Unexpected hydrolytic instability of N-acylated amino acid amides and peptides

Article abstract of DOI:10.1021/jo500273f

Remote amide bonds in simple N-acyl amino acid amide or peptide derivatives 1 can be surprisingly unstable hydrolytically, affording, in solution, variable amounts of 3 under mild acidic conditions, such as trifluoroacetic acid/water mixtures at room temperature. This observation has important implications for the synthesis of this class of compounds, which includes N-terminal-acylated peptides. We describe the factors contributing to this instability and how to predict and control it. The instability is a function of the remote acyl group, R²CO, four bonds away from the site of hydrolysis. Electron-rich acyl R² groups accelerate this reaction. In the case of acyl groups derived from substituted aromatic carboxylic acids, the acceleration is predictable from the substituent's Hammett σ value. N-Acyl dipeptides are also hydrolyzed under typical cleavage conditions. This suggests that unwanted peptide truncation may occur during synthesis or prolonged standing in solution when dipeptides or longer peptides are acylated on the N-terminus with electron-rich aromatic groups. When amide hydrolysis is an undesired secondary reaction, as can be the case in the trifluoroacetic acid-catalyzed cleavage of amino acid amide or peptide derivatives 1 from solid-phase resins, conditions are provided to minimize that hydrolysis.

Full text of DOI:10.1021/jo500273f

Article
pubs.acs.org/joc
Unexpected Hydrolytic Instability of N‑Acylated Amino Acid Amides
and Peptides
J. Geno Samaritoni,† Alexus T. Copes,‡ DeMarcus K. Crews,§ Courtney Glos,† Andre L. Thompson,§
Corydon Wilson,§ Martin J. O’Donnell,*,† and William L. Scott*,†
†Department of Chemistry and Chemical Biology, Indiana University Purdue University Indianapolis, Indianapolis, Indiana 46202,
United States
‡Howard University, Washington, DC 20059, United States
§Morehouse College, Atlanta, Georgia 30314, United States
S
* Supporting Information
ABSTRACT: Remote amide bonds in simple N-acyl amino
acid amide or peptide derivatives 1 can be surprisingly unstable
hydrolytically, affording, in solution, variable amounts of 3
under mild acidic conditions, such as trifluoroacetic acid/water
mixtures at room temperature. This observation has important
implications for the synthesis of this class of compounds,
which includes N-terminal-acylated peptides. We describe the
factors contributing to this instability and how to predict and control it. The instability is a function of the remote acyl group,
R2CO, four bonds away from the site of hydrolysis. Electron-rich acyl R2 groups accelerate this reaction. In the case of acyl
groups derived from substituted aromatic carboxylic acids, the acceleration is predictable from the substituent’s Hammett σ value.
N-Acyl dipeptides are also hydrolyzed under typical cleavage conditions. This suggests that unwanted peptide truncation may
occur during synthesis or prolonged standing in solution when dipeptides or longer peptides are acylated on the N-terminus with
electron-rich aromatic groups. When amide hydrolysis is an undesired secondary reaction, as can be the case in the trifluoroacetic
acid-catalyzed cleavage of amino acid amide or peptide derivatives 1 from solid-phase resins, conditions are provided to minimize
that hydrolysis.
Amide bonds such as those in N-acylated amino acid amides
1 are generally stable to conditions that would readily cause
hydrolysis of an analogous ester bond. Strong acids such as
TFA, with varying amounts of water or triethylsilane (TES), are
often used to cleave peptides from solid-phase resins without
significant hydrolysis of amide bonds.2 The Rink resin was
specifically designed to permit TFA-promoted cleavage of the
resin link (such as that in 2) to provide peptide primary amides
1 (R3 = H) with both the internal amide(s) and C-terminal
primary amide remaining intact.3
In general, when amide bond cleavage does occur, it is
usually under more vigorous conditions or by virtue of a unique
structural feature that permits intramolecular-catalyzed hydrol-
ysis. For example, hydrolysis of a peptide into its constituent
amino acids requires 6 N HCl at 100 °C,4 and cleavage of the
N-terminal amino acid from a polypeptide via the classic
Edman degradation is promoted by an intramolecular reaction
to form a key cyclic intermediate.5
INTRODUCTION
■
In preliminary work generalizing our earlier synthesis1 of N-
acylated unnatural amino acid amides 1 to procedures
compatible with a wide variety of R2 groups, an unexpected
R2-dependent hydrolytic instability was encountered (Scheme
1). During the trifluoroacetic acid (TFA) cleavage of the final
Rink resin-bound intermediate 2 to give 1, varying amounts of
carboxylic acid 3 were produced.
Scheme 1. Unexpected Presence of Acid 3 in the Cleavage of
Resin-Bound Amide 2
It was of concern that the instability of 1 (R3 = H in the
encountered case) could be a more general phenomenon and
potentially affect the synthesis of N-acylated natural and
unnatural amino acid amides, peptides, and proteins that are
targets of organic and peptide chemists. Because of these wider
implications, we sought to understand the origins and nature of
this instability, to develop predictive tools to anticipate it, and
to establish reaction and workup conditions to minimize it.
Several examples have appeared in the literature in which
specific peptidyl amino acid sequences and N-acylated peptides
undergo facile amide cleavage under mildly acidic condi-
tions.6−9 During acid deprotection of a peptide containing
Received: February 4, 2014
Published: March 12, 2014
© 2014 American Chemical Society
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Scheme 2. Hydrolytic Instability of 4 as a Function of the Electronic Nature of G6
consecutive pipecolic acid residues, Ruzza and co-workers
observed unexpected amide bond hydrolysis and provided
experimental evidence supporting the intermediacy of an
oxazolinium ion.10 This hydrolytic sensitivity of peptidyl
pipecolic acid residues was exploited by Zajdel, Subra, and
co-workers in the design of pipecolic resin linkers for solid-
phase applications.11,12
The closest precedent for the observations reported in this
work comes from Goodman and co-workers, who observed
amide hydrolysis under mild conditions in a series of N-
benzoyl-N-methylAib-Phe-OMe analogues (4; Scheme 2).6
It was demonstrated that this instability is a function of the
remote benzoyl groups on the N-terminal amino acid of this
unusual dipeptide and that the rate of hydrolysis was correlated
with the Hammett σ constants of the benzoyl substituents, G.
Analysis of the data established the existence of a linear free-
energy relationship and supported a mechanism involving the
intermediacy of an oxazolinium ion that proceeded from the
tertiary amide.
alkylation step. Therefore, R2 was limited to the Fmoc group
for all subsequent work quantifying the conditions and success
of the alkylation chemistry, and the hydrolysis of the primary
amide in the naphthyl case was mentioned only parentheti-
cally.1
However, when recently seeking to develop Scheme 3 further
into a robust Distributed Drug Discovery (D3)14−16 lab that
would permit the synthesis of large numbers of new
compounds through widespread variation of both R1 and R2,
it became apparent that the amide hydrolysis observed earlier1
was not an anomaly but a clear function of the nature of the R2
group and that it would be important to understand the factors
involved in this R2-dependent hydrolytic instability so that its
occurrence could be anticipated, predicted, and minimized.
This report describes the achievement of that goal.17
RESULTS AND DISCUSSION
■
Creating D3 laboratories involves developing robust, detailed
procedures that enable students, in the course of their regular
undergraduate organic chemistry lab, to reproducibly synthesize
many new biomimetic molecules as potential drug leads for
neglected diseases.14−16 These procedures are often adap-
ted15,16 from published chemistry.1,15,18 We were developing a
new D3 laboratory, The Combinatorial Synthesis of N-Acylated
Unnatural Amino Acid Amides, based on the published
reaction sequence shown in Scheme 3.1 It would be compatible
with a wide variety of R2 groups obtained from carboxylic acids
R2CO2H. For this adaptation to be successful, it would soon
become apparent that understanding and addressing our earlier,
parenthetical observation of R2-dependent hydrolytic instability
would be critically important.1
To our knowledge, there are no reports of mild hydrolyses of
the simple N-acylated primary amides or peptides represented
by 1. We were surprised, then, in our earlier published work1
synthesizing 1 (Scheme 3, R3 = H) from the benzophenone
Scheme 3. Synthesis of N-Acylated Unnatural Amino Acid
Amides 1 (R3 = H)
This became clear when, in the course of this adaptation, two
students conducted independent replicated syntheses of six new
molecules, 1a−f (Scheme 3 and Table 1). The molecules were
made through combinatorial reactions carried out in a 2 × 3
combinatorial grid19 that utilized two different carboxylic acids,
R2CO2H, in rows A and B and three different alkylating agents,
R1X, in columns 1−3.14−16
The replicated and combinatorial nature of this study
provided a concise set of compelling observations, revealing
an R2-dependent primary amide instability. If any one of the
results for row A (A1−A3) of this grid had been obtained
through a single, isolated experiment, then the presence of acid
3 might have been viewed as an anomaly. The fact that every
reaction in row A gave significant amounts of carboxylic acids
3a−c as side products, that none of the reaction mixtures in
row B contained hydrolyzed material, and that these results
were independently repeated by two researchers suggested the
presence of a systematic effect worthy of further inquiry. In
addition, the combinatorial nature of these six experiments
made it possible to narrow the important variable quickly to the
acylating group R2 (row A vs row B rather than columns 1−3)
and to narrow further the common determining variable to the
nature of the para substituent on the aromatic acylating agent.
imine of Rink MBHA glycine amide 7 to observe small
amounts of amide bond hydrolysis products 3 under TFA
cleavage conditions. This occurred when R2 was naphthyl but
not when it was an Fmoc group.13a−f
Our focus in that report was to develop chemistry to
incorporate unnatural side chains, R1, into the α-position of
glycine with the acyl group, R2, being used only to enable
isolation and quantification of 1 (R3 = H) produced in the key
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Table 1. Initial Observation of Hydrolytic Instability of 1 (Z = NH2) as a Function of R2
On the basis of these observations, it was hypothesized that
hydrolysis was a function of the electronic properties of the
remote aromatic acyl group, R2, and that electron-donating
groups would facilitate the hydrolysis, whereas electron-
withdrawing substituents would suppress it. Accordingly,
follow-up experiments were designed to explore this
assumption systematically.
continuing formation of 11a−g from 10a−g in solution at
increasing times postcleavage and postfiltration (2, 4, 8, and 24
h). The results from all time points are shown in Table 2 and
are reported as the percent of acid 11 present. That the amount
of acid increases with time of exposure to the cleavage cocktail
after cleavage and filtration from the resin (all time points after
0.5 h) indicates that hydrolysis proceeds primarily in solution
and comes directly from 10.
The data shows that electron-withdrawing substituents
(10a−d, high σ values) suppress hydrolysis, whereas electron-
donating substituents with the lowest Hammett σ values (10e−
g) are associated with the most rapid hydrolyses to acids. This
is readily apparent at all time points and mirrors the results
obtained from the acylated N-methylAib-Phe-OMe dipeptides
studied by Goodman and co-workers (Scheme 2).6 Plotting the
percent acid present versus the Hammett σ constant for the
data obtained at 24 h exposure revealed a linear relationship,
which allowed qualitative predictions for other substituents to
be made (see the Supporting Information).
Predictive Nature of R2 Hammett σ Values on
Hydrolytic Instability. To undertake a systematic inves-
tigation of the R2-dependent hydrolytic instability, compounds
10a−g, featuring a representative range of Hammett σ
constants20,21 at the para position of the aromatic benzoyl
group, were prepared on Rink Amide MBHA resins (2a−g),
cleaved, and studied in solution (Scheme 4). After subjection to
Scheme 4. Study of Hydrolysis of 10 (Cleaved from 2) in
95:5 TFA/H2O
This study was expanded (Table 3) to include various alkyl
groups represented by R2. Resin precursor 12 was prepared and
cleaved to 13, and time-dependent hydrolysis was studied. The
results support those of our initial study: electron donation
from R2 enhances production of N-acylated phenylalanines 14.
Comparison of the results from 13d, 13e, 13h, and 13i
illustrates how hydrolysis increases in proceeding from primary
to secondary to tertiary alkyl. The rate-enhancing substituent
effect of the p-methoxy group can be blocked or retained by
carbonyl group insulation (see 13b: R2 = 4-MeO-C6H4CH2 vs
13j: R2 = 4-MeO-C6H4) or conjugation (see 13g: R2 = 4-MeO-
C6H4CHCH vs 13j: R2 = 4-MeO-C6H4), respectively.
Optimization of Cleavage Conditions to Minimize
Hydrolysis. Having demonstrated that R2-dependent amide
hydrolysis can occur under typical resin-cleavage conditions,
various cleavage methods were investigated to identify
conditions that would maximize compound yield while
minimizing subsequent hydrolysis. In the earlier study of the
Hammett constant-dependent hydrolysis of 10 to 11 (Table 2),
the original cleavage cocktail (95:5 TFA/water, 0.5 h, rt), all
samples were filtered from resin so that subsequent time points
(2, 4, 8, and 24 h) would reflect the instability of 10 solely in
solution.
The study was designed to follow the initial formation of
hydrolysis products 11a−g (either directly from 2a−g or
through 10a−g during the 0.5 h cleavage) along with
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Table 2. Percent of Acid 11 from 10 at Various Exposure Times to 95:5 TFA/H2O
a
b
c
starting material
X
σ
0.5 h
0
2 h
4 h
8 h
24 h
t1/2 (h)
d
10a
10b
10c
10d
10e
10f
CN
NH2
CF3
Cl
0.66
0.60
0.54
0.23
0.00
trace
trace
1
trace
trace
1
<1
1
1
nd
e
trace
0
3
495
247
42
3
7
1
4
7
13
31
54
80
33
66
90
99
H
6
12
18
16
Me
OMe
−0.17
−0.27
12
19
26
35
7.5
3.8
10g
43
58
a
b
c
Linear regression analysis assigns a rho value, ρ, of −2.3 to this reaction; see the Supporting Information. Resin still present at this time point. See
d
e
+
the Supporting Information. Not determined. Value for NH3 .
Table 3. Percent of Acid 14 Formed from 13 at Various
Exposure Times to 95:5 TFA/H2O
12k to 13k and hydrolysis to 14k (Table 4) was used as the test
case in evaluating a series of cleavage methods. Resin 12k was
prepared and subjected to the varied cleavage conditions. The
percent yield of 13k was determined in each case by
quantitative liquid chromatography calibrated with authentic
13k (QLC, expressed as the percent yield of 13k), and the ratio
of 13k to hydrolysis product 14k was determined by proton
NMR. Included in the list of methods are mixtures composed
of lower percentages of TFA, shorter reaction times, and
mixtures in which water was replaced by triethylsilane as a
carbocation scavenger.22 Hydrolysis was substantially reduced
(higher 13k:14k ratios) by using cleavage cocktails consisting
of 65 or 35% TFA or by elimination of water from the cocktail
(entries 3 and 4). However, reducing the percentage of TFA
(method E, entries 9 and 10) or reducing the time of cleavage
(30 min vs 1 or 2 h) runs the risk of incomplete cleavage from
the resin.
The data indicates that reducing cleavage time to 30 min
results, without exception, in lower yields of 13k. The use of
35:60:5 TFA/DCM/water (method D, entries 7 and 8)
resulted in the lowest yields, but, interestingly, when
triethylsilane was substituted for water (method E, entries 9
and 10), this composition performed significantly better. The
improved yields associated with triethylsilane may reflect its
ability to irreversibly scavenge the benzhydryl carbocation
derived from the resin.22 Scavenging by water to give the
diarylcarbinol would be reversible under the TFA conditions,
potentially resulting in reattachment of the cleaved product to
a
compound 14
R2
0.5 h
6 h
23 h
a
b
c
d
e
f
9-fluorenylmethyloxy
4-MeO-C6H4CH2
Me
0
0
0
0
trace
0
3
0
3
Me2CHCH2
c-propyl
3
3
15
59
60
80
84
92
96
trace
3
15
19
54
40
59
53
2-naphthyl
g
h
i
4-MeO-C6H4CHCH
8
Me3C
10
16
10
1-adamantyl
4-MeO-C6H4
j
a
Resin still present at this time point.
we had already determined that if solutions of 10 were
immediately stored at −20 °C after the 0.5 h cleavage time at
room temperature then subsequent hydrolysis to 11 was
completely suppressed (see the Supporting Information).
However, for a robust and uncomplicated D3 procedure, it
was necessary to develop optimized cleavage methods that
would not require the use of low temperatures. The cleavage of
Table 4. Optimization of Cleavage Conditions of 12k as a Function of the Cleavage Method and Time
a
b
c
entry
method
time
pecent yield 13k
13k:14k
1
2
A
A
B
B
C
C
D
D
E
30 min
2 h
62
76
73
88
55
80
19
32
55
73
86:14
90:10
96:4
95:5
97:3
97:3
96:4
96:4
99:1
98:2
3
30 min
1 h
4
5
30 min
1 h
6
7
30 min
1 h
8
9
30 min
1 h
10
E
a
Methods: (A) 95:5 TFA/H2O; (B) 95:5 TFA/Et3SiH; (C) 65:30:5 TFA/CH2Cl2/Et3SiH; (D) 35:60:5 TFA/CH2Cl2/H2O; and (E) 35:60:5 TFA/
b
c
1
CH2Cl2/Et3SiH. Measured by QLC. Measured by H NMR.
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Scheme 5. Instability of N-Acylated Peptides as a Function of the Acyl Group
the resin via the primary amide or by way of Friedel−Crafts
alkylation of the aromatic ring.23 The combination of a 1 h
cleavage, replacement of water with triethylsilane, and use of a
lower percentage of TFA represents the preferred methods that
are described by C and E (entries 6 and 10).
Scheme 6. Proposed Mechanism for Formation of
Hydrolysis Products 3
R2-Dependent N-Acylated Peptide Instability. Our
observation and analysis of the mild hydrolysis promoted by
remote aromatic acyl groups R2 on the N-terminus of simple N-
acylated amino acid amides 1 (R3 = H) prompted an additional
experiment documenting its implications for N-acylated peptide
instability. Rink Amide MBHA and Wang N-acylated dipeptide
resins 15 and 16, respectively, were prepared and subjected to
cleavage (95:5 TFA/water, 0.5 h, rt, Scheme 5). Analysis by
LC/MS was performed after 0.5, 6, and 24 h (Table 5).
Table 5. Ratio of Dipeptide 17 or 18 to Hydrolysis Product
14j at Various Times
a
source of 14j
0.5 h
6 h
24 h
17:14j (from Rink resin)
18:14j (from Wang resin)
∼99:1
∼99:1
89:11
88:12
60:40
49:51
a
Resin still present at this time point.
from R2 (alkyl or aryl) might be expected to be operative in the
cyclization of 19 to 20 or in the acid-catalyzed ring opening of
21 to 3.30
Although negligible hydrolysis occurred in the initial 30 min
exposure, by 6 h, 10−15% of the resin-free mixture was
composed of hydrolysis product 14j,24 increasing to nearly 50%
by 24 h.
Mechanistic Considerations. Observations from the
above studies are consistent with hydrolysis proceeding through
a rate-limiting step in which a positively charged transition state
is stabilized by an electron-donating R2 group. A proposed
mechanism25,26 is depicted in Scheme 6. Initial protonation at
the C-terminal amide carbonyl27 to give 19 is followed by
cyclization to the tetrahedral intermediate 20. Collapse of the
tetrahedral intermediate affords an oxazolinium ion (21) of the
type originally proposed by Urban et al.7 and subsequently put
forward by others.6,10,28,29 Hammett substituent effects arising
Possible Utility of N-Acyl Group-Dependent Lability.
In the future, the substituent-dependent amide hydrolytic
lability we have observed could be advantageously used. With
the appropriate choice of the aryl group, a peptide temporarily
acylated with ArCO-AA-OH (Ar = electron-rich aryl group, AA
= amino acid residue) could be converted back to the
unacylated parent peptide under mild, perhaps even physio-
logical, conditions. For example, if the peptide’s physical
properties require modification to improve its pharmacological
profile, then a highly electron-rich Ar group might permit
ArCO-AA-OH to be temporarily attached and then removed in
a prodrug strategy.31,32
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General Procedure for the Preparation and Distribution of
Isopycnic Suspensions of Resins. Preparation. Approximately
80−100 mL of isopycnic solvent per gram of resin was used. To the
resin contained in a 150 mL beaker with stirrer bar was added 60 mL
of NMP followed by 30 mL of DCM. The mixture was briefly stirred
and was then allowed to stand. If resin showed a tendency to
accumulate downward, then DCM was added until there was no
overall movement upward or downward. If resin showed a tendency to
rise, then NMP was added until neutral buoyancy was achieved.
Distribution. Distribution of equal quantities of resin to multiple 3.5
mL glass reaction vessels in a Bill-Board was accomplished with the
use of an Eppendorf repeater pipet fitted with a 50 mL tip. An aliquot
of 2 mL was dispensed to each vessel followed by an aliquot of 1 mL
to each vessel. This procedure was repeated until the number of
milliliters of isopycnic suspension remaining in the beaker was judged
to be less than the number of vessels, n (the smallest volume needed
to dispense 1 mL into all vessels in a single pass). Isopycnic solvent
was added to the beaker to give a volume of (2n + 10) mL. A 2 mL
aliquot was dispensed to each vessel. Isopycnic solvent was added to
the beaker again to give a volume of (2n + 10) mL. Aliquots of 2 mL
were distributed to each vessel again. The resin in each vessel was then
washed with 4 × 2 mL of the solvent to be used in the next step.
General Procedure of Resin Cleavage. This procedure is
associated with the results presented in Tables 1 and 4. The DCM-
washed resins, contained in 3.5 mL reaction vessels, were treated with
1.5 to 2 mL of cleavage reagent. The vessels were rotated at room
temperature for the required time period (0.5, 1, or 2 h) and were then
drained into 5-dram, tooled-neck vials. Each resin was then washed
with 2 mL of the cleavage cocktail followed by 2 mL of DCM. Aliquots
of 100 μL were removed and immediately evaporated to dryness under
a stream of nitrogen, and the residue was dissolved in acetonitrile for
LC/MS analysis.
CONCLUSIONS
■
This report shows that amide bonds in simple N-acylated
amino acid amide or peptide derivatives can be surprisingly
unstable. When an electron-rich aromatic carboxylic acid is
acylated to the amino nitrogen of a simple amino acid
carboxamide or the N-terminus of a peptide, room-temperature
trifluoroacetic acid/water mixtures result in hydrolysis of the
amide bond four bonds away from the NH-acyl group. Even
simple aromatic acyl derivatives, when appropriately substituted
with electron-donating substituents, can promote facile remote
amide cleavage. In particular, this hydrolysis has been found to
be sensitive to the substituent effects of R2 located on a remote
N-terminal aromatic acyl group. In the case of N-benzoyl
amides, the relative rate of hydrolysis can be predicted from the
substituent’s Hammett σ value. After 24 h at room temperature,
compounds having benzoyl acyl substituents with negative σ
values are almost completely hydrolyzed, those with values
between 0 and 0.60 are moderately hydrolyzed, and those with
values ≥0.60 show minimal amide cleavage. Acyl groups
derived from secondary or tertiary alkyls are electron-rich and
also promote substantial hydrolysis after 6 h at room
temperature.
This acyl group-dependent acid-catalyzed lability occurs
under acidic conditions commonly employed when cleaving
products from solid-phase resins. When these products are N-
acylated peptides, the hydrolysis reaction could become
problematic. To address this issue, a systematic evaluation of
cleavage conditions was conducted. It was found that even with
hydrolysis-promoting substituents postcleavage hydrolysis can
be minimized by the use of cleavage cocktails containing 65%
or less TFA, replacing water with triethylsilane.
LC/MS Analyses of Products. Analyses were performed using an
Agilent Eclipse XDB-C18 5 μm column, 4.6 × 150 mm length, with 5
μL injections and a flow rate of 1.0 mL/min. A linear gradient from
20% 1:1 MeCN/MeOH (5 mM NH4OAc) and 80% water (5 mM
NH4OAc) to 100% 1:1 MeCN/MeOH (5 mM NH4OAc) over 10 min
was used. Detection was performed at 254, 214, and 210 nm. Percent
amide and/or percent acid were calculated from the reported peak
areas observed at 254 nm unless otherwise noted.
It is important to note that in the current study N-capped
dipeptides are also hydrolyzed under typical cleavage
conditions. This suggests that unwanted peptide truncation
may occur during synthesis or storage when dipeptides or
longer peptides are acylated with electron-rich aromatic groups
on the N-terminus. The combined ability to predict hydrolytic
instability based on acyl group Hammett σ values with the
availability of cleavage conditions to minimize the predicted
hydrolysis should help chemists working in this field to design
appropriate synthetic strategies to N-acylated amino acid
amides and peptides.
Combinatorial Preparation of Resins 2 (Scheme 3, R1 = 4-
Methylbenzyl, Ethyl, or Allyl; R2 = 4-Methylphenyl or 4-
Trifluoromethylphenyl). These resins were prepared for the
cleavage study reported in Table 1. A 25 mL SPPS vessel was charged
with 1.36 g (0.598 mmol, 0.44 mmol/g) of Fmoc-Gly-NH-Rink
MBHA resin. The resin was washed with 3 × 15 mL of NMP and was
then treated with 5 × 10 mL washes of 20% (v/v) piperidine in NMP
delivered over a 35 min period. The deprotected resin was then
washed with 3 × 15 mL of NMP and was treated with 1.09 g (6.01
mmol, 10.0 equiv) of benzophenone imine in 9 mL of NMP followed
by 0.313 g (5.22 mmol, 8.72 equiv) of acetic acid in 1 mL of NMP.
The vessel was rocked on an orbital shaker for 22 h and then drained,
and the resin was washed with 3 × 10 mL each of NMP, THF, 3:1
THF/H2O, THF, and DCM to give resin 7. The resin was transferred
to a 150 mL beaker using NMP, was made into an isopycnic
suspension, and was equally distributed to twelve 3.5 mL reaction
vessels contained in two 2 × 3 Bill-Boards to provide 50 μmol to each
vessel (see the General Procedure for the Preparation and Distribution
of Isopycnic Suspensions of Resins). The resins were then washed with
3 × 2 mL of NMP. Each Bill-Board was then treated identically as
follows: Phosphazene base tert-butylimino-tri(pyrrolidino)-
phosphorane (BTPP, 1.0 M, 0.50 mL, 0.50 mmol, 10 equiv) in
NMP was added to each vessel and allowed to stand for 5 min. 4-
Methylbenzyl bromide, ethyl iodide, and allyl bromide (0.50 mL of 1.0
M solutions in NMP, 0.50 mmol, 10 equiv) were added, respectively,
to column 1 vessels A1/B1, column 2 vessels A2/B2, and column 3
vessels A3/B3. The Bill-Boards were rotated for 22 h, the vessels were
drained, and the resins were washed with 3 × 2 mL each of NMP,
DCM, and THF to give resins 8 (Scheme 3, R1 = 4-methylbenzyl,
ethyl, or allyl). To each vessel was added 2 mL of 1.0 N HCl/THF
EXPERIMENTAL SECTION
■
General Methods. All reagents were commercially available and
were used without further purification. Fmoc-protected amino acid-
bound Rink Amide MBHA and Wang resins were purchased from
Peptides International (Louisville, KY). Boc-protected amino acid-
bound Merrifield resins were purchased from Novabiochem (EMD
Millipore) or Polymer Laboratories (now Agilent). Combinatorial and
parallel synthetic sequences and resin cleavage/hydrolysis experiments
were performed in custom-made 3.5 mL glass reaction vessels
containing a sintered glass frit and screw-cap ends fitted with
poly(tetrafluoroethylene) (PTFE) septa all purchased from Chemglass
(Vineland, NJ). The reaction vessels were secured in polypropylene
Bill-Boards consisting of a 2 × 3 or 4 × 6 array of position holes fitted
with rubber O-rings and were supplied by Leads Metal (Indianapolis,
IN). Synthetic sequences for the preparation of advanced intermediate
resins were performed in 25 or 50 mL solid-phase peptide synthesis
vessels (SPPS) purchased from Chemglass. Rotation of Bill-Boards
was accomplished using a spit rod fitted with three 6-brackett metal
assemblies custom-made by Leads Metal and was driven by a Jenn-Air
rotisserie motor or using brackets to fit 2 × 3 or 4 × 6 Bill-Boards held
in a rotovap assembly.
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(1:2), and the Bill-Boards were rotated for 20 min, the vessels were
drained, and the resins were washed with 3 mL of THF, 2 × 2.5 mL of
0.2 M diisopropylethylamine in NMP, and 2 × 2.5 mL of NMP to give
resins 9 (Scheme 3, R1 = 4-methylbenzyl, ethyl, allyl). 4-
Methylbenzoic acid and 4-trifluoromethylbenzoic acid (0.25 M, 1.0
mL, 0.25 mmol, 5.0 equiv) in 0.25 M 1-hydroxybenzotriazole (HOBt)
in NMP were added, respectively, to row A vessels A1−A3 and row B
vessels B1−B3. Each vessel was then treated with 0.50 mL (0.25
mmol, 5.0 equiv) of 0.50 M diisopropylcarbodiimide in NMP, and the
Bill-Boards were rotated for 22 h. The vessels were drained, and the
resins were washed with 2 × 3 mL each of NMP, THF, and 3 × 3 mL
of DCM to afford resins 2 (Scheme 3). Cleavage of each resin with
95:5 TFA/water for 2 h was performed as described above (General
Procedure of Resin Cleavage).
bromide in 14.3 mL of NMP. The vessel was rocked overnight for 24 h
and then drained, and the resin was washed with 3 × 20 mL each of
NMP, DCM, and THF. Resin 8 was dried under a slow stream of
argon for 45 min and was then stored overnight.
Preparation of Resin 9 (R1 = 4-Methylbenzyl) and Distribution to
3.5 mL Reaction Vessels. Resin 8 was treated with 30 mL of 2:1 THF/
1.0 N HCl, and the vessel was rocked for 40 min and then drained.
The resin was washed with 3 × 20 mL of THF, neutralized with 3 ×
20 mL washes of 0.2 M diisopropylethylamine (DIEA) in NMP, and
then washed with 3 × 20 mL of NMP to give resin 9. Resin 9 was then
quantitatively transferred to a 400 mL beaker using 100 mL of NMP to
facilitate the transfer. Dichloromethane (50 mL) was then added, and
the composition was adjusted to achieve neutral buoyancy. A final
volume of 200−250 mL was then equally dispensed 2 mL at a time
into a 5 column × 7 row array of reaction vessels with uncapped
bottoms held in two 4 × 6 Bill-Boards (see General Procedure for the
Preparation and Distribution of Isopycnic Suspensions of Resins).
Each vessel was then washed with 3 × 2 mL of NMP and then capped.
Preparation of Resins 2a−g (R1 = 4-Methylbenzyl). The array of
35 vessels each containing 50.0 μmol of resin 9 was treated in the
following manner: 1.0 mL of a 0.25 M solution of row 1 carboxylic
acid 1RCO2H (250 μmol, 5.0 equiv) dissolved in 0.25 M 1-
hydroxybenzotriazole (250 μmol, 5.0 equiv, HOBt) in NMP was
added to each of the five vessels in row 1 followed by 0.5 mL of a 0.50
M solution of diisopropylcarbodiimide (250 μmol, 5.0 equiv, DIC) in
NMP. The five vessels in each of the remaining six rows were treated
in an identical manner with its assigned carboxylic acid, HOBt, and
DIC. The Bill-Board was rotated for 2 days, and the vessels were
drained and then washed with 3 × 1.5 mL each of NMP, THF, and
DCM to give resins 2a−g.
1
LC/MS and H NMR Results of TFA-Mediated Cleavage of 2
× 3 Combinatorial Bill-Boards (Scheme 3 and Table 1).
Cleavage of 2 gave the crude products in Table 1 (see General
Procedure of Resin Cleavage). Analytical data for crude products 1 (Z
= NH2) and 3 (Z = OH) include LC/MS and diagnostic peaks in the
1H NMR.
α-[(4-Methylbenzoyl)amino]-4-methylbenzenepropanamide
(1a) and N-(4-Methylbenzoyl)-4-methylphenylalanine (3a). For
1
1a: H NMR (500.13 MHz, CDCl3): δ 2.30 (s), 2.38 (s), 3.19 (dd),
3.29 (dd), 5.01 (dd), 7.55 (d); LC/MS (ESI): 8.70 min, m/z 297 [M
1
+ H]+. For 3a: H NMR (500.13 MHz, CDCl3): δ 3.13 (2dd), 4.94
(dd), 7.59 (d); LC/MS (ESI): 6.36 min, m/z 298 [M + H]+.
N-(2-Amino-1-ethyl-2-oxoethyl)-4-methylbenzamide (1b)
and 2-[(4-Methylbenzoyl)amino]butanoic Acid (3b). For 1b:
1H NMR (500.13 MHz, CDCl3): δ 1.02 (t), 1.90 (septet), 2.08
(septet), 4.75 (dd), 7.68 (d); LC/MS (ESI): 6.08 min, m/z 221 [M +
1
H]+. For 3b: H NMR (500.13 MHz, CDCl3): δ 1.83 (septet), 1.99
TFA-Mediated Cleavage of Resins 2a−g to 10a−g and 11a−
g (Scheme 4, Hammett Study, Table 2). Each of the 35 vessels in
the 5 × 7 array (50 μmol each) was treated with 1.5 mL of 95:5 TFA/
H2O and was rotated at room temperature for 30 min. All vessels were
then drained into collection vials, and the resins were washed with 1
mL of 95:5 TFA/H2O. A 90 μL aliquot from the combined filtrates of
each acylated amide 10a−g in column 1 (representing the 0.5 h
exposure) was removed and immediately evaporated to dryness under
a stream of nitrogen. These samples were diluted with acetonitrile and
analyzed by LC/MS. The remainder of each filtrate in column 1 (0.5 h
exposure) was then stored at −20 °C and assayed by LC/MS again
after 48 h. These column 1 filtrates, stored at −20 °C, would be
worked up for the purpose of isolating primary amides 10a−g. Filtrates
in columns 2−5 were allowed to stand at room temperature for 1.5,
3.5, 7.5, and 23.5 h, respectively, and were then assayed by LC/MS
(Table 2). After sampling for analysis, filtrates were stored at −20 °C.
Complete conversion of amides 10e−g to acids 11e−g (X = H, Me,
and OMe), for the purpose of isolation and characterization, was
accomplished by allowing their column 5 filtrates to stand at room
temperature for 5 days.
(septet), 4.68 (dd); LC/MS (ESI): 3.64 min, m/z 222 [M + H]+.
N-[(1-Aminocarbonyl)-3-buten-1-yl]-4-methylbenzamide
(1c) and 2-[(4-Methylbenzoyl)amino]-4-pentenoic Acid (3c).
1
For 1c: H NMR (500.13 MHz, CDCl3): δ 2.40 (s), 2.60−2.78 (m),
4.81 (dd), 5.18−5.23 (m), 5.74−5.82 (m), 7.24 (d), 7.65 (d); LC/MS
(ESI): 6.66 min, m/z 233 [M + H]+. For 3c: 1H NMR (500.13 MHz,
CDCl3): δ 4.77 (dd); LC/MS (ESI): 4.02 min, m/z 234 [M + H]+.
α-[(4-Trifluoromethylbenzoyl)amino]-4-methylbenzenepro-
1
panamide (1d). H NMR (500.13 MHz, CDCl3): δ 2.32 (s), 3.10
(dd), 3.19 (dd), 4.85 (m), 7.70 (d), 7.86 (d); LC/MS (ESI): 9.39 min,
m/z 351 [M + H]+.
N-(2-Amino-1-ethyl-2-oxoethyl)-4-trifluoromethylbenza-
mide (1e). 1H NMR (500.13 MHz, CDCl3): δ 1.02 (t), 1.80 (septet),
2.02 (septet), 4.62 (m), 7.72 (d), 7.93 (d); LC/MS (ESI): 7.39 min,
m/z 275 [M + H]+.
N-[(1-Aminocarbonyl)-3-buten-1-yl]-4-trifluoromethylben-
zamide (1f). 1H NMR (500.13 MHz, CDCl3): δ 2.64 (m), 4.74 (m),
5.19−5.23 (m), 5.78−5.85 (m), 7.71 (d), 7.90 (d); LC/MS (ESI):
7.85 min, m/z 287 [M + H]+.
Preparation of Resins 2a−g from Resin 7 (Scheme 3).
Preparation of Resin 7 from Fmoc-Gly-NH-Rink MBHA Resin. A 50
mL SPPS vessel was charged with 3.43 g (1.75 mmol, 0.51 mmol/g) of
Fmoc-Gly-NH-Rink MBHA resin. The resin was washed with 3 × 15
mL of NMP and was then treated with 5 × 20 mL washes of 20% (v/
v) piperidine in NMP delivered over a 45 min period. The deprotected
resin was then washed with 4 × 15 mL of NMP and was treated with
3.16 g (17.4 mmol, 9.94 equiv) of benzophenone imine in 15 mL of
NMP followed by 910 mg (15.2 mmol, 8.70 equiv) of acetic acid. The
vessel was rocked on an orbital shaker for 20 h and then drained, and
the resin was washed with 3 × 20 mL each of NMP, THF, 3:1 THF/
H2O, THF, and DCM. Resin 7 was dried under a gentle stream of
nitrogen gas for 2 h and then under vacuum for 3 h.
Isolation of Amides 10a−g. After storage at −20 °C for 2 days,
each solution from column 1 was added to 100 mL of cold 1.0 N
sodium hydroxide. The mixture was then extracted with two 20 mL
portions of DCM. The combined extracts were dried over Na2SO4 and
were concentrated to give crude amides 10a−g as amorphous solids.
α-[(4-Cyanobenzoyl)amino]-4-methylbenzenepropanamide
(10a). Yield 7.6 mg (49%), mp 235−238 °C; 1H NMR (500.13 MHz,
DMSO-d6): δ 2.22 (s, 3H), 2.93 (dd, J = 13.6 and 10.9 Hz, 1H), 3.09
(dd, J = 13.7 and 3.9 Hz, 1H), 4.62 (ddd, J = 10.8, 8.5, and 4.1 Hz,
1H), 7.05 (d, J = 7.8 Hz, 2H), 7.13 (br s, 1H), 7.22 (d, J = 8.0 Hz,
2H), 7.61 (br s, 1H), 7.94 (s, 4H), 8.80 (d, J = 8.5 Hz, 1H); 13C NMR
(125.77 MHz, DMSO-d6): δ 20.5, 36.7, 55.0, 113.5, 118.2, 128.1,
128.6, 128.9, 132.2, 135.0, 135.2, 138.0, 164.7, 173.0; HRMS (ESI-
TOF): m/z [M + Na]+ calcd for C18H17N3O2Na, 330.1219; found,
330.1215.
Preparation of Resin 8 (R1 = 4-Methylbenzyl). Resin 7 (1.75
mmol) was swelled in the SPPS vessel with 30 mL of NMP for 30 min.
The vessel was drained, and the resin was treated with 5.49 g (17.6
mmol, 10.0 equiv) of phosphazene base tert-butylimino-tri-
(pyrrolidino)phosphorane (BTPP) in 9.5 mL of NMP. The contents
were gently agitated by hand to affect mixing, and the mixture was
treated with 3.26 g (17.6 mmol, 10.0 equiv) of 4-methylbenzyl
α-[(4-Aminobenzoyl)amino]-4-methylbenzenepropanamide
1
(10b). Yield 4.8 mg (32%), mp 208−211 °C (MeOH); H NMR
(500.13 MHz, DMSO-d6): δ 2.22 (s, 3H), 2.92 (dd, J = 13.6 and 10.4
Hz, 1H), 3.01 (dd, J = 13.7 and 4.2 Hz, 1H), 4.53 (ddd, J = 10.2, 8.5,
and 4.2 Hz, 1H), 5.60 (br s, 2H), 6.51 (d, J = 8.6 Hz, 2H), 7.02 (br s,
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1H), 7.04 (d, J = 7.9 Hz, 2H), 7.18 (d, J = 8.0 Hz, 2H), 7.41 (br s,
1H), 7.53 (d, J = 8.6 Hz, 2H), 7.90 (d, J = 8.4 Hz, 1H); 13C NMR
(125.77 MHz, DMSO-d6): δ 20.5, 36.8, 54.5, 112.3, 120.6, 128.5,
128.8, 128.9, 134.8, 135.5, 151.6, 165.9, 173.7; HRMS (ESI-TOF) m/
z: [M + Na]+ calcd for C17H19N3O2Na, 320.1375; found, 320.1386.
α-[(4-Trifluoromethylbenzoyl)amino]-4-methylbenzenepro-
172 °C; 1H NMR (500.13 MHz, CDCl3): δ 2.32 (s, 3H), 3.23 (dd, J =
14.1 and 6.0 Hz, 1H), 3.32 (dd, J = 14.1 and 5.6 Hz, 1H), 5.03 (m,
1H), 6.51 (br d, J = 7.0 Hz, 1H), 7.11 (m, 4H), 7.42 (t, J = 7.6 Hz,
2H), 7.52 (t, J = 7.4 Hz, 1H), 7.68 (d, J = 7.9 Hz, 2H); 13C NMR
(125.77 MHz, CD3OD): δ 21.1, 37.8, 55.7, 128.4, 129.5, 130.1, 130.2,
132.8, 135.4, 135.6, 137.5, 170.2, 174.9; HRMS (ESI-TOF) m/z: [M +
H]+ calcd for C17H18NO3, 284.1281; found, 284.1285.
1
panamide (10c). Yield 4.1 mg (23%), mp 220−224 °C; H NMR
(500.13 MHz, DMSO-d6): δ 2.22 (s, 3H), 2.94 (dd, J = 13.6 and 10.9
Hz, 1H), 3.09 (dd, J = 13.7 and 3.9 Hz, 1H), 4.63 (ddd, J = 10.9, 8.2,
and 4.1 Hz, 1H), 7.05 (d, J = 7.8 Hz, 2H), 7.12 (br s, 1H), 7.22 (d, J =
7.9 Hz, 2H), 7.58 (br s, 1H), 7.83 (d, J = 8.3 Hz, 2H), 7.99 (d, J = 8.1
Hz, 2H), 8.75 (d, J = 8.4 Hz, 1H); 13C NMR (125.77 MHz, DMSO-
d6): δ 20.5, 36.7, 54.9, 123.8 (q, 1JCF = 272.4 Hz), 125.1 (q, 3JCF = 3.6
Hz), 128.2, 128.5, 128.9, 131.1 (q, 2JCF = 31.7 Hz), 135.0, 135.2, 137.8,
164.9, 173.0; HRMS (ESI-TOF) m/z: [M + H]+ calcd for
C18H18F3N2O2, 351.1315; found, 351.1317.
N-[(4-Methyl)benzoyl]-4-methylphenylalanine (11f). After
standing at room temperature for 5 additional days, the filtrate from
the 24 h postcleavage exposure was evaporated under a stream of
nitrogen gas to give 16.5 mg of a residue that was chromatographed on
a Dynamax Microsorb C-18, 5 μm column (21.4 × 250 mm) using the
isochratic system of 50/50 1:1 MeCN/MeOH (5 mM ammonium
acetate)/water (5 mM ammonium acetate) at a 5 mL/min flow rate to
give 8.6 mg (61%) 11f as an amorphous solid after evaporation, mp
1
184−187 °C; H NMR (500.13 MHz, CD3OD): δ 2.26 (s, 3H), 2.37
α-[(4-Chlorobenzoyl)amino]-4-methylbenzenepropanamide
(10d). Yield 7.5 mg (47%), 225.5−227 °C (EtOH); 1H NMR (500.13
MHz, DMSO-d6): δ 2.22 (s, 3H), 2.92 (dd, J = 13.7 and 10.8 Hz, 1H),
3.06 (dd, J = 13.7 and 4.0 Hz, 1H), 4.59 (ddd, J = 10.8, 8.4, and 4.1
Hz, 1H), 7.05 (d, J = 7.8 Hz, 2H), 7.10 (br s, 1H), 7.20 (d, J = 8.0 Hz,
2H), 7.52 (d, J = 8.6 Hz, 2H), 7.56 (br s, 1H), 7.82 (d, J = 8.7 Hz,
2H), 8.58 (d, J = 8.4 Hz, 1H); 13C NMR (125.77 MHz, DMSO-d6): δ
20.5, 36.7, 54.8, 128.1, 128.5, 128.9, 129.2, 132.8, 135.0, 135.3, 135.9,
165.0, 173.1; HRMS (ESI-TOF) m/z: [M + H]+ calcd for
C17H18ClN2O2, 317.1051; found, 317.1050.
(s, 3H), 3.07 (dd, J = 13.8 and 8.3 Hz, 1H), 3.27 (dd, J = 13.8 and 4.9
Hz, 1H), 4.75 (dd, J = 8.2 and 5.0 Hz, 1H), 7.05 (d, J = 7.9 Hz, 2H),
7.12 (d, J = 8.0 Hz, 2H), 7.23 (d, J = 8.0 Hz, 2H), 7.61 (d, J = 8.2 Hz,
2H); 13C NMR (125.77 MHz, CD3OD): δ 21.1, 21.4, 38.2, 56.5,
128.3, 129.9, 130.1, 130.3, 132.7, 135.9, 137.2, 143.4, 169.7, 176.1;
HRMS (ESI-TOF) m/z: [M + H]+ calcd for C18H20NO3, 298.1438;
found, 298.1441.
N-[(4-Methyoxy)benzoyl]-4-methylphenylalanine (11g).
After standing an additional 3 days at −20 °C, the filtrate from the
24 h postcleavage exposure was evaporated under a stream of nitrogen
gas to give 17.1 mg of a residue that was chromatographed on a
prepacked 500 mg column of Hypersep CN (cyanosilica, Thermo
Scientific) using a step-gradient elution with hexanes (3 mL), 8:2
hexanes/acetone (3 mL), and eluting the desired material with 7:3
hexanes/acetone to give 5.3 mg (36%) 11g as an amorphous solid, mp
α-(Benzoylamino)-4-methylbenzenepropanamide (10e).33
Yield 8.2 mg (58%), mp 216−220 °C; 1H NMR (500.13 MHz,
DMSO-d6): δ 2.22 (s, 3H), 2.93 (dd, J = 13.6 and 10.6 Hz, 1H), 3.06
(dd, J = 13.7 and 4.1 Hz, 1H), 4.61 (ddd, J = 10.6, 8.4, and 4.1 Hz,
1H), 7.05 (d, J = 7.8 Hz, 2H), 7.10 (br s, 1H), 7.22 (d, J = 8.0 Hz,
2H), 7.44 (t, J = 7.5 Hz, 2H), 7.51 (tt, J = 7.4 and 1.3 Hz, 1H), 7.54
(br s, 1H), 7.80 (d, J = 7.1 Hz, 2H), 8.46 (d, J = 8.4 Hz, 1H); 13C
NMR (125.77 MHz, DMSO-d6): δ 20.5, 36.7, 54.8, 127.3, 128.0,
128.5, 128.9, 131.1, 134.0, 134.9, 135.3, 166.0, 173.3; HRMS (ESI-
TOF) m/z: [M + H]+ calcd for C17H19N2O2, 283.1441; found,
283.1440.
α-[(4-Methylbenzoyl)amino]-4-methylbenzenepropanamide
(10f). Yield 8.0 mg (54%), mp 203−206 °C; 1H NMR (500.13 MHz,
CDCl3): δ 2.32 (s, 3H), 2.40 (s, 3H), 3.07 (dd, J = 13.9 and 8.0 Hz,
1H), 3.24 (dd, J = 13.8 and 5.8 Hz, 1H), 4.83 (m, 1H), 5.32 (br s,
1H), 5.73 (br s, 1H), 6.77 (d, J = 7.1 Hz, 1H), 7.12 (d, J = 7.8 Hz,
2H), 7.19 (d, J = 8.0 Hz, 2H), 7.23 (d, J = 8.0 Hz, 2H), 7.63 (d, J = 8.2
Hz, 2H); 13C NMR (125.77 MHz, DMSO-d6): δ 20.6, 20.9, 36.8, 54.8,
127.4, 128.58, 128.60, 129.0, 131.3, 135.0, 135.4, 141.0, 165.9, 173.4;
HRMS (ESI-TOF) m/z: [M + H]+ calcd for C18H21N2O2, 297.1598;
found, 297.1595.
α-[(4-Methoxybenzoyl)amino]-4-methylbenzenepropana-
mide (10g). Yield 6.6 mg (42%), mp 210−212 °C; 1H NMR (500.13
MHz, DMSO-d6): δ 2.21 (s, 3H), 2.93 (dd, J = 13.6 and 10.6 Hz, 1H),
3.03 (dd, J = 13.7 and 4.0 Hz, 1H), 3.70 (s, 3H), 4.57 (ddd, J = 10.5,
8.4, and 4.1 Hz, 1H), 6.97 (d, J = 7.8 Hz, 2H), 7.04 (d, J = 7.9 Hz,
2H), 7.06 (br s, 1H), 7.21 (d, J = 8.0 Hz, 2H), 7.49 (br s, 1H), 7.79 (d,
J = 8.9 Hz, 2H), 8.29 (d, J = 8.4 Hz, 1H); 13C NMR (125.77 MHz,
DMSO-d6): δ 20.5, 36.7, 54.7, 55.2, 113.2, 126.2, 128.5, 128.9, 129.1,
134.9, 135.4, 161.4, 165.4, 173.4; HRMS (ESI-TOF) m/z: [M + Na]+
calcd for C18H20N2O3Na, 335.1372; found, 335.1362.
Isolation and Purification of Acids 11e−g (Hammett Study,
Scheme 4). The benzoyl substituents present in the corresponding
amides 10e−g facilitate hydrolysis at rates high enough to result in
complete conversion to the acids over time, rendering convenient
isolation and purification.
1
168−72 °C; H NMR (500.13 MHz, CD3OD): δ 2.27 (s, 3H), 3.07
(dd, J = 13.9 and 9.3 Hz, 1H), 3.27 (dd, J = 13.9 and 5.0 Hz, 1H), 3.83
(s, 3H), 4.80 (dd, J = 9.4 and 5.1 Hz, 1H), 6.94 (d, J = 8.9 Hz, 2H),
7.08 (d, J = 7.9 Hz, 2H), 7.14 (d, J = 8.0 Hz, 2H), 7.71 (d, J = 8.9 Hz,
2H); 13C NMR (125.77 MHz, CD3OD): δ 21.1, 37.8, 55.7, 55.9,
114.7, 127.4, 130.1, 130.2, 130.3, 135.6, 137.4, 164.1, 169.7, 175.1;
HRMS (ESI-TOF) m/z: [M + H]+ calcd for C18H20NO4, 314.1387;
found, 314.1390.
Preparation of N-Acylated (S)-Phenylalanine-NH-Rink
Amide MBHA Resins 12b−j. A 50 mL SPPS vessel was charged
with 780−940 mg (250−300 μmol) of Fmoc-Phe-NH-Rink Amide
MBHA resin (0.32 mmol/g, Peptides International) and treated with
20 mL of 20% (v/v) piperidine in N-methylpyrolidinone (NMP). The
mixture was rocked for 45 min at room temperature and was then
drained. The resin was washed with 4 × 15 mL of NMP and was then
diluted with 55:45 NMP/DCM to make an isopycnic mixture (neutral
buoyancy). The mixture was then equally distributed to 3.5 mL
reaction vessels using a repeater pipet (see General Procedure for the
Preparation and Distribution of Isopycnic Suspensions of Resins).
Each resin was washed with 4 × 1.5 mL of NMP and was then treated
with a 0.25 M solution of the required carboxylic acid (5.00 equiv) in
0.25 M N,N,N′,N’-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hex-
afluorophosphate (5.00 equivalents, HBTU) in DMF followed by 10.0
equiv of a 0.50 M solution of diisopropylethylamine in DMF. The
vessels were rotated at room temperature for 48 h and were then
drained. Each resin was washed with 5 × 1.5 mL of DMF and 6 × 1.5
mL of DCM and was then stored overnight in its capped vessel.
TFA-Mediated Cleavage of Resins 12a−j to 13 and 14 (Table
3). Each vessel containing 50 μmol of resin 12a−j was treated with 1.5
mL of 95:5 TFA/H2O and was rotated at room temperature for 0.5 h.
All vessels were then drained into collection vials, and the resins were
washed with 1.5 mL of 95:5 TFA/H2O followed by 1.5 mL of DCM.
Three 200 μL aliquots from each vial were removed and placed in sets
of three vials representing the three time exposures of 0.5, 6, and 23 h,
at which point they were evaporated under a stream of nitrogen gas
and analyzed by LC/MS (214 nm) as solutions in acetonitrile. To
prevent further hydrolysis of primary amides 13 after 30 min exposure
to the TFA solution, the vials containing the bulk of the filtrates were
stored at −20 °C.
N-Benzoyl-4-methylphenylalanine (11e).34 After standing at
room temperature for 5 additional days, the filtrate from the 24 h
postcleavage exposure was evaporated under a stream of nitrogen gas
to give 14.4 mg of a residue that was chromatographed on a prepacked
500 mg column of Hypersep CN (cyanosilica, Thermo Scientific)
using step-gradient elution with hexanes (3 × 3 mL), 8:2 hexanes/
acetone (3 mL), and eluting the desired material with 7:3 hexanes/
acetone to give 6.9 mg (51%) 11e as an amorphous solid, mp 171−
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9 H - F l u o r e n - 9 - y l m e t h y l N - [ 2 - A m i n o - 2 - o x o - 1 -
(phenylmethyl)ethyl]carbamate [Fmoc-Phe-NH2] (13a).35 The
filtrate was evaporated to dryness under a stream of nitrogen to give
6.6 mg of crude material. Trituration under diethyl ether afforded 6.1
°C; 1H NMR (500.13 MHz, DMSO-d6): δ 3.03 (dd, J = 13.7 and 10.7
Hz, 1H), 3.15 (dd, J = 13.7 and 4.1 Hz, 1H), 4.72 (ddd, J = 10.7, 8.6,
and 4.3 Hz, 1H), 7.14−7.15 (m, 2H), 7.26 (t, J = 7.6 Hz, 2H), 7.37 (d,
J = 7.2 Hz, 2H), 7.58−7.62 (m, 3H), 7.88 (dd, J = 8.6 and 1.7 Hz,
1H), 7.96 (d, J = 8.3 Hz, 2H), 8.01 (d, J = 7.6 Hz, 1H), 8.42 (br s,
1H), 8.66 (br d, J = 8.4 Hz, 1H); 13C NMR (125.77, DMSO-d6): δ
37.2, 54.7, 124.2, 126.1, 126.6, 127.48, 127.55, 127.6, 127.9, 128.7,
129.0, 131.3, 131.9, 134.0, 138.5, 166.0, 173.2 (18 13C signals are
predicted, but only 17 were observed); HRMS (ESI-TOF) m/z: [M +
H]+ calcd for C20H19N2O2, 319.1441; found, 319.1447.
1
mg (33%) of 13a as an amorphous solid, mp 192−194 °C (dec); H
NMR (500.13 MHz, DMSO-d6): δ 2.78 (dd, J = 13.5 and 10.6 Hz,
1H), 3.00 (dd, J = 13.6 and 4.0 Hz, 1H), 4.16−4.19 (m, 4H), 7.08 (br
s, 1H), 7.18 (t, J = 7.1 Hz, 1H), 7.24−7.33 (m, 6H), 7.39−7.43 (m,
2H), 7.45 (br s, 1H), 7.53 (d, J = 8.8 Hz, 1H), 7.63 (d, J = 7.6 Hz,
1H), 7.65 (d, J = 7.6 Hz, 1H), 7.88 (d, J = 7.5 Hz, 2H); 13C NMR
(125.77 MHz, DMSO-d6): δ 37.4, 46.4, 55.9, 65.5, 120.0, 125.17,
125.25, 126.1, 126.9, 127.5, 127.9, 129.1, 138.2, 140.5, 143.6, 143.7,
155.7, 173.3 (Note that two extra aromatic signals are present,
indicative of the diastereotopic nature of the aromatic carbons, which
display identical atom connectivity. Two extra signals are also present
in the 13C NMR of commercial Fmoc-Phe-OH); HRMS (ESI-TOF)
m/z: [M + Na]+ calcd for C24H22N2O3Na, 409.1528; found, 409.1535.
4-Methoxy-α-[(2-phenylacetyl)amino]benzenepropanamide
(13b). The filtrate was added carefully to 25 mL of cold 2 M NaOH,
and the mixture was extracted with 2 × 20 mL of DCM. The
combined extracts were dried (Na2SO4) and concentrated to give 7.7
mg of crude material. Trituration under 2 mL of diethyl ether afforded
α-[(2E)-3-((4-Methoxyphenyl)-1-oxo-2-propenyl)amino]-
benzenepropanamide (13g). The filtrate was immediately
evaporated to dryness with a stream of nitrogen gas to give 10.5 mg
of crude material that was purified by reverse-phase HPLC on a
Dynamax Microsorb C-18, 5 μm column (21.4 × 250 mm) eluting
isochratically with 60:40 1:1 MeCN/MeOH (with 5 mM ammonium
acetate)/water (with 5 mM ammonium acetate) at 5 mL/min and
detection at 254 nm to afford 3.5 mg (21%) of 13g (95:5 mixture of
1
E/Z isomers) as an amorphous solid, mp 191−201 °C (dec); H
NMR (500.13 MHz, CDCl3): δ 3.10 (dd, J = 13.8 and 8.0 Hz, 1H),
3.22 (dd, J = 13.8 and 5.9 Hz, 1H), 3.83 (s, 3H), 4.82 (dd, J = 13.8 and
7.3 Hz, 1H), 5.33 (br s, 1H), 5.77 (br s, 1H), 6.23 (br s, 1H), 6.26 (d, J
= 15.5 Hz, 1H), 6.88 (d, J = 8.5 Hz, 2H), 7.26−7.34 (m, 5H), 7.44 (d,
1
5.7 mg (36%) of 13b as an amorphous solid, mp 169−70 °C; H
1
NMR (500.13 MHz, DMSO-d6): δ 2.75 (dd, J = 13.7 and 9.6 Hz, 1H),
2.98 (dd, J = 13.7 and 4.7 Hz, 1H), 3.28 (d, J = 14.0 Hz, 1H), 3.34 (d,
J = 13.9 Hz, 1H), 3.71 (s, 3H), 4.43 (ddd, J = 9.3, 8.5, and 4.7 Hz,
1H), 6.77 (d, J = 8.7 Hz, 2H), 7.00 (d, J = 8.6 Hz, 2H), 7.05 (br s,
1H), 7.18−7.24 (m, 5H), 7.45 (br s, 1H), 8.11 (br d, J = 8.5 Hz, 1H);
13C NMR (125.77 MHz, DMSO-d6): δ 39.2, 39.6, 53.6, 54.9, 113.4,
J = 8.6 Hz, 2H), 7.58 (d, J = 15.5 Hz, 1H); H NMR (500.13 MHz,
CD3OD): δ 2.99 (dd, J = 13.9 and 8.9 Hz, 1H), 3.22 (dd, J = 13.9 and
5.8 Hz, 1H), 3.84 (s, 3H), 4.77 (dd, J = 8.9 and 5.8 Hz, 1H), 6.52 (d, J
= 15.8 Hz, 1H), 6.95 (d, J = 8.7 Hz, 2H), 7.22−7.24 (m, 1H), 7.30−
7.31 (m, 4H), 7.46 (d, J = 15.7 Hz, 1H), 7.51 (d, J = 8.7 Hz, 2H); 13C
NMR (125.77 MHz, CD3OD): δ 39.2, 55.87, 55.94, 115.4, 118.8,
127.8, 128.8, 129.5, 130.3, 130.6, 138.6, 142.1, 162.8, 168.8, 176.3;
HRMS (ESI-TOF) m/z: [M + H]+ calcd for C19H21N2O3, 325.1547;
found, 325.1552.
126.0, 127.9, 128.1, 129.1, 129.8, 137.9, 157.6, 170.0, 173.0; HRMS
(ESI-TOF) m/z: [M + Na]+ calcd for C18H20N2O3Na, 335.1372;
found, 335.1375.
α-(Acetylamino)benzenepropanamide (13c).36 LC/MS (210
nm/ESI): 33.7 min, m/z 207 [M + H]+. YMC-Pack Pro C18, 150 ×
4.6 mm i.d., 3 μm, 0.75 mL/min, 10% 50:50 acetonitrile/methanol
with 5 mM ammonium acetate/90% water with 5 mM ammonium
acetate.
α-[(2,2-Dimethyl-1-oxopropyl)amino]benzenepropanamide
(13h).38 The filtrate was immediately evaporated to dryness with a
stream of nitrogen gas to give crude material that was purified by
reverse-phase HPLC on a Dynamax Microsorb C-18, 5 μm column
(21.4 × 250 mm) eluting isochratically with 60:40 1:1 MeCN/MeOH
(with 5 mM ammonium acetate)/water (with 5 mM ammonium
acetate) at 5 mL/min and detection at 254 nm to afford 8.0 mg (43%)
of 13h as an amorphous solid, mp 94−96 °C; 1H NMR (500.13 MHz,
CDCl3): δ 1.12 (s, 9H), 3.09 (m, 2H), 4.69 (dd, J = 14.4 and 7.2 Hz,
1H), 5.59 (br s, 1H), 6.12 (br s, 1H), 6.30 (br d, J = 7.2 Hz, 1H),
7.22−7.25 (m, 3H), 7.28−7.31 (m, 2H); 13C NMR (125.77 MHz,
CDCl3): δ 27.3, 38.0, 38.7, 53.8, 127.1, 128.7, 129.3, 136.6, 173.4,
178.7; HRMS (ESI-TOF) m/z: [M + H]+ calcd for C14H21N2O2,
249.1598; found, 249.1597.
α-[(3-Methyl-1-oxobutyl)amino]benzenepropanamide
(13d). The filtrate was added carefully to 25 mL of cold 2 M NaOH
and the mixture was extracted with 2 × 20 mL of DCM. The
combined extracts were dried (Na2SO4) and concentrated to give 5.7
mg of crude material. Trituration under 2 mL of diethyl ether afforded
1
4.0 mg (32%) of 13d as an amorphous solid, mp 182−183 °C; H
NMR (500.13 MHz, DMSO-d6): δ 0.67 (d, J = 6.5 Hz, 3H), 0.75 (d, J
= 6.5 Hz, 3H), 1.83 (septet, J = 6.6 Hz, 1H), 1.90 (d, J = 6.3 Hz, 2H),
2.72 (dd, J = 13.7 and 10.1 Hz, 1H), 2.99 (dd, J = 13.7 and 4.5 Hz,
1H), 4.46 (ddd, J = 10.1, 8.7, and 4.5 Hz, 1H), 7.01 (br s, 1H), 7.15−
7.18 (m, 1H), 7.22−7.24 (m, 4H), 7.38 (br s, 1H), 7.91 (br d, J = 8.6
Hz, 1H); 13C NMR (125.77 MHz, DMSO-d6): δ 22.0, 22.1, 25.3, 37.5,
44.4, 53.5, 126.0, 127.8, 129.0, 138.1, 171.2, 173.3; HRMS (ESI-TOF)
m/z: [M + Na]+ calcd for C14H20N2O2Na, 271.1422; found, 271.1427.
α-[(Cyclopropylcarbonyl)amino)]benzenepropanamide
(13e). The filtrate was added carefully to 25 mL of cold 2 M NaOH,
and the mixture was extracted with 2 × 20 mL of DCM. The
combined extracts were dried (Na2SO4) and concentrated to give 6.6
mg of crude material. Trituration under 2 mL of diethyl ether gave 4.1
α-[(Tricyclo[3.3.1.13,7]decyl)amino]benzenepropanamide
(13i). The filtrate was immediately evaporated to dryness with a
stream of nitrogen gas to give crude material that was purified by
reverse-phase HPLC on a Dynamax Microsorb C-18, 5 μm column
(21.4 × 250 mm) eluting isochratically with 75:25 1:1 MeCN/MeOH
(with 5 mM ammonium acetate)/water (with 5 mM ammonium
acetate) at 5 mL/min and detection at 254 nm to afford 8.1 mg (33%)
1
of 13i as a glass: H NMR (500.13 MHz, CDCl3): δ 1.64−1.67 (m,
3H), 1.71−1.74 (m, 3H), 1.76 (m, 6H), 2.01 (br s, 3H), 3.08 (m, 2H),
4.69 (dd, J = 14.2 and 6.9 Hz, 1H), 5.58 (br s, 1H), 6.06 (br s, 1H),
6.24 (br d, J = 7.1 Hz, 1H), 7.22−7.25 (m, 3H), 7.28−7.31 (m, 2H);
13C NMR (125.77 MHz, CDCl3): δ 28.0, 36.4, 38.0, 39.0, 40.6, 53.6,
1
mg (35%) of 13e as an amorphous solid, mp 200−202 °C; H NMR
(500.13 MHz, DMSO-d6): δ 0.53−0.60 (m, 4H), 1.60−1.63 (m, 1H),
2.76 (dd, J = 13.7 and 9.5 Hz, 1H), 2.96 (dd, J = 13.7 and 5.0 Hz, 1H),
4.45 (ddd, J = 9.2, 8.5, and 5.0 Hz, 1H), 7.00 (br s, 1H), 7.17−7.20
(m, 1H), 7.23−7.28 (m, 4H), 7.42 (br s, 1H), 8.22 (br d, J = 8.5 Hz,
1H); 13C NMR (125.77 MHz, DMSO-d6): δ 6.1, 6.2, 13.3, 37.7, 53.7,
126.0, 127.9, 129.0, 138.0, 172.3, 173.1; HRMS (ESI-TOF) m/z: [M +
Na]+ calcd for C13H16N2O2Na, 255.1109; found, 255.1102.
127.1, 128.7, 129.3, 136.6, 173.5, 178.2; HRMS (ESI-TOF) m/z: [M +
H]+ calcd for C20H27N2O2, 327.2067; found 327.2064.
α-[(4-Methoxybenzoyl)amino]benzenepropanamide (13j)
and N-(4-Methoxybenzoyl)phenylalanine (14j). In a 25 mL
solid-phase peptide synthesis (SPPS) vessel, Fmoc-Phe-NH-Rink
Amide MBHA resin (576 mg, 190 μmol, 0.33 mmol/g, Peptides
International) was deprotected using 30% piperidine in NMP.1 The
amine resin was then treated with 3.77 mL of a 0.25 M solution of 4-
methoxybenzoic acid (143 mg, 942 μmol, 4.96 equiv) in 0.25 M
N,N,N′,N’-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluoro-
phosphate (HBTU) (361 mg, 952 μmol, 5.01 equiv) in NMP
followed by 3.77 mL of a 0.50 M solution of diisopropylethylamine
N-[2-Amino-2-oxo-1-(phenylmethyl)ethyl]-2-naphthalene-
carboxamide (13f).37 The filtrate was added carefully to 25 mL of
cold 2 M NaOH and the mixture was extracted with 2 × 20 mL of
DCM. The combined extracts were dried (Na2SO4) and concentrated
to give 7.3 mg of crude material. Trituration under 2 mL of diethyl
ether gave 5.2 mg (33%) of 13f as an amorphous solid, mp 203−205
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(1880 μmol, 9.91 equiv) in NMP. The vessel was rocked at room
temperature on an orbital shaker for 22 h and was then drained. The
resin gave a negative Kaiser test39 and was then washed with 4 × 10
mL × 2 min each successively of NMP, THF, and DCM. Acylated
resin 12j was treated with 6 mL of 95:5 (v/v) TFA/H2O, and the
vessel was rocked for 30 min at room temperature. The filtrate was
collected, and the resin was washed once with 6 mL of the cleavage
cocktail and once with 6 mL of DCM. The filtrate was allowed to
stand at room temperature for 5 h to allow for hydrolysis to proceed
partially and was then evaporated under a stream of nitrogen gas over
a 3 h period (effluent gases were scrubbed with a solution of sodium
hydroxide) to give 64 mg of a sticky white solid that was passed
through a 500 mg cartridge of cyanosilica gel using 7:3 hexanes/
acetone to elute the amide/acid mixture. After solvent removal by
evaporation, the mixture was dissolved in DCM and was washed with
2 × 10 mL of 1.0 N NaOH and once with 10 mL of water and was
then dried (Na2SO4). Concentration gave 19.6 mg (35%) of 13j as an
with a few milliliters of cold 1:1 acetone/water to afford 97 mg of
crude 13k, which was triturated under hot methanol to give 29.5 mg
(9%) of the QLC calibration sample. 1H NMR (500.13 MHz, DMSO-
d6): δ 2.33 (s, 3H), 2.98 (dd, J = 13.6 and 10.7 Hz, 1H), 3.10 (dd, J =
13.6 and 4.0 Hz, 1H), 4.62 (ddd, J = 10.7, 8.6, and 4.2 Hz, 1H), 7.09
(br s, 1H), 7.15 (t, J = 7.3 Hz, 1H), 7.24 (m, 4H), 7.32 (d, J = 7.5 Hz,
2H), 7.52 (br s, 1H), 7.70 (d, J = 8.2 Hz, 2H), 8.37 (d, J = 8.4 Hz,
1H); 13C NMR (125.77 MHz, DMSO-d6): δ 20.8. 37.1, 54.6, 126.0,
127.3, 127.9, 128.6, 129.0, 131.2, 138.5, 141.0, 165.9, 173.3. LC/MS
(ESI): 8.19 min, m/z 283 [M + H]+.
N-(4-Methoxybenzoyl)-Phe-Ala-NH-Rink Amide MBHA Resin
(15) and N-(4-Methoxybenzoyl)-Phe-Ala-O Wang Resin (16,
Scheme 5). Two 50 mL SPPS vessels were charged, respectively, with
566 mg (300 μmol) of Fmoc-Ala-O Wang resin (0.53 mmol/g,
Peptides International) and 698 mg (300 μmol) of Fmoc-Ala-NH-
Rink MBHA resin (0.43 mmol/g, Peptides International). To each
vessel was added 20 mL of 20% (v/v) piperidine in NMP. The vessels
were rocked at room temperature for 45 min and were then drained.
Each resin was washed with 4 × 10 mL of NMP and was treated with
3.0 mL (750 mmol, 2.5 equiv) of 0.25 M HBTU in NMP, 3.0 mL (750
mmol, 2.5 equiv) of 0.25 M Fmoc-Phe-OH in NMP, and 3.0 mL
(1500 μmol, 5.0 equiv) of 0.50 M diisopropylethylamine in NMP. The
vessels were rocked overnight at room temperature. To each vessel
was again added 3.0 mL (750 mmol, 2.5 equiv) of 0.25 M HBTU in
NMP, 3.0 mL (750 mmol, 2.5 equiv) of 0.25 M Fmoc-Phe-OH in
NMP, and 3.0 mL (1500 μmol, 5.0 equiv) of 0.50 M diisopropylethyl-
amine in NMP. The vessels were rocked overnight at room
temperature. After 48 h, Kaiser39 test on both resins was negative.
Each resin was then washed with 4 × 10 mL of NMP followed by 4 ×
10 mL of DCM and then 3 × 10 mL of NMP. Piperidine (20% in
NMP, 20 mL) was added to each resin, and the vessels were rocked for
45 min. The resins were washed with 4 × 10 mL of NMP and were
then treated with 6.0 mL of 0.25 M (1500 μmol, 5.0 equiv) HBTU in
NMP, 6.0 mL of 0.25 M (1500 μmol, 5.0 equiv) 4-methoxybenzoic
acid in NMP, and 6.0 mL (3000 μmol, 10.0 equiv) of 0.50 M
diisopropylethylamine in NMP. The vessels were rocked overnight at
room temperature. After 24 h, each resin gave a negative Kaiser39 test.
The vessels were drained, and the resins were washed with 3 × 10 mL
of NMP, 3 × 10 mL of THF, and 3 × 10 mL of DCM and then capped
for storage for 3 days.
Cleavage/Hydrolysis of Resins 15 and 16 to 17 and 18
(Scheme 5). Isopycnic suspensions of Rink resin 15 (300 μmol) and
Wang resin 16 (300 μmol) were each equally distributed, respectively,
to six 3.5 mL reaction vessels in rows A and B of two 2 × 3 Bill-Boards
(50 μmol of resin per vessel). All vessels of one Bill-Board were then
treated with 1.5 mL of 95:5 TFA/water and were rotated for 30 min at
room temperature. The vessels were drained into 4-dram, tooled-neck
collection vials, and each resin was rinsed with 2 mL of 95:5 TFA/
water. Filtrates from A1 and B1 were sampled and evaporated to
dryness and analyzed (0.5 h time point), filtrates from A2 and B2 were
allowed to stand at room temperature for 6 h and were then analyzed
by LC/MS, and then the filtrates from A3 and B3, which had stood for
24 h, were analyzed. Each vial assayed at 30 min was immediately
stored at −20 °C overnight and was concentrated under a stream of
nitrogen gas to afford 10.9 mg of crude 17 and 15.2 mg of crude 18.
N-(4-Methoxybenzoyl)-L-phenylalanyl-L-alaninamide (17).
The crude material (9.3 mg) was purified by cyanosilica gel
chromatography (500 mg prepacked column, eluting with 1:1 and
then 4:6 hexanes/acetone) followed by reverse-phase HPLC on a
Dynamax Microsorb C-18, 5 μm column (21.4 × 250 mm) using the
isochratic system of 55:45 1:1 MeCN/MeOH (5 mM ammonium
acetate)/water (5 mM ammonium acetate) at a 5 mL/min flow rate to
give 2.1 mg (11%) of 17 as an amorphous solid, mp 229−231 °C; 1H
NMR (500.13 MHz, CD3OD): δ 1.36 (d, J = 7.2 Hz, 3H), 3.06 (dd, J
= 13.8 and 9.2 Hz, 1H), 3.26 (dd, J = 13.9 and 6.0 Hz, 1H), 3.84 (s,
3H), 4.34 (q, J = 7.2 Hz, 1H), 4.79 (dd, J = 9.1 and 5.9 Hz, 1H), 6.95
(d, J = 8.8 Hz, 2H), 7.20 (m, 1H), 7.26−7.32 (m, 4H), 7.72 (d, J = 8.9
Hz, 2H); 13C NMR (125.77 MHz, CD3OD): δ 18.2, 38.6, 50.1, 55.9,
56.7, 114.7, 127.2, 127.9, 129.5, 130.37, 130.39, 138.6, 164.1, 169.9,
1
amorphous solid, mp 201−204 °C; H NMR (500.13 MHz, DMSO-
d6): δ 2.97 (dd, J = 13.6 and 10.7 Hz, 1H), 3.10 (dd, J = 13.6 and 4.0
Hz, 1H), 3.79 (s, 3H), 4.61 (ddd, J = 10.8, 8.6, and 4.2 Hz, 1H), 6.97
(d, J = 8.9 Hz, 2H), 7.08 (br s, 1H), 7.15 (t, J = 7.4 Hz, 1H), 7.24 (t, J
= 7.5 Hz, 2H), 7.33 (d, J = 7.1 Hz, 2H), 7.51 (br s, 1H), 7.78 (d, J =
8.9 Hz, 2H), 8.32 (d, J = 8.5 Hz, 1H); 13C NMR (125.77 MHz,
DMSO-d6): δ 37.1, 54.6, 55.2, 113.2, 126.0, 126.3, 127.9, 129.0, 129.1,
138.5, 161.5, 165.5, 173.4; HRMS (ESI-TOF) m/z: [M + H]+ calcd
for C17H19N2O3, 299.1390; found, 299.1392.
The combined basic extracts were adjusted to pH 1 with 3 N HCl
and were extracted with 2 × 25 mL of DCM. The combined extracts
were dried (Na2SO4) and concentrated to give 12.1 mg (21%) of 14j
1
as an amorphous solid, mp 164−167 °C; H NMR (500.13 MHz,
CD3OD): δ 3.11 (dd, J = 13.9 and 9.4 Hz, 1H), 3.32 (dd, J = 5.0 Hz,
1H), 3.83 (s, 3H), 4.83 (dd, J = 9.4 and 4.9 Hz, 1H), 6.94 (d, J = 8.9
Hz, 2H), 7.19 (m, 1H), 7.24−7.28 (m, 4H), 7.70 (d, J = 8.9 Hz, 2H);
13C NMR (125.77 MHz, CD3OD): δ 38.2, 55.6, 55.9, 114.7, 127.4,
127.8, 129.4, 130.3, 138.8, 164.1, 169.7, 175.0 (only 12 of 13 lines
were observed); HRMS (ESI-TOF) m/z: [M + H]+ calcd for
C17H18NO4, 300.1230; found, 300.1229.
Preparation of Resin 12k for Cleavage Method Study (Table
4). A 25 mL SPPS vessel was charged with 1.857 g (0.5478 mmol,
0.295 mmol/g) of Fmoc-Phe-NH-Rink Amide MBHA resin. The resin
was swelled by washing with 3 × 10 mL of NMP followed by 10 mL of
20% (v/v) piperidine in NMP. The resin was then treated with 12 mL
of 20% piperidine in NMP. The vessel was rocked on an orbital shaker
for 40 min and was then drained, and the resin was washed with 5 ×
10 mL of NMP. To the resin was then added a solution of 0.374 g
(2.75 mmol, 5.02 equiv) of 4-methylbenzoic acid and 0.418 g (2.73
mmol, 4.98 equiv) of 1-hydroxybenzotriazole in 10.9 mL of NMP
followed by a solution of 0.346 g (2.74 mmol, 5.00 equiv) of
diisopropylcarbodiimide in 1 mL of NMP. The vessel was rocked on
an orbital shaker for 6 days and was then drained, and the resin was
washed with 3 × 10 mL each of NMP and THF and then with 5 × 10
mL of DCM to give 12k, which was prepared as an isopycnic mixture
using 2:1 NMP/DCM and was equally distributed to eleven 3.5 mL
reaction vessels to provide 50 μmol per vessel (see General Procedure
for the Preparation and Distribution of Isopycnic Suspensions of
Resins). Each resin was then washed with 5 × 2 mL of DCM.
α-[(4-Methylbenzoyl)amino]benzenepropanamide (13k).40
Compound 13k was synthesized in solution phase using the method
of Uchida et al.41 and was purified for use as the authentic standard in
the quantitative LC measurements (optimization of cleavage
conditions, Table 4). A 25 mL, three-necked, round-bottomed flask
fitted with a stirrer bar, thermometer, and rubber septum was charged
with 0.105 g (1.25 mmol) of sodium bicarbonate, 2.0 mL of water, and
2.0 mL of acetone. The contents were cooled with an ice bath, and
0.200 g (1.22 mmol) of L-phenylalanineamide (Chem-Impex, Int.) was
added in one portion. 4-Methylbenzoyl chloride (0.208 g, 1.34 mmol,
1.10 equiv), dissolved in 0.5 mL of acetone, was added dropwise via
syringe. The contents were allowed to gradually warm to room
temperature and were stirred for 18 h. The contents were cooled with
an ice bath, and the solid was collected by suction filtration washing
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173.6, 177.3; HRMS (ESI-TOF) m/z: [M + H]+ calcd for
C20H24N3O4, 370.1761; found, 370.1767.
York, 1961; Vol. 2, Chapter 10. (b) Law, H. D. The Organic Chemistry
of Peptides; Wiley-Interscience: London, 1970; Chapters 3 and 4.
(c) Bodanszky, M.; Ondetti, M. A. Peptide Synthesis; Interscience
Publishers: New York, 1966; Chapters 1, 2, 4, and 6. (d) Carpino, L.
A. Acc. Chem. Res. 1973, 6, 191−198. (e) Kemp, D. S. In The Peptides:
Analysis, Synthesis, Biology; Gross, E., Meienhofer, J., Eds.; Academic
N-(4-Methoxybenzoyl)-L-phenylalanyl-L-alanine (18). The
crude material (12.9 mg) was chromatographed using a 500 mg
prepacked cyanosilica cartridge (Thermo Scientific) and eluted with
hexanes/acetone (7:3 and 6:4) to afford 4.8 mg (26%) of 18 as an
1
amorphous solid, mp 181−184 °C (dec); H NMR (500.13 MHz,
Press: New York, 1979; Vol. 1, Chapter 7. (f) Geiger, R.; Konig, W. In
̈
CD3OD): δ 1.42 (d, J = 7.3 Hz, 3H), 3.03 (dd, J = 14.0 and 9.7 Hz,
1H), 3.29 (dd, J = 14.4 and 4.9 Hz, 1H), 3.83 (s, 3H), 4.39 (q, J = 7.2
Hz, 1H), 4.86 (dd, J = 9.8 and 4.9 Hz, 1H), 6.94 (d, J = 8.9 Hz, 2H),
7.18 (t, J = 7.3 Hz, 1H), 7.25 (t, J = 7.5 Hz, 2H), 7.31 (d, J = 7.1 Hz,
2H), 7.69 (d, J = 8.9 Hz, 2H); 13C NMR (125.77 MHz, CD3OD): δ
18.0, 38.9, 49.9, 55.9, 56.3, 114.7, 127.3, 127.7, 129.4, 130.3, 130.4,
138.7, 164.1, 169.7, 173.5, 176.3; HRMS (ESI-TOF) m/z: [M + H]+
calcd for C20H23N2O5 371.1601; found 371.1604.
The Peptides: Analysis, Synthesis, Biology; Gross, E., Meienhofer, J., Eds.;
Academic Press: New York, 1979; Vol. 3, Chapter 1.
(14) Scott, W. L.; O’Donnell, M. J. J. Comb. Chem. 2009, 11, 3−13.
(15) Scott, W. L.; Alsina, J.; Audu, C. O.; Babaev, E.; Cook, L.; Dage,
J. L.; Goodwin, L. A.; Martynow, J. G.; Matosiuk, D.; Royo, M.; Smith,
J. G.; Strong, A. T.; Wickizer, K.; Woerly, E. M.; Zhou, Z.; O’Donnell,
M. J. J. Comb. Chem. 2009, 11, 14−33.
(16) Scott, W. L.; Audu, C. O.; Dage, J. L.; Goodwin, L. A.;
Martynow, J. G.; Platt, L. K.; Smith, J. G.; Strong, A. T.; Wickizer, K.;
Woerly, E. M.; O’Donnell, M. J. J. Comb. Chem. 2009, 11, 34−43.
(17) Samaritoni, J. G.; Crews, D.; Glos, C. E.; Thompson, A.; Wilson,
C.; O’Donnell, M. J.; Scott, W. L. 246th ACS National Meeting,
Indianapolis, IN, Sept 11, 2013; Poster ORGN 545.
(18) O’Donnell, M. J.; Lugar, C. W.; Pottorf, R. S.; Zhou, C.; Scott,
W. L.; Cwi, C. L. Tetrahedron Lett. 1997, 38, 7163−7166.
(19) Scott, W. L.; Schonegg, R. A.; Cwi, L. U.S. Patent 5,785,927,
July 28, 1998.
ASSOCIATED CONTENT
■
S
* Supporting Information
1H and 13C NMR data and spectra and graphical treatments of
the Hammett and cleavage method studies. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
*E-mail: modonnel@iupui.edu (M.J.O.).
*E-mail: wscott@iupui.edu (W.L.S.).
■
(20) McDaniel, D. H.; Brown, H. C. J. Org. Chem. 1958, 23, 420−
427.
(21) Hansch, C.; Leo, A.; Taft, R. W. Chem. Rev. 1991, 91, 165−195.
(22) Pearson, D. A.; Blanchette, M.; Baker, M. L.; Guindon, C. A.
Tetrahedron Lett. 1989, 30, 2739−2742.
(23) Stewart, J. M.; Young, J. D. In Solid-Phase Peptide Synthesis, 2nd
ed.; Pierce Chemical Company: Rockford, IL, 1984; pp 38−44.
(24) Although it was realized that the potential for partial
racemization13e,42 existed, no attempt was made to establish the
stereochemistry at the α-carbon of 14j.
(25) Jencks, W. P. Acc. Chem. Res. 1980, 13, 161−169.
(26) Cox, R. A. Int. J. Mol. Sci. 2011, 12, 8316−8332.
(27) Yates, K.; Stevens, J. B. Can. J. Chem. 1965, 43, 529−537.
(28) Jiang, W.-Q.; Costa, S. P. G.; Maia, H. L. S. Org. Biomol. Chem.
2003, 1, 3804−3810.
(29) Mannekens, E.; Crisma, M.; Van Cauwenberghe, S.; Tourwe, D.
Eur. J. Org. Chem. 2003, 3300−3307.
(30) Clerin, D.; Kille, G.; Fleury, J.-P. Tetrahedron 1974, 30, 469−
474.
(31) Sinkula, A. A.; Yalkowsky, S. H. J. Pharm. Sci. 1975, 64, 181−
210.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the National Institutes of Health (RO1-GM28193),
the National Science Foundation (NSF/DUE-1140602, NSF/
MRI-CHE-0619254, and NSF/MRI-DBI-0821661), and the
Indiana University Purdue University Indianapolis STEM
Summer Scholars Institute for their generous support of this
work.
REFERENCES
■
(1) Scott, W. L.; Delgado, F.; Lobb, K.; Pottorf, R. S.; O’Donnell, M.
J. Tetrahedron Lett. 2001, 42, 2073−2076.
(2) Wang, S.-S. J. Am. Chem. Soc. 1973, 95, 1328−1333.
(3) Rink, H. Tetrahedron Lett 1987, 28, 3787−3790.
(4) Blackburn, S. Hydrolytic Procedures. In Amino Acid Determi-
nation; Methods and Techniques; Marcel Dekker, Inc.: New York, 1968.
(5) Edman, P. Acta Chem. Scand. 1956, 10, 761−768.
(6) Creighton, C. J.; Romoff, T. T.; Bu, J. H.; Goodman, M. J. Am.
Chem. Soc. 1999, 121, 6786−6791.
(32) Markwardt, F. Pharmazie 1989, 44, 521−526.
(33) Devasia, G. M.; Shafi, P. M. Indian J. Chem., Sect. B: Org. Chem.
Incl. Med. Chem. 1980, 19B, 70−72.
(34) Devasia, G. M.; Pillai, C. R. Tetrahedron Lett. 1975, 46, 4051−
4052.
(35) Moriguchi, T.; Yanagi, T.; Kunimori, M.; Wada, T.; Sekine, M. J.
(7) Urban, J.; Vaisar, T.; Shen, R.; Lee, M. S. Int. J. Pept. Protein Res.
1996, 47, 182−189.
Org. Chem. 2000, 65, 8229−8238.
(36) Izumiya, N.; Francis, J. E.; Robertson, A. V.; Witkop, B. J. Am.
Chem. Soc. 1962, 84, 1702−1709.
(37) Dong, L.; Marakovits, J.; Hou, X.; Guo, C.; Greasley, S.;
Dagostino, E.; Ferre, R.; Johnson, M. C.; Kraynov, E.; Thomson, J.;
Pathak, V.; Murray, B. W. Bioorg. Med. Chem. Lett. 2010, 20, 2210−
2214.
(38) Huber, L.; Klostermeyer, H. Liebigs Ann. Chem. 1977, 1274−
1284; Chem. Abstr. 1978, 88, 38143.
(39) Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. I. Anal.
Biochem. 1970, 34, 595−598.
(40) Altenbach, R. J.; Bai, H.; Brioni, J. D.; Carroll, W. A.;
Gopalakrishnan, M.; Gregg, R. J.; Holladay, M. W.; Huang, P. P.;
Kincaid, J. F.; Kort, M. E.; Kym, P. R.; Lynch, J. K.; Perez-Medrano, A.;
Zhang, H. Q. U.S. Patent Appl. 2002/0028836 A1, March 7, 2002.
(41) Uchida, M.; Tabusa, F.; Komatsu, M.; Morita, S.; Kanbe, T.;
Nakagawa, K. Yakugaku Zasshi 1985, 105, 1040−1050; Chem. Abstr.
1987, 106, 4801.
(8) Spencer, J. R.; Delaet, N. G. J.; Toy-Palmer, A.; Antonenko, V. V.;
Goodman, M. J. Org. Chem. 1993, 58, 1635−1638.
(9) Costa, S. P. G.; Maia, H. L. S.; Pereira-Lima, S. M. M. A. Org.
Biomol. Chem. 2003, 1, 1475−1479.
(10) Rubini, C.; Osler, A.; Calderan, A.; Guiotto, A.; Ruzza, P. J. Pept.
Sci. 2008, 14, 989−997.
(11) Zajdel, P.; Nomezine, G.; Masurier, N.; Amblard, M.;
Pawlowski, M.; Martinez, J.; Subra, G. Chem.Eur. J. 2010, 7547−
7553.
(12) Zajdel, P.; Masurier, N.; Sanchez, P.; Pawlowski, M.; Kreiter, A.;
Nomezine, G.; Enjalbal, C.; Amblard, M.; Martinez, J.; Subra, G. J.
Comb. Chem. 2010, 12, 747−753.
(13) For early discussions of the rich history of peptide chemistry,
the role of amides and carbamates (Cbz, Boc, Fmoc, etc.) in α-
nitrogen protection, the importance of minimizing racemization, and
many practical applications in the field, see: (a) Greenstein, J. P.;
Winitz, M. Chemistry of the Amino Acids; John Wiley & Sons: New
3150
dx.doi.org/10.1021/jo500273f | J. Org. Chem. 2014, 79, 3140−3151

The Journal of Organic Chemistry
Article
(42) Goodman, M.; McGahren, W. J. Tetrahedron 1967, 23, 2031−
2050.
3151
dx.doi.org/10.1021/jo500273f | J. Org. Chem. 2014, 79, 3140−3151