Synthesis of the natural product descurainolide and cyclic peptides from lignin-derived aromatics

Article abstract of DOI:10.1039/c7ob02697h

Alternative sources of potential feedstock chemicals are of increasing importance as the availability of oil decreases. The biopolymer lignin is viewed as a source of useful mono-aromatic compounds as exemplified by the industrial scale production of vanillin from this biomass. Alternative lignin-derived aromatics are available in pure form but to date examples of the use of these types of compounds are rare. Here we address this issue by reporting the conversion of an aromatic keto-alcohol to the anti- and syn-isomers of Descurainolide A. The key step involves a rhodium-catalyzed allylic substitution reaction. Enantio-enriched allylic alcohols were generated via an isothiourea-catalyzed kinetic resolution enabling access to both the (2R,3R) and (2S,3S) enantiomers of anti-Descurainolide A. In addition we show that the lignin-derived keto-alcohols can be converted into unnatural amino acid derivatives of tyrosine. Finally, these amino acids were incorporated into cyclic peptide scaffolds through the use of both chemical and an enzyme-mediated macrocylisation.

Full text of DOI:10.1039/c7ob02697h

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D. Smith and N. J. Westwood, Org. Biomol. Chem., 2017, DOI: 10.1039/C7OB02697H.
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Synthesis of the Natural Product Descuranolide and Cyclic
Peptides from Lignin-derived Aromatics
O. Stephen Ojo†,a Brunello Nardone,a Stefania F. Musolino,a Andrew R. Neal,a Liam Wilson,a Tomas
Lebl,a Alexandra M. Z. Slawin,a David B. Cordes,a James E. Taylor,a James H. Naismith,a,b Andrew D.
Smitha and Nicholas J. Westwood*a
§Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
Alternative sources of potential feedstock chemicals are of increasing importance as the availability of oil decreases. The
biopolymer lignin is viewed as a source of useful mono-aromatic compounds as exemplified by the industrial scale
production of vanillin from this source. Alternative lignin-derived aromatics are available in pure form but to date
examples of the use of these types of compounds are rare. Here we address this issue by reporting the conversion of an
aromatic keto-alcohol to the anti- and syn-isomers of Descurainolide A. The key step involves a rhodium-catalyzed allylic
substitution reaction. Enantio-enriched allylic alcohols were generated via an isothiourea-catalyzed kinetic resolution
enabling access to both the (2R,3R) and (2S,3S) enantiomers of anti-Descurainolide A. In addition we show that the lignin-
derived keto-alcohols can be converted into unnatural amino acid derivatives of tyrosine. Finally, these amino acids were
incorporated into cyclic peptide scaffolds through the use of both chemical and an enzyme-mediated macrocylisation.
pure form from birch lignin. Here, in the first part of the work,
Introduction
we report the use of the renewable keto-alcohol
point in natural product synthesis, specifically, a natural
product that contains an electron-rich aromatic ring (from
via to , Scheme 1B). In a second novel application of
renewable building block and its analogue , we also report
the synthesis of the unnatural tyrosine derivatives and
This section of work culminates in the incorporation of and
into cyclic peptides and
1 as a starting
Lignin is the second most abundant naturally occurring
polymer after cellulose.1 At present, only 1-2% of industrially
produced lignin is converted into commercial products with
the rest being used as a low value fuel.1 It is clear that
1
2
3
1
4
applications
of
this
renewable
material
remain
5
6.
underexplored.2 The large number of aromatic units present in
lignin has inspired methods to obtain substituted-phenolic
building blocks.3,4 Whilst considerable efforts have been
channelled into lignin depolymerisation,5 many of these
processes lead to major separation challenges if pure aromatic
building blocks are required.6,7 An alternative approach
investigates the processing of native lignins8 through the use
of highly selective catalytic transformations.9 The advantage of
this second approach is that it delivers pure monomers albeit
with moderate lignin conversions. However, this approach will
only be viable in an industrial context if the pure monomers
are useful.10,11 Recently we have shown that phenolic
5
6
7
8.
monomer
lignin’s β-O-4 units at the benzylic alcohol and subsequent C-O
bond cleavage (Scheme 1A).11 Monomer
was obtained in
1 can be obtained via the selective oxidation of
1
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obtained in one step from 1 12
.
Whilst electron-rich
substituents in aromatics of the general structure
2 are
tolerated in the rhodium-catalyzed reaction,16 to the best of
our knowledge no examples of the use of trialkoxyaryl-
substituents have been reported.
Scheme 2 Our Retrosynthetic Analysis of 3 from keto-alcohol 1.
In contrast to the relatively small natural product 3, cyclic
peptides are, arguably, most suitable for targeting large sites.17
Extended binding pockets or surfaces, for example protein-
protein interactions,18 are often considered “undruggable” by
small molecules. Natural cyclic peptides, such as cyclosporin A
(an immunosuppressant drug), possess drug-like cell
permeability and oral bioavailability that apparently defy the
physicochemical requirements for bioavailability predicted by
Lipinski’s Rule of Five.19 More recently, synthetic cyclic
peptides have been used to investigate the relationship
between macrocycle structure and cell permeability.20
Scheme 1A: Key steps in the depolymerisation of lignin to give the aromatic
monomer 1. 1B: Planned application of
via 1C: Planned application of and
acids which are subsequently incorporated into cyclic peptide scaffolds.
1
in the synthesis of a natural product
3
2.
1
4 in the formation of an unnatural amino
Descurainolide A (3) is a natural product isolated from the
seeds of the plant Descurainia Sophia.12 It contains a five-
membered lactone ring and a 2,6-dimethoxy-phenol ring
analogous to that in 1 12
.
The absolute stereochemistry of
currently not assigned.12 Whilst no biological activity has been
directly associated with to date, extracts from the seeds of
3 is
3
the D. Sophia plant have been used to treat coughs, asthma
and oedema.12 Uses as a cardiotonic or in cancer therapy have
also been proposed.12 The synthesis of highly enantio-enriched
Whilst some naturally occurring cyclic peptides contain
modified amino acids (for example the patellamides21 in which
serine and threonine residues are processed to oxazoline and
thiazoline/thiazole rings), synthetic chemistry has enabled a
number of unnatural amino acids to be built into cyclic
samples of both enantiomers of
it should: i) showcase the use of the renewable monomer
provide both enantiomers of for biological assessment and
3
was viewed as important as
1; ii)
3
peptides. Through the production of the tyrosine analogues
5
iii) enable the assignment of the absolute configuration of the
natural product. We envisaged that the construction of the
and 6 from our keto-alcohols 1 and 4, cyclic peptides
containing these unnatural amino acids were prepared.
Tyrosine analogues of this type are alternatively synthesized
by employing tyrosine phenol lyases (TPL).22a In addition,
unnatural amino acid analogues of Tyr, such as those
containing aromatic methoxy groups are important to help
investigate the role of Tyr in different enzymes (e. g.
lactone ring in
9
, a potential precursor of
3
, could be achieved
by diastereoselective iodolactonisation13 of
a
suitably
protected γ-olefinic ester 10. Compound 10 could be obtained
by hydrolysis of one of the esters in 11 followed by
decarboxylation of the resulting carboxylic acid (Scheme 2).
Compound 11 is the expected product of a transition metal-
catalyzed allylic substitution reaction between dimethyl
cytochrome
c
oxidase).22b The ability to separate
diastereomeric cyclic peptides was also required here and
enabled a detailed NMR study. Overall, through the use of two
different applications, the utility of the lignin-derived keto-
malonate and allylic carbonate 12 14
This reaction would
.
enable the selective formation of the C1-C2 bond, delivering
the required branched allylic system.15 Although, our initial
alcohols 1 and 4 was demonstrated.
study was aimed at racemic
proceed with retention of stereochemistry at the allylic center.
This should enable access to either enantiomer of , provided
highly enantio-enriched forms of 12 are available (vide infra).
The allylic carbonate 12 could be obtained from (Scheme
1B), which in turn could be prepared from our lignin-derived
monomer . Previously, we have shown that 13 can be
3, this reaction is known to
Results and Discussion
3
Part 1: Application of 1 to Natural Product Synthesis
2
Initially, it was decided to use silyl-protection for the
phenol in 13. Treatment of 13 with TBSCl, followed by 1,2-
reduction of the enone using NaBH4 with CeCl3.7H2O23
1
2 | J. Name., 2012, 00, 1-3
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afforded allylic alcohol (±)-2a. To install the C1-C2 bond (11
ARTICLE
,
Treatment of purified (±)-16 with LiCl in DMSO/H2O afforded γ-
olefinic ester (±)-17 in 76% yield31 and iodolactonisation using
I2/MeCN at 0 °C furnished (±)-18 in a anti:syn ratio of 19:1 (Figure
S2).32 After careful separation, dehalogenation of anti-(±)-18, using
Pd/H2 and NaOAc in MeOH provided anti-(±)-19 in 86% yield and
removal of the Piv-group with 2M HCl/ 1,4-dioxane gave anti-(±)-
Descurainolide A (3) in a 62% yield. X-ray crystallographic analysis of
anti-(±)-3 confirmed the bond connectivity and relative
stereochemistry (Scheme 4).30
Scheme 2), allylic carbonate (±)-14a was prepared by treating
(±)-2a with LiHMDS and then methyl chloroformate. The
choice of the allylic carbonate over allylic acetate was based
on the observation that oxidative insertion, the first step of the
transition metal catalyzed allylic substitution, is faster for
allylic carbonates than for allylic acetates.24 Also, the influence
of leaving groups on the regioselectivity of the reaction
(branched versus linear) has previously been studied.25
Scheme 5 Synthesis of syn-(±)-Descurainolide A: (a) 4 M NaOH, THF/MeOH (1:1), 100
°C, 92% for (±)-15 and 90% for (±)-17 (b) NaH, MeI, DMF, rt, 1 h, 95% (c) NaH, BnBr,
DMF, rt, 1 h, 83% (d) I2, NaHCO3 (aq), DCM, rt, 12 h, 85% combined yield (e) Pd/C, H2,
NaOAc, MeOH, rt, 16 h, 62% syn-(±)-3.
Scheme 3 Synthesis of Allylic Carbonates 14a and 14b: (a) (i) TBSCl, DMAP, Imidazole,
DCM, rt, 1 h, 90% (ii) PivCl, DMAP, Imidazole, DCM, 1 h, 87% (b) NaBH4, CeCl3.9H2O,
MeOH, 1 h, 95% for 2a, 91% for 2b (c) Methyl chloroformate, LiHMDS, THF, 1 h, -10 °C,
15% for 14a, 89% for 14b. See Scheme S1 for more detail.
Attempts to tune the iodolactonisation reaction of (±)-17 to enable
the preparation of sufficient amounts of syn-(±)-18 en route to syn-
(±)-3 proved unsuccessful (Table S1). However, conversion of Piv-
protected ester (±)-17 via (±)-20 and (±)-21 to benzyl protected acid
(±)-22, and subsequent iodolactonisation using I2 and aqueous
NaHCO3 provided (±)-23 in a anti:syn ratio of 1:3 (Figure S3) and
85% combined yield (Schemes 5 and S3). After careful separation,
hydrogenolysis of syn-(±)-23 over palladium on carbon gave syn-(±)-
3 in 62% yield. Detailed comparison of the NMR data associated
with our samples of anti-(±)-3 and syn-(±)-3 with that reported for
the isolated Descurainolide A12 confirmed that the natural material
had the proposed relative stereochemistry12 (Table S2).
It was found that allylic carbonate (±)-14a was unstable on
storage and purification on silica giving numerous compounds,
one of which was the linear isomer 15 (Scheme 3).26,27 It was
therefore decided to use an electron-withdrawing substituent
on the phenolic oxygen which led to the preparation of the
stable pivolate-protected (±)-14b from 13, via alcohol (±)-2b
,
(Scheme 3).28 The exposure of (±)-14b to rhodium-catalyzed
allylic substitution with dimethyl malonate (5 mol% of
RhCl(PPh3)3 and 20 mol% of P(OMe)3 in THF at 40 °C provided
(±)-16 in high yield (87%) as a single regioisomer (Schemes 4,
S2 and Figure S1).29
In an attempt to determine the absolute configuration of natural
anti-3, access to both enantiomers of a protected lignin-derived
allylic alcohol 2 was required. In this regard, acylative kinetic
resolution is an attractive strategy as it would allow access to both
enantiomers from the readily available racemate.33 The Smith group
has reported that the isothiourea HyperBTM 24 is an effective
catalyst for the kinetic resolution of challenging aryl-alkenyl
alcohols in which the catalyst must differentiate between two sp2-
hybridized substituents during the selectivity-determining acylation
step.34 Moreover, this methodology is particularly effective for
alcohols bearing electron-rich aryl rings, such as those derived from
monomers of lignin.
To assess the applicability of this method for the preparation of
enantiomerically enriched anti-3, the selectivity (S=kfast/kslow) for the
kinetic resolution of a range of protected allylic alcohols 2a-f was
investigated (Table 1). Both tri- and disubstituted allylic alcohols 2a-
d bearing either O-silyl or O-pivaloyl protecting groups underwent
highly selective kinetic resolution (S = 29-64) using isobutyric
anhydride (0.6 eq) and HyperBTM 24 (1 mol%) in the presence of
iPr2NEt in toluene at -78 °C (Table 1, entries 1-4).34 Lower, but still
synthetically useful, selectivity was obtained for alcohols 2e and 2f
Scheme
4 Synthesis of anti-(±)-Descurainolide A: (a) NaH, Dimethyl malonate,
RhCl(PPh3)3 (5 mol%), P(OMe)3 (20 mol%), THF, 40 °C, 1 h, 87% (b) LiCl, H2O, DMSO, 140
°C, 16 h, 76% (c) I2, MeCN, 0 °C to rt, 46 h, 70% combined yield (d) Pd/C, H2, NaOAc,
MeOH, rt, 16 h, 86% (e) 2 M HCl/1,4-dioxane (1:1), 100 °C, 12 h, 70%. The thermal
ellipsoid plot for anti-(±)-3 is also shown at 50% ellipsoid probability.30
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conv.
(%)a
2 ee (%)b
(yield, %)
99d
25 ee (%)b
Scheme 6 Preparative kinetic resolution of (±)-2b: (a) (2S,3R)-HyperBTM 24 (1 mol%),
entry
R
R1
Sc
i
(iPrCO)2O (0.6 eq), Pr2NEt (0.5 eq), PhMe, –78 °C, 37% for (S)-2b and 47% for (R)-25b;
(yield, %)
78
(b) 1M
KOH, MeOH/H2O, rt, quant.; (c) (2R,3S)-HyperBTM (ent-24) (1 mol%), (iPrCO)2O
(0.15 eq), iPr2NEt (0.2 eq), PhMe, –78 °C, 75% for (R)-2b.
1
2
TBS
Piv
TBS
Piv
Ts
MeO 2a
MeO 2b
56
57
52
53
56
44
43
64
29
49
20
10
(44)
(50)
99
(30)
89
75
(52)
81
Table 1 Kinetic resolution of allylic alcohols (±)-2a-f
3
H
H
2c
(39)
97
(43)
85
4
2d
(38)
93
(47)
72
5e
6
MeO 2e
MeO 2f
(36)
55
(41)
71
Tf
(48)
(35)
aCalculated by HPLC analysis. bDetermined by HPLC analysis. cCalculated using the
equations developed by Kagan.36 dProof of the absolute configuration of (S)-2a came
from X-ray crystallographic analysis (Figure S4). eReaction performed in THF.
bearing O-tosyl and O-triflyl groups, respectively (Table 1, entries 5
and 6).
As the kinetic resolution of alcohol (±)-2b gave the highest
selectivity, this process was performed on a preparative scale (3.4
mmol, 1.0 g). In this case, leaving the kinetic resolution to reach
57% conversion allowed (S)-2b to be isolated as a single enantiomer
in 37% yield (Scheme 6), with the recovered isobutyrate ester (R)-
Using the previously established protocols (R)- and (S)-16 were
converted to anti-(2R,3R)-3 and anti-(2S,3S)-3 respectively
(Schemes 7 and S4 and Table S3). Chiral HPLC analysis of anti-
(2S,3S) and anti-(2R,3R)-3 and analysis of the two compounds by
circular dichroism (Figure S7) demonstrated that the two samples of
3 were highly optically enriched enantiomers of each other. X-ray
crystallographic analysis of anti-(2R,3R)-3 (Figure S6) confirmed the
allylic substitution reaction occurred with the precedented
retention of configuration and the absolute configuration of anti-
(2R,3R)-3.15
25b (47% yield, 75% ee) readily hydrolysed with 1M KOH to give (R)-
2b (75% ee). The enantiopurity of (R)-2b was further enhanced
through a second kinetic resolution using enantiomeric (2R,3S)-
HyperBTM (ent-24) to acylate selectively the remaining minor
enantiomer, requiring only 20% conversion to obtain (R)-2b in 92%
ee. Overall, highly enantiomerically enriched (S)- and (R)-2b could
be obtained from (±)-2b in a combined 72% yield, demonstrating
the powerful nature of acylative kinetic resolution.
Repeated attempts to obtain a sample of the natural product
from the group that originally isolated it were unsuccessful. This
hindered our attempts to assign the absolute configuration of
native Descuranolide A (3), due to the small values for the optical
rotation of 3 that we observed and that are reported in the
literature (Table S3).12 In summary of part 1, the renewable lignin-
derived keto-alcohol 1 was converted to both of the potential
diastereomers of the natural product 3 enabling confirmation of the
relative stereochemistry. Assignment of the absolute configuration
will require reisolation of the natural material due to the low optical
rotation of 3.
With (R)-2b and (S)-2b in hand, the synthesis of both
enantiomers of anti-3 was achieved (Schemes 7 and S4). Conversion
of (S)-2b into (S)-14b enabled the absolute configuration of (S)-14b
to be confirmed by X-ray analysis (Figure S5). The subsequent Rh(I)-
catalysed allylic substitution reaction was expected to proceed with
retention of stereochemistry (double inversion) when soft
nucleophiles (pKa < 22) are employed,15 and with high conservation
of enantiomeric excess (cee).35 At 40 °C, we observed erosion of ee,
from 99% (S)-14b to 83% of (R)-16. Perhaps at 40 °C the σ-π-σ
isomerisation between the two enantiomers of the π-
allylrhodium(III) complex, which is reported to be slow,15c is slightly
HighlyStereoselective
Allylic Substitution
O
O
O
anti-(2R,3R)-3
O
O
O
OMe
MeO
OMe
c-f
(R)
MeO
PivO
MeO
PivO
MeO
HO
b
a
(S)
(R)
(S)-2b
(R)
Me
99% ee
OMe
99% ee
OMe (R)-16
98% ee
OMe
98% ee
(S)-14b
O
O
O
O
O
anti-(2S,3S)-3
(S)
O
OMe
MeO
OMe
c-f
enhanced in this system. However, at ambient temperature, we
MeO
PivO
MeO
PivO
MeO
b
a
(R)
(S)
(S)
(R)-2b
obtained a high cee (~99%) for both (R)- and (S)-16.
Me
HO
OCOiPr
OH
92% ee
OH
OMe (R)-14b
93% ee
OMe (S)-16
93% ee
OMe
MeO
PivO
MeO
PivO
MeO
PivO
a
b, c
91% ee
(±)-2b
+
Scheme 7 Synthesis of anti-(2R,3R)- and anti-(2S,3S)-Descurainolide A (3): (a) Methyl
chloroformate, LiHMDS, THF, 1 h, -10 °C; 95% for R-14b, 74% for S-14b (b) NaH,
Dimethyl malonate, RhCl(PPh3)3 (5 mol%), P(OMe)3 (20 mol%), THF, 22 °C, 16 h; 79% for
S-16, 85% for R-16 (c) LiCl, H2O, DMSO, 140 °C, 16 h; 47% for R-17, 55% for S-17 (d) I2,
MeCN, 0 °C to rt, 48 h; 45% for anti-(2R,3S)-18, 48% for anti-(2S,3R)-18 (e) Pd/C, H2,
OMe
(R)-25b
75% ee
OMe
(R)-2b
92% ee
OMe
(S)-2b
99% ee
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NaOAc, MeOH, rt, 16 h; 52% for anti-(2R,3R)-19, 59% for anti-(2S,3S)-19 (f) 2 M
HCl/1,4-dioxane (1:1), 100 °C, 12 h; 42% for anti-(2S,3S)-3, 61% for anti-(2R,3R)-3.
(±)-38 and (±)-39 in 93% and 98% yields respectively.
Hydrolysis of the ester and deprotection of the TBS-silyl ether
occurred when (±)-38 and (±)-39 were refluxed in 2 M HCl:1,4-
dioxane (1:1) for 12 h, furnishing N-Fmoc protected amino
acids (±)-5 in 72% yield and (±)-6 in 76% yield.40
Part 2: Application of 1 and 4 to Cyclic Peptide Synthesis
Having demonstrated that renewable aromatic monomer
1
could be used as a starting point in natural product synthesis,
we turned to our second application in the synthesis of
unnatural amino and cyclic peptides. Both building blocks
1
and 4 (Scheme 1) were converted to their corresponding triols
(±)-26 and (±)-27 in three synthetic steps (Schemes S5 and
S7).37
Scheme 9 (a) Et3SiH, BF3.OEt2, DCM, -78 °C, 2 h, 56% for (±)-34 and 66% for (±)-35 (b)
LiHMDS, TsCl, THF, rt, 1 h, 75% and 60% (c) NaN3, DMF, rt, 3 h, 88% for (±)-36 and 78%
for (±)-37 (d) (i) Pd/C (10% wt), H2, EtOAc, 1 h (ii) FmocCl, THF, 1 h, 93% for (±)-38 and
98% for (±)-39 (e) 2 M HCl:1,4-dioxane (1:1), 100 °C, 12 h, 72% for (±)-5 and 76% for (±)-
6.
Scheme
8 3 h (b) 2,2-
aReaction Conditions: (a) K2CO3, BnBr, DMF, rt,
dimethoxypropane, pTSA.H2O (catalytic), DCM; 91% for (±)-28 and 89% for (±)-29 over
2-steps (c) Pd/C (10% wt), H2 gas, NaOAc, MeOH, rt, 12 h (d) Dess-Martin Periodinnane,
DCM, rt, 2 h (e) NaClO2, NaH2PO4, 2-methyl-2-butene, tBuOH/H2O, rt, 12 h (f) SOCl2,
DCM then EtOH; 78% for (±)-30 and 68% for (±)-31 over 4-steps (g) pTSA.H2O, 77% for
(±)-32 and 69% for (±)-33.
At this stage, the application of the S- and G- functionalized
lignin phenolic monomers
1 and 4 in the synthesis of N-Fmoc
protected unnatural amino acids has been demonstrated. In
addition, the treatment of the crude amino-ester (±)-40
(Scheme 10) with LiOH in THF:H2O (1:1) for 1 h at room
temperature gave the amino acid (±)-41
recrystallized to provide suitable quality crystals.30 This
analysis confirmed the assigned structure of (±)-41
, which was
The primary alcohols in (±)-26 and (±)-27 were selectively
protected as benzyl ethers, and the remaining diols were
protected as acetals to give (±)-28 and (±)-29 (Scheme 8). After
careful separation, the major diastereoisomer (relative
stereochemistry unassigned) of (±)-28 and (±)-29 respectively
were subjected to debenzylation,37a Dess-Martin oxidation, 37b
Pinnick oxidation37c and esterification37d to furnish (±)-30 and
(±)-31 in 78% and 68% over four steps. Subsequent removal of
the acetal groups using pTSA.H2O provided diol-esters (±)-32
and (±)-33 in 77% and 69%, respectively.38
.
Exposure of (±)-32 and (±)-33 to Et3SiH and BF3.OEt2 in
DCM at -78 °C for 2 h gave α-hydroxy esters (±)-34 and (±)-35
in moderate yields (Scheme 9).39 This benzylic deoxygenation
Scheme 10 (a) Pd/C (10% wt), H2, EtOAc, 1 h; (b) LiOH, THF:H2O (1:1), rt, 1 h, 88% crude
yield. The thermal ellipsoid plot for anti-(±)-41 is shown at 50% ellipsoid probability.25
protocol was found to be very substrate dependent and this
was the main factor that defined the route taken from
1 and 4
to 5 and 6 respectively. After conversion of the remaining
The linear peptides 42 and 43 (amino acid sequence:
AXIGFPVG where X = (±)-5 or (±)-6 after removal of the Fmoc
group respectively) were synthesised using a solid-phase
alcohol in (±)-34 and (±)-35 to the corresponding tosylate using
LiHMDS and TsCl, subsequent reaction with NaN3 in DMF gave
azido-esters (±)-36 and (±)-37 in 88% and 78% yields
respectively. The reduction of the azide group was
accomplished using Pd/C (10 wt%) in ethyl acetate under an
atmosphere of H2 gas. The treatment of the resulting crude
amines with FmocCl then gave N-Fmoc protected amino esters
peptide synthesis (SPPS) strategy.
A preloaded Gly-2-
chlorotrityl chloride resin was used to couple N-Fmoc
protected amino acids (±)-5 and (±)-6 with seven other
naturally occurring amino acids (Scheme 11).41,42,43 After
washing and swelling of the resin, suitably protected valine (V),
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proline (P), phenylalanine (F), glycine (G) and isoleucine (I)
were successively double coupled using a 4-fold excess of
amino acid for 20 mins at 75 °C making use of (i) DIC (0.5 M in
DMF), Oxyma (1 M in DMF) and (ii) HBTU (0.5 M in DMF), DIEA
(2 M in DMF) coupling reagent protocols. Prior to adding the
next amino acid, the peptide-bound resin was Fmoc-
deprotected with a solution of 20% piperidine in DMF for 10
min.
42
or
(R = OMe)
43 (R = H)
(a)
F
F
G
HN
O
P
G
HN
O
N
P
N
HN
I
O
O
HN
I
O
O
O
O
NH
O
O
O
NH
HN
O
O
+
HN
O
O
NH
V
O
2R
NH
V
N
2S
N
G
NH
H
resin
O
G
NH
H
Gly-2-chlorotrityl chloride resin
A
R
H2N
A
R
O
OMe
OMe
HO
a
HO
2(S)-7 (R =OMe)
or
2(S)-8 (R = H)
Ph
2(R)-7 (R =OMe)
or
O
O
8
2(R)- (R = H)
resin
H
O
O
O
O
H
N
N
H
N
H2N
N
N
O
N
HN
H
H
O
Scheme 12 Synthesis of cyclic peptides 2(S)-7, 2(R)-7, 2(S)-8 and 2(R)-8 (a) (i) PyBOP,
O
DIEA, DMF (ii) RP-HPLC purification enabled separation of diastereomers.
R = OMe
or
R = H
MeO
HO
dr = 1:1
b
R
Interestingly, detailed 1D and 2D NMR analysis of the cyclic
F
peptides showed that each diastereoisomer of
7 and 8
Ph
I
V
G
A
O
consisted of two main conformers in varying ratios (Figures 1,
S14, S23, S30 and S36). The absolute stereochemistry of the
O
O
H
O
O
H
O
N
N
H2N
N
H
N
N
H
OH
N
H
HN
G
O
two diastereomers of
7 were assigned based by comparison of
O
1
the H-15N HSQC spectra with the spectrum of a related cyclic
peptide (S)-44 prepared using enantiomeric pure ˪-(S)-Tyrosine
(Y) (Figure 1). The 1H-15N HSQC analysis of (S)-44 (Figure 1C)
was very similar to that of one of the two diastereomers
(Figure 1A) and very different from the analysis of the second
diastereomer (Figure 1B), For example, the signal
corresponding to the NH of the tyrosine residue in 2(S)-44
(8.01/111.7 ppm, labeled as Yb in Figure 1C) was similar in
chemical shift to the signal for the analogous NH for the
unnatural amino acid residue derived from (±)-5 in what was
P
MeO
HO
42 AXIGFPVG
(
5
R = OMe;
) where X is from
dr = 1:1
R = H; 43 (AXIGFPVG) where X' is from 6
R
Scheme 11 Synthesis of cyclic peptide precursors 42 and 43. (a) suitably protected
natural amino acid or (±)-5 or (±)-6, DIC (0.5 M in DMF), Oxyma (1 M in DMF) and/or
HBTU (0.5 M in DMF), DIEA (2 M in DMF); (b) (i) 20% HFIP in DCM, 30 min and (ii) 94%
trifluoroacetic acid, 4% H2O, 2% triisopropylsilane, 2 hrs.
The unnatural amino acid (±)-5 or (±)-6 was then single
coupled for 30 minutes, followed by Fmoc removal and double
coupling of the terminal alanine residue. The final Fmoc-
deprotection was carried out with a solution of 50% piperidine
in DMF for 3 minutes and the resin was washed extensively.
The linear peptide was cleaved from the resin using a solution
of 20% HFIP (hexafluoro isopropanol) in DCM followed by 94%
trifluoroacetic acid, 4% H2O, 2% triisopropylsilane. Peptides 42
and 43 (as 1:1 mixtures of diastereoisomers) were isolated in
52% and 45% yield respectively. The chemical cyclisation43 of
the linear peptides 42 (dr 1:1, X derived from (±)-5) and 43 (dr
1:1, X derived from (±)-6) was achieved using PyBOP and DIEA
assigned as the 2(S)-
as Xb in Figure 1A), whereas, the analogous signal in the
assigned 2(R)- diastereomer was found at 8.07/124.7 ppm
(Figure 1B). An analogous situation was observed for the two
separated diastereomers of cyclic peptide enabling one of
them to be assigned as the 2(S)- diastereomer due to
7 diastereomer (8.02/111.8 ppm, labeled
7
8
8
1
similarity of its H-15N HSQC analysis to that of (S)-44 (Tables
S8-S11).
to give cyclic peptides 7 and 8 respectively, both as 1:1 mixture
of diastereoisomers (Scheme 12). The use of HATU as the
coupling reagent led to formation of the cycle that had also
been modified by the coupling reagent, presumably through
reaction of the unprotected phenolic OH.44 No major
differences in the rate of cyclisation of the diastereomeric
linear peptides was observed. The two diastereoisomers of
and were separated by semi-preparative RP-HPLC.
7
8
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Figure 1: Assignment of the stereochemistry of the two diastereomers of cyclic peptide
7 by comparison with the 1H-15N HSQC analysis of 2(S)-44.
and Physical Sciences Research Council (grant EP/J018139/1,
EP/K00445X/1 (NJW, ADS, OSO), the doctoral training grant
(ARN) and the CRITICAT Centre for Doctoral Training [Ph.D.
studentship to SFM; Grant code: EP/L016419/1]), the
Leverhulme Trust (JET), European Research Council (ERC-2013-
ADG (JHN)).
In the final part of this study, the enzyme-mediated
cyclisation of linear peptide 42 was explored using the
45
macrocyclase enzyme PatGmac
.
Previous reports have shown
the effectiveness of this enzyme for the cyclization of a wide
range of linear peptides that contain natural and unnatural
amino acids.46 Based on the understanding of the mechanism
45
Notes and references
1
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of PatGmac
,
the precursor linear peptide must have a C-
terminal amino acid motif (AYD) that is recognised by the
enzyme. Therefore, modified linear peptide 45 (VGAXIGFPAYD
where X was derived from (±)-5) was prepared and converted
2
3
to the diastereomeric cyclic peptides 2(S)-7 and 2(R)-7. The
two diastereomers were formed in an approximately 1:1
(Scheme 13, Figures S21 and S22), implying that the rate of
macrocyclisation by PatGmac was not influenced by the
stereochemistry at the unnatural amino acid stereogenic
center in this system.
4
5
6
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10 chemsuschem 2017
Scheme 13 (a) PatGmac enzyme, 20 mM bicine buffer, 500 mM NaCl, and 5% DMSO
solution, pH = 8.1 and incubated at 37 °C (without shaking). No difference in the rates
of formation of the two diastereomeric cyclic peptides was observed (Figure S21).
11 C. S. Lancefield, O. S. Ojo, F. Tran, N. J. Westwood, Angew.
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Conclusions
In conclusion, we have synthesized the (2R,3R)- and (2S,3S)-
13 M. S. Oderinde, H. N. Hunter, S. W. Bremner, Organ, M. G. Eur.
J. Org. Chem. 2012, 175–182.
enantiomers of anti-Descurainolide A
3 from a lignin-derived,
keto-alcohol 1. The allylic alcohol (±)-2b was converted into
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highly enantio-enriched samples of both enantiomers using
the organocatalyst HyperBTM 24. The rhodium catalyzed allylic
substitution reaction proceeded with high conservation of
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step. In addition, we have converted the lignin-derived
phenolic monomers
amino acids and , respectively. These unnatural amino acids
were incorporated into cyclic peptides and . Overall this
1 and 4 into N-Fmoc protected unnatural
5
6
7
8
work has showcased the applicability of these phenolic
monomers in differing areas of synthetic organic chemistry.
(MeO)2C6H3) branched allylic carbonate was used in transition
metal catalysed allylic etherification, see; J. Štambaský, V.
Kapras, M. Štefko, O. Kysilka, M. Hocek, A. V. Malkov, P.
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We would like to acknowledge the EPSRC UK National Mass
Spectrometry Facility at Swansea University for mass
spectrometry analysis. Financial support from: the Engineering
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data. These data can be obtained free of charge from The
Cambridge
Crystallographic
Data
Centre
via
www.ccdc.cam.ac.uk/data_request/cif.
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