Modular incorporation of 1-benzyltryptophan into dipeptide hosts that bind acetylcholine in pure water

Article abstract of DOI:10.1139/V10-100

Proteins that recognize and bind quaternary ammonium ions depend on " aromatic-cage " structural motifs that use multiple aromatic residues to engage the side chain's ammonium cation. We introduce herein the use of 1-benzyltryp- tophan (Trp(Bn)) residues as synthetic, unnatural partial analogues of natural aromatic cages. We demonstrate the modular incorporation of these building blocks into simple dipeptide hosts and show that they are capable of binding quaternary ammonium ions in buffered water and in chloroform.

Full text of DOI:10.1139/V10-100

1009
Modular incorporation of 1-benzyltryptophan into
dipeptide hosts that bind acetylcholine in pure
water
Cory S. Beshara and Fraser Hof
Abstract: Proteins that recognize and bind quaternary ammonium ions depend on ‘‘aromatic-cage’’ structural motifs that
use multiple aromatic residues to engage the side chain’s ammonium cation. We introduce herein the use of 1-benzyltryp-
tophan (Trp(Bn)) residues as synthetic, unnatural partial analogues of natural aromatic cages. We demonstrate the modular
incorporation of these building blocks into simple dipeptide hosts and show that they are capable of binding quaternary
ammonium ions in buffered water and in chloroform.
Key words: molecular recognition, tryptophan, peptides, cation–p interaction, hydrophobic effect.
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Resume : Les proteines qui reconnaissent et qui se lient a des ions ammonium quaternaires dependent sur des motifs
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structuraux « cage aromatique » qui font appel a de multiples residus aromatiques pour s’engager avec le cation ammo-
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nium de la chaıne laterale. Dans ce travail, on introduit l’utilisation des residus 1-benzyltryptophane (Trp(Bn)) comme ana-
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logues partiels de synthese, non naturels, des cages aromatiques naturelles. On demontre que l’incorporation modulaire des
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ces blocs dans des hotes dipeptidiques et on montre qu’ils sont capables de se lier a des ions ammonium quaternaires dans
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l’eau tamponnee et dans le chloroforme.
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Mots-cles : reconnaissance moleculaire, tryptophane, peptides, interaction cation–p, effet hydrophobe.
could be adapted to achieve the far more difficult task of
Introduction
binding cations in pure water by the incorporation of Trp(Bn)
residues. We report here, the synthesis of 1-benzyltryptophan
based reagents and their rapid and modular incorporation
into quaternary ammonium ion binding dipeptide hosts re-
lated to the parent Trp-Trp dipeptide (2).
The cation–p interaction is implicated in many recogni-
tion events in biology.1–5 Its importance for the recognition
of cholinergic agents by their receptors3,6,7 has long been
understood. Its role in the recognition of post-translationally
methylated lysine residues has more recently come to
light,4,8–10 and the protein data bank (PDB) now contains
many examples of trimethyllysine-containing quaternary am-
monium complexes.11 In these structures, the binding of the
quaternary ammonium ion is accomplished by motifs re-
ferred to as ‘‘aromatic cages’’ in which clusters of three to
four aromatic side chains make close cation–p contacts
Results and discussion
We were surprised to find very few reports of the appa-
rently simple 1-benzyltryptophan (1) in the literature. Only
six literature references were found in a Scifinder structure-
based search,13–18 and none of these references report the ap-
pended 1-benzyl group being used as a functional element.
The obvious routes for the synthesis of 1 start with simple
tryptophan analogs and involve deprotonation and alkylation
of the side chain nitrogen atom. We first targeted the treat-
ment of tryptophan methyl ester with 1 equiv. of NaH fol-
lowed by treatment with a benzyl halide. Reactions were
attempted in THF and DMF using a variety of temperatures
and reaction times. While selective benzylation of the side
chain nitrogen could be achieved, we found significant race-
mization of the a-carbon under all conditions that we tested
(racemization is most easily detected by the observation of
+
with the RNMe3 portion of the side chain (Fig. 1). The
most electron-rich aromatic side chain, tryptophan (Trp), is
present as at least one of the binding partners in all trime-
thyllysine-binding aromatic cage motifs currently reported
in the PDB, where it is accompanied by additional phenyla-
lanine (Phe) and (or) tyrosine (Tyr) residues.
Inspired by the use of multiple aromatic rings in the pro-
tein domains that bind quaternary ammonium ions, we con-
sidered the unnatural modified amino acid 1-benzyltryptophan
(Trp(Bn), 1, Fig. 1) as a building block that could be easily
incorporated into peptide-based hosts. A prior report of the
dipeptide Boc-Trp-Trp-OMe (2) as a simple receptor for am-
monium ions in organic solvents12 led us to wonder if it
1
diastereomeric a-CH H NMR resonances once dipeptides
are made from a Trp(Bn) precursor). We next attempted to
Received 9 April 2010. Accepted 27 June 2010. Published on the NRC Research Press Web site at canjchem.nrc.ca on 23 September
2010.
In memory of Michael Pollard.
C.S. Beshara and F. Hof.1 University of Victoria, Department of Chemistry, PO Box 3065, STN CSC, Victoria, BC V8W 3V6, Canada.
1Corresponding author (e-mail: fhof@uvic.ca).
Can. J. Chem. 88: 1009–1016 (2010)
doi:10.1139/V10-100
Published by NRC Research Press

1010
Can. J. Chem. Vol. 88, 2010
Fig. 1. Top: aromatic cage binding motifs that are used by proteins
Scheme 1. Selective 1-benzylation of Trp and isolation as the HCl
that recognize biologically important ammonium ions of the form
RNMe3 . The structure on the right is an example of an aromatic
salt.
+
cage taken from the PDB entry 1PDQ. Bottom: the unnatural amino
acid 1-benzyltryptophan.
To create water-soluble analogs that retained an overall
anionic charge, we doubly deprotected compounds 2, 7, and
8 and created simple N-terminal acetylated derivatives of
the form Ac-Trp-Trp-OH (not shown). Unfortunately, dipep-
tides of this form were insoluble in phosphate-buffered D2O
(pD = 7.4). In an effort to increase water solubility while
leaving the Trp(Bn) binding motifs unchanged, we targeted
structures with N-terminal succinyl groups. Thus, com-
pounds 2, 7, and 8 were Boc-deprotected in HCl–AcOH to
give the dipeptide hydrochloride salts 10, 11, and 12, re-
spectively, as pure white solids after filtration and washing
with Et2O. The suspension of 10–12 in CH2Cl2, neutraliza-
tion with i-Pr2EtN, and treatment with succinic anhydride
gave the succinyl (Suc) derived Suc-Trp-Trp-OMe dipeptides,
which were directly saponified in methanol–water to yield,
after acidification of the aqueous work-up layer, dipeptides
13–15 (40%–56% overall yield over four synthetic steps;
Scheme 2). Compounds 13–15 are all soluble in phosphate-
buffered D2O (pD = 7.4) at concentrations up to 10 mmol/L.
Acetylcholine chloride (AcCh+ Cl–) was used as a proto-
treat the free-acid Trp (3) with two or more equivalents of
NaH to generate the doubly deprotonated carboxylate–indole
dianion, with the idea that the more reactive indole anion
could be reacted selectively with 1 equiv. of benzyl halide.
These efforts effectively prevented racemization, but gener-
ated intractable mixtures and low yields arising from mix-
tures of O- and N-benzylation products. We subsequently
adapted a reported deprotonation of unprotected Trp under
dissolving metal conditions (Na–NH3) and subsequent treat-
ment with BnCl in liquid NH3 (Scheme 1).14 We found that
this method was successful only when performed on ‡5 g of
3, and crude yields of 1, uncontaminated by O-benzyl or
racemized by-products, reached a high of 70% when the re-
action was carried out on a 40 g scale. We found an effi-
cient purification for 1 by first dissolving the crude in a
mixture of MeOH and concentrated aqueous HCl; precipita-
tion by the addition of Et2O gave the pure product as the
HCl salt (1ꢀHCl) in a 51% overall yield.
+
typical RNMe3 guest because of its high solubility in both
water and chloroform, which is a critical feature that we did
not find in halide or BArF salts of other guest candidates tri-
methyllysine, tetramethylammonium, or benzyltrimethylam-
monium. Titration of AcCh+ Cl– into the dipeptide hosts
13–15 in phosphate-buffered D2O (10 mmol/L Na2HPO4–
NaH2PO4, pD = 7.4) revealed guest-induced chemical shifts
that fit to 1:1 binding isotherms to provide Kassoc values
(Fig. 2 and Table 1). Even at the highest host and guest con-
centrations tested (10 and 930 mmol/L, respectively), the
unbenzylated host 13, which is most closely related to the
literature host 2 that can bind AcCh+ in CDCl3,12 showed
no detectable binding of AcCh+ under these competitive sol-
vent conditions.
The monobenzylated host 14 and dibenzylated host 15
both showed evidence of binding that, while weak, was re-
producibly observed over many determinations with the
highest affinity arising from dibenzylated 15. Direct evi-
dence for the interaction of benzyl groups with the bound
cation was given by the large chemical shift changes among
the benzylic methylenes of dipeptide 15 (Fig. 2B), while the
shifts of both benzyl and indolyl resonances in the aromatic
regions (Fig. 2A) further support the participation of both
types of aromatic rings in binding.
Scheme 2 depicts the synthesis of Boc-Trp(Bn)-OH (4)
and H2N-Trp(Bn)-OMe (5) that were used in the subsequent
synthesis of a family of organic- and water-soluble host di-
peptides. Treatment of 1ꢀHCl with thionyl chloride in meth-
anol gives methyl ester 5 in a 87% yield, whereas treatment
of 1ꢀHCl with NaOH in the presence of (Boc)2O under
mixed aqueous-organic conditions gives the Boc-protected
compound 4 in a 78% yield. Compound 5 was coupled with
commercially available Boc-Trp-OH (6) to form the mono-
benzylated dipeptide host (Boc-Trp-Trp(Bn)-OMe, 7) in a
49% yield. Compound 4 was combined with 5 under stand-
ard solution-phase HBTU coupling conditions to yield the
dibenzylated dipeptide host (Boc-Trp(Bn)-Trp(Bn)-OMe, 8)
in a 61% yield. To complete the series, 4 was coupled with
commercially available H2N-Trp-OMe (9) to afford Boc-
Trp-Trp-OMe (2) with free indole side chains in a 76%
yield. Unlike the prior synthesis of host 2,12 all transforma-
tions reported herein were optimized to allow purification
without resorting to flash chromatography.
To examine the role of solvent effects in these hosts, we
carried out binding studies of the chloroform-soluble analogs
2, 7, and 8 using the same titration methodology. In this sol-
vent, the unbenzylated host 2 bound AcCh+ Cl– with a Kassoc
of 85 (mol/L)–1, which is in agreement with the value previ-
ously reported.12 Suprisingly, our new benzylated hosts 7
and 8 bound AcCh+ more weakly than did the unbenzylated
Published by NRC Research Press

Beshara and Hof
1011
Scheme 2. Synthesis of Trp and Trp(Bn)-containing hosts.
parent (Table 1), despite the presence of additional aromatic
binding surfaces. We also found that host 2 is also the most
potent host for the binding of the the noncoordinating
stronger in nonpolar media; the fact that we see the opposite
trend in CDCl3 suggests that it is not a major contributor to
interactions between the benzyl groups and cationic guests.
Instead, it is likely the increase in hydrophobic surface area
provided by the benzyl groups that leads to the higher cation
affinities observed for benzylated hosts in D2O.
BnNMe3 BArF–, suggesting that this result is not an artifact
+
arising from counterion effects in chloroform.19 The cation–
p interaction is an electrostatic attraction that should be
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1012
Can. J. Chem. Vol. 88, 2010
Fig. 2. Concentration-dependent chemical shift changes displayed by the host 15 (10 mmol/L) in its (a) aromatic and (b) methylene regions
upon addition of AcCh+ Cl– in D2O (phosphate buffer, pD 7.4, 10 mmol/L). Concentrations of AcCh+ Cl– are indicated. (c) A plot of titra-
tion data and the 1:1 binding isotherm arising from nonlinear least-squares regression (see Experimental).
Table 1. Kassoc values for cations with hosts 13–16 in D2O and 2, 7, and 8 in
CDCl3.
Kassoc
Host
Guest
AcCh+ Cl–a
AcCh+ Cl–a
Solvent
D2O
D2O
D2O
D2O
CDCl3
CDCl3
CDCl3
CDCl3
CDCl3
CDCl3
((mol/L)–1)
£1b
3 1
14 6
£1b
85 17
22 4
1 0.5
63 12
27 5
47 3
13 (Trp-Trp)
14 (Trp-Trp(Bn))
15 (Trp(Bn)-Trp(Bn)) AcCh+ Cl–a
16 (Trp(Bn))
2 (Trp-Trp)
AcCh+ Cl–a
AcCh+ Cl–a
AcCh+ Cl–a
AcCh+ Cl–a
7 (Trp-Trp(Bn))
8 (Trp(Bn)-Trp(Bn))
2 (Trp-Trp)
7 (Trp-Trp(Bn))
8 (Trp(Bn)-Trp(Bn))
BnNMe3 BArF–c
+
BnNMe3 BArF–c
+
BnNMe3 BArF–c
+
Note: Kassoc was determined using at least three replicate titrations, tracking two to
three proton resonances during each titration. Errors represent the upper and lower
limits of replicate titrations. ‘‘D2O’’ also contained 10 mmol/L NaH2PO4–Na2HPO4
adjusted to pD 7.4.
aGuest solution (ranging from 80 to 930 mmol/L) was titrated into a solution of the
host (10 mmol/L).
bTitration resulted in insignificant Dd values.
cA concentrated solution of the host (80–250 mmol/L) was titrated into the guest
+
(2 mmol/L) due to low solubility of the BnNMe3 BArF– salt in CDCl3.
that only one benzyl indole group and a neighboring car-
To gain some insight into the possible structures of these
host–guest complexes, we carried out Monte Carlo conforma-
tional searches using a representative dibenzylated dipeptide
host (the methyl ester analog of 15) and the truncated guest
Me4N+. These gas-phase calculations are limited in scope
and capabilities; they are subject to unrealistic overemphasis
of electrostatic attractions (hence our use of the methyl ester
host analog) and they are incapable of representing the hy-
drophobic driving forces that are critical in aqueous solu-
tion. However, they are very good at determining
energetically reasonable bond angles and intra- and inter-
molecular contacts, and are therefore useful for gaining a
qualitative sense of what types of structures might be allow-
able for the host–guest complex. The two most common
types of structures among low-energy conformations ob-
tained from our Monte Carlo calculation involve (i) the in-
teraction of the cation with both benzyl indole moieties in a
pocket-like geometry (‘‘pocket’’, Fig. 3A) or (ii) the interac-
tion of the cation with only a single Trp(Bn) side chain
(‘‘cleft’’, Fig. 3B). The cleft structure in Fig. 3B suggests
bonyl might be needed to engage the cationic guest. To test
this hypothesis, we designed compound 16 (Suc-Trp(Bn)-
OH) to see if this monomeric host fragment was a compe-
tent cation binder in water. Compound 16 was synthesized
in a 56% yield by the reaction of succinic anhydride with
precursor 3 (Scheme 2). NMR titrations of 16 with
AcCh+ Cl– in buffered D2O, carried out as for the other
hosts, gave no observable sign of binding, proving that two
Trp residues are required for cation binding in water by
these dipeptides.
Conclusions
These studies with simple dipeptide hosts introduce the
readily synthesized Trp(Bn) building block as a new quater-
nary ammonium ion binding motif that can function in pure,
buffered water. The weakness of the observed binding by
these dipeptides in either medium almost certainly arises
from their lack of preorganization relative to more rigid
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Beshara and Hof
1013
Fig. 3. Representative structures, (A) ‘‘pocket’’ and (B) ‘‘cleft’’, aris-
ing from Monte Carlo conformation searches at the PM3 level of
theory. Structures like that shown in Fig. 3A are consistent with
both of our key experimental observations in water: the N-benzyl
subsitituents play a significant role in binding, and both amino acid
side chains are required for cation binding in water.
minimum were retained, and examination revealed that all
such low-energy structures could be sorted into two groups
by considering the number of aryl side chains making con-
tact with the quaternary ammonium ion (pocket = two
Trp(Bn) side chains in contact, Fig. 3A; cleft = one Trp(Bn)
residue in contact, Fig. 3B).
1-Benzyltryptophan hydrochloride (1ꢀHCl)
Liquid ammonia (1000 mL) was condensed into a 2 L
flask containing Na(s) (10.4 g, 452 mmol) and Fe(NO3)3
(0.6 g, 0.01 mmol) under argon. To facilitate the addition of
ammonia, the flask was cooled in a dry ice – acetone bath
and equipped with a cold finger (dry ice – acetone) via a
Claisen adapter. The resultant mixture turns blue immedi-
ately and slowly fades to a grey color. Tryptophan (40 g,
196 mmol) was introduced to the grey solution as a slurry
in ether, and benzyl chloride (22.5 mL, 196 mmol) was
added immediately thereafter via syringe through the sep-
tum. The reaction refluxed on an ice bath for 1 h, and
evaporated overnight. The crude product dissolved in hot
water and a small amount of solid residue was removed by
filtration. Acetic acid was added until the product precipi-
tated, and filtration yielded the 1-benzyltryptophan as an
off-white solid (41 g, 71%). A portion of this product (24 g,
81 mmol) was suspended in methanol (200 mL) at room
temperature and concentrated aqueous hydrochloric acid
was added dropwise until the starting material was com-
pletely dissolved. The reaction was stirred for 5 min, and
was then concentrated to half volume. The solution was
then poured into 1.6 L of ether and the resultant slurry was
filtered to yield 1ꢀHCl as a white solid that was left to air-
macrocyclic systems.9,10 We are curious to see if rigidifica-
tion of these building blocks by incorporation into small,
folded peptide scaffolds increases the potency of Trp(Bn)
building blocks as cation binders.
Experimental
General procedure
All solvents and reagents were used as purchased from
Sigma-Aldrich and used without further purification, except
solvents that were dried using an MBraun solvent purifica-
tion system. NMR spectra and NMR titration data were ob-
tained on a Bru¨ker Avance 500 MHz NMR spectrometer
with the exception of 19F NMR, which was obtained on a
Bru¨ker Avance 300 MHz spectrometer. Coupling constants
25
dry overnight (19 g, 72%); mp 204–206 8C. ½aꢁD +5.08 (c
1
0.08 in MeOH). H NMR (300 MHz, DMSO-d6) d: 3.32
1
are reported in Hertz. H and 13C spectra were referenced to
(2H, J = 6.3, d), 4.12 (1H, bs), 5.38 (2H, s), 7.03 (1H J = 7
and 1, td), 7.10 (1H, J = 8 and 1, td), 7.18–7.33 (5H, m),
7.37 (1H, J = 8, d), 7.39 (1H, s), 7.63 (1H, JHH = 7, d),
8.31 (3H, bs), 13.77 (1H, bs). 13C NMR (75.5 MHz,
DMSO-d6) d: 26.0, 49.1, 52.6, 106.8, 110.2, 118.7, 118.9,
121.4, 127.0, 127.3, 127.7, 128.5, 128.7, 136.1, 138.1,
170.7. ESI (accurate mass) m/z: 295.1452 (M+ C18H19N2O2
requires 295.1447).
residual solvent signals, and the 19F spectrum was externally
referenced to FCCl3. HR-MS were acquired on a Waters/
Micromass LCT TOF instrument. Melting points were deter-
mined with the use of a GallenKamp melting point appara-
tus. Specific rotations (c = g/100 mL) were aquired with a
Rodolf Research Analytical Autopol I instrument and are
given in units of 10–1 deg cm2 g–1.
The typical titration procedure is as follows: The mole-
cule of interest was dissolved in the appropriate solvent (dis-
pensed with a micropipette) to make a solution (4 mL) of a
predetermined concentration (2–10 mmol/L). The stock
(600 mL) was then introduced into an NMR tube and to the
remaining stock solution was added an excess of host or
guest. The NMR spectrum of the stock solution was used as
a reference for unbound host or guest and the solution ti-
trated with predetermined volumes (micropipette) of titrant,
followed by the collection of new NMR spectra. This
method ensures that the concentration of the host remains
constant throughout the titration. The Dds were recorded
and the data worked-up and fitted to the 1:1 binding iso-
therm using the spreadsheet made freely available by Sand-
erson.20 Initial guesses for host or guest concentrations
during the course of the experiments were obtained by the
method discussed by Hirose.21
N-(tert-Butoxycarbonyl)-1-benzyltryptophan (4)
3 (1.0 g, 3.0 mmol) was suspended in 1:1 dioxane–water
(12 mL) at room temperature. NaOH (aq, 1 mol/L, 6 mL,
6 mmol) was added, followed by the addition of (Boc)2O
(0.84 mL, 3.6 mmol), and this mixture was stirred for 18 h.
The reaction was acidified to pH 2.5 with HCl (2 mol/L),
and diluted further with water (50 mL). Ethyl acetate was
then used to extract the mostly aqueous mixture. The or-
ganic layers were combined, dried, and concentrated to yield
a white solid (0.928 g, 78%); mp 181–182 8C (EtOAc).
25
1
½aꢁ +9.18 (c 0.08 in MeOH). H NMR (300 MHz, DMSO-
d6)Dd: 1.13 (9H, s), 2.99 (1H, J = 15 and 9, dd), 3.14 (1H,
J = 14 and 5, dd), 4.16 (1H, J = 9 and 5, td), 5.36 (2H, s),
6.97–7.1 (3H, m), 7.13–7.16 (2H, m), 7.19–7.31 (4H, m),
7.37 (1H, J = 8, d), 7.55 (1H, J = 7, d), 12.55 (1H, bs). 13C
NMR (75.5 MHz, DMSO-d6) d: 26.7, 28.1, 48.9, 54.4, 77.9,
110.0, 110.3, 118.6, 121.2, 126.9, 127.2, 127.4, 127.8,
128.4, 135.9, 138.3, 155.3, 173.8. ESI (accurate mass) m/z
calcd.: 417.1790; found: 417.1789.
Calculations were carried out at the PM3 level of theory
using the conformational search routine in Spartan’04.22
Structures within 5 kcal/mol (1 cal = 4.186 8 J) of the global
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1014
Can. J. Chem. Vol. 88, 2010
Methyl 1-benzyltryptophanate hydrochloride (5)
J = 9 and 4, td), 4.60 (1H, J = 7 and 6, td), 5.35 (2H, s),
6.72 (1H, J = 8, d), 6.97(1H, J = 7, t), 7.00–7.10 (3H, m),
7.12–7.21 (3H, m), 7.25–7.27 (2H, m), 7.31–7.33 (2H, m),
7.38 (1H, J = 8, d), 7.51 (1H, J = 8, d), 7.60 (1H, J = 8, d),
8.38 (1H, J = 7, d), 10.81 (1H, s). 13C NMR (126 MHz,
DMSO-d6) d: 27.0, 27.7, 28.1, 49.0, 51.8, 53.0, 55.0, 78.0,
109.3, 110.07, 110.12, 111.2, 118.1, 118.4, 118.5, 118.8,
120.8, 121.3, 123.7, 126.9, 127.2, 127.4, 127.5, 127.7,
128.5, 135.8, 136.0, 138.2, 155.1, 172.1, 172.2. ESI (accu-
rate mass) m/z calcd.: 617.2740; found: 617.2754.
3 (5.0 g, 15.1 mmol) was dissolved in methanol (300 mL)
and cooled to 0 8C. Thionyl chloride (1.1 mL, 15.1 mmol)
was added dropwise and the mixture was then allowed to
warm to room temperature before being brought to reflux.
After overnight stirring at reflux, the reaction was cooled
and concentrated. Trituration with diethyl ether provided a
white powder with no further purification needed (4.55 g,
25
87%); mp 115–117 8C (ether). ½aꢁD +11.38 (c 0.15 in
1
MeOH). H NMR (300 MHz, DMSO-d6) d: 3.26 (1H, J =
15 and 7, dd), 3.38 (1H, J = 15 and 5, dd), 3.60 (3H, s),
4.19 (1H, J = 7 and 5, dd), 5.38 (2H, s), 7.04 (1H, J = 7
and 1, td), 7.11 (1H, J = 8 and 1, td), 7.18–7.33 (5H, m),
7.37 (1H, s), 7.41 (1H, J = 8, d), 7.59 (1H, J = 7, d), 8.71
(3H, bs). 13C NMR (75.5 MHz, DMSO-d6) d: 26.1, 49.0,
52.5, 52.7, 106.6, 110.2, 118.5, 119.0, 121.5, 127.0, 127.3,
127.5, 128.4, 128.5, 136.0, 138.1, 169.7. ESI (accurate
mass) m/z calcd.: 309.1603; found: 309.1603.
Methyl N-[N-(tert-butoxycarbonyl)-1-benzyltryptophyl)]-1-
benzyltryptophanate (8)
A white powder was isolated (0.83 g, 61%); mp 149–
25
1
151 8C (EtOAc). ½aꢁD +4.08 (c 0.08 in MeOH). H NMR
(500 MHz, DMSO-d6) d: 1.28 (9H, s), 2.91 (1H, J = 15 and
9, dd), 3.05 (1H, J = 15 and 4, dd), 3.09 (1H, J = 15 and 7,
dd), 3.18 (1H, J = 15 and 6, dd), 3.52 (3H, s), 4.27 (1H, J =
9 and 4, td), 4.58 (1H, J = 7 and 6, td), 5.32 (2H, s), 5.35
(2H, s), 6.74 (1H, J = 8, d), 6.98–7.03 (1H, m), 7.05–7.09
(2H, m), 7.12–7.28 (11H, m), 7.32–7.38 (2H, m), 7.51 (1H,
J = 8, d), 7.61 (1H, J = 8, d), 8.31 (1H, J = 7, d). 13C NMR
(126 MHz, DMSO-d6) d: 27.0, 27.6, 28.1, 48.90, 48.94,
51.8, 53.0, 55.0, 78.0, 109.3, 109.9, 110.1, 110.2, 118.4,
118.5, 118.8, 118.9, 121.1, 121.3, 126.8, 126.9, 127.2,
127.5, 127.7, 128.0, 128.4, 135.8, 135.9, 138.2, 138.3,
155.1, 171.9, 172.2. ESI (accurate mass) m/z calcd.:
707.3209; found: 707.3201.
Dipeptide coupling general procedure
N-terminally protected (Boc) amino acid (2.0 mmol) and
HBTU (0.78 g, 2 mmol) were added to a flask containing
THF (70 mL). The mixture was cooled to 0 8C and 1 equiv.
of DIEA (0.35 mL, 2.03 mmol) was added. The mixture was
stirred for 10 min. C-terminally protected (methyl ester)
amino acid (2 mmol) and 1.2 more equiv. of DIEA
(0.42 mL, 2.4 mmol) were added to the reaction, which was
then warmed to room temperature and stirred for 2 h, re-
gardless of completeness. Once complete, the reaction was
concentrated, taken up in ethyl acetate, and washed with
1 mol/L HCl (ꢂ2), satd. NaHCO3 (ꢂ2), water, and brine.
The organic layer was dried with Na2SO4 and concentrated
to produce a white powder (or foam) after exposure of the
residue to high vacuum overnight.
General procedure for BOC deprotection
Starting material (0.6 mmol) was suspended in acetic acid
at room temperature. In a separate flask, HCl (concd) was
diluted with acetic acid to form a 1.5 mol/L solution of HCl
in acetic acid. The resultant HCl in acetic acid solution
(9.4 mL, 14 mmol HCl) was added in one portion to the
starting material suspension. The suspension was dispersed
upon addition of the HCl solution, followed by the appear-
ance of another precipitate 1 h postaddition. The reaction
was stirred overnight, filtered, and the cake washed with
ether to yield a white solid that needs no further purification.
Methyl N-[N-(tert-butoxycarbonyltryptophyl)]-
tryptophanate (2)
After initial concentration, the product was precipitated
from a minimum volume of CH2Cl2 at –20 8C as a white
25
solid (1.51 g, 71%); mp 162–164 8C (CH2Cl2). ½aꢁD –7.08
1
(c 0.08 in MeOH). H NMR (500 MHz, DMSO-d6) d: 1.26
Methyl N-tryptophyl-tryptophanate hydrochloride (10)
The work-up differed as the product was soluble in acetic
acid. The acetic acid was concentrated and diluted with
ether. A light brown solid resulted, was filtered, and isolated
(0.23 g, 90%); mp (foams) 162–170 8C, mp (melts) 178–
(9H, s), 2.84 (1H, J = 15 and 9, dd), 3.00 (1H, J = 15 and 4,
dd), 3.06 (1H, J = 15 and 7, dd), 3.13 (1H, J = 15 and 6,
dd), 3.52 (3H, s), 4.21 (1H, J = 9 and 4, td), 4.53 (1H, J =
7.0 and 6, td), 6.69 (1H, J = 8, d), 6.92–6.97 (2H, m), 7.00–
7.06 (3H, m), 7.14 (1H, J = 2, d), 7.28–7.33 (2H, m), 7.44
(1H, JHH = 8, d), 7.55 (1H, J = 8, d), 8.23 (1H, J = 8, d),
10.77 (1H, s), 10.86 (1H, s). 13C NMR (126 MHz, DMSO-
d6) d: 27.1, 27.7, 28.1, 51.8, 53.1, 55.0, 78.1, 109.2, 110.1,
111.3, 111.5, 118.0, 118.2, 118.46, 118.51, 118.9, 120.8,
121.0, 123.7, 127.1, 127.4, 136.06, 136.10, 155.1, 172.1,
172.2. ESI (accurate mass) m/z calcd.: 527.2270; found:
527.2271.
25
180 8C (ether). ½aꢁ –4.28 (c 0.08 in MeOH). 1H NMR
(500 MHz, DMSO-Dd6) d: 3.05–3.30 (4H, m), 3.58 (3H, s),
4.03 (1H, J = 8 and 6, dd), 4.62 (1H, J = 7, q), 6.98–7.01
(2H, m), 7.08 (2H, J = 8, q), 7.21 (1H, s), 7.22 (1H, s),
7.35 (2H, J = 8, t), 7.50 (1H, J = 8, d), 7.71 (1H, J = 8, d),
8.1 (3H, bs), 9.11 (1H, J = 7.0, d), 10.96 (1H, s), 11.04 (1H,
s). 13C NMR (126 MHz, DMSO-d6) d: 27.2, 27.3, 52.0, 52.5,
53.3, 106.8, 108.9, 111.4, 111.5, 117.9, 118.4, 118.47,
118.52, 121.0, 121.1, 124.0, 125.0, 127.0, 127.1, 136.1,
136.3, 168.8, 171.6. ESI (accurate mass) m/z calcd.:
405.1927; found: 405.1927.
Methyl N-[N-(tert-butoxycarbonyl)tryptophyl]-1-
benzyltryptophanate (7)
A white powder was isolated (1.16 g, 49%); mp 76–78 8C
25
1
then 99–100 8C (EtOAc). ½aꢁ –15.68 (c 0.09 in MeOH). H
NMR (500 MHz) d: 1.26 (9DH, s), 2.89 (1H, J = 15 and 9,
dd), 3.06 (1H, J = 15 and 4, dd), 3.10 (1H, J = 15 and 7,
dd), 3.18 (1H, J = 15 and 6, dd), 3.54 (3H, s), 4.26 (1H,
Methyl N-tryptophyl-1-benzyltryptophanate hydrochloride
(11)
A white solid was isolated (0.112 g, 69%); mp 222–
Published by NRC Research Press

Beshara and Hof
1015
25
1
t), 7.05 (1H, J = 8, t), 7.06 (1H, J = 8, t), 7.11 (1H, J = 2.0,
d), 7.16 (1H, J = 2.0, d), 7.31 (1H, J = 8, d), 7.33 (1H, J =
8, d), 7.53 (1H, J = 8, d), 7.58 (1H, J = 8, d), 8.01 (1H, J =
8, d), 8.17 (1H, J = 8, d), 10.77 (1H, bs), 10.85 (1H, bs),
12.36 (2H, bs), 12.7 (bs, 1H). 13C NMR (126 MHz) d: 27.0,
27.6, 29.2, 30.0, 53.0, 53.2, 109.7, 110.2, 111.2, 111.4,
118.2, 118.40, 118.44, 120.8, 120.9, 123.5, 123.6, 127.3,
127.4, 136.0, 136.1, 170.9, 171.6, 173.2, 173.9. ESI (accu-
rate mass) m/z calcd.: 489.1774; found: 489.1773.
224 8C (AcOH). ½aꢁ +2.38 (c 0.08 in MeOH). H NMR
(500 MHz, DMSO-dD6) d: 3.08–3.29 (4H, m), 3.55 (3H, s),
4.04 (1H, J = 6, t), 4.62 (1H, J = 7, q), 5.37 (2H, s), 5.37
(2H, s), 7.00 (1H, J = 7, t), 7.03 (1H, J = 7, t), 7.09 (2H,
J = 7, t), 7.16 (2H, J = 7, d), 7.21(2H, J = 7, t), 7.27 (2H, J =
7, t), 7.35 (1H,s), 7.36 (1H, J = 11, d), 7.41(1H, J = 8, d),
7.53 (1H, J = 8, d), 7.70 (1H, J = 8, d), 8.16 (3H, bs), 9.15
(1H, J = 6, d) 11.00 (1H, s). 13C NMR (126 MHz, DMSO-
d6) d: 27.1, 27.3, 48.9, 51.9, 52.4, 53.4, 106.7, 108.9, 110.2,
111.4, 118.4, 118.5, 118.8, 121.1, 121.3, 125.0, 126.9,
127.1, 127.3, 127.6, 127.8, 128.5, 135.9, 136.3, 138.3,
168.6, 171.5. ESI (accurate mass) m/z calcd.: 495.2396;
found: 495.2397.
N-[N-(3-Carboxypropanoyl)-tryptophyl]-1-benzyltryptophan
(14)
Isolated as a white powder (0.074 g, 88%); mp 180 8C
25
dec (EtOAc). ½aꢁD –11.98 (c 0.09 in MeOH). 1H NMR
Methyl N-(1-benzyltryptophyl)-1-benzyltryptophanate
hydrochloride (12)
A white solid was isolated (0.30 g, 83%); mp 219–220 8C
(500 MHz, DMSO-d6) d: 2.25–2.32 (4H, m), 2.89 (1H, J =
15 and 9, dd), 3.06 (1H, J = 15 and 8, dd), 3.11 (1H, J =
15 and 4, dd), 3.21 (1H, J = 15 and 5, dd), 4.50 (1H, J =
8.0 and 5, td), 4.58 (1H, J = 9.0 and 4, td), 5.35 (2H, s),
6.96 (1H, J = 7, t), 7.01 (1H, J = 7, t), 7.04 (1H, J = 7, t),
7.07 (1H, J = 7, t), 7.12 (1H, J = 2, d), 7.16–7.27 (5H, m),
7.31 (1H, J = 8, d), 7.32 (1H, s), 7.35 (1H, J = 8, d), 7.55
(1H, J = 8, d), 7.58 (1H, J = 8, d), 8.01 (1H, J = 8, d), 8.28
(1H, J = 8, d), 10.78 (1H, J = 2, d), 12.3 (1H, bs), 12.7 (1H,
bs). 13C NMR (126 MHz, DMSO-d6) d: 26.8, 27.6, 29.1,
30.0, 49.0, 52.9, 53.3, 109.7, 110.1, 110.2, 111.2, 118.1,
118.4, 118.5, 118.7, 120.8, 121.2, 123.6, 126.9, 127.2,
127.4, 127.6, 127.9, 128.5, 135.8, 136.0, 138.3, 170.9,
171.7, 173.1, 173.8. ESI (accurate mass) m/z calcd.:
579.2244; found: 579.2231.
25
1
(AcOH). ½aꢁD +6.28 (c 0.08 in MeOH). H NMR (500 MHz,
DMSO-d6) d: 3.12–3.28 (4H, m), 3.51 (3H, s), 4.09 (1H, bs),
4.61 (1H, J = 7, q), 5.35 (2H, s), 5.37 (2H, s), 7.03 (2H, J =
7, t), 7.08–7.12 (2H, m), 7.16–7.31 (10H, m), 7.33 (1H, s),
7.37 (1H, J = 8, d), 7.39 (1H, s), 7.41(1H, J = 8, d), 7.53
(1H, J = 8, d), 7.75 (1H, J = 8, d), 8.31 (3H, bs), 9.2 (1H,
J = 7, d). 13C NMR (126 MHz, DMSO-d6) d: 27.7, 27.8,
49.6, 49.8, 52.5, 53.2, 54.1, 107.5, 109.5, 110.7, 110.8,
119.0, 119.4, 119.5, 119.6, 121.97, 122.02, 127.6, 127.7,
127.89, 127.93, 128.2, 128.4, 128.5, 129.1 (ꢂ2), 129.4,
136.6, 136.8, 138.7, 138.9, 169.2, 172.2. ESI (accurate
mass) m/z calcd.: 585.2866; found: 585.2875.
N-[N-(3-Carboxypropanoyl)-1-benzyltryptophyl]-1-
benzyltryptophan (15)
A white solid was isolated (0.106 g, 64%); mp (turns
General method for the sequential installation of the
succinyl group on N-terminal amines and saponification
of C-terminal methyl esters
25
brown) 180 8C, mp (melts) 190–192 8C (EtOAc). ½aꢁD –1.18
Starting material 10, 11, or 12 (0.25 mmol) was sus-
pended in CH2Cl2 (10 mL) at room temperature. Succinic
anhydride (0.036 g, 0.36 mmol) was added to the reaction,
followed by DIEA (0.095 mL, 0.55 mmol), and the mixture
stirred overnight. Dilution of the reaction with CH2Cl2
(50 mL), followed by extraction with HCl (1 mol/L, 2 ꢂ
50 mL), followed by back extraction of the aqueous with
CH2Cl2 was sufficient to isolate the methyl ester of the de-
sired product. The combined organic layers were washed
with brine (1 ꢂ 50 mL) and dried over Na2SO4 before con-
centration. The sticky residue was used without further puri-
fication to produce the de-esterified product. Dissolution in
methanol (3 mL), followed by the addition of NaOH
(1 mol/L, 0.75 mL) furnished the final product after 2 h of
stirring. The reaction was diluted with water (5 mL) and
acidified with HCl (1 mol/L) to pH 2. The suspension was
extracted with ethyl acetate and the organic layer was
washed with brine and dried over Na2SO4 before concentra-
tion.
1
(c 0.09 in MeOH). H NMR (500 MHz, DMSO-d6) d: 2.24–
2.32 (4H, m), 2.88 (1H, J = 15 and 9, dd), 3.07 (1H, J = 15
and 8, dd), 3.12 (1H, J = 15 and 4, dd), 3.21 (1H, J = 15 and
5, dd), 4.49 (1H, J = 8.0 and 5, td), 4.59 (1H, J = 9.0 and 4,
td), 5.32 (2H, s), 5.34 (2H, s), 6.99 (1H, J = 7, q), 7.05–7.08
(2H, m), 7.14–7.29 (10H, m), 7.34 (2H, J = 9, d), 7.35 (2H,
J = 8, d), 7.56 (1H, J = 8, d), 7.60 (1H, J = 8, d), 8.05 (1H,
J = 8, d), 8.31 (1H, J = 8, d), 12.7 (2H, bs). 13C NMR
(126 MHz, DMSO-d6) d: 26.8, 27.5, 29.1, 29.9, 48.9, 49.0,
53.0, 53.2, 109.7, 109.9, 110.0, 110.2, 118.5, 118.6, 118.7,
118.9, 121.1, 121.2, 126.9, 127.2, 127.4, 127.6, 127.9,
128.0, 128.4, 135.8, 138.3, 138.4, 170.8, 171.5, 173.1,
173.8. ESI (accurate mass) m/z calcd.: 669.2724; found:
669.2713.
Benzyltrimethylammonium tetrakis[3,5-
bis(trifluoromethyl)phenyl]borate (BnNMe3 BArF–)
+
Benzyltrimethylammonium chloride (0.107 g, 0.57 mmol)
and NaBArF (0.510 g, 0.57 mmol) were stirred overnight in
CH2Cl2 (40 mL). The resulting suspension was filtered
through a syringe filter with a pore size of 0.45 mm and the
filtrate was concentrated to produce a white solid. The solid
was rinsed with pentane and dried in vacuo for 3 h (0.499 g,
86%); mp 136–137 8C (CH2Cl2). 1H NMR (500 MHz,
CDCl3) d: 2.82 (9H, s), 4.12 (2H, s), 7.22 (2H, J = 10, d),
7.47 (2H, J = 10, t), 7.51 (4H, bs), 7.57 (1H, J = 10, t),
7.67–7.68 (8H, m). 13C NMR (126 MHz, DMSO-d6) d: 53.2
N-[N-(3-Carboxypropanoyl)-tryptophyl]-tryptophan (13)
Isolated as a light brown powder (0.055 g, 44%); mp
(turns dark brown) 88 8C, mp (melts) 154–156 8C (EtOAc).
25
1
½aꢁ –11.68 (c 0.09 in MeOH). H NMR (500 MHz, DMSO-
d6)Dd: 2.25–2.34 (4H, m), 2.88 (1H, J = 15 and 9, dd), 3.08
(1H, J = 15 and 8, dd), 3.12 (1H, J = 15 and 4, dd), 3.19
(1H, J = 15 and 5, dd), 4.50 (1H, J = 8 and 5, td), 4.58
(1H, J = 9.0 and 4, td), 6.97 (1H, J = 8, t), 6.98 (1H, J = 8,
Published by NRC Research Press

1016
Can. J. Chem. Vol. 88, 2010
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Chem. 1999, 42 (16), 3066. doi:10.1021/jm990093z. PMID:
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(JCN = 4, t), 71.9, 117.8, 124.7 (JCF = 273, q), 124.8, 129.2
(JCF = 29, q), 130.4, 132.3, 132.7, 134.9, 161.9 (JCB = 50,
q). 19F NMR (282 MHz) d: –62.3. 11B NMR (160 MHz)
d: –6.8. ESI (accurate mass) m/z calcd. (anion): 862.0685;
found: 862.0700; calcd. (cation): 150.1283; found: 150.1281.
N-(3-Carboxypropanoyl)-1-benzyltryptophan (16)
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5 (0.926 g, 2.8 mmol) was suspended in dry THF and
stirred at room temperature. Succinic anhydride (0.28 g,
2.8 mmol) and DIEA (0.49 mL, 2.8 mmol) were then added
and the mixture refluxed overnight. The volume was re-
duced until a thick syrup formed. Ethyl acetate was added
(300 mL) and the organic was extracted three times with
NaOH (aq, 0.5 mol/L, 3 ꢂ 75 mL). The aqueous extracts
were combined and acidified to pH 2 with HCl (1 mol/L).
The brown oil was extracted with ethyl acetate. Upon drying
(Na2SO4) and concentrating the residue solidifies on expo-
sure to vacuum overnight. Purification with column chroma-
tography (CHCl3, AcOH, MeOH) with gradient elution from
94:5:1 to 93:5:2 was used to elute pure product. Trituration
of the combined and concentrated fractions with ether
yielded a white crystalline solid (0.634 g, 56%); mp 148–
25
150 8C (ether). ½aꢁD +308 (c 0.09 in MeOH). 1H NMR
(500 MHz, DMSO-d6) d: 2.25–2.34 (4H, m), 3.00 (1H, J =
15 and 9, dd), 3.17 (1H, J = 15 and 5, dd), 4.88 (1H, m),
7.01 (1H, J = 8, t), 5.35 (2H, s), 7.07 (1H, J = 8, t), 7.15–
7.16 (2H, m), 7.21–7.23 (1H, m), 7.27–7.30 (3H, m), 7.37
(1H, J = 8, d), 7.56 (1H, J = 8, d), 8.19 (1H, J = 8, d),
12.37 (2H, bs). 13C NMR (126 MHz, DMSO-d6) d: 27.1,
29.0, 29.8, 48.9, 52.9, 109.9, 110.0, 118.6, 118.7, 121.2,
126.9, 127.2, 127.5, 127.8, 128.4, 135.9, 138.3, 170.9,
173.3, 173.8. ESI (accurate mass) m/z calcd.: 393.1456;
found: 393.1450.
Supplementary data
Supplementary data for this article are available on the
journal Web site (canjchem.nrc.ca).
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Acknowledgements
This work was funded by the Michael Smith Foundation
for Health Research (MSFHR). FH is a MSFHR Career
Scholar and Canadian Institutes of Health Research (CIHR)
New Investigator.
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form, and they attributed the relative strength to NH–Cl– hy-
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Published by NRC Research Press

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