Preparation of N-acetyl, tert-butyl amide derivatives of the 20 natural amino acids

Article abstract of DOI:10.1007/s00726-009-0279-y

N-Acetyl-AA(amino acid)-NHtBu derivatives of all 20 naturally occurring amino acids have been synthesized. Syntheses were performed via solution-phase methodology with yields that allow for access to gram quantities of substrates, in most cases. Syntheses include the coupling of a hindered amine, tert-butylamine, with each amino acid, either directly or in two steps using an activated ester isolated as an intermediate. The introduction of protecting groups was necessary in some cases. The development of synthetic sequences to access challenging substrates, such as the one derived from asparagine, are discussed.

Full text of DOI:10.1007/s00726-009-0279-y

Amino Acids (2010) 38:747–751
DOI 10.1007/s00726-009-0279-y
ORIGINAL ARTICLE
Preparation of N-acetyl, tert-butyl amide derivatives
of the 20 natural amino acids
A. R. Ekkati Æ A. A. Campanali Æ A. I. Abouelatta Æ
M. Shamoun Æ S. Kalapugama Æ M. Kelley Æ
Jeremy J. Kodanko
Received: 14 January 2009 / Accepted: 11 March 2009 / Published online: 31 March 2009
Ó Springer-Verlag 2009
Abstract N-Acetyl-AA(amino acid)-NHtBu derivatives of
all 20 naturally occurring amino acids have been synthesized.
Syntheses were performed via solution-phase methodology
with yields that allow for access to gram quantities of sub-
strates, in most cases. Syntheses include the coupling of a
hindered amine, tert-butylamine, with each amino acid,
either directly or in two steps using an activated ester isolated
as an intermediate. The introduction of protecting groups was
necessary in some cases. The development of synthetic
sequences to access challenging substrates, such as the one
derived from asparagine, are discussed.
DMF
Fmoc
HOBt
IBCF
NMM
PNP
Dimethylformamide
9-Fluorenylmethyloxycarbonyl
1-Hydroxybenzotriazole
Isobutylchloroformate
4-Methylmorpholine
p-Nitrophenol
HBTU
O-(Benzotriazol-1-yl)-N,N,N,N0-
tetramethyluronium hexafluorophosphate
Tetrahydrofuran
THF
TFA
Trifluoroacetic acid
trityl or Trt Triphenylmethyl
Keywords Amino acid substrate synthesis ꢀ
N-Boc-amino acid ꢀ N-Fmoc-amino acid ꢀ
Amino acid protection ꢀ Amino acid deprotection
Introduction
Reactive oxygen species, such as singlet oxygen or
hydroxy radical, can damage proteins (Garrison 1987;
Stadtman 1993). Due to the fact that this oxidative dam-
age has a causative role in many disease states, as well as
in aging, protein oxidation has been the focus of intense
investigations and is well understood today (Berlett and
Stadtman 1997). On the contrary, little is known about the
reactivity of proteins with metal-based oxidants, wherein
the reactive oxygen atom is bound to a metal center. In
particular, there is not much information available about
how metal-based oxidants react with individual amino
acid residues (Ekkati and Kodanko 2007). This is far from
ideal, because metal-based oxidants have been implicated
in the targeted oxidation of proteins (Cuenoud et al. 1992;
Brown et al. 1995) and have the potential to be widely
useful in applications such as protein inactivation or
footprinting.
Abbreviations
Ac
Acetyl
AA
Amino acid
OBn
Cbz
Boc
tBu
Benzyl ester
Benzyloxycarbonyl
tert-Butoxycarbonyl
tert-Butyl
DCC
EDAC
1,3-Dicyclohexylcarbodiimide
1-(3-Dimethylaminopropyl)-3-
ethylcarbodiimide hydrochloride
Electronic supplementary material The online version of this
article (doi:10.1007/s00726-009-0279-y) contains supplementary
material, which is available to authorized users.
A. R. Ekkati ꢀ A. A. Campanali ꢀ A. I. Abouelatta ꢀ
M. Shamoun ꢀ S. Kalapugama ꢀ M. Kelley ꢀ J. J. Kodanko (&)
Department of Chemistry, Wayne State University,
5101 Cass Ave., Detroit, MI 48202, USA
In order to study the ability of metal-based oxidants to
modify amino acid residues of polypeptides, we have
e-mail: jkodanko@chem.wayne.edu
123

748
A. R. Ekkati et al.
O
R
varied depending on the particular amino acid. The tert-
butylamide was chosen because it prevents oxidation
adjacent to the nitrogen atom of the C-terminal amide. This
necessity presented an additional challenge in forming
amide bonds with some substrates, due to the steric hin-
drance of tert-butylamine. To overcome this challenge,
four general methods were used to access substrates 1–20,
Methods A, B, C, and D. All methods involve the con-
densation of tert-butylamine with some form of the amino
acid. Method A involves the condensation of an N-acetyl
amino acid with a p-nitrophenol ester. Method B employs
EDAC or DCC/HOBt to achieve condensation. Method C
starts with tert-butoxycarbonyl protected N-terminal amino
acid, instead of an N-acetyl, and utilizes benzyl and tosyl
protecting groups for the side chains. Substrates 19 and 20
required unique syntheses and are discussed separately in
Method D.
O
R
H
N
H
N
N
N
H
H
O
O
Polypeptide
Residue Mimic
1 Ala
2 Val
3 Phe 10 Trp
4 Leu
5 Pro
6 Gly
7 Cys
15 Ser
16 Thr
17 His
18 Arg
19 Asn
20 Ile
8 Met
9 Tyr
11 Gln
12 Lys
13 Glu
14 Asp
Fig. 1 Structure of Ac-AA-NHtBu substrates that mimic amino acid
residues of polypeptides
designed substrates of the general formula Ac-AA-NHtBu
(Fig. 1, AA = amino acid) that mimic amino acid residues
in a polypeptide chain. These substrates mimic residues by
virtue of having their N- and C-termini blocked as amides,
specifically, acetyl and tert-butyl, respectively. In this
paper, we describe the synthesis of the aforementioned
amino acid substrates derived from each of the 20, natu-
rally occurring, amino acids (1–20). The synthetic routes to
access substrates 1–20 rely on solution-phase methodol-
ogy, and can be used to furnish substrates on multi-gram
scale, leading to their ready availability for reactivity
studies with metal-based oxidants.
Method A
N-Acetylated amino acids of Ala, Val, Phe, Leu, Pro, and
Gly were condensed with p-nitrophenol using DCC as the
coupling reagent to form activated p-nitrophenol esters
(Scheme 1). Each activated ester was isolated and purified
by recrystallization before treatment with excess tert-
butylamine, which furnished the corresponding C-terminal
amides. This procedure was advantageous because the salt
(tBuNH3)(OC6H4NO2) was removed easily by filtration
after the condensation was complete, allowing access to the
final substrates in good to moderate yields (Table 1).
Materials and methods
All reagents were purchased from commercial sources and
used as received unless otherwise noted. N-acetyl amino
acids and some protected amino acids were prepared
according to previously reported literature (Chenault et al.
1989; Reddy and Ravindranath 1992; Ray et al. 2006). Other
protected amino acids were purchased: Boc-Asp(OBn)-OH,
Fmoc-Asn(Trt)-OH, Boc-His(Tos)-OH, and Boc-Arg(Tos)-
OH. All experimental procedures can be found in the Sup-
porting Information. Due to the fact that amino acids can
racemize during N-acetylation (Reddy and Ravindranath
1992) and enantiopure substrates were not required for
oxidation studies, optical rotation data, and measurements of
enantiopurity were not collected. NMR spectra were recor-
ded on a Varian FT-NMR Unity-300, Mercury-400 or
500 MHz Spectrometer. Mass spectra were recorded on a
Waters ZQ2000 single quadrupole mass spectrometer using
an electrospray ionization source. IR spectra were recorded
on a Nicolet FT-IR spectrophotometer.
1) DCC, PNP
Ac-AA-OH
Ac-AA-NHtBu
2) H2NtBu
1-6
AA = Ala, Val, Phe, Leu, Pro, Gly
Scheme 1 Syntheses of substrates 1–6 via an isolated p-nitrophenol
ester
Table 1 Conditions and yields for formation of p-nitrophenol esters
and condensation products for 1–6
Ac-AA-NHtBu
AA=
Ac-AA-OPNP
yielda (%)
Condensation
yielda
Ala (1)
Val (2)
Phe (3)
Leu (4)
Pro (5)
Gly (6)
42
71
40
62b
61
66
61
79b
63b
60b
73b
44b
Results and discussion
a
rt unless otherwise stated
Although the synthetic target for substrates 1–20 are
identical, the methods used to access these substrates
b
50°C to rt
123

Preparation of N-acetyl, tert-butyl amide derivatives
749
Method B
the recrystallization process, not from inefficient conden-
sation reactions (Table 2).
In this method, the N-acetyl amino acids of Cys, Met, Tyr,
Trp, Gln, and Lys were converted to substrates 7–12 by
direct condensation with tert-butylamine using DCC,
EDAC, IBCF, or EDAC/HOBt as coupling reagents
(Schemes 2, 3, 4). Synthesis of Cys derived substrate 7
began with the condensation of Ac-Cys(Trt)-OH and tert-
butylamine utilizing the coupling reagent isobutylchloro-
formate (Scheme 2). After amide bond formation, the trityl
protecting group was removed in moderate yield to pro-
duce the desired Ac-Cys-NHtBu substrate 7. Final
substrates 8–11 were purified by recrystallization. The
lower isolated yields observed in some cases resulted from
For Lys derived substrate 12, the amine side chain was
protected with a Cbz group. After condensation of Ac-
Lys(Cbz)-OH with tert-butylamine, the Cbz protecting
group was removed by catalytic hydrogenation over Pd/C
(Scheme 4). It is interesting that low yields were obtained
during the condensation when the base employed was
Et3N, but increased when the base was changed to NMM
(LeTiran et al. 2002).
Method C
Functional groups present in the side chains of Glu, Asp,
Ser, and Thr created the potential for undesired side reac-
tions such as diacetylation or ring formation. For example,
during initial attempts to synthesize 15 and 16 from
N-acetyl derivatives, low yields were observed in the
condensation reaction when the hydroxyl groups of the Ser
and Thr substrates were not protected. This complication
lead to the use of protected amino acids (Boc-AA(OBn)-
OH) that are commercially available as the starting point.
Each protected amino acid in this series was condensed
with tert-butylamine using either EDAC or DCC/HOBt at
0°C to rt (Scheme 5) to form the corresponding C-terminal
amide in good to moderate yields (Table 3). Subsequent
removal of the N-Boc group with either TFA/CH2Cl2 or
8 N HCl/THF followed by treatment with acetyl chloride
furnished the N-acetyl amino acids. To complete the
sequence, the benzyl groups were removed by catalytic
hydrogenation over Pd/C, yielding substrates 13–16. It is
important to note that a single diastereomer of 16 was
formed using this synthetic pathway, consistent with no
epimerization of the a-center occurring under these cou-
pling conditions.
1) IBCF, H2NtBu, THF
6 h, –18 to –20 oC, 38%
Ac-Cys(Trt)-OH
Ac-Cys-NHtBu
2) TFA, Et3SiH
DCM, rt, 4 h
7
63%
Scheme 2 Synthesis of substrate 7 via direct condensation employ-
ing the coupling reagent isobutylchloroformate; starting from
N-acetylated and trityl protected cysteine
EDAC or DCC/HOBt
Ac-AA-NHtBu
Ac-AA-OH
H2NtBu, rt, 18-24 h
8-11
AA = Met, Tyr, Trp, Gln
Scheme 3 Synthesis of substrates 8–11 via direct condensation;
starting from N-acetyl amino acids
1) EDAC/HOBt
H2NtBu, rt, 14 h
Ac-Lys-NHtBu
Ac-Lys(Cbz)-OH
2) Pd/C, H2
MeOH, 3 h
12
Syntheses of substrates 17 and 18 derived from His and
Arg, respectively, began from N-Boc amino acids that
possessed N-toluenesulfonyl protection on the side chains.
These Boc-AA(Tos)-OH amino acid derivatives were
condensed with tert-butylamine using HBTU (Scheme 6)
to provide the C-terminal amide in good to moderate
yields. The N-Boc groups were removed and the resultant
amines were acetylated. Removal of the N-toluenesulfonyl
62%, 2 steps
Scheme 4 Synthesis of 12 via direct condensation of N-acetyl-
Lys(Cbz)-OH with tert-butylamine followed by hydrogenation of the
CBz protecting group
Table 2 Yields and conditions for the synthesis of substrates 8–11
Ac-AA-NHtBu AA=
Condensation
yield (%)
45a
37b
48c
56c
1) EDAC or DCC/HOBt
H2NtBu, 0 oC to rt
Met (8)
Tyr (9)
Ac-AA-NHtBu
Boc-AA(OBn)-OH
2) TFA/DCM or 8N HCl, rt
3) AcCl, Et3N, 0 o
4) Pd/C, H2, rt, 24 h
Trp (10)
Gln (11)
13 - 16
C
a
DCC
AA = Glu, Asp, Ser, Thr
b
EDAC/HOBt
c
EDAC
Scheme 5 Synthesis of substrates 13–16 via Boc-AA(OBn)-OH
123

750
A. R. Ekkati et al.
O
O
O
Table 3 Yields for condensation, acetylation, and removal of side
chain protecting group for the formation of substrates 13–16
1) IBCF, H2NtBu, THF
2 h, –18 to –20 oC, 96%
NHTrt
OH
NH2
NHtBu
AcHN
FmocHN
Ac-AA-
NHtBu
AA=
Boc-AA(OBn) Ac-AA(OBn)-NHtBu Ac-AA(OBn)-
yield, two
stepsc (%)
2) i. Piperidine, 4 h
ii. Ac2O, Et3N, DCM, 2 h
76%, 2 steps
O
NHtBu
yield (%)
NHtBu
deprotection
yield (%)
19
3) 95:5 TFA/H2O, 2 h, 68%
Glu (13) 53a
Asp (14) 53a
61d
60d
42e
62e
96
96
96
95
Scheme 7 Synthesis of Asn derivative substrate 19 via N-Fmoc and
trityl protection
Ser (15)
Thr (16)
56a
87b
1) HBTU, H2NtBu, THF
24 h, –20 to –30 oC, 59%
a
EDAC
OH
NHtBu
b
FmocHN
AcHN
HOBt/DCC
2) i. 20% Piperidine/DCM
25 min
ii. Ac2O, Et3N, DCM
20 min
O
O
c
d
e
Boc deprotection followed by acetylation
TFA/CH2Cl2
20
8 N HCl/THF
34%, 2 steps
Scheme 8 Synthesis of Ac-Ile-NHtBu via N-Fmoc protection
1) HBTU, H2NtBu
THF, overnight, Yield1
Boc-AA(Tos)-OH
Ac-AA-NHtBu
methyl ester in addition to desired amide 19. This methyl
ester gave incomplete conversion to the amide, even upon
subjection to higher temperatures and longer reaction
times. Attempts to isolate 19 from the methyl ester in pure
form were unsuccessful. The third and final route to 19
started from Fmoc-Asn(Trt)-OH (Scheme 7). After careful
optimization, condensation of tert-butylamine with Fmoc-
Asn(Trt)-OH using isobutyl chloroformate, NMM, and
a reaction temperature of -20°C furnished the amide
product Fmoc-Asn(Trt)-NHtBu in 96% yield. Removal of
the N-Fmoc group using piperidine and acetylation of the
resultant primary amine with acetic anhydride furnished
Ac-Asn(Trt)-NHtBu. To complete the synthesis of 19, the
N-trityl group was removed using 95:5 TFA/H2O, which
was advantageous because the triphenylmethanol byprod-
uct was easily removed by precipitation upon the addition
of H2O.
2) i. TFA, DCM, rt, 2 h
ii. Ac2O, Et3N, DCM, 24 h
Yield2, 2 steps
3) Na, NH3 (l)
–78 oC, 15 min
17-18
Yield3
1His 62% and Arg 71%
2His 57% and Arg 62%
3His 53% and Arg 67%
AA = His, Arg
Scheme 6 Synthesis of substrates 17 and 18 via Boc-AA(Tos)-OH
protecting groups of Ac-His(Tos)-NHtBu and Ac-Arg(Tos)-
NHtBu was straightforward using Na and liquid ammonia
to produce 17 and 18 with moderate to excellent yields; 18
was isolated as the HCl salt.
Method D
Syntheses of substrates derived from Asn and Ile, 19 and
20, respectively, proved to be particularly challenging. The
final sequences to these two substrates are included below,
along with a description of dead-end routes that did not
allow access to these substrates.
The second challenging substrate to synthesize was 20,
a derivative of Ile. In the synthesis of 20, amide bond for-
mation was facile, but epimerization at the a-stereocenter
and formation of diastereomeric mixtures proved to be
challenging to suppress. Initial acetylation of Ile was carried
out using acetic anhydride and acetic acid; these reaction
conditions gave mixtures of diastereomers due to epimer-
ization at the a-carbon, presumably proceeding through an
oxazolone intermediate. It is known that under basic
conditions, amino acids can be acetylated without epimer-
ization (Reddy and Ravindranath 1992). Therefore, Ile was
acetylated using acetic anhydride with the addition of
NaOH at low temperature to keep the reaction at alkaline
pH. Although this procedure gave the single diastereomer
Ac-Ile-OH, upon condensation with tert-butylamine, epi-
merization was again noted. Different coupling reagents
and conditions were screened in an attempt to generate
The initial synthetic route for Asn derived substrate 19
followed the same pathway as that of the Gln substrate 11
(Scheme 3). However, the condensation reactions with Ac-
Asn-OH and tert-butylamine using EDAC furnished only
trace amounts of product under a variety of conditions,
presumably due to complications of ring formation (Jones
2002). The second route toward substrate 19 made use of
the availability of Ac-Asp(OBn)-NHtBu, an intermediate
in the synthesis of 14. In this route, transformation of the
benzyl ester into an amide using methanolic ammonia was
attempted (Swayze and Townsend 1995; Ramasamy et al.
1987). It was noted that the benzyl ester also converted to a
123

Preparation of N-acetyl, tert-butyl amide derivatives
751
References
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result in formation of diastereomeric mixtures. Condensa-
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OSu or with Boc-Ile-OH gave similar results (Ray et al.
2006). Fortunately, condensation of Fmoc-Ile-OH and tert-
butylamine using the coupling reagent HBTU at -20°C
furnished, exclusively, a single diastereomer (Scheme 8).
Upon removal of the N-Fmoc group with 20% piperidine/
CH2Cl2, the resultant primary amine was acetylated with
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and deprotonation, which can result in facile epimerization,
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nucleophile tert-butylamine, with respect to other amines
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Conclusion
In summary, synthetic routes to access Ac-AA-NHtBu
derivatives 1–20, where AA represents each of the natural
amino acids, have been developed. Due to the fact that
these derivatives posses amides on the C- and N-termini,
they are good models for residues in a peptide chain.
Unlike solid-phase methods, the solution-phase syntheses
described herein provide ready access to gram quantities of
these amino acid derivatives in most cases as well as single
diastereomers in some cases. With synthetic access to
substrates 1–20 developed, their reactivity with a variety of
metal-based oxidants can now be defined. Studies of this
type are currently underway in our laboratory and will be
reported in due course.
123