The greening of peptide synthesis

Article abstract of DOI:10.1039/c7gc00247e

The synthesis of peptides by amide bond formation between suitably protected amino acids is a fundamental part of the drug discovery process. However, the required coupling and deprotection reactions are routinely carried out in dichloromethane and DMF, b

Full text of DOI:10.1039/c7gc00247e

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The Peptide Synthesis
Stefan B. Lawrenson a, Roy Arava and Michael Northa*
Received 00th January 20xx,
Accepted 00th January 20xx
The synthesis of peptides by amide bond formation between suitably protected amino acids is a fundamental part of the
drug discovery process. However, the required coupling and deprotection reactions are routinely carried out in
dichloromethane and DMF, both of which have serious toxicity concerns and generate waste solvent which constitutes the
vast majority of the waste generated during peptide synthesis. In this work, propylene carbonate has been shown to be a
green polar aprotic solvent which can be used to replace dichloromethane and DMF in both solution- and solid-phase
peptide synthesis. Solution-phase chemistry was carried out with Boc/benzyl protecting groups to the tetrapeptide stage,
no epimerisation occurred during these syntheses and chemical yields for both coupling and deprotection reactions in
propylene carbonate were at least comparable to those obtained in conventional solvents. Solid-phase peptide synthesis
was carried out using Fmoc protected amino acids on a ChemMatrix resin and was used to prepare the biologically
relevant nonapeptide Bradykinin with comparable purity to a sample prepared in DMF.
DOI: 10.1039/x0xx00000x
www.rsc.org/
NMP is a particular concern as these solvents are known to be
reprotoxic,10 cause environmental problems when present in
waste11 and are two of the six chemicals of highest concern
Introduction
Peptides are central compounds in the pharmaceutical
industry, providing both final medications and lead
compounds for the preparation of peptidomimetic or non-
peptidic pharmacauticals.1 Therapeutic peptides are
recognised as being extremely specific in their binding to in
vivo targets, resulting in them often being highly potent and
very selective, whilst having very few negative side effects.2,3
They are therefore expected to play a major role in the future
of drug discovery.4 Currently there are more than 60 peptide
based medicines approved for use by the US Food & Drug
Administration, around 140 peptide drugs currently in clinical
trials and over 500 in preclinical trials.2,5 Consequently, the
peptide medicines market is currently worth an estimated US$
14.1 billion, and is predicted to grow to around US$ 25.4 billion
by 2018.2 As such, there is a resurgence of interest in the
synthesis of synthetic peptides.
under EU REACh (Registration, Evaluation and Authorisation of
Chemicals) regulations.12 As a result, green solvent selection
guides have repeatedly highlighted the need to replace DMF
with a greener alternative in all chemical processes.13,14
As for most chemical transformations,11 by far the largest
source of waste in either solution- or solid-phase peptide
synthesis is the solvent, but only a few attempts have
previously been made to avoid the use of DMF.
Acetonitrile15,16 and THF16 have been used as replacements for
DMF in peptide bond formations and acetonitrile has also
been used as a solvent for Fmoc deprotections in solid-phase
peptide synthesis,15 but neither of these are considered to be
green solvents.13,14 Recently, the use of 2-MeTHF as a solvent
for coupling reactions during solid-phase peptide synthesis
was reported though the Fmoc deprotection steps were still
performed in DMF.17 2-MeTHF is a sustainable but not very
green solvent due to concerns about its effect on health,
safety and the environment.13,14 Whilst this paper was being
written, the use of a mixture of 2-MeTHF and ethyl acetate for
both coupling and Fmoc deprotection steps was reported.18
However, in some cases elevated temperatures were required
and the production of mixed solvent waste is undesirable as it
complicates solvent recycling especially when the solvents
have virtually identical boiling points (77 and 78 oC for ethyl
acetate and 2-MeTHF respectively). There are also a few
reports of use of water as a solvent for solid-phase peptide
bond formations using coupling agents19 or enzymes20 and of
solvent free non-resin supported peptide synthesis by ball-
milling.21 However, the development of a green solvent for
both solution- and solid-phase peptide synthesis remains an
unsolved challenge.
The synthesis of peptides by amide bond formation
between partially protected amino acid derivatives is however,
one of the most wasteful and least green chemical processes.6
Numerous auxiliary reagents in the form of protecting groups
and coupling agents are required,7 giving the process low atom
efficiency. Additionally, peptide synthesis is almost universally
carried out in toxic solvents such as DMF or
DMF/dichloromethane mixtures.8 These issues are amplified in
solid-phase peptide synthesis8,9 where large excesses of
reagents are used and resins are washed multiple times with
toxic solvents. The use of polar aprotic solvents such as DMF or
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Table 1: Physical properties of polar aprotic solvents
Solvent
DMF
Bp (Mp) o
153
C
Dipole moment (D)
Dielectric constant
3.8
4.1
3.4
3.9
4.9
4.9
38
32
38
47
65
90
NMP
204
MeCN
DMSO
PC 1
82
189
242
EC 2
248 (36)
Scheme 2: Synthesis of dipeptides 5-6a,b in PC.
Table 2: Synthesis of dipeptide 5aa
Scheme 1: Synthesis of cyclic carbonates.
Cyclic carbonates, especially propylene carbonate (PC)
1
Entry
Coupling agent / additive
EDC / HOAt
DCC / PFP
T (oC)
20
Base
Solvent
CH2Cl2:DMF
CH2Cl2:DMF
PC
Yield (%)
8634
76
and ethylene carbonate (EC) 2 are remarkably polar aprotic
1
2
3
4
5
6
7
8
9
NaHCO3
Et3N
compounds with dipole moments and dielectric constants
higher than those of conventional polar aprotic solvents22
(Table 1). Unlike conventional polar aprotic compounds, they
contain only carbon, hydrogen and oxygen and so generate no
NOx or SOx on incineration and PC is known to be non-toxic
and is used in topically applied medical products23 and
cosmetics.24 PC is also less expensive than DMF25 and unlike
DMF is not significantly hygroscopic.26 Cyclic carbonates,
including PC and EC, can be prepared by the 100% atom
economical reaction between epoxides and carbon dioxide
20
DCC / PFP
20
Et3N
83
DCC / PFP
40
Et3N
EC
96
EDC / HOBt
EDC / HOBt
EDC / HOBt
EDC / HOBt
EDC / HOBt
20
iPr2EtN
iPr2EtN
iPr2EtN
iPr2EtN
iPr2EtN
PC
93
20
CH2Cl2:DMF
DMF
81
20
83
70
DMF
78
70
PC
82
a) aEDC
=
N-Ethyl-N-(3-dimethylaminopropyl)carbodiimide; DCC
1-Hydroxy-7-azabenzotriazole; HOBt
Hydroxybenzotriazole; PFP = Pentafluorophenol.
= N,N-
(Scheme 1),27
a reaction which is currently attracting
Dicyclohexylcarbodiimide; HOAt
=
=
considerable attention due to its ability to utilize waste carbon
dioxide in chemical synthesis.28 Cyclic carbonates including PC
and EC are already produced commercially29 and have a
number of applications, including as electrolytes in lithium ion
batteries.30 PC was ranked as the greenest solvent in the latest
green solvents guide14 and has previously been used as a
solvent for cycloaddition reactions31 and for metal
(entry 4) gave a significantly higher yield of 5a than that
obtained in the conventional solvent system. However, whilst
other reactions have been successfully carried out in EC at
ambient temperature,33 this peptide coupling had to be carried
out at 40 oC to keep the reaction mixture liquid. Therefore, the
coupling reaction in PC was further optimised (entry 5) and
this allowed the literature yield of dipeptide 5a to be
exceeded. To allow a direct comparison between PC and
conventional solvent systems to be made, the use of EDC,
HOBt and diisopropylethylamine in a dichloromethane and
DMF mixed solvent and in just DMF was also carried out
(entries 6 and 7). In both cases the yield of peptide 5a was
inferior to that obtained in PC.
catalysed24,32
and
organocatalysed33
transformations.
Therefore we decided to investigate the use of PC and EC as
solvents for both coupling and deprotection steps in both
solution- and solid-phase peptide synthesis using a range of
common protecting groups and coupling agents.
Results and Discussion
It is becoming increasingly common to carry out peptide
synthesis at elevated temperatures, often with microwave
heating.35 Therefore, the synthesis of peptide 5a in both DMF
To investigate the feasibility of peptide synthesis in cyclic
carbonate solvents, the known34 coupling reaction between
Boc-alanine 3a and phenylalanine benzyl ester 4a was studied
(Scheme 2) and the results are presented in Table 2. Entry 1
o
and PC was also carried out at 70 C in a microwave reactor
gives the literature conditions34 using
a conventional
(entries 8 and 9). In both cases high yields of peptide 5a were
again obtained. No evidence for reaction of amine 4a with the
propylene carbonate was seen in these reactions even though
there is literature precedent for the reaction of amines with
dichloromethane and DMF solvent system. Entry 2 shows that
changing the coupling agent, additive and base to give a less
costly and homogeneous reaction whilst still using the
conventional solvent system results in a 10% reduction in the
yield of dipeptide 5a. However, as shown in entry 3, simply
changing the solvent to PC virtually restored the yield of
dipeptide 5a to the literature value and use of EC as solvent
cyclic carbonates.36
A control experiment was therefore
carried out in which amine 4a was dissolved in PC and heated
o
to 70 C in a microwave reactor in the absence of any other
reagents. Even under these conditions, only 1% reaction
2 | J. Name., 2012, 00, 1-3
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between the amine and solvent was detected. Thus, reaction hydrogen chloride in dioxane gave the corresponding chloride
of amino acid derivatives with PC does not occur to any salt 6b in just 93% yield. Thus, both coupling and deprotection
significant extent under the reaction conditions used for reactions could be carried out in PC in higher yield than the
peptide synthesis, even at 70 oC and cannot compete with yields obtained using DMF, dichloromethane and dioxane.
peptide synthesis.
To illustrate the generality of this process, five fully
Activated amino acid derivatives generated during peptide protected tetrapeptides 9a-e were prepared as shown in
synthesis are prone to racemisation.8 To demonstrate that no Scheme 3. Peptide sequences 5-9a,b contain only unhindered
such racemisation occurs when the coupling reactions are and unfunctionalised amino acids. However, sequences 5-9c-e
carried out in PC, diastereomeric dipeptide 5a' was prepared were chosen to include the sterically hindered amino acids
from Boc-(R)-alanine 3a and ester 4a in 87% yield using the valine and aminoisobutyric acid (Aib), functionalised amino
conditions of Table 1, entry 5. HPLC analysis of dipeptides 5a acids with their functional groups unprotected (methionine
and 5a' as well as a 1:1 mixture of the two dipeptides and tryptophan) and functionalised amino acids with their
confirmed that they had stereochemical purities of greater functional groups protected using standard protecting groups
than 99:1 even when the peptide synthesis was carried out at (serine, lysine, aspartic acid and glutamine). All five
70 oC.37
tetrapeptides were successfully prepared using PC as the only
The other key step for solution-phase peptide synthesis is solvent. To further prove that no racemisation of the activated
selective removal of the N-terminal Boc protecting group to amino acids occurs during peptide synthesis in PC,
allow subsequent chain elongation. This is usually achieved by diastereomeric dipeptide 5b
use of strong acid such as hydrogen chloride or using Boc-(R)-leucine and HPLC analysis of the diastereomers
trifluoroacetic acid in solvents such as 1,4-dioxane, 5b 5b and 7a 7a again showed stereochemical purities of
' and tripeptide 7a' were prepared
a
/
'
/
'
dichloromethane, THF, acetonitrile or DMF. Therefore, the use greater than 99:1.37 Finally, to show that the methodology was
of PC as solvent for Boc deprotection reactions was also compatible with the palladium on carbon used to remove
investigated. Initially, hydrogen chloride was generated in PC benzyl based protecting groups, tripeptide 7a was subjected to
and used to convert fully protected dipeptide 5a into the hydrogenolysis to give acid 10a in 96% yield and then treated
hydrochloride salt of Ala-Phe-OBn in 76% yield. However, as a with trifluoroacetic acid in PC to give fully deprotected
solution of hydrogen chloride in PC is not commercially tripeptide 11a in quantitative yield.
available, it was more convenient to use trifluoroacetic acid in
Having demonstrated that PC could be used as a solvent
PC and this converted fully protected dipeptide 5a into for solution-phase peptide synthesis, its application to solid-
trifluoroacetate salt 6a in 99% yield. In contrast, use of 4M
phase peptide synthesis was investigated. This presented a
new challenge: the solvent must swell the resin upon which
the peptide is being constructed. We have recently reported a
study of the swelling of various resins used for solid-phase
synthesis in over 20 green solvents including PC and EC.38 This
showed that polystyrene based resins such as 2% cross linked
polystyrene (Merrifield resin39) did not swell adequately in PC.
However, a cross-linked polyethylene glycol (ChemMatrix
resin40) did swell (to 6.0 mL g-1) in PC which is well above the
4.0 mL g-1 required for a resin to be considered as well
swollen.41 If a resin swells too much in a particular solvent, this
can also be a problem for solid-phase peptide synthesis as it
can cause blockages and result in over-filling of resin holding
vessels. It is thus advantageous that whilst PC swelled
ChemMatrix resin to 6.0 mL g-1 this is less than the 7.6–7.8 mL
g-1 swelling observed in DMF and NMP.38 Hence, all solid-phase
peptide synthesis was carried out on ChemMatrix resin.
EDC / HOBt /
iPr2EtN, PC
Boc-AA2-OH + H-AA1-OBn
Boc-AA2-AA1-OBn
70-93%
5a-e
Boc-(R)-Leu-Phe-OBn
CF3CO2H, PC 80-99%
5b'
Boc-AA3-OH +TFA. H-AA2-AA1-OBn
6a-e
EDC / HOBt / iPr2EtN, PC
68-83%
Boc-AA3-AA2-AA1-OBn
Boc-(R)-Leu-Ala-Phe-OBn
7a'
7a-e
84-99%
CF3CO2H, PC
Boc-AA4-OH +TFA. H-AA3-AA2-AA1-OBn
8a-e
EDC / HOBt / iPr2EtN, PC
65-91%
Boc-AA4-AA3-AA2-AA1-OBn
The synthesis of the tripeptide TFA.H-Leu-Ala-Phe-OH 11a
was chosen as a test sequence and its solid-phase preparation
was undertaken in both DMF and PC (Scheme 4). The synthesis
started with commercially available ChemMatrix resin
preloaded with an acid-labile HMPB (4-(4-hydroxymethyl-3-
methoxyphenoxy)butyric acid) linker42 and a phenylalanine
residue with a free amino group. Each coupling reaction was
carried out using three equivalents each of an Fmoc-protected
amino acid, HBTU43 (3-[Bis(dimethylamino)methyliumyl]-3H-
benzotriazol-1-oxide hexafluorophosphate) and HOBt and six
equivalents of diisopropylethylamine. At the end of each
coupling and deprotection reaction, the resin was only washed
9a-e
H2, Pd/C, MeOH
Boc-Leu-Ala-Phe-OH
10a
100%
7a
96%
CF3CO2H, PC
TFA.H-Leu-Ala-Phe-OH
11a
5-11a: AA1 = Phe; AA2 = Ala; AA3 = Leu; AA4 = Gly
5-9b: AA1 = Phe; AA2 = Leu; AA3 = Gly; AA4 = Ala
5-9c: AA1 = Val; AA2 = Met; AA3 = Ser(Bn); AA4 = Lys(Z)
5-9d: AA1 = Val; AA2 = Asp(Bn); AA3 = Trp; AA4 = Gln(Trt)
5-9e: AA1 = Leu; AA2 = Aib; AA3 = Pro; AA4 = Tyr(Bn)
Scheme 3: Synthesis of tetrapeptides in PC.
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H-Phe
O
HMPB
FmocAlaOH (3.0 eq), HBTU (3.0 eq),
HOBt (3.0 eq), DIPEA (6.0 eq),
2x1 h, DMF or PC
Fmoc-Ala-Phe
H-Ala-Phe
O
HMPB
20% piperidine in DMF or PC,
10+20 min.
O
O
HMPB
FmocLeuOH (3.0 eq), HBTU (3.0 eq),
HOBt (3.0 eq), DIPEA (3.0 eq),
2x1 h, DMF or PC
Fmoc-Leu-Ala-Phe
= ChemMatrix
HMPB
1) 20% piperidine in DMF or PC,
10+20 min.
2) TFA, TIPS, H2O (95:2.5:2.5), 2 h
TFA.H-Leu-Ala-Phe-OH
11a
Scheme 4: Solid-phase synthesis of tetrapeptide 11a.
with the solvent being used for the synthesis rather than with
a range of solvents (usually including DMF, dichloromethane
and methanol) to ensure that the use of non-green solvents
was completely avoided. The tripeptide was cleaved from the
resin by treatment with trifluoroacetic acid in the presence of
TIPS (triisopropylsilane) and water as cation scavengers,44 then
precipitated from and triturated with diethyl ether. Analysis of
Figure 1: HPLC-UV traces of bradykinin. A: commercial sample, B: sample prepared in
DMF, C: sample prepared in PC. Chromatograms were eluted with a gradient from 1:9
MeCN:H2O to 1:1 MeCN:H2O.37
H-(Pbf)Arg
O
both samples by 1H NMR spectroscopy showed that TFA.H-
Leu-Ala-Phe-OH 11a had been successfully prepared in both
solvents.
HMPB
FmocPheOH (3.0 eq), A then B
H-Phe-(Pbf)Arg
O
HMPB
FmocProOH (3.0 eq), A then B
H-Pro-Phe-(Pbf)Arg
Having demonstrated that PC could be used as a solvent
O
for solid-phase peptide synthesis, the synthesis of a
HMPB
FmocSer(OBn)OH (3.0 eq), A then B
H-(Bn)Ser-Pro-Phe-(Pbf)Arg
biologically relevant peptide was undertaken. The naturally
occurring nonapeptide bradykinin (H-Arg-Pro-Pro-Gly-Phe-Ser-
Pro-Phe-Arg-OH) which is a vasodilator the concentration of
which is increased by ACE-inhibitors45 was selected as a
suitable test as it is of moderate length and contains a mixture
of functionalised and unfunctionalised amino acids. The
synthesis of bradykinin starting from H-Arg-HMPB-ChemMatrix
was carried out using the coupling and deprotection reactions
given in Scheme 5.37
O
HMPB
FmocPheOH (3.0 eq), A then B
H-Phe-(Bn)Ser-Pro-Phe-(Pbf)Arg
O
HMPB
FmocGlyOH (3.0 eq), A then B
H-Gly-Phe-(Bn)Ser-Pro-Phe-(Pbf)Arg
O
HMPB
FmocProOH (3.0 eq), A then B
H-Pro-Gly-Phe-(Bn)Ser-Pro-Phe-(Pbf)Arg
O
The cleaved peptide was precipitated into cold diethyl
ether, triturated and lyophilised. To allow a comparison with
conventional methodology to be undertaken, the synthesis
was also carried out in DMF and both synthetic samples were
compared with commercially available bradykinin by HPLC-UV-
HRMS (Figure 1).37 The bradykinin samples prepared in PC and
DMF were found to have purities of 77 and 79% respectively,
showing that use of PC as solvent had no significant
detrimental effect on the synthesis. The sample prepared in
DMF (Figure 1B) contained a single impurity peak which
overlapped with the bradykinin peak and so would be difficult
to separate by preparative HPLC. Analysis of the mass
spectrum of this impurity peak showed that it was actually a
HMPB
FmocProOH (3.0 eq), A then B
H-Pro-Pro-Gly-Phe-(Bn)Ser-Pro-Phe-(Pbf)Arg
O
HMPB
FmocArg(Pbf)OH (3.0 eq), A then B
H-(Pbf)Arg-Pro-Pro-Gly-Phe-(Bn)Ser-Pro-Phe-(Pbf)Arg
O
HMPB
TFA, TIPS, H2O (95:2.5:2.5), 3 h
= ChemMatrix
HO Arg-Phe-Pro-Ser-Phe-Gly-Pro-Pro-Arg-H
Bradykinin
A) HBTU (3.0 eq), HOBt (3.0 eq), DIPEA (6.0 eq), 2x1 h, DMF or PC
B) 20% piperidine in DMF or PC, 10+20 min.
Scheme 5: Solid-phase synthesis of bradykinin in PC.
4 | J. Name., 2012, 00, 1-3
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mixture of the premature chain termination sequences H-Ser- was added dropwise and the solution allowed to stir at room
Pro-Phe-Arg-OH and H-Pro-Phe-Arg-OH. In contrast, this temperature for 2h. Then, PC was removed by short path
impurity was not present in the sample prepared in PC (Figure distillation. The residue was suspended in Et2O and stirred for
1C) and the impurity peaks that were present were well 5 minutes before being filtered to give HCl.Ala-Ph-OBn as a
separated from the bradykinin peak, thus facilitating white solid (32.4 mg, 76%).
purification by preparative HPLC if desired.
General procedure for solid-phase peptide synthesis in PC
The synthesis of peptides on a solid-phase was carried out in a
Experimental
5 mL syringe fitted with a polypropylene frit. Preloaded
Details of instrumentation are given in the supplementary
ChemMatrix-HMPB resin (200 mg, 0.6 mmol g-1) was first
information.
washed with PC (3 × 5mL) and then swollen in PC (3.5 mL) for
1h. In a separate vial, an Fmoc-amino acid (0.36 mmol, 3.0
General procedure for peptide coupling reactions in PC
eq.), HBTU (137 mg, 0.36 mmol, 3.0 eq.), HOBt (49 mg, 0.36
To a suspension of an N-Boc-amino acid (1.0 eq.) and an amino
acid or peptide benzyl ester (1.0 eq.) in PC (5 mL mmol-1), at 0
°C, was added a solution of HOBt (1.1 eq.) and iPr2EtN (3.0 eq.)
in a minimal quantity of PC. EDC (1.1 eq.) was added dropwise
and the reaction mixture was allowed to stir at room
temperature for 16h. The reaction mixture was then diluted
using EtOAc (50 mL) and washed with 1M HClaq (3 × 25 mL),
saturated Na2CO3 (3 × 25 mL) and H2O (3 × 25 mL). The organic
layer was dried (MgSO4), filtered and concentrated in vacuo.
Any residual PC was removed via short path distillation.
Purification details for each peptide and characterising data
are given in the supplementary information.
mmol, 3.0 eq.) and DIPEA (125 μL, 0.72 mmol, 6.0 eq.) were
combined in PC (3.6 mL). After 3 min., this preactivated amino
acid solution was added to the resin. Coupling reactions were
performed for 1h. at ambient temperature and carried out in
duplicate. Following each coupling reaction the resin was
washed using PC (3
× 5mL). Fmoc-deprotections were
performed in duplicate (10 min followed by 20 min) using a
freshly prepared solution of 20% (v/v) piperidine in PC (10–12
mL g-1). Following deprotection the resin was washed using PC
(3 × 5mL). Once the full sequence was constructed and the
final Fmoc–deprotection had been performed, the resin was
dried under reduced pressure. The peptide was then cleaved
from the resin using TFA:TIPS:H2O (95:2.5:2.5) (8–10 mL g-1) for
2h. at ambient temperature. The resin was removed by
filtration and the peptide precipitated into cold Et2O,
triturated and lyophilised. Characterising data for the peptides
prepared in this way are given in the supplementary
information.
General procedure for Boc deprotections in PC
An N-Boc-peptide benzyl ester (1.0 eq.) was dissolved in a
minimum amount of PC and trifluoroacetic acid (60 eq.) was
added. The reaction mixture was allowed to stir for 3h. at
room temperature before being concentrated in vacuo. Any
residual PC was removed via short path distillation.
Characterising data for each deprotected peptide are given in
the supplementary information.
Conclusions
Propylene carbonate
1 has been shown to be a green
Synthesis of Boc-Ala-Phe-OBn 5a in PC in a microwave reactor
replacement for reprotoxic amide based solvents which are
widely used in peptide synthesis. Both solution- and solid-
phase peptide synthesis can be carried out in propylene
carbonate using acid and base labile amine protecting groups
respectively. No significant racemisation of the activated
amino acids occurs in propylene carbonate and the viability of
solid-phase peptide synthesis in propylene carbonate was
demonstrated by the synthesis of the nonapeptide bradykinin.
N-Boc-Alanine (189 mg, 1.0 mmol, 1.0 eq), phenylalanine
benzyl ester (292 mg, 1.0 mmol, 1.0 eq) and HOBt (149 mg, 1.0
mmol, 1.1 eq) were combined in a G10 microwave vial. PC (2
i
mL) and Pr2EtN (523 μL, 3.0 mmol, 6.0 eq) were then added.
The vial was cooled to 0 °C and EDC (195 μL, 1.1 mmol, 1.1 eq)
was added dropwise. The reaction mixture was then heated at
70 °C for 3 hours using an Anton Parr Monowave 300
microwave reactor. The reaction was then diluted with EtOAc
(50 mL) and washed with 1M HCl (50 mL), sat. Na2CO3 (50 mL)
and brine (50 mL). The organic fraction was dried (MgSO4),
filtered and concentrated in vauco. Residual PC was removed
via short path distillation. The residue was purified using flash
column chromatography (40:60, EtOAc:petroleum ether) to
give N-Boc-Ala-Phe-OBn as a white solid (352 mg, 82%).
Acknowledgements
The authors thank EPSRC (grant number EP/M506680/1) for a
studentship to SL.
Notes and references
1
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Graphical Abstract
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