Synthesis of peptides employing protected-amino acid halides mediated by commercial anion exchange resin

Article abstract of DOI:10.1007/s10989-012-9337-5

Coupling of protected-amino acid halides (chloride, fluoride) mediated by commercial anion exchange resin for the solution phase synthesis of peptides is described. The reaction was carried out in an organic medium, circumventing the use of an organic base or an inorganic base. The coupling is fast, clean and racemization free. The anion exchange resin functions as a solid-phase basic scavenger, soaking up the HCl produced and allowing the amine to react. The method is extended for the coupling of sterically hindered α,α,- dialkylamino acids. Graphical Abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s10989-012-9337-5

Int J Pept Res Ther (2013) 19:225–229
DOI 10.1007/s10989-012-9337-5
Synthesis of Peptides Employing Protected-Amino Acid Halides
Mediated by Commercial Anion Exchange Resin
•
K. M. Bhaskara Redddy Y. Bharathi Kumari
•
Kuppanna Ananda
Accepted: 23 November 2012 / Published online: 3 January 2013
Ó Springer Science+Business Media New York 2012
Abstract Coupling of protected-amino acid halides
(chloride, fluoride) mediated by commercial anion
exchange resin for the solution phase synthesis of peptides
is described. The reaction was carried out in an organic
medium, circumventing the use of an organic base or an
inorganic base. The coupling is fast, clean and racemiza-
tion free. The anion exchange resin functions as a solid-
phase basic scavenger, soaking up the HCl produced
and allowing the amine to react. The method is extended
for the coupling of sterically hindered a,a,-dialkylamino
acids.
Introduction
The activation of the carboxylic group of Fmoc-amino acid
as its chloride significantly enhances its reactivity with
amino component in peptide synthesis leading to the rapid
and efficient incorporation of acid moiety. The high degree
of activation associated with an acid chloride makes cou-
plings of hindered or weakly nucleophilic systems possible
while other methods give only poor results (Carpino et al.
1996; Hruby 2001; Humphrey and Chamberlin 1997;
Beyermann et al. 2002). The trapping of the liberated
hydrogen chloride during coupling has been accomplished
by using a two-phase system with a mild inorganic base in
the aqueous layer (Carpino et al. 1986, 1991a, b). For
reactions carried out in homogeneous solution the addition
of tertiary amines which is required to capture the hydro-
gen chloride liberated during the coupling process causes
the Fmoc-protected amino acid chlorides to be converted
into oxazol-5 (4H)-ones, and therefore, limits drastically
the application of this method. If reactions of these acid
chlorides were carried out with additives such as silver
cyanide (Perlow et al. 1992; Williams et al. 1992) or
ammonium (Sivanandaiah et al. 1992) and potassium salts
of HOBt (Sivanandaiah et al. 1994a, b; Babu and Gopi
1998), have been successfully used in solution and solid
phase synthesis for coupling of sterically hindered amino
acids. Urethane protected amino acid fluorides have been
shown to be efficient, rapid acting coupling agents for both
solution and solid phase peptide synthesis (Carpino et al.
1991a, b, 1993). Although the acylation of natural amino
acids using acid fluoride derivatives are highly efficient
reagents for peptide assembly in the absence of tertiary
bases (Wenschuh et al. 1994, 1995).
Keywords Anion exchange resin Á Dialkylamino acids Á
Coupling efficiency Á Deprotonation Á Amino acid
scavenging
Abbreviations
IER
Phg
Ionexchange resin
Phenylglycine
DCM Dichlromethane
DIEA Diisopropylethylamine
K. M. Bhaskara Redddy Á K. Ananda (&)
Mylan Laboratories Ltd., Chemical Research Division,
Anrich Industrial Estate, Bollaram,
Hyderabad 502325, India
e-mail: ananda.kuppanna@mylan.in
Y. B. Kumari
Department of Chemistry, College of Engineering, Jawaharlal
Nehru Technological University Hyderabad, Kukatpally,
Hyderabad 500085, India
However, the incorporation of sterically hindered amino
acids into peptide segments does require the use of a base.
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Acylation of urethane protected amino acid fluorides
mediated by zinc was reported earlier (Babu and Ananda
2000). TBDMS-OBt (Tantry and Suresh Babu 2002) has
been found to be advantageous due to its solubility and
separation of side product. Zinc (Gopi and Suresh Babu
1998) and indium (Cho and Jang 2004) mediated coupling
is an alternative reaction pathway for the mediation of
coupling by the acid chloride method. The need for further
mild coupling methods for the solution phase synthesis of
peptides with concomitant purification is addressed in this
communication.
Table 1 Characterization of amino acid esters
Aminoacid
ester
Yield
(%)
MP (°C) I.R (cm-1
)
[a]D
25c = 1,CHCl3
H-Phe-OMe
H-Ala-OMe
H-Aib-OMe
H-Gly-OMe
H-Leu-OMe
80
88
79
83
75
137–138 3,271,
3,234
?21.8
?9.1
-
92–93
Gum
Gum
3,276,
3,240
3,219,
3,260
3,321,
3,249
?9.5
?18.6
142–143 3,322,
3,234
Anion exchange resins are insoluble polymers that con-
tain basic functional group and have ability to exchange
counter ions (Hesham et al. 2010). Anion-exchange resins
are widely used to remove acidic molecules from solution.
In the area of organic synthesis for example, such resins
provide a convenient means for ‘scavenging’ excess of
reagents and byproducts. Over the years, ion exchange
resins have found use as pharmacologically active ingre-
dients in drug formulations (Singh et al. 2007). However,
the ion exchange resins have also been used in pharma-
ceutical manufacturing for the isolation and purification of
drugs and catalysis of reactions (David 2005). They are also
used to scavenging excess reagents and byproducts have
been exploited in the solution phase synthesis of chemical
libraries (Gayo and Suto 1997). A further development was
the synthesis of amides via the mixed anhydride prepared
from the carboxylic acid and ethyl chloroformate in the
presence of amberlyst (Mark et al. 1998). Recently several
groups have reported on the use of solid phase resins as
quench reagents and resin bound reagents to expedite the
synthesis and purification of derivatives (Flynn et al. 1997;
Booth and Hodges 1997). A typical strongly basic anion
exchange resin will have quaternary ammonium groups
attached to a styrene and divinylbenzene copolymer. Here
we report, the use of indion 830-S commercially available
and strongly basic quaternary ammonium anion exchange
resin (Ion exchange India Pvt Limited, Mahalaxmi, Mumbai,
India) for trapping of the liberated hydrogen chloride/fluo-
ride during peptide coupling has been accomplished by using
a homogeneous system.
After deprotonation, the free amino acid ester dissolved
completely in DCM. The pH of the solution was found to
be neutral. IR analysis clearly indicated the presence of
sharp doublet at around 3,260 cm-1 corresponding to the
free amino function in the place of a broad peak at around
2,950 cm-1 of the amino acid salt functionality.
The resins were selected (Table 2) based upon their
availability. The resin that provided the highest purity and
yield was indion 830-S. The conditions used for screening
of suitable resin for coupling of protected amino acid
chlorides to form peptide bond by using tetrahydrofuran at
room temperature.
This encouraged us to extend the procedure to peptide
coupling employing Fmoc-amino acid chlorides. The
Fmoc-amino acid chloride in DCM was added amino acid
ester hydrochloride and anion exchange resin and stirred at
room temperature (Scheme 1). The acylation, as monitored
by TLC and IR, was complete in about 15–30 min. This
was confirmed by the absence of the characteristic
stretching frequency for an acid chloride at around
1,780 cm-1
.
Table 2 Different ion exchange resins were tried during peptide
bond formation
Type of resins
Reaction time (h)
Peptide yield (%)
Amberlyst 21
Amberlyst FPA90RFCl
Amberlyst-A68
Indion 830S
18
24
12
4
70
60
65
90
65
Results and Discussion
Duolite
9
Initially, the deprotonation of hydrochloride salts of ethyl
and methyl esters of amino acids is accomplished using
anion exchange resin. The reaction is neat and quantitative.
Thus, the free amino acid esters (Table 1) have been iso-
lated in good yield and purity. A suspension of amino acid
ester hydrochloride in dichloromethane was stirred with
anion exchange resin. Deprotonation was complete in
*5 min which was revealed by TLC.
O
O
O
R1
THF/ CH2Cl2, IER
HN
HCl.
HN
+
H2N
X
Y
OMe
OMe
X
N
H
R
R
R1
O
X= Fmoc, Boc, Z
Y= F, Cl
Scheme 1 Peptide bond formation
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227
Fig. 1 HPLC chromatograph of Fmoc-Cys(Bzl)-Phe-NH2 synthesized using anion exchange resin
Fig. 2 HPLC chromatograph of Fmoc-Cys(Bzl)-Phe-NH2 synthesized using DIEA
Ion-exchange resin could be used as a scavenger or
quench reagent in peptide bond formation (Scheme 1).
Since the addition of base was avoided, neither oxazol-
5(4H)-one formation nor the premature deblocking of the
Fmoc-group was observed during coupling (as analyzed by
absence of characteristic peaks in IR). The acylation
reactions of acid chlorides in the presence of anion
exchange resin were found to be free from racemization (as
determined by HPLC and 1H NMR of diastereomeric
dipeptides Fmoc-L-Phg-Phe-OMe and Fmoc-D-Phg-Phe-
OMe (Carpino 1988). The method is extended for the
synthesis of peptides containing the two most racemization
prone amino acids such as Cysteine and Histidine. The
HPLC chromatogram (Fig. 1) of the peptide Fmoc-Cys
(Bzl)-Phe-NH2 by using the above conditions suggest that
the racemization levels were reduced to\0.3 %. The same
peptide was synthesized using DIEA as base results in
racemization up to 2–3 % as revealed by HPLC chro-
matogram (Fig. 2).
Due to instability of Boc & Z-amino acid chlorides, the
acid fluorides of Boc- and Z-amino acids were prepared
and used in amide bond formation. Furthermore, the cou-
pling of hindered amino acids, such as a,a-dialkylamino
acids and N-Me amino acids, was also accomplished. As
expected, these couplings are especially difficult when both
the carboxyl and the amino component are a,a-disubsti-
tuted. During the activation of urethane protected Aib,
oxazolone formation is facilitated by the geminal dialkyl
effect. Although number of coupling reagents are able to
generate Aib–Aib peptide bonds in poor to moderate yields
in solution. It is know found that the coupling of Fmoc-
Aib-Cl to Aib-OMe hydrochloride, mediated by anion
exchange resin has been found to be complete in
30–45 min with 85 % yield. The resulting peptide esters
were isolated and characterized as white solids in good
yields. In addition to these, we also extended the coupling
Table 3 Characterization of amino acid dipeptide esters
Thus, the synthesis of several peptides (Table 3) has
been achieved. The yields and purity of the peptides were
good. This illustrates the use of an ion-exchange resin to
purify an amide product without the typical aqueous
extraction. The amino acid ester hydrochloride is reacted
with a slight excess of the amino acid chloride to provide
peptide. After the reaction, a small amount of water was
added to quench any unreacted acid chloride. The car-
boxylic acid and associated HCl is then adsorpted by the
resin, leaving only clean product in solution. In case of
peptides having unreacted amino component the solution
is stirred with a cation exchange resin and filtered. In
reaction that did not contain an anion exchange resin, the
reaction did not go to completion and the peptides were
not isolated.
Peptide
Yield
(%)
MP (°C) [a]D
25c = 1,CHCl3
Fmoc-Val-Ile-OMe
Fmoc-Gly-Phe-OMe
Fmoc-Pro-Pro-OMe
Fmoc-L-Phg-Phe-OMe
Fmoc-D-Phg-Phe-OMe
Boc-Leu-Phe-OMe
Boc-Leu-Ala-OMe
86
85
82
89
88
86
86
138–140 -28.9
131–134 -16.0
118–120 ?40.1
191–192 ?24.0
192–193 -24.0
86–87
115–116 -29.3
97–98 -6.7
-18.6
Z-Glu(OtBu)-Ala-OMe 80
Fmoc-Cys(Bzl)-Phe-
NH2
83
150–151 ?18.1
Fmoc-His(Trt)-Pro-
OMe
80
119–121 -19.6
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dichloromethane (5 ml) and the mixture was stirred at r.t.
After completion of the reaction (monitored by TLC), the
mixture was filtered, the organic layer was stirred with
cation exchange resin and filtered. After the coupling the
CO stretching frequency of acid fluorides at 1,840 cm-1
had disappeared completely. The coupling was clean and
complete in about 10 min. The workup as well as the
isolation of the product was easy.
Table 4 Characterization of a,a-dialkylamino acid containing
peptides
Peptide
Yield (%)
MP (°C)
Fmoc-Aib-Aib-OMe
Boc-Aib-Aib-OMe
Z-Aib-Aib-OMe
86
85
82
89
88
80
78
79
71–72
89–90
107–108
164–165
122–125
Oil
Fmoc-Ac6c-Ac6c-OMe
Fmoc-Deg-Deg-OMe
Fmoc-Sar-Aib-OMe
Boc-Val-Aib-OMe
Boc-Aib-Val-OMe
Racemization Studies
110–11
86–87
Following the general procedure for the synthesis of pep-
tides mediated by IER, mixture of Fmoc-Phg-Cl/Fmoc-
D-Phg-Cl (1 mmol), IER (3 equi) and HCl.H-Phe-OMe
(1.05 mmol) in DCM (10 ml) resulted in Fmoc-Phg-
Phe-OMe (89 %) and Fmoc-DPhg-Phe-OMe (90 %) of the
pure peptide. The HPLC analysis was carried on Waters
analytical HPLC using a Merck RP, C-18, 250 X4.0 mm
column, with detection at 220 nm. The eluent was aceto-
nitrile (50 %) and water (50 %) with a flow rate of 1.0 ml/
min, Rf value for L, L-diastreomer 16.46 min, Rf value for
D, L-diastereomer 17.54 min.
of sterically hindered amino acid fluorides in presence of
anion exchange resin and found that the yields are better
compared to the amino acid chlorides. Thus, the synthesis
of few peptides containing dialkylamino acids have been
achieved (Table 4).
Experimental
General Methods
1
All the peptides were analyzed using H NMR to establish
Conclusion
their structures.
We have demonstrated a novel and facile method for the
synthesis of peptides by using anion exchange resin as
hydrochloride scavenger under mild and neutral conditions.
The acylations of urethane protected amino acid fluorides
can be carried out in presence of anion exchange resin.
Extending the duration of coupling, sterically hindered
amino acids can be incorporated into peptides sequences
without any side reactions. The advantages of the present
method include high yields of the products, simple exper-
imental procedure, the reaction is neat and clean, no tox-
icity of the reagent.
The Fmoc-amino acid chlorides and urethane protected
amino acid fluorides were prepared using the reported
procedures (Carpino et al. 1986; Kaduk et al. 1995). Ure-
thane protected amino acid fluorides were prepared by
using DAST. The formation of acid fluoride was confirmed
by IR at around 1,840 cm-1
.
Typical Procedure
To a solution of amino acid ester hydrochloride salt
(1 mmol) and IER (1 equi) in THF (3 ml) was added a
solution of Fmoc-amino acid chloride (1 mmol) and IER (2
equi) in THF (3 ml) and the reaction mixture stirred for
30 min at r.t. After the completion, of the reaction (mon-
itored by TLC), the mixture was filtered. The organic layer
was stirred with cation exchange resin (1 mmol) for
15 min and filtered. The organic layer was evaporated and
crystallized the residue from suitable solvents gave the
products as white solids.
References
Babu VVS, Ananda K (2000) Synthesis of peptides employing
Fmoc-/Boc-/Z-amino acid fluorides and activated commercial
zinc dust. Lett Pept Sci 7:41–46
Babu VVS, Gopi HN (1998) Rapid and efficient synthesis of peptide
fragments containing a-aminoisobutyric acid using Fmoc-amino
acid chlorides/potassium salt of 1-hydroxybenzotriazole. Tetra-
hedron Lett 39:1049–1050
Coupling of Protected Amino Acid Fluorides
Beyermann M, Bienert M, Carpino LA, Goodman E, Felix A,
Moroder L, Toniolo C (eds) (2002) Houben-Weyl methods of
organic chemistry, synthesis of peptides and peptidomimetics,
vol E 22a. Thieme, New York, pp 475–493
Booth RJ, Hodges JC (1997) Polymer-supported quenching reagents
for parallel purification. J Am Chem Soc 119:4882–4886
To a well stirred solution of an amino acid ester hydro-
chloride salt (1 mmol) and commercial anion exchange
resin (3 equi) in dichloromethane (5 ml) was added a
solution of protected amino acid fluoride in
123

Int J Pept Res Ther (2013) 19:225–229
229
Carpino LA (1988) Methyl ester nonequivalence in the 1H NMR
spectra of diastereomeric dipeptide esters incorporating N-ter-
minal a-phenylglycine units. J Org Chem 53:875–878
Carpino LA, Cohen BJ, Kenton E, Stephen S Jr, Sadat-Aalee SY,
Tien JH, Lengredge DC (1986) (Fluoren-9-ylmethoxy)carbonyl
(Fmoc) amino acid chlorides. Synthesis, characterization, and
application to the rapid synthesis of short peptide segments.
J Org Chem 51:3732–3734
Carpino LA, Chao HG, Beyermann M, Bienert M (1991a) [(9-
Fluorenylmethyl)oxy]carbonyl (Fmoc) amino acid chlorides in
solid-phase peptide synthesis. J Org Chem 56:2635–2642
Carpino LA, Mansour EME, Sadat-Alaee D (1991b) Tert-Butyloxy-
carbonyl and benzyloxycarbonyl amino acid fluorides. New,
stable rapid-acting acylating agents for peptide synthesis. J Org
Chem 56:2611–2614
Kaduk C, Wenschuh H, Beyermann M, Forner K, Carpino LA,
Bienert M (1995) Synthesis of Fmoc-amino acid fluorides via
DAST, an alternative fluorinating agent. Lett Pept Sci 2:285–288
Mark J, Gayo-Fung LM, Moorthy palanki SS, Sullivan R (1998)
Solution-phase parallel synthesis using ion-exchange resins.
Tetrahedron 54:4141–4150
Perlow DS, Erb JM, Gould NP, Tung RD, Freidinger RM, Williams
PD, Veers DF (1992) Use of N-Fmoc amino acid chlorides and
activated 2-(fluorenylmethoxy)-5(4H)-oxazolones in solid-phase
peptide synthesis. Efficient synthesis of highly N-alkylated
cyclic hexapeptide oxytocin antagonists related to L-365,209.
J Org Chem 57:4394–4400
Singh I, Rehni AK, Kalra R, Joshi G, Kumar M, Aboul-Enein HY
(2007) Ion exchange resins: drug delivery and therapeutic
applications. Fabad J Pharm Sci 32:91–100
Carpino LA, Mansour EME, El-Faham A (1993) Bis(BOC) amino
acid fluorides as reactive peptide coupling reagents. J Org Chem
58:4162–4164
Sivanandaiah KM, Babu VVS, Renukeshwar HC (1992) Fmoc-amino
acid chlorides in solid phase synthesis of opioid peptides. Int J
Pept Protein Res 39:201–206
Carpino LA, Beyermann M, Wensehuh H, Bienert M (1996) Peptide
synthesis via amino acid halides. Acc Chem Res 29:268–274
Cho DH, Jang DO (2004) Indium-mediated mild and facile method
for the synthesis of amides. Tetrahedron Lett 45:2285–2287
David PE (2005) Pharmaceutical applications of ion-exchange resins.
J Chem Educ 82:575–587
Flynn DL, Crich JZ, Devraj RV, Hockerman SL, Parlow JJ, South
MS, Woodward S (1997) Chemical library purification strategies
based on principles of complementary molecular reactivity and
molecular recognition. J Am Chem Soc 119:4874–4881
Gayo L, Suto MJ (1997) Ion-exchange resins for solution phase
parallel synthesis of chemical libraries. Tetrahedron Lett
38:513–516
Gopi HN, Suresh Babu VV (1998) Synthesis of peptides employing
Fmoc-amino acid chlorides and commercial zinc dust. Tetrahe-
dron Lett 39:9769–9772
Hesham AE, Allen A, Gary IF (2010) Synthesis and properties of ion
exchangers derived from chloromethyl styrene codivinylbenzene
and their use in water treatment. Int J Polym Sci 2010:1–9
Hruby VJ (2001) Design in topographical space of peptide and
Sivanandaiah KM, Babu VVS, Gangadhar BP (1994a) Synthesis of
peptides mediated by KOBt Indian. J Chem 33B:620–624
Sivanandaiah KM, Babu VVS, Shankaramma SC (1994b) Synthesis
of peptides mediated by KOBt. Int J Pept Protein Res 44:24–30
Tantry SJ, Suresh Babu VV (2002) 1-(t-butyldimethylsilyloxy)
benzotriazole (TBDMS-OBt): a new and novel reagent for the
synthesis of peptides. Lett Pept Sci 9:35–41
Wenschuh H, Beyermann M, Krause E, Brudel M, Winter R,
Schumann M, Carpino LA, Binert M (1994) Fmoc amino acid
fluorides: convenient reagents for the solid-phase assembly of
peptides incorporating sterically hindered residues. J Org Chem
59:3275–3280
Wenschuh H, Beyermann M, Haber H, Seydel JK, Krause E, Binert
M, Carpino LA, El-Faham A, Albericio F (1995) Stepwise
automated solid phase synthesis of naturally occurring peptaibols
using FMOC amino acid fluorides. J Org Chem 60:405–410
Williams PD, Bock MG, Tung RD, Garsky VM, Perlow DS, Erb JM,
Lundell GF, Gould NP, Whitter WL, Hoffman JB, Kaufman MJ,
Clineschmidt BV, Pettibone DJ, Freidinger RM, Verber DF
(1992) Development of a novel class of cyclic hexapeptide
oxytocin antagonists based on a natural product. J Med Chem
35:3905–3918
peptidomimetic ligands that affect behavior.
A chemist’s
glimpse at the mind-body problem. Acc Chem Res 34:389–397
Humphrey JM, Chamberlin AR (1997) Chemical synthesis of natural
product peptides: coupling methods for the incorporation of
noncoded amino acids into peptides. Chem Rev 97:2243–2266
123