Efficient procedure for the preparation of oligomer-free N-fmoc amino acids

Article abstract of DOI:10.1080/00397910802632563

A two-step method is presented for the peptide-free, high-purity, and high-yield synthesis of N-Fmoc amino acids. The first step involves the preparation of stable dicyclohexylammonium-amino acid ionic adduct in acetone. Subsequently, the ionic adducts, on reaction with Fmoc-Nosu under mild alkaline conditions, give dipeptide-free N-Fmoc amino acids. The positive charge of the dicyclohexylammonium counterion in the ionic salt has a longer radius, moderating the nucleophilicity of the carboxylate ion of the amino acid and preventing by-products by arresting the formation of mixed anhydrides, the precursors of oligopeptide impurities.

Full text of DOI:10.1080/00397910802632563

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Efficient Procedure for the
Preparation of Oligomer-Free N
α -Fmoc Amino Acids
Shaik Nowshuddin a , M. N. A. Rao a & A. Ram Reddy
a
a Department of Chemistry, University College of
Science, Osmania University, Hyderabad, India
Version of record first published: 27 Apr 2009
To cite this article: Shaik Nowshuddin, M. N. A. Rao & A. Ram Reddy (2009): Efficient
Procedure for the Preparation of Oligomer-Free N α -Fmoc Amino Acids, Synthetic
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Synthetic Communications1, 39: 2022–2031, 2009
Copyright # Taylor & Francis Group, LLC
ISSN: 0039-7911 print=1532-2432 online
DOI: 10.1080/00397910802632563
Efficient Procedure for the Preparation of
Oligomer-Free Na-Fmoc Amino Acids
Shaik Nowshuddin, M. N. A. Rao, and A. Ram Reddy
Department of Chemistry, University College of Science,
Osmania University, Hyderabad, India
Abstract: A two-step method is presented for the peptide-free, high-purity, and
high-yield synthesis of Na-Fmoc amino acids. The first step involves the prepara-
tion of stable dicyclohexylammonium–amino acid ionic adduct in acetone. Sub-
sequently, the ionic adducts, on reaction with Fmoc-Nosu under mild alkaline
conditions, give dipeptide-free Na-Fmoc amino acids. The positive charge of
the dicyclohexylammonium counterion in the ionic salt has a longer radius, mod-
erating the nucleophilicity of the carboxylate ion of the amino acid and prevent-
ing by-products by arresting the formation of mixed anhydrides, the precursors of
oligopeptide impurities.
Keywords: Adduct, dicyclohexylamine–amino acid, Fmoc, mixed anhydride
Protecting groups are inevitable and essential tools to a successful
multistep synthetic sequence. They can temporarily mask certain inter-
fering functional groups with the prospective reactions. Organic synthesis
involving carbohydrates, proteins, and oligonucleotides is incomplete
without the systematic application of protecting groups.[1,2] The primary
and secondary amino groups are nucleophilic and are easily oxidizable.
Amino groups in the amino acids need protection in the peptide synthesis
involving condensation of amino acids. The most commonly employed
protecting groups, either in solution or solid-phase oligopeptide synth-
esis, are benzyloxycarbonyl (Z),[3–5] tertiary-butyloxycarbonyl (Boc),[6–8]
Received September 5, 2008.
Address correspondence to A. Ram Reddy, Department of Chemistry,
University College of Science, Osmania University Campus, Osmania University,
Hyderabad, Andhra Pradesh 500 007, India. E-mail: aramreddy2008@yahoo.com
2022

Oligomer-Free Na-Fmoc Amino Acids
2023
and 9-fluorenylmethoxycarbonyl (Fmoc)[9,10] with appropriate ortho-
gonal side-chain protection. Fmoc amino acid derivatives are versatile
N-protected intermediates for peptide synthesis, especially when used in
conjunction with acid labile side-chain protecting groups.[11] These are
more readily deblocked under mildly basic nonhydrolytic conditions
following the E1CB mechanism. However, when these compounds were
prepared by a standard method such as the Schotten–Bauman procedure,
a majority of Fmoc amino acids were contaminated by substantial
amounts of impurities.[12–20] The ability of the leaving group and the
solvent and the nucleophilic base employed play a profound role in the
purity of the products.[21] Zalipsky[22] observed that Fmoc succinimidyl
carbonates underwent Lossen rearrangement in aqueous basic medium
to give b-alanine. When an efficient leaving group such as ClÀ or N3 in
Fmoc is present, less product and more side products were obtained,
but with 2-thiomercaptol as the leaving group, an appreciable yield of
Fmoc amino acids was obtained.[23] Barlos and coworkers[24] carried
out a one-pot synthesis of N-trityl amino acids involving silylated esters
as intermediates in good yield. Amino acids exist in zwitter ionic form
and as carboxylates in basic medium. The nucleophilicity of these carbo-
xylates, if not moderated, may attack the electrophilic sites present in the
Fmoc derivatives and give side products such as dipeptides via anhydride
intermediates as proposed in Scheme 1.
There is a growing demand for the preparation of synthetic peptide
pharmaceuticals with a high level of purity. Leonidas and Charalambos[25]
demonstrated that the N-o-nitrophenylsulfenyl (N-NPS) dicylcohexyl-
amine–amino acid adducts are stable. Barcelo et al.[26] isolated stable
N-Boc tyrosine=serine–dicyclohexylamine (DCHA) salts. Therefore, in this
article, we report a simple two-step synthesis of various oligomer-free
Na-Fmoc amino acids involving the respective DCHA–amino acid ionic
Scheme 1. Formation of dipeptides impurities involving anhydride intermediate.

2024
S. Nowshuddin, M. N. A. Rao, and A. R. Reddy
Scheme 2. Synthesis of Na-Fmoc amino acids.
salt intermediates. The scope of the reactions using structurally divergent
side-chain-protected amino acids as substrates giving Na-Fmoc products
is shown in Scheme 2. The first step includes synthesis of a crystalline
DCHA–amino acid salt (compound II) by stirring equimolar amounts of
dicyclohexylamine and the respective amino acid in acetone at room tem-
perature for 4 to 5 h.
All the DCHA–amino acid salts (II) were obtained with good yields.
These are stable, and no physical properties were altered on storage.
In the second step, the DCHA–amino acid salt (II) and Fmoc-Nosu
were mixed between 0 and 5ꢀC and subsequently stirred overnight with
an alkaline pH at room temperature to yield the Na-Fmoc amino acid
(compound III). All the Fmoc amino acids prepared following Scheme 2
are given in Table 1.
It is interesting to notice that the purity and yield of the Fmoc amino
acids are appreciable even when the medium is alkaline aqueous
medium. The purity of the protected amino acids (III) was monitored
by high-performance liquid chromatography (HPLC) and thin-layer
chromatography (TLC), and we found that varies between 99.9% and
98.0%. HPLC could not detect an oligopeptide impurity following the
present method. The developed thin-layer chromatogram of Fmoc-
glycine is shown in Fig. 1.
In the present two-step method, only Na-Fmoc-glycine is obtained
without dipeptide impurities, whereas in the single-step preparation of
Chang and coworkers,[28] Na-Fmoc-glycylglycine (gly-gly) peptide impu-
rities are present. Similar observations are obtained from HPLC studies.
The HPLC of Fmoc-glycine employing a Zorbax CN C18 4.6-mm, 5-mm
column is shown in Fig. 2.
The overall yield obtained varied between 92% (entry 21, Table 1)
and 56 (entry 5, Table 1). From Table 1, it can be noticed that the trifunc-
tional amino acids yield less Na-Fmoc amino acids than bifunctional
amino acids except lysine under the given set of common experimental
conditions. When we tried to reduce the two-step process to a one-step
process [i.e., compound (I) to compound (III) directly by mixing the

2025

2026
S. Nowshuddin, M. N. A. Rao, and A. R. Reddy
Figure 1. Thin-layer chromatoplate of Fmoc-glycine. (a) present method;
(b) previous method.[28]
respective amino acids (I) and the reagents in the same order as in the
two-step method but without isolating the crystalline salt (II)], the overall
yield of (III) was poor. When the reaction was carried out at pH 6–7, 50%
of the starting amino acid was recovered. Similarly when the reaction
temperature was maintained between 0 and 5ꢀC in both steps, only
5–12% of Fmoc amino acids resulted. When the reaction was conducted
at reflux temperature, racemization occurred. Use of tertiary amines such
as tributylamine, triethylamine, or primary amine aniline instead of
Figure 2. High-performance liquid chromatography of Fmoc-glycine. (a) present
method; (b) previous method.[28]

Oligomer-Free Na-Fmoc Amino Acids
2027
DCHA did not yield the crystalline salts (II). This may be due to the
nature of the counterion associated with the salt.
CHARACTERIZATION OF THE ION ADDUCTS (II)
Formation of intermediate salt occurs when the corresponding amino
acid is mixed with an equimolar amount of DCHA in acetone solvent.
Because the pKa value of DCHA is 11, it readily accepts a proton from
the ammonium part of the zwitter ionic amino acid to give DCHA–
amino acid salt as shown in the Scheme 3.
The intermediate (II) obtained is ionic and conducting. The 0.01 M
solution of s-trityl cystein, lysine, and alanine displayed a conductivity
of 31,330 and 5.5 mmhos in methanol at 25ꢀC, and the respective
0.01 M DCHA salts exhibited 460,670 and 8.4 mmhos respectively in
methanol. Further, the conductivity decreases with dilution. The decrease
in conductivity on dilution demonstrates that the adducts formed
between the amino acids and DCHA are ionic in nature as represented
in Scheme 3. Similar evidence of the salt character of the intermediate
adducts is deduced from infrared (IR) studies. The IR spectrum of the
amino acid–DCHA salts displayed a doublet absorption band at 3400
and 3500cmꢂ1, medium-intensity satellite bands at 2700–3000 cmꢂ1, and
strong absorption bands near 1600 and 1400cmꢂ1 among other absorption
bands. The doublet absorption between 3400 and 3500cmꢂ1 can be attrib-
uted to the symmetric and unsymmetric NꢂH stretching modes of
vibrations of a primary amine, tNH2, which indicates that the amino group
of the amino acid in the salt exists as a free amino group but not in ammo-
nium form. The absorption bands at 1600, 1590cmꢂ1 and at 1400cmꢂ1 are
similar to that of sodium salt of amino acids and is attributed to tcooꢂ. The
satellþite bands observed between 2700 and 3000cmꢂ1 are attributed to the
tNH2 of DCHA salt. The conductivity as well as the IR spectral studies
demonstrate that the intermediate (II) obtained by mixing DCHA and
amino acid is a stable ionic salt. Cyclohexyl amine salt of L-arginine
resulted in poor yield.
Carboxylate counterions influence the formation of Na-Fmoc deriva-
tives. As shown in Scheme 1, the crystalline salt intermediates (II) can be
Scheme 3. Synthesis of dicyclohexylammonium-amino acid salt.

2028
S. Nowshuddin, M. N. A. Rao, and A. R. Reddy
secured selectively only when the base employed is DCHA but not
tertiary or primary amines. Formation of such salts would drastically
decrease the dipeptide content while undertaking the synthesis of Fmoc
amino acids.[16] The electrostatic interaction decreases the nucleophilicity
of the carboxylate ion in the salt by encompassing the positive charge
of DCHA over the amino acid, which does not readily form the mixed
anhydride to give a dipeptide by-product. Thus, the stabilization of the
intermediate decreases the reactivity of the carboxylate ion and prevents
the formation of a dipeptide, consuming Na-Fmoc amino acids. In
aprotic solvents such as acetone, the counterions play an important role
in stabilization of the salts.[29] From Table 1, it can be observed that the
electronic and steric effects of the side chain in the amino acids have
considerable influence on the product distribution. Significant contribu-
tion to the yield and purity of the N-Fmoc derivatives comes also from
the regulation of temperature during the mixing of the reagents.
From Table 1, it can be found that whatever, the yield may be the
purity of the Na-Fmoc compounds is good with a minimum of 98.0%.
The maximum purity reaches 99.9% without any oligopeptide impurity,
contrary to the efficient one-pot synthesis.[16,19] The present method
needs two simple steps but offers comparable yields with improved
purity.
CONCLUSION
Formation and the stability of an ionic compound in a nonpolar, aprotic
solvent largely depends on the nature of the counterion. In DCHA–
amino acid ionic adducts, the positive charge on DCHA reduces the
nucleophilicity of the carboxylate ion, preventing the formation of
precursors of by-products. On the other hand, the nucleophilicity of
the amino group in the ionic adduct is either unaffected or marginally
increased as a consequence of stabilization of the carboxylate moiety,
leading to the selective protection of the a-amino group by Fmoc.
EXPERIMENTAL
Typical Example for the Preparation of Side-Chain-Protected
DCHA–Amino Acid Salt
Five g (0.056 mol) of L-Asp(t-Bu)-OH (or 0.026 mol of amino acid),
acetone (50 ml), and DCHA (5.74 g, 0.031 mol) were taken in a 100-ml
round-bottom flask and stirred at room temperature. The turbid solution

Oligomer-Free Na-Fmoc Amino Acids
2029
initially formed gradually changed to a white crystalline salt with stirring.
After 4–5 h, the reaction was complete. The L-Asp(t-Bu)-OH DCHA
crystalline salt was filtered, washed with acetone (10 ml), dried, and
stored. Yield 8.0 g (82%).
General Procedure for the Synthesis of Na-Fmoc Amino Acids
Eight g (0.021 mol) of L-Asp(t-Bu)-OH DCHA salt were taken in a
250-ml round-bottom flask, and 75 ml of water were added to it. The
pH of the solution was adjusted to 8 with 10% Na2CO3 solution.
Fmoc-Nosu (7.28 g) dissolved in 75 ml acetonitrile was added to the
homogeneous alkaline L-Asp(t-Bu)-OH DCHA salt solution while the
temperature was maintained between 0 and 5 ꢀC. After the addition
was complete, the reaction was allowed to reach room temperature and
stirred overnight. The reaction mixture was acidified with 5% KHSO4
to reach a pH of 2 to 3. The Na-Fmoc-L-Asp(t-Bu)-OH solution was
extracted with 100 ml of ethyl acetate. The extracted ethyl acetate was
washed with saturated NaCl solution and distilled water and dried over
anhydrous Na2SO4. The ethyl acetate was removed at reduced pressure.
The oily crude product was obtained. The product was crystallized with
dichloromethane and n-hexane to give the Na-Fmoc-L-Asp(t-Bu)-OH
(5.3 g, 60.5%).
ACKNOWLEDGMENT
One of the authors, S. Nowshuddin, thanks Dr. Murali K. Divi,
chairman and managing director, Divis Laboratories Limited, and
Dr. P. Gundu Rao for their encouragement.
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