14 mmol)
in dry THF (1 mL) was added to a solution of n-BuLi (0.09 mL,
0.14 mmol, 1.6 M) in dry THF (1 mL) at ꢁ78 ꢀC under N2 atmo-
sphere. After stirring at ꢁ78 ꢀC for 40 min, a solution of compound
27 (20 mg, 0.035 mmol) in dry THF (1 mL) was added dropwise. The
reaction mixture was stirring at ꢁ78 ꢀC for another 2 h. MeOH
(0.2 mL) was added to quench the reaction. The mixture was di-
luted with diethyl ether (25 mL), washed with saturated aq NH4Cl
and brine and dried (MgSO4), and concentrated. The residue was
NMR (100 MHz, CDCl3) d 171.5,155.8,147.6,145.3,141.7,135.5,125.4,
122.6, 122.5, 117.8, 112.7, 71.3, 69.4, 56.9, 56.6, 48.0, 46.2, 46.1, 44.7,
38.0, 36.7, 36.4, 36.2, 35.4, 35.1, 34.6, 32.2, 30.0, 29.6, 29.5, 29.5,
29.3, 28.2, 27.4, 25.0, 22.3, 21.1, 19.2, 13.1; ESIMS m/z 686.4
([MþNa]þ); ESIHRMS calcd for C41H62NO6 ([MþH]þ) 664.4578,
found 664.4502.
purified by column chromatography (50:1 n-pentane/EtOAc) to
25
give 29 as colorless oil (23.5 mg, 84%). [
a]
þ14.7 (c 1.18, CH2Cl2);
4.2. Peptide synthesis, purification, and conjugation
D
1H NMR (400 MHz, CDCl3)
d
6.19 (d, J¼11.2 Hz, 1H), 6.03 (d,
J¼11.2 Hz, 1H), 5.03 (s, 1H), 4.80 (s, 1H), 4.15 (q, J¼7.2 Hz, 2H),
3.86e3.81 (m, 1H), 2.87 (dd, J¼13.6, 3.6 Hz, 1H), 2.50 (dd, J¼12.8,
3.6 Hz, 1H), 2.39 (dt, J¼14.0, 4.4 Hz, 1H), 2.33e2.25 (m, 3H, in-
cluding 2.31 (t, J¼7.6 Hz, 2H)), 2.14e1.92 (m, 5H), 1.67e1.26 (m,
30H) (including 1.28 (t, J¼7.2 Hz, 3H)), 1.21 (s, 6H), 0.99 (t, J¼8.2 Hz,
9H), 0.97 (t, J¼8.2 Hz, 9H), 0.95 (d, J¼3.2 Hz, 3H), 0.63 (q, J¼8.0 Hz,
6H), 0.58 (q, J¼8.0 Hz, 6H), 0.57 (s, 3H); 13C NMR (100 MHz, CDCl3)
The peptide 4-D15L8 was synthesized on a Pioneer automated
peptide solid-phase synthesiser (Applied Biosystems) using stan-
dard 9-fluorenylmethoxycarbonyl (Fmoc) chemistry. The synthesis
was performed on a 0.2-mmol scale on an Fmoc-Gly-polyethylene
glycol-polystyrene resin (Applied Biosystems) with a substitution
degree of 0.18 mmol/g. The side-chains of the amino acids (Cal-
biochemeNovabiochem AG) were protected by piperidine-stable
groups: tert-butyl (Asp, Glu), tert-butoxycarbonyl (Lys8), allylox-
ycarbonyl (Lys15), N-trifluoroacetyl (Lys41), S-acetamidomethyl
(Cys), trityl (His, Asn, Gln) and 2,2,4,6,7-pentamethyldihy-
drobenzofuran-5-sulfonyl (Arg). The allyloxycarbonyl group pro-
tection of Lys15 allowed for pre-cleavage incorporation of a dansyl
group, and the N-trifluoroacetamidophenyl protection of Lys41 and
acetamidomethyl protection of Cys24 were selected to allow for
future post-cleavage conjugations. The Fmoc protecting groups
were removed from the amino termini by treatment with 20% pi-
peridine in DMF. A fourfold excess of amino acid was used in each
coupling and amino acids were activated with a mixture of TBTU
(0.5 M in DMF) and DIPEA (1 M in DMF). A standard amino acid
coupling time of 60 min was used, except in the cases of Gln and His
(90 min) and Asn and Arg (120 min). The N terminus of the peptide
was capped with 0.3 M acetic anhydride in DMF. After completed
synthesis, the resin was rinsed with dichloromethane and dried in
vacuum.
Before cleaving the peptide from the resin, Lys15 was depro-
tected over 3 h at room temperature under nitrogen by using [Pd
(PPh3)4] (3 equiv) in a mixture of trichloromethane, acetic acid,
and N-methylmorpholine (17:2:1 v/v; 12 mL per g of polymer).
The resin was washed sequentially (ꢂ3) with 20 mM dieth-
yldithiocarbamic acid in DMF, 30 mM DIPEA in DMF, then with
DMF and dichloromethane, and finally desiccated. Coupling of
dansyl chloride (4 equiv) to the liberated lysine residue was
performed in DMF with gentle stirring at room temperature for
3 h in the presence of DIPEA (8 equiv). The resin was washed with
DMF and dichloromethane and then desiccated. The crude pep-
tide was cleaved from the resin by treatment with a mixture of
TFA, triisopropylsilane, and water (95:2.5:2.5 v/v; 15 mL per g of
d
174.1, 145.6, 141.1, 136.6, 121.6, 118.1, 112.5, 73.7, 70.7, 60.4, 56.9,
56.7, 47.9, 47.2, 46.0, 45.8, 38.1, 36.8, 36.7, 36.4, 36.3, 35.0, 34.7, 33.1,
30.3, 30.1, 30.1, 29.5, 29.4, 28.3, 27.4, 25.3, 22.3, 21.1, 19.2, 14.5, 13.1,
7.4, 7.1, 7.1, 5.2; ESIMS m/z 821.6 ([MþNa]þ); ESIHRMS calcd for
C49H91O4Si2 ([MþH]þ) 799.6457, found 799.6360.
4.1.23. 11a-(7-Ethoxycarbonylhept-1-yl)-25-hydroxy-vitamin
D3
(30). PPTs (2.3 mg, 0.0092 mmol) was added to a solution of
compound 29 (20.5 mg, 0.0257 mmol) in acetone/H2O (v/v 9:1,
3 mL). The reaction mixture was refluxed for 3 h, then cooled down,
diluted with ethyl acetate, washed in turn with saturated aq
NaHCO3, water and brine, dried (MgSO4), and concentrated. The
residue was purified by column chromatography (2:1 n-pentane/
EtOAc) to give 30 as colorless oil (14.5 mg, 100%). [
a
]
24 ꢁ3.4 (c 0.71,
D
CH2Cl2); 1H NMR (400 MHz, CDCl3)
d
6.24 (d, J¼11.2 Hz,1H), 6.02 (d,
J¼10.8 Hz, 1H), 5.04 (s, 1H), 4.81 (s, 1H), 4.12 (q, J¼6.9 Hz, 2H),
3.96e3.93 (m, 1H), 2.86 (dd, J¼13.6, 4.0 Hz, 1H), 2.76 (dd, J¼12.8,
3.6 Hz, 1H), 2.43e2.37 (m, 1H), 2.31e2.27 (m, 3H, including 2.29 (t,
J¼7.6 Hz, 2H)), 2.17 (ddd, J¼4.6, 8.6, 13.4 Hz, 1H), 2.07e1.90 (m, 6H),
1.69e1.19 (cluster of signals, 36H, including 1.25 (t, J¼7.2 Hz, 3H),
and 1.21 (s, 6H)), 0.96 (d, J¼6.4 Hz, 3H), 0.54 (s, 3H); 13C NMR
(100 MHz, CDCl3) d 174.1, 145.4, 141.7, 135.4, 122.6, 117.7, 112.6, 71.4,
69.5, 60.4, 56.9, 56.6, 48.0, 46.2, 46.1, 44.7, 38.0, 36.7, 36.4, 36.2,
35.4, 35.1, 34.7, 32.2, 30.0, 29.5, 29.5, 29.4, 28.2, 27.4, 25.2, 22.3, 21.1,
19.2, 14.5, 13.1; ESIMS m/z 593.4 ([MþNa]þ); ESIHRMS calcd for
C37H63O4 ([MþH]þ) 571.4760, found 571.4669.
4.1.24. 11a-[7-(4-Nitrophenyloxy)carbonylhept-1-yl]-25-hydroxy-vi-
tamin D3 (3). Solid LiOH$H2O (12.6 mg, 0.3 mmol) was added to
a solution of 30 (14.7 mg, 0.026 mmol) in THF/H2O (v/v 1:1, 0.5 mL).

4586
Q. Zhang et al. / Tetrahedron 66 (2010) 4577e4586
polymer) for 2 h at room temperature. After filtration, TFA was
evaporated and the peptide was precipitated by addition of cold
diethyl ether, centrifuged, washed in diethyl ether and
lyophilized.
Supplementary data
Supplementary data associated with this article can be found in
the online version at doi:10.1016/j.tet.2010.04.051.
The crude product was purified by reversed-phase HPLC on
a semipreparative Hypersil C-18 Gold column (150ꢂ20 mm, pore
ꢀ
ꢀ
size 175 A, particle size 5 A), eluted with a shallow 35e55% aceto-
nitrile gradient in water and 0.1% TFA as additive at a flow rate of
10 mL/min. The purified peptide was identified by MALDI-TOF mass
spectrometry, concentrated and lyophilized.
References and notes
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The ligand was conjugated to the peptide by adding a solution of
the active ester 2 (2 equiv) in DMSO (40 mM), to a solution of the
peptide 4-D15L8 (0.568 mmol) in DMSO (2 mM) in the presence of
3. Zhang, J.; Sokal, I.; Peskind, E. R.; Quinn, J. F.; Jankovic, J.; Kenney, C.; Chung, K.
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DIPEA (30 equiv). The reaction was monitored by MALDI-TOF mass
spectrometry. After one day at room temperature, the reaction
mixture was purified by reversed-phase HPLC (conditions as
above). Elution with a 46e81% acetonitrile gradient gave a pool of
pure fractions, as indicated by the peak cluster centered around m/
z¼5789 in the MALDI-TOF mass spectrum (the calculated MþH
value is 5786 for C261H424N77O64S2F3), which was partially con-
centrated and then lyoplilized. The yield of conjugation was 38% (by
weight).
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€
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4.3. Structural analysis by circular dichroism spectroscopy
Circular dichroism spectra were recorded on a JASCO J-810
spectrometer by using 10-mm cuvettes and measuring in the in-
terval 190e260 nm at room temperature. Spectra of 10 mM peptide
in water were obtained by collecting data at 0.5 nm intervals with
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Qobs
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[
Q]222, and was calculated from Eq. 1, where
is the observed
222
ellipticity at 222 nm (deg), mrw is the mean residue weight (g/mol),
c is the peptide concentration (g/mL) and l is the optical path length
of the cell (cm).
Qo2b2s2mrw
½
Q 222
ꢃ
¼
(1)
10lc
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This research was supported by a grant from the Swedish Re-
search Council and by the Uppsala Berzelii Technology Center for
Neurodiagnostics, with financing from the Swedish Agency for In-
novation Systems, the Ahlén Foundation, Uppsala University Hos-
pital and the Swedish Research Council. We also thank Mrs. Sara
Norrehed, Department of Biochemistry and Organic Chemistry, for
generous help with 2D-NMR spectra.