Enzymatic synthesis of C-terminal arylamides of amino acids and peptides

Article abstract of DOI:10.1021/jo900634g

(Chemical Equation Presented) A mild and cost-efficient chemo-enzymatic method for the synthesis of C-terminal arylamides of amino acid and peptides is described. Using the industrial serine protease Alcalase under near-anhydrous conditions, C-terminal arylamides of N-Cbz-protected amino acids and peptides could be obtained from the corresponding C-terminal carboxylic acids, methyl (Me) or benzyl (Bn) esters, in high chemical and enantio- and diastereomeric purities. Yields ranged between 50% and 95% depending on the size of the aryl substituents and the presence of electron-withdrawing substituents. Complete α-C-terminal selectivity could be obtained even in the presence of various unprotected side-chain functionalities such as β/γ-carboxyl, hydroxyl, and guanidino groups. In addition, the use of the cysteine protease papain and the lipase Cal-B gave anilides in high yields. The chemo-enzymatic synthesis of arylamides proved to be completely free of racemization, in contrast to the state-of-the-art chemical methods.

Full text of DOI:10.1021/jo900634g

pubs.acs.org/joc
Enzymatic Synthesis of C-Terminal Arylamides of
Amino Acids and Peptides
Timo Nuijens,†,‡ Claudia Cusan,† John A. W. Kruijtzer,‡ Dirk T. S. Rijkers,‡
Rob M. J. Liskamp,‡ and Peter J. L. M. Quaedflieg*,†
†DSM Pharmaceutical Products, Innovative Synthesis & Catalysis, P.O. Box 18, 6160 MD Geleen,
The Netherlands, and ‡Medicinal Chemistry and Chemical Biology, Utrecht Institute for Pharmaceutical
Sciences, Utrecht University, P.O. Box 80082, 3508 TB Utrecht, The Netherlands
peter.quaedflieg@dsm.com
Received March 26, 2009
A mild and cost-efficient chemo-enzymatic method for the synthesis of C-terminal arylamides of
amino acid and peptides is described. Using the industrial serine protease Alcalase under near-
anhydrous conditions, C-terminal arylamides of N-Cbz-protected amino acids and peptides could be
obtained from the corresponding C-terminal carboxylic acids, methyl (Me) or benzyl (Bn) esters, in
high chemical and enantio- and diastereomeric purities. Yields ranged between 50% and 95%
depending on the size of the aryl substituents and the presence of electron-withdrawing substituents.
Complete R-C-terminal selectivity could be obtained even in the presence of various unprotected side-
chain functionalities such as β/γ-carboxyl, hydroxyl, and guanidino groups. In addition, the use of
the cysteine protease papain and the lipase Cal-B gave anilides in high yields. The chemo-enzymatic
synthesis of arylamides proved to be completely free of racemization, in contrast to the state-of-the-
art chemical methods.
Introduction
substrates in chromogenic, fluorogenic or amperogenic en-
zymatic assays.2 For this application, the arylamides
should be optically pure. p-Nitroaniline (pNA, absorption at
405 nm) is often used for chromogenic assays and 7-amino-
4-methylcoumarin (AMC, emission at 518 nm) for fluoro-
genic assays. For instance, over 1000 C-terminal peptide
pNA and AMC arylamide substrates have been used for the
determination of the activity of enzymes involved in blood
coagulation.3 Chemical methods for their preparation have
Arylamides, a class of carboxamides, are used in a wide
variety of applications such as in polymers, peptidomimetics,
dendrimers, scaffolds, and inhibitors and as cell-signaling
molecules.1 A special class is formed by the C-terminal
amino acid or peptide arylamides, which are often used as
(1) (a) Vemparala, S.; Ivanov, I.; Pophristic, V.; Spiegel, K.; Klein, M. L.
Org. Lett. 2006, 27, 693. (b) Pophristic, V.; Vemparala, S.; Ivanov, I.; Liu, Z.;
Klein, M. L.; DeGrado, W. F. J. Phys. Chem. B. 2006, 110, 3517. (c) Yin, H.;
Frederick, K. K.; Liu, D.; Wand, A. J.; DeGrado, W. F. Org. Lett. 2006, 8,
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Wall, M. J.; Chen, J.; Wilson, K. J.; Ballentine, S. K.; Desjarlais, R. L.;
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€
Chen, M.; Haeggstrom, J. Z.; King, B.; Kochanny, M. J.; Liang, A. M.;
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DOI: 10.1021/jo900634g
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Published on Web 06/17/2009
J. Org. Chem. 2009, 74, 5145–5150 5145
2009 American Chemical Society

Nuijens et al.
JOCFeatured Article
been investigated for decades and require highly reactive
carboxylic acid derivatives for reaction with a free arylamine,
due to its weak nucleophilicity. For example, the preparation
of N-protected amino acid pNA derivatives required the use
of POCl3 in pyridine.4 Often, most conventional coupling
strategies, e.g., the DCC (N,N0-dicyclohexylcarbodiimide)
method, do not result in satisfying yields.5 Very commonly
used in the past was the mixed anhydride method, which gave
higher yields but also a high degree of racemization of the
C-terminal amino acid residue.6 This racemization could
only be partially suppressed by the addition of copper(II)
chloride.7 Other methods based on reactive condensing
agents, i.e., reactive phosphorus or boron derivatives, also
led to significant racemization.8 It appeared especially diffi-
cult to couple electron-deficient arylamines, such as those
bearing a halogen, nitro, or cyano substituent. Very recently,
a three-step protocol for the preparation of amino acid and
peptide C-terminal electron-deficient arylamides was de-
scribed, based on the reaction of an azide with a selenocar-
boxylate.9 Although high yields were obtained, some
racemization proved to be inevitable. Evidently, each of
the known chemical methods for the preparation of
C-terminal arylamides of amino acid and peptides suffers
from some drawbacks. Besides the low yields and the race-
mization issue, the condensing reagents used are often
hazardous, expensive, and consumed in stoichiometric
amounts, leading to significant amounts of waste. Addition-
ally, all reactive amino acid side chain functionalities have to
be protected to prevent side reactions.
SCHEME 1
In the thermodynamically controlled pathway II, the energy
barrier is high and the equilibrium is usually on the side of the
starting materials. Furthermore, reaction rates are generally
low due to the equilibrium between the protonated and
(unreactive) deprotonated carboxylic acid. Manipulation
of the reaction conditions, e.g., by crystallization of the final
product during the amidation, is essential to obtain accep-
table arylamide yields.
Compared to chemical approaches, chemo-enzymatic
syntheses of arylamides may have several advantages such
as the use of mild conditions, high selectivity, and complete
absence of racemization. Moreover, the coupling reactions
are catalyzed by (often recyclable) enzymes, and no stoichio-
metric amounts of expensive coupling reagents are used.10
Furthermore, no amino acid side chain protection is needed.
There are two approaches in chemo-enzymatic amide
synthesis, i.e., the kinetically controlled pathway and the
thermodynamically controlled pathway (pathways I and II,
respectively, in Scheme 1).11
Pathway I, starting from an R-amino ester, is usually
preferred in chemo-enzymatic amide synthesis because the
energy barrier for product formation is low. Reaction of
the amine with the acyl-enzyme intermediate (1d) leads to
the desired amide. However, after formation of 1d, water can
act as a nucleophile leading to undesired hydrolysis, so
ideally k1 . k2. Nevertheless, during the course of the
amidation, the amide product is hydrolyzed to the free
carboxylic compound. Hence, there is an optimum in the
amount of product and for a high product yield the reaction
should be stopped after this optimum has been reached.
The thermodynamically controlled chemo-enzymatic
synthesis of C-terminal R-amino arylamides was disclosed
in the late 1930s.12 High yields were obtained using proteases
in buffered aqueous systems due to rapid precipitation of the
products. However, this method is limited to relatively
strong arylamine nucleophiles such as aniline, and hydro-
phobic amino acids are required to obtain precipitation
of the product. Furthermore, in the case that peptides are
C-terminally arylamidated, simultaneous hydrolysis of the
peptide bonds occurs. Kato et al. describe13 the kinetically
controlled chemo-enzymatic arylamidation with the less
nucleophilic pNA using Alcalase in organic solvents with a
water content of 5%; however, their reported yields do not
exceed 25%.
Herein, we report a versatile, mild, high-yielding, selective,
and racemization-free chemo-enzymatic strategy for the
synthesis of amino acid and peptide C-terminal arylamides
either kinetically (I) or thermodynamically (II) using the
protease Alcalase or the lipase Cal-B in organic solvents or
the protease papain in two-phase systems.
Results and Discussion
Alcalase,14 a cheap industrial serine protease from Bacillus
licheniformis, has been extensively used for various hydrolytic
(4) Rijkers, D. T. S.; Adams, H. P. H. M.; Hemker, H. C.; Tesser, G. I.
Tetrahedron 1995, 51, 11235.
(5) Zimmerman, M.; Yurewicz, E.; Patel, G. Anal. Biochem. 1976, 70,
258.
(6) Benoiton, N. L.; Lee, Y. C.; Steinaur, R.; Chen, F. M. Int. J. Pept.
Protein Res. 1992, 40, 559.
(7) Houben-Weyl, Methods of Organic Chemistry. Synthesis of Peptides
and Peptidomimetics, 4th ed.; Thieme Verlag: Stuttgart, 2002; Vol. E22a,
p 586.
(12) (a) Bergmann, M.; Behrens, O. K. J. Biol. Chem. 1938, 124, 7. (b)
Bergmann, M.; Fruton, J. S. J. Biol. Chem. 1938, 124, 321. (c) Behrens, O. K.;
Doherty, D. G.; Bergmann, M. J. Biol. Chem. 1940, 136, 61. (d) Dikker, C.
A.; Fruton, J. S. J. Biol. Chem. 1948, 173, 471. (e) Hanson, H. T.; Smits, E. L.
J. Biol. Chem. 1949, 179, 115. (f) Doherty, D. G.; Popenoe, E. A. J. Biol.
Chem. 1951, 189, 447.
(8) Kato, T.; Nagatsu, T.; Kimura, T. Experientia 1978, 34, 319.
(9) Wu, X.; Hu, L. J. Org. Chem. 2007, 72, 765.
ꢀ
(10) Guzman, F.; Barberis, S.; Illanes, A. Electr. J. Biotech. 2007, 10, 279.
(11) Bordusa, F. Braz. J. Med. Biol. Res. 2000, 33, 469.
(13) Kato, Y.; Asano, Y.; Nakazawa, A.; Kondo, K. Tetrahedron 1989,
45, 5743.
(14) Available from Novozymes (Bagsvaerd, Denmark).
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TABLE 1. Yields and Enantiomeric Excess (ee) of Synthesized Arylamides Using Alcalase-CLEA as Biocatalysta
a All reactions were performed in MTBE (methyl tert-butyl ether, except entry 4) at 50 °C and stopped after 16 h.
b Isolated yields based on the acyl donor. c Of enzymatically synthesized arylamide. d Of chemically synthesized arylamide
following the literature procedure.19 e Not determined. f Reaction was performed in THF (tetrahydrofuran).
reactions and condensations. This biocatalyst displays a
relatively broad substrate tolerance and has been used
in kinetically controlled enzymatic peptide synthesis.15 High
yields are usually obtained due to the stability of the enzyme
in nearly anhydrous (water contents <1 wt %) organic
solvents, thereby minimizing product and/or ester hydroly-
sis.16 Therefore, we selected Alcalase in a first attempt
to synthesize arylamides via the kinetically controlled path-
way using Cbz-Phe-OMe (1a) as model substrate and aniline
as arylamine.
When Alcalase (commercially available as 10 wt % aque-
ous solution) was isolated and dried by precipitation with
t-BuOH as described by Chen et al.17 followed by addition to
Cbz-Phe-OMe (1a) in pure aniline, the yield of arylamide 2a
did not exceed 27% (based on HPLC analysis). An equili-
brium mixture of arylamide 2a and hydrolyzed starting ma-
terial Cbz-Phe-OH (1c) was observed, and the water content
proved to be critical for the yield of the arylamide. Surpris-
˚
ingly, the addition of 4 A molecular sieves resulted in almost
complete (>95%, HPLC analysis) conversion to arylamide
2a with the enzyme still remaining active. The increased yield
may be explained by the fact that not only water but also
(15) (a) Maurer, K. H. Curr. Opin. Biotechnol. 2004, 15, 330. (b) Smith, E.
L.; DeLange, R. J.; Evans, W. H.; Landon, M.; Markland, F. S. J. Biol.
Chem. 1968, 243, 2184. (c) Siezen, R. J.; Leunissen, J. J. Protein Sci. 1997, 6,
501.
˚
MeOH is trapped by 4 A molecular sieves. After workup,
NMR analysis showed contaminations derived from the
(16) Wang, P.; Hill, T. G.; Wartchow, C. A.; Huston, M. E.; Oehler, L.
M.; Smith, M. B.; Bednarski, M. D.; Callstrom, M. R. J. Am. Chem. Soc.
1992, 114, 378.
(17) Chen, S. -T.; Chen, S. -Y.; Wang, K. -T. J. Org. Chem. 1992, 57,
6960.
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TABLE 2. Yields of Dipeptide Arylamides and ee’s of the Amino Acid Residuesa
entry
precursor
amide product
yieldb (%)
eec,d (%)
1
2
3
Cbz-Phe-Leu-OMe
Cbz-Phe-Leu-OBn
Cbz-Phe-Leu-OH
3a
3b
3c
Cbz-Phe-Leu-anilide
Cbz-Phe-Leu-anilide
Cbz-Phe-Leu-anilide
93
94
93
n.d.e
n.d.
>99.5f, 95.7f
>99.5g, 96.8g
n.d.
4
5
Cbz-Phe-Leu-OH
Cbz-Phe-Leu-OH
3c
3c
Cbz-Phe-Leu-pNA
Cbz-Phe-Leu-AMC
83
77
n.d.
aAll reactions were performed in MTBE at 50 °C using 10 equiv of aniline and stopped after 16 h. bIsolated yields based on the acyl donor.
c Of enzymatically synthesized arylamide. d Of chemically synthesized19 arylamide. e Not determined. f Phe moiety. g Leu moiety.
Alcalase solution. When Alcalase cross-linked enzyme aggre-
gates (CLEA)18 were used, equally high yields of 2a could
be obtained starting from 1a or benzyl ester 1b (Table 1,
entries 1 and 2), without any contaminations in the final
product. Sufficiently dry Alcalase-CLEA was obtained by
washing once with t-BuOH. After the reaction it was
conveniently removed by simple filtration.
was continuously added and evaporated at reduced pressure.
Second, we demonstrated that Alcalase-CLEA, after con-
version of 1c to 2a, could be easily separated from the
reaction mixture by a rapid filtration and washing step.
Of even greater interest than of amino acids are C-terminal
arylamides of peptides since they are pre-eminently used as
substrates for enzymatic assays. Using both the thermody-
namic (II) and the kinetic (I) approach, dipeptides 3a-c were
smoothly converted into the corresponding C-terminal ani-
lides 4a-c (Table 2, entries 1-5, 77-94% yield). No side
products were detected by HPLC indicating that no peptidic
bond hydrolysis nor transamidation had occurred in all
cases. In addition, no detectable racemization of the Leu
residue had taken place as was demonstrated by peptide
hydrolysis followed by chiral HPLC of the resulting amino
acids. This is in sharp contrast to the corresponding chemical
conversions19 (using PCl3) were inevitable racemization
occurs leading to ee values of 96% of each residue in the
dipeptide.
Besides the absence of racemization, another advantage of
the enzymatic arylamidation is that no protection of the
amino acid side chains is required. This is again in contrast to
chemical coupling methods where reactive side chain func-
tionalities such as the β- or γ-carboxyl group of aspartic and
glutamic acid, the hydroxyl function of serine, threonine or
tyrosine and the guanidino function of arginine need protec-
tion. We recently demonstrated that Alcalase-CLEA can be
used for the regioselective esterification20 and hydrolysis21 of
Asp and Glu derivatives. Now we illustrate this advantage by
the smooth and regioselective conversion of Cbz-Arg-OMe
5, Cbz-Asp-OH 6, and Cbz-Ser-OH 7 to the corresponding
C-terminal anilides 8, 9, and 10 (Table 3, entries 1-4).
Very high yields of anilides 9 and 10 (Table 3, entries 3 and
4, 91-93%) were obtained using Alcalase-CLEA. For Cbz-
Asp-OH (6), the R-C-terminal selectivity appeared to be
100% as proven by comparison (HPLC and NMR) with
the chemically synthesized reference compound. In the
In subsequent amidations, the amount of arylamine was
decreased using the cosolvents toluene, THF, or MTBE. To
our surprise, the thermodynamic approach (II) using the free
carboxylic acid Cbz-Phe-OH (1c) and anhydrous aniline in
˚
the presence of 3 A molecular sieves gave equally high yields
of the arylamide 2a (Table 1, entry 3). In addition, reaction
times were comparable to those starting from an amino acid
ester (entries 1 and 2). In order to broaden the scope of these
amidation reactions, we investigated whether the weakly
nucleophilic pNA (pKa=1.0, vs pKa=5.2 for aniline), the
sterically demanding AMC, as well as a number of methoxy-,
fluoro-, and cyano-substituted anilines could be used in the
Alcalase-mediated arylamidations (see Table 1, entries 4-14).
To our satisfaction, high yields of arylamides (2b-l) were
obtained although the efficiency of pNA, and AMC was
clearly lower than that of aniline. To verify the absence of
racemization during the enzymatic arylamidation, ee’s were
determined by chiral HPLC and compared to chemically
synthesized arylamides (see Table 1).19 As expected, no
detectable racemization was observed using the enzymatic
reactions, in sharp contrast to the conventional chemical
arylamidations using PCl3 in pyridine, where significant
racemization is observed (1-5%).
At this point, it was important to address some industrially
relevant aspects of these enzymatic conversions. First, we
proved that, in order to obtain the required anhydrous
conditions, the use of molecular sieves, which are unamen-
able for use at large scale, could be replaced by azeotropic
distillation. For instance, the conversion of 1c to 2a using
1:1 (v/v) aniline/toluene proceeded to >95% when toluene
(18) Alcalase-CLEA, Codexis, 650 AGEU/g; see also: Sheldon, R. A.
Biochem. Soc. Trans. 2007, 35, 1583.
(19) Oyamada, H.; Saito, T.; Inaba, S.; Ueki, M. Chem. Soc. Jpn 1991, 64,
1422.
(20) Nuijens, T.; Cusan, C.; Kruijtzer, J. A. W.; Rijkers, D. T. S.;
Liskamp, R. M. J.; Quaedflieg, P. J. L. M. Synthesis 2009, 5, 809.
(21) Nuijens, T.; Cusan, C.; Kruijtzer, J. A. W.; Rijkers, D. T. S.;
Liskamp, R. M. J.; Quaedflieg, P. J. L. M. Tetrahedron Lett. 2009, 50, 2719.
5148 J. Org. Chem. Vol. 74, No. 15, 2009

Nuijens et al.
JOCFeatured Article
TABLE 3. Yields of Synthesized Arylamides of Cbz-Arg-OMe, Cbz-
Asp-OH, and Cbz-Ser-OHa
in our application since it has a broad substrate tolerance for
aliphatic acids,23 is known to be active in the transesterifica-
tion of amino acids,24 and maintains a high activity in
anhydrous organic solvents.25 Hence, we subjected Cbz-
Ala derivatives 11a and 11b to Cal-B and aniline under
anhydrous conditions. Indeed, the corresponding anilide
12 was obtained in high yield (Table 4, entries 1 and 2).
Conclusions
In this paper, we have shown that N-terminal-protected
amino acids and peptides can be enzymatically converted in
high yields into various C-arylamides under (nearly) anhy-
drous conditions even if the aryl amine has an extremely low
nucleophilicity or is sterically very demanding. Although
Alcalase readily accepted most C-terminal moieties in its
active site, in some cases, for instance with C-terminal
positively charged arginine residues, the use of papain in a
two-phase system gave more satisfactory results. Starting
from either C-terminal free carboxylic acids or C-terminal
alkyl esters, high yields were obtained, and in case of dipep-
tides, no peptide bond hydrolysis or transamidation oc-
curred. In contrast to the state-of-the-art chemical
methods, no racemization occurred at the C-terminal posi-
tion and no amino acid side chain protection was required.
Thus, we feel that this enzymatic method is a significant step
forward in the synthesis of this important class of com-
pounds. Since the (nearly) anhydrous conditions can be
obtained by (azeotropic) distillation, this technology is also
amenable to scale-up.
entry
precursor
enzyme
product
yield (%)
1
2
3
4
Cbz-Arg-OMe 5
Cbz-Arg-OMe 5
Cbz-Asp-OH 6
Cbz-Ser-OH 7
Alcalase-CLEA
papain
Alcalase-CLEA
Alcalase-CLEA
8
8
9
10
54
82
91
93
a Reactions were performed in THF (entries 1 and 2) or MTBE
(entries 3 and 4) at 50 °C using 10 equiv of aniline and were stopped
after 16 h. Isolated yields are based on the acyl donor. Cbz-Arg-OMe
was used instead of Cbz-Arg-OH due to solubility problems with the
latter in anhydrous organic solvents.
TABLE 4. Yields of Synthesized Cbz-Ala Arylamide Using Cal-Ba
Experimental Section
General Procedure for the Synthesis of Arylamides 2a and 4a.
Alcalase-CLEA (500 mg) was added to a mixture containing
MTBE (4.5 mL), aniline (500 μL, 4.48 mmol), 1a-c or 3a-c
˚
(0.45 mmol), and 3 A molecular sieves (200 mg). The mixtures
entry
precursor
product
yieldb (%)
ee (%)
were shaken at 50 °C at 150 rpm for 16 h. The reaction mixture
was filtered over a P4 glass filter, and the solids were washed
with ethyl acetate (EtOAc, 50 mL, 3ꢀ). Distilled water (150 mL)
was added to the combined filtrates, and under vigorous stirring
the pH was adjusted to 3.0 with 1 N aqueous HCl solution. After
an additional 10 min of stirring the two layers were separated.
This procedure of washing the organic layer with an aqueous
phase of pH 3 was repeated twice. Subsequently, the organic
phase was washed with saturated aqueous NaHCO3 (3 ꢀ 100 mL)
and brine (100 mL), dried (Na2SO4), and concentrated in
vacuo. The residue was diluted with n-heptane (2 mL), and the
resulting crystals were isolated by filtration and washed with
n-heptane (2 ꢀ 5 mL).
1
2
Cbz-Ala-OMe
Cbz-Ala-OH
11a
11b
12
12
92
77
>99.5
>99.5
aAll reactions were performed in MTBE at 50 °C using 10 equiv of
aniline and were stopped after 16 h. bIsolated yields based on the acyl
donor.
arylamidation of Cbz-Ser-OH (7) only a small amount (2%)
of the side chain ester was formed, as was demonstrated by
LC-MS. However, Alcalase does not favor positively
charged amino acids at the P1 position22 in the active site,
resulting in only a moderate yield of 8 (Table 3, entry 1, 54%).
To remedy this, the widely employed and commercially avail-
able cysteine protease papain was used, which does favor
positively charged amino acids at its P1 position. The applica-
tion of this enzyme in pure aniline did not result in any
conversion of 5, since papain is not active in nearly anhydrous
organic solvents. Therefore, a two-phase system consisting of
aniline and phosphate buffer (pH=7.5) was used, and a high
yield of anilide 8(Table 3, 82%) was readily obtained after only
10 min.
Cbz-Phe-Leu-anilide (4a). Compound 4a was obtained as a
solid (from: 3a, 93% yield, 204 mg; 3b, 94% yield, 206 mg; 3c,
93% yield, 204 mg): tR (HPLC method 1) 21.08 min; purity
99%; Rf (EtOAc/n-hexane, 1/1, v/v) 0.52; mp 225 °C; [R]20
=
D
16.8 (c 0.5, DMSO); 1H NMR (300 MHz, CDCl3) δ=0.81 (d,
J=6.0 Hz, 6 H), 1.34-1.50 (m, 2 H), 1.65-1.76 (m, 1 H), 3.00 (d,
2 H), 4.30-4.51 (m, 2 H), 5.00 (s, 2 H), 5.21 (d, J=6.6 Hz,
1 H), 6.25 (d, J=7.2 Hz, 1 H), 7.00-7.27 (m, 13 H), 7.47
(d, J=7.5 Hz, 2 H), 8.25 (s, 1 H); 13C NMR (75 MHz, DMSO-d6)
δ=21.7, 22.4, 24.5, 38.4, 41.0, 51.6, 51.9, 55.7, 66.6, 126.5, 127.6,
In order to broaden the scope of this technology beyond
proteases, we also explored the use of lipases. We envisioned
that Candida antarctica lipase-B (Cal-B) would be promising
(23) Otto, R. T.; Scheib, H.; Bornscheuer, U. T.; Pleiss, J.; Syldatk, C.;
Schmid, R. D. J. Mol. Catal. B 2000, 8, 201.
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Nuijens et al.
JOCFeatured Article
127.8, 128.2, 129.2, 136.1, 136.3, 155.9, 171.0, 172.7; FIA-ESI-
(+)-TOF-MS m/z [M+H]+ calcd for C29H34N3O4 488.2543,
found 488.2559; IR (neat/cm-1) 3281, 1692, 1641, 1531, 1495,
1447, 1285, 1258, 1234.
(100 mL). Under vigorous stirring, the pH was adjusted to 3.0
with 1 N aqueous HCl solution. After an additional 10 min of
stirring, the two layers were separated. This procedure of
washing the organic layer with an aqueous phase of pH 3 was
repeated twice. The organic phase was concentrated in vacuo,
and the residue was suspended in distilled water (20 mL).
The resulting crystals were isolated by filtration over a P4
glass filter, washed with distilled water (50 mL, 2ꢀ), and
resuspended in toluene (100 mL), and the mixture was concen-
trated in vacuo.
General Procedure for the Synthesis of Arylamides 2b and 4b.
Alcalase-CLEA (500 mg) was added to a mixture containing
THF (5 mL), pNA (500 mg, 3.6 mmol), 1c or 3c (0.33 mmol), and
˚
3 A molecular sieves (200 mg). The mixture was shaken at 50 °C
with 150 rpm for 16 h. The reaction mixture was filtered over a
P4 glass filter, and the solids were washed with EtOAc (50 mL,
3ꢀ). The combined filtrate was concentrated in vacuo and
purified by preparative HPLC.
Compound 8 was obtained as a hygroscopic solid: (a) 54%
yield, 93 mg; (b) 82% yield, 141 mg; tR(HPLC method 1) 9.46
min; purity 98%; Rf (EtOAc/n-hexane, 1/1, v/v) 0.31; mp 56 °C;
[R]20D=44.8 (c 0.5, DMSO); 1H NMR (300 MHz, DMSO-d6)
δ=1.40-1.90 (m, 4 H), 3.13 (m, 2 H), 4.20 (m, 1 H), 5.04 (s,
2 H), 7.00-7.48 (m, 12 H), 7.64-7.36 (m, 3 H), 7.93 (s, 1 H)
10.28 (s, 1 H); 13C NMR (75 MHz, DMSO-d6) δ=25.1, 28.8,
54.8, 65.3, 119.1, 123.2, 127.6, 127.7, 128.2, 128.5, 136.9, 138.9,
155.9, 156.9, 170.7; FIA-ESI(+)-TOF-MS m/z [M+H]+ calcd
Cbz-Phe-Leu-p-nitroanilide (4b). Compound 4b was obtained
as a solid in 83% yield (145 mg): tR (HPLC method 1) 20.93
minp purity 96%; Rf (EtOAc/n-hexane, 1/1, v/v) 0.60; mp
196 °C; [R]20D = 26.2 (c 0.5, DMSO); 1H NMR (300 MHz,
DMSO-d6) δ=0.98 (dd, J=6.3 and 10.2 Hz, 6 H), 1.55-1.80 (m,
3 H), 2.76-2.84 (dd, 1 H), 3.05-3.11 (dd, 1 H), 4.35-4.43 (m,
1 H), 4.51-4.59 (m, 1 H), 5.01 (s, 2 H), 7.20-7.54 (m, 10 H), 7.52
(d, J=8.7 Hz, 1 H), 7.93 (d, J=9.0 Hz, 2 H), 8.29 (d, J=9.3 Hz,
1 H), 8.36 (d, J=7.5 Hz, 1 H), 10.7 (s, 1 H); 13C NMR (75 MHz,
DMSO-d6) δ=21.5, 22.8, 24.2, 37.2, 52.2, 55.7, 65.0, 118.9,
124.8, 126.1, 127.5, 127.8, 128.1, 129.1, 136.9, 137.8, 142.2,
144.9, 155.7, 171.6, 171.9; FIA-ESI(+)-TOF-MS m/z
[M+H]+ calcd for C29H33N4O6 533.2395, found 533.2411;
IR (neat/cm-1) 3277, 2958, 1691, 1647, 1532, 1515, 1341,
1300, 1254.
for C20H26N5O3 384.2030, found 384.2041; IR (neat/cm-1
)
3292, 3260, 1748, 1597, 1553, 1498, 1488, 1434, 1368, 1321, 1262.
Cbz-Asp-anilide (9).26 Alcalase-CLEA (500 mg) was added
to a mixture containing MTBE (4.5 mL), aniline (500 μL,
4.5 mmol, 10 equiv), Cbz-Asp-OH 6 (120 mg, 0.45 mmol, 1 equiv),
˚
and 3 A molecular sieves (200 mg). The mixture was shaken at
50 °C with 150 rpm for 16 h. The reaction mixture was filtered over
a P4 glass filter, and the solids were washed with EtOAc
(50 mL, 3ꢀ) and aqueous HCl (50 mL, pH = 1, 3ꢀ). The two
phases of the filtrate were separated, and the organic layer was
concentrated in vacuo. After preparative HPLC purification,
compound 9 was obtained in 91% yield as a solid (140 mg).
Cbz-Ser-anilide (10).27 Compound 10 was synthesized as
described for 9 using Cbz-Ser-OH 7 (108 mg, 0.45 mmol,
1 equiv) and obtained in 93% yield as a solid (423 mg).
General Procedure for the Synthesis of Arylamides 2c-l and
4c. Alcalase-CLEA (500 mg) was added to a mixture contain-
ing MTBE (4.5 mL), arylamine (500 mg), 1c or 3c (100 mg,
˚
0.33 mmol), and 3 A molecular sieves (200 mg). The mixture was
shaken at 50 °C with 150 rpm for 16 h and subsequently filtered
over a P4 glass filter. The solids were washed with EtOAc
(50 mL, 3ꢀ). The combined filtrate was concentrated in vacuo,
and the resulting residue was purified by preparative HPLC.
Cbz-Phe-2-methoxyanilide (2d). Compound 2d was obtained
in 87% yield as a solid (116 mg): tR (HPLC method 1) 20.41 min;
purity 99%; Rf (EtOAc/n-hexane, 1/1, v/v) 0.56; mp 160 °C;
[R]20D=86.2 (c 0.5, DMSO); 1H NMR (300 MHz, DMSO-d6)
δ=2.85 (dd, 1 H), 3.12 (dd, 1 H), 3.81 (s, 3 H), 4.55 (m, 1 H), 4.98
(m, 2 H), 6.92 (m, 1 H), 7.20-7.40 (m, 12 H), 7.77 (d, J= 8.4 Hz,
1 H), 8.01 (m, 1 H), 9.24 (s, 1 H); 13C NMR (75 MHz, DMSO-d6)
δ = 37.0, 55.7, 56.7, 65.2, 111.1, 120.2, 121.0, 124.3, 126.2,
126.9, 127.3, 127.6, 127.9, 128.2, 129.2, 136.8, 137.8, 149.1,
155.9, 170.2; FIA-ESI(+)-TOF-MS m/z [M+H]+ calcd for
C24H25N2O4 405.1808, found 405.1783; IR (neat/cm-1) 3291,
1685, 1656, 1598, 1531, 1499, 1444, 1326, 1263, 1246, 1051.
Cbz-Arg-anilide (8). (a) Using Alcalase-CLEA. Alcalase-
CLEA (500 mg) was added to a mixture containing THF
(4.5 mL), aniline (500 μL, 4.5 mmol, 10 equiv), 5 (145 mg,
Cbz-Ala-anilide (12).28 Cal-B (100 mg) was added to a mix-
ture containing MTBE (4.5 mL), aniline (500 μL), 11a or 11b
(100 mg), and molecular sieves (200 mg). The mixture was
shaken at 50 °C at 150 rpm for 16 h. The reaction mixture was
filtered over a P4 glass filter, and the solids were washed with
EtOAc (50 mL, 3ꢀ). Distilled water (150 mL) was added to the
filtrate, and under vigorous stirring the pH was adjusted to
3.0 with 1 N aqueous HCl solution. After an additional 10 min
of stirring, the two layers were separated. This procedure
of washing the organic layer with an aqueous phase of pH 3
was repeated twice. Subsequently, the organic phase was
washed with saturated aqueous NaHCO3 (100 mL, 2ꢀ) and
brine (100 mL), dried (Na2SO4), and concentrated in vacuo. The
residue was diluted with n-heptane (2 mL), and the resulting
crystals were isolated by filtration and washed with n-heptane
(2 ꢀ 5 mL) giving compound 12 (from: 11a, 92% yield, 116 mg;
from 11b, 77% yield, 103 mg).
˚
0.45 mmol, 1 equiv), and 3 A molecular sieves (200 mg). The
mixture was shaken at 50 °C with 150 rpm for 16 h and
subsequently filtered over a P4 glass filter. The solids were
washed with EtOAc (50 mL, 3ꢀ), and the combined filtrate
was concentrated in vacuo. The residue was triturated in
distilled water (20 mL), the resulting crystals were isolated by
filtration over a P4 glass filter, washed with water (50 mL, 2ꢀ),
and resuspended in toluene (50 mL), and the mixture was
concentrated in vacuo.
Supporting Information Available: Experimental proce-
dures for the preparation of compounds 1a,b, 3a-c, 5, 9,
1
and 11a. H NMR, 13C NMR, HR-MS, and HPLC data for
all synthesized compounds. Additional IR absorption spectra
for compounds 2a-l, 4a-c, 6, 9, 10, and 12. This material is
available free of charge via the Internet at http://pubs.acs.org.
(b) Using Papain. Papain (10 mg) was added to a mixture
containing phosphate buffer (2.5 mL, 250 mM, pH 7.5), aniline
(2.5 mL), 1,4-dithiothreitol (1 mg), and 5 (145 mg, 0.45 mmol).
The mixture was shaken at ambient temperature for 10 min
followed by the addition of EtOAc (100 mL) and distilled water
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62, 8821.
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W. W. J. Am. Chem. Soc. 1954, 76, 147.
ꢀ
ꢀ
(28) Kaminski, Z. J.; Kolesinska, B.; Kaminska, J. E.; Gora, J. J. Org.
ꢀ
ꢀ
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5150 J. Org. Chem. Vol. 74, No. 15, 2009