Microwave-assisted solution phase peptide synthesis in neat water

Article abstract of DOI:10.1039/c3ra43040e

An environmentally benign protocol for solution phase peptide synthesis has been developed in neat water using TBTU/HOBt/DIEA as a coupling combination under microwave irradiation. Key features of this procedure are the replacement of commonly used toxic organic solvents like DMF and NMP, the use of lower amounts of reactants, compatibility with both N-α-Boc- and N-α-Fmoc-protected amino acids and all commonly used side-chain protective groups, short reaction time, and racemization-free synthesis in high yield and purity. The Royal Society of Chemistry 2013.

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Microwave-assisted solution phase peptide synthesis in neat water
Amit Mahindra, Karthik Nooney, Shrikant Uraon, Krishna K. Sharma, Rahul Jain*a
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
DOI: 10.1039/b000000x
5
An environmentally benign protocol for the solution phase peptide synthesis has been developed in neat
water using TBTU/HOBt/DIEA as a coupling combination under microwave irradiation. Key features of
this procedure are the replacement of commonly used toxic organic solvents like DMF and NMP, use of
less amount of reactants, compatibility with both NꢀαꢀBocꢀ and NꢀαꢀFmocꢀprotected amino acids and all
commonly used sideꢀchain protective groups, short reaction times, and racemizationꢀfree synthesis in
10 high yield and purity.
desired.
Introduction
The application of ecoꢀfriendly methodologies in peptide
synthesis is not well explored. The first SPPS in water (H2O) was
50 reported by Kawasaki et. al.21 The method used a PEGꢀbased
The condensation of two amino acids to form a peptide bond is
considered as one of the most important reactions for sustaining
life. Peptides play an important role in medicinal chemistry
15 leading to discovery of a number of drugs.1 Over the last few
decades, significant research progress is achieved in identification
of peptides having important therapeutic effects.2ꢀ7 In general,
most of the drugs show bioactivity by binding with the
proteincious receptors or enzymes. Therefore, for significant
20 interactions with receptors or enzymes, ligands, which are
proteincious or peptidic in nature are ideal. For example, some of
the peptides that are known to have specific receptorꢀbased
activity includes, bradykinin,8 gonodotropinꢀreleasing hormone
(GnRH),9 dermorphin,10 enkephalins,11 bombesin,12 and
25 thrombin.13
resin,
a
waterꢀsoluble amino protecting group, and
EDCꢁHCl/HONB as the coupling combination. Grøtli et al.
synthesized peptides in H2O using Bocꢀprotected amino acids,
EDCꢁHCl/HONB and PEGꢀbased resin under microwave (MW)
55 irradiation.22a In both methods, aqueous 0.2% Triton X solution
was added during the reaction to increase the swelling of resin
and solubility of amino acids. More recently, Collins reported
SPPS using amino acids bearing α,βꢀunsaturated sulfoneꢀbased
αꢀamino protective groups in a solvent combination of H2Oꢀ
60 ethanol (EtOH) under microwave irradiation.22b None of these
SPPS methods used neat water as the reaction medium and
appeared limited in terms of exclusive amino protecting groups
and expensive solid supports.
Peptides were synthesized using solution phase protocol till the
discovery of solid phase peptide synthesis (SPPS) by Bruce R.
Merrifield in 1963.14 The SPPS offers several advantages over
solution phase peptide synthesis that include, easy isolation and
30 purification of large peptides. In SPPS, a library of peptides can
be generated in short span of time, but synthesis requires 3ꢀ8 fold
excess of amino acids and coupling reagents, in addition to
expensive and nonꢀreusable solid supports. The solution phase
peptide synthesis requires 1.2ꢀ2.0 fold excess of the amino acids
35 and coupling reagents, thereby offering significant advantage in
the synthesis of short peptides, specifically those containing
expensive and difficult to access modified/synthetic amino acids.
The method could be equally useful for large peptides if synthesis
is achieved using segment or chemical ligation approaches.15 The
40 Fig 1 displays structures of some short biologically active
peptides.16ꢀ20 Both solid phase and solution phase peptide
syntheses generates a large volume of toxic waste, including a
large amount of toxic organic solvents like N,Nꢀ
dimethylformamide (DMF), 1ꢀmethylꢀ2ꢀpyrrolidone (NMP) and
45 dichloromethane (CH2Cl2). Therefore, discovery of alternate
environmentally friendly methods of peptide synthesis are
65 Fig. 1 Short biologically active peptides
Solution phase peptide synthesis has been performed in various
solvents, but the application of H2O as a reaction medium is not
yet explored. Over the past few years water as a environmentally
benign solvent has grabbed considerable interest.23 Water as a
70 solvent offers many advantages because it is abundant, nonꢀtoxic,
nonꢀflammable, and reduces the organic solvent waste in
chemical industry.24 Along with H2O, the MW is another rapid
and greener alternative compared to conventional heating.25 The
MW irradiation is much widely used in SPPS,26 but has limited
75 applicability in solution phase peptide synthesis.27 More recently,
we reported first generalized protocol for solution phase peptide
synthesis under MW irradiation.28 In some recent reports,
formation of amide bond was also achieved using liquidꢀassisted
and solventꢀfree ballꢀmilling.29 In our quest to discover ecoꢀ
80 friendly peptide synthesis protocols, herein, we report solution
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phase peptide synthesis in neat H2O under MW irradiation. To 40 (nꢀBuOH), EtOH and isopropyl alcohol (IPA), and the results are
the best of our knowledge, this is the first report on the synthesis
of peptides in solution phase using neat H2O as a reaction
medium. The reaction employs commercially available Bocꢀ and
Fmocꢀprotected amino acids with commonly used coupling
summarized in Table 1. The preliminary results indicate that the
H2O (entry no. 4) and TBME (entry no. 6) were comparable in
terms of yield and purity. Also it was evident from the entry nos.
1 and 3 that the commonly used but toxic organic solvents (DMF
5
reagents for the formation of peptide bond. This protocol offers 45 and NMP) gave lesser yields compared to H2O and TBME, which
several benefits over the existing solution and solid phase peptide
synthesis methodologies: (a) use of neat H2O as the reaction
medium, (b) full compatibility with both Bocꢀ and Fmocꢀ
are considered greener alternative according to the GSK’s solvent
selection guide.30 According to GSK’s guide, the solvents are
ranked on the basis of their waste, flammability, environmental,
health and safety issues. Solvents such as DMF, NMP and
10 protected amino acids, (c) no need for the special protection on
the reactive sideꢀchain of amino acids, (d) short reaction time, 50 CH3CN come under the category of highly toxic and must be
and (e) and racemizationꢀfree peptide synthesis in high yield and
purity.
avoided. While H2O, TBME, EtOAc, IPA, 2ꢀMeꢀTHF, and 1ꢀ
butanol are considered to be the greener replacements of the toxic
organic solvents.
Results and discussion
Table 1 Effect of various solvents on the yield and purity of BocꢀPheꢀIleꢀ
55 OMe (2a)a
15 To establish the protocol, we first carried out a model reaction
with BocꢀPheꢀOH (1a) and IleꢀOMeꢁ2HCl (1b) using TBTU as a
coupling reagent and HOBt as an auxiliary nucleophile in DMF
as solvent (Scheme 1). The selection of the coupling reagent and
auxiliary nucleophile was based upon their easy access, low cost
20 and wide application in peptide synthesis.
Entry
Solvent
Yield
Purity
1
2
3
4
5
6
7
8
N,NꢀDimethylformamide (DMF)
Acetonitrile (CH3CN)
1ꢀMethylꢀ2ꢀpyrrolidone (NMP)
Water (H2O)
86
87
84
90
85
88
79
65
52
68
97
97
96
98
96
97
95
98
98
95
Ethyl acetate (EtOAc)
tertꢀButyl methyl ether (TBME)
2ꢀMethyltetrahydrofuran (2ꢀMeꢀTHF)
1ꢀButanol (nꢀBuOH)
Ethanol (EtOH)
Isopropyl alcohol (IPA)
9
10
Scheme 1 Generalized scheme for coupling reaction
Reaction conditions: solvent (2.5 mL), 1a (1.2 equiv), 1b (1.0 equiv),
TBTU (1.2 equiv), HOBt (1.2 equiv), DIEA (2 equiv) 60 ˚C, 30 min,
MW.
Optimization of the protocol
Thereafter, we studied the influence of temperature and time on
25 the formation of amide bond under MW irradiation. The graph in
Fig. 2 shows monitoring of the coupling of (1a) with (1b) under
varied sets of temperature and time. We observed that a
temperature of 60 ˚C and reaction time of 30 min provides the
best coupling conditions and gave 86% isolated yield of BocꢀPheꢀ
30 IleꢀOMe (2a). The HPLC analysis indicated 97% purity for 2a,
confirming the effectiveness of protocol.
aIsolated yield.
60
With these initial results in hand, H2O and TBME appeared to be
the promising greener replacement to the commonly used toxic
organic solvents in solution phase peptide synthesis. To
determine the most suitable coupling reagents, we explored the
65 peptide bond formation under MW irradiation by screening
various coupling reagent and auxiliary nucleophile combinations.
The results of the study are shown in Fig. 3. As evident from the
bar graphs, a coupling combination of TBTU/HOBt gave best
results when H2O was used as solvent. While, DIC/HONB
70 emerged as the best coupling reagents combination when TBME
was used as the solvent for the reaction.
Fig. 2 Yield of peptide 2a as a function of time and temperature with
TBTU/HOBt/DIEA under MW irradiation
35 The next step was to investigate the applicability of various
solvents under the optimized conditions. The solvent screened
during the optimization process included, DMF, acetonitrile
(CH3CN), NMP, H2O, ethyl acetate (EtOAc), tert-butyl methyl
ether (TBME), 2ꢀmethyltetrahydrofuran (2ꢀMeꢀTHF), 1ꢀbutanol
Fig. 3 A comparison of yield of 2a in H2O and TBME in various coupling
reagents
75 Finally, the scope and limitation of inꢀwater peptide synthesis
2
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was further explored by synthesizing a number of peptide
sequences in both TBME and H2O (Fig. 4). We observed that the
yield of Bocꢀprotected peptides was higher in H2O. However,
The use of TBME is limited to the dipeptides synthesis, because
of solubility problem of higher peptides in the reaction medium.
In nutshell, experiments conducted indicate that neat H2O can be
slightly higher yields were observed when TBME was used in 10 preferred as a medium of choice for solution phase peptide
5
cases involving Fmocꢀprotected amino acids, which is in
agreement with a substantial increase in their hydrophobicity.
synthesis under microwave irradiation.
Fig. 5 Progress of reaction: (a) beginning of reaction (b) end of reaction
20 With the optimized conditions in hand, the generality of the MWꢀ
assisted solution phase peptide in neat H2O was explored. As
shown in Fig. 5a, all reactants are partially soluble in the
beginning of the reaction and the solution becomes clear on the
exposure of MW irradiation at the end of reaction (Fig. 5b). We
25 then synthesized a wideꢀvariety of structurally diverse dipeptides,
tripeptides and tetrapeptides consisting of Fmocꢀ and Bocꢀ
protected amino acids. The method was also extended to include
amino acids without protection on the reactive sideꢀchain and
results were summarized in the Table 2. In all cases, excellent
30 yields were obtained irrespective of the orthogonality of the αꢀ
amino and sideꢀchain protecting groups. We have scaledꢀup the
method by synthesizing peptides (≤ 1.0 g) without any difficulty.
Fig. 4 Reaction conditions: (a) H2O (2.5 mL), 1a (1.2 equiv), 1b (1.0
15 equiv), TBTU (1.2 equiv), HOBt (1.2 equiv), DIEA (2 equiv), 60 ˚C, 30
min, MW; (b) TBME (2.5 mL), 1a (1.2 equiv), 1b (1.0 equiv), DIC (1.2
equiv), HONB (1.2 equiv), DIEA (2 equiv), 60 ˚C, 30 min, MW.
Table 2 MWꢀassisted solution phase peptide synthesis in water
35
Entry
AA1
AA2
Product
Sequence
Yield
Purity
1
2
3
4
5
6
7
8
BocꢀPheꢀOH
BocꢀValꢀOH
BocꢀPheꢀOH
BocꢀTrpꢀOH
BocꢀMetꢀOH
BocꢀSer(Bzl)ꢀOH
BocꢀTrpꢀOH
BocꢀHisꢀOH
BocꢀHis(Bom)ꢀOH
BocꢀArgꢀOH
BocꢀHis(Bzl)ꢀOH
FmocꢀPheꢀOH
FmocꢀSer(t-butyl)ꢀOH
BocꢀArg(Mtr)ꢀOH
BocꢀValꢀOH
H2NꢀIleꢀOMe
H2NꢀIleꢀOMe
H2NꢀHisꢀOMe
H2NꢀIleꢀOMe
H2NꢀIleꢀOMe
H2NꢀIleꢀOMe
H2NꢀHisꢀOMe
H2NꢀHisꢀOMe
H2NꢀArgꢀOMe
H2NꢀArgꢀOMe
H2NꢀHisꢀOMe
H2NꢀIleꢀOMe
H2NꢀIleꢀOMe
H2NꢀIleꢀOMe
H2NꢀPheꢀIleꢀOMe
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
3a
BocꢀPheꢀIleꢀOMe
BocꢀValꢀIleꢀOMe
BocꢀPheꢀHisꢀOMe
BocꢀTrpꢀIleꢀOMe
BocꢀMetꢀIleꢀOMe
BocꢀSer(Bzl)ꢀIleꢀOMe
BocꢀTrpꢀHisꢀOMe
90
85
82
75
75
75
82
63
69
42
75
71
60
74
86
98
96
97
96
95
99
100
100
99
97
98
98
99
94
94
BocꢀHisꢀHisꢀOMe
9
BocꢀHis(Bom)ꢀArgꢀOMe
BocꢀArgꢀArgꢀOMe
BocꢀHis(Bzl)ꢀHisꢀOMe
FmocꢀPheꢀIleꢀOMe
FmocꢀSer(tꢀbutyl)ꢀIleꢀOMe
BocꢀArg(Mtr)ꢀIleꢀOMe
BocꢀValꢀPheꢀIleꢀOMe
10
11
12
13
14
15
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Table 2 (Contd.)
Entry AA1
AA2
Product
Sequence
Yield
Purityb
16
17
18
19
20
21
22
23
24
25
26
27
28
BocꢀIleꢀOH
H2NꢀPheꢀIleꢀOMe
H2NꢀPheꢀIleꢀOMe
H2NꢀTrpꢀIleꢀOMe
H2NꢀPheꢀIleꢀOMe
H2NꢀSer(Bzl)ꢀIleꢀOMe
H2NꢀPheꢀIleꢀOMe
H2NꢀPheꢀIleꢀOMe
3b
3c
3d
3e
3f
3g
3h
3i
4a
4b
4c
4d
5a
BocꢀIleꢀPheꢀIleꢀOMe
Boc AbuꢀPheꢀIleꢀOMe
BocꢀPhgꢀTrpꢀIleꢀOMe
BocꢀTrpꢀPheꢀIleꢀOMe
BocꢀIleꢀSer(Bzl)ꢀIleꢀOMe
FmocꢀAbuꢀPheꢀIleꢀOMe
FmocꢀLeuꢀPheꢀIleꢀOMe
FmocꢀCys(tꢀbutyl)ꢀSer(t-butyl)ꢀIleꢀOMe
BocꢀPheꢀIleꢀPheꢀIleꢀOMe
BocꢀGlu(OBzl)ꢀPhgꢀTrpꢀIleꢀOMe
80
83
65
58
75
75
83
72
71
68
95
98
95
96
94
95
98
97
99
96
97
98
99
BocꢀAbuꢀOH
BocꢀPhgꢀOH
BocꢀTrpꢀOH
BocꢀIleꢀOH
FmocꢀAbuꢀOH
FmocꢀLeuꢀOH
FmocꢀCys(tꢀbutyl)ꢀOH H2NꢀSer(tꢀbutyl)ꢀIleꢀOMe
BocꢀPheꢀOH
BocꢀGlu(OBzl)ꢀOH
BocꢀAla(2ꢀnaphthyl)ꢀOH H2NꢀTyr(Bzl)ꢀPheꢀIleꢀOMe
H2NꢀIleꢀPheꢀIleꢀOMe
H2NꢀPhgꢀTrpꢀIleꢀOMe
BocꢀAla(2ꢀnaphthyl)ꢀTyr(Bzl)ꢀPheꢀIleꢀOMe 74
FmocꢀPheꢀAbuꢀPheꢀIleꢀOMe
BocꢀβꢀAlaꢀTrpꢀMetꢀAspꢀPheꢀNH2
FmocꢀPheꢀOH
BocꢀβꢀAlaꢀOH
H2NꢀAbuꢀPheꢀIleꢀOMe
H2NꢀTrpꢀMetꢀAsp(Bzl)ꢀPheꢀNH2
65
57
a Reaction conditions: AA1 (1.2 mmol), AA2 (1.0 mmol), DIEA (2 mmol), TBTU (1.2 mmol), HOBt (1.2 mmol), H2O (2.5 mL). bPurity was determined by
HPLC analysis.
5
Lastly, the MWꢀassisted inꢀwater peptide synthesis procedure
was applied to synthesize pentagastrin (5a). Pentagastrin is a
synthetic bioactive pentapeptide having the core active segment
of gastrin.31 The bioactive peptide stimulates the secretion of
10 gastric acid, pepsin, and intrinsic factor, and is used as a
diagnostic aid as the pentagastrinꢀstimulated calcitonin test. The
seven steps total synthesis of pentagastrin (5a) in neat H2O under
MW irradiation is given in Scheme 2. The peptide was
synthesized using NꢀαꢀBocꢀprotected amino acids in 42% overall
15 yield. The HPLC chromatogram of the synthetic peptide is shown
in Fig. 6. It can be safely concluded that peptide is synthesized in
racemizationꢀfree high yield.
25 Scheme 2 Synthetic scheme for pentagastrin (5a)
Conclusion
In conclusion, we report the first MWꢀassisted solution phase
peptide synthesis in neat H2O. This rapid and environmental
benign protocol for the preparation of racemizationꢀfree peptides
30 is equally compatible with Fmocꢀ and Bocꢀprotected amino acids,
and completely eliminates the use of toxic organic solvents in
peptide synthesis. The protocol proceeds smoothly under very
mild reaction conditions in short reaction times, is cost effective
(1.2ꢀfold excess of reactants are used), and has compatibility with
20 Fig. 6 HPLC chromatogram of pentagastrin (5a). Method: Cꢀ18, 300 Å, 5
ꢂm, 250×4.6 mm column, run for 60 min with a flow of 1 mL/min, using
a gradient of 95–5%, where buffer A was 0.1% TFA in H2O and buffer B
was 0.1% TFA in CH3CN and detection at 220 nm
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a wideꢀvariety of sideꢀchain protecting groups. Owing to the great
diversity in amino acids, this protocol would be extremely useful
in the synthesis of libraries of bioactive peptides.
(neat) υ 3308.44, 2968.41, 2929.20, 2879.44, 1752.45, 1683,
1646.03, 1535.98, 1437.33, 1391.70, 1366.80, 1173.53, 1017.9,
60 989.31, 860.33, 716.14, 701; HRMS (ESIꢀTOF) calculated for
[M+H+] 393.2389, found 393.2384; HPLC: tR= 47.80 min, 98%.
Spectral data of Boc-Val-Phe-Ile-OMe (3a): 1H NMR (400
MHz, CD3OD): δ = 7.27ꢀ7.15 (m, 5H), 4.76 (t, J = 7.0 Hz, 1H),
4.35 (d, J = 6.3 Hz, 1H), 3.89 (d, J = 6.5 Hz, 1H), 3.65 (s, 3H),
65 3.06 (dd, J = 6.5, 13.6 Hz, 1H), 2.90 (dd, J = 8.2, 13.7 Hz, 1H),
1.97ꢀ1.89 (m, 1H), 1.86ꢀ1.78 (m, 1H), 1.41 (s, 9H), 1.30ꢀ1.26 (m,
1H), 1.23ꢀ1.14 (m, 1H), 0.89ꢀ0.83 (m, 12H); 13C NMR (100
MHz, CD3OD): δ = 172.7, 171.8, 170.3, 156.4, 136.7, 129.1,
128.0, 126.3, 79.1, 60.0, 56.8, 56.7, 54.1, 37.7, 29.3, 27.4, 24.8,
70 18.4, 17.2, 14.5, 10.3; IR (neat): υ 3277, 2966, 2414, 1648, 1527,
1458, 1366, 1246, 1175, 1017, 928, 878, 741, 697; HRMS (ESIꢀ
TOF): calculated for [M+H+] 492.3073; found 492.3065; HPLC:
tR= 46.29 min, 95.78%.
Experimental Section
5
General experimental procedure for dipeptides (2a-n): In a 10
mL MW vial equipped with a magnetic stir bar, amino acid (AA2)
methyl esterꢁxHCl (1 mmol) and DIEA (2 mmol) was added H2O
(2.5 mL). Fmoc/BocꢀAA1ꢀOH (1.2 mmol) was added, followed
by TBTU (1.2 mmol) and HOBt (1.2 mmol). The reaction
10 mixture was subjected to MW irradiation (CEM Discover®
microwave reactor) with gas cooling (pressure of 40 psi was
maintained during irradiation) for 30 min at 40W with magnetic
stirring, and a temperature limit of 60 ˚C (reaction time refers to
the hold time at the desired set temperature). H2O was evaporated
15 and the reaction mixture was purified on automated flash column
chromatography system (Biotage®) to give Fmoc/BocꢀAA1ꢀAA2ꢀ
OMe.
75 Abbreviations: DIC, 1,3ꢀdiisopropylcarbodiimide; HONB, Nꢀ
hydroxyꢀ5ꢀnorborneneꢀendoꢀ2,3ꢀdicarboximide; Bom, benzylꢀ
oxymethyl; Bzl, benzyl; Mtr, 4ꢀmethoxyꢀ2,3,6ꢀtrimethylꢀbenꢀ
zenesulfonyl; TBTU, 2ꢀ(1Hꢀbenzotriazolꢀ1ꢀyl)ꢀ1,1,3,3ꢀtetraꢀ
methyluronium tetrafluoroborate; HOBt, 1ꢀhydroxyꢀbenzoꢀ
80 triazole; PyBOP, benzotriazolꢀ1ꢀylꢀoxytripyrrolidino phosphꢀ
onium hexafluorophosphate; HATU, 2ꢀ(1Hꢀ7ꢀazabenzotriazolꢀ1ꢀ
yl)ꢀ1,1,3,3ꢀtetramethyluronium hexafluorophosphate; HCTU, 2ꢀ
(6ꢀchloroꢀ1Hꢀbenzotriazoleꢀ1ꢀyl)ꢀ1,1,3,3ꢀtetramethylaminium
hexafluorophosphate; HOAt, 1ꢀhydroxyꢀ7ꢀazabenzotriazole;
General experimental procedure for N-α-Boc-protected
peptides (3a-f, 4a-c): In a 10 mL MW vial equipped with a
20 magnetic stir bar, BocꢀAA1ꢀAA2ꢀOMe (1 mmol) was reacted with
6N HCl (5 mL) at 25 ˚C for 15 min. Dihydrochloride salt of
dipeptide was neutralized with DIEA (2 mmol). The resulting
AA1ꢀAA2ꢀOMe (1 mmol) was dissolved in H2O (2.5 mL). Bocꢀ
AA3ꢀOH (1.2 mmol) was added followed by TBTU (1.2 mmol)
25 and HOBt (1.2 mmol). Mixture was subjected to MW irradiation
(CEM Discover® microwave reactor) with gas cooling (pressure
of 40 psi was maintained during irradiation) for 30 min at 40W
with magnetic stirring, and a temperature limit of 60 ˚C (reaction
time refers to the hold time at the desired set temperature). H2O
30 was evaporated and the reaction mixture was purified on
automated flash column chromatography system (Biotage®) to
afford tripeptides (3a-f). The deprotection and coupling cycle
described above was repeated to obtain tetrapeptides (4a-c).
General experimental procedure for N-α-Fmoc-protected
35 peptides (3g-i, 4d): In a 10 mL MW vial equipped with a
magnetic stir bar, FmocꢀAA1ꢀAA2ꢀOMe (1 mmol) was reacted
with 20% piperidine (2 mL) at 25 ˚C for 10 min followed by
removal of excess piperidine. The resulting AA1ꢀAA2ꢀOMe (1
mmol) was dissolved in H2O (2.5 mL). FmocꢀAA3ꢀOH (1.2
40 mmol) was added followed by TBTU (1.2 mmol), HOBt (1.2
mmol), and DIEA (2 mmol). Mixture was subjected to MW
irradiation (CEM Discover® microwave reactor) with gas cooling
(pressure of 40 psi was maintained during irradiation) for 30 min
at 40W with magnetic stirring, and a temperature limit of 60 ˚C
45 (reaction time refers to the hold time at the desired set
temperature). H2O was evaporated and the reaction mixture was
purified on automated flash column chromatography system
(Biotage®) to afford tripeptide (3g-i). The deprotection and
coupling cycle described above was repeated to obtain
50 tetrapeptide (4d).
85 EDC.HCl,
1ꢀethylꢀ3ꢀ(3ꢀdimethylaminopropyl)carbodiimide
hydrochloride; CDI, 1,1’carbonyldiimidazole; PEG, polyethylene
glycol; DIEA, N,Nꢀdiisopropylethylamine.
Acknowledgment
Amit Mahindra thanks the Council of Scientific and Industrial
90 Research (CSIR), New Delhi for the award of Senior Research
Fellowship.
Notes and references
*Department of Medicinal Chemistry, National Institute of
Pharmaceutical Education and Research, Sector 67, S. A. S. Nagar,
95 Punjab 160 062, India Corresponding author. Tel.: +91 (172) 2292024;
Fax: +91 (172) 2214692; E-mail: rahuljain@niper.ac.in
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Spectral data of Boc-Phe-Ile-OMe (2a): H NMR (400 MHz,
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Microwave-assisted solution phase peptide synthesis in neat water
Amit Mahindra, Karthik Nooney, Shrikant Uraon, Krishna K. Sharma, Rahul Jain*a
Text: An environmentally benign protocol for peptide synthesis is reported in neat
water using TBTU/HOBt/DIEA under microwave irradiation.