A solanesol-derived scaffold for multimerization of bioactive peptides

Article abstract of DOI:10.1021/jo101043m

A flexible molecular scaffold bearing varying numbers of terminal alkyne groups was synthesized in five steps from solanesol. R(CO)-MSH(4)-NH₂ ligands, which have a relatively low affinity for binding at the human melanocortin 4 receptor (hMC4R), were prepared by solid phase synthesis and were N-terminally acylated with 6-azidohexanoic acid. Multiple copies of the azide N₃(CH₂)₅(CO)-MSH(4)-NH₂ were attached to the alkyne-bearing, solanesol-derived molecular scaffold via the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. Control studies showed that the binding affinity of the triazole-containing ligand, CH ₃(CH₂)₃(C₂N₃)(CH ₂)₅(CO)-MSH(4)-NH₂, was not significantly diminished relative to the corresponding parental ligand, CH₃(CO)- MSH(4)-NH₂. In a competitive binding assay with a Eu-labeled probe based on the superpotent ligand NDP-α-MSH, the monovalent and multivalent constructs appear to bind to hMC4R as monovalent species. In a similar assay with a Eu-labeled probe based on MSH(4), modest increases in binding potency with increased MSH(4) content per scaffold were observed.

Full text of DOI:10.1021/jo101043m

pubs.acs.org/joc
A Solanesol-Derived Scaffold for Multimerization of Bioactive Peptides
†
Ramesh Alleti, Venkataramanarao Rao, Liping Xu, Robert J. Gillies, and
†
‡
‡
,†
Eugene A. Mash*
†
Department of Chemistry and Biochemistry, University of Arizona, Tucson, Arizona 85721-0041, and
H. Lee Moffitt Cancer Center & Research Institute, Tampa, Florida 33612
‡
emash@u.arizona.edu
Received May 28, 2010
A flexible molecular scaffold bearing varying numbers of terminal alkyne groups was synthesized in five steps
from solanesol. R(CO)-MSH(4)-NH ligands, which have a relatively low affinity for binding at the human
2
melanocortin 4 receptor (hMC4R), were prepared by solid phase synthesis and were N-terminally acylated
with 6-azidohexanoic acid. Multiple copies of the azide N (CH ) (CO)-MSH(4)-NH were attached to the
3
2 5
2
alkyne-bearing, solanesol-derived molecular scaffold via the copper(I)-catalyzed azide-alkyne cycloaddi-
tion (CuAAC) reaction. Control studies showed that the binding affinity of the triazole-containing ligand,
CH (CH ) (C N )(CH ) (CO)-MSH(4)-NH , was not significantly diminished relative to the correspond-
3
2 3
2
3
2 5
2
ing parental ligand, CH (CO)-MSH(4)-NH . In a competitive binding assay with a Eu-labeled probe based
3
2
on the superpotent ligand NDP-R-MSH, the monovalent and multivalent constructs appear to bind to
hMC4R as monovalent species. In a similar assay with a Eu-labeled probe based on MSH(4), modest
increases in binding potency with increased MSH(4) content per scaffold were observed.
1
Introduction
existence and location by noninvasive molecular imaging.
One strategy for development of such reagents involves
linking imaging agents to molecules that contain multiple
copies of individual binding units, or ligands, targeted to cell
Early detection and diagnosis of many human cancers
would be facilitated by the availability of reagents that could
seek out and selectively bind to cancer cells and report their
2
surface receptors that are displayed by cancer cells. Such
multivalent constructs should display enhanced affinity and
1
-3
*
To whom correspondence should be addressed. Phone: (520) 621-6321.
Fax: (520) 621-8407.
1) (a) Weissleder, R.; Mahmood, U. Radiology 2001, 219, 316–333.
selectivity for cancer cells based on cooperative binding.
The foundation for ligand-guided multivalent attachment of
reporter groups to cell surface receptors was laid in part by studies
that employed a poly(vinyl alcohol) scaffold (PVA) decorated
(
(
(
b) Gillies, R. J.; Hruby, V. J. Expert Opin. Ther. Targets 2003, 7, 137–139.
c) Gillies, R. J.; Hoffman, J. M.; Lam, K. S.; Menkens, A. E.; Piwnica-
Worms, D. R.; Sullivan, D. C.; Weissleder, R. Mol. Imaging 2005, 4, 98–103.
d) Cassidy, P. J.; Radda, G. K. J. R. Soc., Interface 2005, 2, 133–144.
2) (a) Handl, H. L.; Vagner, J.; Han, H.; Mash, E.; Hruby, V. J.; Gillies,
4
with fluorescein and R(CO)-NDP-R-MSH-NH ligands. Such
(
2
(
constructs bound specifically and irreversibly to mouse and
human melanoma cells that expressed melanocortin receptors.
R. J. Expert Opin. Ther. Targets 2004, 8, 565–586. (b) Vagner, J.; Handl,
H. L.; Monguchi, Y.; Jana, U.; Begay, L. J.; Mash, E. A.; Hruby, V. J.;
Gillies, R. J. Bioconjugate Chem. 2006, 17, 1545–1550. (c) Bowen, M. E.;
Monguchi, Y.; Sankaranarayanan, R.; Vagner, J.; Begay, L. J.; Xu, L.;
Jagadish, B.; Hruby, V. J.; Gillies, R. J.; Mash, E. A. J. Org. Chem. 2007, 72,
(3) (a) Mammen, M.; Choi, S.-K.; Whitesides, G. M. Angew. Chem., Int.
Ed. 1998, 37, 2754–2794. (b) Kiessling, L. L.; Gestwicki, J. E.; Strong, L. E.
Angew. Chem., Int. Ed. 2006, 45, 2348–2368. (c) Carlson, C. B.; Mowery, P.;
Owen, R. M.; Dykhuizen, E. C.; Kiessling, L. L. ACS Chem. Biol. 2007, 2,
119–127. (d) Trouche, N.; Wieckowski, S.; Sun, W.; Chaloin, O.; Hoebeke,
J.; Fournel, S.; Guichard, G. J. Am. Chem. Soc. 2007, 129, 13480–13492.
(e) Diestler, D. J.; Knapp, E. W. Phys. Rev. Lett. 2008, 100, 178101/1–
178101/4. (f) Choi, S.-K. Synthetic Multivalent Molecules; Wiley-Inter-
science: Hoboken, NJ, 2004.
1
675-1680 and 3608. (d) Jagadish, B.; Sankaranarayanan, R.; Xu, L.;
Richards, R.; Vagner, J.; Hruby, V. J.; Gillies, R. J.; Mash, E. A. Bioorg.
Med. Chem. Lett. 2007, 17, 3310–3313. (e) Handl, H. L.; Sankaranarayanan,
R.; Josan, J. S.; Vagner, J.; Mash, E. A.; Gillies, R. J.; Hruby, V. J.
Bioconjugate Chem. 2007, 18, 1101–1109. (f) Vagner, J.; Xu, L.; Handl,
H. L.; Josan, J. S.; Morse, D. L.; Mash, E. A.; Gillies, R. J.; Hruby, V. J.
Angew. Chem., Int. Ed. 2008, 47, 1685–1688.
DOI: 10.1021/jo101043m
r 2010 American Chemical Society
Published on Web 08/11/2010
J. Org. Chem. 2010, 75, 5895–5903 5895

Alleti et al.
JOCArticle
10
The PVA-based multimer system was not extended to other
peptide hormone/receptor systems at that time due to problems
with the attachment chemistry and solubility of the multimeric
constructs. Recent advances in polymer-supported multivalent
at the human melanocortin 4 receptor (hMC4R). R(CO)-
MSH(4)-NH was selected for this work because synergistic
2
effects are more easily detected for multivalent constructs of
3
c
low-affinity ligands. Mixture 2 was found to be about 18
times more potent than mixture 1 in a competitive binding
5
binding prompted a reexamination of this earlier approach to
11
ligand multimerization with the intent of using more efficient
attachment chemistry and developing more soluble scaffolds.
With regard to ligand attachment, the copper(I)-catalyzed
azide-alkyne cycloaddition (CuAAC) reaction, which gen-
assay against a europium-labeled derivativeofMSH(4). The
synthetic andbiologicalresultswith1 and2 wereencouraging,
and work toward a polyisoprene-derived polyol scaffold was
begun. However, when solubility problems were encountered
during hydroboration and oxidation of polyisoprene, a study
of the chemistry of a second model system intermediate in size
was deemed appropriate. It was also recognized that study of
a larger model system would permit testing of the biological
effects of higher levels of ligand multimerization. The synthe-
sis of a scaffold derived from the polyterpene solanesol (3),
decoration of the scaffold with copies of MSH(4) via CuAAC,
and results of competitivebinding studies withcells expressing
hMC4R are presented herein.
6
erates 1,4-disubstituted triazole products, is a reasonable
choice to replace the maleimide electrophile/thiol nucleo-
phile and thiol/disulfide exchange attachment chemistries
4
c,d
used previously with PVA.
With regard to scaffold, PVA derived by incomplete hydro-
lysis of poly(vinyl acetate) is often more water-soluble at room
temperature than is more completely hydrolyzed poly(vinyl
7
acetate). This is presumably due to interruption of hydrogen-
bonded microcrystalline domains, and suggested that a polymer
bearing fewer, stereorandom hydroxyl groups might be less
crystalline and more water-soluble. Such a polymer can be
8
prepared from polyisoprene. Since analysis of polymer products
is complicated by high molecular weight and polydispersity, we
elected to employ more tractable model systems for the establish-
ment of the synthetic methodology necessary to this approach.
Previously we described the preparation from squalene of
regiochemically and stereochemically mixed hexol deriva-
tives 1 and 2 which carry one and two copies, respectively, of
2
d
the linear tetrapeptide amide R(CO)-MSH(4)-NH . In
2
9
contrast with CH (CO)-NDP-R-MSH-NH , CH (CO)-
3
2
3
MSH(4)-NH ligands have relatively low affinity for binding
2
(4) (a) Schwyzer, R.; Kriwaczek, V. M. Biopolymers 1981, 20, 2011–2020.
b) Wunderlin, R.; Sharma, S. D.; Minakakis, P.; Schwyzer, R. Helv. Chim.
(
Acta 1985, 68, 12–22. (c) Sharma, S. D.; Granberry, M. E.; Jiang, J.; Leong,
S. P. L.; Hadley, M. E.; Hruby, V. J. Bioconjugate Chem. 1994, 5, 591–601.
Results
(
d) Sharma, S. D.; Jiang, J.; Hadley, M. E.; Bentley, D. L.; Hruby, V. J. Proc.
Natl. Acad. Sci. U.S.A. 1996, 93, 13715–13720. (e) Carrithers, M. D.; Lerner,
M. R. Chem. Biol. 1996, 3, 537–542.
Synthesis. The ready availability of solanesol (3) and
1
2
(5) (a) Cairo, C. W.; Gestwicki, J. E.; Kanai, M.; Kiessling, L. L. J. Am. Chem.
solanesyl bromide (4) made these compounds appealing
starting materials for construction of an intermediate-sized
polyisoprene scaffold (Scheme 1). Double alkylation of
dimethyl malonate with 4 gave 5 in 72% yield. Reduction
of 5 with LAH produced diol 6 in 82% yield. Hydroboration
and oxidation of 6 under standard conditions proved to be
Soc. 2002, 124, 1615–1619. (b) Griffith, B. R.; Allen, B. L.; Rapraeger, A. C.;
Kiessling, L. L. J. Am. Chem. Soc. 2004, 126, 1608–1609. (c) Line, B. R.; Mitra, A.;
Nan, A.; Ghandehari, H. J. Nucl. Med. 2005, 46, 1552–1560. (d) Li, R. C.; Broyer,
R. M.; Maynard, H. D. J. Polym. Sci., Part A: Polym. Chem. 2006, 44, 5004–5013.
(
e) Cilliers, R.; Song, Y.; Kohlmeir, E. K.; Larson, A. C.; Omary, R. A.; Meade,
T. J. Magn. Reson. Med. 2008, 59, 898–902.
6) (a) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed.
001, 40, 2004–2021. (b) Gil, M. V.; Ar ꢀe valo, M. J.; L oꢀ pez, O. Synthesis 2007,
589–1620. (c) Angell, Y. L.; Burgess, K. Chem. Soc. Rev. 2007, 36, 1674–
689. (d) Holub, J. M.; Garabedian, M. J.; Kirshenbaum, K. QSAR Comb.
(
ꢀ
2
1
1
unsatisfactory since the use of BH /THF left an unaccepta-
3
13
bly high percentage of alkene groups in the product polyol.
The use of excess disiamylborane in THF for extended
Sci. 2007, 26, 1175–1180.
7) Moore, W. R. A. D.; O’Dowd, M. In Properties and Applications of
Polyvinyl Alcohol; Finch, C. A., Ed.; Staples Printers Ltd: Kent, England,
968; Chapter 4, pp 77-87.
8) (a) Levesque, G.; Pinazzi, C. Bull. Soc. Chim. Fr. 1971, 1008–1010.
b) Yamaguchi, H.; Azuma, K.; Minoura, Y. Polym. J. 1972, 3, 12–20. (c) Pinazzi,
(
14
periods gave more satisfactory results. For example, hy-
1
droboration of 6 with 1.7 equiv of disiamyborane per alkene
for a period of 72 h left 4% of the alkene groups intact in the
product polyol as determined by mass spectrometry (Figure 1)
and titration with bromine (see the Experimental Procedures
for details). The principal polyols present in the product
(
(
C. P.; Guillaume, P.; Reyx, D. Eur. Polym. J. 1976, 12, 9–15. (d) Perera, M. C. S.;
Elix, J. A.; Bradbury, J. H. J. Appl. Polym. Sci. 1987, 33, 2731–2742.
(
9) The “high-affinity” ligand, NDP-R-MSH, is Ac-Ser-Tyr-Ser-Nle-
Glu-His-DPhe-Arg-Trp-Gly-Lys-Pro-Val-NH see: (a) Sawyer, T. K.;
2
;
Sanfilippo, P. J.; Hruby, V. J.; Engel, M. H.; Heward, C. B.; Burnett, J. B.;
Hadley, M. E. Proc. Natl. Acad. Sci. U.S.A. 1980, 77, 5754–5758. (b) Hadley,
M. E.; Anderson, B.; Heward, C. B.; Sawyer, T. K.; Hruby, V. J. Science
(11) Xu, L.;Vagner, J.;Alleti, R.;Rao, V.;Jagadish, B.;Morse, D. L.;Hruby,
V. J.; Gillies, R. J.; Mash, E. A. Bioorg. Med. Chem. Lett. 2010, 20, 2489–2492.
(12) Hamamura,K.;Yamatsu,I.;Minami,N.;Yamagishi,Y.;Inai,Y.;Kijima,
S.; Nakamura, T. J. Label. Compd. Radiopharm. 2002, 45, 823–829. Solanesol (3)
and solanesyl bromide (4) are commercially available from NetChem.
(13) This presumably was due to temporary intermolecular and intramo-
lecular cross-linking during hydroboration, leading to sterically inaccessible,
unreactive double bonds. Similar behavior was noted in previous work on the
hydroboration of polyisoprene; see refs 8a-8d.
(14) Use of triisopinocampheyldiborane as described in ref 8b gave
mixtures of polyols and isopinocampheol that were difficult to separate.
The byproduct from use of disiamylborane, 3-methyl-2-butanol, was more
easily removed from the polyol product under vacuum.
1
981, 213, 1025–1027.
10) The “low-affinity” ligand MSH(4), Ac-His-DPhe-Arg-Trp-NH
based on the minimal active sequence for full agonist activity of R-MSH; see:
a) Hruby, V. J.; Wilkes, B. C.; Hadley, M. E.; Al-Obeidi, F.; Sawyer, T. K.;
(
2
, was
(
Staples, D. J.; de Vaux, A. E.; Dym, O.; de L. Castrucci, A. M.; Hintz, M. F.;
Riehm, J. P.; Rao, K. R. J. Med. Chem. 1987, 30, 2126–2130. (b) Castrucci,
A. M. L.; Hadley, M. E.; Sawyer, T. K.; Wilkes, B. C.; Al-Obiedi, F.; Staples,
D. J.; de Vaux, A. E.; Dym, O.; Hintz, M. F.; Riehm, J. P.; Rao, K. R.;
Hruby, V. J. Gen. Comp. Endocrinol. 1989, 73, 157–163. (c) Haskell-Luevano,
C.; Hendrata, S.; North, C.; Sawyer, T. K.; Hadley, M. E.; Hruby, V. J.;
Dickinson, C.; Gantz, I. J. Med. Chem. 1997, 40, 2133–2139.
5
896 J. Org. Chem. Vol. 75, No. 17, 2010

Alleti et al.
JOCArticle
SCHEME 1. Synthesis of Polyol/Polyalkyne Scaffold 9a-c
a
3 2 2 2 4 2 2 2 4
Reagents: (a) PBr , ether (ref 12); (b) NaH, CH (CO Me) ,THF;(c) LiAlH , ether; (d) disiamyborane, THF; H O , NaOH; (e) NaH, Br(CH ) CtCH (8).
c
b
Product 7 resulting from anti-Markovnikov hydration is shown. It is assumed that mixtures of all possible stereoisomers of 7 are produced. Structure 9 is a
representative hexaalkyne scaffold. The sites of attachment of the 5-hexyn-1-yl groups shown in 9 are arbitrary.
mixture are formulated as the stereochemically mixed eicosols
based on mass spectral and NMR data (Figure 1). While
hydroboration of 6 with BH should yield products contain-
prepared in 83% yield by reaction of azide 10 with 1-hexyne at
room temperature in methanol in the presence of tetrakis-
(acetonitrile)copper(I) hexafluorophosphate and TBTA.
6-(4-Butyl-1H-1,2,3-triazol-1-yl)hexanoic acid (12) was pre-
pared in 60% yield by reaction of 6-azidohexanoic acid with
1-hexyne at room temperature in a mixture of water and
tert-butanol in the presence of copper sulfate and sodium
ascorbate.
7
18
3
8
d
ing both secondary and tertiary alcohol groups, disiamyl-
borane was expected to be more regioselective and lead mostly
1
3
to secondary alcohol groups. The C NMR spectrum of 7
Figure 1) suggests that this expectation was met. Compound
was obtained in about 70% yield based on diol 6. This result
was shown to be repeatable in three separate runs. Reaction of
with 30 equiv of sodium hydride and 12 equiv of 1-bromo-5-
(
7
7
1
5
hexyne (8) in DMF afforded a mixture of polyol/polyalky-
nes 9. The product distribution was determined by mass
spectrometry (Figure 2). The average number of alkynes was
approximately six per molecular scaffold. The product yield
based on this level of alkyne incorporation was 69%. This
result was shown to be repeatable in three separate runs.
Azide 10 was prepared in 84% yield from serine amide
1
6
17
hydrochloride and 6-azidohexanoic acid. Triazole 11 was
(
(
15) Sharma, S.; Oehlschlager, A. C. J. Org. Chem. 1989, 54, 5064–5073.
16) Hidy, P. H.; Hodge, E. B.; Young, V. V.; Harned, R. L.; Brewer,
G. A.; Phillips, W. F.; Runge, W. F.; Stavely, H. E.; Pohland, A.; Boaz, H.;
Sullivan, H. R. J. Am. Chem. Soc. 1955, 77, 2345–2346. Serine amide is
commercially available from Senn Chemicals USA.
J. Org. Chem. Vol. 75, No. 17, 2010 5897

Alleti et al.
JOCArticle
1
3
FIGURE 1. (a) A portion of the DEPT 135 C NMR spectrum of 7 in CD
3
OD (see ref 8d). The lines from 76 to 79 ppm are due to methine
carbons bearing an OH group. The line at 62.8 ppm is due to the two methylene carbons that bear OH groups. The relatively sharp line at 73.3
ppm is due to residual byproduct 3-methyl-2-butanol in this sample. (b) MS showing the distribution of products in the mixture of polyols 7.
Peaks near 1572, 1590, 1608, and 1626 represent solanesol-derived heptadecaols, octadecadols, nonadecaols, and eicosaols, respectively.
a
SCHEME 2. Solid Phase Synthesis
a
Reagents: (a) piperidine; (b) Fmoc/t-Bu solid phase synthesis; (c)
3 2 5
N (CH ) (CO)OH, Cl-HOBt, DIC; (d) TFA/triisopropylsilane/
thioanisole/water (91/3/3/3).
FIGURE 2. MS showing the distribution of products in the mixture
9
2
6
resulting from alkylation of polyols 7. Peaks near 1888, 1968,
048, 2128, 2208, 2288, and 2368 represent incorporation of 3, 4, 5,
, 7, 8, and 9 alkyne units, respectively, onto the solanesol-derived
17
acid was coupled to the N-terminus of the resin-bound
tetrapeptide. Simultaneous side chain deprotection and clea-
vage of the tetrapeptide from the resin was effected by using a
mixture of trifluoroacetic acid, 1,2-ethanedithiol, thioanisole,
and water (91:3:3:3), producing the desired azide-terminated
ligand, N (CH ) (CO)-MSH(4)-NH (13). Triazole-containing
scaffold.
1
0
Solid Phase Synthesis. The low affinity ligand MSH(4)
was constructed on Rink amide Tentagel S resin (initial
loading 0.62 mmol/g) as depicted in Scheme 2. The product
resin retained all side chain protecting groups. 6-Azidohexanoic
3
2 5
2
1
9
ligand CH (CH ) (C N )(CH ) (CO)-MSH(4)-NH (14) was
3 2 3 2 3 2 5 2
similarly prepared by N-terminal acylation of the resin-bound
tetrapeptide with acid 12 in place of 6-azidohexanoic acid.
(
17) Grandjean, C.; Boutonnier, A.; Guerreiro, C.; Fournier, J.-M.;
Mulard, L. A. J. Org. Chem. 2005, 70, 7123–7132.
18) Chan, T. R.; Hilgraf, R.; Sharpless, K. B.; Fokin, V. V. Org. Lett.
004, 6, 2853–2855.
(19) (a) Merrifield, R. B. J. Am. Chem. Soc. 1963, 85, 2149–2154.
(b) Hruby, V. J.; Meyer, J.-P. In Bioorganic Chemistry: Peptides and Proteins;
Hecht, S. M., Ed.; Oxford University Press: New York, 1998; pp 27-64.
(
2
5
898 J. Org. Chem. Vol. 75, No. 17, 2010

Alleti et al.
JOCArticle
a
SCHEME 3. Synthesis of Mixtures of Multivalent Constructs 15 and 16a-e from Solanesol-Derived Molecular Scaffold 9 via CuAAC
a
Only one set of hexameric products from each mixture is depicted here for illustrative purposes. The sites of side chain attachment shown are arbitrary.
TABLE 1. Mass Spectral and HPLC Characterization of Compounds
3 and 14
compd formula [M]
TABLE 2. Synthesis and Compositions of 15 and 16a-e
1
mg
of 9
mg of 13
(equiv)
mg of 10
(equiv)
yield, mg
(%)
MALDI-
TOF
a
R
calcd mass [ion] mass found (error)
t
product
þ
þ
a
a
a
a
a
a
1
1
3
4
C
C
38
H
H
50
N
N
14
O
O
5
783.4161 [M þ 1] 783.4158 (0.4 ppm) 15.10
15
16a
16b
10
20
20
20
20
10
0
15 (15)
0
0
15 (7)
12 (6)
4 (4)
16 (88)
22 (76)
29 (78)
31 (76)
19 (41)
22 (76)
Figure S12
Figure S13
Figure S14
Figure S15
Figure S16
Figure S17
44
60
14
5
865.4949 [M þ 1] 865.4936 (0.9 ppm) 13.77
8 (1)
16 (2)
8 (1)
16 (2)
16 (4)
a
Linear gradient of from 10% to 90% acetonitrile in water containing
.1% TFA over 50 min.
1
1
1
6c
6d
6e
0
a
Compounds 13 and 14 were purified by reverse-phase C18
preparative HPLC and were characterized by ESI-MS and
MALDI-TOF. Details appear in Table 1.
See the Supporting Information.
Multimer Assembly. Reaction of the polyol/polyalkyne
mixture 9 with an excess of 10 in the presence of copper(I)
and TBTA in methanol under microwave irradiation
gave mixtures of MSH(4)-bearing multivalent constructs 16a
and 16b, respectively (Scheme 3 and Table 2). Reaction
of 9 with 1, 2, or 4 equiv of 13, followed by reaction of the
intermediate MSH(4)-containing multivalent constructs
with an excess of 10 under these same conditions, gave
mixtures of MSH(4)/serine amide multivalent constructs
16c-e. After dilution with water, copper ions were removed
by complexation with dithizone and extraction of the com-
(
temperature held constant at 100 °C) gave a mixture of
20
serine amide multivalent constructs 15 (Scheme 3 and Table 2).
After dilution with water, copper ions were removed by
complexation with dithizone and extraction of the complex
2
1
with CHCl3. Other organic-soluble molecules (TBTA,
excess 10) were also removed by this extraction. The serine
amide-containing product mixture 15 was recovered from
the aqueous solution by lyophilization as a white powder and
was characterized by MALDI-TOF mass spectrometry (see
the Supporting Information). The average number of serine
amide residues per molecule of 15 was determined to be six.
Reaction of 9 with 1 or 2 equiv of 13 in the presence of
2
1
plex with CHCl . Other organic-soluble molecules (TBTA,
3
excess 10) were also removed by this extraction. The resulting
aqueous solutions of multivalent constructs were subjected
to lyophilization, producing 16a-e as white powders in good
to very good yields. These mixtures were characterized by
MALDI-TOF and by UV analysis (see the Supporting
Information).
20
copper(I) and TBTA in methanol under microwave irradiation
Binding Studies. Hek293 cells overexpressing hMC4R
2
were used to assess ligand binding by using two previously
2
(
20) Kappe, C. O.; Dallinger, D.; Murphree, S. S. Practical Microwave
Synthesis for Organic Chemists; Wiley-VCH: Weinheim, Germany, 2009.
21) Caplin, S. Tissue Cult. Assoc. Man. 1976, 2, 439–440. Copper ion
could not be removed by dialysis, presumably due to the strong chelatory
properties of MSH(4).
(
(22) The hMC4R vector was originally received from Dr. Ira Gantz; see:
Gantz, I.; Miwa, H.; Konda, Y.; Shimoto, Y.; Tashiro, T.; Watson, S. J.;
DelValle, J.; Yamada, T. J. Biol. Chem. 1993, 268, 15174–15179.
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TABLE 3. Assays of Competitive Binding of MSH(4), 11, 14, 15, and
spectroscopy, and subjected to testing with two previously
described competitive binding assays.
16a-e to hMC4R
a
b
23a
In assay A, serine amide derivative 11 and the mixture
assay A
assay B
c
c
of serine amide multivalent constructs 15 were both ineffec-
tive at blocking probe 17 from binding to hMC4R over the
range of concentrations tested. The K for the monovalent
compd
K
i
, μM
n
K
i
, μM
n
d
d
e
11
nb
nb
3
3
5
5
nb
nb
3
3
4
3
4
6
4
6
4
e
i
15
control compound 14 was 1.4 times the value for the parental
ligand, indicating that attachment of the triazole-containing
“spacer” to the N-terminus of MSH(4) has a modest detri-
mental effect on ligand binding to hMC4R. In keeping with
prior results obtained with the squalene-derived monovalent
MSH(4)
1.9 ( 0.55
2.7 ( 0.21
0.76 ( 0.04
0.82 ( 0.26
1.7 ( 0.39
1.2 ( 0.38
0.84 ( 0.10
0.52 ( 0.02
0.22 ( 0.06
1
1
1
1
1
1
4
f
6a
6b
6c
6d
6e
a
ndf
nd
f
nd
nd
f
1
1
and divalent MSH(4) constructs 1 and 2, multimer 16e was
shown to be nearly as potent as MSH(4), but not more so, in
assay A. This result suggests monovalent binding of 16e at
available hMC4R. Monovalent binding of 16e would be
expected whenever the off-rate of monovalently bound 16e
is faster than the binding of a second “anchoring” ligand arm
2.2 ( 0.17
4
b
c
This assay employed probe 17. This assay employed probe 18. K
values were calculated with the equation K )),
= EC50/(1 þ ([ligand]/K
where [ligand] refers to the concentration of probe used as the labeled
competed ligand. For probe 17, [ligand] = 10 nM and K = 18.8 nM.
For probe 18, [ligand] = 0.5 μM and K = 9.1 μM. The value given
represents the average of n independent competition binding experi-
ments, each done in quadruplicate. This compound was unable to
prevent probe 17 from binding in the concentration range tested
i
i
D
D
D
9
d
to hMC4R on the cell surface. The NDP-R-MSH ligand,
upon which 17 is based, is a much stronger binder than
2
3a
-
5
-12
e
24
(
10 -10
M in serine amide). This compound was unable to prevent
5
probe 18 from binding in the concentration range tested (10 -10
in serine amide). Not determined.
MSH(4) and is known to have a slow off-rate (∼8 h). Thus,
-
-12
M
f
monopolization of neighboring receptors by the superpotent
probe 17 would disfavor the binding of a second ligand arm
of 16e. Depletion of receptors at the cell surface by cycling
may also have contributed, since internalization results in
fewer neighboring surface receptors to which a second ligand
arm of 16e might bind.
1
1,23
described europium-based competitive binding assays.
2
3a
Assay A employed Eu-DTPA-NDP-R-MSH-NH (17) as
2
1
1
the labeled probe, and assay B employed Eu-DTPA-
PEGO-MSH(4)-NH (18) as the labeled probe. The K values
2
for the serine amide-containing control compounds 11 and
5, for the parental ligand MSH(4), for the triazole-contain-
ing MSH(4) analogue 14, and for MSH(4)-containing multi-
valent constructs 16a-e are given in Table 3.
11
i
Assay B was developed to better balance the off-rates of
competed and competing ligands, probe 18 and 16a-e,
respectively. In this assay, serine amide derivative 11 and
serine amide multivalent constructs 15 were ineffective at
blocking probe 18 from binding to hMC4R over the range of
1
concentrations tested. The K for compound 14 was 1.1 times
i
higher than the value for the parental ligand, indicating that
attachment of the triazole-containing “spacer” to the N-
terminus of MSH(4) had a very modest detrimental effect on
ligand binding to hMC4R. The K values measured in assay B
i
differed among the various solanesol-derived constructs
1
6a-e (Table 3). Construct 16a contained, on average, one
copy of MSH(4) and five unreacted alkyne groups per
scaffold and was 2.2 times less potent than the parental
ligand, MSH(4). Construct 16b contained, on average, two
copies of MSH(4) and four unreacted alkyne groups per
scaffold and was 1.6 times less potent than MSH(4). Con-
struct 16c contained, on average, one copy of MSH(4) and
five serinamide residues per scaffold and was 1.1 times less
potent than MSH(4). Construct 16d contained, on average,
two copies of MSH(4) and four serinamide residues per
scaffold and was 1.5 times more potent than MSH(4). Con-
struct 16e contained, on average, four copies of MSH(4) and
two serinamide residues per scaffold and was 3.4 times more
potent than MSH(4). As demonstrated by comparison of the
Discussion
The four-step synthesis of the mixture of polyols 7 starting
from solanesol was efficient and highly regioselective. The
water-soluble product 7, which is available in gram quanti-
ties, was statistically alkylated by using a 12-fold molar
excess of 1-bromo-5-hexyne to produce a mixture of polyol/
polyalkynes 9 that bore an average of six alkyne residues
per scaffold. Presumably, the identity of the alkylating agent
and the extent of alkylation might be varied. Microwave-
driven CuAAC was used to attach copies of serine amide and
MSH(4) derivatives bearing N-terminal azide groups. In
principle, this method might be used to attach other ligands,
imaging agents, and/or therapeutic agents to scaffold 9. The
constructs so produced were purified from copper and from
small molecules, characterized by MALDI-TOF and by UV
K values for 16a and 16b with those for 16c and 16d,
i
conversion of the remaining hydrophobic terminal alkyne
spacers to the longer, bulkier, but more hydrophilic triazole-
serinamide side chains enhances binding. That this enhance-
ment is not due to specific binding by the serinamide residues
is supported by the inactivity of compounds 11 and 15 in
assay B. Enhanced binding for solanesol-derived constructs
with higher levels of MSH(4) incorporation was observed,
(
23) (a) Handl, H. L.; Vagner, J.; Yamamura, H. I.; Hruby, V. J.; Gillies,
R. J. Anal. Biochem. 2004, 330, 242–250. (b) De Silva, C. R.; Vagner, J.;
Lynch, R.; Gillies, R. J.; Hruby, V. J. Anal. Biochem. 2010, 398, 15–23.
(24) Haskell-Luevano, C.; Miwa, H.; Dickinson, C.; Hadley, M. E.;
Hruby, V. J.; Yamada, T.; Gantz, I. J. Med. Chem. 1996, 39, 432–435.
5
900 J. Org. Chem. Vol. 75, No. 17, 2010

Alleti et al.
JOCArticle
but the small magnitude of the changes in the K values was
i
0.29 mmol) in dry ether (6 mL) was added diester 5 (200 mg,
0.147 mmol) dropwise at room temperature. The suspension
was stirred at rt for 8 h. After completion of the reaction (TLC),
the reaction mixture was quenched with saturated aqueous
unexpected and inconsistent with prior studies with multi-
2
5
valent MSH(4) constructs. The observed binding enhance-
ments for the series 16c-e can be entirely attributed to
statistics and also suggest monovalent binding of 16a-e at
available hMC4R. It may be that mixtures 16a-e are com-
petent binders as monovalent species, but 16b and 16d-e are
incompetent binders as multivalent species due to improper
Na
diethyl ether and the combined organic extracts were washed
with saturated NaHCO (15 mL) and brine (15 mL). The ether
2 4
SO solution and filtered. The filtrate was extracted with
3
layer was dried over MgSO , filtered, and concentrated. Column
4
chromatography on silica gel 60 eluted with 30% ether/hexanes
2
6
ligand spacing or presentation. Incompetence could also be
due to release of the monovalently bound multivalent con-
struct from the cell surface before binding of a second ligand
arm can occur (vide supra). At present, the on-rates and off-
rates of MSH(4) and related constructs, such as 18 and
f
gave 157 mg (0.12 mmol, 82%) of 6 (R 0.60, 10% EtOAc/
1
hexanes) as a colorless, viscous oil: H NMR (500 MHz, CDCl )
3
δ 1.59 (s, 54H), 1.68 (s, 6H), 1.97-2.07 (m, 68H), 3.56 (s, 4H),
1
3
3
5.10 (t, J = 6 Hz, 18H); C NMR (125 MHz, CDCl ) δ 15.9,
1
1
1
7.6, 25.6, 26.6, 29.9, 40.1, 43.4, 68.7, 74.7, 119.4, 124.2, 131.2,
34.8, 137.8. Elemental Anal. Calcd for C H O : C 85.78, H
1
6a-e, are unknown. As before, depletion of receptors at
93 152
2
1.77. Found: C 85.70; H 11.87.
7,37-Bis(hydroxymethyl)-2,6,10,14,18,22,26,30,34,40,44,48,
2,56,60,64,68,72-octadecamethyltriheptacontan-3,7,11,15,19,23,
the cell surface by cycling should reduce the number of
neighboring receptors to which a second ligand arm might
bind and limit opportunities for multivalent binding. In
support of this point, internalization of probes 17 and 18
contributes significantly to the fluorescence measured in
these assays, which may more properly be termed “binding
and uptake” assays. In a preliminary study with probe 17
that compared measured fluorescence at 37 and 4 °C, as
much as 90% of the fluorescence at 37 °C was attributable to
3
5
2
7,31,35,39,43,47,51,55,59,63,67,71-octadecaol (7). [Note: All
reagents and solutions were deoxygenated with argon before
use.] In a three-necked flask (500 mL) a solution of borane in
THF (1 M, 84 mL, 84 mmol) was deoxygenated with argon gas.
The flaskwas immersed inanice bath, anda solution of 2-methyl-
2-butene in THF (2 M, 63 mL, 126 mmol), also deoxygenated
with argon gas, was added to the borane solution dropwise with
stirring at 0 °C. The resulting mixture was maintained at 0 °C for
2
7
internalized probe. Work is presently underway to deline-
ate the details of binding and uptake in the further char-
acterization of the interactions of solanesol-derived multi-
valent constructs with hMC4R.
3
h prior to use. Diol 6 (1.80 g, 1.38 mmol) in THF was then
added dropwise at 0 °C. The mixture was stirred at 0 °C for 5 h,
kept at 4 °C for 3 days, and then 3 N NaOH (26 mL) and 30%
H O (26 mL) were added. After 36 h at rt, the reaction mixture
2
2
was diluted with water and extracted with ether. The ether
extracts were combined, and the volatile materials, including
most of the byproduct 3-methyl-2-butanol, were removed under
reduced pressure. The crude product was then washed twice with
hexane (20 mL). Alternatively, after dilution with water, the
reaction mixture and the aqueous layer were placed in a liquid-
liquid extraction apparatus, the aqueous phase saturated with
NaCl, and the mixture continuously extracted with ether. After
72 h the ether extracts were separated, volatile components
removed under reduced pressure, and the residue washed with
hexane. Yield: 1.58 g (0.97 mmol, 70%). The product was chara-
2
8
Experimental Procedures
Chemical Synthesis (Scheme 1). Dimethyl 2,2-Bis[(2E,6E,10E,
4E,18E,22E,26E,30E)-3,7,11,15,19,23,27,31,35-nonamethylhex-
atriaconta-2,6,10,14,18,22,26,30,34-nonaenyl]malonate (5). To a
suspension of NaH (60 mg, 2.5 mmol) in dry THF (10 mL) at 0 °C
was added dimethyl malonate (80 μL, 92 mg, 0.7 mmol). The
mixture was allowed to warm to rt and was stirred for 3 h. To the
mixture was added a solution of solanesyl bromide (4, 1.00 g,
.45 mmol) in dry THF (10 mL) dropwise over 20 min. After 1 h,
additional NaH (60 mg, 2.5 mmol) and 4 (200 mg, 0.28 mmol)
were added. After 5 h, the reaction was quenched with saturated
aqueous NH Cl solution and the mixture was extracted with
ether (200 mL). The organic extract was washed with water and
1
1
2
1
1
3
cterizedby C NMR andbymass spectral analysis (see Figure 1).
A 10 mM solution of 7 in methanol was titrated with a 10 mM
solution of bromine in methanol. It was found that 3.8% of the
double bonds remained in the sample of polyol 7 produced in the
experiment described above as determined by the amount of
4
brine, dried over anhydrous MgSO
3
4
, and filtered. Approximately
g of NaHCO was added to the filtrate to neutralize any HBr
3
29
bromine decolorized by 7.
Synthesis of Polyol/Polyalkynes 9. To a suspension of NaH
formed during evaporation of volatiles using a rotary evaporator.
Column chromatography on silica gel 60 eluted with 2% ether/
(
60% in oil, 183 mg, 4.59 mmol) in dry DMF (10 mL) at room
temperature was added polyol 7 (250 mg, 0.153 mmol). After
hexanes gave 684 mg (0.50 mmol, 72%) of 5 (R
f
0.45, 5% EtOAc/
hexanes) as a viscous pale yellow oil: H NMR (500 MHz,
CDCl ) δ 1.58 (s, 54H), 1.66 (s, 6H), 1.96-2.09 (m, 64H), 2.59
d, J = 7.2 Hz, 2H), 3.67 (s, 6H), 4.96 (t, J = 7.2 Hz, 2H), 5.10 (t,
1
1
0 min of stirring, a solution of 1-bromo-5-hexyne (8, 298 mg,
5
2
1
3
.84 mmol) in dry DMF (2 mL) was added. The mixture was
(
1
3
stirred under argon for 3 days, then quenched with aqueous
NH
Cl (20 mL) and extracted with EtOAc (3 ꢀ 30 mL). The
EtOAc extracts were washed with water (3 ꢀ 20 mL) and brine
3 ꢀ 15 mL). Volatiles were evaporated and the residue washed
J = 6.6 Hz, 16H); C NMR (125 MHz, CDCl ) δ 16.0, 17.6,
5.7, 26.7, 30.8, 39.7, 52.2, 57.8, 117.7, 123.9, 124.2, 131.1, 134.8,
35.2, 139.1, 171.8; HRMS calcd for C95H
152
3
4
2
1
1
þ
O
4
Na (M þ Na)
(
380.1588, obsd 1380.1505.
2
,2-Bis[(2E,6E,10E,14E,18E,22E,26E,30E)-3,7,11,15,19,23,27,
1,35-nonamethylhexatriaconta-2,6,10,14,18,22,26,30,34-non-
(12 mg,
3
(29) This method was validated by titration of 0.10 M solutions of
aenyl]propane-1,3-diol (6). To a suspension of LiAlH
4
citronellal and geraniol in methanol with a 0.10 M solution of bromine in
methanol. A negative control experiment with hexol A (see ref 2d for the
preparation of A) gave a persistent bromine color upon addition of the first
drop of bromine solution.
(
25) Vagner, J.; Handl, H. L.; Gillies, R. J.; Hruby, V. J. Bioorg. Med.
Chem. Lett. 2004, 14, 211–215.
26) Shewmake, T. A.; Solis, F. J.; Gillies, R. J.; Caplan, M. R. Bioma-
cromolecules 2008, 9, 3057–3064.
(
(
(
27) Xu, L. Unpublished work.
28) The General Experimental section appears in the Supporting
Information.
J. Org. Chem. Vol. 75, No. 17, 2010 5901

Alleti et al.
JOCArticle
with hexanes (2 ꢀ 10 mL) to afford a mixture of polyol/polyalkynes
CH Cl (3 ꢀ 15 mL), DMF (3 ꢀ 15 mL), 0.5 M HOBt in DMF
2
2
9
(218 mg). On the basis of mass spectral analysis (see Figure 2) it
(1 ꢀ 15 mL), 0.5 M HOBt in DMF (1 ꢀ 15 mL) plus a drop of
bromophenol blue, DMF (2 ꢀ 15 mL), and CH Cl (1 ꢀ 15 mL),
was found that an average of six alkylations per molecule had
occurred. On the basis of this average, the calculated yield is
69%. This reaction was repeated twice under similar conditions
with similar results.
2
2
in that order. A solution of Fmoc-Trp(Boc)-OH, the first Fmoc-
amino acid (1.05 g, 2.04 mmol), Cl-HOBt (345 mg, 2.04 mmol),
and DIC (512 mg, 4.08 mmol) in DMF (15 mL) was allowed to
react for 2 min, then added to the resin and the mixture was shaken
for 1 h. The resin was then washed with DMF (3 ꢀ 15 mL), CH Cl
N-(1-Amino-3-hydroxy-1-oxopropan-2-yl)-6-azidohexanamide
10). To a solution of serine amide hydrochloride (1.10 g, 7.9 mmol)
1
6
(
in DMF (20 mL) at room temperature was added triethylamine
2
2
(3 ꢀ 15 mL), and DMF (3 ꢀ 15 mL). Free NH groups were then
2
(
2
0.80 g, 7.9 mmol). To the resulting white suspension was added
capped by addition of a 1:1 mixture of acetic anhydride and pyridine
(6 mL). After the mixture was shaken for 20 min, the resin was
1
,5-dioxopyrrolidin-1-yl 6-azidohexanoate (1.88 g, 7.1 mmol)
7
and the reaction mixture was stirred at room temperature. After
completion of the reaction (disappearance of 2,5-dioxopyrroli-
din-1-yl 6-azidohexanoate as monitored by TLC), volatiles were
evaporated under reduced pressure. Column chromatography
on silica gel 60 (50 g) eluted with 10% EtOAc/methanol gave
washed with DMF (3 ꢀ 15 mL), CH Cl (3 ꢀ 15 mL), and DMF
2
2
(3 ꢀ 15 mL). The absence of free amine groups was confirmed by
the Kaiser test. The same cycle of procedures was repeated for
coupling of the other amino acids in the sequence, and finally for
attachment of the N-terminal 6-azidohexanoic acid residue or
the N-terminal 6-(4-butyl-1H-1,2,3-triazol-1-yl)hexanoic acid
residue, thus producing the resin-bound tetrapeptide derivatives
related to compounds 13 and 14, respectively. Cleavage and
deprotection were achieved by using a 91:3:3:3 mixture of trifluoro-
acetic acid, triisopropylsilane, thioanisole, and water (10 mL).
The mixture of cleavage cocktail and resin was shaken over-
night, the solution was separated from the resin, volatiles were
evaporated, the residue was triturated with ether, and the
crude product was separated by centrifugation. Purification of
the tetrapeptide amides 13 and 14 was accomplished by reverse
phase chromatography with a 19 ꢀ 256 mm X-Bridge Preparative
1
.46 g (6.0 mmol, 84%) of 10 (R
f
0.5, 15% methanol/CHCl
white solid, mp 94-96 °C: H NMR (500 MHz, CDCl , CD
OD) δ 4.36 (t, J = 5 Hz, 1H), 3.79 (dd, 1H), 3.58 (dd, 1H), 3.19
3
) as a
1
3
3
-
(
2
t, J = 7 Hz, 2H), 2.19 (t, J = 7 Hz, 2H), 1.56 (m, 4H), 1.32 (m,
13
H); C NMR (125 MHz, CDCl , CD OD) δ 24.8, 26.1, 28.4,
3 3
3
C
5.7, 51.0, 54.2, 62.2, 173.9, 174.0; HRMS (ESI) calcd for
þ
H
9 17
N
5
O
N-(1-Amino-3-hydroxy-1-oxopropan-2-yl)-6-(4-butyl-1H-1,2,
3
(MH) 244.1411, obsd 244.1412.
3-triazol-1-yl)hexanamide (11). To a solution of azide 10 (100 mg,
0.41 mmol) in methanol (5 mL) were added 1-hexyne (337 mg,
4.11 mmol), TBTA (42 mg, 0.08 mmol), and tetrakis(acetonitrile)-
copper(I) hexafluorophosphate (30 mg, 0.08 mmol). The reac-
tion mixture was stirred for 15 h at room temperature. Volatiles
were removed under reduced pressure and residue chromato-
graphed on silica gel 60 (10 g) eluted with 10% methanol/CHCl3
C18 column. The mobile phase used was 10-90% acetonitrile
and water containing 0.1% TFA within 50 min; the flow rate
was 15 mL/min and the UV detector system operated at 230 nm.
The purity of compounds 13 and 14 was checked by reverse
phase HPLC, using a 4.6 ꢀ 75 mm Symmetry Analytical C18
column. The mobile phase was 10-90% acetonitrile and water
containing 0.1% TFA within 50 min; the flow rate was 1 mL/
min and the UV detector system operated at 230 nm. Com-
pounds 13 and 14 were characterized by MALDI-TOF mass
spectrometry. Both the reflectron and linear techniques were
used for positive ion detection. The matrix, sinapic acid, and the
analyte were dissolved in water:acetonitrile 1:1 containing 0.1%
of formic acid and the solutions mixed in a ratio of 100:1. ESI
was also used to ionize some of the samples. The samples were
dissolved in methanol:water (1:1) at a concentration of ca. 50
μM. Standard ESI conditions were applied to detect positively
charged ions.
toafford11(110mg, 0.34 mmol, 83%) as a solid, mp124-126 °C:
1
3
H NMR (500 MHz, CD OD) δ 0.94 (t, J = 7.5 Hz, 3H), 1.35
(
m, 4H), 1.65 (m, 4H), 1.90 (m, 2H), 2.28 (t, J = 7.5 Hz, 2H),
2
4
.67 (t, J = 7.5 Hz, 2H), 3.76 (m, 2H), 4.35 (t, J = 7 Hz, 2H),
.41 (t, J = 5 Hz, 1H), 7.73 (s, 1H); C NMR (125 MHz,
1
3
CD
1
3
OD) δ 14.1, 23.2, 26.0, 27.0, 30.9, 32.7, 36.5, 51.0, 56.5, 63.1,
þ
23.1, 175.0, 175.8; HRMS (ESI) calcd for C H N O (MH)
15 28 5 3
3
26.2187, obsd 326.2189.
-(4-Butyl-1H-1,2,3-triazol-1-yl)hexanoic acid (12). 1-Hexyne
5.00 g, 63 mmol), CuSO (1.5 g, 6.3 mmol), and sodium
6
(
4
ascorbate (2.5 g, 12.6 mmol) were added to a round-bottomed
flask containing a 1:1 mixture of t-BuOH and water (20 mL).
1
-Azidohexanoic acid (1.00 g, 6.32 mmol) was added to the
7
6
solution with stirring. The flask was purged with argon and
sealed with a glass stopper to avoid evaporation of 1-hexyne.
The reaction mixture was stirred overnight, and then additional
Multimer Assembly (Scheme 3). Serine Amide Multivalent
Constructs 15. A mixture of polyol/polyalkynes 9 (10 mg, 5 µmol),
azide 10 (10 mg, 50 µmol), TBTA (3 mg, 5 µmol), and tetrakis-
(acetonitrile)copper(I) hexafluorophosphate (2 mg, 5 µmol) in
dry methanol (1 mL) was irradiated for 4 h in a Biotage micro-
wave reactor (100 °C). Additional azide 10 (5 mg, 25 µmol) was
then added to the reaction mixture and irradiation was resumed
for another 4 h. After the reaction was complete, water (20 mL)
1-hexyne (1.03 g, 12.6 mmol) was added. After 24 h the reaction
mixture was diluted with EtOAc (100 mL) and washed with 1 N
HCl (2 ꢀ 20 mL) and brine (2 ꢀ 20 mL), then the organic phase
2 4
was separated, dried over Na SO , filtered, and concentrated in
vacuo. Column chromatography on silica gel 60 (40 g) eluted
with 2% methanol/CHCl gave the product 12 (910 mg, 3.79
was added and the mixture was extracted with CHCl contain-
3
3
1
21
ing dithizone (20 mg/L, 3 ꢀ 30 mL) to remove copper. The
mmol, 60%) as a white solid, mp 41-43 °C: H NMR (500 MHz,
CDCl
3
) δ 0.87 (t, J = 6.9 Hz, 3H), 1.33 (m, 4H), 1.58 (m, 4H),
.84 (m, 2H), 2.28 (t, J = 7.2 Hz, 2H) 2.66 (t, J = 7.5 Hz, 2H),
water layer was then washed with CHCl
3
(2 ꢀ 20 mL) to remove
1
4
any remaining 10 and TBTA. After lyophilization, 16 mg (88%
yield) of 15 was obtained as a white powder. Product 15 was
analyzed by MALDI-TOF (see Figure S12 in the Supporting
Information).
1
.27 (t, J = 7.2 Hz, 2H), 7.24 (s, 1H); C NMR (125 MHz,
3
CDCl
3
) δ 13.7, 22.2, 23.9, 25.0, 25.8, 29.9, 31.4, 33.7, 49.9, 120.6,
þ
1
48.2, 178.0; HRMS (ESI) calcd for C12
40.1707, obsd 240.1706.
Solid Phase Synthesis (Scheme 2). In a syringe (polypropylene
22 3 2
H N O (MH)
2
General Procedure for Reaction of Azide-Functionalized
MSH(4) Derivative 13 and Azide-Functionalized Serine Amide
Derivative 10 with Polyol/Polyalkynes 9 To Produce Compounds
16a-e. Mixtures of polyol/polyalkynes 9 (10 or 20 mg, 5 or 10
µmol), MSH(4) azide 13 (variable amount, see Table 2), TBTA
(3 mg, 5 µmol), and tetrakis(acetonitrile)copper(I) hexafluoro-
phosphate (2 mg, 5 µmol) in dry methanol (1 mL/5 µmol of 9)
were irradiated for 6 h in a Biotage microwave reactor (100 °C).
Azide 10 (variable amount, see Table 2) was then added to the
reaction tube equipped with a polypropylene frit) Rink amide
resin (1 g, 0.68 mmol) was allowed to swell in THF for 1 h. THF
was removed and addition of 20% piperidine in DMF (15 mL)
for 2 min led to the deprotection of the Fmoc functionality.
DMF was removed, 20% piperidine solution in DMF (15 mL)
was again added, and the mixture was shaken for 18 min. DMF
was removed and the resin washed with DMF (3 ꢀ 15 mL),
5
902 J. Org. Chem. Vol. 75, No. 17, 2010

Alleti et al.
JOCArticle
reaction mixtures and irradiation was resumed for another 4 h.
After the reactions were complete, water (20 mL/5 µmol of 9)
and each concentration was tested in quadruplicate. Cells were
incubated in the presence of unlabeled and labeled ligands at
was added and the mixtures were extracted with CHCl
3
contain-
2
37 °C and 5% CO for 1 h. The cells were washed with wash
2
1
ing dithizone (20 mg/L, 3 ꢀ 30 mL) to remove copper. The
buffer (50 mM Tris-HCl, 0.2% BSA, 30 mM NaCl), enhance-
ment solution (PerkinElmer 1244-105) was added (100 μL/
well), and fluorescence was measured on a Wallac VICTOR
water layers were then washed with CHCl
3
(2 ꢀ 20 mL) to
3
remove any remaining 10 and TBTA. After lyophilization, white
powders were obtained (yields are given in Table 2). Products
were analyzed by MALDI-TOF and by UV spectroscopy (see
Figures S13-17 in the Supporting Information).
instrument with standard Eu TRF measurement conditions
(340 nm excitation, 400 μs delay, and emission collection for
400 μs at 615 nm). Competitive binding data were analyzed with
GraphPad Prism software using nonlinear regression analysis
and fitted to a classic one site binding competition equation.
Each EC50 value was generated from individual competitive
Formulation of Solutions. Solutions of multivalent constructs
for binding assays were prepared in DMSO (HYBRI-MAX)
based on the average incorporation of MSH(4) and/or serina-
mide as determined by mass spectrometric analysis. Concentra-
tions of MSH(4) ligand in solution were confirmed by measurement
of the UV absorbance at 280 nm, using a standard calibration
plot (see the Supporting Information). In all cases the concen-
trations calculated from the mass spectral analysis and the weight
of the compound used agreed within 5% with the measured UV
absorption.
binding assays and converted to a K
= EC50/(1 þ ([ligand]/K )), where [ligand] refers to the
concentration of the probe used as the labeled competed ligand.
For probe 17, [ligand] = 10 nM and K = 18.8 nM. For probe
18, [ligand] = 0.5 μM and K = 9.1 μM. Results are given in
i
value by using the equation
K
i
D
D
D
Table 3. The value given represents the average of n independent
competition binding experiments, with the error bars indicating
standard error of the mean.
Binding Assays. Quantitative receptor-binding assays were
1
1
carried out following a previously described method. Hek293
cells overexpressing hMC4R were used to assess ligand binding.
Cells were grown in Dulbecco’s Modified Eagle Medium
Acknowledgment. This work was supported by grants
R33 CA 95944, RO1 CA 97360, RO1 CA 123547, and P30
CA 23074 from the National Cancer Institute.
(
9
DMEM) supplemented with 10% FBS. Cells were seeded in a
6-well plate at a density of 15 000 cells per well and were
allowed to reach 80-90% confluence. On the day of the experi-
ment, media was aspirated from all wells, and 50 μL of the
Supporting Information Available: A general experimental
section, a list of nonstandard abbreviations, copies of the H and
C NMR spectra of compounds 5, 6, 10, 11, and 12, the APT of
-
4
1
compounds to be tested (dilutions ranging from 2 ꢀ 10 to 1 ꢀ
-
11
13
1
0
M) and 50 μL of Eu-labeled ligand (assay A, 10 nM probe
1
7; assay B, 0.5 μM probe 18) were added to each well. Ligands
7, MALDI-TOF spectra of multivalent construct mixtures 15
and 16a-e, and a description of UV studies. This material is
available free of charge via the Internet at http://pubs.acs.org.
were diluted in binding media (DMEM, 1 mM 1,10-phenan-
throline, 200 mg/L bacitracin, 0.5 mg/L leupeptin, 0.3% BSA)
J. Org. Chem. Vol. 75, No. 17, 2010 5903