A divergent strategy for attaching polypyridyl ligands to peptides

Article abstract of DOI:10.1021/jo9021953

(Figure Presented) A divergent method for incorporating polypyridyl ligands into peptides is reported. Three N-Fmoc unnatural amino acids (1-3) that contain varying linkers between the α-carbon and a 2-(hydroxymethyl) pyridyl group were synthesized in enantioenriched form. These amino acids were used as anchors for incorporating multidentate ligands onto a peptide chain in a site-specific fashion. Multiple peptide-ligand conjugates were synthesized from single precursors by solution- or solid-phase methods. Peptides containing more than one metal-binding unit can be produced by this method.

Full text of DOI:10.1021/jo9021953

pubs.acs.org/joc
A Divergent Strategy for Attaching Polypyridyl Ligands to Peptides
Nitinkumar D. Jabre, Tomasz Respondek, Selma A. Ulku, Nadiya Korostelova, and
Jeremy J. Kodanko*
Department of Chemistry, Wayne State Universty, 5101 Cass Ave, Detroit, Michigan 48202
jkodanko@chem.wayne.edu
Received October 12, 2009
A divergent method for incorporating polypyridyl ligands into peptides is reported. Three N-Fmoc
unnatural amino acids (1-3) that contain varying linkers between the R-carbon and a
2-(hydroxymethyl)pyridyl group were synthesized in enantioenriched form. These amino acids were
used as anchors for incorporating multidentate ligands onto a peptide chain in a site-specific fashion.
Multiple peptide-ligand conjugates were synthesized from single precursors by solution- or solid-
phase methods. Peptides containing more than one metal-binding unit can be produced by this
method.
Introduction
In addition, peptidescan beusedto deliver metalcomplexes to
specific locations in cells.8,9 Metal-peptide constructs have
been used extensively in de novo protein design10-13 and in
the creation of artificial enzymes that catalyze organic trans-
formations.14,15
A variety of different synthetic methods have been re-
ported for constructing peptide-metal complex conjugates.
In particular, solid-phase methodologies have been sought
that can take advantage of the combinatorial approach in
order to produce large libraries of metal-binding peptides for
screening and optimization purposes. Metal complexes have
been attached to numerous positions on peptides via solid-
phase synthesis, including the N-terminus,9,16 side chains,2,17
The attachment of metal-binding ligands is a powerful
method for creating peptides with remarkable properties.
Besides the obvious enhancement of metal-binding affinity
that a strong ligand confers, the conjugation of unnatural
metal-binding motifs creates peptides for a variety of appli-
cations.1 For instance, peptide-metal complex conjugates
containing radioactive metal isotopes can act as radiophar-
maceuticals, particularly for anticancer applications.2-4 Ap-
pending metal centers that are catalytically active or possess
labile coordination sites has produced peptides with the
ability to selectively bind to or target DNA and proteins.5-7
(1) Dirscherl, G.; Koenig, B. Eur. J. Org. Chem. 2008, 597–634.
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Feringa, B. L. J. Chem. Soc., Perkin Trans. 1 1998, 769–774.
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Published on Web 01/05/2010
DOI: 10.1021/jo9021953
r
2010 American Chemical Society

Jabre et al.
JOCArticle
FIGURE 2. Tetra-, penta-, and hexadentate polypyridyl ligands.
several orders of magnitude, to transition metals than its
tridentate counterpart di(2-pyridylmethylene)amine, the
core element of SAAC (Figure 1).27,28 This trend of greater
affinity continues with ligands of higher denticity to the
hexadentate ligand TPEN, which binds to first-row transi-
tion metals with dissociation constants in the pM to fM
range.28 Besides their greater affinity, ligands such as TPA
and N4Py have been used to create catalysts and active-site
models of enzymes in nature. Complexes derived from these
multidentate ligands and their derivatives containing Fe, Cu,
and Mn bind to and activate O2,29-31 oxidize organic sub-
strates including the strong C-H bonds found in hydro-
carbons,32-37 bind to and release NO,38 and cleave DNA
under oxidizing conditions.39-41 Furthermore, recent work
published from our laboratory demonstrated that the iron-
(IV)-oxo species derived from N4Py oxidizes amino acids
selectively,42,43 and that [FeIV(O)(N4Py)]2þ can be incorpo-
rated into a short peptide.44 The latter publication provided
FIGURE 1. Unnatural amino acids containing bi- or tridentate
polypyridyl ligands.
or even as part of the peptide backbone itself.18,19 Of these
three strategies, attaching metal complexes to side chains via
a metal-binding amino acid is perhaps the most ideal because
ligands can be placed site-specifically at a desired location
anywhere on the peptide chain, rather than just on the
N-terminus or through conjugation with Cys or Lys residues.
Selected examples of amino acids used in the site-specific
strategy include Ala derivatives which contain bidentate
ligands such as 2,2-bipyridyl (Bpa) or 1,10-phenanthroline
(Fen),20-24 as well as the single amino acid chelates derived
from Lys that contain di(2-pyridylmethylene)amine (SAAC)
or di(2-quinolinemethylene)amine (SAACQ) units,2,17,25,26
which act as tridentate chelating groups (Figure 1). Despite
major synthetic efforts directed toward producing peptide-
metal ligand conjugates, there is a paucity of divergent
synthetic strategies that can be used to adorn a single,
resin-bound peptide with multiple metal-binding units in a
site-specific fashion. This is unfortunate because the metal-
binding unit represents an additional handle for achieving
diversity in peptide-metal complex conjugates.
(27) Blackman, A. G. Polyhedron 2005, 24, 1–39.
(28) Anderegg, G.; Hubmann, E.; Podder, N. G.; Wenk, F. Helv. Chim.
Acta 1977, 60, 123–40.
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(31) Karlin, K. D.; Kaderli, S.; Zueberbuehler, A. D. Acc. Chem. Res.
1997, 30, 139–147.
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Karlin, K. D. J. Am. Chem. Soc. 2007, 129, 264–265.
(34) van den Berg, T. A.; de Boer, J. W.; Browne, W. R.; Roelfes, G.;
Feringa, B. L. Chem. Commun. 2004, 2550–2551.
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Lett. 2003, 44, 4581–4584.
(36) Nam, W. Acc. Chem. Res. 2007, 40, 522–531.
In addition to the bidentate and tridentate N-donor
ligands shown in Figure 1, polypyridyl ligands with higher
denticity have properties that make them attractive for
incorporating into peptides (Figure 2). By virtue of the
additional ligating group, the tetradentate ligand TPA
(tri(2-pyridylmethylene)amine) binds more strongly, by
(37) Kaizer, J.; Klinker, E. J.; Oh, N. Y.; Rohde, J.-U.; Song, W. J.;
Stubna, A.; Kim, J.; Muenck, E.; Nam, W.; Que, L., Jr. J. Am. Chem. Soc.
2004, 126, 472–473.
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2000, 56, 4629–4638.
(19) Heinze, K.; Wild, U.; Beckmann, M. Eur. J. Inorg. Chem. 2007,
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Mascharak, P. K. J. Am. Chem. Soc. 2008, 130, 6650.
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2007, 180–182.
(20) Cheng, R. P.; Fisher, S. L.; Imperiali, B. J. Am. Chem. Soc. 1996, 118,
11349–11356.
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J. Am. Chem. Soc. 2000, 122, 11517–11518.
(21) Imperiali, B.; Roy, R. S. J. Org. Chem. 1995, 60, 1891–4.
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1613–16.
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(25) Stephenson, K. A.; Zubieta, J.; Banerjee, S. R.; Levadala, M. K.;
Taggart, L.; Ryan, L.; McFarlane, N.; Boreham, D. R.; Maresca, K. P.;
Babich, J. W.; Valliant, J. F. Bioconjugate Chem. 2004, 15, 128–136.
(26) Stephenson, K. A.; Reid, L. C.; Zubieta, J.; Babich, J. W.; Kung,
M.-P.; Kung, H. F.; Valliant, J. F. Bioconjugate Chem. 2008, 19, 1087–1094.
(41) Mialane, P.; Nivorojkine, A.; Pratviel, G.; Azema, L.; Slany, M.;
Godde, F.; Simaan, A.; Banse, F.; Kargar-Grisel, T.; Bouchoux, G.; Sainton,
J.; Horner, O.; Guilhem, J.; Tchertanova, L.; Meunier, B.; Girerd, J.-J. Inorg.
Chem. 1999, 38, 1085–1092.
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Kalapugama, S.; Kodanko, J. J. Inorg. Chem. 2009, 48, 7729–7739.
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8078–8080.
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SCHEME 1. Retrosyntheses of Unnatural Amino Acids 1-3
FIGURE 3. Schematic diagram for incorporating metal-binding
units into peptide chains using a divergent strategy.
proof of concept that these polypyridyl ligands can be
attached to peptides, bind metal ions, and produce reactive
intermediates much like their parent ligands.
the R-position of an amino acid with a 2-(hydroxymethyl)-
pyridyl group. Retrosynthetic analyses envisioned for the
preparation of these three unnatural amino acids are pre-
sented in Scheme 1. The unnatural amino acid 1 (2-hydroxy-
methyl-5-pyridyl alanine, HPA) was designed so that the
2-(hydroxymethyl)pyridyl unit is separated from the R-posi-
tion by a one-carbon spacer and hence resembles phenyl-
alanine. Retrosynthetically, the unnatural amino acid 1 was
disconnected to protected glycine derivatives 445 or 545 and
bromide 6. Next, to aid the flexibility in designing of peptide-
ligand conjugates, amino acid 2 was envisioned possessing a
three-carbon linker between the R-position and 2-(hydroxy-
methyl)pyridyl group. This amino acid contains norvaline,
and hence it is abbreviated as HPN (2-hydroxymethyl-
5-pyridyl norvaline). Sonogashira coupling between propar-
gylglycine derivative 746 and precursor 847,48 was considered
as the most plausible synthetic route for the preparation of
HPN (3). The third unnatural amino acid 3 [(2-hydroxy-
methyl-5-pyridyl methyloxy)-3-alanine, HPMA] was envi-
sioned as an oxygen-containing variant of HPN. The
synthetic disconnection for HPMA involved O-alkylation
of serine derivative 949 by bromide 6. This route holds the
advantage that the asymmetric center in 3 would be derived
from Ser.
Herein we report a divergent method for preparing
peptide-ligand conjugates based on the polypyridyl amine
scaffold. Recognizing that the pyridyl ring is a core element
in the ligands shown in Figure 2, three unnatural amino acids
containing the pyridylmethyl group were designed to act as
anchors for the site-specific incorporation of polypyridyl
ligands into peptides. The enantioselective syntheses of these
amino acids are discussed, as well as a synthetic strategy that
creates multiple metal-binding peptides with varying denti-
city from a single peptide precursor, both in solution and on
solid phase.
Results and Discussion
The strategy envisioned for incorporating polypyridyl
ligands into peptide chains is shown in Figure 3. Unnatural
amino acids 1-3 were designed so that the 2-pyridylmethyl-
ene group, a common fragment of several nonheme ligands
(Figure 2), is bound at the end of the side chain. After
introducing these amino acids into a peptide chain, trans-
formation of the silyloxy functionality into a leaving group,
followed by N-alkylation with secondary amines, could be
used to attach various ligands to the peptide in a divergent
fashion. In addition, changing the position of the unnatural
amino acid within the peptide chain could facilitate the
insertion of ligands at various positions. Furthermore, avail-
ability of three amino acids with various linkers, between the
R-position and 2-(hydroxymethyl)pyridyl group, would pro-
vide additional flexibility in designing peptide-ligand con-
jugates. This combination of structural, positional, and
metal-binding ligand flexibilities would expedite the synthe-
sis of libraries of metal-binding peptides.
Synthesis of Unnatural Amino Acids 1-3. To begin the
synthesis of unnatural amino acids 1-3, precursors 4,45 5,45
7,46 8,47,48 and 949 were prepared by previously reported
(45) O’Donnell, M. J.; Polt, R. L. J. Org. Chem. 1982, 47, 2663–6.
(46) Middleton, M. D.; Peppers, B. P.; Diver, S. T. Tetrahedron 2006, 62,
10528–10540.
(47) Kodanko, J. J.; Morys, A. J.; Lippard, S. J. Org. Lett. 2005, 7, 4585–
4588.
(48) Guthikonda, R. N.; Cama, L. D.; Quesada, M.; Woods, M. F.;
Salzmann, T. N.; Christensen, B. G. J. Med. Chem. 1987, 30, 871–80.
(49) Nakajima, K.; Takai, F.; Tanaka, T.; Okawa, K. Bull. Chem. Soc.
Jpn. 1978, 51, 1577–8.
Design and Retrosynthesis of Unnatural Amino Acids 1-3.
Three unnatural amino acids were designed to provide
variation in linker composition and length by connecting
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SCHEME 2. Synthesis of Pyridylmethyl Bromide 6
SCHEME 3. Synthesis of Fmoc-HPA(OTBS)-OH (1)
procedures. The route to compound 6 began from alcohol
10, which is a known compound available in one step from
the commercially available starting material dimethyl pyr-
idine-2,5-dicarboxylate (Scheme 2). Compound 10 was
accessed by selective reduction of the diester in the presence
of NaBH4/CaCl2 using a slight modification of the previously
described procedure.50 Upon scale-up using the previously
reported procedure, in which THF/EtOH was used as a
solvent mixture, transesterification to form mixtures of
methyl and ethyl esters was observed. Use of THF/MeOH
as a solvent mixture under otherwise identical reaction
conditions furnished 10 with better yields. After optimizing
conditions to provide 10 on multigram scale, protection
of alcohol 10 with TBSCl, followed by reduction of C-5
ester moiety with sodium bis(2-methoxyethoxy)aluminum
hydride gave the corresponding alcohol 11 in 78% yield over
two steps. Importantly, no chromatography was required in
this three-step sequence. Transformation of alcohol 11 to
pyridylmethyl bromide 6 was achieved using NBS and PPh3,
which provided 6 in 92% yield from 11.44
With all precursors in hand, the synthesis of 1 was devel-
oped using an asymmetric alkylation51 as the key step
(Scheme 3). First, the alkylation reaction was carried out
to furnish racemic product. Reaction of 4 with bromide 6
using the achiral phase-transfer catalyst (PTC) Bu4NHSO4
furnished racemic 12 in 82% yield (Scheme 3).21,52 Incorpor-
ating the benzyl protecting group was advantageous because
deprotection of 12 by hydrogenolysis53 could be used to
access 14, which was converted into Fmoc-HPA(OTBS)-OH
(1) in a separate step. Unfortunately alkylation reactions
between 4 and 6 with chiral PTCs gave 12 in good yield
(74-88%) but low to moderate ee (11-72%) under a variety
of conditions (Table 1, entries 3 and 6). Initial optimization
studies established that use of substrate 5 (R = t-Bu) instead
of 4 (R = Bn)52 along with N-(9-methylanthracenyl)-O-allyl-
TABLE 1. Optimization of Asymmetric Alkylation Reaction
entry
PTCa,b(mol %)
R
temp (°C)
% yield (% eeb,c
)
1
2
3
4
5
6
7
8
9
TBAHd (20)
TBAI (20)
15 (20)
15 (20)
16 (20)
16 (20)
16 (20)
16 (20)
16 (5)
Bn
t-Bu
Bn
Bn
Bn
Bn
t-Bu
t-Bu
t-Bu
0
0
0
82 (0)f
70 (0)f
88 (11)f
10 (nde)g
46 (79)g
74 (72)g
87 (92)g
80 (93)f
70 (93)f
-78
-78
-30
-30
0
0
aPhase-transfer catalyst 15 = N-benzylcinchonidine chloride; 16 =
N-(9-methylanthracenyl)-O-allylcinchonidine bromide. bSee the Sup-
porting Information for structures of 15 and 16 and determination of
ee. cAbsolute configuration of major enantiomers of 12 and 13 is based
on literature precedent (refs 22, 52, and 54,). dTetrabutylammonium
hydrogensulphate (Bu4NHSO4). end = not determined. f50% aq
NaOH, toluene/CH2Cl2 (7:3). gCsOH H2O, CH2Cl2.
3
able to scale up. Toward this goal, the solid CsOH H2O base
3
was replaced with 50% aq NaOH to furnish 13 with similar
yields and ee when the reaction was performed in toluene:
CH2Cl2 (7:3) at 0 °C (entry 8). Using these conditions, the
catalyst loading could be lowered to 5 mol % (Entry 9).
Deprotection of the C- and N-termini of 13 by treatment with
NH2OH HCl55 followed by TMSOTf/2,6-lutidine56 gave 14,
3
albeit in low isolated yield. This required us to adopt an
alternative strategy. Global deprotection of 13 with 6N HCl,
followed by replacement of the TBS group and Fmoc protec-
tion gave 1 in 80% yield over the three steps, without the need
for isolating 14.
Sonogashira coupling was chosen as the key step toward
the synthesis of HPN amino acid 2 because previous studies
indicated that propargylglycine derivatives had been used in
arylation reactions.57,58 In order to minimize cumbersome
protecting group manipulations, alkyne 7 was used directly
as the coupling partner. Although alkyne 7 is available in one
step and 93% ee from propargyl bromide and imine 5,46 two
cinchonidine bromide (16) as a chiral PTC and CsOH H2O
3
as a base at -30 °C furnished 13 in 87% yield and 92% ee
(entry 7).54 Although the reaction conditions could be used to
prepare gram quantities of 13, the reaction was further
optimized to obtain conditions that were more easily amen-
(50) Chong, H.-S.; Torti, S. V.; Ma, R.; Torti, F. M.; Brechbiel, M. W.
J. Med. Chem. 2004, 47, 5230–5234.
(51) O’Donnell, M. J. Acc. Chem. Res. 2004, 37, 506–517.
(52) O’Donnell, M. J.; Bennett, W. D.; Wu, S. J. Am. Chem. Soc. 1989,
111, 2353–5.
(55) Loegers, M.; Overman, L. E.; Welmaker, G. S. J. Am. Chem. Soc.
1995, 117, 9139–50.
(56) Meng, D.; Bertinato, P.; Balog, A.; Su, D.-S.; Kamenecka, T.;
Sorensen, E.; Danishefsky, S. J. J. Am. Chem. Soc. 1997, 119, 10073–10092.
(57) Brea, R. J.; Lopez-Deber, M. P.; Castedo, L.; Granja, J. R. J. Org.
Chem. 2006, 71, 7870–7873.
(53) Corey, E. J.; Noe, M. C.; Xu, F. Tetrahedron Lett. 1998, 39, 5347–
5350.
(54) Corey, E. J.; Xu, F.; Noe, M. C. J. Am. Chem. Soc. 1997, 119, 12414–
12415.
(58) Lopez-Deber, M. P.; Castedo, L.; Granja, J. R. Org. Lett. 2001, 3,
2823–2826.
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Jabre et al.
SCHEME 5. Synthesis of Fmoc-HPMA(OTBS)-OH (3)
JOCArticle
SCHEME 4. Synthesis of Fmoc-HPN(OTBS)-OH (2)
N-Cbz, or N-Trt protected Ser derivatives with 6 in biphasic
solvent mixtures. Optimum yields were obtained when Trt-
Ser-OBn (9) was treated with 6 in the presence of TBAB as a
phase-transfer catalyst in a mixture of benzene and 40%
aqueous NaOH, giving alkylation product 19 in 66% yield
(Scheme 5). The synthetic route for HPMA was advanta-
geous not only because installation of a new chiral center was
not necessary, as in the syntheses of 1 and 2, but also because
hydrogenolysis could be used to remove both the N- and
C-terminal protecting groups in 19 in a single step while
leaving the TBS protecting group intact. Subsequent Fmoc
protection furnished 3 in 55% over the two steps.
Incorporation of Metal-Binding Units into a Peptide via a
Solution-Phase Method. Having developed scalable synthe-
ses of amino acids 1-3, we sought to develop a divergent
method for ligand incorporation. Toward this goal, the
model dipeptide 20 was synthesized in order to develop con-
ditions for attaching ligands using solution-phase methods
(Scheme 6). Dipeptide 20, prepared in racemic form for these
studies, was accessed by the acetylation of 14 followed by
coupling of the resultant acid with H-Gly-OBn using EDAC
and HOBt, which gave 20 in 60% yield over the two steps.
The silyl ether group of 20 was transformed into chloride 21
in a two-step sequence involving deprotection with a mixture
of TBAF and AcOH in THF, followed by treatment with a
combination of TsCl and excess LiCl in MeCN. Attachment
of the amine side chains by N-alkylation was carried out by
heating mixtures of chloride 21, 3.0 equiv of the appropriate
secondary amine (NHR1R2),40,62 and excess i-Pr2EtN in
acetonitrile at 55 °C. These conditions furnished products
22 (TPA derivative) and 23 (Bn-TPEN derivative) in good to
excellent yields (79-95%). However, formation of the N4Py
derivative 24 occurred in low yield under these conditions,
presumably due to the steric hindrance of the amine nucleo-
phile.14 Addition of a catalytic amount of NaI improved the
yield dramatically and provided 24 in 90% yield.
commercially available starting materials, Sonogashira coupl-
ing reactions with 7 had not been reported in the past.
Gratifyingly, the arylation of 7 with bromopyridine 847,48
proceeded smoothly at 60 °C in the presence of catalytic
amounts of CuI (16 mol %) and Pd(PPh3)4 (10 mol %),
furnishing the coupled product (Scheme4). Purificationat this
stage proved difficult due to catalyst decomposition products
that could not be separated from the desired product, so the
benzophenone imine and TBS group wereremoved inone step
by treatment with dilute aqueous citric acid, giving 17 in 73%
yield over the two steps. At this stage, the ee of 17 was
determined to be 93%, identical to compound 7, confirming
that no epimerization took place during the coupling or
deprotection sequence. Subsequent reduction of the alkyne
was carried out using 5% Pd(OH)2 and H2 (100 psi) in MeOH,
giving 18 in 98% yield. To complete the synthesis of Fmoc-
HPN(OTBS)-OH (2), hydrolysis of the tert-butyl ester using
6 N HCl, followed by Fmoc protection and replacement of the
TBS group, furnished 2 in 57% yield over the three steps.59
In order to gain access to the amino acid HPMA, the
O-alkylation of Ser with bromide 6 was chosen because it has
been demonstrated in the literature,60,61 and the correspond-
ing precursors are easily accessed. Initial trials for O-alkyla-
tion of N-Boc-, N-Cbz-, or N-Trt-protected Ser derivatives
with 6 in the presence of a variety of strong or weak bases
such as NaH, NaHMDS, KHMDS, Et3N, i-Pr2EtN, or
K2CO3 gave either decomposition products or no reaction.
With strong bases, bromide 6 was found to decompose.
Alkylation under phase-transfer conditions was pursued as
an alternate method because bromide 6 was demonstrated to
be stable under these conditions during the synthesis of 1.
The O-alkylation reaction was attempted by treating N-Boc,
Extension of the Methodology to Resin-Bound Peptides.
After successful incorporation of metal-binding units into
model dipeptide 20 via solution-phase methods, the transi-
tion to solid-phase synthesis was made. Starting from Fmoc-
Gly bound to Rink amide resin, the sequence began
by deprotection of the Fmoc group, coupling with Fmoc
amino acids 1, 2, or 3 using reagents DCC and HOBt or
alternatively HBTU, followed by Fmoc deprotection and
(59) Attempts were made to deprotect the t-Bu ester under mild condi-
tions that would preserve the TBS group in moving from 7 to 2, but overall
yields were considerably lower.
(60) Palmer, M. J.; Danilewicz, J. C.; Vuong, H. Synlett 1994, 171–2.
(61) Faul, M. M.; Winneroski, L. L.; York, J. S.; Reinhard, M. R.;
Hoying, R. C.; Gritton, W. H.; Dominianni, S. J. Heterocycles 2001, 55,
689–704.
(62) Baffert, C.; Collomb, M.-N.; Deronzier, A.; Kjaergaard-Knudsen,
S.; Latour, J.-M.; Lund, K. H.; McKenzie, C. J.; Mortensen, M.; Nielsen, L.
P.; Thorup, N. Dalton Trans. 2003, 1765–1772.
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SCHEME 6. Incorporation of Metal Binding Units Using Solution-Phase Synthesis
SCHEME 7. Incorporation of Metal Binding Units Using SPPSa
aConditions: (i) 20% piperidine, rt, 20 min (two cycles); (ii) Fmoc-AA-OH, DCC, HOBt or HBTU, i-Pr2EtN, DMF, rt, 3-4 h; (iii) Ac2O, i-Pr2EtN,
DMF, rt, 1 h; (iv) TBAF, AcOH, THF, rt, 1 h (two cycles); (v) LiCl, TsCl, i-Pr2EtN, MeCN, rt, 6 h; (vi) NHR1R2, i-Pr2EtN, MeCN, NaI, 55 °C, 24 h;
(vii) TFA/H2O (9:1), rt, 1 h.
N-acetylation with Ac2O, which gave resin-bound dipeptides
25-27. Next, the silyloxy ether groups of dipeptides (25-27)
were converted into the corresponding chlorides using the
conditions shown in Scheme 6.63 Using the appropriate
secondary amines, five dipeptide derivatives (28-32)
were obtained from these chlorides after cleavage from the
resin with TFA/H2O (95:5). HPLC analysis indicated that
products obtained from the resin were of >80% purity.
(63) In some cases, it was necessary to resubject pyridylmethyl alcohols to
the chlorination conditions (LiCl, TsCl, i-Pr2EtN) to observe complete
conversion to the chloride. See the Supporting Information for further
details.
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SCHEME 8. Synthesis of Heptapeptide 38
TBS protecting group, chlorination, and substitution with
bis(pyridin-2-ylmethyl)amine was used to install the full
metal-binding unit TPA. Global deprotection and cleavage
from the resin using Reagent K furnished the heptapeptide
38. These results confirm that peptides with a rich array of
functional groups can be prepared by this method.
Conclusion
In summary, we have presented a divergent strategy for
attaching polypyridyl ligands to peptides. Three unnatural
amino acids (1-3) were synthesized in enantioenriched form
that can be used in SPPS. All together, the availability of
three unnatural amino acids, flexibility to incorporate them
anywhere into a peptide chain, and the ability to attach a
variety of ligands by varying the secondary amine nucleo-
philes, makes this methodology highly divergent and ideal
for the synthesis of peptide-ligand conjugate libraries.
These studies are now underway in our laboratory.
Experimental Section
General Considerations. All reagents were purchased from
commercial suppliers and used as received. HPLC was per-
formed on a preparative purification system equipped with a
multiwavelength detector. Column purifications were per-
formed using silica gel flash chromatography unless mentioned
otherwise. Compounds 4,45 5,45 6,44 7,46 8,47,48 9,49 16,54 20,44
21,44 and 2444 used in this report were synthesized according to
previously reported literature procedures. All reactions were
performed under ambient atmosphere unless otherwise noted.
Anaerobic reactions were performed in Schlenk tubes. These
reactions were deoxygenated by performing five vacuum-back-
fill cycles with Ar and were run under a constant purge of
Ar. For anaerobic reactions, Et3N and DMF were distilled over
CaH2. THF was deoxygenated by bubbling Ar through a
submerged needle, before use. Toluene and dichloromethane
All combinations, except the N4Py derivative derived from
HPMA, were prepared by this method. Solution-phase
studies (not shown) proved that complex mixtures resulted
from attempts to aminate the chloride derived from HPMA
with hindered amine nucleophiles. Presumably, an elimina-
tion pathway to form dehydroalanine competes with the
substitution reaction with bulky nucleophiles. To illustrate
the utility of our method further, two tetrapeptides (33 and
34) were constructed in a similar manner each containing
two unnatural amino acids (HPA or HPN). Both unnatural
amino acids were converted into TPA ligands using the
aforementioned reaction conditions (35 and 36). All three
steps, (i) TBS cleavage, (ii) chlorination, and (iii) N-alkyla-
tion, proceeded cleanly (Scheme 7). Success in the syntheses
of peptides 35 and 36 confirms that reaction conditions used
for attaching ligands to peptides were not causing epimeriza-
tion. Overall, the divergent methodology described herein is
not limited to attaching one ligand onto a peptide chain but
can be used to link multiple ligands to the same peptide
chain.
In order to demonstrate the scope of this divergent
method, the heptapeptide 37 was prepared (Scheme 8). First
the pentapeptide Fmoc-Leu-Met-Val-Asn(Trt)-Gly bound
to Rink amide resin was synthesized using a standard pro-
tocol on an automated peptide synthesizer. After deprotec-
tion of the Fmoc group, coupling of the amino acids 2 and
Fmoc-Trp(Boc)-OH was carried out manually. Cleavage of
the Fmoc group and capping of the N-terminus with Ac2O
furnished resin-bound peptide 37. Selective removal of the
˚
were dried over 4 A molecular sieves before use.
General Procedures for Preparation of 12 and 13. General
Procedure A. A mixture of 4 or 5 (16.7 mmol), PTC (See Table 1),
and 7:3 toluene/CH2Cl2 (72 mL) was cooled to 0 °C under
a nitrogen atmosphere. A 50% aqueous NaOH solution (24.5
mL) was added at 0 °C. 5-(Bromomethyl)-2-((tert-butyldi-
methylsilyloxy)methyl)pyridine 6 (5.30 g, 16.7 mmol) was dis-
solved in 7:3 toluene/CH2Cl2 (11 mL), and this solution was
added dropwise to the reaction mixture at 0 °C over 10 min. The
reaction mixture was stirred vigorously at 0 °C for 3-12 h. After
consumption of the starting material as judged by TLC analysis
(20% EtOAc/hexanes), the reaction mixture was diluted with
cold H2O (40 mL). The organic layer was separated and the
aqueous layer was extracted using CH2Cl2 (3 ꢀ 20 mL). The
combined organic layer was dried over anhydrous Na2SO4 and
concentrated to obtain a brown viscous oil. The crude product
was purified by silica gel chromatography (5% to 15% EtOAc/
hexanes) to afford corresponding product 12 or 13.
General Procedure B. A mixture of 4 or 5 (0.79 mmol), 15 or 16
(See Table 1), 5-(bromomethyl)-2-((tert-butyldimethylsilyloxy)-
methyl)pyridine 6 (250 mg, 0.79 mmol), and CH2Cl2 (2.3 mL)
was cooled to the appropriate temperature (see Table 1) under a
nitrogen atmosphere. Solid CsOH H2O (1.32 g, 7.89 mmol) was
3
added, and the reaction mixture was stirred vigorously for 3-4
h. After consumption of the starting material as judged by TLC
analysis (20% EtOAc/hexanes), the reaction mixture was
diluted with diethyl ether (20 mL) and then combined with cold
water (30 mL). The organic layer was separated, and the
aqueous layer was extracted with CH2Cl2 (3 ꢀ 15 mL). The com-
bined organic layers were dried over anhydrous Na2SO4 and
656 J. Org. Chem. Vol. 75, No. 3, 2010

Jabre et al.
JOCArticle
concentrated to get brown viscous oil. The crude product was
purified by silica gel chromatography (5% to 15% EtOAc/
hexanes) to afford 12 or 13.
From Compound 13. A solution of compound 13 (2.70 g,
5.09 mmol) and 6 N HCl (13.5 mL) was refluxed for 6 h. The
reaction mixture was combined with H2O (30 mL) and extracted
with diethyl ether (3 ꢀ 10 mL). The aqueous layer was concen-
trated under high vacuum to obtain a crude product. The crude
product was dissolved in H2O (20 mL) and concentrated under
high vacuum (twice) to remove excess HCl. The crude product
was dried on high vacuum yielding an off-white solid, which was
found to be hygroscopic and stored under nitrogen.
(S)-Benzyl 3-(6-((tert-Butyldimethylsilyloxy)methyl)pyridin-
3-yl)-2(diphenylmethyleneamino)propanoate (12). Compound
12 was synthesized in racemic form by general procedure A
while the corresponding enantioenriched form was prepared
using general procedure B: 1H NMR (400 MHz, CDCl3) δ 8.20
(s, 1H), 7.55 (d, J = 7.3 Hz, 2H), 7.38-7.22 (m, 13H), 6.63 (d,
J = 7.3 Hz, 2H), 5.19 (d, J = 13.0 Hz, 1H), 5.12 (d, J = 13.0 Hz,
1H), 4.76 (s, 2H), 4.26 (dd, J = 8.9, 4.9 Hz, 1H), 3.27-3.16 (m,
2H), 0.91 (s, 9H), 0.72 (s, 6H); 13C NMR (100 MHz, CDCl3) δ
171.5, 171.1, 159.3, 149.7, 139.0, 138.0, 135.9, 135.7, 131.4,
130.5, 128.8, 128.5, 128.4, 128.3, 128.2, 128.1, 128.0, 127.4,
119.5, 66.7, 66.6, 65.9, 36.4, 25.9, 18.3, -5.4; IR (thin film)
3061, 3032, 2954, 2928, 2885, 2856, 1741, 1622, 1599, 1574, 1489,
1471, 1446, 1397, 1372, 1314, 1287, 1252, 1163, 1128, 1104, 1029,
1004, 939, 910, 838, 779, 696, 640 cm-1; LRMS (ESMS) calcd
for C35H41N2O3Si (M þ H)þ 565, found 565.
A mixture of the off-white solid (1.10 g, 4.00 mmol), Et3N
(8.3 mL, 60.0 mmol), and DMF (22 mL) was maintained at rt
under nitrogen atmosphere for 5 min. A suspension of TBSCl
(6.20 g, 40.0 mmol) in DMF (6 mL) was added under nitrogen
atmosphere, and the reaction mixture was maintained for 2 h.
The reaction mixture was combined with Na2CO3 (0.1% aq,
150 mL) and washed with diethyl ether (3 ꢀ 20 mL). A colorless
solid formed that was isolated and combined with the aqueous
layer, which was cooled to 0 °C, and a solution of FmocOSu
(1.50 g, 4.50 mmol) in 1,4-dioxane (12 mL) was added over
5 min. The reaction mixture was maintained at 0 °C for 1 h and
then warmed to rt. The reaction mixture was maintained at rt
overnight, combined with H2O (160 mL), and extracted with
hexanes (2 ꢀ 200 mL). The aqueous solution was acidified to pH
4-5 by slow addition of 10% aqueous citric acid. The aqueous
solution was extracted with EtOAc (4 ꢀ 50 mL). The combined
organic layer was dried over anhydrous Na2SO4 and concen-
trated to obtain the product. The crude product was further
purified by silica gel chromatography (0-5% MeOH/CH2Cl2)
(2.00 g, 74%). A small sample was recrystallized from MTBE/
hexanes (1:2) for analysis: mp = 81-83 °C; 1H NMR (400 MHz,
CD3OD) δ 8.34 (s, 1H), 7.76-7.70 (m, 3H), 7.57-7.43 (m, 3H),
7.38-7.24 (m, 4H), 4.72 (s, 2H), 4.46-4.41 (m, 1H), 4.34-4.28
(m, 1H), 4.19-4.07 (m, 2H), 3.30-3.20 (m, 1H), 3.03-2.93 (m,
1H), 0.91 (s, 9H), 0.06 (s, 6H); 13C NMR (100 MHz, CD3OD) δ
175.2, 160.2, 158.2, 149.8, 145.2, 142.5 139.8, 133.7, 128.7, 128.1,
126.3, 126.2, 121.5, 120.9, 67.9, 66.3, 56.6, 35.7, 26.3, 19.2, -5.3;
IR (KBr) 3419 (b), 3065, 2953, 2928, 2887, 2856, 1713, 1606,
1536, 1450, 1401, 1335, 1253, 1106, 1053, 1007, 839, 779,
759, 740, 671, 621 cm-1; [R] = -2.5° (c = 1.1, MeOH); HRMS
(ESMS) calcd for C30H37N2O5Si (M þ H)þ 533.2472, found
533.2481.
(S)-tert-Butyl 2-Amino-5-(6-(hydroxymethyl)pyridin-3-yl)-
pent-4-ynoate (17). (S)-tert-Butyl 2-(diphenylmethyleneamino)-
pent-4-ynoate 7 (2.81 g, 8.44 mmol), 5-bromo-2-((tert-butyl-
dimethylsilyloxy)methyl)pyridine 8 (3.03 g, 10.1 mmol), Pd(PPh3)4
(975 mg, 0.84 mmol), CuI (257 mg, 1.35 mmol), Et3N (8.53 g,
84.4 mmol), and THF (85.0 mL) were combined in a sealed tube
under nitrogen atmosphere in a drybox, resulting the formation of a
dark brown solution. The sealed tube was heated at 60 °C. Over
time, formation of a colorless precipitate was observed. After 48 h,
THF was removed under vacuum. The resulting residue was dis-
solved in water (50 mL) and extracted with EtOAc (3 ꢀ 150 mL).
The combined organic layer was dried over anhydrous Na2SO4 and
concentrated. The crude product was purified by silica gel chroma-
tography (0% to 10% EtOAc/hexanes) to give the product as a
yellow oil (3.75 g, 80%), which contained trace amounts of PPh3
from decomposition of the Pd catalyst.
(S)-tert-Butyl 3-(6-((tert-Butyldimethylsilyloxy)methyl)pyri-
din-3-yl)-2-(diphenylmethyleneamino)propanoate (13). Compound
13 was synthesized in racemic form by general procedure A and in
enantioenriched form using either general procedure A or B (see
Table 1): 1H NMR (400 MHz, CDCl3) δ 8.23 (s, 1H), 7.58 (d, J =
7.3 Hz, 2H), 7.43-7.26 (m, 8H), 6.72 (d, J = 6.5 Hz, 2H), 4.79 (s,
2H), 4.13-4.10 (m, 1H), 3.20-3.14 (m, 2H), 1.43 (s, 9H), 0.92 (s,
9H), 0.08 (s, 6H); 13C NMR (100 MHz, CDCl3) δ 170.4, 170.0,
158.8, 149.4, 138.9, 137.7, 135.9, 131.5, 129.9, 128.4, 128.0, 127.9,
127.6, 127.1, 119.1, 88.0, 66.9, 65.6, 36.1, 27.6, 25.5, 18.0, -5.7; IR
(thin film) 3059, 2955, 2929, 2885, 2856, 1734, 1624, 1599, 1574,
1488, 1472, 1462, 1446, 1394, 1368, 1314, 1288, 1253, 1151, 1104,
1030, 1006, 977, 938, 910, 840, 815, 779, 731, 696, 670, 641 cm-1
;
[R] = -132 (c = 1.0, MeOH); HRMS (ESMS) calcd for C32H43-
N2O3Si (M þ H)þ 531.3043, found 531.3037.
(S)-2-Amino-3-(6-((tert-butyldimethylsilyloxy)methyl)pyridin-
3-yl)propanoic Acid (14). A mixture of 12 (6.15 g, 10.9 mmol),
MeOH (123 mL), and 5% Pd/C (615 mg) was stirred at rt under
H2 (1 atm) for 4 h. After consumption of the starting material, as
judged by TLC analysis, the reaction mixture was filtered
through Celite. The filtrate was concentrated to obtain a sticky
colorless solid, which upon triturating with Et2O for 30 min
furnished 14 as a colorless solid. The colorless solid was isolated
by filtration and washed with Et2O (2.60 g, 77%): mp = 165 °C
dec; 1H NMR (400 MHz, CD3OD) δ 8.38 (s, 1H), 7.81 (dd, J =
8.1, 2.4 Hz, 1H), 7.54 (d, J = 7.3 Hz, 1H), 4.78 (s, 2H), 3.81 (dd,
J = 8.1, 4.9 Hz, 1H), 3.30-3.10 (m, 2H), 0.96 (s, 9H), 0.13 (s,
6H); 13C NMR (100 MHz, CD3OD) δ 173.2, 161.1, 150.0, 139.8,
132.0, 121.9, 66.5, 57.0, 35.0, 26.3, 19.2, -5.3; IR (KBr) 3405 (b),
2956, 2930, 2887, 2858, 1604, 1524, 1492, 1472, 1463, 1442, 1398,
1361, 1333, 1254, 1108, 838, 778 cm-1; LRMS (ESMS) calcd for
C15H27N2O3Si (M þ H)þ 311, found: 311.
(S)-2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-3-(6-((tert-
butyldimethylsilyloxy)methyl)pyridin-3-yl)propanoic Acid
(1). FromCompound 14. A mixture of compound 14 (1.00 g,
3.22 mmol), Na2CO3 (324 mg, 3.06 mmol), and H2O (10 mL)
was cooled at 0 °C. A suspension of FmocOSu (1.09 mg, 3.22
mmol) in acetone (8 mL) was added, and the reaction mixture
was maintained at 0 °C for 1 h. The reaction mixture was
warmed to rt and maintained overnight. The reaction mixture
was maintained at pH 7-8 during this period. After completion
of the reaction, as judged by TLC analysis, the reaction mixture
was combined with H2O (130 mL) and the aqueous layer was
washed with hexanes (2 ꢀ 30 mL). The aqueous layer was
acidified to pH 4-5 by slow addition of 10% aqueous citric
acid and extracted with EtOAc (5 ꢀ 30 mL). The combined
EtOAc layers were washed with NaCl (satd aq). The EtOAc
layer was dried over anhydrous Na2SO4 and concentrated to
obtain crude product (1.14 g, 67%).
A mixture of yellow oil (2.36 g, 4.24 mmol), 15% aqueous
citric acid (14 mL), and THF (27 mL) was maintained at rt for
12 h. The organic layer was concentrated under vacuum, and
the resulting residue was dissolved with 1 N HCl (5 mL). The
aqueous solution was extracted with diethyl ether (3 ꢀ 75 mL).
The aqueous layer was basified using Na2CO3 (aq., pH = 9-10)
and extracted with EtOAc (3 ꢀ 150 mL). The combined orga-
nic layer was dried over anhydrous Na2SO4 and concentra-
ted to give 17 as a yellow oil (1.07 g, 91%): 1H NMR (400
MHz, CD3OD) δ 8.47 (s, 1H), 7.82 (d, J = 8.1 Hz, 1H), 7.50
(d, J = 8.1 Hz, 1H), 4.67 (s, 2H), 3.59-3.30 (m, 1H), 2.85-2.80
J. Org. Chem. Vol. 75, No. 3, 2010 657

Jabre et al.
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(dd, J = 16.2, 4.9 Hz, 2H). 1.47 (s, 9H); 13C NMR (100 MHz,
CD3OD) δ 174.0, 161.6, 151.8, 121.5, 120.4, 89.7, 82.8, 80.5,
65.3, 54.6, 28.3, 26.4; IR (thin film) 3342, 3293, 2977, 1916, 1732,
1701, 1594, 1556, 1487, 1459, 1420, 1393, 1338, 1302, 1238, 1157,
1064, 1028, 954, 846 cm-1; [R] = -6.9 (c = 1.0, MeOH); LRMS
(ESMS) calcd for C15H21N2O3 (M þ H)þ 277, found 277.
(S)-tert-Butyl 2-Amino-3-(6-(hydroxymethyl)pyridin-3-yl)pen-
tanoate (18). A mixture of (S)-tert-butyl 2-amino-5-(6-(hydroxy-
methyl)pyridin-3-yl)pent-4-ynoate 17 (1.12 g, 4.06 mmol), Pd-
(OH)2/C (112 mg, 5% w/w), and MeOH (25 mL) was stirred at
rt under H2 (100 psi) for 3 h. The reaction mixture was filtered
through a Celite bed to remove Pd/C, and the filtrate was
layer was extracted with EtOAc (3 ꢀ 5 mL). The combined organic
layer was dried over anhydrous Na2SO4 and concentrated to obtain
crude product. The resulting crude was purified by silica gel
chromatography (2-10% EtOAc/hexanes) to obtain 19 as a color-
less oil (132 mg, 66%): 1H NMR (400 MHz, CD3OD) δ8.30(d, J=
1.6 Hz, 1H), 7.61 (dd, J = 8.1, 1.6 Hz, 1H), 7.46-7.45 (m, 7H),
7.23-7.13 (m, 14H), 4.78 (s, 2H), 4.74 (d, J = 13.0 Hz, 1H), 4.52
(d, J = 13.0 Hz, 1H), 4.42 (d, J = 7.3 Hz, 2H), 3.70 (dd, J = 8.9,
4.1 Hz, 1H), 3.56-3.47 (m, 2H), 0.96 (s, 9H), 0.12 (s, 6H);13CNMR
(100MHz, CD3OD) δ173.9, 160.3, 147.3, 146.0, 137.1, 135.9, 132.8,
128.7, 128.2, 128.0, 127.9, 127.8, 126.5, 120.4, 72.6, 71.0, 70.0, 66.5,
65.4, 56.7, 25.2, 18.0, -6.4; IR (thin film) 3061, 3032, 2955, 2928,
2887, 2854, 1734, 1601, 1489, 1471, 1456, 1448, 1371, 1362, 1254,
1182, 1174, 1128, 1101, 837, 777, 746, 706 cm-1; [R] = þ23.6° (c =
1.7, MeOH); HRMS (ESMS) calcd for C42H49N2O4Si (M þ H)þ
673.3462, found 673.3459.
(S)-2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-3-((6-((tert-
butyldimethylsilyloxy)methyl)pyridin-3-yl)methoxy)propanoic
Acid (3). A mixture of compound 19 (114 mg, 0.170 mmol),
Pd(OH)2/C (28 mg, 5% w/w), and MeOH (10 mL) was stirred
at rt under H2 (1 atm) for 12 h. The reaction mixture was
filtered through a Celite bed to remove Pd/C, and the filtrate
was concentrated to give a colorless solid.
A mixture of the colorless solid (100 mg, 0.170 mmol),
FmocOSu (69 mg, 0.21 mmol), and pyridine (2.8 mL) was
maintained at rt for 12 h. The reaction mixture was concentrated
to obtain crude product. The crude product was dissolved in
water (10 mL) and acidified with 5% aqueous citric acid (pH =
5-6). The aqueous layer was extracted with CH2Cl2 (3 ꢀ 30
mL). The combined organic layer was dried over anhydrous
Na2SO4 and concentrated to obtain crude product. The result-
ing crude was purified by silica gel chromatography (0-10%
MeOH/CH2Cl2) to obtain 3 as a colorless amorphous solid
(53 mg, 55%): 1H NMR (400 MHz, CD3OD) δ 8.40 (s, 1H), 7.79
(d, J = 8.1 Hz, 1H), 7.76 (d, J = 7.3 Hz, 2H), 7.63 (dd, J = 6.9,
3.2 Hz, 2H), 7.47 (d, J = 8.1 Hz, 1H), 7.36 (t, J = 7.3 Hz, 2H),
7.26 (t, J = 6.9 Hz, 2H), 4.75 (s, 2H), 7.58-7.51 (m, 2H), 4.38
(dd, J = 10.5, 7.3 Hz, 2H), 4.31-4.23 (m, 2H), 4.18 (t, J = 6.5
Hz, 1H), 3.91-3.79 (m, 2H), 0.93 (s, 9H), 0.10 (s, 6H); 13CNMR
(100 MHz CD3OD) δ 175.7, 160.2, 157.2, 147.4, 144.2, 144.1,
141.4, 137.2, 127.6, 127.0, 125.1, 125.0, 120.4, 119.8, 70.9, 70.0,
66.8, 65.3, 47.2, 25.2, 18.0, -6.5; IR (thin film) 3340 (b) 2953,
2856, 1716, 1606, 1506, 1450, 1253, 1105, 910, 839, 779, 759, 738
cm-1; [R] = þ11.3 (c = 0.97, MeOH); HRMS (ESMS) calcd for
C31H37N2O6SiNa2 (M - H þ 2Na)þ, 607.2216, found 607.2209.
General Procedure for N-Alkylation in Solution-Phase Synthe-
sis. A mixture of compound 21, corresponding secondary amine
(see the Supporting Information, part A), MeCN, and i-Pr2EtN
was heated at 55 °C for 18-20 h. The reaction mixture was
evaporated, and crude product was dissolved in CH2Cl2. The
organic layer was extracted with NaHCO3 (5% aq, 2 ꢀ 5 mL).
The organic layer was dried over anhydrous Na2SO4 and
concentrated to obtain the crude product. The yield was calcu-
lated by 1H NMR analysis using methyl m-toluate as an internal
standard. Yields for compounds 22 and 23 are 95% and 79%,
respectively.
1
concentrated to give 18 (1.10 g, 98%) as a colorless solid: H
NMR (400 MHz, CD3OD) δ 8.26 (s, 1H), 7.66 (dd, J = 8.1, 1.6
Hz, 1H), 7.44 (dd, J = 8.1, 1.6 Hz, 1H), 4.61 (s, 2H), 2.64-2.60
(m, 2H), 1.69-1.53 (m, 5H), 1.41 (s, 9H); 13C NMR (100 MHz,
CD3OD) δ 175.9, 159.8, 149.3, 138.8, 137.8, 121.9, 82.2, 65.3,
55.5, 35.2, 33.1, 28.3, 28.1; IR (KBr) 3353, 2976, 2931, 2863, 1726,
1601, 1572, 1479, 1460, 1393, 1368, 1251, 1155, 1068, 846 cm-1
;
[R] = þ3.6 (c = 1.0, MeOH); LRMS (ESMS) calcd for C15H25-
N2O3 (M þ H)þ 281, found 281.
(S)-2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-3-(6-(hydro-
xymethyl)pyridin-3-yl)pentanoic Acid (2). A mixture of (S)-tert-
butyl 2-amino-3-(6-(hydroxymethyl)pyridin-3-yl)propanoate 18
(1.10 g, 3.9 mmol) and 6 N HCl (4 mL) was refluxed for 12 h.
The reaction mixture was combined with H2O (10 mL) and
extracted with diethyl ether (2 ꢀ 50 mL). The aqueous layer was
concentrated under high vacuum to obtain a crude product. The
crude product was dissolved in H2O (10 mL) and concentrated
under high vacuum (twice) to remove excess HCl. The reaction
mixture was concentrated to give the product as a yellow solid
(1.02 g, 88%).
A mixture of the yellow solid (300 mg, 1.01 mmol), aqueous
Na2CO3 (pH = 8-9), Fmoc-OSu, and 1,4-dioxane (2 mL) was
maintained at rt. After 12 h, the reaction mixture was acidified
with 15% aqueous citric acid solution to pH 4-5 and the
aqueous layer was extracted with EtOAc (3 ꢀ 150 mL). The
combined organic layer was dried over anhydrous Na2SO4 and
concentrated. The crude product was purified by silica gel
chromatography (0-10% MeOH/CH2Cl2) to give the corre-
sponding product as a colorless solid (385 mg, 85%).
A mixture of the colorless solid (248 mg, 0.56 mmol), TBSCl
(500 mg, 3.33 mmol), pyridine (482 mg, 6.11 mmol), and CH2Cl2
(5 mL) was maintained at rt under a nitrogen atmosphere. After
12 h, the reaction mixture was diluted with CH2Cl2 (5 mL) and
washed with water (3 ꢀ 50 mL). The aqueous layer was extracted
using EtOAc (3 ꢀ 150 mL). The combined organic layer was
dried over anhydrous Na2SO4 and concentrated. The crude
product was purified by silica gel chromatography (0-10%
MeOH/CH2Cl2) to give 2 as a colorless solid (233 mg, 76%): 1H
NMR (400 MHz, CD3OD) δ 8.29 (s, 1H), 7.77 (dd, J = 7.3, 3.2
Hz, 2H), 7.75-7.55 (m, 3H), 7.48 (d, J = 7.3 Hz, 1H), 7.36-7.27
(m, 4H), 4.89 (s, 2H), 4.39-4.34 (m, 2H), 4.20 (t, J = 6.9 Hz,
2H), 2.75-2.59 (m, 2H), 1.90-1.80 (m, 1H), 1.75-1.65 (m, 3H),
0.95 (s, 9H), 0.13 (s, 6H); 13C NMR (100 MHz, CD3OD) δ 174.4,
157.5, 155.6, 144.2, 143.9, 143.6, 142.8, 141.4, 139.1, 127.6,
126.9, 125.1, 122.7, 119.8, 66.7, 63.1, 53.7, 31.4, 30.9, 27.0,
25.2, 18.1, -6.5; IR (thin film) 3318, 3018, 2953, 2929, 2857,
2360, 2342, 1716, 1610, 1533, 1450, 1399, 1259, 1174, 1106, 1084,
1052, 839, 780, 758, 740, 667 cm-1; [R] = þ16.9 (c = 1.0,
CHCl3); HRMS (ESMS) calcd for C32H40N2O5SiNa (M þ
Na)þ 583.2604, found 583.2598.
Benzyl 2-(2-Acetamido-3-(6-((bis(pyridin-2-ylmethyl)amino)-
methyl)pyridin-3-yl)propanamido)acetate (22). Compound 22
was prepared by general procedure for N-alkylation using 21
(33 mg, 81.6 μmol), MeCN (1.5 mL), i-Pr2EtN (22 μL, 122
μmol), and bis(pyridin-2-ylmethyl)amine (21 mg, 106 μmol). A
sample was purified by column chromatography on alumina
(0-5% MeOH/EtOAc) for analysis: 1H NMR (400 MHz,
CDCl3) δ 8.48 (d, J = 4.0 Hz, 2H), 8.29 (s, 1H), 7.63-7.59
(m, 2H), 7.53-7.46 (m, 4H), 7.33-7.26 (m, 5H), 7.11-7.02 (m,
3H), 6.52 (d, J = 8.1 Hz, 1H), 5.07 (s, 2H), 4.73 (dd, J = 14.6, 6.5
Hz, 1H), 4.06-3.91 (m, 2H), 3.82-3.80 (m, 6H), 3.07 (dd, J =
13.6, 7.3 Hz, 1H), 2.94 (dd, J = 13.6, 7.3 Hz, 1H), 1.88 (s, 3H);
(S)-Benzyl 3-((6-((2,3,3-Trimethylbutan-2-yloxy)methyl)pyridin-
3-yl)methoxy)-2-(tritylamino)propanoate (19). A mixture of com-
pound 9 (95 mg, 0.30 mmol), benzene (1.0 mL), 40% aqueous
NaOH (300 mg, 3.00 mmol), TBAB (97 mg, 0.30 mmol), and
compound 6 (131 mg, 0.30 mmol) was maintained at rt for 24 h. The
reaction mixture was combined with water (2 mL), and the aqueous
658 J. Org. Chem. Vol. 75, No. 3, 2010

Jabre et al.
JOCArticle
13C NMR (100 MHz, CDCl3) δ 171.0, 170.4, 169.2, 159.2, 158.0,
149.6, 149.0, 137.3, 136.4, 135.0, 130.3, 128.6, 128.5, 128.3,
122.9, 122.7, 122.0, 67.2, 60.0, 59.7, 53.6, 41.2, 34.8, 29.6, 23.0;
IR (thin film) 3283 (b), 2926, 1749, 1654, 1590, 1569, 1434, 1370,
1188, 994, 756, 698 cm-1; HRMS (ESMS) calcd for C32H35N6O4
(M þ H)þ 567.2720, found 567.2706.
6.44 (b, 1H), 5.10 (s, 2H), 4.70-4.65 (m, 1H), 3.95 (dd, J =
13.0, 5.7 Hz, 2H), 3.73-3.69 (m, 4H), 3.58-3.52 (m, 4H), 3.05
(dd, J = 13.9, 6.5 Hz, 1H), 2.96 (dd, J = 13.9, 6.5 Hz, 1H),
2.69-2.55 (m, 4H), 1.90 (s, 3H); 13C NMR (100 MHz, CDCl3)
δ 171.0, 170.3, 169.1, 160.2, 159.6, 158.5, 149.5, 148.9, 148.8,
139.1, 137.2, 136.5, 136.4, 135.1, 130.2, 128.6, 128.5, 128.3,
128.2, 126.9, 122.8, 122.6, 121.9, 67.2, 60.8, 60.5, 60.1, 58.9,
53.9, 51.7, 41.2, 34.9, 23.0; IR (thin film) 3286(b), 2917, 2849,
1749, 1653, 1591, 1539, 1435, 1373, 1260, 1188, 1029, 756, 699,
665 cm-1; HRMS (ESMS) calcd for C41H46N7O4 (M þ H)þ
700.3611, found 700.3604.
Benzyl 2-(2-Acetamido-3-(6-(((2-(benzyl(pyridin-2-ylmethyl)-
amino)ethyl)(pyridin-2-ylmethyl)amino)methyl)pyridin-3-yl)pro-
panamido)acetate (23). Compound 23 was prepared by gen-
eral procedure for N-alkylation using compound 21 (20 mg,
49.5 μmol), MeCN (0.9 mL), i-Pr2EtN (35 μL, 198 μmol), and
N1-benzyl-N1,N2-bis(pyridin-2-ylmethyl)ethane-1,2-diamine
(50 mg, 148 μmol). A sample was purified by preparative
HPLC for analysis. HPLC column: Zorbax XDB-C18, 21.2 ꢀ
150 mm, 5 μm equipped with guard column, Zorbax XDB-
C18, 21.2 mm, 5 μm; flow rate = 20 mL/min; gradient elution
0-5 min 5% MeCN in 0.1% aq TFA, 5-15 min 5-95%
MeCN in 0.1% aq TFA, rt = 10.2 min. After HPLC purifica-
tion, desired fractions were combined and the pH of the
mixture was adjusted between 8 and 9 using satd Na2CO3.
The aqueous layer was extracted with CH2Cl2 (3 ꢀ 10 mL).
The combined organic layer was dried over anhydrous
Na2SO4 and concentrated to obtain the product: 1H NMR
(400 MHz, CDCl3) δ 8.45 (d, J = 4.0 Hz, 2H), 8.26 (s, 1H),
7.58-7.53 (m, 2H), 7.46-7.41 (m, 3H), 7.34-7.08 (m, 14H),
Acknowledgment. We thank Prof. Barbara Imperiali for
helpful discussions, Ms. Alyssa Yousif for preparation of
synthetic intermediates, Zhongwu Guo for use of a peptide
synthesizier, and Wayne State University and the American
Chemical Society Petroleum Research Fund (46714-G1) for
their generous support of this research.
Supporting Information Available: Experimental proce-
dures for preparation of 10, 11, 28-32, 35, 36, and 38 including
characterization data. This material is available free of charge
via the Internet at http://pubs.acs.org.
J. Org. Chem. Vol. 75, No. 3, 2010 659