Pyrrolidinone-modified di- and tripeptides: Highly diastereoselective preparation and investigation of their stability

Article abstract of DOI:10.1039/b711349h

Pyrrolidine-2,4-diones have been identified as a novel starting point for the synthesis of peptide analogues. This paper describes a method for the efficient and diastereoselective incorporation of this moiety into peptide chains to furnish di- and tripep

Full text of DOI:10.1039/b711349h

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Pyrrolidinone-modified di- and tripeptides: highly diastereoselective
preparation and investigation of their stability†
Masood Hosseini,a,b David Tanner,a Anthony Murrayb and Janne E. Tønder*a,b
Received 27th July 2007, Accepted 12th September 2007
First published as an Advance Article on the web 19th September 2007
DOI: 10.1039/b711349h
Pyrrolidine-2,4-diones have been identified as a novel starting point for the synthesis of peptide
analogues. This paper describes a method for the efficient and diastereoselective incorporation of this
moiety into peptide chains to furnish di- and tripeptide analogs. The stability of these pyrrolidinone
modified di- and tripeptides was found to be markedly improved when compared to that of a natural
peptide. In addition, solid phase peptide synthesis employing a pyrrolidinone containing tripeptide is
demonstrated.
Introduction
Peptides are very attractive as drug candidates, due to their
selectivity for specific targets and high efficacy as compared to
traditional small molecule based drugs, and there is also less
concern with the formation of toxic metabolites. However, there
are some limitations associated with peptide drugs, the greatest
problem being very low bioavailability upon oral administration.1,2
The main cause of this limitation is pre-systemic enzymatic
degradation and, moreover, the gastrointestinal tract provides an
additional barrier. If a peptide drug manages to pass these barriers,
enzymatic digestion in the blood stream will occur, resulting
in a very low half-life. To overcome these obstacles one must
first identify the problems associated with degradation,3 and in
this respect many research groups have developed a range of
pseudopeptides to aid drug development.4
Fig. 1 Natural products containing pyrrolidinones.
In our group, we have focused our strategy on site-specific
modifications of amino acids in peptides with pyrrolidine-2,4-
dione derivatives, a motif which is found in several biologically
significant natural products5 such as dolastatin 15, which contains
the pyPhe-OMe moiety,6 and mirabimide E, containing a pyAla-
OMe unit (Fig. 1).7
The 5-alkyl-pyrrolidine-2,4-diones (1) have an amide group in-
stead of the amine functionality of amino acids, and an acidic OH
resembling the carboxylic acid of the natural amino acid, hence the
name tetramic acids. The side-chain of the amino acid is retained
by this modification, and likewise the chirality is preserved. The
rigid 5-membered ring system furthermore gives higher levels of
conformational restriction, which can be advantageous in terms
of target selectivity. By these site-specific alterations, we believe
that we can regulate the enzymatic degradation of peptide drugs,
while maintaining the activity which is inherent to the side chain
of amino acids. When introducing a pyrrolidinone as replacement
for an amino acid, the backbone of the peptide will be altered:
the natural amide bond in the peptide chain will be substituted
by an imide bond at the nitrogen, and to an amine or alkoxy
at the 4-carbonyl of the pyrrolidinone. This will have an effect
on the recognition pattern in enzymatic degradation processes as
well as the receptor interaction pattern. The optimal scenario is
naturally one where the enzymatic degradation is disturbed while
the interaction with the receptor target is either unaffected or
increased.
To reach these goals we firstly focused on developing the
chemistry, which will enable us to introduce diastereo- and chemo-
selectively a pyrrolidine-2,4-dione motif in a peptide chain. Our
strategy is the use of N-acylation to funtionalize the amide
nitrogen of the pyrrolidine-2,4-dione8 and a reductive amination
process for functionalization at the 4-carbonyl.9
aDepartment of Chemistry, Technical University of Denmark, Building 201,
DK-2800, Kgs Lyngby, Denmark
This method has enabled us to functionalize pyrrolidinones
with amino acids at nitrogen, giving structures of the type H-
AA-pyAA-OH, at an intermediate position of a small peptide,
giving structures of the type H-AA-pyAA-AA-OH, and at the 4-
carbonyl of pyrrolidine-2,4-diones, giving structures of the type
pyAA-AA-OH.
bMedicinal Chemistry Research & Biopharmaceutical Protein and Peptide
Chemistry, Novo Nordisk A/S, Novo Nordisk Park, DK-2760, Ma˚løv,
Denmark. E-mail: jejr@novozymes.com
† Electronic supplementary information (ESI) available: NMR spectra.
See DOI: 10.1039/b711349h
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Table 2 Synthesis of 4-ethoxy-3-pyrrolin-2-ones
Results and discussion
The synthesis of 5-alkylpyrrolidine-2,4-diones has previously been
described in the literature. The primary routes take advantage of
amino acids as chiral precursors and ring formation by Dieckmann
cyclization, or condensation with Meldrum’s acid followed by
decarboxylative cyclization.10
As our strategy requires enantiopure pyrrolidine-2,4-diones,
we did not attempt to use the Dieckmann procedure, which
previously has been reported to afford epimerization.11 The
procedure using Meldrum’s acid was therefore explored, and
we found that it furnished enantiopure products, but the final
purification was problematic. However, by substituting DCC
with EDC as activating agent and using a modified extraction
procedure, we could obtain pure gram-scale product in high
yield and excellent enantiopurity, without the need for column
chromatography (Table 1).
With this convenient procedure at hand, we were ready to
incorporate the products into peptides. As these pyrrolidinones
possess a weakly acidic a-proton (pKa ∼ 6.4),12 protection for the
5-oxygen was required to allow selective N-acylation. We explored
different protective groups (TIPS, TPS, TBS, Ts, PMB, SEM, Et,
Me) and found that the subsequent acylation was possible in high
yield only when simple alkyl groups were used to protect the
oxygen (Table 2). Furthermore, we found that the deprotection
(10% HBr) of these ethyl enol ethers (pyAA-OEt) was faster
than the naturally occurring methyl enol ethers (pyAA-OMe), and
therefore settled on the ethyl group.
As previous reported routes to pyAA-OAlk were rather tedious
and resulted in low yield,13 we decided to optimize the reaction
with regard to different bases and electrophiles. Conversion of
pyAA-OH to pyAA-OEt was best performed using KHMDS/18-
crown-6 as base and EtOTs as the hard electrophile, ensuring
selective O-alkylation.
Entry
X
R
Base
Solvent Isolated yield (%)
1
2
3
I
Br
Bn
Bn
(2a) KOt-Bu
(2a) KOt-Bu
(2a) KOt-Bu
(2a) KOt-Bu
(2a) KOt-Bu
(2a) K2CO3
(2a) BuLi
DMF
DMF
DMF
THF
THF
THF
THF
—
—
26
—
62
92b
—
—
84
87
84
74
OTs Bn
OTs Bn
OTs Bn
OTs Bn
OTs Bn
OTs Bn
OTs Bn
OTs Me
OTs i-Pr
4
5a
6a
7a
8a
9a
10a
11a
12a
(2a) LHMDS THF
(2a) KHMDS THF
(2b) KHMDS THF
(2c)
KHMDS THF
OTs i-Bn (2d) KHMDS THF
a Crown ether added. b 58% ee.
superior to both KOt-Bu in terms of yield and to K2CO3 in terms
of enantiopurity (Table 2). With this procedure in hand, we now
had access to stable protected 4-ethoxy-3-pyrrolin-2-ones (2a–d)
in high yield without loss of enantiopurity.
To further functionalize these compounds 2a–d at nitrogen, we
were reluctant to use strong base, as deprotonation at C-5 and
formation of an aromatic intermediate leading to racemization of
the product was a concern. Traditional peptide coupling reagents,
which provide milder reaction conditions, have previously been
shown not to facilitate the reaction, as the nucleophilicity of
the amide nitrogen is relatively poor,14c and even at elevated
temperature (60 ◦C) no product was detected in our studies
employing peptide coupling reagents (EDC, HOBt, TEA). We
therefore had no choice but to use strong bases as previously
reported for similar compounds,7,14 while paying close attention
to the problem of racemization.15
To investigate the dependence of racemization on temperature,
we conducted a series of experiments on pyPhe-OEt, measur-
ing the extent of racemization by chiral HPLC. Performing a
deprotonation/quench sequence below −45 ◦C gave no trace
of racemization, while this process was quite fast at higher
temperatures.8
We furthermore found that the use of lithium bases (LiHMDS
and n-BuLi) with the aid of 12-crown-4 did not provide notice-
able amounts of product, which presumably is a result of the
insolubility of the anion, while alkyl halides (EtI, EtBr) did not
provide any product, which can be ascribed to their soft nature.
Among the potassium bases, KHMDS afforded yields in the range
of 74–87%, with no racemization of the final product. This was
Table 1 Synthesis of pyrrolidine-2,4-diones
For further coupling, we were interested in developing a route
that could be used both in solution and on solid support. There-
fore, a procedure that would be compatible with both Boc and
Fmoc chemistry was necessary. We were naturally concerned with
the use of base in the presence of the base-labile Fmoc protecting
group; however, initial experiments and a previous report5h had
shown that the acylation of the activated amino acids was much
faster than the undesired deprotection of the Fmoc group. In initial
experiments, we used the commercially available pyGly-OMe and
Fmoc-Phe-OPfp to optimize the acylation conditions to form the
dipeptide analogue (3a).
Entry
Compound
R
Isolated yield (%)
We explored different bases and found that lithium bases were
superior to potassium bases in terms of reactivity, BuLi providing
the highest yield. However, the highest yield in this series (Table 3,
entries 1–8) was not satisfactory. We therefore warmed the reaction
mixture to a maximum of −45 ◦C, which we previously found to
1
2
3
4
pyAla
pyVal
pyLeu
pyPhe
(1a)
(1b)
(1c)
(1d)
Me
i-Pr
i-Bu
Bn
92
92
93
91
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Table 3 Optimization of acylation conditions
Table 5 Deprotection of dipeptide analoguesa
Temp./◦C
Isolated yield (%)
Product
Protecting group
Isolated yield (%)
Entry
Basea
H-Val-pyVal-OEt
H-Leu-pyVal-OEt
H-Phe-pyVal-OEt
(4a)
(4b)
(4c)
Fmoc
Boc
Fmoc
Boc
Fmoc
Boc
96
97
97
98
95
99
1b
2c
3b
4b
5b
6b
7b
8c
9b
KOt-Bu
KOt-Bu
KOt-Bu
LiHMDS
BuLi
i-PrMgCl
BuLi
−78
8
14
14
62
69
65
76
72
91
−78
−78
−78
−78
−78 → 22
−78
a DBU and TFA were used for deprotection of Fmoc- and Boc-protected
compounds, respectively.
BuLi
BuLi
−78
−55 → −45
a 1.0 eq. base used. b 1.0 eq. nucleophile used. c 2.0 eq. nucleophile used.
on the protecting group) and provided the free-amine dipeptide
analogues (4a–c, Table 5) in high yield and with no trace of
epimerization. Use of standard Fmoc deprotection conditions
(20% piperidine/DMF) caused significant difficulty in purifica-
tion, while treating the substrate with DBU for prolonged times
(>30 min) resulted in polymeric byproducts.
As we recently9 developed a method to access dipeptide ana-
logues by means of reductive amination of pyAA-OH at C-4 with
protected amino acids (Scheme 1), we explored the opportunity to
elongate these further to tripeptide analogues.
be the maximum temperature before racemization was observed,
and found that at these temperatures the yields were now quite
satisfactory (Table 3, entry 9).
With this procedure in hand, we synthesized a range of dipeptide
analogues (3a–u) with pyAA-OEt at the C-terminus. Both Boc-
and Fmoc-protected activated amino acids gave high yields, while
no epimerization was observed by 1H NMR (Table 4).
Deprotection of these dipeptide analogues to enable further
synthesis was accomplished with either TFA or DBU (depending
Table 4 Synthesis of dipeptide analogues
Scheme 1 Reductive amination of dipeptide analogues.
Compounda
Isolated yield (%)b
Our starting point was the use of our optimized acylation
procedure, which had provided high yields in the synthesis of the
dipeptide analogues described above (Table 4). Initial experiments
revealed that the reaction was very sensitive to temperature.
Interestingly, reactions at lower temperature provided higher yield,
whereas for high temperature reactions several byproducts were
formed. We therefore increased the amount of nucleophile (which
was apparently unstable) to 2 eq., and lowered the temperature to
−100 ◦C, which provided the highest yield in this series (Table 6).
However, we were not satisfied with the use of −100 ◦C and 2 eq.
of the non-commercially available nucleophile, as this can be quite
tedious in routine parallel synthesis and even more in scale-up
processes. We therefore attempted to optimize the reaction with
the use of other bases.
Potassium bases (KHMDS), as previously observed (Table 4),
led to a low degree of conversion; however, utilizing a sterically
hindered Grignard reagent as base furnished a much more stable
nucleophile, which even at room temperature led to very clean
product formation with no epimerization. Furthermore, only
1.0 eq. of the nucleophile was now required as a result of the
stability of the anion.
Fmoc-Phe-pyGly-OMe
Fmoc-Val-pyAla–OEt
Fmoc-Leu-pyAla-OEt
Fmoc-Phe-pyAla-OEt
Fmoc-Ala-pyVal-OEt
Fmoc-Val-pyVal-OEt
Fmoc-Leu-pyVal-OEt
Fmoc-Phe-pyVal-OEt
Fmoc-Ala-pyLeu-OEt
Fmoc-Val-pyLeu-OEt
Fmoc-Phe-pyLeu-OEt
Fmoc-Ala-pyPhe-OEt
Fmoc-Val-pyPhe-OEt
Fmoc-Leu-pyPhe-OEt
Boc-Ala-pyVal-OEt
Boc-Val-pyVal-OEt
Boc-Leu-pyVal-OEt
Boc-Phe-pyVal-OEt
Boc-Ala-pyPhe-OEt
Boc-Val-pyPhe-OEt
Boc-Leu-pyPhe-OEt
Boc-Phe-pyPhe-OEt
(3a)
(3b)
(3c)
(3d)
(3e)
(3f)
(3g)
(3h)
(3i)
91
92
88
85
89
76
78
78
84
81
80
84
88
85
85
86
80
94
85
89
88
92
(3j)
(3k)
(3l)
(3m)
(3n)
(3o)
(3p)
(3q)
(3r)
(3s)
(3t)
(3u)
(3v)
a Fmoc-AA-OPfp and Boc-AA-ONp were used as electrophiles. b Only one
diastereomer was observed by 1H NMR (de > 95%).
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Table 6 Optimization of tripeptide synthesis
differences in their degradation. After the chosen reaction time of
200 min, only 20% of the natural peptide was intact, while 53%
of the pyrrolidinone-modified peptide was intact. From LCMS
analysis, we could not determine the degradation products of the
natural peptide, while H-Val-pyAla-OEt was clearly cleaved at the
imide bond.
Entry Eq. nucleophile
Temp./◦C
Isolated yield (%)a
1
2
3
4
5
6
7
8
1.0
1.0
1.0
1.0
1.0
1.0
2.0
2.0
−15
−25
−35
−45
−55
−78
−78
−100
28
33
37
36
32
41
63
76
a Only one diastereomer was observed by 1H NMR (d.e > 95%).
Fig. 2 Degradation curves of selected peptide analogues.‡
We then examined the scope of this reaction, and synthesized a
range of tripeptide analogues with different amino acids protected
as Boc, Fmoc and Cbz derivatives (5a–i, Table 7).
We likewise examined pyVal-Ala-Ot-Bu, for which no degrada-
tion could be observed, indicating the amine bond in this system
was very stable. The proteolytic stability of the tripeptide Z-Val-
pyVal-Ala-Ot-Bu (5a) was found to be even more stable than the
dipeptide H-Val-pyAla-OEt (4a), with 76% of 5a remaining in
solution at the end of the 200 min experiment.
To evaluate the compatibility of 5 with solid phase peptide
synthesis, 5d was deprotected and the resulting 6a was attached
to a 2-ClTrt resin preloaded with H-Ser(t-Bu)-OH. Deprotection
of the amine group and attachment of a phenylalanine residue,
followed by cleavage of the peptide from the resin, provided a
pentapeptide (7a) containing a pyrrolidinone moiety (Scheme 2).
Throughout the series products were diastereoisomerically pure
1
as determined by H NMR and isolated in good yield. Hence,
this method provides a very convenient synthetic route to libraries
of tripeptide analogues. When pyrrolidinones with functionalized
side chains are to be employed, appropriate protecting groups
must be chosen. Thus, for O-protection, t-Bu is preferred to
the Bn group, since Bn groups can be readily cleaved during
PtO2-mediated reductive amination. For N-protection, a double
protection scheme should be employed to avoid NH deprotonation
in the acylation step.
To evaluate the proteolytic stability of the new peptide ana-
logues, we conducted a series of peptide stability tests in 50%
pooled human plasma (Fig. 2).16 Evaluating H-Val-pyAla-OEt and
the corresponding natural peptide H-Val-Ala-OMe showed clear
Conclusion
In conclusion, we have developed a methodology for the incorpo-
ration of pyrrolidine-diones into peptide chains either at the C- or
N-terminus or in an intermediate position in good to excellent
yield and with excellent enantio- and diastereoselectivity. The
pyrrolidinone-containing tripeptides were shown to be applicable
in solid phase peptide synthesis, allowing parallel synthesis
and possible identification of biologically active compounds for
therapeutic and diagnostic purposes. In support of this, we have
examined the proteolytic stability of these, showing markedly
increased stability towards human plasma.
Table 7 Synthesis of tripeptide analogues
Isolated
Entrya
PG
R
yield (%)b
1
2
3
4
5
6
7
8
9
Boc
Boc
Boc
Fmoc
Fmoc
Fmoc
Cbz
Cbz
Cbz
i-Pr
i-Bu
Bn
i-Pr
i-Bu
Bn
i-Pr
i-Bu
Bn
(5a)
(5b)
(5c)
(5d)
(5e)
(5f)
(5g)
(5h)
(5i)
78
90
83
83
85
88
82
83
89
Experimental
General methods and materials
NMR spectra were recorded on 400 MHz or 300 MHz Bruker in-
struments. Chemical shifts are reported in ppm (parts per million)
using the solvent residual peak as the internal reference. NMR
‡ A peptide concentration of 0.5 mg mL−1 in 50% human plasma was
used. Solutions were stirred at 37 ◦C for 200 min and analyzed by HPLC
at appropriate intervals.
a Fmoc-AA-OPfp, Boc-AA-ONp and Cbz-AA-ONp were used as elec-
trophiles. b Only one diastereomer was observed by 1H NMR (d.e > 95%).
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Scheme 2 Solid phase peptide synthesis of a pyrrolidinone-containing peptide (polystyrene resin with a 2-ClTrt linker was used as the solid support).
abbreviations are as follows: quin = quintet, sext = sextuplet,
sept = septuplet, dsept = double septuplet, oct = octuplet.
TLC was performed on aluminium sheets coated with silica gel
60 F254, and visualizing was effected with UV-light and/or 1%
ninhydrin in n-BuOH–AcOH. pyAA-OH (1a–d), pyAA-OEt (2a–
d) and dipeptides (3a–n) were prepared as previously described.8
Synthesis of pyVal-Ala-Ot-Bu using reductive amination was
performed as previously described.9
Boc-Val-pyVal-OEt (3p)
Following the procedure for 3o (86%): 1H NMR (CDCl3,
400 MHz): d 5.43 (dd, J = 9.6, 4.0 Hz, 1H), 5.26 (d, J = 9.1 Hz,
1H), 5.04 (s, 1H), 4.49 (d, J = 3.0 Hz, 1H), 3.97–4.10 (m, 2H), 2.84–
3.10 (m, 1H), 2.55–2.69 (m, 1H), 1.97–2.15 (m, 1H), 1.36–1.49 (m,
12H), 1.12 (d, J = 7.1 Hz, 3H), 1.02 (d, J = 7.1 Hz, 3H), 0.84
(d, J = 7.1 Hz, 3H), 0.76 (d, J = 7.1 Hz, 3H). 13C NMR (CDCl3,
100 MHz): d 14.1, 15.2, 16.4, 18.9, 19.9, 28.3, 28.5, 30.4, 58.2,
64.3, 67.8, 79.4, 94.6, 155.8, 170.2, 172.3, 178.7. [a]2D0 +46 (c 0.71,
MeOH). IR (KBr): 3446(s), 2968(m), 1729(s), 1620(s), 1174(m).
HRMS (EI+): Calcd. for C19H32N2O5 [M + H+] 369.2389. Found
369.2402.
Boc-Ala-pyVal-OEt (3o)
To a solution of pyVal-OEt (35 mg, 0.21 mmol) in THF (3 mL)
was added BuLi (1.6 M, 0.13 mL, 0.21 mmol) and the mixture was
stirred for 10 min whereafter Boc-Phe-ONp (71 mg, 0.23 mmol)
in THF (2 mL) was added dropwise over 15 min. The mixture was
allowed to stir for an additional 10 min and quenched with AcOH
(0.1 mL) and evaporated on silica. Column chromatography over
silica gel (EtOAc/Hep 1:3) provided the pure product (60 mg,
Boc-Leu-pyVal-OEt (3q)
Following the procedure for 3o (80%): 1H NMR (CDCl3,
400 MHz): d 5.42 (dt, J = 10.0, 3.3 Hz, 1H), 4.99–5.21 (m, 2H),
4.49 (d, J = 2.5 Hz, 1H), 4.05 (dt, J = 2.5, 7.5 Hz, 1H), 3.99–
4.10 (m, 2H), 2.36–2.96 (m, 2H), 1.68–1.86 (m, 1H), 1.41–1.48 (m,
12H), 1.11 (d, J = 7.6 Hz, 3H), 1.02 (d, J = 6.6 Hz, 3H), 0.92
(d, J = 6.6 Hz, 3H), 0.77 (d, J = 7.1 Hz, 3H). 13C NMR (CDCl3,
100 MHz): d 14.1, 15.3, 18.9, 21.2, 23.6, 24.9, 28.3, 28.5, 41.1, 52.9,
64.2, 67.7, 79.4, 94.5, 155.5, 170.1, 173.6, 178.6. [a]2D0 +22 (c 0.26,
MeOH). IR (KBr): 3449(s), 2978(m), 1725(s), 1629(s), 1180(m).
HRMS (EI+): Calcd. for C20H34N2O5 [M + H+] 383.2546. Found
383.2531.
1
0.18 mmol, 85%). H NMR (CDCl3, 300 MHz): d 5.21–5.43 (m,
2H), 5.03 (s, 1H), 4.49 (d, J = 2.6 Hz, 1H), 3.95–4.10 (m, 2H), 2.46–
2.65 (m, 3H), 1.36–1.49 (m, 13H), 1.32 (d, J = 6.4 Hz, 3H), 1.10
(d, J = 7.2 Hz, 3H), 0.76 (d, J = 7.2 Hz, 3H). 13C NMR (CDCl3,
75 MHz): d 14.1, 15.4, 18.2, 18.8, 28.3, 28.5, 50.1, 64.2, 67.8, 79.4,
94.5, 155.0, 170.1, 173.4, 178.7. [a]2D0 +5 (c 0.63, MeOH). IR (KBr):
3440(s), 2955(m), 1729(s), 1832(s), 1180(m). HRMS (EI+): Calcd.
for C17H28N2O5 [M + H+] 341.2079. Found 341.2079.
Fmoc-Val-pyVal-OEt (3f)
Following the procedure for 3o (76%): 1H NMR (CDCl3,
400 MHz): d 7.70 (d, J = 7.6 Hz, 2H), 7.56 (d, J = 7.6 Hz, 2H),
7.34 (t, J = 7.3 Hz, 2H), 7.26 (t, J = 7.6 Hz, 2H), 3.81–5.67 (m,
11H), 1.91–3.29 (m, 4H), 1.30–1.67 (m, 6H), 0.35–1.11 (m, 16H).
13C NMR (CDCl3, 100 MHz): d 13.63, 14.06, 14.12, 15.11, 16.28,
17.55, 18.90, 19.08, 19.99, 22.67, 23.02, 26.76, 28.51, 29.29, 29.33,
29.54, 29.57, 29.61, 29.63, 30.00, 30.39, 30.56, 31.37, 31.89, 41.00,
47.18, 47.22, 58.73, 64.36, 65.19, 66.93, 67.17, 67.81, 94.59, 119.93,
125.20, 127.04, 127.62, 127.69, 141.27, 143.78, 143.93, 144.03,
156.23, 156.80, 170.19, 171.63, 178.76. [a]2D0 +24 (c 0.22, MeOH).
IR (KBr): 3435(s), 2927(m), 1727(s), 1619(s), 1240(m). HRMS
(EI+): Calcd. for C29H34N2O5 [M + H+] 491.2546. Found 491.2564.
Boc-Phe-pyVal-OEt (3r)
Following the procedure for 3o (94%): Following the procedure
for 3o (94%)1H NMR (CDCl3, 400 MHz): d 7.13–7.40 (m, 5H),
5.14–5.75 (m, 2H), 5.05 (s, 1H), 4.51 (d, J = 2.5 Hz, 1H), 3.98–4.13
(m, 2H), 3.03–3.26 (m, 1H), 2.50–2.86 (m, 2H), 1.44 (t, J = 7.1 Hz,
3H), 1.22–1.39 (m, 9H), 1.12 (d, J = 7.1 Hz, 3H), 0.77 (d, J =
6.6 Hz, 3H) 13C NMR (CDCl3, 100 MHz): d 14.5, 15.8, 19.2, 28.6,
28.9, 38.3, 55.8, 64.7, 68.2, 79.8, 94.9, 127.0, 128.7, 130.0, 137.3,
155.5, 170.7, 172.3, 179.2. [a]2D0 +50 (c 0.24, MeOH). IR (KBr):
3372(m), 2979(m), 1726(s), 1619(s), 1315(s). HRMS (EI+): Calcd.
for C23H32N2O5 [M + H+] 417.2389. Found 417.2404.
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Boc-Ala-pyPhe-OEt (3s)
3.5 Hz, 1H), 4.55 (s, 1H), 4.06–4.22 (m, 2H), 2.62–2.76 (m, 1H),
1.60–1.84 (m, 3H), 1.43 (t, J = 7.1 Hz, 3H), 1.17 (d, J = 7.6 Hz,
3H), 1.04 (d, J = 6.6 Hz, 3H), 0.98 (d, J = 6.1 Hz, 3H), 0.79 (d, J =
6.6 Hz, 3H). 13C NMR (CDCl3, 100 MHz): d 14.6, 15.5, 19.3, 21.1,
24.0, 25.5, 29.8, 40.6, 53.7, 66.0, 69.9, 95.3, 170.2, 172.4, 181.7. [a]D20
+30 (c 0.16, MeOH). IR (KBr): 3436(s), 1691(s), 1619(s), 1339(m),
1224(m), 757(m). HRMS (EI+): Calcd. for C15H26N2O3 [M + H+]
283.2022. Found 283.2029.
Following the procedure for 3o (85%): 1H NMR (CDCl3,
400 MHz): d 7.22–7.18 (m, 3H), 7.02 (d, J = 6.6 Hz, 2H),
5.40–5.28 (m, 2H), 4.80–4.77 (m, 2H), 4.06–3.89 (m, 2H), 3.55
(dd, J = 5.1, 13.6 Hz, 1H), 3.12 (d, J = 13.6 Hz, 1H), 1.48
(s, 9H), 1.45 (t, J = 7.1 Hz), 1.34 (d, J = 6.6 Hz) 13C NMR
(CDCl3, 100 MHz): d 14.1, 18.3, 28.4, 34.7, 50.1, 60.0, 67.7, 79.5,
94.7, 127.1, 128.2, 129.8, 134.1, 155.2, 169.3, 173.9, 177.2. [a]D20
+120 (c 0.17, MeOH). IR (KBr): 3436(s), 1727(s), 1674(s), 1621(s),
1304(m). HRMS (EI+): Calcd. for C21H28N2O5 [M + H+] 389.2076.
Found 389.2068.
H-Phe-pyVal-OEt (4c). Following the procedure for 4a (99%):
1H NMR (CDCl3, 400 MHz): d 8.39 (br. s, 3H), 7.15–7.36 (m, 5H),
5.34 (dd, J = 8.6, 3.5 Hz, 1H), 5.04 (s, 1H), 4.39 (d, J = 2.0 Hz,
1H), 3.97–4.15 (m, 2H), 3.33 (dd, J = 14.1, 3.5 Hz, 1H), 2.40–2.62
(m, 1H), 1.45 (t, J = 7.1 Hz, 3H), 1.06 (d, J = 7.1 Hz, 3H), 0.68
(d, J = 7.1 Hz, 3H). 13C NMR (CDCl3, 100 MHz): d 14.5, 15.1,
19.1, 28.9, 36.7, 55.8, 65.0, 68.7, 94.5, 128.2, 129.5, 130.0, 134.2,
167.7, 170.8, 180.3. [a]2D0 +96 (c 0.62, MeOH). IR (KBr): 3436(s),
1691(s), 1619(s), 1373(m), 1224(m), 701(m). HRMS (EI+): Calcd.
for C18H24N2O3 [M + H+] 317.1865. Found 317.1861.
Boc-Val-pyPhe-OEt (3t)
Following the procedure for 3o (89%): 1H NMR (CDCl3,
400 MHz): d 7.16–7.26 (m, 3H), 6.99 (d, J = 6.6 Hz, 2H), 5.44
(dd, J = 9.9, 3.8 Hz, 1H), 5.24 (d, J = 10.1 Hz, 1H), 4.73–4.82
(m, 2H), 3.87–4.09 (m, 2H), 3.57 (dd, J = 13.6, 5.1 Hz, 1H), 3.12
(dd, J = 13.9, 2.3 Hz, 1H), 2.04–2.17 (m, 1H), 1.49 (s, 9H), 1.45
(t, J = 7.3 Hz, 3H), 1.03 (d, J = 7.1 Hz, 3H), 0.86 (d, J = 7.1 Hz,
3H). 13C NMR (CDCl3, 100 MHz): d 14.1, 16.1, 19.9, 28.3, 30.1,
34.7, 58.1, 60.0, 67.6, 79.4, 94.8, 127.0, 128.2, 129.7, 134.0, 155.8,
169.4, 172.8, 177.0. [a]2D0 +130 (c 0.21, MeOH). IR (KBr): 3452(s),
2966(m), 1735(s), 1622(s), 1378(s), 1165(m). HRMS (EI+): Calcd.
for C23H32N2O5 [M + H+] 417.2389. Found 417.2381.
Removal of the Fmoc group
H-Val-pyVal-OEt (4a). To a solution of Fmoc-Val-pyVal-OEt
(3f) (38 mg, 0.08 mmol) in DCM (3 mL) at room temperature was
added DBU (0.02 mL, 0.14 mmol) and the mixture was stirred
for 5 min, at which point 3 mL silica was added and the mixture
evaporated at reduced pressure. Column chromatography (0→5%
MeOH in EtOAc) afforded the pure product as a low-melting
Boc-Leu-pyPhe-OEt (3u)
1
solid. Yield (20 mg, 96%). H NMR (CDCl3, 400 MHz): d 8.18
Following the procedure for 3o (88%): 1H NMR (CDCl3,
400 MHz): d 7.18–7.26 (m, 3H), 6.90–7.08 (m, 2H), 5.46 (t, J =
11.6 Hz, 1H), 5.13 (d, J = 8.6 Hz, 1H), 4.68–4.85 (m, 2H), 3.89–
4.09 (m, 2H), 3.56 (dd, 1H), 3.12 (dd, J = 13.6, 2.0 Hz, 1H),
1.74–1.86 (m, 1H), 1.32–1.51 (m, 14H), 1.01 (d, J = 6.6 Hz, 3H),
0.92 (d, J = 7.1 Hz, 3H). 13C NMR (CDCl3, 100 MHz): d 14.1,
21.0, 23.6, 24.9, 28.4, 34.6, 41.0, 53.0, 60.0, 67.6, 79.4, 94.7, 127.1,
128.2, 129.8, 134.1, 155.7, 169.3, 174.3, 177.1. [a]2D0 +73 (c 0.76,
MeOH). IR (KBr): 3336(m), 1732(s), 1688(s), 1386(m), 737(m).
HRMS (EI+): Calcd. for C24H34NO5 [M + H+] 431.2546. Found
431.2554.
(br. s, 4H), 5.15 (d, J = 3.0 Hz, 1H), 5.10 (s, 1H), 4.50 (d, J =
2.5 Hz, 1H), 3.99–4.16 (m, 2H), 2.56–2.69 (m, 1H), 2.23–2.35 (m,
1H), 1.45 (t, J = 7.1 Hz, 3H), 1.16 (d, J = 7.1 Hz, 3H), 1.11 (d, J =
7.1 Hz, 3H), 0.96 (d, J = 7.1 Hz, 3H), 0.70 (d, J = 7.1 Hz, 3H).
13C NMR (CDCl3, 100 MHz): d 14.1, 14.6, 15.1, 18.9, 19.2, 28.4,
29.1, 58.2, 64.7, 68.2, 94.3, 167.8, 170.5, 179.8. [a]2D0 +88 (c 0.37,
CHCl3). IR (KBr): 3437(m), 2976(m), 1698(s), 1614(s), 1318(m),
1203(s). HRMS (EI+): Calcd. for C14H24N2O5 [M + H+] 269.1865.
Found 269.1874.
H-Leu-pyVal-OEt (4b). Following the procedure for 4a (97%):
1H NMR (CDCl3, 400 MHz): d 5.28 (s, 1H), 5.10 (dd, J = 9.6,
3.5 Hz, 1H), 4.55 (s, 1H), 4.06–4.22 (m, 2H), 2.62–2.76 (m, 1H),
1.60–1.84 (m, 3H), 1.43 (t, J = 7.1 Hz, 3H), 1.17 (d, J = 7.6 Hz,
3H), 1.04 (d, J = 6.6 Hz, 3H), 0.98 (d, J = 6.1 Hz, 3H), 0.79 (d, J =
6.6 Hz, 3H). 13C NMR (CDCl3, 100 MHz): d 14.6, 15.5, 19.3, 21.1,
24.0, 25.5, 29.8, 40.6, 53.7, 66.0, 69.9, 95.3, 170.2, 172.4, 181.7. [a]D20
+30 (c 0.16, MeOH). IR (KBr): 3436(s), 1691(s), 1619(s), 1339(m),
1224(m), 757(m). HRMS (EI+): Calcd. for C15H26N2O3 [M + H+]
283.2022. Found 283.2029.
Removal of the Boc group
H-Val-pyVal-OEt (4a). To a solution of Boc-Val-pyVal-OEt
(6) (41 mg, 0.11 mmol) in DCM (3 mL) was added TFA (3 mL)
and the residual mixture was stirred for 15 min at RT. The
mixture was evaporated and residual TFA was removed by solid
phase extraction (PS–H2CO3) to provide the pure product (29 mg,
0.11 mmol, 97%). 1H NMR (CDCl3, 400 MHz): d 8.18 (br. s, 4H),
5.15 (d, J = 3.0 Hz, 1H), 5.10 (s, 1H), 4.50 (d, J = 2.5 Hz, 1H),
3.99–4.16 (m, 2H), 2.56–2.69 (m, 1H), 2.23–2.35 (m, 1H), 1.45 (t,
J = 7.1 Hz, 3H), 1.16 (d, J = 7.1 Hz, 3H), 1.11 (d, J = 7.1 Hz,
3H), 0.96 (d, J = 7.1 Hz, 3H), 0.70 (d, J = 7.1 Hz, 3H). 13C NMR
(CDCl3, 100 MHz): d 14.1, 14.6, 15.1, 18.9, 19.2, 28.4, 29.1, 58.2,
64.7, 68.2, 94.3, 167.8, 170.5, 179.8. [a]2D0 +88 (c 0.37, CHCl3).
IR (KBr): 3437(m), 2976(m), 1698(s), 1614(s), 1318(m), 1203(s).
HRMS (EI+): Calcd. for C14H24N2O5 [M + H+] 269.1865. Found
269.1874.
H-Phe-pyVal-OEt (4c). Following the procedure for 4a (95%):
1H NMR (CDCl3, 400 MHz): d 8.39 (br. s, 3H), 7.15–7.36 (m, 5H),
5.34 (dd, J = 8.6, 3.5 Hz, 1H), 5.04 (s, 1H), 4.39 (d, J = 2.0 Hz,
1H), 3.97–4.15 (m, 2H), 3.33 (dd, J = 14.1, 3.5 Hz, 1H), 2.40–2.62
(m, 1H), 1.45 (t, J = 7.1 Hz, 3H), 1.06 (d, J = 7.1 Hz, 3H), 0.68
(d, J = 7.1 Hz, 3H). 13C NMR (CDCl3, 100 MHz): d 14.5, 15.1,
19.1, 28.9, 36.7, 55.8, 65.0, 68.7, 94.5, 128.2, 129.5, 130.0, 134.2,
167.7, 170.8, 180.3. [a]2D0 +96 (c 0.62, MeOH). IR (KBr): 3436(s),
1691(s), 1619(s), 1373(m), 1224(m), 701(m). HRMS (EI+): Calcd.
for C18H24N2O3 [M + H+] 317.1865. Found 317.1861.
H-Leu-pyVal-OEt (4b). Following the procedure for 4a (98%):
1H NMR (CDCl3, 400 MHz): d 5.28 (s, 1H), 5.10 (dd, J = 9.6,
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Boc-Val-pyVal-Ala-OtBu (5a)
3.31–3.42 (m, 1H), 3.19 (q, J = 6.9 Hz, 1H), 2.57–2.67 (m, 1H),
2.16–2.33 (m, 1H), 1.68–1.83 (m, 1H), 1.48 (s, 9H), 1.27 (d, J =
7.1 Hz, 3H), 0.87–1.09 (m, 12H). 13C NMR (CDCl3, 100 MHz): d
18.7, 19.5, 20.9, 21.6, 21.7, 22.8, 23.5, 24.7, 24.8, 27.7, 28.0, 39.4,
41.2, 41.7, 47.1, 52.3, 53.8, 54.3, 56.2, 63.1, 66.9, 81.7, 119.9, 125.1,
125.1, 127.0, 127.6, 127.7, 141.3, 143.7, 143.9, 156.1, 173.3, 173.7,
175.0, 175.3. [a]2D0 +16 (c2.90, MeOH). IR (KBr): 3351(m), 2960(s),
1726(s), 1518(m), 1151(m). HRMS (EI+): Calcd. for C34H45N3O6
[M + H+] 592.3387. Found 592.3383.
To a solution fo pyVal-Ala-Ot-Bu (50 mg, 0.18 mmol) in THF 3 mL
was added i-PrMgCl (2.0 M in THF, 0.090 mL, 0.18 mmol). The
mixture was allowed to stir for 10 min, at which point a solution of
Boc-Val-ONp (69 mg, 0.20 mmol) in THF (2 mL) was added. The
mixture was stirred for 1 h, and then AcOH (0.1 mL) was added,
and the mixture was diluted with DCM (20 mL), washed with
10% NaHCO3 (20 mL) and evaporated under reduced pressure.
Column chromatography over silica gel EtOAc–heptane (1 : 4)
provided the pure compound (68 mg, 0.14 mmol, 78%). 1H NMR
(CDCl3, 400 MHz): d 5.36 (dd, J = 9.6, 4.0 Hz, 1H), 5.12 (d,
J = 9.6 Hz, 1H), 4.50–4.65 (m, 1H), 3.75–3.91 (m, 1H), 3.63 (q,
J = 6.9 Hz, 1H), 2.90 (dd, J = 16.9, 12.4 Hz, 1H), 2.68 (dd, J =
17.2, 8.1 Hz, 1H), 2.22–2.38 (m, 1H), 1.89–2.05 (m, 1H), 1.47–
1.54 (m, 12H), 1.42 (s, 9H), 1.08 (d, J = 6.6 Hz, 3H), 1.01 (d, J =
6.6 Hz, 3H), 0.98 (d, J = 7.1 Hz, 3H), 0.83 (d, J = 7.1 Hz, 3H).
13C NMR (CDCl3, 100 MHz): d 16.3, 17.2, 18.1, 19.9, 22.1, 27.9,
28.1, 28.3, 30.0, 36.9, 54.0, 56.6, 58.7, 61.7, 80.1, 84.7, 115.7, 126.2,
141.4, 141.4, 156.1, 161.4, 161.7, 161.8, 170.9, 171.5, 174.1. [a]2D0 +6
(c 0.40, MeOH). IR (KBr): 3436(s), 2924(m), 1720(m), 1246(m).
HRMS (EI+): Calcd. for C24H43N3O6 [M + H+] 470.3230. Found
470.3226.
Fmoc-Leu-pyVal-Ala-Ot-Bu (5e)
Following the procedure for 5a (85%): 1H NMR (CDCl3,
400 MHz): d 7.77 (d, J = 7.6 Hz, 2H), 7.61 (d, J = 7.1 Hz, 2H),
7.40 (t, J = 7.6 Hz, 2H), 7.32 (d, J = 7.1 Hz, 2H), 5.52 (s, 2H),
4.20–4.48 (m, 4H), 3.32–3.42 (m, 1H), 3.20 (q, J = 7.1 Hz, 1H),
2.56–2.66 (m, 2H), 2.23–2.34 (m, 1H), 2.00–2.11 (m, 1H), 1.48 (s,
9H), 0.76–1.12 (m, 14H). 13C NMR (CDCl3, 100 MHz): d 14.1,
16.1, 18.8, 19.5, 19.9, 21.6, 22.7, 27.6, 28.0, 29.0, 30.5, 31.8, 39.5,
47.1, 54.4, 56.3, 59.0, 63.3, 67.0, 81.7, 119.9, 125.1, 127.0, 127.6,
141.3, 143.8, 143.9, 156.4, 173.54, 173.46, 175.3. [a]2D0 +16 (c 2.90,
MeOH). IR (KBr): 3435(s), 2967(w), 1723(s), 1697(s), 1452(w),
740(w). HRMS (EI+): Calcd. for C35H47N3O6 [M + H+] 606.3543.
Found 606.3514.
Boc-Leu-pyVal-Ala-Ot-Bu (5b)
Fmoc-Phe-pyVal-Ala-Ot-Bu (5f)
Following the procedure for 5a (90%): 1H NMR (CDCl3,
400 MHz): d 5.38 (dt, J = 10.0, 3.3 Hz, 2H), 4.78–5.09 (m, 1H),
4.32 (dd, J = 7.1, 4.0 Hz, 1H), 3.33 (td, J = 10.2, 7.3 Hz, 1H),
3.15 (q, J = 7.1 Hz, 1H), 2.59 (s, 1H), 2.57 (s, 1H), 2.17–2.30 (m,
1H), 1.63–1.84 (m, 2H), 1.46 (s, 9H), 1.40 (s, 9H), 1.32 (dd, J =
11.1, 4.0 Hz, 1H), 1.24 (d, J = 7.1 Hz, 3H), 1.04 (d, J = 7.1 Hz,
3H), 0.99 (d, J = 6.6 Hz, 3H), 0.93 (d, J = 7.1 Hz, 3H), 0.89 (d,
J = 6.6 Hz, 3H). 13C NMR (CDCl3, 100 MHz): d 18.7, 19.6, 21.0,
21.7, 23.5, 24.8, 27.7, 28.0, 28.2, 39.6, 40.9, 53.3, 54.2, 56.2, 63.0,
79.4, 81.5, 155.5, 173.1, 175.3, 175.8. [a]2D0 −13 (c 6.40, MeOH). IR
(KBr): 3436(s), 2975(m), 1721(s), 1368(m), 1168(m). HRMS (EI+):
Calcd. for C25H45N3O6 [M + H+] 484.3387. Found 484.3383.
Following the procedure for 5a (88%): 1H NMR (CDCl3,
400 MHz): d 7.76 (d, J = 7.6 Hz, 2H), 7.55 (t, J = 7.3 Hz, 2H),
7.40 (t, J = 7.6 Hz, 2H), 7.17–7.34 (m, 7H), 5.71–5.83 (m, 1H),
5.22–5.51 (m, 1H), 3.93–4.37 (m, 4H), 3.06–3.25 (m, 3H), 2.75–
2.91 (m, 1H), 2.43–2.58 (m, 2H), 2.09–2.30 (m, 1H), 1.49–1.53 (m,
9H), 1.26 (d, J = 7.1 Hz, 3H), 1.05 (d, J = 7.1 Hz, 3H), 0.92 (d,
J = 7.1 Hz, 3H). 13C NMR (CDCl3, 100 MHz): d 14.1, 18.7, 19.6,
21.5, 22.7, 27.8, 28.1, 29.0, 31.8, 38.6, 39.4, 47.1, 50.3, 54.2, 55.6,
56.2, 63.2, 65.1, 66.9, 81.5, 119.9, 120.0, 124.7, 125.1, 127.0, 127.6,
128.4, 129.5, 136.0, 141.2, 141.5, 143.8, 143.8, 144.3, 155.5, 173.2,
173.5, 175.3. [a]2D0 +20 (c 1.20, MeOH). IR (KBr): 3436(s), 2968(m),
1727(s), 1368(m), 1222(m). HRMS (EI+): Calcd. for C38H45N3O6
[M + H+] 640.3387. Found 640.3399.
Boc-Phe-pyVal-Ala-Ot-Bu (5c)
Following the procedure for 5a (83%): 1H NMR (CDCl3,
400 MHz): d 7.13–7.34 (m, 5H), 5.61–5.74 (m, 1H), 5.17 (d, J =
8.6 Hz, 1H), 4.19–4.31 (m, 1H), 3.15 (q, J = 7.1 Hz, 2H), 2.74–3.08
(m, 2H), 2.44–2.65 (m, 2H), 2.20–2.28 (m, 1H), 1.48 (s, 9H), 1.25
(d, J = 7.1 Hz, 3H), 1.05 (d, J = 6.6 Hz, 3H), 0.92 (d, J = 7.1 Hz,
3H). 13C NMR (CDCl3, 100 MHz): d 18.7, 19.6, 21.6, 27.8, 28.1,
28.2, 38.5, 39.5, 54.1, 55.2, 56.2, 63.1, 76.7, 77.0, 77.2, 77.3, 79.6,
81.5, 126.8, 128.3, 129.5, 136.3, 155.1, 173.4, 173.9, 175.3. [a]D20
−45 (c 1.20, MeOH). IR (KBr): 3435(s), 1723 (s), 1368(s), 1169(s),
700(w). HRMS (EI+): Calcd. for C28H43N3O6 [M + H+] 518.3230.
Found 518.3244.
Cbz-Val-pyVal-Ala-Ot-Bu (5g)
Following the procedure for 5a (82%): 1H NMR (CDCl3,
400 MHz): d 7.28–7.41 (m, 5H), 5.51 (dd, J = 9.6, 4.0 Hz, 1H),
5.43 (d, J = 9.1 Hz, 1H), 5.10 (s, 2H), 4.23–4.32 (m, 1H), 3.26–3.37
(m, 1H), 3.17 (q, J = 6.7 Hz, 1H), 2.51–2.65 (m, 2H), 2.18–2.33
(m, 1H), 1.95–2.09 (m, 1H), 1.47 (s, 9H), 1.45 (s, 9H), 1.25 (d, J =
7.1 Hz, 3H), 1.06 (d, J = 7.1 Hz, 3H), 1.02 (d, J = 6.6 Hz, 3H), 0.92
(d, J = 7.1 Hz, 3H), 0.80 (d, J = 7.1 Hz, 3H). 13C NMR (CDCl3,
100 MHz): d 16.0, 18.8, 19.6, 20.0, 21.6, 27.6, 28.0, 30.4, 39.6,
54.3, 56.2, 59.0, 63.3, 66.9, 81.5, 128.1, 128.5, 136.4, 156.2, 173.3,
173.5, 175.3. [a]2D0 +17 (c 2.50, MeOH). IR (KBr): 3435(s), 2968(m),
1726(s), 1369(m), 1151(m). HRMS (EI+): Calcd. for C27H41N3O6
[M + H+] 504.3074. Found 504.3086.
Fmoc-Val-pyVal-Ala-Ot-Bu (5d)
Following the procedure for 5a (83%): 1H NMR (CDCl3,
400 MHz): d 7.77 (d, J = 7.6 Hz, 2H), 7.61 (d, J = 7.6 Hz, 2H),
7.40 (t, J = 7.3 Hz, 2H), 7.32 (t, J = 7.3 Hz, 2H), 5.52 (dt, J = 10.0,
2.8 Hz, 1H), 5.15–5.46 (m, 1H), 4.08–4.46 (m, 5H), 3.76 (s, 1H),
Cbz-Leu-pyVal-Ala-Ot-Bu (5h)
Following the procedure for 5a (83%): 1H NMR (CDCl3,
400 MHz): d 7.28–7.39 (m, 5H), 5.48 (dt, J = 10.0, 2.8 Hz, 1H),
3492 | Org. Biomol. Chem., 2007, 5, 3486–3494
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5.33 (d, J = 9.1 Hz, 1H), 5.10 (s, 2H), 4.32 (dd, J = 7.1, 4.0 Hz,
1H), 3.29–3.39 (m, 1H), 3.16 (q, J = 6.7 Hz, 1H), 2.60 (d, J =
10.1 Hz, 2H), 2.19–2.31 (m, 1H), 1.72–1.83 (m, 1H), 1.47 (s, 9H),
1.25 (d, J = 7.1 Hz, 3H), 1.03 (dd, J = 11.9, 6.8 Hz, 6H), 0.86–0.95
(m, 6H). 13C NMR (CDCl3, 100 MHz): d 18.7, 19.6, 21.0, 21.6,
23.5, 24.8, 27.8, 28.0, 39.5, 41.3, 53.8, 54.3, 56.2, 63.1, 66.8, 81.5,
128.0, 128.4, 136.4, 155.9, 173.2, 175.0, 175.3. [a]2D0 −8 (c 1.60,
MeOH). IR (KBr): 3436(s), 1487(s), 1375(s), 1069(w), 846(w).
HRMS (EI+): Calcd. for C28H43N3O6 [M + H+] 518.3230. Found
518.3246.
with DMF (3 × 5 mL), treated with 20% piperidine/DMF (3 ×
5 min) and washed with DMF (3 × 5 mL). To the resin in DMF
(4 mL) was added Fmoc-Phe-OPfp (150 mg, 0.27 mmol) and N,N-
diisopropylethylamine (0.01 mL, 0.06 mmol), and the mixture was
shaken for 1 h at room temperature. The resin was washed with
DMF (3 × 5 mL) and treated with 2% TFA in DCM. The resin
was washed with DCM (3 × 5 mL). The combined DCM fractions
were evaporated and purified by preparative HPLC (MeCN–H2O)
to afford the pure product as a white solid (23 mg, 26 mmol, 26%).
1H NMR (MeOD, 300 MHz): d 0.91 (d, J = 7.16 Hz, 3H), 1.08
(d, J = 6.78 Hz, 3H), 1.18 (s, 9H), 1.43 (d, J = 7.16 Hz, 3H), 2.16
(m, 1H), 2.57 (dd, J = 17.33, 8.29 Hz, 1H), 2.77 (m, 3H), 3.05
(m, 2H), 3.62 (m, 3H), 3.87 (dd, J = 9.42, 4.14 Hz, 1H), 4.26 (m,
5H), 4.60 (t, J = 3.58 Hz, 1H), 5.88 (m, 1H), 7.26 (m, 14H), 7.55
(dd, J = 7.16, 2.26 Hz, 2H), 7.77 (d, J = 7.54 Hz, 2H), 8.35 (d,
J = 7.54 Hz, 1H). 13C NMR (MeOD, 75 MHz): d 18.02, 18.83,
22.74, 27.39, 28.31, 37.83, 38.17, 38.70, 47.95, 54.03, 54.25, 55.48,
57.16, 57.23, 62.48, 63.10, 67.79, 74.30, 120.59, 125.94, 126.03,
127.37, 127.64, 127.87, 128.47, 129.09, 130.09, 130.23, 137.63,
138.15, 142.21, 144.84, 144.93, 157.72, 172.66, 173.51, 173.65,
Cbz-Phe-pyVal-Ala-Ot-Bu (5i)
Following the procedure for 5a (89%): 1H NMR (CDCl3,
400 MHz): d 7.08–7.37 (m, 10H), 5.70–5.85 (m, 1H), 5.41 (d,
J = 8.6 Hz, 1H), 5.00 (s, 2H), 4.14–4.31 (m, 1H), 3.12–3.22 (m,
2H), 3.00–3.10 (m, 1H), 2.83 (dd, J = 13.1, 8.1 Hz, 1H), 2.41–2.57
(m, 2H), 2.11–2.28 (m, 1H), 1.48 (s, 9H), 1.25 (d, J = 6.6 Hz,
3H), 0.95–1.08 (m, 3H), 0.67–0.94 (m, 3H). 13C NMR (CDCl3,
100 MHz): d 18.7, 19.6, 21.5, 27.8, 28.1, 38.6, 39.4, 54.1, 55.6,
56.2, 63.2, 66.7, 81.5, 126.9, 128.0, 128.4, 128.4, 129.4, 136.0,
136.3, 155.5, 173.2, 173.5, 175.2. [a]2D0 +41 (c 2.20, MeOH). IR
(KBr): 3378(s), 2973(m), 1725(s), 1669(s), 1358(m). HRMS (EI+):
Calcd. for C31H41N3O6 [M + H+] 552.3074. Found 552.3068.
◦
173.94. Mp 149–152 C. HRMS (EI+): Calcd. for C50H60N5O9
[M + H+] 874.4386. Found 874.4380.
Acknowledgements
Peptide stability
This work was supported by the Lundbeck Foundation, Cor-
porate Research Affairs at Novo Nordisk A/S, Torkil Holm’s
Foundation, The Danish Natural Science Research Council, The
Augustinus Foundation, and Ib Henriksen’s Foundation.
Peptide or peptide analogue (5 mg) was added to◦a temperature-
equilibriated solution of 50% human plasma (37 C, 10 mL, the
lyophilized material from SigmaAldrich was dissolved in Millipore
water) was added and the mixture stirred vigorously at 37 ◦C. At
appropriate times samples were taken and analyzed by HPLC.
References
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Fmoc-Phe-pyVal-Ala-OH (6a)
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A
solution of Fmoc-Phe-pyVal-Ala-Ot-Bu (5f) (23 mg,
2004, 5, 29.
0.036 mmol) in TFA (2 mL) was stirred at RT for 10 h. Evaporation
of the mixture provided the pure product as a clear oil (20 mg,
0.034 mmol, 95%). H NMR (MeOD, 300 MHz): d 0.92 (d, J =
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1
6.78 Hz, 3H), 1.11 (d, J = 6.78 Hz, 3H), 1.60 (d, J = 7.16 Hz,
3H), 2.13 (s, 1H), 2.80 (m, 3H), 3.09 (dd, J = 13.56, 4.90 Hz, 1H),
4.12 (m, 5H), 4.60 (dd, J = 6.78, 4.14 Hz, 1H), 5.65 (dd, J = 9.80,
4.52 Hz, 1H), 7.28 (m, 9H), 7.54 (d, J = 7.16 Hz, 2H), 7.73 (d, J =
7.54 Hz, 2H). 13C NMR (MeOD, 75 MHz): d 14.9, 18.8, 23.2, 28.6,
35.7, 38.1, 52.6, 56.4, 57.6, 62.4, 67.9, 120.8, 126.2, 127.8, 128.0,
128.1, 128.7, 129.3, 130.5, 138.3, 142.4, 145.0, 158.3, 171.9, 172.2,
175.1 HRMS (EI+): Calcd. for C34H38N3O6 [M + H+] 584.2761.
Found 584.2745.
Fmoc-Phe-Phe-pyVal-Ala-Ser(t-Bu)-OH (7a)
To
a solution of Fmoc-Phe-pyVal-Ala-OH (6a) (58 mg,
0.10 mmol) in DMF (2 mL) was added bromotris(pyrrolidino)-
phosphonium hexafluorophosphate (46 mg, 0.10 mmol),
N-hydroxybenzotriazole (14 mg, 0.10 mmol) and N,N-
diisopropylethylamine (0.03 mL, 0.18 mmol). The mixture was
stirred for 5 min at room temperature, added to a suspension of
H-Ser(t-Bu)-2-ClTrt resin (1.1 mmol g−1, 120 mg) in DMF (2 mL)
and shaken for 1 h at room temperature. The resin was washed
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